Conservation of Begonia Germplasm Through Seeds: Characterization of Germination and Vigor in Different Species

CONSERVATION OF BEGONIA GERMPLASM THROUGH SEEDS: CHARACTERIZATION OF GERMINATION AND VIGOR IN DIFFERENT SPECIES

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

By: Steven Robert Haba, B.S.

Graduate Program in Horticulture and Crop Science

The Ohio State University

2015

Thesis Committee:

Dr. Pablo Jourdan, Advisor

Dr. Mark Bennett

Dr. Claudio Pasian

Dr. Mark Tebbitt

Copyrighted by

Steven Robert Haba

2015

ABSTRACT

Begonia is one of the most speciose genera of angiosperms, with over 1500

species distributed throughout tropical and subtropical regions; it is also a very important

ornamental group of plants displaying a high degree of morphological diversity. This

genus is a priority for conservation and germplasm development at the Ornamental Plant

Germplasm Center located at The Ohio State University, which currently holds

approximately 200 accessions, maintained primarily as clonal plants. In an effort to

expand germplasm work in seed storage of Begonia, and in response to a scarcity of

published information about begonia seed biology we initiated a project to develop

baseline information about germination, dormancy, and stress tolerance of begonia seeds.

Because of the extremely small size of begonia seeds (ca. 200 µm) I adapted germination

and viability testing protocols typical of Arabidopsis research, to develop relatively efficient quantitative protocols for seed studies. Using this methodology seeds can be routinely germinated on 1% agar plates at 25°C and 16 hours light. To examine the variation in seed characteristics among Begonia accessions in the collection, I selected six species from diverse environments and from different sections of the genus for which we had abundant seed and compared their germination patterns in response to temperature and light, tolerance to high humidity/high temperature stress, and dormancy.

I have determined that begonia seeds are desiccation-tolerant (orthodox), require light for germination (photoblastic), germinate under a wide range of temperatures, and mostly ii

appear to lack any strong dormancy — depending on species, and are tolerant of various level(s) of stress. I found that Begonia ulmifolia, B. fischeri, and B. dregei are tolerant to high levels of stress (120 hours at 41°C) whereas B. boliviensis, B. grandis subsp. evansiana and B. subvillosa are less so. In addition, B. dregei appears to have some dormancy, which was observed through extended dark treatments (2-52 weeks) and exposure to low temperatures (12 and 15°C). Furthermore, B. grandis subsp. evansiana appears to show secondary dormancy as a response to extended dark treatments (2-52 weeks). Overall, I was able to establish a quantitative germination protocol that allowed for examination of different species to various environments. Tetrazolium testing was also possible after treatment of seeds with sodium hypochlorite. This work has established baseline information about seeds of Begonia that can be applicable to other physiological and conservation studies and useful for maintaining and characterizing a seed collection at the OPGC.

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ACKNOWLEDGEMENTS

In the beginning of the master’s thesis, it seems as though you’ve taken on a task that is tangible, but it doesn’t take long before the reality sets in. Both the research and the writing have been a tremendous learning experience for my personal growth as well as in how to collaborate and incorporate other viewpoints into a project.

I would like to thank my immediate advisor, Dr. Pablo Jourdan, for his incredible energy and ability to consistently provide “suggestive” guidance, which allows individuals, such as myself, to pursue goals and interests of my own. Pablo’s positivity and fruitful ideas were essential to the success of this project and to the everyday excitement at the Ornamental Plant Germplasm Center. I feel very privileged to have worked under someone who is passionate and intelligent about horticulture, as well as individuality.

I equally would like to thank Dr. Mark Tebbitt, as our mutual interests in plants and horticulture has created a plethora of new information on Begonia seeds, as well as a glance at the overwhelming diversity of Begonia. I was extremely lucky to have a

Begonia systematist and a knowledgeable, friendly botanist to talk to about Begonia only several hours away.

Much gratitude is needed for Dr. Mark Bennett and Dr. Claudio Pasian, both professors were crucial steps of the ladder to get my writing and research skills to where they are today. I greatly appreciate Claudio’s rigorous organization and I thank Mark iv

Bennett for his critical comments and ideas about experimental design and protocol development.

In addition, I would like to thank J.C. Jang, as we would not been able to develop this project if it were not for the adoption of the germination tests used in his lab. His suggestions gave us a platform for which we were able to develop something that works time and time again.

Many thanks are warranted for the staff, students, and interns at the Ornamental

Plant Germplasm Center, as their ability to work with me on various projects, to go above and beyond their immediate responsibilities, have been much appreciated. I would like to specifically thank Fernanda Brunetta Godinho, as her hard work was not only tedious and extreme, but crucial to the rigorous data generated for this project.

None of this would have been possible without the continued love and support of my parents, Pamela and Jim Haba, and willingness to allow me to pursue my interests, as they have since childhood. Last but not least, I would like to thank my girlfriend, Leith

Fava, for her appreciation, reliability, and perpetual love.

VITA

March 31, 1987………………………………………………….….Born, Cleveland, Ohio

2007………………………………..A.S. Horticulture Science, The Ohio State University

2009………………………………………..B.S. Crop Science, The Ohio State University

2009-2010………………………………………...Section Grower, Green Circle Growers, Oberlin, Ohio

2010 to present...... Research Assistant/Greenhouse Coordinator, Ornamental Plant Germplasm Center, Columbus, Ohio

PUBLICATIONS

Haba, Steven R. and P. Jourdan. Begonias at the Ornamental Plant Germplasm Center. The Begonian. March/April 2012.

FIELDS OF STUDY

Major Field of Study: Horticulture and Crop Science

TABLE OF CONTENTS

Page

ABSTRACT ...... ii

ACKNOWLEDGEMENTS ...... iv

VITA ...... vi

LIST OF FIGURES ...... ix

LIST OF TABLES ...... xiv

1. The genus Begonia ...... 1

Introduction ...... 1 Begonias at the Ornamental Plant Germplasm Center ...... 4 Begonia seed ...... 6 Seed anatomy and morphology ...... 8 Desiccation tolerance ...... 9 Germination ...... 9 Study objectives ...... 13

2. A protocol for Begonia seed germination ...... 20

Introduction ...... 20 Material and methods ...... 25 Results ...... 28 Discussion ...... 31

3. Comparison of seed characteristics among six Begonia species ...... 46

Introduction ...... 46 Seed testing ...... 48 Material and methods ...... 55 Results ...... 63 Discussion ...... 69 vii

Conclusions ...... 80

Appendices: Appendix A Begonia Seed Cleaning ...... 106 Appendix B Begonia Seed Production ...... 111 Appendix C Begonia germplasm collection at the OPGC ...... 115 Appendix D Tetrazolium Testing ...... 121 Appendix E Thermal Gradient Temperature Log ...... 125 Appendix F Germination of Begonia roxburghii seed ...... 127 Appendix G Additional notes and comments on the response of individual species to controlled deterioration, temperature, and dark treatments ...... 132

Bibliography ...... 139

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LIST OF FIGURES

Figure Page

1.1. The major cultivar groups of Begonia used in international trade. A. Semperflorens-cultorum group (commonly called wax or bedding begonia) B. ×tuberhybrida group (commonly called tuberous begonia) C. Rex-cultorum (commonly called rex begonia) D. Chiemantha group (commonly called Elatior or Rieger begonia); photo by Ross Bosswell ...... 15

1.2. Relative seed size of Begonia, Arabidopsis, and Capsicum ...... 16

1.3. A-F. Scanning electron micrographs of six Begonia species included in this study (Source: Leona Horst, USDA-ARS, Wooster, Ohio) G. Parts of a typical Begonia seed: hilum micropyle (where seed attaches to fruit wall), operculum (seed lid), collar cells (a feature specific to Begonia seed which split upon germination), testa cell (the common component of a seed coat) ...... 17

2.1. Seed weights of different Begonia species. Approximately 200 seed sampled with a microspoon were used to obtain weights (grams). Seeds had been stored at 4°C, 25%RH for at least 6 months. (c.f. Figure 2.3B) ...... 37

2.2. Tools and materials used in the preliminary setup for the germination protocol; tools include pipettes, micro spoon, microfuge tubes, and materials include 0.1% agarose and Begonia seed. The work must be done in a well-lit location for efficient handling of the seeds ...... 38

2.3. Tools and materials used for the routine Begonia germination protocol. A. Begonia seed lot after processing. B. Microspoon used to quantify approximately 200 seed for each Begonia accession. C. Dissecting needle used to aliquot one seed at a time. D. Plastic grid with 100 dots in four 25 dot squares used under the Petri dish to facilitate placement of seed. E. System for placement of seed onto germination plates; dissecting needle (held in the hand) is first dipped in water within the microfuge tube and then used to pick up a seed one at a time from the white paper towel and placed onto the agar plate. The water drop facilitates adhesion of the seed to the needle. F. A plate with 1.0% agar where 100 Begonia seed have been distributed ...... 39

2.4. Contrasting germination between Arabidopsis and Begonia. A. Germination of Arabidopsis thaliana ecotype ‘Col’ showing emergence of the radicle (red arrow). B. Different stages of Begonia germination, illustrating the emergence of the hypocotyl, which develops from the plumule, the embryonic shoot (red arrow) before any radicle is visible ...... 40

2.5. The germination process in Begonia fischeri. The operculum is pushed open about 3 days after the imbibed seed is exposed to light. Between days 3 and 6 expansion of the hypocotyl follows; between days 6 and 9 a collet and root hairs form; by 9 to 12 days the seed coat is shed, cotyledons emerge, and a radicle develops. Each of these processes is highlighted with a red arrow ...... 41

2.6. Baseline Begonia germination (mean%±SE) defined here as hypocotyl emergence (1/3 the length of the seed coat) of six Begonia species after three weeks under standard conditions (16 hour light/ 8 hour dark 25°C) using agar as substrate ...... 42

2.7. Cytological examination of Begonia fischeri germination from mature imbibed seed to radicle formation (0-12 days). Source: Robert L. Geneve, University of Kentucky, 2014 (used by permission) ...... 43

2.8. Development of a primary root in Begonia fischeri (12-16 days after imbibition). Source: Robert L. Geneve, University of Kentucky, 2014...... 44

3.1. Begonia species used in the study of germination. The description and comments are partly based on information provided by Tebbitt (2005) ...... 83

3.2. Tools and materials used for moisture content determination of Begonia seeds. A. Aluminum dishes, seed lots, scale, microspoon. B. Transfer of seeds to an aluminum dish for initial weight record. C. Oven kept at 105°C where seeds were incubated for 24 hours and then re-weighed to calculate loss of moisture and thus, moisture content ...... 84

3.3. Germination pattern for the six species of Begonia in the study. The process is described in the rightmost column. A seed was considered to have germinated when the hypocotyl had elongated to about 1/3 the length of the seed, indicated by the different color background on the third row ...... 85

3.4. Thermal gradient table with plates containing seeds evaluated for germination at different temperatures. The table was illuminated by t12 cool white flourescent bulbs, yielding an average light level at table height of 50 µmol·m-2·s-1. Five different temperatures at the table positions were clearly marked by numbered blue tape...... 86

3.5. Method to exclude light from germination plates in the dark treatments. Each plate was wrapped in aluminum foil to exclude light and labeled with accession identification number as well as the first two check dates. The plates were kept in the same incubator used for the germination experiments in the light ...... 87

3.6 Close-up view of the system used for controlled deterioration experiments with the small Begonia seeds. The mesh platform is placed inside a standard germination box containing 40 ml of water; cups containing an ultra-fine mesh were used to hold the seeds that had been transferred there with a microspoon (5 µl). Seed does not have direct contact with water on lower surface of germination box, yet became fully hydrated from 100 % RH (saturated) environment ...... 88

3.7. Experimental setup for controlled deterioration experiments. A. Side view of germination boxes containing 40ml of water and the mesh platforms. B. Top view of germination boxes showing the mesh platforms and the individual mesh caps containing seeds. C. Water-jacketed incubators used to maintain a constant 41°C ...... 89

3.8. Germination (mean%±SE) of Begonia species after 70 days at various temperatures; baseline germination is displayed as toggled bar(s) for comparison. Additional temperatures of 12° and 15°C are presented for B. dregei ...... 90

3.9. Germination curves for Begonia species incubated at five different temperatures (17-29°C) and 16 hour photoperiod. Error bars represent the standard error (SE) of the mean ...... 91

3.10. Response of Begonia dregei seeds to germination at 12° and 15°C and 16h photoperiod. Baseline germination was recorded under standard conditions of 25°C and 16 hour light/ 8 hour dark ...... 92

3.11. Response of seeds to freezing. Germination curves (mean%±SE) of six species of Begonia and Arabidopsis seed under standard conditions at 25°C with 16 hour light/ 8 hour dark photoperiod following storage in the freezer (one week @ - 20°C) ...... 93

3.12. Response of imbibed seeds to incubation in the dark. Maximum germination (mean%±SE) after six weeks under standard conditions at 25°C for 16 hour light/ 8 hour dark photoperiod following incubation in the dark for up to 52 weeks; baseline germination (BG) is included as toggled bars for comparison ...... 94

3.13. Response of Arabidopsis thaliana ecotype Columbia (‘Col’) to controlled deterioration at 100%RH and 41°C. All germination tests were done under standard conditions of 25°C for 16 hour light/ 8 hour dark photoperiod following controlled deterioration. The asterisk (*) identifies treatments where seed was pre-incubated at 100%RH at 20°C prior to exposure to high temperature. In the other treatments seeds were placed directly at high temperature/humidity. A. Fungal growth on seed after 120 hour controlled deterioration. B. Final germination (mean%±SE) and T50 values for each treatment; longer duration of treatments led to germination that was so low that T50 could not be calculated (nc). C. Bar graph representation of final germination mean%±SE. D. Germination curves (mean%±SE) for each treatment ...... 95

3.14. Final (maximum) germination (mean%±SE) for each Begonia species obtained after controlled deterioration at 100%RH and 41°C. All germination tests were done under standard conditions of 25°C for 16 hour light/ 8 hour dark photoperiod. The asterisk (*) and hatched bars identify treatments where seed was pre-incubated at 100%RH for 24 hours at 20°C prior to exposure to high temperature/humidity ...... 96

3.15. Comparison of germination curves based on mean%±SE for seeds that had been pre-equilibrated at high humidity for 24 hours at 20°C (*) with those that were incubated directly at 100%RH and 41°C and maintained in the controlled deterioration conditions for 24 hours ...... 97

3.16. Comparison of germination curves based on mean%±SE for seeds that had been pre-equilibrated at high humidity for 24 hours at 20°C (*) with those that were incubated directly at 100%RH and 41°C and maintained in the controlled deterioration conditions for 48 hours ...... 98

3.17. Comparison of germination curves based on mean%±SE for seeds that had been pre-equilibrated at high humidity for 24 hours at 20°C (*) with those that were incubated directly at 100%RH and 41°C and maintained in the controlled deterioration conditions for 72 hours ...... 99

3.18. Response of seeds to controlled deterioration conditions for 96 hours. Germination (mean%±SE) of Begonia after three weeks under standard conditions at 25°C with 16 hour light/ 8 hour dark photoperiod following direct exposure to 41°C at 100%RH for 96 hours. Arabidopsis seeds were included as a control ...... 100

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3.19. Response of seeds to controlled deterioration conditions for 120 hours. Germination (mean%±SE) of Begonia after three weeks under standard conditions at 25°C with 16 hour light/ 8 hour dark photoperiod following direct exposure to 41°C at 100%RH for 120 hours. Arabidopsis seeds were included as a control ...... 101

3.20. Influence of gibberellic acid (GA3) on germination of Begonia dregei. The baseline germination rate (no GA3) is compared to that of seeds that were soaked for 24 hours in three concentrations of GA3 (250 ppm, 500 ppm, 1000 ppm) then sown on 1.0% agar and incubated under standard conditions of 16 hour light/8 hour dark, 25°C; A. Germination curves for the four treatments. B. Final germination (expressed as mean%±SE) for each of the treatments ...... 102

A.1. Tools used for cleaning Begonia seed at the Ornamental Plant Germplasm Center; A. Shaker table B. Transfer of seed to vibrating surface. C. Control for shaker table settings. D. Monitor displaying X-ray imaging E. Cleaned seed lot with minimal debris F. X-ray of processed seed lot ...... 109

A.2. X-ray image of a cleaned seed lot of Begonia: arrows indicate empty shells (no embryo)...... 110

B.1. Examples of A. typical male flowers, B. female flowers, C. maturing fruit following hand pollination, D. seed that has been in constant air flow cabinet for several weeks, ...... 114

D.1. Tetrazolium staining of Begonia seeds. Red-stained seed are considered viable or metabolically active, and therefore capable of germination ...... 124

F.1. Germination of Begonia roxburghii seed that was either freshly harvested or ‘aged’ (stored at 4°C; 25%RH for six months). Standard germination conditions of 25°C and 16 hour photoperiod were used ...... 130

F.2. Germinated seeds and seedlings on excised fruit of the fleshy berries of Begonia roxburghii. The fruit was harvested three months after hand pollination, sectioned, and placed on 1.0% agar ...... 131

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LIST OF TABLES

Table Page

1.1. The Begonia germplasm collection at the Ornamental Plant Germplasm Center as of September 2014 maintained either as clones or seeds ...... 18

1.2. Summary of published methods for Begonia seed germination; chernozem= soil rich in humus; *=light measurement in lux; AOSA/ISTA-derived from McDonald and Kwong (2005) ...... 19

2.1. Germination of Begonia fischeri in three different substrates after 28 days incubation under ‘standard’ conditions (25°C, 16 hour light/ 8 hour dark)...... 45

3.1. Climatic data for likely habitat where the six Begonia species tested may naturally occur; source: Kottek et al. (2006) ...... 103

3.2. Measured moisture content (mean%±SE) of six different Begonia species seeds after storage at 4°C, 25%RH for 6 months. Moisture content was averaged over three replicates after seeds were oven dried at 105°C for 24 hours ...... 104

3.3. Effect of seven temperature regimes on germination (mean%±SE), and time (in days) to 50% germination (T50) for B. dregei, which could not be calculated (nc) for five temperature regimes because germination was so low. BG = baseline germination ...... 104

3.4. Mean germination time (T50) values (in days) corresponding to Begonia germination tests at five temperatures (optimal and sub-optimal). Two replicates of 100 seeds each were used for the experiment. For Begonia dregei the germination from 17°C to 29°C was so low that T50 could not be calculated (nc) . 105

3.5. Germination (mean%±SE) response of fresh and aged seed of Begonia. Fresh seed was directly sown after harvest from plants in greenhouse; aged seed had been stored in a cooler (4°C, 25%RH) for at least 6 months ...... 105

D.1. TZ % staining of seed lots of six species of Begonia compared to the germination rate obtained for the same seed lot ...... 124

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E.1. Record of the actual temperatures (°C) obtained on the thermal gradient table used in the experiments. Temperature was recorded by HOBO® Data Loggers over 72 hours and the results averaged to give the actual temperature at the site. Data loggers were placed on two halves of the rectangular table, northern (N) and southern (S), to determine if the gradient was consistent throughout the surface ... 126

CHAPTER 1

THE GENUS BEGONIA

Introduction

Cultivars of Begonia are among the most popular bedding plants in North

America, an understandable fact when one considers the beauty, diversity and flexibility offered by species and hybrids of this very large genus (Tebbitt, 2005). Worldwide, the use of begonias is extensive, with new cultivars being released continuously in a process that has been ongoing since the mid-1800s. Breeding is very active among the major floriculture companies (Benary, Dümmen, Syngenta Flowers, Ball Horticulture, Takii,

Sakata, TerraNova, and others) because of significant economic value. Begonias are a very valuable ornamental crop; gross sales reported by the USDA for 2013 were approximately $65 million (Census of Agriculture, 2013). This crop has consistently ranked among the top five bedding plants, together with petunia, impatiens, marigolds, and geraniums, in all the major markets of the Northern Hemisphere. The thousands of cultivars that have been named are mostly hybrids of poorly documented parentage, but the international trade is dominated by plants classified into major cultivar groups, each derived by multiple-species hybrids developed over the last 150 years (Fig. 1.1; Tebbitt,

2005). Additional groupings of begonia have been developed, based on their habit and horticultural use (e.g. shrub, cane-like, rhizomatous, etc.); these were created by the 1

American Begonia Society, an organization dedicated to the preservation and use of

Begonia species and their cultivars (American Begonia Society, 2014).

Production of the most commercially important begonias (B. Semperflorens-

cultorum group and B. ×tuberhybrida group) is both by seed and by vegetative means.

The latter is becoming more common because of the rapid dissemination of new genotypes that do not require extensive breeding for optimizing seed production.

Nevertheless, seed production remains an important horticultural practice (McDonald and

Kwong, 2005; Reynolds, 2003). Furthermore, seeds are easily transported (Ludwig,

2001), can be stored for long periods of time with minimal inputs, and lack the problems

of viral or bacterial infections commonly associated with long-term maintenance of

clonal stock.

