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1 THE PRODUCTION OF RED FLESHED GRAPEFRUIT/PUMMELO TETRAPLOID PARENTS TO AID THE GRAPEFRUIT IMPROVEMENT PROGRAM S By DIVYA KAINTH A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DE GREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010
2 2010 Divya Kainth
3 To my late father Dr. Satvinder Singh, my mother Mrs. Surinder Kaur and my beloved sister Kavya
4 ACKNOWLEDGMENTS I would like t o express my sincere gratitude to my advisor Dr. Jude W. Grosser, the chairperson of my graduate committee for giving me an opportunity to work with him on such an interesting project and for giving me encouragement, moral and financial support throughout my degree program. I had a great learning experience under his constant scientific guidance. I would like to extend my gratitude to Dr. Frederick G. Gmitter Dr. Jim H. Graham and Dr. Jose Chaparro for serving on my committee and for their help, academic s upport, and valuable advice and constructive criticism that helped me throughout my research ex perience. Deepest thanks to United States Department of Agriculture / Cooperative State Research, Education, and Extension Service Florida Citrus Production Rese arch Advisory Council, New Varieties Development and Management Corporation/ Florida Department of Citrus and Barney and Harriett Greene for providing me financial support through my entire program. I would like to acknowledge my lab mates including postd octoral research associates, graduate students, biological scientists and OPS employees. Many thanks to Dr. Manjul Dutt for his expert advice, Gary Barthe for providing me with all the equipments and chemicals I needed for my research. Special thanks to Mi lica Calovic who gave me training in somatic hybridization technique as well as helped me finding solutions to the research problems I faced, from her expertise and experience. I am also very thankful to my friends in my lab Monica Vasconcellos Julie Gmit ter, Jamuna, Elaine, Pamela for their support and friendship. I am thankful to Chuck and Ralph Story for taking care of my plants in the greenhouse. I want to thank Katherine Snyder and Paul Weikel for helping me with poster preparations and other technica l things. Great thanks to the entire Citrus Research and Education Center (CREC) community for
5 making me feel at home away from home. I am very grateful to my friends Harsimran gill, Gaurav Goyal, Sunehali Sharma, Amit Kohli, Omar, Tejdeep, Ekta Pathak, As hish and Jagroop Gill for their support through all thick and thin. Finally, I would like to thank my family and friends in India who supported my decision to come here and learn and their unconditional love.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW ................................ ..................... 12 History of Grapefruit ................................ ................................ ................................ 12 Grapefruit Nutrition ................................ ................................ ................................ 14 Importance of Seedless Cultivars ................................ ................................ ........... 14 Problems in the Grapefruit Market ................................ ................................ .......... 17 Introduction of Pummelos in Breeding Programs ................................ .................... 18 History of Tetraploids in Citrus ................................ ................................ ................ 20 Somatic Hybridization ................................ ................................ ....................... 20 Spontaneous Altered Ploidy ................................ ................................ ............. 23 Colchicine Induced Polyploidy ................................ ................................ .......... 26 Objectives ................................ ................................ ................................ ............... 30 2 INDUCTION OF AUTOTETRAPLOIDS IN PUMMELO (CITRUS GRANDIS) THROUGH COLCHICINE TREATMENT OF MERISTEMATICALLY ACTIVE SEEDS IN VITRO ................................ ................................ ................................ ... 32 Introduction ................................ ................................ ................................ ............. 32 Materials and Methods ................................ ................................ ............................ 34 Plant Materials ................................ ................................ ................................ .. 34 Colchicine Treatments ................................ ................................ ...................... 35 Ploidy Analysis ................................ ................................ ................................ 36 Results and Discussion ................................ ................................ ........................... 37 Conclusion ................................ ................................ ................................ .............. 42 3 SELECTION OF ALTERED PLOIDY LEVELS FROM NATURAL POPULATIONS OF MONOEMBRYONIC PUMMELOS (CITRUS GRANDIS) ....... 43 Introduction ................................ ................................ ................................ ............. 43 Materials and Methods ................................ ................................ ............................ 45 Plant Materials ................................ ................................ ................................ .. 45 Methodology ................................ ................................ ................................ ..... 45 Ploidy Analysis ................................ ................................ ................................ 47 Results and Discussion ................................ ................................ ........................... 47
7 Conclusion ................................ ................................ ................................ .............. 49 4 PRODUCTION OF COLCHICINE INDUCED AUTOTETRAPLOIDS IN PUMMELO ( CITRUS GRANDIS ) VIA INDIRECT ORGANOGENESIS .................. 50 Introduction ................................ ................................ ................................ ............. 50 Materials and Methods ................................ ................................ ............................ 52 Plant Materials ................................ ................................ ................................ .. 52 Colchicine Treatments ................................ ................................ ...................... 53 Ploidy Analysis ................................ ................................ ................................ 55 Results and Discussion ................................ ................................ ........................... 55 Conclusion ................................ ................................ ................................ .............. 62 5 COLCHICINE INDUCED POLYPLOIDY IN VIVO IN PUMMELO ( CITRUS GRANDIS L. OSBECK ) ................................ ................................ .......................... 63 Introduction ................................ ................................ ................................ ............. 63 Materials and Methods ................................ ................................ ............................ 64 Plant Materials ................................ ................................ ................................ .. 64 Materials and Methods ................................ ................................ ..................... 64 Colchicine Treatments ................................ ................................ ...................... 65 Ploidy Analysis ................................ ................................ ................................ 67 Results and Discussion ................................ ................................ ........................... 67 Conclusion ................................ ................................ ................................ .............. 71 6 SOMATIC HYBRIDIZATION OF GRAPEFRUIT + PUMMELO TO PRODUCE GRAPEFRUIT/PUMMELO T YPE ALLOTETRAPLOIDS ................................ ........ 72 Introduction ................................ ................................ ................................ ............. 72 Materials and Methods ................................ ................................ ............................ 73 Protoplast Isolation, Fusion, and Culture ................................ .......................... 73 Callus Recovery and Attempted Induction of Somatic Embryogenesis ............ 74 Modification of Protoplast Culture Conditions ................................ ................... 75 Use of in vitro plants ................................ ................................ .................. 75 Nurse culture ................................ ................................ .............................. 75 ....... 75 Results and Discussion ................................ ................................ ........................... 76 7 SUMMARY AND C ONCLUSIONS ................................ ................................ .......... 80 APPENDIX A CITRUS PROTOPLAST MEDIA AND SOLUTIONS ................................ ............... 83 LIST OF REFERENCES ................................ ................................ ............................... 88 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 98
8 LIST OF TABLES Table page 2 1 Average heights of seedlings recovered from treatments with various colch icine concentrations and exposure periods. ................................ ............... 38 2 2 Survival rate, ploidy and tetraploid induction efficiency from treatments with various colchicine concentrations and exposure periods from sel ections 5 1 99 2, UKP1 and C2 5 12 of pummelo ( Citrus grandis ) ................................ ....... 40 4 1 Effect of in vitro application of colchicine to the indirect organogenesis of seedlings from different pummelo selections and ploidy level of the regenerated shoots. ................................ ................................ ............................ 59 5 1 Number of buds sprouts from the colchicine treatment for pummelo selections 5 1 99 2, C 2 5 12 and UKP 1 ................................ .......................... 68 5 2 Percentages of cytochimeras produced from the buds treated by colchicine ..... 70 A 1 Composition of the EME medium. ................................ ................................ ...... 83 A 2 Composition of sucrose and mannitol solutions (CPW salts). ............................ 83 A 3 Composition of 0.6 m BH3 nutrient medium. ................................ ...................... 84 A 4 Composition of protoplast transformation solutions. ................................ ........... 85 A 5 Composition of DBA3 medium. ................................ ................................ ........... 86 A 6 Composition of the enz yme solution used for citrus protoplast isolation. ............ 87
9 LIST OF FIGURES Figure page 2 1 Flow cytometry histograms representing seedlings from selecti on UKP 1. ........ 39 2 2 Dried and successfully recovered seedlings ................................ ...................... 41 3 1 the greenhouse ..... 46 4 1 Indirect organogenesis. ................................ ................................ ...................... 56 4 2 Average Number of shoots produced through indirect organogenesis from colch icine treatments in pummelo seedlings from sele ctions. ........................... 57 4 3 Peak obtained by flow cytometry from a tetraploid shoot of C HBP produced by colchicine treatment in vitro ................................ ................................ .......... 61 4 4 Diploid and tetraploid peaks obtained by flow cytometry from a mixoploid shoot of C HBP produced by colchicine treatment in vitro. ................................ 61 4 5 Brow ning of pummelo seedling stem piece explants caused by higher colchicine concentration and duration. ................................ ............................... 62 5 1 Application of 0.1% colchicine to the cotton balls placed on the grafted seedling p ummelo buds with a surgical syringe and a needle. .......................... 67 5 2 Colchicine treated grafts. ................................ ................................ ................... 69 5 3 Flow cytometry histograms representi ng budsprouts with mixoploid profile (diploid and tetraploid cells) in selection 5 1 99 5. ................................ .............. 71 6 1 cultured for 6 weeks on DOG media containing 1g/l PVP. ................................ ................................ ........................... 78
10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for th e Degree of Master of Science THE PRODUCTION OF RED FLESHED GRAPEFRUIT/PUM MELO TETRAPLOID PARENTS TO AID THE GRAPEFRUIT IMPROVEMENT PROGRAMS By Divya Kainth December 2010 Chair: Jude W. Grosser Major: Horticultural Sciences Seedless and red fleshed grapefruit cultivars have a distinct upper hand in the citrus market and are pre ferred by consumers over white and seedy cultivars. Since seedlessness is the characteristic of triploids, more triploids via different diploid by tetraploid crosses need to be produced to incorporate all the desired traits. Although a large variety of cit rus diploids are available, a broader tetraploid gene pool is still needed. The objective of this research wa s to produce grapefruit/pummelo type tetraploids for use in crosses with diploids to produce seedless triploids. To accomplish this objective, the first approach wa s to somatically hybridize leaf protoplasts from red fleshed pummelo parents with protoplasts from the callus of Ruby Red grapefruit by PEG (polyethylene glycol) induced fusion to obtain allote traploids. The second approach wa s to produce autotetraploids by using colchicine, an anti mitotic agent Both in vitro and in vivo experiments were conducted. Under in vitro experiments, the explants from the etiolated seedlings grown under sterile conditions we re treated with different colchicine co ncentrations 0.01, 0.05 and 0.1% for different durations 4, 8, 16 and 32 hours. The shoots obtained throug h indirect organogenesis from the explants
11 from each treatment we re tested for their ploidy via flow cytometery. Pregerminated seeds were also tre ated with colchicine at 0.1, 0.2 and 0.3% for 12 and 24 hours, in another experiment. For in vivo experiments, ma ture budsticks having 4 5 buds we re cleft grafted onto vigorous rootstocks and treated with 1% colchicine The tr eatment is given for 1 and 2 d ays. The sprouting buds we re tested for their ploidy Thirdly, 600 to 700 seeds per pummelo culti var were planted and seedlings we re selected on the basis of root morphology and screened for natural altered ploidy levels i.e. triploids or tetraploids. The seedling s obtained by these approaches we re tested using flow cytometry. The confirmed tetraploids were micrografted onto vigorous rootstocks to expedite transfer to the greenhouse and subsequently to the field The tetraploids produced in these experiment s will be very valuable starting material for the programs involved in improving hybrid pummelo/ grapefruit quality.
12 CHAPTER 1 INTRODUCTION AND LIT ERATURE REVIEW History of Grapefruit Grapefruit ( Citrus paradisi Macf ) is an evergreen subtropical citrus cultivar that originated as a natural hybrid in Barbados in mid 18 th century. It is a relatively new bados by Griffith Hughes in 1750. G rapefruit was also growing in most parts of Jamaica. Naturalist John cause of its taste resembling that of grapes Tussac on the other hand described its habit of being borne in clusters and hence the name grapefruit. The latter explanation was more logical and accepted in 1943. The Latin classification for grapefruit is C. paradisi Macf It means citrus of paradise and assigned by Macfadyen (1830, 1837) Due to grapefruits resemb lance to pummelo ( C. grandis L. Osbeck ) in the fruit appearance, the latter was named as a probable parent. However, there were conflicting beliefs about how grapefruit originated. It was though either to be a mutant from pummelo or a hybrid from a cross b etween pummelo and sweet orange ( C. sinensis L. Osbeck ). C hemical and m orphological evidences provided b y Scora et al.(1982), support the latter hypothesis. More recently, the analysis of its genetic profile confirmed that grapefruit originated as an inte rspecific hybrid as a result of chance hybridization between pummelo and sweet orange (Scora, 1 975; Barret and Rhodes., 1976; Scora et a l., 1982 ; Gmitter, 1995). This also gives a logical explanation for its size characteristics from the pummelo parent and the nucellar embryony characteristic from sweet orange. William C. Cooper, a citrus scientist (USDA, ARS, Orlando, Florida, 1975), mentions in his book, In Search of the Golden Apple, that
13 grapefruit was first introduced in Florida in 1823 when grapefruit seeds from the Bahama Islands were taken to Safety Harbor near Tampa in Florida. From there, it spread to the surrounding areas and eventually was found all over Florida. At that time, the grapefruit was considered mostly an ornamental novelty. In 1870, J ohn A. MacDonald was drawn towards the cluster of lemon colored fruits on a single grapefruit tree in the Drawdy property at Blackwater Florida He used the seeds from the fruit of this tree as the source to establish the first grapefruit nursery in Flori da. George W. Bowen established the first grapefruit grove for commercial production in Florida in 1875. By 1885, growers in Florida started shipping the fruit to New York and Philadelphia where the people were developing the taste for the fruit and the de mand was starting to rise A single grapefruit tree that repeatedly survived freezing temperatures was observed in Kennedy Ranch in southern Texas and lead to successful production of grapefruit even under relatively cooler climatic conditions prevailing t here. By 1910, grapefruit acquired the status of an important commercial crop in the Rio Grande Valley of Texas, while it was still in the process of getting established as a commercial crop in California and Arizona. Grapefruit continued gaining popularit y for its fruit and juice in countries like Jamaica, Israel, United States, Brazil and other South American countries. By the 1940s, grapefruit was recognized as develo ped a liking for its taste, increasing the grapefruit demand and production to a great extent. At present, the United states of America is the leading producer of grapefruit in the world, followed by China, South Africa, Mexico, Syria, Israel, Turkey, Indi a, Argentina and Cuba respectively according to FAO (Anonymous., 2009) The U.S.
