Dr Abdou Salam Ouedraogo (1957–2000)

Home , Afzelia xylocarpa, Anisoptera costata, Astronium graveolens, Buchanania, Calophyllum brasiliense, Desiccation tolerance

DEDICATION iii

This book is dedicated to the fond memory of our late colleague Dr Abdou Salam Ouédraogo (1957–2000), who died in the Kenya Airways crash on the night of Sunday 30 January 2000. A citizen of Burkina Faso, Abdou was well known throughout Africa and the scientific community as a distinguished scientist and leader in his field. In 1995 he was awarded a PhD in conservation biology by the University of Wageningen, the Netherlands. He was the founding director of the Forest Tree Seed Centre in Ouagadougou and co-ordinator of the regional forest genetic resources programme at the Food and Agriculture Organization/Permanent Interstate Committee for Drought Control in the Sahel (FAO/CILSS). During his tragically curtailed career, he was an active member of several panels and committees for international organisations. He served as a member of the FAO Panel of Experts on forest genetic resources, representing Africa. He was part of the International Tropical Timber Organization (ITTO) Panel of Experts advizing on forest genetic resources while also a member of the programme committees of the World Agroforestry Centre (ICRAF) and the forestry department of the Centre de co- opération Internationale en Recherche Agronomique pour le Développement (CIRAD–Forêt). He fulfilled leadership roles in key conservation bodies as the deputy leader of the Research Group on Biodiversity (for Africa) of the International Union of Forestry Research Organization (IUFRO) and as chair of the Species Survival Commission African Tree Specialist Group, part of the International Union on the Conservation of Nature and Natural Resources (IUCN). He was a member of external review panels for key international forestry organizations such as the Forest Seed Centre of the Danish International Development Agency (DANIDA) in 1996, and IUFRO and CIRAD–Forêt (both in 1999). Abdou joined the International Plant Genetic Resources Institute (IPGRI) in 1993 as Senior Scientist, Forest Genetic Resources, leading IPGRI’s global project on forest genetic resources. These years of ‘hands-on’ experience gave him a thorough understanding of the complex realities and needs of the developing world in the area of plant genetic resources, particularly in sub-Saharan Africa. It was in iv STORAGE BIOLOGY OF TROPICAL TREE SEEDS this capacity that he and others conceived the project ‘Conservation, management and sustainable use of forest genetic resources with reference to Brazil and Argentina’, presented in this book. He also gained an excellent knowledge of the modus operandi of international organizations, including the centres of the Consultative Group on International Agricultural Research (CGIAR), the FAO, IUFRO, CIRAD-Forêt and DANIDA, in addition to experience in leadership and management. In October 1999 he was promoted to the position of Regional Director for sub-Saharan Africa based at IPGRI’s office in Nairobi, Kenya. During the short time that he was in this position, he demonstrated unique vision, leadership, an engaging personality and good humour. We will remember him particularly for his great ability to work with people and instil team spirit, and for his vitality and his positive attitude towards life, even when faced with enormous challenges. He was a unique individual, loved by everyone who knew him. His life was a continuous endeavour to make the world a better place.

May God rest his soul. INTRODUCTION 1

General introduction

1 3 Mohammad E. Dulloo , Dorthe Jøker2, Kirsten A. Thomsen and Weber A. N. Amaral1

¹International Plant Genetic Resources Institute, via dei Tre Denari 472/a, 00057 Maccarese, Rome, Italy 2Forest & Landscape Denmark, Hørsholm Kongevg 11, DK-2790 Hørsholm, Denmark 3The State Forest Tree Improvement Station, Krogerupvej 21, 3050 Humlebaek, Denmark

Biodiversity offers both challenges and opportunities to humankind. Challenges in dealing with this complexity and in developing management regimes for species and ecosystems where there are information gaps. Opportunities in deploying these biological resources to sustain local communities and farmers. Forests and trees play a particularly important role for peoples’ livelihoods, in providing goods and services and contributing to local and regional development and environmental protection. In the tropics, tree species’ diversity is huge. The rate of loss of this diversity is, however, extremely high due to deforestation, ecosystem fragmentation and overexploitation. Despite such diversity only a handful of species are employed in forest restoration, agroforestry systems and planting programmes. In addition, commercially oriented planting schemes are often dominated by fast growing, frequently exotic species to the detriment of well-adapted local or indigenous tree species, thereby causing loss of diversity and, thus, adding a further constraint to the deployment of indigenous species. Insufficient baseline information about the potential of indigenous species and the availability of seeds and seedlings, is a major constraints for the deployment of locally adapted tree species. Access to seeds and seedlings are in general associated with seed handling and storage problems, which limit the use of many potentially high value indigenous species in tree planting and conservation programmes. Little is known about the seed physiology of most tropical forest tree species. Furthermore, a large proportion of tropical forest tree species produces recalcitrant seeds, which are difficult to collect, process and store. Many of these seeds are sensitive to both desiccation and low temperatures and, consequently, do not tolerate being dried to a low moisture content 2 STORAGE BIOLOGY OF TROPICAL TREE SEEDS and cannot be stored at low temperatures for long periods of time. This is a major problem for humid tropical forest tree species, where more than 70% of them have seeds with recalcitrant or intermediate seed storage behaviour. Understanding seed drying and storage behaviour can help ensure a better handling and storage of tropical tree seeds, and hence the availability of these resources for livelihoods. The International Plant Genetic Resources Institute (IPGRI) and Danida Forest Seed Centre (DFSC) (now Forest & Landscape Denmark) have carried out a joint project on the handling and storage of recalcitrant and intermediate tropical forest tree seeds, which was implemented in two phases (1996–1998 and 2000–2002). The project aimed to: a) strengthen the capability of forest tree seed laboratories/institutes in developing countries through research and technology transfer; b) develop effective techniques for seed handling, including collecting, storage, germination and testing; and c) improve the basic scientific knowledge on recalcitrant and intermediate storage behaviour of tropical tree seeds. During the first phase of the project, an informal network of researchers on recalcitrant seeds was established, together with a standard screening protocol and a series of appropriate handling methods. Research capacity was enhanced through training workshops, and participants were trained in data gathering and seed survival when dried and stored at different temperatures. In the follow-up phase (2000–2002), initial efforts were consolidated by enhancing the use and conservation of indigenous tropical forest tree species through the development of optimal seed handling and storage procedures, regional training workshops and strengthening the network. The project was concluded at a final workshop held at Chania, Crete in Sept 2002, and suggestions were made by the project partners for future actions and activities beyond the project scope.

Contributing partners and species

The project involved 15 countries in Africa, America and Asia, and several other replicating institutions in Europe and Australia (see list of Contributors—Appendix 5). The project gathered contributions from 13 Forest Tree Seed Institutions across the three continents, four universities and three research institutes in Europe and Africa. The species to be studied were chosen according to the following criteria: x high socioeconomic importance; x propagation of species mainly via seeds; x nondormant seeds ; INTRODUCTION 3

x availability of distinct provenance or seed sources; x yearly production of seeds in large quantities. Collecting partners and countries of origin used these criteria to (a) draw up a list of priority species in need of immediate action and, (b) seek common patterns of seed behaviour among different tree species. The end product has been the generation of a wealth of data and information on 52 tropical tree species that are the subject of this book.

The screening protocol

At an initial workshop in 1995 in Humlebaek, Denmark, the project partners developed a screening protocol to determine seed tolerance of desiccation. This protocol was then amended and adopted by all partners as a standard methodology, at a workshop in 1996 in Durban, South Africa. The protocol, shown in Appendix 1, contains precise instructions on how to determine minimum safe moisture content and optimal storage conditions to assure its replicability among partners and multiple trials. All procedures are described, from sampling and collection to the exchange of seed materials... To improve the quality of the work and at the same time promote collaboration between institutes, partners were encouraged, using the same standard protocol, to screen seed lots in replication with another partner institution. The results and reports on the species investigated are presented in the chapters of this book.

Capacity building

The initial workshops (Humlebaek 1995 and Durban 1996) planned the project activities and collaboration with partners. Technical information and back up visits were provided by DFSC, over the course of the project. Three further regional training workshops were held to strengthen technical expertise of the participating institutions. The Kenya Forestry Research Institute (KEFRI) in Kenya hosted the workshop for African participants and their replicating partners in March 2000. The Latin American workshop was held at CATIE, Turrialba, Costa Rica in May 2000, while the ASEAN Forest Tree Seed Centre, Thailand was the venue for the Asian regional training workshop held in April 2001. During these workshops participants were trained to use the screening protocol and had the opportunity to exchange information and experience at a regional level. Several other 4 STORAGE BIOLOGY OF TROPICAL TREE SEEDS forest tree seed institutions, which were not formally part of the project, also benefited from these activities. The aforementioned final workshop in Chania, Crete in September 2002, made recommendations with respect to, among others, capacity building that are presented in Appendix 2.

Research through networking

One of the outstanding products of this project was the establishment of an international network of scientists from tropical countries, working on tree seeds with supposed recalcitrant or intermediate behaviour through collaborative screening activities. In this way, the project generated substantial data on optimal storage moisture content and temperature, and desiccation tolerance of 52 important forest tree species from 25 families (see photographs of a selection of species in Appendix 3). Seed handling and moisture content at harvest, fruit and seed sizes, germination temperature and speed, and desiccation and storage results were generated. DFSC, IPGRI and the partners, shared results and findings through short technical articles, and produced jointly 10 issues of the project newsletter during the development of the project. These newsletters proved to be effective in strengthening collaboration between members, and helped to enlarge the network beyond project partners. Other scientists working with tropical recalcitrant seeds also provided short communications. In total, the project newsletters reached more than 650 recipients worldwide and were published electronically (at www.dfsc.dk). In this project, tree seed tolerance of desiccation and storage was explored for priority species in an attempt to understand viability loss and improve handling and storage. Seeds from different sources were used as experimental materials for this purpose. Different combinations of water contents and storage temperatures were employed to identify optimum storage conditions. Furthermore, North–South and South–South collaboration among project participants was used to replicate experiments, an important innovative aspect and strength of the project. To facilitate comprehensive reporting on seeds of a specific species, the authors and editors attempted to avoid repetition and, therefore, the results of collaborative investigations for the same species were combined in a single chapter. In Part I of this publication the project advisers review INTRODUCTION 5 the findings and highlight the key technical breakthroughs of this research project. The 18 chapters of Part I deal with case studies of 25 species investigated in Africa from Burkina Faso, Kenya, Malawi, Senegal, South Africa and Tanzania. Part II gives details of the 8 Asian contributions on 20 species from China, India, Malaysia, Thailand and Vietnam. Part III deals with Latin America with 9 contributions on 10 species from Bolivia, Brazil, Colombia and Costa Rica.

Conclusion

The project successfully achieved its objectives in dealing with the screening by a wide range of institutes with varying facilities and capacities of several important species. Although much remains to be done before these selected tree species can be comprehensively utilized in multiple forest planting schemes in developing countries, this project does show the way forward as it has resulted in a greater understanding of how to handle forest seeds with intermediate and/or recalcitrant behaviour and more importantly how to adapt and adjust laboratory protocols to a large number of species In addition, the participating institutions are now more capable of screening indigenous seed species; such knowledge will contribute to greater use of diversity in the afforestation programmes of their countries. It is hoped that the recommendations for the next steps will be developed further and supported (see Appendix 2). We hope that the results published in this book, and more importantly the synergies created by this project could be of value to a greater number of users in the field of tree seed research and conservation, and could contribute to the supply of forest genetic resources for restoration and reforestation projects and programmes in tropical developing countries worldwide. It is hoped that the information could be of considerable use to beginners and more experienced stakeholders in tree seed handling, and that that it will result in a greater choice of indigenous tree species for the sustainable use of farmers and local communities. 6 STORAGE BIOLOGY OF TROPICAL TREE SEEDS PREFACE ix

Preface

Forest trees are an important element of the landscape and many have great economic and cultural value, providing and supporting the livelihoods of millions of people, especially in the tropics. Tropical forests are rich in species diversity, but little is known about the biology of many of the tropical forest tree species, and, in particular, information on the seed biology is very scanty. Until recently, indigenous trees were seldom used in forest replanting, preference being given to fast growing exotic species. A shortage of good quality indigenous forest tree seeds has contributed to this situation. However, studies on tropical forest tree seeds in general also remain more complex compared to those on annual crops, as a result of dormancy problems and large variations in seed longevity, compounding the handling problems. Acknowledging the important role that tree seeds play in the developing world, the International Plant Genetic Resources Institute (IPGRI) together with Danida Forest Seed Centre (DFSC) (now Forest & Landscape Denmark) initiated a project in 1995 to generate knowledge on the physiology of tropical tree seeds with particular emphasis on priority forest species. This book is the culmination of a six-year project, which aims to improve handling and storage of forest tree seeds. Participants from Africa, Asia and South/Central America were trained, and gained sufficient experience during the execution of the project to ensure the creation of novel, detailed data sets to share with a global audience. A key feature of this project was its collaborative nature, such that investigations of most seed lots were replicated, usually in at least two countries, using the same standard protocols. These joint endeavours were communicated in the project newsletters, which reached many stakeholders worldwide. The end product is this book on the storability and management of seeds of 52 tropical forest tree species. It is noticeable that trends in the forestry sector are changing slowly, as more sustainable forest management is practised, and tree planting is becoming more diversified. A much larger number of economic, local tree species are increasingly being used, reflecting a change in emphasis from exotic species, such as from the genera Eucalyptus and Pinus, towards indigenous species. We have no doubt that this book is a contribution that will further boost the use of local tree species in forest planting programmes all over the tropics through proper x STORAGE BIOLOGY OF TROPICAL TREE SEEDS handling of the seeds and promote tropical tree seed research and conservation. The project would not have been realized and completed without the commitment and contribution of a number of people. A special tribute must be paid to late Abdou Salam Ouedraogo, who together with Dr Karen Poulsen pioneered the development of this project. The project has been unique for its investigative research on tropical tree seeds and in gathering tree seed institutions from Africa, Asia, Latin America and Europe around one research programme and using the same protocols and methods. We dedicate this book to the memory of Dr Ouedraogo. Thanks are also due to all the authors of the various chapters for their hard work and contribution. We are grateful to Dr Moctar Sacandé who spent considerable time reviewing the contributions and bringing them up to a sound scientific standard. We are thankful to Kirsten A. Thomsen and Dorthe Jøker, who have played a crucial role in providing technical support to all the partners and who have been editors of the project newsletter. We are grateful to IPGRI’s forest genetic resources project coordinator Dr Weber Amaral for his continuous support and Dr Ehsan Dulloo who ensured the management of the project. We thank the key resource persons of the project, namely, Profs. Patricia Berjak (University of Natal, South Africa), Hugh W. Pritchard (Royal Botanic Gardens Kew) and Erik N. Eriksen (Danish Royal Veterinary Agricultural University) for their support and also for acting as replicating partners for some of the countries. Finally thanks go to the Danida, for the important support it provided in funding the two phases of this six-year project.

Jan Engels Bjerne Ditlevsen Group Director Director Genetic Resources Science and Danida Forest Seed Centre Technology Humlebaek, Denmark IPGRI, Rome, Italy INTRODUCTION 1

General introduction

1 3 Mohammad E. Dulloo , Dorthe Jøker2, Kirsten A. Thomsen and Weber A. N. Amaral1

Contributing partners and species

The screening protocol

Capacity building

Research through networking

Conclusion

Burkina Faso (CNSF)

Kenya (KEFRI)

Malawi (FRIM)

Senegal (ISRA)

South Africa (UN)

Tanzania (TTSA) 8 STORAGE BIOLOGY OF TROPICAL TREE SEEDS AFRICA 9

Storage behaviour of Khaya senegalensis seeds from Burkina Faso

Christiane S. Gaméné1 and Erik N. Eriksen2

1Centre National de Semences Forestières 01 BP 2682 Ouagadougou 01, Burkina Faso 2The Royal Veterinary and Agricultural University, Horticulture, Agrovej 10, Taastrup, 2630 Denmark

Abstract

Seeds of Khaya senegalensis with initial 3% moisture content collected from Burkina Faso germinated 100% at 25°C. Seeds incubated at different temperatures did not germinate at 10°C or below, while maximum germination occurred at 15, 20 and 25°C, revealing that the threshold temperature for germination of K. senegalensis seeds is between 10 and 15°C. The optimum and fastest germination temperature was at 25°C with a mean germination time of 8.7 days. The sowing positions of seeds (‘on the side’ or ‘on the edge’) did not affect germination capacity. Seeds maintained high levels of viability after storage at –18, 4 and 25°C for 26 months.

Introduction

Khaya senegalensis (Desr.) A.Juss., the dry-zone mahogany, originates from tropical Africa along a belt approximately parallel to the equator from Senegal, Gambia and Mali in the west through Nigeria and Cameroon to northern Uganda and southern Sudan in the east. It has been successfully planted in Southeast Asia, Australia and a few other places in the tropics. It prefers deep alluvial soils, and is often found growing near rivers and in hollow borders, in areas with annual rainfall between 700 and 1300 mm and a dry season of 4–7 months (Sosef et al. 1998). The tree can grow up to 35 m high. The bark is brownish to grey and splintery; the slash is red and exudes red sap. The leaves are 12–25 cm long, feather-like with 2–6 pairs of glabrous leaflets (see Fig. 1). Like other species of the Meliaceae family, K. senegalensis produces hard, valuable timber that is widely used on a commercial scale, particularly in West Africa. The leaves serve as 10 STORAGE BIOLOGY OF TROPICAL TREE SEEDS fodder. Oil can be extracted from the seeds and used for cooking. The bark is used in traditional medicine (FAO 1986). The ashes are used as a protective agent for storing millet grains (von Maydell 1986). The flowers are small (5 mm). The fruits are globular capsules, with a diameter of 5–10 cm, which split into four valves at maturity. Each of the four cells contains 6–18 brown, flat seeds with narrow wings. Flowering occurs shortly before or early in the rainy season and insects carry out pollination. The fruits mature when the colour changes from grey to black, and they remain on the tree during most of the dry season. The species is becoming vulnerable in many countries due to overexploitation and is now listed in the IUCN Red List of Threatened Species (IUCN 2002). In Burkina Faso, an ex situ conservation programme has therefore been set in motion (Tolkamp et al. 1992) to support the planting programmes and to sustain the long-term conservation of this species. However, very little is known about the biology of K. senegalensis seeds. According to von Maydell (1986), the seeds must be sown fresh after harvest. Similarly, two other species in the Khaya genus, i.e. K. ivorensis and K. anthotheca have been reported to have short lived seeds (Mbuya et al. 1994; Baskin and Baskin 1998). The results of our investigations on the optimal storage conditions of K. senegalensis seeds from Burkina Faso are presented in this report.

Materials and methods

Seed collection

Dehiscent fruits of K. senegalensis were collected from at least 20 trees in the Forêt Classée du Barrage, Ouagadougou, on 5–6 Mar 1997 and 16 Apr 1998, in the dry season. A quantity of about 13 kg of seeds was prepared from each fruit collection. Hundred seeds were sampled for initial tests, and the remaining seeds were coated with fungicides (1 g Benomyl and 1 g Thiram per kilogramme of seeds). Half of the seeds from the 1997 collection were dispatched to Copenhagen, where they arrived the same week on 10 Mar 1997.

Initial tests

Weight and moisture content determinations were carried out on 100 individual seeds. Moisture contents were determined gravimetrically by AFRICA 11 weighing before and after drying seeds in an oven at 103°C for 17 h. Moisture content was calculated as a percentage of fresh weight. Germination capacity was determined using four replicates of 25 seeds in experiments in both Burkina Faso and Denmark. In Burkina Faso, the seeds were sown in sand and incubated at 30°C in the laboratory, whilst in Denmark the seeds were sown in vermiculite and incubated in a germination cabinet at 25°C with 10 h light/14 h dark. To determine the optimum temperature for germination, samples of seeds were incubated at five different temperatures of 5, 10, 15, 20 and 25°C for five weeks. Seeds were placed ‘on the edge’ or ‘on the side’ during the germination test at 25°C to evaluate the possible effect of the two sowing positions on germination capacity and rate, because of the flat shape of the seeds.

Storage trials

Storage experiments were performed in Burkina Faso, using seed samples that were stored at ambient temperature in a cupboard at ca. 25°C, in a cold room at ca. 4°C and in a freezer at –18°C. Seeds were packed in sealed aluminium bags. Moisture content and germination were tested every two months for 22 months. In Denmark, samples of seeds were stored at –18, 5 and 25°C for 26 months.

Results Initial tests

Mean weights, initial moisture contents and germination of K. senegalensis seeds were determined. The seed coat was much lighter than the embryo (Table 1). The whole seed weights and their moisture contents were comparable for the two collections in different years. All seeds germinated t95% in Burkina Faso as well as in Denmark.

Germination trials

Samples of K. senegalensis seeds were incubated to determine the optimum temperature for germination. These seeds did not germinate at or below 10°C (Fig. 2). The nongerminated seeds at 5 and 10°C were cut open for examination after the test was ended. They were still fresh after the 28 days of incubation, indicating that they were still viable. However, 12 STORAGE BIOLOGY OF TROPICAL TREE SEEDS maximum germination occurred at 15, 20 and 25°C, showing no significant differences between these conditions (P<0.05). These results revealed that the threshold temperature for germination of K. senegalensis seeds was between 10 and 15°C. It was observed that seeds took longer to complete germination at 15°C than at 25°C. The mean time to complete germination was 37.8 days for seeds incubated at 15°C, 13.1 days for those incubated at 20°C and 8.7 days for seeds at 25°C.

Table 1. Mean weights, initial moisture contents and germination (G%) of K. senegalensis seeds, and locations where measurements were made Embryos Seed coat Whole seeds G (%) Weight MC Weight MC (%) Weight MC (%) (g) (%) (g) (g) Burkina — — — — 0.21r 2.57r 98 (1997) 0.04 0.02 Denmark 0.15r 2.52r 0.06r0. 4.86r 0.20r 3.22r 96 (1997) 0.04 0.97 01 0.97 0.05 1.36 1 Burkina — — — — 0.20r 3.41r 95 (1998) 0.04 0 .06

40 100

80 30

25 60

40 Germination (%) 15 Germination time (days) 10 20

0 0 0 5 10 15 20 25 30 Germination temperature (°) Figure 2. Effect of temperature on germination of K. senegalensis seeds. The rates of germination at 15, 20 and 25°C are also shown. AFRICA 13

The effect of the sowing position of seeds was tested at 25°C. Seeds germinated similarly at both positions ‘on the side’ or ‘on the edge’. There was no effect of the two different positions on the germination capacity or the rate of germination of these seeds.

Storage trials

Table 2 shows the experimental results of K. senegalensis seeds stored at three temperatures in Burkina Faso. Seeds of the first collection in 1997 were stored up to 22 months and all seeds germinated more than 85% after storage. There was no significant difference (P<0.05) between germination capacity after storage under these three conditions. The seed moisture contents varied slightly, although on average they maintained ca. 3% MC over the storage period. For material stored in Denmark, germination was 99, 96 and 98% after 26 months at ca. 3% MC and –18, 5 and 25°C, respectively (data not shown).

Table 2. Effect of storage temperature on viability (germination %) of K. senegalensis seeds collected in Burkina Faso and tested at CNSF in 1997 Storage Freezer (–18°C) Cold room (4°C) Ambient (25°C) duration G (%) MC (%) G (%) MC (%) G (%) MC (%) (months) 1997 98 2.6 98 2.6 98 2.6 0 95 2.5 95 2.5 95 2.5 2 99 4.0 99 4.1 96 3.6 4 98 3.9 100 4.1 97 4.5 6 99 5.9 94 5.7 99 5.8 8 — — 98 3.9 98 3.0 10 98 3.8 97 3.9 98 4.1 12 96 3.3 94 3.6 95 3.3 14 97 3.8 99 4.2 99 3.9 16 96 4.0 98 4.0 96 3.6 18 97 4.0 97 4.3 95 3.8 20 98 4.5 97 5.2 97 4.8 22 98 4.6 85 4.8 91 4.2 1998 95 2.7 95 2.7 95 2.7 0 85 3.4 85 3.4 85 3.4 3 98 4.1 99 3.4 99 4.3 6 90 3.5 91 4.9 97 3.3 9 100 4.8 72 6.0 97 4.8 14 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Discussion

The seeds of Khaya senegalensis tested in this study had low moisture contents at harvest and initially germinated to more than 95%. They were tolerant to further drying to 2.5% MC, maintaining their initial viability. They did not lose viability at any of the tested temperatures below and above 0°C, after storage for up to 22 months (Table 2), and their moisture content remained constant at approx. 3%. Seeds that tolerate desiccation would be expected to store easily at low temperatures. Thus, it was not surprizing that K. senegalensis seeds maintained viability after storage at both the low moisture contents and low temperatures. While dry seeds (3% MC) were apparently not sensitive to low temperatures (–18 and 5°C) during storage, hydrated seeds did not tolerate chilling temperatures, being unable to germinate at d10°C. Maximum germination occurred when they were incubated at t15°C, indicating that the threshold germination temperature lies between 10 and 15°C. However, seeds took 37.8 days to complete germination at 15°C, compared to 8.7 days at 25°C (Fig. 1). A similar phenomenon has been found with other tropical species like neem (Sacandé et al. 2001). The hypothesis is that the cause of this chilling sensitivity may lie in the high intrinsic melting transition temperature (Tm—ranging from 10–15°C) of membranes in these tropical seeds, as is also the case for vegetative tissues of tropical plants (Crowe et al. 1989; Sacandé et al. 2001). This implies that at these temperatures or below, membranes tend to be in the rigid gel phase, and as a result germination capacity decreases or even annuls. The optimum and fastest germination was obtained at 25°C. The results therefore indicate orthodox storage behaviour for Khaya senegalensis, its seeds tolerating desiccation to low moisture content and being able to maintain viability for long periods in the dry state.

References

Baskin, C.C. and J.M. Baskin. 1998. Seeds: Ecology, Biogeography and Evolution of Dormancy and Germination. Academic Press, New York, Pp. 666. Crowe, J.H., F.A. Hoekstra, L.M. Crowe, T.J. Anchordoguy and E. Drobnis. 1989. Lipid phase transitions measured in intact cells with Fourier transform infrared spectroscopy. Cryobiology 26:76–85. FAO. 1986. Some Medicinal Forest Plants of Africa and Latin America. FAO Forestry Paper No. 67. FAO, Rome, Italy. IUCN. 2002. 2002 IUCN Red List of Threatened Species [also at http://www.redlist.org]. AFRICA 15 von Maydell, H.J. 1986. Trees and Shrubs of the Sahel. GTZ, GmbH, Eichsborn, RoEdorf. Mbuya, L.P., H.P. Msanga, C.K. Ruffo, A. Birnie and B. Tengnäs. 1994. Useful trees and shrubs for Tanzania. Identification, propagation and management for agricultural and pastoral communities. Technical Handbook No 6. Regional Soil Conservation Unit, SIDA. Sacandé, M., E.A. Golovina, A.C. Van Aelst and F.A. Hoekstra. 2001. Viability loss of neem (Azadirachta indica) seeds associated with membrane phase behaviour. J. Exp. Bot. 52:919–931. Sosef, M.S.M, L.T. Hong and S. Prawirohatmodjo. 1998. Plant Resources of South-East Asia No 5(3) – Timber Trees: Lesser-known Timbers. Bachuys Publishers, Leiden. Tolkamp, G.W., R. Balima, B. Belem and L.G. Ouedraogo. 1992. Evaluation d´une premiére sélection de Khaya senegalensis (cailcédrat) au Burkina Faso. Rapport No. 2. Centre National de Semences Forestieres. 16 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Desiccation and storage of Kigelia africana, Lophira lanceolata, Parinari curatellifolia and Zanthoxylum zanthoxyloides seeds from Burkina Faso

Mathurin D. Sanon1, Christiane S. Gaméné1, Moctar Sacandé2 and Oblé Neya1

1Centre National de Semences Forestières 01 BP 2682 Ouagadougou 01 Route de Kaya, Burkina Faso. E-mail: [email protected] 2Current address: Seed conservation Department, Royal Botanic Gardens, Kew, Ardingly, Wakehurst Place, RH17 6 NT, UK

Abstract

Four native species from Burkina Faso were studied for desiccation tolerance and storage behaviour of their seeds. The results showed that Kigelia africana seeds tolerated drying to about 7% MC, maintaining c. 70% germination after desiccation and after storage for 18 months at 4 to 25°C. Seeds of Lophira lanceolata initially germinated 49%, but lost viability after drying to 3% MC, and less than 5% germinated after three months storage. No seed of Parinari curatellifolia germinated either fresh or dried. Zanthoxylum zanthoxyloides seeds germinated poorly reaching only 2% initially, but maintained this viability when dried to 3% MC. The low germination of P. curatellifolia and Z. zanthoxyloides seeds might be due to seed immaturity or dormancy problems, indicating the need to investigate stages of maturity and optimum conditions for germination in addition to the storage of these seeds.

Introduction

Burkina Faso is a Sahelian country with regular droughts and limited energy resources. As a solution to the desertification problems, important reforestation programmes have been set up to meet the need for firewood and timber, to protect dry lands, and to help conserve genetic resources. The seedlings used in plantations, are mostly produced from seed in nurseries. However, seed conservation requires that large numbers of seeds of many species be stored over long periods of time (FAO 1992). Many species of great socioeconomic importance in Burkina Faso are difficult to conserve and to promote AFRICA 17 because of the short lifespan of their seeds (Ouédraogo et al. 1996). Among these species, we have chosen to investigate seeds of Kigelia africana, Lophira lanceolata, Parinari curatellifolia and Zanthoxylum zanthoxyloides because they have multiple uses to rural communities. Despite these many uses, there is little information about their propagation, germination and storage, making it difficult to grow and plant these species. Kigelia africana (Lam.) Benth., belongs to the Bignoniaceae family. The species is mostly found in tropical Africa, from Senegal to Cameroon and South Africa. The tree reaches 10–12 m high with a characteristic shape when the flowers and fruits are hanging from the branches. The wood is white with brown heart, and is used to make mortars, canoes and stools. The roots are used in the treatment of syphilis, dysentery and snake-bite. The bark is used against epilepsy and leprosy. The leaves and fruits treat dysentery, rheumatism and asthenia, and are purgative. It flowers in November–December, at the end of the dry season. The inflorescence is a raceme of about 1.2 m long and the flower is 5 cm long. The fruit looks like a big grey sausage of 15–25 × 30–90 cm (Arbonnier 2000; Von Maydell 1983). Lophira lanceolata Van Tiegh. ex Keay is a member of the Ochnaceae family. It thrives in stony soils from Senegal to Cameroon, and to Sudan. It is a gregarious, locally common tree, which reaches 8–10 m high. The wood is pinkish with a red heart, very hard and heavy. It is used for making sleepers and bridges, and also for building and for making mortars, charcoal and as firewood. The roots treat sterility, constipation, diarrhoea and vomiting. The bark is used in the treatment of bronchitis. Branches are used as toothpicks, and to treat tooth decay. The leaves treat fever, hypertension, dysentery and syphilis. The fruit oil is used as body balm and in local food preparation. The white flowers are used by bees for honey production, and occur during the dry season before new leaves grow. The leaves are gathered at the end of short branches. The inflorescence is a terminal panicle of 10–15 cm. The fruit is hard and measures 2.5–3×0.8–12 cm, becoming red to brown at maturity (Arbonnier 2000; Von Maydell 1983). Parinari curatellifolia Planch. ex Benth., belongs to the Chryso- balanaceae family and is found from Senegal to Cameroon, to central and eastern Africa. It is a small to large tree up to 20 m in height. Its roots are used in the treatment of tooth decay, ear infection and malaria. The bark is used against fever, bronchitis and malaria. The pulp and the seed are eaten and also used for making 18 STORAGE BIOLOGY OF TROPICAL TREE SEEDS drinks. The hard wood is heavy and resistant to insects, and is used in construction and for making mortars and dugouts. The inflorescence is a 20 cm panicle and the flowers are white and rose- like. The fruit is an ovoid yellow to brown drupe (Arbonnier 2000; Von Maydell 1983). Zanthoxylum zanthoxyloides (Lam.) B.Zepernick & F.K.Timler belongs to the Rutaceae family. The species is gregarious and is found from Senegal to Nigeria. The roots are used for the treatment of haemorrhoids and rheumatism. The bark treats high fever and tooth decay. The seeds are used to make jewels. This thorny tree of 6–8 m often flowers twice a year, in the dry season (November to April) and during the rainy season (May to October). The inflorescence is a glabrous panicle of 5–25 cm long. The flower is greenish white and the fruit is a capsule of 5–6 mm diameter. The fruit at maturity becomes brown and opens by two valves. The seeds are black or blueish (Arbonnier 2000; Von Maydell 1983). For all these four species, a knowledge of seed germination and storage behaviour are essential for planting and conservation. The investigations undertaken by CNSF were aimed at determining the optimum conditions that maintain the viability of these seeds over periods of time.

Materials and methods

Seed collection and processing

Fruits of K. africana were collected on 23–24 Aug 2000 at Balla, in the western part of Burkina Faso. L. lanceolata fruits were collected on 20 March at Niangoloko, western Burkina. P. curatellifolia fruits were collected on 5 Nov 2001. Fruits of Z. zanthoxyloides were harvested on 13 Dec 2000 at Mina-Bandougou, west Burkina Faso. All fruits were transported to CNSF in jute bags by car. All seeds were manually prepared. The winged components of L. lanceolata fruits were removed and the normal seeds were selected. Seeds of K. africana were extracted and dried in the shade for a day before selecting normal seeds. The pulp of P. curatellifolia was removed and the seeds were washed and dried in the shade for a day before laboratory experiments. Seeds of Z. zanthoxyloïdes were extracted, then washed and shade-dried before use for experiments. AFRICA 19

Desiccation trials and seed germination

After initial moisture content and germination determinations, seed lots were dried to several target moisture contents chosen according to the IPGRI screening protocol on intermediate and recalcitrant seeds (IPGRI/DFSC 1999). Five replicates of five seeds were used to determine moisture content. Once a target moisture content was reached (IPGRI/DFSC 1999), seeds were germinated to determine the effect of drying on their viability. Two replicates of 50 seeds were used for each germination test. All germination experiments were carried out in the laboratory at about 25°C.

Storage trials

Samples of dry seeds to different moisture contents were stored at different temperatures. Seeds of K. africana and Z. zanthoxyloides were enclosed in aluminium bags, while those of L. lanceolata and P. curatellifolia were put in plastic bags. Seeds were then stored at –18°C in a freezer, at 4°C in a cool room, at 16°C in a cupboard and at 25°C in an incubator, following the method of Gaméné et al. (1994) and Gaméné (1995). Germination and moisture content were assessed every three months, as described above.

Results

Initial characteristics

Details of the large quantities of fruits collected and the initial characteristics of fruits and seeds of the four native species from Burkina Faso are shown in Table 1. K. africana and L. lanceolata seeds with >20% MC initially germinated t50%. However, seeds of P. curatellifolia and Z. zanthoxyloides had initially <20% MC and poor germination (0 and 2%, respectively). Table 1. Initial characteristics of fruits and seeds of the four native species to Burkina Faso K. africana L. lanceolata P. curatellifolia Z. zanthoxyloïdes Collection 23/08/2000 20/03/2001 5/11/ 2001 13/12/2000 Fruit harvested (kg) 236.125 95.3 44.21 27.65 Seed prepared (kg) 3.721 51.7 6.49 6.19 Initial MC (%) 26.98 21.30 17.49 13.85 Germination (%) 71 49 0 2 20 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Desiccation and germination

K. africana seeds tolerated desiccation to about 7% MC, maintaining the initial viability of 72% (Table 2). There were only slight variations in germination percentages during drying to low moisture contents. L. lanceolata seeds initially germinated to 49%, but decreased in viability during desiccation to low moisture contents. Less than 5% germinated at 6 and 9% MC and no seed germinated at 3% MC (Table 2). No seed of P. curatellifolia germinated either fresh or dried. Z. zanthoxyloides seeds germinated poorly reaching only 2% initially, but maintained this viability when dried to 3% MC (Table 2).

Table 2. Germination capacity (G%) after drying seeds to different moisture contents (MC%) K. africana L. lanceolata P. curatellifolia Z. zanthoxyloïdes MC G (%) MC G (%) MC G (%) MC G (%) (%) (%) (%) (%) 6.63 72 3 0 2.18 0 3 2 8.95 78 6 4 3.63 0 6 0 11.9 66 9 2 7.11 0 9 3 14.8 72 12 13 16.3 0 14 2 20 74 15 22 25 68 20 47

Storage trials

K. africana seeds with 27% MC or dried to 15, 20 and 25% MC and stored at 16°C did not germinate after 6 months (Table 3). All the other seed samples dried to 7, 9 and 12% MC or higher survived storage at 4, 16 and 25°C for 16 months, maintaining >50% germination. There was no significant difference between seeds stored at 4, 16 and 25°C. L. lanceolata seeds initially germinated 49%. However, they germinated <5% after six months storage at 16°C, but did not germinate at all at any other storage conditions (Table 4). P. curatellifolia seeds, fresh or dried, did not germinate at all (0%). This may be due to collection of immature seeds or dormancy problems in these seeds. Z. zanthoxyloïdes maintained the initial viability of 2% germination when dried to 3% MC. However, these seeds with 6 and 9% MC germinated up to 14 and 37% after nine months of storage at 16 and 4°C, respectively (Table 5). No seed germinated after 12 months or longer. AFRICA 21

Table 3. Germination of K. africana seeds after desiccation and storage at 4, 16 and 25°C MC 6.6% 9.0% 11.9% 14.8% 20% 25% 27% 4°C—initial 72 78 66 72 74 68 71 3 months 72 62 58 58 60 68 65 6 months 69 81 71 56 44 55 36 9 months 48 62 65 42 47 37 41 12 months 55 48 48 33 53 35 48 15 months 85 69 68 50 53 40 38 18 months 68 71 50 46 59 48 52 16°C—initial 72 78 66 72 74 68 71 3 months 77 84 77 52 43 43 25 6 months 61 69 63 20 2 12 70 9 months 55 78 59 1 0 7 0 12 months 61 56 48 0 1 2 0 15 months 77 65 59 0 0 0 0 18 months 70 42 53 0 0 0 0

25°C—initial 72 78 66 72 74 68 71 3 months 79 55 53 69 59 69 56 6 months 73 71 64 55 63 68 0 9 months 51 66 52 49 57 39 63 12 months 40 41 37 44 44 51 61 15 months 63 74 50 33 81 48 56 18 months 73 73 40 9 60 42 60

Table 4. Germination of L. lanceolata seeds after desiccation to different moisture contents and storage at –18, 4 and 16°C MC 3% 6% 9% 12% 15% 20% 21.3% –18°C—initial 0 4 2 13 22 47 49 3 months - 0 0 0 0 0 0 6 months - 0 0 0 0 0 0 9 months - 0 0 0 0 0 0

4°C—initial 3 4 2 13 22 47 49 3 months - 0 0 0 0 0 0 6 months - 0 0 0 0 0 0 9 months - 0 0 0 0 0 0

16°C—initial 3 4 2 13 22 47 49 3 months - 0 0 3 1 5 2 6 months - 0 0 0 0 0 2 9 months - 0 0 0 0 0 0 22 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Table 5. Germination (%) of Z. zanthoxyloïdes seeds after desiccation to different moisture contents and storage at –18, 4, 16 and 25°C –18°C MC 3% 6% 9% 14% Initial 2 0 3 0 3 months 0 1 0 0 6 months 0 0 0 0 9 months 9 0 0 0 12 months 0 0 0 0 15 months 0 0 0 0

4°C 3% 6% 9% 14% Initial 2 0 3 0 3 months 0 1 0 0 6 months 0 0 0 0 9 months 1 0 37 0 12 months 0 0 0 0 15 months 0 0 0 0

16°C 3% 6% 9% 14% Initial 2 0 3 0 3 months 5 5 0 0 6 months 0 0 1 0 9 months 9 14 4 2

12 months 0 0 1 0 15 months 0 0 0 0

25°C 3% 6% 9% 14% Initial 2 0 3 0 3 months 9 4 1 1 6 months 2 0 0 0 9 months 0 0 0 0 0 0 0 0 12 months 0 0 0 0 15 months AFRICA 23

Discussion

Seeds of Kigelia africana were desiccation tolerant, maintaining initial viability after drying to 7% MC and storage for 18 months at 4 to 25°C (Tables 2 and 3). There was no significant difference between the viability at 4, 16 and 25°C. Lophira lanceolata seeds were sensitive to desiccation down to 3% MC and maintained only <5% after six months storage at 16°C (Table 4). The low germination of P. curatellifolia and Z. zanthoxyloides seeds might be because of seed immaturity or dormancy problems. Improvements (2 to 37%) in seed germination of Z. zanthoxyloides under various storage conditions (moisture content and temperature—Table 5) suggest that removal of dormancy may have occurred. All these results indicate that there is a need to investigate in detail the stages of maturity and optimum conditions for germination and storage of these seeds.

Acknowledgements

We thank IPGRI and DFSC for allowing us to contribute to this project and Mr Mamadou Sidibé for technical assistance.

References

Arbonnier, M. 2000. Arbres, arbustes et lianes de zones sèches d’Afrique de l’Ouest. CIRAD, MNHN, IUCN, Pp. 542. FAO. 1992. Guide de manipulation des semences forestières. Etude FAO 20/2, Rome, Italy, Pp. 444. Gaméné, C.S. 1995. Etude de la conservation des semences forestières. Rapport No 14, CNSF, Pp. 25. Gaméné, C.S., M. Sacandé, H.L. Kraak, J.G. van Pijlen and C.H.R. de Vos. 1994. Storage of neem (Azadirachta indica) seeds from Burkina Faso. Abstracts, Technological Advances in Variety and Seed Research. ISTA/ISHS CPRO- DLO, Wageningen, The Netherlands. IPGRI/DFSC. 1999. The project on handling and storage of recalcitrant and intermediate tropical forest tree seeds. Danida Forest Seed Centre. Newsletter 5:23–39. Von Maydell, H.J. 1983. Arbres et arbustes du Sahel. Leurs Caractéristiques et leurs Utilizations. GTZ, Pp. 583. Ouédraogo, A.S., K. Poulsen and F. Stubsgaard. 1996. Intermediate/Recalcitrant tropical forest tree seeds. Proceedings of a Workshop. Humlebaek, Denmark, Pp. 169. 24 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Effect of neem (Azadirachta indica) fruit development on seed quality

Oblé Neya 1,2, Christiane S. Gaméné 1, 3 and Moctar Sacandé 1,4

¹Centre National de Semences Forestières, Ougadougou, Burkina Faso ²Department of Plant Sciences, Wageningen University, Laboratory of Plant Physiology, Arboretumlaan 4, 6703 BD Wageningen, Netherlands 3 Current address: Université Agricole de Gembloux, Gembloux, Belgium 4Current address: Seed Conservation Department, Royal Botanic Gardens, Kew, Ardingly, Wakehurst Place, RH17 6 TN, UK

Abstract

Formation and development of neem fruits and the physiological quality of the seed were monitored over the fruiting period. For 20% of the inflorescences there was a ‘synchronized’ fruiting mode, such that all fruits were formed within a week. For the remaining 80% of inflorescences fruiting was spread out over 2–5 weeks. Mean number of fruits per inflorescence increased from 7 to 34 fruits in the first five weeks after which it decreased to 2 fruits at the end of the fruiting period in the 10th week. Seed quality was estimated by changes in dry weight, moisture content and germination capacity during development. Seeds of eight weeks old fruits had a MC of 46% and germinated maximally to 82%, indicating physiological maturity. Thus, it is suggested that neem seeds for immediate sowing may be collected in reasonable quantity (before fruit falls) and with higher quality (>80%) at around 8 weeks after pollination.

Introduction

Neem, Azadirachta indica A.Juss., originates from the dry areas of India (Cartiana, Dekkam, Siwalik) and Myanmar (Toup 1921, cited by CTFT 1988). It was introduced into the English colonies of the African continent early in the twentieth century (1918–1921), probably in Nigeria first (Dewaulle 1977). The species spread to Burkina Faso almost 60 years ago, through the border provinces of Ghana (Guinko 1984; Devernay 1994). Neem is an important species whose by-products have multiple uses in Asia, as well as in the Sahelian regions of Africa (Bellefontaine 1992). It is used to protect soils against erosion and to improve microclimate. Neem provides shade, firewood and valuable timber. Extracts of its AFRICA 25 leaves and seeds are intensively used in medicine and also as insecticides (von Maydell 1983; National Research Council 1992). Neem propagates via seeds, although the seeds are well known to be short-lived in ex situ storage (Gaméné et al. 1996). Considering the multiple functions this species offers mankind and the environment, it is important to study the biology of its seeds, starting with the phenology of fruiting, in order to increase the quality of seeds at harvest. The research on neem seed development presented in this paper aims to help determine the minimum age for the collection of high quality fruits and seeds, by analysing changes in moisture content and germination capacity during seed development.

Materials and methods

Formation and development of neem fruits

Developmental studies on neem seeds were carried out by adapting the methods of Sacandé and Groot (1997). Two sites of urban plantations in the city of Ouagadougou were selected and a total of 43 trees were monitored in September 1998. These locations are found at 01q50’W and 12q50’N, where the average annual precipitation is between 650 and 750 mm (Sacandé et al. 1998). The trees and inflorescences were paint-marked and labelled. Numbers were marked with paint on the trunks of the trees, and at least four limbs were marked on each tree, depending on the accessibility and availability of inflorescences. These limbs were labelled with different letters in alphabetical order. Phenological changes and quantitative data were recorded regularly during the whole fruiting period. Between 10 and 15 inflorescences were selected for regular and frequent observations to determine the onset of fruit formation, as characterized by the withering of flowers and the first visibility of small fruits. This corresponded to Week 0, after which observations were carried out weekly. Quantitative and qualitative changes of the fruits were recorded on monitoring sheets.

Moisture content of developing seeds

Samples of 100 seeds for each lot were used to determine initial seed weight and moisture content, based on weighing before and after 26 STORAGE BIOLOGY OF TROPICAL TREE SEEDS drying in an oven at 103°C for 17 h (ISTA 1985). The moisture content (MC) of each individual seed was calculated using the formula %MC=(FW–DW)/FW×100, where FW=(fresh) weight before drying and DW=(dry) weight after drying. The mean moisture content was then calculated for each seed-lot.

Germination of developing seeds

Seeds from different collections of fruits were sown in boxes of clean moistened river sand for germination. Four replicates of 25 seeds were used for each germination test. The seeds were sown covered with a thin layer of sand, the boxes tightly closed to maintain moisture and then put on a germination table at ambient temperature (25–28°C) in the laboratory. Germination was recorded every two days up to 28 days, when all seeds had germinated. Germination data were statistically analyzed using analysis of variance (ANOVA) in Genstat.

Results

Neem fruit formation

There were two patterns of neem fruit formation. In ‘synchronized’ mode, fruits were formed within a week on 20% of all observed inflorescences and developed almost homogeneously. By contrast, fruit formation on the remaining 80% of the inflorescences was spread over 2–5 weeks. As a result, there was a great variation of fruits of different ages, which also developed sequentially, the young fruits being at the top of the inflorescences. The mean number of fruits per inflorescence progressively increased from 7 to 34 fruits between Week 0 and Week 5, and then decreased to 2 fruits at the end of the 10 weeks monitoring (Table 1). These numbers were means of total fruits counted weekly on the selected 10–15 inflorescences for each tree. When the mean number of fruits per inflorescence were compared over the fruiting period, ANOVA showed great variations as indicated by the coefficients of variation of 74% within fruits of a single tree (e.g. tree 15) and 41% between fruits of two trees (e.g. trees 1 and 2). However, CVs of 28% and 50% were calculated for fruits at 6 weeks and at 9–10 weeks of development, respectively. AFRICA 27

Table 1. Means of total number of fruits recorded weekly per inflorescences on 20 individual trees over 10 weeks of development. Means with the same letter in the last row are not significantly (P>0.01) different Tree no. Weeks after fruit formation 0 1 2 3 4 5 6 7 8 9 10 1 10 19 26 29 34 35 34 33 27 21 4 2 10 16 19 19 23 17 22 18 11 9 3 3 8 14 13 15 17 22 21 22 8 6 0 4 4 7 10 18 29 31 33 30 24 7 3 5 6 11 14 18 20 22 22 18 16 6 1 6 5 15 34 38 39 41 41 41 33 24 3 7 4 12 15 20 20 21 20 19 13 10 2 8 5 16 23 25 29 28 30 27 21 13 6 9 4 18 24 25 36 38 30 17 18 12 2 10 12 26 34 33 42 46 38 21 19 7 0 11 11 23 30 39 43 43 35 31 23 10 4 12 6 11 17 28 40 37 29 24 18 8 1 13 5 15 19 24 26 24 20 16 16 10 1 14 9 39 51 59 59 59 34 29 27 15 4 15 8 37 46 51 65 59 47 13 12 8 1 16 4 18 18 17 32 33 31 30 17 7 3 17 8 16 24 29 36 29 30 27 25 11 2 18 11 26 23 25 26 30 23 24 19 9 3 19 5 6 10 11 11 12 11 13 11 4 0 20 7 24 22 26 29 34 25 17 14 13 4 Means 7 ± 18 ± 24 ± 27 ± 33 ± 34 ± 29 ± 24 ± 18 ± 10 ± 2 ± 3ac 8abc 11abc 12ab 13b 12b 8ab 7abc 6abc 5ac 1c

Moisture content and germination of developing seeds

Seed moisture content and germination could only be determined on samples from 4 weeks onward (Fig. 1). Before this period, fruits and seeds were not differentiated enough for handling and experimentation. During development, moisture content decreased gradually from >70% at 4 weeks to 50% MC at 6 weeks and 42% MC at 9 weeks (Fig. 1). This was concomitant with a gradual decline in fresh weight. By contrast, there was a slight increase in dry weight between Weeks 4 and 5, after which there was no further change. Germination tests were used to estimate the physiological maturity and quality of seeds. Germination capacity was first observed 6 weeks after flowering, when 20% of the seeds germinated. This then increased during seed development and maximum germination occurred between 7 and 9 weeks, when >80% of seeds germinated. ANOVA showed that 28 STORAGE BIOLOGY OF TROPICAL TREE SEEDS the coefficient of variation was 0.21 and 18% the smallest significant difference between two germination percentages. The germination data were significantly different (P<0.01) for seeds of 6 and 7 weeks, but not for seeds of 7 and 9 weeks old.

100 100 germination moisture content fresh weight 80 dry weight 80

60 60

40 40 Germination (%)

20 20

0 0 (%) content (g)/Moisture Weight 0145678910 Weeks after flower opening

Figure 1. Weights, moisture content and germination capacity of neem seeds at different stages of development.

Discussion

Fruit formation

Neem fruits always form at the top of the most external panicles of an inflorescence. According to FAO (1992), fruit formation in an inflorescence is related to species and mainly depends on the mode of pollination, which varies from one pollinator to another. The observed two patterns of fruit formation is interesting although no explanation has been found for such phenomena so far. This should be further investigated, as it may affect the quality of seeds and the handling after harvest. However, seeds from both patterns of formation behaved similarly in this study. Neem is a polygametic species producing bisexual flowers on the same individual (Schmutterer 1995, cited by Sacandé et al. 1998). The ‘synchronized’ mode of pollination may be due to a massive AFRICA 29 fall of mature pollen grains on the stigma at that period of fruiting. Further investigations will elucidate the pollination (or fertilization) systems of this species. Fruit quantity varied greatly from one inflorescence to another on the same tree and from one tree to another (Table 1). Both the quantity of flowers per inflorescence and the rate of successful fertilization of flowers may account for these differences. During development, the mean number of fruits per tree increased from 7 up to 34 in five weeks. Because new fruits were continuously produced, there was a time lag of approximately 4 weeks between the first fruits and the last fruits. From 6 weeks onward, corresponding to the maturation period, the number of fruits decreased, partly due to fruit fall (Fig. 1). The peak of fruit formation indicates that there are almost no more new fruits forming on the inflorescences at the start of fruit fall, itself a sign of seed maturing (FAO 1992). Mature seeds and fruits are often of interest to insects, rodents, birds and other animals. Thus, these factors together with weather conditions, play a part in fruit fall, particularly at maturity when fruits become heavier (dry weight is at its maximum).

Moisture content and germination of seeds

An accepted measure of maturity is when the seed reaches its maximum dry weight or its minimum moisture content and becomes physiologically mature (FAO 1992). Neem fruits and seeds were not differentiated enough for experiments before 4 weeks old. From this stage onward, seed moisture content decreased together with fresh weight to 40–50% MC at maturity (Fig. 1). Neem seeds did not exhibit maturation drying, such as that observed during the final event in the development of desiccation tolerant seeds (Côme and Corbineau 1996). These results corroborated other studies on development, that found that neem seed matures at about 50% MC at 10 weeks after pollination (Sacandé et al. 1998). By contrast, there was an increase in the dry weights from Week 4 to Week 5, remaining at this maximum level until Week 9. The rapid gain of dry weights is a result of the synthesis and deposition of stored reserves in the developing seeds (Bewley and Black 1994), after which they attain the capacity to germinate. Germination tests were used to estimate the physiological maturity and quality of seeds during development. Germination capacity was 30 STORAGE BIOLOGY OF TROPICAL TREE SEEDS first observed at 6 weeks, when 20% of seeds germinated. Germination then increased and reached a maximum of > 80% after 9 weeks. The maturation stage is thus characterized by the attainment of maximum germination at least 7 weeks after pollination and a stable, relatively high moisture content of >40% MC (Fig. 1). Therefore, for immediate use in planting programmes we suggest collection of seeds at least 8 weeks after fruit formation, before there is a drastic decrease in the number of fruits per tree (Table 1 and Fig. 1). Many other factors may mean that seeds are collected before total maturity, including limited fruit production, reduction of time between maturation and the dispersal of fruits or seeds (Turnbull 1975, cited in FAO 1992). Prolonging the collection period therefore gives more time to a Seed Centre to better organize operations for collecting more seeds, before predation by insects and other animals.

Acknowledgements

We thank Drs Lambert G. Ouédraogo, director of CNSF and Jean-Baptiste Ilboudo, Université Polytechnique de Bobo Dioulasso for their useful advice and comments during the implementation of this project, and Mr Mamadou Sidibé for technical assistance.

References

Bellefontaine, R. 1992. L'avenir du neem en zone tropicale sèche est-elle menacée? Le Flamboyant 21:24–26. Bewley, J.D. and M. Black. 1994. Seeds: Physiology of Development and Germination (2nd edn.). Plenum Press, New York. Côme, D. and F. Corbineau. 1996. Metabolic damage related to desiccation sensitivity. Pp. 107–121 in Proceedings of a Workshop on Improved Method for Handing and Storage of Intermediate and Recalcitrant Tropical Forest Tree Seeds (A.S. Ouédraogo, K. Poulsen and F. Stubsgaard, eds.). IPGRI, Rome, Italy. Devernay, S. 1994. L'introduction du neem, arbre exotique au Burkina Faso; bilan socio-économique. ORSTOM, Ouagadougou. Pp. 59. Dewaulle, J.C. 1977. Plantations forestières en Afrique tropicale sèche. Centre Technique Forestier Tropical, Nogent-sur-Marne, France. Pp. 177. FAO. 1992. Guide de manipulation des semences forestières. Etude FAO 20/2, Rome, Italy. Pp. 444. Gaméné, C.S., H.K. Kraak, J.G. van Pijlen and C.H.R. De Vos. 1996. Storage of neem seeds from Burkina Faso. Seed Sci. Technol. 24:124–132. Guinko, S. 1984. Végétation de la Haute Volta. These de Doctorat d’Etat. University Bordeaux III. Pp. 318. AFRICA 31

ISTA. 1985. International rules for seed testing. Seed Sci. Technol. 13:299–355. von Maydell, H.K. 1983. Arbres et arbustes du Sahel, leurs utilizations et leurs caractéristiques. Edition Eschorn. Pp. 531. National Research Council (NRC, USA). 1992. Neem: A Tree for Solving Global Problems. National Academy Press, Washington, DC. Sacandé, M. and S.P.C. Groot. 1997. Proposal for a standard protocol for studies on neem (Azadirachta indica A. Juss.) seed development. IPGRI/DANIDA project on handling and storage of recalcitrant and intermediate tropical forest tree seeds. IPGRI-DANIDA Newslett.er, 2: 15–18. Sacandé, M., F.A. Hoekstra, J.G. van Pijlen and S.P.C. Groot. 1998. A multifactorial study of conditions influencing longevity of neem (Azadirachta indica) seeds. Seed Sci. Res. 8:473–482. 32 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Desiccation and storage of Lannea microcarpa seeds from Burkina Faso

Matthew I. Daws1, Christiane S. Gaméné2, Moctar Sacandé2,3,4, Hugh W. Pritchard1, Steven P.C. Groot3 and Folkert Hoekstra4

1Royal Botanic Gardens, Kew, Wakehurst Place, Ardingly, West Sussex RH 17 6TN, UK 2Centre National de Semences Forestières (CNSF), 01 BP 2682 Ouagadougou 01, Burkina Faso 3DLO – Centre for Plant Breeding and Reproduction Research (CPRO), PO Box 16, 67700 AA Wageningen, Netherlands 4Wageningen Agricultural University, Department Biomolecular Sciences, Arboretumlaan 4, 6703 BD, Netherlands

Abstract

Seeds of Lannea microcarpa collected from Burkina Faso in 1997 and 1998 were subjected to desiccation and a range of storage conditions. Seeds were able to tolerate desiccation to low moisture contents (ca. 5%) with little loss of viability. In addition, it was possible to store seeds at 25°C at reduced moisture contents (approx. 6%), for up to 14 months. However, seeds stored at –20 or 4°C exhibited a reduction in viability within three months of storage. This indicates that seeds of L. microcarpa may exhibit nonorthodox seed storage behaviour.

Introduction

Lannea microcarpa Engl. & K.Krause is a tree, which belongs to the Anacardiaceae family. The species is found in all the Sudanian zones of West Africa. The Northern limit of its habitat is the Sahelo- sudanian zone and the southern limit is the Guinean zone. It likes deep soil but can also withstand uncultivated and lateritic soil. The tree can reach 16 m high. The bark is grey white, smooth when the tree is young, and becomes splintery when getting old, and the slash is red. The compound leaves, with 2 to 9 leaflets, are alternate and measure up to 25-cm long. The flowers are small, green yellowish with glabrous sepals. The fruit is a drupe, which becomes purple AFRICA 33 black at maturity. The fruits are edible and are used for cooking and production of wine. The bark is used for making cords. Preliminary studies on L. microcarpa indicate that the seeds are difficult to store (Gaméné, pers. comm.). The purpose of this investigation was to detail the response of L. microcarpa seeds, collected in Burkina Faso, to desiccation and storage.

Materials and methods

Initial tests

Fruits were collected on 18–20 June 1997 and 4–6 July 1998, from Bissiga (12°40cN 01°10cW), Burkina Faso. After harvest, 100 individual fruits were sampled and weighed. The remaining fruits were soaked in water and immediately de-pulped, polished with sand to remove the mesocarp tissue and finally washed with water. The seeds were then soaked in 1% NaOCl solution for 10 minutes, dried with a cloth and then coated with fungicide: 1 g of Benomyl and 1 g of Thiram per 1 kg of seeds. Following this treatment one sample of seeds was sent by airmail to the Royal Botanic Gardens, Kew, and another to Wageningen: the remaining seeds were retained at CNSF, Burkina Faso.

Desiccation and germination trials

Desiccation trials were undertaken at both Kew and CNSF. Seeds were desiccated by mixing them with silica gel (1 kg of seeds for 1 kg of silica gel) or smaller quantities of seeds mixed on a 1:1 ratio (wt) with silica gel. During desiccation, a seed sample was regularly weighed which allowed seeds to be dried to target moisture contents of 20, 16, 13, 10, 8 and 5%. As a control for the desiccation experiment, samples of seeds were mixed with sawdust or vermiculite and held at 25–26°C: bags were regularly vented. Germination at each moisture content was assessed by sowing 100 seeds (4 × 25) at 26 (Kew), 25 (CPRO) or 30°C (CNSF). A photoperiod of 8 h a day was applied at CPRO and Kew, on seeds sown on filter paper and a gel of 1% agar in water, respectively. Seeds were sown in sterilized sand and at constant light at CNSF. 34 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Moisture content vs. relative humidity

The relationship between moisture content and equilibrium relative humidity (eRH) was established to construct a water sorption isotherm for L. microcarpa. Seeds (2 × 3 at each target relative humidity) were incubated above silica gel (4% eRH) and a range of saturated salt solutions (lithium chloride (15%), calcium chloride (33%), zinc nitrate (42%), sodium bromide (59%) and sodium chloride 76% eRH) at 21°C, until they reached constant weight. Equilibrium relative humidity of the seeds was then determined using a Rotronic WA-14P water activity measuring station (Rotronic Instruments UK, Horley) set up with a DMS 100H humidity sensor. Following relative humidity determinations, the moisture content of the seeds was determined by drying the seeds at 103°C for 17 h. The data of moisture content were then plotted against those of eRH.

Storage trials

In 1997 storage trials were undertaken at CNSF and CPRO with nondried and dried seeds with different moisture contents ranging from 3 to 25%, as well as their controls, at –20, 5, 15, 16, 20 and 25°C. To reach the desired moisture level, the seeds were mixed with silica gel (CNSF) whereas at CPRO, the fresh seeds were gradually dried in a cabinet with fixed 32% relative humidity. Seeds of each treatment sample were sealed in aluminium foil at CPRO and enclosed in plastic bags at CNSF for their storage. Two to four replications of 25 seeds were used for the assessment of the germination while 25 seeds were used for the MC. At CPRO, germination and moisture content, of stored seeds, were assessed every two or three months, up to 24 months, as described above. After storage, the aluminium sachets representing each seed treatment were equilibrated overnight at 25°C before germination tests were commenced the next day. In 1998, storage trials at CNSF were undertaken with seeds dried to four target moisture contents (3, 6, 9 and 12%) and stored at –18, 4 and 25°C for up to 6 months. AFRICA 35

Results

Initial tests

L. microcarpa seeds harvested in 1997 and 1998 had similar characteristics for fruit and seed weights (Table 1). However, their initial moisture contents for the whole seeds, and seed parts varied. Whole seeds from the 1997 collection had a high initial moisture level (28.4%), while their embryos had the lowest moisture content (10.5%). Seeds collected in Burkina in 1998 had a lower moisture content and germination when tested at CNSF compared to when assessed at Kew (Table 1).

Table 1. Initial characteristics of seeds of L. microcarpa from Burkina Faso

Fruit Seed Initial MC (%) Germination weight (g) weight (%) (g) Whole Seed coat Embryo seed CNSF 1.14±0.29 0.20±0.03 28.43 14.74±8.64 10.5±7.5 94±5 (1997) CNSF 1.04±0.16 0.19±0.03 12.56±4 8.89±4.27 15.2±5.9 78 (1998) Kew – 0.18±0.03 22±2 18±1 25±3 88±3 (1998)

Seed desiccation and germination

Seeds of L. microcarpa collected in both 1997 and 1998 survived desiccation to moisture contents of approximately 5% with little loss of viability (Fig. 1). Seeds desiccated gradually from >20% MC to 5% MC retained high germination, which did not alter (P<0.05) with drying. Based on the plot of seed moisture content against relative humidity, it was observed that seeds that had tolerated drying to 5% MC, had equilibrated to a whole seed relative humidity of approximately 35% (Fig. 2). 36 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

100

40 Kew 1998 CNSF 1997

Germination (%) 30 CNSF 1998

0 0 5 10 15 20 25 30 Whole seed moisture content (%) Figure 1. Effect of desiccation on viability of L. microcarpa seeds. The trials were carried out at CNSF, Burkina Faso (1997 and 1998) and at Kew (1998).

Moisture content - fresh weight basis (%) Moistureweight basis content - fresh 0 0 20406080100 Relative humidity (%)

Figure 2. Desorption (water lost from initial fresh seeds) isotherm measured at 21°C, for L. microcarpa whole seeds. AFRICA 37

Seed storage

Table 2 indicates that seeds collected in 1997 could be stored at 25°C for up to 14 months at 5.5 and 5.8% mean moisture content (5 and 8% target moisture contents) while retaining about one-third of the initial germinability. However, it should be noted that during storage the seed moisture contents fluctuated from the initial value (note the quite large SDs for MCs), which might have affected seed viability.

Table 2. Effect of storage at 25°C and two different moisture contents, on viability of L. microcarpa (trials carried out in 1997 at CNSF) Storage period (months) Germination (%) post storage 5.48±2.4% MC 5.81±2.1% MC 0 73 77 2 49 44 4 31 18 6 15 22 8 43 31 10 31 17 12 20 14 14 27 18 16 2 5 18 3 4 20 0 0

Seeds collected in 1998 were stored at four moisture contents (18, 17, 16 and 11%) at –18, 4 and 25°C for up to 6 months. Seeds survived desiccation to all the target moisture contents, but viability at –18 and 4°C was lost by the first sampling interval at three months, irrespective of moisture content. However, at 25°C up to 50% viability was retained for three months (data not shown). Storage of seeds (1997 batch) at CPRO revealed a similar pattern (Table 3). Seed retained some viability for up to three months when stored at 25°C, particularly at lower moisture contents. However, viability was slightly lower after –20 and 5°C storage even at low (3–6%) moisture contents. 38 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Table 3. Effect of storage at different temperatures and moisture contents on viability of L. microcarpa seeds (trials undertaken at CPRO in 1997). MC Temperature 3 months 12 months 25% 25°C 0 0 20% 25°C 0 0 12% 15°C 16 0 9% 15°C 70 0 6% 25°C 84 25 15°C 84 69 5°C 58 54 –20°C 56 44 3% 25°C 90 55 15°C 84 70 5°C 62 52 –20°C 50 44

Discussion

Desiccation trial

Seeds of L. microcarpa survived desiccation to moisture contents of approximately 5% (Fig. 1). Thus, seeds of these two batches from Burkina Faso have seeds that are desiccation tolerant. However, these results are in contrast to earlier work, which reported seeds of L. microcarpa as being recalcitrant (CNSF, cited in Hong et al. 1996).

Seed storage

This study found that seeds of L. microcarpa can be stored, at 25°C, for up to 14 months. This is in contrast to the work of Kamra (1990) who found that viability at room temperature was lost within one month. Work undertaken at CNSF found that viability was lost at –18°C. This loss of viability may have resulted from ice crystal formation within the seeds since the seeds were stored at high (>11%) moisture contents, which correspond to a relative humidity in excess of 80% (Fig. 2). At relative humidities of 80% or more, ice crystal formation would be expected to occur upon freezing (Vertucci and Farrant 1995). In addition, some seed viability was also lost at 4–5°C after three months storage. This loss of viability may reflect chilling sensitivity of these seeds. Chilling sensitivity has been observed in the seeds of a number of species of tropical origin (Corbineau and Côme 1988). A reduction in seed viability of the seeds stored at –20°C, at CPRO, even AFRICA 39 at low (3–6%) moisture contents suggests that seeds of L. microcarpa may exhibit intermediate (sensu Ellis et al. 1990) seed storage behaviour. Clearly, further work is required to fully clarify the seed storage behaviour of this species.

Conclusions

Seeds of L. microcarpa can be dried to low moisture contents with little or no affect on viability. Furthermore, seeds can be stored, at reduced moisture contents, for up to 14 months at 25°C.

Acknowledgements

The authors thank Ms Odilie Bastidas at CPRO and Ms Caroline A. Howard and Catherine Harris at RBG Kew, Wakehurst Place, and Mr Mamadou Sidibé at CNSF, for their technical assistance.

References

Corbineau, F. and D. Côme. 1988. Storage of recalcitrant seeds of four tropical species. Seed Sci. Technol. 16:97–103. Ellis, R.H., T.D. Hong and E.H. Roberts. 1990. An intermediate category of seed storage behaviour? I. Coffee. J. Exp. Bot. 41:1167–1174. Hong, T.D., S. Linington and R.H. Ellis. 1996. Seed storage behaviour: a compendium. Handbooks for Genebanks: No. 4. International Plant Genetic Resources Institute, Rome, Italy. Kamra, S.K. 1990. Improving the forest seed situation in some African countries. Pp. 126–131 in Tropical Tree Seed Research – ACIAR Proceedings No 28. Vertucci, C.W. and J.M. Farrant. 1995. Acquisition and loss of desiccation tolerance. Pp. 237–271 in Seed Development and Germination (J. Kigel and G. Galili, eds.). Marcel Deckker, New York. 40 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Desiccation, germination and storage of Sclerocarya birrea seeds from Burkina Faso

Christiane S. Gaméné1,3, Deon Erdey2, David Baxter2, Nthabiseng Motete2 and Patricia Berjak2.

1Centre National de Semences Forestiéres, 01 BP 2682 Ouagadougou 01, Burkina Faso 2Plant Cell Biology Research Unit, School of Biological Sciences, University of KwaZulu-Natal, Durban 4041, South Africa 3Unité de Biologie Végétale. Faculté Universitaire des Sciences Agronomiques. Passage des Déportés 2, B-5030 Gembloux, Belgium

Abstract Seeds of Sclerocarya birrea collected in Burkina Faso, were dispatched to South Africa for replicating experiments to determine their desiccation sensitivity and storage behaviour. Initial seed viability varied between 25% and 70–75% germination, while desiccation of seeds to 5% MC had little effect on germination. Experiments in South Africa revealed that moisture content changed mostly in the endocarp, with very little variation in seed moisture content. As drying probably hardened the endocarp, it is recommended that the opercula be removed for all germination trials with this species. Based on the 1998 storage trial in Burkina Faso, it is apparent that S. birrea seeds require an after- ripening period to achieve maximum germination, which was greatest for those seeds stored for 18 months at 25°C. If well dried, the seeds can survive 18 months’ storage even at –18°C, which would suggest that this species is orthodox.

Introduction

Sclerocarya birrea (A. Rich.) Hochst. (or marula in English) is member of the Anacardiaceae family that grows to a medium size of about 15 to 20 m high. It is a deciduous tree, which is widely distributed in Africa, mainly in warm, dry and frost-free mixed bush-veld regions (Coates– Palgrave 1977; Pooley 1993). Recent efforts to domesticate the tree have resulted in its introduction to Israel (Nerd and Mizrahi 1993; Nerd and Mizrahi 2000). The stem is straight, branching high up with a spreading round crown (Pooley 1993). The bark is grey and splintery and the slash is red and fibrous. The wood is used for carvings and fuel AFRICA 41

(Pooley 1993). The compound leaves are alternate and glabrous, measuring up to 200 mm with 5 to 8 pairs of leaflets (see Fig. 1). The bark is widely used in the treatment of dysentery and diarrhoea (Coates–Palgrave 1977; Galvez et al. 1993; Pooley 1993) due to its high antibacterial activity (Eloff 2001). The flowers are reddish or greenish, with male and female flowers occurring on separate trees (Coates–Palgrave 1977; Pooley 1993). The fruit is a highly specialized drupe or stone fruit, measuring 30–40 mm. The fruits drop from the tree when still green, ripening to pale yellow on the ground (Pooley 1993; Nerd and Mizrahi 2000). The stone is embedded in an edible fibrous, juicy flesh (mesocarp, sensu lato) which is covered by the peel (exocarp, sensu lato). The fruit contains high levels of vitamin C (Jaenicke and Thiong’o 2000) and a jelly preserve can be made from the juice, which is not only used fresh, but also commercially utilized to produce a liqueur (Pooley 1993). One to four fruit locules occur in the hard-lignified endocarp. Each locule usually contains a single seed, and appears to be a hermetically sealed unit with an orifice tightly closed by a relatively small lid, the operculum (see Fig. 2). During germination, each embryo emerges through this orifice. The diameter of the operculum lid is always smaller than that of the seed and it is therefore impossible to remove intact seeds from the fruit (Shone 1979). The seeds are covered with a thin papery seed coat. The protective function of the seed coat is therefore taken over by the stony endocarp (von Teichman et al. 1986). The seeds of S. birrea contain about 56% oils (Weinert et al. 1990; Glew et al. 1997; Maundu et al. 1999; Jaenicke and Thiong’o 2000; Zharare and Dhlamini 2000), consisting mostly of unsaturated fatty acids (70% oleic acid and 8% linoleic acid) and only 11% saturated fatty acids (Weinert et al. 1990; Ogbobe 1992). Marula oil has potential use in salads and cooking (Zharare and Dhlamini 2000). Rich in proteins, i.e. 29 to 37% (Ogbobe 1992; Glew et al. 1997; Maundu et al. 1999; Jaenicke and Thiong’o 2000), the seeds are eaten by people either raw or cooked with porridge (Pooley 1993). The seeds have also been described as a potential nutrient source for free grazing animals in Botswana during times of drought (Aganga and Mosase 2001). Published reports are conflicting regarding the desiccation tolerance and storage behaviour of S. birrea seeds; the term seed refers to both the seed components (testa, cotyledons and axis) as well as the endocarp. Seeds of this species have been reported to be recalcitrant (Kamra 1990), losing viability after only one month of storage 42 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

(Ouédraogo and Verwey 1987), and/or intermediate (Were and Munjuga 1999). Others have reported orthodox storage behaviour, as the seeds tolerate desiccation to 10% moisture content or even less, retaining their initial viability for up to 4 years in storage (Msanga 1998; Ouédraogo et al. 1999). This study aimed to investigate the desiccation tolerance and storage behaviour of S. birrea seeds and to determine how handling and transport affected these responses.

Figure 1. Leaves, fruit and depulped fruit of Sclerocarya birrea (Drawing by S. Kambou, CNSF). AFRICA 43

operculum endocarp

embryonic axis locule cotyledon septum

Figure 2. Diagrammatic representation of a seed of Sclerocarya birrea.

Materials and methods

Seed collection and processing

Fruits were collected on 14–16th May 1997 and 22–24th May 1998 from a S. birrea stand in Saponé, Burkina Faso and transported in jute bags to the laboratory at CNSF, Ouagadougou. The pulp was manually removed by soaking fruits in water, then polishing with sand. After re- washing with water, the clean seeds were then soaked in 1% NaOCl solution for 10 min, dried with a cloth and coated with Benomyl mixed with Thiram (1:1 g per kg of seeds). Part of the seed sample was dispatched to Durban for replicating experiments. The seed consignment was received at the University of Natal, Durban (UND) on 3rd June 1997, two weeks after it had been sent. Approximately 25% of the seeds had germinated (pre-sprouted) in transit, and were set aside. The seeds were surface sterilized with 1% sodium hypochlorite solution as outlined in the protocol, dried overnight on paper towel, and coated with Benlate. The second consignment of seeds was received in Durban on 6th of June 1998. On initial inspection the seeds appeared to be in good condition and were maintained in their original packaging for a short period until used. 44 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Initial tests

Burkina Faso

100 fruits were sampled and weighed individually, after which the seeds were depulped manually and weighed again. Moisture content of the seed coat, endocarp and embryo were assessed for the 100 individual seeds, by drying them in an oven at 103°C for 17 h. The moisture content was then expressed on a percentage fresh mass basis. Four replicates of 25 seeds were sown in sand and incubated at 25°C and/or 30°C to assess their initial germination capacity.