In spite of the popularity and widespread production of begonias, there is

remarkably little published information about many fundamental features of these plants, especially in areas relevant for conservation and germplasm management, such as seeds

(Hu et al., 2012). A major focus of research has been in taxonomic and phylogenetic

analysis (Dewitte et al., 2011; Hughes and Hollingsworth, 2008; Neal et al., 2006; West

and Lott, 1991), but as a genus with numerous species distributed primarily in tropical

and subtropical regions of the world, and with limited commercial use outside of the

ornamentals industry, it is perhaps not too surprising that there is much yet to be

discovered. Research which increases our broad understanding of Begonia to ultimately

enhance its commercial use is vital, especially for a group with so much horticultural

potential given its great morphological diversity. A particularly important area of

research is seed biology, where limited work has been done in Begonia plants.

Begonia species are usually herbaceous terrestrial (occasionally epiphytic) plants that typically occur in partially shaded, moist and humid conditions mainly in the tropics

(Tebbitt, 2005). These species occur naturally in many forms, such as shrubs, herbs, or lianas, and are frequently characterized by fleshy stems, large stipules, typically asymmetrical leaves, unisexual flowers, and seeds which have a collar of cells below an operculum (Clements et al., 2004; Gregório et al., 2014). The genus is highly diverse,

consisting of over 1500 species classified into 63 sections (Doorenbos et al., 1998).

Together with Hillebrandia, Begonia makes up the family Begoniaceae within the order

Cucurbitales (Watson and Dallwitz, 1992). Our understanding of the phylogenetic

relationships of Begonia is still being refined as new information arises. It is theorized

that Begonia originated in sub-continental Africa and then migrated to a variety of

environments around the world (Goodall-Copestake et al., 2010). Species richness is

highest in southeastern Asia and the northern and southeastern portions of South

America. Among the various traits used to distinguish species is seed morphology (De

Lange and Bouman, 1992; 1999); comparatively speaking, species from Africa showed a

considerable amount of diversity in seed size and structure, supporting the concept of this

region as a center of origin. Asian and Neotropical species, while still exhibiting

diversity in seed size and shape, were less diverse than the African species examined.

However, intercontinental relationships could not be readily established based on seed

micromorphology.

Begonias at the Ornamental Plant Germplasm Center

The large diversity and potential for further development have made Begonia one

of the priority genera for conservation and germplasm enhancement at the Ornamental

Plant Germplasm Center (OPGC), an institution located at The Ohio State University that

conserves germplasm in the public domain (http://opgc.osu.edu). The Begonia collection

at the OPGC (Appendix C) consists of approximately 180 accessions representing about

150 taxa and 35 cultivars or interspecific hybrids. The collection represents a sampling

of some of the more distinctive morphological variants that are cultivated but otherwise

lacks a distinct identity. As a consequence, the collection is being developed towards

greater emphasis in the conservation of species (as opposed to complex hybrids that may

be sterile) where the germplasm can be stored as seed. Emphasis is given to species that

may have contributed to the development of the more important hybrid groups in the

horticultural trade.

Nearly all of the accessions in the collection have been kept as clones although about half of them also are kept as seed (Table 1.1). In the thirteen years since the founding of the OPGC, 92 Begonia clonal accessions have been inactivated or lost as a result of improper care of plant material, or poor growing environment(s), and inability to meet the different growing requirements that this highly diverse genus demands.

Keeping clones long-term is highly laborious, justifying an effort to shift the germplasm into something more manageable, specifically as seed populations. Maintenance of clones is also very expensive requiring large amounts of greenhouse space, regular irrigation, pest/disease management, frequent propagation, and the clones are at risk of

4 disease buildup, such as from Xanthomonas and different viruses. Thus, clonal collections should be pursued only if there are no alternatives, such as the need to preserve specific genotypes that do not breed true, or where the plants are sterile.

Preservation of germplasm as seeds permits the conservation of individual genes in populations rather than unique genotypes (unless the plants are inbred lines, with 99.7% similarity, or are apomictic) and the economics of seed storage are much more affordable.

There is very little published information about the general biology of Begonia seed that can be applied in a practical way to facilitate germplasm conservation (Hu et al.,

2012). Of particular relevance for germplasm preservation is the fact that there is little known about the longevity of Begonia seed in storage. Similarly, important details, such as desiccation tolerance, seed vigor characteristics, or response to freezing are lacking, especially for diverse species. There is undoubtedly a great deal of knowledge about how to handle commercial Begonia seed within the industry and even among some hobbyists

(e.g., members of the American Begonia Society) but there is scant published documentation on relevant protocols (Geneve, 2005). Therefore, generation of baseline information on seed biology that can facilitate management of Begonia seed and that may lead to improved handling and production of these plants for conservation and germplasm enhancement is essential. The diversity of Begonia in the OPGC germplasm collection presents challenges to insure that the seed is of high quality and long-lived. Little is known about the physiological and functional attributes of the seed of begonia and given that the genus occurs in a wide diversity of environments it is likely that a range of conditions would need to be met in order to properly conserve the genus as a whole.

Understanding the biology of Begonia seed in order to create conditions that allow the

OPGC to conserve and maintain germplasm collections in the most efficient and effective

way possible is not an option, but mandatory, if this collection is to be expanded to meet future needs.

Begonia seed

The majority of begonias are monoecious juicy-stemmed herbs with easily distinguishable male and female flowers. The ovaries are inferior and usually form a winged loculicidal capsule, but on occasion they may form berries. Fruit shape and form appear to relate to dispersal methods usually representing three syndromes employing wind, water, or animal vectors which correlate with specific habitats (Tebbitt et. al,

2006). Most species produce dry fruits upon maturity, thus relying on the abiotic factors of wind and water for seed dispersal. The “typical” fruit of Begonia is characterized as loculicidal 3-winged capsules, that dehisce by cracks or pores from a papery pericarp

(Gregório et al., 2014). Seed dispersal by animal vectors is common in consistently moist, dense environments, where wind is not a factor, and therefore consumption by animals or transport in feet or fur may be the adapted method (De Lange and Bouman,

1992). Because of their low nutrient content and less desirability to seed predators, small seeds may have been an adaptation that contributes to the pan-tropical distribution of

Begonia (Burtt, 1976). Small seeds are also more likely to be dispersed by rain-wash or by being picked up by the feet and hair of animals (Burtt, 1976). Interestingly, seedlings are rarely observed in natural habitats (Burtt, 1976; M. Tebbitt, personal communication).

Seeds of begonias are described as “tiny” or “minute” (Boesewinkel and De

Lange, 1983) though it would not be unfair to describe them as microscopic (Fig. 1.2);

their production per fruit has been noted as 50-200 seeds or “many” (De Lange and

Bouman, 1992; West and Lott, 1991). An examination of several fruits of Begonia

schmidtiana ‘Chauncy’ at the OPGC yielded approximately 6500 ovules per capsule (M.

Hansen, personal communication). All ovules within a given Begonia fruit may not develop into seed, but there is obvious potential for plants to produce an abundance of progeny from one flowering cycle.

Most Begonia seeds are reported to measure between 300-600 µm but they can

range from 220 µm to 2240 µm (De Lange and Bouman, 1999). Working with such small

seeds (Fig. 1.2) either in laboratories or in greenhouses requires special handling; it is a

common industry practice to pelletize begonia seeds (an expensive procedure) to increase

their size for easier planting (Bruggink, 2005). The typical weight of 500 seed among

various OPGC accessions is 8-10 mg, so each seed may weigh 20 µg (S. Haba,

unpublished data). This is consistent with reports from the industry that describe 88,000

seeds per gram for Begonia Semperflorens-cultorum group or 35,000 seeds per gram for

B. ×tuberhybrida cultivars (Reynolds, 2003). Others have reported that approximately

70,000 seeds have a total mass of 1 gram (Harthum, 1975; West and Lott, 1991). The

Seed Storage Database (Royal Botanic Gardens Kew, 2014) has 1000 seed weight

information for 26 species of Begonia. The average weight per seed for the 26 species is

9.7 ± 7.6 µg; the range in weight reported was from 2.4 µg for B. thiemei to 40.4 µg for

B. nyassensis. A seed producer can generate a very large amount of seed on only a few

7 plants; a typical begonia seed crop for wax begonia cultivars consists of only a few greenhouse benches (F. Kwong, personal communication).

Seed anatomy and morphology

Begonia seed has been described as non-endospermic, with an embryo that is weakly to well differentiated (Watson and Dallwaitz, 1992). However, Boesewinkel and

De Lange (1983) indicated that the endosperm is present but it has been reduced to a single layer surrounding the embryo. Recent cytological work has shown that indeed a layer of endosperm is present, and encompasses the developing embryo (R. Geneve, personal communication). Only one study has examined the chemical composition of

Begonia seeds; West and Lott (1991) determined through histological analysis that the major seed storage substances are lipids sequestered in lipid vesicles and proteins concentrated in protein bodies.

Morphological differences among seeds of Begonia species have been used in taxonomic studies because different species vary in overall seed size, shape of testal cells, undulation of anticlinal walls, bulging of the outer periclinals, and the pattern and roughness of the cuticle (Fig. 1.3; De Lange and Bouman, 1999; West and Lott, 1991).

In evolutionary terms, larger differences were observed between seeds of different sections from the phylogenetically basal African species, as compared to the more derived Asian and American species.

Desiccation tolerance

It is generally established that most Begonia seeds are desiccation tolerant. This

statement is based on the experience of many growers as well as that at the OPGC. The

Seed Storage Database (Royal Botanic Gardens Kew, 2014) labels as ‘orthodox’ nine

species of Begonia based on “75 % viability following drying to mc's [moisture content]

in equilibrium with 15 % RH and freezing for 59 days at -20C at RBG Kew, WP.” It is

likely that most other species of Begonia will also have desiccation tolerant seed, but

given some of the diverse habitats that such species encounter and the general diversity in

other physiological features of the species, it is prudent to routinely assess the tolerance

to desiccation, particularly for material that is to be conserved in seed banks.

Germination

The germination of very small seeds such as those of Begonia is particularly

challenging in standard commercial production practices. One practice to facilitate

handling has been the pelleting of seeds, but even with high quality pelletized seed that under laboratory conditions are described as having 90% germination, exacting environmental conditions are still required. These conditions include high humidity, low light intensity, and high temperature, the same conditions that stimulate the growth of algae and encourage the presence of fungus gnats and shore flies. Strategies for minimizing problems have been proposed; for example, Hvoslef-Eide and Munster

(2007) germinated seeds of Begonia under red light to prevent algae growth and therefore improve germination. Even successful growers have challenges in germinating Begonia

9 seed and in turn developing a representative plant in a desired time period. An experienced Ohio grower in the spring of 2014 was able to produce only three seedlings from 100 pelleted seed, with expected germination in the 90% range (C. Pasian, personal communication).

In laboratory settings, germination tests for Begonia have been carried out using different substrates, containers, and environmental conditions (Table 1.2). Over the last

50 years only a handful of research reports on Begonia seed germination have been published (Table 1.2). Some of the work (Carpenter et al., 1995; Reynolds, 2003;

Shoemaker and Carlson, 1992) has involved seeds of complex hybrids that form the bulk of cultivars in the trade, domesticated plants that may behave differently than wild species as they have probably been selected for high germination and lack of dormancy

(Black et. al, 2006; Hayashi et al., 2008; Weiss et al., 2013). A few studies have looked at species, all from Asia (Hu et al., 2012, Ma et al., 2005; Nagao et al., 1959). Seeds can be germinated on filter paper, blotters, or even by floating on water. The containers for germination have been Petri dishes and glass tubes. These studies provide a mixed picture of germination requirements, but in general, begonia seeds are germinated in moist conditions under light and at room temperature. Germination in the laboratory generally occurs within 3 weeks and germination rates are in the range of 0-90%.

McDonald (2005) provides a summary of the standards and conditions recommended for Begonia germination testing at seed testing laboratories and commercial grower facilities. Such standards are very limited for most flower seeds, including Begonia. The germination standard for fibrous and tuberous nonstop begonias

are 88% and 85% respectively; where laboratory count days are given as 21 days for the

tuberous nonstop, but no days are specified for the fibrous types; the reverse is the case

for greenhouse count day. This means that the fibrous begonias are mainly tested for

germination after 21 days in the greenhouse whereas the tuberous nonstop are tested for

the same time in the lab. The temperatures for the lab test are defined as 77°F (25°C) and

for the greenhouse as 76-80°F (24-27°C). The moisture for all stages of germination are

defined as ‘wet’ and the seeds are not covered either in lab conditions or the greenhouse.

It is important to note that these are not Association of Official Seed Analysts (AOSA) or

International Seed Testing Association (ISTA) standards.

Dormancy in Begonia has been reported for some species (Hu et al., 2012) but not

for the cultivars. The dormancy appears to be a relatively weak non-deep physiological

dormancy that is overcome by moist chilling, dry after-ripening, KNO3, or GA3

treatments. In general, commercial seed of Begonia is non-dormant. The extent to which

the range of wild species of Begonia exhibit dormancy is not known (Hu et al. 2012).

This is an area that merits further study.

Other factors affecting Begonia seed germination include photoperiod, light

quality, and gibberellic acid (Nagao et al., 1959; Shoemaker and Carlson, 1992;

Carpenter et al., 1995). In general, it appears that light is required for germination but that

both duration of light exposure (photoperiod) and intensity (µmol·m-2·s-1) may be variable (relative to species origin); low light intensity may favor germination over high light intensity (Ma et al., 2005). Shoemaker and Carlson (1992) reported that light is necessary for germination showing that seed will germinate if exposed for less than 10

seconds revealing a very low total irradiance requirement. Nagao et al. (1959) concluded

that light enhances germination at 29˚C and that GA3 may intensify the light action or

substitute for part of it. The GA3 treatment not only increased the germination under continuous light, but also reduced considerably the critical day length for germination.

The photoperiodic behavior Nagao et al. (1959) observed in seed germination of B. grandis subsp. evansiana is similar to that of typical long-day plants in their flowering pattern. These authors also found that no seeds germinated unless they were exposed to light at least for 48 hours, and illumination of 3 to 4 days’ duration was sufficient to give maximum germination. Carpenter et al. (1995) recommended germination of B.

Semperflorens-cultorum group seeds with light at 150 µmol·m-2·s-1 continuously for 1-2

days at 27˚C after moistening the germination substrate (presumably leading to

imbibition); seed were then placed in complete darkness until cotyledon emergence.

According to the authors, this method “will give high total germination and more rapid

and uniform seedling emergence.” However, germination of seed for commercial plug

production does not expose seed to darkness and yet achieves high germination rates (F.

Kwong, personal communication). It is possible that once a light threshold is reached,

seed will germinate regardless of subsequent dark or light conditions. In addition,

Carpenter et al. (1995) report that Begonia ‘Scarlanda’ germinates at a faster rate (lower

T50 and T90-10) at lower photosynthetically active radiation (PAR), approximately 1.5

µmol·m-2·s-1 , than at 150 µmol·m-2·s-1. Thus, the Begonia species examined thus far

require exposure to light following imbibition and germination may then proceed either

in the light or in the dark. While the role of phytochrome in mediating seed germination

12 in Begonia has not been specifically studied, it is likely that the response will be similar to that of other light-requiring species (see review by Casal and Sanchez, 1998).

The few studies on seed germination have shown that seeds can germinate at temperatures as low as 15˚C and as high as 30˚C (Table 1.2). It is quite possible that temperature requirement could vary greatly for species depending on natural habitat.

Some Begonia are found in cooler climates than others, e.g. it snows in parts of the natural habitat of B. boliviensis, but the same conditions would kill other species from lowland tropical rainforest (M. Tebbitt, personal communication). Thus it is important to consider in more detail the response of different species’ seeds to temperature.

In contrast to laboratory studies, the time it takes for begonia seed to emerge from the soil (or to complete germination) is not well documented; anecdotal reports suggest it varies from within a week of sowing to a few months. A comment heard from Begonia growers and breeders is that seeds “are slow to germinate” but quantitative comparisons are lacking. Thus, there are many facets to understanding seed germination of Begonia both under laboratory and greenhouse conditions that need to be better defined.

Study objectives

The overall objective of this study was to develop protocols and information for routine Begonia seed germination at the OPGC. In addition, a comparative analysis of various seed attributes for different species was undertaken to generate more detailed information about the seed biology of Begonia. Specific objectives of this study were to develop a standardized protocol for seed germination of Begonia held in germplasm

collections and to apply this protocol to a diverse group of species representing different

habitats as a means to explore potential differences between these species at the seed

level. Important factors addressed include: quantification of desiccation tolerance,

presence of dormancy, assessment of requirement for light (photoblasticity), and response

to environmental stresses (e.g. controlled deterioration). In addition, laboratory protocols for standard practices such as seed conditioning and tetrazolium testing have been examined. This information should be made publically available to facilitate further studies in understanding the seed biology of this diverse genus. More in-depth investigation is necessary to have a better understanding of the biology behind small seeded crops like Begonia (Hu et al., 2012; West and Lott, 1991). There is limited information about how Begonia from various regions of the world and habitats will store or behave under routine germination testing. By examining seeds of species from various natural habitats, I propose to clarify how the seeds of a wide range of Begonia species behave, and therefore should be maintained in the future. Availability of high-quality public domain germplasm can benefit the entire industry. Furthermore, results of this research may also provide guidelines for germination protocols of other small seeded crops such as Campanula, Eustoma, Petunia, Mimulus, etc.

A B

Semperflorens-cultorum group ×tuberhybrida group (wax begonia) (tuberous begonia) C D

Chiemantha group Rex-cultorum (a group grown mainly for their (foliage Rex begonias) showy flowers produced in mid- winter)

Figure 1.1. The major cultivar groups of Begonia used in international trade. A. Semperflorens-cultorum group (commonly called wax or bedding begonia) B. ×tuberhybrida group (commonly called tuberous begonia) C. Rex-cultorum (commonly called rex begonia) D. Chiemantha group (commonly called Elatior or Rieger begonia); photo by Ross Bosswell.

Figure 1.2. Relative seed size of Begonia, Arabidopsis, and Capsicum.

A B

B. grandis subsp. evansiana B. ulmifolia C D

B. fischeri B. boliviensis E F

B. subvillosa B. dregei G

Figure 1.3. A-F. Scanning electron micrographs of six Begonia species included in this study (Source: Leona Horst, USDA-ARS, Wooster, Ohio) G. Parts of a typical Begonia seed: hilum micropyle (where seed attaches to fruit wall), operculum (seed lid), collar cells (a feature specific to Begonia seed which split upon germination), testa cell (the common component of a seed coat). 17

Number of Clonal Seed Category accessions Inventory Inventory Species 146 58 88 Interspecific 27 27 hybrids Cultivars 8 8 Total by category 181 93 88

Table. 1.1. The Begonia germplasm collection at the Ornamental Plant Germplasm Center as of September 2014 maintained either as clones or seeds.

CHAPTER 2

A PROTOCOL FOR BEGONIA SEED GERMINATION

Introduction

Small seeded plants are a challenge to handle in many respects, but specifically in

germplasm maintenance. Issues often arise concerning seed production, seed cleaning

procedure(s), viability testing, transplanting, and subsequent growth of seedlings.

Begonia seeds have often been referred to as being in the “dust” category, small seed that

can travel long distances by wind dispersal, or anemochorously (Clement et al., 2004).

Perhaps because of the difficulty in handling the minute seeds, studies dealing with the

biology of Begonia seeds have been scant (Table 1.2); there are no standardized protocols

specifically dealing with begonia seed even though the genus is listed in the Association

of Official Seed Analysts (AOSA) and International Seed Testing Association (ISTA)

manuals. The information presented in these manuals is very limited. Efficient, routine

protocols for seed germination would be beneficial. More extensive studies examining

various aspects of Begonia seed biology are especially needed and would benefit from

availability of such protocols.

One of the challenges when handling multiple germplasm accessions of small- seeded crops like Begonia is that the chance of cross contaminating different seed lots,

and consequently mislabeling them, is made more likely. For example, several seed

accessions of Begonia produced under controlled conditions in a greenhouse in 2005 and

held at the OPGC were sown in 2011 and the resulting seedlings turned out to be a

mixture of types. When seeds labelled as B. thelmae were sown, the seed population yielded B. thelmae, B.heracleifolia, and B. hydrocotylifolia, species that have very different growth habits and are not easily confused as adult plants. Similarly, seed labelled as B. paleata when grown yielded no B.paleata but the very distinct B. holtonis.

Whether this contamination or mislabeling occurred during the harvesting process or during the cleaning process is unclear. Similarly, some seed packets I have received from

amateur, but highly accomplished and careful Begonia breeders and collectors have

yielded mixed species populations. Regardless, handling of Begonia seed in situations,

such as germplasm centers where multiple species are maintained, needs to be done in a

very particular way to ensure that populations remain pure and free of contamination.

The procedures used at the OPGC for processing seeds of Begonia are described in

Appendix A.

An additional challenge in working with small seeds is the difficulty in using

standard germination protocols for seeds that cannot be handled even with a pair of fine- tipped forceps; such tiny seeds need different strategies for establishing quantitative tests.