14 also ranks first in grapefruit export and export value. The main grapefruit producing states in the U.S. are Florida, California, Texas and Arizona. Over 30% of the world grape production goes to the fresh fruit market and over 55% goes for juice processing. Grapefruit Nutrition Nutritionally, grapefruit is considered a very rich fruit. It is a very ri ch source of many nutrients and phytochemicals and an excellent source of Vitamin C that supports the immune system and helps fight against colds. The ri ch pink and red grapefruits get their color from the presence of the hytonutrient. Lycopene is a powerful antioxidant with a high capaci ty to fight oxygen free radicals that cause damage to the cells. Grapefruit also have liminoids which help to prevent tumor formation. Compounds called glucarates in the pulp helps in breas t cancer prevention. Grapefruit consumption has also shown to reduce cholesterol levels due to the presence of a form of soluble fiber called pectin (Cerda et al., 1988). Being highly nutritious, low in calories, having anti carcinogenic properties and all the above mentioned health benefits, grapefruit has found a niche in healthy diet plans. Importance of Seedless C ultivars Challenges to increase the demand and popularity of grapefruit have led citrus breeding programs to improve the quality and productio n of grapefruit type fruit s. At present, the major concern for the grapefruit breeders is to develop cultivar s which would be resistant to diseases such as citrus canker and to produce cultivars which would be more attract ive to more consumers. Today, the trend in consumer preference is to wards seedless fruits. This has been best exemplified by the increased demand of seedless watermelons and grapes in the recent past. In citrus, seedless cultivars were
15 recognized in 1943 by Krug and Bacchi. Seedless fruits are of significance to the juice industry also, as the seeds in the fruit can be associated with unfavorable aromatic compou nds and bitterness in the juice Seediness can be a problem for a variety to be released commercially. In citrus, a number of seedy citru s cultivars could not attain commercial importance although they had all the other desired horticultural traits (Fatta Del Bosco et al., 1992). In developed countries, maturity season gaps are filled wit h seedy cultivars that exhibit other superior t raits just because there are not enough seedless cultivars available at that time of the year. A nother very desirable trait in grapefruits is its pink or red colored flesh. Out of the three flesh colors available in grapefruits, white grapefruit varieties app ear to be least appealing to consumers and fetch lower prices in the commercial markets than those from pigmented grapefruits. This has led grapefruit breeders to devote more effort toward development of colored cultivars. Incorporation of the traits li ke seedlessness and pink/red flesh color into single cultivars would make it highly desirable in the consumer market. Seedlessness in diploid citrus is related to self incompatibility, male or female sterility, or e arly embryo abortion ( Recupero et al. 200 5). Scientists have investigated different wa ys to produce triploid plants Triploid callus lines have been produced by tissue culture techniques (Zhang, 1985), in vitro endosper m cultures (Gmitter et al., 1990 ), selection of spontaneous triploid embryos from 2x X 2x crosses (Ollitrault et al., 2010 ), inducing seedlessness by induced mutations via gamma irradiation (Hearn e 1984; Hensz, 1977) and by interploid hybridization, using tetraploids as the female (seed) parent (Cameron and Soost, 1969; Esen and S oost, 1972; Cameron and Burnett, 1978; Oiyama and Kobayashi, 1990).
16 Male or female sterility is generally associated with triploidy (Geraci et al., 1975; Esen et al., 1978 ). The diploid x diploid crosses with parents having genes for male/female sterility, pollen incompatibility and parthenocarpy are being made to produce seedless citrus fruits in Australia (Sykes and Lewis, 1996; Koltunow et al., 2000). More recently, triploid breeding has proven to be very successful in obtaining the seedless trait in ci trus. The p rocess involves either a cross between two diploid parents resulting from the fertilization of 2n megagametophyte (Luro et al., 2004) or via interploidal crosses between a diploid and a tetraploid parent (Esen and Soost, 1973). Elite diploid spe cies selected on the basis of important horticultural traits such as fruit color and size, flavor, program to produce a quality hybrid (Grosser et al., 1998; Grosse r et al., 1992a).The sterility in the triploid seedless progeny is caused by the odd number of chromosomes that impair the normal meiotic division process and results in chromosomally unbalanced gametes (Reforgiato Recupero et al., 2005). In general, tripl oids can be obtained from crosses between three types of parent combinations 4x X 2x, 2x X 4x and 2x X 2x. Out of these, 4x X 2x have shown to give maximum triploid recovery (Esen and Soost, 1971; Cameron and Burnett, 1969; Cameron and Burnett, 1978). Th is high triploid recovery rate is due to the successful fertilization between a diploid female gamete and a haploid male gamete (18n + 9n) (Cameron and Soost, 1969; Cameron and Burnett, 1978), followed by the development of triploid zygotic embryos T he hy brids resulting from 2x X 4x crosses have much poorer triploid recovery rates (Cameron a nd Soost, 1969; Furusato, 1957; Tachikawa, 1971 ; Esen et al., 1978). Seeds from such crosses are normally smaller or shriveled. Many of the seeds from
17 such crosses we re found to be empty, shriveled or poorly developed. An a bortion rate of 92 99% was observed for triploid embryos from the 2x X 4x crosses (Esen and Soost, 1973b, 1973c). It was thought that the smaller size and higher abortion rate was due to the early te rmination of the pentaploid endosperm development which leads to early initiation and termination of the triploid embryo development Later it was found that a low triploid hybrid recovery rate is due to the 3:5 embryo to endosperm ratio in the seeds which characteristic of non viability (Esen and Soost, 1973a) The ratio for normal diploid seeds is 2:3. A ratio below this impairs the embryo vialbility (Esen and Soost, 1972a). This problem has been overcome by the application of embryo rescue techniques, a nd at present triploid recovery from 2x X 4x crosses is quite efficient (Viloria et al., 2005). Problems i n the Grapefruit Market There are some other fa ctors in grapefruit that limits fruit consumption. Grapefruit contains a flavanoid called naringin, whi ch is the cause of its distinctive bitterness. The bitter taste in the fruit or juice from grapefruit is not necessarily liked by everyone. Despite the cholesterol lowering effects of naringins (Silva et al., 2001), some people especially children do not c onsume the juice because o f the presence of the bitter taste it imparts to the juice, which they would happily consume otherwise. This factor reduces the consumer market to some extent. Another problem in grapefruit market is its interaction with some dru gs when taken together. It is called and it is the result of t he presence of furancoumarins like bergamottin and dihydroxybergamottin as well as naringin These compounds make some of the pharmaceutical drugs more potent by i ncreasing their bio availability when they are combined with grapefruit juice. T hese interactive drugs
18 include the immunosuppressant cyclosporine and calcium channel blocker drugs, such as felodipine, nifedipine a nd verapamil (Bailey et al. 1994 ) Other dr ugs whose bioavailability is enhanced by grapefruit juice are the antihistamine terfenadine, the hormone estradiol and the antiviral agent saquinavir. The furanocoumarins and naringin interfere with the normal detoxification and metabolism processes in the intestines and These compounds cause inactivation of enzyme cytochrome P450 3A4 in the small intestine that is responsible for the metabolism of the s tatin drugs (Veron ese et al. 2003 ). Another mechanism occurs simultaneously which involves inhibition of P glycoprotein which is carrier molecule transporting s t atin drugs back to the gut. Both these mechanisms cause accumulation of these drugs which may result into severe muscle damage or paralysis or even death in certain instances. The drug interactions and its deleterious effects furth er narrow the consumer market. Improved g rapefruit like cultivars having a lower level of t hese compounds sh ould have more consumer appeal especially to the elderly population. Introduction of P ummelos in Breeding P rograms Considering all the desirable traits, breeders have to face some challenges in breeding cultivars to meet all the consumer expectations Grapefruits have a comparativel y narrow gene pool since it is a group of related cultivars that have arisen through mutation from a single hybrid genotype This gives little latitude for the breeders to find desirable traits and incorporate them in the commercial cultivars in order to m ake them more improved and marketable. Grapefruit offers less variation that can be exploited to aid grapefruit improvement programs, as compared to the other citrus types.
19 Pummelo ( C. grandis ), an ancestor of the grapefruit, can be utilized for its much larger gene pool and is thus involved in the breeding of pummelo x grapefruit hybrids. Cultivars M elogold (Soost and Cameron, 1985 ), Oroblanco (Soost and Ca meron, 1980 ) and Wheeny grapefruit are some of the examples of successful pummelo grapefruit hyb rids. The red fleshed pummelo selections/cultivars can serve as excellent breeding parents to produce red flesh ed hybrids. Pummelos, being true species are a good source for adding variation for incorporation in to the grapefruit gene pool. Pummelos are mo noembryonic and produce zygotic seedlings each of a diffe rent genotype, which allows expression to a high degree of phenotypic variation s Grapefruit is polyembryonic and almost all the seedlings are identical to the mother geno type, limiting its variation potential. Many traits like fruit quality, resistance to various pathological and environmental stresses, monoembryony, etc. can be transferred from pummelos to commercial grapefruit cultivars via sexual hybridization. Pummelo being monoembryonic facilit ates the breeding process. Usually, in polyembryonic cultivars the hybrid produced by the cross gets suppress ed by number s of vigorous nucellar embryos surrounding it. In general, in order for it to survive, the embryo has to be rescued under sterile condi tion which requires skill and is a very labor intensive technique (Rangan et al 1978), or biochemical techniques have to be used to distinguish zygotic embryos from nucellar ones (Geraci et al., 1981). However, it depends on percentage of zygotic embryos w hich vary with genotype. On the contrary, when a monoembryonic tetraploid is used as a seed/female parent in the inter ploidal crosses, it eliminates the need of embryo rescue, saving time and labor. Each zygotic
20 embryo gives rise to a single triploid seedl ing without having to deal with any suppressing effects of nucellar seedlings. Another advantage of using pummelos is that in general, they have less naringin content in comparison to grapefruits and are thus taste sweeter and less bitter than the latter. A hybrid with one pummelo parent would help dilute the naringin content and help in development of sweeter and more appealing cultivars. Similarly, the lesser furanocoumarin content in pummelos make them ideal to be included as parents in breeding program s as that would help lower the non desirable furanocoumarins, widening the consumer market for grapefruits. Thus, producing tetraploid parents of such red fleshed pummelo/grapefruit types would aid the grapefruit improvement program tremendously by providi ng superior tetraploid breeding parents for use in interploid crosses. History of Tetraploids in C itrus Breeders have used the following methods to produce tetraploids: Somatic Hybridization C onventional plant breeding in higher plants like citrus is limit ed to the crosses between phylogenetically related plant species. This is due to problems like sexual incompatibility, heterozygosity, male or female sterility, polyembryony, difference s in ploidy levels etc (Gros ser and Gmitter, 1990). T he prolonged juve nile period make s cultivar development much more time consuming. To overcome these impediments, a technique involving fusion of somatic cells to produce hybrids, known as somatic hybridization, has been developed. Many of these impediments can be overcome to a great extent using this technique (Grosser and Gmitter, 1990). The somatic hybridization is a nonconventional breeding technology which involves fusion of
21 protoplasts derived from two cultivars which may be distantly to closely related at intraspecifi c, interspecific, intergeneric, and interfamily levels. The fused protoplasts form heterokayons which undergo several cell divisions and colonize to form an embryo, ultimately growing into a hybrid plant. Somatic hybridization has diversified the tetraplo i d gene pool available for hybridization with diploid cultivars. These somatic hybrids when used as parents in interploidal crosses have produced many triploids. The first somatic hybrids, using somatic hybridization were produced from Nicotinia glauca and N. langsdorffii (Carlson et al., 1972). Since then, a number of hybrids have been produced in various crops via somatic hybridization (Grosser et al., 2000; Johnson and Veilleux, 2001; Orczyk et al., 2003). This technique did not prove to be very successf ul in most crops but in citrus, steps from protoplast isolation to plant regeneration have become routine (Vardi et al., 1982; Grosser, 1994a; Grosser and Gmitter 1990 & 2005; Grosser et al. 2000). The allotetraploids produced from protoplast fusions, whe n used as breeding parents in interploid crosses, produce seedless triploids (Grosser and Gmitter, 2005). In citrus, the first somatic hybridization was done between C sinensis and Poncirus trifoliata (Ohgawara et al., 1985).This technique has been widely used in citrus, w here it s most important application is to produce tetraploid breeding parents and increase genetic diversity in the parental gene pool. During the past two decades, over 300 intergeneric and interspecific somatic hybrids have been produce d worldwide (Guo and Deng, 2001; Grosser et al., 2000). Out of these, more than 200 somatic hybrids have been developed at the Citrus Research and Education Center (CREC) in Lake Alfred, FL At the CREC, a primary objective of these somatic hybrids is to s erve as parents for
22 use in interploidal crosses to produce triploid see dless cultivars. Thousands of triploids have been produced and are now in trials at the CREC. The increase in ploidy level from diploid to tetraploid has proven beneficial in some case s with respect to the horticultural traits of the somatic hybrids. Some of the somatic hybrids produced and evaluated were found to have improved horticultural performance in respect to pest, disease, nematode resistance and other abiotic stresses (Grosser et al., 1996a; Ollitrau l t et al., 1998a). Some of the tetraploid hybrids can be directly used as rootstocks as they have good potential for tree size control (Grosser et al., 1995; Grosser and Chandler, 2003), better horticultural performance or improve d disease resistance (Louzada et al., 1992). This technique has been successfully used to develop ger m plasm from sexually incompatible or difficult to hybridize citrus relatives to expand the citrus germplasm ( Grosser et al. 1994; Grosser et al., 1996b; M otomura et al., 1997). A somatic hybrid from the fusion of C. sinensis cv. Hamlin (sweet orange) protoplasts isolated from an embryogenic suspension culture with Severinia disticha (Philippine box orange) protoplasts isolated from epicotyl derived callus wa s the first example of a somatic hybrid produced between sexually incompatible woody species (Grosser et al., 1987). In seedless scion breeding, the hybrid allotetraploids produced by protoplast fusion can be used as males or females in interploid cross es. The crosses where diploids are used as female parent have some limitations. In early work, the number of triploid hybrids produced was often very low and sometimes triploids remain undetected. It was d ifficult to isolate them from the nucellar seedling s in polyembyonic cultivars (Esen et al. 1978). The difficulty in recovery of the triploid hybrids due to
