Journal of Experimental Biology and Agricultural Sciences, February - 2015; Volume – 3(1)

Journal of Experimental Biology and Agricultural Sciences

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ISSN No. 2320 – 8694

ADDRESSING CONSTRAINTS IN PROPAGATION OF spp. THROUGH SEED SECTIONING AND AIR LAYERING

Ofori D A1,*, Asomaning J M2, Peprah T2, Agyeman V K2, Anjarwalla P1, Tchoundjeu Z3, Mowo J G1 and Jamnadass R1

1World Agroforestry Centre, United Nations Avenue, Gigiri , Box 30677-00100, Nairobi, Kenya 2 CSIR-Forestry Research Institute of Ghana, P. O. Box UP63, KNUST, Kumasi, Ghana 3World Agroforestry Centre, WCA Regional Office, Box 16377, Yaounde, Cameroon

Received – December 18, 2014; Revision – January 05, 2015; Accepted – February 23, 2015 Available Online – February 23, 2015

KEYWORDS ABSTRACT

Seed germination Allanblackia are indigenous to West, East and Central African regions with the potential of providing an alternative source of income for rural populations through sale of seed oil. The species Vegetative propagation therefore needs to be domesticated to support sustainable production of Allanblackia seed oil. Propagation of Allanblackia parviflora through seed germination and air layering was therefore Allanblackia undertaken. Stratified and non-stratified seeds or seed sections were germinated in sand, plain and black polythene bags. Air layering was undertaken on pollarded shoots growing on 1.5, 4 and 6m stumps Seed oil using coir dust as the medium. At 90 days, germination of un-stratified seeds showed significant differences for the following treatments; proximal sections (0 – 54%; p < 0.0001), longitudinal sections Marcotting (0 – 44%, p < 0.0001) and whole seed (13 - 31%, p < 0.0001). Germination of stratified seeds and seed sections also showed significant differences; proximal sections (37 - 53%, p < 0.0001), whole seed (21 -

32%, p < 0.0001). Distal seed sections did not germinate but only produced seed roots. For air layering, rooting success (P < 0.05) declined with increasing stump height from 72% to 21.4% for 1.5m and 6m stumps respectively. The results are discussed in relation to mass propagation of Allanblackia spp. to support large scale cultivation.

* Corresponding author E-mail: [email protected] (Ofori D A)