A common response of seed analysts to testing begonia seeds is to describe germination tests as “challenging and laborious” (P. Conine, personal communication). This difficulty hampers accurate records of longevity in storage, a critical factor in germplasm preservation. Difficult and inaccurate tests may provide a misleading picture of the condition of particular seed lots because the reliability of such tests is questionable. In order to examine many aspects of Begonia seed biology that focus on assessing germination response to various treatments, it was imperative to have a simple, reliable,

efficient, and effective germination test. Most of the currently used germination methods have been unsatisfactory.

Germination of Begonia seed in the few laboratories that have reported such work has been done using paper substrate in Petri dishes or by floating seeds in water (Table

1.2), (Carpenter et al., 1995; Hu et al., 2012; Ma et al., 2005; Nagao et al., 1959;

Shoemaker and Carlson, 1992). A major challenge is the accurate counting of seeds. For

example, previous germination tests at the OPGC involved a rough count of 500 seeds

that were then scattered on a paper substrate (blotter) that had been soaked in double-

distilled water (ddH2O). After incubation for up to two months, seedlings were teased

from one another as they were scored for germination, based on cotyledon emergence and

growth of a primary root. Moisture management in this system was a challenge as the

tests extended over multiple weeks, leading to more opportunity for drying out of the

blotter, fungal growth, and possible environmental variability within the germination box.

This germination protocol was inefficient, and did not provide a reliable quantitative

analysis of Begonia germination. It is possible that much of the previously gained

information about seed viability of begonia at the OPGC and within the National Center

for Germplasm Preservation (NCGRP) may be potentially inaccurate due to unreliable

seed germination tests. Therefore, different strategies are required to properly handle and

preserve this unique genus in a gene/seed bank setting.

The NCGRP in Ft. Collins, Colorado backs up established seed lines from the

OPGC, and performs viability testing on Begonia at 20°C in germinator boxes or Petri

plates, utilizing blotter or filter paper as a substrate. Germination count days are extended

compared to other seeds at the center, because of apparent low viability. Of the 16

Begonia accessions held at the NCGRP, fourteen did not reach the 85% germination threshold required for cryopreservation, thus they are kept at -20°C only (P. Conine, personal communication). The germination procedures are based on those outlined by

AOSA or ISTA (summarized for Begoniaceae by Stephenson and Mari, 2005):

Temperature - either constant at 20°C or alternating 20-30°C; substrate - covered Petri dishes with two layers of blotters or three layers of filter paper, or on top of blotters or paper; light/dark - AOSA indicates light, ISTA does not specify; first count - 7-14 days; final count - 21 days; dormancy-breaking treatments - ISTA indicates prechill. ISTA provides the following evaluation guidelines: “A dicotyledon with epigeal germination.

The shoot system consists of an elongated hypocotyl and two cotyledons with the terminal bud lying between. There is no epicotyl elongation within the test period and the epicotyl and terminal bud are usually not discernible. The root system consists of a primary root, usually with root hairs, which must be well developed, as secondary roots cannot be taken into account.”

The Millennium Seed Bank at the Royal Botanic Gardens Kew, England, utilizes agar as a germination medium for all its viability determinations, although this substrate is not in a standard protocol for commercial seed analysis nor for seeds in germplasm centers worldwide that tend to use standard commercial protocols as defined by ISTA or

AOSA (Millennium Seed Bank, 2014). The small-seeded model plant Arabidopsis thaliana has been routinely germinated in research laboratories on agar media and various modifications of this method have been developed that could be adapted to other small seeded species like Begonia.

The use of agar media in a standardized germination test provides distinct

advantages over paper-based substrates. Agar provides a more uniformly moist surface

that does not require periodic addition of water that may potentially dislodge seeds or

young seedlings. It is difficult to make careful observations of root development on the

textured surface of filter papers or blotters. It is equally difficult to remove seedlings

from blotters without damaging the roots, whereas transfer of seedlings from agar is

achieved with relative ease. Agar also provides an improved option for digital imaging

and evaluation of Begonia seed germination.

An alternative substrate for germination is organic growing media such as a peat

or coir based media routinely used in commercial production. Begonia breeders and

growers, such as Benary in Germany, only test germination in organic media under

appropriate greenhouse conditions because they are developing products for greenhouse

production, making such tests logical for them. However, this kind of substrate makes it

difficult to control the light environment around the seeds and makes observation of root

development equally challenging. Although germination in greenhouse medium is an

ultimate test for commercial production, it is not a practical laboratory test strategy.

A simple and efficient germination protocol for Begonia would facilitate study of

the basic germination process including the influence of light and temperature on germination rate. Thus, it is critical to establish a standardized, quantitative germination protocol. A standard protocol could be used for evaluation of various seed biology parameters such as controlled deterioration (CD) that can permit comparison of seed vigor and stress tolerance of different begonia seed lots. The optimization of a germination protocol is crucial for assessing viability of begonia seed lots not only from a

germplasm standpoint, but also for production of seeds sold throughout the world. Here I report on the development of such a germination protocol.

Material and methods

Seed sources

Begonia fischeri (OPGC 2737) was used as the source of seeds for protocol development. Seeds were produced on plants grown at the greenhouse facilities of the

Ornamental Plant Germplasm Center (OPGC) in compartments maintained at an average daily temperature (ADT) of 25°C and approximately 50% relative humidity (RH). The compartments where seed were produced were shaded with 40% black shade cloth, giving an average irradiance of 350 µmol·m-2·s-1 (Quantum Light Meter, Spectrum®

Technologies, Inc., Aurora, IL). Female flowers were pollinated with pollen from male flowers from the same plant or from other plants that had been vegetatively propagated.

In effect, the seeds were all the products of selfing. The processes for producing and processing seed are described in Appendices A and B. It took approximately one month after pollination for B. fischeri fruit to mature. Seeds were stored at 4°C, 25%RH until used in experiments. Prior to a specific use, the seeds were equilibrated at room temperature (20-25°C) for 1-2 hours.

Germination substrate

Seeds were germinated on 1% agar (PhytoTechnology Laboratories, Shawnee

Missions, KS) prepared by dissolving 10 grams of agar with 1L of double-distilled water

(ddH2O) and microwaved until the agar was completely dissolved. Upon cooling but before solidifying, the agar was dispensed with an automatic dispenser (Wheaton

Omnispense PLUS) in the amount of 20 ml per Petri plate. This was routinely performed to assure that enough plates were available for the experiments. Other substrates tested for comparison included blue blotters (Anchor Paper, St. Paul, MN) or Fafard 3B Mix (a multi-use growing medium consisting of Canadian sphagnum peat moss, bark, perlite, vermiculite, dolomitic limestone, wetting agent) obtained from Sungro Horticulture,

Agawam, MA.

Seed placement

Because Begonia seeds cannot be individually handled with forceps, alternative methods of placement on the germination substrate were required. Initially, seeds were suspended in a viscous solution of 0.1% agarose (Sigma Chemical, St. Louis, MO) and then delivered onto germination substrate with a pipet. In each case, triplicate samples containing 50-100 seeds per 1% agar plate, germination boxes lined with moistened blue blotter (11x11x4 cm, Hoffman Manufacturing, Jefferson, OR), or plug trays containing

Fafard 3B Mix (48 cell pack; i.e., 1204 Trays, Landmark Plastic, Akron, OH) were used.

Seeds that had been equilibrated at room temperature after storage at 4°C were quantified with a micro spoon (S014-PhytoTechnology Laboratories), which can hold approximately 200 seeds (1 mg) of Begonia (Fig 2.1) and either placed in a 1.5 ml microfuge tube or a dry paper towel resting on top of a small platform (Fig. 2.2). Both methods were illuminated with sufficient overhead lighting (provided by t8 fluorescent tubes) to facilitate visualization of the seeds. The seeds in the microfuge tube received

0.8 ml of 0.1% agarose and the tube shaken gently to distribute the seeds evenly in the viscous solution (J. Jang, personal communication). The seed/agarose mixture was then taken up with an Eppendorf® single-channel pipet equipped with either a 200 µl or 500

µl tip and transferred to the 1% agar substrate, blotter or greenhouse media, dispensing

one seed at a time. To facilitate placement of the seeds on the Petri plates in a regular

pattern, a plastic grid was placed under the plate and used as a guide.

The seeds placed on the paper towel were processed differently. Approximately

200 Begonia seed were gently scattered on the paper towel. One at a time, seed was

picked up from the paper towel with a dissecting needle (Fig. 2.3) that has been dipped in

water to assist in adhesion of seed onto the 1% agar substrate. This needle transfer

method was only used with the agar substrate and not with blotter paper or greenhouse

media. When each plate received 100 seeds, it was wrapped with wax strips (Parafilm

M® Bemis, Neenah, WI) to secure the lid.

Germination environment

Plates, germination boxes or flats containing seeds were placed in a seed

germination chamber (Percival Scientific, Model GR-36L, Perry, IA) maintained at 25°C constant temperature with 16/8 h photoperiod, light at 150 µmol·m-2·s-1 from cool white

t8 fluorescent tubes for the duration of the treatment and/or viability test(s).

Anatomical observations during germination

Examination of the internal structure of seeds and seedlings was kindly done by

Prof. Robert L. Geneve of the University of Kentucky. The procedures for fixing,

sectioning and staining begonia seedlings were the standard methods used in plant

anatomy, and a representative procedure is described by Finniseth et al. (1998).

Statistical Analysis

In order to minimize variation due to sampling or experimental conditions (testing

method, lab environment, or seed analyst), calculations were performed according to the

‘germination tool box’ provided on-line by ISTA (2014) that include calculators for tolerance and confidence interval for germination tests, following statistical procedures developed by Miles (1963) to determine if results are ‘reportable.’ These are international

standard testing procedures that provide statistically valid assessments of germination

data. Average percent germination and standard error (SE) were calculated in Excel

(Microsoft Corporation, Redmond, WA).

Results

The seed production and processing protocols described in Appendices A and B

yielded clean, fully embryonic, and likely “viable” seed populations of B. fischeri that

could be used for development of a standardized germination protocol. This production

and processing system has been applied to other species with success and has become the

standard for all Begonia species grown at the OPGC. Depending on the species, yields of

1-5 million seeds per batch are possible.

The substrate used for germination, either 1% agar in Petri dishes, blue blotter

paper in germination boxes, or greenhouse-growing medium (Fafard 3B) in multicell

trays did not influence the overall germination of B. fischeri seeds. After 28 days at 25°C

with a 16/8 h photoperiod, the total germination under the three conditions was nearly

identical (Table 2.1). For these tests, germination was based on the presence of expanded

cotyledons. A seed was considered germinated when two clearly visible cotyledons had

expanded. Percent germination, expressed as (mean%±SE) on an agar substrate was comparable (94.5±1.2%) to other common media types.

The most time consuming part (rate-limiting step) in the set-up portion of seed germination protocols for Begonia is the placement of the tiny seeds on the substrate.

Initially, I used a suspension of seeds in 0.1% agarose to aliquot each seed on the substrate. This method took approximately 10-15 minutes and was relatively laborious.

Most of the effort was needed to re-distribute the seeds that accidently dislodged from the agarose suspension so that a single seed remained per site on the substrate. An alternative method was ultimately adopted where the seeds were first scattered on a paper towel and transferred individually to the agar substrate with a dissecting needle. This method proved to be more efficient, requiring only 8-10 minutes to aliquot 100 seeds on a plate; it was therefore used in all subsequent manipulations (Fig. 2.3).

Germination was assessed with the aid of a stereo-microscope (Zeiss DV4, Jena,

Germany) that permitted easy visualization of the developing seedlings. Each germination plate could be examined within approximately 3-5 minutes. Various details of the germination process could be observed, such as the opening of the operculum, the splitting of the anticlinal walls and the expansion of the embryo axis. In addition, abnormal seeds were easily identified. Thus germination on an agar substrate provided a clear background where details of seedling development could be noted.

Since the agar method does not use sterile substrate or seeds, on occasion, fungal growth could be seen on the seeds and substrate. This was generally dependent on the quality of the seed lot. Overall, the fungi did not seem to interfere with germination of healthy, viable seed as germination occurred regardless of infection on Petri plates. When

fungi persists on an individual seed and no germination occurs, the seed is likely dead, and will not germinate.

In observing the germination of Begonia seeds, I noted that the process did not follow the typically described ‘radicle emergence’ as the first step in germination, that occurs in seeds of Arabidopsis thaliana ecotype Columbia (Fig 2.4). Instead, I noticed that what emerged from the operculum was not a radicle but the hypocotyl (Fig. 2.5). The hypocotyl is the embryonic stem and assists in support and uptake of water once germination has occurred (Fig. 2.4). Once the hypocotyl emerges from the seed, it makes contact with a substrate and then sends out root hairs, which anchor the hypocotyl/seedling for further development. A true root is not noticeable until the 12th day after imbibition.

Careful observation of developing seedlings allowed me to define germination, for the purpose of various physiological studies, as elongation of the embryo axis

(hypocotyl) extending for about one third the length of the seed. In my experience with

Begonia seed, if the hypocotyl extends to about a third to a half of the seed length, then development continues into a full seedling. Using this definition and under the incubation conditions selected for this protocol, it was possible to generate germination curves for B. fischeri as well as for another five species of Begonia (Fig. 2.6).

Germination for five of the six species was quite similar and relatively quick, reaching maximal germination within 8-10 days. Three of the species, B. fischeri, B. subvillosa and B. boliviensis, had greater than 80% germination within 3 days. In contrast, Begonia dregei had a much lower maximal germination rate of 72%.

Anatomical studies of the germinating seed and developing seedling provide more details on the process of germination. The mature seed consists of a testa, a one cell layer endosperm, two well-developed cotyledons and a very small shoot apical meristem.

However, there is no pre-formed root meristem in the mature seed (Fig. 2.7).

Germination involves elongation of the hypocotyl and the production of root hairs from a structure called a collet (hypocotyl/radicle juncture). The root meristem appears to arise de novo below the collet when the cotyledons have expanded. This is followed by radicle elongation and development of a root (Fig. 2.8).

Discussion

Germination of Begonia seeds on agar substrate results in percentages that are equivalent to those obtained with the traditional blotter or filter paper typical of seed laboratory tests or in media used for greenhouse production. The agar method offers distinct advantages for the routine assessment of seed lot viability of the OPGC begonia collection. The overall method is not necessarily easier or less time consuming, but it allows for an easier and more thorough visual evaluation of the seed and seedlings to identify, for example, the presence of abnormal seedlings.

The somewhat translucent substrate facilitates observation and documentation of root hair and root development. The seedlings can be more easily monitored with a stereo/dissecting microscope and the translucent substrate makes it easier to examine the developing seedlings in detail. If a particular seed lot has a higher incidence of fungal growth, as sometimes happens, and the fungus tends to cover the entire seed, it is still possible to observe the germination by looking at the underside of the plate and assess

whether a normal seedling still develops in the presence of the fungus. Another advantage of this method is that seeds can be scored for germination shortly after the seed coat splits

(operculum rupture) because of the ease of determining exactly when the hypocotyl has

first emerged. Therefore, the data collection actually represents what is happening

biologically on a real time scale (Fig 2.4).

The agar substrate also provides a more consistently moist environment, whereas

the paper and growing media can be variable, often requiring addition of water, which

may disrupt developing seedlings. Germination of seeds in agar also makes it easy to

transplant seedlings for subsequent development in greenhouse media, should that be

necessary. Such transplanting can be done without significant root disruption as often

happens when roots are attached to paper substrates, i.e. filter paper, blotter. I have

transplanted seedlings from agar routinely with consistently good results that include low

mortality and virtually no delay in growth after transplant. Such transplanting may be a

useful practice in breeding work where wide hybrids may produce few viable seeds that

can be more readily nurtured into plants.

The very small size of Begonia seeds suggested that a viscous suspension of seeds

would be easier to distribute on germination surfaces. A thick solution of agarose was

initially used as the carrier for seed, following protocols routinely used in Arabidopsis

research. However, there were routine challenges and delays in setting up germination

plates with the pipetting method (Fig. 2.2), so after the first year of trials and much

feedback from individuals operating the pipets, I looked at alternatives. Sampling seeds

with a microspoon and placing them on a dry paper towel provided an even spread of

seeds that could subsequently be picked up with the water droplet that adheres to the tip

of a dissecting needle. Seed was much easier to work with on a dry surface and placement of seed onto the agar plates could be readily accomplished with the aid of a dissecting needle. This alternative method of handling the seed was much simpler and quicker, and required less dexterity than careful handling of a viscous solution in a pipet.

This method may also be applicable to other small seeded crops.

Use of greenhouse media and growing environment is impractical for routine use in a laboratory setting because of the inevitable variation in temperature and humidity that is likely to occur in such situations. From a commercial standpoint, it is appropriately logical to test seed performance in a typical production setting, but from a germplasm preservation and management standpoint, where seed viability is determined by germination tests, such greenhouse germination systems are too cumbersome.

This improved methodology has also been used for viability testing of Begonia as well as other small seeded species at the OPGC for several years, amounting to hundreds of tests with consistent results (data not shown). Similar advantages, such as translucent of media aiding in observation of root morphology has been noted in germination tests of other genera, such as Rudbeckia, Coreopsis, Dianthus, Antirrhinum, and Ratibida (E.

Renze, personal communication).

Germination process

Many general Botany or Plant Science textbooks define the first step in germination as ‘radicle emergence,’ an accurate term for most crop plants and plant model systems such as Arabidopsis, soybean, tobacco, etc. However, in a more precise and nuanced description, Black et al. (2006, pg.259) define germination, “as those events occurring between the start of uptake of water by a seed (imbibition) and the emergence

of the embryonic axis through its surrounding structures (usually the radicle penetrating the testa or pericarp). A seed from which the radicle has emerged is regarded as having germinated. In the seeds of some species it is not the radicle which emerges first to complete germination, but instead the cotyledons or the hypocotyl. This occurs particularly in some members of the families Bromeliaceae, Chenopodiaceae,

Onagraceae, Palmae, Saxifragaceae and Typhanaceae.” This more accurate definition of germination is particularly appropriate for Begonia because the first structure to emerge from the seed upon germination is not a radicle.

Careful observation of germinating Begonia seed revealed that there is initially no radicle present, and in its place is a hypocotyl (Fig. 2.5), which provides the pressure to break through the seed coat (operculum) and then continues to expand. Soon after hypocotyl extension, root hairs begin to form (Figs 2.5, 2.7). These hairs appear to anchor the seedling to the substrate. The elongated hypocotyl is then followed by cotyledon expansion and presumably, photosynthetic activity. It appears that once cotyledons perceive light and begin to photosynthesize, then the radicle emerges, and penetrates the substrate (soil) to establish a more extensive root system (Fig 2.8). Growth of Begonia seedlings increases in rate, probably in response to the roots beginning to translocate nutrients and water to the shoot system. This may have evolved as means to protect the seedlings from developing in inhospitable environments. Begonia seeds in laboratory tests germinate freely in the presence of fungus, suggesting tolerance of fungi during establishment in moist environments, a favorable environment for pathogenic fungi (Wick, 1998). This difference in germination pattern by hypocotyl seems to allow

seedlings to become established in a variety of environments (moist, damp, dry, slopes, forest floors, moist tree nooks, floating in water).

The collar cell/anticlinal walls of Begonia seed which split upon germination and that distinguish the genus from other plants at the family level taxonomically (Bouman and de Lange, 1983; Clements et al., 2004, De Lange and Bouman, 1992; 1999; Gregório et al., 2014), can potentially be explained physiologically, by this newly described pattern of germination. The collar cell/anticlinal wall structure is likely to have been formed as a means to accommodate a larger embryonic axis, a hypocotyl.

Another genus in which hypocotyl emergence occurs first in germination is

Impatiens. Observations of germination in I. capensis and I. pallida, showed that adventitious roots develop at the base of the hypocotyl followed soon by radicle emergence and root elongation (Nozzolillo and Thie, 1983). This growth patterns gives the seedlings an appearance of having a fibrous root system. The formation of these adventitious roots or root hairs in both Impatiens and Begonia may represent an ecological adaptation to their moist natural habitat. These species are typically found in areas of running water where an anchoring mechanism to secure the seedling in place quickly may be advantageous. Not only would this adaptation provide mechanical support but also a larger surface area for nutrient and water absorption, and possibly for establishing myccorhizal relationships early on in seedling development. An additional benefit could be establishment on steep slopes, such as next to a waterfall or on a moist cliff, common habitats for Begonia in nature.

A similar situation where the first structure to protrude from the seed upon germination is a hypocotyl and not a radicle is Theobroma grandiflorum (Ferraz et al.,

2012). Soon after hypocotyl emergence, adventitious roots form and continue to expand, then a primary root forms. The seeds have hypogeal germination and an epicotyl elongates after the primary root has expanded. This species, related to cacao, is native to the tropical Amazonian region. The significance of this germination system from an adaptive/environmental standpoint is not clear, but it demonstrates that alternative mechanisms involving the early stages of germination occur in woody plants as well.