23 embryo abortion was also rep orted (Esen et al., 1979; Okudai et al., 1981). In such cases, the immature triploid embryos have to be rescued 12 15 w eeks after pollination and culture d in vitro (Satarrantio and Resupero, 1981 ; Oiyama and Kobayashi, 1990). An alternative and easier way of producing triploid hybrids is using tetraploid hybrids as the female parents in the interploid crosses (Cameron and Burnett, 1978 ; Esen et al., 1979). Such crosses have high seed set compared to the reciprocal crosses and triploids obtained are generally more vigorous (Esen and Soost., 1972; Cameron and Burnett., 1978). Another way to produce triploids involving somati c hybridization is by fusing protoplasts isolated from a haploid and a diploid parent ( Ollitrault et al. 1998b, 2000a). Somatic hybridization has been used for the production of hybrids which are better adapted to climatic conditions or have improved dis ease resistance. Mand a rin + pummelo hybrids with potential tolerance/resistance to sting nematode have been produced (Grosser et al., 2007). Mand a rin and sweet orange with pummelo fusions have been made to produce hybrids with citrus tristeza virus resista nce in efforts to replace sour orange rootstock (Grosser et al., 2007). Somatic hybrids resulting from the fusion of Cleopatra mandarin ( C. reticulata Blanco) with Rangpur lime ( C. limonia L. Osbeck) and with sour orange have potential tolerance agains t blight and citrus tristeza virus (CTV), as they had combined complimentary characteristics from both parental sources ( Mendes et al., 2001). Spontaneous Altered Ploidy Polyploidy has a lot of potential to contribute in crop improvement programs (Frost, 1 925b, 1926 ) Polyploidy in citrus such as triploids, tetraploids, pentaploids, hexaploids and octaploids can occur spontaneously in nature. Most cultivars of citrus were found as
24 spontaneous seedlings. Tetraploid triphasia desert lime (Esen and Soost., 197 2), Clausena excavata (Froelicher et al., 2000), tetraploid Hongkong wild kumquat (Longley, 1925) and triploid Tahiti lime (Bachi, 1940) are some of the excellent examples of spontaneously occurring polyploidy genotypes. Naturally occurring polyploids whi ch arise from nucellar embryos are considered to be somatic in origin (Barret and Hutchinson, 1978). Somatic tetraploids occur very commonly in nucellar seedling progenies (Iwamasa, 1966). Such polyploids were classified as autopolyploids (Esen and Soost, 1972b). However in order to avoid the problem in distinction between autopolyploids and allopolyploids, the preferable term used for them is spontaneous polyploids. However, they are few in number, because the diploids generally are more vigorous and pro vide competition for endosperm nourishment in the seed. The frequencies of polyploids may also depend on certain external environmental factors including temperature and light intensity (Barret t and Hutchinson, 1978). It is possible to detect ploidy varia nts on the basis of morphological characteristics for certain cultivars in the past. Tetraploid seedlings typically have thicker lea ves than diploids nucellar seedlings progenies (Frost., 1 925,1926 ). Frost found that 2.5% of the cultivated citrus species w ere tetraploids. Work done in Russia had similar results with 34 tetraploids found from an apomitic population of 1379 seedlings (2.5 % ) in two cultivated citrus species and P. trifoliata (Lapin, 1937). Lapin also reported that maximum percentage of triplo ids occurring naturally in citrus was observed in the cultivar Kaghzi (15.45%), followed by Foster (14%), Kinnow (11.33%), Musambi
25 confirmed for some of these cultivars (U sman et al., 2006). Two spontaneous tetraploids have been obtained in Japan in the Citrus spe cies observed (Nakamura, 1942). Tetraploids among nucellar seedlings of C. aurantium L. and C. l imon were reported in Italy (Russo and Torrisi, 1951). In Japan, 7 tetraploids out of 4,196 plants (0.2%) were derived from a c itrus hybrid and 2 other citrus species, on the basis of thicker roots and fewer lateral roots in comparison to diploids (Furusato, 1953a). Furusato noticed underdeveloped trees growing on P. trif oliata rootstocks in Japanese citrus groves. He determined that 0.7% of the rootstocks to be tetraploid. Later, spontaneous tetraploid seedling percentage among P. trifoliata seedlings was determined to be 1.5% (Iwasa and Shiraishi, 1957). Occurrence of te traploids has also been reported from nucellar seedlings of Citrus Poncirus and other hybrids by Barret t and Hutchinson in 1978. Development of zygotic embryos in polyembryonic cultivars is the cause of spontaneous triploid seedlings (Geraci et al. 1975; Wakana et al., 1982 ). Therefore the frequency of triploids is much less in polyembryonic cultivars as compared to monoebryonic cultivars (Geraci et al., 1977). Different ploidy levels in a plant would produce distinct morphological characteristics whic h can be used to identify these polyploids. In many plant species, increased ploidy generally causes reduction in the growth rate (Lindstorm, 1936). In Citrus Frost first reported (1925) distinct appearance and reduced growth as characteristic of a citrus tetraploid. Tetraploids in citrus have distinctive vegetative morphology which can be used to identify them (Frost, 1938; Frost and Krug, 1942, Fu rusato, 1953a; Tachikawa, 1 97 1 ; Barret t and Hutchinson, 1978), as well as reproduc tive morphology (Tachikawa, 1971 ; Barret and Hutchinson, 1978). Tetraploids
26 have lower fruit yields and number of seeds per fruit. Shriveled or underdeveloped seeds are found to have a higher ploidy level comparatively in sweet orange and tangor cultivars (Wakana et al., 1982 ). The underdeveloped characteristic of these seeds may be due to intrinsic capability of the ovule due to polyploidy, number of pollinations or delayed pollination which results in shortage of endosperm required for growth and development of the embryo (Usman et al., 2006). Roots of citrus autotetraploids are found to be shorter and stouter with fewer lateral roots (Furusato, 1953a; Barret t and Hutchinson, 1978). Tetraploids were recently obtained from Hudson grapefruit by planting a large population of seeds. The selected seedlings were screened via flow cytometry to confirm the tetraploids (Grosser et al., personal communication). Selection of the seedlings was based on the thicker root morphology, thicker leaves and dark green color and slow er growth rate. Th e quality of fruit from autotetraploids is generally poorer in comparison to the diploid counterparts and is usually not used for commercial purposes. Tetraploids produce a smaller tree size and vigour than diploids (Frost, 1938; Frost and Krug, 1942; Barr et and Hutchinson, 1978; Lee, 1988). Also, tetraploids used as rootstocks impart dwarf ing to the normal scion cultivars grafted on them (Russo and Torrisi, 1951; Furusato, 1953b). Variabi li ty in the vigor between diploid and tetraploid rootstocks was notic ed by Mukherjee and Cameron (1958). The dwarfing effect of these tetraploid rootstocks is beneficial for the growers as dwarf trees lower the cost of pruning and harvesting. Colchicine Induced Polyploidy Since there is a limited number of tetraploids ava ila ble in citrus, representing a minor section of the total gene pool available (Barrett, 1974), different methods have been employed to induce increased ploidy levels, ultimately aiming towards triploidy.
27 Doubling the chromosome number of the plant itself has been a very successful approach to produce autotetraploids. Autotetraploids serve as important breeding parents in inteploidal crosses. However, the process to obtain autotetraploids from the respective diploids has been quite challenging. Various tec hniques and chemicals have been tried over the past, each attempting to improve the ease, safety and efficiency of the method. Treatment of plant parts with compounds which have potential to cause chromosome doubling, like colchicine, trifluralin, oryzalin 2,4 dichlorophenoxyacetic acid and amiprophosmethyl (AMP) have resulted in elevated levels of ploidy in various plant species in the past. These compounds inhibit the spindle formation during mitosis and interfere with normal polar segregation of sister chromatids to form a restitution nucleus (Blakeslee and Avery, 1937). As a result, duplicate chromosomes stay in one cell, doubling the ploidy of the cell. Co l chicine is the most commonly used chemical for doubling the chromosomes in plants. However, other chemicals have also been tried but fail to produce tetraploids with the same efficiency as colchicine. Colchicine is an alkaloid derived from meadow saffron ( Colchicum autumnale L.). It has been used successfully for doubling of chromosomes in fruit crops like banana (Hamill et al., 1992), cherry (James et al., 1987), grapes (Notsuka et al., 2000) and blueberry (Lyrene and Perry, 1982). Some success has been seen with oryzalin on lily, nerine and apples (Van Tuyl et al., 1992; Bouvier et al., 1994). In cit rus, oryzalin has been used in efforts to produce autotetraploids by treating the embyogenic callus of the citrus cultivars such as 'Umatilla' and 'Dweet' tangors ( C. reticulata Blanco x C. sinensis ), 'Caffin' Clementine ( C. clementina Hort ex Tan.) and 'W heeny' grapefruit ( C. paradisi Macf) (Wu and
28 Mooney., 2002). Unfortunately, oryzalin did not produce any autotetraploids because it is more toxic than colchicine, and pr obably should be used at a lesser concentration. However colchicine was successfully u sed to produce autotetraploids in the same cultivars Nucellar callus cultures of lemon cultivars were successfully induced to polyploidy when treated with 2,4 dichloro phenoxyacetic acid (Vardi., 1982 ). The use of chemicals other than colchicine for induci ng polyploidy has remained limited because of their comparatively lower efficiency. There are two important factors that determine the effectiveness of these mutagenic agents. These factors are the concentrations in which they are applied and the du ration for which the plant part to be mutated is exposed to the chemicals. Excessive concentration and exposure causes the death or necrosis of the exposed tissue due to the toxic effect. Initially, lower concentrations with longer exposure periods in vitr o were tried to produce autotetraploids (Gmitter et al., 1990; Gmitter and Ling, 1991). More recently, autotetraploids have been produced in sweet orange ( Citrus s inensis ) at a higher frequency using higher concentrations with shorter exposure periods (Zha ng et al., 20 07). Zhang suggested an efficient method to produce a high frequency of autotetraploids by first determining the peak period of division in ca llus in terms of days after sub culturing, and then administering the treatment at that peak period. Colchicine treatments have been tried on various plant parts. In citrus, Frost and Lapin (1937) have obtained spontaneous tetraploids from several varieties. Two tetraploid citrus clones were obtained by colchicine treatments (Tachikawa et al., 1961). Cit rus embryogenic callus has been a very common and successful target to initiate chromosome doubling in the cells, followed by regenerati on of plants with doubled
29 chromosome number (Wu and Mooney, 2002; Zhang et al., 2007). Treatments on cell suspension cul tures have regenerated complete tetraploid plants in Po nkan mandarin (Dutt et al., 2010 ). Cell suspension cultures have increased chances of producing stable, nonchimeric tetraploid plants as the embryos originate from single cells (Stewart et al., 1958) Callus or cell suspensions regenerate complete autotetraploids more efficiently as the subsequent plant regeneration is from single cell and a single mutated cell is able to produce a complete tetraploid plant. Treatment of callus from monoembryonic cult ivars with colchicine has produced autotetraploid tangor plants (Wu and Mooney, 2002). However, the frequency of autotetraploids obtained was quite low, and in general, it is extremely difficult to produce embryogenic callus from monoembryonic genotypes. Non chimeric autotetraploids have been produced successfully via treatment of underdeveloped ovules from the immature fruits of 'Orlando' tangelo and 'Valencia' sweet orange with colchicine (Gmitter and Ling, 1991). Polyploids have also been recovered by treating the axillary buds and shoot tips with colchicine under sterile conditions followed by an in vitro micrografting technique (Juarez et al., 2004; Yahata et al., 2005; Oiyama, 1992; Oiyama and Okudai, 1986). However, treating the b uds or shoot tips often produce plants composed of tissues wit h varying chromosome numbers that are called cytochimeras (Barrett, 1974; Jaskani et al., 1996) These chimeras are mostly unstable and sterile, and do not have any application in breeding programs. Many of these chimeras revert back to their original ploidy level because of their instability. These chimeras can be of different types and can be classified on the basis of the histogenic layers ( LI, LII or LIII ) mutated. A chimera can be periclinal (one mutated laye r) mer iclinal (part of the apical dome mutated) and
30 sectorial (section of the apical meristem mutated) The cause of these chimeras from the treated bud is basically the fact that the buds are muticellular. In muticellular tissue, colchicine may not be ab le to affect all the cells and the unaffected cells remain diploid. When such type of source is used as explants for colchicine treatments, a large proportion of chimeric tetraploids are produced (Kadota and Niimi, 2002). Treatment of shoot tips in vitro to induce tetraploids is very common in monoembryonic citrus cultivars as production of callus lines, used for treatment otherwise, is very difficult. Stable tetraploid plants have been produced in the tip grafting (Juarez et al., 2004). Efforts have been made to produce tetraploids from monoembryonic cultivars by techniques ex vitro Barrett (1974) tried treatment of axillary buds of monoembryonic diploid clones with 1.0% colchicine solution ex vitro H e could not produce a complete and stable tetraploid but he induced eight types of cytochimeras based on the three histogenic layer mutations. He used large plant s for his experiment; however, in large, broadly differentiated meristems, it is very difficul t to affect the entire tissue to produce stable tetraploids (Sanford, 1983). Later, smaller explants young axillary buds were used for colchicine treatments which were then micrografted onto the vigorous rootstocks to produce complete autotetraploid plan ts (Oiyama and Okudai, 1986). There are no reports of artificially producted autotetraploid plants from monoembryonic pummelos. New autotetraploid pummelo cultivars are needed which can be hybridized with grapefruit cultivars to increase genetic diversity for grapefruit cultivar improvement. Objective s The overall objective of this research project is the generation of red fleshed grapefruit/pummelo tetraploid parents with various fruit quality or disease resistance
31 characteristics to facilitate the grapef ruit improvement breeding program. In order to achieve this objective various approaches were applied which are as follows. Somatic hybridization to produce allotetraploids. Screening for naturally occurring triploids and tetraploids. Induction of tetraplo idy by the in vitro (pregerminated seeds, stem segments) and in vivo (axillary buds).
