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Indirect Shoot Organogenesis and Selection of Somaclonal Variation in Dieffenbachia


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1 INDIRECT SHOOT ORGANOGENESIS AND SELECTION OF SOMACLONAL VARIATION IN Dieffenbachia By XIULI SHEN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Xiuli Shen

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3 To my parents

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4 ACKNOWLEDGMENTS For my accomplishments, I thank my mother, We njin Sun, my father, Haifu Shen, my two younger brothers, Qi Shen and Bin Shen for th eir unconditional love an d unreserved support. I also own my deepest thanks to my son, Hao Xu, for bringing me the happiness and encouragement during years of my study. I would also like to sincerely thank Dr. Mich ael E Kane and Dr. Jianjun Chen for their mentoring over the last 4 years; Thanks also is given to th e other committee members, Dr. Richard J Henny, Dr. David J Norman, Dr. Sloane M Scheiber, for their support. Without them it would have been impossible to su ccessfully complete this project. Acknowledgement also goes to the College of Agricultural and Life Sciences, Mid-Florida Research and Education Center, and Dr. Chens program for providing a gr aduate assistantship to support this research. I must also thank the people in the Pl ant Restoration, Conservation and Propagation Biotechnology program: Nancy Philman, Carmen Valero-Aracama, Pete Sleszynski, Scott Stewart, Phil Kauth, Tim Johnson, Chris Dudding, Dani ela Dutra, for their help in many ways to this project. Thanks also go to people in the Environmenta l Horticultural Department as well as those outside of the department who contributed to th e completion of this study Special thanks go to all my friends in China, Canada and the U. S. for sharing all of my ups and downs.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .........9 LIST OF ABBREVIATIONS........................................................................................................10 ABSTRACT....................................................................................................................... ............11 CHAPTER 1 LITERATURE REVIEW.......................................................................................................13 Introduction................................................................................................................... ..........13 Dieffenbachia Genus..............................................................................................................13 Botany......................................................................................................................... .....13 Propagation.................................................................................................................... ..14 Breeding....................................................................................................................... ...15 Developmental Pathways for Plant Regeneration..................................................................16 Organogenesis.................................................................................................................17 Definition.................................................................................................................17 Advantages of Shoot Organogenesis........................................................................17 Phases of Shoot Organogenesis................................................................................17 Factors Affecting Shoot Organogenesis..........................................................................19 Genotypes.................................................................................................................19 Explants....................................................................................................................20 Plant Growth Regulators..........................................................................................21 Somatic Embryogenesis..................................................................................................22 Definition.................................................................................................................22 Stages of Somatic Embryogenesis...........................................................................22 Morphology and Physiology of Somatic Embryos..................................................24 Histological Studies on Shoot Orga nogenesis/Somatic Embryogenesis..................25 Somaclonal Variation........................................................................................................... ..26 Origin of Somaclonal Variation......................................................................................27 Cultivars...................................................................................................................27 Explant Sources........................................................................................................27 Callus........................................................................................................................28 Duration of the Tissue Culture Phase.......................................................................28 Measurement of Somaclonal Variation...........................................................................28 Phenotypic Variations..............................................................................................28 Changes at Chro mosomal Levels.............................................................................29 Variations at DNA Levels........................................................................................30

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6 2 PROTOCOL ESTABLISHMENT FOR I NDIRECT SHOOT ORGANOGENESIS FROM LEAVES OF Dieffenbachia CV. CAMOUFLAGE...................................................31 Introduction................................................................................................................... ..........31 Materials and Methods.......................................................................................................... .32 Media and Sterilization Conditions.................................................................................32 Plant Materials and Establis hment of Shoot Cultures.....................................................32 Indexing of Established Cultures.....................................................................................33 Callus Induction...............................................................................................................33 Indirect Shoot Organogenesis..........................................................................................33 Histological Analysis.......................................................................................................34 Acclimatization................................................................................................................35 Statistical Analysis..........................................................................................................35 Results........................................................................................................................ .............36 Callus Induction...............................................................................................................36 Indirect Shoot Organogenesis..........................................................................................36 Acclimatization................................................................................................................37 Histological Analysis.......................................................................................................37 Discussion..................................................................................................................... ..........38 Callus Induction and Shoot Formation............................................................................38 Acclimatization................................................................................................................39 Histological Analysis.......................................................................................................40 3 Factors affecting indirect shoot organogenesis in Dieffenbachia ...........................................46 Introduction................................................................................................................... ..........46 Materials and Methods.......................................................................................................... .47 Plants Materials, Medi a and Culture Conditions.............................................................47 Callus Induction...............................................................................................................47 Shoot Induction...............................................................................................................48 Acclimatization................................................................................................................49 Statistical Analysis..........................................................................................................49 Results........................................................................................................................ .............49 Explant Effects................................................................................................................49 Genotypic Effects............................................................................................................49 PGR Effects.................................................................................................................... .50 Acclimatization................................................................................................................52 Discussion..................................................................................................................... ..........52 Genotype Effects.............................................................................................................53 PGR Effects.................................................................................................................... .54 Carry-Over Effect of PGRs.............................................................................................55 Explant Effects................................................................................................................55 Acclimatization................................................................................................................56 4 ASSESSMENT OF SOMACLONAL VARIATION IN Dieffenbachia PLANTS REGENERATED FROM INDIRECT SHOOT ORGANOGENESIS AT PHENOTYPIC LEVEL..........................................................................................................60

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7 Introduction................................................................................................................... ..........60 Materials and Methods.......................................................................................................... .61 Callus Induction...............................................................................................................61 Sustained Callus Culture.................................................................................................62 Shoot Induction...............................................................................................................62 Acclimatization................................................................................................................62 Determination of Somaclonal Variation..........................................................................63 Examination of the Duration of Callus Cu lture Affecting Somaclonal Variation..........64 Experimental Design and Statistical Analysis.................................................................64 Results........................................................................................................................ .............64 Somaclonal Variant Identification...................................................................................64 Genotype Differences......................................................................................................65 Culture Duration Effects on Somaclonal Variation.........................................................66 Effects of Callus Subculture Number on Shoot Regeneration........................................66 Discussion..................................................................................................................... ..........66 Somaclonal Variation and Genotypes.............................................................................66 Callus Culture Duration...................................................................................................68 REFERENCES..................................................................................................................... .........77 BIOGRAPHICAL SKETCH.........................................................................................................87

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8 LIST OF TABLES Table page 2-1 Effect of TDZ and 2,4-D supplementation to BM on frequency of callus formation from leaf explants of Dieffenbachia cv. Camouflage cultured for 8 weeks in dark and 4 weeks in a 16 h light photoperiod at 40 mol m-2 s-1.....................................................42 2-2 Effect of 2iP and IAA concentrations and combinations on shoot regeneration from Dieffenbachia cv. Camouflage calli cultured for 8 weeks in a 16 h light photoperiod at 40 mol m-2 s-1...............................................................................................................43 3-1 Effects of TDZ and 2,4-D on the frequency of callus formation on leaf explants and shoot number per callus of Dieffenbachia 4 cultivars Camouflage, Camille, Octopus and Star Bright................................................................................................................ ...58 4-1 Quantitative evaluation of somaclonal variants of Dieffenbachia cv. Camouflage regenerated by indirect s hoot organogenesis and grow n in the greenhouse for 8 months......................................................................................................................... .......71 4-2 Quantitative evaluation of somaclonal variants of Dieffenbachia cv. Camille regenerated by indirect s hoot organogenesis and grow n in the greenhouse for 8 months......................................................................................................................... .......73 4-3 Effects of genotype on the number a nd rate of somaclonal variation among Dieffenbachia plants regenerated by indirect sh oot organogenesis and grown in the greenhouse for 8 months....................................................................................................74 4-4 Effects of the duration of callus cult ure on the number and rate of somaclonal variation of Dieffenbachia cv. Camouflage regenerated by indirect shoot organogenesis and grown in the greenhouse for 8 months................................................75

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9 LIST OF FIGURES Figure page 2-1 Indirect shoot organogenesis in Dieffenbachia cv. Camouflage.......................................44 2-2 Histological evidence of indirect shoot organogenesis in Dieffenbachia cv. Camouflage at different developmenta l stages when cultured on BM medium supplemented with 5 M TDZ and 1 M 2, 4-D for callus induction and on BM medium supplemented with 40 M 2iP and 2 M IAA for shoot induction.....................45 3-1 Characterization of calli cultured on BM supplemented with 5 M TDZ and 1 M 2,4-D after 8 weeks culture in dark for cv s. Camouflage, Camille and Octopus (12 weeks for cv. Star Bright) and 4 week s culture in a 16 h light photoperiod......................57 3-2 Indirect shoot organogenesis in Dieffenbachia cv. Camille..............................................59 4-1 Plants of Dieffenbachia cv. Camouflage regenerated by indirect shoot organogenesis showing variation in leaf variegation and color.................................................................70 4-2 Plants of Dieffenbachia cv. Camille regenerated by i ndirect shoot organogenesis showing variation in leaf shape..........................................................................................72 43 Effects of subculture number on shoot regeneration from calli of the 3 Dieffenbachia cultivars...................................................................................................................... ........76

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10 LIST OF ABBREVIATIONS 2,4-D 2,4-dichlorophenozyacetic acid 2iP N6-( 2 isopentenyl) adenine BA 6-benzyladenine BM basal medium CPPU [N-(2-chlor o-4-pyridyl)-N-phenylurea GA3 gibberellic acid NAA 1naphthalene acetic acid PGR plant growth regulator TDZ thidiazuron

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INDIRECT SHOOT ORGANOGENESIS AND SELECTION OF SOMACLONAL VARIATION IN Dieffenbachia 11 By Xiuli Shen May 2007 Chair: Michael E Kane Cochair: Jianjun Chen Major: Horticultural Science A series of experiments were conducted to i nvestigate the feasibil ity of selecting of somaclonal variants for new cultivar development in Dieffenbachia In the first set of experiments, a protocol for indirect shoot organogenesis was established for Dieffenbachia cv. Camouflage. Maximum 96% callus formation frequency was observed on a basal medium supplemented with 5 M TDZ and 1 M 2,4-D. The maximum shoots regenerated per callus (7.9) was obtained on a basal medium supplemen ted with 40 M 2ip and 2 M IAA. In the second set of experiments, 4 Dieffenbachia cultivars were examined for the capacity for indirect shoot organogenesis and effects of genotypes, explant sources and plant growth re gulators were investigated. There were significant genotypic effects on both callus formation and shoot regeneration. Cultivar Camouflage exhibited the gr eatest ability for indirect shoot organogenesis, while cv. Octopus had no capacity for shoot rege neration from calli. Only leaf explants taken from in vitro shoot cultures were capable of callus fo rmation. Root explants failed to undergo indirect shoot organogenesis, rega rdless of cultivar. In the third set of experiments, somaclonal variation at the phenotypic level among Dieffenbachia plants regenerated via indirect shoot organogenesis was evaluated. Three types of so maclonal variations wi th different leaf variegation and color were observed in cv. Camouf lage with a total somaclonal variation rate of

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12 40.4%. Somaclonal variation in l eaf shape was observed in c v. Camille with a somaclonal variation rate of 2.6%. No somacl onal variation was observed in re generated plants of cv. Star Bright. The duration of callus cultu re of cv. Camouflage had no e ffect on somaclonal variation as variation rates between plants regenerated from 8 months and 16 months callus culture were similar. Our results indicated that selection of somaclonal variation has great potential for new cultivar development in Dieffenbachia

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13 CHAPTER 1 LITERATURE REVIEW Introduction The genus Dieffenbachia belongs to the family Araceae a nd is one of the most popular ornamental foliage plants. Its popularity is largel y attributed to its attr active foliar variegation, which is generally manipulated throu gh breeding. Traditional breeding in Dieffenbachia however, is hindered by its na turally-occurring dichogamy an d long breeding cycle (Henny, 1988). High demand for new cultivars with novel fo liar variegation has created a need for developing more efficient br eeding methods. Selection of somaclonal variants from in vitro generated population could be an alternative means for new cultivar development. This study aimed at establishing regeneration methods for Dieffenbachia through indirect shoot organogenesis and examining the feasibility of selection of somaclonal variants from the regenerated population for new cultivar development. It is hypothesized th at plants regenerated through indirect shoot organogenesis were associat ed with high somaclonal variation because of intermediary callus formation. Si nce indirect shoot organogenesis has not been established in Dieffenbachia the first experiment was to establish a pr otocol for indirect s hoot organogenesis in Dieffenbachia The second experiment was to determ ine factors affecting indirect shoot organogenesis. The third experiment was to assess somaclonal variation among regenerated plants for potential new cultivar development. Dieffenbachia Genus Botany Dieffenbachia genus, a monocot and a member of th e family Araceae, is native to tropical regions of Central and South America (Chen et al., 2003b). It is a herbaceous perennial evergreen with thick stems bear ing alternate leaves (Black, 2 002). Flowers are unisexual and

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14 consist of spadix and spathe. The spadix is a central fleshy spike, covered with many small staminate and pistillate flowers. The spathe is a modified bract and envelops the spadix until anthesis (Henny, 1988). Self-pollination is prev ented by the naturally occurring dichogamy in this genus as female flowers mature earlier than male flowers. The leaves are broad and variegated with white marking or different patterns which are sh eathed petioles and have a very striking appearance (Henny and Chen, 2003). Thus, the value of Dieffenbachia lies in its attractive foliar variegation. Additionally, Dieffenbachia requires low light levels for its growth. As a result, Dieffenbachia is widely used as living speci mens for interior decoration and interiorscaping. Dieffenbachia is among the most popular ornament al foliage plants in the United States, continually ranking in th e top five for annual wholesal e value (Chen et al., 2002). Propagation Dieffenbachia can be propagated by both sexual and as exual methods. Seed propagation is not usually used in the production because of lim ited seed production and poor seed germination. Dieffenbachia can be easily propagated by asexual met hods, by tip, cane cutti ngs and divisions, but such methods only produce a few cuttings or plants from each stock plant and require significant labor input. Asexual propagation can also carry over pathogens. The advantage of propagation by cuttings or di visions is that plants pr opagated are true-to-type. Plant tissue culture technique can potentially overcome all these limitations of traditional approaches to Dieffenbachia In vitro culture is becoming an important method for propagation of Dieffenbachia since 1980s. An initial application of tissue cu lture in propagation of Dieffenbachia was to eliminate systemic viral and bacterial pathogen. Dieffenbachia cv. Perfection derived from lateral bud and shoot tip culture were indexed 3 separate times on each of 4 different media, and plantlets lines free of te sted fungi and bacteria were released. This line showed vigorous growth and branched more freel y due to the depletion of fungi and bacteria

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15 (Knauss, 1976; Chase et al., 1981). Later, the tissue culture was ma inly used to produce a large number of uniform and true-t o-type healthy plants for co mmercial production on a year-round basis. Through modification of media and culture conditions, the propagation rate of Dieffenbachia had been greatly increased (Voyviatzi and Voyiatzis, 1989). Tissue culture also reduces greenhouse space needed for stock plan t production and produces salable plants in a greater range of pot sizes. In addition to providing true-totype liners, tissue culture also produces off type plants. In the early application of tissu e culture for propagation of Dieffenbachia all off-type plants were rouged out to maintain the genetic fidelity of plants produced. It wa s later realized that these offtype plants could be a source for selection of somaclonal variation for new cultivar development. Since Larkin and Scowcroft (1981) advocated th at somaclonal variation could be used as a promising tool for breeding to produce novel genetic variation, some soma clones with desirable features have been obtained by this method (C hen and Henny, 2006). It generally requires about 2 to 3 years for a new cultivar development co mpared to 7 to 10 years through traditional breeding (Henny et al., 2000). Breeding Progress in breeding of Dieffenbachia has been slow due to th e long breeding cycles. The first hybrid Dieffenbachia Bausei was released in 1870 (Bir dsey, 1951), since then only about 100 new cultivars have been developed. Hybridiz ation is the most co mmon method in producing new cultivars in Dieffenbachia but this requires many crosses and careful selection. Naturally occurring dichogamy in this genus also makes th is process very laborious and time consuming. It usually takes about 7-10 years for a new cultivar to be released (Henny et al., 1986; Henny et al., 1987a, b; Henny et al., 1988a, b; Henny et al., 1991a, b, c; Henny, 1995a, b). In addition to hybridization, new cultivars are al so developed from sports or from somaclonal variation. For

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16 example, cv. Camouflage is a mutant of Panthe r, and cv. Camille is a sport of cv. Marianne. Selection of somaclonal variation from in vitro cultures has become an important method for new cultivar development. So far 20 cultivars of Dieffenbachia derived from selection of somaclonal variants have been released (Chen and Henny, 2006). As the value of Dieffenbachia lies in its aesthetic appearance, any changes in plant form, size, color or variega tion pattern can be desirable traits for Dieffenbachia Developmental Pathways for Plant Regeneration There are generally 3 developmental pathways for in vitro plant regeneration: 1) propagation from pre-existing meristems (shoot culture or nodal culture), 2) shoot organogenesis, and 3) somatic embryogenesis (n on-zygotic embryogenesis). Proliferation from pre-existing meristems refers to production of shoots from shoot meristems (shoot tips) or axillary meristems (axillary buds) followed by ro oting of individual shoot (Kane, 2000a). Shoot organogenesis is propagation from explants without pre-exis ting meristems through production and subsequent rooting of adventitious shoots (Schwarz and Beaty, 2000). Somatic embryogenesis regards to production of embryos th at are not the result of gametic fusion (Gray, 2000). Any in vitro regeneration system is associated with a certain degree of somaclonal variation and selection of somaclonal variants can be used as an important method for new cultivar development. The degree of somaclonal va riation is highly relate d with the regeneration pathway. Plants regenerated from pre-existing meristems are usually true-to-type and this method is used for clonal micropropagation. Methods involving adventitious meristem formation or a callus phase result in plants with higher levels of somaclonal variation (Bouman and De Klerk, 1997).

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17 Organogenesis Definition Shoot organogenesis refers to propagation from explants wi thout pre-existing meristems through production and subsequent rooting of adventitious shoots. There are 2 types of shoot organogenesis, direct and indir ect. Direct shoot organogenesis is the production of shoots from explants directly, while indirect refers to the formation of shoots indirectly from an intermediary callus that first develops on th e explants (Kane et al., 1994). Advantages of Shoot Organogenesis Shoot organogenesis has the following advant ages compared to traditional propagation and other in vitro propagation systems: 1) shoot organogene sis is more efficient than propagation from pre-existing meristems; 2) shoot produc tion is more synchronous than somatic embryo formation; 3) indirect shoot orga nogenesis is associated with hi gher genetic variability and have more chance for selection of somaclonal variants ; 4) survival rate is higher among plantlets produced via shoot organogenesis than those via somatic embryogenesis after transferred to greenhouse; 5) shoot organogenesi s is more easily achieved in most species than somatic embryogenesis. Phases of Shoot Organogenesis A series of cellular events o ccur in the de novo shoot merist em formation process, and it can be demonstrated in the following model: Explant competence determination shoots dedifferentiation inducti on differentiation There are generally 3 phases of shoot orga nogenesis, namely dedi fferentiation, induction and differentiation. Dedifferentiation refers to differentiated cells in explants that dedifferentiate

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18 and revert to a less committed, more flexible/plastic developmental state that may or may not involve callus formation. In the direct shoot organogenesis, some ce lls from explant tissues dedifferentiate directly to produce a new primordi al, while in the indirect shoot organogenesis, cells from explants dedifferentiate to give rise to callus forma tion from which the new shoots are produced. As a result of dedifferentiation, some of these dedifferentiated cells in explants may become competent and capable of responding to specific inducing signals. Plant growth regulators are one of these induci ng signals through which cells become competent. It has been noticed in the induction of adve ntitious shoots from immature leaves of cassava that no adventitious shoots could be obtained on regenera tion media if leaf explants were cultured on induction media for 1-3 days, indicat ing that cells has not become competent in such short time. However, after culture for 4 or more days on in duction media, explants showed regeneration (Ma and Xu, 2002). The span between the time cells become competent and when they become fully determined for primordial production refers to as the induction phase. During the induction phase, a competent cell or a group of competent cells become committed to a unique developmental fate on the stimulus of induci ng signal. At the end of induction phase, cells become fully determined and capable of shoot organogenesis even with the removal of inducing signals (Schwarz and Beaty, 2000). During the differentiation phase, morphological differentiation and development of the nascent organ occur. In their study on mo rphogenesis in petiole derived callus of Amorphophallus rivieri Durieu, Hu et al ( 2005) observed that compact callus consisted of 3 components: epidermis, subepidermis and inner parenchyma cells; cells in subepidermis started to divide to gave rise to the formation of a long and narrow meristematic zone after 1 week of

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19 culture on differentiation medium; the meristem atic masses then became more organized and formed meristematic domes by continuous anticlinal and periclinal division; the periclinal cell divisions on the flanks of the meristems resulted in leaf primordial formation after culture for 4 weeks. Through scanning electron microscopy, a gr oup of regenerating shoot buds with distinct epidermal layer, unicellular trichomes, and stom ata has been observed in callus cultures of Flacourtia jangomas (Lour.) Raeusch The presence of stom ata on the epidermal layer is thought to be a striking feature for shoot forma tion (Chandra and Bhanja, 2002). In a study on organogenesis induced from mature zygotic embryoderived leaflets of peanut, Chengalrayan et al (2001) reported that during th e initial 4 days of culture, ce lls of the explants enlarged; mesophyll cells underwent anticlin al division giving rise to gl obular cell cluster on day 7 of culture; subsequent periclinal and anticlinal di visions resulted in dome-shaped shoot meristems in 15 days; well-developed shoot buds formed after 21 days of culture. Factors Affecting Shoot Organogenesis There are many factors which affect explant tissues becoming competent, and eventually determined to complete shoot formation. Genotypes Genotype has the most influence on capacity for shoot organogenesis in vitro Some genotypes are easily cultivated via shoot organogenesis while others are either recalcitrant or exhibit no capacity for shoot organogenesis (T hao et al., 2003; Landi and Mezzetti, 2005). Significant differences in growth rate, color a nd structure of calli we re found among 4 carnation genotypes tested and these differenc es were also responsible for differentiation of calli into a certain organogenic pathway (caulogenesis, shoo t organogenesis or rhiz ogenesis). Callogenesis was not only a result of dedifferentiation of expl ant tissues, but also an essential preparatory

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20 stage for in vitro morphogenesis. Callus type can be a prediction for its organogenic potential (Kallak et al., 1997). Among 38 carnation cultivars tested for shoot organogenesis from nodal explants, 81.6% of them showed a high regeneration ability (> 50% and 4 shoots per node segment), and 18.4% showed low shoot regeneration (< 50% and 4 shoot per node segment)(Nontaswatsri et al., 2002). Phillips (2004) concluded that specific genes were involved in dedifferentiation, acquisition of competence and i nduction stages of shoot organogenesis. Some genotypes may lack the genes required for shoot organogenesis or these genes may not be functional. Plants of different genotypes may possess di fferent endogenous plant growth regulator levels and this might be responsible for differences in response in vitro Explants The developmental stage and type of explants play a critical role in the success of shoot organogenesis. The capacity of cells to become competent for regeneration may be completely lost once they mature to a point due to the elimina tion of totipotency of cells In general, explants taken from less differentiated, immature ti ssues are easily induced and regenerated via direct shoot organogenesis. Explants derived from highly differentiated tissues are less responsive in vitro and usually undergo indi rect shoot organogenes is. In study on the effect of explant types on shoot organogenesis of Vigna unguiculata Choi et al (2003) reported that the highest frequency of shoot organogenesi s was obtained from single whol e cotyledon explants (67.5%); the proximal halves of cotyledon (50.1%) showed better regenerati on than distal halves (30.1%); no shoots formed from one or two cotyledon halves with embr yo axis or embryo axis alone. Explants taken from stock plants of different developmental stages possess different endogenous hormonal level which affects their response in vitro Differences in the ability to produce adventitious shoots among differe nt explant types were also not ed in shoot organogenesis in