In conclusion, the use of agar as a substrate for seed germination of Begonia and the development of a simple and effective method to distribute seeds on the surface provides a relatively simple system to test a variety of environmental conditions for their effect on seeds. Such a method has been used to compare various seed biology characteristics of different species of Begonia (Chapter 3) demonstrating its applicability to obtain fundamental information that may facilitate the germplasm preservation of this large and diverse genus.

0.0040 B. grandis subsp. evansiana 0.0035 B. ulmifolia 0.0030 B. fischeri 0.0025

0.0020 B. boliviensis

0.0015 B. subvillosa Weight of 5 µl (g) 5 µl of Weight 0.0010 B. dregei 0.0005 0.0000 Species

Figure 2.1. Seed weights of different Begonia species. Approximately 200 seed sampled with a microspoon were used to obtain weights (grams). Seeds had been stored at 4°C, 25%RH for at least 6 months. (c.f. Figure 2.3B).

Figure 2.2. Tools and materials used in the preliminary setup for the germination protocol; tools include pipettes, micro spoon, microfuge tubes, and materials include 0.1% agarose and Begonia seed. The work must be done in a well-lit location for efficient handling of the seeds.

A B Microspoon used to measure Processed seed lot approximately 200 seed

C D

Dissecting needle for placement Grid for organization

E F Typical set-up Completed replicate of 100 seed

Figure 2.3. Tools and materials used for the routine Begonia germination protocol. A. Begonia seed lot after processing. B. Microspoon used to quantify approximately 200 seed for each Begonia accession. C. Dissecting needle used to aliquot one seed at a time. D. Plastic grid with 100 dots in four 25-dot squares used under the Petri dish to facilitate placement of seed. E. System for placement of seed onto germination plates; dissecting needle (held in the hand) is first dipped in water within the microfuge tube and then used to pick up a seed one at a time from the white paper towel and placed onto the agar plate. The water drop facilitates adhesion of the seed to the needle. F. A plate with 1.0% agar where 100 Begonia seed have been distributed.

Figure 2.4. Contrasting germination between Arabidopsis and Begonia. A. Germination of Arabidopsis thaliana ecotype ‘Col’ showing emergence of the radicle (red arrow). B. Different stages of Begonia germination, illustrating the emergence of the hypocotyl, which develops from the plumule, the embryonic shoot (red arrow) before any radicle is visible.

Day 0-imbibed mature seed Day 3-operculum(seed lid) shed

Day 3-6-expansion of hypocotyl Day 6-9-collet visible- root hairs emerge

Day 9-12 cotyledons emerge, radicle has formed and begins to expand

Figure 2.5. The germination process in Begonia fischeri. The operculum is pushed open about 3 days after the imbibed seed is exposed to light. Between days 3 and 6 expansion of the hypocotyl follows; between days 6 and 9 a collet and root hairs form; by 9 to 12 days the seed coat is shed, cotyledons emerge, and a radicle develops. Each of these processes is highlighted with a red arrow.

100 100 80 80 B. fischeri B. grandis subsp. evansiana 60 60 40 40

% Germination 20 % Germination 20 0 0 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 Days Days

100 100 80 80 B. ulmifolia B. boliviensis 60 60 40 40

% Germination 20 % Germination 20 0 0 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 Days Days

100 100 80 80 B. subvillosa 60 60 B. dregei 40 40

% Germination 20 % Germination 20 0 0 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 Days Days

Figure 2.6. Baseline Begonia germination (mean%±SE) defined here as hypocotyl emergence (1/3 the length of the seed coat) of six Begonia species after three weeks under standard conditions (16 hour light/ 8 hour dark 25°C) using agar as substrate.

Figure 2.7. Cytological examination of Begonia fischeri germination from mature imbibed seed to radicle formation (0-12 days). Source: Robert L. Geneve, University of Kentucky, 2014 (used by permission).

Figure 2.8. Development of a primary root in Begonia fischeri (12-16 days after imbibition). Source: Robert L. Geneve, University of Kentucky, 2014.

Substrate Germination (%±SE) Blue blotter 93.3±1.00 Greenhouse media (Fafard 3B) 94.5±0.50 Agar (1%) 94.5±0.50

Table 2.1. Germination of Begonia fischeri in three different substrates after 28 days incubation under ‘standard’ conditions (25°C, 16 hour light/ 8 hour dark).

CHAPTER 3

COMPARISON OF SEED CHARACTERISTICS AMONG SIX BEGONIA SPECIES

Introduction

The germplasm collection of Begonia at the Ornamental Plant Germplasm Center

consists of taxa that originate from different parts of the world and from different types of

habitats. The collection represents a sampling of some of the more distinctive

morphological variants that are cultivated but otherwise lacks a distinct identity. To

maximize available genetic diversity and facilitate management, the collection is being

developed towards greater emphasis in the conservation of species (as opposed to

complex hybrids that may be sterile) where the germplasm can be stored as seed rather

than as clonal accessions. As a consequence, it is becoming increasingly important to

understand as many aspects of Begonia seed biology as possible. Certain seed attributes

such as desiccation tolerance, dormancy, responses to temperature and light, tolerance to

various stress tolerances (heat, humidity, freezing) and longevity in storage may vary for

species from different geographical regions as well as geographically similar species. The

development of a standardized germination protocol for Begonia seed (Chapter 2) makes it possible to do comparative studies on seed behavior among different species to generate information that may facilitate management of seed populations over time.

Research on Begonia seeds has been focused on their morphology as an aid in taxonomic classification schemes (De Lange and Bouman, 1992; 1999). Little is known 46

about other aspects of seed biology, some of it undoubtedly influenced by the very small

size of the seeds that makes their use in experiments very challenging. It is known that

Begonia seeds are endospermic, with the principal storage compounds being oil and

protein (West and Lott, 1991). The seeds require light for germination, and some Asian

species have non-deep physiological dormancy (Carpenter et al., 1995; Hu et al., 2012;

Ma et al., 2005; Nagao et al., 1959: Shoemaker and Carlson, 1992). The complex

hybrids commonly used in the ornamentals industry appear to germinate over a wide

range of temperature (Reynolds, 2003). Nevertheless, more systematic analysis of seed

behavior among diverse species is lacking. Such work is needed to determine the degree

of variability that may exist in seed attributes within this diverse genus.

Begonia is native to mostly tropical and subtropical regions, and species occur in

moist, mesic, and even a few semi-arid habitats such as the eastern coastal forest of South

Africa. One species, B. grandis subsp. evansiana, occurs in the temperate deciduous forests of China. Many species from the humid tropics can only be grown in high humidity environments such as terrariums, while others prefer to be grown in relatively dry conditions. In fact, many horticultural forms of begonia behave like succulents that are easily overwatered under ‘standard’ growing conditions such as those in greenhouse production of bedding plants. It is likely that seeds of species requiring such different environments for optimal plant growth will exhibit similarly variable response to the storage and handling typical in seed banks.

Seed testing

In seed banks, viability of accessions is primarily determined by germination tests. Such tests must accurately reflect the characteristics of a seed lot so decisions about regeneration of accessions showing declining viability can be made. Thus, germination protocols should be standardized so that they can be used as a reliable tool for maintenance of diverse genera and species. Such protocols are scarce for wild species, and the lack of accurate viability measurements (i.e. percent germination) for some species may reduce the utility of important genetic sources in a gene bank like the

Ornamental Plant Germplasm Center (Wada and Reed, 2011). Therefore, a germination protocol that can be used with a wide array of Begonia species will make accessions more useful to plant breeders.

It is generally accepted within the floriculture industry that begonia seed are orthodox or desiccation tolerant (F. Kwong, personal communication), but thorough documentation is lacking. The Seed Storage Database (Royal Botanic Gardens Kew,

2014) labels as ‘orthodox’ nine species of Begonia. Seeds at the OPGC have been stored for at least 10 years at 3-5°C and 25% RH; such seeds are expected to equilibrate to a moisture content of 5-12%, typical of desiccation tolerant seed (McDonald and Kwong,

2005) and retain their viability over extended periods of time (possibly decades). In a preliminary assessment of viability of Begonia germplasm at the OPGC, seeds of B. cardiocarpa have retained germination capacity after storage for 10 years (data not shown); thus, it is very likely indeed that they are orthodox. However, it is desirable to have accurate measurements of seed moisture content (MC) for different Begonia accessions in order to determine whether such attributes are characteristic of the genus as

a whole or whether it varies by species, especially for those that occur in very different

habitats. Seed described as orthodox retain viability at a MC of 6-10 % whereas

desiccation intolerant seeds are killed by MC <30% (Harrington, 1973). The small size of

Begonia seeds has contributed to the difficulty in obtaining accurate moisture content

values (West and Lott, 1991) so that routine measurement of this important parameter has

not been previously reported for this taxon. Like members of the Orchidaceae (Hay et al.,

2010) Begonia is a primarily tropical genus whose species produce large quantities of

minute seed that appear to exhibit desiccation-tolerant behavior in environments where moisture may be relatively constant.

Of particular importance for a seed bank or germplasm center is the potential longevity of seeds. The two main factors that contribute to the deterioration of seed in storage are temperature and relative humidity (Black et al., 2006). Storage of seed for commercial purposes (usually for no more than 5 years) often adheres to a ‘rule of thumb’ first described by Harrington (1973): seed storage conditions when reported as degrees Fahrenheit and percent relative humidity should not exceed 100 when added together. At the OPGC, the combination of temperature (ca. 40 °F) and relative humidity

(ca. 25 % RH) is approximately 65. While these conditions provide for sufficient longevity of many different kinds of seed, it is simply not known how long Begonia seed will remain viable under such conditions. There are indications that Begonia seed may be persistent and potentially long-lived. For example, Begonia is listed as a genus with seeds that persist in natural seed banks (Baskin and Baskin, 1998). Such persistence in soil seed banks provides a reasonable indication for potential longevity in long-term storage.

The National Center for Genetic Resources Preservation (NCGRP) maintains seeds of thousands of species for the National Plant Germplasm System of the USDA.

Among these seeds are some of Begonia that are maintained either at -20°C or in liquid nitrogen (-196°C). Unfortunately the difficulty in obtaining accurate germination counts for Begonia (Chapter 1) has made information about the performance of the seeds at the

NCGRP difficult to interpret. Thus, it is not altogether clear whether the Begonia seeds in storage are of low viability to begin with, or are adversely affected by the freezing conditions, or they are not accurately reflected in the germination tests used (Chapter 2).

Avid Begonia hobbyists, members of the American Begonia Society, have stated that they keep seed inventories (unknown species) in the freezer for periods of five to ten years, and are able to germinate these seeds without any difficulty other than a ‘minor’ loss in seed viability (C. Jaros, personal communication). While these claims suggest that long-term storage of Begonia seed may be feasible, there is a need for empirical data to support them.

Although it is not yet possible to accurately predict seed storage behavior, there are various factors that may be correlated with longevity. Seed structure and climate of origin appear to be indicators of longevity (Probert et al., 2009). In general, species from cool, moist environments tend to have a smaller embryo and appear to be shorter lived than seeds from dry environments that also have larger embryos. Begonia seeds are small, fully embryonic, and the species usually occur in moist habitats; these attributes would suggest that the seeds may be shorter lived, but assessment of other seed attributes, such as stress tolerance, is needed.

Assessment of longevity in storage depends on appropriate germination tests. The

protocol described in Chapter 2 provides a basic system where different parameters that influence germination of seeds of different species of Begonia can be systematically examined. An analysis of published works dealing with Begonia seed germination

(Table 1.2) shows that both complex hybrid cultivars and species from Asia can germinate under ‘standard’ germination test conditions, but that the details of such conditions present a less-than-clear picture of the ideal environment for germination, if indeed a single ideal environment exists. Suggestions for optimum temperature vary, depending both on sources of data as well as type of Begonia. Testing the effect of temperature on germination of seeds of species from geographically and ecologically distinct areas will allow us to identify differences, if they exist, and perhaps optimize conditions for routine germination testing of the species in the collection.

Small seeds often lack extensive food reserves, and generally require light for germination as an indication (environmental signal) of a favorable environment for seedling establishment (Black et al., 2006). Similarly, seeds that require light for germination tend to be small in size (Fenner and Thompson, 2005; Koutsovoulou et al.,

2014; Pons, 2000). While it is evident that Begonia seeds studied thus far require light for germination, a comparative analysis of different species from diverse habitats for response to light may also be informative. Thus, examination of the requirement of light for germination (photoblasticity) in different Begonia species may be warranted.

If seeds fail to germinate under a favorable set of conditions, it is important to

determine if they are dead or dormant. The presence of dormancy in Begonia has been

described and it appears that some species have non-deep physiological dormancy (Hu et

al., 2012; Ma et al., 2005). The extent to which dormancy occurs throughout the genus is not known. Most commercial begonia seed is considered to lack dormancy (Hayashi et al., 2008; Weiss et al., 2013) but since these are highly bred cultivars, it is possible that any dormancy that may have been present in the progenitors has been selected out.

Recently, Hu et al. (2012) utilized two species from low-latitude, monsoon climate regions of China to test hypotheses of dormancy. The authors found that species, B. lithophila and B. guishanensis, possess non-deep physiological (PD) dormancy that can be broken by dry after-ripening treatments, moist chilling, GA3, and KNO3 additives.

This dormancy may provide adaptability to the environment where these species originated so that at time of dispersal, seeds are dormant and overcome dormancy through some after ripening process once the monsoon rains return. A requirement for dormancy studies is the use of various biochemical/physiological tests (Black et al.,

2005) to assess viability of non-germinated seed. A common test uses tetrazolium (TZ) to identify viable, dormant seed; such a test has been reported for Begonia (McDonald and

Kwong, 2005), but its routine use has not been described probably because of difficulty in examining small seeds. Developing a strategy that would make it possible to routinely test begonia seeds with TZ is necessary (Appendix D).

The diversity in Begonia seed morphology as well as the presence of dormancy in some species but perhaps not in others indicates that morphological and structural differences may also be accompanied by physiological differences that could ultimately affect viability in storage. It would not be surprising if Begonia species from diverse habitats differ in response to temperature, requirement for light, rate of germination, and seed longevity. Differences in seed physiology are common in the many agricultural

crops; a key difference is that reflected between seed germination tests and seed vigor tests. Seed germination tests are done under ideal conditions in laboratory settings and provide an indication of seed quality/physiological status (Geneve, 2005). However, such tests are not good predictors of field performance; invariably, seed germination rates in the greenhouse or field and are lower than those achieved in the laboratory. This occurs because the environment is more variable and seeds, especially in the field, may be exposed to various stresses such as moisture extremes (too dry or too wet) or temperature fluctuations (too cold or too warm). Seed vigor is thus a more important attribute of seed lots because it provides a more likely indicator of seed performance (Geneve, 2005). The value of seed vigor tests is that they identify seed lots that are able to germinate across diverse environments. The ISTA defines seed vigor as “the sum total of those properties of the seed which determine the level of activity and performance of the seed or seed lot during germination and seedling emergence” (ISTA, 2014). Vigor of seed lots is assessed in the laboratory by accelerated ageing or controlled deterioration (CD) tests or by germination under low temperature in native (non-sterile) soil (Geneve, 2005).

Differences in response to CD conditions may be due to conditions during seed production and conditioning, to improper storage environments, or to inherent genetic characteristics of different cultivars (Geneve, 2005). While CD is most commonly used to compare individual seed lots of a cultivar, it follows that the stress conditions used for

CD could be used to examine differences between species. Controlled deterioration has been used as a predictor for field emergence and seed storage potential, at least for short- term storage (Hampton and TeKrony, 1995; Tesnier et al., 2002). Similar controlled deterioration has been used to generate viability equations that can be applied to different

species and predict long-term storage (Ellis and Roberts, 1980; Pritchard and Dickie,

2003). The challenge in generating such equations is the species-specific constant that

must be generated and that requires large quantities of seed as well as extensive

experimentation. Probert et al. (2009) have used controlled deterioration to explore

ecological correlates of ex situ seed longevity for a wide range of plant families. To date,

there have been no controlled deterioration studies of Begonia seed that could provide

some indication of species differences and that could help in devising more exacting

experiments for generation of viability equations. The ‘stress tests’ provided by CD

conditions may be useful tools to explore potential differences in tolerance to heat and

high moisture among different species of Begonia and give an indication of relative

health and vigor of seed lots, as well as physiological state.

The germination protocol described in Chapter 2 provides a means to efficiently

test germination of different seeds under variable conditions. In routine germination tests

now performed at the OPGC, many Begonia species have shown >90% germination at 25

˚C utilizing the newly adopted method on 1% agar plates. It is now possible to systematically apply this protocol in a comparative study of seed germination and response to controlled deterioration for six different species of Begonia. The goal was to assess whether seeds of these six species, adapted to different habitats, respond in unique ways to altered environments (temperature, light, and humidity/heat). The information generated may provide some guidelines for long-term storage conditions of Begonia and

tell us whether taxonomically diverse species respond similarly to ex-situ conservation.

At the same time, these experiments may provide some insight into the adaptation of

these species to diverse environments and whether seed characteristics (such as

dormancy, germination rate, tolerance to high temperature/high humidity) reflect this

adaptation. Thus my research may provide both fundamental and practical information that may inform gene banks, Begonia enthusiasts, and breeders of the unique seed properties from this widespread group of plants.

Material and methods

Plant material

The six species chosen for this study (Fig 3.1) represent a combination of diverse

growth forms, habitats, and geographic origins, which all form dry, winged fruit upon

maturation. These species originate in South Africa, China and South America and are

classified within 5 of the 63 sections of the genus (Doorenbos et al., 1998). These

species were used to produce seeds after self-pollination under standard greenhouse

conditions at the OPGC. Seeds were produced in 2011 and 2012 and were processed as

described in Appendices A and B. Begonia ulmifolia seed were produced from cross-

pollinations of seedlings and selfings in a full sun compartment at an average radiance of

550 µmol·m-2·s-1 ((Quantum Light Meter, Spectrum® Technologies, Inc., Aurora, IL) in

the winter of 2012. Seed of all other test species were produced by selfing under 40%

black shade cloth at an average radiance of 350 µmol·m-2·s-1. Seeds of B. boliviensis

were generated from plants vegetatively propagated as B. boliviensis ‘Bonfire’ (Cross,

2004). The six species used in these experiments represented a compromise between my

ability to produce sufficient seed under greenhouse conditions for given taxa in the

OPGC collection and a reasonably diverse sampling of genotypes that originate from

diverse habitats. Moreover, five of the six species are contributors to Begonia hybrids

that dominate the present floriculture market.

Species descriptions

A general description of the six species in this study and their habitats, expanding

upon that provided on Fig 3.1 is presented below. The information is mostly derived from

the work of Tebbitt (2005) unless specific reference is made to an alternative source.

Table 3.1 provides a summary of the climatic parameters typical of the area where these species occur.

Begonia grandis subsp. evansiana. A perennial native to parts of subtropical and temperate China, occurring as far north as Beijing. In its native range, summer months are dominated by heavy precipitation and humidity whereas winters tend to be cold and dry. This species has been utilized in recent years as a hardy begonia for landscapes of the Northeastern United States, being grown in USDA Winter Hardiness Zone 6 (M.

Tebbitt; C. Pounders; personal communications). Due to its hardiness, it is being included

in breeding programs to create tropical-looking plants able to grow in places like the

Midwest and New England. Mature plants form perennating tubers from which new growth is made once the winter season is over. The habitat for this species is unlike others in this study as conditions can be very cold, with average winter temperatures approaching -20°C/-4°F (Kottek et al., 2006).

Begonia ulmifolia. A perennial commonly described as weedy, a trait that may account

for its wide distribution. It is known to occur in lowland moist habitats such as disturbed

areas and often colonizes open spaces. It is native to Guyana, Brazil, and Venezuela, and

has become naturalized in several places outside of its native range (M. Tebbitt, personal

communication). Typical climatic conditions include a pronounced dry season, with consistent high temperatures, generalized as tropical.

Begonia fischeri. An annual to perennial native to the Atlantic Coastal Forest of Brazil,

commonly referred to as Mata Atlántica, where temperatures remain relatively constant

(approximately 23°C) and rainfall decreases in the summer months (Table 3.1). It has

also become naturalized in several places outside of its native range (M. Tebbitt, personal

communication). This species is important to the industry in that is has been involved in

the hybridization(s) of the B. Semperflorum-cultorum group (Tebbitt, 2005). This species is different from the others in that it often produces seed without any pollination and the resulting seedlings are superficially identical. In addition, when the species is used as a female in interspecific crosses, all resulting progeny are phenotypically identical to the mother plant (Steven Haba, unpublished data). Such behavior is suggestive of apomixis, but I have not done rigorous experiments to confirm this observation.