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1 Introduction and incubation in plain and black polythene bags at 23-30oC, leading to seed germination of 75% and 68% in 10 months Allanblackia parviflora belonging to the family is respectively (Ofori et al., 2011). This calls for new methods an indigenous species in the West African sub-region. It like seed sectioning that has proved to be effective in hastening has a potential of providing a source of household income for seed germination in spp. (Joshi et al., 2006; rural populations and can also contribute to forest landscape Asomaning et al., 2011) to be tested. Based on the fact that restoration by counteracting the impacts of climate change, due Allanblackia is a dioecious species, seedlings produced may to its ability to grow in varying environmental conditions either be male or female (Peprah et al., 2009; Ofori et al., (Peprah et al., 2009). Other uses of this species include shade, 2014- Unpublished), and also have long gestation period ≥ 6 timber, medicine and seed oil for food and cosmetic products years, there was a need to explore for asexual propagation (Irvine 1961; Ofori et al. 2006).Traditionally oil extracted from method for production of true-type planting stocks (Hartman et the seed has been locally used for cooking and soap making al., 2011) that would also have short gestation period (Ofori et (Ofori et al., 2006). The seed oil is the most economic product al., 2008; Hartman et al., 2011). of the species and pressed crude oil from the dry seeds (at 3.1- 5% moisture content) is about one-third of seed dry weight 2 Materials and methods (Attipoe et al., 2006; Sefa et al., 2010). In the international market over 100,000 tons of Allanblackia oil is annually 2.1 Seed germination required but only about 200 tons are supplied annually on average from Ghana (A. parviflora= 40 tons), Nigeria (A. Seed segments namely: distal sections (DS), proximal sections floribunda= 20 tons) and Tanzania (A. stuhlmannii= 150 tons) (PS) and longitudinal sections (LS) were cut from freshly (Oppong, 2008; Kattah, 2010). Therefore, there is a need to extracted seeds of A. parviflora without seed coat using a sharp domesticate Allanblackia to sustain the supply of Allanblackia knife. Fifty seed segments each of the above named sections seeds to feed both the local and foreign markets. and / or whole seeds were prepared in four replicates and placed in plain polythene, black polythene or black polythene In an effort to domesticate a new tree crop like A. parviflora bag further enclosed in plain polythene bag without adding any and promote its cultivation on a large scale, there is a need to germination media. Seed sections/seeds were sparingly sprayed develop methods for propagation for sustainable supply of with fine mist of tap water and the open end of the polythene adequate quantity of quality planting stocks for development of bags sealed. All samples were kept in a house at Forestry agroforestry systems and plantations. Previous studies have Research Institute of Ghana, Kumasi. An ambient temperature shown that Allanblackia species were difficult to propagate of 23-30°C was maintained. Samples were opened and through traditional methods of propagation and thus efforts sparingly sprayed with water at intervals of 2 weeks during the must be harnessed to overcome the challenges of period of observation. domestication of the species (Anegbeh et al., 2006; Atangana et al., 2006; Ofori et al., 2008; Asaah et al., 2011; Ofori et In another set, freshly extracted whole seeds were placed in al.,2011; Ofori et al., 2013). A. parviflora seed germination moistened sand in sealed plastic bags with some perforations. rate is very slow. For instance, a seed lot usually take a long The plastic bags with seeds were stored in an incubator at period to complete germination, normally over three years temperature 25±2ºC for the seeds to undergo a period of warm (Ofori et al., 2008). Seed dormancy in A. parviflora, partly stratification for 28 days after that seeds were removed, associated with a thick and hard seed coat, contributes to the washed in tap water and prepared into various sections as lack of uniform seed germination and subsequent plant growth described above and placed for germination in polythene bags (Ofori et al., 2011). Thus the use of seeds and seedlings in A. as done for seeds that were not stratified. Treatments were parviflora domestication is therefore limited by the long seed arranged in randomized complete block design with 4 dormancy period, its recalcitrant nature, and limitations in seed replications at the plant house. Seed sections were counted storage technology of the species (Ofori et al., 2011). Current after germination when radicle and plumule attained at least best practice for seed germination is the removal of seed coat 1cm long.

Table 1 Number of pollarded, height of pollarding and layers set

Height of pollarding (m) No. of Pollarded trees No. of layers set 1.5 7 20 4.0 4 20 6.0 4 14 Total 15 54

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Addressing constraints in propagation of Allanblackia spp. through seed sectioning and air layering 91 2.2 Air layering soil (forest soil + soil collected under Allanblackia tree at a ratio of 3:1) and placed in a weaning chamber that maintains Fifteen (15) trees were pollarded at three different heights (1.5, high humidity (RH ≥ 75%) to reduce transpiration in order to 4 and 6 m high) and allowed to coppice. Air layering was help the plant to recover from shock and to develop extensive undertaken on the coppiced shoots by removing 10cm of the root system to absorb enough soil nutrient solution. bark of each selected shoot growing on the pollarded trees (Figure 1).

Figure 2a Whole seed showing epicotyl at proximal ends and seed roots at the distal ends.