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21 eggplants. On MS basal medium supplemented w ith 0.2 M TDZ, the percentage of explants that developed buds and the number of buds per explant was 100% (75-100 buds per explant) for leaf, 100% (75-100 buds per explant) for cotyle don, 5% (1-25 buds per explant) for hypocotyl, 20% (25-50 buds per explant) for epicotyl, and 65% (1-25 buds pe r explant) for node explants respectively (Magioli et al., 1998). Plant Growth Regulators Plant growth regulators are the most importa nt inducing signal for shoot organogenesis. Dedifferentiation, induction and development of shoots or roots are regulated by both endogenous and exogenous growth regulators. The type of plant growth regulators and their interaction play an important role in dedifferentiation, inductio n and development of shoots or roots (Khanam et al., 2000). It is generally believed that cytoki nins are beneficial for shoot formation, while auxins stimulate callus forma tion, root formation and somatic embryogenesis. The ratio of cytokinins to auxins is also critical in determinin g shoot versus root formation. A high cytokinin to auxin ratio promotes shoot meri stem formation, while callus or root meristems are formed when the cytokinin to auxin ratio is low. Adventitious shoots were induced on media containing only BA or BA as the major PGR, while somatic embryos were produced on 2,4-D containing media in morphogenesis from leaves of cassava (Ma and Xu, 2002). It has been reported that different combination of auxins (NAA, IAA, 2,4-D) a nd cytokinins (BA, kinetin) at 0, 1, 2 mg l-1 levels resulted in either direct adven titious shoot formation, callus formation, or indirect adventitious shoot formation (Makunga et al 2005). Both exogenous cytokinin and auxin were required for shoot organo genesis from petiole explants in Nymphoides indica BA was most effective in stimulati ng adventitious shoot formation compared to 2iP and kinetin (Jenks et al., 2000). In their study on the effects of cytokinin/auxin combination on organogenesis in Duboisia myoporoides Khanam et al (2000) reporte d that the auxins 2,4-D and

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22 NAA were more effective than IAA or IBA for callus formation, the greatest number of shoots were induced with 10 M BA and 1 M NAA among the combination of BA (0, 0.1, 1, 10 ) and NAA (0, 1, 10) examined. Somatic Embryogenesis Definition Somatic embryogenesis is the production of embryos not resulting from gametic fusion (Merkle et al., 1990). Asexual embr yogenesis is known to occur na turally in many plant species and many different names are used by different au thors to describe this phenomenon in different species, such as apomixes, polyembryony, adve ntive, sporophytic and nucellar embryony. Many plant tissues, such as the nuce llus, inner integument, synergids, antipodals, endosperm, and suspensor have been observed to naturally give rise to asexual embryos (Tisserat et al., 1979). Asexual embryogenesis can also be obtained in vitro culture and this pro cess is usually called somatic embryogenesis. Since the first report of somatic embryogenesis in carrot cell culture in 1958 (Steward et al., 1958a, b), in vitro somatic embryogenesis has been reported in over 100 species (Krishnaraj and Vasil, 1995; Merkle et al., 1995). There are 2 types of somatic embryogenesis, direct or indirect. Direct so matic embryogenesis refers to somatic embryos produced directly from cells of explant tissues with no intervening ca llus. Indirect somatic embryogenesis is characterized by th e production of callus from expl ant tissues first, from which somatic embryos form (Merkle, 1997). Somatic embryogenesis has been achieved in a number of economically and ornamentally impor tant plant species, but studies on Dieffenbachia has not been reported. Stages of Somatic Embryogenesis There are generally 3 developmenta l stages in somatic embryogenesis in vitro : induction, development and maturation (Zimmerman, 1993). E xplants are composed of heterogeneous cell

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23 types. During the induction stage, undifferentiated cells from expl ants can be induced to become embryogenically determined and somatic embryos can be obtained directly from these cells via direct embryogenesis. Differentiated cells from explants undergo dedifferentiation, meristemation and redifferentiation into embryogeni c cells. In some cases, callus is formed first from explant tissues, and somatic embryos subsequen tly form. This process is referred to indirect somatic embryogenesis (Kohlenbach, 1985; Merkle 1997). Explants may also contain a portion of pre-embryogenic determined cells derived fr om ovular or embryo tissues that may develop into embryos under a suitable condition (Bhoj wani and Razdan, 1996). Somatic embryogenic cells can be distinguished from non-embryogeni c cells by the following features. Embryogenic cells usually contain dense cytoplasm, prom inent nuclei, thickened cell wall and are less vacuolated (Mooney and Van Staden, 1987). During the development stage, a single embr yogenic cell undergoes a se ries of transverse and longitudinal divisions, passe s through globular, heart, torpe do, and cotyledonary stages for dicots or globular, scutellar a nd coleoptilar stages for monocot s, and finally forms a bipolar structure with root and shoot meristems on opposite end which has the capacity to reproduce entire plants (Arnold et al., 2002). During the maturation stage, the somatic embryos accumulate reserve and achieve desiccation tolerance (Ammirato, 1974). This is very important for successful germination and subsequent growth. The maturation stage in so matic embryogenesis has just been realized recently as an important stage because the degr ee of maturation can sign ificantly affect the germination capability of somatic embryos (Bhojwan and Razdan, 1996). Normal looking somatic embryos may be physiologically immature. A period of reversible arrested growth is

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24 necessary for proper germination and regenerati on. Without such developm ental stages, somatic embryos usually germinate precociously and finally die (Gray and Purohit, 1991). Morphology and Physiology of Somatic Embryos Although somatic embryos share many histologi cal and morphological characteristics of zygotic embryos, they differ in several aspects. Unlike a zygotic embryo which has a suspensor and nourished via a suspensor, a somatic embryo may not have a suspensor or has an abnormal suspensor. Somatic embryos are typically formed from a proembryonal complex. A somatic embryo may grow directly from a proembryonal complex without the fo rmation of suspensor (Thomas et al., 1972). In other cases, somatic embryos may be supported above explants by either a narrowed, not easily observed suspenso r, or a very broad suspensor (Hakman et al., 1985). Cotyledons may develop abnormally, including multiple cotyledons, fused cotyledons or unequally sized pairs (Rashid and Street, 1973). Somatic embryos have a less compressed and narrowed appearance than zygotic embryos due to the lack of protective seed coats (Gray and Purohit, 1991). Somatic embryos tend to develop asynchronously. Several stages are presen t in cultures at any time thus somatic embryos do not mature altogether (Profumo et al ., 1986). Additionally, somatic embryos usually germinate continuously after th ey are formed without a quies cent resting phase typically observed in zygotic embryos. This leads to precoc ious germination and plants usually die after a certain period of time (Gray, 1989). Gray et al (1993) also noticed that certain parts of embryos might precociously germinate before full embryo maturation, such as trichome development on hypocotyls and cotyledons, hypocotyl elongati on, cotyledon elongation, premature root emergency in their study on somatic embryogenesis in Cucumis melo

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25 Histological Studies on Shoot Or ganogenesis/Somatic Embryogenesis Plantlets regenerated in vitro from adventitious meristems ma y originate either from shoot organogenesis or somatic embryogenesis. In shoo t organogenesis, shoots are usually formed first, then roots are induced fr om the base of shoots, with a well-developed vascular connection between shoots and maternal explants In somatic embryogenesis, shoots and roots are derived from embryogenic shoots and roots on bipolar st ructure of somatic embryos. There is no vascular connection between somatic embryos and pa rental explants (Puigderrajols et al 2000; Chengalrayan, 2001). Before conc luding that regenerants from in vitro culture have a somatic embryogenic origin, it is necessary to provide co nvincing histological evidence to prove it. A visual similarity between the morphological a ppearance of the regenera ting structures and somatic embryos is insufficient to draw the conc lusion of somatic embryogenesis. In his studies on morph-histological study of so matic embryo-like structures in hypocotyl cultures of Pelargonium (ornamental plants), Haensch (2004a, b) found that although somatic embryo-like structures were produced from hypocotyls, hist ological examination confirmed the lack of bipolarity and revealed vascular connections to the explan ts and concluded that there was no proof of somatic embryogenesis. Like zygotic embryogenesis, somatic embryoge nesis is assumed to pass through the same characteristic stages, and the demonstration of these key stages can be used as the primary criteria for confirming somatic embryogenesis. The ontogeny of somatic embryogenesis can be revealed by scanning electron microscopy or light microscopy. The normal somatic embryo development from globular, to heart-, to torp edo-shaped and finally cotyledon stage embryo has been described through scanning elec tron microscope in safflower ( C. tinctorius L.)(Mandal and Gupta, 2002). Histological events during soma tic embryo development in Serbian spruce ( Picea omorika ) have been recorded. Each somatic embr yo initials formed on the cotyledon surface

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26 after an unequal transversal division of a cell. Th e smaller, apical cell gave rise to apical dome and later on filamentous suspensor (Budimir, 2003). Somatic embryos with plumule and root pole have been documented in Musa spp. after 50 days of cultu re by passing through globular stage (Lee et al., 1997). The attainme nt of a globular appearance is regarded as one of the first key features of somatic embryo development. The presence of protoderm, the outmost layer of a developing somatic embryo, is also consider ed as a unique feature of somatic embryo development, and it is believe d that protoderm has a function of regulation of further embryo development by applying physical and cell di vision limitation (Sharma and Millam, 2004). The developing somatic embryos had no detectab le vascular connection with the mother explants (Quiroz-Figueroa et al., 2002). Two cell types can be observed u nder light microscope, embryogenic and nonembryogenic cells. Embryogenic cells are smaller, compact, and possess relatively dense cytoplasma, densely stained nuclei, and small invisible vacuoles. Nonembryogenic cells usually have large vacuoles and unclear cytoplasma. Embryogenic cells may form embryogenic masses (EMS) by continued cell division. These activ ely dividing cells may develop into globular masses, forming proembryoi ds, but only a very limited number of these embryogenic cells may give rise to meristem-like structure (meristemoid), and differentiate into different staged embryos (Onay, 2000). Somaclonal Variation Variation among plants regenerated from tissue culture has b een called somaclonal variation (Larkin and Scowcroft, 1981). From its origin, it can be deduced that somaclonal variation occurs in the period before the formati on of meristematic tissues and terminated with formation of meristematic tissues (Bouman a nd De Klerk, 1997). Somaclonal variation is a random phenomenon that can occur at any locati on in the genome (De Schepper et al., 2003). Assessment of somaclonal variation can be ach ieved by analysis of phenotype, chromosome

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27 number and structure, proteins, or direct DNA evaluation of plants (Bouman and De Klerk, 1997). The extent of phenotypic vari ation is usually determined as the percentage of plants showing aberrations from the pare ntal plant for one or more defi ned characteristics. Plants with the deviant phenotypes are known as so maclones or somaclonal variants. Origin of Somaclonal Variation Cultivars The potential for somaclonal variation varies among cultivars (genot ypes). In their study on chromosomal instability in regenerants from Medicago media Pers, Najaran and Walton (1987) found there was significant difference in the ability to produce somaclones among cultivars, being 9.4%, 10%, 20% and 64% for the 4 cultivars tested respectively. Explant Sources As mentioned previously, explan ts usually are composed of many cell types and these cells may vary in differentiation and even ploidy levels Plants regenerated from a single explant may originate from different cells. Generally speaking, explants with pre-existing meristematic tissues such as axillary buds, shoot tips and meristems s how less variation than explants that have no pre-existing meristematic tissues su ch as leaves, roots or stems (Ski rvin et al., 1994). Piccioni et al (1997) reported that no somacl onal variants were observed in al falfa plants regenerated from axillary branching propagation, bu t plants regenerated from callus derived from petioles showed greater variation (23%) analyzed by RAPD markers. In chrysanthemum ( Chrysanthemum morifolium ), plants regenerated from pedicel explants showed little variation from control plants, but considerable varian ts were found among plants regenerated from petals. These variants flowered earlier and produced more flowers than control plants (De Jong and Custers, 1986). Tubers produced on potato plants regenerated fr om leaf, stem, rachis and tuber pieces had different skin color. Regenerants derived from st em, leaf, tuber piece pro duced tubers with pink-

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28 red color as control plants while regenerants from rachis produ ced white, red-splashed white and mixed colored tubers (Wheeler et al., 1985). Callus Somaclonal variation is quite of ten associated with indirect in vitro regeneration involving an interviewing callus stage. During this period, differentiate d cells undergo dedifferentiation, induction, redifferentiation (Rout, 1999). Bouman and De Klerk (2001) showed that the rate of somaclonal variation among Begonia regenerated via somatic embryogenesis was 1.5% but 10.6% for regenerants derived from the callus stage. Duration of the Tissue Culture Phase There is a general trend that somaclonal variat ion increases with the length of time that a culture has been maintained in vitro. In a study on the genetic instability during embryogenic cloning of celery, Orton (1985) not iced that if calli, derived fro m immature petiole segment, were maintained for 6 months by a series of tran sfer, 84% of the callus cells were karologically indistinguishable from the control, the remain ing16% exhibited chromoso me loss or fusion, only 1 regenerated plant out of 95 showed an abnor mal phenotype. After 12 months in culture, 97% callus cells were ka rologically distinguishable from the c ontrol, most were aneuploidy and all callus cells lost the capac ity to produce embroids. Measurement of Somaclonal Variation Phenotypic Variations Since Skirvin and Janick (1976) advocated that somaclonal variation could be used as a promising tool for breeding, some somaclones with desirable features have been obtained. The variation could be change in fo liar variegation pattern, modifica tion of flower color and size, alteration of leaf shape, increase in lateral shoots, and change in overall plant form (Griesbach et al., 1988; Khalid et al., 1989). New somaclonal variants with desirable dwarf and bushy

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29 phenotype of lisianthus ( Eustoma grandiflorum ) have been obtained from leaf, stem culture. Naturally, all cultivars of lisianthus have a long stem with only apical br anches. Application of growth retardants and pinching are required to stimulate basal branch formation for its ornamental values (Griesbach and Semeni uk, 1987). Khalid et al (1989) produced new somaclonal variants of chrysanthemum with nove l lilac-colored ray florets using petals as explants. Dwarf daylily ( Hemerocallis ) has been selected from ca llus derived from shoot tip culture (Griesbach, 1989). Arene et al (1993) obtained rose varian ts with altered petal number, color and having dwarf grow th habit from callus of Rose hybrida cv. Meirutral. Tremblay et al (1999) reporte d that several morphological changes including dwarfism, fascination, variegate patte rns, height alternation, modified branch angle, and bushy shape were observed in somatic embryogenesis of spruce sp ecies. Griga (2000) noted that morphological alternations including altered leaflet shape, changed stipule morphology, shortened internode, irregular leaf position on th e stem, shortened flower stalk were found in pea ( Pisum sativum ) plants regenerated from immature zygotic embr yo culture. Somaclonal variants with increased disease resistance and higher yiel d traits have been obtained from wheat somatic embryogenesis (Arun et al., 2003). Changes at Chromosomal Levels Somaclonal variation can be assayed at the chromosomal level. Some somaclonal variations result from changes in the chromoso me number. Polyploidy and aneuploidy are quite often associated with morphologi cal modification. Moyne et al ( 1993) reported that tetraploid, aneuploid and polyploidy cells we re found in cultures of rose. Diploidization, aneuploidization and polyploidization have been observed in cultures of Larix decidua initiated from megagametophyte explants (Von Aderkas and A nderson, 1993). Except changes in chromosome number, alternation in chromosome structure, such as point mutation, chromosome breakdown

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30 and reunion, deletion, and insertion have been observed in plants derived from somaclonal variation (Bouman and De Klerk, 1997). Variations at DNA Levels Changing DNA sequence is a fundamental event responsible for much of the reported somaclonal variation among regenerated plants. DNA sequence changes or variations include mutations involving one or a fe w nucleotides, deletions and insertions caused by unequal crossover, or activity of transposable elements The precise way to determine mutations at DNA levels is to sequence the gene of interest. An al ternative approach is to detect variation at DNA levels by electrophoresis of protei ns and/or by PCR amplification of nucleic acid segments. The protein electrophoresis implies the existence of mutant alleles by virtue of changes in gene products. Isozyme analysis is a frequently used method of studying certain gene product changes during in vitro culture (Mangolin et al., 1997; Chen et al ., 1998; Azeqour et al., 2002; Feuser et al., 2003). A series of PCR-base d techniques have been developed over the years for genotyping and fingerprinting purposes. Among the PCR-base d methods, RAPD and AFLP have been used to analyze genetic variation among regenerants of a wide range of plant species (Chen et al., 2006). Somaclone specific fragments can actually be cloned, and nucleotide sequences can be analyzed to determine exactly what changes oc cur at DNA sequence level. Fragment cloning was performed in variants regene rated from maize, pea, and tomato (Gostimsky et al., 2005).

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31 CHAPTER 2 PROTOCOL ESTABLISHMENT FOR INDI RECT SHOOT ORGANOGENESIS FROM LEAVES OF Dieffenbachia CV. CAMOUFLAGE Introduction The genus Dieffenbachia a member of the family Araceae, is composed of 30 species native to South and Central America (Mayo et al., 1997). Dieffenbachia has been produced as an ornamental foliage plant for interiorscaping since 1864 (Birdsey, 1951) and consistently ranks among the top five most popular foliage plan t genera based on annual wholesale value (McConnell et al., 1989; USDA, 1998). In addition to its tolerance to low light levels, another factor contributing to its popularity is the increasing release of ne w and attractive cultivars that provide consumers with a wide range of selection for novelty. Interspecific hybridization has been the primary method of developing new cultivars (Henny and Chen, 2003). Hybridization of Dieffenbachia however, has been hampered by naturally occurring dichogamy, long breeding cy cle, and limited seed production (Henny, 1988). With the commercial application of in vitro propagation of Dieffenbachia new cultivars have been released following selection of so maclonal variants (Chen and Henny, 2006). The frequency of somaclonal variants is generally high, and the time required for a new cultivar release can be only 2 to 3 years compared to 7 to 10 years required using traditional breeding methods (Skirvin et al., 1994; Chen et al., 2003a). Chen et al (2004) anal yzed genetic relatedness of some cultivated Dieffenbachia using amplified fragment le ngth polymorphism (AFLP) and found that cultivars selected from somaclonal vari ants differ genetically from their parents. Successful use of in vitro techniques for producing somaclonal variants depends on the establishment of an efficient method for regenerati ng a large number of plants indirectly from an intervening callus stage (Maralappanavar et al., 2000; Niwa et al., 2002; Hossain et al., 2003; Anu et al., 2004; Arce-Montoya and RodriguezAlvarez, 2006; Hammerschlag et al., 2006).

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32 Although plant regeneration via indirect shoot organogenesis has b een achieved in a vast array of plant species, a protocol for indirect shoot organogenesis has not been developed in Dieffenbachia Currently commercial in vitro propagation of Dieffenbachia is through shoot culture (Knauss, 1976; Chase et al., 1981; Voyiatzi and Voyiat zis, 1989; Henny et al., 2000). The objective of this study was to establish a protocol for indu cing indirect shoot organogenesis in Dieffenbachia cv. Camouflage. Materials and Methods Media and Sterilization Conditions A basal medium (BM) consisting of MS (M urashige and Skoog, 1962) mineral salts, 0.4 mg l-1 thiamine, 2.0 mg l-1 glycine, 100.0 mg l-1 myo-inositol, 0.5 mg l-1 pyridoxine, 0.5 mg l-1 nicotinic acid, and 25 g l-1 sucrose in combination with pl ant growth regulators was used. Medium was adjusted to pH 5.8 with 0.1 N KOH prior to the addition of 6 g l-1 TC agar (PhytoTechnology Laboratories, Shawnee Mi ssion, KS) and autoclaved at 1.2 kg cm-2 for 20 min. Plant Materials and Establishment of Shoot Cultures Stem segments about 10 mm long, containing la teral buds, were cut from stock plants of Dieffenbachia cv. Camouflage. The stock plants were ma intained in a shaded greenhouse with a maximum irradiance of 345 mol m-2 s-1 under natural photoperiod (10 to 14.5 h light) and a temperature range of 20 to 31C. Lateral buds were excised, rinsed under running water for about 10 min and used as explants to initiate in vitro shoot cultures. After su rface sterilization in aqueous 1.2% sodium hypochlorite (20% v/v Clorox U ltra) containing seve ral drops of Tween 20 for 10 min on a shaker and rinsing 3 times, 5 mi n each, with sterile water, lateral buds were further trimmed by removing the outermost one or two bud scales. Lateral buds still attached to a 2 mm2 thick square of stem tissue were cut a nd placed individually into 25 150 mm culture

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33 tubes containing 15 ml BM supplemented with 80 M 2iP and 2 M IAA. Cultures were maintained under a 16 h light photoperiod at 40 mol m-2 s-1 provided by cool white fluorescent lamps (General Electric F20WT12CW) at 22 3C. Shoot clusters were divided and transferred to the same fresh medium every 8 weeks to increase in vitro stock shoot cultures. Indexing of Established Cultures Following the establishment of in vitro shoot cultures, cultures w ith visually detectable contamination were immediately discarded. Cultur es with no symptom of contamination were routinely indexed for the presence of cultivable bacterial and fungal contamination using the procedure developed by Kane (2000b). Callus Induction Leaves obtained from the in vitro shoot cultures served as explant sources for callus induction. Leaf explants were cut into 5 mm2 sections with mid-vei n, and the leaf margins removed with a scalpel. Leaf explants were cu ltured with abaxial surface in contact with the callus induction medium which were BM supplem ented with TDZ at 0, 1, 5, 10 M and 2,4-D at 0, 0.5, 1 M. Explants were cultured in 100 15 mm sterile Petri plat es containing 20 ml medium. There were 5 explants pe r Petri plate and 5 replicate plat es per treatment. Cultures were initially maintained in dark for 8 weeks and th en transferred to the 16 h light photoperiod at 40 mol m-2 s-1 for another 4 weeks. The number of explants forming calli was scored after 12 weeks of culture. Callus formation frequency was calculated as the percentage of leaf explants forming calli. Indirect Shoot Organogenesis To evaluate medium for inducing shoot form ation from callus, calli produced on callus induction medium containing 5 M TDZ and 1 M 2,4-D (the me dium yielding optimal callus induction) were cultured on BM supplemented with factorial combinations of (1) 2iP at 0, 20, 40,

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34 80 M and IAA at 0 and 2 M; (2) kinetin at 0, 1, 2, 4 M and GA3 at 0, 5, 10 M. Plant growth regulator-free medium served as the control. Calli were separated from leaf explants after 12 weeks callus production. Calli were cut into small pieces with a fresh weight of approximately 150 mg, and then transferred to glass baby f ood jars (4.5 7 cm) containing 40 ml medium. There were 5 callus clumps per jar and 5 replic ates per treatment. The number of shoots formed per callus was determined after 8 weeks cult ure under the 16 h photoperiod at 40 mol m-2 s-1. Histological Analysis To verify the occurrence of indirect shoot or ganogenesis, callus samples were collected for histological examination 4 weeks after the initia tion of culture and the 9 additional samples of 3 day intervals. For shoot differe ntiation, callus samples were ta ken every 3 days after callus clumps were transferred onto shoot induction me dium until 42 days after culture. For optical light microscopy (OLM), the following proce dure was used. Callus specimens weighting approximately 150 mg were fixed in Trumps fixative (McDowell and Trump, 1976). Fixative infiltration was achieved under vacuum until a ll samples of calli sank to the bottom of scintillation vials. Calli were rinsed 3 times in phosphate buffer (pH 7.2) for 10 min each. Calli were post-fixed in a 1% buffered osmium tetroxid e solution for 1 h, and then rinsed 3 times in phosphate buffer (pH 7.2) for 10 min each, followed by 3 times wash in distilled water. Calli were dehydrated in a series of ascending aqueous ethanol solutions at 25%, 50%, 75% for 30 min each, at 95%, 100% for 1 h each, followed by dehydration in 100% acetone for 1 h. Calli were then embedded in Spur resin (Spurr, 1969). Call us sections (10 m) were cut using a Leica Ultracut rotary ultramicrotome R (Leica Microscopy and Scientific Instruments, Deerfield, IL), and mounted on glass slides. Sections were stai ned with 0.2% toludine blue and examined under an Olympus BH-2 Epifluorescent Microscope (Olympus America Inc., Melville, NY). Photographs were taken using a Pixera 120C di gital camera. For scanning electron microscopy

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35 (SEM), callus samples of approximately 150 mg were immersed in 100% methanol. Samples were lyophilized using a Bal-Tec 030 critical poi nt drier (ICMAS Inc., Alcoa, TN) with liquid CO2 sputter coated with gold-palladium usi ng a Denton Vacuum Desk II (Denton Vacuum, Moorestown, NJ) for approximately 50 s and vi ewed with a Hitachi S-4000 scanning electron microscope (Hitachi Scientific Instruments, Danbury, CT) operating at 6 KV. Digital images were processed using SEMages 16 software (A dvance Database System, Inc., Denver, CO). Acclimatization Shoots, some with roots, were removed from the baby food jars and excised individually from the callus clumps. Medium was carefully rins ed off shoots. Shoots longer than 20 mm with 2-3 leaves were planted individually in 60-cell plug trays (4.5 4 5 cm3 each cell, REB Plastics Inc, Apopka, FL) containi ng a 2:1:1 (v/v) soilless mixture of Canadian peat: vermiculite: perlite. All plantlets were maintained in a greenhouse under shade cloth with a maximum irradiance of 345 mol m-2 s-1, natural photoperiod (10 to 14.5 h light), and a temperature range of 20 to 31C. Plugs were hand watered twice a week. Peters 20N-10P-20K liquid fertilizer (200 mg l-1 N; The Scotts Company, Marysville, OH) was applied weekly following 2 weeks acclimatization. Statistical Analysis All experiments were established in a co mpletely randomized design. The experiments showing treatment responses were repeated once. Da ta were subject to analysis of variance using SAS (SAS Institute, Inc., 1999). Mean separation was achieved by the least significant difference (LSD) test at 95% level.