Begonia boliviensis. A group of plants widely known for its horticultural attributes, B.

boliviensis is a parental contributor to the B. ×tuberhybrida group. The species is native

to the Andean Mountains of southern Bolivia and northern Argentina where temperatures

tend to be mild and cooler than lowland tropical climates. In several cases, snow has

been observed in its natural setting (M. Tebbitt, personal communication). Common

habitats are cool, high elevation (ca. 2000 m) areas, with abundant irradiation; found on

consistently wet cliffs. Mature plants will form tubers from which new growth occurs

following dry or winter-like conditions. The seeds generated for this study were

produced by selfing the cultivar ‘Bonfire’ that is vegetatively propagated. The cultivar

originated from northern Argentina where seeds were collected from a wild population

and an individual grown in New Zealand was selected based on horticultural attributes

and propagated (Cross, 2004). Thus, the seeds used in this study need not be

representative of the species as a whole since they originated by selfing of a single

genotype.

Begonia subvillosa. A species which has contributed to the B. Semperflorens-cultorum

group, perennial in nature and native to the Atlantic Coastal Forests of Brazil, Begonia

subvillosa, like B. fischeri exhibits seed production systems and characteristics consistent

with either apomictic or pure-line breeding, since seed set is plentiful and occurs without

any hand pollination or insect visits. Several cross-pollination attempts have led to progeny (F1 and F2) resembling the maternal parent and all seedlings are superficially

homogenous (Steven Haba, unpublished data). Habitat and climatic data are the same as

described for B. fischeri.

Begonia dregei. This species is unique among those tested in that it is native to semi-arid

conditions. It is a perennial native to subtropical forests along the southeastern coast of

Africa in a habitat commonly referred to as xeric forest (Hilliard, 1976; Matolweni et al.

2000; McLellan, 1990, 1993, 2005). The species is self-compatible and seed production

is very straightforward. When Begonia dregei is grown from seed, a caudex forms,

which is a swollen stem that acts as a water reservoir in its native habitat.

Seed storage conditions

Once seed was produced and cleaned properly, following standardized procedure

(Appendix A,B), they were stored in 15 ml polypropylene, conical centrifuge tubes at 3-

5°C (40°F); 25%RH.

Moisture content

Moisture content was determined on seed that had been stored at 4°C and 25%RH

for at least six months. Approximately 2-4 mg of seed was placed onto each of 3 pre-

weighed aluminum dishes and the exact weight of seed recorded in milligrams. The trays

were placed in an oven at 105°C for 24 hours and the dried seeds were re-weighed (Fig.

3.2). Moisture content was calculated by subtracting the weight of oven-dried seeds from

that of the seeds prior to drying; the difference was divided by the original weight to get

% moisture.

Standard germination conditions

The protocol described in Chapter 2 was followed for the ‘standard’ germination

test (Fig. 2.3): 2 agar plates, each containing 100 seeds of each of the six species were

incubated at 25°C and a 16 h photoperiod. An examination was also done of alternating

temperatures, where plates were incubated at 23°C day/20°C night with a 16 h

photoperiod. To standardize assessment of germination, a seed was considered

germinated when the embryo axis extended about one-third the length of the seed (Fig.

3.3). In my experience, if a seedling reaches this stage of development, it will invariably continue on with production of normal seedlings.

Temperature effects on germination

Seeds of each Begonia accession were assessed for germination at different

temperatures (17˚ to 30˚C) on a thermal gradient table (Seed Technologies, Holland).

Two replicate Petri plates of each accession were evaluated at each temperature (Fig 3.4).

Germination was assessed every other day for up to 2 months; plates were never removed from the table to minimize internal temperature fluctuations. In addition, plates were not

sealed with parafilm in this experiment to avoid condensation issues throughout the experiment. The test was repeated to ensure that the data was consistent throughout time.

Percent germination (%G), expressed as mean %± standard error (SE) and time to 50% germination (T50) were then calculated. To verify the temperature gradient on the table, temperatures were logged with HOBO® Data Loggers (Onset Computer Corporation,

Bourne, MA);(Appendix E) for a period of 72 hours prior to the experiments. Additional tests with the thermal gradient table included temperatures of 15°C and 12°C used only with B. dregei. In order to exclude after-ripening as a causal agent for differences in germination of B. dregei, viability was periodically tested throughout the course of the experiments under standard conditions.

In order to assess the response of seeds to freezing conditions, seeds of each test species along with Arabidopsis that had been stored at 4˚C and 25%RH were placed into microfuge tubes (5 tubes/species, 35 tubes total) and kept in a -20˚C freezer (Isotemp

Plus; Fisher Scientific, Waltham, MA). The seed (one tube per species; approximately

300 seed) were initially removed after one week of incubation, and for a second time after 29 weeks, and evaluated for viability by a germination test under standard conditions.

Influence of light on germination

The requirement of light for germination was assessed under standard conditions that provided the baseline germination rates as described in Chapter 2. Light was excluded by carefully wrapping each plate with aluminum foil immediately after sowing the seeds (Fig. 3.5). Wrapped plates were incubated under the same standard conditions used for uncovered plates (25°C, 16 h photoperiod) and kept in the incubator for a period

of 2, 3, 6, 12, 36, and 52 weeks prior to exposure to light. After each of these intervals,

foil was removed to assess whether germination had occurred. This was done in ambient

light for a brief period (approximately 5-10 min); this light exposure was deemed a

“flash.” The plates were again covered with foil and incubated for another week. After

this additional week, the foil was removed, and germination was assessed to see if the

“flash” was sufficient for germination, thereafter plates were incubated under standard,

lighted conditions for a period of up to seven weeks to observe germination that was

assessed once a week.

Controlled deterioration

Controlled deterioration was done by exposing seeds to 100% RH at 41˚C for

different lengths of time; this method is often described as ‘accelerated ageing’

(TeKrony, 1993) but here I will use the more general term of controlled deterioration. A

germination box (11x11x4 cm, Hoffman Manufacturing, Jefferson, OR) was filled with

40 ml of distilled water and a mesh platform (Hoffman Manufacturing, OR) was placed in the box. Four permeable fine-filter tops (BD Falcon REF352235, San Jose, CA) were

placed on the mesh platform, two containing Begonia seeds and two containing

Arabidopsis thaliana ecotype Columbia (Col) seeds (Fig. 3.6). The Arabidopsis seed,

also small but 2-3× larger than Begonia, was included as a reference to verify that the

treatment conditions had an effect on the seeds; this small-seeded species served, in essence, as an internal ‘standard’ for the environment being manipulated.

Two different approaches were taken for controlled deterioration: in one, the

seeds were first incubated at 100% RH and at room temperature (20°C) for 24 hours prior

to exposure to 41˚C for 24, 48 and 72 hours (Fig. 3.7). In the other, the seeds were placed

in the 100% RH boxes and immediately incubated at 41˚C for 24, 48, 72, 96, and 120 hours. Following each treatment, the seeds were transferred to agar plates and incubated under standard conditions where germination was scored every three days for approximately three weeks (24 days). There were a total of eight treatments and two replicates for each species. Percent germination (mean%±SE) and time to 50% germination (T50) were calculated. In one case, B. ulmifolia and B. fischeri seed were incubated at temperatures of 50°C for 48 hours.

Assessment of dormancy

Presence of dormancy in seeds was assessed by comparing germination of either freshly harvested or aged seed that had been stored at 4˚C and 25% RH for up to six months. Only three of the species could be used for examination of fresh seed because of limitations on plants that were in bloom at the appropriate time for the experiment: B. grandis subsp. evansiana, B. boliviensis, and B. subvillosa. Standard germination tests were performed on these two types of seed. All dormancy tests were averaged over four replicates to account for variation in the fresh seed populations, which did not go through the elaborate seed cleaning process established at the OPGC since (Appendix A).

An additional test for the presence of dormancy was done with seed of B. dregei.

Seeds soaked in three concentrations of GA3 (grade III; Phytotechnology Laboratories) at

250, 500, and 1000 mg·L-1 (0.72, 1.44, and 2.88 mM respectively) for 24 hours, then sown and incubated under standard conditions to assess the effect of gibberellic acid on overall germination.

Statistical analysis

In order to minimize variation due to sampling or experimental conditions (testing method, lab environment, or seed analyst), calculations were performed according to the

‘germination tool box’ provided on-line by ISTA (2014) that include a calculator for tolerance and confidence intervals for germination tests, following statistical procedures developed by Miles (1963) to determine if results are ‘reportable.’ These are international standard testing procedures that provide statistically valid assessments of germination data. Average percent germination and standard error (SE) were calculated in Excel

(Microsoft Corporation, Redmond, WA).

Results

Moisture content

The seed moisture content of all six species was consistent with that of typically orthodox seed and ranged from 9.6 to 12.6 % moisture (Table 3.2). Begonia subvillosa had the lowest MC whereas B. dregei had the highest. These levels are similar to those of other desiccation tolerant seed (Harrington, 1973). The six species examined for this study form typical dry fruit upon maturation, and all have seed that are orthodox.

Germination process

All six species followed the same germination pattern that was described in

Chapter 2. The process starts by elongation of the hypocotyl without radicle emergence, followed by anchoring root hairs, further elongation of the hypocotyl, cotyledon expansion and finally radicle and root development (Fig. 3.3).

Baseline germination rates

Germination under standard conditions showed that five of the species reached

maximum germination of approximately 95% within about 9 days (Fig. 2.6). Only B.

dregei showed maximum germination of approximately 70%. This germination rate is considered descriptive of the seed lot being examined and was used as the ‘baseline germination’ for comparison with germination under different environmental conditions.

A brief assessment of germination under constant temperature or at alternating

temperatures of 23°C day/20°C night at 16 h light/ 8 h dark photoperiod showed no

difference in overall germination (data not shown); therefore, all subsequent germination

experiments were done at constant temperatures.

Influence of temperature on germination

Varying the temperature of germination from 17°C to 29°C did not alter the maximum germination for the six Begonia species examined (Fig. 3.8). Five of the species had maximum %G > 85%, only B. dregei showed maximum %G of about 50%.

This low maximum was lower than the initial baseline germination of about 70% (Fig

2.6). When seeds of B. dregei were exposed to lower temperatures (12°C and 15°C), the maximum germination increased to nearly 100%, but it required nearly 70 days to reach that level. Thus, this lot of B. dregei seed differs markedly from the other species. The

Arabidopsis ‘control’ seed included in this experiment achieved a maximum germination

of 96.5 % ± 1.5 across the 17-29°C temperature range (data not shown).

The generally similar maximum germination rates achieved for the five species

required different lengths of time to be reached. Figure 3.9 shows the germination curves

for each of the 6 species at different temperatures. The time to the onset of germination

(lag phase) decreased as the temperature increased. Except for B. dregei, this lag phase

lasted 6-12 days at 17°C, but at 26°C it was as little as 2 days. Similarly, the time to

reach 50% germination (T50) decreased from 17°C to 23°C, but it did not decrease much more as the temperature increased (Table 3.3). It was not possible to calculate a T50 for

B. dregei as this seed lot did not even reach 50% germination in this experiment. The germination at different temperatures demonstrates that the five remaining species of

Begonia could germinate at relatively equivalent rates in the approximately 10° temperature range between 20°C and 29°C. However, below 20°C, differences between the species were accentuated, with B. fischeri, B. grandis and B. boliviensis having a T50 of 10-11 days while B. subvillosa and B. ulmifolia having a T50 of 15-17 days (Table 3.4).

It thus appears that the latter two species are more sensitive to cold temperatures than the

former three.

B. dregei presents an altogether different picture in response to germination and

temperature. Baseline germination in the first set of experiments was 72%. When

examining the effect of temperature, the seeds germinated in the range of only15-49%. In

an attempt to re-evaluate these results, an additional germination test was carried out for

this species and the incubation period extended until germination reached its maximum,

or at least comparable to baseline germination rate, or the plates had dried. This

additional test included lower temperatures of 12°C and 15°C (Fig. 3.8; Fig 3.10). At

12°C, B. dregei can reach nearly 100 % (92.5±1.50) germination if given a sufficient

period of time, such a. 68 days (Table 3.3).

These data show that species differences in response to temperature do exist and

demonstrate that each species has unique seed biology attributes, at least with respect to

response to temperature and seed germination. While these experiments use a single seed lot of each species, and the seeds were produced in artificial conditions in a greenhouse, they provide a measure of comparison between the different genotypes involved.

Response to freezing

Exposing seeds of the six species to -20°C for one week did not seem to affect their viability, since all germinated readily when sown under standard conditions and the germination rates are comparable to the baseline germination (Fig. 3.11). A similar result was obtained when the seeds were kept at -20°C for 29 weeks (data not shown).

Influence of light/dark on germination

Seeds plated on 1% agar and incubated in the dark for up to 52 weeks did not germinate until exposed to light (60 µmol·m-2·s-1) under the standard germination conditions (Fig. 3.12). Begonia ulmifolia, B. fischeri, B. boliviensis, and B. subvillosa all had germination rates similar to the baseline for all intervals of incubation in the dark.

There was no decline in germination potential over the year-long incubation in darkness.

However, B. grandis subsp. evansiana seeds did show lower overall germination when kept in the dark; the decline in germination was noticeable after 2 weeks, but the response did not seem to be a progressive loss of viability since incubation for 12 weeks had lower germination than after 52 weeks in the dark. To determine whether the seeds that failed to germinate were still viable, the plates that had been incubated for 12 weeks in the dark and that had the germinated seeds removed were transferred to 4°C for a period of four weeks. After this time they were then placed in standard conditions to see if the remaining seeds would germinate. An additional 18 seeds of the approximately 70 remaining in each of the plates did germinate, indicating they were viable, but perhaps

had a secondary dormancy imposed by the prolonged dark treatment. Such a possibility needs to be more carefully assessed.

In contrast to the decline in germination after incubation in the dark shown by B. grandis, B. dregei seed showed increased germination, consistently above the baseline, when incubated in the dark prior to exposure to light (Fig. 3.12). The increase was between 9 and 27% germination above the baseline. It is evident that the species differ in their response to prolonged incubation in the dark once imbibed; such conditions may mimic their potential for persistence in a soil seed bank.

Controlled deterioration

The conditions chosen for controlled deterioration (100% RH and 41°C) had the intended effect of decreasing seed viability. Exposure of Arabidopsis thaliana Col seeds for up to 120 hours to these conditions resulted in rapid loss of viability (Fig 3.13).

Within 72 hours, germination potential of the seeds had decreased by 50% and by 120 hours, all seeds had died and abundant fungal growth was noted. There were differences in the rate of viability decline if the Begonia seeds were first equilibrated at high moisture for 24 hours followed by incubation at high temperature as opposed to immediate exposure to both high humidity and temperature. Simultaneous exposure to high temperature and high humidity seemed to cause faster deterioration. For example, pre- equilibrated seeds kept for 72 hours at 41°C showed 51% germination whereas the non- equilibrated seeds showed 16% germination, a difference of 35%. Regardless of this differential response, decrease in viability was clearly established in Arabidopsis and

Begonia by the stress treatments set up in the laboratory (Fig 3.13, 3.14).

All six species showed decline in seed viability as the length of exposure to high

temperature increased. All six also showed a differential response when pre-incubated at

high humidity. However, each species had a distinct pattern of decline in viability (Figs.

3.15 – 3.17). Except for B. dregei, the species showed more rapid decline in viability when placed directly in high humidity and high temperature. The larger differences in decline in viability between pre-equilibration at high humidity and simultaneous exposure to stress were noted for B. boliviensis, B. subvillosa and B. dregei (Fig. 3.15). In the first

two species, pre-equilibration seemed to delay the decline in viability whereas in the

latter it led to faster decline although the differences were very small. B. grandis subsp.

evansiana showed similar patterns of decline for both treatments while B. ulmifolia and

B. fischeri showed relatively little decline in viability under either regime, at least for the

first 72 hours.

After 96h of controlled deterioration, B. ulmifolia and B. fischeri retained >80%

germination; B. grandis and B. dregei had declined to 30-50% germination; and B. subvillosa and B. boliviensis had less than 10% (Fig. 3.18). When the deterioration conditions were extended to 120 hours, B. grandis subsp. evansiana, B. boliviensis, and

B. subvillosa had either died or showed only 10% viability; B. ulmifolia, B. fischeri, and

B. dregei, on the other hand, still showed >30% germination. In fact, B. ulmifolia seeds

germinated at >80% and B. fischeri at nearly 40 % (Fig. 3.19). Only after increasing the

incubation temperature to 50°C for 48 hours did both of these species die.

Assessment of dormancy

Begonia boliviensis and B. subvillosa germinate at similar rates independent of

whether freshly-harvested seed or 6-month old seed were used, indicating the absence of

dormancy (Table 3.5). Begonia grandis subsp. evansiana showed a 17% difference in germination between freshly harvested seed (75.8% ±5.38) and aged seed (93% ±1.47).

Seed of B. dregei responded to treatment with GA3 at concentrations of 250, 500, and 1000 ppm by increasing the maximum germination to 80.5%, 86%, and 90% respectively (Fig. 3.20). Such germination potentials were above the baseline germination of approximately 70%.

Discussion

The goal of this study was to compare some physiological characteristics of seeds from different Begonia species to gain a better understanding of parameters that may influence the longevity of such seeds stored at a seed bank. Because of a dearth of studies on the physiology of Begonia seeds, my focus has been on generating first, basic information about conditions that influence germination and second, determining how much variation there is among the species for response to controlled deterioration. The results of this study suggest that Begonia seeds are amenable to long-term storage and may in fact be quite tolerant of stresses that indicate potential for longevity. This study was facilitated by the development of a relatively straightforward, if not rapid, germination protocol (Chapter 2) that is likely applicable to all Begonia species.

Seeds of the six species used in this study are clearly orthodox. The moisture content of seeds equilibrated at 25% RH was measured at between 9% and 12%. The small size and limited availability of seeds made it difficult to use a large mass of seeds for moisture content determination so the results may have a lower accuracy than desired because only milligram quantities of seed were available. Access to a micro-balance may

have permitted more accurate weight measurements but nevertheless, the current estimation of moisture content fully support reports, both anecdotal and more carefully documented (Royal Botanic Gardens Kew, 2014), that Begonia seeds are desiccation tolerant. Such tolerance is further indicated by the ability of seeds to withstand -20°C without loss of viability. Thus, the long-term storage of Begonia seeds, at least from species that produce dry, dehiscent fruits, represents a sound strategy for germplasm conservation at the OPGC. However, preliminary studies of seeds from B. roxburghii, a species that produces berry-like fruit, suggests that not all Begonia species may be equally desiccation tolerant (Appendix F). Begonia species that produce fleshy fruits, like B. roxburghii may have seed whose viability decreases as storage time increases.

Comparative studies of Begonia species that differ in fruit characteristics are warranted, to determine if the different seed dispersal strategies found in the genus influence the potential longevity of the seeds.

Light is clearly necessary for germination of the six species I examined. This study did not assess the role of either light quality or intensity on germination. Such studies have been done with cultivars of B. Semperflorens-cultorum group. Carpenter et al. (1995) described the seeds as being ‘photodormant’ and that photosynthetically active radiation of 1.5 – 150 µmol·m-2·s-1 for up to 4 days was sufficient to overcome such

‘photodormancy’ and permit subsequent germination even in the dark. There has been debate in the seed biology literature whether photodormancy is an accurate term; the current consensus is that in such cases light is simply an essential component of the environment conducive to germination and that the inability to germinate in the dark is

not a dormancy state but rather the absence of the appropriate environment (Baskin and

Baskin, 1998, 2004).

The requirement for light appears to be absolute within Begonia, but more studies to evaluate this condition are needed. Nagao et al. (1959) reported that GA3 treatment would not substitute for irradiance in the germination of B. grandis subsp. evansiana.

These published reports on light and germination, and my own experiments indicate that

Begonia seed are positively photoblastic and that 50 µmol·m-2·s-1 is a sufficient amount of light for germination of the six species examined (Fig. 3.12). The requirement for light is consistent with the habitats (Table 3.1) where these species are found which typically involves some disturbance and include open areas near water such as rivers and waterfalls, where pockets of sunlight shine through. Behavior of Begonia seeds suggests adaptation to survival beneath green canopies as they positively respond to a long-day photoperiod, and to a high R: FR ratio for germination, and are less dependent on temperature (Yanes-Vazquez and Orozco-Segovia, 1990).

The requirement of light for germination is commonly associated with small seeds and various small-seeded families, including Campanulaceae, which exhibit this requirement (Pons, 2000; Koutsovoulou et al., 2014). Common flower seeds that require light for germination include Achillea, Alyssum, Antirrhinum, Calceolaria, Coleus,

Exacum, Ficus, Gaillardia, Gerbera, Gloxinia, Kalanchoe, Nicotiana, Petunia,

Saintapulia, Streptocarpus, etc (McDonald, 2005). Weed ecology studies have shown that small-seeded weeds often require light for germination and remain un-germinated in the soil for many years until exposed to light (Pons, 2000). Seeds that require light for germination must be imbibed before they can perceive the light signal (McDonald, 2005)

and my studies confirm this since dry seeds were routinely exposed to light when handled in the laboratory and yet failed to germinate when incubated in the dark. Light requirements are complex and include both the quality and intensity to influence the onset of germination, and the rate or the uniformity in germination of a population of seeds (McDonald, 2005). The phytochrome system, mediated through gibberellins, is involved in the germination of lettuce seeds, for example, and such system may be operating in all light-requiring seeds (Black et. al, 2006).

