UNIVERSITY OF HAWAI'I LIBRARY IN VITRO PROPAGATION OF BREADFRUIT [ARTOCARPUS ALTILIS (pARKINSON) FOSBERG] FOR GERMPLASM CONSERVATION AND EXCHANGE A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI'I IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN HORTICULTURE (ECOLOGY, EVOLUTION AND CONSERVATION BIOLOGY) AUGUST 2003 By Cynthia S. Nazario Thesis Committee: Robert Paull, Chairperson Diane Ragone Yoneo Sagawa DEDICATION This thesis is dedicated to my parents who have always encouraged me to pursue my dreams. For my Mother who gently reminds me to go within and follow my intuition and for my Father who gave me the gift ofreason and wisdom, I am eternally grateful. III ACKNOWLEDGMENTS National Tropical Botanical Garden James Leary IV TABLE OF CONTENTS Acknowledgements iv List ofTables xi L1st' 0 fF'Igures xu" Chapter 1: Breadfruit l Taxonomy 1 Botanical Description 2 Ecology , 4 Origin and Distribution 5 Uses ofBreadfruit.. 8 Food 8 Wood 8 Fiber 9 Leaves 9 Latex 10 Medicinal. 10 Nutritional Composition 11 Propagation 11 Agronomy 13 Pest and Diseases 14 Production 15 Breeding 16 Genetic Resources 17 v Conservation 19 Chapter 2: In Vitro Propagation 28 Definition 28 History '" '" '" 29 General 29 Woody Plants 31 Breadfruit and Related Species , 32 Applications '" 33 Micropropagation 34 Benefits and Constraints 34 Culture Medium '" 36 Inorganic Salts '" 36 Micronutrients 38 Vitamins '" '" 39 Hexitols 39 Plant Growth Regulators 40 Auxins '" '" 40 Cytokinins 41 Gibberellins '" .42 Abscisic Acid 42 Ethylene 43 Carbohydrates 43 Gelling Agents 44 Vi pH 44 Culture Vessels 45 Micropropagation Stages .46 Stage 0 - Preparation ofStock Plants 46 Stage 1 - Initiation ofExplants .48 Disinfestation .48 Explant 50 Production ofPhenolic Compounds 52 Stage 2 - Multiplication ofPropagules 57 Adventitious vs. axillary bud formation 58 Single-node culture 58 Subculture 58 Stage 3 - Elongation of Shoots and Induction ofRooting 59 Rooting Procedure (in vitro vs. ex vitro) 60 Stage 4 - Acclimatization 60 In Vitro Techniques for Germplasm Conservation 61 Genetic Stability 62 Chapter 3: In Vitro Propagation ofTwelve Pacific Island Breadfruit (Artocarpus altilis) Cultivars 70 Introduction 70 Materials and Methods 72 Explant Source 72 VB Culture Media 74 Culture Environment 74 Culture Vessels 74 Culture Room 74 Stage 0 - Preparation of Stock Plants '" 76 Stage 1 - Establishment ofAseptic Cultures 77 Disinfestation ofExplants 78 Excision ofExplants 79 Culture Initiation 79 Experiment 1 79 Experiment 2 81 Stage 2 - Multiplication and Elongation ofShoots 84 Stage 3 - Rooting 84 Experiment I 84 Experiment 2 85 Stage 4 - Acclimatization ofRegenerated Plantlets 85 Results and Discussion 87 Stage 1 - Experiment 1.. 87 Contamination 87 Mortality 89 Survival. 93 Stage 1 - Experiment 2 95 Bud Break '" 95 Vlll Multiple Bud Break 98 Stage 2 - Multiplication and Elongation ofShoots 101 Stage 3 - Rooting " 105 Experiment 1 105 Experiment 2 105 Number ofRoots 107 Length ofRoots 108 Stage 4 - Acclimatization ofRegenerated Plantlets 112 Conclusion 114 Chapter 4: Effects ofBenzyladenine on Phenolic Compound Production in Tissue Culture ofBreadfruit.. 117 Introduction 117 Materials and Methods 118 Explant Source and Sterilization 118 Culture Media 120 Culture Environment 120 Culture Vesse1s 120 Culture Room 120 Culture Initiation '" '" 120 Phenolic Compound Production 121 Results 122 Culture Initiation '" 122 Phenolic Compound Production 123 IX BA Treatment 0-0 125 BA Treatment 1-1.. 125 BA Treatment 1-12 125 BA Treatment 12-1 126 BA Treatment 12-12 126 Discussion , , 126 Phenolic Compound Production 126 Bud Break " 127 Conclusion 128 x LIST OF TABLES 1. Composition ofMineral Salt Media for Tissue Culture ofBreadfruit 75 2. Twelve Pacific Island Breadfruit Cultivars used in Tissue Culture 77 3. Distribution ofBreadfruit Explants on BA Treatments 83 Xl LIST OF FIGURES Figure 1. Breadfruit Shoot Tip 80 2. Excision ofBreadfruit Explant 80 3. Percent Contamination ofTissue Cultured Breadfruit Cultivars 88 4. Percent Mortality in 12 Tissue Cultured Breadfruit Cultivars 91 5. Percent Mortality ofTissue Cultured Breadfruit Cultivars 91 6. Contamination in Breadfruit Explant 92 7. Breadfruit Explants After One Week in Culture 92 8. Percent Survival ofTissue Cultured Breadfruit Cultivars 94 9. Percent Survival in 12 Tissue Cultured Breadfruit Cultivars 94 10. Percent Bud Break ofBreadfruit Tissue Cultured on 12 mg-l BA 97 11. Percent Bud Break ofBreadfruit Tissue Cultured on WPM 97 12. Average Number ofBud Breaks in Breadfruit Tissue Culture 100 13. Average Number ofBud Breaks in Breadfruit Cultivars 100 14. Apical Cutting ofMa'afala Breadfruit Cultivar. 102 15. Nodal Cutting ofMa'afala Breadfruit Cultivar. 102 16. Apical Cuttings ofMa'afala Breadfruit Cultivar. 104 Xli 17. Apical Cuttings ofUlu fiti Breadfruit Cultivar. 