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36 Results Callus Induction The frequency of callus occurrence from medium containing TDZ and 2,4-D differed significantly based on their con centrations. No callus occurred on medium devoid of TDZ and 2,4-D or with 2,4-D only at 0.5 and 1.0 M. Howeve r, TDZ alone at concentrations of 1, 5, and 10 M induced 4, 10, and 26% of explants to pr oduce calli respectivel y. TDZ was required for callus formation, but the higher fr equency of callus formation occurred in medium supplemented with both TDZ and 2,4-D. Induction medium cont aining 5 M TDZ and 1 M 2,4-D resulted in the maximum of 96% of explants to produce calli (Table 2-1). Indirect Shoot Organogenesis A low number of shoots devel oped from calli cultured on BM alone (1.6 shoots/callus) or BM supplemented with 2 M IAA (1.2 shoots/ca llus). Basal medium supplemented with 2iP alone at concentrations of 20, 40, and 80 M elevated shoot numbers to 3.6, 6.3, and 4.0 per callus respectively. Combining 2iP with IAA furthe r increased shoot numbers compared to 2iP at respective concentrations alone, but the increase was not statistic ally significant. Highest shoot number (7.9 shoots/callus) occurr ed on BM supplemented with 40 M 2iP and 2 M IAA (Table 2-2). No shoot formation was observed on BM s upplemented with combinations of kinetin and GA3. Green nodular calli were observed on leaf e xplants after 12 weeks of culture on BM supplemented with 5 M TDZ and 1 M 2,4-D (Fi gure 2-1A). When calli were separated from primary leaf explants and transferred onto BM supplemented with 40 M 2iP and 2 M IAA, small green meristems were visible on the surface of calli within 4 weeks (Figure 2-1B) and later developed into shoot buds (Figure 2-1C). Leaf formation and shoot elongation occurred in the

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37 following 2 weeks (Figure 2-1D). Single shoot or shoot clusters with l eaves and roots were developed by the end of 8 weeks of culture (Figure 2-1E). Acclimatization Dieffenbachia cv. Camouflage plants were very easily acclimatized (Figure 2-1F). Ex vitro survival rate of 100% was observed. Histological Analysis Indirect shoot organogenesis was confirme d by histological sectioning. Calli were observed from leaf explants on BM medium supplemented with 5 M TDZ and 1 M 2,4-D after 28 days of culture. Two types of cells were observed in calli under light microscopy: regenerative cells which were smaller in size and more compact with more densely stained cytoplasm, thinner cell walls, more prominent nuc lei and no visible vacu oles (Figure 2-2A); nonregenerative cells which were larger and less compact with less cytoplasm and smaller nuclei, thicker cell walls and larger vacuoles (Figure 22B). Early mitotic activity was observed after 31 days of culture (Figure 2-2C). The first cell division was usually anticlinal followed by periclinal cell division (Figure 2-2D). After several mitoti c division, the differentia tion of a meristematic zone occurred (Figure 2-2E). By continuous an ticlinal and periclinal cell division, bigger meristematic cell masses composed of actively dividing cells were formed by 43 days culture. Each meristematic mass was characterized by cells with thick walls, and the individual cell was separated by thinner walls (Fi gure 2-2-F). Meristematic cell masses may also develop into globular shapes, assuming an appearance similar to globular somatic embr yos (Figure 2-2G and 2-2H). Initial cell divisions usually were initiated from a superficial callus cell or cells (Figure 22D), but a cell or a group of cells in inner ce ll layers in the callus may also give rise to meristematic mass (Figure 2-2I). The meristem atic mass became progressively more organized and formed a meristematic dome which represented an apical meristem of a bud after 12 days of

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38 culture on shoot induction medium (Figure 2-2J). The cell division along the flanks of the bud meristem resulted in leaf primordia formation after 18 days shoot induction (Figure 2-2K). A well-developed adventitious bud with apical shoot meristem and leaf was formed after 27 days of culture (Figure 2-2L). Sometimes multiple shoots were formed (Figure 2-2M). Root formation occurred after 39 days of culture (Figure 2-2N). A complete pl antlet was regenerated after 8 weeks culture on shoot induction medium. Vascular connection between a developing shoot and callus tissue was detected at day of 24 (Figur e 2-2O). Scanning electron microscopy showed stomata were present on the epidermis of developing shoots at day 36 (Figure 2-2P). Discussion A plant regeneration system via indirect shoot organogenesis was established in this study. To our knowledge, this is the first repor t of indirect shoot organogenesis in Dieffenbachia Our observations indicated that Dieffenbachia in general was recalcitra nt in regard to shoot organogenesis or somatic embryogenesis (attempt s were made to induce somatic embryogenesis failed to a wide range of PGR types, concentr ations and combinations). This may, in part, explain why in vitro regeneration via organogenesis or somatic embryogenesis in Dieffenbachia has not been previously reported. Callus Induction and Shoot Formation The concentration and combina tion of PGR are the key factor s influencing indirect shoot organogenesis in Dieffenbachia A similar PGR effect on callus formation has been reported in other species (Khanam et al., 2000; Reddy et al., 2001; Ma and Xu, 2002; Giridhar et al., 2004; Azad et al., 2005; Datta and Majumder, 2005; Zhou and Brown, 2005). 2,4-D has been shown to be most effective for callus induction in many species. On the contrary, our study showed that 2,4-D was not a prerequisite for callus initiation as calli were induced on BM without 2,4-D. In contrast, TDZ was required for callus formation in Dieffenbachia cv. Camouflage. There have

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39 been several reports of significant TDZ effects on callus formation and shoot organogenesis in other species (Gurriaran et al ., 1999; Mithila et al., 2003; Datt a and Majumder, 2005; Landi and Mezzetti, 2005). TDZ, a cotton defoliant, has both auxin-like and cytokinin-like activity and can be substituted for auxins or the combination of auxins and cytokini ns (Singh et al., 2003). Hutchinson et al (1996) reported that TDZ could result in an increase in endogenous levels of auxins. It could be possible that TDZ might have fulfilled both auxin-like and cytokinin-like roles in callus induction of this Dieffenbachia Cytokinin type and concentration have signifi cant effects on subseque nt shoot regeneration from calli. Results from this study indicated that 2iP was more effective than kinetin because no shoot regeneration was observed on BM supplemented with kineti n alone or combination with GA3. Voyiatzi and Voyiatzis (1989) also found that 2iP was more effective in inducing lateral shoot multiplication in Dieffenbachia than kinetin and 80 M 2iP with 2 M IAA was optimal for shoot formation of D. exotica cv. Marianna. The present resu lts suggested that 40 M 2iP with 2 M IAA was optimal for shoot organogenesis of cv. Camouflage. This difference might be due to the fact that different cultivars, expl ants were used. This study used calli derived from leaves, while axillary buds and shoot tips were used for shoot multiplication of cv. Marianna (Voyiatzi and Voyiatzis, 1989). Di fferential responses of genotype s and explant sources to PGR requirements have been well documented (M agioli et al., 1998; Choi et al., 2003). Acclimatization Dieffenbachia shoots regenerated in vitro proved to be easily adaptable to ex vitro conditions. No separate in vitro rooting stage was required as shoot survival rate was 100% in a soilless substrate. This is different from many ot her species where rooting is an obstacle for plant establishment (Reed, 1995; Gavidia et al., 1996; Pruski et al., 2000). Dieffenbachia cv. Camouflage belongs to an easy-to-root type, wh ich is in contrast to its very limited shoot

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40 formation ability. It was conceivable that endogenous level of auxin in Dieffenbachia might be sufficient for root formation. A promotive carry ov er effect of the IAA in the shoot induction medium on rooting was also possible. Histological Analysis In vitro shoot organogenesis and somatic embryoge nesis are the most used methods for a large-scale propagation and producti on of somaclonal variants. Sometimes it is very difficult to detect the true nature of in vitro regeneration especially when a callus phase is involved in the regeneration process and when the appearance of regenerating structures looks very much like somatic embryos. In our study, nodul ar calli were induced from cv. Camouflage. The appearance of these nodular calli superficia lly resembled globular somatic em bryos and they also could be easily detached from leaf explants. When these nodular calli were separate d from leaf explants and transferred onto BM medium supplemented w ith the combination of different plant growth regulators, they either degenerate d or simply proliferated. Others (Haensch, 2004a, b; Salaj et al., 2005) have reported embryo-like stru ctures were produced in their st udies. However, histological analysis revealed in their studies that all these somatic embryo-lik e structures were composed of parenchymatous and vacuolated cells, and had no re generation capacity. In contrast, histological examination in our study showed that these nodular calli consisted of actively dividing cells, therefore having regeneration potential. The re asons why these promising cells passed through organogenesis instead of somatic embryogenesis are not quite clear, but the f act that the route of morphogenesis could be changed by the manipulation of plant growth regulators in the culture medium has been demonstrated in other species. It seemed that we have not found an optimal combination of the genotype, explant tissue, type, concentration of plant growth regulators, or culture condition for inducing somatic embryogenesis in Dieffenbachia It is worthy of further investigation. Apart from the influence of PGRs, adventitious shoot regeneration in vitro must

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41 involve interrupting a genetically determined developmental pathway and reprogramming a new developmental pathway at gene level (Schwarz and Beaty, 2000). Our study indicated that we should evalua te critically public ations on somatic embryogenesis. More and more papers on somatic embryogenesis from different species have been published in recent years. Some of these papers did not provide histological support, only based on morphological similarities of regenerating structures w ith somatic embryos. Most of them did complete a histological examination, but verifications of bipolar structures were not provided. Usually shoot meristem and root merist em were shown in separate pictures. Only a few publication presented histologic al evidence of bipolarity and th e lack of vascular connection. The ability to regenerate shoots from calli has several advantages. A great number of shoots can be produced from an explant through callus induction and shoot formation. Indirect shoot organogenesis has a gr eater potential for regenera ting somaclonal variants. In Dieffenbachia cv. Camouflage, phenotypic variations in leaf variega tion and color of acclimatized plants were observed. The feasibilit y of inducing indirect shoot organogenesis in other Dieffenbachia cultivars, and then evaluating this regeneration protocol for potential isolation of somaclonal variants wi ll be described in Chapter 3 and 4.

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42 Table 2-1. Effect of TDZ and 2,4-D supplementati on to BM on frequency of callus formation from leaf explants of Dieffenbachia cv. Camouflage cultured for 8 weeks in dark and 4 weeks in a 16 h light photoperiod at 40 mol m-2 s-1. TDZ(M) 2,4-D (M) Shoots/callus SE* 0 0 0 a 0 0.5 0 a 0 1 0 a 1 0 4 0.1 a 1 0.5 46 0.3 b 1 1 66 0.2 c 5 0 10 0.3 a 5 0.5 60 0.3 c 5 1 96 0.1 d 10 0 26 0.3 e 10 0.5 76 0.2 f 10 1 78 0.2 f Means followed by the same letter are not significan t at 0.05 level. Data represent means of two repeated experiments, each with 5 rep licates and 5 subsamples per replicate.

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43 Table 2-2. Effect of 2iP and I AA concentrations and combinati ons on shoot regeneration from Dieffenbachia cv. Camouflage calli cultured for 8 weeks in a 16 h light photoperiod at 40 mol m-2 s-1. 2iP (M) IAA (M) Shoots/callus SE* 0 0 1.6 0.3 a 0 2 1.2 0.4 a 20 0 3.6 0.9 b 20 2 4.2 2.0 b 40 0 6.3 2.2 c 40 2 7.9 1.5 c 80 0 4.0 0.1 b 80 2 3.8 0.4 b *Means followed by the same letter are not significan t at 0.05 level. Data represent means of two repeated experiments, each with 5 rep licates and 5 subsamples per replicate.

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44 Figure 2-1. Indirect shoot organogenesis in Dieffenbachia cv. Camouflage. A) Induction of calli on leaf explants on BM supplemented with 5 M TDZ and 1 M 2,4-D after 8 weeks culture in dark and 4 weeks culture in a 16 h light photoperiod. Bar = 1 mm. B) Small green meristems formed on th e surface of calli on BM containing 40 M 2iP and 2 M IAA within 4 weeks. Bar = 1mm. C) Shoot buds formed from these meristems 2 weeks later. Bar = 1 cm. D) Shoots and leaf development. Bar = 1 cm. E) Welldeveloped shoots with roots within 8 weeks of culture. Bar = 1 cm. F) Acclimatized plants in the greenhouse exhibi ting variation in leaf vari egation and color. Bar = 5 cm.

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45 Figure 2-2. Histological evidence of indirect shoot organogenesis in Dieffenbachia cv. Camouflage at different developmenta l stages when cultured on BM medium supplemented with 5 M TDZ and 1 M 2, 4-D for callus induction and on BM medium supplemented with 40 M 2iP and 2 M IAA for shoot induction. A) regenerative cells in calli originated from leaf explants. Bar = 250 m. B) nonregenerative cells in calli. Bar =250 m. C) early mitotic activity observed at day 37 on callus induction medium. Bar = 167 m. D) initial anticli nal division on the surface of calli. Bar = 250 m. E) initiation of meristematic zone by continued anticlinal and periclinal cell division. Bar = 250 m. F) development of meristematic mass at day 43. Bar =250 m. G) formation of globular shaped meristematic mass. Bar = 500 m. H) appearance of a globular shaped meristematic mass shown by SEM. Bar = 1 mm. I) meristematic cell mass formed w ithin calli. Bar = 250 m. J) meristematic dome formation after 12 da ys of culture on s hoot induction medium. Bar = 250 m. K) development of shoot meri stem and leaf primordia at day 18. Bar = 500 m. L) a well-developed shoot bud su rrounded by leaves at day 27. Bar = 500 m. M) multiple shoot formation. Bar = 250 m. N) root formation by day 39. Bar = 250 m. O) vascular connection between a developing shoot and callus tissue. Bar = 500 m. P) SEM showed a developing shoot with stomata on the epidermis at day 36 on shoot induction medium. Bar = 750 m.

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46 CHAPTER 3 FACTORS AFFECTING INDIRECT SHOOT ORGANOGENESIS IN Dieffenbachia Introduction Dieffenbachia, a monocot belonging to the family Ar aceae, noted for its a ttractive, striking foliage, is one of the most important ornament al plant genera (Chen et al., 2004). Traditional breeding in Dieffenbachia can be hampered by its naturally -occurring dichogamy, long breeding cycle, poor seed production and germination (Henny, 1988). Selec tion of somaclonal variants from in vitro regenerated populations is an alterna tive means for new cultivar development (Henny and Chen, 2003). Due to the intervening callus phase, regeneration through indirect shoot organogenesis has been shown to produce more somaclonal variants (Chen et al., 2003a). Selection and subsequent in vitro propagation of variants with desired phenotypes can facilitate the release of new cultivar in 2-3 years compar ed to 7-10 years through the traditional breeding. Among factors influencing in vitro regeneration via indirect shoot organogenesis, genotype, explant source, and plant growth re gulators in media are the most important. Genotypic differences in shoot or ganogenesis have been observed in a wide range of species. Some genotypes exhibit high regene rative capacity, while others are either recalcitrant or exhibit no capacity at all (Kuehnle and Sugil, 1991; Nont aswatsri et al., 2002). E xplant type may also influence capacity for in indirect shoot organogenesis, which may be related to the totipotency of cells at their developmental stages (Aga rwal and Ranu, 2000; Bacchetta et al., 2003). Additionally, the type, c oncentration and combination of plant growth regulators in the media can greatly affect callus formation and subsequent shoot induction (Mithila et al., 2003; Thao et al., 2003). To date, successful in vitro regeneration in Dieffenbachia has been largely limited to shoot culture from shoot tip or axil lary bud explants (Knauss, 1976; Voyviatzi and Voyiatzis, 1989).

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47 We have established a protocol fo r indirect shoot organogenesis in Dieffenbachia cv. Camouflage (Chapter 2), but information on th e effects of genotype, e xplant source and plant growth regulators on callus-m ediated shoot organogenesis in Dieffenbachia cultivars is lacking. The objectives of this study we re to: 1) examine differences in capacity for indirect shoot organogenesis in 4 Dieffenbachia cultivars and 2) investigate the effects of explant type and plant growth regulators (type, con centration and combination) on indi rect shoot organogenesis. It is expected that information obtained from this study will help establish a more efficient protocol for new cultivar development in Dieffenbachia through selection of somaclonal variants. Materials and Methods Plants Materials, Media and Culture Conditions In vitro shoot cultures of the 4 Dieffenbachia cvs. Camouflage, Camille, Octopus and Star Bright served as the explant source. Previous ge netic analysis demonstrated that these cultivars are genetically different (Chen et al., 2004). For callus induction and shoot organogenesis, a basal medium (BM) consisting of MS (Muras hige and Skoog, 1962) mineral salts, 0.4 mg l-1 thiamine, 2.0 mg l-1 glycine, 100.0 mg l-1 myo-inositol, 0.5 mg l-1 pyridoxine, 0.5 mg l-1 nicotinic acid, and 25 g l-1 sucrose supplemented with pl ant growth regulato rs was used in all experiments. Medium was adjusted to a pH of 5.8 with 0.1 N KOH prior to the addition of 6 g l-1 TC agar (PhytoTechnology Laboratories, Shawnee, Mission, KS) and autoclaving at 1.2 kg cm-2 for 20 min. Cultures were maintained either in dark or under a 16 h light phot operiod at 40 mol m-2 s-1 provided by cool white fluorescent lamps (Gen eral Electric F20WT12CW) at 22 3C. Callus Induction Leaves and roots from 4 week-old in vitro shoot cultures of the 4 Dieffenbachia cultivars Camouflage, Camille, Octopus and Star Bright se rved as explants. Leaves were cut into 5 mm2 sections with mid-vein, and the leaf margins remove d with a scalpel. Leaf explants were cultured

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48 with the abaxial surface in contact with the cal lus induction media. Root s were cut into 10 mm long segments, and then cultured horizontally on media. A series of 8 screening experiments were completed to select the most effective concentration and combination of PGRs. Each experiment consisted of factorial PGR combin ation with PGR-free medium serving as the control: (1) BA at 0, 1, 10, 50 and 2,4-D at 0, 1, 10, 50 M; (2) CPPU at 0, 1, 2.5, 5 and 2,4-D at 0, 2, 4, 8, 10 M; (3) CPPU at 0, 1, 2.5, 5 NAA at 0, 2, 4, 8, 10 M ; (4) kinetin at 0, 1, 5, 10 and IAA at 0, 1 M; (5) dicamba at 0, 1, 3, 9 and 2,4-D at 0, 1 M; (6) picloram at 0, 1, 3, 9 and 2, 4D at 0, 1 M; (7) TDZ at 0, 1, 10, 50 and NAA at 0, 1, 10, 50 M; (8) TDZ at 0, 1, 5, 10 and 2,4D at 0, 0.5, 1 M. Explants were cultured in 100 15 mm sterile Petri plat es containing 20 ml medium. There were 5 explants per Petri plates and 5 replicate plat es per treatment. Petri plates were sealed with one layer of Nescofilm (K arlan Research Products Corp., Cottonwood, AZ). Cultures were initially maintained in dark for 8 weeks for all cultivars except Star Bright that were in dark for 12 weeks (no a ny response observed by 8 weeks cult ure) and then transferred to a 16 h light photoperiod for another 4 weeks. The number of explants forming calli was scored after 12 weeks culture for cvs. Camouflage, Cam ille, Octopus and 16 weeks for Star Bright. The frequency of callus formation was calculated as the percentage of leaf explants forming calli. Shoot Induction Calli were excised from leaf explants, cut into pieces with a fresh weight of approximately 150 mg and then transferred to the shoot induc tion medium which was BM supplemented with 80 M 2iP and 2 M IAA. Calli were cultured in baby food jars containing 40 ml media with 5 callus clumps per vessel and 5 re plicates per treatment. Culture vessels were sealed with one layer of Nescofilm. The number of shoots form ed per callus was dete rmined after 8 weeks culture under a 16 h light photoperiod.