Prolonged periods in darkness following imbibition (up to 52 weeks) did not have an adverse effect on germination of 5 of the 6 species. The response of B. ulmifolia, B. fischeri, B. boliviensis, and B. subvillosa suggests seeds of these species may remain in a quiescent state in the dark for an indefinite period, until proper levels of moisture, light, and sufficient temperature create a hospitable environment for the seed to develop into young plants. This behavior is consistent with the listing, in Baskin and Baskin (1998), of Begoniaceae as a family with seeds that persist in the soil bank (although no specific reference was cited). In situ studies of Begonia seed in soil banks are likely to be extremely difficult given the minute size of the seeds. Although keeping seeds in 1% agar in the dark is rather artificial, it nevertheless provides an indication of the tendency of the seeds to remain in a quiescent state. In contrast, the response of both B. grandis subsp. evansiana and B. dregei to prolonged darkness is a bit more difficult to interpret.

A possible explanation for the decline in germination of B. grandis is the onset of secondary dormancy, which is supported by the subsequent germination of seeds after a chilling treatment. Another possible cause of the different response of B. grandis is a genetically heterogeneous population of seed. The seed generated for this study was the

product of self-pollinations of clonal material. If the original clone was highly

heterozygous, it is possible that the seed population may include many different

genotypes. Nevertheless, the different response of B. grandis to incubation in the dark

merits more thorough examination.

The increase in germination of B. dregei seeds following dark incubation may be

attributable to an after-ripening effect. This species showed lower maximum germination

than the other species unless germination tests were done at lower temperatures and for

extended periods of time (Fig. 3.10). The seed lot used in these experiments had the

potential for nearly 100% germination, but such levels were not achieved by using fresh

or partially aged (dry) seed. Seeds appeared to respond to prolonged periods in an

imbibed state either at 12°C in the light (Fig. 3.10) or a minimum of 2 weeks in the dark

(Fig. 3.12). Similarly, the enhanced germination obtained by treating seeds with GA3

(Fig. 3.20) suggests that physiological processes, perhaps related to some kind of

dormancy, were at play.

A sporadic one or two seeds of different species were noted to have germinated in

the dark, but this outcome was not reproducible. Such germination may be the result of

technical error in sufficiently excluding light from the seeds, or such seeds had initiated

imbibition prior to plating and handling in the light, or may represent some genetic

variation in the seed population for genotypes with either no or lower light requirements.

It would be interesting to grow plants from seeds that germinate in darkness to determine

if the response can be reproduced and the trait is heritable.

Seeds of Begonia species native to Asia have been reported to have non-deep physiological dormancy (PD), the most prevalent dormancy mechanism in angiosperms

(Hu et al., 2012) although general assessment of dormancy in angiosperms often list the

Begoniaceae as having non-dormant seed (Baskin and Baskin, 1998, 2004; Finch-Savage

and Leubner-Metzger, 2006). In the study by Hu et al. (2012), freshly harvested seed of

B. lithophila did not germinate above 30% in the 15-30°C unless KNO3 was present in

the media. When this nutrient was added, germination increased from <5% to about 70-

75% at 25°C. Treatments such as moist chilling, incubation in GA3 and dry after

ripening enhanced germination to a maximum of 90% indicating that the dormancy

mechanism was primarily dependent on the nutrient. Thus, the PD in this Begonia

species appears to be relatively weak, but the study is the only significant assessment of

dormancy in the genus to date.

Four of the six species in my study had non-dormant seed, B. boliviensis, B.

fischeri, B. subvillosa, and B. ulmifolia. Only B. grandis subsp. evansiana and B. dregei

displayed behavior that was suggestive of dormancy, although the response was not

clear-cut. The increase in germination following prolonged exposure to darkness (Fig

3.12) or to 12°C (Fig. 3.10) for B. dregei suggest a physiological alteration must take

place in some of the seeds within the population for them to germinate. This is further

supported by the response to GA3, which also led to a slight increase in germination (Fig.

3.20). These data indicate that the B. dregei seed lot had a variable population with some seeds being dormant and others not dormant.

The seeds of B. grandis subsp. evansiana may also represent a heterogeneous

population because overall germination was reduced by prolonged exposure to darkness

and could be partly restored by stratification. In addition, freshly harvested seed

germinated at a lower rate than aged seed (Table 3.5) although the majority of freshly

harvested seed (ca. 75%) were able to germinate. These observations suggest that a low-

level of dormancy may be present in seeds of some species of Begonia and that low

germination rates of freshly produced seed, for example, after regeneration at the OPGC,

may not be due to poor seed quality. The general message from the behavior of B. dregei

and B. grandis seed is that an after-ripening treatment may be prudent for most Begonia in the OPGC collection prior to assessment of seed lot viability by germination tests.

The environments in which Begonia species occur differ somewhat in temperature, elevation, precipitation, and exposure to light; both in amount (day length a.k.a. photoperiod) and intensity (µmol·m-2·s-1) (Table 3.1). These parameters could explain differences in seed behavior following dark treatments. Seed of Begonia from temperate climates appear to have non-deep PD that is overcome by incubation in the dark, and it is likely in nature that seed will remain in this dormant state, in a soil seed bank, until sufficient light is present, possibly by canopy opening or habitat disturbance.

Once germination occurs, seedlings need light in order to photosynthesize and thus become self-sufficient; thus this strategy prevents seed from germinating at deep soil depths (Black et. al, 2006). Furthermore, fluctuating temperatures, which stimulate germination in larger seeds (Koutsouvoulou et al. 2014), did not have a positive effect on germination of Begonia (data not shown). In nature, seeds of B. grandis subsp. evansiana are likely dispersed at the end of the wet season, which will not germinate until conditions are favorable after the cold, dry winter (Kottek et al., 2006).

Temperature

The Begonia species examined are able to germinate over a 10-15°C range in temperature. In general, the highest germination was recorded at about 25°C (Fig. 3.9).

The germination curves between 20°C and 29°C were quite similar, except for B. dregei

which, as indicated above, may have had a mixed population that included some dormant

seed. The B. dregei seed lot used in these experiments seemed to be insensitive to temperature except for those in the lower range, when germination, though extended over a long time, reached maximum levels (Fig. 3.10). These germination curves are indicative of fairly uniform seed populations (again, except for B. dregei) because the interval from minimal germination (e.g. 10%) to maximal germination (e.g. 90%) was within 8-10 days; in the case of B. fischeri, the interval was even shorter, only about 2-4

days. Such a rapid shift from barely germinated to nearly fully germinated indicates that

most seeds have the same response to the environment and are, indeed, quite similar

(identical?) to each other. As mentioned earlier, progeny of B. fischeri, B. subvillosa, and

B. ulmifolia are morphologically extremely uniform and the behavior of their seeds support the idea of either highly inbred or apomictic populations. On the other hand, B. boliviensis and B. grandis showed somewhat more variation in response to temperature,

again suggesting perhaps a more heterogeneous seed population. Rapid and uniform

germination is an attribute of ‘high vigor’ seed, which also implies a distinct

physiological state for these fast-germinating species as opposed to the others. The

‘standard’ conditions for germination described in Chapter 2 appear to be adequate for

assessment of seed viability (i.e. germination) of the species within the OPGC Begonia

collection.

Variation in the response to temperature may be reflective of the environment to which each species is adapted and also of how broadly adaptive these species may be

(Table 3.1). Hu et al. (2012) found that optimum germination varied by 10°C for two

Begonia species from the same geographical area. The species from more humid tropical

areas (B. fischeri, B. subvillosa, and B. ulmifolia) may be less sensitive to temperature

fluctuations because such variation is not encountered and therefore selection for optimal

germination at one temperature versus another has not occurred. Species where the

temperature may be seasonally more variable (B. boliviensis, B. dregei, and B. grandis) may be more ‘attuned’ to temperature as a germination signal. In general, temperature does not appear to adversely affect the potential for maximum germination, and Begonia seeds appear to be tolerant of a wide range of temperatures, another indication that these tiny seeds may have significance environmental plasticity and adaptability.

Higher maximum germination in B. dregei at lower temperatures (12°C) may be reflective of the environment that seed experience in their native habitat (Table 3.1). Seed is likely produced by the beginning of the summer/drier months. Temperatures during the summer in South Africa are low (average of 13.4°C), due to cool ocean currents, and this temperature along with precipitation increase, favors establishment and subsequent growth of seedlings. The slow germination of B. dregei ex- situ over the course of time, suggests a natural adaptation to germinate throughout the course of the year, or in a staggering fashion to safeguard against herbivory and possible competition (Fenner and

Thompson, 2005).

Stress tolerance

The controlled deterioration or accelerated (artificial) ageing tests used in this study have been developed to test seed vigor in various crops. Seed vigor is a more

‘sensitive’ measure of seed quality than standard germination tests because vigor declines more rapidly than ability to germinate upon seed storage (Geneve, 2005). As a

consequence, vigor tests may expose more subtle differences in the physiological characteristics of the Begonia seeds in this study and this is exactly what I found. Seed vigor is affected by aspects of seed production and conditioning, as well as by genetics, but I anticipate that the seeds produced for this study reflect inherent species differences and not just conditions of the way they were produced and processed. Since vigor tests have not been previously reported for Begonia, it was important to include an internal control to verify that the conditions in the laboratory were indeed stressful to seeds. The response of Arabidopsis seed (Fig 3.13) confirmed that adequate stressful conditions were imposed on seeds and that difference in the response of the Begonia species may indeed reflect inherent physiological differences of the lots being examined.

Even though species respond in similar patterns to temperature and light; differences appear to be present in terms of stress tolerance and ultimately physiology.

Initially I predicted that Begonia species may not tolerate high temperature stress, as the species in this study occur in tropical areas, as well as temperate forests and semi-arid climates where temperatures around 40°C may occur, but are not common. Although the interspecific variation in stress tolerances observed could be due to the seeds not being produced within the same year, and in different environments (full sun to partially shaded, see Materials and Methods; plant material), affecting seed quality (Baskin and

Baskin,2004;Contreras et al., 2008; Tesnier et al., 2002) i.e. viability and vigor. Black et al. (2006) state that deterioration rates are likely to vary among individuals in a heterogeneous population because factors that contribute to seed longevity are under genetic control (i.e. desiccation tolerance, maturity, dormancy, morphology, composition, and stress tolerance).

The two approaches used for controlled deterioration were based on experiences

described by Dr. Francis Kwong, a seed scientist for PanAmerican Seed who routinely

assesses seed vigor of flower crops. Dr. Kwong indicated that he typically separated the

hydration component from the high temperature component (F. Kwong, personal

communication). This is not common practice for accelerated aging tests of agronomic

crops such as corn and soybean (A. Evans, personal communication), but may be

followed because small seeds may be too sensitive to high humidity and temperature and

thus be killed within 24 hours. Alternative tests such as salt-saturated accelerated ageing have been developed to provide a less intense stress environment for small seeds (Jianhua and McDonald, 1997). Nevertheless, since no previous controlled deterioration tests have been done with Begonia, I decided to examine both approaches.

Separating the hydration step from the high temperature exposure in controlled deterioration did show a difference in the rate of viability decline. However, the patterns were ultimately similar. The significance of this difference and possible underlying mechanisms for this difference is not clear and possible explanations are beyond the scope of my work. Regardless of the protocol for controlled deterioration, the test revealed substantial differences in the response of the species; such differences are clearly demonstrated on Fig 3.18. Begonia fischeri and B. ulmifolia are clearly much more ‘stress tolerant’ than the remaining species as evidenced by their high germination after 96h of high temperature/humidity. An additional 24 hours of exposure revealed that

B. ulmifolia is the more ‘stress tolerant’ of the first two (Fig 3.19). Begonia subvillosa and B. boliviensis were the least tolerant.

Because of both genetic differences and seed production conditions affect seed vigor tests like controlled deterioration, the relevance of the observed differences between the species to specific environmental adaptations is more limited. In a few seed production cycles, some test plants did not set seed from crosses perhaps due environmental variability; this may hamper the ability to produce fresh seed for dormancy studies in the future. Specifically, there has been no seed produced on B. ulmifolia, B. fischeri, or B. dregei for potential dormancy comparison(s) utilizing fresh seed. The environment in which the pollinations were made may have not provided proper conditions for viable seed set, and may have been variable during production times, thus making it difficult to compare data from one seed lot to that of one produced in a different time frame (Tesnier et al., 2002). Lighting conditions differed for species during seed bulking based on available space in the greenhouse. In spite of these limitations, the value of these CD tests is that they reveal physiological differences that, more than likely, have a genetic (species-specific) component. The fact that all six

Begonia species could still germinate at >40% rates after 48 hours of stress implies a degree of ‘robustness’ for these very small seeds.

Conclusions

The experiments performed in this study demonstrate that the germination protocol I developed is useful and practical for examination of various factors that influence seed behavior in Begonia. Seeds of a diverse group of species can be readily germinated in the ‘standard’ conditions that have been defined and thus it is possible to adopt this protocol for routine germination tests of the Begonia germplasm accessions

maintained as seed. The test may also be applicable in commercial testing of Begonia

seed viability; this may require additional standardization. While the standard

germination test is a good starting point for assessing seed lot viability, it is prudent to

consider alternative treatments and conditions for species that initially show low levels of

germination. Achieving >90% germination in Begonia appears to be a reasonable

expectation, even for seed produced in artificial environments such as greenhouses of

variable conditions of light and humidity. Freshly harvested seed tested for viability may

show low percentages of germination if non-deep physiological dormancy is prevalent in a species or seed lot. Some level of such dormancy can now be expected for species from subtropical or temperate areas, but it is less likely in species from tropical climates.

Thus, either an after-ripening treatment or inclusion of GA3 in the germination media may be a prudent practice for such species.

The response to temperature, light, and prolonged exposure to darkness indicate that the different begonia species share some common attributes as well as unique features. Not surprisingly, all the species tested follow the characteristic germination pattern described in Chapter 2, which is probably typical of all species in the genus. For tropical species, germination at 23-25°C may be optimum. For subtropical and temperate species, germination in the 20-23°C range may be more appropriate. It does not appear necessary to germinate these seeds at higher temperatures as I did not find significantly increased germination rates at 29°C. Germination in the light is mandatory for Begonia seed.

The combination of seed attributes such as: desiccation tolerance, ability to withstand freezing conditions, persistent viability in an imbibed state in the dark,

relatively high levels of ‘stress tolerance’ (for some species) and the high germination of seeds that have been in storage for at least 10 years, all present a situation that suggests

Begonia seed longevity in storage may be high. At least for seeds from species that produce dry fruits. The preliminary observations made on B. roxburghii introduce a note of caution about over-generalization of seed traits to all species in such a highly diverse genus. Nevertheless, the diversity in growth forms of the species studied (tuberous, fibrous-rooted, thick-stemmed, shrub-like) that parallel phylogenetic diversity, lends some support to the idea that Begonia seed may be readily conserved in a seed bank.

This potential for longevity supports the direction of the germplasm collection at the

OPGC to focus on species and seed and less so in clonal material.

In summary: Begonia species germinate in a unique manner (by hypocotyl emergence) under a wide array of temperatures, seeds are likely to be desiccation tolerant

(pending further tests of seeds from berry-type fruit), produce large number of offspring, which are expected to live for long periods in seed banks due to their high tolerance to stress and ability to sit in an imbibed state in the dark for long periods of time (at least 52 weeks). Due to these distinct characteristics of Begonia, seed are likely very competitive among other small seeded plants when new hospitable environments become available in nature. Some dormancy appears to be present in species which naturally occur in more temperate areas. This is likely an adaptation to increase survival following, colder, inhospitable temperatures.

Habit Taxonomic Rank Provenance /Description / Comments Section and Species Identifier Section: Begonia Native to the Atlantic Coastal forest of Brazil and eastern parts of Paraguay. B. subvillosa One of the parents in the B .Semperflorens-cultorum group OPGC 435 Fibrous-rooted

Section: Barya Native to Andean Mountains of South America. Occurs in wet cliffs, cool climates. B. boliviensis One of the parents in the B. ×tuberhybrida group Tuberous growth habit. OPGC 2761 “Moist habitat at high elevations (ca. 2000 m)”

Section: Diplocinum Native to a large part of subtropical and temperate China, including forested hills of central China. B. grandis subsp. Adapted for cold-hardiness - tolerates USDA Winter evansiana Hardiness Zone 6. A tuberous begonia OPGC 2758

Section: Begonia Native to Atlantic Coastal Forest, Brazil One of the parents in the B Semperflorens-cultorum B. fischeri group

OPGC 2737 Shrub-like

Section: Augustia Native to South Africa (eastern coast) growing in xeric environments. B. dregei Contributor to Cheimantha types OPGC 1734 Thick-stemmed group; produces caudex

Section: Donaldia Native to Guyana, Brazil, and Venezuela growing in lowland moist habitats. B. ulmifolia Has been hybridized with one other species OPGC 1717 Thick-stemmed group

Figure 3.1. Begonia species used in the study of germination. The description and comments are partly based on information provided by Tebbitt (2005).

A B C

Figure 3.2. Tools and materials used for moisture content determination of Begonia seeds. A. Aluminum dishes, seed lots, scale, microspoon. B. Transfer of seeds to an aluminum dish for initial weight record. C. Oven kept at 105°C where seeds were incubated for 24 hours and then re-weighed to calculate loss of moisture and thus, moisture content.

Figure 3.3. Germination pattern for the six species of Begonia in the study. The process is described in the rightmost column. A seed was considered to have germinated when the hypocotyl had elongated to about 1/3 the length of the seed, indicated by the different color background on the third row.

Figure 3.4. Thermal gradient table with plates containing seeds evaluated for germination at different temperatures. The table was illuminated by t12 cool white flourescent bulbs, yielding an average light level at table height of 50 µmol·m-2·s-1. Five different temperatures at the table positions were clearly marked by numbered blue tape.

Figure 3.5. Method to exclude light from germination plates in the dark treatments. Each plate was wrapped in aluminum foil to exclude light and labeled with accession identification number as well as the first two check dates. The plates were kept in the same incubator used for the germination experiments in the light.

Figure 3.6. Close-up view of the system used for controlled deterioration experiments with the small Begonia seeds. The mesh platform is placed inside a standard germination box containing 40 ml of water; cups containing an ultra-fine mesh were used to hold the seeds that had been transferred there with a microspoon (5 µl). Seed does not have direct contact with water on lower surface of germination box, yet became fully hydrated from 100 % RH (saturated) environment.

B C

Figure 3.7. Experimental setup for controlled deterioration experiments. A. Side view of germination boxes containing 40ml of water and the mesh platforms. B. Top view of germination boxes showing the mesh platforms and the individual mesh caps containing seeds. C. Water-jacketed incubators used to maintain a constant 41°C.

B. grandis subsp. evansiana B. ulmifolia

100 100 80 80 60 60 40 40

% Germination 20 % Germination 20 0 0 BG 17 20 23 26 29 BG 17 20 23 26 29 Temperature (°C) Temperature (°C)

B. fischeri B. boliviensis

100 100 80 80 60 60 40 40

% Germination 20 % Germination 20 0 0 BG 17 20 23 26 29 BG 17 20 23 26 29 Temperature (°C) Temperature (°C)

B. subvillosa B. dregei

100 100

80 80

60 60

40 40

% Germination 20 % Germination 20

0 0 BG 17 20 23 26 29 BG 12 15 17 20 23 26 29 Temperature (°C) Temperature (°C)

Figure 3.8. Germination (mean%±SE) of Begonia species after 70 days at various temperatures; baseline germination is displayed as toggled bar(s) for comparison. Additional temperatures of 12° and 15°C are presented for B. dregei.

100 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30 % Germination % Germination 20 20 10 10 0 0 0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 1012141618202224 Days 17°C Days 20°C 100 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30 % Germination % Germination 20 20 10 10 0 0 0 2 4 6 8 1012141618202224 0 2 4 6 8 1012141618202224 Days Days 23°C 26°C 100 90 80 70 60 50 40 30 % Germination 20 10 0 0 2 4 6 8 10 12 14 16 18 20 22 24 Days 29°C

Figure 3.9. Germination curves for Begonia species incubated at five different temperatures (17-29°C) and 16 hour photoperiod. Error bars represent the standard error (SE) of the mean.

100

50 Baseline

40 15 °C

% Germination 12 °C 30

0 0 3 6 9 1215182124273033363942454851535557596163656769 Days

Fig. 3.10. Response of Begonia dregei seeds to germination at 12° and 15°C and 16h photoperiod. Baseline germination was recorded under standard conditions of 25°C and 16 hour light/ 8 hour dark.

100 90 Arabidopsis 'Col'

80 B. grandis subsp. 70 evansiana B. ulmifolia 60 50 B. fischeri 40 B. boliviensis % Germination 30 20 B. subvillosa

10 B. dregei 0 0 7 14 21 28 35 42 49 56 Days

Figure 3.11. Response of seeds to freezing. Germination curves (mean%±SE) of six species of Begonia and Arabidopsis seed under standard conditions at 25°C with 16 hour light/ 8 hour dark photoperiod following storage in the freezer (one week @ -20°C).