Figure 1 Air layering on shoots growing on a tree pollarded at 2.3 Data analysis 1.5 m high. For seed germination, data per replicate were presented in Due to the differences in coppicing ability of Allanblackia percentages. The percentage data were arcsine transformed to trees, the number of layers set for the different treatments conform to normal distribution before subjecting them to varied (Table 1). Another set of air layers was set up on the analysis of variance using SAS 9.2 edition. Mean separation same trees during the same season in the following year and was done by using least significant difference (LSD). Results these two sets of air layers were treated as replicates. The were presented graphically and LSD values inserted on the rooting medium used was coir dust, which was applied at the graphs to depict significant differences among treatments. place of bark removal and tied securely with a string. Layering experiment was analysed by transforming number of Assessment was done at four weeks after layering and then two rooted layers per treatment (pollarding height) into weeks intervals afterwards. The distal ends of the layered percentages, as (x/y)*100, where x = number of successful shoots were removed to enhance production of fresh shoots layers and y = total number of layers set. The transformed data just after root development. Successful layers were harvested were then used for analysis of variance, using Microsoft Excel for potting at four weeks after severance of the distal ends. 2010. Results were presented graphically and error bars These were potted in polythene pots containing sandy loam inserted to depict significant differences.

Figure 2b Germinated A. parviflora seed showing two types of roots and the epicotyl.

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(A) (B) Figure 3 (A) Longitudinal sections showing epicotyl at proximal ends and seed roots at the distal ends (B) embryo filling the seed from the proximal ends to the last third of the seed.

3 Results of distal end sections however, only seed roots grew from the distal extremities without further production of shoots and 3.1 Seed Germination therefore seedlings could not be produced from them (Figure 5). All seed sections sown in sand got rotten and did not Seed germination pattern of A. parviflora is summarised in produce either root or shoots. Table 2. Generally, germination of whole seeds of A. parviflora began with the emergence of the seed root at the distal end of the seed followed by the appearance of the epicotyl from the proximal end and then hypocotyl at the base of the epicotyl (Figures 2a & 2b). This type of germination pattern was also observed in longitudinal sections of the seed (Figure 3).

Figure 5 Distal end sections showing seed roots.

3.2 Proximal sections of un-stratified seeds

Proximal sections placed in plain polythene material (PPM) recorded the highest germination percentage (53.9%). This was Figure 4 Proximal sections showing epicotyl developing into significantly different (P = 0.0001) from germination shoots. percentage of the proximal sections placed in black polythene material (PBPM) and proximal sections placed in black and Proximal sections also produced both epicotyl and hypocotyl at then in plain polythene materials (PBPPM). Proximal sections the proximal end but without seed root (Figure 4). In the case placed in sand (PSD) recorded zero germination (Figure 6).

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Addressing constraints in propagation of Allanblackia spp. through seed sectioning and air layering 93

Figure 6 Germination percentages (%) of proximal sections Figure 7 Germination percentages of longitudinal sections placed in black polythene material (PBPM), black polythene in placed in plain polythene material (LSPPM), black polythene plain polythene material (PBPPM), plain polythene material material (LSBPM) and in sand (LSD). Bar = ± LSD. (PPM) and in sand (PS). Bar = ± LSD.

Figure 8 Germination percentages of whole seed placed in Figure 9 Germination percentages of stratified proximal black polythene material (WSBPM), black in plain polythene sections placed in black polythene (STPBP) and stratified material (WSBPPM), plain polythene material (WSPPM) and proximal sections placed in plain polythene (STPPP). Bars = ± sand (WSSD). Bar = ± LSD. LSD.

Figure 10 Germination percentages of stratified whole seed Figure 11 Effect of pollarding height on the success of air placed in black polythene material (STWSBPM), stratified layering. Error bars = ±SE. whole seed placed in plain polythene material (STWSPPM) and stratified whole seed placed in sand (STWSSD). Bars = ± LSD.

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Table 2 Germination pattern observed from seed/seed sections of A. parviflora.