32 CHAPTER 2 INDUCTION OF AUTOTETRAPLOIDS IN PUMMELO (CITRUS GRANDIS) THROUGH COLCHICINE TREATMENT OF MERISTEMATICALLY ACTIVE SEEDS IN VITRO Introduc tion Grapefruit ( Citrus paradisi ) which originated in Barb a dos is cultivated worldwide and is among the most popular fresh citrus cultivars The United States followed by China and South Africa s growing demand and p opulari ty exerts pressure on breeders to come up with improved cultivars with traits to such as dark red flesh color and seedlessness. Excessive number of seeds in citrus makes it unappealing to the consumers and unacceptable f or local and international markets. Breeders worldwide are in creasing efforts to come up with quality seedless cultivars. Since, triploidy is associated with sterility, s eedlessness has been successfully achieved in the past in many cultivars through ploid y manipulations. Several approaches from traditional hybridization to molecular biotechnology have been ap plied in order to induce seedless fruits in citrus. These include selection of spontaneous triploids from a natural population (Geraci et al., 1975; W akana et al., 1981) or those from 2x X 2x crosses (Esen and Soost, 1971; Esen et al., 1978), somatic variation (Deng et al., 1985), en dosperm culture (Wang and Chang, 1978; Gmitter et al., 1990; Chen et al., 1991), somatic hybridization between a diploid an d a haploid (Kobayashi et al., 1997); genetic transformation (Koltunow et al., 1998), and interploidal hybridization between a diploid and a tetraploid parent (Esen and Soost, 1972) Out of these, the interploidy hybridization has been the most effective a nd is commonly used approach to produce seedless triploids. However, scarcity of tetraploids in the citrus gene pool has given rise to the need to induce tetraploidy in breeding lines that could be used as parent al material for the interploidal crosses.
33 Cr osses where the tetraploid is used as the female parent to produce triploids are more efficient because of the normal sexual fertilization between a diploid female gamet e and a haploid male gamete (18 + 9) (Esen and Soost, 1972; Soost and Cameron, 1975; Ca meron and Burnett, 1978). In such crosses, when tetraploid polyembryonic cultivars are used as a female parent, there arises a need to rescue the embryo via tissue culture techniques. This is to avoid the suppression and subsequent abortion of the zygotic hybrid embryo by the more vigorous nucellar seedlings. Though the identification and selection of the zygotic seedling is possible through morphological, isozyme, histochemical, cytological and molecular techniques, it becomes a costly, labor intensive and time consuming process. Use of a tetraploid monoembryonic female parent instead eliminates the need for embryo rescue making the process much simpler and efficient. Considering the present market demands, scarcity of superior tetraploid parents in grapef ruit and factors slowing triploid breeding, p ummelos are beginning to be included as germplasm sources in grapefruit breeding programs. Pummelo, being the ancestor of grapefruit and monoembryonic, has a comparatively greater diversity in its gene pool whic h makes it an ideal candidate to contribute towards grapefruit improvement. s l ower naringin and furanocoumarin content could help balanc e out higher contents present in grapefruits. Use of such red fleshed tetraploid pummelo types as a female pare nt in the interploidal crosses c ould produce hybrids which are more appealing and consumer friendly. Colchicine an alkaloid obtained from meadow saffron ( Colchicum autumnale ), is a mitotic inhibitor (Blakeslee and Avery, 1937) and is commonly used to indu ce tetraploidy
34 in breeding lines in Citrus Barret t (197 4 ) attempted to p r oduce autotetraploids in monoembryonic cultivars by treating the axillary buds with colchicine i n vivo However he was not able to produce any stable tetraploid plant s The most prob able explanation for this was that he used bigger plants and it is difficult to double the chromosomes in large and broadly differentiated meristems (Sanford, 1983). Stable autotetraploids in monoembryonic cultivars have been produced by cochicine treatmen t of the axillary buds (Oiyama and Okudi, 1986), shoot tips (Juarez et al., 2004) and somatic embryogenic callus (Wu and Mooney, 2002). Lehrer et al (2008) induced tetraploidy by treating germinated seeds of Japanese barberry ( Berberis thunbergii var. atro purpurea ) with colchicine and oryzalin i n vivo This technique has also been used to produce tetraploids in ornamental plants Syringa spp L. ( lilac ) (Fiala 1988) Rhododendron spp L. ( rhododendron ) (Leach, 1961), Iris spp. L. ( iris ) (McEwen, 1990 ). Howev er, there is no report of tetraploid induction from seed treatment in Citrus Also, there has not been any report on tetraploid induction in p ummelo cultivars to date. This study reports an efficient method for induction of tetraploids by treatment of germ inating seeds from elite p um melo selections with colchicine i n v itro It compares the effect of different colchicine concentrations and exposure durations of seed ling growth and developmen as well as tetraploid induction. Materials and M ethods Plant M ateri als Seeds were extracted in 2008 2009 from the fruits of red fleshed p ummelo selections 5 1 99 2, C2 5 12 and UKP 1 located at the Citrus Rese arch and Education Center (CREC, Lake Alfred) All selections were open nk pummelo selected for high fruit quality The extracted seeds were washed
35 under running water 3 4 times and were treated with 0.6% (v/v) sodium hydroxide for 8 minutes to remove slime. The seeds were dried, peeled and sterilized using 10% Cl orox bleach before they were placed on seed germination medium under steril e conditions. The seeds were placed on this medium for 12 14 days to germinate and for hypocotyl about 5 8 mm in length to emerge out of the cotyledons. At this point, the seeds were undergo ing high meristematic activity and were ready for treatment Colchicine Treatments The experiment design was a two way factorial consisting of 3 colchicine concentrations and 2 exposure periods. There were three replication s of each treatment. The Colchic ine stock solution was prepared by dissolving colchicine in dimethylsulfoxide (DMSO) at a final concentration of 1g/ml. This solution was filter sterilized. Per treatment 15 pre germinated seeds were placed in conical Falcon tubes containing 10 ml of liqu id seed germination media with final colchicine concentrations of 1, 2 and 3g/L. Seeds immersed in liquid seed germination media without colchicine were used as controls. The seeds exposed to different concentration s of colchicine were expos ed for periods of 12 or 24 hours each. F lasks were put on the rotary shaker at 30 rpm for the respective exposure periods to facilitate the contact and penetration of colchicine in the m eristems which were covered by the cotyledons. The rotary shaker was contained in a dark chamber maintained at 25 2 respective exposure time, the seeds were taken out and placed on solid seed germination media and placed under dark conditions to facilitate seedling elongation. The seedling s were transferre d to rooting media supplemented wi th naphthalene acetic acid (NAA) after about 2 weeks an d were placed under 16 h light / 8 h dark conditions
36 for further growth. Emergent seedlings were analyzed for their ploidy via flow cytomet ry at a stage when the seed ling had at least 3 fully expa nded leaves. The seedlings confirmed to be tetraploid were micrografted on to vigorous trifoliate rootstock seedlings The micrografted tetraploids were put under shade for 10 14 days prior to moving them to the greenhouse wit h set points of 21 17 C day / night temperatures. Ploidy A nalysis Ploidy was analyzed using a tabletop Ploidy Analyser flow cytometer (Partec GmbH, Germany) This technique makes it possible to analyze 150 200 genotypes per day. Flow cytometry works by estim ating the volume and florescence of isolated nuclei. The ploidy is presented in form of a histogram of integral fluorescence with the peaks depicting the ploidy level of the respective sample. The p rotocol is a series of steps starting with excis i on of a 0.2 0.3 cm 2 piece of fully expanded leaf tissue and placed in a 6 0 X15 mm plastic P etri dish Each sample was chopped with a sharp razor blade after adding few drops of Nuclei Extraction Buffer. After chopping, 6 7 more drops of Nuclei Extraction Buffer w ere added and the samp le was filtered through a 50m filter into a 3.5 mL (55mmX12mm) Sarstedt tube. The fluorescent dye Diaminidino 2 phenylindole (DAPI) was added drop by drop through the filter to infiltrate the remaining cells, until the half of the tube was filled. Each sample was incubated for 10 15 seconds at room temperature before running it on the flow cytometer The sample moves as a very narrow, laminar flowing sample stream through the flow cuvette When the cells labeled with fluorescen t dye pass through the measuring area one after the other, t hey get illuminated by the excitation light of a defined wavelength. The light activates the fluorescent molecules so that they emit light
37 back. The f luorescent light intensity is proportional to the D NA content in labled cells depict s the respective number of chromosomes and hence the ploidy level of the sample. Results and Discussion The colchicine treatment induce d tetraploidy in seedlings from all three p ummelo selections. However, the frequenc y of tetraploids varied among treatments. Some treatments also produced cytogenic chimeric plants having tetraploid and diploid nuclei in var ying proportions of cells Chimeric plants have been recovered in similar in vitro studies conducted by Wu and Moon ey (2002); Kodota and Niimi (2002). Data was assessed by calculating the survival rate of the seedlings and tetraploid induction efficiency (TIE) for each treatment. Tetraploid induction efficiency was computed by the formula given below by Bouvier et al ( 1994). TIE = % Seedling survival X % tetraploid seedlings The most important factors that determine the tetraploid induction efficiency are colchicine concentration and the exposure period for which seeds were exposed to colchicine. Higher colchicine co ncentrations and longer duration period hampers seedling growth, causes hyperploidy, browning, necrosis in the meristematic tissue and death of the seedling (Sanford, 1983). In this study, all colchicine treatments greatly decreased the growth rate of the treated seedlings from all three selections. Five weeks after the treatment, the control counterparts grew up to a height ranging from 5 to16 cm long whereas all the surviving treated seedlings remained stunted with a height no more than 2.5 cm. A verage h eight s of the seedlings at x days after colchicine treatments were 12.4, 13.5 and 12.8 cm for selections 5 1 99 2, UKP1 and C2 5 12 respectively (Table 2 1).
38 Table 2 1 Average heights of seedlings recovered from treatments with various colchicine conce ntration s and exposure periods. Treatment 5 1 99 2 UKP 1 C2 5 12 Control 12.4 2.6 13.5 2.21 12.8 2.67 0.1% X 12 hours 1.2 0.63 1.6 0.5 1.5 0.4 0.1% X 24 hours 1.2 0.56 1.3 0.51 1 0.95 0.2% X 12 hours 0.9 0.5 1.4 0.35 1.2 0.38 0 .2% X 24 hours 1.1 0.56 1.3 0.6 1.1 0.4 0.3% X 12 hours 0.5 0.1 1.8 0.64 X 0.3% X 24 hours 1.1 0 13.5 2.21 1.6 0 9 5% level H igh mortality was observed in all the treatments. O verall seedling survival including all treatments was highest for selection 5 1 22 9 which had 30% of seedling survival rate followed by C2 5 12 having 25.6% and UKP1 with 21.1% (Table 2 2 ). The difference in the survival rates among different selections m ay be attributed to genotype effect or just random variation The control treatments without any colchicine exposure had 93 to 100% seedling survival rates. Higher mortality rate in the treated seedlings was due to the toxicity of cochicine. This explains decreasing seedling survival when cochicine concentrations were increased or when seeds were given longer exposures to the chemical. At the lowest concentration of cochicine ( 0.1% ) the surviving seedling percentage was around 50%. The survival rate droppe d to 0 to 20% at 0.3% colchicine concentration. The seedling survival rate dropped to almost half when the exposure period was increased from 12 hours to 24 hours for every concentration. These results indicate that the seedling survival rate is inversely proportional to the concentration and exposure periods of colchicine.
39 A B Fi gure 2 1. Flow cytometry histograms representing seedlings from selection UKP 1 A) tetraploid profile. B) mixoploid profile Figure 2 1 shows the histograms obtained from the ploidy analyzer for a non chimeric tetraploid and chimeric samples. The most effective concentration at which the tetraploids were regenerated was 0.1%. This in agreem ent with Oiyama and Okudai (1986 ), who reported that 0.1% of colchicine was the best concentration for tetraploid induction in shoot tips in citrus. Most of the tetraploids produced were derived from the treatments with 0.1% colchic i ne concentration. Concentration of 0.2% at 12 hours of exposure period also produce d 2 stable tetraploids, 1 each for selection s 5 1 99 2 and C2 5 12, respectively. Table 2 2 lists number of tetraploids and mixoploids obtained from each treatment as well as the corresponding tetraploid induction eff i ciencies. There were a total of 5 tetraploids regenerated from the 3 selections. Different selections generated varying number s of tetraploids and mixoploids. Three tetraploids were produced in selection UKP1 two in 5 1 99 2 and one in C2 5 12. There were also three mixoploids obtained from UKP1, one from 5 1 99 2 a nd two from C2 5 12. Mixoploids are commonly found when the targeted tissue is muticellular. In such cases, a few cells are muta genized and the others remain diploid. When these partially mutated
40 meristems differentiate to form plant organs, a mixture of t etraploid and diploid tissue is observed. Table 2 2 Survival rate, ploidy and tetraploid induction efficiency from treatments with various colchicine concentrations and exposure periods from selections 5 1 99 2, UKP1 and C2 5 12 of p ummelo ( Citrus grand is ) Treatment Seedling survival (%) Mixoploid Number Tetraploid Number Tetraploid Induction Efficiency 5 1 99 2 Control 93 0.1% X 12 hours 53 1 1 35 0.1% X 24 hours 47 0 0 0 0.2% X 12 hours 27 0 1 18 0.2% X 24 hours 20 0 0 0 0.3% X 12 hou rs 20 0 0 0 0.3% X 24 hours 7 0 0 0 UKP 1 Control 100 0.1% X 12 hours 50 2 2 67 0.1% X 24 hours 44 1 0 0 0.2% X 12 hours 31 0 1 21 0.2% X 24 hours 19 1 0 0 0.3% X 12 hours 13 0 0 0 0.3% X 24 hours 6 0 0 0 C2 5 12 Control 100 0.1% X 12 hours 53 1 0 0 0.1% X 24 hours 40 0 1 27 0.2% X 12 hours 33 1 0 0 0.2% X 24 hours 20 0 0 0 0.3% X 12 hours 0 0 0 0 0.3% X 24 hours 7 0 0 0 Tetraploid induction efficiency is a good measure to find out the most effective treatment as it ta kes into account both seedling survival rate and number of tetraploids produced The highest TIE of 67% was obtained from treatment with 0.1% concentration of colchicine and 12 hour exposure period in selection UKP1.