South Africa

100 seeds were weighed individually. Moisture contents of whole seeds, as well the individual seed components, were then determined gravimetrically, by weighing before and after drying them in an oven at 80°C for 48 h. Germination capacity was assessed by partially burying 25 seeds in trays in moist vermiculite at 27 to 30°C, and scored as positive on the basis of radicle protrusion of approximately 1 mm.

Desiccation and germination trials

Burkina Faso

Six samples of seeds were mixed with silica gel (1 kg seeds: 1 kg silica gel) and dried to 20, 16, 13, 10, 8 and 5% target moisture contents. Concurrently, six controls were mixed with sawdust, and aerated periodically. Five replicates of five seeds each were used to assess moisture contents, while four replicates of 25 seeds each were sown for germination tests, as described above.

South Africa

The desiccation trial commenced on 7th June, after the initial moisture content had been determined. Desiccation to 16, 13, 10, 8 and 5% MC was carried out using activated silica gel mixed with seeds and sealed in plastic bags. The samples were weighed daily, at which time the silica gel was changed to ensure a rapid rate of drying. Control seeds were mixed with vermiculite in plastic bags and maintained for equivalent periods. Germination was also assessed as described previously. AFRICA 45

From the 1997 trials, it became apparent that the endocarp represents a formidable barrier to germination. To ensure rapid germination, and thereby decrease the proliferation of seed-associated mycoflora, the endocarp of each seed was first cracked using a vice. The seeds were then placed in seedling trays in moist vermiculite and set to germinate at 27–30°C. The second observation was that as the endocarp represents a large component of the seed, when determining moisture content at any sampling on a whole seed basis, the actual moisture content of the germinable parts may be obscured. In order to determine if this was the case, the moisture contents of whole seeds were compared with those of the individual seed components at every sampling.

Storage trials

Burkina Faso

Samples of seeds at 10, 8 and 5% MC were packed in aluminium foil bags and stored at –18°C in the freezer, at 4°C in the cool room and at 25°C in ambient room temperature. Moisture content and germination of seed samples were determined every two months. A second lot of seeds dried to 9, 6 and 3% MC were also stored in aluminium foil bags at –18, 4 and 25°C. Germination and moisture content were assessed every 3 months for 18 months.

South Africa

The seeds remaining after the first trials were kept in an unsealed plastic bag for six months at 25°C. Samples of seeds from the second lot of 1998 at 12, 9, 6 and 3% MC were stored for 3 and 6 months at –20, 5 and 15°C.

Results

Initial tests

The initial tests indicated comparable mass ratios of seed to fruit weight (0.20–0.19) for the collections in 1997 and 1998 (Table 1). The embryonic axes had higher moisture contents than the whole seeds. There was no 46 STORAGE BIOLOGY OF TROPICAL TREE SEEDS significant difference in the moisture contents of different components between the two harvests. However, seed germination varied between 25% and 75%, initially for experiments in Burkina, while initial viability was similar between harvests (45 and 49%) for seeds received in 1997 and 1998 respectively, when germinated in South Africa. The seeds of the 1998 collection were also characterized by drier moisture contents than those collected in 1997. The variation around the means of the component moisture contents of the 1997 seed lot showed that it consisted of a mixture of wetter and dryer seeds. In contrast, the 1998 seed lot was homogenous (Table 1). In both cases, however, it is apparent that representing moisture contents for S. birrea on a whole seed basis only can be misleading as this corresponds almost entirely with that of the endocarp only, while moisture contents of the axis and cotyledons were very different.

100 100

90 1997 (A) 90 1998 (A) 80 80 70 70 60 60 50 50 40 40 30 30 Germination (%) 20 Germination (%) 20 10 10 0 0 16 16 1997 (B) 1998 (B) 14 14

12 12

10 10

8 8

6 6

4 4 Moisture content (%) content Moisture Moisture content (%) CONTROL 2 2 DRIED 0 0 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 TMC (%) TMC (%)

Figure 3. Germination (A) of S. birrea seeds from Burkina Faso, desiccated to different moisture contents (B). In experiments at UND, South Africa, the seed components were also dried to various moisture contents (1998 – B). Table 1. Results of the initial tests of S. birrea seeds in Burkina Faso and South Africa. Data points are mean values of 100 individuals tested (r standard deviation) Weight (g) Germination Initial moisture content (%) (%) Fruit Seed Seed Endocarp Testa Embryo Cotyledon Axis 1997 18.08r4.7 3.66r1.0 21.6r10.9 – 30.11r4.9 – – 70 (at 25°C) Burkin 9 8 31.9r14.99 3 0 75 (at 30°C) a Faso South – 2.9r0.92 – 26.4r11.46 33.2r12. 19.7r2.76 20.0r4.14 32.2r6.96 45 (at 30°C) Africa 8 1998 20.92r3.7 4.01r0.9 11.9r5.82 – 28.77r8.3 – – Burkin 6 9 31.02r9.27 25 (at 30°C) 9 a Faso South – 2.4r0.49 – 7.9 r 1.03 12.3 0.67 12.9 2.17 9.8 5.01 8.6 1.17 49 (at 30°C) Africa r r r r AFRICA 47 AFRICA 48 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

100 100 90 1997 (A) 90 1998 (A) 80 80 70 70 60 60 50 50 40 40 30 30

Germination (% ) 20 Germination (% ) 20 10 10 0 0 25 25 WHOLE 1997 (B) 1998 (B) ENDO 20 20 TESTA COTY AXIS 15 15

10 10

5 5 CONTROL Moisture content (% content ) Moisture Moisture content (% ) DRIED 0 0 0 2 4 6 8 101214161820 02468101214161820 TMC (% ) TMC (% )

Figure 4. Germination (A) of S. birrea seeds from Burkina Faso that were desiccated to different moisture contents (B). The experiments at CNSF were carried out on whole seeds including endocarps (1997 – B) or endocarps only (1998 – B), which were not dried ({) or dried to TMC (• ).

South Africa, 1997

Overnight drying after washing resulted in moisture contents of the experimental seeds falling below that of the initial moisture content and hence the moisture contents at each target moisture content (TMC) were lower than would have been predicted by calculation. The seeds were dried down from 19.72 to 1.94% but the viability varied little (Fig. 3). South Africa, 1998: Drying the seeds to TMCs 10, 8 and 5% decreased the percentage germination to 47, 28 and 9%, respectively (Fig. 3). This was correlated with a small loss of water from the embryonic axes (i.e. 8% down to 5% moisture, Fig. 3, 1998B). AFRICA 49

Storage trials

The viability of S. birrea seeds was monitored over 18 months storage at CNSF in Burkina Faso and at the University of Natal, South Africa. In the first experiments in Burkina Faso (1997), seed viability declined after 4 months storage in all cases, irrespective of seed moisture content or storage temperature (Fig. 5). For the same lot investigated in South Africa, although the effects of seed storage could not be assessed, an interesting observation was made on the seeds that were maintained in plastic bags for six months at 25°C. Whereas the moisture content of the seeds had dropped to 4.73%, the germinability of the seeds increased to 57% (data not shown). Seeds of the second consignment were stored for 6 months in South Africa in 1998. Some of the seed samples with 9 and 12% MC that did not germinate after 3 months storage, germinated after 6 months (Fig. 6A). The mishandling of these seeds at three months might have caused such a decrease. Only seeds with 6% MC at 5°C lost viability completely after 6 months. While viability remained 20% or more for seeds with 3, 9 and 12% MC at all three storage temperatures of –20, 5 and 15°C, it was less than 10% for seeds with 6% MC (Fig. 6A) after 6 months. However, there was little change in moisture content over the storage period, when expressed on a whole seed basis (Fig. 6B). Axis moisture content decreased mostly in seeds stored at 9 and 12% MC (Fig. 6C). After 18 months of storage of the 1998 lot in Burkina Faso, viability of seeds with 3, 6 and 9% MC remained higher at 25°C than at –18°C or at 4°C (Fig. 7). However, germination varied over the months, with no clear trend over time. Seeds at –18°C had increased germination after three months then decreased to its initial level of ca. 30% from 6 to 12 months, before increasing again to 80% after 15 months. A similar trend was observed in seeds at all three moisture contents stored at 4°C storage. They maintained about 40% initial germination level up to 12 months, before increasing to 80% and then decreasing to that initial level (Fig. 7). Seeds had constant increment viability during storage at 25°C over the 18 months. No seeds lost viability during storage in all these conditions. From these investigations, the optimal storage temperature was 25°C, with whole seeds having between 8 to 10% MC. 50 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Discussion

Seeds of S. birrea appear to be desiccation tolerant. Seeds collected in 1997 could be dried to moisture contents (expressed on a whole seed basis, including the endocarps) of 5.74 to 1.94%, with little or no change in initial viability (Figs. 3 and 4, respectively). However, those seeds maintained in sawdust at their harvest moisture contents, for the same duration, exhibited a much greater decline in germination capacity, to 31% (Fig. 4, 1997A). Thus while the seeds appear to be desiccation tolerant, maintaining them at their harvest moisture contents for even a short period of time (2 days) appears to be detrimental. This might have well been the consequence of continuing activity of fungi located within the endocarp. In addition, 25% of the seeds harvested in 1997 germinated during transport to South Africa, thus drying the seeds by a predetermined extent is essential prior to transport and distribution. Seeds harvested in 1997 were of a good quality (70–75% germination) and were characterized by high moisture contents of 33.17% of the embryos (Table 1). In contrast, only 25 to 49% of the seeds collected in 1998 germinated, and embryonic moisture content was much lower (11.93 to 8.61%) (Table 1). Seed viability remained low following desiccation of these seeds in Burkina Faso (Fig. 3). Similar results have been reported for seed lots with low initial viability (Were and Munjuga 1999). In contrast, subsequent desiccation of this seed lot in South Africa decreased viability to 9%, with little change in axis moisture content (Fig. 3, 1998A, B). von Teichman et al. (1986) have shown that although the endocarp does not restrict water uptake, it does provide a substantial mechanical resistance to germination. Removing the opercula significantly increases germination (von Teichman et al. 1986; Msanga 1998), recorded as 70% after one week and 85% after two weeks of sowing (Msanga 1998). In contrast, germination of seeds with opercula still closed is sporadic, with only 50% of the seeds germinating after nine months (Msanga 1998). The endocarp may restrict germination by minimizing leaching of germination inhibitors and also serve as a barrier to oxygen diffusion (von Teichman et al. 1986). In the experiments done in South Africa, it was not always possible to locate the opercula, as the endocarps were dry on receipt and encased by dried pulp and copious amounts of fungicide. In many cases, the cracking of seeds in a vice to alleviate the mechanical resistance did not prove effective, probably due to injury of the axes. If the opercula were still intact, even after the endocarps were cracked, the seeds did not 100 100 100 25 C (A) 90 -18 qC (A) 90 4 qC (A) 90 q 80 80 80 70 70 70 60 60 60 50 50 50 40 40 40 30 30 30 Germination (%) Germination (%) 20 Germination (%) 20 20 10 10 10 0 0 0 12 12 12 25 C (B) -18 qC (B) 4 qC (B) q 10 10 10

8 8 8

6 6 6 4 4 4 5% 8% 2 Moisture content(%) 2 Moisture content (%) content Moisture 2 (%) content Moisture 10% 0 0 0 51 AFRICA 012345 012345012345 Storage time (months) Storage time (months) Storage time (months)

Figure 5. Germination (A) and moisture contents (B) after storage of S. birrea seeds from Burkina Faso, 1997. Whole seeds were dried to 5% ({), 8% (•) or 10% () MC (fresh mass basis) and stored at –18, 4°C or 25°C for 4 months. 52 100 100 100 0 0 -20 0C (A) 5 C (A) 15 C (A) 90 90 90 80 80 80 70 70 70 STORAGE BIOLOGY OF TROPICAL TREESEEDS OF TROPICAL BIOLOGY STORAGE 60 60 60 50 50 50 40 40 40 30 30 30 Germination (%) Germination (%) Germination Germination (% ) 20 20 20 10 10 10 0 0 0 14 14 14 -20 0C (B) 5 0C (B) 15 0C (B) 12 12 12

10 10 10

8 8 8

6 6 6

4 4 4 Moisture content(% ) Moisture content(% ) Moisture content (% ) 2 2 2

0 0 0 14 14 14 0 0 -20 C (C) 5 0C (C) 15 C (C) 12 12 12

10 10 10

8 8 8

6 6 6 3% 4 4 4 6% 9% Moisture content(% ) Moisture content(% ) Moisture content (% ) 2 2 2 12% 0 0 0 012345678 012345678 012345678 Storage time (months) Storage time (months) Storage time (months)

Figure 6. Germination (A) and moisture contents (B and C) of S. birrea seeds from Burkina Faso replicated at Natal University, South Africa, in 1998. Whole seeds, including the endocarp (B) or axes (C) were dried to 3% ({), 6% (), 9% () or 12% (■) MC (fresh mass basis), and then stored at –20, 5 or 15°C. • 100 100 100 -18 qC (A) 4 qC (A) 25 C (A) 90 90 90 q 80 80 80 70 70 70 60 60 60 50 50 50 40 40 40 30 30 30 Germination (%) Germination (%) Germination 20 20 (%) Germination 20 10 10 10 0 0 0 16 16 16 14 -18 qC (B) 14 4 qC (B) 14 25 qC (B) 12 12 12 10 10 10 8 8 8 6 6 6 3% 4 4 4 6% Moisture content (%) content Moisture Moisture content (%) content Moisture 2 9% (%) content Moisture 2 2 0 0 0 0 2 4 6 8 101214161820 02468101214161820 0 2 4 6 8 101214161820 AFRICA 53 AFRICA Storage time (months) Storage time (months) Storage time (months)

Figure 7. Germination (A) and moisture contents (B) of S. birrea seeds after storage at CNSF, Burkina Faso, in 1998. Seeds were initially dried to 3% ({), 6% (• ) or 9% () MC (fresh mass basis), and then stored at –18, 4°C or 25°C for 18 months. 54 STORAGE BIOLOGY OF TROPICAL TREE SEEDS germinate. According to Leger (1997), cracking of the seeds used, followed by soaking for 24 h in cold water allowed for only 27% germination totality. The decrease in seed viability following desiccation (Fig. 4, 1998) is interpreted to be (at least partially) the result of increased mechanical resistance, as the endocarps became drier, rather than a result of desiccation damage to the axes themselves, which showed little change in water content. It was also observed that profuse fungal proliferation occurred on, and in, those seeds that did not germinate. Other studies aimed at improving the germination performance of S. birrea seeds have shown that while soaking the seeds in hot water has little effect (Shone 1979), HCl acid scarification (in older seeds) greatly improves seed germination (Page, pers. comm). From an ecological perspective, elephants are the principal seed- dispersing agents. Seeds that passed through the elephant digestive tract had a much higher germination rate than those that were hand harvested from trees and placed to germinate under the same conditions (Lewis 1987). For the 1997 Burkina Faso and 1998 South Africa storage trials, seed viability decreased following 4 to 6 months of storage, with little or no change in whole seed moisture content (Figs. 5 and 6, respectively). Following storage over a longer period of 18 months (Fig. 7); however, two things were apparent: first, seed germination was highly variable, particularly for the initial 6 months. This variability may, in part, be ascribed to setting the seeds to germinate with the opercula still attached. Thus failure to germinate following storage under any of the conditions tested may be due to increased mechanical resistance (particularly at the lower water contents) rather than physiological death. Second, while the seeds were able to tolerate storage at –20°C, seed germination increased after 18 months storage at 25°C, suggesting that postharvest development may have taken place. According to Shone (1979), the seeds are immature when dropped and will germinate only following 7 to 8 months of storage, when 100% germination can be achieved. An increase in germination totality has also been reported following only 3 months storage under ambient conditions (Were and Munjuga 1999) while germination rate has been shown to increase following storage for one year at 21°C (von Teichman et al. 1986). Germination totality also increased following 6 months storage under ambient conditions for the 1997 seed lot in South Africa (results not shown). Thus, while seed after-ripening (i.e. postshedding seed development) does appear to contribute to the response of these seeds to storage, a decrease in mechanical resistance AFRICA 55 of the lignified plug over time if stored under ambient conditions may be involved, or a combination of both these factors. The combined results of these experiments seem to suggest that these seeds are orthodox, as they can withstand substantial water loss and low storage temperatures. In fact, if well dried (to whole seed moisture contents lower than 10%), seeds can be stored for up to four years (Msanga 1998). In addition, analysis of target moisture contents on a whole seed basis in this species shows a bias towards changes in the moisture content of the endocarp, while there is very little change in the actual moisture content of the embryonic axes during desiccation (Fig. 4). This emphasises the need to clarify the use of target moisture contents in these experiments on a species basis, and that it may be more beneficial to consider the moisture content of the axis and cotyledons separately, rather than that of the whole seed only.

References

Aganga, A.A. and K.W. Mosase. 2001. Tannin content, nutritive value and dry matter digestibility of Lonchocarpus capassa, Zizyphus mucronata, Sclerocarya birrea, Kirkia acuminata and Rhus lancea seeds. Anim. Feed Sci. Technol. 91:107– 113. Coates–Palgrave, K. 1977. Trees of Southern Africa. Struik Publishers, Cape Town, Republic of South Africa. Eloff, J.N. 2001. Antibacterial activity of Marula (Sclerocarya birrea (A. rich.) Hochst. Subsp. caffra (Sond.) Kokwaro)(Anacardiaceae) bark and leaves. J. Ethnopharmacol. 76:305–308. Galvez, J., M.E. Crespo, A. Zarzuelo, P. Dewitte and C. Spiessens. 1993. Pharmacological activity of a procyanidin isolated from Sclerocarya birrea bark – Antidiarrheal activity and effects on isolated guinea-pig ileum. Phytother. Res. 7:25–28. Glew, R.H., D.J. VanderJagt, C. Lockett, L.E. Grivetti, G.C. Smith, A. Pastuszyn and M. Millson. 1997. Amino acid, fatty acid, and mineral composition of 24 indigenous plants of Burkina Faso. J. Food Composit. Anal. 10:205–217. Jaenicke, H. and M.K. Thiong’o. 2000. Preliminary nutritional analysis of marula (Sclereocarya birrea).Acta Horticult. 531:245–249. Kamra, S.K. 1990. Improving the forest seed situation in some African countries. Trop. Tree Seed Res. 28:126–131. Leger, S. 1997. The hidden gifts of nature. A Description of Today’s Use of Plants in West Bushmanland (Namibia). German Development Service, Berlin, Germany. 56 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Lewis, D.M. 1987. Fruiting patterns, seed germination, and distribution of Sclerocarya caffra in an elephant-inhabited woodland. Biotropica 19:50–56. Maundu, P.M., Ngugi, G.W. and C.H.S. Kabuye (eds.). 1999. Traditional Food Plants of Kenya. Kenya Resource Centre for Indigenous Knowledge (KENRIK). National Museums of Kenya, Kenya. Msanga, H.P. 1998. Seed germination of indigenous trees in Tanzania. Including notes on seed Processing, Storage and Plant Uses. Canadian Forest Service, Alberta, Canada. Nerd, A. and Y. Mizrahi. 1993. Domestication and introduction of marula (Sclerocarya birrea subsp. Caffra) as a new crop for the Negev desert of Israel. Pp. 496–499 in New Crops (J. Janick and J.E. Simon, eds.). Wiley, New York. Nerd, A. and Y. Mizrahi. 2000. Introduction of marula, an unexploited fruit tree from southern Africa, to the Israeli Negev. Isr. J. Plant Sci. 48:217–222. Ogbobe, O. 1992. Physico-chemical composition and characteristics of the seed and seed oil of Sclerocarya birrea. Plant Foods Hum Nutr. 42:201–206. Ouedraogo, A.S. and J.A. Verwey. 1987. Forest tree seed problems in Burkina Faso (Sahelian and Soudanian regions). Pp. 238–249 in Proceedings of the IUFRO International Symposium on Forest Seed Problems in Africa, Harare, Zimbabwe. Ouedraogo, A.S., K. Thomsen, J.M.M. Engels and F. Engelmann. 1999. Challenges and opportunities for the enhanced use of recalcitrant and intermediate tropical forest tree seeds through improved handling and storage. Pp. 227–234 in IUFRO Seed Symposium 1998 “Recalcitrant Seeds”: Proceedings of the Conference (M. Marzalina, K.C. Khoo, N. Jayanthi, F.Y. Tsan and B. Krishnapillay, eds.). Forest Research Institute, Kuala Lumpur, Malaysia. Pooley, E. 1993. The Complete Field Guide to Trees of Natal, Zululand & Transkei. Natal Flora Publications Trust, c/o Natal Herbarium, Durban, Republic of South Africa. Shone, A.K. 1979. Notes on the Marula. Bulletin 58. Department of Forestry, Pretoria, Republic of South Africa. von Teichman, I., J.G.C. Small and P.J. Robbertse. 1986. A preliminary study on the germination of Sclerocarya birrea subsp. caffra. South Afr. J. Bot. 52:145–148. Weinert, I., P.K. van Wyk and L.C. Holtzhausen. 1990. Marula. In Fruits of Tropical and Subtropical Origin (S. Nagy, P.E. Show and W.F. Nardowsky, eds.). Florida Science Scource, Florida, USA. Were, J. and M. Munjuga. 1999. Preliminary findings on the storage behaviour of Prunus africana and Sclerocarya birrea seed in Kenya. Pp. 431–437 in IUFRO Seed Symposium 1998 “Recalcitrant Seeds”: Proceedings of the conference (M. Marzalina, K.C. Khoo, N. Jayanthi, F.Y. Tsan and B. Krishnapillay, eds.). Forest Research Institute, Kuala Lumpur, Malaysia. Zharare, P. and N. Dhlamini. 2000. Characterization of Marula (Sclerocarya caffra) Kernel Oil and assessment of its potential use in Zimbabwe. J. Food Technol. Afr. 5:126-128. AFRICA 57

Effects of desiccation and storage on Vitellaria paradoxa seed viability

Christiane S. Gaméné1,3, Hugh W. Pritchard2 and Matthew I. Daws2

1Centre National de Semences Forestières, 01 BP 2682 Ouagadougou 01, Burkina Faso 2Royal Botanic Gardens, Kew, Wakehurst Place, Ardingly West Sussex RH17 6 TN, UK 3Unité de Biologie Végétale, Faculté Universitaire des Sciences Agronomiques Passage des Déportés, 2. B-5030 Gembloux, Belgium

Abstract

Seeds of Vitellaria paradoxa are economically important in sub- Saharan Africa because they contain 40–60% oil. However, little is known regarding the seed biology of this species. In particular, it is not known how best to germinate and store seeds of this species. This study showed that germination rate was most rapid at 36°C and failed to occur at temperatures below 11°C. Furthermore, seeds of V. paradoxa are sensitive to desiccation to whole seed water contents below 20% and are therefore unsuitable for long-term ex situ storage under conventional (15% relative humidity and –20°C) genebank conditions. However, it is possible to store fully imbibed seeds, at least in the short term, at 16 to 21°C, viability remaining for at least several months.

Introduction

Vitellaria paradoxa Gaertn.f. , the shea-butter tree or karité is a forest tree which belongs to the Sapotaceae family. It is widely distributed, being found throughout the dryland regions of West Africa. V. paradoxa is the second most important oil seed crop in Africa after oil-palm, because of its adaptation to the dryland region. The seed contains 40–60% of its dry matter as oil (Eckey 1954), which is used in cosmetics and cooking. The tree can reach 10 to 15 m high. Its bark is thick and splintery, which protects old trees from bush fires. The leaves are 12–25 cm long and 4–7 cm wide with undulate edges. The flowers are green-yellowish 58 STORAGE BIOLOGY OF TROPICAL TREE SEEDS and are gathered in a bunch of 30 to 40 individuals (see Fig. 1). The fruits are berries, which are either green or turn to green-yellow at maturity. V. paradoxa is an important species because of its multiple uses by local communities (Boussim 1991). The seeds are also exported to the cosmetic industry in Asia, America and Europe. Considerable exploitation has made V. paradoxa a vulnerable species. Consequently it has been classified as having a high risk of becoming endangered if no conservation action is taken now (IUCN 2002). To contribute to a conservation programme for this species, we studied and report here the effects of desiccation and storage conditions on the viability of V. paradoxa seeds.

Figure 1. A drawing of the leaves, fruit, seed and inflorescence of V. paradoxa (Source: S. Kambou, B. Faso, 1996). AFRICA 59

Materials and methods

Seed collection

Two collections were made on 29–31 July 1996 and 7–9 July 1997 from the same seed source at Poa, a village located about 65 km from Ouagadougou, on the southwest road to Koudougou. Fruits were soaked in water and immediately de-pulped and cleaned. In 1996, 70 kg of seeds were prepared from 225 kg of fruits collected from 30 adult trees, and 92 kg of seeds were obtained after extraction from 325 kg of fruits collected in 1997.

Initial tests

Before and after manually de-pulping, 100 fruits and seeds from the 1996 collection were sampled and weighed individually to determine their fresh weight. The moisture contents of seeds and seed components were determined separately and gravimetrically by drying them in an oven at 103°C for 17 h. The cleaned seeds were soaked in 1% NaOCl solution for 10 min. They were dried with a cloth and then coated with fungicides in the proportions of 1 g of Benomyl mixed to 1 g of Thiram for 1 Kg of seeds. After this treatment about half of the seeds were dispatched to the Royal Botanic Gardens Kew (RBG Kew), UK. A sample of 100 seeds was taken to assess the initial germination capacity of the seed lot. The seeds were incubated in a germination cabinet at 25°C. Apart from weight determinations, the same initial tests were carried out on the seed lot collected in 1997.

Desiccation trials

At CNSF, after the initial determinations of germination and moisture contents, seeds were dried by mixing with silica gel to obtain target moisture contents of 20, 16, 13, 10, 8 and 5%. The controls were mixed with sawdust. On arrival at RBG Kew, five replicates of 20 seeds were dried in silica gel for 2, 4, 5, 7 and 14 days at 26°C and put on 1% agar, in clear plastic boxes to germinate at 26°C (12 h light d–1). 60 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Storage trials

In 1996, the storage trials were undertaken with nondried seeds stored at 25°C at CNSF and at 16°C at RBG Kew. In 1997, storage experiments were carried out with seeds dried to whole seed moisture contents of 41, 36 and 31% and stored at 25°C at CNSF. At RBG Kew the nondried seeds were maintained in vermiculite at a range of temperatures (6, 11, 16, 21 and 26°C) for 184 days to assess longevity.

Results

Initial tests

The mean fruit and seed mass of V. paradoxa was 36.71 g and 9.24 g, respectively. Table 1 presents initial moisture contents and germination capacity of seed lots from 1996 and 1997. The moisture contents of embryos were always higher than those of the whole seeds or seed coats. All seed lots had high initial germination percentage (97%). At receipt on the 6th of Aug 1996 at Kew, 10.4% of the seeds had presprouted, but there were no signs of insect or fungal infection. The average seed was 3.4r0.3 cm long and 2.4r0.2 cm wide.

Table 1. Initial moisture contents and germination capacity of V. paradoxa seeds Year Moisture content (%) Initial Whole seed Embryo Seed coat germination (%) 1996 (at CNSF) 41 47 18 97 1997 (at CNSF) 41 — – 97 1997 (at RBG Kew) 48 55 23 97

Desiccation trials

Before desiccation, seeds had a whole seed moisture content of 41–48% and germinated to approximately 99% both at CNSF and in replicating experiments at Kew (Table 2). Seed viability declined with decreasing moisture contents (Table 2) and more than 50% of seeds were killed by a whole seed moisture content of approximately 20%. AFRICA 61

Effect of temperature on germination

The effect of temperature on seed germination is shown in Table 3. High levels of germination (93–98%) were obtained at all temperatures between 16 and 36°C. The optimum temperature for seed germination and radicle expansion was 36°C. The estimated minimum temperature for growth was close to 11°C. Presprouted seeds, which were held at 6°C for just 1 week only, exhibited 2 mm of radicle growth 2 weeks after transfer to 26°C, compared to 7 mm for nonchilled material at the same temperature. Thus germinated seeds at 6°C appear to be affected by chilling stress.

Table 2. Effect of desiccation (MC %) on the viability (G %) of V. paradoxa seeds studied at CNSF and at RBG Kew. These seeds were collected in 1996 CNSF Control Dried Target MC (%) Seed MC G (%) Drying Seed MC G (%) (%) time (%) (days) 20 40 99 5 20 35 16 39 97 6 16 20 13 47 98 8 12 5 10 38 98 10 9 0 8 38 98 11 8 0 5 38 99 13 6 0 RBG Kew Initial 39 99 0 39 99 30 42 99 1 29 92 20 36 98 3 20 48 16 40 99 3 19 39 13 39 99 4 17 24 10 40 99 5 13 6 8 40 98 7 12 3 5 40 97 14 6 0

Storage trials

Fully hydrated storage at 16 and 25°C was possible for at least six months for seeds collected in 1996 (Table 4). In 1997, the effect of differing storage temperatures on longevity was investigated. In this experiment the average embryo moisture content during storage was 52 r 3% across the 62 STORAGE BIOLOGY OF TROPICAL TREE SEEDS temperature treatments. Seed viability loss lines (for each temperature) were subjected to probit analysis and the longevity parameters (the slopes of the regression line through the data) compared. Longevity increased in the following sequence of temperatures: 6°C <11°C <26°C <16°C <21°C. At 21°C, a value of 164 days to lose one probit of viability indicates that to drop from >99% to approximately 2% viability would take an estimated 820 days. In comparison for the same loss of viability to occur at 6°C would take an estimated 65 days.

Table 3. Effects of temperature on germination and radicle growth of presprouted seeds (1996, RBG Kew) Temperature Final Time to 50% Increase in (°C) germination germination (days) presprouted radicle (%) length after 14 d (mm) 11 3 – — 16 98 11 2.5 21 98 9 5.5 26 98 6 7 31 95 5 7 36 93 4 12

Table 4. Effect of storage at 16°C (RBG Kew) and 25°C (CNSF) on viability of V. paradoxa seeds collected in 1996 Nominal Storage Storage Whole seed MC Germination whole seed temperature (°C) duration (days) after storage (%) after MC (%) storage (%) 41 25 6 39 97 25 21 43 98 25 40 38 98 25 70 39 96 25 140 38 22 46 16 0 46 98 16 31 43 98 16 56 40 95 16 84 38 93 16 114 41 78 16 150 38 65 16 191 34 42

Further, the effect of partially hydrated storage, at 25°C, was investigated. Seeds fully hydrated had all germinated after five months of storage (Fig. 2). In contrast, seeds that were reduced to whole seed moisture content of 32 or 28%, exhibited no presprouting during storage. In addition, they retained some AFRICA 63 viability for the 11 months of the storage experiment. Subjecting the mortality curves to probit analysis revealed that at 32 and 28% moisture content 280 and 200 days, respectively, were required for viability to drop by one probit. Thus for viability to decrease from a starting level of 82% should theoretically take 27 and 18 months, respectively (extrapolation from the regression lines in Fig. 2).

100

7 90

80 6 70

60 5 50

4 40

30 Moisture content (%) content Moisture Germination (probits) Germination 3 20

10 2 0

024681012 Storage period (months)

Figure 2. Viability and whole seed moisture content of V. paradoxa seeds stored with initial moisture contents of 28% (triangles), 32% (squares) and 50% (circles) and at 25°C for 11 months at CNSF. Seeds were from the 1997 collection. Open symbols are germination data and closed symbols are moisture content data.

Discussion

Germination

Germination of V. paradoxa was most rapid at 36°C. This is typical for seeds of many tropical species (see Tompsett and Kemp 1996; Pritchard et al. 1995a) and is probably explained by the fact that in the natural environment, dispersed seeds are likely to experience soil temperatures of 36°C or more (Daws et al. 2002). In addition, 64 STORAGE BIOLOGY OF TROPICAL TREE SEEDS germination was almost completely inhibited at 11°C and seeds failed to germinate after transfer to 26°C. Thus like many tropical species (Corbineau and Côme 1988), seeds of Vitellaria paradoxa are chilling sensitive.

Desiccation

Seeds of V. paradoxa are recalcitrant. Desiccation to relatively high moisture content (approximately 20%) results in a loss of seed viability. V. paradoxa seeds are large (ca. 9 g), which is typical for recalcitrant species (Hong and Ellis 1996), and large seed size may reduce the rate of seed desiccation. Subsequently, this would reduce the likelihood of desiccation induced death of seeds in the natural environment.

Storage

The results of this study indicate that V. paradoxa seeds can be stored fully hydrated at 25°C while retaining some viability for at least 11 months. Recalcitrant seeds are generally thought to be short-lived and the potential longevity we report here is comparatively high (Danthu et al. 2000). Nonetheless, Pritchard et al. (1996) reported that recalcitrant seeds of Aesculus hippocastanum could be stored fully hydrated at 16°C for up to three years. The fact that all V. paradoxa seeds stored fully hydrated at 25°C germinated in storage (Fig. 2) and the sensitivity of the seeds to temperatures <11°C suggests that short-term storage, in this species, is a balance between reducing germination rates by reducing the temperature while avoiding chilling injury. In this species temperatures between 16 and 21°C seem optimal. It has been suggested that the storage of recalcitrant seeds at reduced, nonlethal, water contents (‘sub-imbibed’ storage) may enhance longevity by reducing the rate at which seeds progress towards germination in storage (King and Roberts 1980). This study indicates that sub-imbibed storage maybe beneficial for V. paradoxa seeds. Seeds stored fully hydrated at 26°C were predicted to lose one probit of viability in 44 days (and all presprouted within five months of storage) (Table 5). However, when stored at reduced moisture contents (at 25°C), seeds lost one probit of viability every 200 (28% moisture content) or 280 days (32% moisture content) (Fig. 2). It has AFRICA 65 been suggested that any desiccation of recalcitrant seeds will result in an accumulation of desiccation induced damage and hence a shortening of potential longevity. This has been observed in seeds of Araucaria hunsteinii and Trichilia dregeana (Pritchard et al. 1995b; Drew et al. 2000). However, unlike these two studies where sub-imbibed storage resulted in a significant decrease in seed longevity, sub- imbibed storage of V. paradoxa does not appear to be particularly damaging and may in fact be beneficial for extending longevity. Similarly, partial drying improves some aspects of seed quality in recalcitrant Aesculus hippocastanum (Tompsett and Pritchard 1998). Clearly further work on the effect of partial drying on recalcitrant seed viability and storability is required.

Conclusion

The seeds of Vitellaria paradoxa do not tolerate desiccation; they are recalcitrant, losing viability below 20% moisture content. However, hydrated storage is possible for >6 months at temperatures around 16– 26°C. Reducing temperature from 25 to 16–21°C increased longevity while storage at temperatures below 16°C accelerates viability loss through chilling injury.

Table 5. Effect of temperature on the longevity of V. paradoxa seeds. Seed moisture content=48%, embryo moisture content=50–55% (1997, RBG Kew) Storage temperature (°C) Longevity (probits d–1)1 6 13 11 26 16 114 21 164 26 44 1The values represent the time taken for viability to fall by one probit (area of a normal curve corresponding to one standard deviation of the mean), for example, from 84 to 50% germinability.

References

Boussim, I. 1991. Contribution à l'étude des Tapinanthus Parasites du karité au Burkina Faso. Thèse de Doctorat 3è Cycle, Option Biologie et Ecologie Végétales, ISN/ IDR, Univ., Ouagadougou. Pp. 131. 66 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Corbineau, F. and D. Côme. 1988. Storage of recalcitrant seeds of four tropical species. Seed Sci. Technol. 16:97–103. Danthu, P., A. Gueye, A. Boye, D. Bauwens and A. Sarr. 2000. Seed storage behaviour of four Sahelian and Sudanian tree species (Boscia senegalensis, Butyrospermum parkii, Cordyla pinnata and Saba senegalensis). Seed Sci. Res. 10:183–187. Daws, M.I., D.F.R.P. Burslem, L.M. Crabtree, P. Kirkman, C.E. Mullins and J.W. Dalling. 2002. Differences in seed germination responses may promote coexistence of four sympatric Piper species. Funct. Ecol. 16:258– 267. Drew, P.J., N.W. Pammenter and P. Berjak. 2000. ‘Sub-imbibed’ storage is not an option for extending longevity of recalcitrant seeds of the tropical species, Trichilia dregeana. Seed Sci. Res. 10:355–363. Eckey, E.W. 1954. Vegetable Fats and Oils. Reinhold Publishing Corporation, New York. Hong, T.D. and R.H Ellis. 1996. A protocol to determine seed storage behaviour. IPGRI Technical Bulletin No. 1. Rome, Italy. IUCN. 2002. 2002 IUCN Red List of Threatened Species [also at http://www.redlist.org]. King, M.W. and E.H Roberts. 1980. Maintenance of recalcitrant seeds in storage. Pp. 53–89 in Recalcitrant Crop Seeds (H.F. Chin and E.H. Roberts, eds.). Kuala Lumpur, Malaysia, Tropical Press SDN. BHD. Pritchard, H.W., P.B. Tompsett and K.R. Manger. 1996. Development of a thermal time model for the quantification of dormancy loss in Aesculus hippocastanum seeds. Seed Sci. Res. 6:127–135. Pritchard, H.W., A.J. Haye, W.J. Wright, and K.J. Steadman. 1995a. A comparative-study of seed viability in Inga species – desiccation tolerance in relation to the physical characteristics and chemical composition of the embryo. Seed Sci. Technol. 23:85–100. Pritchard, H.W., P.B. Tompsett, K. Manger and W.J. Smidt. 1995b. The effect of moisture content on the low temperature responses of Araucaria hunsteinii seed and embryos. Ann. Bot. 76:79–88. Tompsett, P.B. and R. Kemp. 1996. Database of Tropical Tree Seed Research. Royal Botanic Gardens, Kew, Australia. Tompsett, P.B. and H.W. Pritchard. 1998. The effect of chilling and moisture status on the germination, desiccation tolerance and longevity of Aesculus hippocastanum L. seed. Ann. Bot. 82:249–261. AFRICA 67

Desiccation and storage of Prunus africana seeds (Hook. f.) Kalkm

James Were1, Nioses Munjuga1, Matthew I. Daws2, Nthabiseng Motete3, Deon Erdey3, David Baxter3, Patricia Berjak3, Hugh W. Pritchard2, Catherine Harris2 and Caroline A. Howard2

1International Centre for Research in Agroforestry (ICRAF) in Kenya 2Royal Botanic Gardens, Kew, Seed Conservation Department, Wakehurst Place, West Sussex RH17 6TN, UK 3Plant Cell Biology Research Unit, School of Biological Sciences, University of KwaZulu-Natal, Durban 4041, South Africa

Abstract

Prunus africana is a highly utilized tree species, which is currently threatened by overexploitation. However, little is known about the seed biology of this species. Consequently it is not clear whether seeds can tolerate desiccation and subsequent storage at sub-zero temperatures. Here we report the results of desiccation as well as storage at various moisture contents and temperatures on seed viability of P. africana. The results indicate that seeds can tolerate desiccation to approximately 5% moisture content and can be stored, at least for three months, at –20°C. Consequently seeds of P. africana are probably orthodox in seed storage behaviour.

Introduction

Recently, the demand for seedlings of Prunus africana (Hook. f.) Kalkm., has increased in Kenya and elsewhere due to the high value of its timber and the increasing importance of the medicinal properties of its bark (Simons et al. 1998). The ‘Over The Counter’ (OTC) value of preparations based on Prunus bark extract, for the treatment of benign prostatic hyperplasia, is estimated at US$220 million annually. Large quantities of seeds need to be collected and stored for future uses, as P. africana is a mast fruiter, only producing seed in certain years. Observations on the forest floor one or two months after seed dispersal reveal high levels of germination with thousands of seedlings (wildings) under fruiting trees. Wildings are shade intolerant and most die unless 68 STORAGE BIOLOGY OF TROPICAL TREE SEEDS habitat disturbance occurs. In addition, wildings do not transplant well. Thus they do not contribute significantly to regeneration of the species. This calls for seed collection in order to raise seedlings in tree nurseries. An additional problem with the propagation of P. africana is that little is known about the seed biology of this species, and what information there is suggests that seed viability is rapidly lost post shedding. Here we report results of desiccation and storage trials on seeds of P. africana from two regions of Kenya. The results are discussed in the context of improving current methods of handling and storing P. africana seeds.

Materials and methods Seed lot details and processing

Fruits were collected in the Cherangani Hills, Kenya and sent to the University of Natal, Republic of South Africa (RSA; batch 1) and Wakehurst Place, United Kingdom (UK). Further fruits were collected from the Kakamega and South Nandi forests and sent to RSA (batch 2) and used at ICRAF in Kenya. The seeds were extracted from the fruits by rubbing the fruits against a wire mesh under running water (RSA and UK) or by mixing the fruits with sand and rubbing against a wire mesh (ICRAF). Seeds used at ICRAF and RSA batch 2 contained seeds at varying stages of maturity. Seeds were consequently sorted into the following developmental stages (based on fruit colour): green (immature; RSA and ICRAF), purple-green (intermediate; ICRAF) and purple (mature; RSA and ICRAF).

Seed desiccation

Cleaned seeds were dried, at room temperature, to a range of moisture contents, using silica gel, and sown either on sterilized sand (ICRAF) or filter paper (RSA) or 1% agar in water (UK) in Petri dishes. Seeds were germinated in temperature controlled incubators at between 25 and 30°C. Both maturity classes of batch 2 (RSA) were subjected to desiccation. However, only fully mature seeds were desiccated at ICRAF.

Seed storage

Seeds of RSA batch 2 (from both green and purple fruits) were dried to a water content, which corresponded to approximately 90% of the initial AFRICA 69 whole seed moisture content. These seeds and nondried control seeds were subsequently stored at either 16 or 25°C for up to 16 weeks. Furthermore, seeds (from both green and purple fruits) were dried to four target moisture contents (12, 9, 6 and 3%) and stored at either 15, 5 or –20°C for three months. In this experiment fungal infected seeds were removed post desiccation (but before storage) and discarded.

Results Initial seed lot details

Table 1 presents some initial characteristics of the various seed lots. It should be noted that for RSA batch 2 and the seed lot used at ICRAF the whole seed moisture content of immature seeds was greater than for mature seeds.

Table 1. Initial seed lot details Seed lot Fruit colour Fresh seed weight Whole seed MC (%) (g) RSA batch 1 — 0.28 42.2 RSA batch 2 Green 0.20 68.5 Purple 0.19 56.6 UK — 0.46 49 ICRAF Green — 49 Purple-green — 47 Purple — 46

Seed desiccation tolerance

Seed desiccation to approximately 10% MC resulted in an increase in seed germination for both immature (RSA 2) and mature (RSA 2) seeds. Further desiccation of these two seed lots (to ca. 5% MC) resulted in no germination. Desiccation of seeds of RSA batch 1 to moisture content as low as 5% had little or no effect on germination: germination remained greater than 80%. Desiccation of the ICRAF seed lot to 5% resulted in a gradual reduction in germination with germination falling to ca. 5% at this moisture level (Fig. 1). 70 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

100 RSA 1 RSA 2 Immature RSA 2 Mature ICRAF 80

40 Germination (%)

0 10203040506070 Whole seed moisture content (%)

Figure 1. The effect of desiccation, at room temperature, on seed germination of P. africana seeds.

Seed storage

Figure 2 indicates that mature seeds of P. africana can be stored for up to three months at moisture contents ranging from 3 to 30%. Viability is best retained at 15°C, although some viability is retained (especially at the lower moisture contents) at –20°C. It is interesting to note that seeds of this batch (RSA 2) were able to tolerate desiccation to 2.92% MC unlike in Figure 1. Immature seeds of P. africana can also be stored for up to three months at 15°C, but no viability is retained following storage at –20°C (Fig. 3). Mature seeds can also be stored both fully hydrated, or at 90% of full hydration at 16 or 25°C for up to 16 weeks although at both temperatures viability does decline during storage (Fig. 4). Immature seeds stored less well at 16 and 25°C, than mature seeds with viability reaching low levels by eight weeks storage. In addition, partial drying appears to have an adverse affect on storability (Fig. 5) although there was little adverse effect for mature seeds (Fig. 4). AFRICA 71

100 30.54 %MC 4.03 %3.60 % MC 2.92 % MC

40 (%)

20 Germination 0 C 15 5 -20 C 15 5 -20 C 15 5 -20 C 15 5 -20 Treatment Figure 2. The effect of desiccation to various moisture contents and storage at 15, 5 and –20°C for three months on germination of mature P. africana seeds (RSA batch 2). Also shown are germination results for desiccated, but nonstored controls.

100 6.53 % mc 7.52 % mc 5.38 % mc 3.42 % mc

40 Germination (%) Germination

0 C 15 5 -20 C 15 5 -20 C 15 5 -20 C 15 5 -20 Treatment Figure 3. The effect of desiccation to various moisture contents and storage at 15, 5 and –20°C for three months on germination of immature P. africana seeds (RSA batch 2). Also shown are germination results for desiccated, but nonstored controls. 72 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

70 16 °C 25 °C 60

30 Germination (%) 20

0 Storage time 0 2 4 8 16 2 4 8 16 (wks)

Figure 4. The effect of storage at 16 and 25°C on germination of mature P. africana seeds (RSA batch 2). Shaded and open bars correspond to seeds at moisture contents of 55 and 48%, respectively.

45 16 °C 25 °C

20 Germination (%) 15

0 Storage time 0 2 4 8 2 4 8 (wks)

Figure 5. The effect of storage at 16 and 25°C on germination of immature P. africana seeds (RSA batch 2). Shaded and open bars correspond to seeds at moisture contents of 69 and 63%, respectively. AFRICA 73

Discussion

Desiccation

Figure 1 suggests that seeds of P. africana are at least partially tolerant of desiccation. This is in agreement with previous studies which found that seeds retained at least some viability at a MC of 15% (Schaefer 1990a,b). In this current study, even immature seeds (RSA batch 2) survived substantial desiccation to levels lower than would be expected for recalcitrant seeds. Surprizingly there were somewhat different results between seeds of RSA 2 dried for the desiccation screen and for the storage work. In one experiment desiccation reduced viability (see Fig. 1) and in the other viability was maintained (Fig. 2). This indicates that while seeds can be at least classified as nonrecalcitrant they must nonetheless be handled carefully or viability loss may occur. An increase in germinability was observed for two seed lots following desiccation. This pattern has been observed in several recalcitrant species including Aesculus hippocastanum (Tompsett and Pritchard 1998) and has been interpreted as allowing a period of further seed maturation. Thus, it appears that seeds of P. africana may require a further period of maturation post shedding for full germination competence to be achieved.

Storage

Mature seeds of P. africana can be successfully stored, for at least three months, at a range of moisture contents and storage temperatures (Figs. 2 and 4). Immature seeds store less well at all temperatures and moisture contents and do not survive storage at –20°C. The mature seeds survive –20°C at moisture contents between 30 and 3% suggesting that sub-zero temperatures maybe potentially useful for longer term storage of this species. Intermediate species (sensu Ellis et al. 1990) are tolerant of, at least, partial desiccation, but rapidly lose viability at sub-zero temperatures. Thus Ellis et al. (1991) found that seeds of Carica papaya tolerated desiccation to ca. 8% MC, but further desiccation or storage at –20°C resulted in a loss of viability. In contrast, the fact that comparatively 74 STORAGE BIOLOGY OF TROPICAL TREE SEEDS little viability is lost after three months at –20°C suggests that seeds of P. africana are probably not intermediate, sensu Ellis et al. (1990).

Conclusions

The results of this study indicate that it is important to either collect only mature seeds or to sort seeds into mature and immature seed classes. In addition, seeds of P. africana can tolerate at least some desiccation and show some promise for storage at sub-zero temperatures. However, further work is needed to determine for how long seeds can be stored at sub-zero temperatures.

References

Ellis, R.H., T.D. Hong and E.H. Roberts. 1990. An intermediate category of seed storage behaviour? I. Coffee. J. Exp. Bot. 41:1167–1174. Ellis, R.H., T.D. Hong and E.H. Roberts. 1991. Effect of storage temperature and moisture on the germination of papaya seeds. Seed Sci. Res. 1:69–72. Schaefer, C. 1990a. Seed testing research on species indigenous to Kenya. Pp. 132–139 in Tropical Tree Seed Research – ACIAR Proceedings No. 28. Schaefer, C. 1990b. Processing, storage and germination of Prunus africana seeds. Technical Note No. 10. Kenya Forestry Research Institute. Simons, A.J., I.K. Dawson, B. Duguma and Z. Tchcoundjeu. 1998. Passing Problems: Prostrate and Prunus. Herbal Gram No. 43. Tompsett, P.B. and H.W. Pritchard. 1998. The effect of chilling and moisture status on the germination, desiccation tolerance and longevity of Aesculus hippocastanum L. seed. Ann. Bot. 82:249–261. AFRICA 75 Desiccation sensitivity of seeds of four tree species of economic importance in Kenya

William Omondi

Kenya Forestry Research Institute, Forest Seed Centre, PO Box 20638 City Square, Nairobi, Kenya

Abstract

The objective of this paper was to assess the desiccation tolerance and storage potential of seeds of Dovyalis caffra, Kigelia africana, Melia volkensii and Syzygium guineense, all of which are tree species of socioeconomic importance in Kenya. Seeds were collected when mature and desiccated using silica gel. Desiccation responses varied between species. Both D. caffra and K. africana tolerated drying to about 10% MC, maintaining viability of ca. 80 and 53%, respectively. In contrast, seeds of S. guineense lost their viability upon drying to 10% MC. M. volkensii seeds had a very low initial viability and exhibited a high level of desiccation sensitivity. The present result indicates potential for dry storage of the first two species.

Introduction

Kenyan forests exist as various fragile ecosystems, i.e. moorland on mountains, moist highland forests, dry forests, evergreen and semi- evergreen woodlands, savanna, coastal forests, riverine vegetation and mangroves. The Forest Department of Kenya controls, manages and develops all forest resources of the country. Forests contribute to the economic development process through industrial and domestic uses (Ongugo 2000). The Kenya National Policy published in 1994 principally aims to (a) increase the forest and tree cover in the country, (b) conserve the remaining natural habitats and to rehabilitate and conserve their biodiversity, and (c) manage the forest resources for maximum sustainable benefits. The Arid and Semi Arid lands of Kenya are endowed with trees with potential for economic and social development. However, these trees are currently threatened with extinction through overexploitation. 76 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Ex situ conservation of plant genetic resources through seed storage in Kenya is undertaken by the National Genebank, which holds over 40 000 accessions of seeds mostly of crops and their wild relatives. The total cold storage capacity at the gene bank is 150 m3 divided into two chambers operating at –20 and 5°C. The Kenya Forestry Seed Centre collects and distributes tree seeds for planting. Seeds are stored at the Centre’s cold facilities operating at 1, 3 and 10°C, with a maximum capacity of 12 tonnes that are principally used to hold stocks of seeds prior to their distribution to users. In addition, about 15 forest species are conserved under a conservation programme in 10 natural stands, and 5 seed orchards for indigenous species have been established. The Forest Department has long been the major recipient of seeds from the KFSC and the choice of species for the afforestation programmes was mainly biased towards exotics for plantations, e.g. Pinus and Cupressus species. However, during the last decade, demand for indigenous species has increased significantly due to awareness and promotion, and demand for nontimber tree products for medicines, soil improvement, edible fruits and forage. Under this changing scenario, the KFSC had to redesign its strategy in seed production strategy to meet diversified demands. It was during this intervention that problems associated with handling of seeds of several species of economic potential became apparent. The main threats to indigenous dryland species are charcoal production by local communities. Most of these species are slow growing and are not included in planting programmes. Furthermore, technical limitations in seed handling, storage and seedling production are the main problems encountered in promoting these species. The high cost of commercial timber for building and furniture has similarly driven rural communities and traders to indiscriminate tree felling, both for local uses and for sale. Most of these species are products of natural regeneration over many years and only in rare circumstances are they planted. The production and handling of their seeds is hampered by factors, which are yet to be overcome. Similarly the conversion of natural vegetation into agricultural lands and settlements poses a real threat to biodiversity. Today, forest cover accounts for less than 2.8% of Kenya’s total area. Developing protocols and supplying high quality seeds, which can be used to raise seedlings in sufficient quantities to meet demand, are therefore necessary. To this end, basic studies on seed production, collection and handling need to be devised for a range of species for which the demand for planting material is increasing. AFRICA 77

Species information

Dovyalis caffra (Hook. f. & Harv.) Hook. f. of the Flacourtiaceae family is native to southern and eastern Africa. The species is widely cultivated and establishes well over a wide geographical and altitudinal range (200–2000 m). The shrub can attain heights of 10–15 m. Flowers are insect pollinated and the young green fruits turn yellow and later orange before natural fruit fall occurs. The physiology of its seeds is confusing because they have been classified as both orthodox and recalcitrant by different sources (Albrecht 1993). D. caffra is the most sought after species for live hedges of homes and Institutions. Melia volkensii Gurke belongs to the Meliaceae family. The species occurs naturally in semi arid to arid zones of southern Somalia through Kenya to Tanzania (Dale and Greenway 1961). It grows to a height of 15 m. Its main uses include fuelwood, construction timber, medicine, fodder, bee forage, shade, mulch, green manure and erosion control (Teel 1985; Milimo 1986; Dale and Greenway 1961). It coppices readily, is fast growing and sheds its leaves in the dry season to provide mulch. These characteristics make it a popular species for dry areas where it is intercropped with agricultural crops. Although farmers use various methods, the most common method of propagation is from seeds. The seeds, however, exhibit physical dormancy, which have slowed the rate of its adoption and utilization by many farmers and conservation efforts. In the laboratory the seeds germinate when soaked and nicked as described by Milimo (1986). As an alternative, there is a recent initiative to promote large-scale distribution of M. volkensii seedlings for on-farm planting and this seems to be a viable option. Syzygium guinense (Willd.) DC. (ssp. guineense) of the Myrtaceae family is a dense leafy tree, usually 10–15 m but can reach up to 25 m. The species is widely distributed in Africa with several subspecies occurring from coastal areas to 2100 m requiring rainfall of over 1000 mm a year. It prefers moist soils with a high water table along rivers but will also grow in open woodland. Its main uses are charcoal, timber, tool handles, fruit/food, medicine (bark and roots), fodder, bee forage and tannin/dye (ICRAF 1992). The crown is rounded and heavy, with drooping small branches. Leaves are dark green in opposite pairs, smooth on both surfaces. Flowers are white in dense heads, the honey-sweet smell attracting many insects. Its edible fruits are one seeded and occur in bunches. They are oval, green when immature and dark purple and shiny when mature. No conservation 78 STORAGE BIOLOGY OF TROPICAL TREE SEEDS initiatives have been undertaken, perhaps due to its abundant status. Difficulties associated with seedling production and distribution, have inhibited its large-scale planting in many areas where the fruits are sold. This is attributed to the nonviability of dried seeds and the logistics of handling and distributing fresh seeds as planting materials. Kigelia africana (Lam.) Benth., the sausage tree, is a member of the Bignoniaceae family. The species is widely distributed in sub-Saharan Africa. According to the Survey of Economic Plants for Arid and Semi Arid Lands (SEPASAL) Database (2002) there are 21 synonyms of the same species. Morphological and size variations occur in East Africa with obvious differences in tree and fruit size. The tree can easily be recognized by the large sausage-like hanging fruits. Bats are the principal pollinators. Fruits are between 0.5 and 1 m long, 12 cm thick and weigh up to 10 kg when mature. Various parts of the tree possess medicinal properties used by communities in its region of occurrence. In Kenya, slices of the baked ripe fruits are used to flavour and make potent traditional beer. Natural regeneration occurs during the rainy season when the seeds are washed away by water and dumped in sites where available moisture levels are high enough to facilitate the germination process. No organized seed collection and storage has been undertaken in Kenya, and seedling production is rare perhaps due to the technical difficulties in collecting seeds from the large fruits. However, given the potential medicinal uses and the declining numbers of the individuals within its natural range, it is necessary to develop an appropriate protocol for seed and seedling handling, procurement and long-term conservation.

Materials and methods

Seed collection and processing

Fruits were collected during their peak maturity in Kenya. Seeds were manually extracted from the mature fruits. The pulpy fruits of D. caffra and S. guinense, were first soaked in water to facilitate softening prior to seed preparation by squeezing and continuous washing with water. Seeds of K. africana were extracted by pounding the fruits with heavy sticks. Seeds were then scraped out of the fibrous inner parts, cleaned by rubbing over a wire mesh and washed with water to remove excess fibrous materials. Fruits of M. volkensii were depulped using a pestle and mortar. Seeds were further extracted from the nuts, using the method described by Milimo (1986). The stony endocarp was horizontally placed on a tree AFRICA 79 stump. A sharp pocket knife was put across the endocarp and several hammer blows were gently applied until the nut was cracked. The cracked nuts were then gently separated and the seeds were pulled out.

Desiccation, storage and germination trials

Samples of fresh seeds of all the species were used to determine weights, moisture contents and germination capacity. Seeds were desiccated, by mixing them with silica gel, as described in the project protocol (IPGRI/DFSC 1999). D. caffra seeds were placed in mosquito nets over silica gel in a plastic container. The silica gel was frequently changed to ensure rapid drying. Moisture contents of at least two replicates of five seeds were determined by weighing them before and after drying in an oven at 103°C for 17 h (ISTA 1999). Seeds with different moisture contents were drawn for both germination and storage trials. Samples of seeds were then sealed in aluminium foil bags and stored under different temperatures of 20±2, 3 and –20ºC. Short-term storage trials at different conditions were carried out basically to determine seed survival when exposed to varying temperatures. Germination tests were conducted after 3, 6 and 12 months of storage. Seeds of M. volkensii and S. guinense were sown on 1% water-agar and then incubated in germination cabinets at 26–28°C, while those of K. africana and D. caffra were germinated in sterile river sand contained in a glass house with alternating temperatures of 20 and 35°C. Seeds were recorded as germinated when their radicles emerged. The calculated germination value (GV), which combined both the germination speed and the total germination, was evaluated using the formula: GV=(DG/N)yGP/10 (where: DG=daily germination speed obtained by dividing the cumulative germination percent by the number of days since sowing; N=number of counts starting from the date of first germination; GP=germination percent and 10=constant). Means of GV were compared using the Duncan Multiple range (P=0.05).

Results

D. caffra seeds initially germinated 98% (Table 1). Desiccation results showed that seeds dried down to 4% MC, germinated 97%, maintaining initial viability. There were slight variations but not 80 STORAGE BIOLOGY OF TROPICAL TREE SEEDS significant in the germination percentages during desiccation. For seeds at 11% MC, the moisture content of the embryo was 19.6%, while that of the seed coat and endosperm was ca. 33.4%. A fruit weighed on average 16.6 g and a seed 0.04 g (data not shown). The germination values (GV) of the treatments decreased with increased desiccation. Retaining fresh seeds with 52% MC overnight before initiating the desiccation process increased their mean germination value from 9 to 15 days. The germination value of the seeds significantly decreased when seeds were desiccated below 24% MC (Table 1).

Table 1. Germination (G) of D. caffra seeds after desiccation in silica gel to different moisture contents (MC). Values with the same letters are not significant different (p=0.05). MC (%) G (%) Days to maximum germination GV 53 97 15 8.6a 52 97 9 20.9b 35 98 14 8.6a 32 98 12 8.4a 29 98 15 7.5a 24 87 14 9.7a 22 97 15 5.7c 17 83 14 5.5c 11 84 14 5.6c 4 97 18 4.4c

Seed survival after three months storage at 3ºC remained high, ranging between 89% germination for 11% MC and 95% germination for 32% MC, whilst survival at ambient storage for seeds with 11 and 17% MC was 79 and 81% germination, respectively. All seeds stored at –20ºC lost viability. After six months, all seeds lost viability under all storage conditions (Table 2). An average mature fruit and seed of Kigelia africana weighed 5r2 kg and 0.35r0.1 g, respectively. The initial moisture content of fresh seeds was 42%, while that of the fruit pulp was 73%. Fresh seeds germinated to 67%. There was only a slight reduction in total germination values with desiccation. Seeds dried to 8.4% MC germinated 53% (Fig. 1). This change was, however, not significant given that the initial viability of the seed lot was 70%. AFRICA 81

Table 2. Survival (% germination) of D. caffra seeds after 3 months’ storage under different conditions. MC (%) Storage temperatures 25ºC 3ºC –20ºC 11 79 89 — 17 81 89 — 22 0 98 0 24 0 92 0 29 0 98 0 32 0 95 0 53 0 42 0

100

Germination (%) 20

0 42 40 35 30 20 10 5 seed moisture content (%)

Figure 1. Germination capacity during desiccation of K. africana seeds.

A whole seed of Melia volkensii weighed between 0.35 and 0.37 g. Dark brown mature seeds with 42% initial MC germinated 38% (Table 3). Few seeds germinated (4%) when dried down to 10.3% MC, but no seeds survived below this moisture level, indicating their desiccation sensitivity. Although not part of this study, seeds of different maturity stages based on colour (brown and light brown) were also tested. The light brown seeds had lower initial germination percentage and did not germinate at higher moisture levels. 82 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Table 3. Germination after drying dark brown mature seeds of M. volkensii Drying time (h) MC (%) Germination (%) 0 42 38 6 16.4 11 12 12.6 6 24 10.3 4 30 6.4 0 36 5.2 0

Under normal conditions, the initial viability of freshly harvested M. volkensii seeds attained 40–80%. However the accessions germinated on agar-water in this study had low viability (<40%) due to heavy seed infestation by fungi. The fungi proliferated more during germination. However, a similar test carried out in sand in a glasshouse at relatively high temperature yielded 60 and 65% germination for dark brown and brown seeds, respectively when sown at an initial MC of 42%. The desiccation rate of immature seeds (r2=0.75) was higher than those of mature seeds (r2=0.92) (data not shown). S. guineense seeds were shed at >45% MC and initially germinated to ca. 90%. By reducing the moisture content from 45 to 22.5%, 41% of viability was lost. The embryos had 80.4% MC at maturity as compared to 42% MC for the seeds. The mass ratio of embryo to seed was 0.005. Fresh seeds and embryos were desiccated to various target moisture contents. Subsequently, viability decreased from 87% germination for seeds at 39% MC to 8% germination for seeds at 22% MC. This reduction of viability to 8% occurred at 27.2% MC for embryonic axes. No seeds germinated below this moisture level (Table 4).

Table 4. Moisture contents (%) of S. guineense seeds and embryos, and their germination capacity (G%) after desiccation

Target MC (%) Actual MC (%) Embryo MC (%) G (%) 35 38.6r1.60 66.2r0.42 87 30 28.7r0.77 39.4r1.06 76 25 28.0r1.36 34.3r1.04 38 20 21.6r0.21 27.2r1.08 8 10 14.6r1.34 13.0r0.64 0 5 6.3r0.37 6.3r0.37 0 AFRICA 83

Discussion

Dovyalis caffra seeds were able to withstand desiccation to 10% MC and storage at 4°C, indicating that these seeds can be dried and distributed in this state for effective sowing and seedling production. The results also provide an indication that relatively moist (11% MC) seeds of this species tolerate chilled (3°C) storage (Table 2). There was an increase in the seed moisture contents due to the processing, as shown in our previous report (Omondi et al. 2000). Such a variation, however, did not affect the germination capacity of the seeds during any of the experiments. The morphological structure and nondehiscent fruits of Kigelia africana don’t provide distinct maturity indicators, making it difficult to select mature fruits. K. africana seeds are nonetheless desiccation tolerant and have the ability to survive low temperatures in the short term (Omondi et al., unpublished data). Further medium and long- term storage trials under low temperature conditions are underway. This study has established that K. africana can be propagated via seeds that can be dried and distributed effectively. This provides opportunity for its domestication in many areas from which its products and services will be easily accessible. Seeds of Melia volkensii exhibited a low initial level of viability and a high level of desiccation sensitivity. Under natural conditions, the stony endocarp reduces seed desiccation upon maturity. Presently, in Kenya, conservation of this species is mainly undertaken through intensive farm planting with assistance from tree planting projects whose major role is to distribute seedlings raised at Institutional nurseries. Attempts by farmers to raise their own seedlings are still hampered by difficulties with breaking the seed coat dormancy. Mass production of seedlings by experienced workers at central sites is therefore the best way of ensuring the planting of more trees of the species. More investigations need to be undertaken on this species. Syzygium guineense (ssp. guineense) seeds exhibited a high level of desiccation sensitivity. Attempts to sow the fruits without depulping them have been tried in many nurseries without success. These results suggest that the seeds can best be maintained in the hydrated state. Fungal proliferation was evident particularly on the desiccated seeds. The most practical alternative, however, is the promotion of on-farm planting and field genebanks in designated areas, e.g. marketplaces, schools and other trust lands. More research needs to be done on 84 STORAGE BIOLOGY OF TROPICAL TREE SEEDS alternative methods of conservation, for example, cryopreservation of embryos as has been suggested for Warburgia salutaris seeds by Kioko et al (2000). However, due to its potential for production of fruit-based products, on-farm domestication is the most feasible option.

Conclusions

Desiccation screening provides a first line management tool to determining seed storability. Besides, it is useful in the determination of high moisture limits for seed survival. Although limited in extent, these studies have proved to be important for the management and practical handling of seeds of all four species studied. For conservation purposes, field gene banks should be improved to give access not only to germplasm but also to ensure the long-term sustainable uses of trees themselves.

Collaboration within the project

The networking within the project and the replicating partnership has provided an opportunity for capability building and quality assurance of staff and equipment. The exchange of data and seeds has allowed partners to undertake research on seeds of species from other regions. In this respect the project has given scientists a better understanding of various test methodologies. The linkages between partners and the co- ordinators will be sustained even beyond the project period. Problems associated with seed dispatch for some of the species become evident when some freshly extracted seeds packaged in wet media arrived at their destination with visible radicle emergence. This was noticeable with seeds of K. africana and D. caffra. Some seeds tested in this study required slight adjustments of the screening protocol. For instance, separating seeds whose sizes were equal to those of silica gel required longer time, and this was made easier by using mosquito-netting bags as described for D. caffra in this study. The rate of desiccation of large seeds was too slow, exposing them to more infection by pathogens. The protocol still needs to be reviewed to take into consideration the physiological and morphological variations of other seed categories. AFRICA 85

Future activities

Given the prevailing rate of environmental degradation it is no longer possible to guarantee the conservation of many indigenous plants within their local habitats, thus making it necessary to resort to ex situ conservation. However, for those species whose seeds are desiccation sensitive and therefore cannot be stored ex situ in genebanks, field genebanks are the only practical option of ensuring their survival. Such banks should be established under defined ownership and management to guarantee their sustainable existence. The management of farm owned trees, botanical gardens and institutional trees in the form of landscaping and orchards for commercial production of specified products all need to be addressed. Other areas that require future attention include studies on natural regeneration and soil seed banks for species with desiccation sensitive seeds and investigating further community-based contributions. Establishing a ‘Recalcitrant Seed Newsletter’ would help to exchange information between and with other scientists and partners.

References

Albrecht, J. 1993. Tree Seed Handbook of Kenya. Forestry Seed Centre Muguga, Nairobi, Kenya. Pp. 264. Dale, I.R. and P.J. Greenway. 1961. Kenya Trees and Shrubs. London, Hatchards and Buchanans Kenya Estates. International Centre for Research in Agroforestry (ICRAF). 1992. A Selection for Useful Trees and Shrubs for Kenya. Vol. 2. Nairobi, Kenya. International Rules for Seed Testing (ISTA). 1999. Supplement to Seed Sci. Technol. 27:1–333. IPGRI/DFSC. 1999. The project on handling and storage of recalcitrant and intermediate tropical forest tree seeds. Danida Forest Seed Centre. Newslett. No. 5:23–39. Kioko, J., P. Berjak, H. Pritchard and M. Daws. 2000. Seeds of the African Pepper-bark (Warburgia salutaris) can be cryopreserved after rapid dehydration in silica gel. Pp. 371–377 in Cryopreservation of Tropical Plant Germplasm: Current Research Progress and Application (F. Engelmann and H. Takagi, eds.). Japan International Research Centre for Agricultural Sciences, Tsukuba, Japan; International Plant Genetic Resources Institute, Rome, Italy. Milimo, P.B.W. 1986. The Control of Germination in Melia volkensii Seeds. M.Sc. Thesis. University of Alberta, Canada. Pp. 73. 86 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Omondi, W., K. Thomsen and S. Diklev. 2000. Screening of Dovyalis caffra at Kenya Forestry Seed Centre. IPGRI/DFSC Newsletter No. 6. Ongugo, P.O. 2000. Policy and legislative constraints and opportunities for development of forest genetic resources for conservation and sustainable utilization. In Recent Research and Development in Forest Genetic Resources (Eyog-Matig et al., eds.). IPGRI, Rome, Italy. Survey of Economic Plants for Arid and Semi Arid (SEPASAL). 2002. Centre for Economic Botany, Royal Botany Gardens, Kew, Richmond, Survey TW9 3AE, UK. Teel, W. 1985. A Pocket Directory of Trees and Seeds in Kenya. Kenya Energy Non-Governmental Organizations (KENGO), Nairobi, Kenya. AFRICA 87

Desiccation and storage of Sterculia quinqueloba seeds from Malawi

Tembo Chanyenga

Forest Research Institute, PO Box 270, Zomba, Malawi

Abstract

A study on the desiccation and storage of Sterculia quinqueloba seeds was conducted to determine conditions in which its viability could be maintained for periods of time. Water floatation followed by cut tests demonstrated that 80% of seeds that floated were infected and a further 7% were empty. Of the seeds that sank, 96% were full and normal in appearance. Both flotation and cut tests were suitable for preliminary tests of S. quinqueloba seed quality. Seeds desiccated to 8% MC maintained 80% germination (shoot), while those dried to 10–20% MC retained 78% germination (shoot), showing no significant differences in seed viability at these moisture contents. S. quinqueloba seeds with 10% MC were stored optimally both in the cold-room and under ambient conditions, retaining ca. 70% germination after six months. It is suggested that S. quinqueloba seeds can be desiccated, stored and dispatched while maintaining high viability.

Introduction

Sterculia quinqueloba (Garcke) K. Schum, which is native to hot and dry regions of Malawi, is an important species because of its potential to produce ‘Karaya’ gum, which is used for many purposes in industry and by local communities. The species is also used as an ornamental tree and its grey bark provides fibre for rope and mats (Msanga 1999). However, this species is facing rapid deforestation of fully grown stems despite its poor wood quality for timber, fuelwood and charcoal (Chapola 1996), which are the major causative factors of deforestation in Malawi. Chanyenga and Lowore (1999) reported a stocking density of less than four stems per hectare of mature trees of 25 cm diameter at breast height in the study area. The sawn timber is mainly used for coffin making and other light constructions (Chapola 1996). Tapping 88 STORAGE BIOLOGY OF TROPICAL TREE SEEDS studies have revealed that S. quinqueloba has the potential to produce gum of high commercial value (Munthali 2000) with similar properties to Streculia urens gum produced in India (Chapola 1996). As a nontimber forest product, karaya gum provides local people with additional income. In developing countries such as Malawi, a lack of understanding of seed biology and appropriate technologies hinder long-term seed storage work. Early studies indicated that it is difficult to store S. quinqueloba seeds. Msanga (1999) showed that these seeds are recalcitrant and only retain viability for up to two months at room temperature. Recent studies revealed that the seeds are intermediate, not recalcitrant and can be stored at 10% MC in the cold-room for six months, attaining 66–75% germination (Munthali and Gondwe 2001). This study also demonstrated that seeds with greater than 10% MC lost their viability when kept at 25–28°C under ambient conditions (Munthali and Gondwe 2001). The objective of the present study therefore, is to establish how best the seeds of S. quinqueloba can be stored.

Figure 1. S. quinqueloba trees in the forest at Liyenda. AFRICA 89

Materials and methods

Seed collection and processing

Fruits were collected from Liyenda in Mwanza, which is located between 15°23’ S–34°50’E and 15°23’S–34°5’E, with an annual rainfall of 800–1000 mm. The dominant associated miombo species are Acacia hockii, Acacia nigrescens, Combretum imberbe, Pterocarpus rotundifolia, Sclerocarya birrea, Sterculia apendiculata, Terminalia sericea and Vangueria infusta. The fruits were harvested, from 13 adult trees with an average height of 12 m, on the 15–17 October 2001 and transported to the laboratory in polythene bags in a closed Land Rover vehicle. A total of 19.6 kg of fruits were collected and immediately weighed to determine their fresh weight (Fig. 2). At the laboratory, the fruits were spread on the floor in a room at 27–29°C. Seeds were manually extracted from fruit pods. Seeds coloured black were mature and immature seeds were white (see Fig. 3). Only the mature black seeds were used in the experiments. In total, 2.58 kg of mature seeds were prepared within 6 h and selected for this study. Extracted seeds were soaked for 10 min in 1% sodium hypochlorite solution, rinsed and dried using blotting paper. Excess gum was removed from the seeds using toilet tissue.

Figure 2. Weighing S. quinqueloba fruits after collection. 90 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Figure 3. Picture of mature (black) and immature (white) seeds.

Desiccation and germination experiments

Seeds were mixed with silica gel to desiccate them to different target moisture contents of 8, 10, 25 and 35%. After drying, samples of 25 seeds (as stipulated by the Seed Desiccation and Storage Protocol) were tested to determine their moisture content, by weighing them before and after oven drying at 103°C for 17 h. The moisture content was then calculated as a percentage of the fresh mass. During preparation, it was observed that most seeds were floating. Thus, samples of floating seeds were separated from the sinking seeds for a small comparative experiment. Seeds were thoroughly dried under shade. Cut tests were carried out on 100 individual seeds of both floating and sinking lots to check viability (ISTA 1999). Germination capacity was assessed for seeds of all target moisture contents. Four replicates of 25 seeds were sown in sterilized sand used as a rooting medium in containers that were kept moist with 8 h light at 26.5°C, and 16 h dark at 21°C. Germination was recorded based on root and shoot development on the seedlings. A seed was considered to have germinated if its root and shoot had grown up to 1 cm in the container. Seeds that did not germinate were cut to observe their status.

Storage trials

Following the seed storage protocol (IPGRI/DFSC 1999), seeds desiccated to 8, 10, 25 and 35% MC were randomly sampled and hermetically sealed in aluminium foil laminate bags, and stored at AFRICA 91

25–28°C in an ambient room and at 4°C in a cold-room. After storage, seeds were tested for their germination capacity by observing radicle protrusion and shoot development.

Results

Initial tests

The results of the preliminary tests showed that 13% of seeds that floated were full. Of those seeds that sank, 96% were full and normal (Table 1). A high proportion of the floating seeds (80%) were infected by insects, compared to only 4% of seeds that sank, as revealed by cut tests. The remaining 7% of the floating seeds were empty. The 100 floating seeds weighed 0.697 kg, whilst nonfloating seeds weighed 0.981 kg. No initial moisture content studies were carried out on the fruits, because they appeared to be dry at harvest.