Germination material Germination Pattern Observed (Seed/Seed Section) Whole seed Emergence of root and shoot from the distal and proximal ends respectively. An adventitious root later comes from the base of shoot at the proximal end. Successfully gives rise to complete seedling. Distal section Emergence of seed root from the distal end. No shoot develops from this seed section therefore seed piece degenerates without any production of seedling. Proximal section Emergence of shoot from the proximal end. Adventitious root later develops from the base of the shoot. Seed piece successfully gives rise to complete seedling. Longitudinal section Emergence of root and shoot from the distal and proximal ends respectively. An adventitious root later comes from the base of shoot at the proximal end. Successfully gives rise to complete seedling

3.3 Longitudinal sections of un-stratified seeds higher than germination percentage of seeds placed in sand Germination percentages recorded for longitudinal sections (STWSSD = 22.0%). placed in plain polythene material (LSPPM), black polythene material (LSBPM) and in sand (LSD) are presented in Figure 3.7 Air layering 7. Longitudinal sections placed in plain polythene material gave significantly (P = 0.0001) higher germination percentage The results of air layering experiment are shown in Figure 11. (43.9%) than longitudinal sections placed in black polythene The success of rooting of layers declined with increasing material (LSBPM = 38.1%). Longitudinal sections placed in height of pollarding. Percentage success of layers was highest sand (LSD) did not germinate. on 1.5 m stumps (72%), followed by trees pollarded at 4m high (50%) and lastly trees pollarded at 6 m high (21.4%). 3.4 Un-stratified whole seeds Discussions Germination percentages recorded when whole seeds were placed in black polythene material (WSBPM), black polythene The results showed that whole seeds of A. parviflora without material plus plain polythene (WSBPPM), plain polythene seed coat had lower germination ability than seed sections material (WSPPM) and in sand (WSSD) showed significant without seed coat; hence seed sectioning could be a method for differences (P = 0.001) and are presented in Figure 8. The seed enhancing imbibition of water and / or seed germination. germination percentages for WSBPM (29.3%) and WSPPM Preliminary studies have showed that moisture content of (30.6%) were not significantly different, but these two Allanblackia seed embryo is twice that of the cotyledon and treatments were significantly higher than WSBPPM (24.4%), any loss of water from the embryo causes a disruption of the which was also significantly higher than WSSD (12.8%). embryo leading to loss of viability (Muthoka, unpublished data), probably justifying the higher germination percentage of 3.5 Stratified proximal sections seeds in polythene bags that maintains high humidity compared to sand that has low moisture holding capacity. This Germination percentages of proximal sections prepared from presupposes that when the moisture level of sand drops, water stratified seeds and placed in black and plain polythene may be drawn from the embryo into the cotyledon, thereby materials are presented in Figure 9. The germination causing a disruption of the embryo. Nonetheless the high percentage of stratified proximal sections placed in plain pathogen load in sand may also be a factor contributing to the polythene (STPPP = 52.5%) was significantly (P = 0.0001) high rate of rotting of seeds sown in sand. higher than stratified proximal sections placed in black polythene (STPBP = 36.8%). Furthermore, the high seed germination percentages recorded from seeds and seed sections placed in plain polythene 3.6 Stratified Whole Seed compared to those placed in black polythene bag and black polythene further enclosed in plain polythene bag could be due Germination percentages of stratified whole seed placed in to the presence of optimum temperature conditions generated black polythene material (STWSBPM), plain polythene in the plain polythene bag compared to the black bag. This is material (STWSPPM) and sand (STWSSD) are presented in supported by an earlier study in Ghana, reporting that Figure 10. The germination percentage of stratified whole seed temperatures generated in plain and black polythene bags in plain polythene material (STWSPPM = 32.0%) was during incubation of seeds for germination ranged from 23- significantly higher (P = 0.0001) than seeds placed in black 27oC and 27-30oC respectively (Ofori et al., 2011). polythene material (STWSBPM = 27.1%), which was also