41 A B C Figure 2 2 Dried an d successfully recovered seedlings. A) Browning of the epico tyl before the leaves emerged. B ) N ecrosis of the seedling and subsequent death afte r the emergence of the leaves. C ) A stable tetraploid confirmed by flow cytometry micrografted on to a vigorous r ootstock Treatments with higher concentrations all with 0.3% and most with 0.2% and some of 0.1% were lethal. The seedlings showed necrosis with subsequent death even before the new flush emerged (Figure 2 2 A ). Some of the surviving seedlings from
42 c oncentrations of 0.2 and 0.3% showed mixed hyperploidy. Such plants were unstable and ceased to grow after a certain point of time. One tetraploid obtained from selection UKP1 and a mixoploid from 5 1 99 2 reverted back to diploid after 8 weeks. The revers ions of the tetraploids into diploids show that they were not stable over time. The tetraploids produced need to be tested at multiple time intervals to confirm their stability. The stable tetraploid plants confirmed by flow cytomet ry were micrografted ont o vigorous trifoliate rootstocks for further growth and were transferred to the greenhouse (Figure 2 2C ). Conclusion The tetraploids from elite monoembryonic pummelo selections selected for their red flesh and superior quality may be of significant value i n grapefruit triploid breeding programs. In this study, a method to induce tetraploidy in seedlings by treating pre germinated seeds with colchicine at various concentrations and exposure periods is described. Stable tetraploids were successfully produced in all three selections and were confirmed by f low cytometry. This method facilitates treatment of large number of seeds at the same time considering safety concerns when working with colchicine as opposed to the shoot tip grafting where individual shoot t ips that have to be treated separately.These tetraploids are potential female parents in interploidy crosses for triploid breeding and will be used to produce red fleshed seedless pummelo/grapefruit types.
43 CHAPTER 3 SELECTION OF ALTERED PLOIDY LEVELS FROM NATURAL POPULATIONS OF MONOEMBRYONIC PUMMELOS (CITRUS GRANDIS) Introduction Seedlessness is one of the most important commercial traits in citrus cultivar s Seedlessness is associated with triploidy. Triploid breeding programs attempt to incorporate seedl essness trait in to commercial cultivars by natural and artificial ploidy manipulations. Seedless cultivars have been successfully produced by crosses between diploid and tetraploid parent s These days, seedlessness is becoming popular and seedless cultivar s have been produced in many fruit crops like grapes, watermelon, banana and c itrus. In c itrus, because grapefruit is very popular for its taste and nutritional benefits, there is a demand for improved seedless cultivars for fresh fruit in commercial grape fruit market s Even in the juice industry, seedy cultivars are not desirable as they are associated with unfavorable aroma and bitterness. However, production of triploids has been limited by the scarcity of suitable tetraploid parents in the grapefruit ge ne pool, which could be used as parents in interploidal crosses. Polyploids can occur spontaneously in nature. The Hong Kong kumquat i s the first naturally occurring tetraploid to be discovered (Longley, 1925). Since then m any tetraploids have been sele cted and identified from natural populations in different citrus species (Frost, 1943; Lapin. 1937; Nakamura, 1942; Russi and Torrisi, 1951). However the frequency of naturally occurring tetraploids varies with the cultivar and species In general, tetrap loidy ranges from <1 to 6%. Spontaneous tetraploids have also been found in c itrus relatives including Poncirus trifoliata (Lapin, 1937). In grapefruits, tetraploids were re cently obtained by selecting seedling s of Hudson grapefruit on the basis of thick er roots, thicker and darker leaves, and slow er growth rate, followed by
44 confirmation of tetraploidy by flow cytometry (Grosse r et al, personal communication ). In polyembryonic genotypes, tetraploidization seems to occur more frequently as a result of chro mosome doubling in the nucellar tissue (Cameron and Frost, 1968). There has not been any report of naturally occurring tetraploid s in monoembryonic cultivars. Monoembryonic tetraploids are very significant in interploidal crosses when used as female parent s, as they eliminate the need to rescue the hybrid embryo saving time and labor. Pummelo being monoembryonic and an ancestor of grapefruit is of great interest to grapefruit breeders who are trying to incorporate the diversity from pummelos in to improved grapefruit type hybrid s. Triploids occur with comparatively higher frequencies in c itrus cultivars Triploids with frequencies of 15.45%, 14%, 11.33% and 9.33% were found in Kaghzi, Foster, Kinnow and Musambi respectively (Lapin, 1937; Usman et al., 2006 ). The smaller and abnormal seeds in monoembryonic cultivars selected from 2x X 2x crosses have higher probability for triploidy (Esen and Soost, 1977; Wakana et al., 1981; Soost, 1987). Th e triploidy is such seeds originate from 2n megagametophytes fertil ized by 1n (Esen and Soost, 1971, 1973; Geraci et al., 1975). Although the tetraploids and triploids can be identified with cytogenic, biochemical and molecular techniques, the screening of large populations is difficult, time consuming, expensive and ine fficient. Selection on the basis of ploidy characteristics like thicker roots and l eaves, darker color, and slow growth rate reduces the number of plants to be screened. This avoids screening of major portion of population with low probability of being tet raploid Screening of the selected seedl ings via flow cytometry is
45 an efficient way to confir m specific ploidy level. In this study, the natural populations of five p ummelo selections were screened for triploids and tetraploids. Materials and M ethods Plant Materials Five p ummelo selections 5 1 99 2, HBJL 9, HBJL 7, 8 1 99 2 and 5 1 99 3 located at the Citrus Research and Education Center (CREC in Lake Alfred, FL ) All are from open pollinated seedlings 1 99 2 which on the basis of their superior quality and red flesh color. The seeds were extracted from the mature fruit from these selections in 2008 2009. The extracted seeds were washed under running water 3 4 times and were treated with 0.6% (v/v ) sodium hydroxide for 8 minutes to remove pectin and parts of flesh attached to the seeds. The seeds from each selection were dr ied and stored separately at 8 C until used. Methodology The study w as divided into 2 experiment s. The f irst experiment involved finding natural triploid embryos in smaller seeds. Small or abnormal seeds were selected from each selection. From s elections 5 1 99 2, HBJL 9, HBJL 7, 8 1 99 2 and 5 1 99 3; the number of smaller/abnormal seeds selected w ere 11, 21, 26 and 17 respectively. The seeds were dried, peeled and sterilized using 10% Clorox bleach and 1 2 drops of Tween 20 for 8 minutes followed by 3 4 rinses with sterile water. The sterile seeds were dissected aseptically under a stereomicroscope with 100 X magnification to extract the embryo E xcised embryo s were cultured on EME nutrient medium (Grosser and Gmitter, 1990) and incubated at 25 2C under continuous light conditions.
46 Figure 3 1. Pummelo seedlings plante d in placed in the greenhouse In the second experiment, the objective was to identify tetraploids from natural po p ulation s based on morphological markers. The normal seeds extracted from each trays filled with commercial potting mix (Figure 3 1) The trays were placed in a greenhouse at 28C and 80% relative humidity. Irrigation was given three times a week or as required After, 8 10 weeks of growth of seedlings in the greenhouse, the seedlin gs were pulled out and were screened for potential tetraploids. The selection of the seedlings was done on the basis of thicker roots, slow growth rate and relatively dark color of the leaves. The seedlings were uprooted and roots were washed with running tap water prior to examination For selection 5 1 99 2, 117 out of 470 seedlings were chosed for screening via flow
47 cytometry ; for HBJL 9, 129 out of 837; for HBJL 7, 143 out of 682; for 8 1 99 2, 92 out of 422; and for 5 1 99 3, 130 out of 725 Ploidy A n alysis Ploidy level of the selected seedlings was analyzed using a f low cytometer. This technique makes it possible to analyze 150 200 genotypes per day. Flow cytometry works by estimating the volume and florescence of isolated nuclei. The ploidy is re pres ented by a histogram of integral fluorescence with the peaks depicting the ploidy level of the respective sample. The p rotocol is a series of steps starting with excision of a 0.05 0.1 cm 2 piece of fully expanded leaf tissue. S amples of 3 4 leaf pieces were pooled in a 50mm plastic P etri dish and chopped with a sharp razor blade after adding few drops of n uclei e xtraction b uffer. After chopping, 6 7 more drops of n uclei e xtraction b uffer were added and the samp le was filtered through a 50m filter into a 3.5 mL (55mmX12mm) Sarstedt tube. The nuclei staining buffer was added drop by drop through the filter to infiltrate the remaining cells, until half of the tube was filled. Each sample was incubated for 10 15 seconds at room temperature before running it through the flow cytomet er Observation of two different peaks from a pooled sample represented those samples with altered ploidy. Results and Discussion In the first experiment, embryos rescued from the smaller seeds of all the selections gave all diploi d plants as confirmed by flow cytometry. V ery few embryos were found in abnormal and shriveled seeds. In the shriveled and abnormal seeds, the embryos if present were brown in color and smaller as compared to the normal ones. These embryos when put on the EME nutrient media, failed to germinate and ultimately
48 showed high rates of diploid megagametophytes which resulted in formation of triploid embryos (Esen and Soost, 1977 ; Wakana et al., 1981; Soost, 1987). However, these studies were done with tangors and mandarins. There are no report s of such study in Pummelo. Difference in species of citrus probably is the reason these results differ from the previous findings. The rat es of 2n gametophytes production varies with species and is probably lower for p ummelo. In the second screening e xperiment, no tetraploid s were found in any of the se edling populations However, a triploid seedling was found from selection HBJL 7. The desc ribed morphological characteristics are not specific to tetraploid identification, but potentially increase the efficiency of identifying actual tetraploids following flow cytometry analysis. In polyembryonic genotypes, tetraploidization occurs more frequ ently as a result of doubling of chromosomes in the nucellar tissue. The s ame seed can contain a diploid seedling from a zygotic embryo and one or more tetraploid seedling s from the nucellar tissue surrounding that embryo. The re is a low prob ability that a zygotic embryo will produce a monoembryonic genotypes, since no nucellar tissue is present the chances of tetraploidization of the zygotic embryo is extremely low. For this to happen, an unreduced megagametophyte would need to be fertilized by diploid po llen. Rates of unreduced megagametophy tes vary with genotype Even if they are present at high rates, it would be an extremely unlikely that diploid pollen from a tetraploid or a diploid would be able to fertilize an unreduced gametophyte considering the c ompetition the diploid pollen has with haploid pollen. Also, under natural conditions, tetraploid plants
49 which must act as the pollen donors in this case are not growing commercially as scion cultivars. Thus the availability of such 2n tetraploid pollen w ould be very limited The triploid found from the cultivar HBJL 7 is likely the result of haploid pollen from a diploid plant fertilizing an unreduced mega gametophyte. The frequency of such events would depend on the rates at which unreduced gametophy t es a re pro duced in a genotype Conclusion The monoembryonic p ummelo can rarely produce naturally occurring triploids. However the frequency varies with genotype Monoembryony limits the occurrence of tetraploids in the natural populations in absence of a trip loid block Hence, Screening for tetraploid s in natural populations is fruitful only for polyembryon ic genotypes in citrus
50 CHAPTER 4 PRODUCTION OF COLCHICINE INDUCED AUTOTETRAPLOIDS IN PUMMELO ( CITRUS GRANDIS ) VIA INDIRECT ORGANOGENESIS Introduction G rapefruit ( Citrus paradisi ) is an important crop in Florida and is grown for fresh fruit and juice. Florida contributes almost 3 0% to the world grapefruit production Grapefruit is believed to have originated in Barb a dos as an accidental cross between a p u mmelo ( C grandis ) and a sweet orange ( C sinensis ) (Scora, 1 975; Barre t t and Rhodes., 1976; Scora et al., 1982; Gmitter, 1995). The increasing competition and popularity of grapefruit in internati onal markets have stimulated gra pefruit breeders to develop new Grapefruit vary in flesh color with w hite, pink and red cultivars being available. The pink and red flesh ed varieties are most popular and earn higher prices in the markets compared to the white ones. Anot her highly desired trait in commercial grapefruit cultivars these days is seedlessness. Seedless varieties of fruits like banana, watermel on, grapes, plantain are available in the market and sell more than the ir seedy counterparts. Seedlessness has gained importance in citrus, in the recent past. Seediness is causing problems in the acceptance of the fruit in the local or international markets and can even act as a barrier towards release of a variety. Breeders worldwide are trying to genera te seedless cult ivars with improved quality and disease resistance. Seedlessness has been achieved in the past through approaches ranging from traditional hybridization to biotechnology. The s eedlessness has been long associated with triploidy. Seedless triploids have be en resulted from selection of spontaneously occurring triploids in natural populations (Geraci et al., 1975; Wakana et al., 1981) from somaclonal variation (Deng et al., 1985), from diploid X diploid crosses (Esen and
51 Soost, 1971; Geraci et al., 1977; Ger aci, 1978) somatic hybridization between a haploid and a diploid (Kobayshi et al., 1997), from endosperm culture (Wang and Chang, 1978; Gmitter et al., 1990; Chen et al., 1991), from genetic transformation (Koltunow et al., 1998), and from interploidal hy bridization between a tetraploid and a diploid (Esen and Soost, 1972). Interploidal hybridization is the most c ommon and efficient way for breeders to generate triploid cultivars. However, triploid breeding programs always face a shortage of quality tetrap loids for use as parents in such crosses. Crosses where tetraploids are used as female parents are more efficient and have much higher triploid recovery than the reciprocal cross This is due to normal fertilization between female dipl oid and male haploid gametes (2n + 1 ) (Esen and Soost, 1972; Soost and Cameron, 1975; Cameron and Burnett, 1978). However, when the tetraploid female parent is a polyembryonic cultivar, the hybrid embryo needs to be rescued under sterile conditions and has to be grown in vitro This is performed to avoid the suppression of the zygotic hybrid triploid embryo by the dominating nucellar embryo/s that are present This technique is not cost or labor effective and lowers the effectiveness of triploid breeding programs. On the other hand, use of a monoembryonic cultivar as a female parent in such crosses eliminates the need of embryo rescue and is more efficient in terms of ease, labor and cost inputs. One approach to overcome the limitation polyembryony imparts, is to utilize the pum melo gene pool in grapefruit breeding. Pummelo makes an ideal candidate to be included in grapefruit breeding programs as it is one of the ancestor s of grapefruit and is a true species and presents much more genetic diversity It is monoembryonic and would eliminate the need of embryo rescue when used in interploidal crosses as a
52 female parent. A large range of red fleshed pummelo selections is available in the CREC germplasm that can be used as potential paren ts. Pummelos have lower quantities of undesirab le compounds like naringin and f uranocoumarins. Thus, when used in crosses, it should be possible to generate desirable hybrids with reduced levels of these compounds Colchicine is an alkaloid obtained from Colchicum autumnale which acts as a mitotic inhi bitor (Blakeslee and Avery, 1937) and induces tetraploidy in the target cells by interfering with spindle formation at metaphase. Colchicine is commonly used to induce tetraploidy in breeding lines in Citrus Early attempts were performed by Barrett (1978) in monoembryonic cultivars to generate autotetraploids by treating the axillary buds with colchicine e x v itro However, his technique did not produce a non chimeric tetraploid plant Later, autotetraploids were produced in monoembryonic cultivars from co lchicine treatment of axillary buds i n vitro (Oiyama and Okudi, 1986), somatic embryogenic callus (Wu and Mooney, 2002) and shoot tip s (Juarez et al., 2004). However, there are no reports of t etraploid induction in pummel o at present. This study reports an efficient method to induce tetraploids by i n vitro treatment of cut stem explants from pummelo selections with colchicine followed by shoot induction via indirect organogenesis. The effect of different colchicine concentrations and exposure durations were compared for efficiency of indirect organogenesis and tetraploid induction. Materials and M ethods Plant M aterials Pummelo selections c ybrid Hurado Buntan (C HBP), 5 1 99 3, HBJL 7 and HBJL 5 selected for this study on the basis of their red flesh and othe r quality characteristics.