B. grandis subsp. evansiana B. ulmifolia

100 100

80 80

60 60

40 40 % Germination % % Germination % 20 20

0 0 BG 2 3 6 12 36 52 BG 2 3 6 12 36 52 weeks in dark weeks in dark

B. fischeri B. boliviensis

100 100 80 80 60 60 40 40 % Germination % Germination % 20 20 0 0 BG 2 3 6 12 36 52 BG 2 3 6 12 36 52 weeks in dark weeks in dark

B. subvillosa B. dregei

100 100 80 80 60 60 40 40 % Germination % % Germination % 20 20 0 0 BG 2 3 6 12 36 52 BG 2 3 6 12 36 52 weeks in dark weeks in dark

Figure 3.12. Response of imbibed seeds to incubation in the dark. Maximum germination (mean%±SE) after six weeks under standard conditions at 25°C for 16 hour light/ 8 hour dark photoperiod following incubation in the dark for up to 52 weeks; baseline germination (BG) is included as toggled bars for comparison. 94

Arabidopsis %G SE(±) T50 Arabidopsis *24@41 96.5 3.19 3 C 100 *48@41 93.5 0.50 3 *72@41 51 6.03 12 80 24@41 90 6.03 3 48@41 56 3.01 10 60 72@41 16 3.01 nc 40 96@41 6 1.00 nc % Germination 120@41 0 0 nc 20

0 B *24 *48 *72 24 48 72 96 120 A treatment

effect of 120 hour exposure 100

90 D

70 *24h *48h 60 *72h 50 24h 40 48h % Germination 30 72h

20 96h 120h 10

0 0 3 6 9 12 15 18 21 24 Days

Figure 3.13. Response of Arabidopsis thaliana ecotype Columbia (‘Col’) to controlled deterioration at 100%RH and 41°C. All germination tests were done under standard conditions of 25°C for 16 hour light/ 8 hour dark photoperiod following controlled deterioration. The asterisk (*) identifies treatments where seed was pre-incubated at 100%RH at 20°C prior to exposure to high temperature. In the other treatments seeds were placed directly at high temperature/humidity. A. Fungal growth on seed after 120 hour controlled deterioration. B. Final germination (mean%±SE) and T50 values for each treatment; longer duration of treatments led to germination that was so low that T50 could not be calculated (nc). C. Bar graph representation of final germination mean%±SE. D. Germination curves (mean%±SE) for each treatment. 95

B. grandis subsp. evansiana B. ulmifolia 100 100

80 80

60 60

40 40

%Germination 20 %Germination 20

0 0 *24*48*72 24 48 72 96 120 *24*48*72 24 48 72 96 120 treatment hours treatment hours

B. fischeri B. boliviensis 100 100

80 80

60 60

40 40

%Germination 20 %Germination 20

0 0 *24*48*72 24 48 72 96 120 *24*48*72 24 48 72 96 120 treatment hours treatment hours

B. subvillosa B. dregei

100 100

80 80

60 60

40 40 %Germination %Germination 20 20

0 0 *24*48*72 24 48 72 96 120 *24*48*72 24 48 72 96 120 treatment hours treatment hours

Figure 3.14. Final (maximum) germination (mean%±SE) for each Begonia species obtained after controlled deterioration at 100%RH and 41°C. All germination tests were done under standard conditions of 25°C for 16 hour light/ 8 hour dark photoperiod. The asterisk (*) and hatched bars identify treatments where seed was pre-incubated at 100%RH for 24 hours at 20°C prior to exposure to high temperature/humidity.

B. grandis subsp. evansiana B. ulmifolia 100 100

80 80

60 60

40 40

% Germination 20 % Germination 20

0 0 0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24 Days Days

B. fischeri B. boliviensis 100 100

80 80

60 60

40 40

% Germination 20 % Germination 20

0 0 0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24 Days Days

B. subvillosa B. dregei 100 100

80 80

60 60

40 40

% Germination 20 % Germination 20

0 0 0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24 Days Days

Figure 3.15. Comparison of germination curves based on mean%±SE for seeds that had been pre-equilibrated at high humidity for 24 hours at 20°C (*) with those that were incubated directly at 100%RH and 41°C and maintained in the controlled deterioration conditions for 24 hours. 97

B. grandis subsp. evansiana B. ulmifolia 100 100

80 80

60 60

40 40

% Germination 20 % Germination 20

0 0 0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24 Days Days

B. fischeri B. boliviensis 'Bonfire 100 100

80 80

60 60

40 40

% Germination 20 % Germination 20

0 0 0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24 Days Days

B. subvillosa B.dregei 100 100

80 80

60 60

40 40

% Germination 20 % Germination 20

0 0 0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24 Days Days

Figure 3.16. Comparison of germination curves based on mean%±SE for seeds that had been pre-equilibrated at high humidity for 24 hours at 20°C (*) with those that were incubated directly at 100%RH and 41°C and maintained in the controlled deterioration conditions for 48 hours. 98

B. grandis subsp. evansiana B. ulmifolia 100 100

80 80

60 60

40 40

% Germination 20 % Germination 20

0 0 0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24 Days Days

B. fischeri B. boliviensis 100 100

80 80

60 60

40 40

% Germination 20 % Germination 20

0 0 0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24 Days Days

B. subvillosa B. dregei 100 100

80 80

60 60

40 40

% Germination 20 % Germination 20

0 0 0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24 Days Days

Figure 3.17. Comparison of germination curves based on mean%±SE for seeds that had been pre-equilibrated at high humidity for 24 hours at 20°C (*) with those that were incubated directly at 100%RH and 41°C and maintained in the controlled deterioration conditions for 72 hours.

100 90 80 70 60 50 40 % Germination 30 20 10 0 0 3 6 9 12 15 18 21 24 Days

B. subvillosa B. ulmifolia B. dregei B. fischeri B. grandis subsp. evansiana B. boliviensis Arabidopsis 'Col'

Figure 3.18. Response of seeds to controlled deterioration conditions for 96 hours. Germination (mean%±SE) of Begonia after three weeks under standard conditions at 25°C with 16 hour light/ 8 hour dark photoperiod following direct exposure to 41°C at 100%RH for 96 hours. Arabidopsis seeds were included as a control.

100

100 90 80 70 60 50 40 % Germination 30 20 10 0 0 3 6 9 12 15 18 21 24 Days

B. subvillosa B. ulmifolia B. dregei B.fischeri B. grandis subsp. evansiana B. boliviensis Arabidopsis 'Col'

Figure 3.19. Response of seeds to controlled deterioration conditions for 120 hours. Germination (mean%±SE) of Begonia after three weeks under standard conditions at 25°C with 16 hour light/ 8 hour dark photoperiod following direct exposure to 41°C at 100%RH for 120 hours. Arabidopsis seeds were included as a control.

101

100 90 80 70 60 Baseline 50 250 ppm 40 500 ppm % Germination 30 1000 ppm 20 10 0 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 53 A Days

100 90 80 70 60 Baseline 50 250 ppm 40 500 ppm

% Germination 30 1000 ppm 20 10 0 B B. dregei

Figure 3.20. Influence of gibberellic acid (GA3) on germination of Begonia dregei. The baseline germination rate (no GA3) is compared to that of seeds that were soaked for 24 hours in three concentrations of GA3 (250 ppm, 500 ppm, 1000 ppm) then sown on 1.0% agar and incubated under standard conditions of 16 hour light/8 hour dark, 25°C; A. Germination curves for the four treatments. B. Final germination (expressed as mean%±SE) for each of the treatments.

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Ave. Countr Low Ave. Annual Begonia y of Geographical Tem High Altitude Rainfal Climate species Origin City p Temp (m) l (mm) description (°C) (°C) continental B. grandis Beijing monsoon with subsp. (Hebei 100 - cold, dry evansiana China Province) -3.7 26.2 2900 570 winters, and hot, humid summers tropical, constant high temperatures, B. ulmifolia Guyana Lethem 26.4 28.7 89 1599 pronounced dry season tropical, Rio de significant B. fischeri Brazil Janeiro 20.6 26.1 8 1278 rainfall throughout year subtropical highland, mild B. boliviensis Bolivia Vallegrande 13.8 18.8 2030 667 summers and cooler winters, in some cases, snowfall occurs tropical, Rio de significant B. subvillosa Brazil Janeiro 20.6 26.1 8 1278 rainfall throughout year warm and temperature, with significant B. dregei South Lusikisiki 13.4 20.1 550 1016 rainfall Africa throughout the year

Table 3.1. Climatic data for likely habitat where the six Begonia species tested may naturally occur; source: Kottek et al. (2006).

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Moisture Species Content%±SE B. grandis subsp. evansiana 9.8±0.17 B. ulmifolia 12.0±2.11 B. fischeri 10.9±1.24 B. boliviensis 10.8±0.66 B. subvillosa 9.53±1.24

B. dregei 12.6±0.72

Table 3.2. Measured moisture content (mean%±SE) of six different Begonia species seeds after storage at 4°C, 25%RH for 6 months. Moisture content was averaged over three replicates after seeds were oven dried at 105°C for 24 hours.

Temperature Treatment (°C) %G SE T50 25 (BG) 72 0 5 29 46.5 0.50 nc 26 49 3.01 nc 23 49 2.01 nc 20 48.5 3.51 nc 17 43.5 4.01 nc 15 66.5 0.50 45 12 92.5 1.50 50

Table 3.3. Effect of seven temperature regimes on germination (mean%±SE), and time (in days) to 50% germination (T50) for B. dregei, which could not be calculated (nc) for five temperature regimes because germination was so low. BG = baseline germination.

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Species 17°C 20°C 23°C 26°C 29°C Baseline(25°C) B. grandis subsp. evansiana 11 7 6 5 6 3

B. ulmifolia 15 10 6 5 5 2

B. fischeri 11 7 5 3 3 2

B. boliviensis 10 7 6 5 5 3

B. subvillosa 17 10 7 6 5 3

B. dregei nc nc nc nc nc 5

Table 3.4. Mean germination time (T50) values (in days) corresponding to Begonia germination tests at five temperatures (optimal and sub-optimal). Two replicates of 100 seeds each were used for the experiment. For Begonia dregei the germination from 17°C to 29°C was so low that T50 could not be calculated (nc).

Accession Fresh Aged Seed Seed B. grandis subsp. evansiana 75.8± 5.38 93±1.47 B. boliviensis 97.5±1.56 96±0.71 B. subvillosa 98.5±0.65 99±0.41

Table 3.5. Germination (mean%±SE) response of fresh and aged seed of Begonia. Fresh seed was directly sown after harvest from plants in greenhouse; aged seed had been stored in a cooler (4°C, 25%RH) for at least 6 months.

105

APPENDIX A

BEGONIA SEED CLEANING

106

Cleaning Begonia seed is a complex process because of the minute size of the

seed. Common devices used to clean seed of other species at the OPGC, such as blowers

and thrashers, are not a workable means to clean seed lots of Begonia. The following

alternative process has been developed and improved by individuals at the OPGC over the course of many years of trial and error.

First, mature Begonia fruit capsules are removed from the parent plant and placed

in empty square plastic containers (i.e. germination boxes). The containers are left open

within a constant air-flow drying rack for several weeks. This drying period may act as a

dry after ripening treatment for species with dormant seed. After the drying period,

capsules are separated one at a time over a sieve (No.45, Seedburo Equipment Company,

Chicago, IL) allowing the seed to fall through and insuring that all seed is removed from

the fruit before it is discarded. Once the capsules are fully cleaned, the seeds are

examined through a dissecting scope (Zeiss Stemi DV4) to gain an initial idea of what the

population generally looks like. Seeds that are brown/tan colored and plump are likely to

be viable and healthy, while seed which reacts rapidly to static is usually considered bad.

Depending on the appearance of the seeds, the lot is further processed on a shaker table

(Fig. A.1.A).

In order to insure that no seed remains on the shaking surface after prior use and

prevent contamination between seed lots, the shaker table is thoroughly cleaned with

repeated blasts of compressed air. The seed is gently dropped onto the vibrating plate

(Fig. A.1.B) that separates seed based on weight. The table is inclined (45° angle) and

under constant vibration (setting of 3-4.5; Fig. A.1.C). Viable seed is heavier and vibrates to the lowest collection cup. Deformed non-viable seed and debris often drifts to the

107

upper two collection cups. The seed that remains on the table and does not move into

cups on their own are coaxed with a fine paintbrush. A sample of the seed that has fallen

into each collection cup is then examined with X-rays (Model MX-20-DCA2; Faxitron

X-ray Corporation, Lincolnshire, Illinois) to check for the presence of an embryo (Fig.

A.1.D). Filled seeds revealing a fully developed embryo are considered viable seed; non-

viable seeds are empty (Fig. A.2). The X-ray imaging also allows the technician to assess

whether the incline and/or vibration setting on the shaker table need to be modified in

order to differentiate good from bad seed depending on observed good/bad seed ratios

within each collection cup (Fig. A.1.A). If necessary, the seed is put back onto the shaker

table, with adjusted settings, and collection cups are again examined by X-ray images.

Depending on sample size, seeds that vibrate into the middle and highest collection cup

are examined by x-ray in order to harvest and bulk as many good seed as possible.

When seed is fully processed (Fig. A.1.E, F), the population is carefully placed in

a plastic test tube, then in a manila envelope where all accession information is written.

Thereafter, seed is stored at 4°C, 25%RH until viability testing is performed to establish a

germination rate. Otherwise, seed is maintained in the seed cooler for periods of 5-10 years before germination is checked for possibilities of regeneration of seed lots.

108

A B Shaker table, lower cup (yellow tape) is Seed is slowly poured onto top platform of expected to fill with full embryonic seed, due table; vibration and angle settings determine to weight the rate at which seed is separated

C D 3.5 setting of vibration is a practical starting Typical appearance of first run from shaker point; modifications depend on seed lots, i.e. table, both viable and non-viable seed different Begonia species present in equal amount

E F Processed seed lot Close-up of processed seed lot

Figure A.1. Tools used for cleaning Begonia seed at the Ornamental Plant Germplasm Center; A. Shaker table B. Transfer of seed to vibrating surface. C. Control for shaker table settings. D. Monitor displaying X-ray imaging E. Cleaned seed lot with minimal debris F. X-ray of processed seed lot.

109

Figure A.2. X-ray image of a cleaned seed lot of Begonia: arrows indicate empty shells (no embryo).

110

APPENDIX B

BEGONIA SEED PRODUCTION

111

Within gene/seed banks such as the Ornamental Plant Germplasm Center

(OPGC), and organizations such as the American Begonia Society (ABS); members are

interested and invested in the long-term maintenance of species, especially in the form of

seed. Not only are Begonia species diverse, but many in cultivation are either no longer

present or very rare in their native environments because of deforestation, natural

disasters, etc. (Ludwig, 2001). In order to maintain these valuable species, generation of

seed requires effective record keeping and a plan or protocol that all individuals involved

can follow in a routine and repeatable manner.

The OPGC is located in Columbus, Ohio, and therefore all seed generation on

Begonia occurs within environmentally controlled greenhouse compartments. Over the

past several years it has been observed that seed set is negligible without induced pollinations, either by hand or by greenhouse pests that may act as pollinators.

Therefore, the first step in generating seed on Begonia is to be familiar with the flower structure (Fig. B.1.A, B), as the female and male flowers are separately borne for all

Begonia, both dioecious and monoecious types. Male flowers typically have four tepals and a cluster of multiple stamens in the center. Female flowers appear similar to male,

but possess an inferior ovary, which contains ovules (undeveloped seed), that need pollen

in order to be fertilized. The pollen adheres to the “sticky” stigmatic surface of the

female.

Once familiar with flower morphology, pollination can be achieved by transfer of

pollen to the stigmatic surface. Individual male flowers can be removed, and the anthers

brushed upon the stigma of the pistillate flowers to release pollen onto the stigmatic

surface. One male flower contains enough pollen to pollinate 3-5 female flowers. After

112

transfer of pollen, females are labeled with the appropriate cross (parents), or selfing information, time, and date of pollination. If pollinations and fertilization are successful

(Fig. B.1.C), fruits develop and reach maturity within one to two months, depending on species, and are deemed so when the fruit dries. When this occurs, fruits are removed and placed in an empty germination box, where they are left to air dry in a constant flow rack for a period of two to four weeks. Following this dry after ripening treatment, seed is cleaned as explained in Appendix A (Fig. B.1.D).

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A B

Typical male Begonia flower Typical female Begonia flower C D

Developing fruit, approximately three Harvested seed, set in drying rack to weeks after pollination remove all extra moisture

Figure B.1. Examples of A. typical male flowers, B. female flowers, C. maturing fruit following hand pollination, and D. seed that has been in constant air flow cabinet for several weeks.

114

APPENDIX C

BEGONIA GERMPLASM COLLECTION AT THE OPGC

115

The list below provides an inventory of both clonal (CT) and seed populations (SD) of Begonia at the OPGC. Clonal material is maintained in duplicate while seed counts may vary by accession; where (1) appears under SD form, packet of seed has not been quantified.

ID Nr. TAXON CT SD Grif 13914 Begonia grandis subsp. sinensis 0 666 Grif 13917 Begonia grandis subsp. sinensis 0 3606787 NSSL 30737 Begonia cucullata var. cucullata 0 9900 OPGC 422 Begonia heracleifolia 2 753296 OPGC 423 Begonia convolvulacea 2 0 OPGC 426 Begonia echinosepala var. elongatifolia 2 90034 OPGC 427 Begonia nelumbiifolia 2 4608 OPGC 428 Begonia dichotoma 2 805 OPGC 429 Begonia thelmae 2 1 OPGC 433 Begonia friburgensis 2 0 OPGC 434 Begonia fernandoi-costae 2 0 OPGC 438 Begonia sanguinea 2 10946 OPGC 446 Begonia carolineifolia 2 1 OPGC 447 Begonia thiemei 2 295693 OPGC 711 Begonia dichroa 2 101542 OPGC 712 Begonia holtonis 2 334161 OPGC 714 Begonia herbacea 2 549 OPGC 715 Begonia plebeja 2 10503 OPGC 720 Begonia tayabensis 2 12193 OPGC 721 Begonia sizemoreae 2 12104 OPGC 844 Begonia ulmifolia 2 1942 OPGC 847 Begonia integerrima 2 305 OPGC 851 Begonia foliosa var. miniata 2 0 OPGC 878 Begonia imperialis 2 0 OPGC 884 Begonia nelumbiifolia 2 0 OPGC 896 Begonia hydrocotylifolia 2 93176 OPGC 910 Begonia acetosa 2 0 OPGC 914 Begonia masoniana 2 7800 OPGC 921 Begonia convolvulacea 2 0 OPGC 936 Begonia brevirimosa 2 0 OPGC 1029 Begonia hatacoa var. hatacoa 2 1 OPGC 1031 Begonia listada 2 0 Continued 116

Appendix C continued OPGC 1032 Begonia deliciosa 2 1 OPGC 1033 Begonia dregei 2 105102 OPGC 1035 Begonia bowerae 2 106676 OPGC 1036 Begonia manicata 2 5662 OPGC 1039 Begonia wollnyi 2 288740 OPGC 1040 Begonia soli-mutata 2 4187 OPGC 1041 Begonia hatacoa var. silver 2 0 OPGC 1708 Begonia x angularis 2 0 OPGC 1710 Begonia eminii 2 0 OPGC 1712 Begonia juliana 2 17670 OPGC 1714 Begonia popenoei 2 0 OPGC 1715 Begonia robusta var. robusta 2 0 OPGC 1717 Begonia ulmifolia 2 3050193 OPGC 1734 Begonia dregei 2 124519 OPGC 1736 Begonia johnstonii 2 8035 OPGC 1737 Begonia glabra 2 136617 OPGC 1738 Begonia tenuifolia 2 0 OPGC 1739 Begonia strigillosa 2 0 OPGC 1740 Begonia tayabensis 2 50063 OPGC 1742 Begonia sericoneura 2 21796 OPGC 1743 Begonia x angularis 2 0 OPGC 1778 Begonia foliosa 2 169 OPGC 1779 Begonia nelumbiifolia 2 11425 OPGC 1783 Begonia hirtella 2 1 OPGC 2733 Begonia roxburghii 2 1 OPGC 2734 Begonia aconitifolia 2 0 OPGC 2735 Begonia dregei 'Glasgow' 2 399683 OPGC 2736 Begonia mazae 2 0 OPGC 2737 Begonia fischeri 1 1552562 OPGC 2739 Begonia nelumbiifolia 2 0 OPGC 2740 Begonia hatacoa var. viridifolia 2 0 OPGC 2743 Begonia descoleana 2 382465 OPGC 2744 Begonia coccinea 2 7566 OPGC 2744 Begonia coccinea 2 12466 OPGC 2745 Begonia coccinea 2 38994 OPGC 2746 Begonia odorata 2 41392 OPGC 2758 Begonia grandis subsp. evansiana 2 97244 OPGC 2759 Begonia grandis 2 63666 Continued 117