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Addressing constraints in propagation of Allanblackia spp. through seed sectioning and air layering 95 The Allanblackia seed germination behaviour summerised in mature gradient from the base of a tree to the top and its the present study suggests that A. parviflora exhibits root-shoot relationship with reduction in totipotency was exhibited polarity as observed in (Malik et al., 2005) and through air layering. For difficult-to-root species like A. (Joshi et al., 2006; Asomaning et al., 2011), parviflora, re-invigoration through pollarding and the use of where proximal and half proximal seed sections produced coppiced shoots should be adopted to address problems with complete seedlings, but distal end sections produced only roots vegetative propagation. Harnessing these seed germination and and failed to develop into seedlings. On the other hand, middle vegetative propagation techniques could enhance the artificial sections of the seed neither produced roots nor shoots. On the regeneration, management and productivity of A. parviflora contrary, the middle sections of the seed of Garcinia gummi- gutta developed into complete seedlings in addition to distal Acknowledgements and proximal end sections (Joshi et al., 2006). We thank Unilever, IFAD and EC for supporting this study This type of seed germination behaviour was reported in financially. The support from the nursery men of CSIR- Garciniagummi-gutta where and every section or seed segment Forestry Research Institute (Francis Owusu and Simon developed into a complete seedling was attributed to the fact Asumadu) in data collection is also highly appreciated. that mature embryo is a swollen hypocotyl, completely filling the space within the seed. However, for Garcinia kola, the References embryo is rudimentary at the time the seed is matured and it is not well differentiated into cotyledons and embryonic axis Anegbeh PO, Tchoundjeu Z, Simons AJ, Roy-Macauley H (Watson & Dallwitz, 1992) and this may be the reason why (2006) Domestication of : vegetative distal sections of Garcinia kola could give rise to only a root propagation by leafy stem cuttings in the Niger Delta region of whilst the middle section produced neither root nor shoot. The Nigeria. Acta Agronomica Nigeriana 7: 11–16. same principle may explain the failure of distal end sections of A. parviflora seeds to produce shoot, since a longitudinal Asaah E, Tchoundjeu Z, Ngahane W, Tsobeng A, section through A. parviflora seed (Figure 3) showed that the Kouodiekong L, Jamnadass R, Simons A (2011) Allanblackia embryo does not completely fill the whole length of the seed floribunda: a new oil tree crop for Africa: amenability to up to the distal end. Furthermore, seed stratification was grafting. New Forests 41:389-398. observed to have no positive effect on A. parviflora seed germination. Probably, the type of A. parviflora seed dormancy Atangana AR, Tchoundjeu Z, Asaah EK, Simons AJ, Khasa is much more related with the barriers imposed by seed coat DP(2006) Domestication of Allanblackia floribunda: and / or chemical inhibition rather than embryo maturation. amenability to vegetative propagation. Forest Ecology and Management 237: 246–251. Success rate of air layering showed a sturdy decline as the pollarding height increased. The differences in rooting may be Attipoe L, van Andel A, Nyame SK (2006) The Novella due to differences in physiological age along the tree from the project: developing a sustainable supply chain for Allanblackia base to the top, since juvenile-mature gradient has been oil. In: Ruben R, Slingerland M, Nijhoff H (Eds.), Agro-food observed in trees from base to the top (Nketiah et al., 1999; chains and networks for development. Springer, Amsterdam, Hartman et al., 2011). The 1.5m stump height might be within The Netherlands Pp. 179-189. the juvenile root–shoot junction, which is said to be physiologically juvenile with high rooting potential although Asomaning JM, Olympio NS, Sacande M (2011) Desiccation chronologically old (Nketiah et al., 1999; Hartman et al., sensitivity and Germination of Recalcitrant Garcinia kola 2011). This conforms with the earlier work on Milicia spp. Heckel Seeds. Research Journal of Seed Science 4: 15-27. (Ofori et al., 1997) and Triplochiton scleroxylon (Nketiah et al., 1999) where stem cuttings propagated from re-invigorated Joshi G, Kumar ANA, Gowda B, Srinivasa YB(2006) shoots grown on 1 m stumps rooted better than cuttings taken Production of supernumerary from seed fragments in from other parts of the tree. Garcinia gummi – gutta: evolutionary implications of mammalian frugivory. Current science 91:372-376. In conclusion, the removal of the thick and hard seed coat and seed sectioning are forms of mechanical scarification and have Hartmann HT, Kester DE, Davies Jr FT, Geneve RL (2011) great potential for breaking A. parviflora seed dormancy. Hartmann & Kester's Plant Propagation: Principles and However, other forms of dormancy (chemical and embryo Practices, Eighth Edition, Pearson Education, Inc., Publishing maturation) need to be investigated. The germination of as Prentice Hall, One Lake Street, Upper Saddle River, NJ proximal end sections in plain polythene bags may be 07458. recommended for raising seedlings of A. parviflora, since this was the best in terms of percentage as well as speed of Irvine FR (1961) Woody Plants of Ghana with special germination. Germination of longitudinal seed sections also reference to their uses. Oxford University Press, 143-144. requires further development as it has the potential for producing two seedlings from a single seed. The juvenile-