Table 1. Floating test on S. quinqueloba seeds Condition of seeds Floating seeds (%) Sinking seeds (%) Full 13 96 Infected 80 4 Empty 7 0 Total weight 697 g 981 g

Germination tests

Initial germination and development of normal seedlings with roots and shoots was more than 60% (Table 2). Seeds dried to 9.0 and 12.4% MC normally germinated to 80%. Germination tests also revealed that 1–2% of seeds developed roots without shoot and vice versa, suggesting abnormal germination as far as seed development is concerned. However, there were no significant differences in root and shoot development between the germinated seeds at 8, 10 and 25% MC. However, there were significant variations in germination percentages between seeds at 34 and 47% MC compared with those dried to 22, 12 and 9% MC (Table 2). 92 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Table 2. Germination (± sd) of S. quinqueloba seeds after desiccation to four target moisture contents

Target MC (%) Actual MC (%) Germination (%) Germinated Roots Shoots Abnormal Control 47.0 63 62±3.3 63±3.4 1 35 34.0 71 71±1.2 70±1.7 1 25 21.6 78 78±2.3 78±2.3 0 10 12.4 80 80±0.8 78±1.9 2 8 9.0 80 78±1.2 80±1.8 2

Storage trials

Seeds at all tested moisture levels stored better at 4°C in the cold-room compared to those stored under ambient conditions after both three and six months (Table 3). Seeds with 34 and 47% MC germinated to less than 50% after three months in both conditions, and no seeds germinated after six months in ambient conditions. Seeds at 22% MC exhibited more than 50% germination after three months at 4°C, but did not germinate after six months at ca. 25°C. Drier seeds at 9 and 12% MC maintained high germination capacity of ca. 70% after three or six months storage (Table 3). There was no significant difference in germination percentages between the two storage periods at 9% MC both in the cold-room and under ambient conditions.

Table 3. Germination (G%±sd) of S. quinqueloba seeds at different moisture contents after storage at 4°C (cold-room) and ca. 25°C (ambient) for three and six months MC (%) Storage at 4°C (G%) Storage at ca. 25°C (G%) 3 months 6 months 3 months 6 months 47.0 17±3.8 19±3.8 16±5.8 0 34.0 37±12.8 28±9.2 — — 21.6 66±6.9 26±10.5 29±10.5 0 12.4 82±8.3 78±14.8 72±4.6 64±5.7 9.0 70±6.9 71±6.0 69±9.6 69±10

Discussion

In this study, floatation (water) and cut tests demonstrated that both methods were quick effective ways of testing S. quinqueloba seed viability. Although initial (63%) or maximum germination (80%) was high, it did not reach the level of 96% observed for sinking seeds AFRICA 93

(Tables 1 and 2). Seeds withstood desiccation to low moisture contents, germinating 80% after drying to 9–12% MC. This is higher than at the initial moisture content of 47% MC. Thus, desiccation seemed to increase the germination capacity of these seeds, which could be a postharvest maturation process or internal cellular repair (Bonner 1994). A low germination percentage (51%) of S. quinqueloba seeds sown soon after processing without desiccation, had also been reported in another study, using a similar provenance (Munthali and Gondwe 2001). The storability of S. quinqueloba seeds increased with seed desiccation; seeds desiccated to 9% MC survived longer (ca. 70% in both the cold-room and under ambient conditions after six months (Table 3)). This study showed that S. quinqueloba seeds tolerated desiccation up to 9–12% MC and maintained high viability after storage at 4–25°C for six months. However, seeds with >12% MC decreased in viability over the six months period, but they were not particularly chilling sensitive (see 4°C results). Like Msanga’s results (1999), S. quinqueloba seed seems to retain viability for only a few months at room temperature. However, we show that S. quinqueloba seeds can be dried and stored for six months at 25°C with 70% viability and are thus, not recalcitrant. Thus, it can be recommended that the National Tree Seed Center (NTSC) should insure that S. quinqueloba seeds are desiccated before distributing to its customers, as these seeds would retain a higher germination percentage.

Acknowledgements

I am grateful to IPGRI and DANIDA for funding this study, as well as training in handling and storage of tropical forest tree seeds. Contributions of senior officers at FRIM are greatly appreciated, and acknowledgements are due to E. Makawa, M. Namoto and H. Chapama, and E. Kantukule for technical assistance.

References

Bonner, F.T. 1994. Predicting seed longevity for four forest tree species with orthodox seeds. Seed Sci. Technol. 22:361–370. Chanyenga, T. and J. Lowore. 1999. Composition and Abundance of Sterculia quinqueloba for Karaya Gum Tapping at Kamwamba in Mwanza East. FRIM Report No. 99007. 94 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Chapola, G.B.J. 1996. Factors Influencing Karaya Gum Yield of Sterculia quinqueloba. FRIM Report No. 96003. IPGRI/DFSC. 1999. The project on handling and storage of recalcitrant and intermediate tropical forest tree seeds. Danida Forest Seed Centre. Newsletter No. 5:23–39. ISTA. 1999. International Rules for Seed Testing Rules 1999. International Seed Testing Association, Zurich, Switzerland. Pp. 333. Msanga, H.P. 1999. Seed Germination of Indigenous Trees in Tanzania. UBC Press, University of British Columbia, British Columbia, Canada. Munthali, C.R.Y. 2000. Preliminary Results of Karaya Gum Yield of Steculia quinqueloba (Garke) K at Kamwamba in Mwanza, Malawi. FRIM Report No. 00009. Munthali, C.R.Y. and D. Gondwe. 2001. Desiccation and Storage of Sterculia quinqueloba Seed From Liyenda Provenance in Malawi. FRIM Report No. 01009. AFRICA 95

Storage of recalcitrant seeds of Cordyla pinnata from Senegal

Ismaïla Diallo, M. Wade, A.S. Sarr and A. Gaye

Institut Sénégalais de Recherches Agricoles (ISRA),Centre National de Recherches Forestières (CNRF) BP 2312 Dakar Hann, Sénégal

Abstract

Determining optimum conditions for extending storage longevity of recalcitrant seeds is very important for the conservation of biodiversity, particularly for degrading forest resources. Despite both their economical and ecological importance, parklands of Cordyla pinnata in Senegal are increasingly ageing and their seeds are difficult to store. This study showed that seeds with 40% MC decreased from 98% germination to 10% germination after three months storage at 15°C, while no seeds germinated after one month at 5 or 25°C. The initial harvest moisture content of 42% was lower than the 50% MC found in other studies. Further investigations are still needed on the quality of seed collections and optimum storage conditions of C. pinnata seeds.

Introduction

The contribution of the forestry sector to the national economy of Senegal has always been difficult to estimate, although recognized as important. This is essentially due to the fact that the forestry administration controls no more than 30% of the whole production of the forestry sector, mostly close to the big cities (DEF 1992). The official figures for the forestry sector contribution are less than 1% of the GDP and 5% of the total primary sector. Investments in the forestry sector were only 10% of the primary sector in the VIIth Plan 1985–1989 of Senegal. Thus, the importance of the forestry sector is not well reflected in the economic data, or in national priority actions. However, the role played by forests is fundamental to the country’s economy. Forests help to maintain and improve soil fertility, saving chemical fertilizers. They satisfy 53% of energy needs, against 40% for oil products and 5% for electricity. They serve as a food source for people, give them shelter and provide medicines. In addition, forests 96 STORAGE BIOLOGY OF TROPICAL TREE SEEDS increase the pastoral potential for animals and maintain environments favourable for development. However, much work is needed to maintain and sustainably manage forest genetic resources.

Threats to forests in Senegal

All over the country, forest ecosystems and their production potentials are quantitatively and qualitatively decreasing. Overstocking of domestic animals, overgrazing and felling of forage trees (or pruning) mainly in the North, bush-fires, and collection of fuelwood, have been estimated to overexploit potential resources by approximately two million cubic metres per year (DEF 1980). This represents about 100 000 ha of woody savanna in the Sahelian-Soudanian zone, or the equivalent of 200 to 250 000 ha of forest savanna, an estimated 1–2% of the total woody forest resources of Senegal. Qualitatively, the decrease in forest genetic resources is principally due to climatic factors, resulting in a great mortality of vulnerable forest species. Thus, genetic erosion and change in the floristic composition maybe occurring. The extent of such degradation varies according to the ecological zones. The most threatened genetic resources are stands of Acacia nilotica in the river valleys, and also stands of Acacia senegal, Pterocarpus lucens, Sclerocarya birrea and Dalbergia melanoxylon in the sylvo-pastoral zone. These species are threatened, and D. melanoxylon is now endangered and listed on the Red List of IUCN (IUCN 2002). D. melanoxylon is in danger of being wiped out because of the combined effects of drought and overexploitation. In contrast, Calotropis procera is widely thriving in the proximity of humid zones, close to streams and in depressions. The palm tree stands of Cayor, as well as those of Casamance are shrinking due to drought and salination of sea water. In the ‘Basin arachidier’ (Senegal’s great peanut production basin), parkland species like Acacia albida, Cordyla pinnata, Sterculia setigera, Parkia biglobosa and Tamarindus indica are ageing without sustainable replacement. They are also more vulnerable because of the land management systems in the region, which include mechanized farming, burning and clearing of forests, uncontrolled livestock, etc., all of which remains incompatible with subsequent regeneration of these species. In the Guinea-sudanian zone, species used for amenity such as Khaya senegalensis, Pterocarpus erinaceus, Daniella oliveri and Prosopis africana are progressively disappearing from the landscape because of uncontrolled and intensive exploitation. AFRICA 97

Because of all these constraints, it is urgent and important to define national priorities for the conservation of forest genetic resources. To this end, strengthening in situ conservation of parklands and protected forests, and defining a participative policy for in and ex situ conservation, are important measures to undertake Immediate actions should include (a) carrying out national inventories to better know the production potential of the forests, (b) encouraging sustainable utilization of genetic resources based on equitable sharing of benefits among stakeholders, (c) setting up appropriate and consensual regulatory tools (rules) and (d) reinforcing capacity actions and management by local communities and technical administration.

Criteria for selecting species

The results of a study in the ‘Bassin arachidier’ (Ndour and Gaye 1996) have been used to select priority species (Table 1). The criteria for selection were mainly multiple uses as food, medicines, forage and firewood (ICRAF/SALWA 1990). Senegal basin communities prefer different forest species with multiple uses, such as A. albida, A. digitata, B. aegyptiaca, C. pinnata, T. indica and Z. mauritiana. These are among the main six species all over the basin. Primarily, the communities locally use products and by-products, i.e. fruit, leaves, bark and wood. However, fruits of A. digitata, B. aegyptiaca and C. pinnata are sometimes sold to increase family revenues. Because quality and quantity improvements are needed, these investigations can be used as a basis for genetic improvement programmes, for sustainable use of forest products and for germination and establishment of plantations.

Study of Cordyla pinnata seeds

Cordyla pinnata (A.Rich.) Milne-Redhead is a member of the Leguminosae family. It is an important species with multiple uses in Senegal. Approximately 713 kg of C. pinnata fruits were collected on 5–7 Aug 2001 from Sokone region. The replicating partners were the Centre National de Recherches Forestières (CNRF) of Senegal and the Centre National de Semences Forestières (CNSF), Burkina Faso. The mass ratio of seeds to fruits after preparation was 0.12. An average seed of 98 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

C. pinnata weighed about 13 g (Table 2). Initial moisture contents were 42% for whole seeds, 40% for cotyledons and 50% for embryonic axes.

Table 1. Main uses of selected species in the South and North ‘bassin arachidier’ of Senegal (values represent the percentage of those asked)

Species Food Medicine Forage Firewood/other s South North South North South North South North Acacia albida — — — — 24.7% — — — Acacia nilotica — — — 77% — — — — Adansonia 20.2% 89% — 40% — 38% 12.3% — digitata Azadirachta indica — — — 41% — 21% — 23% Balanites 12.1% 86% 13.9% 25% — 25% — 28% aegyptiaca Cordyla pinnata 18.1% 96% 10.7% 21% — 21% 12.3% 25% Detarium 15.3% — — — — — — — microcarpum Ficus ichteophylla — 44% — — — — — — Ficus ichteophylla — — — 39% — 55% — — Parinari — 100% — — — — — — macrophylla Parkia biglobosa 16.5% — — — — — — — Tamarindus indica 10.5% 97% — 44% — 28% — — Zizyphus 20.7% 88% — — 13.6% 31% — — mauritiana

Table 2. Initial characteristics of fruits and seeds of C. pinnata Collected fruits 713 kg Prepared seeds 86 kg Thousand seed weight (TSW) 13 kg Initial MC (average) 41.9% Seed MC after depulping 42.1% Cotyledons MC 40.2% Embryonic axes MC 50.4%

Storage trials

Fresh seeds with 40% MC were stored for six months at 5, 15 and 25°C. The results showed that seeds lost their germination capacity after a month at 5°C (Fig. 1). AFRICA 99

Figure 1. Germination capacity (G) of C. pinnata seeds with 40% MC (moisture content) after storage at 5°C for 6 months.

By contrast, the germination capacity of these seeds was maintained up to two months at a temperature of 15°C, decreasing from 98% at the start to 10% after three months (Fig. 2). For seeds stored at ambient temperature of 25°C, their viability decreased to below 20% germination after only a month (Fig. 3), and no germination occurred after this period.

(%) 120 100 80 60 MC 40 G

Germination 20 0 Initial 1 2 3 4 5 6 Storage periods (months)

Figure 2. Germination (G) of C. pinnata seeds with 40% MC after storage for periods of time at 15°C. 100 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Figure 3. Seed germination capacity (G) at 40% MC (moisture content), after storage for periods of time at 25°C.

Discussion and conclusions

These results showed that C. pinnata seeds with similar moisture content of ca. 40% when stored at different temperatures of 5, 15 and 25°C revealed different germination capacities after storage for six months (Figs. 1, 2 and 3). Only seeds at 15°C maintained about 10% germination after three months (Fig. 2). This moisture content is too high to allow seeds to maintain their viability in these storage conditions. Although desiccation trials were not undertaken in this study, seeds of C. pinnata lose viability when moisture contents drop below 22% (Danthu et al. 2000). Longevity of these seed in wet and airtight storage did not exceed a few months, and temperatures close to zero elicit symptoms of chilling injury leading to rapid seed death. The optimum storage temperature was 15°C, supporting the view that desiccation sensitive tropical seeds are often sensitive to low temperatures (Corbineau and Côme 1998). The initial moisture content of seeds of C. pinnata was 42% MC, which was lower than the 50% MC reported by Danthu et al. (2000). Such a difference maybe due to both seed source and the stage of harvest. Further investigations are still needed on quality of seed collections and optimum storage conditions of C. pinnata seeds. AFRICA 101

Acknowledgement

The authors thank Dr Moctar Sacandé for the English translation and the useful comments on this article.

References

Corbinerau, F. and D. Côme. 1998. Storage of recalcitrant seeds of four tropical species. Seed Sci. Technol. 16:93–100. Danthu, P., A. Gueye, A. Boye, D. Bauwens and A. Sarr. 2000. Seed storage behaviour of four Sahelian and Sudanian tree species (Boscia senegalensis, Butyrospermum parkii, Cordyla pinnata and Saba senegalensis). Seed Sci. Res. 10(2):183–187. Direction des Eaux et Forêts (DEF). 1980. Plan Directeur pour le Développement Forestier (PDDF) 1980. Direction des Eaux et Forêts. Dakar, Sénégal, Pp. 86. Direction des Eaux et Forêts (DEF). 1992. Rapport Annuel. Dakar, Sénégal, Pp. 72. ICRAF/SALWA. 1990. Propositions de Recherches Agroforestières pour le Système du Bassin Arachidier du Sénégal. Rapport AFRENA No. 37. ICRAF/SALWA, Pp. 88. IUCN. 2002. 2002 IUCN Red List of Threatened Species. [also at http://www.Redlist.org]. Ndour, B. and A. Gaye. 1996. Prioritization et Utilization des Ligneux à Usages Multiples dans le Bassin Arachidier. Dakar, Sénégal, Pp. 20. 102 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Desiccation trials on Neem, Azadirachta indica A. Juss., seeds

David Baxter and Patricia Berjak

Plant Cell Biology Research Unit, School of Biological Sciences, University of KwaZulu-Natal, Durban 1041, South Africa

Introduction

Neem is a fast-growing, small to medium-sized evergreen tree originating from Northeast India and Myanmar. The tree is used predominantly for shade and afforestation and is suitable for the improvement of degraded and nutrient-poor soils. The leaves and seeds contain substances with insecticidal properties and the bark, leaves, fruits, oil and sap all have medicinal applications. The wood, which is resistant to termites and other insects, is used for construction, fencing, as fuel and for producing charcoal. The tree is heat-resistant but sensitive to cold temperatures (Albrecht 1993). Because of all these uses there is great pressure on neem trees in the tropics that needs to be lessened through planting and conservation. In order to conserve the seeds, more information is needed on their ability to withstand desiccation. This report details the results of desiccation trials carried out on neem seeds originating from Marigat, Kenya, as a replicating partner for KEFRI.

Materials and methods

Seed details and processing

Neem fruits were collected on 15 May 1996 from Marigat in Kenya and sent by air cargo to South Africa. The seed consignment was received at the laboratory in Durban, South Africa, on 22nd May 1996. Mature, ripe fruits with a yellow-green exocarp were selected for investigation. All rotten fruits were discarded. Processing involved soaking the fruits in water to soften the pulp, after which rubbing with rough mesh was performed to remove excess pulp. The clean seeds were subsequently dried with paper towel and coated with Benlate as indicated by the screening protocol (IPGRI/DFSC 1996). AFRICA 103

Drying seeds and assessing germination

Initial moisture content (MC) was determined on each component seed tissue separately before drying. Desiccation was carried out using silica gel in plastic jars, which were later sealed. For each target moisture content (TMC), approximately 150 g of activated silica gel were mixed with seeds. Isolated embryos were used as the unit for testing germination. Four replicates of 25 seeds each, were placed on moist filter paper in closed plastic Petri dishes in an incubator (at 25°C). A seed was marked as germinated if the radicle had emerged to ca. 1 cm in length.

Results

The average mass of a single neem fruit was 1.1 g of which about 25% (0.3 g) was the actual seed (Table 1). The initial MC of the fruit pulp was ca. 70% fresh weight; this was close to the embryo’s 65% MC. The cotyledons were at 52% MC, while the endocarp was drier, with less than 10% MC.

Table 1. Characteristics of neem fruit, seed and seed components Tissue Weight (g) % MC (fresh mass) Fruit 1.1 ± 0.19 – Seed 0.3 ± 0.04 – Pulp – 69.5 ± 2.6 Endocarp – 9.6 ± 3.4 Cotyledon – 52.4 ± 4.4 Embryo – 65.1 ± 3.1

After desiccating to six different moisture contents ranging from 21 to 8%, seeds were assessed for their germination capacity (Table 2). Compared with the 99–100% germination of control (nondried) seeds, there was, at most, a 11% reduction in the germination of seeds dried to 13, 10, 8 and 5% MC. 104 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Table 2. Whole seed moisture content and viability before and after drying (%, fresh mass) Control Dried Target MC (%) G (%) MC (%) G (%) initial 41.4 r 4.34 99 – – 20 41.7 r 2.83 100 21.3 r 7.70 100 16 44.8 r 3.33 100 20.4 r 7.67 100 13 41.9 r 3.19 100 12.9 r 6.40 92 10 41.9 r 1.91 100 10.9 r 2.52 98 8 41.7 r 1.79 100 9.6 r 1.99 95 5 43.8 r 3.22 100 7.6 r 1.74 89

Discussion

The results obtained seem to indicate intermediate seed storage behaviour, with a relatively small (11%) decrease in seed viability with drying down to 7.6% MC (Table 2). However, these are tentative conclusions and it is not known how long the seeds will survive at low moisture contents. It is assumed that the moisture content, when expressed on a whole seed basis, is closer to that of the cotyledons than that of the embryo, as the cotyledons comprise the bulk of the seeds. Considering the large degree of variation for both the control moisture content values for the cotyledons (Table 1) and whole seeds (Table 2), these almost overlap at r 45–47% MC. A preliminary study carried out within our laboratory, independent of this screening investigation, found that neem seeds from Gede were chilling-sensitive in the hydrated state, suggesting a more recalcitrant behaviour compared with the intermediate-type behaviour shown by these seeds from Marigat.

References

Albrecht, J. 1993. Tree Seed Handbook of Kenya. GTZ Forestry Seed Centre Muguga, Nairobi, Kenya. IPGRI/DFSC 1996. Screening Protocol for Desiccation and Storage of Intermediate and Recalcitrant Tropical Forest Tree Seeds. IPGRI, Rome, Italy. AFRICA 105

Germination of Dovyalis caffra (Hook. F. et Harv.) Warb. seeds

Deon Erdey and Patricia Berjak

Plant Cell Biology Research Unit, School of Biological Sciences, University of KwaZulu-Natal, Durban 1041, South Africa

Introduction

Dovyalis caffra (Hook. F. et Harv.) Warb. , kei apple, is a small (3–9-m tall), densely crowned evergreen shrub or tree, which is widely distributed in southern Africa, mainly along forest margins, in woodland and valley bushveld regions (Pooley 1993; Venter and Venter 1996). This is an important species in Africa, where it is widely used for hedges (Omondi et al. 2000). In addition, the leaves are used as fodder for cattle and goats (Pooley 1993; Venter and Venter 1996). The fruit is spherical (60 mm in diameter), consists of a velvety pericarp, which when ripe turns yellow, and a fleshy mesocarp containing 10 to 20 seeds (Pooley 1993; Venter and Venter 1996). The fruits are edible, with a high vitamin C content (83 mg/100 g) (Venter and Venter 1996), and are used for making jam, juices and other food products (Pooley 1993; Venter and Venter 1996; Omondi et al. 2000). The seeds are small of c. 10 mm in length (Venter and Venter 1996), containing an oily endosperm and an embryo with thin, flat cotyledons (Corner 1976). The species can be successfully propagated from seed (Venter and Venter 1996) as well as by cuttings (Mudge et al. 1995; Venter and Venter 1996). The seeds have been described as orthodox, as viability of mature seeds dried to 6–10% moisture content was maintained following hermetic storage at 3°C for several years (Albrecht 1993).

Materials and methods

Seed details and processing

Fruits were collected on 13 Oct 1999 from 30 trees in Nakuru, Njoro Kenya. Only those fruits that fell to the ground following vigorous shaking of the trees were selected. Fruits were transported in sisal bags to KEFRI. The seeds were extracted by squeezing the fruits; at this 106 STORAGE BIOLOGY OF TROPICAL TREE SEEDS stage the mean moisture content of the seeds was 35% fresh weight. The seeds were then cleaned, by soaking and washing with water a couple of times and manually separated from the pulp. Following this cleaning treatment mean seed moisture content had increased to 53% fresh weight. Seeds at this moisture level were then packaged in bags containing sawdust and dispatched to the replicating partners. Upon arrival in Durban on the 21 Oct 1999, seeds were placed at 16°C in their original packaging until separated from the sawdust within a few days. At the University of Natal, it was found that most of these D. caffra seeds had already started to germinate. Thus, only a very small proportion of received seeds could be used for measuring seed moisture content and assessing germinability, both on filter paper and a 1% water-agar medium. There were insufficient seeds for assessing desiccation tolerance.

Results and discussion

The mean moisture content of the cotyledons upon arrival in Durban was 44% (Table 1). Since the cotyledons make up a significant proportion of D. caffra seed mass and this value is somewhat lower than that measured at KEFRI for whole seeds (53%), it appears that the cotyledons had lost moisture. The mean moisture content of the embryonic axis was higher, at 60% (Table 1). With such high moisture, it was not surprizing that most of the seeds had germinated upon arrival in Durban. While it is possible that there was preferential movement of water from the cotyledons to the embryonic axis during transit (Tompsett and Pritchard 1998), the whole seed moisture content had already been considerably increased during extraction in Kenya. This raises an important, yet often overlooked, point when testing seed species in this programme. Although extracting and cleaning seeds using water may prove to be easier, if the amount of water taken up by the seeds is too great, then germination processes could be set in motion or accelerated, if already initiated. Thus the degree of intolerance to desiccation may be due also to the progress of germination rather than as a consequence of inherent factors. Of the seeds tested for germination, 90 and 92% of the seeds germinated on filter paper and water-agar, respectively (Table 1). AFRICA 107

Table 1. Moisture content of seed components and viability assessment of D. caffra seeds, upon arrival in Durban

Moisture content Seed viability (% germination) Seed tissues (%, fresh mass) Filter paper 1% water-agar Cotyledons 44.1 ± 3.36 92 90 Embryonic axis 60.2 ± 5.86 – –

From our experience with these D. caffra (and other) seeds screened during this programme, it is proposed that material be transported as intact fruits, as soon as possible after harvest. Although this may be considerably more expensive because of the weight and volume of the packages, such a practice would make partner screening a far more meaningful exercise.

References

Albrecht, J. 1993. Tree Seed Handbook of Kenya. GTZ Forestry Seed Centre, Nairobi, Kenya. Corner, E.S.H. 1976. Seeds of Dicotyledons. Vo. 1. Cambridge University Press, Cambridge, UK. Mudge, K.W., V.N. Mwaja, F.M. Itulya and J. Ochieng. 1995. Comparison of 4 moisture management-systems for cutting propagation of Bougainvillea, Hibiscus, and Kei Apple. J. Am. Soc. Horticult. Sci. 120:366–373. Omondi, W., K. Thomsen and S. Diklev. 2000. Screening of Dovyalis caffra at Kenya Forestry Seed Centre. The project on handling and storage of recalcitrant and intermediate tropical forest tree seeds. Newsletter 6:9–11. Pooley, E. 1993. The Complete Field Guide to Trees of Natal, Zululand and Transkei. Natal Flora Publications Trust, c/o Natal Herbarium, Durban, Republic of South Africa. Tompsett, P.B. and H.W. Pritchard. 1998. The effect of chilling and moisture status on the germination, desiccation tolerance and longevity of Aesculus hippocastanum L. seed. Ann. Bot. 82:249–261. Venter, F. and J.-A. Venter. 1996. Making the Most of Indigenous Trees. Briza Publications, Pretoria, South Africa. 108 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Conservation of Ekebergia capensis seeds from South Africa

Deon Erdey, Zama Mbatha and Patricia Berjak

Plant Cell Biology Research Unit, School of Biological Sciences, University of KwaZulu-Natal, Durban 1041, South Africa

Abstract

Species information and screening results are presented for Ekebergia capensis seeds. The results of the desiccation trials indicated that seeds of Ekebergia capensis germinated 100% at an axis MC of 20%. Maintaining seeds within hard, impermeable fruit- or seed-derived coats, such as endocarps prolonged survival of E. capensis seeds during short-term storage by minimizing water loss. Seeds retained 100% viability after 20 weeks storage at 16°C. However, viability was dramatically reduced during sub-imbibed storage. Thus E. capensis seeds can be categorized as recalcitrant.

Introduction

South Africa has among the highest recorded species diversity in the world (Cowling et al. 1989) of which indigenous forests constitute the smallest biome, covering only 0.33% of the surface area (Thompson 1999). These forests, most of which have been designated as Primary Conservation Areas (PCAs), are currently monitored and managed by the Department of Water Affairs and Forestry, DWAF (Neil 2000). Although what little remains of these areas is considered to be relatively well preserved (Von Maltitz and Fleming 2000), land clearing for formal and informal agriculture, commercial forestry, erosion, settlement of the ever-burgeoning population, and indiscriminate harvesting of traditionally-utilized species are placing ever-increasing pressure on South Africa’s natural plant resources (Scott-Shaw 1999). Despite the vast number of plant species found in the region, little is known about their reproductive biology and, consequently, about ex situ conservation of the resources. However, in common with tropical and subtropical species, especially trees species in Africa (Seme et al. 1994), a significant proportion of the local flora is expected to produce nonorthodox seeds. These seeds are very short-lived and heavily predated in the natural environment, leading to a reduction of natural AFRICA 109 regeneration, and erosion of the resources and genepools (Berjak 2000). While very little information is available concerning reforestation efforts by government in South Africa, numerous community based programmes, funded and managed either by autonomous organizations, such as WWF SA, or by commercial forestry (e.g. SAPPI), aimed at sustainable resource utilization and ecotourism, are in progress. At present, ex situ collections of forest tree species in South Africa occur in botanic gardens, or, as is the case for some species (e.g. Ekebergia capensis and Trichilia dregeana), as street trees in cities. The only genebanks in the country, maintained by the Agricultural Research Council (ARC), consist of indigenous vegetables, subtropical crops, deciduous fruits, grapes, oil seeds and small grains. However, due to the lack of information available about the storage behaviour and handling of seeds indigenous to South Africa, 20 local species were, or are in the process of being, screened by the university of Natal. Of these, five are gymnosperms representing the cycads (Encephalartos ferox, E. villosus and Stangeria eriopus), gnetales (Welwitschia mirabilis) and conifers (Podocarpus henkelii), and 15 are angiosperms, which include, among others, members from the palm, sisal, mango and mahogany families. Ekebergia capensis Sparrm (Meliaceae), commonly known as the Cape Ash or dog plum, occurs in a variety of habitats, from scrub to high altitude evergreen forest and riverine forest (Coates Palgrave 1984; Pooley 1995; Venter and Venter 1996). The species is widely distributed in eastern Africa, from Ethiopia in the North to the western Cape Province in South Africa (Tadesse 1995; Venter and Venter 1996). It is protected in South Africa (Venter and Venter 1996) where the species is heavily used for medicinal purposes (Netshiluvhi 1999). Preparations of bark are used for coughs and of roots against dysentery (Coates Palgrave 1984; Pooley 1995; Venter and Venter 1996). The tree also provides firewood, poles and timber (Msanga 1998). Mature fruits are readily consumed by monkeys and birds (Venter and Venter 1996). The fruit consists of a thin, waxy exocarp, a fleshy mesocarp and a hard, fairly impermeable endocarp. When ripe, just before shedding, the fruits turn from green to a deep red colour. About 5 kg of fruit produces 1 kg of approximately 6000 seeds (Venter and Venter 1996; Msanga 1998). Upon removal of the endocarp, mature seeds consist of a thin brown testa enclosing the green cotyledons and axis. The seed is approximately 6–8 mm in length and about 4 mm in diameter. 110 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

E. capensis seeds are non-dormant, germinating rapidly in both light and dark conditions and the optimal temperature for germination is 25°C (Teketay and Granstrom 1997a). Germination has been described as delayed, with fresh seeds attaining 60% after 4–6 weeks (Msanga 1998; Netshiluvhi 1999). However, cracking the endocarps or soaking in concentrated H2SO4 improves seed germination from 60 to 90–100% (Netshiluvhi 1999). According to Venter and Venter (1996), collection should be from the tree, as only 40–50% of seeds extracted from fruits collected on the ground germinate, while seeds harvested from the trees attain 90% germination. In the dry Afromontane forests of Ethiopia, E. capensis lacks a persistent soil seed bank (Teketay and Granstrom 1995). Seeds buried in bags with soil were not viable when sampled 18 months later. Depletion of the seeds in the soil bank was found to be the consequence of germination, establishing large populations of suppressed juveniles on the forest floor (Teketay 1997; Teketay and Granstrom 1997b). These seedling populations persist until a suitable gap becomes available (Teketay 1997). Very little information is available about the storage behaviour of this species. It has been suggested that the seeds should be sown within four weeks after harvesting (Msanga 1998), 23% of seeds (moisture content not given) remaining viable following 3 months storage at 20°C (Teketay and Granstrom 1997a). This viability decreased to 4% after 24 months (Teketay and Granstrom 1997a). Thus, it has been concluded that E. capensis seeds are recalcitrant (Msanga 1998; Pammenter et al. 1998), and cannot tolerate freezing temperatures (Msanga 1998). A detailed account of the biology and screening results for E. capensis are presented.

Materials and methods

Collection and processing

Fruits were collected from Phoenix, Durban in July 2001. The fruit pulp was removed by squashing, then the seeds (endocarp intact) rinsed to remove excess pulp. What has been described as the ‘seed’ in this study, and most other cases, was not the seed sensu stricto. Botanically, after removing the pulp (mesocarp) the remaining entity is the pyrene, which still encloses the seed. This emphasises the need to clarify that the propagules under investigation are AFRICA 111 accurately described and where possible, that all hard covering structures (fruit- or seed-derived) be removed prior to germination assessments.

Moisture content determination and germination

Seed moisture content (MC) was determined gravimetrically, on either a whole seed basis or separately for embryonic axes and cotyledons from individual whole seeds. Seeds or seed tissues were weighed before and after oven drying at 80°C for 48 h, and the moisture content was expressed on a percentage fresh mass (% fmb) basis. Seeds without endocarp (removed using pliers) were soaked in fungicide and antibiotic cocktail (2.5 ml/l Previcure N (AgrEvo South Africa (Pty)Ltd, active ingredientprompamocarb-HCl 722 g l), -1 0.2mll-1 Early Impact (Zeneca Agrochemicals SA (Pty) Ltd, active ingredients triazole 94 g l-1 and benzamidazole 150 g l-1), and 50 ug ml-1 kanamycin) for 10 min. Seeds were then plated onto a 1% water-agar in 90 mm Petri dishes (five seeds per plate) and incubated at 25°C. A seed was scored as germinated upon 5 mm radical emergence. Germination rate was expressed as the time (days) taken for 50% of the seed sample to germinate (T50).

Desiccation and storage trials

Whole seeds (endocarps removed) were buried in silica gel, and sampled after 2, 4, 6, 12 and 24 h. Moisture contents of samples were determined as described above. Fresh seeds with high moisture content were stored with intact endocarps for 18 weeks and without endocarps for 8 weeks (hydrated storage). In addition to the storage conditions prescribed by the protocol (IPGRI/DFSC 1999), E. capensis seeds were also stored following partial dehydration with silica gel (sub-imbibed storage). Partially dried seeds without endocarp, were then stored for 8 weeks. In all cases, seeds were stored in honey jars at 16 or 25°C. 112 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Results

Initial trials

The initial moisture contents of Ekebergia capensis seed components were 60% for axes and 31% for cotyledons, and all of the seeds germinated 100% (Table 1). Seed viability was retained when seeds were dehydrated rapidly (for 12 h) to axis moisture content of 20%. Further reduction in axis moisture content to 16.6% resulted in a 20% decline in germination totality (Table 1). In contrast, however, there was a gradual decline in the rate of germination, as indicated by an increase T50, during desiccation (Table 1).

Table 1. Desiccation of seed components and germination of E. capensis seeds. Seed initial moisture content was 30.79% Drying time (h) MC (%) Germination Axes Cotyledons Totality (%) T50 (d) 0 60.0 31.0 100 4.5 2 42.8 26.4 100 6 4 37.5 23.0 100 7.5 6 23.0 13.0 100 10 12 20.0 9.0 100 8 24 16.6 6.5 80 8.5

Storage trials

Axis moisture content decreased only slightly, from a mean value of 60% to 58.3% after 18 weeks for seeds with their endocarps stored at 16°C, with no loss in seed viability (Table 2). In contrast, a gradual decrease in axis moisture content to 41.1% was observed for those seeds stored at 25°C after 12 weeks. While viability was retained during this period, germinability had declined precipitously to zero by week 14 (Table 2). Storage of seeds without endocarp showed a gradual decrease to 47.4 and 44.4% MC at 16 and 25°C, respectively for axes after 8 weeks (Table 3). While seeds maintained 90% viability for 8 weeks at 16°C, only 75% of those seeds germinated after storage at 25°C for the same duration. In addition, at both temperatures, vigour as indicated by the germination rate declined progressively (Table 3). Partial desiccation for 2 h, to 42.9% MC for axes and 26.5% MC for cotyledons, had no immediate effect on germination totality (Table 4). While there was little change in axis moisture content during subsequent storage of the partially dried seeds (sub-imbibed storage), AFRICA 113 viability declined to 60 and 40% by 8 weeks at 16 and 25°C, respectively. While there was little change in the germination rate of seeds stored at 16°C, the period to reach 50% germination increased from 6 to 9 days for seeds stored at 25°C.

Discussion

Mature seeds of Ekebergia capenis were characterized by moisture contents, greater than 23%, fmb, at harvest, which according to Berjak and Pammenter (2001) is suggestive of the recalcitrant category in some species. All the freshly harvested E. capensis seeds germinated within 4–5 days (Table 1). In contrast, previous work has reported that fresh seeds attain only 60% germination after 4–6 weeks (Msanga 1998; Netshiluvhi 1999). This emphasises the need to clarify what propagules (seed sensu stricto or pyrene) are under investigation for germination tests. Our experience with E. capensis as well as other species, including Stangeria eriopus, Encephalartos ferox and E. villosus, has shown that removal of such structures not only increases the speed at which these seeds germinate (which may otherwise take months, e.g. in the case of S. eriopus), but also minimizes the effects of seed-associated microorganisms during germination. This is achieved by allowing for the selection of healthy, uninfected seeds from the start, and maximizing the efficacy of surface sterilization for in vitro germination assays, both of which would normally be obscured by such structures.

Table 2. Germination (G%) of E. capensis seeds with endocarp after storage at 16 and 25°C for 18 weeks. The whole seeds were initially at 30.79% MC and moisture contents of axes and cotyledons (Coty%) are also shown

Storage time 16°C 25°C (weeks)

Axis MC Coty MC G T50 Axis MC Coty MC G T50 (%) (%) (%) (%) (%) (%) (%) (%) 0 60.0 31.0 100 4.5 60.0 31.0 100 4.5 2 56.5 28.5 100 2.6 56.5 28.5 100 3 4 58.3 28.5 100 3.9 58.3 33.3 100 3.5 6 54.5 28.5 100 – 50.0 28.5 100 – 8 58.3 28.5 100 – 54.5 28.5 100 3.9 10 58.3 28.5 100 3.2 54.5 23.0 100 4 12 58.3 28.5 100 3.0 41.1 16.6 100 4.6 14 62.9 33.3 100 3.0 – – 0 – 16 52.3 25.9 100 1.5 – – 0 – 18 58.3 28.5 100 – – – 0 – 114

Table 3. Germination (G%) of E. capensis seeds without endocarp after storage at 16 and 25°C for 8 weeks. Moisture contents TREESEEDS OF TROPICAL BIOLOGY STORAGE of whole seeds (Seed%), axes and cotyledons (Coty%) are also shown

Storage time 16°C 25°C (weeks)

Seed MC Axis MC Coty G T50 Seed MC Axis MC Coty MC G T50 (%) (%) MC (%) (%) (%) (%) (%) (%) (%) (%) 0 30.79 59.18 31.03 100 4.5 30.79 59.18 31.03 100 4.5 2 25.90 45.94 25.92 100 4.3 29.19 53.48 28.57 100 4 4 27.17 53.48 27.00 80 3.4 27.95 56.52 27.53 85 3 6 24.68 45.94 24.24 80 6 26.57 53.48 25.92 95 5.6 8 24.78 47.36 21.25 90 6 22.37 44.44 21.25 75 6.3

Table 4. Germination (G %) of E. capensis seeds without endocarp after sub-imbibed storage at 16 and 25°C for 8 weeks. Endocarps were removed and the seeds were then partially dried in silica gel for 2 h, before storage

Storage time 16°C 25°C (weeks) Seed MC Axis MC Coty MC G T Seed MC G T 50 Axis MC (%) Coty MC (%) 50 (%) (%) (%) (%) (%) (%) (%) (%) 0 27.03 42.85 26.47 100 6 27.03 42.85 26.47 100 6 2 20.69 45.94 20.00 100 6.6 23.66 39.39 23.66 95 7 4 20.33 41.17 20.29 80 5.5 23.77 42.85 24.24 80 8 6 20.67 42.85 20.00 70 – 23.92 48.71 24.24 50 9 8 18.14 33.33 18.03 60 6.5 21.00 37.50 21.25 40 – AFRICA 115

Desiccation trials

The results of the desiccation trials indicate that fresh seeds of E. capensis germinated 100% at axis moisture contents of 20% (Table 1). Drying whole seeds of E. capensis rapidly in silica gel had no immediate effect on germination totality as all germinated, until a 20% decrease occurred when axis moisture content was reduced to 16.6% (Table 1). In a study undertaken with the same species, it was reported that seeds that were dried slowly (i.e. dried in silica gel, with their endocarps intact) started to lose viability at an axis moisture content of about 56.5% while no seeds germinated when dried to 37.5% MC. In contrast, seeds that were dried rapidly (similar to those in the present study) retained 90% viability to a moisture content of 33.3% and viability was completely lost only at 16.6% (Pammenter et al. 1998). Dehydration of E. capensis seeds for relatively short intervals, stimulated germination rate. Similar stimulatory effects of shorter-term dehydration have been reported for seeds of E. capensis (Pammenter et al. 1998). While the basis for the stimulation is not yet known, the phenomenon is presently being investigated in our laboratory.

Storage trials

The results from the various storage trials employed in these investigations indicate that conventional storage of these seeds, as prescribed by the protocol, was possible in the short-term only. During seed processing, E. capensis seeds are removed from the fruit pulp still enclosed by the endocarp. As previous reports imply that storage was in this condition (Msanga 1998; Netshiluvhi 1999), seeds were presently stored at 16 and 25°C either with the surrounding endocarp, but also following removal of the endocarps. Storing seeds with their endocarps intact seemed to have a different outcome compared with that observed for those stored without endocarps. At both temperatures (16 and 25°C), 100% germination was achieved until week 12 of storage for those seeds stored with their endocarps intact (Table 2), whereas those stored without the endocarp at 25°C had lost viability completely by week 8 (Table 3). Seeds with endocarp stored at 25°C lost viability precipitously after week 12 as opposed to those stored at 16°C, which retained 100% 116 STORAGE BIOLOGY OF TROPICAL TREE SEEDS germinability for up to 18 weeks. In contrast, if the endocarps had been removed, a decline of seed viability was apparent already after 2 weeks at either temperature (Table 3). After endocarp removal, stored seeds lost water slowly and steadily at both 16 and 25°C, which is suggested to be the underlying cause of the decline in viability (Table 3). This was ultimately also the case for endocarp-enclosed seeds at 25°C, with an abrupt further decline after 10 weeks in storage (Table 2). Msanga (1998) stated that E. capensis seeds should be sown within four weeks after harvesting. The presently-demonstrated ability for the seeds to germinate following 12 weeks storage in the experiments may be accounted for, in part, by the fact that the germination assays involved the removal of the endocarps prior to germination testing. If the seeds had become somewhat debilitated during storage (as is presently indicated by the increasing time taken for germination), then they may not have been sufficiently vigorous to rupture the endocarp. This may explain the observations of Msanga (1998). The present results indicate that storing the seeds with their endocarps intact was a better option than storing them without endocarps. This can generally be interpreted as a matter of water retention. The enclosed seeds maintained viability, as long as there was no lethal decline in their water content—which was also the case, but occurred considerably earlier in seeds after endocarp removal. It must, however, be emphasized that water loss and particularly the rate of water loss emerge as critical factors: experimentally, enclosure by the endocarp merely takes advantage of the properties of a natural structure to maintain moisture content. Pammenter et al. (1998) used this same natural device to slow the dehydration of E. capensis seeds buried in silica gel, compared with those similarly treated, but after endocarp removal. In that case, the prolonged time taken for water loss of the endocarp-enclosed seeds was unequivocally associated with profound deterioration of the seeds, while still relatively highly hydrated, compared with the maintenance of quality of rapidly-dehydrated unenclosed seeds at similar moisture contents. On the basis of these storage results, it appears the survival of E. capensis seeds during short-term storage is extended at lowered temperatures (i.e. 16°C), but reduced under conditions close to ambient (i.e. 25°C). While storage of these seeds at temperatures lower than 16°C is not presented here, there is some evidence to suggest chilling-sensitivity. Other (unpublished) results for E. capensis seeds in our laboratory indicate that while viability is retained for 12 weeks at 6°C, germination declines significantly after only 8 weeks when storage is at 3°C. Sub-imbibed AFRICA 117 storage, following partial dehydration of fresh (imbibed) E. capensis seeds, proved not only ineffective in extending the storage lifespan of these species in the short term, but actually curtailed longevity. E. capensis seeds dried in silica gel for 2 h, then stored at 16 and 25°C for 8 weeks had only 60 and 40% germination, respectively (Table 4). This was lower than the germination capacity (100%) of hydrated seeds stored (with their enclosing endocarps) for the same duration and under the same conditions (Table 2). In theory, partial dehydration to levels permitting continuing vital metabolism, but curtailing germinative metabolism (and hence germination per se) could increase the storage lifespan of recalcitrant seeds (Berjak and Pammenter 2001). However, based on these results, as well as the results of other recalcitrant species, including T. dregeana (Drew et al. 2000), the contrary is the case, as storage lifespan is actually diminished by initial partial dehydration. In summary, under the conditions of the dehydration protocol for this project, the lower moisture contents tolerated during desiccation by the seeds described above agree with the proposal that the variation of desiccation tolerance across species represents a continuum of behaviour (Farrrant et al. 1988; Berjak and Pammenter 1997; Pammenter and Berjak 1999). However, from these, and other, investigations, it is apparent that the ability of any one of these species to tolerate desiccation is dependent on the rate of drying, and, possibly, the maturity of the seed at harvest, and, therefore, cannot be quantified on the basis of a ‘critical moisture content’ value per species. Seed viability was retained to higher levels at 16°C, than 25°C. This is suggested to be the outcome of more rapid, germinative metabolism occurring in storage at the higher temperature, thus imposing the requirement for additional water—and therefore a mild, but sustained water stress— earlier at 25 than 16°C. Concomitantly, however, metabolism of any seed-associated mycoflora would have been enhanced at 25°C, thereby exacerbating seed deterioration. Sub- imbibed storage of nonorthodox seeds was not successful for this species, as viability was dramatically reduced. Seeds of Ekebergia capensis survived desiccation to an axis MC of 20%. Seeds, with endocarp intact, remained viable following 18 weeks storage at 16°C. Removal of the endocarps prior to storage, or storage with intact endocarps at 25°C greatly reduced seed viability in 2 and 12 weeks, respectively. 118 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

The collaboration within the project

Of the seven species targeted for replication by UND, only 2 species were received and screened. An additional species, Lophira lanceolata, was collected and dispatched by the collecting partner. However, the consignment was sent via air freight to South Africa and, despite our efforts, was delayed at Johannesburg International Airport Customs for over 1 month, resulting in the seeds being unusable for screening. On the whole, the lack of communication from some collecting partners was disappointing, but we are confident that this will be improved upon in future collaborations. The protocol employed in these investigations has proved very useful as a standard measure for determining desiccation and storage behaviour of, as yet, unclassified tropical forest tree seeds. We propose, however, that the following be taken into consideration when utilizing this protocol. (a) Consignments should be dispatched via courier, which also ensures that the seeds arrive at their destination in the shortest possible time. This is essential, particularly for those species where rapid viability loss at ambient RH is known or suspected. (b) Dispatch should be as fruits, rather than seeds. Under these conditions, providing the time taken to reach the destination partner is short, the seeds are more likely to maintain the physiological state at harvest. (c) Analysis of target moisture content (TMC) on a whole seed basis, due to the small size of the embryonic axes (e.g. in E. capensis and S. cuminii), will often show a bias towards changes in the moisture content of the storage tissues, while axis moisture content remains considerably higher during desiccation. This emphasises the need to clarify the use of TMCs in these experiments on a species basis, and that it may be more beneficial to consider the moisture content of the axis and cotyledons separately, rather than only that of the whole seed. In addition, while a useful indication in desiccation experiments, the relationship between TMC and actual seed moisture content is not always precise, and should be evaluated on a species basis. (d) Use of viability indicators, such as the float test method, prior to initial seed germination assessments in some species (e.g. cycads and Harpephyllum caffrum), as methods to separate out poor quality seeds from the start, contributes to more accurate and solid data sets. (e) Some species (e.g. H. caffrum) are characterized by poor initial germination either as a consequence of inherent factors, or because of the nature of the germination assay applied during assessment. In the latter, germination conditions should be optimized prior to the initiation of the screening protocol. Where possible, AFRICA 119 information regarding specific germination requirements should be shared between partners prior to the exchange of seeds. In view of the well-known differential effects of drying rate on viability of recalcitrant seeds, any investigation of tropical forest tree seeds should include both silica gel drying, and drying under ambient conditions (shade or bench-top drying).

Future activities

In those species where recalcitrance has been established, cryostorage protocols should be developed for long-term conservation of germplasm. In addition, continued collaboration with partners in terms of information, seed and resource exchange should take place. In this regard, it would be most useful if a multi-user, single site electronic discussion forum were available, enabling the network members to remain in constant contact, thereby improving the exchange of ideas and information.

Acknowledgements

We thank Ms Sthandiwe Shange, Mr Joseph Kioko, Ms Sharon Eggers, Ms Gundula von Fintel, Mr Errol Dowes, Ms Princess Khuzwayo, Ms Claire Whittaker and Ms Zama Mbatha, for their involvement in carrying out this research.

References

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Teketay, D. and A. Granstrom. 1995. Soil seed banks in dry Afromontane forests of Ethiopia. J. Vegetat. Sci. 6:777–786. Teketay, D. and A. Granstrom. 1997a. Germination ecology of forest species from the highlands of Ethiopia. J. Trop. Ecol. 14:793–803. Teketay, D. and A. Granstrom. 1997b. Seed viability of Afromontane tree species in forest soils. J. Trop. Ecol. 13:81–95. Thompson, M.W. 1999. South African national land cover database project. Data Users Manual Final Report (Phases 1, 2 and 3). CSIR Report Env/P/C 98136. CSIR, Pretoria, South Africa. Venter, F. and J.-A. Venter. 1996. Making the Most of Indigenous Trees. Pp. 52–53. Briza Publications, Pretoria, South Africa. 122 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Sensitivity of Syzygium cuminii (L.) Skeels seeds to desiccation

David Baxter, Deon Erdey and Patricia Berjak

Plant Cell Biology Research Unit, School of Biological Sciences, University of KwaZulu-Natal, Durban 1041, South Africa

Abstract

The response of Syzygium cuminii seeds to desiccation was assessed following burial in silica gel. Seeds with initial 46.2% MC all germinated (100%). Following slow drying (17 d) to ca. 14 to 16% seed moisture content, however, none of the seeds germinated, suggesting that S. cuminii seeds are desiccation sensitive. In addition, the germination of control seeds (maintained in vermiculite at their initial moisture content for the same duration) decreased by 20%. This suggests that the seeds of this species exhibit recalcitrant storage behaviour. The marked proliferation of seed- associated mycoflora during all the germination assessments in this study, however, makes unequivocal conclusion about the effects of desiccation only, difficult.

Introduction

Syzygium cuminii (L.) Skeels, the jambolan-plum tree belongs to the Myrtaceae family. It was introduced to Africa from India and tropical Asia for its edible and attractive purple-red fruits (Mbuya et al. 1994; Pooley 1995; Coates Palgrave 1984). S. cuminii seeds are recalcitrant (Patil et al. 1997; Rawat and Nautiyal 1997) and viability under ambient conditions is lost soon after shedding (Mbuya et al. 1994; Patil et al. 1997). Storage of these seeds, therefore, poses a significant challenge, which is compounded by the fact that the seed is known to be susceptible to fungal infection. We investigated seeds received from Tanzania, and report the results in the present paper. AFRICA 123

Materials and methods

Seed details and processing

S. cuminii fruits were collected on 6 Mar 1997 from (MU 042 I) Morogoro Urban. Upon arrival at the laboratory in Tanzania the seeds were reported as being very warm. The pulp was removed manually in water. No anti-fungal treatment was administered. The seed consignment was received from Tanzania on 7 March, and appeared to be in good condition. The seeds were surface sterilized with 1% sodium hypochlorite solution as outlined in the protocol (IPGRI/DFSC 1996), dried overnight on paper towel, and coated with Benlate.

Drying and germination assessment

Desiccation was carried out using activated silica gel mixed with the seeds in plastic jars, which were then sealed. Control seeds, mixed with vermiculite, were maintained in a similar manner. The desiccation trial commenced on 14 March, after the initial moisture content of 100 seeds had been determined. Moisture content was determined gravimetrically on 25 seeds at each sampling, following oven drying at 80°C for 48 h. At each sampling, germination (four replicates of 25 seeds each) was assessed by placing the seeds in trays with moist vermiculite at 25°C. Germination was scored as positive on the basis of radicle protrusion of approximately 1 mm. Seeds with target moisture contents (TMCs) lower than 15% were imbibed above water in a sealed container for 72 h, prior to the germination assessment.

Results

The average seed weight obtained for S. cuminii was 1.32±0.26 g. Moisture contents of the seed components were as follows: cotyledon 50.2% and embryo 74.7% (Table 1). The initial moisture content of the seed batch was 46.2%, the seeds being 100% viable (Table 2). 124 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Drying was undertaken to five target moisture contents ranging between 16.11 and 9.12%. Whereas the moisture contents of the control treatments showed no significant variation, remaining at an average 45%, the percentage viability fluctuated. At the sampling of both 16.11% and 9.12%, the viability of the controls was 79% germination. However, this was as low as 24% germination for a control treatment at 13% target MC (Table 2). The viability following all drying treatments was zero—possibly as a result of the long period of desiccation (large seed mass in proportion to silica gel used) coupled with increased susceptibility to fungal infestation particularly at lowered moisture contents. Once set to germinate, seeds from all treatments eventually showed fungal proliferation. Fungi included Penicillium spp. (predominantly P. oxalicum), Fusarium spp. and Aspergillis niger.

Table 1. Seed weight and moisture contents of seed components S. cuminii Weight (g) Moisture content (% fresh mass) (g.g–1dry weight) Seed 1.32±0.26 46.20r4.26 0.87r0.16 Cotyledon – 50.21±6.03 1.04±0.27 Embryo – 74.69±4.36 3.06±0.68

Table 2. Moisture content (MC) and viability (G%) assessment of whole seeds of S. cuminii

Target MC Control Dried (%) MC (%) MC (g.g–1) G (%) MC (%) MC (g.g–1) G (%) Initial 46.20 r 4.26 0.868 r 0.16 100 - - - 20 44.41 r 2.85 0.803 r 0.09 73 14.39 r 4.14 0.171 r 0.06 0 16 45.47 r 1.88 0.836 r 0.06 79 16.11 r 5.79 0.198 r 0.091 0 13 46.58 r 1.67 0.874 r 0.06 24 12.20 r 2.09 0.140 r 0.028 0 10 45.96 r 2.81 0.856 r 0.10 37 12.05 r 2.02 0.138 r 0.026 0 8 46.38 r 3.03 0.871 r 0.10 79 9.12 r 0.723 0.100 r 0.009 0

Discussion

Seeds of S. cuminii appear to be desiccation sensitive, as none of the seeds dried in silica gel to moisture contents below 16.1% germinated (Table 2). The drying rate applied here was slow, as the first TMC of AFRICA 125

20% was achieved after only 17 days (results not shown). The deleterious effect of slow drying on S. cuminii seed viability has been previously described by Rawat and Nautiyal (1997), who recorded that seeds dried slowly, under ambient conditions in the shade, lost viability after 21 days at 27% seed moisture content. The germination of control seeds, maintained in vermiculite under ambient conditions for the same duration (17 d), decreased by 20%, despite little or no decline in initial seed moisture content of ca. 46% (Table 2). This supports previous suggestions that S. cuminii seeds exhibit recalcitrant storage behaviour (Patil et al. 1997; Rawat and Nautiyal 1997). However, it is difficult to draw conclusions based on the current data, as the results were confounded by heavy fungal proliferation. For future trials to produce unequivocal results in terms of desiccation sensitivity, it is essential that the mycoflora be inactivated or eliminated.

References

Coates Palgrave, K. 1984. Trees of Southern Africa. Struik Publishers, Cape Town, South Africa. IPGRI/DFSC. 1996. Desiccation and storage protocol. The Project on Handling and Storage of Recalcitrant and Intermediate Tropical Forest Tree Seeds. Newsletter No. 1. Mbuya, L.P., H.P. Msanga, C.K. Ruffo, A. Birnie and B. Tengnäs. 1994. Useful Trees and Shrubs for Tanzania. Regional Soil Conservation Unit, Swedish International Development Authority, Nairobi, Kenya. Patil, V.S., G.K. Halesh and K.V. Janardhan. 1997. Recalcitrant behaviour of Jamun seeds. Plant Physiol. Biochem. 24:106–107. Pooley, E. 1995. The Complete Field Guide to Trees of Natal, Zululand and Transkei. Natal Flora Publications Trust. c/o Natal Herbarium, Durban, South Africa. Rawat, D.S.C. and A.R. Nautiyal. 1997. Seed viability in Syzygium cuminii in response to drying. Pp. 59–61 in Proceedings of the IUFRO Symposium on Innovations in Forest Tree Seed Science and Nursery Technology (S.C. Naithani, B. Varghese and K.K. Sahu, eds.). Raipur, India, November 22–25, 1997, Jai Printers, Raipur, India. 126 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Sensitivity of Trichilia emetica Vahl. seeds to desiccation

David Baxter, Deon Erdey and Patricia Berjak

Plant Cell Biology Research Unit, School of Biological Sciences, University of KwaZulu-Natal, Durban 1041, South Africa

Abstract

Trichilia emetica (Vahl.) seeds, collected in Kenya, were dispatched to Durban for a replication trial aimed at assessing the tolerance of these seed to desiccation. After discarding the infected seeds, only a small sample of good seeds was available for screening. All fresh seeds with 39.6% MC germinated (100%). The germination capacity decreased to 50% following desiccation to 14.3% MC, while none of those seeds dried to lower moisture content levels germinated. This suggests that seeds of T. emetica are desiccation sensitive. In addition, the germination of fresh seeds maintained in vermiculite for the same period of time as those dried to 14.3% MC, decreased by 25%, with little change in the initial moisture content, indicating a recalcitrant storage behaviour. The persistence of fungi, present upon receipt of the seeds and during subsequent handling, may have exacerbated the effects of the screening treatments on seed germination.

Introduction

Trichilia emetica Vahl., a member of Meliaceae family, is an evergreen or semi-evergreen tree approximately 8–25-m high, growing in coastal, riverine and gallery forest, and is widely distributed in tropical and subtropical Africa, from Sudan in the North to the eastern-coast of South Africa (Venter and Venter 1996). Almost every component of the tree is highly utilized, so much so that T. emetica is protected in South Africa. The wood, which is pinkish to light brown, can be used as timber and fuelwood, for indoor furniture, poles and posts. The leaves and shoots are used as fodder, and the bark and roots used medicinally as an emetic. The bark is also used to produce a pinkish dye. The seeds are commercially used for the production of oil and soap. The oil extracted from seeds is used cosmetically and to treat broken bones. In addition, seeds can be soaked in water and the milky ‘soup’ is eaten with spinach (Coates Palgrave 1984; Albrecht 1993; Pooley 1995; Venter AFRICA 127 and Venter 1996). In addition to the above uses, T. emetica is valued for shade and planted in public places (Venter and Venter 1996). The black seeds are almost completely surrounded by a scarlet aril and are produced in spherical capsules, each containing approximately six to eight seeds (Coates Palgrave 1984; Venter and Venter 1996). There are about 200 (Venter and Venter 1996) to 1340 seeds per kilogramme (Msanga 1998), which presumably depends on their water contents at the time of determination. The demand for parts of the tree and their wide-ranging uses necessitates a greater understanding of the behaviour of seeds of T. emetica, especially as difficulty has been encountered storing the seeds, as they exhibit recalcitrant storage behaviour (Msanga 1998), losing viability within a very short time.

Materials and methods

Seed details and processing

T. emetica fruits were collected on 13 June 1997 from Siaya, Kenya. The fruits were separated from seeds manually, placed in open plastic containers and transported to the laboratory. The seeds were dispatched on 14 June, and the consignment was received from Kenya on 20 June (after a delay during transportation). Presumably, as a result they were in very poor condition. The fungal infestation persisted even after surface sterilization, approximately 80% of the seeds being affected. An investigation, with limited numbers of probably contaminated seeds, was nevertheless undertaken. The seeds were surface sterilized with 1% sodium hypochlorite solution as outlined in the protocol (IPGRI/DFSC 1996). As the fungi persisted after sterilization, this step was repeated after 10 h. The seeds were subsequently dried overnight on paper towel, and coated with Benlate.

Drying and germination assessment

Seed desiccation was carried out using activated silica gel mixed with the seeds in plastic jars, which were later sealed. The desiccation trial commenced on 24 June, after the initial moisture content had been determined gravimetrically by weighing seed samples before and after oven drying at 103°C for 17 h. The silica gel was changed each day to 128 STORAGE BIOLOGY OF TROPICAL TREE SEEDS try and ensure a rapid rate of drying. Fewer target moisture contents than outlined in the protocol were attempted owing to the low seed numbers. Germination was assessed by placing the seeds in trays in moist vermiculite at 25°C, and scored as positive on the basis of radicle protrusion of approximately 1 mm. There were sufficient seeds for only 2 replicates of 50 seeds for each target moisture content.

Results

The average seed weight obtained for T. emetica was 2.92 ± 0.92 g and they had 39.6% initial moisture content (Table 1). Moisture content was also determined for the seed components, and the cotyledons had initially 26.4% and the embryos 33.2% MC (Table 1). The initial viability obtained for the seed consignment, from which obviously infected seed had been removed, was 100% (Table 2). Drying was undertaken to five target moisture contents, equivalent to the range between 14.3 and 2.4% MC. Results obtained showed considerable fluctuation which could be attributed to the poor state of the seeds from the start. The viability of the dried seeds decreased with decreasing moisture content. Seeds with 14.3% MC germinated 50%, but no germination occurred below this moisture level. The controls for the dried treatments showed a decrease from 39.6 to approximately 30% MC. The corresponding capacities of these control seeds to germinate were erratic but generally showed a progressive decrease in viability to 15% germination at 30.9% MC (Table 2). The predominating fungal species were Fusarium spp., Penicillium spp. and Aspergillis niger, and these were manifested especially in the dried seeds. As a result of the initial fungal infection, the seed quality was variable, with correspondingly variable results being obtained.

Table 1. Seed weight and moisture contents of seed components of T. emetica Weight (g) Moisture content (% fresh mass) (g.g–1dry weight) Seeds 2.92 ± 0.92 39.57 r 6.85 0.685 ± 0.098 Cotyledon – 26.41 ± 11.46 0.396 ± 0.267 Embryo – 33.17 ± 12.80 0.547 ± 0.248 AFRICA 129

Table 2. Whole seed moisture content and viability assessment in T. emetica seeds

Control Dried Target MC (%) MC (g.g–1) G (%) MC (%) MC (g.g–1) G (%) MC (%)

Initial 39.57 r 6.85 0.685 r 0.098 100 20 30.85 r 6.59 0.460 r 0.015 75 14.33 r 5.54 0.173 r 0.087 50 16 32.52 r 6.88 0.500 r 0.192 35 12.44 r 3.94 0.144 r 0.059 0 13 29.64 r 4.88 0.428 r 0.106 95 3.87 r 0.70 0.04 r 0.007 0 8 30.87 r 4.30 0.451 r 0.091 15 2.41 r 0.25 0.025 r 0.003 0

Discussion

The consignment T. emetica seeds received from Kenya, with 39.6% MC was initially highly viable (100% germination). Drying these seeds to 14.3% MC resulted in a loss of 50% germination, and no seeds germinated below this moisture level (Table 2). Similarly, Msanga (1998) has reported that T. emetica seeds cannot tolerate desiccation below 20%, findings that have been confirmed by recent studies in our laboratory. The germination capacity of the controls for the dried treatments were erratic but generally showed a progressive decrease in viability to 15% germination at 30.9% MC. Predominance of fungi within the seed material severely hindered the drawing of conclusions regarding the storage behaviour of T. emetica. Upon arrival of the seeds at the laboratory in Durban they were highly contaminated with fungi. The severely affected seeds were discarded while the rest were washed thoroughly and subsequently surface sterilized with 1% hypochlorite solution. However, after 10 h fungal proliferation was once again obvious. Sterilization was repeated and the drying experiments undertaken. The lack of germination after drying could be attributable to the desiccation sensitivity of the species. However, the poor germinability appeared to be also influenced by the fungal infection predominating before the seeds arrived in the laboratory. This may have contributed to the variability of the germination of the controls. It would appear that there is an increased susceptibility of dried seeds to fungal proliferation and in this case it was exacerbated by the inoculum already being present within the seed consignment. 130 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

References

Albrecht, J. 1993. Tree Seed Handbook of Kenya. GTZ Forestry Seed Centre Muguga, Nairobi, Kenya. Coates Palgrave, K. 1984. Trees of Southern Africa. Struik Publishers, Cape Town, South Africa. IPGRI/DFSC. 1996. Desiccation and storage protocol. The Project on Handling and Storage of Recalcitrant and Intermediate Tropical Forest Tree Seeds. Newsletter No. 1. Msanga, H.P. 1998. Seed germination of indigenous trees in Tanzania. Including Notes on Seed Processing, Storage and Plant Uses. Canadian Forest Service, Alberta, Canada. Pooley, E. 1995. The Complete Field Guide to Trees of Natal, Zululand and Transkei. Natal Flora Publications Trust, c/o Natal Herbarium, Durban, South Africa. Venter, F. and J-A. Venter. 1996. Making the Most of Indigenous Trees. Briza Publications, Cape Town, South Africa. AFRICA 131

Tolerance to desiccation and storability of Warburgia salutaris (ugandensis) seeds from Kenya

Joseph Kioko, David Baxter and Patricia Berjak

Plant Cell Biology Research Unit, School of Biological Sciences, University of KwaZulu-Natal, Durban 1041, South Africa

Abstract

Warburgia salutaris is one of the most highly utilized medicinal plants in tropical and subtropical Africa and is now highly endangered in the wild. The seeds are reputedly sensitive to desiccation, but the response is not well characterized. W. salutaris seeds from Kenya with initially 46% MC (0.85 g g–1) were dried to 7.7% MC (0.084 g g–1). This resulted in their germination capacity decreasing from 93 to 10%. Fresh embryonic axes with 1.6 g g–1 (61.1% MC) maintained viability when seeds were (slow) dried to 0.1 g g–1 (ca. 9.1% MC) in silica gel. With rapid dehydration in laminar airflow, viability was lost by a water content of 0.3 g g–1 (ca. 23.1% MC). Seed quality was generally poor, which seems to be an ever-present problem with W. salutaris, where maggot infestation occurs regularly. Only seeds dehydrated to 0.1 g g–1 water content could withstand cryopreservation with 30% survival after freezing in liquid nitrogen. Since the ability to withstand desiccation and subsequent cryopreservation may depend on the seed’s developmental stage, further studies are being conducted to investigate the effect of harvesting time on both desiccation and cryopreservation.

Introduction

Warburgia salutaris (Bertol.f.) Chiov. has two synonyms, Warburgia breyeri Pott and Warburgia ugandensis Sprague. The species is a member of the Canellaceae family and commonly known as the pepper-bark tree or the East African green-heart. The wood can be used as timber and fuelwood, and for the production of tool handles (Albrecht 1993). It is one of the most highly utilized medicinal plants in tropical and subtropical Africa. The bark contains at least five sesquiterpenoid dialdehydes, including warburganal and polygodial. Warburganal has broad anti-microbial activity against various yeasts and moulds and is 132 STORAGE BIOLOGY OF TROPICAL TREE SEEDS a potent anti-feedant against Spodoptera exempta, the African armyworm, whilst polygodial has been shown to enhance significantly the antibiotic activities of actinomycin D, rifampicin and maesanin. Bark from roots and stems is used by local communities as an expectorant, smoked for colds and coughs, and also used for relief of gastro-intestinal disorders and skin complaints (Pooley 1993), among other ethnobotanical applications. The bark is so much in demand that many trees in the southern Africa have been completely destroyed (Palgrave 1977). The high demand, for example over 15 000 kg of bark per year in one market in Durban alone (Cunningham 1988) and attendant overexploitation, have made the species highly endangered and consequently almost extinct in the wild. It is now listed as a species at high risk of extinction on the IUCN Red List of Threatened Species (IUCN 2002) that needs urgent and priority conservation actions. While seed storage would offer an efficient method for the conservation of this species, mature seed production in the wild is virtually unknown. Furthermore, the seeds are parasitized by fungi and insects, and predated by birds and primates to a great extent in some countries like South Africa. There is also little and inconclusive information on the postshedding physiology and storability of the seeds (Albrecht 1993; Hutchings 1996). However, seeds of W. ugandensis (salutaris) are reputedly sensitive to desiccation and hence problems have been encountered in attempting to dry and store these seeds. The importance of understanding the seed behaviour and successfully storing them is vital in terms of long-term conservation and lessening the heavy utilization of the tree’s by-products. Thus, the present study was aimed at understanding pertinent aspects of postshedding seed behaviour and developing a method for the long- term storage of the germplasm of this species.

Materials and methods

Seed collection and processing

Fruits were harvested in Kenya during Feb 1997 and in 1998. The seed consignments received in Durban were mostly immature being green and very firm to touch. Fruits were thus stored at least for four weeks at 16ºC, until they were relatively soft and considered mature. A large AFRICA 133 number of fruits were discarded because they were either infested by insect larvae, or were rotten. The soft mature fruits were opened and seeds scooped out and gently wiped clean using paper towel and blotted dry. Seeds were coated with Benlate, and desiccated in sealed plastic jars containing approximately 150 g of activated silica gel.

Desiccation trials

Desiccation experiments involved first drying seeds down to 5% target moisture content (TMC), after which attempts were made to achieve other TMC, once seeds became available through ripening. To investigate the effect of the rate of drying on germination capacity, embryonic axes were excised from the seeds and placed on dry filter paper in a Laminar Air Flow cabinet for up to 2 h, for rapid drying. Slower drying was carried out by using whole seeds that were buried in activated silica gel at 25°C for up to 72 h. Moisture content was determined (ISTA 1999) either on the fresh mass basis or on the dry weight basis in some of the experiments.

Viability assessment

Whole seeds were germinated on moist filter paper in plastic Petri dishes or in vermiculite at 25°C, while embryonic axes were assessed for viability by tetrazolium staining. Red coloration of the axes was indicative of their viability (ISTA 1999). Seeds were scored as germinated on the basis of radicle protrusion of approximately 1 cm. Twenty seeds or axes were used for viability tests.

Cryopreservation

Seeds were enclosed in 1 ml cryotubes (four cryotubes with five seeds each per treatment), and plunged into liquid nitrogen (–196°C). They were maintained at –196°C for 1 h, after which they were rapidly plunged into a water bath for thawing at 35°C for 2 min. 134 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Results and discussion

Seed characteristics

The average weight of W. salutaris fruits was 33.20±7.87 g, while the seed weight was 0.22±0.04 g (Table 1). Initially, fruits, seeds and components had on average 68.7% MC for fruits with pulp, 46% MC for whole seeds, 56.5% MC for cotyledons and 61.1% MC for embryonic axes (Table 1).

Table 1. Initial characteristics of fruits, seeds and seed components of the 1997 collection Weight (g) Moisture content (% fresh mass) (g g–1 dry weight) Fruit plus pulp — 68.70±1.69 2.20±0.16 Fruit 33.20±7.87 — — Seed 0.22±0.04 45.97r4.61 0.86±0.17 Cotyledon — 56.47±6.47 1.35±0.37 Embryonic axis — 61.11±3.91 1.60±0.28

Effect of dehydration on seed germination

Drying to the target moisture content of 5%, resulted in seeds with 7.7% MC. Such drying decreased the initial germination of 93 to 10% (Table 2). The viability of seeds maintained as a control also decreased to 53% germination. Sufficient seeds were available for later drying to two other target moisture contents of 16 and 13%. No germination occurred in these samples, and the seeds rapidly became overrun with fungi (Table 2). Seeds of both experimental and control treatments showed zero viability, and great fungal proliferation (Rhizopus nigricans) occurred. Possibly, this was a result of the long delay between harvesting and commencement of the second drying experiment. Furthermore, the differential ripening of the fruits resulted in the desiccation trials being staggered using seeds from available ripe fruits for a particular target moisture content. This may have also affected the viability and capacity of seeds to withstand the desiccation stress. Fresh seeds (1998) of W. salutaris had average axis water content of 1.6 g g–1 (61.1% MC), and maintained viability to a water content as low as 0.1 g g–1 (ca. 9.1% MC) following drying in silica gel, although their performance was less than optimal (Fig. 1). The rate of AFRICA 135 germination, represented in this figure by the slope of the curve, generally increased directly with the degree of dehydration, reaching a maximum at 0.3 g g–1 (ca. 23.1% MC). This response has been observed for several recalcitrant seed species, in which limited dehydration stimulates germination (e.g. Pammenter et al. 1998). On the other hand, with rapid dehydration of excised embryonic axes, viability was lost by a water content of 0.3 g g–1 (Fig. 2) with the cells suffering extensive ultrastructural damage (data not shown). These results indicate that seeds of W. salutaris may possess mechanisms, which modulate against desiccation damage, but that during rapid axis dehydration there is insufficient time for these mechanisms to come into operation. Tolerance to slow, rather than rapid, dehydration is atypical of recalcitrant seeds (Berjak et al. 1993), but is observed in developing orthodox-seed embryos that have acquired the ability to withstand dehydration (Bochicchio et al. 1994). This, coupled with the ability of the seeds to withstand desiccation to 0.1 g g–1, might imply that W. salutaris seeds are either fully desiccation-tolerant or that they undergo indeterminate development (Finch-Savage and Blake 1994), in which the degree of desiccation tolerance depends on the developmental stage at which the seeds are shed. As optimal performance of these seeds occurred at 0.3 g g–1, they are most likely not to be typically orthodox. This aspect will be the subject of further studies in the coming seasons. As a result of the lack intermediate target moisture contents and shortage of seeds, one cannot draw accurate conclusions from the data available. But the marked susceptibility of the seeds to fungus, particularly at lowered moisture contents was noteworthy—this susceptibility appearing to increase with length of time the seed remained within the fruit pulp. This seems to be an ever-present problem with W. ugandensis (salutaris), where maggot infestation appears to be a regular occurrence. In South Africa, none of the trees ever seem to yield viable seed, because of this problem.As a result of the lack intermediate target moisture contents and shortage of seeds, one cannot draw accurate conclusions from the data available. But the marked susceptibility of the seeds to fungus, particularly at lowered moisture contents was noteworthy—this susceptibility appearing to increase with length of time the seed remained within the fruit pulp. This seems to be an ever-present problem with W. ugandensis (salutaris), where maggot infestation appears to be a regular occurrence. In South Africa, none of the trees ever seem to yield viable seed, because of this problem. 136 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

100 2 g/g 0.5 g/g 0.3 g/g 80 0.1 g/g

40 Germination (%)

024681012 Weeks after sowing Figure 1. Germination capacity of W. salutaris seeds (1998 collection) after drying axes to different axis water contents (g g–1) by mixing them with silica gel. (This chart has been also published in Kioko et al. 1999, 2000).