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49 Acclimatization Shoots, some with roots, were removed from the vessels and excised individually from the callus clumps. Medium was carefully rinsed o ff the shoots. Shoots longer than 20 mm with 2-3 leaves were planted individua lly in 60-cell plug trays (cell dimensions: 4.5 4 5 cm3, REB Plastics Inc, Apopka, FL) containi ng a 2:1:1 (v/v) soilless mixture of Canadian peat: vermiculite: perlite. All plantlets were main tained in a shaded greenhouse under natural photoperiod (10 to 14.5 h light) with a maximum irradiance of 345 mol m-2 s-1 and a temperature range of 20 to 31C. Plugs were hand watered twice a week. Pete rs 20N-10P-20K liquid fe rtilizer (200 mg/l N; The Scotts Company, Marysvil le, OH) was applied weekly. Statistical Analysis All experiments were established in a completely randomized design. Experiments showing responsive treatments were repeated once. Data were subject to analysis of variance using SAS (SAS Institute, Inc., 1999). Mean separation was achieved by least significant difference (LSD) test at the 95% level. Results Explant Effects A distinct difference in callus formation was observed between leaf a nd root explants. No callus was induced on root explants regardless of cultivar and PGR combination. However callus formation occurred on leaf explants of all the 4 cultivars cultured on BM containing TDZ and 2,4-D at appropriate concentrations. Genotypic Effects Leaf explants of different cultivars exhibite d different responsiveness for callus formation. Callus initiation occurred from the leaf margins of cvs. Camoufla ge, Camille and Octopus after 4 weeks of culture; but no ca llus was noted on cv. Star Bright expl ants until 8 weeks culture. Leaf

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50 explants first exhibited elongation and expansion, then became curved and swollen prior to callus proliferation. The effect of genotype on callus morphology wa s evident. Four morphologically distinct callus types varied in structure and color were observed. Green nodular (Figure 3-1A), brown nodular (Figure 3-1B), yellow friable, mucilagi nous (Figure 3-1C) and light green compact (Figure 3-1D) calli were produced from leaf expl ants of cvs. Camouflage, Camille, Octopus and Star Bright respectively. Base d on visual observation, callus gr owth rate also differed among cultivars. Octopus produced the most calli covering almost the entire surface of the leaf explants. Least calli was produced on Star Bright with callus pr oduction only limited to the cut ends of leaf explants. Moderate growth rate of calli was observed in cvs. Camouflage and Camille. Differences in the frequency of callus formation among cultivars were significant. Maximum frequency of callus formation (96%) wa s obtained from Camouflage leaf explants, followed by 66% from Octopus, 62% from Ca mille and 52% from Star Bright on BM supplemented with 5 M TDZ and 1 M 2,4-D (Table 3-1). A significant effect of genotype on shoot i nduction was also observ ed. After calli were transferred onto BM supplemented with 80 M 2iP and 2 M IAA, maximum shoot production (6.7shoots/callus) was obtained from cv. Camo uflage, followed by 4.4 shoots/callus from cv. Camille, 3.5 shoots/callus from cv. Star Bright. Cultivar Octopus displayed no capacity for indirect shoot regeneration (Table 3-1). PGR Effects PGR type, concentration and combination ha d significant effects on callus induction on cultured leaf explants. Among the PGRs screen ed, callus formation was only observed on leaf explants cultured on BM supplemented with factorial combinations of 0, 1, 5, 10 M TDZ and 0.5 and 1 M 2,4-D. Other PGRs or their combina tions failed to induce callus formation from

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51 either leaf or root explants. Additionally, the effect of TDZ and 2,4-D on the frequency of callus formation varied significantly. No callus could be induced on the medium without any PGRs. TDZ was required for callus induction for cvs. Camouflage, Camille and Star Bright. No callus formation was observed on media without TDZ in these 3 cultivars. For cv. Octopus, a 2% frequency of callus formation was noted on medi um without TDZ. However, 2,4-D was required for callus production on Camille explants. Although callus formation was observed on media without 2,4-D for the other 3 cultivars, the frequency of callus formation was lower (4% to 26%). Callus production increased with incr easing 2,4-D concentrations. Consequently, uniformed callus formation was obtained on BM supplemented with both TDZ and 2,4-D for all the 4 cultivars. In general callus formation wa s promoted with increasing levels of both TDZ and 2,4-D, but high TDZ concentration ( 10 M) inhibited callus fo rmation (Data not shown). The combination of 5 M TDZ and 1 M 2,4-D was optimal for callus production among the 4 cultivars tested (Table 3-1). A distinct carry-over effect of TDZ and 2,4D from callus induction media on subsequent shoot organogenesis was noted. Calli produced on callus induction medium with higher levels of 2,4-D displayed reduced capacity for shoot develo pment. Conversely, calli produced on medium supplemented with higher levels of TDZ were more highly shoot organogenic (Table 3-1). When cultured on BM supplemented with 80 M 2iP and 2 M IAA, calli derived from BM supplemented with 10 M TDZ alone exhibi ted the highest shoot regeneration (6.7 1.3 shoots/callus) in cv. Camouflage. The developmental process of indirect shoot organogenesis was illustrated in cv. Camille. Brown nodular calli were produced on leaf explan ts of cv. Camille on BM supplemented with 5 M TDZ and 1 M 2,4-D after 8 weeks in dark and 4 weeks cultures in a 16 h light photoperiod

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52 (Figure 3-2A). After calli were separated fr om leaf explants and transferred onto BM supplemented with 80 M 2iP and 2 M IAA for shoot induction, small green meristems appeared on the surface of calli within 4 weeks (Figure 3-2B). Shoot buds developed from these meristems by the 6th week (Figure 3-2C). Leaf formation and shoot elongation occurred within 8 weeks (Figure 3-2D). Shoot clus ters with well-developed leaves and roots were formed by the end of 8 weeks culture (Figure 3-2E). Acclimatization Shoot and root formation was observed in most Dieffenbachia cultures. Shoots longer than 20 mm with 2 to 3 leaves we re easily acclimatized in a sh aded greenhouse (Figure 3-2F). Ex vitro survival 100% was obtained in all the cultivar s acclimatized. Discussion The present study demonstrates the difficu lties in the induction of indirect shoot organogenesis in Dieffenbachia Attempts to use leaf explants taken directly from plants grown in the greenhouse to initiate callus production failed due to significant (> 70%) culture contamination. Leaf explants obtained from greenhouse plants displayed no response regardless of media tested. We demonstrated that using leaf explants from in vitro -produced shoots was a viable, alternative method to reduce contamin ation and increase explant responsiveness. However, the responsiveness of leaf explants from in vitro plants on callus induction media was slow. At least 12 weeks (16 weeks for cv. Star Bright) were required for callus induction. The shoot regeneration capacity of Dieffenbachia from callus was low with a maximum 6.3 shoots/callus. Slow responsiveness to PG R treatments, high contamination rate in vitro and recalcitrant nature for in vitro culture may explain why indirect shoot organogenesis has not been reported in Dieffenbachia by others.

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53 Other factors may account for high contamination rate. Dieffenbachia is mainly propagated vegetatively by cuttings and divi sions, and is maintained und er shaded condition which may allow bacteria and fungi to accumulate on the plant surface. The presence of endophytes can not be ruled out (Knauss, 1976). This might be one reason for such high cont amination rate in our attempts to establish in vitro culture. Additionally, Dieffenbachia is a naturally-slowgrowing plant and this may also be manifested in vitro Genotype Effects Dieffenbachia is a monocotyledonous genus and is am ong the most recalcitrant plants for regeneration. In the present study, 4 cultivars evaluated are of di fferent genetic origin. Camille, one of the most popular cultivars in the foliage plant industry, is a s port selected from cv. Perfection, while cv. Star Bright is an interspe cific hybrid selected fr om crosses of several parents. Camouflage was selected from somaclona l variants of cv. Panther, while Octopus was isolated from somaclonal variants of Camouflage (Chen et al., 2004). Genotype remains a key determinant of capacity for indirect shoot organogenesis. There were distinct differences in callus morphology, callus forming ability and subsequent shoot differentiation among the 4 Dieffenbachia cultivars. In the pres ent study, 4 morphologically distinct types of calli have been observed. Am ong them, three types were organogenic and one type non-organogenic. The frequenc y of callus formation was also genotype-dependent with the highest (96%) observed in cv. Camouflage. Genot ypic differences were still obvious after calli were transferred onto shoot induction medium. Ca lli of cv. Octopus did not exhibit any shoot formation while Camouflage exhibited the hi ghest shoot formation (6.7 shoots/callus). Variation in callus morphology a nd callus forming ability of different genotypes has been previously reported in other sp ecies (Thao et al., 2003; Landi and Mezzetti, 2005). Our results were consistent with these repor ts. Kallak et al (1997) concluded that callogenesis was not only a

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54 result of dedifferentiation of explant tissues, but also an es sential preparatory stage for in vitro morphogenesis and callus type may be an indicat or for shoot organogeni c potential. In their analysis of gene tic relatedness of Dieffenbachia cultivars using AFLP, Chen et al (2004) found that cv. Camouflage significantly differed from cvs. Camille, Octopus and Star Bright as it was positioned in one genetic cluster and the other three shared another cluster. This might partially explain why cv. Camouflage performed differently in terms of the frequency of callus formation and shoots/callus compared to the other 3 cultiv ars. Phillips (2004) reported that specific genes involved in each stage of shoot organogenesis (d edifferentiation, induction and differentiation). In some genotypes genes involved in shoot organogenesis may be suppressed due to inappropriate culture condition. PGR Effects The type, concentration and combination of PGRs in media are another key factor regulating shoot organogenesis. Th e present study showed that PG Rs have significant effects on the induction of calli. 2,4-D has been shown to be the most effective for callus induction in a variety of species (Ma and Xu, 2002; Thao et al ., 2003). In contrast, we observed that 2,4-D was not required for callus initiation in 3 of 4 cultivars. Among PGRs screened, TDZ was most effective in stimulating callus formati on. TDZ has also been found effective in in vitro regeneration in a variety of species, such as pelargonium (Haensch, 2004b), African violet (Mithila et al., 2003), geranium (Robichon et al., 1997). Hutchinson et al (1996) reported that TDZ treatment could result in an increase in endogenous auxin levels. This may explain why TDZ was most effective in callus induction in present study because auxins usually stimulate callus formation. TDZ also has cytokinin-like activity (Zha ng et al., 2001; Landi and Mezzetti, 2006). Induction of indirect shoot organogenesis in Dieffenbachia cultures supports this claim. It was

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55 noted that many small shoot meristems formed on the surface of calli on media with TDZ, but these shoots did not elonga te. Attempt to include GA3 in media to elongate these stunted shoots failed. Bacchetta et al (2003) repor ted that TDZ induced multiple s hoots with stunted growth in Lilium. Orlikowska et al (1995) noted 4.5 M TDZ in combination with 5.4 M NAA induced direct shoot organogene sis on petiole explants in an unidentified Dieffenbachia cultivar. Carry-Over Effect of PGRs Depending on the type and concentration, PGRs in the callus induction media may have had either a negative or positive carry-over effe ct on subsequent shoot formation (Vardja and Vardja, 2001). In our study, we observed that shoot forming ability was higher from calli cultured on media containing TDZ alone. Sim ilar result was found in indirect shoot organogenesis in Duboisia myoporoides in which the combination of cytokinin/auxin used for callus induction had effect on subsequent shoot induction (Khanam et al., 2000). Calli derived from TDZ-supplemented media may have accumulated TDZ or a metabolite in their cells during the period of culture. When these calli were transferred to shoot induction media, high TDZ levels in callus cells may have stimulated shoot proliferation. Explant Effects The effect of explant sources on indirect shoot organogenesis has been reported by other authors (Orlikowska et al., 1995; Kallak et al., 1997; Berrios et al., 1999; Mithila et al., 2003). This study showed that only leaf not root explants, exhibited the capacity for indirect shoot organogenesis. Root explants failed to indirect shoot organogenesis. An explanation for this difference would be that cells from root explants were organogeni cally less competent than those of leaf explants. Another po ssible reason could be that PG R combinations and/or their concentrations screened were in appropriate for callus induction on root explants. Orlikowsika et al (1995) also reported that Dieffenbachia root explants were non-regenerative in vitro

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56 Acclimatization In contrast to their recalcitrant nature for callus induction and shoot regeneration, microcuttings of Dieffenbachia cultivars were easy-to-root and 100% ex vitro survival of shoots was achieved in all the 3 cultivar s exhibiting the capacity for indi rect shoot organogenesis. Root formation occurred concurrently with shoot formation on shoot i nduction medium. It was conceivable that endoge nous auxin levels in Dieffenbachia microcuttings were sufficient for root formation. Variations in leaf variegation and color (in cv. Camouflage) and leaf morphology (in cv. Camille) were observed among acclimatized plants. The evaluation of this regeneration protocol for potential isolati on of somaclonal variants will be described in Chapter 4.

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57 Figure 3-1.Characterization of calli cultured on BM supplemented with 5 M TDZ and 1 M 2,4-D after 8 weeks culture in dark for cv s. Camouflage, Camille and Octopus (12 weeks for cv. Star Bright) and 4 weeks cu lture in a 16 h light photoperiod. A) Green nodular calli of cv. Camouflage. Bar = 1 mm. B) brown nodular calli of cv. Camille. Bar = 1 mm. C) Yellow, friable calli of cv. Octopus. Bar = 5 mm. D) green compact calli of cv. Star Bright. Bar = 5 mm.

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58 Table 3-1. Effects of TDZ and 2,4-D on the frequency of callus formation on leaf explants a nd shoot number per callus of Dieffenbachia 4 cultivars Camouflage, Camille, Octopus and Star Bright. Camouflage Camille Octopus Star Bright TDZ (M) 2,4-D (M) Callus formation frequency (%) SE Shoots/ callus SE Callus formation frequency (%) SE Shoots/ callus SE Callus formation frequency (%) SE Shoots/ callus SE Callus formation frequency (%) SE Shoots/ callus SE 0 0 0 a 0 a 0 a 0 a 0 a 0 0 a 0 0 0.5 0 a 0 a 0 a 0 a 2 0.2 a 0 0 a 0 0 1.0 0 a 0 a 0 a 0 a 0 a 0 0 a 0 1 0 4 0.1 a 0 a 0 a 0 a 6 0.3 a 0 0 a 0 1 0.5 46 0.3 b 3.7 0.5 b 8 0.2 a 0 a 30 0.4 bc0 0 a 0 1 1.0 66 0.2 c 2.7 0.5 c 28 0.2 bd 0 a 30 0.3 bc0 4 0.1 a 0 5 0 10 0.3 a 0 a 0 a 0 a 14 0.4 ab0 0 a 0 5 0.5 60 0.3 c 4.4 0.5 b 22 0.2 b 0 a 36 0.4 c 0 4 0.1 a 0 5 1.0 96 0.1 d 3.5 0.5bc 62 0.3 c 3.7 0.5 b 66 0.3 d 0 52 0.3 c3.5 1.6 10 0 26 0.3 e 6.7 1.3 d 0 a 0 a 24 0.3 b 0 4 0.1 a 0 10 0.5 76 0.2 f 5.3 0.7 e 36 0.3 d 0 a 54 0.3 de0 28 0.2 b 0 10 1.0 78 0.2 f 3.8 0.4 b 56 0.3 c 4.4 0.5 c 40 0.2 ce0 32 0.1 b 0 Callus formation frequency was evaluated afte r 8 weeks for cv. Camouflage, Camille and Octopus (12 weeks for cv. Star Bright) i n dark and 4 weeks in a 16 h light pho toperiod on BM supplemented with differe nt combination of TDZ and 2,4-D. Shoots per callus were scored after 8 weeks in a 16 h light ph otoperiod on BM supplemented with 80 M 2iP and 2 M IAA.

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59 Figure 3-2. Indirect shoot organogenesis in Dieffenbachia cv. Camille. A) Induction of calli on leaf explants on BM supplemented with 5 M TDZ and 1 M 2,4-D after 8 weeks culture in dark and 4 weeks culture in a 16 h photoperiod. Bar = 500 m. B) Small green meristems formed on th e surface of calli on BM containing 80 M 2iP and 2 M IAA within 4 weeks. Bar = 1cm. C) Shoot buds formed from these meristems by the 6th week. Bar = 1cm. D) Leaf form ation and shoot elongation occurred. Bar = 1cm. E) Well-developed shoots with r oots within 8 weeks of culture. Bar = 1 cm. F) Acclimatized plants in the greenhouse. Bar = 1 cm.

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60 CHAPTER 4 ASSESSMENT OF SOMACLONAL VARIATION IN Dieffenbachia PLANTS REGENERATED FROM INDIRECT SHOOT OR GANOGENESIS AT PHENOTYPIC LEVEL Introduction Phenotypic variation observed among plants rege nerated from tissue culture is referred to as somaclonal variation (Larkin and Scowcroft, 1981). Somaclonal variation can be assessed by analysis of phenotype, chromosome number and stru cture, proteins, or di rect DNA evaluation of plants (Bouman and De Klerk, 1997). The extent of phenotypic variation is usually determined as the percentage of plants showing aberrations from the parental plant for one or more defined characteristics. These characteristics may in clude change in foliar variegation pattern, modification of flower color and size, alteration of leaf shape, increase in lateral shoots, and change in overall plant form (Griesbach et al., 19 88; Khalid et al., 1989). Plants with the deviant phenotypes are known as somaclones or somaclonal variants. Commerciall y, visual phenotypic evaluation is paramount since the value of the pl ants, especially floric ulture crops, lies in appearance. Chen and Henny (2006) documented th e occurrence of somacl onal variation in 58 genera across 33 families of floriculture crops and proposed that somaclonal variation is an important source for cultivar develo pment of floriculture crops. Many factors, including genotype growth regulators, and ti ssue source are involved in somaclonal variation (Karp, 1991; Chen and He nny, 2006). Among these factors, plant genotype is probably the most important determinant of variation. Some cultivar s show higher variation rate while others are highly stable (Najaran and Walton, 1987; Skirvin et al., 1994; Bouman and de Klerk, 1997). In addition, the duration of tis sue culture also affects somaclonal variation. Somaclonal variation generally in creases with the time that a culture has been maintained in vitro especially for ca llus culture; th e longer a culture remains in vitro the greater somaclonal variation (Orton, 1985; R odrigues et al., 1998).

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61 Dieffenbachia belongs to the family Araceae and consistently ranks among the top five most popular ornamental foliage plant genera. Du e to its naturally occu rring dichogamy, limited seed production, development of a new Dieffenbachia cultivar through traditional breeding usually requires 7 to 10 years (Henny a nd Chen, 2003). With the establishment of in vitro culture techniques for Dieffenbachia propagation in the 1980s, new cultiv ars have been selected from somaclonal variants and released in the foliage plant industry (Chen and Henny, 2006). However, there is no report on the ev aluation of somaclonal variation among Dieffenbachia plants regenerated from i ndirect shoot organogenesis. Our previous studies established methods for regenerating the 3 Dieffenbachia cultivars Camouflage, Camille, and Star Bright through in direct shoot organogenesis (Chapter 3). The objectives of the present study were to phenot ypically assess somacl onal variation among the regenerants of the 3 Dieffenbachia cultivars and determine the effects of genotype and the duration of callus culture on shoot regeneration and somaclonal variation. Materials and Methods Regeneration of the 3 Dieffenbachia cultivars Camouflage, Camille, and Star Bright through indirect shoot organogenesis was described in Chapter 3, which can be briefly outlined as follows: Callus Induction Leaves obtained from in vitro shoot culture of the 3 cultivars were cut into 5 mm2 sections containing mid-vein. A callus induction medium c onsisted of a basal medium (BM) containing MS (Murashige and Skoog, 1962) mineral salts, 0.4 mg l-1 thiamine, 2.0 mg l-1 glycine, 100.0 mg l-1 myo-inositol, 0.5 mg l-1 pyridoxine, 0.5 mg l-1 nicotinic acid, and 25 g l-1 sucrose which was supplemented with 5 M TDZ and 1 M 2,4-D. Medium was adjusted to pH 5.8 with 0.1 N KOH prior to the addition of 6 g l-1 TC agar (PhytoTechnology Laboratories, Shawnee Mission,

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62 KS) and autoclaved at 1.2 kg cm-2 for 20 min. Leaf explants were cultured in 100 15 mm sterile Petri plates containing 20 ml medium. There were 5 explan ts per plate inoculated with abaxial surface in contact with th e medium. The culture was initially maintained in the dark for 8 weeks for cvs. Camouflage and Camille, and 12 w eeks for Star Bright (due to no responses by 8 weeks culture), and then transferred to a 16 h light photoperiod at 40 mol m-2 s-1 provided by cool white fluorescent lamps (General Electric F20WT12CW) at 22 3C for another 4 weeks. Sustained Callus Culture Calli were excised from leaf explants at th e end of callus induction, and cut into pieces weighing approximately 150 mg (fresh weight). Half the calli were transfe rred to the same fresh callus induction medium for sustai ned callus production. The other ha lf was transferred to shoot induction medium to determine effects of subculture number on the capacity for shoot regeneration from calli. Calli were subcultured at 8 week intervals by this manner. Shoot Induction Five callus pieces, each weighing approximately 150 mg, were transferred into individual baby food jar containing 40 ml shoot induction medium. The shoot induction medium was composed of BM supplemented with 40 M 2iP and 2 M IAA and solidified with 6 g l-1 TC agar. Cultures were maintained under a 16 h ligh t photoperiod with a light intensity of 40 mol m-2 s-1 at 22 3C for 8 weeks. The number of s hoots per callus piece was recorded for each subculture. Acclimatization After 8 weeks on shoot induction medium, shoots, some with roots, were removed from the vessels and excised individua lly from the callus clumps. Medium was carefully rinsed from the shoots. Shoots longer than 20 mm with 2-3 leav es were planted individually in 60-cell plug trays (cell dimensions: 4.5 4 5 cm3, REB Plastics Inc, Apopka, FL) containing a 2:1:1 (v/v)

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63 soilless mixture of Canadian peat: vermiculite: perl ite. All plantlets were maintained in a shaded greenhouse under natural photoperiod (10 to 14.5 h light) with a maximum irradiance of 345 mol m-2 s-1 and a temperature range of 20 to 31C. Plugs were hand watered twice a week. Peters 20N-10P-20K liquid fert ilizer (200 mg/l N; The Scotts Company, Marysville, OH) was applied weekly following 2 weeks acclimatization. Determination of Somaclonal Variation Plants grown in plug trays were regularly checked for the presence of variant phenotypes. Emphasis was primarily placed on foliar characteristics as Dieffenbachia is valued for its leaf variegation. Novel variegation pa tterns were described, photogra phed, and grouped into different types of somaclonal variant. Numbers of plants pe r variant type were counted, and percentage of such type in relation to the total numbe r of regenerated plants was calculated. Parental and variant types of plants regenerated from Dieffenbachia cvs. Camouflage and Camille were then transplanted from plug trays to 15-cm diameter plastic pots containing Vergo Container Mix A (Verlite Co. Tampa, FL). The co mponents of the substrate were 2:1:1 (v/v) of Canadian peat: vermiculite: perlite. Five gr ams of an 18.0N-2.6P-10.0K controlled-released fertilizer with microele ments (Multicote 18-6-12, Haifa Chemicals Ltd., Haifa Bay, Israel ) was applied to the soil surface in each pot. Plants were grown on raised benches in a shaded and evaporated pad cooled greenhouse under a maxi mum photosynthetically ac tive radiation of 350 mol m-2 s-1. Temperatures ranged from 20 to 30oC and relative humidity from 50 to 100%. Plants were overhead irrigated through sprinkler s one to two times a w eek. Plant height, canopy height and width, length and widt h of the largest leaf, number of basal shoots were recorded 8 months after growing in the shaded greenhouse. Both shoot and cane cuttings were then made fr om parental plants and identified variants and rooted in Vergo Containe r Mix A in the aforementioned shaded greenhouse. Morphological

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64 characteristics of cutting-propagated progenies were compared to respective parents for determining if the varian t phenotypes were stable. Examination of the Duration of Callus Culture Affecting Somaclonal Variation Two populations of regenerated plants of Dieffenbachia cv. Camouflage were used to examine the duration of callus culture phase influencing som aclonal variation. One population was derived from 8 months callus culture in vitro The other population was derived from 16 months callus culture in vitro Plants with novel foliar varieg ation patterns were grouped as mentioned above. Numbers of plants per variant t ype were counted and calculated in relation to the total number of regenerate d plants in each population. Experimental Design and Statistical Analysis All experiments in this study we re established in a completely randomized design. Data for plant growth were subject to analysis of va riance using SAS (SAS institute Inc., 1999). Leastsquares mean test was used to compare means at the 95% level of probability. Results Somaclonal Variant Identification A total of 2,248 plants from cv. Camouflage, 384 of cv. Camille and 62 of cv. Star Bright were regenerated from indirect shoot orga nogenesis. Novel phenotypes were observed from regenerants of cvs. Camouflage and Camille, but not from cv. Star Bright. Three types of variants, called SV1, SV2, and SV 3, were identified among regenera ted Camouflage plants; each had distinct phenotype (Figure 41). The parental plant had ca mouflaged leaves with random green batches. Leaves of SV1, however, were gr een with whitish variegation along the midvein. SV2 had light green leaves with many yellowish spots, and connections among the spots resulted in large yellowish blotches. Leaf color of SV3 wa s similar to SV2 but had fewer yellowish spots. The other differences between SV2 and SV3 were that SV2 had some white spots on leaves and

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65 sparkled petioles. The leaf shape of the 3 variants was comparable to that of their parent. In addition to these morphological differences, anal ysis of variance for plant growth parameters showed that SV1 significantly differed in plant he ight, canopy height and width, leaf length from the parental plant and SV2. SV1 was taller with larger canopy and longer leaves than parental plants and SV2. SV2 and SV3 had no basal shoo ts (single stem) but basal shoot numbers between SV1 and parental plants were similar ra nging from 3 to 4 (Table 4-1). No statistical analysis could be done with SV3 because only 1 plant was available. One variant type was identified among the regene rants of cv. Camille. This variant had the same foliar variegation pattern as its parent but possessed lanceolate leaves compared to the oblong leaves of the parent (Figure 4-2). The rati o of leaf length and leaf width was 3.4 for the variants compared to 1.5 for pa rental plants. The variant also exhibited significant difference from the parent in growth parameters measured except for basal shoot number. Variants grew taller with larger canopy and had longer, narrow er leaves than the parent (Table 4-2). The morphological characteristics of all variants, 3 types from cv. Camouflage and 1 from cv. Camille, were stable in shoot and cane cutting propagated population. Genotype Differences Among the 2,248 regenerated plants of cv. Camouf lage, a total of 908 va riant plants were isolated, of which 904 plants belonged to the SV 1 type, 3 plants belonged to the SV2 type, and one being the SV3 type. Correspondingly, the occu rrence of SV1, SV2, and SV3 was at rates of 40.2%, 0.13%, and 0.04%, respectively. Thus, somacl onal variation rate at the phenotypic level for regenerated cv. Camouflage plants was 40.4% (Table 4-3). Of the 384 regenerated plants of cv. Camille, 10 variants had lanceolate leaves resulting in a somaclonal variation rate of 2.6% (Table 3). No variation was observed among the 62 regenerated plants of cv. Star Bright.