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Kattah C (2010) Developing a sustainable Allanblackia species, Allanblackia parviflora A. Chev.in Ghana. Genetic business in Africa. Proceeding of the first Allanblackia Resources and Crop Evolution (Unpublished data). Conference held on2-3 February 2010 in Monrovia, Liberia. Ofori DA, Kehlenbeck K, Munjuga M, Jamnadass R, Asaah Malik SK, Chaudhury R, Kalia RK (2005) Rapid in vitro EK, Kattah C, Rutatina F (2013) Allanblackia species: a model multiplication and conservation of Garcinia indica; A tropical for the domestication of high potential tree crops in Africa. medicinal tree species. Scientia Horticulturea 106: 539 -553. Acta Horticulturae (ISHS) 979: 311-318 available on http://www.actahort.org/books/979/979_32.htm. Nketiah T, Newton AC, Leakey RRB (1999) Vegetative Propagation of Triplochiton scleroxylonin Ghana; Effects of Ofori DA, Newton AC, Leakey RRB, Grace J (1997) cutting origin. Journal of Tropical Forest Science. 11:512-515. Vegetative propagation of Milicia excelsa by leafy stem cuttings: Effects of maturation, coppicing, cutting length and Ofori DA, Peprah T, Siaw D, Cobbinah JR (2006) position on rooting ability. Journal of Tropical Forest Science Domestication of Allanblackia in Ghana. In: Muasya S, 10: 115-129. Rutatina F, Munjuga M, Simons AJ (Eds.) Status and Prospects for Allanblackia Domestication in Tanzania. CD- Oppong K (2008) Allanblackia seed oil supply chain ROM Compilation, ICRAF, Nairobi, Kenya, ISBN 92 9059 management in Ghana. Allanblackia stake holders’ workshop 196 X. held on 30th July 2008 at Sanaa Lodge, Cape Coast.

Ofori DA, Peprah T, Cobbinah JR, Atchwerebour HA, Peprah T, Ofori DA, Siaw DEKA, Addo-Danso SD, Osabutey F, Tchoundjeu Z, Simons AJ, Jamnadass R (2011) Cobbinah JR, Simons AJ, Jamnadass R (2009) Reproductive Germination requirements of Allanblackia parviflora seeds and biology and characterization of Allanblackia parviflora A. early growth of seedlings. New Forests 41:337-348. Chev. in Ghana. Genetic Resources and Crop Evolution 56: 1037-1044. Ofori DA, Peprah T, Henneh S, Von Berg JB, Tchoundjeu Z, Jamnadass R, Simons AJ (2008) Utility of grafting in tree Sefa W, Adubofuor J, Oldham JH (2010) Optimum conditions domestication programmes with special reference to for expression of oil from Allanblackia floribunda seeds and Allanblackia parviflora A. Chev. Ghana Journal of Forestry 23 assessing the quality and stability of pressed and solvent &24: 42–48. extracted oil. African Journal of Food Science 4: 563-570.

Ofori DA, Peprah T, Koech G, Anjarwalla P, Munjuga M, Watson L and Dallwitz MJ (1992). The families of flowering Jamnadass R (2014) Survival, growth performance and plants: descriptions,illustrations, identification and information reproductive biology of high valued indigenous fruit tree retrieval. Version 19th August 2014. http://delta-intkey.com

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