3.0 100

2.5 80

2.0

60 1.5 Viability (%) Viability 40 Water content (g/g) 1.0

20 0.5

0 0.0 0 20406080100120 Drying time (min)

Figure 2. Dehydration of embryonic axes (1998 collection) in laminar airflow, to water contents amenable to cryopreservation resulted in complete loss of viability by 0.3 g g–1. Axes were assessed for viability by tetrazolium staining. AFRICA 137

As a result of the lack intermediate target moisture contents and shortage of seeds, one cannot draw accurate conclusions from the data available. But the marked susceptibility of the seeds to fungus, particularly at lowered moisture contents was noteworthy—this susceptibility appearing to increase with length of time the seed remained within the fruit pulp. This seems to be an ever-present problem with W. ugandensis (salutaris), where maggot infestation appears to be a regular occurrence. In South Africa, none of the trees ever seem to yield viable seed, because of this problem.As a result of the lack intermediate target moisture contents and shortage of seeds, one cannot draw accurate conclusions from the data available. But the marked susceptibility of the seeds to fungus, particularly at lowered moisture contents was noteworthy—this susceptibility appearing to increase with length of time the seed remained within the fruit pulp. This seems to be an ever-present problem with W. ugandensis (salutaris), where maggot infestation appears to be a regular occurrence. In South Africa, none of the trees ever seem to yield viable seed, because of this problem.

Cryopreservation of whole seeds

Only seeds dehydrated to 0.1 g g-1 water content could withstand cryopreservation in liquid nitrogen (Table 3). The seeds germinated within six weeks to a give final germination of 30%. The resultant seedlings appeared normal and similar to those from fresh nonfrozen seeds.

Table 2. Whole seed moisture content (1997 collection) and viability assessment by germination in Petri dishes. (*= denotes the presence of fungi) Moisture content Seed viability (%) Target (% fresh mass) (g.g–1 dry weight) Initial (1997) 45.97 r 4.61 0.865 ± 0.175 93 5% TMC 7.74 r 0.63 0.084 ± 0.007 10 Control 51.86 r 3.42 1.087 ± 0.145 53 16% TMC 13.54 r 1.91 0.157 ± 0.026 0* Control (1998) 35.99 r 3.90 0.568 ± 0.098 0* 13% TMC 12.06 r 1.23 0.137 ± 0.016 0* Control 32.81 r 5.50 0.498 ± 0.131 0* 138 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Table 3. Survival of whole seeds of W. salutaris (1998 collection) after drying and cryopreservation in liquid nitrogen for 1 h. (This Table has been published in Kioko et al. 1999, 2000) Dehydration (h) MC (g g–1) % survival after Drying Drying and cryostorage 0 2.3 90 0 6 1.0 85 0 10 0.9 80 0 18 0.4 90 0 24 0.5 80 0 48 0.3 100 0 72 0.1 80 30

The survival of only 30% of the seeds after freezing maybe due to the variability in the maturity stages of the seeds on harvest, which was observed by ultrastructural studies (Kioko et al. 1999, 2000). It is presently hypothesized that ongoing development not only increases the frequency of subcellular organelles, but also depletes lipid reserves. Both these factors might contribute to the increasing vulnerability of the seeds to cryopreservation. It should be noted though, that despite optimal germination performance of seeds dried to 0.3 g g–1, none survived cryopreservation at this water content. Thus, for effective conservation of the germplasm of W. salutaris as cryopreserved whole seeds, it seems that the water content must be below this presently indicated optimal of 0.3 g g–1. However, the range of water contents between 0.3 and 0.1 g g–1 needs to be explored to ascertain that which will optimise survival after cryopreservation.

Conclusion

Seeds of Warburgia salutaris can withstand dehydration and freezing, so far with a recovery of 30% being attained. Since the ability to withstand desiccation and subsequent cryopreservation may depend on the stage of development at which the seeds are shed, further studies are being conducted to investigate the effect of both desiccation and cryopreservtion on seeds harvested at different maturity stages. Because of the nearly extinct status of the species, there is an urgent need to develop appropriate methods for seed storage. Thus, there are ongoing investigations into whether the seeds can be stored at lower moisture contents, using conventional seed storage methods. AFRICA 139

References

Albrecht, J. 1993. Tree Seed Handbook of Kenya. GTZ Forestry Seed Centre Muguga, Nairobi, Kenya. Berjak, P., C.W. Vertucci and N.W. Pammenter. 1993. Effects of developmental status and dehydration rate on characteristics of water and desiccation- sensitivity in recalcitrant seeds of Camellia sinensis. Seed Sci. Res. 3:155–166. Bochicchio, A., C. Rizzi, P. Vernieri and C. Vazzana. 1994. Sucrose and raffinose contents and acquisition of desiccation tolerance in immature maize embryos. Seed Sci. Res. 4:123–126. Cunningham, A.B. 1988. An Investigation into the Herbal Medicine Trade. Report 29, Institute of Natural Resources, University of Natal, Pietermaritzburg, South Africa. Finch-Savage, W.E. and P.S. Blake. 1994. Indeterminate development in desiccation-sensitive seeds of Quercus robur L. Seed Sci. Res. 4:127–133. ISTA (International Seed Testing Association). 1999. International rules for seed testing. Annexes. Seed Sci. Technol. 27:27–35, 47–50. IUCN. 2002. IUCN Red List of Threatened Species 2002. Kioko, J., P. Berjak, H. Pritchard and M. Daws. 1999. Studies of post-shedding behaviour and cryopreservation of seeds of Warburgia salutaris, a highly endangered medicinal plant indigenous to tropical Africa. Pp. 265–371 in Recalcitrant Seeds. IUFRO Seed Symposium 1998 (M. Marzalina, K.C. Khoo, N. Jayanthi, F.Y. Tsan and B. Krishanpillay, eds.). Kuala Lumpur, Malaysia. Kioko, J., P. Berjak, H. Pritchard and M. Daws. 2000. Seeds of the African Pepper-bark (Warburgia salutaris) can be cryopreserved after rapid dehydration in silica gel. Pp. 371–377 in Cryopreservation of Tropical Plant Germplasm: Current Research Progress and Application (F. Engelmann and H. Takagi, eds.). Japan International Research Centre for Agricultural Sciences, Tsukuba, Japan; International Plant Genetic Resources Institute, Rome, Italy. Palgrave, K. 1977. Trees of Southern Africa. Struik Publishers, Cape Town, South Africa. Pammenter, N.W., V. Greggains, J.I. Kioko, J. Wesley-Smith, P. Berjak and W.E. Finch-Savage. 1998. The time factor during dehydration of nonorthodox (recalcitrant) seeds: effects of differential drying rates on viability retention of Ekebergia capensis. Seed Sci. Res. 8:463–472. Pooley, E. 1993. The Complete Field Guide to Trees of Natal, Zululand & Transkei. Natal Flora Publication Trust, c/o Natal Herbarium, Publication Trust, Durban, South Africa. 140 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Seed desiccation and storability of Strychnos cocculoides, Ximenia americana and Warburgia salutaris

Ludovick O.N. Uronu and Heriel P. Msanga

Tanzania Tree Seed Agency, P.O.Box 373, Morogoro, Tanzania

Abstract

Seeds of Strychnos cocculoides, Ximenia americana and Warburgia salutaris, three indigenous species from Tanzania were investigated. Germination capacity of W. salutaris seeds was reduced from 78% at 45% MC to 0% at 8% MC. However, both, S. cocculoides and X. americana seeds retained 59 and 80% germination, respectively, after desiccation to 5% MC. Seed viability was reduced significantly (P<0.05) during storage; after three months at 4 and 16°C, S. cocculoides seeds with 10 and 30% MC were 60% viable, while none germinated at –20°C after a month. Seeds of X. americana stored at >9% MC, were severely affected by fungi, while at <5% MC seeds retained 68% germination after nine weeks of storage at 16°C. S. cocculoides seeds appear to have intermediate-type storage behaviour and should be dried to 10–30% MC and stored at temperatures above 0°C but not exceeding 25°C for at most three months. Seeds of X. americana are orthodox and can be stored at 5% MC for at least nine months at 4 or 16°C. W. salutaris seeds are recalcitrant and should not be dried below 30% MC.

Introduction

Tanzania has about 33.3 million hectares of forest and woodlands, of which two thirds consists of woodlands on public land (National Forest Policy 1998). Most valuable trees occur in natural forest, including miombo woodland ecosystems, which are threatened with extinction due to pressures such as continuous clearing of land for agriculture, overgrazing and annual forest fires (Wood 1966; Mnzava 1980). It is estimated that deforestation in Tanzania, as for other tropical forests, is occurring at a rate of between 130,000 and 500,000 ha (ca. 1%) per annum (National Forest Policy 1998). Consequently, many indigenous trees are becoming scarce in natural forests and woodlands. AFRICA 141

In Tanzania, the rural population derives a significant part of their food, energy and other essential requirements from wild trees. These trees play an important role as food sources, thus improving nutritional status, providing cash from the sale of fresh fruits or processed products. In addition, they provide materials for building and construction (FAO 1983; Palgrave 1992). It is therefore crucial to promote regeneration of these species, in particular, by using artificial regeneration techniques. Thus, there is an urgent need for the domestication of natural species. Strychnos cocculoides, Ximenia americana and Warburgia salutaris are three species of particular interest. Although natural regeneration is inadequate, these species do regenerate naturally by seed, coppice and root suckers (FAO 1983; Palgrave 1992; Mbuya et al. 1994). Domestication of indigenous tree species is hindered by a lack of knowledge of handling and storage of seeds, particularly for those species where fruit setting is unpredictable and a ready supply of seeds is not always available at the right time. Knowledge of seed storage behaviour is also required in order to conserve plant biodiversity through seed banking. According to Nkang et al. (2000), seeds of many tropical tree species do not undergo maturation drying and can only be stored short term. Due to their high moisture content, they germinate soon after shedding. The environment under which seed is stored is an important factor in determining how long the seed can be stored and maintain an acceptable level of germination and vigour. Roberts (1973) classified species as either ‘recalcitrant’ or ‘orthodox’ depending on their seed storage behaviour. Recalcitrant species are those species whose seeds cannot survive drying below relatively high moisture content and cannot be successfully stored for long periods. Orthodox species are those species whose seeds can survive drying to relatively low moisture content and can be successfully stored for long periods. Ellis et al. (1990) introduced a third category of species with ‘intermediate’ seed storage behaviour; intermediate seeds survive some desiccation but become damaged during dry storage at low temperature. There is limited information on the desiccation tolerance and storage behaviour of seeds of S. cocculoides, X. americana and W. salutaris. Msanga (1998) reported that S. cocculoides seeds are intermediate whilst seeds from X. americana and W. salutaris are recalcitrant and cannot retain viability for long periods under ambient conditions. However, more detailed information is needed to be of practical use in a programme of 142 STORAGE BIOLOGY OF TROPICAL TREE SEEDS domestication. This report describes the results of experiments carried out by the National Tree Seed Programme and International Plant Genetic Resource Institute, in collaboration with Danida Forest Seed Centre, aimed at investigating the desiccation tolerance and optimum storage conditions of seeds from these three species.

Species selection

Strychnos cocculoides Baker (known as Monkey orange and corky bark) is a member of the Loganiaceae family. It is a semi-deciduous shrub growing up to 3–8 m high. The branches have recurved stipular spines, sometimes ending in a straight spine. Its leaves are ovate or elliptic, base cunneate to round, upper surface shiny, lower surface dull with five veins from the base. It is indigenous to Tanzania and occurs in areas with annual rainfall between 500 and 1500 mm. It is widely distributed in the Zambezian regional centre of endemism, which is mainly dominated by miombo woodland. In Tanzania the species is found from sea level up to more than 2000 m (FAO 1983; Mbuya et al. 1994). In its natural habitat, there are about 4 to 20 stems with diameter >15 cm per ha of wildings (FAO 1983). S. cocculoides flowers during the rainy season (Oct to Feb) and fruits during the dry season (July to Dec) (FAO 1983), ripening within eight months of pollination. The fruit is a large, round, hard woody berry about 7 cm in diameter, dark green sparkled with white when young, becoming yellow (or orange) when ripe (Beentje 1994). Each fruit contains 25 to 30 seeds embedded in a pulp (Msanga 1998). The seeds are creamy white, flat and circular in shape, with a diameter of ca. 2 cm. The fruit pulp is edible, has a pleasant taste, and contains 135.3 mg/100 g vitamin C, 36.9 mg/100 g reducing sugar, 0.6% protein and 1.3% fat (Ndabikunze pers. comm.) and also yields 0.85% of reddish fixed oil. The roots maybe chewed as a treatment for eczema (skin disease) (Watt and Breyer- Brandwijk 1962) and are also alleged to cure gonorrhea. Ximenia americana L. (known as Wild plum) is a member of the Olacaceae family. It is a pan-tropical tree found in African savannah, America and tropical Asia. In Tanzania, it occurs in coastal regions and in the Rift valley, in open sand woodland, on stony slopes, scattered thorn bush, arid and semi-arid zones, at up to 2000 m above sea level (FAO 1983; Mbuya et al. 1994). It is a spiny shrub or small tree up to 5 m. The bark is brown-black with small scales. Leaves are alternate, simple or tufted, oblong, 2–4 cm in length, blue-grey-green; they fold upwards along the midrib and have a round or notched tip. The fruit is an ovoid AFRICA 143 drupe up to 2.5 cm long, yellow-pink-red, with one large seed (Mbuya et al. 1994; Msanga 1998). Natural regeneration of this species is sparse and inadequate. It flowers in January and again in June, fruiting respectively from Mar to May and from Oct to Nov (FAO 1983). The fruit has an edible, sweet-sour pulp. The seed is highly oily with oil making up 49– 60% of its dry weight (Beentje 1994). The oil is used for example, as body and hair lotion, to oil bows and bowstrings and is also mixed with butter and used in the preparation of traditional leather skirts in Tanzania. A maceration liquid obtained from boiled roots of X. americana treats chronic gonorrhea. The wood is used to make tool handles and for fuel (Msanga 1998). Warburgia salutaris (Bertol. F.) Chiov. (known as East African green heart) belongs to the Canellaceae family. It is an evergreen tree up to 25 m high, with a dense leaf canopy. It has rough brown-black bark, cracked into rectangular scales. Leaves are up to 10 cm long, shiny dark green above, with very clear midrib below and a wavy edge (Mbuya et al. 1994). The fruit is a capsule, green at first, turning purplish when ripe, almost round and about 1 cm in diameter. Each fruit contains about five seeds (Msanga 1998). The species is widely distributed in lower rainforest and drier highland forest areas, 1000–2000 m above sea level in Tanzania (Mbuya et al. 1994). W. salutaris is fairly slow growing, and regenerates by stem cutting and seed (Mbuya et al. 1994). It flowers in April, fruiting from Oct to Dec (Uronu 1999). The wood of W. salutaris is used for cabinet making, turnery and furniture; it also yields aromatic oil used in perfumes. The resin is used locally to fix tool handles. Dried bark is chewed and the juice swallowed as remedy for stomach-ache, constipation, coughs, cold, fever, muscle pains, weak joints and general body pains; the pounded bark is mixed with water and used for toothache. Fresh roots are boiled and the decoction mixed into soup, which is drunk for the prevention of diarrhoea; leaves prepared in the same way are used as a topical treatment for several skin diseases (Msanga 1998).

Materials and methods

Fruit collection and processing

Ripe fruits of S. cocculoides, X. americana and W. salutaris were picked from the crowns of 30 trees per species (Table 1). S. cocculoides was collected in 144 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Nov 2001; X. americana in Nov 1999; W. salutaris in Jan 2001. After collection, the fruits were immediately transported to the laboratory for extraction and experimentation. Seeds of S. cocculoides were manually extracted by cracking fruits with a wooden stick. The pulp was scooped out, the seeds squeezed out by hand and then cleaned using a paper towel. Fruits of X. americana were squeezed by hand to release the seeds from the pulp. The seeds were then washed in water and any remaining pulp gently wiped off using a paper towel.

Table 1. Location of seed sources Species Locality Location Rainfall Longitude Latitude Altitude (mm yr–1) (E) (S) (m) Strychnos Kwatanga 34° 46’ 8° 23’ 1240 818 cocculoides Ximenia Kigwe 35° 18’ 5° 56’ 800 540 americana Warburgia Viti 38° 17’ 4°43’ 1680 900 salutaris

Initial trials

Fresh fruit and seed weight was determined by measuring 100 individuals using a laboratory balance (ISTA 1993). Moisture contents were determined on whole seeds, seed coats and embryos (cotyledons plus embryonic axis). Samples were weighed and dried in an oven at 103°C for 17 h (ISTA 1993). Dried samples were weighed again and MC expressed as a percentage of fresh weight (f.wt). In order to assess the initial viability of the seeds, four replicates of 25 seeds each were sown in clean sand that had been sieved to 0.8 mm (ISTA 1993) in plastic boxes (20 cm diameter u 8 cm deep). Water was carefully added to ensure that the medium remained moist. The boxes were kept in a germination room at ambient temperature (25–30°C) with continuous artificial daylight and relative humidity maintained at 80% (ISTA 1993). Germination was recorded daily until no further germination occurred. Seeds were scored as germinated when visible protrusion of leaves or cotyledons on the surface of the sand was observed. For all species, data for cumulative germination were recorded daily. All seed viability estimates were subsequently related to this initial seed germination percent. AFRICA 145

Seed desiccation sensitivity

Seed desiccation sensitivity was carried out for each species at 25°C using silica gel according to the International Plant Genetic Resources Institute screening protocol for recalcitrant and intermediate tropical forest tree seeds (IPGRI/DFSC 1996). Whole seed moisture content and germination after drying for different periods were determined as before.

Seed storage

Storage experiments were conducted for S. cocculoides and X. americana. A split plot experimental design with four replications was used. Factor 1 was seed moisture content: 36, 30, 10 and 5% MC for S. cocculoides and 17, 12, 9, 6 and 5% for X. americana. Factor 2 was storage temperature: –20, 4 and 16°C for S. cocculoides and 4, 16 and 25°C for X. americana. Seeds were stored for three months for S. cocculoides and tested after two and three months; X. americana seeds were stored for nine months and were also regularly tested.

Data analysis

Plot means and standard error for cumulative germination percentages of seeds for all treatments (initial, desiccated and stored) were calculated and used as the basis for comparison. Prior to analysis of variance (ANOVA), data were arc sine transformed in order to normalise the data. ANOVA of a split plot design in a Randomized Complete block were performed using the General Linear Models procedure of SAS (SAS 1991). Mean and standard deviation were calculated for each treatment, and significant differences were estimated at Pd0.05. Duncan’s multiple range test was used to analyse differences between means.

Results

Initial trials

Although fruit size varied greatly between these three species, ranging from 3.5 g for X. americana to 138 g for S. cocculoides, mean seed weights 146 STORAGE BIOLOGY OF TROPICAL TREE SEEDS were similar (Table 2). For each species, embryo moisture content was similar to whole seed moisture content; seed coats were drier than the embryo in the case of S. cocculoides and W. salutaris. For all three species, initial germination of seeds was high, between 78% for W. salutaris and 96% for X. americana (Table 2). Fungi identified as Fusarium spp. were observed during all germination tests.

Table 2. Initial characteristics of fruits and seeds Species Fruit weight Seed Moisture content (%, f.wt) Germination (g) weight (g) (%) Whole Seed Embryo seed coat S. 138.0r3.5 0.6r0.1 36.6 12.6 39.2 80r2.6 cocculoide s X. 3.5r1.6 0.8r0.1 17.7 24.8 12.5 96r1.3 Americana W. 19.7r3.6 0.5r0.1 45.2 36.0 48.0 78r3.1 salutaris

Desiccation sensitivity

For all three species, drying resulted in a significant reduction in germination (P<0.05; Fig. 1). The moisture content of S. cocculoides seeds was reduced to 10% after eight days drying with silica gel and to 5% after 15 days. There were ca. 65% of germinated seeds at this low moisture content. X. americana seeds had dried to 5% MC within six days but maintained viability as high as 80%. W. salutaris seeds were at 25% MC after three days of drying, and at 8% MC after five days, however, the declining moisture content was accompanied by declining viability.

Storage experiments

Storage temperature and seed moisture content significantly (P<0.05) influenced the germination of both S. cocculoides and X. americana seeds (Tables 3 and 4). Seeds of S. cocculoides with 36, 10 and 5% MC stored better after three months at 4 and 16°C, than at –20°C. More than 50% germination was maintained for the three moisture contents after storage at higher temperatures, while it decreased to 6% germination after three months storage at –20°C (Table 3). AFRICA 147

100 40 S. cocculoides 35 80 30

60 25 20 40 15 10 20 5 0 0 0 2 4 6 8 10 12 14 16 100 40 35 80 30

60 25 X. americana 20 40 15 10 Germination (%) 20 Moisture content (%) 5 0 0 0246810 100 50 W. salutaris 45 80 40 35 60 30 25 40 20 15 20 10 5 0 0 0246810 Desiccation time (days)

Figure 1. Effect of moisture contents and desiccation periods on germination capacity of S. cocculoides, X. americana and W. salutaris seeds. 148 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Table 3. Effect of storage temperature on germination capacity of Strychnos cocculoides after three months at –20, 4 and 16°C. * = Means along the same column followed by the same letter are not significant (P>0.05) different Storage Target MC Actual MC Germination (%) after storage (%) (%) 0 2 months 3 months Fresh 36.2 36.2 80a* — — –20°C 36.2 36.2 80a 46c 10d 30 30.1 79a 40c 33d 10 9.9 68b 45c 35d 5 5.2 59b 15d 6d 4°C 36.2 36.2 801 63b 54c 30 30.1 79a 75a 64b 10 9.9 68b 60b 60b 5 5.2 59b 49c 47c 16°C 36.2 36.2 80a 76a 61b 30 30.1 79a 73a 65b 10 9.9 68b 53b 54b 5 5.2 59b 46c 31c

X. americana seeds dried to 5% MC maintained high viability (>50%) at all three storage temperatures (4, 16, and 25q&), with t ca. 50% viability after nine months storage (Table 4). Viability of seeds with 12 and 18% MC significantly decreased within three months (P<0.05) and no seeds germinated after nine months of storage.

Table 4. Effect of storage temperature on germination capacity of X. americana with different moisture contents after three months at 4, 16 and 25°C. * = Seeds heavily attacked by fungi. Means along the same column followed by the same letter are not significantly different (P>0.05)

Storage Target MC (%) Actual MC (%) Germination after storage (months) 0 1 2 3 6 9 Fresh 17 17.7 96a — — — — — 4°C 17 17.7 96a 71b 56c 41c 20d * 12 11.9 94a 76b 53c 30d 33d 04 9 9.1 85a 77b 66b 59b 39d 30d 6 6.2 85a 80a 74b 68b 64b 52c 5 5.1 80a 75b 75b 75b 68b 68b 16°°C 17 17.7 96a * * * * * 12 11.9 94a * * * * * 9 9.1 85a 73b * 73b 21d 6 6.2 85a 84a 84a 84a 43d 41d 5 5.1 80a 84a 84a 84a 71b 55c 25°C 17 17.7 96a * * * * * 12 11.9 94a * * * * * 9 9.1 85a * * * * * 6 6.2 85a 76b 76b 76b 44d 42d 5 5.1 80a 79b 79b 79b 53c 49d AFRICA 149

Discussion

Seed moisture content

High initial moisture content at harvest is one of the characteristics of recalcitrant seeds (e.g. Thomsen 1998; Msanga et al. 2000; Martins et al. 2000). Differences in moisture content between different seed tissues, as seen here for W. salutaris and S. cocculoides (Table 2), is also a characteristic of recalcitrant seeds (Berjak et al. 1993). However, seeds of X. americana had a lower initial moisture content (17%) compared with the other species. This maybe due to the high lipid contents of X. americana seeds.

Effect of seed desiccation

The response to desiccation, both in terms of time to dry and germination after drying, differed between species. Drying seeds to 5% MC took about five days for X. americana and W. salutaris, but 15 days for S. cocculoides (Fig. 1). The germination of X. americana seeds remained high (t80%) with drying to this moisture content; there was a greater reduction in the viability of S. cocculoides seeds (falling to ca. 60%); there was no germination of seeds of W. salutaris after drying to this level. This suggests that X. americana maybe orthodox; S. cocculoides still needs further investigation, however, the data indicates intermediate-type storage behaviour; W. salutaris appears to be recalcitrant.

Effect of seed storage

Storage conditions significantly (P<0.05) influenced germination capacity for both S. cocculoides and X. americana (Tables 3 and 4). For S. cocculoides, the loss of viability in seeds with high moisture content stored at –20° C could be due to freezing injury. Since they survived 5% MC for a period of time, they should be categorized as intermediate between orthodox and recalcitrant. Storage at 4 and 16°C maintained high viability over at least three months (Table 3), although it is suggested that these conditions provide optimal environments for cell enzyme activation, depending on moisture content (Bewley and Black 1994). 150 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Seeds of X. americana, which were highly affected by fungi (data not shown), were also reported to have high oil content of about 60% dry weight (Beentje 1994). This could have been controlled by application of fungicide to prevent fungal attack, which is however, a big problem in the tropics. However, this did not hamper X. americana seeds retaining high viability at different moisture contents and after storage (Tables 2 and 4), confirming that this species is orthodox (Roberts 1973; Bilia et al. 1999; Nkang et al. 2000).

Conclusions

The results of this study, allow the conclusion that (a) seeds of W. salutaris from Tanzania do not survive drying below 30% MC. (b) Seeds of S. cocculoides can be dried down to 5% and can be stored for at least three months with high viability, suggesting intermediate- type behaviour in this species. (c) Seed of X. americana can be dried down to 5% and retain viability for nine months storage. This study investigated desiccation tolerance and storage conditions (temperature) that maintain germination, we recommend that: (1) for a period not exceeding three months, seeds of S. cocculoides should be stored with <30% MC at a temperature above freezing point but less than 25°C; (2) seeds of X. americana should be stored at 5% MC at 4 and 16°C; (3) seeds of W. salutaris should be kept fresh and not be dried below 30% MC. Since viabilities for both S. cocculoides and X. americana seeds were still high at the termination of this study, we will record more data to determine the maximal storage periods.

References

Beentje, H.J. 1994. Kenya Trees Shrubs and Lianes. National Museums of Kenya, Nairobi, Kenya. Pp. 722. Berjak, P., J.M. Farrat, N.W. Pammenter, C.W. Vertucci and J. Wesley-Smith. 1993. Current understanding of desiccation sensitive (recalcitrant) seeds: development, states of water and responses to dehydration and freezing. Pp. 705–714 in Fourth International Workshop on Seed: Basic and Applied Aspects of Seed Biology (D. Come and F. Corbineau, eds.). 3 Paris, ASFIS. Bewley, J.D and M. Black (eds.). 1994. Seeds. Physiology of Development and Germination. Plenum Press, New York. Pp. 445. Bilia, D.A.C., J. Marcos-Filho and A.D.C.L. Novembre. 1999. Desiccation tolerance and seed storability of Inga uruguruensis (Hook.et.Arn.). Seed Sci. Technol. 27:77–89. AFRICA 151

Chin, H.F. 1990. Storage of recalcitrant seeds. Past, present and future. In Tropical Tree Seed Research: Proceedings of International Workshop held at the Forestry Training Centre (Turnbul, ed.). Cympie, Qld Australia, 21–24 August 1989. IUFRO ACIAR Proceedings No. 28. Ellis R.H., T.D. Hong and E.H. Roberts. 1990. An intermediate category of seed storage behaviour? I. Coffee. J. Exp. Bot. 41:1167–1174. FAO. 1983. Food and Fruit-bearing Forest Species. Example from Eastern Africa. FAO Forestry Paper No.44/1. Rome, Italy. Pp. 172. IPGRI/DFSC 1996. Screening Protocol for Desiccation and Storage of Intermediate and Recalcitrant Tropical Forest Tree Seeds. IPGRI, Rome, Italy. ISTA. 1993. International rules for seed testing. Seed Sci. Technol. 21(Suppl.):183. Martins, C.C., J. Nakagawa and M.L. Alves Bovi. 2000. Desiccation tolerance of four seed lots from Euterpe edulis mart. Seed Sci. Technol. 28:101–113. Mbuya, I., H.P. Msanga, C.K. Ruffo, A. Birnie and B. Tengnas (eds.). 1994. Useful trees and shrubs for Tanzania: identification, propagation and management for agricultural and pastoral communities. Technical Handbook No. 6. SIDA’s Regional Soil Conservation Unit, Nairobi, Kenya. Pp. 542. Mnzava, E.N. 1980. Village Afforestation: Lesson and Experience in Tanzania. FAO, Rome, Italy, Pp. 61. Msanga, H.P. 1998. Seed germination of indigenous trees in Tanzania. Including Notes on Seed Processing, Storage and Plant Uses. Natural Resources Canada, Canadian Forest Service, Northern Forest Centre, Edmonton, Canada. Pp. 292. Msanga, H.P., A.G. Mugasha, L.O.N. Uronu and S.A.O. Chamshama. 2000. Effect of temperature and moisture content on storability of Sorindeia madagascariensis seed. Paper presented at a Workshop on Annual Forest Research at Arusha, Tanzani, 3–4 September 1998. Tanzania Journal of Forest and Nature Conservation. National Forest Policy. 1998. United Republic of Tanzania, Ministry of Natural Resources and Tourism, Dar es Salaam. Pp. 59. Nkang, A., D. Omokaro and A. Egbe. 2000. Effects of desiccation on the lipid peroxide and activities of peroxides and polyphenoloxidase in seeds of Telfairia occidentalis. Seed Sci. Technol. 28:1–9. Palgrave, K.C. 1992. Trees of Southern Africa. Struk publishers, Cape Town, South Africa. Pp. 959. Roberts, E.H. 1973. Predicting the storage life of seeds. Seed Sci. Technol. 1:499– 514. SAS (Statistical Analysis Systems). 1991. Statistical Analysis Systems Institute, Cary, NC. Pp. 846. Thomsen, K. 1998. Screening Protocol for Project on Handling and Storage of Recalcitrant and Intermediate Tropical Forest Tree Seeds. Vol. 4. IPGRI, Rome, Italy. Pp. 4–9. 152 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Uronu, L.O.N. 1999. Seed Collection Calendar for Some Useful Tree Species in Tanzania. Technical Note No. 8. National Seed Programme, Morogoro, Tanzania. Pp. 34. Watt, J.M and M.G. Breyer-Brandwijk (eds.). 1962. Medicinal and Poisonous Plants of Southern and Eastern Africa. E&S. Livingston, London. Pp. 457. Wood, P.J. 1966. A Guide to Growing Trees in Sukuma Land, Tanzania Silvicultural. Technical Note No. 2. Lushoto, Tanzania. Pp. 5. AFRICA 153

Storage behaviour of Podocarpus falcatus (Thunb.) Mirb. and Hagenia abyssinica J.F.Gmel. seeds

Karen M. Poulsen1, Leuleseged Demalash2 and Kirsten A. Thomsen1

1The State Forest Tree Improvement Station, Krogerupvej 21, 3050 Humlebaek, Denmark, 2National Tree Seed Project, P.O.Box 5580, Addis Abeba, Ethiopia

Abstract

Seeds of Podocarpus falcatus with 4% MC (fresh weight basis), were stored at –20, 4 and 20°C for two and a half to four years. Viability was maintained for four years without reduction in germination capacity for seeds stored at 4°C, while at 20°C no seeds germinated after two years. At –20°C germination capacity was significantly reduced after two and half years indicating intermediate storage behaviour. Seeds of Hagenia abyssinica showed very poor initial germination of 2%. However, storing at 8.8% MC and at –20, 4 and 20°C did not change this initial level of germination capacity, and orthodox storage behaviour is suspected. Poor pollination (fertilization) is suspected to cause poor germination in this species.

Introduction

Podocarpus, Podocarpus falcatus (Thunb.) Mirb., is a member of the Podocarpaceae family and Hagenia, Hagenia abyssinica J.F.Gmel., belongs to the Rosaceae family. Podocarpus falcatus is native to central and southern Africa, where it grows at altitudes of 1500 to 3000 m above sea level and at annual rainfalls of 1200 to 1800 mm (Hong et al. 1996; Kabera 1990). Hagenia abyssinica is native to East Africa growing at altitudes of 1850 up to 3700 m above sea level and at mean annual rainfalls of 1000 to 1500 mm (Mbuya et al. 1994). Both P. falcatus and H. abyssinica are important species used in Ethiopia and other African countries. They produce high quality wood. However, for both species the seed quality poses problems. P. falcatus fruit has an outer fleshy layer (mesocarp). Beneath this is a stony layer, or endocarp covering the embryo, which is made up of 154 STORAGE BIOLOGY OF TROPICAL TREE SEEDS two large cotyledons, with an embryonic axis in between. The stony coat including the embryo is referred to as the seed. This is the unit stored and sown by nurseries. The fruit is almost round and 1.5–2 cm in diameter. The seed is also round, about 1 cm in diameter and the stony coat is around 2 mm thick. H. abyssinica is a dioecious species, female and male flowers occurring on separate trees. Pollination is suspected to take place by wind, and poor pollination could be the major cause of low production of seeds in this species. The trees produce numerous closely clustered flowers, and apparently only a few are pollinated. Flower heads are often considered as pure seed, and this unit is stored and sown, since it is extremely tedious to isolate the very tiny and rarely present seed from flowers. One kilogram held 350,000 flower heads. This study aimed to investigate the storage behaviour of P. falcatus and H. abyssinica seeds from Ethiopia. A screening protocol for determining storage behaviour was tested, as outlined under the project on recalcitrant/ intermediate seeds (DFSC/IPGRI 1996), coordinated by the International Plant Genetic Resources Institute (IPGRI) and Danida Forest Seed Centre (DFSC).

Materials and methods

Collecting and processing

Seeds of Podocarpus falcatus and Hagenia abyssinica were collected in Ethiopia in collaboration with the National Tree Seed Project of Ethiopia. P. falcatus fruits were collected on the 3rd March 1997, at Debre Zeit around 40 km East of Addis Abeba, Ethiopia. About 19 kg of only yellow (i.e. ripe) fruits were collected from the crowns of four different trees. After collection, fruits were put in cotton bags and stored overnight at 20–25°C. Fruits were sampled the next day and inspected. All fruits were mature and about 36% had already turned brown and had become slightly shrunken during the night. Some larvae in the fruit flesh were observed. The fruits were soaked in running water for 3 h and the flesh was removed manually. Seeds (4.1 kg) were finally washed and left to desiccate on nets in the shade for 2.5 days before use. H. abyssinica fruits were collected on 28 Feb and on 1 Mar at Addis Alam—a few hours drive from Addis Abeba. Fruits were harvested by climbing the trees and cutting down the large, dry and crisp AFRICA 155 inflorescences. The fruits were left in cotton bags for a couple of days and then placed on net trays in the shade for three days. Fruits were squashed by hand, and twigs and other impurities were removed. In total 5 kg of inflorescences were collected, after removal of twigs 2.22 kg remained for machine processing. After cleaning in the Kimseed Clarke Seed Cleaner (Kimseed manufacturer, Australia), 1.32 kg remained, with a purity of 54%, i.e. the percentage of flower heads containing seeds. The unit stored and sown is thus the flower heads. One kilogram held ca 350 000 flower heads, thus the seed is very tiny. After processing, a sample of seeds of both species were carried to Denmark by hand and thus exposed to only ambient temperature during all transport steps. Upon arrival the seeds were sampled for testing.

Desiccation and germination tests

Seeds were desiccated by mixing them with an equal amount of silica gel in a plastic bag (DFSC/IPGRI 1996). The silica gel was changed daily, or when necessary. In Ethiopia, seed moisture contents were determined by oven drying for 16 h at 105°C using two replicates of 20 seeds. At DFSC moisture contents were determined by drying at 103°C for 17 h using two replicates of 15 seeds. Moisture contents of the stony layer (endocarp) and the embryos were determined separately. Both tissues were manually cut into pieces and weighed before and after drying, and moisture contents were reported on a fresh weight basis. For H. abyssinica, moisture content was measured using two replicates of 1 g of flower heads that were oven-dried at 103°C for 17 h at DFSC. In Ethiopia, moisture content was determined on isolated seeds as well as on flower heads, using two replicates of 20 seeds or flower heads and oven-drying them at 105°C for 16 h. Germination capacity in P. falcatus was determined using four replicates of 25 seeds after removal of the stony layer by cracking in a vice. Seeds were sown in a mixture of sand and vermiculite, and incubated at 25°C for 12/12 h in light and dark. Germinated seeds were scored when the radicle reached the length of the entire seed. Samples of germinated seeds were transplanted, and developed into normal plants. The speed of germination was determined as the Mean Length of Incubation Time (MLIT) using the method of Czabator (1962) and six weeks germination was included in the index. 156 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

For H. abyssinica, four replicates of 100 flower heads were germinated on top of a paper at 25°C with 12/12 h in light and dark. There was only one seed per flower head and germination was scored, when the radicle was visible.

Storage trials

For each of the two species, desiccated seeds were split into three samples, sealed in plastic bags and stored at –20, 4 and 20°C and tested every six months, regarding moisture content and germination capacity over 30 months. Seeds stored at 4°C were also tested at 36 and 48 months.

Statistical analysis

The General Linear Models Procedure of the SAS programme was used on storage data and for each storage temperature, testing the model by plotting residuals versus predicted values.

Results

Initial characteristics

Moisture content of P. falcatus fruits/seeds was determined at various stages of ripeness and after drying (Table 1). Initial moisture contents of fresh seeds, just released from the flesh, were 25.2% for the stony coat and 21.4% for the embryo. Seeds were dried to around 7% MC, the embryo having 3–4% MC and the stony coat 7–11% MC (Table 1), before being packed into a plastic bag and stored at room temperature until the storage trial was initiated at DFSC, Denmark. Because the stony layer was readily permeable to water, it was possible to desiccate seeds from an initial value of 20.7% MC to 10% MC in just 16 h when mixed with silica gel. This was an efficient method of obtaining rapid desiccation of smaller amounts of seeds. At 14.5% MC the 1000 seed weight was 516 g. AFRICA 157

Table 1. Initial moisture contents (MC%) of P. falcatus seeds and fruit parts at various stages of ripeness and after drying Fruit part MC (%) Seed from yellow fruit MC (%) Green Green/ Yellow Sample 1 Sample 2 yellow Flesh (mesocarp) 68.7 69.0 68.8 – – Seed – – – 8.4 5.9 Stony coat 27.2 27.4 25.2 10.7 7.4 (endocarp) Embryo 26.4 21.7 21.4 4.1 3.2

Separately processed H. abyssinica seeds had an initial MC of 6.3%, whilst the flower heads including seeds showed 7.2% MC. Since flower heads and seeds had similar moisture contents it was concluded, that the tedious procedure of isolating seeds from flower heads could be abandoned. Only 5.9% of a sample of 1400 flower heads contained seeds before processing, this number increased to 8.1% after processing (sample of 1300 flower heads).

Germination and storage

Germination declined significantly for seeds stored at 20°C (Fig. 1). This decrease was already observed after six months storage and no seeds germinated after two years storage. The decrease also affected the speed of germination, the MLIT increasing from 17 to 30 days (data not shown). The seed tolerated storage at 4°C for 4 years maintaining initial germination capacity (>70%). The transient low germination of 40% observed at 2.5 years was not readily explained, the standard deviation of the model however is relatively high (16%). At –20°C the germination capacity was reduced to 43%, but not significant, after two years storage and then to 29% after 2.5 years, which was a significant decrease (Fig. 1). H. abyssinica seeds germinated 2% in initial experiments at DFSC, which was half of the 4% germination obtained from the tests in Ethiopia. The poor germination percentages made the storage trial relatively inconclusive. When only 8.1% of the flower heads held a seed, the above result indicates that only 25–50% of seeds were viable (Fig. 1). During processing of Podocarpus falcatus, it was noticed that around 10% of fruits were floating in water, and thus floaters and sinkers were sampled for cutting tests. The result showed no difference. Based on this, 158 STORAGE BIOLOGY OF TROPICAL TREE SEEDS removal of floaters as a processing step cannot be recommended. It is noted that the moisture content of the embryo at shedding is similar to that of other orthodox seeds, e.g. beech nuts (Table 1). The embryos maintained moisture contents varying from 3.6 to 4.7% during storage (data not shown). The initial germination capacity of P. falcatus seeds after processing and transport to Denmark was 64% at DFSC, while 72% was obtained in Ethiopia. The cutting test before processing also indicated 72% germinability for the same seed lot (data not shown).

100 P. falcatus

0 6 12 18 24 30 36 42 48

H. abyssinica Germination (%) -20 °C 4 4 °C 20 °C

0 6 12 18 24 30 36 42 48

Storage period (months)

Figure 1. Germination of P. falcatus and H. abyssinica seeds after four years storage. Initial germination capacity was 64% for P. falcatus seeds with 8.4% MC and was 2% for H. abyssinica seeds with 8.8% MC. AFRICA 159

Discussion

For many years seed could be classified into two groups of storage behaviour i.e. orthodox or recalcitrant (Roberts 1973), but since then a number of species have shown not to conform to either of the two patterns. These species, tentatively termed ‘intermediate’ include mainly woody, tropical species e.g. coffee (Ellis et al. 1990), papaya (Ellis et al. 1991), Araucaria sp. (Tompsett 1984a,b), various dipterocarps (Tompsett 1987), neem (Sacandé et al. 1998) and others (Hong et al. 1996). The intermediate character shows up either as better storage at temperatures above zero than below (chilling sensitivity) or as only a moderate desiccation tolerance, typically to around 10–12% MC (see review by Pammenter and Berjak 1999). It is thus suspected that a number of tropical tree species with recorded poor storage capacity, have intermediate characters. Literature on Podocarpus seeds is scanty, however Dodd and Staden (1981) report that Podocarpus henkelii (Stapf.) is recalcitrant. Obviously P. falcatus seed tolerates desiccation to embryo moisture content of 4%, and are not recalcitrant, and in addition, the moisture content at shedding is also low, similar to that of some orthodox seeds. Furthermore, 4% MC corresponded to 15% equilibrium relative humidity for oily seeds like peanut (Walters and Hill 1998), meaning that the seed tolerates moisture contents in the range of bound water level in orthodox seeds. However, there was a significant reduction in germination capacity after two and a half years storage at –20°C (Fig. 1). These results indicated that the seeds showed an intermediate storage behaviour, being tolerant to fairly low moisture contents and being fairly long-lived at reduced temperatures, but possibly not at very low freezing temperatures. However, seed handling (e.g. imibibition damage) after storage may have been part of problems, since the thick endocarp was removed before germination, but this would have equally affected seeds at all storage temperatures. It should be emphasized that this species is widely distributed and seed source apparently influences storage behaviour, as for example for neem (Azadirachta indica A.Juss.) seeds (see review by Poulsen 1996). The stony coat of P. falcatus seed is suspected to impose a mechanical dormancy, although the fresh, nondried stone does not show as much dormancy as the desiccated stone. It is therefore highly relevant for practical use to test storage of fresh stones at temperatures around 4°C, and priority should be given to the development of large-scale practical methods for dormancy release. 160 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Hagenia abyssinica seeds and flower heads had similar moisture contents of 6.3–7.2%, and were already very dry upon shedding. Flower heads had been used for moisture content determination of the seeds, although processing increased the proportion of flowers bearing seeds from 5.9 to 8.1%. The tedious procedure of isolating seeds from flower heads was abandoned since this did not make a significant difference. H. abyssinica seeds had a germination percentage of 2% in initial experiments upon receipt at DFSC. In other words, from the 8.1% flower heads containing a seed only 25% would germinate. The poor initial germination percentages made the storage trials relatively difficult to interpret. However, during the two and a half years storage, the initial germination capacity before storage was maintained at all temperatures (Fig. 1), indicating an orthodox storage behaviour. It is suspected that the very low initial germination percentage is due to poor pollination (or fertilization) of the flowers. Female and male flowers of H. abyssinica are found on separate trees, and pollination which is suspected to take place by wind, could be scanty. Thus the reproduction strategy of the tree may be to produce many flowers and even with germination percentages of just 4%, a kilogram of flower heads (ca. 350 000 individuals) would contain 14 000 viable seeds (4%). It is reported that seeds usually germinate maximum 10% at the National Tree Seed Project, Ethiopia. “Seed cleaning” thus remains a major problem and so far no method of separating full and empty flower heads is available. This species could have enormous difficulties regenerating, and thus further investigations on its reproduction system are needed.

Acknowledgements

The National Tree Seed Project, Ethiopia is kindly acknowledged for facilitating the studies including collection of seeds and making laboratory facilities and staff available.

References

Czabator, F.J. 1962. Germination value: an index combining speed and completeness of pine seed germination. Forest Sci. 8:386–396. DFSC/IPGRI 1996. The Project on Handling and Storage of Recalcitrant and Intermediate Tropical Forest Tree Seeds. Newsletter No. 1. July 1996. Pp. 5–20. Dodd, M.C. and J. van Staden. 1981. Germination and viability studies of the seeds of Podocarpus henkelii stapf. South Afr. J. Sci. 77:171–174. Ellis, R.H., T.D. Hong and E.H. Roberts. 1990. An intermediate category of seed storage behaviour. J. Exp. Bot. 41:1167–1174. AFRICA 161

Ellis, R.H., T.D. Hong and E.H. Roberts. 1991. Effect of storage temperature and moisture on the germination of papaya seeds. Seed Sci. Res. 1:69–72. Hong, T.D., S. Linnington and R.H. Ellis. 1996. Seed storage behaviour: a compendium. Handbooks for Genebanks: No. 4. IPGRI, Rome, Italy. Kabera, I. 1990. Podocarpus falcatus: a monograph and appraisal, with special reference to Rwanda School Agric. Forest Sc., Univ. Coll. North Wales, Bangor; UK. .Mbuya, L.P., H.P. Msanga, C.K. Ruffo, A.B. Birnie and B. Tengnäs. 1994. Useful trees and shrubs for Tanzania. Identification, Propagation and Management for Agricultural and Pastoral Communities. Regional Soil Conservation Unit, RSCU. Pammenter, N.W. and P. Berjak. 1999. A review of recalcitrant seed physiology in relation to desiccation-tolerance mechanisms. Seed Sci. Res. 9:13–37. Poulsen, K.M. 1996. Case study: neem (Azadirachta indica) seed research. Pp. 14– 26 in Intermediate/Recalcitrant Tropical Forest Tree Seeds (A.S. Ouédraogo, K. Poulsen and F. Stubsgaard, eds.). Proceedings of a Workshop on Improved Methods for Handling and Storage of Intermediate/Recalcitrant Tropical Forest Tree Seeds. Humlebaek, Denmark, 8–19 June 1995. Roberts, E.H. 1973. Predicting the storage live of seeds. Seed Sci. Technol. 1:499–514. Sacandé, M., F.A. Hoekstra, J.G. van Piljen and S.P.C. Groot. 1998. A multifactorial study of conditions influencing longevity of neem (Azadirachta indica) seeds. Seed Sci. Res. 8:473–482. Tompsett, P.B. 1984a. The effect of moisture content and temperature on the seed storage life of Araucaria columnaris (Forst.). Seed Sci. Technol. 12:801–816. Tompsett, P.B. 1984b. Desiccation studies in relation to the storage of Araucaria seed. Ann. Appl. Biol. 105:581–586. Tompsett, P.B. 1987. Desiccation and storage studies on Dipterocarpus seeds. Ann. Appl. Biol. 110:371–379. Walters, C. and L.M. Hill. 1998. Water sorption isotherms of seeds from ultradry experiments. Seed Sci. Res. 8(Suppl. 1):69–73 162 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

. 2 Asia

China (SCAU)

India (PRSU)

Malaysia (FRIM)

Thailand (ASEAN-FTSC)

Vietnam (FSIV) 164 STORAGE BIOLOGY OF TROPICAL TREE SEEDS ASIA 165

Desiccation and storage of Vatica astrotricha, Hopea hainanensis, Podocarpus nagi and Acmena acuminatissma seeds

X. F. Wang, T. Zhao, M. Yan and C. J. Huang

College of Life Science, South China Agricultural University, Guangzhou 510642, People’s Republic of China

Abstract

Seeds of four tropical tree species, Vatica astrotricha, Hopea hainanensis, Podocarpus nagi and Acmena acuminatissma from China were screened for their tolerance to desiccation and storability. Germinability of P. nagi seeds increased when seeds were dried from 26.8% initial MC to 11.12% MC. In contrast, seeds of V. astrotricha, H. hainanensis and A. acuminatissma, decreased in viability during desiccation. Their critical moisture contents were between 38.8 and 33.1%, 25.4 and 23.0% and 44.8 and 33.2%, respectively. V. astrotricha seeds were very sensitive to temperatures below 25°C, while seeds of H. hainanensis and A. acuminatissma were sensitive to temperatures below 15°C. These seeds were thus, classified as recalcitrant. Temperatures of 25°C were suitable for V. astrotricha seeds, and 15°C for both H. hainanensis and A. acuminatissma seeds for their medium- term storage.

Introduction

China is the fifth largest forest country in the world, with a total forest area of about 0.146 billion hm2 (FAO 1997; Tang 1998). The forest genetic resources in China are very abundant and diversified. Different types of forest, from tropical rainforests to cold temperate zone needle- leaved forests, are spread from south to north of the country. There are 115 families, 302 genera and about 7000 woody plant species (including about 2800 species of macrophanerophytes) in the forests of China (Tang 1998), among which three families, 196 genera and about 1000 species are native to China. The most widely used species in the reforestation scheme of China come from families such as Araucariaceae, Pinaceae and Podocarpaceae of gymnosperms, and Casuarinaceae, Dipterocarpaceae, Leguminosae and Verbenaceae of 166 STORAGE BIOLOGY OF TROPICAL TREE SEEDS angiosperms. However, China is also one of the poorest forest countries in the world, with a forest area per person of only about 0.1 hm2. The forest cover in China is only about 14%, and is declining (Tang 1998). Forests are not evenly distributed, mainly in northeast, southwest and tropical and subtropical areas of southeast of China. For the tropical and subtropical tree species in south China, some problems limiting the utilization of their seeds in plantation programs are: shortage of seed, difficulties in collection, short viability etc. For example, Vatica astrotricha and Hopea hainanensis have unpredictable fruit production from year to year. Thus it can be difficult to collect enough seeds. Viability of many tropical tree seeds is short and immediate sowing after harvest or collection is necessary. For this research, four tropical or subtropical tree species in south China were selected according to their socioeconomical importance. Little is known on the biology of their seeds, and hardly any studies have been carried out on these species, despite their importance and problems related to the difficult handling of their seeds. Vatica astrotricha Hance is a species of Dipterocarpaceae family, macrophanerophytes, which grows up to 7–25-m tall with straight bole. Leaves are corious. Fruits are globular with two large and several small wings. It is distributed mainly in the tropical rainforests of Hainan province, south China. There are some small ex situ collections in the botanic gardens of Tropical and Subtropical Forest Research Institutes in Hainan and Guangdong provinces. The wood is hard and heavy, and is very good for indoor construction and furniture. Drying is thought to reduce seed viability and damages the ultrastructure of cells. For the storage of these seeds, relatively high moisture contents should be used (South China Institute of Botany 1964; Song et al. 1983). Hopea hainanensis Merr. & Chun, belongs to the Dipterocarpaceae family, macrophanerophytes, growing up to 10–20 m tall with straight bole. This species is critically endangered due to its exploitation, and is listed in the IUCN Red List of Threatened Species (IUCN 2002). Leaves are corious. Fruits are oviform with two large wings. It is distributed mainly in the tropical rainforests of Hainan province, south China. The wood is hard and heavy, and is very good for indoor construction and furniture. Seeds rapidly lose their viability. Seeds lose viability after drying (South China Institute of Botany 1964). For the storage of this seed, relatively high moisture contents were recommended. Suitable storage conditions for this seed are: moisture content of 33–38%, at temperatures between 15 and 20°C. Seeds with 36–38% moisture still had 80% viability after storage at 18°C for a year (Song et al. 1984). ASIA 167

Podocarpus nagi (Thunb.) Makino is a species of the Podocarpaceae family, evergreen tree up to 20–30 m tall with straight bole. The bark is thin, about 4 mm. Leaves are opposite, thick and corious. The leaf shape is very alike that of ordinary bamboo. Seeds are globular with a diameter about 1.3 cm. It is mainly distributed in the rainforests of Hainan and Guangdong provinces and also planted as ornamental trees along the road or in the garden. It grows mainly at altitudes under 1000 m. The wood has a fine and straight texture with specific weight of 0.47–0.52 g cm–3. The wood is good for indoor construction and furniture. Immediate sowing is necessary for this seed (South China Institute of Botany 1964). No literature was related to the physiology and storage behaviour of this seed. Acmena acuminatissma (Blume) Merr. & L.M. Perry, belongs to the Myrtaceae family and is an evergreen tree up to 20-m tall with straight bole. The bark is thin, about 5–6 mm. Leaves are simple and opposite, thin and corious. Flowers are bisexual. Berry fruits are globular; with a diameter about 1–1.5 cm. Internal structure of the seed is not obvious. In south China, it is distributed mainly in Hainan, Guangdong and Guangxi provinces. It grows mainly in the tropical or subtropical evergreen or monsoon forests from altitudes between 400 and 1000 m. The wood has fine texture, hard and heavy, and good for indoor construction and furniture. Immediate sowing is necessary for this seed (South China Institute of Botany 1964). No literature was related to the physiology and storage behaviour of this seed.

Materials and methods

Seed collection and processing

Fruit collection and seed preparation were carried out according to the IPGRI/DFSC protocol (1999). Fruits of V. astrotricha and H. hainanensis were collected on 15 Aug 2001 and 15 Apr 2002, respectively, from trees in the forests of Jian Feng Ling, Hainan province and transported in about 2 days to the laboratory by express post (V. astrotricha arrived at the laboratory on 18 Aug, and H. hainanensis on 17 Apr). Fruits of P. nagi were collected on 14 Feb 2001, and A. acuminatissma were collected on 14 Feb 2001 and 7 Jan 2002 from trees in the garden of Guangdong Forest Science Institute, Guangzhou and brought to the laboratory in the same day. Seed processing was not necessary for P. nagi. Wings of V. astrotricha and H. hainanensis seeds were removed, while for A. acuminatissma, seeds were depulped. 168 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Desiccation, germination and storage trials

All procedures of desiccation and storage trials of IPGRI/DFSC protocol were followed. For the desiccation trail, 500 g of silica gel were used for each desiccation plastic bag.

Results

Initial characteristics

Initial weights of fruits and seeds of the species were measured. A. acuminatissma seeds were the biggest (1.35 g) and had the lowest mass ratio of seed to fruit weights (Table 1). Of the four species, seeds of V. astrotricha were the smallest (0.12 g) and H. hainanensis had the highest seed to fruit ratio of 0.83.

Table 1. Mean weights of fruits and seeds of the four studied species

V. astrotricha H. hainanensis P. nagi A. acuminatissma Fruits (g) 0.26 ± 0.09 1.17 ± 0.21 – 3.91 ± 0.82 Seeds (g) 0.12 ± 0.07 0.97 ± 0.20 0.55 ± 0.15 1.35 ± 0.30 Mass ratio (S/F) 0.46 0.83 – 0.35

At harvest, seeds of V. astrotricha and A. acuminatissma had >50% initial MC, while H. hainanensis and P. nagi seeds had 34 and 27% initial MC, respectively (Table 2). Initial germination was 97% for H. hainanensis and 94% for A. acuminatissma seeds. P. nagi seeds had a very low initial germination of 2%. Seeds of V. astrotricha initially germinated to 56%. However, the high standard deviation value may indicate that seeds at harvest were not uniform in maturity (Table 2).

Table 2. Initial moisture contents and germination of seeds after harvest and preparation.

V. astrotricha H. hainanensis P. nagi A. acuminatissma

Initial MC (%) 55.21 ± 5.15 33.53 ± 1.11 26.84 ± 3.06 58.58 ± 0.77 Germination 56 ± 16.33 97 ± 2 2 ± 2.31 94 ± 2.31 (%) ASIA 169

Desiccation trials

Seeds were desiccated to test their capacity to germinate after drying (Fig. 1). Viability of V. astrotricha and A. acuminatissma seeds gradually decreased to 0% below 10 and 15% MC, respectively. Seeds of H. hainanensis germinated to high percentages (95%) when dried to 25% MC, but germination dropped to 0% below 20% MC, indicating a threshold moisture content for maintaining viability between 20 and 25% MC (Fig. 1). Seeds of P. nagi, however, gradually increased during desiccation from 27% (2% germination) to ca. 10% MC (20% germination).

Storage trials

Moisture contents of V. astrotricha and H. hainanensis seeds varied slightly during storage (Fig. 2). V. astrotricha seeds did not germinate after a month of storage at 5 and 15°C. Seeds survived better at 25°C, but no seeds germinated after 6 months at this temperature (Fig. 2). For H. hainanensis, no seeds germinated after 6 months at 5 and 25°C, while seeds maintained >50% germination after 6 months storage at 15°C, and some seeds germinated after 9 months at this temperature (Fig. 2). A. acuminatissma seeds lost their viability after 3 months storage at 5 and 25°C, whereas seeds stored at 15°C retained their initial level of germination after 3 months, but did not germinate after 6 months (Fig. 3). Seed moisture content varied during storage (Fig. 3), similarly to the other previous species (Fig. 2).

Discussion

Seeds of V. astrotricha did not have uniform maturity at harvest. As a result, there were great variations in their germination percentage (Table 2). This would have affected the viability after desiccation and storage. Storage trials showed that seeds of V. astrotricha are sensitive to low temperatures of 5 and 15°C (Fig. 2), indicating chilling sensitivity. For the short term (at least 3 months), it would be suitable to keep V. astrotricha seeds at their initial moisture contents under 25°C conditions. The moisture content and germination of the control seeds changed only a little (data not shown). It was concluded that seeds of V. astrotricha are recalcitrant, although mature and good-quality seeds should be collected in the first place for any such investigations. 170 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Seeds of H. hainanensis were sensitive to desiccation, as they germinated maximally at 25.4% MC, whereas no germination occurred below that level, indicating that their critical moisture content lies between 25.4 and 23% MC (Fig. 1B). Although there was a little variation in the moisture content and germination of control seeds (data not shown), stored seeds could survive at 15°C for at least 9 months. P. nagi seeds with initial 27% MC only germinated to 2% (Table 2). However, germination gradually increased to 20% germination during desiccation to ca. 10% MC (Fig. 1C). This unexpected improvement of viability upon drying may be due to postharvest ripening or release of dormancy, or both. Storage trials were not carried out due to limited number of seeds. This species need further investigations to better understand its seed physiology at maturity and during postharvest handling. A. acuminatissma seeds had high initial viability, which gradually decreased during desiccation (Fig. 1). Seeds of A. acuminatissma were sensitive to desiccation below 40% MC and are thus regarded as recalcitrant. The moisture content and germination percentage of control seeds changed only a little (data not shown). Storage at 15°C allowed seeds to maintain their initial germination level for 3 months. However, these seeds did not survive after 6 months. Thus, for short-term storage (at least 3 months), 15°C is a relatively suitable temperature that maintains viability.

Conclusions

The protocol (IPGRI/DFSC 1999) is a useful tool as standard procedures for screening tropical and subtropical seeds for their tolerance to desiccation and storability, which can be used by different institutes in different contexts and countries. Future activities should focus on the studies of mechanisms of desiccation sensitivity or tolerance, and of a better understanding of the morphology of tropical tree seeds. ASIA 171

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References

FAO. 1997. State of the World Forests 1997. FAO, Rome, Italy. IPGRI/DFSC. 1999. The project on handling and storage of recalcitrant and intermediate tropical forest tree seeds. Danida Forest Seed Centre. Newsletter No. 5:23–39. IUCN. 2002. 2002 IUCN Red List of Threatened Species [see also at http://www.redlist.org]. Song, X.Z., Q.D. Chen, D.F. Wang and J. Yang. 1983. A study of ultrastructural changes in radicle-tip cells and seed vigor of Hopea and Vatica in losing water process. Scientia Silvae Sinicae 19(2):122–125. Song, X.Z., Q.D. Chen, D.F. Wang and J. Yang. 1984. Studies on the storage of Hopea hainanensis seeds. Scientia Silvae Sinicae 20(3):225–235. South China Institute of Botany. 1964. Flora of Hainan. Vol. 1. Science Press, Beijing, China. Tang, S.Z. 1998. Forest genetic resources of China and its influence on the environment. Biol. Bull. 33(11):2–6. 174 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Conservation of four tropical forest tree seeds from India

Subash C. Naithani, Ranjana Naithani, B. Varghese, J.K. Godheja and K.K Sahu

Seed Biology Laboratory, School of Life Sciences, Pt. Ravishankar Shukla University, Raipur– 492010 (Chattisgarh), India

Abstract

Seed storage physiology of Buchanania lanzan, Diospyros melanoxylon, Gmelina arborea and Madhuca indica, four tropical tree species, were evaluated following the DFSC/IPGRI protocol. All the seeds were harvested with relatively high moisture contents between 17 and 63% and showed 100% initial germination. Drying the seeds over silica gel revealed desiccation tolerance in B. lanzan, D. melanoxylon and G. arborea, and desiccation-sensitivity in M. indica. Seeds of M. indica lost viability when dried below 18.3% MC, but could be stored for 55 days at 25°C, during which time 25% germinated. Desiccation-tolerant seeds of B. lanzan, D. melanoxylon and G. arborea were also tolerant of low temperature (–20 and 0°C) storage, retaining their initial viability over 60 to 150 days. Germinability was gradually lost with further storage (ca. 280 days). Rapid drying of B. lanzan, D. melanoxylon and G. arborea seeds improved their storage, maintaining higher viability over longer periods.

Introduction

India with over 45000 plants and 81000 animal species, is one of the world’s top 12 ‘mega-biodiversity’ nations. The recorded forest area of India is 76.52 million ha which constitutes almost 20% of the total geographic area. Of these forest areas, 11% are considered closed with more than 40% of canopy coverage, while about 8% of the geographical area are open forests with 10–40% canopy coverage (Tewari 1981). Forests in India have declined by nearly half in the twentieth century, and the rate of deforestation is still increasing. Further, serious threat from timber smuggling, overexploitation by industry, deteriorating law enforcement, ravaging forest fires, uncontrolled grazing, agricultural encroachment, urbanization, unmanaged exploitation for ASIA 175 firewood and other basic needs has put an immense pressure on the Indian forests (Hocking 1993). The estimated annual deficit in tropical forestry plantations in the country has increased to 36.4 million ha/year in the new millennium. In recent years, tree planting has become an intensive activity for soil conservation and to meet the multifarious demands of timber, fuel, fodder and non-wood forest produce. This reinforces a series of in situ and ex situ measures and the legal and policy reforms needed to implement and enforce the National Biodiversity Action Plan (MoEF 2000). Industrial and commercial plantations today constitute 45% of all plantations, with Tectona grandis, Gmelina arborea, Dalbergia sissoo, Acacia nilotica, Populus, Eucalyptus and Pinus sp. the main species used. However, apart from the few timber species largely preferred for plantations, there are other numerous tree species which have promizing national and international markets for fruits (Report of Committee of Forests and Tribals of India 1982), medicine (Jain 1968) and lumber products (Murty et al. 1989). These species have not yet gained enough attention in afforestation programmes and remained under- utilised. If appropriate cultivation techniques and markets were developed, these species could greatly assist in the economic development of the country. There is little information available on the seed biology of Indian forest trees, and availability of quality seed limits the management of forests (afforestation and plantation activities). Seed still remains the primary source of planting material in tropical countries and the annual demand for forest tree seeds in India, is about 10 000 metric tonnes. However, seed is often in short supply, of low quality and variable maturity and has limited storage life. Plantings have been limited to species where basic knowledge of seed collection, processing and seed storage physiology is available. Desiccation-sensitivity and nonstorability of many of the tropical forest tree seeds adds to these problems (Gunn 1991). Thus there is an urgent need to establish and strengthen seed programmes in India to ensure continuous and adequate supplies of well-adapted indigenous seeds. The use of indigenous species in afforestation and plantation programmes also has the advantage of using readily available reservoirs of genetically diverse stock. We have chosen to investigate the desiccation and storage behaviour of four important indigenous species from India. Buchanania lanzan Spreng., also referred to as B. latifolia Roxb., is a member of Anacardiaceae family and is locally known as ‘chironji’, ‘cuddapah almond’ or almondette tree. It is a common tree in the 176 STORAGE BIOLOGY OF TROPICAL TREE SEEDS deciduous forests of India, widely distributed in dry forests from the Sutlej and all through the central to south India (Prakash 1991). It is extensively found in moist and dry deciduous forests of south and north India. The tree plays an outstanding role in the rural and tribal economy of India, especially for its edible fruits and seeds (Hocking 1993). The kernels, which are nutritious, are eaten raw or roasted and serve as a substitute for almonds. The dry fruit is expensive, costing US$4–5/kg. The wood provides poor quality timber and is used for firewood, boxes, cheap furniture and doors, etc. (Prakash 1991). The leaves and bark of the tree are of high medicinal value for the treatment of bleeding wounds, jaundice, throat ulcer and toothache, etc. (Hocking 1993). It is a small to moderate-sized tree with a straight trunk, often of a considerable height up to 18 m and girth up to 7.5 m. The seeds have been shown to lose viability significantly within the first year of storage (Prakash 1991). Diospyros melanoxylon Roxb. also known as the Coromandel Ebony or locally as ‘tendu’, is a member of the Ebenaceae family. A small to moderate size tree usually not exceeding 12 m in height, it is widely distributed in the dry deciduous forests of India. Black heartwood from the tree is considered to be a valuable substitute for true ebony wood, which is heavy and durable. The leaves are a major source of income for the rural and tribal populace, being used for the wrapping of tobacco to prepare local cigarettes or bidis. Superior quality leaves of large size, papery texture and inconspicuous veins fetch up to five times the price of inferior quality leaves. Around 300 000 tons of bidi leaves are produced annually in India, valued at 4515 million Rupees. There is, however, vast opportunity for propagating better clones or strains. The fruit is edible and the dried fruit powder has medicinal properties. The dried flowers are used for the treatment of urinary, skin, blood diseases and dyspepsia. The species has very high potential national market for various non-wood forest products (NWFP). It is highly suitable for afforestation in dry and barren hill slopes. Gmelina arborea Roxb., belongs to Verbenaceae family and is extensively used for mass plantations, locally referred as ‘Khamar or Gamhar’. This medium-sized tree (30 m/1.2–4.5 m) with clear bole of 9–15 m is generally used for pulp production for the paper industry. It occurs in many parts of India and develops best in moist, fertile, well- drained valleys and in moist deciduous forests. It is highly recommended for plantings in skeletal soils in medium rainfall areas, field boundaries and for agro- and social forestry plantations. The timber is extensively used for furniture, musical instruments, plywood, ASIA 177 shipbuilding and paper making, etc. The bark, root, fruit, flowers and leaves are used by local people as medicine in blood diseases, ulcers and fever. Seeds are known to lose their viability within a year (Prakash 1991). Madhuca indica J.F. Gmel., is part of the Sapotaceae family and is native to southern Asia, especially India. It is commonly known as ‘mahua’ in India and also referred to as M. longifolia (Koeing.) Macbride and Bassia latifolia Roxb. (Hocking 1993; Sastry and Kavathekar 1994). It is the lifeline of the tribal belt in central India, and culturally the tree most identified with Indian life in the plains. The tree has been used extensively for its multifarious properties, especially by various tribal communities and therefore hardly cut down though it is a good wood. It is large, much branched deciduous tree with a short bole and rounded crown, and is found throughout the greater part of India, up to 1200 m. The tree is valuable for its timber, flowers and seeds (Hocking 1993). The fleshy edible corollas have high economic value and are a rich source of sugars, vitamins and calcium and are used in the manufacture of country liquor and vinegar (Sastry and Kavathekar 1994; Bhanja 2000). The kernel contains a high percentage (20–43%) of fatty oil, known as mahua oil or butter. It is used in commerce as nontraditional oil (Sastry and Kavathekar 1994). The seeds of M. indica are generally referred to as short-lived (Prakash 1991; Hocking 1993; Sastry and Kavathekar 1994). The DFSC/IPGRI protocol (1999, 2000) is a systematic and scientific approach to improving the postharvest handling and storage of tropical tree seeds. The application of the protocol for these selected four tree species is important in designing the ex situ storage methods for short- and long-term conservation. Hence, an attempt was made to (a) establish the storage physiology of M. indica, B. lanzan, G. arborea and D. melanoxylon seeds and (b) to maximize their storage potential by determining their lowest safe moisture content and most appropriate storage conditions. Additionally, we analyzed lipid and phenol contents in B. lanzan and G. arborea seeds. The B. lanzan seeds manifest rancidity, thus taste pungent almost within a period of one year after harvest. Looking to the commercial and food value of the seeds, an attempt was made to estimate the changes in the lipid content and FFA in response to slow or fast drying. The contents of phenols, known as inhibitors to germination, were also estimated in view to uncover their effects on fresh seeds of G. arborea. 178 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Materials and methods

Seed collection and extraction

Mature fruits of Madhuca indica, Buchanania lanzan, Diospyros melanoxylon and Gmelina arborea were plucked from 20 to 25 marked plus trees. The protocol developed in the IPGRI/DFSC project (DFSC/IPGRI 1999, 2000) was followed to determine the seed desiccation tolerance. The collected fruits were transported in jute bags to the laboratory within 1 to 4 h of collection. The fruits were sorted and infected, mechanically damaged and very small fruits were discarded. The total weight of the harvest and the weight of 100 fruits were determined and the average weight of all these fruits was calculated. Moisture contents were determined after manually extracting seeds. The fleshy pulp was removed and traces of pulp adhering to the extracted seeds were also removed by rubbing them with sand. The seeds were then washed under running water for 2–3 minutes and gently surface dried. One hundred individual seeds were then weighed to determine the average weight of seeds (DFSC/IPGRI 1999). The seed length and breadth was also measured for fifty individuals. Desiccation and storage trials were performed following fungicidal treatment of extracted seeds. The seeds were first soaked in a 1% solution of sodium hypochlorite for 10 minutes and then rinsed and blotted dry. They were coated with 1 g Thiram per kg seed and were kept under ambient conditions (25–27°C and 35–40% RH) until further use. The seed samples were ready for various analyses within 6–10 h of their harvest from the trees.

Germination

Seeds were surface sterilized with 1% sodium hypochlorite solution for 15 min, thoroughly washed with distilled water 4–5 times. Germination tests were performed with 5 replicates of 10 seeds each. M. indica seeds were allowed to imbibe distilled water and to germinate in the dark at 26–28°C on filter paper towels rolled up inside two plastic sheets. The seeds of other three species, i.e. B. lanzan, D. melanoxylon and G. arborea, were germinated in vermiculite inside plastic boxes. During incubation, seeds were supplied with distilled water as necessary. Germination was scored after every 24 h, as radicle emergence to 5 mm (Varghese and Naithani 2002). The germination ASIA 179 test was terminated when no seed had germinated for a week or when seeds blackened and/or showed fungal manifestations.

Moisture content and desiccation trials

Moisture contents (MC) of whole fruit, seed coat, embryo, embryonic axes and the cotyledons were determined following the methods of the International Seed Testing Association (ISTA 1985). Self-indicating silica gel was used for the desiccation trials following the DFSC/IPGRI (2000) protocol. Seed samples at the desired MC (using the target MC as a reference) were retrieved at various intervals and were tested for actual MC and germination. Another lot of seeds was allowed to dry at ambient conditions of 25–27°C and 35–40% relative humidity (natural drying) by placing them on perforated plastic trays.

Storage trials

Seeds of all species at different moisture contents were stored at four different temperatures of 25, 15, 0 and –20°C. The seeds were regularly retrieved to determine their survival and moisture content. Seeds of G. arborea were also tested for survival at liquid nitrogen (LN2) temperature (–196°C) after rapid drying to various moisture contents. The seeds were packed in polypropylene cryovials and plunged into LN2. After 24 h these seeds were thawed rapidly by immersing in a water bath maintained at 37°C. Survival was assessed by germination testing (Varghese and Naithani 2001).

Total lipid and free fatty acid contents and total phenol determinations

The total lipid content of seeds of B. lanzan was estimated using the method of Raheja et al. (1973). Seeds were weighed and ground with LN2 and total lipid content was calculated gravimetrically after distillation of the seed powder 1 h in petroleum benzene at its boiling point (40–60°C). The oil content (mean of five determinations) was expressed as mg lipids per g fresh weight of the seed. The free fatty acid (FFA) content in the lipid was estimated following the method described by Itaya and Ui (1965). In four replicates, the total lipid extracted in petroleum benzene from B.lanzan seeds, was mixed with 0.66 M phosphate buffer (pH 6.2) (v/v) and 180 STORAGE BIOLOGY OF TROPICAL TREE SEEDS incubated at room temperature to separate the aqueous and chloroform layers. Copper triethanolamine solution was added to the chloroform layer, the mixture shaken in a cyclomixer and then incubated for 15–30 min to complete the reaction. Finally, to 3 ml of the chloroform layer pipetted out was added 11 mM diethyl dithio carbamate, and the absorbance was read at 440 nm. The standard curve was plotted using 0.2 mg ml-1 stearic acid. The phenol content was estimated by the method of Swain and Hills (1959). In four replicates, funicular tissue of 500 mg of G. arborea were homogenized in extraction buffer (made of 80% ethyl alcohol in 0.2 M borate buffer, pH 7.6) with silica. The homogenate was centrifuged at 8000 g for 5 minutes and the supernatant was collected. The residue was washed twice with extraction buffer. Finally, all the ethanolic supernatants were mixed and evaporated in vacuum to yield concentrated aqueous layers at 4°C. The aqueous layer was used as the source of phenolic compounds. A standard curve was prepared using different concentrations of chlorogenic acid.

Results

Initial tests

Fruit and a seed of B. lanzan weighed on average 0.71 g and 0.21 g, respectively (Table 1). The seed was globular with a mean diameter of 0.95 cm and thickness of 0.69 cm. The initial moisture contents of both manually extracted, and processed seeds were similar at ca. 17% MC, showing that the processing did not affect initial seed moisture content. The fruits in contrast, had very high moisture contents. About 6.8 kg of seeds were extracted out of 31.5 kg of D. melanoxylon fruits. The mean seed dimensions were 2.0-cm long by 1.1 cm breadth and the average seed weight was 1.3 g (Table 1). The fresh seed embryonic axes had 53.6% MC and the cotyledons 36.3% MC, while the whole seed moisture content was 38.4%. Botanically after removing the fleshy mesocarp from G. arborea fruits, the remaining stony entity is the pyrene. However, the pyrene has been used as ‘seed’ in this study, although extracted seeds (without endocarp) were used in some of the germination tests for comparison. The mean fruit and seed weights were 9.76 g and 0.97 g, respectively (Table 1). The initial moisture contents were 26.1% for the whole ‘seed’ (the pyrene) and 24.5% for the excised seeds. ASIA 181

The berry fruits of M. indica weighed 16 g each (Table 1). The mean weight of an entire seed was 3.5 g with average length and thickness of 3.1 and 1.5 cm. The initial moisture content of manually extracted seeds was 63.2%, with embryonic axes and cotyledons having respectively 66.6 and 47.6% MC.