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66 Culture Duration Effects on Somaclonal Variation Duration of callus culture phase had no effect on somaclonal variation of cv.Camouflage. The somaclonal variation rates for plants regene rated from 8 and 16 months callus culture were 41.2% and 39.5% respectively (Table 4-4). Effects of Callus Subculture Nu mber on Shoot Regeneration The 3 Dieffenbachia cultivars displayed si gnificant differences in sustained capacity for shoot regeneration in long-term callus culture (Figure 4-3). Cultivar Camouflage showed a greater capacity for shoot regeneration than cvs. Camille and Star Brig ht. For cv. Camouflage, the number of shoots per callus increased from 4.7 at the first subculture to 6.3 by the sixth subculture, and then gradually de creased to 2.9 by the twelfth s ubculture. For cv. Camille, the number of regenerated shoots per callus increased from 2.6 at the first subculture to 4.0 by the fifth subculture, then decreased to 2.8 at se venth subculture, and fi nally decreasing to no regeneration by the eighth subculture (Figure 4-3). Cultivar Star Bright exhibited no capacity for sustained callus culture as calli lo st shoot regeneration ability afte r the first subculture. Calli of cv. Camille maintained the capacity for regeneration in vitro up to 16 months while cv. Camouflage calli retained rege nerative after 24 months culture in vitro Discussion Somaclonal Variation and Genotypes The present study demonstrated that somaclonal variation occurred among Dieffenbachia plants regenerated through indir ect shoot organogenesis from l eaf-derived calli. The variants showed distinct phenotypes from their parents. Th ese variants, particularly those isolated from regenerated cv. Camouflage, could potentially be new commercial cultivars as their phenotypes are novel and stable as demonstrated by their pr ogenies after cutting propagation. Additionally, the growth rates of these varian ts were either higher than or comparative to those of their

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67 parental plants in our shaded greenhouse evalua tion. Reserach thus far has primarily emphasized phenotypic evaluation. This is beca use in the absence of reliable genetic markers of somaclonal variation and consider ing the fact that Dieffenbachia is primarily propagated through asexual means, phenotype represents the fastest and the most convenient way to identify somaclonal variants. In addition, ornamental plants are prized for their vi sual phenotypic appearance. As long as a novel phenotype has ornamental value and is stable, it could potentially become a new cultivar. The rate of somaclonal variation differed great ly, ranging from none for cv. Star Bright and 2.6% for cv. Camille to 40.4% for cv. Camouflage The low number of regenerated plants, 62 and 384 for cvs. Star Bright and Camille respec tively, may be a factor for the low rates of variation observed. It is believe d, however, that the genetic makeup of the cultivars plays an important role. First, cvs. Camouflage, Camille, and Star Brigh differed in callus formation frequencies, 96%, 62%, and 52% respectively (Table 3-1). Second, the cultivars also varied with respect to in vitro shoot formation (Figure 4-3). Calli of cv. Star Bright had no capacity for subculture, and calli of cv. Camille gradually lost the ability to form shoots at approximately 16 months subculture. In contrast, calli of cv. Camouf lage maintained their capability to form shoots for at least 24 months after being subcultured. Cultivar differences in the frequency of callus and shoot formation frequencies resulted in low numb ers of regenerated plants for cvs. Camille and Star Bright. Thus, it is possibl e that a prerequisite for produc ing somaclonal variants through indirect shoot organogenesis is that calli maintain a high an d prolonged capacity for shoot organogenesis. Chen et al (2004) reported that cv. Camouflage si gnificantly differed from cvs. Camille and Star Bright genetically as the form er was positioned in one genetic cluster while the latter two shared a common cluster. This might partially explain why cv. Camouflage exhibited

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68 greater ability to form calli and shoots in vitro than cvs. Camille and Star Bright. High multiplication rate is associated with rapid m itosis, thus more error may have occurred. Somaclonal variation rates varying among sp ecies and cultivars have been widely documented. Skirvin et al (1994) stated that the somaclonal varia tion rate expected in vitro was probably about 1% to 3%. Our result with cv. Camille was similiar to this rate since 2.6% somaclonal variation occurred in this cultivar. The rate of somaclonal variation in cv. Camouflage was high (40.4%) but not unique. High rate s have been reported in other plants as well. Legkobit and Khadeeva (2004) noted 50% to 80% somaclonal variation from Stachys (betony) species. Bairu et al ( 2006) reported somaclonal variation in Cavendish banana cultivars was high as 72%. The high somaclonal variation rate may also be due to the multiplication of variant cells already prod uced in the previous cycle since re generated plants were derived from calli subcultured up to 8 cycles at 8 weeks interval for cv. Camouflage. Xie et al (1995) reported that a particular labile portion of th e rice genome was susceptible to stress ( in vitro culture condition) and showed higher rearrangement and mutation rates than other portion during in vitro culture. Further research is warranted to determine if the deviant phenotypes resulted from genetic or epigenetic changes and why such a high rate occurred in cv. Camouflage. Callus Culture Duration It is generally believed that the rate of somaclonal variati on increases with the time a culture has been maintained in vitro The longer a culture is maintained in vitro the greater the somaclonal variation (Rodrigues et al., 1998; Kuznetsova et al ., 2006). However, the present study showed that for Dieffenbachia cv. Camouflage there was no difference in the rate of somaclonal variation between plan ts regenerated from 8 and 16 m onths callus cultu re. It is not known at this time if shortening the duration of each subculture (less than an 8 week interval)

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69 and increasing time in maintenance or changing gr owth regulator combinations in media could result in any difference in somaclona l variation rates of cv. Camouflage. Both the type and the rate of somalconal variation are crucial factors for determining the feasibility of using somaclonal variation for ne w cultivar development. In this study, wide variation in leaf variegation, co lor, shape and plant forms were observed, and these traits are novel and stable demonstrated by their progenies after cutting propagation. These results showed that selection of somaclonal variants has great potential for new cultivar development in Dieffenbachia A future study will focus on analysis of genetic changes of the somaclonal variants using molecula r marker techniques.

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70 Figure 4-1. Plants of Dieffenbachia cv. Camouflage regenerated by indirect shoot organogenesis showing variation in leaf variegation and color. A) Parental plant: creamy, camouflaged leaves with random green batc hes of different size. Bar = 10 cm. B) SV1: solid dark green leaves with whitish variegation along the midvein. Bar = 10 cm. C) SV2: light green leaves with ma ny yellowish spots, and connections among spots resulted in large yello wish blotches. Bar = 10 cm. D) SV3: green leaves with few scattered yellowish spots. Bar = 10 cm.

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71 Table 4-1. Quantitative evaluation of somaclonal variants of Dieffenbachia cv. Camouflage regenerated by indirect shoot organogenesis and grown in the greenhouse for 8 months. No. of plants Plant height (cm) SE Canopy height (cm) SE Canopy width (cm) SE Largest leaf length (cm) SE Largest leaf width (cm) SE Basal shoot number SE Parental t yp e 114 22 0.9 a 39 1.0 a 47 1.0 a 30 0.4 a 11 0.2 a 3.7 0.1a SV1 34 30 1.4 b 44 1.6 b 53 1.6 b 33 0.6 b 12 0.3 a 3.9 0.2 a SV2 3 15 4.7 a 35 5.3 a 41 5.2 a 26 2.0 c 12 1.0 a 1.0 0.0 b SV3 1 26 17 24 22 8 1 Means followed by the same letter in each column are not significant at 0.05 level. Parental type: regenerated plants exhibited the same leaf variegation as the parent. SV1, SV2, SV3: three types of somaclonal variation observed in cv. Camouflage.

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72 Figure 4-2. Plants of Dieffenbachia cv. Camille regenerated by i ndirect shoot organogenesis showing variation in leaf shape. A) Parent al type plants with oblong shaped leaves. Bar = 1 cm. B) Somaclonal variants with lanceolate leaves. Bar = 1 cm.

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73 Table 4-2. Quantitative evaluation of somaclonal variants of Dieffenbachia cv. Camille regenerated by indirect shoot organogenesis and grown in the greenhouse for 8 months. No.of plants Plant height (cm) SE Canopy height (cm) SE Canopy width (cm) SE Largest leaf length (cm) SE Largest leaf width (cm) SE Leaf length/ leaf width SE Basal shoot number SE Parental t yp e 30 6.0 0.2 a 11 0.3 a 10 0.2 a 5.1 0.2 a 3.4 0.1 a 1.5 0.1 a 3.6 0.2 a Variant type 10 6.7 0.3 b 13 0.6 b 12 0.4 b 7.0 0.3 b 2.1 0.3 b 3.4 0.1 b 3.8 0.4 a Means followed by the same letter in each column are not significant at 0.05 level. Parental type: regenerated plants exhibi ted the same leaf shape as the parent. Variant type: regenerated plants exhibited different leaf shape.

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74 Table 4-3. Effects of genotype on the numbe r and rate of somaclonal variation among Dieffenbachia plants regenerated by indirect shoot organogenesis and grown in the greenhouse for 8 months. No. of somaclonal variants % of somaclonal variants Cultivars No. of plants regenerated Total SV1 SV2 SV3 Total SV1 SV2 SV3 Camoufla g e 2248 908 904 3 1 40.4 40.2 0.13 0.04 Camille 384 10 2.6 Star Bright 62 0 0 SV1, SV2, SV3: three types of somaclonal variation observed in cv. Camouflage.

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75 Table 4-4 Effects of the duration of ca llus culture on the number and ra te of somaclonal variation of Dieffenbachia cv. Camouflage regenerated by indirect shoot organogenesis and grown in the greenhouse for 8 months. Duration (months) No. of plants regenerated No. of variants Parental type SV1 SV2 SV3 8 1171 483 ( 41.2 ) 688 ( 58.8 ) 479 ( 40.9 ) 3 ( 0.26 ) 1 ( 0.09 ) 16 1077 425 (39.5) 652 (60.5) 425 (39.4) 0 (0) 0 (0) *Figures in parenthesis are the rate of somalconal variation. Parental type: regenerated plants exhibited the same leaf variegation as the parent. SV1, SV2, SV3: three types of somaclonal variation observed in cv. Camouflage.

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76 0 2 4 6 8 10 12 14 123456789101112 SubcultureShoots/callus cv. Camouflage cv. Camille cv. Star Bright Figure43. Effects of subculture number on shoot regeneration (shoots/callus SE) from calli of the 3 Dieffenbachia cultivars. Calli were subcultu red at 8 week intervals on the callus induction medium under a 16 h light photoperiod. Shoots/callus were recorded after 8 weeks culture on shoot induction medium under a 16 h light photoperiod.

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78 Black RJ (2002) Florida 4-H horticulture iden tification and judging st udy manual: flowers and foliage plants. Circular 4HEHL 21, Florida C ooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida Bouman H & De Klerk G (1997) Somaclonal vari ation. In: Geneve RL, Preece JE, Merkle SA (eds) Biotechnology of Ornamental Plants. CAB International, p165-183 Bouman H & De Klerk G (2001) Measurement of the extent of somaclonal variation in begonia plants regenerated under various conditions, co mparison of three assays. Theor Appl Genet 102:111-117 Budimir S (2003) Developmental histology of organogenic and embryogenic tissue in Picea omorika culture. Biol Plant 47:467-470 Chandra I & Bhanja P (2002) Study of organogenesis in vitro from callus tissue of Flacourtia jangomas (Lour.) Raeusch through scanning el ectron microscopy. Curr Sci 83:476-479 Chase AR, Zettler FW & Knauss JF (1981) Perf ection-137B: a pathogen-free selection of Dieffenbachia maculata derived through tissue culture. Ci rcular S-280, Florida Agricultural Experiment Stations, Institute of Food and Agri cultural Sciences, University of Florida, p 1-7 Chen J, Devanand PS, Norman DJ, Henny RJ & Chao CT (2004) Analysis of genetic relatedness of Dieffenbachia cultivars using AFLP markers. J Amer Soc Hort Sci 129:81-87 Chen J & Henny RJ (2006) Somaclonal variation: an important source for cultivar development of floriculture crops. In: Teix eira da Silva JA (ed) Floric ulture, Ornamental and Plant Biotechnology. Global Science Books, London, UK, p 244-253 Chen J, Henny RJ & Chao CT (2003a) Somacl onal variation as a source for cultivar development of ornamental aroids. Recent Res Devel Plant Sci 1:31-43 Chen J, Henny RJ, Devanand PS & Chao CT (2006) AFLP analysis of nephthytis ( Syngonium podophyllum Schott) selected from somaclona l variants. Plant Cell Rep 24:743-749 Chen J, Henny RJ & McConnell DB (2002) Devel opment of new foliage plant cultivars. In: Janick J, Whipkey A (eds) Trends in New Cr ops and New Uses. ASHA Press, Alexandria, VA, p 466-472 Chen J, McConnell DB, Henny RJ & Everitt KC (2003b) Cultural guidelines for commercial production of interiorscape Dieffenbachia ENH880, Environmental Horticulture Department, Institute of Food and Agricu ltural Sciences, University of Florida Chen WH, Chen TM, FuYM, Hsieh RM & Chen WS (1998) Studies on somaclonal variation in Phalaenopsis Plant Cell Rep 18:7-13

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87 BIOGRAPHICAL SKETCH Xiuli Shen was born in Harbin city, Heil ongjiang province, China, on October 2, 1964. She grew up in a happy family with loving parents and two younger brothers. From a very young age, she showed her love for science and nature She was an excellent student and did very well in every subject in school. After graduating from high school in 1982, she moved to Changchun and enrolled at Jinlin University and received a Bachelor of Science degree in July1987 with a major in molecular biology. Then she moved back to her home city and worked in the Department of Biotechnology in the Northeast Ag ricultural University as assistant professor from 1987 to 1996, and as an associate professor from 1996 to 2000. To fulfill her dream to look at the world outside of China and to be able to read a novel written in a foreign language, she came to Sask atoon, Saskatchewan, Canada with her 9-year-old son in May 2000. After 3 years of studying at the Univer sity of Saskatchewan, she earned her Master of Science degree in plant science in 2003. She worked as intern in the Plant Biotechnology Institute, Nationa l Research Council of Cana da during summer 2003. In August 2003, she entered graduate school at the University of Florida to work on a doctoral program in horticultural science. She exp ects to earn her Ph.D. in May 2007. She has one son, Hao Xu, living with her in Gainesville.


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INDIRECT SHOOT ORGANOGENESIS AND SELECTION OF SOMACLONAL
VARIATION IN Dieffenbachia



















By

XIULI SHEN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007


































2007 Xiuli Shen



































To my parents









ACKNOWLEDGMENTS

For my accomplishments, I thank my mother, Wenjin Sun, my father, Haifu Shen, my two

younger brothers, Qi Shen and Bin Shen for their unconditional love and unreserved support. I

also own my deepest thanks to my son, Hao Xu, for bringing me the happiness and

encouragement during years of my study.

I would also like to sincerely thank Dr. Michael E Kane and Dr. Jianjun Chen for their

mentoring over the last 4 years; Thanks also is given to the other committee members, Dr.

Richard J Henny, Dr. David J Norman, Dr. Sloane M Scheiber, for their support. Without them it

would have been impossible to successfully complete this project.

Acknowledgement also goes to the College of Agricultural and Life Sciences, Mid-Florida

Research and Education Center, and Dr. Chen's program for providing a graduate assistantship

to support this research.

I must also thank the people in the Plant Restoration, Conservation and Propagation

Biotechnology program: Nancy Philman, Carmen Valero-Aracama, Pete Sleszynski, Scott

Stewart, Phil Kauth, Tim Johnson, Chris Dudding, Daniela Dutra, for their help in many ways to

this project.

Thanks also go to people in the Environmental Horticultural Department as well as those

outside of the department who contributed to the completion of this study. Special thanks go to

all my friends in China, Canada and the U. S. for sharing all of my ups and downs.









TABLE OF CONTENTS

page

A CK N O W LED G M EN T S ................................................................. ........... ............. .....

L IST O F T A B L E S ......................................... ................................................... . 8

LIST OF FIGURES .................................. .. ..... ..... ................. .9

L IST O F A B B R E V IA TIO N S ...................... .. .. .......... ............................................ 10

A B S T R A C T ......... ....................... ............................................................ 1 1

CHAPTER

1 L IT E R A TU R E R E V IE W ............................................................................... .................. 13

In tro d u c tio n ............................................ .......................................................................... 1 3
Dieffenbachia Genus ................................... .. .......... ............... 13
B otany ................................... .. ........................................13
P ro p a g a tio n ................................................................................................................ 1 4
B reed in g ............................ ... ......................................15
Developmental Pathways for Plant Regeneration ...................................... ...............16
O rg a n o g e n e sis ........................................................................................................... 1 7
D efin itio n .....................................................................................17
Advantages of Shoot Organogenesis.......................... ...... .......... 17
Phases of Shoot O rganogenesis .......................................................................... ...... 17
Factors A affecting Shoot Organogenesis ................................................................... 19
G enotypes .............................................................................................................19
E x p la n ts ........................................................................................2 0
Plant G row th R regulators ............................................... ............... 21
Som atic E m bryogenesis .............................................................22
D efin itio n ......................................................................2 2
Stages of Som atic Em bryogenesis ....................................................... 22
Morphology and Physiology of Somatic Embryos ..................................... 24
Histological Studies on Shoot Organogenesis/Somatic Embryogenesis ...............25
Som aclonal V aviation ....................................................... 26
Origin of Som aclonal V aviation .............................................. ............... 27
C u ltiv a rs .............................................................................2 7
E plant Sources .............................................................................................. ........27
C allu s .................. ........................................................................2 8
Duration of the Tissue Culture Phase ........................ ........................................28
M easurem ent of Som aclonal V ariation ................................................................... 28
P henotypic V variations ...........................................................28
Changes at Chrom osom al Levels.................................. .................. 29
V ariations at DN A Levels ................................ ......................... ..............30









2 PROTOCOL ESTABLISHMENT FOR INDIRECT SHOOT ORGANOGENESIS
FROM LEAVES OF Dieffenbachia CV. CAMOUFLAGE........................ ..................... 31

In tro d u ctio n ......... ..... .. ......... ............................................................................................ 3 1
M materials and M ethods ................ .... ...................................................... ..... 32
M edia and Sterilization Conditions.............................................................................32
Plant Materials and Establishment of Shoot Cultures ............................................... 32
Indexing of E established C ultures...................... .. .. ......... .. .....................................33
C a llu s In d u ctio n ......................................................................................................... 3 3
Indirect Shoot O rganogenesis............................................................... .....................33
H istological A naly sis............ ... ............................................................ ......... ....... 34
A cclim atization ......................................... 35
Statistical A n aly sis ................................................................3 5
Results .......................................................................36
C a llu s In d u ctio n ......................................................................................................... 3 6
Indirect Shoot Organogenesis.................................. 36
A cclim atization ......................................... 37
H isto lo g ical A n aly sis................................................................................................. 3 7
D iscu ssion .................. .... ...... ............................................38
Callus Induction and Shoot Form action .................................................................... 38
A cclim atization ......................................... 39
H isto lo g ical A n aly sis................................................................................................. 4 0

3 Factors affecting indirect shoot organogenesis in Dieffenbachia ........... ........................46

Introduction ................ ...... ........... .............. ...............46
M materials and M ethods ..........................................................................................................47
Plants Materials, Media and Culture Conditions.............................. ............... 47
C a llu s In d u ctio n ......................................................................................................... 4 7
S h o o t In d u ctio n ............................................................................................................... 4 8
A cclim atization ......................................... 49
Statistical A n aly sis ................................................................4 9
R e su lts ......... ... .. ....... ............................................4 9
E x plant E effects ................................................................4 9
G enotypic E effects ................................................................49
P G R E ffe cts .......................................................................................................5 0
A cclim atization ......................................... 52
D iscu ssion .............. .. .............. ...................................................52
G enotype E effects ....................................................... 53
P G R E effects ........... ....................................................................... 54
Carry-Over Effect of PGRs ............................................. ........................55
E plant E effects ..........................................................................................55
A cclim atizatio n ............................................................................................... 5 6

4 ASSESSMENT OF SOMACLONAL VARIATION IN Dieffenbachia PLANTS
REGENERATED FROM INDIRECT SHOOT ORGANOGENESIS AT
PH EN O TYPIC LEVEL ................. ............................................ ..........60


6









In tro d u c tio n ....................................................................................................................... 6 0
M materials an d M eth o d s ...........................................................................................................6 1
C a llu s In d u ctio n ......................................................................................................... 6 1
Su stained C allu s C culture ............................................................................... ..62
Shoot Induction .................................. .. .......... .. ............62
A cclim atization................ ..... .......... .. .. ................ 62
D eterm nation of Som aclonal V ariation................................................. ................... ... 63
Examination of the Duration of Callus Culture Affecting Somaclonal Variation ..........64
Experim ental Design and Statistical Analysis...................................... ............... 64
Results ..... ............. ............................................... ............... 64
Somaclonal Variant Identification...................... ....... .............. ..............64
G enotype D differences .................. ... ................................................. .............65
Culture Duration Effects on Somaclonal Variation............................... ..............66
Effects of Callus Subculture Number on Shoot Regeneration .......................................66
D isc u ssio n .................... ... ..... ..................................................................6 6
Som aclonal V ariation and G enotypes ........................................ ......... ............... 66
C allus C culture D uration .............................................. ..................... ............... 68

R E F E R E N C E S ..........................................................................77

B IO G R A PH IC A L SK E T C H .............................................................................. .....................87
































7









LIST OF TABLES


Table page

2-1 Effect of TDZ and 2,4-D supplementation to BM on frequency of callus formation
from leaf explants of Dieffenbachia cv. Camouflage cultured for 8 weeks in dark and
4 weeks in a 16 h light photoperiod at 40 mol m2 s-1. ...................................42

2-2 Effect of 2iP and IAA concentrations and combinations on shoot regeneration from
Dieffenbachia cv. Camouflage calli cultured for 8 weeks in a 16 h light photoperiod
at 40 m ol m 2 S-1. .....................................................................43

3-1 Effects of TDZ and 2,4-D on the frequency of callus formation on leaf explants and
shoot number per callus of Dieffenbachia 4 cultivars Camouflage, Camille, Octopus
and Star B right. .............................................................................58

4-1 Quantitative evaluation of somaclonal variants of Dieffenbachia cv. Camouflage
regenerated by indirect shoot organogenesis and grown in the greenhouse for 8
m o n th s ................... ........................................................... ................ 7 1

4-2 Quantitative evaluation of somaclonal variants of Dieffenbachia cv. Camille
regenerated by indirect shoot organogenesis and grown in the greenhouse for 8
m o n th s ................... ........................................................... ................ 7 3

4-3 Effects of genotype on the number and rate of somaclonal variation among
Dieffenbachia plants regenerated by indirect shoot organogenesis and grown in the
greenhou se for 8 m month s ........................................................................... ...................74

4-4 Effects of the duration of callus culture on the number and rate of somaclonal
variation ofDieffenbachia cv. Camouflage regenerated by indirect shoot
organogenesis and grown in the greenhouse for 8 months.........................................75









LIST OF FIGURES


Figure page

2-1 Indirect shoot organogenesis in Dieffenbachia cv. Camouflage ...............................44

2-2 Histological evidence of indirect shoot organogenesis in Dieffenbachia cv.
Camouflage at different developmental stages when cultured on BM medium
supplemented with 5 [iM TDZ and 1 [iM 2, 4-D for callus induction and on BM
medium supplemented with 40 [iM 2iP and 2 [iM IAA for shoot induction.................45

3-1 Characterization of calli cultured on BM supplemented with 5 [tM TDZ and 1 itM
2,4-D after 8 weeks culture in dark for cvs. Camouflage, Camille and Octopus (12
weeks for cv. Star Bright) and 4 weeks culture in a 16 h light photoperiod...................57

3-2 Indirect shoot organogenesis in Dieffenbachia cv. Camille. ...........................................59

4-1 Plants of Dieffenbachia cv. Camouflage regenerated by indirect shoot organogenesis
showing variation in leaf variegation and color........................................................70

4-2 Plants of Dieffenbachia cv. Camille regenerated by indirect shoot organogenesis
show ing variation in leaf shape............................................................................ .. ..... 72

4- 3 Effects of subculture number on shoot regeneration from calli of the 3 Dieffenbachia
cultivars ..................... ...... .... ......... ..............................................76









LIST OF ABBREVIATIONS

2,4-D 2,4-dichlorophenozyacetic acid

2iP N6-(A2 isopentenyl) adenine

BA 6-benzyladenine

BM basal medium

CPPU [N-(2-chloro-4-pyridyl)-N-phenylurea

GA3 gibberellic acid

NAA 1- naphthalene acetic acid

PGR plant growth regulator

TDZ thidiazuron








Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

INDIRECT SHOOT ORGANOGENESIS AND SELECTION OF SOMACLONAL
VARIATION IN Dieffenbachia

By

Xiuli Shen

May 2007

Chair: Michael E Kane
Cochair: Jianjun Chen
Major: Horticultural Science

A series of experiments were conducted to investigate the feasibility of selecting of

somaclonal variants for new cultivar development in Dieffenbachia. In the first set of

experiments, a protocol for indirect shoot organogenesis was established for Dieffenbachia cv.