53 These selections are all derived from Hirado Buntan pink pummelo and are located at the Citrus Research and Education Center (CREC). The seeds were extracted from the mature fruit from each selection in 2008 2009. The seeds we re treated with sodium hydroxide for 4 5 minutes for slime removal and rinsed 3 4 times with water. The seed s were dried and refrigerated at 4 C u ntil used. For the experiment, the seeds from each selection were peeled and sterilized separately with 10% C lorox bleach and 2 drops of Tween 20. Individual seed was put into 15 cm long test tubes containing 15 ml of solid MS medium consisting of MS salts and vitamins (Murashige and Skoog 1962) supplemented with 30 g/l sucrose and 7 g/l agar, pH 5.8 These were t hen placed in the dark for about 4 weeks. The etiolated conditions were provided to facilitate and h asten stem elongation and suppress lateral branching. At about 4 weeks, when the seedlings attained a height of about 12 15 cm they were put under continuo us light conditions for 4 5 days to allow greening and hardening of the stems. At this time, the seedlings were ready to be treated. Colchicine Treatments After hardening of the seedlings, the leaves were removed and the stem was cut into about 1cm long ex plants by making a diagonal cut at both ends. For each selection, colchicine treatments were applied separately. The experiment was designed in a 4 x 4 two way factorial design with 4 colchicine concentrations including the control and 4 exposure periods. Five replications were performed for each treatment. A Colchicine stock solu tion was prepared by dissolving the reagent in dimethylsufoxide (DMSO) in sterile water to a concentration of 1g/ml. This solution was filter sterilized and stored at 20 C The cu t explants were incubated in conical Falcon tubes containing 10 ml of liquid DBA 3 medium. Colchicine dissolved in DMSO was
54 added to each flask at concentrations of 0.01, 0.05 and 0.1%. DBA 3 shoot induction medium con tains the cytokinin growth regulator 6 b enzylaminopurin e (BAP) which stimulates cell divis ion and hence increases the frequency of dividing cells for the colchicine to act on. The untreated c ontrol treatment consisted of explants immersed in DBA 3 media without colchicine. Each treatment at di fferent concentrations was performed for exposure periods : 4, 8, 16 and 32 hours. These flasks were p laced on the rotary shaker at 30 rpm under cond i tions of 25 2C and darkness for the respective treatment duration to facilitate contac t and penetration of colchicine in the cells at the cut end. After the explants were exposed for the re quisite treatment they were p laced in 100x20mm P etri dishes on solid DBA 3 medium and were incubated in the dark for 2 weeks to induc e callus from the cut ends. The exp lants were then moved from dark to the continuous light conditions to facilitate shoot induction via indirect organogenesis. The explants with emerging shoots from the cut ends were transferred to RMB+ medium supplemented with gibberellic acid (GA3) which promotes shoot elongation. When shoots were about 1 2 cm long they, individual elongated shoots were moved to the rooting medium for further hardening and growth. Emergent shoots were analyzed for their ploidy level through a flow cytometer at a stage when the shoots had about 3 expanded leaves. The tetraploid and mixoploid shoots confirmed by flow cytometry were propagated by m icrografting technique onto vigorous rootstocks. The micrografted mixoploids and tetraploids were put under shade for 10 14 days af ter which they were moved to the greenhouse with 21 17 C day night temperatures for acclimatization.
55 Ploidy A nalysis Ploidy was analyzed using a tabletop Ploidy Analyser flow cytometer (Partec GmbH, Germany) This technique makes it possible to analyze 150 200 genotypes per day. Flow cytometry works by estimating the volume and florescence of isolated nuclei. The ploidy is presented in form of a histogram of integral fluorescence with the peaks depicting the ploidy level of the respective sample. The p rot ocol is a series of steps starting with excis i on of a 0.2 0.3 cm 2 piece of fully expanded leaf tissue and placed in a 50mm plastic P etri dish The sample was chopped with a sharp razor blade after adding few drops of Nuclei Extraction Buffer. After chopp ing, 6 7 more drops of Nuclei Extraction Buffer were added and the samp le was filtered through a 50m filter into a 3.5 mL (55mmX12mm) Sarstedt tube. The staining buffer Diaminidino 2 phenylindole (DAPI) was added drop by drop through the filter to infiltrate the remaining cells, until the half of the tube was filled. Each sample was incubated for 10 15 seconds at room temperature before running it on the flow cytometry. Results and Discussion Colchicine had a negative effect on the number of mutate d shoots at all concentrations and all exposure periods. This is attributed to the toxic nature of colchicine. The colchicine concentration and the exposure time of the explants were the key factors affecting indirect organogenesis as well as generation of tetraploids. These two factors have been recognized previously as two main parameters affecting tetraploidization by Sanford (1983). Colchicine treatments had also affected indirect organogenesis adversely. The explants from treatments with higher concent rations of colchicine or longer exposure periods did not produce as much callus at the cut ends as
56 compared to the control and the lower concentration and duration treatments. Callus production at the cut ends is a healing response of the tissue to the inj ured cells at the cut. Colchicine seemed to impair the ability of the stem tissue to heal by inhibit ing callus production at the injury sites. A B Figure 4 1. Indirect organogenesis. A ) Cut explants placed on DBA3 shoot induction medium to induce shoo ts via indirect organogenesis. B ) C loser look of the shoots emerging from the callus produced from the cut ends of the stem pieces. The rate of s hoot induction via indirect organogenesis decreased progressively with the increase in the concentration a nd duration of the colchicine treatments. Treatments with higher concentrations of colchicine or longer exposure periods had significantly fewer shoots as compared to those from control treatments or less severe treatments (Figure 4 2) The decrease in sho ot i nduction is probably due to decreased callus production in the severe treatments. Reduced callus production provides less active surface area for shoot formation Also, colchicine toxicity caused mortality of the cells which resulted in less embryo for mation and subsequent shoot induction. In all the
57 selections, the treatments with concentration of 0.01% and 4 hours of exposure duration produced the maximum average number of shoot s ranging from about 25 to 50. The h ighest number of shoots for this treat ment was produced for selection C HBP. Lowest numbers of shoots were produced from treatments with highest colchicine concentration of 0.1% and maximum exposure of 32 hours. This treatment produced 1 5 shoots emerging from the explants in all the selection s except in selection HBJL 5 where it totally suppressed shoot induction. A B Figure 4 2 Average Number of shoots produced through indirect organogenesis from colchicine treatments in p ummelo seedlings from selections A) 5 1 99 3. B)
58 C H BP. C) HBJL 7 D ) HBJL 5 (mean separations at each hour by calculating standard error of mean at 5% level). C D Figure 4 2. Continued Indirect organogenesis from the cut ends of the explants allowed for induction of polyploidy in the target cells more efficientl y. Colchicine concentrations of 0.05% and 0.1% induced maximum tetraploidy (Table 4 1). Out of the 19 total tetraploids produced
59 from the 4 pummelo selections, 10 were produced from the treatment with a concentration of 0.1% and 6 were produced from treatm ent with a concentration of 0.05%. Table 4 1. Effect of in vitro application of colchicine to the indirect organogenesis of seedlings from different pummelo selections and ploidy level of the regenerated shoots Pummelo Selection Conc. Exposure Period A vg # of shoots Tetra ploids Mixo ploids C HBP 0.01% 4 50 0 0 C HBP 0.01% 8 19.6 0 0 C HBP 0.01% 16 3.4 0 0 C HBP 0.01% 32 20.4 0 0 C HBP 0.05% 4 14.25 1 0 C HBP 0.05% 8 20.4 0 1 C HBP 0.05% 16 2.75 2 1 C HBP 0.05% 32 8.8 0 0 C HBP 0.10% 4 3.8 1 0 C HBP 0.10% 8 6 0 0 C HBP 0.10% 16 2 0 0 C HBP 0.10% 32 0.6 2 0 5 1 99 3 0.01% 4 25.33 0 0 5 1 99 3 0.01% 8 16 0 0 5 1 99 3 0.01% 16 43.67 3 0 5 1 99 3 0.01% 32 14.67 0 0 5 1 99 3 0.05% 4 1.67 0 0 5 1 99 3 0.05% 8 7.33 0 0 5 1 99 3 0.05% 16 12 0 0 5 1 99 3 0.05% 32 10.3 1 2 5 1 99 3 0.10% 4 1.25 4 1 5 1 99 3 0.10% 8 5 2 0 5 1 99 3 0.10% 16 7 0 0 5 1 99 3 0.10% 32 4 1 1 HBJL 5 0.01% 4 25.67 0 0 HBJL 5 0.01% 8 15.67 0 1 HBJL 5 0.01% 16 2.67 0 0 HBJL 5 0.01% 32 29 0 0 HBJL 5 0.05% 4 21.33 0 0 HBJL 5 0.05% 8 4 1 0 HBJL 5 0.05% 16 21 1 1 HBJL 5 0.05% 32 7.67 0 0 HBJL 5 0.10% 4 0.33 0 0 HBJL 5 0.10% 8 12 0 0 HBJL 5 0.10% 16 0.67 0 0 HBJL 5 0.10% 32 0 0 0
60 Though few tetraploids were produced from treatments with the lowest concentration of 0.01%, these treatments were more effective at longer durations. Apparently, colchicine applied at 0.1% is most efficient for tetraploid induction by this technique. This colchicine concentration was also recognized as optimal for treatment of citrus sh oot tips for tetraploid induction by Oiyama and Okudai (1986). The number of tetraploids and mixoploids produced in the 4 selections varied. Selections 5 1 99 3, C HBP, HBJL 7 and HBJL 5 produced 11, 6, 0 and 2 tetraploids and 4, 2, 1 and 2 mixoploids resp ectively. HBJL 7 failed to produce any non chimeric tetraploid plants The differential response of the selections in the number of mutated plants can be explained by the variation in the susceptibility of the genotypes to colchicine. Such genotypic effect s leading to variable results have been reported previously (Aleza et al., 2009; Ganga and Chezhiyan, 2002; Stanys et al., 2006). Figure 4 3 displays the histogram obtained from the ploidy analyzer via Flow cytometry for a tetraploid sample showing a singl e tetraploid peak, whereas Figure 4 4 displays a histogram for a mixoploid sample showing two peaks depicting the presence of two ploidy levels in the same sample. The tetraploids were propagated by micrografting to vigorous trifoliate rootstock seedlings. Mixoploids were discarded as they are unstable, often sterile and of little use for the breeding process.
61 Figure 4 3. Peak obtained by flow cytometry from a tetraploid shoot of C HBP produced by colchicine treatment in vitro Figure 4 4 Diplo id and tetraploid p eaks obtained by flow cytometry from a mixoploid shoot of C HBP produced by colchicine treatment in vitro
62 Figure 4 5. Browning of pummelo seedling stem piece explants caused by higher colchicine concentration and duration The ex plants from the treatments with higher concentrations and longer exposure periods showed browning as well as necrosis (Figure 4 5) Browning prevented the explants from producing callus and subsequent shoots. Such explants turned from light brown to dark b rown in color and eventually died. Conclusion The results from this study demonstrated successful production of non chimeric tetraloid plants from monoembryonic pummelo selections selected for their red flesh and superior quality. In this study, a method t o induce tetraploidy in the non apomictic seedlings by treating cut stem sections with colchicine at various concentrations and exposure periods is described. The shoot induction was through indirect organogenesis i.e. from the callus produced from the cut ends of the treated explants. Stable tetraploids were successfully produced in 3 out of 4 selections and were confirmed by flow cytometry. The tetraploids obtained could be of significant value as parents in grapefruit triploid breeding programs.