Table 1. Initial characteristics of fruits and seeds of B. lanzan, D. melanoxylon, G.arborea and M. indica from India

B. lanzan D. melanoxylon G.arborea M. Indica Fruit weight (g) 28 r 3.1 9.76 r 2.52 16.0 r 2.9 0.71 r 0.68 No. of fruits/kg – 40 r 5 – – Seed weight (g) 0.21 r 0.02 1.30 r 2.6 0.97 r 0.17 3.5 r 0.8 No. of seeds/kg – 625 r 5 – – Seed diameter (cm) 0.95 r 0.07 – – – Seed thickness (cm) 0.69 r 0.03 – 1.14 r 0.14 – Seed length (cm) – 2.0 r 0.8 1.69 r 0.19 3.1 r 0.35 Seed breadth (cm) – 1.1 r 0.36 – 1.5 r 0.18 Seed shape globular ellipsoidal, flat Ovoid Ellipsoidal Initial MC% (manually extracted) 17.1 r 1.16 38.4 r 1.0 27.82 r 1.76 63.2 r 1.0

Seed MC after processing 16.3 r 0.42 38.0 r 1.0 26.07 r 2.33 – MC whole fruit (%) 62.2 r 3.3 58.0 r 2.7 75.03 r 1.0 73.4 r 3.6 MC seed coat (%) not separable not separable 27.9 r 1.29 63.4 r 3.1 MC embryos (%) 15.4 r 0.6 – 24.5 r 1.8 57.8 r 0.8 MC cotyledons (%) 14.0 r 0.55 36.3 r 0.5 18.48 r 2.97 47.6 r 0.4 MC axes (%) 16.7 r 0.56 53.6 r 2.4 28.79 r 0.81 66.6 r 3.5 MC endocarp (%) 10.3 r 0.46 – – –

Desiccation trials

B. lanzan seeds were dehydrated from 16% MC to 4% MC over 48 h using silica gel. Germinated seeds remained at 93–100%, indicating that there was no adverse effect of drying on germination (Table 2). Total lipids of B. lanzan declined gradually from 66.4% to 23.6% (fresh weight) in seeds slowly dried from 18.9 to 5.4% MC over 310 days at ambient conditions of 25°C and 35–40% relative humidity (Table 3). This decrease occurred with simultaneous enhancement in free fatty acid (FFA) levels from 12 to 219 Pmol g-1 fw (Table 3). In contrast, silica gel dried seeds exhibited comparatively higher levels of lipids with very low levels of FFA. The loss of total lipids in slow dried seeds was accompanied by a fall in germination from 100 to 35%. In 182 STORAGE BIOLOGY OF TROPICAL TREE SEEDS contrast, rapid drying of B. lanzan seeds to 4.8% MC did not lead to a significant reduction in germination.

Table 2. Effect of desiccation using silica gel on germination (G%) of B. lanzan seeds

Control Dried Drying time (h) MC (%) G (%) Target MC (%) Actual MC (%) G (%) – 16.3 r 0.4 100 Initial – – 20 min 17.8 r 2.1 100 15 15.6 r 0.9 100 40 min 18.1 r 2.2 100 12 13.4 r 0.7 100 1 h 50 min 18.1 r 2.2 100 10 10.6 r 0.2 100 2 h 20 min 17.0 r 1.8 100 9 10.0 r 0.3 100 3 h 20 min 17.0 r 1.6 100 8 9.2 r 0.3 100 3 h 50 min 17.3 r 1.6 100 7 8.3 r 0.7 100 4 h 50 min 17.2 r 1.1 100 6 7.1 r 0.5 100 24 h 18.3 r 0.4 100 3 4.1 r 0.3 100 48 h 23.4 r 0.7 100 <3 3.8 r 0.2 93

Table 3. Total lipid and free fatty acid (FFA) contents in B. lanzan seeds when dried using silica gel or at ambient conditions, and their viability (G%)

Ambient drying Silica gel drying MC (%) G (%) Total FFA MC (%) G (%) Total FFA lipids (%) (Pmol g-1 fw) lipids (%) (Pmol g-1 fw) 18.9 r 1.2 100 66.4 12 18.9 r 1.2 100 66.4 10 14.5 r 0.9 100 61.0 28 12.0 r 0.6 100 67.2 9 8.8 r 0.5 88 52.4 63 7.9 r 0.4 100 64.0 17 5.4 r 0.8 35 23.6 219 4.8 r 0.3 90 59.0 54

The seeds of D. melanoxylon were desiccated to 4.2% MC without reduction in viability (Table 4).

Table 4. Desiccation and germination of D.melanoxylon seeds

Control Dried Drying time (h) MC (%) G (%) Target MC (%) Actual MC (%) G (%) 0 37.2 r 0.63 97 Initial 37.2 r 0.63 97 3 38.4 r 0.26 90 30 30.7 r 0.51 97 6 37.3 r 0.35 90 25 21.8 r 1.37 97 12 38.5 r 0.87 86 20 17.8 r 0.45 97 18 39.6 r 0.62 90 15 15.3 r 0.45 93 30 38.7 r 0.29 93 10 10.6 r 0.20 97 67 40.5 r 0.72 90 5 6.9 r 0.75 100 95 41.3 r 0.75 93 3 4.4 r 0.15 100 119 41.4 r 0.69 90 2 4.2 r 0.50 100 ASIA 183

G. arborea whole seeds (pyrene) with 27% MC showed 60% germination (Table 5). Drying these seeds (with endocarp) from 23 to 3% MC over 150 h improved the germination capacity from 60 to 96%. Control seeds maintained in the vermiculite, showed a slight loss of moisture content but improved germination from 60 to 95% after the same period (Table 5). Total phenol extracted from funicular tissue, which remained tenaciously attached within the cavity of the stone or endocarp after depulping, showed a gradual decline from 7.21 to 0.08 mg g-1 fw when the pyrenes were dried from 28 to 4% MC (Table 6). Excised seeds of G. arborea at 28% MC treated with phenol extracted (5 ml) from funicular tissue of the pyrene exhibited germination of only 62%. As the phenol content of the extract fell, any inhibition effect on excised seed germination was lost.

Table 5. Desiccation and germination of G.arborea seeds with endocarp (pyrene) and without endocarp (excised seeds)

Control Dried Drying time (h) MC (%) Germination (%) Target MC (%) Germination (%) MC (%) With Excised With Excised endocarp seeds endocarp seeds 0 min 27.3 r 1.6 60 100 Initial 27.3 r 1.6 60 100 1 h 15 min 28.19 r 1.2 60 100 22 23.0 r 0.5 65 100 4 h 26.87 r 1.1 62 100 17 17.0 r 0.8 72 100 11 h 27.12 r 1.2 60 100 12 11.9 r 1.3 86 100 15 h 10 min 29.22 r 0.8 64 100 8 8.1 r 0.2 90 100 21 h 20 min 26.87 r 1.1 65 100 6 6.3 r 0.4 96 100 42 h 10 min 27.48 r 1.7 85 100 5 4.3 r 0.1 100 100 54 h 30 min 24.19 r 1.8 85 100 4 4.1 r 0.4 100 100 72 h 30 min 23.13 r 1.3 94 94 3 3.4 r 0.1 100 100 150 h 20.65 r 2.0 95 95 2 2.7 r 0.3 96 95

M. indica seeds desiccated with silica gel over a period of 200 h at 25°C reached a moisture content of 9.4% (Table 7). Initially seeds with 63.2% MC germinated 100%. However, germinability declined gradually to 10% with desiccation and no seed germinated below 18% MC (Table 7). Seeds kept as the control in vermiculite showed 100% over the same treatment time. 184 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Table 6. Total phenol (80% alcoholic extract) from funicular tissue attached within the cavity of endocarp and germination of G. arborea excised seeds treated with 5 ml of extracted phenol

MC whole seeds (%) Total phenol (mg phenol g-1 fw) Germination (%)

28 r 0.9 7.2 62 15.5 r 0.9 6.2 78 7.1 r 0.6 1.9 100 4.0 r 0.2 0.08 100 4.0 r 0.2 0 100

Table 7. Desiccation and germination (G%) of M. indica seeds Control Dried Drying time (h) MC (%) G (%) Target MC (%) MC (%) G (%) 0 h 63.2 r 1.0 100 Initial 63.2 r 1.0 100 20 h 59.6 r 1.5 100 55 53.3 r 2.9 100 30 h 60.0 r 2.7 100 50 51.7 r 0.9 100 46 h 60.9 r 2.2 100 45 43.6 r 2.2 95 80 h 60.1 r 1.8 100 40 37.2 r 2.6 80 105 h 59.7 r 1.7 100 35 31.3 r 3.0 75 130 h 60.2 r 2.2 100 30 30.5 r 2.5 60 150 h – – 25 26.2 r 1.8 40 174 h – – 20 18.3 r 0.9 10 200 h 58.1 r 1.6 100 10 9.4 r 2.9 0

Storage trial

Hydrated seeds of B. lanzan with t13% MC could not tolerate low temperatures of –20, 0 and 15°C, but these seeds germinated 35–68% after storage at 25°C for 280 days (Fig. 1). Further drying of seeds to 7–10% MC improved their chilling tolerance and they better survived during storage. These seeds exhibited 95–100% survival after 90 days at all storage temperatures and gradual loss in germinability (58–88%) after 280 days of storage. B. lanzan seeds dried to ca. 4% MC showed 95–100% germination after 180 days of storage at all tested temperatures. Further storage for 280 days led to a slight reduction in seed viability (85–92% germination). Initially all hydrated and dehydrated seeds of D. melanoxylon germinated 100% (Fig. 1). Seeds with high moisture contents of 27– 37% could not tolerate storage at –20 and 0°C, but further ASIA 185 desiccation to 18–22% MC resulted in a gradual increase in survival, 7–23% germination after 20 days at these temperatures (Fig. 1). However, seeds with 15–37% MC were storable at 15°C for 150 days and at 25°C for 240 days, though with a gradual loss in viability. Seeds desiccated to 10.5, 6.9, and 4.5% MC survived storage at all tested temperatures, germinating in the range of 80–100% after 150 days (Fig. 1). Dried seeds with 6.9 and 4.5% MC germinated highly (85–95%) after 250 days of storage at all tested temperatures (–20, 0, 15 and 25°C). Although G. arborea seeds with endocarp (pyrene) of various moisture contents were stored, germination tests were performed using excised seeds (after removing the endocarp) in order to avoid the inhibitory effect of the endocarp. Seeds with 18–26% MC could not survive storage at –20 and 0°C and became nonviable within 90 days (Fig. 1). Further drying to 12.4% MC improved longevity as the seeds germinated 30–45% after 150 days of storage at –20 and 0°C. Seeds with t12.4% MC showed similar survival after storage at 15 and 25°C (Fig. 1). Seeds dehydrated to 3.9% MC exhibited highest survival (72–95%) at –20, 0 and 15°C after 270 days of storage. In a separate cryo-storage trial, all seeds with endocarp (silica gel) dried from 34.3 to 2.4% MC could not survive cryo-temperature when stored for 24 h (data not shown). M. indica seeds lost viability within 10 days when stored at zero and sub-zero temperatures (Table 6). However, best results were obtained at 25°C in seeds with 43.6% MC, which showed 25% germination after 55 days of storage. Seeds with higher or lower than >43.6% MC lost germinability within 40 days (Table 8). The seeds stored at 15°C also lost viability within 40 days. It has been observed that these seeds at high moisture contents required a lot of care during storage to avoid fungal infestation and suffocation.

Discussion

Initially, mature seeds of all the four tree species studied exhibited almost 100% germination. Unlike some desiccation tolerant (or orthodox) seeds, the seeds of M. indica, D. melanoxylon, G. arborea and B. lanzan are shed at high (63.2%) or relatively high (17.1–38.4%) moisture contents (Table 1). 186 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

B. lanzan D. melanoxylon G. arborea

100 100 100

80 80 80

16.3 % MC 37.2 % MC 15.6 % MC 27.3 % MC 60 13.4 % MC 60 21.8 % MC 60 10.0 % MC 17.8 % MC 7.1 % MC 15.3 % MC 40 4.1 % MC 40 10.5 % MC 40 3.8 % MC 6.9 % MC 4.5 % MC 20 -20°C 20 -20°C 20 -20°C

0 0 0

100 100 100

80 80 80

60 60 60

26 % MC 40 40 40 18.5 % MC 12.4 % MC 0°C 0°C5.9 % MC 0°C Germination (%) Germination (%) 20 20 Germination (%) 20 3.9 % MC 1.9 % MC

0 0 0

100 100 100

80 80 80

60 60 60

40 40 40

20 20 20 15°C 15°C 15°C

0 0 0

100 100 100

80 80 80

60 60 60

40 40 40 Germination (%) Germination (%) 20 20 Germination (%) 20 25°C 25°C 25°C 0 0 0

0 30 60 90 120 150 180 210 240 270 300 0 30 60 90 120 150 180 210 240 270 300 0 30 60 90 120 150 180 210 240 270 300

Storage period (days) Storage period (days) Storage period (days)

Figure 1. Germination of B. lanzan, D. melanoxylon and G. melina seeds at different moisture contents after storage at four temperatures of –20, 0, 15 and 25°C for about 9 months. ASIA 187

Table 8. Germination after days (d) of storage of M. indica seeds at 15 and 25°C

MC (%) Storage Germination (%) after storage temperature (°C) 0 d 10 d 20 d 30 d 40 d 55 d 63.2 r 1.0 15 55 15 0 – – 25 100 75 40 10 0 – 53.3 r 2.9 15 50 20 0 – – 25 100 50 20 10 0 – 51.7 r 1.0 15 65 35 10 0 – 25 100 70 40 20 0 0 43.6 r 2.2 15 80 50 25 0 – 25 95 90 75 55 35 25 37.2 r 2.6 15 40 0 – – – 25 80 65 40 20 5 0 31.3 r 3.0 15 35 0 – – – 25 75 45 20 0 – – 30.5 r 2.5 15 35 0 – – – 25 60 30 0 – – – 26.2 r 1.8 15 0 0 – – – 25 40 10 0 – – – 18.3 r 0.9 15 0 0 – – – 25 10 0 0 – – –

Seeds of B. lanzan, D. melanoxylon and G. arborea tolerated desiccation over silica gel to as low as 4% MC (Tables 2, 4 and 5), maintaining their initial viability. On the other hand, M. indica seeds showed a rapid loss of viability when dried to 43.6% MC and did not tolerate dehydration below 18% MC (Table 5). This result corroborates another study on this species showing a decline in seed viability with a reduction in moisture content (Varghese et al. 2002). M.indica seeds, like other desiccation-sensitive seeds of Shorea robusta (Chaitanya and Naithani 1998), Avicienia marina (Berjak et al. 1989) and Quercus robur (Finch–Savage 1992), showed high critical moisture content (43.6% MC) and can be categorized as true recalcitrant. The seeds of B. lanzan exhibited acute rancidity when dried to 5.4% MC at ambient temperature of 25°C (Table 3). The seeds with 19% MC were rich in oil content (66.4% fw total lipids) with less FFA (12 Pmol g-1 fw). However, they registered a gradual loss of total lipids (23.6% fw total lipids) with a simultaneous increase in FFA and decrease in viability when dried to 5.4% MC (Table 3). Our results corroborate findings on L. kirki and S. membranaceous (Farrant et al. 1989) which concluded that desiccation-sensitive seeds retain higher viability to a lower moisture content, if dried rapidly as compared to the seeds desiccated slowly in ambient conditions. Pammenter et al. (1991) suggested that rapid drying permits survival to lower water contents 188 STORAGE BIOLOGY OF TROPICAL TREE SEEDS because the water is removed sufficiently rapidly to prevent aqueous- based deleterious reactions occurring. It is suggested that the rapidly dried seeds of B. lanzan maintained higher percentage of viability by not allowing the peroxidation of lipids and production of FFA. The lipid peroxidation has been shown to be a prime cause of membrane perturbations leading to loss of viability during desiccation-induced damage or accelerated ageing in seeds. The peroxidized products of lipids and FFA are cytotoxic by nature (Varghese and Naithani 2002). The germination pattern was slightly different in G. arborea seeds, when seeds with endocarp were compared to excised seeds, i.e. those without endocarp (Table 5). Initially the hydrated seeds (27.3% MC) showed 60% germination, which was enhanced to 100% when the moisture content was reduced to 3–4%. In contrast, the excised seeds extracted from hydrated seeds (27.3% MC) germinated 100% and maintained such viability when dried to 3% MC. High levels of total phenols estimated in the funicular tissue attached within the cavity of stone or endocarp of hydrated seeds have been found to inhibit the germination of hydrated seeds (Table 6). Nearly 38% inhibition in germination was recorded when excised seeds were treated with phenols extracted from funicular tissue of hydrated seeds. The phenolic compounds are known as potent inhibitors of germination and are well documented for their role in inducing dormancy in seeds (Bewley and Black 1994). The seeds of all the four species with >15% MC were extremely sensitive to low temperatures and were killed when exposed to –20 and 0°C during storage (Fig. 1 and Table 8). The failure of high moisture content seeds to survive freezing temperatures is in close agreement with the results obtained for both desiccation sensitive (Wesley–Smith et al. 1992) and desiccation tolerant (Hong and Ellis 1992) seeds. Seeds of D. melanoxylon, G. arborea and B. lanzan tolerated both desiccation and low temperatures (–20, 0°C) when dried below 11% MC (Fig. 1). B. lanzan and G. arborea seeds may be classified as intermediate as gradual loss of viability was distinct after 180 and 90 days of storage respectively, at all storage temperatures. Further experiments are necessary to define the intermediate nature of these seeds as the definition for desiccation- sensitivity remains elusive with ill-defined boundaries. D. melanoxylon seeds also survived for 250 days with 85–95% when dried to 6.9 and 4.5% MC. These seeds are also not strictly following the orthodox storage physiology per se. It was apparent that although these seeds demonstrated strong desiccation tolerance ASIA 189 when rapidly dried to sufficiently low moisture contents, like orthodox seeds, they were not storable at freezing or other temperatures (15 and 25°C) without loss of viability within a short period of (90–180 days) storage. Dried seeds of G. arborea, for example, were desiccation and chilling tolerant, but showed extreme sensitivity during cryo-storage for 24 h (data not shown). M. indica seeds also showed best survival of 25% at 43.6% MC, but further desiccation resulted in reduction of viability and longevity, as other desiccation-sensitive seeds that are damaged if desiccated below a critical moisture content (Chaitanya and Naithani 1998; Chaitanya et al. 2000).

Conclusions

Buchanania lanzan and Diospyros melanoxylon seeds were desiccation and chilling temperature tolerant, and showed no significant loss of viability when stored for 250 days. Thus these seeds can be classified as having orthodox storage behaviour. Rapid drying of B. lanzan seeds with silica gel before storage can be recommended. Seeds of Gmelina arborea were both desiccation and low temperature tolerant at least up to 150 days, although they were extremely sensitive to cryo- temperature, indicating that long-term storage may be difficult. Because total phenols inhibited germination of viable G. arborea seeds, reduced germination in the nursery beds can be avoided by using excised seeds. Madhuca indica seeds were both desiccation and chilling temperature sensitive. The critical moisture content for M. indica seeds was 43.6%. The seeds demonstrate true recalcitrant storage behaviour.

Achievements of the DFSC/IPGRI Project

The successful completion of the first year of the DFSC/IPGRI project paved the way for another research project on ‘Seed technology of Forest tree: Postharvest handling and ex situ storage’. The project goal is to generate a Database for ex situ conservation for large number (nearly 25) of socioeconomically important forest tree species, using the protocol developed by DFSC/IPGRI (1999, 2000), which are so far not commercially exploited due to the lack of sufficient knowledge on their handling and storage. The Ministry of Environment and Forest, Delhi, 190 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

India funded this project, in 2001. Data obtained on storage of Madhuca indica has already been published (Varghese et al. 2002). Two other papers (‘Seed storage behaviour in Buchanania lanzan Spreng.’ and ‘Seed storage behaviour in Gmelina arborea Roxb.’) are under preparation. The Indian partners benefited from working in the DFSC/IPGRI project and the use of a systematically well-developed scientific protocol. This allowed the evaluation of storage physiology of four important tree species of tropical forest of India. Second, attending the workshop organized by DFSC/IPGRI in 2001 at ASEAN Tree Seed Centre was an opportunity to learn and understand various difficulties faced in the execution of the protocol. The application of unified protocol by various partners associated in the project offered a unique platform for comparing the data produced in different parts of the world. Further, four research scholars were trained in the execution of the protocol during 2000–2002.

References

Berjak, P., J.M. Farrant and N.W. Pammenter. 1989. Homoiohydrous (recalcitrant) seeds: the enigma of their desiccation-sensitivity and the state of water in axes of Landolphia kirkii dyer. Planta 186:249–261. Bewley, J.D. and M. Black. 1994. Seeds: Physiology of Development and Germination. Plenum Press, New York. Pp. 210. Bhanja, M. 2000. Polyembryony in Madhuca indica J.F. Gmel. (Sapotaceae). Indian Forester 126:91–92. Chaitanya, K.S.K. and S.C. Naithani. 1998. Kinetin-mediated prolongation of viability in recalcitrant sal (Shorea robusta Gaertn.f.) seeds at low temperature: role of kinetin in delaying membrane deterioration during desiccation-induced injury. J. Plant Growth Regul. 17:63–69. Chaitanya K.S.K., S. Keshavkant and S.C. Naithani. 2000. Changes in total protein and protease activity in dehydrating recalcitrant sal (Shorea robusta) seeds. Silva Fennica 34:71–77. DFSC/IPGRI. 1999. The Project on Handling and Storage of Recalcitrant and Intermediate Tropical Forest Tree Seeds. Humlebaek, Denmark. IPGRI/Danida Forest Seed Centre Newsletter 5:23–40. DFSC/IPGRI. 2000. The Project on Handling and Storage of Recalcitrant and Intermediate Tropical Forest Tree Seeds. Humlebaek, Denmark. IPGRI/Danida Forest Seed Centre Newsletter 7:23–26. Farrant, J.M., N.W. Pammenter and P. Berjak. 1989. Germination associated events and the desiccation-sensitivity of recalcitrant seeds: a study on the three unrelated species. Planta 178:189–198. Finch–Savage, W.E. 1992. Embryo water status and survival in the recalcitrant species Quercus robur L.: evidence for a critical moisture content. J. Exp. Bot. 43:663–669. ASIA 191

Gunn, S. 1991. Banking on the Future. Kew Spring, UK. Pp. 16–21. Hocking, D. 1993. Trees for Dryland. Oxford & IBH Publishing, New Delhi, India. Pp. 238–241. Hong, T.D. and R.H. Ellis. 1992. The survival of germinating orthodox seeds after desiccation and hermetic storage. J. Exp. Bot. 43:239–247. ISTA. 1985. Determination of moisture content. Seed Sci. Technol. 13:338– 341. Itaya, K. and M. Ui. 1965. Colorimetric determination of free fatty acids in biological fluids. J. Lipid Res. 6:16–20. Jain, S.K. 1968. Medicinal Plants. National Book Trust, New Delhi, India. Ministry of Environment and Forests (MoEF). 2000. National Biodiversity Strategy and Action Plan India: Guidelines and Concept Papers. Ministry of Environment and Forests, New Delhi, India. Murty, A.V.S. and N.S. Subramanyan. 1989. A Text Book of Botany. Wiley Eastern Ltd, New Delhi, India. Pammenter, N.W., C.W. Vertucci and P. Berjak. 1991. Homoiohydrous (recalcitrant) seeds: dehydration the state of water and viability characteristics in Landolphia kirkii. Plant Physiol. 96:1093–1098. Prakash, R. 1991. Madhuca longifolia. Pp. 265–269 in Propagation Practices of Important Indian Trees. International Book Distributors, Dehradun, India. Raheja, R.S., C. Kaur, A. Singh and I. Bhatia. 1973. New colorimetric method for the quantitative estimation of phospholipids without acid digestion. J. Lipid Res. 14:695–697. Report of Committee of Forests and Tribals of India. Tribal Development Division, M.H.A., Government of India, New Delhi, India, 1982. Sastry, T.C.S. and K.Y. Kavathekar. 1994. Madhuca. Pp. 300–304 in Plants for Reclamation of Wastelands. CSIR, New Delhi, India. Swain, T. and W.E. Hills. 1959. The phenolic constituents of Prunus domestica. I. The quantitative analysis of phenolic constituents. J. Sci. Food Agric. 10:63–68. Tewari, D.N. 1981. State Trading of Forest Produce in India. Jugal Kishore & Co, Dehradun, India. Varghese, B and S.C. Naithani. 2001. Desiccation stress in neem seeds: physiological and biochemical considerations. Danida Forest Seed Centre Newsletter 8:16–19. Varghese, B. and S.C. Naithani. 2002. Desiccation-induced changes in lipid peroxidation, superoxide level and antioxidant enzymes capacity in neem (Azadirachta indica A.Juss.) seeds. Acta Physiol. Plantarum 24:79–87. Varghese, B., R. Naithani, M.E. Dulloo and S.C. Naithani. 2002. Seed storage behaviour in Madhuca indica J.F. Gmel. Seed Sci. Technol. 30:107–117. Wesley–Smith, J., C.W. Vertucci, P. Berjak, N.W. Pammenter and J. Crane. 1992. Cryopreservation of desiccation-sensitive axes of Camellia sinensis in relation to dehydration, freezing rate and thermal properties of tissue water. J. Plant Physiol. 140:596–604. 192 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Handling and storage of Hopea odorata seeds

Nadarajan Jayanthi and Baskaran D. Krishnapillay

Forest Research Institute Malaysia (FRIM), Kepong, 52109 Kuala Lumpur, Malaysia

Abstract

Hopea odorata seeds with an initial moisture content of 48.5% and 100% germination, were desiccated to 42.5, 31.4, 22.9, 11.0, 9.3 and 6.4% MC. A moderate decrease in germination percentage was observed with drying to 42.5% and 31.4% MC; no seeds survived desiccation to 22.9% MC. Seeds with 48 and 37% MC were stored at 16 and 25°C for 24 months. Pre-germination during storage was highest for seeds with 48% MC stored at 25°C. The storage viability stayed relatively high after the first eight weeks. Then, there was a gradual decline in viability, which dropped to approximately 50% germination after 16 weeks. Viability and moisture contents decreased dramatically between 16 and 18 weeks storage and no seed germinated after 22 weeks.

Introduction

Hopea odorata Roxb. is locally known as ‘merawan siput jantan’, and it belongs to the family of Dipterocarpaceae. The species is distributed throughout Bangladesh, Myanmar, southern Vietnam, Cambodia, Thailand, the Andaman Islands and northern Peninsular Malaysia. The timber is suitable for rollers in the textile industry, piles and bridge constructions. Because of its multiple uses and its heavy exploitation, H. odorata is now listed as a vulnerable species, with high risk of becoming endangered (IUCN 2002). A most important characteristic of the family of Dipterocarpaceae is its flowering behaviour. Flowering does not occur annually, but at irregular intervals and then of varying intensity. H. odorata flowers, on average, once every two years. Flowering is sporadic and often restricted to a few branches. Wildings from natural regeneration of this species probably take many years to reach flowering age under forest conditions. To show the cause of irregular flowering in dipterocarps, Ashton et al. (1988) demonstrated a highly significant correlation between gregarious flowering and preceding periods with both large ASIA 193 diurnal temperature ranges and high maximum values. Appanah and Chan (1981) reported that Meliponid bees (Trigona spp.) and Thrips (Thysanoptera spp.) are the most common pollinators for many dipterocarp species. The fruit of H. odorata is a nut surrounded by the persistent calyx. Two of the calyx lobes are extended into wings with extensive air spaces allowing the fruit to gyrate and fall obliquely and be carried with the wind. The majority of the fruits fall within 100 m of the parent tree under forest conditions. The seeds have been reported to exhibit recalcitrant behaviour (Tang and Tamari 1973). This study aimed at defining desiccation sensitivity and optimizing storage conditions for the seeds.

Materials and methods

Seed collection and processing

Hopea odorata seeds were collected on 19 May 1997 from two trees within the FRIM grounds in Malaysia. The trees were climbed in order to collect mature seeds with brown wings, directly from the branches. Although fallen fruits were not collected from the ground, insects nonetheless infested the cotyledons of about 20% of the seeds. The fruits were put in loosely tied plastic bags (56 × 90 cm) and transported from the field by an open-windowed vehicle (not air-conditioned). Transportation took less than an hour and the maximum and minimum temperatures during transportation were 32 and 25°C, respectively. The fruits were de-winged manually on the day of collection. Moisture content determination and desiccation trials were also initiated immediately. Mechanically damaged, infected and infested fruits were removed manually. The de-winged fruits (hereafter called seeds) were treated with fungicides (Benlate and Thiram) and subsequently rinsed with distilled water. They were then surface dried by spreading them out on a bench in an air-conditioned (ca. 25qC) laboratory for 2–3 h. Trials were not replicated due to the poor fruiting season in 1997. 194 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Initial tests

The weights of fruits and their seeds were determined on 100 individuals. Seed moisture content was determined using 4 replicates of 10 seeds that were dried in an oven at 103°C for 17 h. Moisture contents were then calculated as a percentage on a fresh weight basis (IPGRI/DFSC 1996).

Germination tests

Four replicates of 50 seeds were sown in heat-sterilised (at 130°C) sand and moistened tissue paper for germination at 25–30°C with 10-h light/14-h dark. Germination assessment was done daily since the seed are known to germinate rapidly. Hopea odorata seeds are very small and polyembryonic. Since there were several embryos in one seed, a seed was scored as germinated when a radicle started to emerge and elongate.

Desiccation and storage trials

Because the initial moisture content was high, target moistures of 40, 30, 10, 8, and 5% MC were selected for desiccation trials. Seeds were mixed with silica gel that was changed every 2 h during desiccation, and their moisture contents were determined as described above. Since H. odorata seeds are believed to be recalcitrant, they were subjected to the storage condition for recalcitrant seeds as proposed in the protocol (IPGRI/DFSC 1996). Therefore, seeds with 48% MC (i.e. fresh seeds) and seeds with 38% MC (dried using silica gel) were stored at 25°C and 16°C. Seeds were sampled every two weeks and then moisture content and germination tests were regularly carried out.

Results

Initial tests

The weights of fruits and its seeds were determined on 100 individuals and were respectively 2.77 r 0.03 g and 2.55 r 0.07 g. The shedding ASIA 195 moisture content of the seeds (de-winged fruits) was 48.5% (Table 1). The moisture content of the embryo was higher than that of the cotyledon. There was 100% germination of fresh seeds within 45 days.

Table 1. Moisture contents of different components of H. odorata fruits and seeds

Component MC (%) f.w.b. Fruit (seed + wing) 51.28 Seed 48.50 Wing 11.27 Endocarp 7.48 Cotyledon/storage tissue 49.97 Embryo 63.18

Desiccation trials

There were slight variations between targeted seed moisture contents and the actual measured moisture contents after desiccation using silica gel (Table 2). Seeds desiccated from 48% MC to 6% MC within about 36 h. The moisture contents of seeds in the control treatments were almost constant throughout the experiment and there was 98– 100% germination of these seeds. Drying seeds to 31% MC after 7 h in silica gel reduced their viability to 74%. There was no germination of seeds dried to less than 31% MC.

Table 2. Germination (G) after desiccation of H. odorata seeds mixed with silica gel. Data with the same superscript letter are not significantly different at p=0.05 based on Duncan Multiple Range Tests

Control seeds Dried seeds Target MC (%) MC (%) G (%) Drying time MC (%) G (%) 40 48.5 100a 3 h 20 min 42.5 86b 30 50.3 98a 7 h 15 min 31.4 74c 20 49.3 100a 19 h 11 min 22.9 0d 10 47.9 98a 23 h 29 min 11.0 0d 8 46.5 98a 28 h 17 min 9.3 0d 5 45.7 100a 32 h 57 min 6.4 0d

Storage trials

The effect of moisture content and storage temperature on the germination capacity of H. odorata seeds was evaluated over 24 weeks 196 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

(Fig. 1). For seeds stored at 48% MC and at both 25qC and 16qC, there was a gradual decline in germination from >85% after 2 weeks storage down to ca. 50% after 16 weeks. Similarly, for seeds stored at 37% MC, there was a gradual decline in viability during the first 16 weeks of storage. This rate of decline appeared to be similar for the two storage temperatures; however, the germination of seeds stored at 25qCwas consistently ca. 10% lower than that of seeds stored at 16qC. There was no significant difference (P>0.05) between these two temperatures. The MC of seeds was maintained during the first 16 weeks of storage; however, it subsequently rapidly decreased. By 24 weeks, the final moisture content was less than 10% for seeds with both initial moisture contents at both temperatures (Fig. 1). Pre-germination has been also observed, particularly for the high moisture content (>40%) at 25°C (data not shown), but all the germination tests were carried out only with non-germinated seeds at the time of different tests.

Discussion

The desiccation trial showed that the critical moisture content for these H. odorata seeds was between 31 and 23% (Table 2). Seeds should therefore be dried to a range of moisture contents between these two values in order to more precisely determine the moisture level below which they will be unable to germinate. However, the data from the storage trials can also help identify the critical moisture content. There was no germination of seeds stored at 25qC for 18 weeks by which time the MC had fallen to 24.8% but there was 8% germination of seeds stored at 16qC which had an MC of 25.6% (Fig. 1). Thus, 25% MC is the approximate threshold moisture for desiccation survival of H. odorata seeds. Seeds stored at both 37 and 48% moisture content did not show any significant difference in germination. However, viability was maintained slightly longer at 16°C compared with seeds stored at 25°C. There was a high percentage of seeds pre-sprouted at 25°C, compared to seeds stored at 16°C (data not shown), which might have been due to the initiation of germination process at 25°C. This pre-germination problem was observed for seeds at the highest moisture contents during the desiccation trials. Another critical problem faced during high temperature storage was fungal contamination of seeds. All these storage data confirm that the seeds are recalcitrant and cannot be stored for more than 16–18 weeks. Two relatively high storage temperatures (16 and 25°C) were selected in this study, as it ASIA 197 was reported that recalcitrant seeds can only be stored at relatively warm conditions, because chilling injury is a common phenomenon (Chin and Roberts 1980). The data reported here are in agreement with our previous study, which shows that 29 and 35% MC were the critical and optimum moisture contents respectively for H. odorata at 16°C where 70% germination was recorded after 2 months storage (Jayanthi 1999).

100 100 48% MC viability at 25°C 90 viability at 16°C 80 MC at 25°C 80 MC at 16°C 70

60 60

40 40

20 20

0 0 100 100

37% MC 90 Moisture content (%) content Moisture Germination (%)

80 80

60 60

40 40

20 20

0 0

0 4 8 12 16 20 24 Storage period (weeks)

Figure 1. Germination of H. odorata seeds with initial moisture contents of 48 and 37% stored at 16 and 25°C for 24 weeks. 198 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Conclusion

The most favourable conditions for storing these seeds would be at 37% MC and at a temperature of 16°C, since there was fewer seeds presprouted, and less fungal contamination. High initial moisture contents and warm temperature of 25°C favoured pre-germination of seeds and damaging fungal growth on seeds.

References

Appanah, S. and H.T. Chan. 1981. Thrips: the pollinators of some dipterocarps. Malaysian Forester 44:234–252. Ashton, P.S., T.J. Givinish and S. Appanah. 1988. Staggered flowering in the Dipterocarpaceae: new insights into floral induction and evolution of mast fruiting in the aseasonal tropics. Am. Nat. 132:44–66. Chin, H.F. and E.H. Roberts (eds.). 1980. Recalcitrant Crop Seeds. Tropical Press, Kuala Lumpur, Malaysia. IPGRI/DFSC. 1996. The project on handling and storage of recalcitrant and intermediate tropical forest tree seeds. Newsletter No. 1. July 1996. Pp. 5–20. IUCN. 2002. 2002 IUCN Red List of Threatened Species [see also at http://www.redlist.org]. Jayanthi, N. 1999. The Effects of Moisture Content and Storage Temperature on Storage of an Orthodox, Intermediate and a Recalcitrant Tropical Forest Tree Seed. MSc Thesis. University Putra Malaysia. Pp. 125. Tang, H.T. and C. Tamari. 1973. Seed description and storage tests of some dipterocarps. Malaysian Forester 36:38–53. ASIA 199

Handling and storage of Shorea leprosula Miq. seeds

Nadarajan Jayanthi and Baskaran D. Krishnapillay

Forest Research Institute Malaysia (FRIM), Kepong, 52109 Kuala Lumpur, Malaysia

Abstract

Shorea leprosula seeds with an initial moisture content of 42.1% were 100% viable. Seeds desiccated to 31.6% MC had 84% germination, whereas no seeds survived desiccation to d 20.9% MC. Seeds with 42.3 and 31.7% MC, stored at two temperatures of 16°C and 25°C were dead after 24 weeks of storage. Viability gradually decreased to 50% after about 13 to 15 weeks of storage. Pre-sprouting during storage was highest for seeds with 42.3% initial MC stored at 25°C.

Introduction

Shorea leprosula Miq. locally known as ‘meranti tembaga’, is one of the economically important tropical lowland timber species belonging to the Dipterocarpaceae family. Like other members of the Dipterocarpaceae, S. leprosula also exhibits irregular flowering. It flowers on average once every two years, however, a mass fruiting season only occurs once every six to eight years. The tree flowers from May until as late as August in the lowland and until November in the mountains. In general, the flowers occur heavily over the whole crown of the trees. Young trees of S. leprosula, growing following selective felling were reported to set viable fruits/seeds after 7 years (Ashton et al. 1998). Only few fruits develop on each of the many-flowered inflorencences that are often heavily infested by parasitic weevils, e.g. Nanophyes, Scolitid poecileps and some Lapidoptera spp. The most common pollinators for Shorea leprosula are meliponid bees (Trigona spp.) and thrips (Thysanoptera) species (Appanah and Chan 1981). The seeds have prolonged wings with extensive air spaces and are dispersed by a parachute mechanism. Shorea leprosula is an erratic seed bearer, which makes it impossible to get regular supply of planting materials, and rapid germination is one of the specific characteristics of these seeds. It has been reported that Shorea species are difficult to store for the long term in general due to their intolerance to desiccation (Tompsett 1987). For such recalcitrant seeds, slight desiccation to a 200 STORAGE BIOLOGY OF TROPICAL TREE SEEDS lower level than its relatively higher moisture is sufficient to cause injury. Their sensitivity to desiccation was reported to be variable with the stage of postharvest seed development (Farrant et al. 1988; Hong and Ellis 1992). Storage temperature is also an important factor affecting seed longevity, although seed moisture generally has a greater effect than temperature. Sasaki (1980) reported that S. leprosula seeds at an initial 32% MC could be stored at 21°C with 45% germination, although no mention was made of the storage moisture content and duration for these seeds in those conditions. We report here, the results of investigations on S. leprosula seeds, in a attempt to improve their handling.

Materials and methods

Seed collection and processing

Shorea leprosula seeds were collected on 18 Dec 1997 from Ulu Teranum Reserved Forest, Pahang, located about 100 km from FRIM. Mature seeds were harvested directly from five trees using climbers. No seeds were collected from the ground. About 10 to 20% of seeds were already infected by insects at harvest. The seeds in plastic bags (size 56 × 90 cm) were transported from the field in a non-air-conditioned vehicle with open windows, to the laboratory within 3 h. The plastic bags were tied loosely to avoid moisture loss of the collected seeds during transportation. The maximum and minimum temperatures during transportation were 32°C and 25°C, respectively. The freshly collected seeds were manually de-winged. Moisture content determination and desiccation trials were initiated the same day as the seeds were processed. Mechanically damaged, infected and infested seeds were discarded. Seeds were treated with fungicides (Benlate™ and Thiram™) and subsequently rinsed with distilled water. They were then surface dried by spreading them out on a laboratory bench under air condition temperature of 25°C for about 2–3 h. 3 kg of de-winged seeds were dispatched to our replicating partner at ASEAN–FTSC in Thailand on 20 Dec 1997. Sterilisation of the seeds and fungicide application was carried out as mentioned in the protocol (IPGRI/DFSC 1996). The seeds were not soaked in the sodium hypochlorite (NaOCl) solution, since they tend to germinate immediately in the presence of high humidity. However, they were ASIA 201 just coated using a mixture of 2 g of active Benomyl per 1 kg of seeds, and our replicating partner received them in good condition on 27 Dec 1997.

Initial desiccation and germination tests

Initial tests determined S. leprosula seeds at 42.1% MC, and the target moisture contents were 40, 30, 10, 8, and 5%. Silica gel was used to dry seeds and was changed every 2 h during desiccation. Seeds were weighted before and after oven drying at 103°C for 17 h, and the moisture content was calculated as a percentage of a fresh weight (IPGRI/DFSC 1996). The initial test was initiated 2 days after collection. Four replicates of 50 de-winged seeds were germinated in sand that had been sterilized at 130°C for 1 h and on moistened tissue papers at 25–30°C for 10 h light/14 h dark. Germination assessment was performed daily.

Storage trials

Since the seeds were suspected to possess the typical characteristic of recalcitrant seeds, they were subjected to two moist storage conditions: at the shedding moisture content of 42% and at 32% MC at both 25°C and 16°C.

Results

Initial tests

The average weight of 100 individual fruits and seeds were respectively 5.93 r 0.09 g and 5.73 r 0.07 g. Moisture contents of fruit, whole seed and different seed components can be found in Table 1. The initial germination was 100% and the duration of the test was 30 days. 202 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Table 1. Initial moisture contents (MC) of different components of S. leprosula fruits

Part MC (%) Fruit (seed + wing) 47.6 Seed 42.1 Wing 25.7 Endocarp 37.4 Cotyledon 38.8 Embryonic axis 66.5

Desiccation trial Table 2 shows the target moisture contents, the actual moisture contents after desiccation, the duration of drying and the percentage of germination after desiccation.

Table 2. Desiccation with silica gel and germination (G%) of S. leprosula seeds. Data with the same superscript letter are not significantly different at p=0.05 based on Duncan Multiple Range Tests

Control Dried Target MC (%) Actual MC (%) G (%) Duration of Actual MC G (%) drying (%) Fresh 42.1 100.0a – – – 40 41.8 98.0a 30 min 39.9 100.0a 30 42.2 96.0a 4 h 34 min 31.6 84.0b 20 39.4 100.0a 17 h 50 min 20.9 0c 10 37.4 96.0a 26 h 45 min 11.7 0c 8 38.2 96.0a 31 h 15 min 8.8 0c 5 40.1 100.0a 32 h 57 min 4.3 0c

The moisture contents of the seeds in the control treatments were almost constant throughout the experiment and the seeds retained their germination capacity. After drying to 40% and 32% the percentages of germination recorded were 100% and 84%, respectively. However, no germination was recorded for seeds dried to 20% MC or below.

Storage trial The changes in moisture content and germination during storage at 16 and 25°C are shown in Figures 1 and 2. Germination gradually decreased to approximately 50% after 12–14 weeks, irrespective of the initial moisture content or storage temperature. After 12 weeks, there was a rapid decline in germination and all seeds were dead after 20 weeks of ASIA 203 storage at 25°C. However, 6% germination was recorded after 22 weeks of storage at 16°C for the lowest moisture content. Seed moisture contents also dropped after 12 weeks of storage. Pre-germination was a problem for the highest moisture content and temperature, but no data was recorded for pre-germination. The pre-sprouted seeds were excluded in the sampling for germination test, and all the tests were therefore, carried out only with the non-germinated seeds at the time of the experiment.

100 mc. 25C 80 Germ. 25C mc. 16C 60 Germ. 16C 40 20

Germination/MC (%) 0 0 4 8 12162024 Storage (weeks)

Figure 1. Germination capacity of S. leprosula seeds with an initial 42.34% MC during storage at 16°C and 25°C for 24 weeks. The moisture content varied over the storage period.

100 mc. 25C 80 Germ. 25C mc. 16C 60 Germ. 16C 40

20 Germination/seed

moisture content (%) 0 04812162024 Storage (weeks)

Figure 2. Germination capacity of S. leprosula seeds with an initial 31.67% MC during storage at 16°C and 25°C for 24 weeks. The moisture content slightly varied over the storage period. 204 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Discussion

The relationship between moisture content and germination capacity revealed that Shorea leprosula seeds did not germinate at or below 21% MC (Table 2). These seeds can therefore be categorized as recalcitrant seeds, and the critical moisture content lies between 20 and 30% MC. Tompsett and Kemp (1996) determined the lowest-safe moisture content at 26%. The sensitivity of S. leprosula to desiccation is typical of many recalcitrant seeds. 10% reduction in initial moisture content (42 to 32% MC) caused only 16% decrease in germination (100 to 84%). Song et al. (1983) reported that in recalcitrant seeds of Hopea hainanensis, clear injurious effects of desiccation was observed even at high moisture contents. King and Roberts (1980) suggested that seed death resulting from desiccation occurs at or below a critical moisture and is caused by membrane related physiological damages or an accumulation of by-products of biochemical enzymatic breakdown. Hanson (1984) postulated that in desiccation tolerant seeds, membrane permeability and structure remains intact during desiccation, while in the desiccation sensitive seeds some membrane dysfunction occur during desiccation. There was no significant effect of the storage temperature and initial moisture content on longevity. During storage, viability S. leprosula seeds gradually decreased to 50% half way, after 12–14 weeks (Figs. 1 and 2). These results were an improvement of the storage of S. leprosula seeds, as compared to previous report that seeds at 32% MC could be stored at 21°C, resulting in 45% germination after 30 days (Tompsett and Kemp 1996). However, fewer seeds pre-sprouted at the lowest moisture content and 16°C compared to 25°C. Fungal contamination was an additional problem observed at high storage temperature of 25°C.

Conclusion

Seeds of S. leprosula show recalcitrant behaviour, but retaine about 50% germination after 12–14 weeks at cool to warm storage temperatures. A reduction of 10% MC (from 40 to 30% MC) and storage at 16°C is recommended to limit pre-sprouting and for at least six months storage. ASIA 205

Acknowledgements

The authors thank the directora of the Forest Research Institute Malaysia (FRIM), IPGRI and DFSC for financial and technical support. Thanks are also due to FRIM’s seed collection team and the Seed Technology Laboratory staff.

References

Appanah, S. and H.T. Chan. 1981. Thrips: the pollinators of some dipterocarps. Malaysian Forester 44:234–252. Ashton, P.S., T.J. Givinish and S. Appanah. 1998. Staggered flowering in the Dipterocarpaceae: new insights into floral induction and evolution of mast fruiting in the aseasonal; tropics. Am. Nat. 132:44–66. Farrant, J.M., N.W. Pammenter and P. Berjak. 1988. Recalcitrance – a current assessment. Seed Sci. Technol. 16:155–166. Hanson, J. 1984. The storage of seeds of tropical tree fruits. Pp. 53–62 in Crop Genetic Resources: Conservation and Evaluation (J.H.W. Holden and J.T. Williams, eds.). Allen and Unwin, London. Hong, T.D. and R.H. Ellis. 1992. Optimum air-drying seed storage enviroments for Arabica coffee. Seed Sci. Technol. 20:547–560. IPGRI/DFSC. 1996. The project on handling and storage of recalcitrant and intermediate tropical forest tree seeds. Newsletter No. 1. July 1996. Pp. 5–20. King, M.W. and R.H. Roberts. 1980. Maintenance of recalcitrant seeds in storage. Pp. 53–89 in Recalcitrant Crop Seeds (H.F. Chin and E.H. Roberts, eds.). Tropical Press, Kuala Lumpur, Malaysia. Sasaki, S. 1980. Storage and germination of dipterocarp seeds. Malaysian Forester 43:290–308. Song, X., Q. Cheng, D. Wang and J. Yang. 1983. A study of ultrastructural change in radical-tip cells and seed vigour of Hopea and Vatica in loosing water process. Scientia Silvae Sinicae 19:121–125. Tompsett, P.B. 1987. Desiccation and storage studies on dipterocarp seeds. Ann. Appl. Biol. 110:371–379. Tompsett, P.B. and R. Kemp. 1996. DABATTS, Database of Tropical Tree Seeds Research with Special Reference to the Dipterocarpaceae, Meliaceae and Araucariaceae. Royal Botanic Gardens, Kew, Australia. Pp. 1–263. 206 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Handling and storage of four tropical forest tree seeds from Malaysia

Nadarajan Jayanthi, Baskaran D. Krishnapillay and Siti Hasanah Mat Said

Forest Research Institute Malaysia (FRIM), Kepong 52109, Kuala Lumpur, Malaysia

Abstract

Seeds of Neobalanocarpus heimii, Shorea assamica, Shorea macroptera and Shorea roxburghii were screened for their survival of desiccation and storage. There were differences in the tolerance to desiccation between the species. Comparing these species, their sensitivity to loss of moisture increased and the critical moisture contents become progressively higher. All the seeds at both their initial moisture content and dried to 5% MC less than this initial moisture content were stored at 16 and 25°C. There was no significant difference between the two temperatures in prolonging the storability. However, seeds stored at 16°C showed both less fungal contamination and percentage of sprouted seeds. The seeds could be stored for 16 to 18 weeks at these two temperatures with germination ranging from 17 to 50%. The results showed that these four species are recalcitrant. However, their critical moisture contents were relatively low compared to the two other species, i.e., Hopea odorata and Shorea leprosula, previously studied.

Introduction

Malaysia is endowed with a relatively large tract of rich and diverse tropical rainforests, which has been acknowledged to be among the most complex ecosystem in the world (Alang and Normah 1991). The total forested area in Malaysia as at the end of 1995 was estimated to be 18.91 million hectares or 57.5% of the total land area (Malaysian Forest Sector Review 1995). Of this total, it is estimated that some 16.41 million hectares are the inland dipterocarp forests, with the remaining 1.69, 0.62 and 0.19 million hectares being freshwater swamp, mangrove swamp and plantation forests, respectively. The inland dipterocarp forests could be further categorized into lowland dipterocarp, hill ASIA 207 dipterocarp, upper dipterocarp, lower montane and upper montane forests, respectively. While sustainable development and sustainable forest management have been the centre of attention to many international and regional initiatives, there are a bewildering number of definitions for sustainable forest management (FAO 1993; Krishnapillay et al. 1995). Importantly, Malaysia is committed to manage her forest in a sustainable manner not only for economic reasons but also encompasses the maintenance of environmental stability and ecological balance. Systematic and sustainable yield policies with regards to the management of her forests have been adopted. Other than the natural forests, Malaysia has also ventured into forest plantation, albeit on a small scale. Considering the manner in which forest plantations have developed so far, their current status may appear to be an eclectic mixture of species with different management objectives, sizes, species, locations and ownership. The tree plantations in Peninsular Malaysia can be divided into three principal types. The commercial establishment of forest plantation dates back to the 1950s with the planting of teak (Tectona grandis) in the northern states of Peninsular Malaysia, and sentang (Azadirachta excelsa) which is now in the same class as teak. The pine plantations were originally intended for producing pulp. The general utility timber plantations with fast growing species like Acacia mangium, Gmelina arborea, Paraserianthes falcataria being the major species and Eucalyptus deglupta, and Araucaria species are planted on a limited scale. Besides the three types of plantations, there are a few hundred species trials, many concentrated within FRIM, Kepong and some scattered throughout the country. The species include hardwoods, softwoods, indigenous and exotics from all over the tropics. The information contained in the plots is very valuable, for evaluating the potential of over 150 tree species. Inadequate supply of forest tree seeds is a serious problem, which is hampering enrichment planting efforts especially with indigenous species. Many of the reforestation plans were postponed due to lack of nursery-raised seedlings at the required times. As an alternative, wildings are often collected from primary forests and used in enrichment planting of logged-over areas. However, surveys by the Silviculture Unit of the Forest Research Institute of Malaysia (FRIM) indicated that the transplanted wildings suffer much higher rates of mortality than nursery-raised stock. Therefore, it becomes important to look into problems of seed supply, especially prediction of fruiting, 208 STORAGE BIOLOGY OF TROPICAL TREE SEEDS harvesting and storage of seeds. Efficient seed storage plays an important role in a good forest management system and in the past, different methods of storage have been proposed for tropical seeds but many were unsuccessful, as many seeds could not be stored for more than a few months. For such seeds, even small improvements in the maintenance of viability would mean a significant step forward for commercial production and supply of valuable tree seeds, as well as contribute to more effective conservation genetics. The major problems to be addressed are the lack of effective methods to maintain and use indigenous tropical tree species and seeds for forest management and plantation programmes.

Species for the study

The species selected for this study belong to the Dipterocarpaceae family, which make up the major component of the Malaysian rainforests. Problems with handling seeds of this family are a major constrains in cultivating these species. The four species were selected based on their high socioeconomic importance and their propagation mainly by seeds (PROSEA 1994). In addition, the species have several distinct seed sources within the country, and produce seeds yearly that readily germinate. Neobalanocarpus heimii (King), synonym: Balanocarpus heimii, is also known as ‘chengal, penak’ in Malaysia, ‘takhian-chan’ or ‘chi-ngamat’ in Thailand. The species occurs in evergreen or semi-evergreen forest on fertile clay soils on well-drained flat or hilly country up to 1000 m altitude. It is distributed throughout Peninsular Malaysia and in the most southern part of peninsular Thailand, where it may be extinct. The species is found in numerous protected areas in Malaysia, Singapore and Thailand. It produces a very durable and heavy timber and suitable for many heavy-duty purposes. It used to be the standard timber for durable heavy construction in Peninsular Malaysia. It is suitable for railway sleepers, piles, bridges, telegraph and power-line poles, flooring and joinery, also used for constructing wharves, ships and boats both in fresh and saltwater conditions. N. heimii is now a vulnerable species (IUCN 2002). It is a large tree that now only extends in Peninsular Malaysia. Populations in the southernmost part of Thailand and Singapore are believed to be extinct, thus the export of logs has been banned. ASIA 209

Shorea assamica Dyer. (synonyms: S. philippinensis Brandis, S. koordersii Brandis, S. globifera Ridley) is also known as ‘lemsa kulat’ or ‘meranti pipit’ in Malaysia, ‘damar mesegar’ in Indonesia, ‘saya- khao’ in Thailand and ‘danlig’ in Philippines. It is a large tree of up to 55-m tall with bole up to 150 cm in diameter. Its leaves are ovate, elliptical or rarely obovate. Fruits are pedicle of 2-mm long, larger fruit calyx lobes up to 11 cm × 2 cm. This species occurs in evergreen or semi-evergreen forest on fertile clay soils on well-drained flat or hilly country up to 1000 m altitude and is distributed in India, Peninsular Thailand, Peninsular Malaysia, Sumatra, Borneo, the Philippines, Sulawesi and southern Moluccas. Timber is the major source of white meranti. The resin, called ‘damar tenang’, was once collected on a commercial scale in North Sulawesi (PROSEA 1994). The tree is gregarious and occurs on slopes and along rivers in lowland evergreen forest. The Chinese population is confined to Yingjiang and is threatened by conversion of the forest to agriculture. In India, the species is known from healthy populations, which are regenerating well. The trees are cut for the commercially valuable plywood. Populations of the species are known to occur in some forest reserves. S. assamica is listed as a critically endangered species (IUCN 2002). Shorea macroptera Dyer. (synonyms: S. bailloni, S. sandakanensis) is also known as ‘meranti melantai’ in Brunei and Malyasia, ‘meranti kenuing’ in Indonesia or ‘chanhoi’ in Thailand. It is a medium sized to very large tree of up to 60-m tall with bole branchless for 18–27 m and up to 135 cm in diameter. The leaves are narrowly elliptical to oblong. Larger fruit calyx lobes are up to 12 cm × 2.3 cm. The species occurs in well-drained clay soils undulating land and hillsides up to 900 m altitude, and is distributed in Peninsular Thailand, Peninsular Malaysia, eastern Sumatra and Borneo. The timber is a valuable source of light-red meranti. The bark favoured locally for walls, floors and roofs. S. macroptera is listed as a critically endangedred species (IUCN 2002). Some populations of this tree occur in forest reserves. Shorea roxburghii (synonyms: S. talura, S. floribunda, S. cochin- chinensis) is also known under various local names in Malaysia, Myanmar, Cambodia, Laos, Thailand and Vietnam. It is a small to fairly large tree of up to 40-m tall, bole up to 95 cm in diameter. Larger fruit calyx lobes are up to 9 cm × 1.2 cm. It occurs in dry evergreen, deciduous or bamboo forest with a preference for sandy soils up to 1200 m altitude. The species is distributed in Eastern India, Myanmar, Thailand, Cambodia, Laos, Vietnam and Peninsular Malaysia. The timber is used as white meranti. A low quality resin is obtained from 210 STORAGE BIOLOGY OF TROPICAL TREE SEEDS the tree, which also produces a dye. S. roxburghii is a widespread dipterocarp, unusual for its adaptation to withstand adverse climatic conditions and soil types, but is now listed as endangered species (IUCN 2002).

Materials and methods

Seed collection and processing

All fruits were collected from adult trees (crown collection). S. roxburghii was received from Thailand three weeks after harvest (Table 1). Brown wings were indicative of mature fruits, and 10–20% of these fruits was infected by insects. Fruits were put in loosely tied or fold plastic bags and transported within 1 to 6 h (maximum) in a temperature-controlled van at 20°C. Freshly collected fruits were de- winged manually and mechanically damaged, infected and infested seeds were removed. Clean seeds were selected for different experiments and also dispatched to the replicating partners, as stated in the protocol (IPGRI/DFSC 1999).

Table 1. Species sources and collection dates

N. heimii S. assamica S. macroptera S. roxburghii Seed source Malaysia Malaysia Malaysia Thailand No. of trees 3 (1999); 4 (2001) 7 1 25 Collection date 23/8/1999; 5/12/2001 12/4/2000 3/1/2002 31/3– 1/4/2000 Reception in lab. 25/8/1999; 6/12/2001 13/4/2000 7/1/2002 24/4/2000 Processing date 26/8/1999; 6/12/2001 13/4/2000 8/1/2002 – Dispatch date 7/12/2001 14/4/2000 – – Initial test date 26/8/1999; 6/12/2001 15/4/2000 8/1/2002 25/4/2000

Initial testing

A range of trials was carried out on the selected species (Table 1) as specified in the original protocol (IPGRI/DFSC 1999). Initial germination tests were carried out using heat sterilised (130°C) sand as germination medium. Replicates of seeds were incubated at 28°C in a controlled room with 10 h light and 14 h dark. Germination assessments were carried out daily since all the seeds were readily germinating.

Desiccation and storage trials

The actual protocol for potentially desiccation intolerant seeds was followed (IPGRI/DFSC 1999). Seeds of each species at two moisture ASIA 211 contents, i.e., initial and 10% below initial were selected for storage at 16°C and 25°C for 20 weeks. Samples of these seeds were periodically removed for germination and moisture content determination during storage.

Results

Initial trials

All fruits were collected at t40% MC, which was also close to the whole seed moisture contents (Table 2). In all cases, the embryos had higher moisture content than the rest of the other tissues. All the seeds initially germinated t90%. However, seeds of S. roxburghii showed 4% of pre-sprouted seeds at arrival. This might have been caused by the three weeks delay between the harvest and the reception in the laboratory (see Table 2).

Desiccation and storage trials

Seed viability decreased with drying (Fig. 1). No seeds of S. roxburghii, N. heimii and S. assamica germinated at or below 7.3, 9.3 and 11.3% MC, respectively. However, seeds of S. macroptera germinated about 20% when dried to 13.2% MC (Fig. 1).

Table 2. Initial characteristics of seeds of the four species

N. heimii S. assamica S. macroptera S. roxburghii Seed source Lentang, Ulu Tranum Negeri Sembilan Sakaerat Pahang Forest, Pahang (Malaysia) Research (Malaysia) (Malaysia) Station (Thailand) Collection date 23/8/1999 12/4/2000 3/1/2002 31/3–1/4/2000 Arrival date – – – 24/4/2000 Fruit weight (g) – – 1.8 – Seed weight (g) – – 1.4 – MC whole fruit (%) 52.9 63.0 54.3 38.9 MC whole seeds (%) 52.9 57.0 53.9 38.0 MC seed coat (%) 31.5 31.2 51.4 21.7 MC embryo (%) 70.7 76.1 73.1 52.3 MC cotyledons (%) 55.1 45.2 52.7 46.6 MC processed seeds 51.7 47 54 – (%) Initial germination (%) 96 100 96 90 Pre-sprouted seeds – – – 4 (%) 212 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

N. heimii S. assamica 100 100 80 80 60 60 40 40 20 20

Germination (%) 0 Germination (%) 50.3 45 41.6 36.4 26.7 9.3 0 45.1 43.6 31.3 28.8 19.6 11.3 Moisture content (%) Moisture content (%)

S. macroptera S. roxburghii 100 100 80 80 60 60 40 40 20 20 Germination (%) 0 Germination (%) 0 34.4 29.4 26.1 23.1 18.6 13.2 39 31 25.4 21.2 9 7.3 Moisture content (%) Moisture content (%)

Figure 1. Germination of fresh seeds of the four studied species after desiccation to different moisture contents.

The germination of seeds started to decline within 10 weeks of storage (Table 3). The results of 20 weeks storage also showed that all viability was reduced to ca. 20% for all species at the two storage conditions (Table 3). There was no significant difference between the two temperatures in prolonging the storability. However, seeds stored at 16°C showed both less fungal contamination and percentage of sprouted seeds. The seeds could be stored for 16 to 18 weeks at these two temperatures, with germination ranging from 17 to 50% (Table 3).

Discussion

The viability of seeds of these four species, Neobalanocarpus heimii, Shorea assamica, Shorea macropreta and Shorea roxburghii, started to decrease at ca. 30% MC, and they were sensitive to desiccation down to about 9 to 15% MC. Hence these seeds could be classified as recalcitrant seeds. The viability of the seeds was extended when they were stored at 16°C compared to storage at 25°C. This may be due to slow fungal growth at 16°C, which allowed seeds to further extend ASIA 213 their longevity. The sensitivity of the seeds to slight desiccation is typical of many recalcitrant seeds that have been widely reported (Tompsett 1985). Injurious effects of desiccation were observed in the recalcitrant seeds of Hopea hainannensis, even at high moisture (Song et al. 1983). On desiccation to 26% MC, which severely reduces germination, various detrimental changes were observed in the ultrastructure. However, the level of moisture content below which seeds don’t germinate, varies depending on species.

Table 3. Germination (%) of seeds of the four studied species with different moisture contents (%) after storage at 16 and 25°C for 20 weeks Storage duration (weeks) 2 4 6 8 10 12 14 16 18 20 N. heimii 25°C, 45 % MC 93 80 70 60 53 50 40 37 27 17 16°C, 45 % MC 100 90 77 70 67 57 50 43 33 27 25°C, 40 % MC 93 87 83 77 64 57 40 30 20 0 16°C, 40 % MC 97 87 80 73 67 57 47 30 23 7

S. assamica 25°C, 47 % MC 94 90 92 82 78 62 54 28 0 0 16°C, 47 % MC 96 92 86 82 70 62 58 44 32 0 25°C, 42 % MC 92 84 80 78 76 66 58 28 0 0 16°C, 42 % MC 96 90 88 86 76 68 60 34 0 0

S. macroptera 25°C, 40 % MC 86 80 76 70 66 58 32 20 0 0 16°C, 40 % MC 88 88 74 72 68 64 60 48 28 14 25°C, 35 % MC 78 74 70 64 60 56 34 18 0 0 16°C, 35 % MC 82 80 76 70 66 62 56 50 24 18

S. roxburghii 25°C, 33 % MC 88 82 76 62 64 48 25 18 5 0 16°C,33 % MC 90 86 82 74 64 58 42 34 15 0 25°C, 28 % MC 86 84 80 82 78 62 58 44 28 12 16°C, 28 % MC 88 86 82 76 74 60 48 38 26 16

We were unable to test all the storage temperatures specified in the protocol due to seed limitation. Moreover, some existing knowledge indicates that dipterocarp species do not survive extreme temperatures. These four species behaved similarly to Hopea odorata seeds, which show optimum MC and storage temperatures of 32% and 15–20°C, respectively, retaining 70% viability after 3 months (Jayanthi 214 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

1999). King and Roberts (1980), reported that recalcitrant seeds can only be stored at relatively warm conditions, because chilling injury is a common phenomenon. The embryonic axes have higher water content and were readily injured at lower temperatures resulting in poor germination or death. The onset of such chilling stress may vary considerably between species (Yap 1981). Temperatures below 10–15°C were lethal for all dipterocarp seeds evaluated by Maury-Lechon et al. (1981). One interesting finding from this study was the seeds stored together with vermiculite retain their moisture content at a higher level for a longer period. Therefore, the longevity was extended. Though some seeds were sprouted in the vermiculite, most of the seeds were vigourous even after 4 months of storage. This method has been adopted at FRIM, to dispatch recalcitrant seeds to various countries, and the seeds were received in good condition. Storage of dipterocarp seeds in such media has been carried out by various researchers to maintain high moisture content for prolonging of the seeds during storage (Tang 1971; Tompsett 1989). During storage the seeds were well mixed and regularly ventilated to allow a good aeration. Studies on Shorea curtisii and Shorea ovalis showed that regular ventilation prolonged the viability from 5 to 92 days (Sasaki 1980). Song et al. (1984) showed that Hopea haimensis seeds were able to germinate 80% after up to 365 days by maintaining oxygen level above 10%.

Benefits from the project

This project has helped in understanding our tropical rainforest seeds and how to handle them, and was a good opportunity for training in planning seed collections. The collaboration work with replicating partners was an opportunity to discuss the problems and exchange ideas especially on the same species. It also ensured a sustained commitment and high motivation among participants and consistent cooperative efforts. The project newsletter has been a good mechanism for disseminating the relevant information between participants to the project, and an encouragement especially for young scientist to publish our findings in the form of progress report, notes and articles. This has encouraged and gave us the confidence to write scientific papers in journals. This project overall has furthered our knowledge on storage physiology of many tropical tree seeds and has promoted the wider use of these species in forest management and plantation programmes. ASIA 215

Future activities

From our experience, the maximum period of about a year can be achieved using conventional storage methods for truly recalcitrant seeds. Although these methods are inadequate to ensure continued supplies of planting materials through the periods when the mother trees are not fruiting and especially when fruiting occurs once in every three to 6 years. To enhance the wider usage and better conservation of these species further research on alternative techniques is needed, including micro-propagation, cryopreservation, biochemical inhibition of the metabolic pathways and storage of seedling instead of seeds of recalcitrant and intermediate species. Furthermore, some of these studies are already ongoing at FRIM. It is hoped that these new approaches may yield some useful methods for the prolonged storage and usage of tropical seeds.

Acknowledgements

We thank DANIDA and IPGRI for providing funding, materials and training to carry-out this project. Thanks also go to the seed collection team in FRIM and all the Seed Technology Laboratory staffs, who helped in the collection, transportation and processing of the seeds.

References

Alang, Z.C. and M.N. Normah. 1991. In vitro techniques for conservation and utilization of underexploited tropical species. Pp. 155–169 in Conservation of Plant Genetic Resources through in vitro Methods. ISBN 967–99915–2–0. C, FRIM/MNCPGR. FAO. 1993. Forest Resources Assessment 1990. Tropical Countries. FAO Forest Paper 112. Rome, Italy. IPGRI/DFSC. 1999. The project on handling and storage of recalcitrant and intermediate tropical forest tree seeds. Danida Forest Seed Centre.Newsletter No. 5:23–39. IUCN. 2002. 2002 IUCN Red List of Threatened Species [also at http://www.redlist.org]. Jayanthi, N. 1999. The Effects of Moisture Content and Temperature on the Storability of an Orthodox, Intermediate and Recalcitrant Forest Tree Species. M.Agric.Sc. Thesis. UPM. King, M.W. and E.H. Roberts. 1980. Maintenance of recalcitrant seeds in storage. Pp. 53–89 in Recalcitrant Crop Seeds (H.F. Chin and E.H. Roberts, eds.). Tropical Press, Kuala Lumpur, Malaysia. 216 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Krishnapillay, B., C. Schroeder and M. Marzalina. 1995. Towards the setting up of a forest tree seed center in Malaysia: possibilities and constraints. Proceedings of the IUFRO Symposium of the Project Group P.2.040.00 ‘Seed problems’. Arusha, Tanzania. Malaysian Forest Sector Review. 1995. Kuala Lumpur, Malaysis. Pp. 10. Maury–Lechon, G., A.M. Hassan and D.R. Bravo. 1981. Seed storage of Shorea parvifolia and Dipterocarpus humeratus. Malaysian Forester 44:267–280. PROSEA No 5 (1). 1994. Plant Resources of South East Asia. Timber Trees: Major Commercial Timbers (I. Soerianegara and R.H.M.J. Lemmens, eds.). Backhuys Publishers, Leiden, Netherlands. Sasaki, S. 1980. Storage and germination of dipterocarp seeds. Malaysian Forester 43:290–308. Song, X., Q. Chen, D. Wang, and J. Yang. 1983. A study of ultrastructural changes in radical-tip cells and seed vigor of Hopea and Vatica in loosing water process. Scientia Silvae Sinicae 19:121–125. Song, X., Q. Chen, D. Wang, and J. Yang. 1984. A study on the principal storage indications of Hopea hainanensis seeds. Scientia Silvae Sinirae 20:225–236 Tang H.T. 1971. Preliminary tests on the storage and collection of some Shorea seeds. Malaysian Forester 34:84–98. Tompsett, P.B. 1985. The influence of moisture content and temperature on the viability of Shorea robusta and S. roxburghii seeds. Can. J. Forest Res. 15:1074–1079. Tompsett, P.B. 1989. A review of the literature on storage of Dipterocarp seeds. Pp. 427–446 in Proceedings of the Fourth Round Table Conference on Dipterocarps (I. Soerianegara, S.S. Tjitrosomo, R.C. Umaly and I. Umboh, eds.). Southeast Asian Regional Centre for Tropical Biology, Bogor, Indonesia. Yap, S.K. 1981. Collection, germination and storage of Dipterocarp seeds. Malaysian Forester 44:281–300. ASIA 217

Neem (Azadirachta indica A. Juss. var. siamensis Valeton) seed development in relation to desiccation tolerance and storage properties

S. Saelim, P. Pukittayacamee and J. Bhodthipuks

ASEAN Forest Tree Seed Centre, Muak-Lek, Saraburi 10180, Thailand

Abstract

The development of fruits and seeds of neem (Azadirachta indica A. Juss. var. siamensis Veleton) in relation to desiccation tolerance and storage potential was examined in this study. The number of fruits formed per flowers on racemes varied between trees. Fruits collected at different developmental stages from Wat Haopang and Kangkoi, Thailand, were processed and dried over silica gel desiccant. Mean seed dry weight increased during seed development; being 12.2, 18.2, 14.7, 19.5 and 23.8 mg for fruits at 5, 6, 7, 8 and 9 weeks of flower opening, respectively. Seed moisture content declined during seed development; for seeds from fruits harvested at the same stages as above, seed moisture content was 78.4, 72.8, 63.9, 58.4 and 52.4%, respectively. Seeds from fruits of different ages exhibited different levels of desiccation tolerance. At similar moisture contents, seeds from yellow fruits had a higher germination capacity than those from green fruits. The highest germination after four months storage at 20qC with 28–30% MC was obtained for seeds harvested at 9 weeks or more after flowering when the fruits were yellow-green with a firm mesocarp.

Introduction

The neem tree has multiple uses throughout its tropical distribution with many useful products derived from nearly every part of the plant. The extracts from its bark, roots, leaves and seeds are used for their insecticidal properties and for medicine. Neem wood is used for making furniture, tools and in general construction. Branches and twigs are good sources of fuelwood and young leaves or flowers are used for food and medicine. Neem is considered as a priority species for community and social forest plantings including along roadsides, farm land boundaries and in home gardens. However, low seed viability and problems with storage of seeds for long periods (Ezumah 218 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

1986; Chaisurisri et al. 1986; Maithani et al. 1989; Ponnuswamy et al. 1990; Pukittayacamee et al. 1995) are major constraints limiting the conservation, propagation and genetic improvement of this species. The storage characteristics of seeds from both varieties, the Thai neem, Azadirachta indica A. Juss. var. siamensis Veloton, and the Indian neem, Azadirachta indica A. Juss., have recently been variously described as either intermediate (Sacandé et al. 1996; Pukittayacamee 1997; Omondi 1997) or tending towards the orthodox (Bellefontaine and Audinet 1993; Pritchard and Daws 1997). However, the germination capacity is reduced when the moisture content is below 10%, and the prognosis for storage is still uncertain for most seed lots. Sensitivity to low temperature storage has also been reported in the Indian neem, which seems to store better than the Thai neem. Because the maturation of seed may play an important role in desiccation tolerance and the storage properties of neem, a study of seed maturation is required, particularly for the Thai neem, since this has little been investigated. Moreover, the information of the storage behaviour of the seeds during seed development is required. The aims of this study were therefore to monitor seed development, to determine the desiccation tolerance of seeds at different stages of maturation, and to investigate their storage longevity after desiccation to different levels of moisture content.

Materials and methods

Flower and fruit development

Neem trees were selected from a 10- to 15-year-old plantation located at Wat Hoapang, Muak-Lek, Saraburi, Thailand (14°35’ N, 101°35’ E, 240 m above sea level). Initial tree spacing was 4 m × 4 m but this was later altered to accommodate a maize field. Five branches of each selected tree were marked with plastic tags. They were regularly observed and phenological changes from flower buttons through to fruit formation and complete development of each branch were recorded weekly starting on 15 Jan 1998. Because of their small size, it was difficult to tag individual flowers, which is why fruit development was observed for whole branches rather than for individual flowers. The flowers were counted when flower buds were fully developed and phenological changes of the inflorescences described and classified as stages 1 to 7 as follows: ASIA 219

(1) Green flower buds (2) A few flowers open (3) More flowers open (less than 50%) (4) t50% flowers open (5) Flowers wilting, a few young fruits visible (6) Visible young fruits on <50% of the branch (7) No flowers left, >50% of young fruits visible

The first visible fruits were tied with a different coloured thread and the number of set fruits on each branch was recorded. The age of seeds was estimated from the day when flower blooming peaked. The number of fruits was counted from the fourth week of formation onward.

Fruit maturation and seed development

Two replicates of five marked fruits from each tree were collected weekly from 5 weeks old until the time of shedding for determination of seed water content and dry weight. The ratio of embryonic axis length to fruit length was also calculated for 25 fruits from each tree. After measurement, embryonic axes were sown for germination. Further samples of seeds from each tree were taken at random every 3 days. Two replicates of five seeds each were used for moisture content determination; 25 whole seeds per tree were sown for germination; embryonic axes were also isolated and tested for germination. For all moisture content determinations, seeds (including endocarp) were cut in half and dried in an oven at 103q& for 17 h (ISTA 1999). Moisture contents are expressed on a percentage fresh weight basis. For germination of whole seeds, seeds were sown in sand at room temperature with 8 h fluorescent light and 16 h dark per day. Isolated embryonic axes were placed for germination at 30q& (8 h light, 16 h dark) on wet filter paper in Petri dishes. Embryonic axes were recorded as germinated when their radicle had elongated to double their original length.