Camouflage. Maximum 96% callus formation frequency was observed on a basal medium

supplemented with 5 [M TDZ and 1 [M 2,4-D. The maximum shoots regenerated per callus

(7.9) was obtained on a basal medium supplemented with 40 [M 2ip and 2 [M IAA. In the

second set of experiments, 4 Dieffenbachia cultivars were examined for the capacity for indirect

shoot organogenesis and effects of genotypes, explant sources and plant growth regulators were

investigated. There were significant genotypic effects on both callus formation and shoot

regeneration. Cultivar Camouflage exhibited the greatest ability for indirect shoot organogenesis,

while cv. Octopus had no capacity for shoot regeneration from calli. Only leaf explants taken

from in vitro shoot cultures were capable of callus formation. Root explants failed to undergo

indirect shoot organogenesis, regardless of cultivar. In the third set of experiments, somaclonal

variation at the phenotypic level among Dieffenbachia plants regenerated via indirect shoot

organogenesis was evaluated. Three types of somaclonal variations with different leaf

variegation and color were observed in cv. Camouflage with a total somaclonal variation rate of









40.4%. Somaclonal variation in leaf shape was observed in cv. Camille with a somaclonal

variation rate of 2.6%. No somaclonal variation was observed in regenerated plants of cv. Star

Bright. The duration of callus culture of cv. Camouflage had no effect on somaclonal variation as

variation rates between plants regenerated from 8 months and 16 months callus culture were

similar. Our results indicated that selection of somaclonal variation has great potential for new

cultivar development in Dieffenbachia.









CHAPTER 1
LITERATURE REVIEW

Introduction

The genus Dieffenbachia belongs to the family Araceae and is one of the most popular

ornamental foliage plants. Its popularity is largely attributed to its attractive foliar variegation,

which is generally manipulated through breeding. Traditional breeding in Dieffenbachia,

however, is hindered by its naturally-occurring dichogamy and long breeding cycle (Henny,

1988). High demand for new cultivars with novel foliar variegation has created a need for

developing more efficient breeding methods. Selection of somaclonal variants from in vitro

generated population could be an alternative means for new cultivar development. This study

aimed at establishing regeneration methods for Dieffenbachia through indirect shoot

organogenesis and examining the feasibility of selection of somaclonal variants from the

regenerated population for new cultivar development. It is hypothesized that plants regenerated

through indirect shoot organogenesis were associated with high somaclonal variation because of

intermediary callus formation. Since indirect shoot organogenesis has not been established in

Dieffenbachia, the first experiment was to establish a protocol for indirect shoot organogenesis in

Dieffenbachia. The second experiment was to determine factors affecting indirect shoot

organogenesis. The third experiment was to assess somaclonal variation among regenerated

plants for potential new cultivar development.

Dieffenbachia Genus

Botany

Dieffenbachia genus, a monocot and a member of the family Araceae, is native to tropical

regions of Central and South America (Chen et al., 2003b). It is a herbaceous perennial

evergreen with thick stems bearing alternate leaves (Black, 2002). Flowers are unisexual and









consist of spadix and spathe. The spadix is a central fleshy spike, covered with many small

staminate and pistillate flowers. The spathe is a modified bract and envelops the spadix until

anthesis (Henny, 1988). Self-pollination is prevented by the naturally occurring dichogamy in

this genus as female flowers mature earlier than male flowers. The leaves are broad and

variegated with white marking or different patterns which are sheathed petioles and have a very

striking appearance (Henny and Chen, 2003). Thus, the value of Dieffenbachia lies in its

attractive foliar variegation. Additionally, Dieffenbachia requires low light levels for its growth.

As a result, Dieffenbachia is widely used as living specimens for interior decoration and

interiorscaping. Dieffenbachia is among the most popular ornamental foliage plants in the United

States, continually ranking in the top five for annual wholesale value (Chen et al., 2002).

Propagation

Dieffenbachia can be propagated by both sexual and asexual methods. Seed propagation is

not usually used in the production because of limited seed production and poor seed germination.

Dieffenbachia can be easily propagated by asexual methods, by tip, cane cuttings and divisions,

but such methods only produce a few cuttings or plants from each stock plant and require

significant labor input. Asexual propagation can also carry over pathogens. The advantage of

propagation by cuttings or divisions is that plants propagated are true-to-type.

Plant tissue culture technique can potentially overcome all these limitations of traditional

approaches to Dieffenbachia. In vitro culture is becoming an important method for propagation

of Dieffenbachia since 1980s. An initial application of tissue culture in propagation of

Dieffenbachia was to eliminate systemic viral and bacterial pathogen. Dieffenbachia cv.

Perfection derived from lateral bud and shoot tip culture were indexed 3 separate times on each

of 4 different media, and plantlets lines free of tested fungi and bacteria were released. This line

showed vigorous growth and branched more freely due to the depletion of fungi and bacteria









(Knauss, 1976; Chase et al., 1981). Later, the tissue culture was mainly used to produce a large

number of uniform and true-to-type healthy plants for commercial production on a year-round

basis. Through modification of media and culture conditions, the propagation rate of

Dieffenbachia had been greatly increased (Voyviatzi and Voyiatzis, 1989). Tissue culture also

reduces greenhouse space needed for stock plant production and produces salable plants in a

greater range of pot sizes.

In addition to providing true-to-type liners, tissue culture also produces off type plants. In

the early application of tissue culture for propagation of Dieffenbachia, all off-type plants were

rouged out to maintain the genetic fidelity of plants produced. It was later realized that these off-

type plants could be a source for selection of somaclonal variation for new cultivar development.

Since Larkin and Scowcroft (1981) advocated that somaclonal variation could be used as a

promising tool for breeding to produce novel genetic variation, some somaclones with desirable

features have been obtained by this method (Chen and Henny, 2006). It generally requires about

2 to 3 years for a new cultivar development compared to 7 to 10 years through traditional

breeding (Henny et al., 2000).

Breeding

Progress in breeding of Dieffenbachia has been slow due to the long breeding cycles. The

first hybrid Dieffenbachia Bausei was released in 1870 (Birdsey, 1951), since then only about

100 new cultivars have been developed. Hybridization is the most common method in producing

new cultivars in Dieffenbachia, but this requires many crosses and careful selection. Naturally

occurring dichogamy in this genus also makes this process very laborious and time consuming. It

usually takes about 7-10 years for a new cultivar to be released (Henny et al., 1986; Henny et al.,

1987a, b; Henny et al., 1988a, b; Henny et al., 1991a, b, c; Henny, 1995a, b). In addition to

hybridization, new cultivars are also developed from sports or from somaclonal variation. For









example, cv. Camouflage is a mutant of Panther, and cv. Camille is a sport of cv. Marianne.

Selection of somaclonal variation from in vitro cultures has become an important method for

new cultivar development. So far 20 cultivars of Dieffenbachia derived from selection of

somaclonal variants have been released (Chen and Henny, 2006). As the value of Dieffenbachia

lies in its aesthetic appearance, any changes in plant form, size, color or variegation pattern can

be desirable traits for Dieffenbachia.

Developmental Pathways for Plant Regeneration

There are generally 3 developmental pathways for in vitro plant regeneration: 1)

propagation from pre-existing meristems (shoot culture or nodal culture), 2) shoot

organogenesis, and 3) somatic embryogenesis (non-zygotic embryogenesis). Proliferation from

pre-existing meristems refers to production of shoots from shoot meristems (shoot tips) or

axillary meristems (axillary buds) followed by rooting of individual shoot (Kane, 2000a). Shoot

organogenesis is propagation from explants without pre-existing meristems through production

and subsequent rooting of adventitious shoots (Schwarz and Beaty, 2000). Somatic

embryogenesis regards to production of embryos that are not the result of gametic fusion (Gray,

2000). Any in vitro regeneration system is associated with a certain degree of somaclonal

variation and selection of somaclonal variants can be used as an important method for new

cultivar development. The degree of somaclonal variation is highly related with the regeneration

pathway. Plants regenerated from pre-existing meristems are usually true-to-type and this

method is used for clonal micropropagation. Methods involving adventitious meristem formation

or a callus phase result in plants with higher levels of somaclonal variation (Bouman and De

Klerk, 1997).









Organogenesis

Definition

Shoot organogenesis refers to propagation from explants without pre-existing meristems

through production and subsequent rooting of adventitious shoots. There are 2 types of shoot

organogenesis, direct and indirect. Direct shoot organogenesis is the production of shoots from

explants directly, while indirect refers to the formation of shoots indirectly from an intermediary

callus that first develops on the explants (Kane et al., 1994).

Advantages of Shoot Organogenesis

Shoot organogenesis has the following advantages compared to traditional propagation and

other in vitro propagation systems: 1) shoot organogenesis is more efficient than propagation

from pre-existing meristems; 2) shoot production is more synchronous than somatic embryo

formation; 3) indirect shoot organogenesis is associated with higher genetic variability and have

more chance for selection of somaclonal variants; 4) survival rate is higher among plantlets

produced via shoot organogenesis than those via somatic embryogenesis after transferred to

greenhouse; 5) shoot organogenesis is more easily achieved in most species than somatic

embryogenesis.

Phases of Shoot Organogenesis

A series of cellular events occur in the de novo shoot meristem formation process, and it

can be demonstrated in the following model:

Explant competence determination shoots


dedifferentiation induction differentiation

There are generally 3 phases of shoot organogenesis, namely dedifferentiation, induction

and differentiation. Dedifferentiation refers to differentiated cells in explants that dedifferentiate









and revert to a less committed, more flexible/plastic developmental state that may or may not

involve callus formation. In the direct shoot organogenesis, some cells from explant tissues

dedifferentiate directly to produce a new primordial, while in the indirect shoot organogenesis,

cells from explants dedifferentiate to give rise to callus formation from which the new shoots are

produced. As a result of dedifferentiation, some of these dedifferentiated cells in explants may

become competent and capable of responding to specific inducing signals. Plant growth

regulators are one of these inducing signals through which cells become competent. It has been

noticed in the induction of adventitious shoots from immature leaves of cassava that no

adventitious shoots could be obtained on regeneration media if leaf explants were cultured on

induction media for 1-3 days, indicating that cells has not become competent in such short time.

However, after culture for 4 or more days on induction media, explants showed regeneration (Ma

and Xu, 2002).

The span between the time cells become competent and when they become fully

determined for primordial production refers to as the induction phase. During the induction

phase, a competent cell or a group of competent cells become committed to a unique

developmental fate on the stimulus of inducing signal. At the end of induction phase, cells

become fully determined and capable of shoot organogenesis even with the removal of inducing

signals (Schwarz and Beaty, 2000).

During the differentiation phase, morphological differentiation and development of the

nascent organ occur. In their study on morphogenesis in petiole derived callus of

Amorphophallus rivieri Durieu, Hu et al (2005) observed that compact callus consisted of 3

components: epidermis, subepidermis and inner parenchyma cells; cells in subepidermis started

to divide to gave rise to the formation of a long and narrow meristematic zone after 1 week of









culture on differentiation medium; the meristematic masses then became more organized and

formed meristematic domes by continuous anticlinal and periclinal division; the periclinal cell

divisions on the flanks of the meristems resulted in leaf primordial formation after culture for 4

weeks. Through scanning electron microscopy, a group of regenerating shoot buds with distinct

epidermal layer, unicellular trichomes, and stomata has been observed in callus cultures of

Flacourtiajangomas (Lour.) Raeusch The presence of stomata on the epidermal layer is thought

to be a striking feature for shoot formation (Chandra and Bhanja, 2002). In a study on

organogenesis induced from mature zygotic embryo-derived leaflets of peanut, Chengalrayan et

al (2001) reported that during the initial 4 days of culture, cells of the explants enlarged;

mesophyll cells underwent anticlinal division giving rise to globular cell cluster on day 7 of

culture; subsequent periclinal and anticlinal divisions resulted in dome-shaped shoot meristems

in 15 days; well-developed shoot buds formed after 21 days of culture.

Factors Affecting Shoot Organogenesis

There are many factors which affect explant tissues becoming competent, and eventually

determined to complete shoot formation.

Genotypes

Genotype has the most influence on capacity for shoot organogenesis in vitro. Some

genotypes are easily cultivated via shoot organogenesis while others are either recalcitrant or

exhibit no capacity for shoot organogenesis (Thao et al., 2003; Landi and Mezzetti, 2005).

Significant differences in growth rate, color and structure of calli were found among 4 carnation

genotypes tested and these differences were also responsible for differentiation of calli into a

certain organogenic pathway (caulogenesis, shoot organogenesis or rhizogenesis). Callogenesis

was not only a result of dedifferentiation of explant tissues, but also an essential preparatory









stage for in vitro morphogenesis. Callus type can be a prediction for its organogenic potential

(Kallak et al., 1997).

Among 38 carnation cultivars tested for shoot organogenesis from nodal explants, 81.6%

of them showed a high regeneration ability (> 50% and 4 shoots per node segment), and 18.4%

showed low shoot regeneration (< 50% and 4 shoot per node segment)(Nontaswatsri et al.,

2002). Phillips (2004) concluded that specific genes were involved in dedifferentiation,

acquisition of competence and induction stages of shoot organogenesis. Some genotypes may

lack the genes required for shoot organogenesis or these genes may not be functional. Plants of

different genotypes may possess different endogenous plant growth regulator levels and this

might be responsible for differences in response in vitro.

Explants

The developmental stage and type of explants play a critical role in the success of shoot

organogenesis. The capacity of cells to become competent for regeneration may be completely

lost once they mature to a point due to the elimination of totipotency of cells. In general, explants

taken from less differentiated, immature tissues are easily induced and regenerated via direct

shoot organogenesis. Explants derived from highly differentiated tissues are less responsive in

vitro, and usually undergo indirect shoot organogenesis. In study on the effect of explant types

on shoot organogenesis of Vigna unguiculata, Choi et al (2003) reported that the highest

frequency of shoot organogenesis was obtained from single whole cotyledon explants (67.5%);

the proximal halves of cotyledon (50.1%) showed better regeneration than distal halves (30.1%);

no shoots formed from one or two cotyledon halves with embryo axis or embryo axis alone.

Explants taken from stock plants of different developmental stages possess different endogenous

hormonal level which affects their response in vitro. Differences in the ability to produce

adventitious shoots among different explant types were also noted in shoot organogenesis in









eggplants. On MS basal medium supplemented with 0.2 iM TDZ, the percentage of explants

that developed buds and the number of buds per explant was 100% (75-100 buds per explant) for

leaf, 100% (75-100 buds per explant) for cotyledon, 5% (1-25 buds per explant) for hypocotyl,

20% (25-50 buds per explant) for epicotyl, and 65% (1-25 buds per explant) for node explants

respectively (Magioli et al., 1998).

Plant Growth Regulators

Plant growth regulators are the most important inducing signal for shoot organogenesis.

Dedifferentiation, induction and development of shoots or roots are regulated by both

endogenous and exogenous growth regulators. The type of plant growth regulators and their

interaction play an important role in dedifferentiation, induction and development of shoots or

roots (Khanam et al., 2000). It is generally believed that cytokinins are beneficial for shoot

formation, while auxins stimulate callus formation, root formation and somatic embryogenesis.

The ratio of cytokinins to auxins is also critical in determining shoot versus root formation. A

high cytokinin to auxin ratio promotes shoot meristem formation, while callus or root meristems

are formed when the cytokinin to auxin ratio is low. Adventitious shoots were induced on media

containing only BA or BA as the major PGR, while somatic embryos were produced on 2,4-D

containing media in morphogenesis from leaves of cassava (Ma and Xu, 2002). It has been

reported that different combination of auxins (NAA, IAA, 2,4-D) and cytokinins (BA, kinetin) at

0, 1, 2 mg 1-1 levels resulted in either direct adventitious shoot formation, callus formation, or

indirect adventitious shoot formation (Makunga et al., 2005). Both exogenous cytokinin and

auxin were required for shoot organogenesis from petiole explants in Nymphoides indica. BA

was most effective in stimulating adventitious shoot formation compared to 2iP and kinetin

(Jenks et al., 2000). In their study on the effects of cytokinin/auxin combination on

organogenesis in Duboisia myoporoides, Khanam et al (2000) reported that the auxins 2,4-D and









NAA were more effective than IAA or IBA for callus formation, the greatest number of shoots

were induced with 10 [iM BA and 1 [iM NAA among the combination of BA (0, 0.1, 1, 10 ) and

NAA (0, 1, 10) examined.

Somatic Embryogenesis

Definition

Somatic embryogenesis is the production of embryos not resulting from gametic fusion

(Merkle et al., 1990). Asexual embryogenesis is known to occur naturally in many plant species

and many different names are used by different authors to describe this phenomenon in different

species, such as apomixes, polyembryony, adventive, sporophytic and nucellar embryony. Many

plant tissues, such as the nucellus, inner integument, synergids, antipodals, endosperm, and

suspensor have been observed to naturally give rise to asexual embryos (Tisserat et al., 1979).

Asexual embryogenesis can also be obtained in vitro culture and this process is usually called

somatic embryogenesis. Since the first report of somatic embryogenesis in carrot cell culture in

1958 (Steward et al., 1958a, b), in vitro somatic embryogenesis has been reported in over 100

species (Krishnaraj and Vasil, 1995; Merkle et al., 1995). There are 2 types of somatic

embryogenesis, direct or indirect. Direct somatic embryogenesis refers to somatic embryos

produced directly from cells of explant tissues, with no intervening callus. Indirect somatic

embryogenesis is characterized by the production of callus from explant tissues first, from which

somatic embryos form (Merkle, 1997). Somatic embryogenesis has been achieved in a number of

economically and ornamentally important plant species, but studies on Dieffenbachia has not

been reported.

Stages of Somatic Embryogenesis

There are generally 3 developmental stages in somatic embryogenesis in vitro: induction,

development and maturation (Zimmerman, 1993). Explants are composed of heterogeneous cell









types. During the induction stage, undifferentiated cells from explants can be induced to become

embryogenically determined and somatic embryos can be obtained directly from these cells via

direct embryogenesis. Differentiated cells from explants undergo dedifferentiation,

meristemation and redifferentiation into embryogenic cells. In some cases, callus is formed first

from explant tissues, and somatic embryos subsequently form. This process is referred to indirect

somatic embryogenesis (Kohlenbach, 1985; Merkle, 1997). Explants may also contain a portion

of pre-embryogenic determined cells derived from ovular or embryo tissues that may develop

into embryos under a suitable condition (Bhojwani and Razdan, 1996). Somatic embryogenic

cells can be distinguished from non-embryogenic cells by the following features. Embryogenic

cells usually contain dense cytoplasm, prominent nuclei, thickened cell wall and are less

vacuolated (Mooney and Van Staden, 1987).

During the development stage, a single embryogenic cell undergoes a series of transverse

and longitudinal divisions, passes through globular, heart, torpedo, and cotyledonary stages for

dicots or globular, scutellar and coleoptilar stages for monocots, and finally forms a bipolar

structure with root and shoot meristems on opposite end which has the capacity to reproduce

entire plants (Arnold et al., 2002).

During the maturation stage, the somatic embryos accumulate reserve and achieve

desiccation tolerance (Ammirato, 1974). This is very important for successful germination and

subsequent growth. The maturation stage in somatic embryogenesis has just been realized

recently as an important stage because the degree of maturation can significantly affect the

germination capability of somatic embryos (Bhojwan and Razdan, 1996). Normal looking

somatic embryos may be physiologically immature. A period of reversible arrested growth is









necessary for proper germination and regeneration. Without such developmental stages, somatic

embryos usually germinate precociously and finally die (Gray and Purohit, 1991).

Morphology and Physiology of Somatic Embryos

Although somatic embryos share many histological and morphological characteristics of

zygotic embryos, they differ in several aspects. Unlike a zygotic embryo which has a suspensor

and nourished via a suspensor, a somatic embryo may not have a suspensor or has an abnormal

suspensor. Somatic embryos are typically formed from a proembryonal complex. A somatic

embryo may grow directly from a proembryonal complex without the formation of suspensor

(Thomas et al., 1972). In other cases, somatic embryos may be supported above explants by

either a narrowed, not easily observed suspensor, or a very broad suspensor (Hakman et al.,

1985). Cotyledons may develop abnormally, including multiple cotyledons, fused cotyledons or

unequally sized pairs (Rashid and Street, 1973).

Somatic embryos have a less compressed and narrowed appearance than zygotic embryos

due to the lack of protective seed coats (Gray and Purohit, 1991). Somatic embryos tend to

develop asynchronously. Several stages are present in cultures at any time thus somatic embryos

do not mature altogether (Profumo et al., 1986). Additionally, somatic embryos usually

germinate continuously after they are formed without a quiescent resting phase typically

observed in zygotic embryos. This leads to precocious germination and plants usually die after a

certain period of time (Gray, 1989). Gray et al (1993) also noticed that certain parts of embryos

might precociously germinate before full embryo maturation, such as trichome development on

hypocotyls and cotyledons, hypocotyl elongation, cotyledon elongation, premature root

emergency in their study on somatic embryogenesis in Cucumis melo.