63 CHAPTER 5 COLCHICINE INDUCED POLYPLOIDY IN VIVO IN PUMMELO ( CITRUS GRANDIS L. OSBECK ) Introduction Considering the increasing po p ularity and trend of seedless fruits in fruit market, breeding programs are increasing efforts to produce seedless fruit. In seedless grapefruits ( Citrus paradise Macf. ), the most appealing characteristic for commercial markets is flesh color. Today, there is a need of grapefruit varieties that are seedless and red fleshed with their other quality characteristics intact. Triploid breedin g is one of the most efficient way s to produce seedless cultivars. This process can involve an int e rploidal cross between a tetraploid and a diploid parent. Since most citrus cultivars are diploid, there are very few tetraploids available to be used for su ch interploidal crosses. Production of superior tetraploids i s first step in the process of breeding triploid lines. Grapefruit has a narrow gene pool since it is a group of related cultivars that have arisen through mutation from a single hybrid genotype Inclusion of pummelos ( C. grandis L. Osbeck) in grapefruit breeding program s is being considered as an approach to increase the genetic diversity of grapefruit since it is one of the proposed ancestors of grapefruit Pummelos being monoembryonic, when use d as female parent in interploidal crosses, eliminate the need to rescue hybrid emb r yo s, with saving s in cost and labor inputs. Pummelos have l ower levels of undesirable chemicals such as furanocoumarins and naringin than in grapefruits. Their inclusion as parents in crosses should help reduce these compounds in the subsequent triploid hybrids Numerous pummelo selections with dark red flesh and superior quality are available in the CREC germplasm, which can be used for tetraploid induction.
64 The m ost common ly used method to produce tetraploids is by treatment with colchicine. Colchicine is a mitotic inhibitor that interferes with spindle formation during metaphase and hinders chromosome separation. Colchcine has been used in a number of ways and on various p lant parts to induce tetraploidy. Complete autotetraploids in monoembryonic cultivars have been produced via the i n v itro application of colchicine to the axillary buds (Oiyama and Okudi, 1986), shoot tips (Juarez et al., 2004) and somatic embryogenic call us (Wu and Mooney, 2002). Barret t (1974) attempted to induce tetraploidy using in vivo techniques but failed to produce a complete stable te t raploid. There has been no report of tetraploid production in mature citrus tissue using colchicine. However, tetra ploid s from mature tissue and the technique involved would be highly significant for breeding programs as it reduces the total time taken for triploid product i on by 4 5 year s and such a tetraploid sh ould be true to type. Materials and Methods Plant Materi als Pummelo selections used for this study were 5 1 99 5, UKP 1 and C 2 5 12. All these selections are derived from Hirado Buntan pink pummelo and are located at the Citrus Research and Education Center (CREC in Lake Alfred, FL ). These selections were ch osen on the basis of their dark pink to red flesh color and other quality characteristics. Materials and Methods Fresh budwood from each pummelo selection was harvested from the 6 10 year old trees in the field early in the morning. Leaves and thorns were removed using clipper s and the budwood was wash ed 3 4 times with soap and rinsed with water. The ideal size of the budwood for this experiment is from stems 0.3 0.5 cm in diameter. The
65 stems were cut into bud sticks each containing 4 5 buds. The bud stick s were immediately stored in o n ice until used. Otherwise, bud sticks can be stored under refrigerat ion for 1 2 week s. However, for this experiment, fresh budwood was preferred and the bud sticks were grafted on the same day they were harvested from the fi eld. These bud sticks were further prepared by placing an absorbent cotton ball on each bud followed by ti ght wrapping of bud stick with N escofilm keeping the lower cut end unwrapped. The bud sticks were then cleft grafted onto the experimental rootstoc k O range 1 Clepatra+Argentine trifoliate orange). In the c left grafting technique the rootstocks were prepared by making a horizontal cut perpendicular to the main axis of the stem to be grafted. Then a vertical slit of about 1 2 cm was made down the axis of the stock. Scion was prepared by making two 1 2 cm opposing smooth cuts tapering towards the lower end of the bud stick. The freshly prepared bud sticks were inserted in the split created in the rootstock. The scion was not inserted to the center axis but to one side to allow cambium of the rootstock and the scion to be in good contact. Finally, t he graft union was wrapped tightly with N escofilm Colchicine Treatments The experime nt was designed as a 1 X 2 two way factorial design where two factors were considered. The treatments consisted of one colchicine concentration of 0.1% and two exposure durat ions of 24 hours and 48 hours. The c olchicine stock solution was prepared by disso lving it in dimethylsulfox ide (DMSO) to a concentration of 1g/ml. This solution was filter sterilized. The final concentration of 0.1% was prepared using distilled water. This solution was sucked into a surgical disposable syringe with a needle attached to it (Figure 5 1) In the greenhouse, the colchicine application was
66 applied by injecting this solution o n cotton balls placed up on each bud through the N escofilm For control treatments, distilled water was injected in to the cotton balls in a similar mann er. Considering the hazardous nature of colchicine, the application was done very cautiously by covering the applicator body with protective clothing, gloves and a face shield. Colchicine was appli ed twice a day to keep the cotton moist in contact with the bud. Application was done once in the morning at around 9 a.m. and second t ime in the evening at around 5 p m. for the respective treatment durations. After the specifi ed treatment duration, N escofilm was removed with the help of a sharp razor blade and t he cotton balls were discarded. However, N escofilm at the graft union was not removed. The bud sticks were sprayed with the anti transpirant C loudcover to avoid drying and desiccation of the bud sticks immediately after unwrapping, and before any of the buds sprout ed The grafted plants were irrigated twice a week or as need ed Observations were made for number of buds sprouting from each treatment. The sprouted buds were tested for their ploidy to determine the number of mixoploids and tetraploids obtain ed from each treatment.
67 Figure 5 1 Application of 0.1% colchicine to the cotton balls placed on the grafted seedling pummelo buds with a surgical syringe and a needle Ploidy A nalysis Ploidy level of the sprouting shoots from the treated buds was determined by flow cytometery usi ng a Partec Flow cytometer GmbH Minster Germany. Flow cytometry works by estimating the volume and florescence of isolated nuclei. The ploidy is presented in form of a histogram of integral fluorescence with the peaks dep icting the ploidy le vel of the respective sample. Each s ample was taken by cutting a small piece of tissue of about 0.2 0.3 cm 2 from each sprouted leaf from each shoot. For this study, the samples were not pooled and ploidy was determined for each ind ividual sample. The tissue was put in a 50mm plastic petri dish and chopped with a sharp razor blade after adding few drops of n uclei e xtraction b uffer. After chopping, 6 7 more drops of n uclei e xtraction b uffer were added and the samp le was filtered throu gh a 50m filter into a 3.5 mL (55mmX12mm) Sarstedt tube. The staining buffer was added drop by drop through the filter to infiltrate the remaining cells, until the half of the tube was filled. Each sample was incubated for 10 15 seconds at room temperatur e before running it on flow cytometry. The amount of staining buffer absorbed by the tissue was directly proportional to DNA content of the sample. Results and Discussion The sprouting of the shoots from the buds was delayed by colchicine in all 3 selecti ons for all treatments. B uds in control treatments started sprouting in about 4 7 days after r emoval of the cotton balls and N escofilm wrap. In contrast for the treated buds, shoots did not emerge for about 3 4 weeks after being exposed to colchicine. Th e rate of sprouting and growth was slower for the treated buds than controls.
68 Colchcine seemed to affect the rate and frequency of sprouting of the treated buds. Each graft had 4 buds treated with colchicine. The a verage number of buds that sprouted from each graft for selections 5 1 99 2, C2 5 12 and UKP 1 was 1.77, 2, 1.69 respectively for the treatment with 24 hours of exposure and was 1.54, 2.06 and 1.62 respectively for the treatment with exposure period of 48 hours. Bud sprouts from control treat ments were significantly higher than those from any of the colchicine treatments for all the three pummelo selections (Table 5 1 ). The buds that did not sprout became necrotic. In i tially, t he meristem die d and then the entire bud collapsed T here was no di fference between the effects of exposure duration on the number of buds that sprout ed Table 5 1. Number of buds sprouts from the colchicin e treatment for pummelo selections 5 1 99 2, C 2 5 12 and UKP 1 Cultivar Treatment Avg number of sprouts per graft ( 24 hrs) Avg number of sprouts per graft (48 hrs) 5 1 99 2 Control 4 0 4 0 5 1 99 2 0.1% 1.77 1.05 1.54 0.84 C2 5 12 Control 4 0 3.67 0.47 C2 5 12 0.1% 2 0.88 2.06 0.73 UKP1 Control 4 0 3.67 0.47 UKP1 0.1% 1.69 0.72 3.67 0.92
69 A B Figure 5 2 Colchicine treated grafts. A ) sprouting of the shoot s from colchicine treated buds B ) B ud sticks with cotton balls on the buds grafted onto the rootstocks No ne of the treatment s induce d complete tetraploidy in any of the selectio ns. Barret t (1974) did a similar study where he tried to induce tetraploidy i n vivo by treating the axillary buds from mature tissue of monoembryonic cultivar with 0.1% colchicine but did not produce any complete tetraploid shoot s from the bud sprouts. He induced many cytogenic chimeras in the three histogenic layers. Although a different technique was used in the present study tetraploidy was not induce d Use of larger tissues is a limitin g factor and the most probable reason for lack of success ( Sanford. 1983). Larger plants as opposed to plants grown in vitro have broadly differentiated meristematic tissue making i t difficult to mutagenize the entire tissue by applying colchicine on the surface only.
70 The chimeras induced in all the three selections were only from the treatments which had exposure duration of 2 days. The percentage of chimeras produced by colchicine at 0.1% concentration and 48 hours of expo sure period are shown in Table 5 2 for the 3 pummelo selections. Table 5 2 Percentages of cytochi meras produced from the buds treated by colchicine Cultivar Cytocjimeras at 0.1% 24 hrs Cytochimeras at 0.1% 48 hrs 5 1 99 2 0 26.30 C2 5 12 0 12.5 UKP1 0 18.75 The differential response for chimeras of the pummelo selections is most probably due to genotypic susceptibility to colchicine. The cytochimeras produced varied in their number of diploid cells and tetraploid cells. Chimeras which had higher tetraploid proportion confirmed by flow cytometry were from the lower buds which did not sprout until upper buds were clipped to remove apical dominance. The axillary buds on these shoots were allowed to sprout and give further shoots. One of such chimera from selection 5 1 99 5 produced a shoot which was tetraploid. However, this shoot was not stable ove r time at this ploidy level and subsequently reverted back to diploid.
71 Figure 5 3 Flow cytometry histograms representing budsprouts with mixoploid profile (diploid and tetraploid cells) in selection 5 1 99 5 Conclusion In this study, tetraploidy in duction in the mature tissue of pummel o selections was studied. Colchicine treatments did not result in the production of a complete tetraploid plant but instead induced cytochimeras Apparently it is very difficult to induce tetraploidy in mature and bro adly differentiated tissues. Improved techniques should be trie d in future for similar studies
72 CHA PTER 6 SOMATIC HYBRIDIZATION OF GRAPEFRUIT + PUMMELO TO PRODUCE GRAPEFRUIT/PUMMELO TYPE ALLOTETRAPLOIDS Introduction Seedless fruits have gained great comme rcial importance in local and international markets over the recent pas t. The trend has continued for c itrus as well. Higher demand for seedless citrus varieties has led to increased efforts towards the breeding of seedless triploids Improvement of red g rapefruit ( Citrus paradisi Macf .) being widely produced in the U.S., has also been included in triploid breeding efforts. Red or pink flesh color is another desirable trait popular in commercial grapefruit cultivar s. A commonly used way to produce triplo id cultivars is through interploidal crosses between tetraploid and diploid parents (Esen and Soost, 1973) However, the challenge in using this method is limited by availability of tetraploid parents in grapefruit. Grapefruit has a narrow gene pool since it is a group of related cultivars that have arisen through mutation from a single hybrid genotype In general, most citrus cultivars are diploid but there are a few rare naturally occurring triploids and tetraploids Most breeding programs induce tetrapl oidy in established lines to produce superior tetraploid pa rents to be used for interploidal crosses. Another challenge in grapefruits is its polyembryony characteristic that complicates interploidal crosses by requiring embryo rescue for triploid embryo recovery, adding extra cost and labor inputs Pummelo ( Citrus grandis L. Osbeck ), being an ancestor of grapefruit, should be a good parent to facilitate the production of improved grapefruit/pummelo like triploid varieties. When used as a female parent i n interploidal crosses, pummelos have an advantage that no embryo r escue is required because of their monoembryony.
73 Somatic Hybridization offers a unique way of producing tetraploids where two diploid genotypes can be combined in to one allotetraploid This technique can also circumvent biological problems including nucellar polyembryony and pollen/ovule sterility, faced during conventional breeding processes. Several tetraploid somatic hybrids produced in the past few decades (Guo and Deng, 2001; Grosser et al., 2000) have been successfully used as breeding parents to pr oduce seedless triploids (Grosser et a l., 1998). Some of the reports where protoplast fusion has successfully produced new allotetraploid somatic hybrids involving pummelo include Ananthakris hnan et al. (2006) and Grosser et al. (2007). Somatic hybrids produced using grapefruit embryogenic suspension cultures were reviewed in Grosser et al. (2000), including 9 lo. Materials and Methods Protoplast Isolation, Fusion, and Culture Suspension cultures of ed grapefru it from the friable callus line initiated in Texas (kindly provided by E. S. Louzada, Weslaco Citrus Center, Texas A&M) were maintained in citru s callus collection of Citrus Research and Education Center (CREC in Lake Alfred, FL ). The c allus lines were maintained for several years and stability of the embryogenic potential was not determined However, since this was the only red fleshed grapefrui t callus line available, the protoplast fusion experiment s were carried out using this line Suspension cultures were established and maintained in H+H medium on a subculture cycle of 2 weeks and the protoplasts were isolated during the period from 4 to 1 2 days (Grosser and Gmitter, 1990) Grafted plants of p ummelo selections 8 1 99 2, 5 1 99 2, UKP 1, 5 1 99 5, were maintained in the greenhouse.