Desiccation and storage

Seeds from six trees were collected weekly from 5 weeks old until the fruits were shed. Initial seed moisture content and germination were determined as before, immediately after collection. Green fruits were left in collection 220 STORAGE BIOLOGY OF TROPICAL TREE SEEDS containers for a few days to soften the flesh before processing. The fruits were de-pulped using a Dybvig Seed Cleanser (Amata–archachai and Wasuwanich 1986) and left at room temperature until the surface was dry. Dry seeds were placed in a plastic box on top of indicator silica gel that was replaced when the colour turned pink, i.e., humidified. Seeds desiccated to approximately 30, 20 and 10% MC were stored at 20°C and 10°C in transparent plastic bags (10 cm×20 cm). The stored seeds were randomly sampled monthly and tested for germination (four replicates of five seeds for each tree and storage moisture content).

Results

Flower and fruit development

With the opening of neem flower buds, the compound-leaves and an inflorescence that is arranged as a panicle with more than 10 secondary peduncles per stalk is revealed. The number of inflorescences for each developing bud ranged from 0 to 7 and the length of the inflorescences that developed from a single bud ranged from 10 to 30 cm. Flower bloom occurred 20 to 27 days after buds burst and, following anthesis, fruits would develop and become apparent within 3–5 days. Subsequent full development of all young fruits from the same branch was complete within 7–12 days. Although flower opening and fruit set varied within the same inflorescence, the overall age (mean) of fruit in the same tree may be roughly estimated starting from the day of peak flower opening on each tree. Fruit development on most of the trees in this study took only 1 week to progress from stage 2 to stage 6. The percentage of fruit-set ranged from 2 to 4% for each individual tree (Fig. 1). During the development stages, dry weight of seeds increased gradually and stabilized after the seeds were 8 weeks old (Fig. 2). In contrast, moisture content gradually declined as the seeds matured, from 78.4% at 5 weeks down to 52.4% at 9 weeks (Fig. 2). Fruit length increased sharply after 7 weeks (Table 1). By 9 weeks after anthesis, the embryonic axes were fully mature (100% germination). The length ratio of embryonic axes to fruits decreased between 7 and 9 weeks after anthesis indicating that the embryos developed after the fruits had reached full size. ASIA 221

1 No. of fruits/No. of flowers (%) flowers of fruits/No. No. of

0 12345

Tree No.

Figure 1. Average quantity of fruits set per number of flowers on neem trees expressed as a percentage. The results of five trees are presented.

Desiccation trials

For each of the 6 studied trees, similar trends in seed moisture content and germination with desiccation were observed for seeds harvested at 7, 8, 9, and/or 10 weeks after anthesis (Fig. 3). The germination percentage of 7 and 8 weeks old seeds increased after drying for a few days while the germination percentage of 9 and 10 weeks old seeds gradually declined from initial germination percentages. The critical moisture contents (where there was a sharp decline in germination) of seeds from different stages of development were ca. 40% for 7 weeks old seeds and ca. 20% for 8, 9, and 10 weeks old seeds. Overall, seeds from 8-and 9-week-old fruits from each tree had higher germination percentages than seeds from 7 and 10-week-old fruits during the desiccation trial. The moisture content of 10- week-old seeds dropped below 15% within only 20 days and the viability also declined. There was a quadratic relationship between seed moisture content and germination for seeds harvested at different times during development (Fig. 4). Desiccation tolerance increased during seed development; the moisture content at which viability fell to 50% progressively decreased from 28% at 7 weeks down to 15% at 10 weeks. Furthermore, the maximum germination achieved during development increased and the moisture content at which the maximum occurred decreased. For example, maximum germination for seeds from 7-week-old fruit was ca. 222 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

70% at about 50% moisture content which compares with a maximum germination of ca. 85% at about 32% moisture content for seeds from 10- week-old fruit.

100 30

Tree No. 1 25 80

20 60 15 40 10

20 5

0 0 04 6 8 10 100 30

Tree No. 5 25 80

20 60 15 40

10 weightDry (mg) Moisture content (%) 20 5

0 0 04 6 8 10 100 30

Average of 5 trees 25 80

20 60 15 40 10

20 5

0 0 046810

Weeks after flower opening Figure 2. Moisture content and dry weight changes during development of neem seeds. Data for two individual trees and the means of five trees are presented. ASIA 223

100 100 Tree No. 6 Tree No. 6 80 80 7 weeks 8 weeks 60 60 9 weeks 10 weeks 40 40

20 20

0 0 100 100 Tree No. 7 Tree No. 7 80 80

60 60

40 40

20 20 Germination (%) Germination

0 0 Moisture(%) Content 100 100 Tree No. 8 Tree No. 8 80 80

60 60

40 40

20 20

0 0 100 100 Tree No. 9 Tree No. 9 80 80

60 60

40 40

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0 0 Moisture(%) Content 100 100 Tree No. 10 Tree No. 10 80 80

60 60

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20 20

0 0 100 100 Tree No. 11 Tree No. 11 80 80

60 60

40 40

20 20 Germination (%) Germination

0 0 Moisture Content (%) 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Drying period (days)

Figure 3. Germination capacity of neem seeds, harvested at 7, 8, 9, and 10 weeks after drying to different moisture contents. 224 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

100 7-week-old seeds 8-week-old seeds 100 2 2 Y= -14.272 + 3.1124X - 0.0296 X Y= -28.535 + 4.6280X - 0.0483 X 80 2 2 80 R = 0.588 R = 0.783

60 60

40 40

20 20

0 0 100 9-week-old seeds 10-week-old seeds 100 2 2 Y= -22.090 + 4.8526X - 0.0542 X Y= -18.272 + 5.9451X - 0.0858 X 2 2 80 R = 0.781 R = 0.615 80 Germination (%) Germination Germination (%) Germination 60 60

40 40

20 20

0 0 0 1020304050607080900 102030405060708090

Moisture content (%)

Figure 4. Correlation between decreasing moisture contents and germination capacity of neem seeds from different developmental stages between 7 and 10 weeks.

Storage performance

In every test, seeds that were stored at 10°C did not survive regardless of moisture content (data not shown). The best storage conditions for the different stages of seed maturation are summarised in Table 2. Seeds from 9-week-old fruits could be stored at 20°C for up to three months at 30–35% moisture content and seeds from 10-week-old fruits could be stored for 2 months at ca. 25% moisture content. However, seeds from the other stages of development had generally lost viability within 1 month. Storage results for seeds that were collected from a natural stand (Kangkoi source) at the end of the season are summarised in Table 3. Green fruits could be stored for 4 months and possibly longer with 28% initial moisture content. However, yellow fruits could not be stored for longer than 3 months with the same moisture content. Both green and yellow fruits could not be stored when they were dried to less than 10% moisture content. In conclusion, the storage of neem fruit ASIA 225 was affected by the stage of fruit maturation and by seed moisture content. Fruits with yellowish-green colour (9 weeks old) had better storage properties than fruits of other ages.

Table 2. Germination of neem seeds from different developmental stages desiccated for different periods to moisture contents as shown and then stored at 20°C for up to 4 months

Tree Age Drying Moisture Germination after storage No. (weeks) period content (%) (days) 0 1 2 3 months 4 months months months 7 8 21 18.21 30 0 0 0 0 9 15 31.88 80 75 20 5 0 26 12.75 4 0 0 0 0 10 18 23.19 68 52 5 0 0 25 8.92 10 0 0 0 0 11 11 28.64 72 100 65 0 –

8 8 21 34.85 30 45 0 – – 9 15 34.85 60 35 65 45 – 26 20.09 32 5 0 0 0 10 18 23.36 32 31 10 10 0 25 12.61 10 3 0 0 – 28 7.58 5 0 0 0 – 9 9 15 37.29 64 – 40 17 – 26 19.91 0 0 0 0 0 10 18 28.41 80 33 13 0 0 25 11.58 10 3 0 0 – 28 9.8 10 0 0 – – 10 8 21 32.13 30 25 20 0 – 9 15 68 0 10 0 – 26 37.19 24 15 7 0 0 10 18 16.95 80 60 8 3 0 25 22.67 60 14 0 0 – 28 9.98 20 0 0 – – 15.69 12 9 26 10.24 16 0 0 – 18 26.76 80 50 0 0 10 25 10.17 20 3 0 – 28 10.37 15 5 – – 226 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Table 3. Germination capacity of neem seeds (Kangkoi source) from different fruit colours desiccated to different moisture contents and then stored at 20°C for 4 months

Tree Fruit colour Drying (days) MC (%) After months of storage 0 1 2 3 4 1 Green 10 28.60 36 85 37 44 23 20 14.20 12 0 0 0 – Yellow 10 27.16 56 70 65 45 0 20 14.20 8 25 0 0 – 2 Green 10 29.29 64 80 45 17 30 20 15.61 12 0 0 0 – Yellow 10 26.92 84 – – 18 0 20 5.98 8 5 0 0 – 3 Green 10 27.39 64 90 45 25 0 20 11.75 8 5 0 0 – Yellow 10 27.99 80 – 22 0 0 20 11.38 68 75 0 0 0 4 Yellow 10 21.98 80 71 10 0 0 20 8.00 4 0 0 0 0 Brown (bulk 10 22.39 76 40 30 5 5 seeds) 20 8.23 28 0 0 0 0

Discussion

The degree of fruit maturation varies greatly within the same inflorescence of Thai neem (Ponoy et al. 1997). A period of three weeks was required for all of the flowers in the same inflorescence to develop into fruits. In this study we observed variations in the time required for fruit development among trees. In some trees, only 2 weeks was required for all flowers to develop into small fruits. The time required for fruit development may be affected by many factors such as the health of the mother tree, environmental stresses, pollinators and the tree’s genetic make-up. In this study we observed that trees which flowered earlier in the season appeared to have slower fruit development, though this observation is only preliminary. Some marked fruits were attacked by insects and as a result ripened earlier than expected, turning yellow and becoming soft within only 6 weeks of anthesis. Seeds from these fruits also had lower germination percentages compared with seeds from green fruits of the same chronological age. The level of seed maturation, the effects of desiccation, and the storage properties of neem are normally estimated by the colour of fruits. Some studies reported that for Indian neem, yellow fruits stored better than green fruits when initial germination percentage was the same (Elteraify 1995; Sacandé et al. 1996). In contrast, our study showed ASIA 227 that Thai neem fruits from both sources (Muak Lek and Kangkoi sources) with green (or yellowish-green) colour stored better than yellow fruits, similar to that reported by La-Ongpant (1996). In this study, green fruits were left in containers for a few days to make depulping using the dybvig machine easier and to allow ripening , possibly affecting the storage of seed from green fruits. Another consideration that should be taken into account is how green the neem fruit is at harvest. As mentioned earlier, neem fruit development is variable within the same inflorescence. With current neem harvesting practices green and yellow, young and mature fruits are mixed together. We suggest that only mature fruits with light green or yellowish-green colour and firm flesh be selected for harvest. As the age of the fruits increased, seed storage properties improved (Maithani et al. 1989) as long as they were not over-matured. Colour of neem fruits can normally be used as a simple tool to estimate the level of seed maturation. In this study, we showed that moisture content of seeds could be used as a better indicator of fruit maturation. Similar results were also obtained by La-Ongpant (1996). However, more study is needed using fruit from other seed sources to evaluate the use of moisture content as an indicator of fruit maturation. Indian neem has recently been variously classified as having orthodox or intermediate seed storage physiology (Hegde 1993; Lauridsen and Souvannavong 1993; Sacandé et al. 1996). Thai neem appears to behave closer to the intermediate category than orthodox category since the seeds are sensitive to low temperatures (susceptible to chilling and freezing injury) and the seeds remained viable after the moisture content was dropped to 10–15%. Thai neem remains difficult to store for long periods with low moisture content.

Conclusions

We conclude that the developmental stage of neem fruit has an affect on desiccation tolerance of the seeds. At the same moisture content, the germination percentage was different when seeds were collected from fruits at different developmental stages. Neem fruit should be collected between 8 and 9 weeks after flower opening or when the seed moisture content is approximately 50–55%. In practice, we recommend that neem fruit be collected when it turns yellowish-green in colour and has firm mesocarps. Best storage conditions were obtained using 9-week-old seeds with 30–35% moisture content and storage at 20°C. Under these conditions, seeds could be stored for 3–4 months. 228 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Acknowledgements

The authors are grateful to the IPGRI and DFSC for their kind cooperation and DANIDA for funding of this project. We also thank Sacandé et al. for providing protocols and guidelines and Dr Thomsen for her suggestions. Finally, we express our thanks to all of our staff at the Seed Technology Section at ASEAN Forest Tree Seed Centre, for their technical help throughout this project.

References

Amata–archachai, P. and P. Wasuwanich. 1986. Mechanical extraction and cleaning of nuts of some tropical species. Embryon 2:1–8. Bellefontaine, R. and M. Audinet. 1993. La conservation de graines de neem. In Les Problèmes de Semences Forestières Notamment en Afrique IUFRO Symposium (L.M. Somé and M. de Kam, eds.). Ouagadougou, Burkina Faso, November 1992. Chaisurisri, K., B. Ponoy and P. Wasuwanich. 1986. Storage of Azadirachta indica A.Juss. seeds. Embryon 2(1):19–27. Elteraify, I.E. 1995. Studies on Seed Conservation and Propagation of the Neem Tree (Azadirachta indica A. Juss) in Sudan. Abstract of MSc Thesis. Ezumah, B.S. 1986. Germination and storage of neem (Azadirachta indica A. Juss.) seed. Seed Sci. Technol. 14:593–600. Hegde, N.G. 1993. Neem production and the small farmer. Pp. 85–94 in Genetic Improvement of Neem: Strategies for the Future (M.D. Read and J.H. French, eds.). Winrock International Forestry/Fuel wood Research and Development Project, Kasetsart University, Thailand. ISTA. 1999. International Rules for Seed Testing Rules 1999. International Seed Testing Association, Zurich, Switzerland. Pp. 333. La-Ongpant, S. 1996. Study on the Physiological Maturity and Seed Storage of Neem (Azadirachta indica var. siamensis Valleton). Thesis, Kasetsart University, Bangkok, Thailand. Lauridsen, E.B. and S. Souvannavong. 1993. Neem improvement in Bangladesh. Genetic improvement of neem: strategies for the future. Proceedings of International Consultation on Neem Improvement. Bangkok, Thailand, 18–22 January 1993. Maithani, G.P., M.M.S. Rawat, O.P. Sood and V.K. Bahuguna. 1989. Fruit maturity and interrelated effects of temperature and container on longevity of neem (Azadirachta indica ) seeds. Indian Forester 115:89–97. Omondi, W. 1997. The project on handling and storage of recalcitrant and intermediate tropical forest tree seeds. DFSC/IPGRI Newsletter No. 2, March 1997. Pp. 4. Ponnuswamy, A.S., R.S. Vinaya Rai, C. Surendran and T.V. Karivaratharaju. 1990. Studies on maintaining seed longevity and the effect of fruit grades in neem (Azadirachta indica). J. Trop. Forest Sci. 3:285–290. ASIA 229

Ponoy, B., N. Semsuntud and P. Kuerkool. 1997. Variation in stages of maturation and germination of seeds in the same inflorescence of neem (Azadirachta indica var siamensis). Technical Notes, Silviculture Research Division, Forest Research Office, RFD, Bangkok, Thailand. Pp. 8. Pritchard, H.W. and M. Daws. 1997. The project on handling and storage of recalcitrant and intermediate tropical forest tree seeds. DFSC/IPGRI Newsletter No. 2, March 1997:4–5. Pukittayacamee, P. 1997. The project on handling and storage of recalcitrant and intermediate tropical forest tree seeds. DFSC/IPGRI Newsletter, October 1997:5. Pukittayacamee, P., B. Boontawee, P. Wasuwanich and P. Boonarutee. 1995. Effects of Fruit Maturity, Depulping Techniques, and Drying Conditions on Germination of Azadirachta indica var. siamensis Seed. ASEAN Canada Forest Tree Seed Centre Project. Muak-Lek, Saraburi, Thailand. Sacandé , M., J.G. van Pijlen, C.H.R. de Vos, F.A. Hoekstra, R.J. Bino and S.P.C. Groot. 1996. Intermediate storage behaviour of neem tree (Azadirachta indica) seeds from Burkina Faso. Pp. 14–22 in Proceedings of a Workshop on Improved Methods for Handling and Storage of Intermediate/Recalcitrant Tropical Forest Tree Seeds (A.S. Ouedraogo, K. Poulsen and F. Stubsgaard, eds.). Humlebaek, Denmark, 8–10 June 1995 . 230 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Desiccation and storage trial of Shorea henryana Pierre seeds

Suomal Lait, Komsan Ruengritsarakul, Jutitep Bhodthipuks and Kowit Chaisurisri

ASEAN Forest Tree Seed Centre, Muak-Lek, Sarabur i 18180, Thailand

Abstract

Germination capacity after desiccation and storage was assessed using Shorea henryana seeds collected from mixed deciduous forest at Khao Ang Rue Nai Wildlife Sanctuary, Chachoengsao Province. At ambient temperature, seeds equilibrated at 11% MC after two weeks. Seeds dried to 6% MC using silica gel maintained initial viability. After 3 months storage, seeds with 15% initial moisture content were nonviable regardless of storage conditions. However, seeds with 6% initial MC sealed in plastic bags could be stored for up to 12 months with only a 20–25% reduction of germination percentage when stored at 5 and 15°C. Seeds could be stored successfully in sealed plastic bags at ambient temperature. Results of desiccation tolerance and storability studies suggest that these seeds are likely to be orthodox.

Introduction

Shorea henryana Pierre (synonym Shorea sericeiflora Fisch. & Hutch.) is a medium to large size tree, generally about 20–40 meters tall. Trees are distributed throughout the dry evergreen forests of southeast Thailand (Phengklai and Niyomdham 1999). Wood from this tree is very hard and is used in general construction, boat building and other outdoor applications. Locally, this species is referred to as Keim–ka-nong and its trade name is white meranti or phayom. Because they are believed to be recalcitrant, seeds are sown immediately after harvest to produce normal seedlings. However, the actual characteristics of these seeds have not been studied extensively. Here we have investigated the seed desiccation sensitivity and storage behaviour of S. henryana. ASIA 231

Materials and methods

Seed collection and extraction

Seeds were collected from Khao Ang Ruenai Wildlife Sanctuary located at latitude 13q24c53s N and longitude 101q56c33s E in Sanam Chaikhet District, Chachoengsoa Province, at an altitude of 180–200 m central Thailand. Trees were distributed in small groups in disturbed dry evergreen forest mixed with Lagerstroemia spp., Afzelia xylocarpa, Peltophorum dasyrachis and Anisoptera costata. Most trees were adult trees of more than 60 years old and 25–35-m tall. Fruits were collected between 11 Apr and 17 Apr 2001, immediately after branches were cut from trees. Fruits that had brown colour on at least two-thirds of their wings were selected from 22 trees. On average 1–3 kg of fruits per tree were collected. Nylon net bags were used during seed collection and transportation. One hundred fruits per tree were packed separately during the collection for comparison of their moisture loss during collection and transportation. Dewing fruits including pericarp and calyx as shown in Figure 1 were used in storage experiments. Seeds were soaked in a 1% solution of sodium hypochlorite (NaOCl) for 10 minutes and wiped dry. Seeds were gently mixed in a cement mixer for uniformity before experiments.

Initial moisture content and germination tests

For moisture content, 5 replicates of 5 seeds each were used chopped into small pieces, weighed before and after they were oven-dried at 103 r 1°C for 17 h. The moisture content was calculated on a fresh weight basis. Germination tests were carried out at a constant temperature of 30°C with an 8 h photoperiod using white fluorescent light. Seeds were sown in moistened sand in plastic germination boxes. Four replicates of 25 seeds each were used in this study. The testing period did not exceed 30 days. Germinated seeds as completely developed seedlings were counted and removed every two to three days until 30 days of complete germination. Abnormal seedlings were left in the boxes until the end of the testing period. 232 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Desiccation and storage of seeds

Seeds were desiccated under three different conditions: 1) On top of vermiculite in germination boxes maintained at 15°C as control samples; 2) Mixed in germination boxes with moisture-indicating silica gel that was replaced when necessary; 3) At room temperature in a mono layer. Seeds dried to 15% MC and 6% MC were sealed in plastic bags with or without ventilation holes, and stored at five different temperatures of –10, 5, 10 and 15°C, and at ambient temperature of 23–31°C. Seeds were randomly sampled to test for moisture content and germination capacity after 3, 6, and 12 months.

Results and discussion

Seed characteristics

S. henryana fruits consisted of 3 long wings (8.7 r 1.04 cm) and 2 short wings (5.6 r 1.02 cm). Wings were light green to white when young and changed to brown as they matured. Mean fruit weight was 1.30 r 0.23 g. On average, mature seed dimensions were 1.23 r 0.01-cm long and 2.10 r 0.2-cm wide. Initially seeds had 18.62 r 3.26% MC after collection. The seeds lost 3–4% MC during transportation in nylon net bags as compared with transport in plastic bags. Germination percentages were similar for seeds kept in both plastic and nylon bag containers. However, the nylon bags allowed better ventilation of fruits during transport and likely reduced fermentation by heating of seeds. The embryonic axis had 25.6 r 5.29%, the highest moisture content (Table 1), while the moisture content of cotyledons was about 16.30 r 4.57%. The driest component was the wings with 10% MC (Table 1). Seeds started to germinate 5 days after sowing. However, the first count was delayed until 12–14 days to allow normal seedlings development to be assessed. The germination was epigeal type with the cotyledons emerging above the ground level. Germination was completed within 21 days. ASIA 233

Table 1. Mean fresh weights and moisture contents (r se) of fruit and seed components of S. henryana measured immediately after harvest

Parts Fresh weight (g) Moisture content (%) Wing 0.26 r 0.07 10.22 r 1.37 Calyx 0.33 r 0.10 14.26 r 2.18 Pericarp 0.11 r 0.03 12.80 r 2.72 Cotyledon and seed coat 0.55 r 0.11 16.30 r 5.25 Embryonic axis 0.01 r 0.001 25.63 r 5.29 Storage tissue (dewing fruit) 1.00 r 0.19 15.40 r 3.89

Desiccation trials

It was interesting and exceptional that S. henryana species was able to tolerate desiccation to low moisture contents without losing viability. Seeds of other Shorea species such as S. siamensis, S. roxburghii, S. leprosula have been reported, however, to be recalcitrant (Phukittayacamee et al. 1994; Panochit et al. 1986, 1984; Purohit et al. 1982). The low moisture content toleration of S. henryana seeds is similar to those of a number of legume and Dipterocarp seeds, including Dipterocarpus alatus and D. intricatus that are classified as orthodox (Tompsett 1987). Cotyledons and embryonic axes were milky white at harvest (Fig. 1), which would be indicative of maturity of seeds, says close to shedding and thus tolerant to desiccation stress. Seeds from this species had only 19% MC at maturity and at harvest were low compared to MCs of other Shorea species, which normally contained 40–60% initial MC (Corbineau and Come 1986, Sasaki 1980, Yap 1981). Fresh seeds were dried to different moisture contents and their germination responses tested. Seeds with initial 15% MC dried to 11% MC at ambient temperature within a few days, remaining constant at this level after 15 days (Fig. 2). They quickly reached equilibrium moisture content at ambient conditions. Seed viability remained similar, 75% germination for freshly collected seeds with 6% MC and for desiccated seeds (Fig. 2). The germination percentage of these seeds dropped only slightly from 88 r 7.30% to 73 r 6.83%. Seeds that were kept on top of moist vermiculite as controls were infected with fungi and consequently their germination capacity gradually declined from 87 to 31%, while moisture content remained at 19% (Fig. 2). These results indicate that seeds of S. henryana are desiccation tolerant. 234 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Figure 1. Storage tissue (left– cut open) of S. henryana seeds.

Storage trials

Seeds with 15% MC initially germinated 75%, but less than 10% of viable seeds were obtained after 3 months storage at 5°C and at ambient temperature. Seeds with 6% initial MC sealed in plastic bags maintained high viability at 5 and 15°C after 12 months storage (Table 2). However, the germination capacity of seeds in plastic bags with ventilation holes decreased to nil when the moisture contents of these seeds increased to ca. 10% over the 12 months storage period at all temperatures. In these conditions, no seeds germinated at ambient temperature after 6 months, whereas viability was maintained with seeds in plastic bags without ventilation holes (Table 2). The viability of seeds was maintained at a minimum of 33% germination for all tested storage temperatures where moisture contents remained constant. However, seeds stored in plastic bags with ventilation holes at –10, 10°C and at ambient conditions, decreased dramatically in germination capacity. This could be partially due to an increase in moisture content that occurred during low temperature storage since the relative humidity in the storage unit could not be accurately controlled. The moisture content of seeds stored at –10, 10°C, and at ambient temperature increased by 3–6%, while the moisture content of seeds stored at 5 and 15°C changed only slightly. ASIA 235

100 100 Ambient

80 80

60 60

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20 20

0 0 100 100

Silica gel e (%) g 80 80

60 60

40 40

20 20 Moisture Moisture content (%, FW) Germination percenta

0 0 100 100 Control Moisture content 80 Germination percentage 80

60 60

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Figure 2. Moisture content and germination percentage of S. henryana seeds after desiccation by different methods. 236 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Seeds, however, could be stored in plastic bags at ambient temperature for 6 months, without a decrease in viability. However, after 12 months the germination percentage of these seeds decreased from 75 to 57%, on average. Despite this decrease, storability was comparable to the other temperature regimes tested.

Table 2. Germination (G) of S. henryana seeds after drying to 6 and 15% moisture content (MC). Seeds were stored at –10, 5, 10 and 15°C or at ambient temperature for 3, 6, and 12 months, sealed in plastic bags with ventilation holes (1) or without ventilation holes (2)

Initial Storage Container Duration of storage(months) MC temperatur e (°C) 3 6 12 G (%) MC G MC (%) G MC (%) (%) (%) (%) 15.37 –10 1 0 12.51 0 11.96 – – % (G=75 2 0 19.31 0 19.77 – – %) 5 1 3.0 6.16 0 6.32 – – 2 0 18.35 0 19.78 – – 10 1 0 14.51 0 13.44 – – 2 0 21.20 0 19.30 – – 15 1 0 4.24 0 4.5 – – 2 0 18.81 0 20.34 – – Ambient 1 8.0 11.36 0 12.45 – – 2 0 19.60 0 20.37 – –

6.03% –10 1 37.0 8.8 37.0 9.74 4.0 12.96 (G=65 2 42.0 5.16 49.0 5.21 33.0 6.00 %) 5 1 56.0 5.18 46.0 5.20 49.0 6.82 2 50.0 4.97 30.0 5.54 59.0 6.92 10 1 3.0 12.26 48.0 11.53 0 9.35 2 45.5 5.39 56.0 5.46 35.0 8.00 15 1 57.5 4.77 34.7 4.87 56.0 6.20 2 52.0 4.76 62.0 4.93 64.0 3.98 Ambient 1 45.0 10.3 0 11.35 0 11.71 2 79.0 6.81 75.0 7.61 57 10.13

Conclusions

Results of this study suggest that S. henryana seeds exhibit orthodox storage behaviour, as seeds could be dried to 6% MC without affecting viability. Seeds were storable at –10°C for up to 1 year, sealed in plastic bags, maintaining high germination percentage (33%). Thus, S. henryana seeds can be dried to 6% MC and stored at low temperatures of 5 to 15°C. Like any other orthodox seeds, S. henryana ASIA 237 seeds will lose their viability faster if the seeds reabsorb moisture from the storage environment. Therefore, seeds should be enclosed in hermetic containers to maintain constant moisture content. Seeds can also be stored at ambient conditions for about 6 months. However, more studies utilizing different seed sources and provenances would consolidate their tolerance to desiccation and storage. Further investigations on the causes of viability loss during storage of seeds are also warranted.

Acknowledgements

The authors thank IPGRI, DFSC and Royal Forest Department for financial support. Special thanks go to Khao Hin Son Botanical Garden’s staff for assistance with seed collection and Seed Technology Section’s staff for data collection.

References

Corbineau, F. and D. Come. 1986. Experiments on germination and storage of the seeds of two dipterocarps Shorea roxburghii and Hopea odorata. Malaysian Forester 49(3–4):371–381. Panochit, J., P. Wasuwanich and A.K. Hellum. 1984 . Collection, germination and storage of Shorea siamensis Miq. seeds. Embryon 1:1–13. Panochit, J., P. Wasuwanich and A.K. Hellum. 1986. Collection and storage of seeds of Shorea roxburghii G. Don. Embryon 2:62–67. Phengklai, C. and C. Niyomdham. 1999. The Dipterocarpaceae of Thailand. Pp. 131–132. Proceedings on Dipterocarpus alatus Roxb. and Dipterocarpaceae. Vol. III, Kasetsart University, Bangkok, Thailand, 17–18 November 1999. Phukittayacamee, P. S. Saelim, J. Bhothipuks and S. Kijkar. 1994. Dipterocarp seed behavior and storage. Pp. 11–34. Proceedings Fifth Round-Table Conference on Dipterocarps. Chiang Mai, Thailand, 7–10 November 1994. Purohit, A. N., M.M. Sharman and R.C. Thapliyal. 1982. Effect of storage temperatures on the viability of Sal (Shorea robusta) and talura (Shorea talura) seeds. Seed Sci. Technol. 1:499–514. Sasaki, S. 1980. Storage and germination of dipterrocarps seeds. Malaysian Forester 43(3):290–308. Tompsett, P.B. 1987. Desiccation and storage studies on dipterocarp seeds. Ann. Appl. Biol. 110:371–379. Yap, S.K. 1981. Collection, germination and storage of diptercarp seeds. Malaysian Forester 44(2–3):281–300. 238 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Seed storage methods of star anise (Illicium verum), cinnamon (Cinnamomum cassia) and michelia (Michelia mediocris)

Le Dinh Kha, Nguyen Huy Son, Tran Ho Quang, Nguyen Tuan Hung

Research Centre for Forest Tree Improvement, Forest Science Institute of Vietnam, Tuliem, Hanoi, Vietnam

Abstract

Star anise, cinnamon and michelia are economically important tree species in Vietnam, but their seeds cannot maintain viability for long during storage. Studies on seed storage of these species were undertaken by the Forest Science Institute of Vietnam in order to improve their storage longevity. The results showed that seeds of these species differ in morphological characters such as fruit shape, initial moisture content and initial germination rate. The initial moisture contents of seeds of star anise, cinnamon and michelia were 39.1, 45.5 and 33.6%, and they germinated 38.5, 87.7 and 31.8%, respectively. After seeds were desiccated down to 25% for star anise, 30% for cinnamon and 15% for michelia, they maintained 20.3%, 85.5% and 33.9% germination, respectively. Seeds of star anise and cinnamon with moisture contents of 25–40% and 30–40% respectively had the highest germination percentage after storage at 5°C for 9 and 12 months. Seeds of michelia with initial moisture content of 20–35% could be stored for 9 months at 5 and 15°C, maintaining their initial viability, but not at room temperature.

Introduction

Star anise, Illicium verum Hook.f., is an evergreen broad-leaved tree belonging to the Illiaceae family (Smith 1947). However, this species had been first classified in Magnoliaceae family and then in Winteraceae family (Hou Kuanzhao 1958). It occurs mainly in Vietnam and China, between 22° and 23° N latitude and is able to grow up to 12-m high. In Vietnam the species can be found in Cao Bang and Lang Son regions at altitudes of 500 to 700 m above sea level. In China it is distributed in the South and Southwest of Guangxi, and in some areas of Guangdong and Hunan provinces at 1000 to 1700 m above sea level (China Botanical Council 1978). ASIA 239

Star anise fruits are used mainly as aromatic spice, medicine, digestive treatment and anis alcohol production. Dry fruits of star anise contain 9 to 10% of essential oils. The main oil chemical components are anethol (80–90%), pinen, limonene and terpineol. Its leaves also have essential oils with the same chemical components as its fruits (Do Tat Loi 1995). The fruits are a valuable export product of Vietnam (Do Tat Loi 1995), and are considered economic perennial crops for farmers in Lang Son and Cao Bang regions. Actually, star anise in Vietnam is only found in plantations, but not in natural forest as it is believed that most of the natural stands of this species no longer exist in both countries. Furthermore, flowering and fruiting of star anise is erratic, occurring every 2–3 years. Thus, there is a need for identifying appropriate methods for long-term storage of seeds and supporting planting programmes. Consideration should also be given to conservation either by long-term storage of seed or planting conservation stands. Star anise seed contains high proportion of oil, which possibly would make it difficult to store for a long period. Local farmers store seeds of star anise with 12% MC in sand at a cool place, maintaining viability for 2–3 months. Previous studies on storage techniques of star anise showed that seeds with 30–34% MC initially germinated 76–78% (Bui Nganh and Tran Quang Viet 1980). However, when these seeds were dried to 14% MC, the germination capacity dropped to 9%. There was 20% germination after 7 months storage of seeds kept in nylon bags at 10°C, whereas no seed germinated after storage at 20°C. Cinnamon, Cinnamomum cassia Blume, tree is an evergreen species belonging to the Lauraceae family, which includes 21 genera and 245 species (Nguyen Tien Ban 1997). Cinnamon is the most economically valuable tree among the 40 species of Cinnamomum genus in Vietnam (MARD 2000). The cinnamon tree is medium-sized, up to 18–20 m in height. It is naturally distributed in high mountains from 500 to 700 m above sea level and found in many provinces from the Centre to the North of Vietnam (between 16° and 22°30’ N latitude) in regions with annual rainfall of 2000–2500 mm. In China it is naturally distributed in Guangxi and Guangdong, but it has been planted in Fujian, Yunnan, Jiangxi, Hunan and Zhejiang provinces, extending to 24°30' N and 800 m above sea level (Chinese Botanical Council 1978). Cinnamon bark contains 1–1.5% of essential oils, which are composed of 80–85% cinnamic aldehyde and some eugenols (Do Tat Loi 1995; Vo Van Chi 1997). Our preliminary results indicated 0.9 and 1.9% oils in cinnamon leaves and bark, respectively (unpublished data). The seeds also contain a high proportion of oils so it is very difficult to store for a 240 STORAGE BIOLOGY OF TROPICAL TREE SEEDS long time. The bark is used as spice in cooking, as medicine to cure indigestion, improve blood circulation, and in making incenses. The timber is used for construction and furniture. In recent years about 4500– 6200 tonnes of cinnamon bark were exported to many countries including Taiwan, China, Korea, Japan and USA. Thus, the cinnamon tree is an economically important species particularly for minority people living in the mountains and remote areas of northern and central Vietnam. The tree is also used for greening and improving soils in bare hills and denuded lands of Vietnam. The cinnamon tree flowers in October–November and the seeds mature in the following January to March (FIPI 1996). The only known storage technique is from local farmers’ experiences, which show that seeds mixed with moist sand and kept under shade can be stored for 2–3 weeks. Michelia, Michelia mediocris Dandy, synonym Talauma gioi A.Chev., is a medium to large evergreen tree up to 30 m in height and 70–80 cm in diameter at breast height. It belongs to the Magnoliaceae family, which includes 13 genera with 210 species (Nguyen Tien Ban 1997). There are about 15 species in the Michelia genus (MARD 2000). M. mediocris is endemic to Vietnam and is distributed at 400–1000 m above sea level in the northern and central provinces (16°–22° N latitude), where the mean annual rainfall is 1900–2500 mm. Michelia flowers in April and the fruit matures in October (FIPI 1996) Michelia timber is yellow and aromatic, easy to work so it is used in construction, furniture making and fine art goods. The timber is fine in texture, rarely curved or split after seasoning and resistant to termites and insects. As a result, M. mediocris is heavily exploited and today, it is found only in conservation areas and natural conservation parks. This makes Michelia one of the target important species for planting in Vietnam. However, there is little information on storage methods of M. mediocris seeds, so that seeds are sown for planting immediately after collection. Star anise, Illicium verum Hook.f., cinnamon, Cinnamomum cassia Blume, and michelia, Michelia mediocris Dandy are economically valuable tree species that are exploited to provide a range of products. They are in high demand in plantation programmes, but there is little information on the biology of their seeds. Studies on storage of recalcitrant seeds for native species in Vietnam were therefore undertaken as part of an IPGRI project with support and cooperation of DANIDA Forestry Seed Centre. Other partners in the studies of these three species were the Australian Tree Seed Centre (ATSC) and the Southern Research Station of the United States Forest Service in America. ASIA 241

Materials and methods

Seed materials

Mature fruits of star anise, cinnamon and michelia were collected directly from 25 to 40 trees each in winter during 1999, 2000 and 2001. The seeds were extracted immediately at room temperature (10–20°C). After that, seeds of cinnamon and michelia were soaked in water overnight in order to clean their coats. Star anise seeds were immediately handled, desiccated and stored. Different characteristics of fruits and seeds were studied and the weight of 1000 seeds was measured.

Germination tests

During storage seeds were regularly tested for germination capacity and their moisture contents were determined after 1, 3, 6, 9 and 12 months. Four replicates of 25 seeds were incubated at ambient room temperature of 20–28°C for each germination test. The data were statistically analyzed using MicroSoft Excel Software and statistical methods by Nguyen Hai Tuat (1982).

Desiccation and storage of seeds

To determine initial weights and moisture contents, 100 cleaned seeds of each of the three species were used. Samples of seeds were then desiccated by silica gel with 1:1 ratio (by weight), according to the IPGRI/DFSC screening protocol (1999), and their moisture contents were determined by weighing before and after oven drying at 103°C for 17 h. Seeds were kept in aluminium bags and stored in refrigerators at 5°C, 15°C and in ambient room temperature (RT). Germination capacity and moisture contents of all seeds were determined immediately after desiccation and before storage. 242 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Results

Seed characteristics

Fruits of star anise are compound with 6–12 stars, becoming woody when ripened. The outer coat of the seeds is a cuticle type. Michelia fruits are also composite with a cone shape. Fruits of cinnamon are single-seeded. The outer coat of both cinnamon and michelia seeds is fleshy, thus their cleaning was necessary before experiments. Other characteristics of fruits and seeds of these species are shown in Table 1.

Table 1. Characteristics of fruits and seeds of star anise, cinnamon and michelia species, collected in 1999 and 2000

I. verum C. cassia M. mediocris Fruit Date of collection: 20-11-1999 10-1-2000 25-2-1999 Shape Composite fruit, star single fruit Composite fruit, cone shape shape Fruit coat Woody Fleshy Semi-woody 1000 fruit weight (g) 6400 771 – Seed Shape Flat oval oval flat heart shape Weight of 1000 seeds (g) 85.0 391.4 258.6 Number of seeds in 1 kg 11760 2250 3870 Initial moisture content 31.9 44.5 33.6 (%) Initial germination rate (%) 38.5 87.7 31.8

Desiccation and germination of seeds

Seeds were initially desiccated to various moisture contents and then tested for their germination capacity. I. verum seeds with initial 39.1% MC germinated 38.5% (Table 2). When they were dried to 6.7% MC, they retained 5.6% germination. Seeds of C. cassia having 45.5% MC germinated 87.7%. However, they did not germinate when they were dried down to below 13.7% MC. M. mediocris seeds with 33.6% MC, initially germinated 31.8%. They could be dried to 15.5% MC and germinated 34%, but below that moisture level they completely lost viability (Table 2). ASIA 243

Table 2. Seed initial germination (G%) after being dried to different moisture contents (MC%)

I. verum C. cassia M. mediocris Target MC (%) MC (%) G (%) MC (%) G (%) MC (%) G (%) Control 39.1 38.5 45.5 87.7 33.6 31.8 40 – – 40.2 71.5 – – 35 36.0 40.3 36.2 84.5 – – 30 30.9 39.0 30.9 85.5 28.4 48.0 25 23.7 20.3 – – 24.4 41.5 20 – – – – 20.1 42.0 15 16.5 10.3 13.7 21.7 15.5 33.9 10 7.2 11.2 7.2 0 9.3 0 5 6.7 5.6 6.7 0 – –

Storage of seeds

Moisture contents of seeds of the three species were measured during storage at different temperatures (Table 3). There were greater variations for the higher moisture contents (>20%) than for the lower moisture contents (<10%). For example, these changes were more that 10% MC in I. verum with 30.9% initial MC that increased to 43.5% final MC at room temperature. Similar processes occurred in C. cassia seeds where, e.g., the 40.2% initial MC resulted in 54.1% final MC, while 30.9% initial MC increased to 53.2% final MC. The initial 14.9% MC of M. mediocris seeds increased also to 31.9% MC after 12 months storage. The germination capacity of seeds at five different moisture contents was determined after storage at 5, 15°C and at ambient temperature. Germination decreased depending on the storage conditions (Fig. 1). I. verum seeds survived better at 5°C compared to storage at 15°C or at ambient temperature. After 12 months, germination percentage was maintained for seeds at t30% MC stored at 5°C and at ambient temperature. For seeds at 23.7% MC, germination percentage decreased after 6 months, then stayed at the same level for 12 months. Seeds at 16.5% MC maintained germination only after 3 months of storage. No seed germinated after 9 months storage at 15°C. High survival was maintained for M. mediocris seeds with t20% MC stored at 5 and 15°C for 9 months (Fig. 1). No seeds germinated after 6 months at ambient temperature. After surviving with ca. 30% germination, desiccated seeds to 15.5% MC could not be stored for more than a month at 15°C. C. cassia seeds with high moisture contents, 30 to 40%, could only be stored at a low temperature of 5°C for 9 months, germination 244 STORAGE BIOLOGY OF TROPICAL TREE SEEDS decreasing from 90 to 14% (Table 4). These seeds did not survive storage conditions at 15°C or at room temperature for more than a month or survive drying below 30% MC.

Table 3. Changes of seed moisture contents (MC) during storage at different temperatures. (RT=room temperature)

Species Initial MC (%) MC (%) after 12 months storage 5°C 15°C RT 39.1 40.3 48.3 48.3 36.0 34.1 42.4 42.0 30.9 30.5 38.3 43.5 I. verum 23.7 25.7 32.1 31.4 16.3 15.0 25.9 28.6 7.2 10.2 13.5 8.1 6.7 8.1 6.0 10.0 40.2 47.7 50.9 54.1 36.2 41.8 45.9 41.9 30.9 45.4 49.9 53.2 C. cassia 13.7 28.5 21.7 21.8 7.2 11.1 11.6 16.2 6.7 10.6 10.0 15.8 33.1 34.0 36.7 41.1 28.4 32.6 33.2 38.5 24.4 28.6 27.3 36.4 M. mediocris 20.1 24.1 29.3 33.5 16.0 22.7 32.3 29.9 14.9 19.3 30.7 31.9 9.3 9.1 9.9 8.8

Discussion

Seed characteristics

Fruit and seed characteristics of these three species differed in structure, shape and size, and also in initial moisture contents and germination. The seed initial moisture content and quality of cinnamon were the highest, 45.5% MC and 87.7% germination. Initial moisture of star anise and michelia was 39.1% and 33.6% respectively, and they germinated 38.5% and 31.8% respectively (Table 1). However, initial moisture contents of seeds between 30 and 50%, or higher, is likely to belong to recalcitrant type of seeds (Schmidt 2000). The initial increase in germination patterns after the first month of storage of star anise and michelia seeds suggested after-harvest ripening of these seeds (Schmidt 2000; Willan 1985), then followed a decrease of viability due to storage stress (Fig. 1). ASIA 245

I. verum M. mediocris

100 39.1 % MC 100 36.0 % MC 5°C 5°C 80 30.9 % MC 80 23.7 % MC 16.5 % MC 60 60

40 40

20 20

0 0 100 100 15°C 15°C 80 80

60 60

40 40 Germination (%) Germination Germination (%) Germination 20 20

0 0 100 100 33 % MC ambient 28 % MC ambient 80 24 % MC 80 20 % MC 60 16 % MC 60

40 40

20 20

0 0 024681012 0246810

Storage period (months)

Figure 1. Germination of I. verum and M. mediocris seeds after storage at 5, 15°C and at ambient temperature. Standard error is present when larger than symbol. 246 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Table 4. Germination of C. cassia seeds after storage at 5, 15°C and at ambient temperature. Seeds were collected in 2000 and 2001. (RT=room temperature)

Initial MC (%) Storage Germination (%) after storage temperature 1 months 3 months 6 months 9 months 5°C 90.5 75.0 37.5 25.0 40 15°C 10.5 0 0 0 RT 89.5 0 0 0 5°C 71.0 48.2 22.0 14.0 35 15°C 49.7 0 0 0 RT 23.7 0 0 0 5°C 80.2 28.2 5.5 1.5 30 15°C 35.5 0 0 0 RT 8.7 0 0 0 5 – 20 5°C and RT 0 0 0 0

Desiccation survival

Star anise germination decreased to 5.6% and 11.2% when seeds were dried to 5 and 10% MC. Germination of C. cassia was 85% when they were at 30% MC. However, desiccation decreased the seed capacity to germinate and no seeds germinated at 10% MC, or below. M. mediocris seeds with 20–30% MC, in contrast, germinated 41.5–48%. At 15% MC, the germination was 33.9% and seeds completely lost germination capacity at 10% MC. This indicates that michelia seeds were tolerant to desiccation to the level higher than the minimum moisture content frame suggested for recalcitrant seeds (Schmidt 2000). There were increases in seed moisture contents of all three species during storage (Table 3). Such increases were similar for seeds that were stored at 15°C and at ambient temperature. These seeds may not have been hermetically sealed, thus re-equilibrating with the relative humidity of their immediate surrounding. At similar moisture content, the increase was higher at low temperature than at high temperature (Vertucci and Roos 1993).

Storage trials

Two important factors affecting seed viability were the initial moisture content and the storage temperature. This has also been shown in previous studies on seed storage of Vietnamese species, including media and containers for storage as well (Le Dinh Kha 1999). In this study, recalcitrant seeds of three tropical species have shown to be storable at 5°C (or at 10°C) with reduced moisture contents, e.g., 20% MC. However, storage at 15°C only preserved their high ASIA 247 germination percentage in the first month for cinnamon and 3–6 months for star anise and michelia seeds, then viability decreased due to storage stress (Fig. 1 Table 4). For storage at ambient temperatures of 20–30°C, all seed lots preserved relatively high germination only in the first three months, and after that all seed lots completely lost their viability. In conclusion, seeds of star anise, cinnamon and michelia species are sensitive to desiccation. We suggest the target MC could follow a general scale of 5, 10, 15, 20 and 25% for all seeds with an initial 21–45% MC and seeds with an initial 46–50% MC could be desiccated to the lowest target MC of 10%, but not to 8% MC alone. This project should be extended to other tropical seeds of the Dipterocarpaceae and Lauraceace families, important to Vietnam.

References

Bui Nganh and Tran Quang Viet. 1980. Final Report of the Research Subject on Seed Production of Illicium verum. FSIV. (in Vietnamese). China Botanical Council. 1978. Reforestation Technology of Main Tree Species in China. Agricultural Publishing House, Hanoi, Vietnam, Tome 2. Pp. 1156– 1167 (in Chinese). DFSC/IPGRI. 1999. Desiccation and storage protocol. The project on handling and storage of recalcitrant and intermediate tropical forest tree seeds. Newsletter No 5:23–39. Do Tat Loi. 1995. Medicinal Plants and Pharmaceutical Prescriptions of Vietnam. Science and Technological Publishing House. Pp. 1485 (in Vietnamese). FIPI (Forest Inventory and planning Institute). 1996. Vietnam Forestry. Agricultural Publishing House, Hanoi, Vietnam. Pp. 787. Hou Kuanzhao. 1958. Dictionary of Families and Genera of Chinese Plants. Science Publishing House, Beijing, China. Pp. 553 (in Chinese). Le Dinh Kha. 1999. Storage of recalcitrant and intermediate seeds and pollen storage for some forest tree species in Vietnam. Pp. 397–403 in IUFRO Seed Symposium 1998 "Recalcitrant Seeds" (M. Marzalina, K.C. Khoo, N. Jayanthi, F.Y. Tsan and B. Krishnapillay, eds.). Proceedings of the Conference. Kuala Lumpur, Malaysia. MARD (Ministry of Agriculture and Rural Development). 2000. Names of Vietnam Forest Trees. Agriculture Publishing House. Pp. 460 (in Vietnamese). Nguyen Hai Tuat. 1982. Statistics in Forestry. Agricultural Publishing House, Hanoi, Vietnam. Pp. 289. Nguyen Tien Ban. 1997. Handbook to Reference and Identification of the Families of Angiospermae Plants in Vietnam. Agricultural Publishing House, Hanoi, Vietnam. Pp. 531 (in Vietnamese). 248 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Schmidt, L. 2000. Guide to Handling of Tropical and Subtropical Forest Seed. Danida Forest Seed Centre, Humlebaek, Denmark. Pp. 511. Smith, A.C. 1947. The Families Illiaceae and Schisandraceae. Sargentia No. 7:1–79. Vertucci, C.W. and E.E. Roos. 1993. Theoretical basis of protocols for seed storage. II. The influence of temperature on optimal moisture levels. Seed Sci. Res. 3:201–213. Vo Van Chi. 1997. Dictionary of Vietnamese Medicinal Plants. Medical Publishing House. Pp. 1468 (in Vietnamese). Willan, R.L. 1985. A Guide to Forest Seed Handling with Special Reference to the Tropics. DANIDA, FAO. FAO Forestry Paper 20/2. Pp. 379. 3 South and Central America

Bolivia (BASFOR, Univ. Gabriel)

Brazil (ERGB)

Colombia (CONIF)

Costa Rica (CATIE) 250 STORAGE BIOLOGY OF TROPICAL TREE SEEDS SOUTH AND CENTRAL AMERICA 251

Desiccation and storage of Anadenanthera colubrina seeds

Edilberto Rojas Espinoza

BASFOR – Centro de Semillas Forestales,ESFOR/UMSS-IC/COSUDE, Av. Atahuallpa Final Norte s/n (ESFOR), P.O. Box 5453 Bolivia

Abstract

IPGRI/DFSC protocol has been used to study A. colubrina seeds. The results showed that these seeds could be dried to 4% MC and maintained high viability of 98%. Seeds with >37% MC did not survive longer than 3 months at all tested storage temperatures. However, seeds with <13% MC remained highly viable after storage for 12 months at 18, 4 and –20°C. Their germination capacity decreased during 23 months storage, and only drier seeds with 4 and 8% MC stored at 4 and –20°C maintained about 20% viability.

Introduction

Bolivia is located in the centre of South America with 8 million inhabitants and a surface of 1 098 581 km2. Tropical forests cover more than 50% of the country. There are three different geographical areas. The highlands are in altitudes of an average of 3500 m above sea level, with a mean temperature of 5 to 10°C. The valleys are found at 2500 m above sea level, with mean temperature between 15 and 25°C (Pinto 1982). The oriental plains are in altitudes of 350 m above sea level, where the mean temperature is about 30°C. In the highlands, forestry is not well developed because of climatic factors. The most utilized native species are Buddleja coriacea and Polylepis besseri; exotic Pinus radiata, Cupressus macrocarpa and Eucalyptus globules are also grown. Domestic plans for forestation in the valleys were developed with the help of the international co-operation in the 1970s, ‘80s and ‘90s. Several species were planted, but in different proportions, i.e. Eucalyptus (47%), Pinus (44%) and other species (9%). In the oriental plains, until the last decade, most attention was given to precious species like Swietenia macrophylla, Cedrela odorata and Amburana cearensis. From 1996, the Forest Law reduced their use. In the last five years, forest plantations have increased in these areas, with private initiative and with species of quick growth like Schizolobium amazonicum, S. macrophylla, Tectona grandis and C. odorata. However, the exploitation of the forest resources has been indiscriminate, to such extend that species 252 STORAGE BIOLOGY OF TROPICAL TREE SEEDS like Podocarpus parlatorei are greatly endangered and are now listed in the IUCN Red List of Threatened species (IUCN 2002). The forestry tradition has been to plant well-known species of quick growth, for which there is a guaranteed market for the products. Most of species correspond to exotic species such as Pinus, Eucalyptus and T. grandis and others important species like S. macrophylla, C. odorata and A. cearensis. However, there is a deficiency of knowledge about how to appropriately handle most native forest species. To provide reforestation programs with quality seeds, forest seed banks, such as BASFOR in Cochabamba or the Centro de Investigación en Agricultura Tropical (CIAT) in Santa Cruz, have been established and the new laws have also forced the use of only native species as replacements in natural forests. However, problems were quickly discovered with the germination and storage of forest seeds. Anadenanthera colubrina (Vell.) Brenan, a native species to Bolivia has been selected to study because of its multiple uses. Its common names are willca, cebil, curupari and curupaú (Killeen et al. 1993). This species is dominant and is found in association with Dodonaea viscosa, Schinus molle and Myroxylon peruiferun. The tree reaches 6 m in height and 30 cm in diameter. It is distributed naturally in the central area of the Peru, north of Argentina, Paraguay, northeast of Brazil. In Bolivia it thrives in Cochabamba, Chuquisaca, La Paz, Santa Cruz and Tarija where it can be seen on the hillsides next to the rivers. It grows in secondary forests, on dry and semi-humid inter-andean hills. It grows generally in superficial ground, on drained stony or rocky soil, on steep pending hillsides in altitudes from 315 to 2200 m above sea level. It grows with about 250 to 600 mm per year rainfall with an average temperature of 21°C. The wood has a high calorific value. It is used in construction and for making door and window frames, barrels, mooring masts, hedges, platforms, floors, agricultural implements and railway sleepers. Its by-products are used in medicine and as tannin to harden leathers. It is also known that this species is of quick growth (1–1.5 m per year under favourable conditions). It is recommended as a high-priority species for reforestation programmes. A. colubrina produces small creamy white flowers, gathered in spherical shapes. The fruits are legumes with flat brown sheaths. The seed is dark brown, flat and circular with two cotyledons covered by a brown testa. Seeds of this species, according to Lorenzi (1992), cannot be stored for more than 4 months, rapidly losing their viability (Torrico et al. 1997). SOUTH AND CENTRAL AMERICA 253

Materials and methods

Fruit collection and processing

The fruits of A. colubrina were collected in July 2000 and 2001, from Tín Tín, Mizque, Cochabamba located at 18º22' S latitude and 65º02' W longitude, between 1990 and 2100 m above sea level (Jimenez 1991). Fruits were harvested from 25 trees in a dominant secondary forest and were transported the same day to BASFOR in open polyethylene sacks. The average ambient temperature during transport was 21ºC. Seeds were extracted in the shade the second day of collection and immediately dispatched to the replicating partner.

Initial trials

Upon arrival at BASFOR, the initial moisture content was determined and the first samples of seeds were tested for germination. The weights and dimensions of the fruits were determined. Samples of seeds were desiccated to target MCs and also tested for germination (IPGRI/DFSC 1999, 2000). The moisture content was determined by weighing before and after oven drying at 103°C for 17 h, and then calculated as a percentage of fresh mass. Only seeds from the 2000 harvest were treated for 10 min with 5.25% hypochlorite of sodium (20 ml NaOCl for 1 l of water). Seed weights were monitored until they reached the target moisture contents. Samples were then taken and sown using four replications of 25 seeds in sifted sand, washed and sterilized at 103ºC during 17 h. Seeds were sown in rectangular polystyrene boxes with a cover [180×120 mm (base)×70 mm (height)] and put in a germination room at 28–30ºC with 8 h light and 60–100% relative humidity.

Results

Initial characteristics

Initial characteristics of A. colubrina seeds collected in 2000 and 2001 were similar. Seeds from both lots had initial moisture contents of ca. 41% and germination of ca. 97% (see Table 1). 254 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Table 1. Initial characteristics of A. colubrina seeds harvested in 2000 and 2001

2000 seed lot 2001 seed lot Fruit size: —length 19.9±2.6 18±3.3 —width (cmrSD) 2.0±0.2 2.1±0.2 Fruit weight (g r SD) 7.91±2.2 7.23±2.3 Seed weight (g r SD) 0.32±0.08 0.27±0.7 Seed diameter (cm r SD) 1.72±0.19 1.66±0.15 MC (%) before processing 41 41.3 MC (%) after processing 37.0 38.2 Initial germination (%) 97 98

Desiccation trials

A. colubrina seeds with 36% MC from both the 2000 and 2001 lots, were desiccated down to ca. 7% MC. They all maintained high viability of 98% at this moisture content, showing that these seeds were not desiccation sensitive (Table 2).

Table 2. Germination capacity (G%) of A. colubrina seeds after drying to different moisture contents (MC%)

2000 seed lot 2001 seed lot MC (%) G (%) MC (%) G (%) 36.2 97 35.8 97 32.4 97 33.2 96 27.5 94 26.4 97 23.3 97 22.8 97 13.8 95 12.9 98 6.9 98 6.7 98

Storage trials

The storage results showed that seeds with >37% MC did not survive longer than 3 months at all tested temperatures (Fig. 1). However, seeds with <13% MC remained highly viable after storage for 12 months at 18, 4 and –20°C. In a separate trial, seeds from 2001 lot that were not treated with sodium hypochlorite, had better recovery (76–90%) than the treated seeds of 2000 lot, at all tested temperatures after 11 months storage (Fig. 1). This indicated that NaOCl might have affected the seeds during storage. The germination capacity decreased during storage up to 23 months, and only drier seeds with 4 and 8% MC stored at 4 and –20°C maintained about 20% viability after this time. SOUTH AND CENTRAL AMERICA 255

Discussion

This study showed that seeds of A. colubrina were not desiccation sensitive as previously suggested (Lorenzi 1992). They were dried to 4% MC and maintained high viability of 98%, and were stored for more than a year at 18, 4 and –20°C (Table 2 and Fig. 1). It was suspected in the first year (2000) that sodium hypochlorite damaged and affected viability, maybe due to the high concentration. Thus seeds of the 2001 collection were not treated, and the 11 months storage results were highly improved, 76–90% against ca. 50% (Fig. 1). After 23 months storage, only drier seeds with 4 and 8% MC stored at 4 and –20°C maintained about 20% viability. Additionally, this study allowed collection of data on the phenology, ecology, uses and yields in laboratory and in nursery. These results will help produce seedlings for reforestation and provide another choice of species for planting. There is therefore a potential for ex situ conservation of this species, as well as in situ conservation. Such a study can be extended to other important species like the Aspidosperma quebracho blanco or endangered species like Podocarpus parlatorei (IUCN 2002), for which there is hardly any knowledge of the biology of their seeds. This would raise BASFOR expertise in the handling of forest seeds as a leading institute in Bolivia.

Collaboration within the project

The project of recalcitrant seeds has been well implemented in BASFOR. The training workshop for the protocol held in Costa Rica in 2000 was useful. The workshop gave a good opportunity to meet other scientists from different countries and to build institutional collaborations. The modus operandi was very efficient from all the participating institutions within the project and no serious problem was encountered with logistics and finances. The co-ordination with the replicating partner within Bolivia was good because of the training workshop that put together all the contributors. 256 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

100 2000 seed lot 37.0 % MC 13.5 % MC 80 18°C 10.3 % MC 8.0 % MC 4.3 % MC 38.2 % MC 60 12.8 % MC 10.2 % MC 7.1 % MC 40 18°C 4.3 % MC 2001 seed lot 20

100 4°C

60 4°C 40

20 Germination (%)

100 -20°C

60 -20°C 40

0 0 3 6 9 1215182124 036912

Storage period (months)

Figure 1. Germination of A. colubrina seeds after storage for 23 months (2000 seed lot) and 11 months (2001 seed lot that was not treated with NaOCl).

The protocol is simple and easy to implement. It has been translated to allow studies of other species. For future activities, it is important that many other species be investigated. Establishing a network on recalcitrant seeds would continue to benefit the exchange of information and the publication of results for all the participating countries.

References

IPGRI/DFSC. 1999. The project on handling and storage of recalcitrant and intermediate tropical forest tree seeds. Danida Forest Seed Centre. Newsletter No. 5:23–39. SOUTH AND CENTRAL AMERICA 257

IPGRI/DFSC. 2000. Protocolo para la desecación y almacenamiento de semillas. The Project on Handling and Storage of Recalcitrant and Intermediate Tropical Forest Seeds. Pp. 6. IUCN. 2002. 2002 IUCN Red List of Threatened Species [also at http://www.Redlist.org]. Jimenez, F. 1991. Zonas agroecológicas y tipos de agricultura en el departamento de Cochabamba (zona andina). Cochabamba, Bolivia. Informen ecológicos. Tomo 9. Pp. 40. Killeen, T., E. García and S.y Beck. 1993. Guía de árboles de Bolivia. Herbario Nacional de Bolivia y Missouri Botanical Garden, La Paz Bolivia. Pp. 430– 432. Lorenzi, H. 1992. Árvores Brasileiras: Manual de Identificação e Cultivo de Plantas Arbóreas do Brasil. Nova Odessa, Ed. Plantarum. Pp. 352. Pinto, E. 1982. Estudio Pluviométrico del Departamento de Cochabamba. Tesis para Ing. Agrónomo. Universidad Mayor de San Simón. Cochabamba, Bolivia. Pp. 94. Torrico, G., S. y Beck and E. García. 1997. Estudio sobre los arboles y arbustos nativos de uso múltiple en los departamentos de Cochabamba, Chuquisaca (valles interandinos). Cochabamba, Bolivia. Pp. 41–44. 258 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Desiccation and storage of lucuma, Pouteria macrophylla seeds

Jaime Magne Ojeda and Luis Gonzales Saucedo

Universidad Autonoma Gabriel Rene Moreno, Facultad de Ciencias Agrícolas, Carrera de Ingeniería Forestal, Casila No. 1356, Santa Cruz, Bolivia

Abstract

Pouteria macrophylla seeds from Bolovia have been investigated for their tolerance to desiccation and storage. The maximum weight of the fruits was three times their minimum dry weight of ca. 41 g, while the big seeds weigh about five times compare to the minimum weight of 3.5 g of small seeds. Seeds completed germination over 60 days. While 73% germination was retained after drying seeds to 32% MC, no germination occurred at 19% MC, indicating the desiccation limit for these seeds. These results showed that P. macrophylla seeds were desiccation sensitive and that these seeds seemed to require higher temperature of t30°C.

Introduction

Pouteria macrophylla (Lam) Eyma, belongs to the Sapotaceae family, with synonyms like Chrysophyllum macrophyllum Lam., Lucuma rivicoa (Gaertn.) and Vitellaria rivicoa (Gaertn.) Radlk.. It is also called Canistel (English), Jaune d’oeuf (French Guayana), yema de huevo (Spanish), Uititiriba (Brazil) (FAO 1986), and Lucuma in Bolivia. It is a small to medium forest tree up to 20–25 m high with a dense crown. P. macrophylla is a common species that thrives in moist tropical and subtropical regions of Bolivia. It also naturally occurs in the tropical region of Peru and the Amazonian region of Brazil (Cavalcante 1988). In Bolivia, according to Killeen (1993) it occurs in the northern region of Santa Cruz City, but can also be found in the tropical low land region of Bení, La Paz and Cochabamba. P. macrophylla grows in secondary forests, near the disturbed areas of rural homes, in the moist low land region, generally in deeply and well-drained soils, at altitudes between 315 and 450 m above sea level. It grows well in areas with annual rainfall between 1200 and 2800 mm and a mean temperature of 24°C. The trunk is straight, and attains up to 50 cm in diameter, with 259 STORAGE BIOLOGY OF TROPICAL TREE SEEDS deep crevices near their base. The wood is soft and gray-yellow. It is used in construction and in door and window frames and agricultural implements. The bark exudes white latex when it is cut. Branches in juvenile trees are ascending, becoming more horizontal at maturity. The leaves are simple alternated without stipules, with petioles of 2.5–3.5 cm long and blades that are oblong lanceolate and 10–20 cm long times 4–8 cm wide. In Bolivia, especially in Santa Cruz, flowering of P. macrophylla occurs from Sept to Nov, the last flowers persisting until Jan. The flowers are small and organized in fascicles, with a greenish corolla of up to 10 mm long. The tree fruits from Feb to Mar. The fruits are ovoid and up to 6 cm diameter. When there is a single seed per fruit, the seeds are also ovoid and when there are two or three seeds per fruit, the seeds are flat. The seeds are covered with a dark brown hard testa embedded in a starchy yellow pulp. P. macrophylla reproduces by seed, germinating start about 50 to 60 days after sowing (FAO 1986). We have selected the lucuma species because of its great interest in agroforestry systems, its potential use for reforestation in secondary forests, and for its edible fruits. Compared with other native species, it is rapid growing under good conditions (1–1.5 m yr–1). The starchy mesocarp of the fresh fruits has an agreeable and generally sweet flavour, which is the part directly consumed by people mainly in rural areas. The objective of this work was to study the desiccation behaviour of P. macrophylla seeds.

Materials and methods

Collection of fruits and desiccation of seeds

Fruits were directly collected from trees in Buena Vista, 120 km North from Santa Cruz City in a low land region of Bolivia, near the Andean mountains. Its climate is subtropical, with 1400 mm annual rainfall and 24°C mean annual temperature. Mature fruits of P. macrophylla were collected 16–18 Feb 2001, from 20 trees within the dense young secondary forest, which had been re-established from natural regeneration. The fruits were transported in two permeable bags of 10 kg each to Santa Cruz City the following day. The collected fruits were put into bamboo sieves in shade, for 2–3 days to avoid an excessive humidity loss. 260 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

The fruits were manually and tediously processed, by removing the pulp from seeds. Seeds were then washed with tap water, and immediately soaked in 10% sodium hypochlorite for 10 min, as indicated in the protocol (IPGRI/DFSC 1999, 2000). Soon after preparation, seeds were sent to Cochabamba for laboratory experiments.

Moisture content and germination of seeds

Seed samples were dried down to target moisture contents of 40, 35, 30 and 20%, using silica gel. To determine the moisture content of seeds, five replicates of 20 seeds each were weighed before and after drying at 103°C for 17 h. The moisture content was then calculated using the formula: (IW–FW)/IW×100, where IW=initial weight and FW=final weight. The germination of seeds was designed in a random block. Seeds were sown in sterilized sand into rectangular polystyrene boxes with cover [30×25 cm (base)×8 cm (height)]. The boxes were then put in a room at constant 28°C and 60% relative humidity. In a second trial, P. macrophylla seeds were incubated in fluctuating temperatures between 15 and 38°C, for germination. Artificial light was provided for 8 h each day.

Results

Initial characteristics

There was large variation in weights of the fruits and seeds of P. macrophylla. The maximum weight of the fruits was three times their minimum weight, whilst the maximum compared to the minimum weight for the seeds was about five times (Table 1). These results are based on 100 fruits and their seeds.

Table 1. Fruit and seed characteristics of P. macrophylla.

Parameter Fruits Seeds Weight Length Width Weight Length Width (g) (cm) (cm) (g) (cm) (cm) Maximum 126.06 7.01 5.54 15.14 4.21 2.72 Minimum 40.85 3.08 2.68 3.46 2.07 1.68 Average±S 67±17.2 4.07±0.61 3.42±0 9.15±2.5 3.06±0.33 2.26±0.2 D .48 1 1 CV (%) 26 15 14 27 11 9 261 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Moisture content and germination

Seeds were desiccated to various target MCs and then tested for their germination capacity at 28°C. The time to complete all germination was 60 days. Whilst germination of 73% was obtained after drying to 32% MC, no germination occurred at 19% MC, indicating the desiccation limit for these seeds (Table 2). In a second test, P. macrophylla seeds were incubated at 15–38°C for germination. Seeds at all tested moisture levels germinated better at 15–38°C, which was out side the germination chamber, than at constant 28°C in the germination chamber (Table 2), showing that P. macrophylla seeds needed high temperature to germinate.

Table 2. Germination (%) of seeds after drying to four moisture contents and at varying (15–38°C) and constant (28°C) temperatures.

Treatments Target Actual Germination (%rSD) MC (%) MC (%)

28°C 15–38°C A Initial 46.32 67r4.4 78r 2.8 B 40 41 82r2.3 72r0 C 35 36 74r3 60r5.7 D 30 32 73r3.8 46r19.8 E 20 19 0 —

Discussion

Fruits and seeds of P. macrophylla had big variations in shapes and sizes, and also in their weights (Table 1). In a separate test in Cochabamba, the testa was removed and the seeds did not germinate (data not shown). It is thus recommended that the seed testa is not separated or broken prior to the start of the germination tests. Although the mean temperature out side the germination chamber was 26°C, which was close to the 28°C within the chamber, germination of these seeds seemed to require higher temperature of >30°C. Thus, there is still a need to further investigate seed germination temperature, the flowering system and period of fruiting of this species. The seeds tolerated desiccation to ca. 30% MC, but not lower, indicating that P. macrophylla seeds are desiccation sensitive. This is a first work in the Gabriel Rene Moreno University, related to a systematic study of the collection and storage of forest seeds, using the IPGRI/DFSC protocol. We expect to set up new trials on other species, using this protocol as a principal guide. 262 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Acknowledgements

We thank Dorthe Jøker from DFSC, Dr Ehsan Dulloo, IPGRI Coordinator and Dr Julio Salek, Vice Rector of the University, for their commitments and approval of the project, and Luis Gonzales, Forestry student for collecting good seeds.

References

Cavalcante, P.B. 1988. Fruitas Comestiveis da Amazonia (4th edn). Belem Para, Brasil. FAO. 1986. Food and Fruit-Bearing Forest Species Examples from Latinamerica. Forestry Paper No. 44/3. IPGRI/DFSC. 1999. The project on handling and storage of recalcitrant and intermediate tropical forest tree seeds. Danida Forest Seed Centre. Newsletter No. 5:23–39. IPGRI/DFSC. 2000. Protocolo para la desecación y almacenamiento de semillas. The Project on Handling and Storage of Recalcitrant and Intermediate Tropical Forest Seeds. Pp. 6. Killeen, T. 1993. Guía de Arboles de Bolivia. Herbario Nacional de Bolivia y Missouri Botanical Garden, La Paz, Bolivia. Pp. 430–432. 263 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Desiccation, storage and germination of Genipa americana seeds

Antonieta N. Salomão

Embrapa Recursos Genéticos e Biotecnologia – PqEB W5 Norte, C.P. 02372 CEP 70849-970 Brasília – DF Brazil

Abstract

Germination temperature dependency, tolerance to desiccation and sensitivity to exposure to low and sub-zero temperatures were investigated on Genipa americana seeds. The optimum temperature for germination of fresh seeds was found to be 30q&. The critical moisture content (seed viability reduced to 50%) for the seeds was between 9 and 6%. Germination capacity was maintained for 12 months during storage at 5, 10 and 15ºC, when seeds were at ca. 11% moisture content. A drastic reduction of seed viability occurred after –20ºC exposure. These results suggest that Genipa americana seeds have intermediate behaviour.

Introduction

Genipa americana L. (local name: jenipapo) is a member of the Rubiaceae family that occurs in humid Brazilian ecosystems. The wood of the species is used for many purposes. The Indians use the blue juice made from immature fruits to dye their skin. The mature fruits are edible and used to prepare ice cream, pudding, juice, wine and liquor (Villachica et al. 1996). The seed has a flat and irregular, sometime rectangular, shape. The seed coat is thin and yellowish-brown and the endosperm is yellowish-white. Classifying these seeds into the correct storage behaviour and establishing appropriate conditions for germplasm conservation are important for the long-term conservation of G. americana. The seeds have been found to have a short lifespan, however, there are conflicting reports of actual seed storage category with suggestions of both recalcitrant and intermediate seed storage behaviour (Lorenzi 1992; Carvalho and Nascimento 2000). In this study, the responses to germination temperature, desiccation and storage conditions were investigated. 264 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Materials and methods

Seed collection and extraction

Fruits were collected in 1998 from three trees 1 km apart, in the savannah vegetation at Mangabeira farm (access road BR 080) in the Mato Seco region, state of Goiás. The seeds were extracted by hand. After removing the fleshy pulp by rubbing the fruits in a sieve, the seeds were washed in tap water. The same day, moisture content determinations and desiccation trials were initiated. Fruit and seed weights were determined for 100 individual fruits and their seeds. Initial moisture content was determined for 100 individual seeds and for five replicates of each of five whole seeds, 20 isolated embryonic axes, and 20 isolated endosperms plus seed coats.

Effect of temperature on seed germination

Four replicates of 25 fresh (nondried) seeds were germinated on moistened paper towel, at a range of constant temperatures between 5 and 40°C with 12 h light per day. Samples of seeds were dried at room temperature (25r2qC) by mixing with equal amounts of silica gel. Control samples were placed in similar containers with vermiculite instead of silica gel. The desiccation periods were from 0 up to 72 h. After each desiccation period, MC was determined on five replicates of five whole seeds, and germination tests were carried out with four replicates of 25 seeds each, at 30°C with 12 h light per day.

Seed desiccation and response to –20qC

Further samples of t225 seeds each were dried at room temperature with silica gel, as before, for between 24 and 72 h. After desiccation, moisture content (MC) was determined using five replicates of five whole seeds each and germination tests were carried out with four replicates of 25 seeds, incubating them at 30q& with 12 h light per day. The remaining 100 dried seeds were placed at –20°C for 24 h before sowing for germination at 30°C, as described above. 265 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Storage trials

Seeds treated with fungicide were dried to four different moisture contents. Sub-samples were then mixed with vermiculite, sealed in impermeable bags, and stored at 5, 10 and 15°C for 2, 4, 6, 8, 10 and 12 months. Seeds were regularly taken to test their germination capacity as described above.

Results

Mean fruit and seed weights were 175.5r53.2 g and 0.09r0.01 g, respectively. The initial MC of whole seeds was ca. 44–46% fresh weight (Table 1). For seed tissues, the initial MC was higher for embryonic axes (76%) compared with seed coats and endosperms (37%; Table 1).

Table 1. Initial moisture contents of seeds and seed tissues

Material MCrs.d. (%) Mean of 100 individual seeds 43.8r2.60 Mean of five replicates of five whole seeds 46.0r5.31 Mean of five replicates of 20 embryonic axes 76.5r0.69 Mean of five replicates of 20 endosperms plus seed coat 38.0r0.69

Effect of temperature on seed germination

Maximum germination was achieved at temperatures between 15 and 30°C, while no seeds germinated at 5 and 40°C (Fig. 1). At 10°C, radicles protruded from 97% of the seeds, but only 31% developed into seedlings; at 15, 20, 25 and 30°C, normal seedlings developed within 84, 46, 26 and 22 days, respectively. Radicles of germinated seeds at 35°C were necrosed.

Desiccation trials

High germination percentages were maintained for seeds dried to moisture contents between 47 and 19% (Table 2). A slight decrease in viability (to 78%) was observed for seeds dried to 9% MC, whereas viability was reduced to 29–43% germination in seeds dried to approximately 7% MC. 266 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

100

40 Germination (%)

0 0 5 10 15 20 25 30 35 40 45 50 Germination temperature (°C)

Figure 1. Effect of temperature on the germination of G. americana seeds. Each data point represents mean of four replicates of 25 seeds.