Histological Studies on Shoot Organogenesis/Somatic Embryogenesis

Plantlets regenerated in vitro from adventitious meristems may originate either from shoot

organogenesis or somatic embryogenesis. In shoot organogenesis, shoots are usually formed

first, then roots are induced from the base of shoots, with a well-developed vascular connection

between shoots and maternal explants. In somatic embryogenesis, shoots and roots are derived

from embryogenic shoots and roots on bipolar structure of somatic embryos. There is no vascular

connection between somatic embryos and parental explants (Puigderrajols et al., 2000;

Chengalrayan, 2001). Before concluding that regenerants from in vitro culture have a somatic

embryogenic origin, it is necessary to provide convincing histological evidence to prove it. A

visual similarity between the morphological appearance of the regenerating structures and

somatic embryos is insufficient to draw the conclusion of somatic embryogenesis. In his studies

on morph-histological study of somatic embryo-like structures in hypocotyl cultures of

Pelargonium (ornamental plants), Haensch (2004a, b) found that although somatic embryo-like

structures were produced from hypocotyls, histological examination confirmed the lack of

bipolarity and revealed vascular connections to the explants and concluded that there was no

proof of somatic embryogenesis.

Like zygotic embryogenesis, somatic embryogenesis is assumed to pass through the same

characteristic stages, and the demonstration of these key stages can be used as the primary

criteria for confirming somatic embryogenesis. The ontogeny of somatic embryogenesis can be

revealed by scanning electron microscopy or light microscopy. The normal somatic embryo

development from globular, to heart-, to torpedo-shaped and finally cotyledon stage embryo has

been described through scanning electron microscope in safflower (C. tinctorius L.)(Mandal and

Gupta, 2002). Histological events during somatic embryo development in Serbian spruce (Picea

omorika) have been recorded. Each somatic embryo initials formed on the cotyledon surface









after an unequal transversal division of a cell. The smaller, apical cell gave rise to apical dome

and later on filamentous suspensor (Budimir, 2003). Somatic embryos with plumule and root

pole have been documented in Musa. spp. after 50 days of culture by passing through globular

stage (Lee et al., 1997). The attainment of a globular appearance is regarded as one of the first

key features of somatic embryo development. The presence of protoderm, the outmost layer of a

developing somatic embryo, is also considered as a unique feature of somatic embryo

development, and it is believed that protoderm has a function of regulation of further embryo

development by applying physical and cell division limitation (Sharma and Millam, 2004).

The developing somatic embryos had no detectable vascular connection with the mother

explants (Quiroz-Figueroa et al., 2002). Two cell types can be observed under light microscope,

embryogenic and non- embryogenic cells. Embryogenic cells are smaller, compact, and possess

relatively dense cytoplasma, densely stained nuclei, and small invisible vacuoles. Non-

embryogenic cells usually have large vacuoles and unclear cytoplasma. Embryogenic cells may

form embryogenic masses (EMS) by continued cell division. These actively dividing cells may

develop into globular masses, forming proembryoids, but only a very limited number of these

embryogenic cells may give rise to meristem-like structure (meristemoid), and differentiate into

different staged embryos (Onay, 2000).

Somaclonal Variation

Variation among plants regenerated from tissue culture has been called somaclonal

variation (Larkin and Scowcroft, 1981). From its origin, it can be deduced that somaclonal

variation occurs in the period before the formation of meristematic tissues and terminated with

formation of meristematic tissues (Bouman and De Klerk, 1997). Somaclonal variation is a

random phenomenon that can occur at any location in the genome (De Schepper et al., 2003).

Assessment of somaclonal variation can be achieved by analysis of phenotype, chromosome









number and structure, proteins, or direct DNA evaluation of plants (Bouman and De Klerk,

1997). The extent of phenotypic variation is usually determined as the percentage of plants

showing aberrations from the parental plant for one or more defined characteristics. Plants with

the deviant phenotypes are known as somaclones or somaclonal variants.

Origin of Somaclonal Variation

Cultivars

The potential for somaclonal variation varies among cultivars genotypess). In their study

on chromosomal instability in regenerants from Medicago media Pers, Naj aran and Walton

(1987) found there was significant difference in the ability to produce somaclones among

cultivars, being 9.4%, 10%, 20% and 64% for the 4 cultivars tested respectively.

Explant Sources

As mentioned previously, explants usually are composed of many cell types and these cells

may vary in differentiation and even ploidy levels. Plants regenerated from a single explant may

originate from different cells. Generally speaking, explants with pre-existing meristematic tissues

such as axillary buds, shoot tips and meristems show less variation than explants that have no

pre-existing meristematic tissues such as leaves, roots or stems (Skirvin et al., 1994). Piccioni et

al (1997) reported that no somaclonal variants were observed in alfalfa plants regenerated from

axillary branching propagation, but plants regenerated from callus derived from petioles showed

greater variation (23%) analyzed by RAPD markers. In chrysanthemum (C/ i/hl),l hi/,nnu

morifolium), plants regenerated from pedicel explants showed little variation from control plants,

but considerable variants were found among plants regenerated from petals. These variants

flowered earlier and produced more flowers than control plants (De Jong and Custers, 1986).

Tubers produced on potato plants regenerated from leaf, stem, rachis and tuber pieces had

different skin color. Regenerants derived from stem, leaf, tuber piece produced tubers with pink-









red color as control plants while regenerants from rachis produced white, red-splashed white and

mixed colored tubers (Wheeler et al., 1985).

Callus

Somaclonal variation is quite often associated with indirect in vitro regeneration involving

an interviewing callus stage. During this period, differentiated cells undergo dedifferentiation,

induction, redifferentiation (Rout, 1999). Bouman and De Klerk (2001) showed that the rate of

somaclonal variation among Begonia regenerated via somatic embryogenesis was 1.5% but

10.6% for regenerants derived from the callus stage.

Duration of the Tissue Culture Phase

There is a general trend that somaclonal variation increases with the length of time that a

culture has been maintained in vitro. In a study on the genetic instability during embryogenic

cloning of celery, Orton (1985) noticed that if calli, derived from immature petiole segment,

were maintained for 6 months by a series of transfer, 84% of the callus cells were karologically

indistinguishable from the control, the remainingl6% exhibited chromosome loss or fusion, only

1 regenerated plant out of 95 showed an abnormal phenotype. After 12 months in culture, 97%

callus cells were karologically distinguishable from the control, most were aneuploidy and all

callus cells lost the capacity to produce embroids.

Measurement of Somaclonal Variation

Phenotypic Variations

Since Skirvin and Janick (1976) advocated that somaclonal variation could be used as a

promising tool for breeding, some somaclones with desirable features have been obtained. The

variation could be change in foliar variegation pattern, modification of flower color and size,

alteration of leaf shape, increase in lateral shoots, and change in overall plant form (Griesbach et

al., 1988; Khalid et al., 1989). New somaclonal variants with desirable dwarf and bushy









phenotype of lisianthus (Eustoma grandiflorum) have been obtained from leaf, stem culture.

Naturally, all cultivars of lisianthus have a long stem with only apical branches. Application of

growth retardants and pinching are required to stimulate basal branch formation for its

ornamental values (Griesbach and Semeniuk, 1987). Khalid et al (1989) produced new

somaclonal variants of chrysanthemum with novel lilac-colored ray florets using petals as

explants. Dwarf daylily (Hemerocallis) has been selected from callus derived from shoot tip

culture (Griesbach, 1989). Arene et al (1993) obtained rose variants with altered petal number,

color and having dwarf growth habit from callus of Rose hybrida cv. Meirutral.

Tremblay et al (1999) reported that several morphological changes including dwarfism,

fascination, variegate patterns, height alternation, modified branch angle, and bushy shape were

observed in somatic embryogenesis of spruce species. Griga (2000) noted that morphological

alternations including altered leaflet shape, changed stipule morphology, shortened internode,

irregular leaf position on the stem, shortened flower stalk were found in pea (Pisum sativum)

plants regenerated from immature zygotic embryo culture. Somaclonal variants with increased

disease resistance and higher yield traits have been obtained from wheat somatic embryogenesis

(Arun et al., 2003).

Changes at Chromosomal Levels

Somaclonal variation can be assayed at the chromosomal level. Some somaclonal

variations result from changes in the chromosome number. Polyploidy and aneuploidy are quite

often associated with morphological modification. Moyne et al (1993) reported that tetraploid,

aneuploid and polyploidy cells were found in cultures of rose. Diploidization, aneuploidization

and polyploidization have been observed in cultures ofLarix decidua initiated from

megagametophyte explants (Von Aderkas and Anderson, 1993). Except changes in chromosome

number, alternation in chromosome structure, such as point mutation, chromosome breakdown









and reunion, deletion, and insertion have been observed in plants derived from somaclonal

variation (Bouman and De Klerk, 1997).

Variations at DNA Levels

Changing DNA sequence is a fundamental event responsible for much of the reported

somaclonal variation among regenerated plants. DNA sequence changes or variations include

mutations involving one or a few nucleotides, deletions and insertions caused by unequal

crossover, or activity of transposable elements. The precise way to determine mutations at DNA

levels is to sequence the gene of interest. An alternative approach is to detect variation at DNA

levels by electrophoresis of proteins and/or by PCR amplification of nucleic acid segments. The

protein electrophoresis implies the existence of mutant alleles by virtue of changes in gene

products. Isozyme analysis is a frequently used method of studying certain gene product changes

during in vitro culture (Mangolin et al., 1997; Chen et al., 1998; Azeqour et al., 2002; Feuser et

al., 2003). A series of PCR-based techniques have been developed over the years for genotyping

and fingerprinting purposes. Among the PCR-based methods, RAPD and AFLP have been used

to analyze genetic variation among regenerants of a wide range of plant species (Chen et al.,

2006). Somaclone specific fragments can actually be cloned, and nucleotide sequences can be

analyzed to determine exactly what changes occur at DNA sequence level. Fragment cloning was

performed in variants regenerated from maize, pea, and tomato (Gostimsky et al., 2005).









CHAPTER 2
PROTOCOL ESTABLISHMENT FOR INDIRECT SHOOT ORGANOGENESIS FROM
LEAVES OF Dieffenbachia CV. CAMOUFLAGE

Introduction

The genus Dieffenbachia, a member of the family Araceae, is composed of 30 species

native to South and Central America (Mayo et al., 1997). Dieffenbachia has been produced as an

ornamental foliage plant for interiorscaping since 1864 (Birdsey, 1951) and consistently ranks

among the top five most popular foliage plant genera based on annual wholesale value

(McConnell et al., 1989; USDA, 1998). In addition to its tolerance to low light levels, another

factor contributing to its popularity is the increasing release of new and attractive cultivars that

provide consumers with a wide range of selection for novelty.

Interspecific hybridization has been the primary method of developing new cultivars

(Henny and Chen, 2003). Hybridization of Dieffenbachia, however, has been hampered by

naturally occurring dichogamy, long breeding cycle, and limited seed production (Henny, 1988).

With the commercial application of in vitro propagation of Dieffenbachia, new cultivars have

been released following selection of somaclonal variants (Chen and Henny, 2006). The

frequency of somaclonal variants is generally high, and the time required for a new cultivar

release can be only 2 to 3 years compared to 7 to 10 years required using traditional breeding

methods (Skirvin et al., 1994; Chen et al., 2003a). Chen et al (2004) analyzed genetic relatedness

of some cultivated Dieffenbachia using amplified fragment length polymorphism (AFLP) and

found that cultivars selected from somaclonal variants differ genetically from their parents.

Successful use of in vitro techniques for producing somaclonal variants depends on the

establishment of an efficient method for regenerating a large number of plants indirectly from an

intervening callus stage (Maralappanavar et al., 2000; Niwa et al., 2002; Hossain et al., 2003;

Anu et al., 2004; Arce-Montoya and Rodriguez-Alvarez, 2006; Hammerschlag et al., 2006).









Although plant regeneration via indirect shoot organogenesis has been achieved in a vast array of

plant species, a protocol for indirect shoot organogenesis has not been developed in

Dieffenbachia. Currently commercial in vitro propagation of Dieffenbachia is through shoot

culture (Knauss, 1976; Chase et al., 1981; Voyiatzi and Voyiatzis, 1989; Henny et al., 2000).

The objective of this study was to establish a protocol for inducing indirect shoot organogenesis

in Dieffenbachia cv. Camouflage.

Materials and Methods

Media and Sterilization Conditions

A basal medium (BM) consisting of MS (Murashige and Skoog, 1962) mineral salts, 0.4

mg 1- thiamine, 2.0 mg 1- glycine, 100.0 mg 1- myo-inositol, 0.5 mg 1- pyridoxine, 0.5 mg 1-

nicotinic acid, and 25 g 1- sucrose in combination with plant growth regulators was used.

Medium was adjusted to pH 5.8 with 0.1 N KOH prior to the addition of 6 g 1- TC agar

(PhytoTechnology Laboratories, Shawnee Mission, KS) and autoclaved at 1.2 kg cm-2 for 20

min.

Plant Materials and Establishment of Shoot Cultures

Stem segments about 10 mm long, containing lateral buds, were cut from stock plants of

Dieffenbachia cv. Camouflage. The stock plants were maintained in a shaded greenhouse with a

maximum irradiance of 345 imol m-2 s-1 under natural photoperiod (10 to 14.5 h light) and a

temperature range of 20 to 3 10C. Lateral buds were excised, rinsed under running water for

about 10 min and used as explants to initiate in vitro shoot cultures. After surface sterilization in

aqueous 1.2% sodium hypochlorite (20% v/v Clorox Ultra) containing several drops of Tween

20 for 10 min on a shaker and rinsing 3 times, 5 min each, with sterile water, lateral buds were

further trimmed by removing the outermost one or two bud scales. Lateral buds still attached to a

2 mm2 thick square of stem tissue were cut and placed individually into 25 x 150 mm culture









tubes containing 15 ml BM supplemented with 80 iM 2iP and 2 iM IAA. Cultures were

maintained under a 16 h light photoperiod at 40 imol m-2 s-1 provided by cool white fluorescent

lamps (General Electric F20WT12CW) at 22 + 3C. Shoot clusters were divided and transferred

to the same fresh medium every 8 weeks to increase in vitro stock shoot cultures.

Indexing of Established Cultures

Following the establishment of in vitro shoot cultures, cultures with visually detectable

contamination were immediately discarded. Cultures with no symptom of contamination were

routinely indexed for the presence of cultivable bacterial and fungal contamination using the

procedure developed by Kane (2000b).

Callus Induction

Leaves obtained from the in vitro shoot cultures served as explant sources for callus

induction. Leaf explants were cut into 5 mm2 sections with mid-vein, and the leaf margins

removed with a scalpel. Leaf explants were cultured with abaxial surface in contact with the

callus induction medium which were BM supplemented with TDZ at 0, 1, 5, 10 [M and 2,4-D at

0, 0.5, 1 iM. Explants were cultured in 100 x 15 mm sterile Petri plates containing 20 ml

medium. There were 5 explants per Petri plate and 5 replicate plates per treatment. Cultures were

initially maintained in dark for 8 weeks and then transferred to the 16 h light photoperiod at 40

mrnol m-2 S-1 for another 4 weeks. The number of explants forming calli was scored after 12

weeks of culture. Callus formation frequency was calculated as the percentage of leaf explants

forming calli.

Indirect Shoot Organogenesis

To evaluate medium for inducing shoot formation from callus, calli produced on callus

induction medium containing 5 iM TDZ and 1 iM 2,4-D (the medium yielding optimal callus

induction) were cultured on BM supplemented with factorial combinations of (1) 2iP at 0, 20, 40,









80 pM and IAA at 0 and 2 iM; (2) kinetin at 0, 1, 2, 4 iM and GA3 at 0, 5, 10 riM. Plant growth

regulator-free medium served as the control. Calli were separated from leaf explants after 12

weeks callus production. Calli were cut into small pieces with a fresh weight of approximately

150 mg, and then transferred to glass baby food jars (4.5 x 7 cm) containing 40 ml medium.

There were 5 callus clumps per jar and 5 replicates per treatment. The number of shoots formed

per callus was determined after 8 weeks culture under the 16 h photoperiod at 40 .imol m-2 s-1

Histological Analysis

To verify the occurrence of indirect shoot organogenesis, callus samples were collected for

histological examination 4 weeks after the initiation of culture and the 9 additional samples of 3

day intervals. For shoot differentiation, callus samples were taken every 3 days after callus

clumps were transferred onto shoot induction medium until 42 days after culture. For optical

light microscopy (OLM), the following procedure was used. Callus specimens weighting

approximately 150 mg were fixed in Trumps fixative (McDowell and Trump, 1976). Fixative

infiltration was achieved under vacuum until all samples of calli sank to the bottom of

scintillation vials. Calli were rinsed 3 times in phosphate buffer (pH 7.2) for 10 min each. Calli

were post-fixed in a 1% buffered osmium tetroxide solution for 1 h, and then rinsed 3 times in

phosphate buffer (pH 7.2) for 10 min each, followed by 3 times wash in distilled water. Calli

were dehydrated in a series of ascending aqueous ethanol solutions at 25%, 50%, 75% for 30 min

each, at 95%, 100% for 1 h each, followed by dehydration in 100% acetone for 1 h. Calli were

then embedded in Spur resin (Spurr, 1969). Callus sections (10 [im) were cut using a Leica

Ultracut rotary ultramicrotome R (Leica Microscopy and Scientific Instruments, Deerfield, IL),

and mounted on glass slides. Sections were stained with 0.2% toludine blue and examined under

an Olympus BH-2 Epifluorescent Microscope (Olympus America Inc., Melville, NY).

Photographs were taken using a Pixera 120C digital camera. For scanning electron microscopy









(SEM), callus samples of approximately 150 mg were immersed in 100% methanol. Samples

were lyophilized using a Bal-Tec 030 critical point drier (ICMAS Inc., Alcoa, TN) with liquid

CO2, sputter coated with gold-palladium using a Denton Vacuum Desk II (Denton Vacuum,

Moorestown, NJ) for approximately 50 s and viewed with a Hitachi S-4000 scanning electron

microscope (Hitachi Scientific Instruments, Danbury, CT) operating at 6 KV. Digital images

were processed using SEMages 16 software (Advance Database System, Inc., Denver, CO).

Acclimatization

Shoots, some with roots, were removed from the baby food jars and excised individually

from the callus clumps. Medium was carefully rinsed off shoots. Shoots longer than 20 mm with

2-3 leaves were planted individually in 60-cell plug trays (4.5 x 4 x 5 cm3 each cell, REB

Plastics Inc, Apopka, FL) containing a 2:1:1 (v/v) soilless mixture of Canadian peat: vermiculite:

perlite. All plantlets were maintained in a greenhouse under shade cloth with a maximum

irradiance of 345 .imol m-2 s-1, natural photoperiod (10 to 14.5 h light), and a temperature range

of 20 to 310C. Plugs were hand watered twice a week. Peters 20N-10P-20K liquid fertilizer (200

mg 1- N; The Scotts Company, Marysville, OH) was applied weekly following 2 weeks

acclimatization.

Statistical Analysis

All experiments were established in a completely randomized design. The experiments

showing treatment responses were repeated once. Data were subject to analysis of variance using

SAS (SAS Institute, Inc., 1999). Mean separation was achieved by the least significant difference

(LSD) test at 95% level.









Results


Callus Induction

The frequency of callus occurrence from medium containing TDZ and 2,4-D differed

significantly based on their concentrations. No callus occurred on medium devoid of TDZ and

2,4-D or with 2,4-D only at 0.5 and 1.0 iM. However, TDZ alone at concentrations of 1, 5, and

10 .iM induced 4, 10, and 26% of explants to produce calli respectively. TDZ was required for

callus formation, but the higher frequency of callus formation occurred in medium supplemented

with both TDZ and 2,4-D. Induction medium containing 5 iM TDZ and 1 iM 2,4-D resulted in

the maximum of 96% of explants to produce calli (Table 2-1).

Indirect Shoot Organogenesis

A low number of shoots developed from calli cultured on BM alone (1.6 shoots/callus) or

BM supplemented with 2 iM IAA (1.2 shoots/callus). Basal medium supplemented with 2iP

alone at concentrations of 20, 40, and 80 iM elevated shoot numbers to 3.6, 6.3, and 4.0 per

callus respectively. Combining 2iP with IAA further increased shoot numbers compared to 2iP at

respective concentrations alone, but the increase was not statistically significant. Highest shoot

number (7.9 shoots/callus) occurred on BM supplemented with 40 [iM 2iP and 2 [iM IAA (Table

2-2). No shoot formation was observed on BM supplemented with combinations of kinetin and

GA3.

Green nodular calli were observed on leaf explants after 12 weeks of culture on BM

supplemented with 5 |M TDZ and 1 iM 2,4-D (Figure 2-1A). When calli were separated from

primary leaf explants and transferred onto BM supplemented with 40 iM 2iP and 2 iM IAA,

small green meristems were visible on the surface of calli within 4 weeks (Figure 2-1B) and later

developed into shoot buds (Figure 2-1C). Leaf formation and shoot elongation occurred in the









following 2 weeks (Figure 2-1D). Single shoot or shoot clusters with leaves and roots were

developed by the end of 8 weeks of culture (Figure 2-1E).

Acclimatization

Dieffenbachia cv. Camouflage plants were very easily acclimatized (Figure 2-1F). Ex vitro

survival rate of 100% was observed.

Histological Analysis

Indirect shoot organogenesis was confirmed by histological sectioning. Calli were

observed from leaf explants on BM medium supplemented with 5 [iM TDZ and 1 [iM 2,4-D

after 28 days of culture. Two types of cells were observed in calli under light microscopy:

regenerative cells which were smaller in size and more compact with more densely stained

cytoplasm, thinner cell walls, more prominent nuclei and no visible vacuoles (Figure 2-2A); non-

regenerative cells which were larger and less compact with less cytoplasm and smaller nuclei,

thicker cell walls and larger vacuoles (Figure 2-2B). Early mitotic activity was observed after 31

days of culture (Figure 2-2C). The first cell division was usually anticlinal followed by periclinal

cell division (Figure 2-2D). After several mitotic division, the differentiation of a meristematic

zone occurred (Figure 2-2E). By continuous anticlinal and periclinal cell division, bigger

meristematic cell masses composed of actively dividing cells were formed by 43 days culture.

Each meristematic mass was characterized by cells with thick walls, and the individual cell was

separated by thinner walls (Figure 2-2-F). Meristematic cell masses may also develop into

globular shapes, assuming an appearance similar to globular somatic embryos (Figure 2-2G and

2-2H). Initial cell divisions usually were initiated from a superficial callus cell or cells (Figure 2-

2D), but a cell or a group of cells in inner cell layers in the callus may also give rise to

meristematic mass (Figure 2-21). The meristematic mass became progressively more organized

and formed a meristematic dome which represented an apical meristem of a bud after 12 days of









culture on shoot induction medium (Figure 2-2J). The cell division along the flanks of the bud

meristem resulted in leaf primordia formation after 18 days shoot induction (Figure 2-2K). A

well-developed adventitious bud with apical shoot meristem and leaf was formed after 27 days of

culture (Figure 2-2L). Sometimes multiple shoots were formed (Figure 2-2M). Root formation

occurred after 39 days of culture (Figure 2-2N). A complete plantlet was regenerated after 8

weeks culture on shoot induction medium. Vascular connection between a developing shoot and

callus tissue was detected at day of 24 (Figure 2-20). Scanning electron microscopy showed

stomata were present on the epidermis of developing shoots at day 36 (Figure 2-2P).

Discussion

A plant regeneration system via indirect shoot organogenesis was established in this study.

To our knowledge, this is the first report of indirect shoot organogenesis in Dieffenbachia. Our

observations indicated that Dieffenbachia in general was recalcitrant in regard to shoot

organogenesis or somatic embryogenesis (attempts were made to induce somatic embryogenesis

failed to a wide range of PGR types, concentrations and combinations). This may, in part,

explain why in vitro regeneration via organogenesis or somatic embryogenesis in Dieffenbachia

has not been previously reported.