Appendix C continued OPGC 2760 Begonia grandis subsp. sinensis 2 4571 OPGC 2761 Begonia boliviensis 'Bonfire' 2 98787 OPGC 2762 Begonia grandis 2 250 OPGC 3143 Begonia grandis 2 0 OPGC 3144 Begonia heracleifolia 2 61327 OPGC 3158 Begonia sutherlandii 2 0 OPGC 3547 Begonia microsperma 1 0 OPGC 3548 Begonia prismatocarpa 1 0 OPGC 3549 Begonia prismatocarpa 1 0 OPGC 3550 Begonia quadrialata 1 0 OPGC 3551 Begonia quadrialata subsp. nimbaensis 1 0 OPGC 3552 Begonia scapigera 1 0 OPGC 3553 Begonia scutifolia 1 0 OPGC 3556 Begonia staudtii 1 0 OPGC 3558 Begonia obliqua 'White' 1 0 OPGC 3561 Begonia schmidtiana 'Chauncy' 2 358999 OPGC 3562 Begonia subvillosa 2 117683 OPGC 3564 Begonia egregia 2 0 OPGC 3650 Begonia paleata 1 4000 OPGC 3651 Begonia foliosa var. miniata 1 9256 OPGC 3652 Begonia foliosa var. miniata 1 0 OPGC 3653 Begonia foliosa var. miniata 1 0 OPGC 3654 Begonia luxurians 1 0 OPGC 3655 Begonia foliosa 1 0 OPGC 3656 Begonia hispida var. cucullifera 1 0 OPGC 3657 Begonia foliosa var. miniata 1 0 OPGC 3658 Begonia diadema 1 0 OPGC 3659 Begonia fernandoi-costae 2 1 OPGC 3660 Begonia luxurians 2 1 OPGC 3661 Begonia bipinnatifida 1 0 OPGC 3662 Begonia conchifolia 1 0 OPGC 3663 Begonia cubensis 1 0 OPGC 3664 Begonia acetosa 1 0 OPGC 3666 Begonia griffithiana 2 1 OPGC 3667 Begonia barkeri 6 0 OPGC 3668 Begonia oaxacana 1 1 OPGC 3669 Begonia palmata 1 0 OPGC 3671 Begonia acetosella 12 0 Continued 118

Appendix C continued OPGC 3673 Begonia burkillii 7 0 OPGC 3675 Begonia rex 2 1 OPGC 3676 Begonia polygonoides 1 0 OPGC 3677 Begonia turrialbae 1 0 OPGC 3678 Begonia tayabensis 1 14483 OPGC 3679 Begonia crassicaulis 1 0 OPGC 3680 Begonia roxburghii 1 0 OPGC 3681 Begonia lancangensis 1 0 OPGC 4158 Begonia rubrifolia 2 5487 OPGC 4159 Begonia wallichiana 0 1 OPGC 4160 Begonia cucullata 0 1 OPGC 4161 Begonia cucullata 0 1 OPGC 4161 Begonia cucullata 0 1 OPGC 4162 Begonia cucullata 0 1 OPGC 4163 Begonia cucullata 0 1 OPGC 4164 Begonia cucullata 0 564 OPGC 4165 Begonia cucullata 0 3510 OPGC 4166 Begonia cucullata 0 1 OPGC 4166 Begonia cucullata 0 1 OPGC 4167 Begonia cucullata 0 1 OPGC 4168 Begonia cucullata 0 1 OPGC 4168 Begonia cucullata 0 14523 OPGC 4169 Begonia cucullata 0 4138 OPGC 4169 Begonia cucullata 0 13613 OPGC 4170 Begonia cucullata 0 10833 OPGC 4171 Begonia grandis subsp. evansiana 0 15650 OPGC 4172 Begonia emeiensis 1 0 OPGC 4173 Begonia grandis 1 0 PI 292731 Begonia cucullata var. cucullata 2 0 PI 667309 Begonia cucullata var. cucullata 2 119375 PI 667310 Begonia albopicta 2 19396 PI 667311 Begonia subvillosa 'Teddy Bear' 2 894499 PI 667312 Begonia kellermanii 2 231059 PI 667313 Begonia venosa 2 38945 PI 667318 Begonia hydrocotylifolia 2 140460 PI 667319 Begonia coriacea 2 315 PI 667320 Begonia dipetala 2 289934 PI 667372 Begonia cardiocarpa 2 358813 Continued 119

Appendix C continued OPGC 845 Begonia 'Castaway' 1 0 OPGC 848 Begonia 'Richmondensis' 1 0 OPGC 853 Begonia 'Alba' 1 0 OPGC 856 Begonia 'Concord' 1 0 OPGC 857 Begonia 'Pinafore' 1 0 OPGC 859 Begonia 'Snow Capped' 1 0 OPGC 864 Begonia 'Withlacoochie' 1 0 OPGC 865 Begonia 'Exotica' 1 0 OPGC 866 Begonia 'Eldora' 1 0 OPGC 868 Begonia 'Nokomis' 1 0 OPGC 890 Begonia 'Jim Wyrtzen' 1 0 OPGC 899 Begonia 'Little Brother Montgomery' 1 0 OPGC 904 Begonia 'Morocco' 1 0 OPGC 906 Begonia 'Lopse II' 1 0 OPGC 915 Begonia 'Cowardly Lion' 1 0 OPGC 923 Begonia 'Art Hodes' 1 11258 OPGC 1780 Begonia 'Riehii' 0 525 OPGC 3554 Begonia 'Gold Coast' 1 0 OPGC 3555 Begonia 'Buttercup' 1 0 OPGC 3563 Begonia 'Florida' 1 0 OPGC 3565 Begonia 'Looking Glass' 1 0 OPGC 3665 Begonia 'El Valle' 1 0 OPGC 905 Begonia 'Streaky Jeans' 2 29350 OPGC 893 Begonia spp. 1 0 OPGC 3649 Begonia spp. 2 0 PI 667280 Begonia spp. 2 953698 PI 667325 Begonia spp. 2 336023

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APPENDIX D

TETRAZOLIUM TESTING

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Tetrazolium

If dormancy is present in Begonia seeds it is necessary to demonstrate that viable

seed fail to germinate when given ideal conditions. A standard protocol for such a

viability test is the Tetrazolium Test (Peters, 2000) although this is not routinely applied

to Begonia. One of the challenges is that the test requires that the interior of the seed be

exposed to the tetrazolium (TZ) solution. However, such manipulation is impractical with the tiny begonia seeds, but because the seed coat of begonias is relatively translucent, it may be possible to observe staining through it, if the TZ can diffuse through the seed coat. However, preliminary observations showed that direct exposure of seeds to TZ solution did not yield any color reaction, suggesting that the compound could not penetrate the seed coat. A similar observation had been made with Arabidopsis seeds, but prior treatment of seeds with sodium hypochlorite apparently permitted the TZ to diffuse and react with living tissue (Tesnier et al., 2002). Therefore an assessment was made of this approach with Begonia seeds.

Material and methods

Initially, 100 seeds of each of six Begonia species were soaked for 10 minutes in

10% sodium hypochlorite. This treatment cleared the testa cell that allowed for the TZ staining to be taken up by the living tissue of the seed embryo. The samples were then triple rinsed with ddH2O, decanted to remove remaining sodium hypochlorite, followed

by the addition of 0.1 % tetrazolium chloride solution. The samples were wrapped in

aluminum foil to exclude light and placed in an incubator at 30°C for 24 hours. Seeds

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were subsequently examined with a dissecting microscope (Zeiss Stemi DV4) and scored as viable if any staining had occurred, according to ISTA recommendations (Moore,

1985; Tesnier et al., 2002).

Results & discussion

Tetrazolium staining was observed in all six Begonia species (Fig. D.1) and compared to baseline germination (BG) obtained under standard conditions (Chapter 2,

Table D.1). Tetrazolium staining and germinated rates were comparable; however, this experiment was carried out to determine if a simple staining procedure was possible and was not repeated. It appears that TZ staining of begonia seed is possible without having to physically remove the seed coat. The accuracy and validity of this method needs to be determined by repeated application with seed lots of known differences in germination either from natural variation or from mixtures of live and killed seed. A challenge of any

TZ test is the need for interpretation of the coloring pattern on the embryo. The mere presence of color may not be reliably indicative of a viable, germinable seed. However, with the very small seed of Begonia, it may be possible to correlate intensity of color with subsequent germination. It is obvious that additional work is needed on this method, but the preliminary results of TZ staining after exposure of the seeds to sodium hypochlorite holds promise as an approach that may help dormancy studies in this genus.

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Figure D.1. Tetrazolium staining of Begonia seeds. Red-stained seed are considered viable or metabolically active, and therefore capable of germination.

% Colored %Germination Date Accession (TZ) (light 25°C)

1/8/2013 B. boliviensis 87 97.5 1/8/2013 B. dregei 70 72 8/8/2013 B. fischeri 92 96 2/8/2013 B. grandis subsp. evansiana 79 91.5 8/8/2013 B. subvillosa 93 98.5

1/8/2013 B. ulmifolia 75 94

Table D.1. TZ % staining of seed lots of six species of Begonia compared to the germination rate obtained for the same seed lot.

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APPENDIX E

THERMAL GRADIENT TEMPERATURE LOG

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Hobo Recorder Expected Temp. Actual Temp. Reported Temp. 1.1N 13.5 17.42 17 1.2N 15.3 18.65 1.3N 16.8 19.91 20 1.4N 18.4 21.56 2.1N 19.9 22.73 23 2.2N 21.3 26.45 2.3N 22.7 25.09 2.4N 24.3 26.33 26 3.1N 26.4 27.60 3.2N 29.6 28.82 29 4.1S 13.5 17.23 17 4.2S 15.3 18.27 4.3S 16.8 20.06 20 4.4S 18.4 21.51 5.1S 19.9 22.8 23 5.2S 21.3 35.19 5.3S 22.7 24.22 5.4S 24.3 26.30 26 6.1S 26.4 27.80 6.2S 29.6 29.19 29

Table E.1. Record of the actual temperatures (°C) obtained on the thermal gradient table used in the experiments. Temperature was recorded by HOBO® Data Loggers over 72 hours and the results averaged to give the actual temperature at the site. Data loggers were placed on two halves of the rectangular table, northern (N) and southern (S), to determine if the gradient was consistent throughout the surface.

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APPENDIX F

GERMINATION OF BEGONIA ROXBURGHII SEED

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The six species of Begonia used in this research (Fig. 3.1) produce winged fruits that become dry and papery upon maturity. Another species in the OPGC collection, B. roxburghii, yields fruits that are berries which do not become dry upon maturity, and remain on the mother plant for up to six months (unpublished data). I wanted to see if seeds of such different fruit germinated and developed like the other species. Preliminary observations suggested that seeds removed from the fruit would not germinate and that only when fruits were placed on greenhouse growing media would seedlings develop.

This kind of response suggests the seeds may be recalcitrant and that processing them like seeds of the other species would be problematic. To examine this in more detail,

Begonia roxburghii seed that had been removed from the mature fruits and stored at 4°C,

25%RH as well as seed that was directly removed from moist fruit was sown under standard conditions described in Chapter 2, and sown to test for germination.

Germination of fresh seed, directly removed from moist fruit, and incubated on

1% agar plates for thirty days, reached 79%±0.5, whereas seed that had been stored in the cooler (4°C; 25%RH) for six months germinated at only 14.3%±1.38, a difference of approximately 65% (Fig F.1). This single experiment suggests that seed viability declines rapidly in storage under conditions that lead to desiccation (25%RH). When mature fruits were opened and placed in a Petri dish containing 1% agar and incubated at 25°C and 16h photoperiod (standard germination conditions), seeds were able to germinate readily (Fig.

F.2), indicating the presence of sufficient moisture within the fruit walls to permit germination.

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These initial experiments indicate that fruit type may be an important determinant

of seed characteristics in Begonia. The possibility that fleshy fruited species may have desiccation intolerant seed has important implications for the management of such germplasm within the OPGC. Therefore, additional studies are needed to more thoroughly examine the characteristics of seeds from species with different fruit structure.

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100 90 B. roxburghii 80 70 60 50 Aged 40 Fresh % Germination 30 20 10 0 0 7 14 21 30 Days

Figure F.1. Germination of Begonia roxburghii seed that was either freshly harvested or ‘aged’ (stored at 4°C; 25%RH for six months). Standard germination conditions of 25°C and 16 hour photoperiod were used.

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Figure F.2. Germinated seeds and seedlings on excised fruit of the fleshy berries of Begonia roxburghii. The fruit was harvested three months after hand pollination, sectioned, and placed on 1.0% agar.

131

APPENDIX G

ADDITIONAL NOTES AND COMMENTS ON THE RESPONSE OF INDIVIDUAL SPECIES TO CONTROLLED DETERIORATION, TEMPERATURE, AND DARK TREATMENTS

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Begonia grandis subsp. evansiana

After dark treatments, germination did not begin (lag phase) until at least 7 days

after the foil was completely removed. A large peak in germination was noticeable 14

days after seeds were first exposed to light for the initial check. As B. grandis subsp.

evansiana seeds spent more weeks in the dark, it was noticeable that the seeds were not

germinating in comparison to the baseline rate (Fig.3.12). The 36 week dark treatment had 48% germination, which began 14 days after the initial “flash”, although germination was not high, this percentage is reflective of the potential secondary dormancy that is induced by the dark treatment. These replicates were also placed in the cooler for stratification to increase the maximum germination rate. As plants set seed before the

onset of winter in nature, seeds will likely be distributed by wind and water and will

likely not germinate unless there is an opening in the forest canopy, light (irradiation

requirement unknown; at least 50 µmol·m-2·s-1); or if a natural disturbance were to occur,

leading to increased sunlight, where seed will sense the change in light and temperature,

leading to new plants colonizing an area. Nagao at el. (1959) found that seed of B. grandis subsp. evansiana respond to long day treatments. Extended dark periods (2-52 weeks) without light appear to put seeds into a state of dormancy to prevent germination until, long photoperiods are again present, following winter (Yanes-Vazquez and Orozco-

Segovia, 1990).

In terms of temperature, it is likely that B. grandis subsp. evansiana will not germinate in nature until after the ground has thawed and a proper environment is available to support seedling establishment, at least 17°C, similar to temperatures in its

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natural habitat (Table 3.1). Light is also necessary. Lower temperature adds an additional

four days to reach T50 without any decrease in overall germination percentage.

Begonia grandis subsp. evansiana is considered not tolerant to stress. Moisture content of seeds based on differences from pre-incubation versus direct oven placement in controlled deterioration tests appeared not to affect the decay rate from stress (CD) treatments.

Begonia ulmifolia

Begonia ulmifolia likely germinates freely throughout the year in nature as long as sufficient moisture exists, and light is present. Germination appears to be enhanced with higher temperatures, and delayed by lower temperatures (Fig. 3.8, 3.9).

Begonia ulmifolia is known to be a weedy species, likely taking advantage of openings in the forest floor or in newly disturbed areas. The stress factor of temperature led to the discovery that this species was quite vigorous, germinating at 85.5%±0.50 after

120 hours at 41°C; 100% RH (Fig. 3.19). This is supportive of the fact that B. ulmifolia is weedy in nature, and has a wider distribution than typical Begonia species. Therefore, there was an attempt to stress the seed further, by treating the seed to 50°C for 48 hours.

No seed germinated after this final treatment. More testing would need to be conducted at temperatures between 41-50°C at various lengths of time to determine the point at which

B. ulmifolia decays rapidly. Time in controlled deterioration treatments had no effect on

T50.

Within the CD treatments it is likely that when seed is pre-incubated, moisture is taken up and therefore is more stress tolerant than seed directly exposed to high temperature (41°C) at 100%RH. Furthermore, this tolerance to stress in a more hydrated

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state, could possibly explain longevity in nature on terrestrial moist terrain, where the

native habitat for many Begonia species may be present. Seeds which require light for

germination, such as Begonia, can form soil seed banks in nature which can in turn

germinate at a subsequent stage, after soil disturbance, canopy opening(s), etc. (Grime et

al., 1981; Koutsovoulou et al., 2014). Species which form long-lived seed banks in

temperate forests are commonly colonizers of newly disturbed areas (Pickett and

McDonnell, 1989; Whigham et al., 2006), and for Begonia, likely are in an imbibed state,

possibly affecting ABA/GA regulation within the seed, and therefore effecting overall

germinability of seeds.

Begonia fischeri

For germination following dark treatments it appears that there is no dormancy within B. fischeri; seed is expected to sit for long intervals of time, imbibed, and in complete darkness (at least 52 weeks) with no ill effects of viability (Fig. 3.12).

Generally speaking, B. fischeri is a very vigorous plant, at least in terms of seedling growth and lack of phenotypic response to changes in temperature or light, useful traits for improved germination in domestication and hybridization schemes.

Begonia fischeri is suspected of apomixes (seed set occurs without pollination), therefore it would be predicted that the population is homogenous, and consequently lacking genetic variation to adapt to environmental shifts, specifically stress tolerance. Begonia fischeri is also weedy in nature, has a larger than typical distribution and therefore the observed vigor may play a role in its competiveness and ability to occupy open disturbed habitats as they become available. The additional treatment of 50°C for 48 hours caused total necrosis of the seed population; similarly to B. ulmifolia, further determination of

135

the temperature and exposure time will need to be fine-tuned to see the “tipping point” for decay.

Begonia boliviensis

B. boliviensis germinated to similar percentages at all temperatures examined. It does not appear that there is dormancy within B. boliviensis; seed is expected to sit for long intervals of time imbibed and in complete darkness (at least 52 weeks) with no ill effects of viability (Table 3.12).

Seeds of B. boliviensis overall showed a decrease in %G with increasing intervals of high temperature and humidity. The decrease in vigor with the longer duration of stress correlates with the observed decay of Hordeum and Arabidopsis (Hampton and

TeKrony, 1995; Tesnier et al., 2002). Pre-incubation of B. boliviensis during CD treatments, led to a much higher germination (Fig. 3.15-.17), likely due to the fact that in nature, seeds are in a constant, moist, state (Table 3.1).

Begonia subvillosa

It appears that there is no dormancy within B. subvillosa; seed can sit for long intervals of time imbibed and in complete darkness (at least 52 weeks) with no ill effects on viability (Table 3.12). Germination is relatively high under all temperature regimes applied during the thermal gradient experiment. Even so, increased duration under high humidity and elevated temperature led to decrease in germination of B. subvillosa, but pre-incubation (Fig. 3.15-.17) led to higher %G, suggesting that seed is in a moist state in its native habitat (Table 3.1).

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Begonia dregei

Colder temperatures, such as 12°C and 15°C, may have acted as a form of stratification, and therefore broke dormancy in B. dregei, with dark treatments acting in the same way, which is with an increase of germination after such treatments. In fact,

Tesnier et al. (2002) reported that temperatures of 10 and 15°C mimicked partial stratification, resulting in increased germination of Arabidopsis. Even though time to T50 was extended, after 52 weeks of no light exposure under a completely humid environment (100% RH); the dark treatments lead to a higher %G than what is observed for the baseline rate (no dark treatment, etc.). In nature, seed is likely formed towards the end of the warm season, then thereafter seed is shed and sits in a seed bank until cold moist conditions have broken dormancy. Seed of B. dregei can sit for a period of up to

52 weeks in the dark, then germinate close to 100% if given light and constant moisture.

In terms of tolerance to elevated temperatures and relative humidity (i.e. controlled deterioration), B. dregei would be classified as medium tolerant to the stress test that was applied (Fig. 3.14). The rate at which B. dregei germinates is much lower, in all treatments, in comparison to the other begonia being tested. Pre-incubation had no effect on B. dregei suggesting that seed is not in a saturated environment, at least not consistently. Similar results from CD treatments suggest that B. grandis subsp. evansiana seeds persist in similar, non-saturated environments.

Wet season rains may assist in transporting seed to favorable environment(s). It is possible that if Begonia were to sit in some type of seed bank (Baskin and Baskin, 1998) for extended periods of time, there may be an effect of enzymatic synchronization occurring within the seed embryo before germination can occur (Fenner and Thompson,

137

2005). Seeing as dark treatments increased %G for B. dregei, it suggests possible

dormancy, likely non-deep physiological (PD) dormancy that can at least be broken by

extended periods of moist incubations of 2-52 weeks in the dark (Baskin and Baskin,

2004). Over time in its natural habitat, B. dregei may have selected for dormancy upon

maturity to increase the chance that seeds germinate when the seasonal rains return, along

with higher temperatures and therefore conditions are more favorable for germination.

Preliminary data suggests that exogenous GA3 increases the rate at which seed lots of B.

dregei reaches maximum germination (Fig 3.20). Increases in germination percent and

GA3 concentration are parallel. Hu et al. have shown that dormancy (non-deep PD) of two species from monsoon temperate forests of China is improved with application of

GA3. Gibberellic acid treatments have shown to alleviate dormancy; yet exogenous

application of GA3 had no effect on germination of B. grandis subsp. evansiana, during

dark treatments (Nagao et al., 1959).

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