104 18. Rooted Explant on Y2 MS + 0.5 IlM IBA. 106 19. Rooted Explant on Y2 MS + 0.0 IlM IBA. 106 20. Average Number ofRoots in Breadfruit Tissue Culture 108 21. Average Length ofRoots in Breadfruit Tissue Culture 110 22. Average Length ofRoots in Breadfruit Tissue Culture in Two Replications of Y2 MS 110 23. Rooted Breadfruit Plantlet in Moistened Sphagnum 113 24. Rooted Breadfruit Plantlet in Moistened Perlite/Vermiculite Mix 113 25. Phenolic Content ofBA Treatments for the Ma'afala Breadfruit Cultivar 124 26. Number ofBud Breaks in BA Treatments for the Ma'afala Breadfruit Cultivar. 124 Xlll CHAPTER 1 BREADFRUIT [ARTOCARPUS ALTILIS (PARKINSON) FOSBERG] 1.1 Taxonomy Breadfruit (Artocarpus altiUs (Park). Fosberg) [Syn. A. communis Forst.; A. incisa L.f] is a member ofthe Moreaceae family and includes trees such as the mulberrys (Morus and Broussonetia spp.), the figs (Ficus spp.) and the rubber tree (Hevea brasiliensis). Within this family breadfruit belongs to the genus Artocarpus, which contains approximately 50 species oftrees that grow in the tropical regions ofSoutheast Asia and the Pacific Islands (Barrau, 1976; Popenoe, 1920; Purseglove, 1968). Some of its closest relatives within this genus include A. heterophyllus, jackfruit, and A. integer, champedak. Breadfruit is generally divided into two types - those with seeds and those without. Artocarpus altilis is the name generally given to seedless breadfruit types typical ofPolynesia, however, fruit ofA. altilis can also contain seeds. Artocarpus camansi Blanco, or breadnut, has fruit containing many seeds and has been distinguished as a separate species due to distinct morphological features, such as shape ofanthocarp, shape offruit base, male spike, stigma, and leaves (Coronel, 1983). There is another species ofbreadfruit found in the Pacific Islands known as A. mariannensis Trecul. Known as dugdug and chebiei in its native islands, A. mariannensis is a wild, seeded breadfruit that is morphologically distinct from A. altiUs. AFLP (Amplified Fragment Length Polymorphisms) data suggests that A. camansi and A. mariannensis are distinct species and are both involved in the evolution ofA. altilis as a 1 cultivated crop (Zerega and Motley, 2001). In Micronesia, hybridization between A. mariannensis and A. a/titis has occurred creating numerous cultivars, which share characters ofboth species (Coenan and Barrau, 1961; Ragone, 1991b, 1997). 1.2 Botanical Description Breadfruit is a large evergreen tree that can reach heights up to 25 meters. The trunk is straight and can reach widths of0.5 to 2.0+ m, with some cultivars buttressing at the base. The tree has many spreading branches with pronounced leaf and stipule scars and lenticels. Leaves cluster at the end ofthe branches and are alternately and spirally arranged. Foliage, male flowers, and fruits originate from a terminal bud at the end ofthe branches, enclosed by two large stipules, ranging in size from 15 - 30 cm. Breadfruit is monoecious, with the male inflorescence emerging first, followed by the female inflorescence. Milky latex is found in all parts ofthe plant. Leaves are dark green and often glossy on the upper side with a lighter green underside that has a raised midrib and main veins. Leaves can vary in texture, size, outline, and dissection. They are large and range in size from 22 to 90 cm long and 20 to 50 cm wide with most having a broadly obovate or ovate outline. Leaves are usually leathery and smooth but some cultivars have short, dense white hairs on the underside, giving them a Velcro-like texture. Lobing can vary among cultivars and even among leaves on the same tree. Most often leaves are dissected into 5 to 11 lobes with depth of dissection ranging from 2/3 to 4/5 the distance from the edge ofthe leafto the midrib (Ragone, 1997). 2 Roots are shallow and wide spreading, with a network ofadventitious feeding roots developing below the soil surface (Purseglove, 1968; Rowe-Dutton, 1976). According to Gunarto (1992), breadfruit trees in Cilicap, Central Java, have roots proportionally longer in length compared to the height ofthe tree, with a breadfruit tree of7 m having root lengths ofmore than 200 m. Inflorescences are axillary with the male inflorescence a club-shaped, spongy spike 12 to 45 cm long and 2.5 to 5 cm thick. It is made up ofnumerous tiny flowers that first appear as green buds, which turn yellow upon development, and fade to an orange/brown as they senesce. Female flowers, numbering 1500 to 2000, are attached to the fruit axis or core forming a rounded or elliptic and green prickly head. The fruit is known as a syncarp (Jarrett, 1976). The edible portion or bulk ofthe fruit is formed by the persistent perianth ofeach flower. The rind is thin and patterned with irregular 4­ sided to 6-sided flat, raised or conical polygons, each the surface ofan individual flower. Stigmas protrude from the center ofthese polygons and often leave a distinct black point or spine when they wither. Fruit are often smooth to the touch but some can have a sand­ papery, rough, or bumpy texture. They also vary in their shape, size, and color. Depending on the cultivar, the fruit may be oblong, ovoid, cylindrical or pearshaped. They range in size from 12 to 30 cm in diameter and 9 to 45 cm in length.