Table 2. Effect of desiccation (MC%) on seed germination capacity (Germ.%)

Desiccation (h) Controls (vermiculite) Desiccated seeds (silica gel) MCrs.d. (%) Germ. (%) MCrs.d. (%) Germ. (%) 0 56.48r1.46 96 — — 3 51.48r1.12 99 47.43r0.90 91 5 53.88r 0.65 97 45.41r1.29 87 8 53.33r2.87 97 43.95r2.16 93 12 54.01r0.24 94 36.79r1.75 97 16 53.12r1.08 100 34.84r1.79 97 20 52.61r0.79 99 25.20r2.29 96 24 50.84r1.70 99 19.23r2.51 92 36 54.49r1.07 99 9.34r1.03 78 48 53.12 r1.39 96 6.88r0.24 43 72 48.31r0.45 100 6.72r0.25 29

Effect of desiccation and exposure to –20°C on seed viability

Low germination percentages (between 0 and 15%) were obtained for seeds at all moisture contents placed at –20°C for 24 h (Table 3). The best result of 15% germination was observed for seeds dried to 8.6% MC, while viability was no more than 3–4% at moisture contents above or below this level. 267 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Table 3. Effect of desiccation and exposure to –20°C for 24 h on seed viability Desiccation (h) MC (%) Germination (%) Before –20qC After 24 h at –20°C 0 51.5r1.10 99 — 24 13.2r2.20 66 3 28 8.6r0.23 61 15 48 7.1r0.24 41 4 52 6.5r0.65 31 1 72 6.3r0.09 33 0

Storage trials

Seed viability decreased during storage at 5, 10, and 15qC (Fig. 2). Rate of loss of viability appeared to be fastest for seeds at 38% MC. However, for seeds at both 42% and 38% MC there was no germination after 12 months storage at 5, 10 or 15q&. High levels of germination (>80%) were maintained in seeds dried to 11% MC and stored for 12 months at 5, 10 or 15q&.

Discussion

Drying seeds of G. americana resulted in a decrease in germination percentage, the critical MC being somewhere between 9 and 6% fresh weight (Tables 2 and 3). Although the seeds showed sensitivity to – 20°C (Table 3), germinability was maintained during 12 months storage at 5, 10 and 15ºC, when seeds were stored with ca. 11% moisture content. These results show that G. americana seeds are not recalcitrant and may have intermediate storage behaviour. Unfortunately, the survival of seedlings in the greenhouse was compromised by Fusarium oxysporum contamination. A preliminary test showed that regeneration of G. americana embryonic axes after desiccation and exposure at –196°C was unaffected by bacteria contamination. Therefore, it is suggested to develop a cryopreservation protocol for embryonic axes of this species. 268 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

100 5 °C 42% MC 10 °C 80 15 °C

02468101214 100 38% MC 80

Germination (%) Germination 20

02468101214 100

60 11% MC 40

02468101214

Storage periods (months)

Figure 2. Germination response of G. americana seeds dried to 42, 38 and 11% MC and stored at 5, 10 and 15°C for 12 months.

References

Carvalho, J.E.U. de and W.M.O. do Nascimento. 2000. Sensibilidade de sementes de jenipapo (Genipa americana L.) ao dessecamento e ao congelamento. Rev. Brasileira Fruticult. 22:53–56. Lorenzi, H. 1992. Árvores Brasileiras: Manual de Identificação e Cultivo de Plantas Arbóreas do Brasil. Nova Odessa, Ed. Plantarum. Pp. 352. 269 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Villachica, H., J.E.U. de Carvalho, C.H. Müller, S.C. Siaz and M. Almanza. 1996. Frytales y hortalizas promisorios de la Amazonia. Lima: Tratado de Cooperación Amazonica – Secretaria Pro-Tempore. Pp. 367 p. 270 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Desiccation, storage and germination of Hancornia speciosa seeds

Antonieta Nassif Salomao

Embrapa Recursos Genéticos e Biotecnologia – PqEB W5 Norte, C.P. 02372 CEP 70849-970 Brasília – DF Brazil

Abstract

When seeds of Hancornia speciosa were incubated at 5 up to 40°C, they did not germinate at 5°C, while maximum germination was obtained at 10°C, indicating chilling sensitivity below this temperature in these seeds. The highest (100%) germination was obtained at 25ºC. Seeds could be partially desiccated to 33% MC without significant reduction in germination and be further dried to 9% MC with great decrease in germination capacity. However, seedling vigour was affected by desiccation, when seeds were dried to or below 26% MC. In the storage experiments, seed viability was not maintained for longer than 2 months at 5 and 10ºC. H. speciosa seed responses to dehydration and storage at low temperatures confirmed its classification as a recalcitrant species.

Introduction

Hancornia speciosa Gomez (Apocynaceae), named locally as mangaba, mangabeira, occurs in low and high frequency in semi-arid and savannah regions of Brazil. The species produces an edible fruit, which can be consumed in natura or used to prepare ice cream, pudding, juice, jam, wine, vinegar and liquor. The seed is a flat and irregular discoid with a central hilum. The seed coat is thin and yellowish- brown and the endosperm is white (FAO 1986; Lorenzi 1992). The seed classified as recalcitrant has a short lifespan (Oliveira and Valio 1992). Recently, the species has been included in breeding programmes, due to its nutritional and commercial values. The establishment of conditions for germplasm conservation becomes a priority to meet breeders´ demands. 271 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Materials and methods

Seed collection and extraction

Fruits were collected in Oct and Nov 1996, and in Nov 1997 and 1998, each harvest composing a seed lot. As shown in Table 1, collections were made from three different locations to obtain enough seeds in 1996 and from only one location in the other years. The collection was made from the ground in grazing-lands in each of the locations. The seeds were extracted from fruits by hand. After removing the fleshy pulp by rubbing the fruits in a sieve, the seeds were washed in tap water. Moisture content determination and desiccation trials were initiated the same day of seed processing.

Initial tests

Fruit and seed weights were determined on 100 individuals. Seeds of lots 1 and 4 were used to determine seed weights, and fruit weights were measured using lot 4. Initial moisture contents were measured on individual seeds (1×100 seeds) of lots 1 and 4, and on samples of whole seeds (5×3 to 5 seeds) of lots 1, 2, 3 and 5. Seed components were also used to measure moisture contents of a sample of 10 excised embryonic axes and endosperms from lots 1 and 4.

Table 1. Seed lots used in the trials Collection Seed lot Provenance 1 54.5 km from Brasília (route to Unaí) 1996 2 Mozondó farm, near Maranhão river, between District Federal and the State of Goiás 3 Vãozinho de dentro farm, 55 km from São João da Aliança municipality, State of Goiás 1997 4 Mozondó farm, near Maranhão river, between District Federal and the State of Goiás 1998 5 Near to Mutuca farm, 60 km from São João da Aliança municipality, State of Goiás

Effect of temperature on seed germination

Germination tests were performed by placing two replicates of 25 seeds on a layer of cotton wool moistened with distilled water, over a 272 STORAGE BIOLOGY OF TROPICAL TREE SEEDS range of constant temperatures between 5 and 40°C (lots 1 and 2), and a photoperiod of 12 h light per day.

Desiccation trials

Seeds were desiccated, mixed with silica gel (4 g silica/1 g seed) at room temperature (25r2ºC), for 0 and 100 h (lot 1), for 0 and 48 h (lot 2), and for 0 up to 92 h (lot 4). After each desiccation period, moisture content was determined on five replicates of five seeds (lots 1 and 2) and on 10 individual seeds (lot 4). Germination tests were carried out using two replicates of 25 seeds of lots 1 and 2, and 4 replicates of 25 seeds of lot 4, at a constant temperature of 25°C, and a photoperiod of 12 h light per day. In a separate trial, seeds from lot 5 were desiccated, mixing them with an equal amount of silica gel. Controls were placed in similar containers with vermiculite in place of the silica gel. Dehydration periods of 0 up to 63 h were determined in line with the results of preliminary desiccation trials. After each desiccation period, moisture content was determined with five replicates of five whole seeds, and germination tests were carried out using four replicates of 25 seeds, at a constant temperature of 25°C and with a photoperiod of 12 h light per day.

Storage trials

After desiccation and fungicide application, samples of seeds of lot 5 were mixed with vermiculite and sealed in impermeable bags. Seeds desiccated to 52.5, 50.7 and 47.7% MC were stored at 5°C, and seeds with 49.0 and 38.5% MC were stored at 10°C, storage at both temperatures lasted for 2, 6 and 12 months.

Results

Initial tests

Fruit weights varied greatly within the same population, whereas there was a smaller variation in seed weights (Table 2). Initial moisture contents were high, around 50% for all seed lots (Table 3). 273 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Effect of temperature on germination

Of all tested temperatures, H. speciosa seeds did not germinate at 5°C, while maximum germination was obtained at 10°C. High germination percentages of 88 to 100% were obtained for seeds incubated at 10 to 30°C, above which temperature viability declined to 58 and 4% (Fig. 1). However, it has been observed that seeds initiated germination at 10, 15, 35 and 40°C, with only radicle protrusion but not normal development of seedlings. This occurred only with seeds germinating at 20, 25 and 30°C.

Table 2. Mean weights of 100 individual seeds and fruits from lots 1 and 4

Material Weight (grsd) 100 seeds ( lot 1) 0.228r0.052 100 seeds ( lot 4) 0.184r0.063 100 fruits ( lot 4) 42.188r18.192

Table 3. Mean initial moisture contents of seeds and seed components

Material Moisture contentrsd (%) 100 individual seeds ( lot 1) 51.13r3.61 100 individual seeds ( lot 4) 55.69r8.23 5×5 whole seeds ( lot 1) 52.90r1.54 5×3 whole seeds ( lot 2) 51.53r0.99 5×3 whole seeds ( lot 3) 53.74r1.80 5×5 whole seeds ( lot 5) 50.63r1.10 10 individual embryonic axes ( lot 1) 78.08r3.51 10 individual endosperms ( lot 1) 48.68r4.60 10 individual embryonic axes ( lot 4) 77.53r4.22 10 individual endosperms ( lot 4) 45.57r6.33

Desiccation trial

Table 4 and Figure 2 present the effect of desiccation of H. speciosa seeds from different lots. Germination percentage decreased after drying seeds to ca. 25% MC, and no seed germinated at 7% MC and below. The critical moisture content for the onset of viability loss seemed to be around 30%. Although some seeds germinated at lower moisture contents, reduced vigour was observed in seedlings from seeds dried to 25% MC and below (see Table 4). High moisture content was maintained in the control seeds in vermiculite, which also germinated over 80% on average. 274 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Storage trial

Storage at 5°C led to approximately 50% germination or less, whereas more than 70% were obtained at 10°C after 2 months storage. Only 5% of seeds with 46% MC germinated after 6 months at 10°C. No other seed germinated after storage for 12 months, irrespective of conditions (see Table 5).

100

40 Germination (%) Germination

0 10203040 Germination temperature (°C)

Figure 1. Effect of germination temperature on H. speciosa fresh seeds from lot 1.

Table 4. Effect of desiccation using an equal amount of silica gel (g g–1 seed), on the viability of seeds from lot 5

Desiccation Vermiculite (control) Silica gel (drying) Period (h)

MCrsd (%) Germination MCrsd (%) Germination Observations (%) (%) on seedling vigour 0 50.6r1.10 80 — — — 4 48.8r0.88 88 46.7r1.75 86 — 12 45.3r1.98 90 32.9r1.95 93 — 20 47.1r0.85 86 25.6r2.44 75 Reduced 24 47.1r1.99 89 19.2r1.40 62 Reduced 28 45.5r2.20 85 18.9r2.36 70 Reduced 44 45.8r2.91 83 9.1r1.27 23 Reduced 51 46.9r1.23 78 7.2r0.84 0 — 68 44.7r3.88 81 5.9r0.37 0 — 275 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

100 seed lot1 seed lot2 seed lot3 80

40 Germination (%)

0 102030405060 Moisture content (%)

Figure 2. Relationship between moisture content and germination of H. speciosa seeds.

Table 5. Seed germination (G) response to storage conditions at 5 and 10°C for 12 months

Storage MCrsd Initial G Storage period temp. (%) (%) 2 months 6 months 12 months G (%) MC (%) G (%) MC (%) G (%) 52.54r3.8 80 40 9.52± 0.27 0 38.70± 1.14 0 6 5°C 51.12r3.8 84 58 9.52± 0.27 0 36.88± 0.99 0 6 47.69r2.6 86 36 39.55± 0.93 0 13.90± 1.47 0 3 10°C 48.99r2.9 84 90 46.27± 3.12 5 45.46± 2.87 0 7 38.46r2.9 82 73 40.29± 3.88 0 40.76± 3.61 0 1 276 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Discussion

Seeds of H. speciosa did not germinate at 5°C, while maximum germination was obtained at 10°C, indicating chilling sensitivity in these seed species. Seeds could be partially desiccated to ca. 30% MC without significant reduction in germination and be further dried to 9% MC with great decrease in germination capacity. However, seedling vigour was affected by desiccation, when seeds were dried to or below 26% MC. In the storage experiments, seed viability was not maintained for longer than 2 months at 5 and 10°C. During germination, fungal infection compromised the capacity of seeds. The principal identified fungi were Fusarium oxysporum, Penicillium sp, Periconia sp, Rhizopus sp and Torula sp. However, a preliminary test showed that seed tissues were not affected by bacteria contamination during regeneration of embryonic axes after desiccation or desiccation followed by exposure at –196°C. It is therefore suggested to develop a cryopreservation protocol for the ex situ conservation of this species.

Conclusion

Hancornia speciosa seeds cannot withstand desiccation below 30% MC, which should be avoided. Storage conditions, including temperatures above 10°C need to be further investigated. On the basis of the present results, it must be recommended to avoid germination or storage below 10°C.

References

FAO. 1986. Food and Fruit-Bearing Forest Species 3: Examples from Latin America. Forestry Paper 44/3. Rome, Italy. Pp. 149–151. Lorenzi, H. 1992. Árvores Brasileiras: Manual de Identificação e Cultivo de Plantas Arbóreas do Brasil. Nova Odessa, Ed. Plantarum. Pp. 352. Oliveira, L.M.Q. and I.F.M. Valio. 1992. Effects of moisture content on germination of seeds of Hancornia speciosa Gom. (Apocynaceae). Ann. Bot. 69:1–5. 277 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Desiccation and storage of Cariniana pyriformis seeds

Javier Rodríguez Romero

Corporación Nacional de Investigación y Fomento Forestal – CONIF, Paseo Bolivar No.16-20 (Detrás del Instituto Roosevelt) Bogota, Colombia

Abstract

Cariniana pyriformis seeds from Colombia have been desiccated and stored at different conditions. The results showed that the initial germination of seeds at 12.9% MC was 42%. When these seeds were dried down to 3% MC, germination increased to 54%. Seeds with different moisture contents maintained viability after storage at different temperatures. C. pyriformis seeds with 12.8% MC retained a constant 35% germination after storage at 18°C for 1 and 2.5 months. The lowest germination capacity was around 7% for seeds stored at 30°C. Although the storage period was short, it seems clear that these seeds maybe storable for the long term in dry conditions and at low (cool) temperatures.

Introduction

Cariniana pyriformis Miers, or abarco belongs to the Lecythidaceae (Brazil-nut family). It is naturally distributed in Panama and Colombia. In Colombia the species grows in the three mountain ranges that extend through the country. Other species within the same genus are found in Costa Rica, Brazil, Peru, Paraguay, Bolivia and Trinidad— Tobago. It is also called Colombian mahogany, and its timber is highly valued in Colombia because of its good finish and multiple uses. The timber is very durable and resistant to fungi and insects. Being easy to work, it is used for construction, furniture, pencils and boards. The exploitation of its timber has led to genetic erosion to such an extent that it is now considered endangered. The species is qualified for a vulnerable status on the IUCN Red List of Threatened Species (IUCN 2002). It has become necessary to take action to conserve the species and its genetic resources, and in addition, to find improved methods for its propagation. The abarco reaches up to 30–40 m in height and grows to a diameter of up to 2 m. The trunk is fissured and dark brown, with 278 STORAGE BIOLOGY OF TROPICAL TREE SEEDS bark flaking off in large patches. The crown is umbrella-shaped. The species is deciduous, i.e. the leaves are shed at the beginning of the cold or dry season. Flowers are white, hermaphroditic, with 5–6 petals and 5–6 sepals and borne in terminal or axillary panicles. The androecium has numerous fertile stamens that are fused with the petals. Ovary inferior is composed of three locules each containing several ovules. The fruit is woody, pear-shaped or oval, opening with a lid. The species propagation is possible vegetatively as well as by seed. Cuttings taken from the middle part of the crown are used for vegetative propagation, rooting to about 75%. When propagated by seeds, a germination of about 50% is easily achieved. The species is a semiheliophyte growing in primary and late secondary forests. It is planted at a distance of 4×4 m to achieve an average growth of 6 m3 ha–1 y–1 (Lastra 1971). In Colombia, flowering takes place in Nov–Dec, while fruiting is in Jan–Mar. However, with great variations in fruit production in this species, it can be difficult to predict when collection should take place. Seeds are mature when the fruit has turned dark brown and the lid (operculum) begins to come loose. Six seed production areas have been identified in Colombia, all in the northern part of the country. When the capsules open, the seeds are widely dispersed making it necessary to collect the fruits before they open. However, collection from the tree is difficult because of the high height of trees and the fruits are situated at the end of the branches. Thus, fresh fruits are often collected from the ground.

Materials and methods

Seed collection and processing

Fruits were collected from an identified source of one ha within a 600 ha forest dominated by C. pyriformis. The ground under the 20 trees was carefully cleaned before collecting fruits on the 13 Nov 2001. The fruits were packed in linen bags and transported to the laboratory by air within two days of collection. The containers were perforated to allow exchange of air during transport and temporary storage. Temperatures during (air) transport were estimated to fluctuate between 8 and 25°C. Fruits were processed by half shade drying until the capsules opened to release seeds. The fruits that did not open were considered 279 STORAGE BIOLOGY OF TROPICAL TREE SEEDS as immature and discarded. As far as it was possible, fruits and seeds were protected from excessive temperatures and mechanical damage.

Diagram of seed processing:

Drying under Fruit collection Pre-cleaning half sun, half shade

Cleaning and selecting

Seed extraction

After extraction, all damaged or infected seeds were discarded. Finally the seeds were treated with 1% NaOCl for 50 seconds and then rinsed with water. Processed seeds were then kept in hermetically closed containers at ambient temperature (8–14°C) for 15 days, the delay caused by lack of staff to carry out experiments immediately. Seed characteristics were examined and seed weight was determined on eight replicates of 100 seeds. The unit for all trials was the winged seed (including the testa, which is difficult to remove without damaging the seed). Initial determinations of seed purity and seed weight were carried out according to ISTA rules (1999)

Desiccation and germination

Seeds were mixed with silica gel to desiccate in plastic bags. The controls were put in plastic bags without silica gel. After desiccation, seed samples were weighed before and after drying in oven at 103°C for 17 h. The moisture content was then calculated as a percentage of the fresh weight (IPGRI/DFSC 1999, 2000). Four replicates of 25 seeds were used for each germination tests. Seeds were sown in soil and river sand (1:1) at a photoperiod of 12/12 hours light/dark and at constant 25°C. Germination data were analyzed for their differences using Duncan’s probability test at 5%. 280 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Storage trials

Seeds dried to different moisture contents were sealed in aluminium bags and stored at 4, 18 and 30°C and at ambient temperature of an average 12°C (between 8 and 14°C). Controls at their initial MC were divided into four samples for storage in closed containers at ambient temperature (12°C), 4, 18 and 30°C.

Results and discussion

Initial trials

The mean weight of C. pyriformis fruit was 100 g and there were on average 15 seeds per fruit. After processing, seed purity was determined to be 99.5% (Table 1). There were 8568 seeds per kg, making a thousand seed weight (TSW) about 116.7 g, which is within the range of that found in previous studies (Betancur and Raigosa 1973).

Table 1. Initial characteristics of C. pyriformis fruits and seeds Parameters Values Mean fruit weight 101 g No. seeds/kg fruits 150 seeds Purity 99.4% Thousand seed weight 116.7 g Number of seeds/kg 8568 Initial MC 12.92%

The winged seeds were black when moist and coffee-coloured when dried. Their coat consisted of a layer of testa, which was hard and dry, and of a thin and semi-transparent tegument. Seeds had low initial moisture content of 8.3% (Table 2), although immediately after leaving in the shade, mature fruits had between 10 and 20% MC. Green fruits that were not used in these trials had a higher initial moisture content (36%). The 8.3% MC increased to 12.9% MC (Table 1) after the NaOCl treatment, which was the initial moisture content for all experiments. Seeds with 12.9% initial MC were then dried to 7.8, 6 and 3% MC. 281 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Table 2. Initial moisture content and mean weights of 100 seeds and excised components (embryo and testa) Fresh weight (g) Dry weight (g) MC (%) Seed 11.67±0.58 — 8.3±0.27 Embryo 9.02 8.44 6.4 Testa 3.20 2.77 13.4

Effect of moisture content on germination

Several initial samples were desiccated to different moisture contents (Fig. 1). The moisture content of 7.8 and 6% were reached within about 24 h, while drying seeds down to 3% MC required 4 days (Fig. 1). C. pyriformis seeds initially germinated to 42%. However, there was a significant increase of the germination percentage with reduction of the seed moisture content, attaining 53–54% when seeds were dried to 6 and 3% MC (Table 3). The initial germination percentage of ca. 50% is in the range reported by others studies (Rodríguez 2000). The effect of moisture content on germination was significant, after 1 and 2.5 months of storage (Table 3). In general, there was a decrease of germination after storage, but not complete loss of viability. Germination was maintained at 21%, the same level in seeds with 3% MC after 1 and 2.5 months. Seeds with 12.9% MC germinated to 24%, the highest percentage of all conditions after 2.5 months (Table 3).

Storage trials

All seed samples maintained viability after storage at different conditions. Seeds with 6% MC germinated 49% after one month storage at 18°C, but decreased to about 30% in the second month. C. pyriformis seeds with 12.8% MC retained constant 35% germination after storage at 18°C (Table 4). Seed stored at ambient temperature (average 12°C) and at 4°C maintained their viability during the 2.5 months storage. The lowest germination capacity was around 7% for seeds stored at 30°C. Although the storage period was short, it seems clear that these seeds maybe storable for the long term in dry conditions and at low temperatures. 282 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Table 3. Effect of moisture content on seed germination capacity after storage. Data followed by the same letter in column are not significantly different (Duncan probability test at 5%)

MC (%) Germination Onset (days) End (days) Initial (%) 1 month (%) 2.5 months (%) 12.92 13 24 42 26.251 24.501 7.89 12 23 28 17.502 14.002 6 10 21 53 28.0011 19.252 3 8 20 54 21.001 21.001

10 7.8 % MC 6 % MC 3 % MC 8

4 Moisture content (%)

0 0 102030405060708090100 Drying time (hours)

Figure 1. Desiccation time of C. pyriformis seeds to target moisture contents (MC)

Conclusions

C. pyriformis seeds tolerate desiccation to 3% MC and low (cool) temperature storage. Although the results presented are an evaluation over a short period, they show significant indications as to possible longer-term storage conditions of this species. For short-term storage (at least 3 months), seeds with low moisture content can be stored at a range of temperatures below 20°C. It is recommended that further investigations assess the effects of desiccation and storage conditions on C. pyriformis seeds from other provenances. 283 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Table 4. Germination capacity after storage of C. pyriformis seeds at four different conditions of moisture contents and temperatures for 2.5 months. Data followed by the same letter in column are not significantly different (Duncan probability test at 5%)

MC (%) Storage (°C) Germination (%) 1 month 2.5 months 4 14.4a 14.0b 3 18 35.1a,b 32.0a,b 30 14.4b 7.14a Ambient (~12) 21.2a,b 20.9a,b 4 14.3b 14.2a 6 18 49.0b 29.3a,b 30 14.4b 7.3a Ambient (~12) 35.0a,b 28.5a,b 4 21.1a,b 21.0a,b 7.8 18 21.2a,b 14.0b 30 21.2a,b 7.0a Ambient (~12) 7.5a 7.21a 4 21.0a,b 20.1a,b 12.8 18 35.1a,b 35.0a 30 35.2a,b 7.0a Ambient (~12) 14.3b 14.0b

Acknowledgement

The author thanks Mrs Dorthe Jøker and Dr Moctar Sacandé for the English translation and the useful comments on this article.

References

Betancur, P.G. and E.J. Raigosa. 1973. Características y Propiedades Germinativas de las Semillas de Abarco (Cariniana pyriformis). Revista Nacional de Agronomía, Medellín, Colombia. International Seed Testing Association (ISTA). 1999. International rules for seed testing, 1999. Seed Science and Technology. Zurich, Switzerland. Pp. 333. IPGRI/DFSC. 1999. The project on handling and storage of recalcitrant and intermediate tropical forest tree seeds. Danida Forest Seed Centre. Newsletter No. 5:23–39. 284 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

IPGRI/DFSC. 2000. Protocolos de Desecación y Almacenamiento de Semillas. IUCN. 2002. 2002 IUCN Red List of Threatened Species [also at http://www.Redlist.org]. Lastra, R.H.A. 1971. Revisión de Literatura Sobre el Abarco, Cariniana pyriformis. Miers, Bogotá. INDERENA. Pp. 13. Rodríguez, R.J. 2000. Protocolos de Germinación de Especies Forestales. CONIF, Bogotá. Pp. 75. 285 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Desiccation and storage of seeds of Astronium graveolens and Calophyllum brasiliense, two native species of Costa Rica

William Vasquez1, Kirsten A. Thomsen2 and Dorthe Jøker2

1Banco de Semillas Forestales, CATIE, 7170 Turrialba, Costa Rica 3Forest & Landscape Denmark, Hørsholm Kongevej 11, DK-2970 Hørsholm, Denmark 2The State Forest Tree Improvement Station, Krogerupvej 21, 3050 Humlebaek, Denmark

Abstract

Effects of desiccation and storage were investigated for Astronium graveolens and Calophyllum brasiliense seeds. A. graveolens seeds desiccated to 6.6% MC germinated 89%. Seeds with d8.4% MC stored well for 12 months at –18, 5 and 15°C, whereas seeds with t9.9% MC lost viability fast at –18 and 5°C. Fresh A. graveolens seeds should therefore be dried below 9% MC and stored at low temperatures. C. brasiliense seeds were sensitive to desiccation and low temperatures. Drying seeds with an initial 40% MC and 68% germination to 4.8% MC reduced germination capacity to 4%. Best storage result, 70% germination after three months, was obtained at 15°C for seeds at 32% MC. After 6 months of storage all seeds were dead. It is thus recommended to sow these seeds as soon as possible, or store them for a few months at >30% MC at 15°C.

Introduction

Costa Rica is a small country, which covers an area of 52 100 km2, localized in the middle of Central America between 10qN latitude and 84qW longitude. At the Atlantic coast the rainfall varies from 1500 to 5000 mm, with 2 to 3 dry months (less than 50 mm) per year, while at the Pacific coast the annual rainfall varies from 1500 to 3000 mm, with a drier period of 4 to 6 months. About 23% of Costa Rica is under protection by national parks. Astronium graveolens Jacq. and Calophyllum brasiliense Cambess., were selected for this study because they are used and grow well in plantations and produce good timber (CATIE 2000). Both species are difficult to reproduce and better methods for seed handling are needed. Flores (1993, 1996) and Sanchez (1995) described these species as being recalcitrant. 286 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Astronium graveolens Jacq. from the Anacardiaceae family, is known as ‘ron ron, jocote or jobillo’ in Costa Rica. The species grows naturally from Mexico through Central America and south to Brazil, Bolivia and Paraguay. In Costa Rica, it grows from 100 to 600 m above sea level in areas with annual rainfall from 1300 to 3000 mm. The wood is very durable, fairly easy to work and has a specific gravity of 0.85–1.28g cm–3. It is used for furniture, floors, tool handles, cabinets and paper production (DFSC 2000a). The flowers are hermaphrodite, small, with five green-yellow petals; grouped in 10–25 cm long terminal or auxillary panicles. In Costa Rica they appear during the drier season between December and March. It is difficult to time the collection of seeds as the fruits mature rapidly, three to four weeks after pollination. The fruits are drupe-like nuts, blue to black at maturity. The single seed is enclosed in a bitter-sweet pulp. The seeds lack endosperm and have high oil content. There are about 18 000 clean seeds per kg (DFSC 2000a). Calophyllum brasiliense Cambess. from the Clusiaceae family is called ‘maría’ or ‘santa maría’ in Costa Rica. The natural range includes southern Mexico, Central America and northern South America. It is also found in the Antilles from Cuba to Jamaica and Trinidad – Tobago. In Costa Rica, this species grow naturally at the Atlantic cost from sea level to 900 m, where annual rainfall reaches over 2500 mm and mean temperature varies between 24 and 28°C. The tree is up to 45 m tall, with straight bole without buttresses and branchless for about two thirds of the height. The bark is thick and contains a yellow-green latex. The wood of C. brasiliense is used for both outdoor and indoor construction and is durable in contact with soil and water. The species is andromonoecious, i.e. each tree has both male and bisexual flowers (DFSC 2000b). The trees flower twice a year, between June and July and between November and December. The fruits are green at maturity, but the colour becomes less bright as they ripen. The fruits are, more or less round, berries 2.5 to 3 cm long. Each fruit contains one seed, the pericarp is leathery and dotted with numerous laticifers containing yellow latex. The seed is 1.8–2.3 cm long with large oily cotyledons. There are 415–440 seeds per kg (DFSC 2000b). Despite their uses, little is known on the biology of A. graveolens and C. brasiliense seeds. We report the results obtained from our investigations on these two species since 1999. 287 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Materials and methods

Seed collection and processing

At the beginning of 1999, CATIE Tree Seed Bank (CTSB) identified seed stands, collected and made seeds of A. graveolens and C. brasiliense available for desiccation and storage trials for both CTSB and DFSC as the replicating partner. Fruit collection occurred in accordance with the screening protocol (DFSC/IPGRI 1999). A. graveolens fruits were collected from eight trees of the Volcán, P. Zeledón (BL095) source in Costa Rica on April 13–14 1999. The fruits were manually collected from the tree by cutting off branches with pruning shears. C. brasiliense fruits were collected in the same way in Buenos Aires, Puntarenas (BL096) source in Costa Rica on 15 April 1999. A second collection of this species was made on 12 May 2000. Seeds were manually extracted. The calyx of A. graveolens fruits was removed manually, and the exocarp of C. brasiliense fruits was removed by rubbing the fruits over a wire mesh (see photo). After extraction, part of the seeds was used for experiments in the laboratory of CATIE in Costa Rica and another part was sent to DFSC in Denmark, arriving the same week.

Moisture content and germination

For both species, seed moisture content was determined on two to five replicates of 20–25 seeds. Seeds were weighed before and after drying in an oven at 103°C for 17 h. Moisture contents were expressed on fresh weight basis [r standard deviation (sd)]. At CATIE, four replicates of 25 seeds were germinated in sand at 30°C with constant light. At DFSC, four replicates of 50 A. graveolens seeds were germinated on top of blotter paper at 30°C with 12 h light and 12 h dark, while four replicates of 25 C. brasiliense seeds were germinated on vermiculite at 28°C, with 10 h light and 14 h dark. At the end of these tests, seeds that did not germinate were cut to determine whether they were rotten, fresh or empty. Germination percentages and standard deviations of the means were then calculated for each test. 288 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Desiccation trials

To determine the response to desiccation, seeds were mixed with silica gel to dry to different moisture contents and then tested for germination. After determination of initial moisture contents and weights, seeds were monitored for target weights corresponding to specific moisture contents that were calculated following the protocol (DFSC/IPGRI 1999). A. graveolens seeds were spread out in one layer in net bags containing silica gel to dry. C. brasiliense seeds were mixed with silica gel (2:l) in plastic containers. After desiccation and moisture content determination, samples of seeds were germinated.

Storage trials

At CATIE, samples of desiccated seeds of A. graveolens were sealed in plastic and aluminium bags and stored at 15, 5 and –17°C for 2, 4 and 6 months. Samples of dry seeds were stored at 15°C for 12 months. At DFSC, samples of seeds with different moisture contents were sealed in aluminium bags and stored at 15, 5 and –18°C for 2, 4, 6 and 12 months. After each storage period the moisture contents were again determined and seeds were germinated. For the first trials on C. brasiliense at CATIE, seeds with various moisture contents were stored in perforated plastic bags at ambient temperature of 24–28°C, at 15 and 5°C for 1, 3 and 6 months. The second storage trials were performed with seeds at two high moisture contents. These were also stored in plastic bags for 6 months.

Results

Desiccation and germination

Astronium graveolens. Initial germination of A. graveolens seeds after collection in Costa Rica was 89.0r3.3%, and the moisture content after extraction was 36.6r1.2%. These seeds were dried down to 9.9, 6.4, 3.3 and 1.4% MC at approx. 28°C. Desiccated seeds generally germinated less than the controls, percentages ranging from 55 to 83%. Seeds dried to 1.4% MC, the lowest moisture level germinated 83%, the highest percentage in this trial (Table 1). However, great variations expressed by high standard deviations (3–17%) were observed between the means of replications. On arrival in Denmark, the seeds germinated 289 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

65% and had 22% MC (Table 1). The seeds were then dried down to 18.3, 13.0, 8.4, 5.8 and 4.2% at 23°C. Seeds desiccated to 4.2% MC germinated 59%, and there was no significant difference (see standard deviation) between germination percentages in the DFSC trials.

Table 1. Germination capacity (G%rsd) after desiccation of A. graveolens seeds to various moisture contents at CATIE and DFSC CATIE DFSC MC (%) Drying (h) G (%) MC (%) Drying (h) G (%) 36.6r1.2 Initial 89r3.3 21.9r1.0 Initial 64.8r11.5 13.4 — 55r9 18.3r1.5 2 69r5 9.9 — 60r17 13.0r1.1 3.8 63r6 9.4 — 76r11 8.4r0.5 10.6 61r12 6.4 7.5 72r11 5.8r0.5 23.7 69r13 3.3 32.0 68r13 4.2r0.3 101.6 59r10 1.4 — 83r16 — — —

Calophyllum brasiliense. Initial germination of C. brasiliense seeds for the 1999 collection in Costa Rica was 60% and the moisture content after extraction was 43.5% (Table 2). Seeds desiccated to 4.8% MC resulted in a decrease of their germination capacity from 60% to only 4%. This was an indication of great sensitivity to desiccation (Table 2). On arrival in Denmark, seed germination had dropped to 0% with 34% MC. No further action was undertaken on this seed lot.

Table 2. Germination capacity of C. brasiliense seeds (collection 1999) after desiccation to various moisture content (%rsd) at CATIE

Control Dried Target MC (%) Actual MC (%) G (%) Drying to MC (h) Actual MC (%) G (%) Initial 43.5r0.9 60r22.4 — — — 25 40.3r0.6 53r15.1 72 26.3r0.6 37r3.8 20 39.2r0.4 68r14.2 144 21.3r0.9 22r4.0 10 40.0r1.0 64r18.8 216 10.5r0.5 16r14.2 5 41.7r0.8 76r8.0 360 4.8r0.6 4r0.0

For the second desiccation trial of C.brasiliensis (collection 2000), seeds with 34% MC for the second trials had >90% initial germination. There were no significant differences (Pd0.05) in viability, when seeds were dried to 28% MC, germinating 92% (Table 3). C. brasiliense seeds started germinating 3–4 weeks after sowing. Peak germination was observed between 5 and 10 weeks and the total germination period covered 3 months at CATIE and 290 STORAGE BIOLOGY OF TROPICAL TREE SEEDS five months at DFSC (Table 3, Fig. 1). Drying delayed the germination onset, starting 3 weeks later for 28% MC seed compared with the control at 34% MC (Fig. 1).

Table 3. Germination (G% r sd) after desiccation of C. brasiliense seeds (collection 2000). Mean germination time and germination of controls (*) are also given for the experiments at DFSC CATIE DFSC Drying (h) MC (%) G% MC (%) Mean G time G% (weeks) Initial 56.0r1.3 86r7.6 33.6r1.1 9.09r0.8 95r3.8* 1.00 31.8r1.2 91r8.9 30.9r1.6 8.79r0.9 91r5.0 3.67 29.8r0.9 91r3.8 34.0r0.3 8.11r0.7 98r2.3* 5.33 28.4r0.9 94r5.2 27.6r0.7 10.57r0.6 92r3.3 Control 33.0r0.9 94r5.2 34.4r0.8 7.57r0.4 94r5.4*

100

40 Germination (%) 30 33.6% (fresh) 30.9% 20 Control (30.9%) 27.6% 10 Control (27.6%) 0 0 5 10 15 20 Time (weeks)

Figure 1. Germination of C. brasiliense seeds at different moisture contents at DFSC (the 2000 collection). 291 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Storage

In the CATIE trials, A. graveolens seeds at 9.9% MC generally lost viability faster than those with d6.4% MC (Fig. 2). Seeds with 1.4% MC stored at 15°C for 12 months at CATIE germinated 41% after storage in plastic bags, and 56% after storage in aluminium bags (data not shown). In the DFSC trials, storage results of A. graveolens seeds could be separated into two groups. Seeds with d8.4% MC stored relatively well, with only a slight decrease in their germination capacity, and other seeds with t13.0% MC that rapidly lost viability, particularly at 5 and –18°C (Fig. 2). Storage experiments of the first collection of C. brasiliense resulted in a better germination of seeds with >26% MC and at ambient temperature (ca. 26°C), but they were heavily attacked by fungi already after one month. It was clear that C. brasiliense seeds were sensitive to desiccation and to low temperatures (Fig. 3).

Discussion

A. graveolens seeds showed great tolerance to desiccation. They could be desiccated to 1.4% MC, maintaining initial viability (Table 1). They also retained similar viability after 6 months storage at CATIE and 12 months at DFSC (Fig. 2). Poor storage results for seeds with the highest MC at low temperatures, indicate MC dependent sensitivity to low temperatures. Recommendations for optimal storage conditions are therefore to dry below 9% MC and store at low temperatures. C. brasiliense seeds had a very slow germination, covering 5 months (Fig. 1, Table 3) and they were sensitive to drying and low temperatures. Seeds desiccated to 4.8% MC resulted in a decrease of their germination capacity from 60 to only 4% (Table 2). Lines (2001) reported that control seeds germinate faster than dried seeds. This could be due to the difference in initial moisture contents and/or a reduction of vigour in the desiccated seeds. With a safety MC around 30%, the seeds are difficult to store because fungi will attack them. To prevent heavy fungi attack as those observed in trials at both DFSC and CATIE, it would be necessary to improve the disinfecting procedures and/or to store small quantities of seeds in order to reduce the heating by respiration. Transport conditions should be also controlled to avoid receiving dead seed, as was the case for the first collection of C. brasiliense, which arrived at DFSC with 0% germination, perhaps due to exposure to low temperatures. 292 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Storage at CATIE Storage at DFSC 100 100 9.9% - P 21.9% 15°C 6.4% - P 15°C 18.3% 80 3.3% - P 80 13.0% 1.4% - P 8.4% 9.9% - A 60 60 5.8% 6.4% - A 4.2% 3.3% - A 40 1.4% - A 40 Germination% 20 Germination% 20

0 0246 0 024681012 Storage (months) Storage (months) 100 100 5°C 5°C 80 80

60 60

40 40 Germination % Germination 20 Germination% 20

0 0246 0 024681012 Storage (months) Storage (months)

100 100 -17°C -18°C 80 80

60 60

40 40 Germination% Germination % 20 20

0 0 0246 024681012 Storage (months) Storage (months)

Figure 2. Germination of A. graveolens seeds with different moisture contents stored in plastic (P) and aluminium bags (A) for 6 months (CATIE trials). Seeds with different moisture contents were also stored in aluminium bags for 12 months at DFSC (right side graphs). SOUTH AND CENTRAL AMERICA 293

100 Amb. 31.8% 90 Amb. 29.8% 80 15 °C 31.8% 15 °C 29.8% 70 5 °C 31.8% 5 °C 29.8% 60

Germination (%) 30

0 01234567 Storage (months)

Figure 3. Germination after storage of C. brasiliense seeds with 30 and 32% MC at three temperatures for 6 months at CATIE, Costa Rica (the 2000 collection).

Collaboration and future activities

Working within this project was a great experience for CATIE Tree Seed Bank, which gave opportunity to collaborate with seed scientists around the world. Today there are trained people, e.g. a graduate student (see Lines 2001), for screening tree seeds, and the IPGRI/DFSC protocol (1999) is taught as a module to other seed biologists and technicians in Latin America. However, we suggest to include scientific report writing in a future project. The partnership with DFSC has been very enriching. Practice has improved the implementation of the screening protocol (DFSC/IPGRI 1999), which will be necessary to include studies on maturity determination procedure. For some recalcitrant species, it will be necessary to continue the research activities as a part of conservation strategies. For many endangered native species, it will be necessary to start ex situ conservation strategies, establishing seed stand and cryo-conservation research. We propose to create a Recalcitrant tree seed Network that will maintain research communication between scientists and produce scientific publications. 294 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

References

CATIE. 2000. Manejo de semillas de 100 especies forestales de América Latina. Pp. 204. Manual Técnico No. 41. (R. Salazar, ed.). CATIE, Turrialba, Costa Rica. DFSC/IPGRI. 1999. The project on handling and storage of recalcitrant and intermediate tropical forest tree seeds. Danida Forest Seed Centre. Newsletter No. 5:23–39. DFSC. 2000a. Astronium graveolens Jacq. Seed Leaflet No. 32. Danida Forest Seed Centre. DFSC. 2000b. Calophyllum brasiliense Cambess. Seed Leaflet No. 46. Danida Forest Seed Centre. Flores, E.M. 1993. Calophyllum brasiliense. Trees and Seeds from the Neotropics. Vol. 3, No. 1. Museo National de Costa Rica, Costa Rica. Flores, E.M. 1996. Recalcitrant tree seed species of socio-economic importance in Costa Rica: state of knowledge of physiology. Pp. 136–143 in Intermediate/Recalcitrant Tropical Forest Tree Seeds (A.S. Ouédraogo, K. Poulsen and F. Stubsgaard, eds.). IPGRI, Rome, Italy. Lines, G.K. 2001. Estudio de valoración de semillas de cedro maría (Calophyllum brasiliense) y de aspectos asociados a su germinación. Thesis Bach. Universidad de Costa Rica, Turrialba, Costa Rica. Pp. 56. Sanchez, J.J. 1995. Aspectos de fisiología de la germinación y almacenamiento de semillas de importancia forestal. Pp. 165–168 in Memorias del II Simposio Avances en la Producción de Semillas Forestales en América Latína. Managua, Nicaragua, del 16 al 20 de Octubre de 1995. SOUTH AND CENTRAL AMERICA 295

Drying and storing Hieronyma alchorneoides fruits

William Vasquez1, Rodolfo Salazar1 and Kirsten A. Thomsen2

1CATIE, 7170 Turrialba, Costa Rica 2The State Forest Tree Improvement Station, Krogerupvej 21, 3050 Humlebaek, Denmark

Abstract

Hieronyma alchorneoides fruits with an initial moisture content of 52.2r0.3% and an initial viability of 64r11.8% were desiccated to 3.3% moisture content without loss of viability. Fruits with four moisture contents between 10.8 and 3.3% were sealed in plastic and aluminium containers and stored at 15, 5 and –18°C. Best results were obtained at 5 and 15°C for fruits stored in sealed plastic bags. After 3 months of storage approximately 50% germination was obtained, however, by 6 months, viability had decreased significantly.

Introduction

In Latin American countries, more attention is now given to indigenous forest species than previously. One species of interest is Hieronyma alchorneoides Allemao (pilon) (family Euphorbiaceae) from the very humid tropical forest. It is found up to 900 m above sea level, with an annual rainfall between 2000 and 6000 mm and a mean temperature of 20 to 26°C (Franco 1990). Its distribution extends from Mexico to the Amazonian region and the Antilles. The trees reach up to 45 m tall and 1.2 m of diameter at breast height. The wood is hard (0.63 g/cm3) and is mainly used for construction. The fruits are small (ca. 26 500 per kg; 2.5–5.5 mm diameter) oily drupes. The seeds are very small, endospermic and enclosed in a stony endocarp (Flores 1993; Salazar 1997; DFSC 2000). There are few reports about the storage physiology of H. alchorneoides fruits. According to Flores (1993) they can be kept for at least 10 days if moisture and temperature are adequate (not specified). COSEFORMA (1998) recommends moisture contents between 5 and 10% at 4°C for storage of the fruits (50% viability at 6 months). The purpose of this study was to define the storage physiology of the fruits and to identify optimal storage conditions. 296 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Materials and methods

Fruit collection and processing

Fruits were collected on Jan 8 and 9 1998 from six trees in PINDECO farm (source No. BL083), Volcan, Puntarenas province (Table 1). At the time of collection, the fruits had changed in colour from green to purple and red, and some had already fallen from the trees. The fruits were removed manually from the clusters and the pulp extracted by manually rubbing the fruits in a sieve. The fruit of H. alchorneoides can be depulped, but usually, including most of the present experiments, this has not been done. Although each fruit contains 3 to 6 seeds, the whole fruit was regarded as one germinative propagule. The fruits were treated with a fungicide (Vitavax) and 1 kg of fruits was sent to Danida Forest Fruit Centre (DFSC), arriving on Jan 20 1998.

Table 1. Site characteristics and quantities of H. alchorneoides fruit (source No. BL083)

Site Lat. Long. Altitude Rain Temp. Collectio No. Fruit (m asl) (mm yr–1) (°C) n date trees (kg)

Volcan, 9°13’N 83°26’W 445 3630 27 8/01/98 6 22 Buenos Aires

Initial tests

In Costa Rica, initial moisture content of fruits including pulp was determined (fresh weight basis). After processing, a sample of 20 depulped fruits was taken and used to measure fruit weight. A fruit containing 3–6 seeds inside was regarded as one entity and measured as only one plant. The initial moisture contents of the fruits were determined on samples of two times 5 g each. Before germination tests, the fruits were rubbed with sandpaper for 30 sec and then soaked in water for 24 h. Four replicates of 25 fruits were incubated for germination in sand at 30°C and in 24 h light. In Denmark, mean weight and moisture content were determined using five replicates of 50 fruits (IPGRI/DFSC 1996). Cut and TZ tests were carried out on a sample of 100 fruits. Parts of the fruits were dried to estimate their tolerance to desiccation. The germination tests were SOUTH AND CENTRAL AMERICA 297 carried out using fruits that had been rubbed with sandpaper for 30 sec. Four replicates of 100 rubbed fruits were incubated in vermiculite at 28°C and 12 h light.

Desiccation trials

To determine the minimum moisture content without loss of viability, the fruits were desiccated with silica gel to a range of target moisture contents and tested for germination as before. Controls were kept under similar conditions, but without silica gel, during the desiccation period and a sample sown at the same time as each dried sample.

Storage trials

Fruits with 3.3, 6.8, 9.9 and 11.2% MC were sealed in plastic (P) and aluminium (A) bags (in order to evaluate the effect of packaging material on seed viability), and stored at 15, 5 and –17°C for 3, 6 and 12 months, in Costa Rica. A replicating storage trial was performed on fruits having 4, 7, 10 and 15% MC, and then stored at 15, 5 and –18°C, in Denmark. Germination capacity was tested after 3 and 16 months.

Results

Initial tests

Costa Rica: The initial moisture content of the fruits with pulp was 52.2%, but after manually processing (cleaning), it decreased to 31.6r0.3% (Table 2). The mean weight of twenty fruits was 7.05r1.96 mg per fruit. Denmark: The moisture content on arrival at DFSC was 19.2%. Of the 100 fruits that were cut on arrival, 69 appeared fresh and 60 fruits were stained and estimated as viable in the TZ test. Most of the discarded fruits were empty. After 8 months, 9% of the fruits had germinated. The mean weight was 6.8r0.1 mg per fruit.

Desiccation trials

H. alchorneoides seeds could be dried to 3.3–4.5% MC and germinated more than 70% (Table 2). These results from CATIE contradicted what had been found at DFSC, where seeds were received with low initial moisture content (19%). The germination percentage was also low for 298 STORAGE BIOLOGY OF TROPICAL TREE SEEDS experiments carried out at DFSC. However, drying these seeds to about 8% MC improved germination capacity up to 21%. Germination started after four weeks of incubation and ran for a period of 3 months at CATIE and for more than 8 months at DFSC.

Table 2. Germination (G%) of H. alchorneoides fruits after desiccation (MC%) under laboratory conditions at CATIE, Costa Rica and at DFSC, Denmark CATIE DFSC MC (%) G (%) MC (%) G (%) 31.6r0.3 (initial) 64r11.8 19.2r1.2 8.8r1.0 16.6r0.4 (control) 70r10.6 — — 15.7r0.3 81r18.3 — — 12.4r0.3 (control) 94r7.7 — — 11.7r0.2 (control) 96r 6.0 11.5r0.8 8.8r4.1 10.8r0.2 65r6.8 8.3r1.9 10.5r5.2 8.6r0.3 53r10.0 7.8r0.9 21.5r7.3 4.5r0.2 83r 16.8 — — 3.3r0.3 70r12.4 — —

Storage trials

Clean fruits with four different moisture contents were stored at three different temperatures for 12 months. Starting with high viability (t70%) after desiccation (Table 2), germination decreased during the first three months of storage at all temperatures (Fig. 1). In the case of fruits stored at 5 and 15°C, after 12 months, there was ca. 20% germination for seeds stored in either plastic (P) or aluminium (A). The viability of fruits stored at –17°C was already less than 20% after 3 months and, in general had fallen further by 12 months storage. In all cases, fruits with lower moisture contents (3–7%) seemed to preserve better compared with those at higher moisture contents (10–11%). There were no significant differences between fruits stored in the plastic bags and those stored in aluminium. Duplicate storage trials were carried out at DFSC (Table 3). Although starting with low viability at reception, all fruits survived the storage conditions. Germination capacity was maintained for 10–13 months of storage at 5 and 15°C. Fruits with 15% MC did not germinate after storage at –18°C for 7 months. However, the highest germination of 29% was obtained with fruits at 9% MC and stored for 13 months at –18°C. SOUTH AND CENTRAL AMERICA 299

60 50 15°C 40 30 20 10 Germination (%) 0 36912

Storage (months)

60 50 5°C 40 30 20 10

Germination (%) 0 36912 Storage (months)

30 3.3-P 25 -17°C 6.8-P 20 9.9-P 15 11.2-P 3.3-A 10 6.8-A 5 9.9-A Germination (%) 0 11.2-A 36912 Storage (months)

Figure 1. Germination capacity of H. alchorneoides fruits with different moisture contents of 3.3 to 11.2% after storage in plastic (P) or aluminium (A) at 15, 5 and –17°C for 12 months. 300 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Table 3. Germination capacity (G%) after desiccation (MC%) and replication of storage trials of H. alchorneoides fruits at DFSC

Target MC 15% MC 10% MC 7% MC 4% MC MC (%) 15.49±0.42 9.62± 0.51 7.81± 0.46 7.49± 0.62 15°C G (%) 7.50±4.80 8.75± 3.40 10.25± 2.06 8.00± 2.00 Period 10 10 10 10 (months) MC (%) 16.21± 0.17 9.49± 0.55 8.63± 0.14 7.24± 0.63 5°C G (%) 5.50± 2.38 6.75± 2.36 16.75± 3.20 11.00± 6.22 Period 10 10 13 10 (months) MC (%) 15.04± 0.44 9.18± 0.67 8.07± 0.51 6.49± 0.39 –18°C G (%) 0.25± 0.50 29.25± 8.50 16.00± 7.83 13.75± 6.40 Period 7 13 13 13 (months)

Discussion

Desiccation trial

The viability of Hioronyma alchorneoides fruits dried to moisture contents as low as 3.3% remained high (t70%). This is in accordance with previous experiments where H. alchorneoides seeds could survive drying to 7.5% MC (Trivino et al. 1990). However, replicating experiments at DFSC showed very low percentages (9 to 22%) of germination, although the TZ test performed on the fruits on arrival indicated 60% viability of the seed lot. Sub-optimal conditions at DFSC might have been the reason for the very poor germination. Because they could be dried to very low moisture content and still maintained high viability, it can be concluded that the fruits of H. alchorneoides are tolerant to desiccation. Even with pretreated fruits, germination first started after four weeks and was spread overlong periods. The germination window varied from 3 months at CATIE to more than 8 months at DFSC, possibility due to some dormancy induced during the transit to Denmark. COSEFORMA (1998) presented similar results with germination starting 17 days with pretreated fruits and 24 days without pretreatment. However, in a separate trial, when fruits from this same seed lot were sown in mixed sand and loam (1:1) in a greenhouse, they completed germination within only 30 to 40 days. Because these fruits were covered with a very thin mix of sand and loam, we suspect light sensitivity may play a role in the germination process of this species. SOUTH AND CENTRAL AMERICA 301

Storage trial

After 3 months storage, the best results of up to 50% germination were obtained for fruits stored in plastic bags at 5 and 15°C (Fig. 1). However, their viability decreased significantly after 6 and 12 months. The poor storage results at –17°C at CATIE (<20%) seemed to indicate sensitivity of H. alchorneoides seeds to low temperatures. This was not supported by the results obtained from DFSC (Table 3), where the germination of fruits was at the highest after 13 months storage at –18°C. A preliminary conclusion of these trials is that the fruits are fully desiccation tolerant, but short-lived. However, more investigations are needed to establish optimal conditions for germination and storage.

Acknowledgements

We thank Danida for supporting the IPGRI/DFSC project on handling and storage of recalcitrant and intermediate tropical forest tree seeds, and Alfonso Gonzalez and Sigrit Diklev, both staff of seed laboratories at CATIE and DFSC for technical assistance.

References

COSEFORMA. 1998. Pilón en la Zona Norte de Costa Rica. Cooperación en los sectores forestal y maderero; convenio Costarrecense, Alemán. Pp. 20. Danida Forest Seed Centre (DFSC). 2000. Hyeronima alchorneoides Allemao. Seed leaflet No. 47. Flores, E.M. 1993. Hyeronima alchorneoides. Arboles Semillas Neotrop. 2:53–73. Franco, P. 1990. The genus Hyeronima (Euphorbiacea) in South America. Bot. Forhbuches System. Pflanzengeogr. 111(3):297–346. IPGRI/DFSC. 1996. The project on handling and storage of recalcitrant and intermediate tropical forest tree seeds. Screening protocol. IPGRI/DFSC Newsletter 5. Salazar, R. 1997. Hieronyma Alchorneoides. Nota Técnica No. 16. PROSEFOR, CATIE, Turrialba, Costa Rica. Pp. 1. Trivino, D.T., R.S. De Acosta and A. Castillo. 1990. Tecnicas de Manejo de Semillas Para Algunas Especies Forestales Neotropicales en Colombia. Bogota, Colombia: CONIF-INDERENA-CIID. Serie de Documentacion No. 19. 302 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Drying and storage of Vochysia ferruginea seeds

William Vásquez1, Rodolfo Salazar1 and Erik N. Eriksen2

1Tropical Agricultural Research and Higher Education Centre (CATIE) 7170 Turrialba, Costa Rica 2The Royal Veterinary and Agricultural University, Horticulture, Agrovej 10, 2630 Taastrup, Denmark

Abstract

Desiccation tolerance and the effects of moisture content and storage temperature on seed longevity were investigated on Vochysia ferruginea from two sources of Costa Rica. The initial moisture content of seeds at harvest was 39%, and they had high initial viability, greater than 90% germination. Seeds tolerated drying to 6.4% MC, maintaining their initial viability. Fungal growth was important during the germination tests, which greatly contributed to the reduction of the germination percentage. Viability declined faster for seeds stored at low temperatures and no seeds survived storage at –17°C. Seeds with 9.6 and 12.3% MC germinated 51% after 6 months of storage at 15°C. However, none of these seeds survived after 12 months. It was recommended to store seeds of V. ferruginea with 8–10% MC at 15°C for a few months.

Introduction

Vochysia ferruginea Mart. tree from the Vochysiaceae family grows up to 35 m and 80 cm diameter at breast height. It has a straight trunk, free of branches to half its height. It grows in low areas, in less fertile, acidic but well-drained soils, usually with a slope. The tree easily colonizes abandoned lands. The wood of V. ferruginea is of great interest to loggers because of its high timber value. It is light with a specific weight of 0.38 g cm–3, dries easily and is easy to work. It is used in construction, furniture making and craftwork (Salazar 1997). When planted, the initial growth of seedlings is relatively slow, but accelerates in the second or third year. In the North Huetar region of Costa Rica, the trees can grow 3.4 m in 2 years after planting (Rodriguez 1997). Flowering usually occurs from Mar to Apr, and the trilocular fruits ripen from June to Sept, turning from green to brown. Mature fruits should be collected directly from the trees when they change SOUTH AND CENTRAL AMERICA 303 colour and before they open (Salazar 1997). V. ferruginea tree has a regular peak production of fruits every 2 years. There is little and contradictory information on the physiology of V. ferruginea seeds. According to Flores (1993), seeds must not be dried and should be kept at 24–26°C to maintain viability. However, seeds from different sources have been found to tolerate desiccation, but stored best nondried at 15°C, resulting in 64% germination after 3 months, and 23% germination after 4 months (Müller 1997). The purpose of this study was to establish optimal storage conditions with regard to seed moisture content and storage temperature.

Materials and methods

Seed maturity and collection

In order to identify the best time for collection, fruits were harvested every 2 weeks and seed samples were examined to determine their level of maturity that was also associated with recorded fruit colour. It was then determined that mature fruits should be collected when their colour turned from light green to dark green with marked divisions between the locules. The fruits matured irregularly throughout the tree crowns. Fruits were collected from 10 trees of Volcán, Buenos Aires, Puntarenas (Bl082) in Sept 1997 and from three trees of Cajón de San Pedro, San Isidro Puntarenas in March 1998. These sites are located in 9° N latitude and 83° W longitude, at more than 500 m above sea level, where the mean temperature was 23°C and the annual rainfall attains nearly 3000 mm. Fruits in sacks were transported to the Tree Seed Bank at CATIE 1 day after collection, and were kept in the shade for 2 days before the seeds were manually extracted. For replicating experiments, half of the seeds were sent by courier to both DFSC and the Agricultural University in Copenhagen, arriving 3 to 9 days after.

Initial tests

Seeds collected from Volcán were used in the desiccation trials and the other seeds from San Pedro were used in the storage trials. Fruit and seed characteristics were determined using 100 individuals, mainly from the seed lot of 1997 collection. The fruits and seeds were weighed and measured, and the mean values were calculated. 304 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Desiccation and storage trials

Seeds were mixed with silica gel in plastic bags and the bags placed at ambient conditions (24 to 28°C and 70 to 90% RH). Samples of seeds were used to determine the moisture content of the seeds, gravimetrically by weighing them before and after oven drying at 103°C for 17 h. The moisture content was then determined as a percentage of the seed fresh weight. Seeds were dried to five moisture contents between 21.1 and 6.4% and were tested for germination capacity. Desiccated seeds with four moisture contents between 12 and 8% were stored at 15, 5 and at –17°C for 3, 6 and 12 months. The seeds were treated with NaOCl before the storage trials. For germination, all seeds were sown in sterilized sand and put to germinate at 30°C in constant light.

Results

Initial tests

The seeds sent to the Agricultural University of Copenhagen had very low viability on arrival, thus there were not enough seeds for the replication of all experiments. Only, the results of tests carried out at the Tree Seed Bank at CATIE are presented. Fruit and seed characteristics were determined using 100 individuals. The seed composed about 5% of the fruit (Table 1). There were ca. 30 000 seeds per kg and more than two seeds on average per fruit.

Table 1. Initial characteristics and quantities of V. ferruginea fruits and seeds from Costa Rica. The mean values of weights and dimensions are for 100 individuals Year Fruit Harvest Weight Length Width Seeds (kg) (g) (cm) (cm) /fruit 1997 145.2 1.46r0.20 2.99r0.21 1.17r0.08 2.64r0.59 1998 27.0 — — — — Seed MC (%) Harvest Weight Length Width Seeds (kg) (g) (cm) (cm) /kg 1997 4.5 0.08r0.02 2.67r0.32 0.55r0.25 30 000 39.0r0.3 1998 0.50 — — — — — SOUTH AND CENTRAL AMERICA 305

Desiccation and storage trials

V. ferruginea seeds had 39% initial moisture content (Table 1). All experiments started with seeds at 21.1% MC, and drying these seeds to the first target moisture content took approximately 6 h in silica gel to reach 12.1% MC, whereas the driest moisture content of 6.4% was obtained after 12 h. Seeds with 21.1% MC initially germinated to 94%. However, desiccating V. ferruginea seeds to 6.4% MC did not result in any loss of viability; seeds retained 93% germination (Table 2).

Table 2. Germination (G%) of V. ferruginea seeds from Bl082 (1997) after desiccation to different moisture contents (MC%) Control Desiccated Target MC (%) MC (%) G (%) MC (%) G (%) Initial 21.1 94 21.1 94 20 17.8 92 12.1 98 15 16.6 85 11.7 93 10 17.3 94 8.9 96 5 14.7 89 6.4 93

Table 3 shows the results of the storage trials after 3 and 6 months. There was a great increase from 31% after 3 months to 51% germination after 6 months storage at 15°C for seeds with 12% MC. This maybe due to fungal contamination during the germination tests. At drier moisture contents (<12%), viability of seeds was relatively constant over 6 months storage at 15 and 5°C. No seeds germinated after 12 months of storage. All seeds did not withstand storage at –17°C, losing viability already within 3 months. The analyses of variances showed that there were significant differences (P<0.001) between seeds with higher moisture content (11–12%) and those with lower moisture content (7.9%), after 6 months at 15 and 5°C.

Discussion

Monitoring fruit/seed development of V. ferruginea allowed to identify peak maturity and to harvest high quality seeds germinating 100% for this study. The seeds were small, c. 30 000 seeds per kg, and composed about 5% of the fruit mass, which was also reflected on the huge quantity of fruits harvested (Table 1). The transport conditions should therefore be revised to avoid losses of such valuable materials, by shortening its duration and/or controlling travel conditions in such a way that seeds can be received with less deterioration. Replication of present data would have helped confirm or infirm some of the findings. 306 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Table 3. Germination of V. ferruginea seeds from Cajón, San Pedro (1998) after desiccation and storage. * = different letters indicate significant differences (at P=0.1% level, Least Squares Means) per storage period

MC (%) Germination Storage Germinationrsd (%) after (%) (°C) storage Target Actual 3 6 12 months months months 12 12.3 93 15 31r82,3* 51r 61 0 5 30r53 26r43,4 0 –17 0 0 0 9 11.2 98 15 43r72 42r71,2 0 5 30r73 18r74 0 –17 0 0 0 6 9.6 99 15 60r51 51r81 0 5 38r22 32r52,3 0 –17 0 0 0 3 7.9 100 15 59r91 35r82,3 0 5 42r42 35r82,3 0 –17 0 0 0

V. ferruginea seeds tolerated desiccation to 6.4% MC, maintaining their initial viability of ca. 93% germination. Thus, these seeds were fully desiccation tolerant and would be expected to store for long periods of time at low temperatures. However, the storage results showed that all seeds did not withstand storage at –17°C, losing viability already within 3 months, and no seeds germinated after 12 months at 5 and 15°C (Table 3). The handling of seeds after storage at –17°C might have not been suitable to allow their survival, particularly at low MCs (see Table 3). After 3 months of storage, the viability dropped markedly, particularly for seeds with 11–12% MC, which was partly due to fungal contamination during the germination test. Dry seeds to 7.9 and 9.6% MC maintained ca. 60% germination, the highest percentage after storage at 15°C for 3 months, but not during the 12 months storage. Although, seeds looked fresh after the 12 months storage, they did not germinate, but were completely covered by opportunistic fungi. These seeds have a similar behaviour to V. guatemalensis (Salazar et al. 1996). The mean germination after 6 months was 45% for all the 15°C treatments and 28% for all the 5°C treatments, and these two results were significantly (P<0.01) different (Table 3). Thus, V. ferruginea seeds, stored better at 15°C for at least 6 months. Other studies did not provide good survival of seeds at 7 and 12% MC, and therefore suggested also that nondried V. ferruginea seeds be stored at 15°C SOUTH AND CENTRAL AMERICA 307

(Müller 1997). However, due to a great variation between different seed sources (Müller 1997), it is difficult to recommend a single optimal moisture content for this species. For these studied sources, seeds can be dried to 8–10% MC for the short-term storage at 5 or 15°C.

Conclusions

Although, V. ferruginea seeds were tolerant to desiccation, they were also relatively short lived and sensitive to low temperatures (below 0°C). It is recommended that seeds are stored at 8–12% MC and 15°C for a few months. More investigations are needed on the effect of moisture content and storage temperature, as well as germination conditions controlling fungi.

Acknowledgements

We thank Danida for supporting this IPGRI/DFSC project and Alfonso Gonzalez, CATIE and Sigrit Diklev, DFSC, in seed laboratories for technical assistance.

References

Flores, E.M. 1993. Vochysia ferruginea. Trees and Seeds from the Neotropics. 2:29– 52. Müller, E. 1997. Investigationes en Frutos y Semillas de Árboles Individuales de Cinco Especies Forestales de la Región Huetar Norte de Costa Rica, con especial consideración de 1997. Tesis de Doctorado. Pp. 123–125. Rodriguez, E. 1997. Evaluación del comportamiento, adaptabilidad y crecimiento de siete especies forestales nativas den la Región Huetar Norte de Costa Rica. Pp. 123–125. Memorias III Congreso Forestal Nacional. San José, Costa Rica, del 27 al 29 de agosto de 1997. Salazar, R. 1997. Vochysia ferruginea. Nota Técnica No.4. PROSEFOR, CATIE, Turrialba, Costa Rica. Salazar, R., A. Ramírez and A. González. 1996. Respuesta de las semillas de Vochysia guatemalensis a la desecación. In Memorias IV Taller Nacional de Investigación Forestal y Agroforestal. EARTH, Guácimo, Costa Rica. Pp. 9–16. 308 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Seed storage behaviour of Vochysia guatemalensis

Rodolfo Salazar1, William Vasquez1 and Kirsten A. Thomsen2

1Tropical Agricultural Research and Higher Education Centre (CATIE), 7170 Turrialba, Costa Rica 2The State Forest Tree Improvement Station, Krogerupvej 21, 3050 Humlebaek, Denmark

Abstract

Desiccation tolerance and optimal conditions of moisture content and temperature during storage were investigated on Vochysia guatemalensis seeds collected from two sources in Costa Rica in 1996 and 1997. Seeds with an initial 43% moisture content germinated 100%. They tolerated drying to 5% MC, maintaining more than 90% germination. Seeds desiccated to 10.4% MC stored at 5°C and 15°C, retained 68 and 58% germination after 6 months. However, a maximum of 29% of seeds survived storage at 15°C after 12 months. Although the seeds were desiccation tolerant, storability still posed problems, as most of the seeds lost viability within 1 year storage. Furthermore, even very dry seeds could not be stored at –17°C. It is recommended that the seeds are stored at ca. 10% MC and at 15°C and that the relationship between moisture content and temperature is investigated further.

Introduction

Vochysia guatemalensis Donn. Sm. is a tall tree with yellowish, soft wood that grows in the humid tropical forest, up to 800 m above sea level along the Atlantic Coast from Mexico to Panama. The trees grow up to 40 m in height and 180-cm diameter at breast height. The trunk is straight, cylindrical and free of branches. The species grows well on different soils like flooded to loamy sand, loamy clay and compact soils. It forms pure stands in abandoned pasture and agricultural lands. The grain is straight and the wood, which is easy to work, is used for construction and furniture (Salazar 1997). Good establishment of experimental plantings has made the species highly attractive in forest plantations. However, the lack of knowledge is still a problem for better handling of the seeds. SOUTH AND CENTRAL AMERICA 309

Flowering begins when the trees are six to eight years old and usually occurs between March and June, and the fruits mature from August to October. The winged seeds are flat and pubescent and should be collected from the tree before the trilocular capsule fruit opens (Salazar 1997). According to Flores (1993), fresh seeds with 32% MC stored at 5°C, rapidly lose their viability. At 10 and 25% MC, they germinated 70 and 75%, respectively, after six months storage. Müller (1997) found that drying seeds to 11% MC (45% relative humidity) and storing them at 20°C were the best conditions, as approximately 75% germinated after four months and 30% after six months of storage. For seeds dried to 5% MC and stored at 4 and 15°C, their viability was maintained above 50% germination after three months. Only 15% of seeds with 5% MC germinated after six months at –15°C. The present investigations aimed to determine the best storage conditions for V. guatemalensis seeds and thereby prolonging the storage period.

Materials and methods

Seed collection and extraction

Fruits of V. guatemalensis were collected from 10 to 14 trees of natural stands and plantations in La Argentina de Pocora, Limón (BL063) and San Rafael, Pérez Zeledón (BL077) on the 27 of June 1996 and the 13 of Aug 1997 (see Table 1). Before collection, the fruits were monitored every two weeks and it was determined that mature fruits should be collected when their colour turned from light green to dark green with marked divisions of the locules. Fruits were transported to the Tree Seed Bank at CATIE in sacks the day after collection. The ambient temperature varied between 24 and 29°C and the relative humidity between 60 and 80%. The fruits were dried for two days in the shade and the seeds were extracted manually (Table 1). Extracted seeds, from both years, 1996 and 1997, were split into two, to be used for replicating experiments at CATIE and at Danida Forest Seed Centre (DFSC). 310 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Table 1. Site characteristics and quantities of V. guatemalensis seed lots collected in 1996 and 1997

Site Latitude Longitude Altitude Rainfall Fruits Seeds Ratio (N) (E) (m) (mm) (kg) (kg) (S/F) Limón (BL063) – 1996 09°18’ 83°31’ 740 2934 192.7 7.0 0.036 Pérez Zeledón 10°07’ 83°38’ 250 3722 66.7 1.5 0.022 (BL077) – 1997

Desiccation and germination trials

Costa Rica

Seeds were desiccated in silica gel at 27°C (ambient temperature) to reach seven target moisture contents of 30, 20, 16, 13, 10, 8 and 5%. After desiccation, replicates of seeds were used to determine moisture content gravimetrically by weighing them before and after oven drying at 103°C for 17 h. Moisture content was then calculated as a percentage on a fresh weight basis. Four replicates of 50 seeds, for each treatment, were germinated in sterilized sand (treated with 5% Formalin), in a germination cabinet at 30°C, 80% RH and constant light.

Denmark

The desiccation trials were replicated at DFSC using the same methods except that the seeds were germinated in vermiculite, instead of sand. Temperature and light were similar to the conditions at CATIE.

Storage trials

Costa Rica: Seeds with 3, 6, 9 and 12% MC were packed in transparent plastic bags and stored at –17°C, 5°C and 15°C where after, the seeds were germinated (same conditions as above). Seeds sent to DFSC were dead on arrival.

Results

Initial tests

Initial characteristics of fruits and seeds were similar for 1996 and 1997 collections. Fruits and seeds from Limón—BL063 were bigger (9 and SOUTH AND CENTRAL AMERICA 311

0.45 g, on average) than those from Pérez Zeledón—BL077 (7.8 and 0.39 g, on average), resulting in 5800 seeds per kg for the former source compared to 6139 seeds per kg for the latter source. Both seed lots had high initial MCs of 42–45% (Table 2).

Table 2. Initial characteristics of V. guatemalensis fruits and seeds Source Fruit Weight Length Width (cm) Seeds/fruit (g) (cm) BL063 9.02 4.70 1.02 2.50 (1996) BL077 7.80 5.76 1.96 2.76 (1997) Seeds MC (%) Weight Length Width Seeds/kg (g) (cm) (cm) BL063 0.45 5.67 1.95 5800 45.3 (1996) BL077 0.39 –– –– 6139 42.3 (1997)

Desiccation and germination trials

Seed samples were dried at CATIE to eight moisture contents and tested for germination capacity. Seeds began to germinate 8–9 days after sowing, and germination took another 4 to 11 days. Seeds with an initial 43% MC and 100% germination, tolerated drying to 5% MC with only a slight decrease in germination (Table 3). Similar results were obtained at DFSC, where germination percentage remained the same for non dried and dried seeds, although the MC of these seeds was reduced to 27% at arrival. The energy of germination was calculated as the percentage of germinated seeds 3 days from the onset of the test to the total number of seeds that germinated. Seeds with <15% MC germinated faster (high energy) than seeds with higher moisture contents (low energy) (Table 3). Experiments at DFSC showed similar onset of germination after sowing, but germination finished after 15–19 days. Analysis of variance (ANOVA) confirmed that there were no significant differences between the treatments at both laboratories. 312 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Storage trials

Seeds were desiccated to 12.1, 10.4, 6.7 and 2.2% MC, resulting in, respectively, 93, 95, 90 and 84% germination, and were stored at –17, 5 and 15°C for 3, 6 and 12 months. No seeds germinated after storage at –17°C (Fig. 1). Most seed samples stored at 5 and 15°C already showed a significant decrease in viability after three months storage (Fig. 1A). Germination was between 48 to 76% for seeds with <12% MC at 5°C after 3 and 6 months. At 12% MC, seeds germinated ca. 30% after 3 months, but 48% after 6 months (Fig. 1A and 1B). For seeds with >3% MC, germination percentages after storage at 15°C for 3 and 6 months were between 41 to 86 (Fig. 1A and 1B). At 2.2% MC seeds germinated respectively 13, 20 and 20% after storage at 15°C for 3, 6 and 12 months. After 12 months of storage at 15°C, between 18 and 29% germination was obtained, the best result being for seeds with 10.4% MC (Fig. 1C).