74 They were placed in the shade for few days before healthy and fully expanded leaves were taken for protoplast i solation. Suspension cultures were incubated in 0.6M BH3 protoplast culture medium and enzyme solution according to Grosser and Gmitter ( 1990). Leaves from the pummelo selections were sterilized by immersion in 20% C hloro x bleach w ith 2 drops of Tween T wenty surfactant for 15 minutes. They were rinsed with sterile distilled water 3 4 times and feathered with a sharp scalpel before overnight incubation in 0.6M BH3 protoplast culture medium and enzyme solution. Protoplasts from both s uspension cultures and leaves were passed through 45 m stainless steel mesh screen and centrifuged thereafter on a 25% sucrose/ 13% mannitol gradient (Grosser and Gmitter, 1990). Protoplast fusion was performed in 60 X 15 mm polystyrene petri dishes using the standard method described by Grosser and Gmitter (1990). PEG (40%polyethylene glycol) was used to induce fusion of the suspension cultur e derived and the leaf derived protoplasts to form heterokaryons. After fusion, p rotoplast s were initially cultured in a 1:1 (v:v) mixture of 0.6M BH3 and 0.6M EME prot oplast culture media The petri dishes were sealed with Nescofilm and placed under low light conditions. Osmoticum reduction and transfer to solid EME mannitol medium for somatic embryo induction were pe rformed according to Grosser and Gmitter (1990). Callus R ecovery and Attempted Induction of Somatic Embryogenesis Although the somatic fusion experiments were conducted numerous times, micro calli recovery was inconsistent and successful somatic embryo in duction from recovered calli was had lost its totipotency. Several additional experiments were conducted
75 (troubleshooting) in efforts to improve micro calli recovery and to ac hieve somatic embryo induction as necessary for somatic hybrid plant regeneration, and are described as follows. Modification of Protoplast Culture Conditions Following fusion, the protoplasts were routinely cultured in a 1:1 (v:v) s olution of 0.6M BH3 an d 0.6M EME in a volume of approximately 2 ml per 60x15mm petri dish. After unsuccessful somatic embryo induction, t he quantity of this protoplast culture media used in later experiments was reduced to half in an effort to improve gas exchange Subsequent experiments also tested the culture of protoplasts following fusion directly in either 0.6M BH3 or 0.6M EME protoplast culture media separately. Use of i n v itro p lants Protoplast isolation and fusion was tried with in vitro grown pummelo selections in con trast to those maintained in the greenhouse. This eliminated the step of leaf sterilization prior to protoplast isolation and reduced the possibility of contamination in the leaf protoplast preparations. Nurse c ulture Following fusion, unfused protoplasts isolated from vigorous and totipotent embryogenic suspension cultures of W. Murcott a dded to the fusion protoplast cultures in the 1:1 (v:v) 0.6M BH3 and 0.6M EME protoplast culture media in efforts to stimulate growth and embryogenesis Tre nitiation PVP (polyvinylpyrrolidone) at 1g/l was added to the DOG embryogenic callus induction medium (Gross er and Gmitter, 1990) on which the callus lines were cultured prior to suspension culture initiation in H+H medium (Grosser and Gmitter, 1990). In
76 another experiment, neutralized activated charcoal c harcoal at 1g/l was added to DOG media (Grosser and Gmitter, 1990) on which the callus lines were cultured prior to suspension culture initiation. This was done in an effort to improve the health of the callus and to remove potential impurities. Results and Discussion Numerous somatic hybridization experiments were conducted in efforts to combine protoplast s isolated from suspension cultures of leaf protoplasts isolated from various pummelo selections ; however, no somatic embryos or plants were recovered following protoplast culture. The callus line from which the suspension cultures were established had been maintained for many years, and had yellow/ brown color in contrast to the white color of normal embryogenic callus callus lines. Initially, the protoplast isolation and protoplast fusion process was normal and the fused heterokaryons were visible when observed using an inverted microscope. The protop l ast s underwent normal mitotic division 2 3 d ays following fusion. However, later cell divisio n seemed to be arrested and microcalli often started to shrink. A light brown layer of some unknown compound was formed on top of the protoplast culture media. It appeared that the protoplast s suspensio n cultures were releasing some unknown brown exudates, which might have been inhibitory to the growth of the regenerating protoplast derived microcalli In efforts to improve callus recovery following fusion, various approaches were tested as described abo ve. Reducing the volume of protoplast culture in the petri dishes to increase protoplast density and improve gas exchange did not improve callus
7 7 recovery. Protoplasts cultured in 0.6M BH3 or 0.6M EME separately (rather than the usual mix) also failed to i mprove microcalli growth The brown exudates were still produced on the surface of the liquid media. Experiments to determine if the greenhouse source of pummelo leaf protoplasts was the problem, by using in vitro grown pummelo leaves as an alternative, al so failed to improve the results, and the brown exudate was still observed. Finally, the use of unfused W. Murcott nurse protopalsts isolated from a totipotent vigorous suspension culture were added to cultures following fusion still did not signi ficantly improve the results. The idea was to provide nurse cells for the better growth of the fused protoplasts. However, the exudate seemed to inhibit the growth of protoplasts from both the fusion protoplasts and the nurs e protoplasts All protoplasts shrank and died eventually. Since, it was clear that the protoplast derived microcalli growth was inhibited by unknown compounds fro ; PVP and activated charcoal were added to the DOG media (sepa rately) on which callus lines were maintained prior to suspension culture initiation. PVP and activated charcoal adsorb excessive phenolic as well as non phenolic compounds released in the media by plant cells and can prevent these compounds from affecting cell growth However, addition of these compounds did have any health or growth promoting effect on the callus. slow growing, non embyogenic and continued to exhibit the light yello w /brown color (Figure 6 1)
78 Figure 6 1. allus after being sub cultured for 6 weeks on DOG media containing 1g/l PVP. It is extremely difficult to recover somatic embryos from callus lines which have been maintained for several years, probably due to the build up of cytological mutations Gmitter et al (1991 ) treated cultures exhibiting low embryogenic capacity with colchicine in order to induce tetraploidy. They also observed that colchicine treatment recovered the embryogenic capacity of the callus. However, colchicine was not tested to improve the totipotency of the agen and c ould have altered the ploidy level of the callus line. Conducting somatic hybridization with protoplasts of varying ploidy levels would probably not enhance the results Future efforts to obtain somatic hybrids between grapefruit and pummelo, as necessary to achieve the goals of this project, wo uld be to start with a new freshly initiated totipotent or other suitable grapefruit callus line However, the time
79 period available for this study could not accommodate this process as embryogenic callus induction and habituation is a long process, especially with grapefruit (J.W. Grosser, personal communication).
80 CHAPTER 7 SUMMARY AND CONCLUSIONS The successful pro duction of tetraploid pummelo plants from diploid counterparts was achieved through mutating the ploidy with the mitotic inh ibitor colchicine. Stable tetraploid plants were recovered from zygotic seedlings from several high quality red pummelo selections From numerous somatic hybridization experiments conducted to generate allotetraploid hybrids combining grapefruit and pummelo, none were successful. Although the somatic hybridization via protoplast fusion technique is well established and the experime nt s were well planned, the failure was attributed to the unavailability of totipotent friable embryogenic callus lines from a red fleshed grapefruit. Vario us methods were tried to improve the existing non totipotent but all were unsucces sful. For future attempts at somatic hybridization it will be more beneficial to utilize a freshly initiated totitpotent e mbryogenic callus line of grapefruit for suspension culture initiation. Treating pummelo seedling stem segments with colchicine successfully induced tetraploidy in the tissue of induced adventitious shoots Regeneration of complete and stable tetraploid plants from this technique proved to be superior, as the shoots emerge from a single cell. A mutated tetraploid s ingle cell c ould produce a non chimeric tetraploid plant Colchicine at 0.1% was observed to be the optimum concentration for this technique. This technique produced 16 tetraploid plants which were confirmed to be stable 6 months after micrografting. Treatm ent of pre germinated seeds with colchicine also successfully resulted in induced tetrapl oidy. However, this technique was more likely to produce mixoploids shoots, as the ta rget tissue in this case is a multi celled meristem Higher concentrations of colc hicine were tried in this experiment which
81 caused higher mortality. The i deal colchicine concentration for inducing tetraploids was however the same 0.1%. This technique produced 3 tetraploid plants which were confirmed to be stable 6 months after micro grafting. It should also be pointed out that treating the pregerminated seeds is much less laborious tha n treating the in vitro stem segments with respect to setting up the experiment as well as screening of the treated seedlings/shoots. In the in vivo exp eriment tetraploidy induction in the mature tissue of pummel o selections was studied. Colchicine treatments did not result in the production of any stable tetraploid plant s, but induced cytochimeras instead. Apparently it is very difficult to induce tetra ploidy in mature and broadly differentiated tissues. Improved techniques should be trie d in future for similar studies. The o ccurrence of natural triploids in monoembryonic p ummelo s was apparently very low and varies with genotype as only one triploid pla nt was recovered from pummelo HBJL 7 Monoembryony limits the occurrence of tetraploids in the natural seedling populations in absence of a triploid block The selection of tetraploids in a natural population of seedling plants is not practical in monoembr yonic pummelo. H ence, s creening for tetraploid s in natural seedling populations is fruitful only for polyembryon ic genotypes that produce nucellar seedlings. The final conclusion is that the overall project can be considered a success, because more than 20 stable tetraploid plants were recovered from zygotic seedlings of the various parents. These autotetraploid pummelos have been successfully micrografted to vigorous rootstocks and can now be grown in the field for subsequent use as parents in the CREC triploid breeding program. Efforts to top work these new
82 autotetralpoid pummelos to existing mature field trees, to expedite their flowering and fruiting, are underway.
83 APPENDIX CITRUS PROTOPLAST ME DIA AND SOLUTIONS Table A 1. Compo sition of the EME medium. Component mg/L NH 4 NO 3 1,650 KNO 3 1,900 KH 2 PO 4 170 MgSO 4 .7H 2 O 370 CaCl 2 .2H 2 O 440 Na 2 EDTA 37.30 FeSO 4 .7H 2 O 27.80 MnSO 4 .H 2 O 22.30 ZnSO 4 .7H 2 O 8.60 H 3 BO 3 6.20 KI 0.63 Na 2 MoO 4 .2H 2 O 0.25 CuSO 4 .5H 2 O 0. 025 CoCl 2 .6H 2 O 0.025 Thiamine.HCl 10 Pyridoxine.HCl 10 Myo inositol 100 Malt extract 500 Nicotinic acid 5 50 g/L sucrose was added for 0.146 M EME and 205.38 g/L sucrose for 0.6 M EME. For 1500 EME malt extract was added at 1500 mg/L an d sucrose at 50 g/L. Solid medium contains 8 g/L agar Table A 2 Composition of sucrose and mannitol solutions (CPW salts). Component mg/L MgSO 4 .7H 2 O 250 KNO 3 100 KH 2 PO 4 27.20 KI 0.16 CuSO 4 .5H 2 O 0.00025 CaCl 2 .2H 2 O 150
84 Table A 3. Com position of 0.6 m BH3 nutrient medium. Component mg/L KH 2 PO 4 170 MgSO 4 .7H 2 O 370 CaCl 2 .2H 2 O 440 Na 2 EDTA 37.30 FeSO 4 .7H 2 O 27.80 MnSO 4 .H 2 O 22.30 ZnSO 4 .7H 2 O 8.60 H 3 BO 3 6.20 KCl 1,500 KI 0.63 Na 2 MoO 4 .2H 2 O 0.25 CuSO 4 .5H 2 O 0.025 CoCl 2 .6H 2 O 0.025 Glutamine 3,100 Thiamine.HCl 10 Pyridoxine.HCl 10 Myo inositol 100 Malt extract 500 Casein hydrolysate 250 Nicotinic acid 1 Mannitol 81,990 Sucrose 51,350 (85,560 for 0.7 M) Coconut water 20 mL Fructose 250 Ribose 250 Xylose 250 Mannose 250 Rhamanose 250 Cellobiose 250 Galactose 250 Glucose 250 Sodium pyruvate 20 Citric acid 40 Malic acid40 40 Fumaric acid 40 Vitamin B12 0.02 Calcium pantothene 1 Ascorbic acid 2 Choline chloride 1 p aminobezoic acid 0.02 Folic acid 0.40
85 Table A 3. Continued Component mg/L Riboflavin 0.20 Biotin 0.01 Vitamin A (retinol) 0.01 Vitamin D3 (cholecalciferol) 0.01 Table A 4 Composition of protoplast tr ansformation solutions. 40% polyethylene glycol (PEG) (MW = 8000) 0.3 M Glucose 66 mM CaCl 2 pH = 6 II Solutions A and B Solution A Solution B Component g/100 mL Component g/100 mL Glucose (0.4 M) 7.20 Glycine (0.3 M) 2.2 CaCl2 (66 m M) 0.97 DMSO 10 mL pH 6.0 pH 10.5
86 Table A 5 Composition of DBA3 medium. Component mg/L NH 4 NO 3 1,485 KNO 3 1,710 KH 2 PO 4 153 MgSO 4 .7H 2 O 333 CaCl 2 .2H 2 O 440 Na 2 EDTA 37.30 FeSO 4 .7H 2 O 27.80 MnSO 4 .H 2 O 21.40 ZnSO 4 .7H 2 O 7.70 H 3 BO 3 5.58 KI 0.567 Na 2 MoO 4 .2H 2 O 0.225 CuSO 4 .5H 2 O 0.0225 CoCl 2 .6H 2 O 0.0225 Thiamine.HCl 9 Pyridoxine.HCl 9 Myo inositol 90 Nicotinic acid 4.5 Coconut water 20 mL Malt extract 1,500 2,4 D 0.01 DAP 3 Sucrose 25,000 Agar 8,000
87 Table A 6 Composition of the enzyme solution used for citrus protoplast isolation. Mannitol 0.7 M CaCl2 12.0 mM MES1 (buffer) 6.0 mM NaH2PO4 1.4 mM Onozuka RS cellulose 1% Macerase or macerozyme 1% Pectolyase Y 23 0.2% pH = 5.6 )
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98 BIOGRAPHICAL SKETCH Divya Kainth was born in Chandigarh, India in 1985. She graduated from Gyan Jyoti Public School in 2003. She received a bache lors of science in a griculture from Punjab Agricultural University, Ludhiana, Punjab, India, with an honors degree with a at University of Florida and completed her d egree in December 2 010.