Table 3. Germination (G% and energy) responses of V. guatemalensis seeds to desiccation Target MC (%) DFSC CATIE MC (%) G (%) G energy MC (%) G (%) (%) Control 43.0 100 40 27.9 96 30 33.3 100 43 –– –– 20 17.8 100 52 21.5 94 16 14.9 100 80 17.2 92 13 12.0 100 82 13.7 96 10 9.1 100 94 10.2 92 8 8.0 99 99 9.9 93 5 5.0 97 59 5.9 96

Discussion

Fresh seeds of V. guatemalensis were harvested at peak maturity, which was determined after preliminary observations and tests. The seeds had high initial 43% moisture content and high viability of 100% germination. They, however, tolerated desiccation down to 5% MC, maintaining initial viability. This also confirmed the results of Salazar et al. (1996). Despite the small delay of about a week in receiving seeds in Denmark, these results were reproduced at DFSC (see Table 3), and SOUTH AND CENTRAL AMERICA 313 there were no significant differences between the treatments at both laboratories. The energy of germination calculated at 3 days of the germination onset, allowed us to demonstrate that drying increased the rate of germination (Table 3), seeds with <15% MC germinating faster than those with higher moisture contents. To estimate their longevity, desiccated seeds were stored at different temperatures for 12 months at CATIE. Most seed samples stored at 5 and 15°C showed a significant decrease in viability after three and six months storage, and a maximum of 29 r 3 and 24 r 5% germination was obtained for seeds with 10.4 and 6.7% MC stored at 15°C (Fig. 1). Seeds with 12% MC may have been chill-injured at 5°C, reducing their germination to <50%. By contrast seeds dried to 2.2% MC had reduced but constant germination of 13–20% over 12 months at 15°C. This indicated that drying did not improve storage of V. guatemalensis seeds at 15°C. However, the combination of 10.4% MC and 15°C resulted in the highest germination at all three storage durations. Furthermore, Flores (1993) and Müller (1997) recommended storage of these seeds at more than ca. 10% MC and at 15°C. One might predict that seeds tolerating desiccation to 5% MC, would also be storable at low temperature, as usually found in many desiccation tolerant seeds. However, no seeds germinated after storage at –17°C (Fig. 1), this is in accordance with another study of V. guatemalensis from several seed sources, where the seeds also showed poor storage survival at –15°C (Müller 1997). As for neem, another tropical species, dry seeds must be handled with caution when rehydrating them for germination, to avoid imbibitional damage (Sacandé et al. 1998). In addition, seeds taken from sub-zero temperatures should be ‘acclimated’ at room temperature for at least 24 h before germination tests. This fluidises cell membranes and avoids cell death by rigid membrane rupture (Hoekstra et al. 1999). Lack of such precautions may have affected the germination capacity of these seeds. Sensitivity to low temperatures may explain why the seeds arrived dead at DFSC in 1997, as they may have been exposed to freezing temperatures at the plane. Hence, taken together, it was still unclear from the present study, what the optimal storage conditions of V. guatemalensis seeds are. The relationship between moisture content and storage temperature should be investigated more thoroughly, as well as types of seed storage containers. 314 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Figure 1. Viability of V. guatemalensis seeds with 2.2, 6.7, 10.4 and 12.1% MC, after storage at –17, 5 and 15°C. Seeds were stored for 3 (A), 6 (B) and 12 (C) months at CATIE. SOUTH AND CENTRAL AMERICA 315

Conclusions

Although the seeds of V. guatemalensis were tolerant to desiccation to 5% MC and maintained a high initial viability, storability still posed problems, as seeds could not be stored for more than six months, losing greatly their germination capacity. The seeds do not tolerate – 17°C. On basis of the results and literature it is recommended to store the seeds with ca. 10% MC and at 15°C.

Acknowledgements

We thank Danida for supporting this IPGRI/DFSC project and Alfonso Gonzalez, CATIE, and Sigrit Diklev, DFSC, in seed laboratories for technical assistance.

References

Flores, E.M. 1993. Vochysia guatemalensis. Trees & Seeds from the Neotropics. Vol. 2. 2:1–27. Hoekstra, F.A., E.A. Golovina, A.C. Van Aelst and M.A. Hemminga. 1999. Imbibitional leakage from anhydrobiotes revisited. Plant Cell Environ. 22:1121–1131. Muller, E. 1997. Investigationes en Frutos y Semillas de Árboles Individuales de Cinco Especies Forestales de la Región Huetar Norte de Costa Rica, con especial consideración de 1997. Tesis de Doctorado. Pp. 123–125. Sacandé, M., F.A. Hoekstra, J.G. Van Pijlen and S.P.C. Groot. 1998. A multifactorial study of conditions influencing neem (Azadirachta indica) seed storage longevity. Seed Sci. Res. 8:473–482. Salazar, R. 1997. Vochysia guatemalensis. Nota Técnica No.4. PROSEFOR, CATIE, Turrialba, Costa Rica. Salazar, R., A. Ramírez and A. González. 1996. Respuesta de las semillas de Vochysia guatemalensis a la desecación. In Memorias IV Taller Nacional de Investigación Forestal y Agroforestal. EARTH, Guácimo, Costa Rica. Pp. 9–16. 316 STORAGE BIOLOGY OF TROPICAL TREE SEEDS Review

An overview of the experimental chapters in this volume 318 STORAGE BIOLOGY OF TROPICAL TREE SEEDS REVIEW 319

Biological aspects of tropical tree seed desiccation and storage responses

Hugh W. Pritchard1, Moctar Sacandé1 and Patricia Berjak2

1Seed Conservation Department, Wellcome Trust Millennium Building, Royal Botanic Gardens Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK 2Plant Cell Biology Research Unit, School of Biological Sciences, University of KwaZulu-Natal, Durban 4041, South Africa

Introduction

The importance of biodiversity conservation compels sustainable management of natural forests, reforestation and the development of rational agroforestry and agro-silvi-pasture systems. Forests are not boundless resources and the present systematic investigations on tropical forest trees reported in this volume are aimed at ensuring the more efficient and effective handling of their seeds. The availability of such improved knowledge will promote conservation, management and sustainable use of the remaining forests, and the implementation of successful reforestation programmes. In a wider context, the urgent need for the enhanced application of preservation technologies in support of species conservation and sustainable use is emphasized by the recent adoption of the Global Strategy for Plant Conservation by the Conference of the Parties to the Convention on Biological Diversity (CBD 2002). Ex situ conservation of seeds is probably the most effective and widely practiced means of species conservation, with ca. 6 million specimens stored globally (FAO 1996; Linington and Pritchard 2001; Engelmann and Engels 2002). To enable the rapid commitment of more species to this type of conservation, studies are needed on the relative tolerance of seeds to genebank conditions, specifically drying and cold storage. Such assessments can progress at various levels of complexity. Small seed lots can be screened rapidly for dehydration tolerance (Pritchard et al. 2004a). In addition, a more detailed quantification of desiccation and storage responses can be made (e.g. Hong and Ellis 1996). Finally, the full seed viability constants, which detail the dependence of seed survival on both moisture content and temperature, can be determined in desiccation tolerant seeds (see Roberts 1973; Pritchard and Dickie 2003). 320 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

The detailed, systematic screening of species for seed storage behaviour using a range of storage conditions, including an assessment of desiccation tolerance, has not been attempted before using a network approach. Thus the presentation in this volume of information for 52 species, many of which are new to seed conservation science, is a remarkable achievement. The purpose of this review is to briefly highlight some of the main features of the findings in a wider biological context. For the detailed description of findings on each species, the reader is directed to the specific chapters elsewhere in this volume.

Germination and dormancy

The maximum germination level for seed lots of the 52 species studied (see Table 1) were generally (ca. 60%) in excess of 90% (Fig. 1). This suggests a good working knowledge of the biology of the species with respect to timing of seed harvest by the participants in the project. Nonetheless, some seed lots yielded much lower germination levels, for which there are a number of possible causes as discussed below, but the many aspects of seed collection, handling and transport (especially for recalcitrant types) from the field to the laboratory, could all have been contributory factors (Berjak and Pammenter 2004). First, sub-optimal thermal conditions may have been used in the germination test. The recommendation for this study was to use germination temperatures around 25°C. This advice was clearly adopted as our analysis of the reports contained herein for 47 species reveals that 35 of them were germinated at constant temperatures in the range 25–30°C. Eight species were germinated at alternating temperatures fluctuating between about 20–25°C to 28–30°C; often these treatments involved exposure to ambient conditions. The application of a wider temperature fluctuation of 35°C/20°C resulted from the use of a glasshouse for two species (one of these was also tested by another laboratory at a more constant temperature, and thus considered above), whilst alternating temperature incubators generated three other temperature combinations (27°C/21°C, 30°C/25°C and 38°C/15°C). For only one species, Pouteria macrophylla, was a direct comparison made between constant (28°C) and alternating temperature (AT; 38°C/15°C), with the AT treatment giving marginally better germination. Overall, the environments used had an average temperature quite close to 25°C and this is probably close to the optimum for the vast majority of species investigated here. REVIEW 321

35 30 25 20 15 10

Number of species 5 0 0 < 9 10 < 20 < 30 < 40 < 50 < 60 < 70 < 80 < 90 - 19 29 39 49 59 69 79 89 100 Germination range (%)

Figure 1. Maximum germination for seed lots of the 52 species under investigation.

For a few species, the effects of a range of temperatures on germination were assessed. For Hancornia speciosa, radicle emergence to 88–100% was observed at temperatures from 10 to 30°C. However, normal seedling development was restricted to 20–30°C. Similarly, Genipa americana seeds only produced normal seedlings between 15 and 30°C; seedlings being produced four times quicker at the upper temperature (22 d cf. 84 d). Khaya senegalensis also benefited from germination at warmer compared with cooler temperatures. Although germination in this species was in the range 92–98%, the mean time to germinate was only 9 d at 25°C cf. 38 d at 15°C. Finally, Vitellaria paradoxa seed germination was 93–98% at temperatures from 16 to 36°C, but radicle extension was four times faster (12 mm d–1) at the warmest temperature. Clearly then, temperatures in the region of 25–30°C stimulate both high germination levels and rapid rates of seedling development in many species suggesting a general lack of seed dormancy in the species investigated. In general, the results are in line with other studies on tropical tree species with nondormant seeds (e.g. see Tompsett and Kemp 1996; Pritchard 2004). Second, physical manipulation of the seeds, i.e. intervention prior to the germination test may not have been optimized or the physical environment of the test, with respect to substrate, may not have been 322 STORAGE BIOLOGY OF TROPICAL TREE SEEDS ideal. For some species, the possibility of there being a physical barrier to germination was known and steps taken to eliminate this effect. For example, in two species of the Meliaceae, Azadirachta indica and Ekebergia capensis, and in Gmelina arborea (Verbenaceae) the ‘endocarps’ were removed prior to seed sowing. Similarly, the 2 mm thick coat of Podocarpus falcatus (Podocarpaceae) and thicker endocarp of Sclerocarya birrea (Anacardiaceae) were removed before sowing. In these cases, ‘coat’ removal reduced the physical restraint above the embryo and facilitated its growth, as opposed to being needed to ensure water uptake. With respect to the substrate used for germination, this is specified here for 45 species. The most popular substrate was vermiculite, followed by sand, and then agar-water and paper. Cotton wool was used for one species. This variation in substrate used reflects the preference of the individual laboratories involved in the network. Even so, the generally high germination level of most species (Fig. 1) suggests such substrate variation probably had a relatively small effect on the results. Third, poor germination in two species with desiccation tolerant seeds was probably because of the presence of physiological dormancy. This was most evident in Zanthoxylum zanthoxyloides and Sclerocarya birrea during storage at noncold temperatures. In Z. zanthoxyloides, seed germination increased to a maximum of nearly 40% during nine months relatively dry storage. Similarly, S. birrea seeds exhibited a systematic increase in germination to about 80% during 18 months dry storage. S. birrea and Lannea microcarpa (both members of the Anacardiaceae) seeds have also been observed to germinate to higher levels following dehydration to low moisture contents (Pritchard et al. 2004b). Fourth, low germination could be a consequence of low viability of the seed lots. This problem was noted for a number of species, although usually remedial action was taken to improve the quality of the seed lot before the experimentation started. However, seeds of Trichilia emetica were heavily fungally infected by the time the collection reached the replicating partner. Also some fruits of Warburgia ugandensis had fungal infection, and the problem was accentuated by insect infestation. Fungal contamination was also a contributory factor in the poor storage capability of desiccation sensitive seeds when held in moist storage (see later discussion). Fifth, seed immaturity could contribute to poor seed performance. For Sclerocarya birrea, the fruits drop when still green and immature and then ripen when on the ground. Similarly, the collected fruits of Warburgia ugandensis were green and very firm and these were left for REVIEW 323 four weeks to ripen before some of the experiments were performed. For Sterculia quinqueloba, white immature seeds were excluded from the experiments. As a consequence of these actions, selected seeds of these species all germinated to quite high levels. In contrast, Prunus africana fruits tended to be even more heterogeneous in development, varying from green (immature), to purple-green and purple (mature). This variation impacted clearly on their germination level, being much lower initially for the immature seeds. Germination of less-than-mature seeds of the palm, Phoenix reclinata, has also been shown to be depressed relative to the mature seeds—and interestingly, also to the seeds when they first become germinable (Berjak et al. 2004). Those authors suggested that an inhibitor may be involved, ensuring that these seeds, that retain relatively high embryo water contents, do not germinate prematurely. With such seeds there can be both a positive effect of dehydration and a higher onset of desiccation stress compared with more mature seeds (see later discussion on partial drying). Indeed, during the development of seeds there is a systematic acquisition of physiological traits: firstly, germinability; secondly, tolerance of rapid artificial drying; and thirdly, maximum storability (Sacandé 2000; Hay and Smith 2003). So, even if germination is observed to be high in immature seeds, the seeds may not exhibit maximum desiccation and storage responses.

Partial drying and seed maturity

For eight species, there were positive effects of partial drying on germination level and/or rate, prior to the onset of desiccation stress. The moisture content limits to such a response was species dependent, but could be separated into two broad categories: drying to ca. 1 % or ca. 30–45% moisture content. In Prunus africana, ‘immature’ seeds had a >80% increase in germination after drying to ca. 10% moisture content; the increase was much less (ca. 20% germination) in ‘mature’ seeds. Similar, small increases in germination were observed in ‘mature’ Sterculia quinqueloba and Podocarpus nagi seeds when dried to ca. 10% moisture content. In contrast, a similar level of desiccation resulted in an increase of around 40% germination for Gmelina arborea. However, in this species there was a similar effect in the controls (undried), suggesting time was also a factor in this response. 324 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

In the other group of species, the onset for this type of desiccation response occurred at much higher moisture contents. In Azadirachta indica var siamensis, seed lots from many of the individual trees sampled showed improved germination after short-term drying (a few days) to around 45– 55% moisture content, the effect being most obvious with seeds harvested at about 8 weeks. Similarly, drying of Pouteria macrophylla seeds below this moisture content range raised germination by about 15%. More subtle effects of drying were evident in Warburgia ugandensis and Ekebergia capensis seed lots, in which partial drying increased germination rate by 3–5 d without any great effect on germination total. The result for Ekebergia capensis supports an earlier observation that short-term drying (hours) speeded up germination by about one day (Pammenter et al. 1998). The partial drying response has been well characterized previously in Aesculus hippocastanum seeds, such that the onset of desiccation stress at 40% moisture content is preceded, during artificial drying, by an increase in germination level and rate (Tompsett and Pritchard 1998). The authors suggested that this was due to the further development of the less mature seeds in the batch, with artificial drying of a few days substituting for what may have happened naturally on the parent plant if the seeds had not been naturally, yet prematurely, abscised. More recent work on this species has revealed that natural seed shedding occurs across Europe when the seeds are at different stages of maturity, thus indicating a phenotypic plasticity in the seed response to desiccation (Daws et al. 2004b). The findings suggest the need for caution when interpreting seed responses for species being grown outside their natural distribution range.

Seed desiccation tolerance

Of the 52 species investigated, it was possible to estimate the critical moisture content (CMC) under the particular desiccating conditions used (Pammenter et al. 1998) for viability loss, i.e. the midpoint (50% of the initial viability), for 48 species. Because the completeness of the data sets varied between studies, it would not have been possible to subject the data to probit analysis for more than a handful of species. Rather, estimates of CMC were made by two other methods: (1) linear interpolation of the data as viability was decreasing; or (2) adoption of the lowest moisture content reached when seed lots retained high levels (i.e. >50%) of viability. REVIEW 325

Using these methods, we observed that 23 species appeared to have seeds with considerable desiccation tolerance, i.e. survived drying to d9%, and usually as low as 4%, moisture content, with little loss of viability (Fig. 2). Conversely, the other 25 species showed some sensitivity to dehydration below higher moisture contents. Such sensitivity was mainly concentrated in the moisture content range 20–<30%. A midpoint for viability loss in recalcitrant seeds has often been observed in this moisture content zone, e.g. for Vitelleria paradoxa (Danthu et al. 2000; Daws et al. 2004a). In addition, a few species had seeds that were sensitive to dehydration at higher and more intermediate moisture contents (Fig. 2). Although it has been suggested that there are CMC (or water potential) zones for desiccation stress (Walters 1998; Sun and Liang 2001), it is important to note that no specific mechanism of desiccation stress is inferred from the presentational use of moisture zones in Fig. 2. For four species (Hagenia abyssinica, Podocarpus nagi, Zanthoxylum zanthoxyloides and Cordyla pinnata) no estimate of the midpoint for desiccation stress was possible. For the first three species, germination levels were either too low to make a rational decision and/or the seeds responded to desiccation with an increase in germination level. For the remaining species, Cordyla pinnata, a desiccation experiment is not reported here, although desiccation sensitivity is known in this species (Danthu et al. 2000).

Number of species 5

0 < 10 < 20 < 30 < 40 Estimated CMC range (%)

Figure 2. Distribution of seed desiccation tolerances (see text for explanation) for 48 species. 326 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Seed desiccation tolerance in relation to mass

One characteristic of the seeds that is recorded in this volume for the vast majority of species is seed mass. Often, the value reported is that taken after the seeds have been hand cleaned and can thus be directly related to their mass, and seed moisture content, at harvest. However, for many species, seed lots were preprocessed (including soaking) before these initial determinations were made. Although one intention of this project was to assess whether there was any relationship between seed moisture content at harvest and seed storage category (Hong and Ellis 1998), the differences in seed preparation has precluded such an analysis here. Alternatively, an assessment has been made of the general association between seed desiccation tolerance and seed mass, following an earlier approach using data for >1000 species (Dickie and Pritchard 2002). Both seed mass information and estimates of the CMC (see above for explanation) for seed desiccation sensitivity were available for 44 species. As shown in Figure 3, there is a very weak association (R2=0.21) between these two parameters. In other words, it is not possible to predict, on the basis of this data set, the CMC of a species on the basis of its seed mass. This is not surprizing, given that there are a range of factors that impact on the maximum desiccation tolerance level in a species, e.g. environment under which the seeds developed (see Daws et al. 2004b for a recent discussion of this point). However, the results do suggest that species with larger (heavier) seeds are more likely to have a higher CMC for desiccation stress (Fig. 3). Such a general association can be envisaged more clearly when the distribution of seed masses for highly desiccation tolerant seeds (i.e. d9% moisture content) is plotted separately from the other species for which a greater level of desiccation sensitivity was observed. The reason for making this gross distinction may seem rather arbitrary but, as will become clear later, few of the data sets would enable a greater level of analysis, for example, in relation to seed storage category. REVIEW 327

10 Estimated critical MC (%) critical Estimated 5

0 1 10 100 1000 10000 Seed mass (mg, log scale)

Figure 3. Relationship between the estimated critical moisture content for desiccation tolerance and seed mass (log scale) for 44 species investigated.

In all, 47 species could be analyzed in this way, including some species for which additional information was obtained from the Seed Information Database (Tweddle et al. 2003). Primarily the latter source of information was used for seed mass data, enabling the calculation of a mean value for some species. As mentioned earlier, seed weights were estimated for seed lots at various stages of preparation, making direct comparison subject to some error. For example, the weight of a 50% moisture content-seed will be approximately double its value after drying with silica gel or in an oven. But such variability in data tends not to influence the general analysis as the convention is to plot seed mass on a log10 scale (Harper et al. 1970). There was about a 4 orders of magnitude variation in seed mass across all species investigated, with overlapping distributions for the species with higher and lower desiccation tolerances (Fig. 4). The average mass for these two classes of seeds was 465 mg (23 species) and 2482 mg (24 species), respectively. These values are very similar to those reported in an analysis of >1000 species, in which the most desiccation tolerant seeds weighed 329 mg on average, compared to 900–3958 mg for less desiccation tolerant species (Dickie and Pritchard 2002). 328 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Number of species 2

0 < 3.2 < 10 < 32 < 100 < 320 < 1000 < 3200 < 10000 < 32000 Seed mass (mg, log scale)

Figure 4. Distribution of seed masses for highly desiccation tolerant species (closed bars) and less desiccation tolerant species (open bars). Data is presented for 47 species.

Seed quality in relation to processing

It is relatively common practice to soak fleshy fruits in water to enable better separation of fruit tissue from the seeds. For example, fruits of Podocapus falcatus were soaked in running water for 3 h, and partially cleaned seeds of Cinnamomum cassis and Michelia mediocris soaked overnight. However, such treatments may stimulate general metabolism and the advancement of seeds towards germination, especially in species with rapidly germinating, nondormant seeds. This may have been the case for Dovyalis caffra, as by the time seeds had reached the replicating partner in the experiments the seed moisture content had increased from 35 to 53% and much of the seed lot had presprouted by receipt. Although not noted here, a consequence of advancing seeds towards emergence could be a reduction in the relative level of desiccation tolerance (Pammenter and Berjak 1999), for example, as previously observed in long-term cool-stored Aesculus hippocastunum (Tompsett and Pritchard 1998). REVIEW 329

Wet storage responses in relation to partial drying and temperature

In addition to possible effects on ‘maturity’ (see earlier discussion), partial drying may contribute to better retention of seed quality during the wet storage of desiccation sensitive seeds. In particular, there may be less presprouting during storage. This was the case for Hopea odorata when stored at ca. 40% compared to ca. 50% moisture content, for Shorea leprosula held at ca. 30% compared to ca. 40% moisture content, and for Vitellaria paradoxa (ca. 30% versus 40% moisture content). In these three species, a reduction in temperature from 25 to 16°C also reduced presprouting and proliferation of fungi during storage. Cool (16°C) storage also reduced presprouting in four other dipterocarps (Neobalanus heimii, Shorea assamica, S. macroptera and S. roxburghii), and in Vitellaria paradoxa. In contrast, there was little difference in retention of seed viability per se in Prunus africana and Hopea odorata during wet storage at just below and above 50% and 40% moisture content, respectively, and temperatures of 16°C and 25°C. However, there are documented cases where partial drying is markedly deleterious (Corbineau and Côme 1986a,b; 1988; Drew et al. 2000). In general, cool storage at around 15°C was observed to be the best temperature for the storage in 14 (of 21) species with desiccation sensitive seeds (Table 1). At this temperature, longevity was often in the region of 3–6 months. A positive effect of lower temperature was observed, however, in Illicium verum, Cinnamomum cassia and Michelia mediocris. In these species, all with desiccation sensitive seeds, storage at 5°C was better than at warmer temperatures (15°C and room temperature). Although a similar response has been observed previously in desiccation sensitive seeds of Araucaria angustifolia and A. hunsteinii (Tompsett and Kemp 1996), this is unusual for tropical seeds. In the case of the three species studied in this project, this relative level of cold tolerance may relate to the altitudinal range in which the species are found, which range from a maximum of 700– 1700 m above sea level. Storage at ca. 16°C has been found to afford far better conditions than either 25°C (when metabolism is heightened and fungi proliferate rapidly) or 6°C, which has proved lethal for certain tropical species, for example Trichilia dregeana (Berjak et al. 2004). Nevertheless, when seeds of T. dregeana harboured internal fungi, even 16°C-storage did not afford a useful storage period. One of the problems frequently encountered during wet storage of seeds is the maintenance of seed moisture content at the prescribed 330 STORAGE BIOLOGY OF TROPICAL TREE SEEDS level. In the case of Hopea odorata and Vatica astrotricha, an increase in the rate of seed viability loss coincided with a rapid fall in seed moisture content below the CMC identified in the drying experiments. Presumably, these effects were a manifestation of water loss from bags used for storage in association with regular ventilation to ensure the seeds did not become anoxic. The opposite may have been the case for Illicium verum, Cinnamomum cassia and Michelia mediocris when stored in aluminium bags at relatively high moisture contents (ca 15–40%). During storage, these seeds all increased in moisture content by about 10%, probably as a result of the onset of general metabolism. However, the effects of fungal metabolism that would elevate the moisture content, cannot be precluded.

Dry storage responses

Data is presented in this volume on the dry storage of seeds from 24 species (Table 1). Generally, these results were generated over a relatively short timescale; exceptionally, Hagenia abyssinica seed was stored for 48 months. Because seeds of all the species were not stored under the same moisture content conditions, it has only been possible to draw some general observations, especially in relation to temperature. For 12 species, cold temperature storage (close to that used in stores for long-term seed conservation) was either better than warmer temperatures or no worse. In addition, cooler temperature (ca. 4°C) was clearly better than warmer temperatures in two species (Ximenia americana and Dovyalis caffra) or appeared to pose no particular problem for storage (Kigelia africana, Genipa americana, Shorea henryana and Sterculia quinqueloba). In addition, it was shown that the partially desiccation tolerant seeds of Warburgia ugandensis could be cryopreserved. Thus, the majority of tropical species with desiccation tolerant seeds responded reasonably well to cold/cool storage. However, it is possible that the responses of tropical seeds to desiccation and cold- (or sub-zero-) storage might relate to provenance. In the case of the gymnosperm, Welwitschia mirabilis, which grows well within the tropical zone, but under extremely arid, desert conditions, seeds have been found to be remarkably tolerant to extreme desiccation and tolerate storage at both –20°C and liquid nitrogen immersion without any adverse effects: in fact, maximum germination was achieved in 3 d, which was half the time taken for seeds that had not been frozen (Whitaker et al. 2004). REVIEW 331

Table 1. General patterns in seed storage survival (often high, sometimes low) under wet and dry conditions in relation to temperature Family Species Wet storage Dry storage (temp./time) (temperature) Anacardiaceae Astronium graveolens ND –17°C Anacardiaceae Buchanania lanzan ND –20°C > other°C Anacardiaceae Lannea microcarpa ND Some indication 15>5> –20°C Anacardiaceae Sclerocarya birrea ND –18°C Apocynaceae Hancornia speciosa 10°C / 2 ND months Bignoniaceae Kigelia africana ND 4°C Canellaceae Warburgia salutaris ND ND Canellaceae Warburgia 16°C/4 weeks Cryopreservation ugandensis (salutaris) (in fruit) possible Clusiaceae Calophyllum 15°C / 3 ND brasiliense months Dipterocarpaceae Hopea hainanensis 15°C / 9 ND months Dipterocarpaceae Hopea odorata 16°C / 4 ND months Dipterocarpaceae Neobalanocarpus 16°C / 5 ND heimii months Dipterocarpaceae Shorea assamica 16°C / 4 ND months Dipterocarpaceae Shorea henryana ND 5°C and 15°C Dipterocarpaceae Shorea leprosula 16°C / 5 ND months Dipterocarpaceae Shorea macroptera 16°C / 5 ND months Dipterocarpaceae Shorea roxburghii 16°C / 5 ND months Dipterocarpaceae Vatica astrotricha 25°C / 3 ND months Ebenaceae Diospyros ND –20°C > other°C melanoxylon Euphorbiaceae Hieronyma ND –18>5 15°C alchorneoides Fabaceae Anadenanthera ND –20°C to 18°C colubrina Fabaceae Cordyla pinnata 15°C (3 ND months) Flacourtiaceae Dovyalis caffra ND 3°C > 25°C (3 months) Illiaceae Illicium verum 5°C / 12 ND months Lauraceae Cinnamomum cassia 5°C / 9 months ND Lecythidaceae Cariniana pyriformis ND 18°C (2.5 months) Loganiaceae Strychnos cocculoides ND 4>16>–20°C (3 months) Magnoliaceae Michelia mediocris 5°C / 9 months ND 332 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Family Species Wet storage Dry storage (temp./time) (temperature) Meliaceae A. indica var. 20°C / 4 ND siamensis months Meliaceae Azadirachta indica ND ND Meliaceae Ekebergia capensis 16°C / 4 ND months Meliaceae Khaya senegalensis ND –18°C (22 months) Meliaceae Melia volkensii ND ND Meliaceae Trichilia emetica ND ND Myrtaceae Acmena 15°C / 3 ND acuminatissma months Myrtaceae Syzygium cuminii ND ND Myrtaceae Syzygium guinense ND ND Ochnaceae Lophira lanceolata 16°C / 6 ND months Olacaceae Ximenia americana ND 4>16>25°C Podocarpaceae Podocarpus falcatus ND –20°C; 4°C (30 months) Podocarpaceae Podocarpus nagi ND ND Rosaceae Hagenia abyssinica ND –20°C (48 months) Rosaceae Prunus africana ND –20° (3 months) Rubiaceae Genipa Americana ND 5 to 15°C Rutaceae Zanthoxylum ND –18°C zanthoxyloides Sapotaceae Madhuca indica 25°C / 2 ND months Sapotaceae Pouteria macrophylla ND ND Sapotaceae Vitellaria paradoxa 16°C / 6 ND months Sterculiaceae Sterculia quinqueloba ND 4°C Verbenaceae Gmelina arborea ND –20 0>15>25°C Vochysiaceae Vochysia ferruginea ND 15 5>–17°C Vochysiaceae Vochysia ND 15>–17°C guatemalensis For wet storage, seeds were mostly held close to their harvest moisture content or that achieved after processing, i.e. it varies with species. Dry storage relates to moisture contents close to 10% or lower and for storage periods of 6–18 months, unless indicated. ND=not determined.

In contrast, in the studies presented here, four species exhibited some sensitivity to cold storage at around –18°C (i.e. Lannea microcarpa, Strychnos cocculoides, Vochysia ferruginea and Vochysia guatemalensis), and Cariniana pyriformis seeds stored better at 18°C compared to a cooler temperature (4°C). Recent findings of exceptional longevity of Coffea arabica seeds at –20°C (Hong and Ellis 2002), which was previously thought to be impossible, suggest the need for caution in assigning species to the ‘cannot cold store’ category, and support the notion that the traditional categories of REVIEW 333 seed storage should be redefined with respect to a moving temperature scale (Pritchard 2004).

Species diversity

Aspects of species diversity have already been referred to above in the context of variable seed mass. In addition, seed shapes were recorded for many, but not all, species, which unfortunately precludes an analysis of the how well seed shape correlates with seed storage category (Tompsett and Kemp 1996; Hong and Ellis 1998). Another aspect of species diversity that may directly impinge on our interpretation of seed viability following drying or storage relates to the nature of ‘propagule’ used in the germination test. Multiseeded units pose a particular problem. In the case of Sclerocarya birrea, there are about two seeds per dispersal unit (a fruit with a hard endocarp). Thus, an assessment of germination could relate to the percentage of seeds growing or the percentage of dispersal units showing growth. In one study reported in this volume, complete extraction of Sclerocarya birrea seeds was achieved, whereas in other studies on this species this has not been the case (Pritchard et al. 2004b). ‘Polyembryony’ in the dispersal unit is also a feature of Syzygium cuminii, Hieronyma alchorneoides and Hopea odorata. In germination tests on H. alchorneoides and H. odorata, first emergence from the dispersal unit was used as the measure of viability. A different problem was encountered in Hagenia abyssinica. Although there was only one seed per flower head, the proportion of empties was exceptionally high. In the case of Welwitschia mirabilis, 83% germination was achieved if the tenacious dry bracts surrounding the seeds were first removed, in contrast to 48%, when seeds were set to germinate with these coverings intact (Whitaker et al. 2004). All these examples emphasise the importance of understanding and describing clearly the anatomy of the ‘propagule’ sown in the germination test. At a taxonomic level, the diversity of the 52 species studied in this project is considerable, being drawn from 42 genera and 27 families. Overall in seed conservation biology, certain families are known to represent seed desiccation sensitivity ‘hotspots’, including Fagaceae (80% of species studied are recalcitrant), Lauraceae (77%) and Sapotaceae (65%) (Dickie and Pritchard 2002). Representatives of both Lauraceae and Sapotaceae were investigated here with many other families. Although only a relatively small number of species were investigated per family, the data has added significantly to baseline 334 STORAGE BIOLOGY OF TROPICAL TREE SEEDS data. For example, for 11 families the percentage of species’ seed storage records increased by 10% as a result of this project. In the case of Lophira lanceolata (Ochnaceae) and Illicium verum (Illiciaceae), these appear to be the first records for the families. In many instances, the data reported here reinforces perceptions of earlier data. For example, only one of nine species of dipterocarps investigated here had some semblance of desiccation tolerance (Table 2), supporting the overall figure of 98% of species investigated having desiccation sensitive seeds (Tweddle et al. 2003). In Anacardiaceae, the four species investigated here all had desiccation tolerant seeds, as do >80% of species so far studied. Finally, one third of Meliaceae species seeds studied here were desiccation tolerant, compared to ca. 40% recorded across the family so far.

Conclusion

Over the last 30 years there has been a significant effort to better understand seed storage responses in a wide range of material. Because, initially at least, the data sets for many species have been rather limited, there have been several occasions where the storage classification of the species could be considered to be ‘provisional’. Experience has taught us that as more information on a species becomes available, there is often an indication of greater opportunities for seed storage than previously thought. In other words, it is often the case that seed biologists learn to handle the seeds more optimally and as a consequence survival of desiccation and storage is improved. The information presented in this volume will ensure improved practice for many of the species investigated during the project. One of the main scientific objectives of this study was for collaborating groups to produce data sets of sufficient quality that they could be merged and that a clear impression could be gained of how best to handle seeds of many species new to seed conservation science. On the one hand, the range of collaborative chapters in this volume indicates that this was achieved on many occasions. On the other hand, however, for some species the findings of the work were not as conclusive as we would have hoped for. This is particularly the case when it comes to separating subtle differences in the storage response of some desiccation tolerant seeds. But this just means that further detailed studies should be conducted on some of the species in the future, and argues for further attempts at improving the diagnosis of seed storage behaviour. Table 2. Taxonomy and general features of the dispersal unit for the 27 families containing the species studied in relation to seed desiccation tolerance at the family level. Parinari curatellifolia (Chrysobalanaceae) is excluded, as the seed lot did not germinate Family Family Fruit/seed features1 No. Species No. No. species taxonomy1 species in with fully species studied here family1 desicc. studied2 (no. highly tol. seeds (% of desicc. tol.) (%)2 family) Anacardiaceae Dicots – fruit usually a drupe; seed with oily 875 81 73 (8) 4 (4) Rosidae- embryos Sapindales Apocynaceae Dicots – fruit diverse (follicle, berry or drupe); 1900 79 33 (2) 1 (0) Asteridae – seeds flattened; seed ±oily endosperm Gentianales Bignoniaceae Dicots – fruit a bivalved capsule, rarely fleshy 750 96 48 (13) 1 (1) Asteridae – and indehiscent; seeds usually flat, Scrophulariales winged Canellaceae Dicots – fruit a berry; seed with oily endosperm 13 0 2 (15) 2 (2) Magnoliidae – Magnoliales Clusiaceae Dicots – fruit a drupe or berry or septicidal 1350 65 46 (5) 1 (0) Dilleniidae – capsule; seeds often arillate Guttiferales Dipterocarpaceae Dicots – dry indehiscent fruit; seeds without 680 2 94 (<1) 9 (1) Dilleniidae – endosperm or dormancy

Malvales REVIEW Ebenaceae Dicots – fruit a berry, rarely dehiscent; seeds 485 41 22 (5) 1 (1) Dilleniidae – large with thin testa and hard oily Theales endosperm and embryo with leafy (usually emergent) cotyledons 335 336

1 Family Family Fruit/seed features No. Species No. No. species TREESEEDS OF TROPICAL BIOLOGY STORAGE taxonomy1 species in with fully species studied here family1 desicc. studied2 (no. highly tol. seeds (% of desicc. tol.) (%)2 family)

Euphorbiaceae Dicots – fruit often a capsular schizocarp or 8100 84 76 (<1) 1 (1) Delleniidae – drupe, samara or berry; seeds with Euphorbiales embryo embedded in oily endosperm Fabaceae Dicots – fruit usually dry and dehiscent or 18 000 98 1335 (7) 2 (1) Rosidae – indehiscent and samaroid or a drupe; Fabales seeds with little endosperm and an aril (often vestigial). Flacourtiaceae Dicots – Fruit usually a berry; seeds often 875 75 8 (<1) 1 (1) Dilleniidae - aril1ate with oily endosperm Violales Illiaceae Dicots – Fruit a head of radiating follicles; 42 0 1 (2) 1 (0) Magnoliidae - embryo very small, endosperm oily Illiciales Lauraceae Dicots – Fruit a berry (rarely dry dehiscent) often 2850 5 38 (1) 1 (0) Magnoliidae - enclosed in fleshy to woody Magnoliales hypanthium; seed (often) with oily embryo Lecythidaceae Dicots – Fruit a capsule, often very large or a 285 33 11 (4) 1 (1) Dilleniidae - drupe or berry; seeds often nut-like, Lecythidales winged or often with aril; large oily embryo with thickened hypocotyl Loganiaceae Dicots – Fruit a capsule, berry or drupe; seeds 570 100 8 (1) 1 (1) Asteridae - sometimes winged Gentianales Magnoliaceae Dicots – Fruit a follicle or indehiscent and berry- 165 53 19 (12) 1 (0) Magnoliidae - like or samaroid; seeds usually large Magnoliales with sarcotesta; embryo small in oily Family Family Fruit/seed features1 No. Species No. No. species taxonomy1 species in with fully species studied here family1 desicc. studied2 (no. highly tol. seeds (% of desicc. tol.) (%)2 family) endosperm Meliaceae Dicots – Fruit a capsule, berry or drupe; seeds 565 39 64 (11) 6 (2) Rosidae - winged or with corky outer layers or Sapindales with fleshy sarcotesta or aril, usually without endosperm Myrtaceae Dicots – Fruit a berry, capsule, drupe or nut; 4620 84 178 (4) 3 (0) Rosidae - seeds often polyembryonous initially Myrtales Ochnaceae Dicots – Fruits often of drupelets, sometimes a 370 0 1 (<1) 1 (0) Dilleniidae – capsule, nut or berry; seeds often Theales winged with oily endosperm

Olacaceae Dicots – Fruit a capsule, berry, drupe or samara; 615 100 3 (<1) 1 (1) Asteridae - seed with oily endosperm Scrophulariales Podocarpaceae Gymnospermae Mature cones drupe-like 168 15 13 (8) 2 (1) Rosaceae Dicots – Fruit a head of follicles or achenes or 2825 92 247 (9) 2 (2) Rosidae – enclosed in swollen hypanthium or a Rosales head of drupelets or a pome; seeds usually without endosperm Rubiaceae Dicots – Fruit a capsule, berry, drupe or 10 200 70 101 (1) 1 (1) Asteridae - schizocarp; seeds usually with oily Rubiales endosperm REVIEW Rutaceae Dicots – Fruit schizocarps, berries, drupes; 1800 51 68 (4) 1 (1) Rosidae - seeds ± endosperm (oily) Sapindales

337 338

Sapotaceae Dicots – Fruit fleshy, indehiscent; seed large, 975 10 30 (3) 3 (0) Delleniidae - embryo with flat cotyledons in oily Sapotales endosperm or thick embryo and no endosperm Sterculiaceae Dicots – Fruit dehiscent or not, fleshy to leathery 1500 84 56 (4) 1 (1) Dilleniidae – or woody; seed embryos usually in oily Malvales endosperm Verbenaceae Dicots – Fruit a head of nutlets, a drupe or 950 100 18 (2) 1 (1) Asteridae - valved capsule; seeds with oily embryo Lamiales Vochysiaceae Dicots – Fruit a loculicidal capsule or samara 210 100 3 (1) 2 (2) Rosidae - winged; seeds often winged or hairy Polygalales 1Mabberley (1997). 2To the extent that they are probably orthodox (see Tweddle et al. 2003), and fit the Type I seed storage class (Pritchard, 2004). Includes species from this study already in Tweddle et al. (2003), as extracted from the Project Newsletters, and other species included in this volume. REVIEW 339

References

Berjak, P. and N.W. Pammenter. 2004. Recalcitrant seeds. In Seed Physiology: Applications To Agriculture (R.L. Benech-Arnold and R.A. Sánchez, eds.). Haworth Press (in press). Berjak, P., J.I. Kioko, A. Makathini and M.P. Watt. 2004. Strategies for field collection of recalcitrant seeds and zygotic embryonic axes of the tropical tree, Trichilia dregeana Sond. Seed Sci. Technol. 32:825–836 (in press). Convention on Biological Diversity (CBD). 2002. Global strategy for plant conservation. The Conference of the Parties 6 (Decision VI/9). The Secretariat of the CBD, Montreal, Canada. Corbineau, F. and D. Côme. 1986a. Experiments on the storage of seeds and seedlings of Symphonia globulifera L.f. (Guttiferae). Seed Sci. Technol. 14:585–591. Corbineau, F. and D. Côme. 1986b. Experiments on germination and storage of the seeds of two dipterocarp: Shorea roxburghii and Hopea odorata. The Malaysian Forester 49:371–381. Corbineau, F. and D. Côme. 1988. Storage of recalcitrant seeds of four tropical species. Seed Sci. Technol. 16:97–103. Danthu, P., A. Gueye, A. Boye, D. Dauwens and A. Sarr. 2000. Seed storage of four Sahelian and Sudanian tree species (Boscia senegalensis, Butyrospermum parkii, Cordyla pinnata and Saba senegalensis). Seed Sci. Res. 10:183–187. Daws, M.I., C.S. Gaméné, S.M. Glidewell and H.W. Pritchard. 2004a. Seed mass variation potentially masks a single critical water content in recalcitrant seeds. Seed Sci. Res. 14:185–195. Daws, M.I., E. Lydall, P. Chmielarz, O. Leprince, S. Matthews, C.A. Thanos and H.W. Pritchard. 2004b. Developmental heat sum influences recalcitrant seed traits in Aesculus hippocastanum L. across Europe. New Phytol. 162:157–166. Dickie, J.B. and H.W. Pritchard. 2002. Systematic and evolutionary aspects of desiccation tolerance in seeds. Pp. 239–259 in Desiccation and Survival in Plants: Drying without Dying (M. Black and H.W. Pritchard, eds.). CABI Publishing, Wallingford, UK. Drew, P.J., N.W. Pammenter and P. Berjak. 2000. 'Sub-imbibed' storage is not an option for extending longevity of recalcitrant seeds of the tropical species. Trichilia dregeana Sond. Seed Sci. Res. 10:355–363. Engelmann, F. and J.M.M. Engels. 2002. Technologies and strategies for ex situ conservation. Pp. 89–103 in Managing Plant Genetic Diversity (J.M.M. Engels, V. Ramanatha Rao, A.H.D. Brown and M.T. Jackson, eds.). CABI Publishing, Wallingford, UK. FAO. 1996. Report on the State of the World’s Plant Genetic Resources for Food and Agriculture. Food and Agriculture Organization of the United Nations, Rome, Italy. von Fintel, G.T., P. Berjak and N.W. Pammenter. 2000. Seed behaviour in Phoenix reclinata Jacquin, the wild date palm. Seed Sci. Res. 14:197–204. 340 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Harper, J.L., P.H. Lovell and K.G. Moore. 1970. The shapes and sizes of seeds. Annu. Rev. Ecol. Syst. 1:327-356. Hay, F.R. and R.D. Smith. 2003. Seed maturity: when to collect seeds from wild plants. Pp. 97–133 in Seed Conservation: Turning Science into Practice (R.D. Smith, J.B. Dickie, S.H Linington, H.W. Pritchard and R.J. Probert, eds.). Royal Botanic Gardens, Kew, UK. Hong, T.D. and R.H. Ellis. 1996. A Protocol to Determine Seed Storage Behaviour. IPGRI Technical Bulletin No. 1, IPGRI, Rome, Italy. Hong, T.D. and R.H. Ellis. 1998. Contrasting seed storage behaviour among different species of Meliaceae. Seed Sci. Technol. 26:77–95. Hong, T.D. and R.H. Ellis. 2002. Optimum moisture status for the exceptional survival of seeds of arabica coffee (Coffea arabica L.) in medium-term storage at –20°C. Seed Sci. Technol. 30:131–136. Linington, S.H and H.W. Pritchard. 2001. Genebanks. Pp. 165-181 in Encyclopaedia of Biodiversity, Vol. 3 (S. Levin, editor-in-chief). Academic Press, New York. Mabberley, D.J. 1997. The Plant-Book (2nd edn). Cambridge University Press, UK. Pammenter, N.W. and P. Berjak. 1999. A review of recalcitrant seed physiology in relation to desiccation-tolerance mechanisms. Seed Sci. Res. 9:13–37. Pammenter, N.W., V. Greggains, J.I. Kioko, J. Wesley-Smith, P. Berjak and W.E. Finch-Savage. 1998. Effects of differential drying rates on viability retention of recalcitrant seeds of Ekebergia capensis. Seed Sci. Res. 8:463–471. Pritchard, H.W. 2004. Classification of seed storage ‘types’ for ex situ conservation in relation to temperature and moisture content. Pp. 136–161 in Ex situ Plant Conservation: Supporting Species Survival in the Wild (E.O. Guerrant, K. Havens and M. Maunder, eds.). Island Press, California, USA. Pritchard, H.W. and J.B. Dickie. 2003. Predicting seed longevity: the use and abuse of seed viability equations. Pp. 653–721 in Seed Conservation: Turning Science into Practice (R.D. Smith, J.B. Dickie, S.H Linington, H.W. Pritchard and R.J. Probert, eds.). Royal Botanic Gardens, Kew, UK. Pritchard, H.W., M.I. Daws, B.J. Fletcher, C.S. Gaméné, H.P. Msanga and W. Omondi. 2004b. Ecological correlates of seed desiccation tolerance in tropical African dryland trees. Am. J. Bot. 91:863–870. Pritchard, H.W., C.B. Wood, S. Hodges and H.J. Vautier. 2004a. 100-seed test for desiccation tolerance and germination: a case study on eight tropical palm species. Seed Sci. Technol. 32:393–403. Roberts, E.H. 1973. Predicting the storage life of seeds. Seed Sci. Technol. 1:499– 514. Sacandé, M. 2000. Stress, Storage and Survival of Neem Seed. PhD thesis, Wageningen University, The Netherlands. Sun, W.Q. and Y. Liang. 2001. Discreet levels of desiccation sensitivity in various seeds as determined by the equilibrium dehydration method. Seed Sci. Res. 11:317–323. REVIEW 341

Tompsett, P.B. and H.W. Pritchard. 1998. The effect of chilling and moisture status on the germination, desiccation tolerance and longevity of Aesculus hippocastanum L. seed. Ann. Bot. 82:249–261. Tompsett, P.B. and R. Kemp. 1996. Database of Tropical Tree Seed Research. Royal Botanic Gardens, Kew, UK. Tweddle, J.C., R.M. Turner and J.B. Dickie. 2003. Seed Information Database (release 5.0, July 2003). http://www.rbgkew.org.uk/data/sid. Walters, C. 1998. Levels of recalcitrance in seeds. Pp. 1–13 in Recalcitrant Seeds (IUFRO Seed Symposium, 1998) (M. Marzalina, K.C. Khoo, N. Jayanthi, F.Y. Tsan and B. Krishnapillay, eds.). FRIM, Malaysia and IUFRO. Whitaker, C., P. Berjak, H. Kolberg and N.W. Pammenter. 2004. Responses to various manipulations, and storage potential, of seeds of the unique desert gymnosperm, Welwitschia mirabilis Hook. fil. South Afr. J. Bot. 70:621–629. 342 STORAGE BIOLOGY OF TROPICAL TREE SEEDS Appendixes 344 STORAGE BIOLOGY OF TROPICAL TREE SEEDS APPENDIXES 345

Appendix 1

The Desiccation and Storage Protocol1

IPGRI-DFSC

International Plant Genetic Resources Institute, via dei Tre Denari 472/a, 00057 Maccarese, Rome, Italy; and Danida Forest Seed Centre, Krogerupvej 21, 3050 Humlebaek, Denmark

The following gives details on the procedures to be followed, and data to be recorded.

1. Floral biology and ecology Existing information on floral biology and ecology should be recorded and additional supplementary data should be collected.

2. Seed source Complete Appendix A (Seed Source Description)

3. Seed collection 3.1. Date 3.2. Harvest method. 3.2.1. Collection from trees. Note ripeness (e.g., colour, firmness). 3.2.2. Collection from the ground. Suspected recalcitrant seeds should be collected within 24 h of shedding. Note whether collection was from bare ground or if ground cover was present. 3.2.3. Note other details, e.g., insect predation. 3.2.4. Tree numbers and selection. Note the number of trees from which the seeds were collected. (Fill in Appendix B). Collect the same amount of fruits from at least 25 trees with an average appearance for that of the stand and producing a good crop. Bulk the fruits and mix them properly. (If the number of trees is lower than 25, the trial must be repeated later with a larger number of trees).

1 Please note that the protocol has not been thoroughly applied for all experimental chapters in this volume and this is the last revised version in June 2000. 346 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

3.2.5. Fruit numbers. If possible, collect double the number of seeds required for the trial to allow for discarding infected seeds.

4. Details of fruit transport 4.1. How were the seeds transported to the laboratory? 4.2. Containers for transport 4.2.1. Nature and size 4.2.2. Would these containers maintain moisture content? (this is important because the initial moisture content is determined only after transport). 4.3. Note minimum and maximum temperature during transport. 4.4. Duration of transport (Fill in Appendix B).

5. Fruit weight Before processing, measure fruit weight, average plus standard deviation. Measure 100 individual fruits (whenever possible). (Note that this is nondestructive and the seeds can be used for further studies). 6. Seed processing 6.1. If processing of seeds is necessary (e.g., cleaning, depulping, extraction, removal of specific parts), describe the procedures. 6.2. Note the number of days between processing and the start of moisture content and desiccation trials in the lab. 6.3. Duration of processing (fill in Appendix B). 6.4. Take a sample of seed before processing for moisture content determination (8A) the seeds should be processed manually without water. 6.5. Avoid desiccation during processing. 6.6. Remove mechanically damaged, infected and infested seeds. 6.7. Soak the seeds for 10 minutes in a 1% solution of sodium hypochlorite (NaOHCl) 6.8. Rinse and blot dry

7. Preparing for dispatch and desiccation 7.1. Take samples for moisture content testing (8A), germination testing (9) and seed characteristics (10). 7.2. Immediately after sampling for moisture content determination, seeds for the replicating partner are packed and dispatched (7.3) and the rest of the seed lot is divided into portions for desiccation (11) and control storage (12), where after the individual portions are weighed. 7.3. Pack seeds for dispatch in plastic bags with a little vermiculite or APPENDIXES 347 similar material to absorb excess moisture. (Germination rate may be increased if the seed is exposed to free water). Bags must then be loosely sealed. For details see Appendix G.

8A. Initial moisture content testing 8.1. Determine the moisture of whole seeds. The moisture content is determined before extraction on manually extracted seed (6.4) and another determination is made after processing. Use 25 seeds in 5 replicates of 5 seeds each. Cut large seeds into smaller pieces. Dry at 103°C for 17 h. Express moisture content on a fresh weight basis. 8.2. Determine the variation in moisture content within the seed by measuring the moisture content of individual components, i.e., axis versus storage tissue and covering structures or other relevant parts. Use 100 individual seeds. Weigh immediately after excising components (to avoid desiccation). In some cases the seed may be too small for separating, thus moisture content determination cannot be performed on the separate component parts.

8B. Subsequent moisture content testing (during/after desiccation) As in 8.1.

9. Seed characteristics 9.1. Weight. Measure the weight of 100 individual seeds calculating the average and standard deviation. (Nondestructive) 9.2. Anatomy. Sketch and describe seed/fruit components, i.e., axis, cotyledons, endosperm, testa and pericarp. 9.3. Note the days elapsed between collection and these tests.

10. Initial germination capacity 10.1 Note the number of days after collection that the germination test was initiated (Fill in Appendix B). 10.2 Describe the unit for testing (i.e., is the unit for testing the seed, part of the seed, or does it include parts of the fruit?). 10.3. Use a minimum of 4 replicates of 25 seeds. 10.4. Use an appropriate germination medium, moistened but not flooded. Sterilize medium by heating (e.g., expose to 130°C for 1 h). 10.5. Germination temperature 10.5.1. For tropical seeds use a germination temperature of 25-30°C 10.5.2. If seeds are of a warm temperate origin, use 20–25°C. 10.5.3. If seeds are of a cold temperate origin, use 15–20°C. 10.5.4. Monitor temperature during germination. 348 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

10.6. Germinate at 8–12/16–12 h light/dark (imitate natural conditions). 10.7. Germination assessment 10.7.1. Assess daily for quickly germinating species. For other species check at least weekly. Specify criterion used to score positive germination. 10.7.2. Assess seedling establishment. Score as positive when cotyledons have emerged (not for hypogeal germination) and first set of leaves show signs of normal development. 10.7.3. Score number of seedlings with abnormal appearance, and describe abnormality. 10.7.4. Continue the test until seeds either germinate or are rotten. 10.7.5. At the end of the test, non-germinating seeds are cut open to determine whether they are still in good condition or rotten preferably using the Tetrazolium test (optional). Note also whether any radicle extension has occurred within the seed. 10.7.6. Pre-sprouted seeds are counted, recorded and not included in trials. 10.7.7. In cases of polyembryony both the number of ‘seeds’ that have germinated with at least one seedling + the total number of seedlings are counted and recorded.

11. Desiccation sensitivity (Appendix C) 11.1. Desiccate seeds by mixing with an equal amount of silica gel and enclose in 3–6 (see table 1 below) containers (e.g., plastic bags), i.e., a separate bag for each target moisture content. 11.2 Place containers under ambient temperature (25–30°C). If ambient temperature is below or above this range, an incubator must be used. 11.3. Controls to determine if the time factor affects the results are placed in similar containers with vermiculite in place of the silica gel (see 12). 11.4. Additionally, less rapid methods of desiccation may be used. 11.5. Change the silica gel as required, and always in all containers at the same time. 11.6. Aerate seeds by mixing once or twice daily to avoid anoxia, as well as when weighing and/or changing silica gel. 11.7. Periodically monitor water loss by weighing seeds (sieve to remove silica gel) and note duration of drying. The frequency of this monitoring should be higher in the beginning. 11.8.1. Target moisture calculation APPENDIXES 349

Initial MC (before processing) (%) Target MC (%)

10 9, 6, 3 11–15 12, 9, 6, 3 16–20 15, 12, 9, 6, 3 21–25 20, 15, 9, 6, 3 26–30 25, 20, 15, 12, 9, 6 31–35 30, 25, 20, 15, 10, 5 36–40 35, 30, 25, 20, 10, 5 41–45 40, 35, 30, 20, 10, 5 46–50 45, 40, 35, 25, 15, 8 51–55 50, 45, 40, 35, 25, 10 56–60 55, 50, 45, 35, 25, 10 >60 60, 50, 40, 30, 20, 10

Calculate corresponding target weight by using the following formula:

Weight of seed (g) at TMC = =(100–MC after processing)/(100–TMC) × initial seed weight (g)

Example: 1500 g of seed at MC after processing of 50%; what is the weight when the seed is dried to a target moisture content of 30%?

(100–50)/(100–30) × 1500 g = 1071 g

11.8.2. Calculate the target weight for the seed batch from each of the containers and take a sample for germination and moisture content testing when target weight is reached. 11.9 To prevent imbibition damage of seed samples at moisture contents below 15%, seeds should be humidified before they are germinated. Humidify seeds by placing above the water surface in a closed container at germination temperature for the time necessary for the seed weight to increase by 10–15%. 11.10. When very few seeds are available, a preliminary investigation may be performed by drying to 12% and 5% levels only. 11.11. If the relationship between moisture content and germination capacity reveals a critical moisture content at 15–20% or higher, the seed is considered recalcitrant. 11.12. Note the daily maximum and minimum temperatures during the drying period, possibly by using a thermohydrograph. 350 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

11.13. A small pilot sample can be started before the desiccation trial to test whether target and actual moisture contents correspond.

12. Control stock of seed during desiccation 12.1. A control stock of seed is maintained at the initial moisture content to determine if the time factor affects the result (see 11.3.). 12.2. Sampling is done simultaneously with the seed undergoing desiccation. 12.3 Storage is therefore carried out in 6 or more containers under exactly the same conditions, except that the silica gel is not included. Avoid direct seed contact with bottom of container. 12.4. Aeration takes place simultaneously with that of seeds undergoing desiccation (see 11.6). 12.5. Note that moisture loss must be avoided during storage. 12.6. Note the number of seeds that are fungally contaminated, and the number that have germinated during storage.

13. Storage behaviour The desiccation trial (11.11) will determine whether or not the seed is recalcitrant. If the seed is recalcitrant refer to 16 if the seed is not recalcitrant refer to 14.

14. Determination of orthodox/intermediate behaviour If a seed shows evidence of reduced vigour at moisture contents 8–9% and above, it is probably not orthodox (assuming that it was fully mature when subjected to desiccation). The term “intermediate” has been used to cover all nonorthodox and nonrecalcitrant storage responses. Note that sometimes confusing indications can be obtained e.g., chilling tolerant seeds that cannot be desiccated below 8–9%, or seeds that do not tolerate low temperatures, but may withstand relatively extreme desiccation in the short term. 14.1. Store samples hermetically at 3, 6, 9 and 12% moisture content at 20, 15, 5 and –20°C in a factorial combination. 14.2. Samples for testing moisture content and germination capacity are drawn at 3, 6, 12, 18 and 24 months. 14.3. In the case of very little seed being available, store seed in a factorial combination at three moisture contents close to the lowest tolerated (e.g., 6, 9 and 12%) at 5 and 15°C and sample after 3, 6, and 12 months only. 14.4. Use sealed aluminium foil packages for storage. If these are not available, they will be supplied by the project. APPENDIXES 351

14.5. Leave the seed package sealed at room temperature for 1 day prior to sampling, in order to avoid condensation on the cold seed. Rehydrate as described in 11.9 if moisture content is below 15%. 14.6. If results show an orthodox response to storage, the seed should be stored at the lowest moisture content tolerated at sub-zero temperatures to achieve the best longevity. In the event of a nonorthodox response refer to points 15 and 16 below.

15. Optimizing storage conditions for intermediate seeds It is not possible to provide strict guidelines regarding intermediate seed, which will therefore depend on the results found in 14. Initiate additional storage trials using factorial combinations of moisture content and temperature in a narrow range around optimal values determined in 11 and 14.

16. Optimizing storage conditions for recalcitrant seed 16.1. Results from the first phase of the project have demonstrated that it is not possible to provide strict guidelines for recalcitrant seed either. Therefore, make additional storage trials using factorial combinations of moisture contents, in a narrow range around optimal values determined in 11, and °C, 1°C and ambient (20–2°C). 16.2. Pack seeds in loosely folded plastic bags on top of some netting to avoid direct seed contact with the bottom of the bag where water may accumulate. The seeds should be positioned in the same way and not stacked on top of each other. 16.3. Draw samples for testing germination capacity and moisture content (simultaneous testing) on a regular basis according to your experience with seed storability, e.g., after 1, 3, 6, 9 and 12 months.

17. Supplementary trials The range of factors to test should not be limited by these recommended procedures as unexpected results are occasionally reported. The following are examples of what could be tested: removal of covering structures, alginate encapsulation, fungal dressing, controlled atmosphere storage. 352 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Appendix 2

Recommendations from the project final workshop at Crete, 2002

Strategy for future collaboration

One of the outputs of the project was the development of a strategy for continuation of the research activities and the establishment of a research network. Initial discussions on future collaboration were held between IPGRI and the main partner DFSC, as well as with the country partners during the regional training workshops held in Kenya, Costa Rica and Thailand. The subject was then thoroughly discussed at the final workshop held in Crete in Sept 2002. In the light of new information generated during the project, participants at the final workshop identified and discussed the bottlenecks that still hamper a large-scale use of high value tropical forest tree species in planting programmes and re-afforestation. They agreed on a strategy having three major objectives: 1. Carry out new research to improve the use of high value tropical forest tree species. 2. Scale-up the use of target species in planting programmes with focus on seed supplies, nursery and silvicultural practices. 3. Promote the broader use of high value tropical forest species through a public awareness campaign and the dissemination of the knowledge gained to users and the public at large. Each of these objectives was discussed in separate working groups, namely, Research Needs, Applied Issues and Use and Dissemination. The main recommendations of each working group are presented below.

1 Recommendations of the Research Needs Working Group

Participants recognized that additional research is still required to ensure quality seeds are made available. Moreover, seeds harvested should be accompanied by the technical information that is essential for successful establishment of afforestation and plantation programmes. In this respect, a number of research gaps were identified relating to the ecological characteristics of the target species, seed health and provenance trials. Recommendations for further research are presented below: APPENDIXES 353 x Investigations on seedling establishment for the target species, studies of the reproductive biology, the identification of seed pollinators and predators, the analysis of the relationship between flowering and environmental conditions (as adverse weather conditions may have a direct impact on fruit and seed production), and the monitoring of irregular flowering. For most of the species tested so far, information on these aspects has not yet been generated. x Seed and embryo development processes have serious implications for germination tests, seed desiccation tolerance, and optimal storage conditions. Pre-handling events and factors related to seed development and productions should be also further investigated. x For most of the species tested during the project, germination successes have been undermined by fungi attacks. Studies need to be carried out on the identification of fungi species and their influence of growth and development conditions on the severity of the attack. Furthermore, studies on the identification of effective systemic fungicide and the evaluation of the effect of fungicides and heat treatment sensitivity on germination and seed quality during storage should be undertaken. x Cryopreservation was identified as the only long-term method available for storing of seeds from species producing recalcitrant seeds. This technique requires extensive research, especially with regard to: the sterilization of explants, the effect of dehydration to different moisture contents on in vitro culture trials, the impact of freezing on regeneration, the process of seed rehydration, the genetic changes of cryopreserved materials. x To improve the use of forest tree seeds in plantation programmes, studies on species-site matching are needed. Provenance trials should be undertaken to evaluate the performance of given species.

2 Recommendations of Applied Issues Working Group

The discussion on the potential applications of the research results obtained during the project was centred on four themes referring to the major steps involved in scaling up the use of the selected species. Specific suggestions were put forward for each different phase, from availability and access to seed sources, to silvicultural practices. More specifically, the major issues tackled were: (a) how to secure seed sources; (b) how to make seeds (or genetic material) available to nurseries; (c) how to improve nursery practices, and finally (d) how to improve silvicultural knowledge on the target species. The group’s recommendations were as follows: 354 STORAGE BIOLOGY OF TROPICAL TREE SEEDS x The creation of an information network on seed sources (both in situ and ex situ collections), after having ensured sources are well distributed geographically to cover most of the range of the target species and include a proper sample of species’ provenances. x Information on seed handling procedures to be adopted prior to the arrival of seed to nurseries should be generated and transferred to seed collectors. Seed users should receive proper documentation attached to seed lots, with information on provenance characteristics, geographical co-ordinates of seed sources, altitude, purity, viability, etc. x Nursery staff should also be trained in best practices for seed handling before and during sowing. Nursery practices should also be improved to minimize seed losses. x Finally, the silviculture of the target species should be supported by a better knowledge of species responses to different treatments; this information could either be gathered or generated to ensure the success of plantations with native species. Permanent sample plots should be established to analyze and monitor the impact of both seed sources and silvicultural treatments on species performance.

3 Recommendations of the Use and Dissemination Group

An optimized dissemination of research results was identified as a crucial step to be taken in order to amplify the benefits of having tested seed tolerance on a large set of species. x The production of different types of information material was proposed. This would consist of seed leaflets, updated seed catalogues, Video/DVD on appropriate seed handling and desiccation protocols, and extension material (e.g., posters, flow charts, etc.). x The publication of research results in peer-reviewed journals was recommended, as this would considerably increase the visibility of the work carried out. x Demonstrations and training on seed handling should be offered to seed users. x Public awareness initiatives should be implemented (e.g., production of promotional T-shirt), involving the media (TV, national radio, newspaper), in order to increase interest in the use of indigenous species, as opposed to the introduction of exotics in large-scale plantations. APPENDIXES 355

Appendix 3

A selection of photos showing the variations of fruit and seed materials, some extraction procedures and scientists at work in their laboratories

Photo 2. Sclerocarya birrea. Stone (pyrene) with Photo 1. Khaya senegalensis seeds. A grid opercula removed to facilitate germination. [Photo: has 1 cm divisions. (Photo: Dorthe Jøker). Andrew McRobb (copyright RBG Kew, with permission)].

Photo 3. Melia volkensii fruit (top left), stones Photo 4. Mature (yellow) and immature (green) (pyrenes) after depulping (top right) and seeds fruits of Dovyalis caffra and fruits cut opened at the bottom. (Photo: William Omondi, showing seeds inside, photo taken at Muguga, KEFRI). KEFRI, Kenya in Oct 1999. (Photo: Kirsten Thomsen). 356 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Photo 5. Seeds of Podocarpus falcatus from Photo 6. Germinating seeds (without endocarp) on Tanzania. The seed on the left has been cut agar plate of Vitellaria paradoxa from Burkina through. A grid has 1 cm divisions. (Photo: Faso. The experiment was carried out in Sept Dorthe Joker). 2002, at the Seed Conservation Department, RBG Kew.

Photo 7. Seeds and fruits of. Acmena Photo 8. Seeds and fruits of Podocarpus nagi. acuminatissma (Photo: Xiaofeng Wang). (Photo: Xiaofeng Wang).

Photo 9. Mature fruits, cut-opened fruits and embryos of Neobalanocarpus heimii. (Photo: Jayanthi Nadarajan, FRIM). Photo 10. A fruit and seeds of Illicium verum. (Photo: Toil d’Epices). APPENDIXES 357

Photo 11. An open fruit of Anadenanthera Photo 12. Pouteria macrophylla seeds, from colubrina exposing its seeds. (Photo: Bolovia. (Photo: Dorthe Jøker). BASFOR).

Photo 13. Preparation of seeds of Syzygium cumini from Tanzania (1997) that are manually extracted from the fruits by two NTSC staff. (Photo: Kirsten Thomsen). Photo 14. Two scientists from the Forestry Seed Centre, Muguga, Kenya, working on Dovyalis caffra seeds to determine their moisture contents, Oct 1999. (Photo: Kirsten Thomsen).

Photo 15. Extraction of Calophyllum brasiliense seeds during Latin America training workshop at CATIE, Costa Rica 2000. (Photo: Dorthe Jøker). 358 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Photo 16. A Vietnamese scientist preparing seeds of Cinnamomum cassia for moisture determination, desiccation and storage trials at FSIV, Vietnam. Jan 2000. (Photo: Sigrit Diklev).

Photo 17. Group photo of Africa training workshop, Kenya 2000, showing participants from Burkina Faso, Denmark, Kenya, Malawi, Senegal, South Africa, Tanzania and Zimbabwe. Ehsan Dulloo (IPGRI), Kirsten Thomsen (DFSC), Hugh Pritchard (RBG Kew) and Deon Erdey (Univ. Natal) facilitated the workshop. (Photo: KEFRI). APPENDIXES 359

Photo 18. Group photo during Latin America training workshop, Costa Rica 2000. Participants were from Bolivia, Brazil, Costa Rica, Colombia, Denmark, Honduras and United States. Ehsan Dulloo (IPGRI) and Dorthe Jøker (DFSC) facilitated the workshop. (Photo: Dorthe Jøker).

Photo 19. Group photo of Asian training workshop, Thailand 2001. Participants were from China, India, Indonesia, Laos, Malaysia, Papua New Guinea, Tonga, Thailand, Vanuatu and Vietnam, Ehsan Dulloo (IPGRI), Dorthe Jøker and Kirsten Thomsen (both DFSC) facilitated the workshop. (Photo: Dorthe Jøker). 360 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Appendix 4 Species and partner institutes

Species Family Collecting partner Replicating partner Acmena acuminatissma Myrtaceae SCAU, China (Blume) Merr. & L.M. Perry Anadenanthera colubrina Fabaceae BASFOR, Bolivia Uni. Gabriel, (Vell.) Brenan Bolivia Astronium graveolens Jacq. Anacardiaceae CATIE, Costa Rica DFSC, Denmark Azadirachta indica var. Meliaceae FTSC, Thailand - siamensis Valeton.

Azadirachta indica A.Juss. Meliaceae CNSF, Burkina Faso - Buchanania lanzan Spreng Anacardiaceae Raipur Univ., India - Calophyllum brasiliense Clusiaceae CATIE, Costa Rica DFSC, Cambess. Denmark Cariniana pyriformis Miers Lecythidaceae CONIF, Colombia CENARGEN, Brazil Cinnamomum cassia Blume Lauraceae RSIV, Vietnam AFTSC, Australia Cordyla pinnata Leguminosae ISRA, Senegal CIRAD Foret, France Diospyros melanoxylon Roxb. Ebenaceae Raipur Univ., India - Dovyalis caffra (Hook. f. & Flacourtiaceae KEFRI, Kenya Uni. Natal, Harv.) Hook. f. RSA Ekebergia capensis Sparrm Meliaceae Uni. Natal, RSA - Genipa americana L. Rubiaceae CENARGEN, Brazil - Gmelina arborea Roxb. Verbenaceae Raipur Univ., India - Hagenia abyssinica J.F.Gmel. Rosaceae NTSP, Ethiopia DFSC Denmark Hancornia speciosa Gomez Apocynaceae CENARGEN, Brazil CATIE, Costa Rica Hieronyma alchorneoides Euphorbiaceae CATIE, Costa Rica DFSC, Allemao Denmark Hopea hainanensis Merr. & Dipterocarpaceae SCAU, China - Chun Hopea odorata Roxb. Dipterocarpaceae FRIM, Malaysia - Illicium verum Hook.f. Illiaceae FSIV, Vietnam AFTSC, Australia Khaya senegalensis (Desr.) Meliaceae CNSF, Burkina Faso Kew /ISRA A.Juss. Senegal Kigelia africana (Lam.) Benth. Bignoniaceae KEFRI/CNSF BF - Lannea microcarpa Engl. & Anacardiaceae CNSF, Burkina Faso Wageningen/R K.Krause BG Lophira lanceolata Van Tiegh. Ochnaceae CNSF, Burkina Faso Univ. Natal, Ex Keay RSA Madhuca indica J.F. Gmel. Sapotaceae Raipur Univ., India - Melia volkensii Gurke Meliaceae KEFRI, Kenya - Michelia mediocris Dandy Magnoliaceae FSIV, Vietnam - Neobalanocarpus heimii (King) Dipterocarpaceae FRIM, Malaysia R&D, Indonesia Parinari curatellifolia Planch. Chrysobalanaceae CNSF, Burkina Faso - ex Benth. Podocarpus falcatus (Thunb.) Podocarpaceae NTSP, Ethiopia DFSC Mirb. Denmark APPENDIXES 361

Podocarpus nagi (Thunb.) Podocarpaceae SCAU, China - Makino Pouteria macrophylla (Lam) Sapotaceae Uni. Gabriel, Bolivia BASFOR, Eyma Bolivia Prunus africana (Hook. f.) Rosaceae KEFRI/ICRAF Kew/Univ. Kalkm. Natal Sclerocarya birrea (A. Rich.) Anacardiaceae CNSF, Burkina Univ. Natal, Hochst. RSA Shorea assamica Dyer. Dipterocarpaceae FRIM, Malaysia - Shorea henryana Pierre Dipterocarpaceae FTSC, Thailand - Shorea leprosula Miq. Dipterocarpaceae FRIM, Malaysia - Shorea macroptera Dyer. Dipterocarpaceae FTSC, Thailand FRIM, Malaysia Shorea roxburghii G.Don Dipterocarpaceae FRIM, Malaysia - Sterculia quinqueloba (Garcke) Sterculiaceae FRIM, Malawi - K. Schum Strychnos cocculoides Baker Loganiaceae NTSP, Tanzania ICRAF/RBG Kew Syzygium cuminii (L.) Skeels Myrtaceae NTSP Tanzania Uni.Natal/RBG Kew Syzygium guinense (Willd.) Myrtaceae KEFRI, Kenya - DC. Trichilia emetica (Vahl.) Meliaceae KEFRI, Kenya Univ. Natal, RSA Vatica astrotricha Hance Dipterocarpaceae SCAU, China Vitellaria paradoxa Gaertn.f. Sapotaceae CNSF, Burkina Faso Kew/ISRA Senegal Vochysia ferruginea Mart. Vochysiaceae CATIE, Costa Rica U-Taastrup Denmark Vochysia guatemalensis Donn. Vochysiaceae CATIE, Costa Rica DFSC, Sm. Denmark Warburgia salutaris (Bertol. F.) Canellaceae KEFRI, Kenya Univ. Natal, Chiov. RSA Warburgia ugandensis( Canellaceae NTSP, Tanzania - salutaris) Sprague Ximenia americana L. Olacaceae NTSP, Tanzania RBG Kew, UK Zanthoxylum zanthoxyloides Rutaceae CNSF Burkina Faso - (Lam.) B.Zepernick & F.K.Timler 362 STORAGE BIOLOGY OF TROPICAL TREE SEEDS

Appendix 5 Contributors Baxter, David University of Natal, South Africa Berjak, Patricia University of Natal, South Africa Bhodthipuks, Jutitep ASEAN FTSC, Thailand Chaisurisri, Kowit ASEAN FTSC, Thailand Chanyenga, Tembo FRIM, Malawi Daws, Matthew I. SCD, RBG Kew, UK Demalash, Leuleseged NTSP, Ethiopia Diallo, Ismaïla ISRA, Senegal Dulloo, Ehsan IPGRI, Italy Erdey, Deon University of Natal, South Africa Eriksen, Erik N. Agri. Univ. Taastrup, Denmark Espinoza, Edilberto Rojas BASFOR, Bolivia Gaméné, Christiane S. CNSF, Burkina Faso Gaye, A. ISRA, Senegal Godheja, J.K. Raipur University, India Groot, Steven P.C. PRI Wageningen, NL Harris, Catherine SCD, RBG Kew, UK Hoekstra, Folkert University Wageningen, NL Howard, Caroline A. SCD, RBG Kew, UK Huang, C. J. SCAU, China Hung, Nguyen Tuan FSIV, Vietnam Jayanthi, Nadarajan FRIM, Malaysia Jøker, Dorthe DFSC, Denmark Kha, Le Dinh FSIV, Vietnam Kioko, Joseph University Natal, South Africa Krishnapillay, Baskaran FRIM, Malaysia Lait (nee Saelim), Suomal ASEAN FTSC, Thailand Mat said, Siti Hasanah FRIM, Malaysia Mbatha, Zama University Natal, South Africa Motete, Nthabiseng University Natal, South Africa Msanga, Heriel P. NTSP, Tanzania Munjuga, Nioses ICRAF, Kenya Naithani, Ranjana Raipur University, India Naithani, Subash C. Raipur University, India Neya, Oblé CNSF, Burkina Faso Ojeda, Jaime Magne University Gabriel, Bolivia Omondi, William KEFRI, Kenya Poulsen, Karen M. DFSC, Denmark Pritchard, Hugh W. SCD, RBG Kew, UK Pukittayacamee, Prapan ASEAN FTSC, Thailand Quang, Tran Ho FSIV, Vietnam Romero, Javier Rodríguez CONIF, Colombia Ruengritsarakul, Komsan ASEAN FTSC, Thailand Sacandé, Moctar SCD, RBG Kew, UK Sahu, K.K. Raipur University, India APPENDIXES 363

Salazar, Rodolfo CATIE, Costa Rica Salomão, Antonieta N. CENARGEN, Brazil Sanon, Mathurin D. CNSF, Burkina Faso Sarr, A.S. ISRA, Senegal Saucedo, Luis Gonzales University Gabriel, Bolivia Soetisna, Usep R&D Biotech., Indonesia Son, Nguyen Huy FSIV, Vietnam Thomsen, Kirsten A DFSC, Denmark Uronu, Ludovick O.N. NTSP, Tanzania Varghese, Bobby Raipur University, India Vasquez, William CATIE, Costa Rica Wade, M. ISRA, Senegal Wang, Xiaofeng F. SCAU, China Were, James ICRAF, Kenya Yan, M. SCAU, China Zhao, T. SCAU, China