Callus Induction and Shoot Formation

The concentration and combination of PGR are the key factors influencing indirect shoot

organogenesis in Dieffenbachia. A similar PGR effect on callus formation has been reported in

other species (Khanam et al., 2000; Reddy et al., 2001; Ma and Xu, 2002; Giridhar et al., 2004;

Azad et al., 2005; Datta and Majumder, 2005; Zhou and Brown, 2005). 2,4-D has been shown to

be most effective for callus induction in many species. On the contrary, our study showed that

2,4-D was not a prerequisite for callus initiation as calli were induced on BM without 2,4-D. In

contrast, TDZ was required for callus formation in Dieffenbachia cv. Camouflage. There have









been several reports of significant TDZ effects on callus formation and shoot organogenesis in

other species (Gurriaran et al., 1999; Mithila et al., 2003; Datta and Majumder, 2005; Landi and

Mezzetti, 2005). TDZ, a cotton defoliant, has both auxin-like and cytokinin-like activity and can

be substituted for auxins or the combination of auxins and cytokinins (Singh et al., 2003).

Hutchinson et al (1996) reported that TDZ could result in an increase in endogenous levels of

auxins. It could be possible that TDZ might have fulfilled both auxin-like and cytokinin-like

roles in callus induction of this Dieffenbachia.

Cytokinin type and concentration have significant effects on subsequent shoot regeneration

from calli. Results from this study indicated that 2iP was more effective than kinetin because no

shoot regeneration was observed on BM supplemented with kinetin alone or combination with

GA3. Voyiatzi and Voyiatzis (1989) also found that 2iP was more effective in inducing lateral

shoot multiplication in Dieffenbachia than kinetin and 80 giM 2iP with 2 giM IAA was optimal

for shoot formation ofD. exotica cv. Marianna. The present results suggested that 40 giM 2iP

with 2 giM IAA was optimal for shoot organogenesis of cv. Camouflage. This difference might

be due to the fact that different cultivars, explants were used. This study used calli derived from

leaves, while axillary buds and shoot tips were used for shoot multiplication of cv. Marianna

(Voyiatzi and Voyiatzis, 1989). Differential responses of genotypes and explant sources to PGR

requirements have been well documented (Magioli et al., 1998; Choi et al., 2003).

Acclimatization

Dieffenbachia shoots regenerated in vitro proved to be easily adaptable to ex vitro

conditions. No separate in vitro rooting stage was required as shoot survival rate was 100% in a

soilless substrate. This is different from many other species where rooting is an obstacle for plant

establishment (Reed, 1995; Gavidia et al., 1996; Pruski et al., 2000). Dieffenbachia cv.

Camouflage belongs to an easy-to-root type, which is in contrast to its very limited shoot









formation ability. It was conceivable that endogenous level of auxin in Dieffenbachia might be

sufficient for root formation. A promotive carry over effect of the IAA in the shoot induction

medium on rooting was also possible.

Histological Analysis

In vitro shoot organogenesis and somatic embryogenesis are the most used methods for a

large-scale propagation and production of somaclonal variants. Sometimes it is very difficult to

detect the true nature of in vitro regeneration especially when a callus phase is involved in the

regeneration process and when the appearance of regenerating structures looks very much like

somatic embryos. In our study, nodular calli were induced from cv. Camouflage. The appearance

of these nodular calli superficially resembled globular somatic embryos and they also could be

easily detached from leaf explants. When these nodular calli were separated from leaf explants

and transferred onto BM medium supplemented with the combination of different plant growth

regulators, they either degenerated or simply proliferated. Others (Haensch, 2004a, b; Salaj et al.,

2005) have reported embryo-like structures were produced in their studies. However, histological

analysis revealed in their studies that all these somatic embryo-like structures were composed of

parenchymatous and vacuolated cells, and had no regeneration capacity. In contrast, histological

examination in our study showed that these nodular calli consisted of actively dividing cells,

therefore having regeneration potential. The reasons why these promising cells passed through

organogenesis instead of somatic embryogenesis are not quite clear, but the fact that the route of

morphogenesis could be changed by the manipulation of plant growth regulators in the culture

medium has been demonstrated in other species. It seemed that we have not found an optimal

combination of the genotype, explant tissue, type, concentration of plant growth regulators, or

culture condition for inducing somatic embryogenesis in Dieffenbachia. It is worthy of further

investigation. Apart from the influence of PGRs, adventitious shoot regeneration in vitro must









involve interrupting a genetically determined developmental pathway and reprogramming a new

developmental pathway at gene level (Schwarz and Beaty, 2000).

Our study indicated that we should evaluate critically publications on somatic

embryogenesis. More and more papers on somatic embryogenesis from different species have

been published in recent years. Some of these papers did not provide histological support, only

based on morphological similarities of regenerating structures with somatic embryos. Most of

them did complete a histological examination, but verifications of bipolar structures were not

provided. Usually shoot meristem and root meristem were shown in separate pictures. Only a

few publication presented histological evidence of bipolarity and the lack of vascular connection.

The ability to regenerate shoots from calli has several advantages. A great number of

shoots can be produced from an explant through callus induction and shoot formation. Indirect

shoot organogenesis has a greater potential for regenerating somaclonal variants. In

Dieffenbachia cv. Camouflage, phenotypic variations in leaf variegation and color of

acclimatized plants were observed. The feasibility of inducing indirect shoot organogenesis in

other Dieffenbachia cultivars, and then evaluating this regeneration protocol for potential

isolation of somaclonal variants will be described in Chapter 3 and 4.






















Table 2-1. Effect of TDZ and 2,4-D supplementation to BM on frequency of callus formation
from leaf explants of Dieffenbachia cv. Camouflage cultured for 8 weeks in dark and
4 weeks in a 16 h light photoperiod at 40 rmol m-2 s-1


2,4-D (EM)
0
0.5


Shoots/callus SE*


0 1 Oa
1 0 4 0.1 a
1 0.5 46 0.3 b
1 1 66 0.2 c
5 0 10 + 0.3 a
5 0.5 60 + 0.3 c
5 1 96 0.1 d
10 0 26 0.3 e
10 0.5 76 0.2 f
10 1 78 0.2 f
Means followed by the same letter are not significant at 0.05 level. Data represent means of two
repeated experiments, each with 5 replicates and 5 subsamples per replicate.


TDZ(PM)
0
0




















Table 2-2. Effect of 2iP and IAA concentrations and combinations on shoot regeneration from
Dieffenbachia cv. Camouflage calli cultured for 8 weeks in a 16 h light photoperiod
at 40 tmol m-2 S-1.

2iP (FiM) IAA (FiM) Shoots/callus SE*
0 0 1.6 0.3 a
0 2 1.2 0.4 a
20 0 3.6 0.9 b
20 2 4.2 2.0 b
40 0 6.3 2.2 c
40 2 7.9 1.5 c
80 0 4.0 + 0.1 b
80 2 3.8 0.4 b
*Means followed by the same letter are not significant at 0.05 level. Data represent means of two
repeated experiments, each with 5 replicates and 5 subsamples per replicate.



































METRIC 1
MT!TI1TT1


METRIC 1
Trrrrrrrr


Figure 2-1. Indirect shoot organogenesis in Dieffenbachia cv. Camouflage. A) Induction of calli
on leaf explants on BM supplemented with 5 [iM TDZ and 1 [iM 2,4-D after 8 weeks
culture in dark and 4 weeks culture in a 16 h light photoperiod. Bar = 1 mm. B) Small
green meristems formed on the surface of calli on BM containing 40 [iM 2iP and 2
IM IAA within 4 weeks. Bar = Imm. C) Shoot buds formed from these meristems 2
weeks later. Bar = 1 cm. D) Shoots and leaf development. Bar = 1 cm. E) Well-
developed shoots with roots within 8 weeks of culture. Bar = 1 cm. F) Acclimatized
plants in the greenhouse exhibiting variation in leaf variegation and color. Bar = 5
cm.


~
-- ;a~d~tL


METRIC 1
. .l I 1 I T I I


r^- .--


































Figure 2-2. Histological evidence of indirect shoot organogenesis in Dieffenbachia cv.
Camouflage at different developmental stages when cultured on BM medium
supplemented with 5 jiM TDZ and 1 jiM 2, 4-D for callus induction and on BM
medium supplemented with 40 jiM 2iP and 2 jiM IAA for shoot induction. A)
regenerative cells in calli originated from leaf explants. Bar = 250 jim. B) non-
regenerative cells in calli. Bar =250 jim. C) early mitotic activity observed at day 37
on callus induction medium. Bar = 167 jim. D) initial anticlinal division on the
surface of calli. Bar = 250 jim. E) initiation of meristematic zone by continued
anticlinal and periclinal cell division. Bar = 250 jim. F) development of meristematic
mass at day 43. Bar =250 jim. G) formation of globular shaped meristematic mass.
Bar = 500 im. H) appearance of a globular shaped meristematic mass shown by
SEM. Bar = 1 mm. I) meristematic cell mass formed within calli. Bar = 250 jim.
J) meristematic dome formation after 12 days of culture on shoot induction medium.
Bar = 250 jim. K) development of shoot meristem and leaf primordia at day 18. Bar =
500 tm. L) a well-developed shoot bud surrounded by leaves at day 27. Bar = 500
ltm. M) multiple shoot formation. Bar = 250 tm. N) root formation by day 39. Bar =
250 tm. 0) vascular connection between a developing shoot and callus tissue. Bar =
500 tm. P) SEM showed a developing shoot with stomata on the epidermis at day 36
on shoot induction medium. Bar = 750 [im.


UOr7









CHAPTER 3
FACTORS AFFECTING INDIRECT SHOOT ORGANOGENESIS IN Dieffenbachia

Introduction

Dieffenbachia, a monocot belonging to the family Araceae, noted for its attractive, striking

foliage, is one of the most important ornamental plant genera (Chen et al., 2004). Traditional

breeding in Dieffenbachia can be hampered by its naturally-occurring dichogamy, long breeding

cycle, poor seed production and germination (Henny, 1988). Selection of somaclonal variants

from in vitro regenerated populations is an alternative means for new cultivar development

(Henny and Chen, 2003). Due to the intervening callus phase, regeneration through indirect

shoot organogenesis has been shown to produce more somaclonal variants (Chen et al., 2003a).

Selection and subsequent in vitro propagation of variants with desired phenotypes can facilitate

the release of new cultivar in 2-3 years compared to 7-10 years through the traditional breeding.

Among factors influencing in vitro regeneration via indirect shoot organogenesis,

genotype, explant source, and plant growth regulators in media are the most important.

Genotypic differences in shoot organogenesis have been observed in a wide range of species.

Some genotypes exhibit high regenerative capacity, while others are either recalcitrant or exhibit

no capacity at all (Kuehnle and Sugil, 1991; Nontaswatsri et al., 2002). Explant type may also

influence capacity for in indirect shoot organogenesis, which may be related to the totipotency of

cells at their developmental stages (Agarwal and Ranu, 2000; Bacchetta et al., 2003).

Additionally, the type, concentration and combination of plant growth regulators in the media

can greatly affect callus formation and subsequent shoot induction (Mithila et al., 2003; Thao et

al., 2003).

To date, successful in vitro regeneration in Dieffenbachia has been largely limited to shoot

culture from shoot tip or axillary bud explants (Knauss, 1976; Voyviatzi and Voyiatzis, 1989).









We have established a protocol for indirect shoot organogenesis in Dieffenbachia cv.

Camouflage (Chapter 2), but information on the effects of genotype, explant source and plant

growth regulators on callus-mediated shoot organogenesis in Dieffenbachia cultivars is lacking.

The objectives of this study were to: 1) examine differences in capacity for indirect shoot

organogenesis in 4 Dieffenbachia cultivars and 2) investigate the effects of explant type and

plant growth regulators (type, concentration and combination) on indirect shoot organogenesis. It

is expected that information obtained from this study will help establish a more efficient protocol

for new cultivar development in Dieffenbachia through selection of somaclonal variants.

Materials and Methods

Plants Materials, Media and Culture Conditions

In vitro shoot cultures of the 4 Dieffenbachia cvs. Camouflage, Camille, Octopus and Star

Bright served as the explant source. Previous genetic analysis demonstrated that these cultivars

are genetically different (Chen et al., 2004). For callus induction and shoot organogenesis, a

basal medium (BM) consisting of MS (Murashige and Skoog, 1962) mineral salts, 0.4 mg 1-

thiamine, 2.0 mg 1- glycine, 100.0 mg 1- myo-inositol, 0.5 mg 1- pyridoxine, 0.5 mg 1- nicotinic

acid, and 25 g 1- sucrose supplemented with plant growth regulators was used in all experiments.

Medium was adjusted to a pH of 5.8 with 0.1 N KOH prior to the addition of 6 g 1- TC agar

(PhytoTechnology Laboratories, Shawnee, Mission, KS) and autoclaving at 1.2 kg cm-2 for 20

min. Cultures were maintained either in dark or under a 16 h light photoperiod at 40 imol m-2 s-1

provided by cool white fluorescent lamps (General Electric F20WT12-CW) at 22 + 3C.

Callus Induction

Leaves and roots from 4 week-old in vitro shoot cultures of the 4 Dieffenbachia cultivars

Camouflage, Camille, Octopus and Star Bright served as explants. Leaves were cut into 5 mm2

sections with mid-vein, and the leaf margins removed with a scalpel. Leaf explants were cultured









with the abaxial surface in contact with the callus induction media. Roots were cut into 10 mm

long segments, and then cultured horizontally on media. A series of 8 screening experiments

were completed to select the most effective concentration and combination of PGRs. Each

experiment consisted of factorial PGR combination with PGR-free medium serving as the

control: (1) BA at 0, 1, 10, 50 and 2,4-D at 0, 1, 10, 50 riM; (2) CPPU at 0, 1, 2.5, 5 and 2,4-D at

0, 2, 4, 8, 10 .iM; (3) CPPU at 0, 1, 2.5, 5 NAA at 0, 2, 4, 8, 10 tM ; (4) kinetin at 0, 1, 5, 10 and

IAA at 0, 1 riM; (5) dicamba at 0, 1, 3, 9 and 2,4-D at 0, 1 riM; (6) picloram at 0, 1, 3, 9 and 2, 4-

D at 0, 1 riM; (7) TDZ at 0, 1, 10, 50 and NAA at 0, 1, 10, 50 riM; (8) TDZ at 0, 1, 5, 10 and 2,4-

D at 0, 0.5, 1 riM. Explants were cultured in 100 x 15 mm sterile Petri plates containing 20 ml

medium. There were 5 explants per Petri plates and 5 replicate plates per treatment. Petri plates

were sealed with one layer ofNescofilm (Karlan Research Products Corp., Cottonwood, AZ).

Cultures were initially maintained in dark for 8 weeks for all cultivars except Star Bright that

were in dark for 12 weeks (no any response observed by 8 weeks culture) and then transferred to

a 16 h light photoperiod for another 4 weeks. The number of explants forming calli was scored

after 12 weeks culture for cvs. Camouflage, Camille, Octopus and 16 weeks for Star Bright. The

frequency of callus formation was calculated as the percentage of leaf explants forming calli.

Shoot Induction

Calli were excised from leaf explants, cut into pieces with a fresh weight of approximately

150 mg and then transferred to the shoot induction medium which was BM supplemented with

80 iM 2iP and 2 [iM IAA. Calli were cultured in baby food jars containing 40 ml media with 5

callus clumps per vessel and 5 replicates per treatment. Culture vessels were sealed with one

layer ofNescofilm. The number of shoots formed per callus was determined after 8 weeks

culture under a 16 h light photoperiod.









Acclimatization

Shoots, some with roots, were removed from the vessels and excised individually from the

callus clumps. Medium was carefully rinsed off the shoots. Shoots longer than 20 mm with 2-3

leaves were planted individually in 60-cell plug trays (cell dimensions: 4.5 x 4 x 5 cm3, REB

Plastics Inc, Apopka, FL) containing a 2:1:1 (v/v) soilless mixture of Canadian peat: vermiculite:

perlite. All plantlets were maintained in a shaded greenhouse under natural photoperiod (10 to

14.5 h light) with a maximum irradiance of 345 .imol m-2 s-1 and a temperature range of 20 to

310C. Plugs were hand watered twice a week. Peters 20N-10P-20K liquid fertilizer (200 mg/l N;

The Scotts Company, Marysville, OH) was applied weekly.

Statistical Analysis

All experiments were established in a completely randomized design. Experiments

showing responsive treatments were repeated once. Data were subject to analysis of variance

using SAS (SAS Institute, Inc., 1999). Mean separation was achieved by least significant

difference (LSD) test at the 95% level.

Results

Explant Effects

A distinct difference in callus formation was observed between leaf and root explants. No

callus was induced on root explants regardless of cultivar and PGR combination. However callus

formation occurred on leaf explants of all the 4 cultivars cultured on BM containing TDZ and

2,4-D at appropriate concentrations.

Genotypic Effects

Leaf explants of different cultivars exhibited different responsiveness for callus formation.

Callus initiation occurred from the leaf margins of cvs. Camouflage, Camille and Octopus after 4

weeks of culture; but no callus was noted on cv. Star Bright explants until 8 weeks culture. Leaf









explants first exhibited elongation and expansion, then became curved and swollen prior to callus

proliferation.

The effect of genotype on callus morphology was evident. Four morphologically distinct

callus types varied in structure and color were observed. Green nodular (Figure 3-1A), brown

nodular (Figure 3-1B), yellow friable, mucilaginous (Figure 3-1C) and light green compact

(Figure 3-1D) calli were produced from leaf explants of cvs. Camouflage, Camille, Octopus and

Star Bright respectively. Based on visual observation, callus growth rate also differed among

cultivars. Octopus produced the most calli covering almost the entire surface of the leaf explants.

Least calli was produced on Star Bright with callus production only limited to the cut ends of

leaf explants. Moderate growth rate of calli was observed in cvs. Camouflage and Camille.

Differences in the frequency of callus formation among cultivars were significant.

Maximum frequency of callus formation (96%) was obtained from Camouflage leaf explants,

followed by 66% from Octopus, 62% from Camille and 52% from Star Bright on BM

supplemented with 5 [tM TDZ and 1 [tM 2,4-D (Table 3-1).

A significant effect of genotype on shoot induction was also observed. After calli were

transferred onto BM supplemented with 80 [iM 2iP and 2 [iM IAA, maximum shoot production

(6.7shoots/callus) was obtained from cv. Camouflage, followed by 4.4 shoots/callus from cv.

Camille, 3.5 shoots/callus from cv. Star Bright. Cultivar Octopus displayed no capacity for

indirect shoot regeneration (Table 3-1).

PGR Effects

PGR type, concentration and combination had significant effects on callus induction on

cultured leaf explants. Among the PGRs screened, callus formation was only observed on leaf

explants cultured on BM supplemented with factorial combinations of 0, 1, 5, 10 [tM TDZ and

0.5 and 1 [iM 2,4-D. Other PGRs or their combinations failed to induce callus formation from









either leaf or root explants. Additionally, the effect of TDZ and 2,4-D on the frequency of callus

formation varied significantly. No callus could be induced on the medium without any PGRs.

TDZ was required for callus induction for cvs. Camouflage, Camille and Star Bright. No callus

formation was observed on media without TDZ in these 3 cultivars. For cv. Octopus, a 2%

frequency of callus formation was noted on medium without TDZ. However, 2,4-D was required

for callus production on Camille explants. Although callus formation was observed on media

without 2,4-D for the other 3 cultivars, the frequency of callus formation was lower (4% to

26%). Callus production increased with increasing 2,4-D concentrations. Consequently,

uniformed callus formation was obtained on BM supplemented with both TDZ and 2,4-D for all

the 4 cultivars. In general callus formation was promoted with increasing levels of both TDZ

and 2,4-D, but high TDZ concentration (> 10 |M) inhibited callus formation (Data not shown).

The combination of 5 iM TDZ and 1 iM 2,4-D was optimal for callus production among the 4

cultivars tested (Table 3-1).

A distinct carry-over effect of TDZ and 2,4-D from callus induction media on subsequent

shoot organogenesis was noted. Calli produced on callus induction medium with higher levels of

2,4-D displayed reduced capacity for shoot development. Conversely, calli produced on medium

supplemented with higher levels of TDZ were more highly shoot organogenic (Table 3-1). When

cultured on BM supplemented with 80 iM 2iP and 2 iM IAA, calli derived from BM

supplemented with 10 iM TDZ alone exhibited the highest shoot regeneration (6.7 + 1.3

shoots/callus) in cv. Camouflage.

The developmental process of indirect shoot organogenesis was illustrated in cv. Camille.

Brown nodular calli were produced on leaf explants of cv. Camille on BM supplemented with 5

iM TDZ and 1 iM 2,4-D after 8 weeks in dark and 4 weeks cultures in a 16 h light photoperiod









(Figure 3-2A). After calli were separated from leaf explants and transferred onto BM

supplemented with 80 iM 2iP and 2 iM IAA for shoot induction, small green meristems

appeared on the surface of calli within 4 weeks (Figure 3-2B). Shoot buds developed from these

meristems by the 6th week (Figure 3-2C). Leaf formation and shoot elongation occurred within 8

weeks (Figure 3-2D). Shoot clusters with well-developed leaves and roots were formed by the

end of 8 weeks culture (Figure 3-2E).

Acclimatization

Shoot and root formation was observed in most Dieffenbachia cultures. Shoots longer than

20 mm with 2 to 3 leaves were easily acclimatized in a shaded greenhouse (Figure 3-2F). Ex

vitro survival 100% was obtained in all the cultivars acclimatized.

Discussion

The present study demonstrates the difficulties in the induction of indirect shoot

organogenesis in Dieffenbachia. Attempts to use leaf explants taken directly from plants grown

in the greenhouse to initiate callus production failed due to significant (> 70%) culture

contamination. Leaf explants obtained from greenhouse plants displayed no response regardless

of media tested. We demonstrated that using leaf explants from in vitro-produced shoots was a

viable, alternative method to reduce contamination and increase explant responsiveness.

However, the responsiveness of leaf explants from in vitro plants on callus induction media was

slow. At least 12 weeks (16 weeks for cv. Star Bright) were required for callus induction. The

shoot regeneration capacity of Dieffenbachia from callus was low with a maximum 6.3

shoots/callus. Slow responsiveness to PGR treatments, high contamination rate in vitro and

recalcitrant nature for in vitro culture may explain why indirect shoot organogenesis has not been

reported in Dieffenbachia by others.









Other factors may account for high contamination rate. Dieffenbachia is mainly propagated

vegetatively by cuttings and divisions, and is maintained under shaded condition which may

allow bacteria and fungi to accumulate on the plant surface. The presence of endophytes can not

be ruled out (Knauss, 1976). This might be one reason for such high contamination rate in our

attempts to establish in vitro culture. Additionally, Dieffenbachia is a naturally-slow- growing

plant and this may also be manifested in vitro.

Genotype Effects

Dieffenbachia is a monocotyledonous genus and is among the most recalcitrant plants for

regeneration. In the present study, 4 cultivars evaluated are of different genetic origin. Camille,

one of the most popular cultivars in the foliage plant industry, is a sport selected from cv.

Perfection, while cv. Star Bright is an interspecific hybrid selected from crosses of several

parents. Camouflage was selected from somaclonal variants of cv. Panther, while Octopus was

isolated from somaclonal variants of Camouflage (Chen et al., 2004).

Genotype remains a key determinant of capacity for indirect shoot organogenesis. There

were distinct differences in callus morphology, callus forming ability and subsequent shoot

differentiation among the 4 Dieffenbachia cultivars. In the present study, 4 morphologically

distinct types of calli have been observed. Among them, three types were organogenic and one

type non-organogenic. The frequency of callus formation was also genotype-dependent with the

highest (96%) observed in cv. Camouflage. Genotypic differences were still obvious after calli

were transferred onto shoot induction medium. Calli of cv. Octopus did not exhibit any shoot

formation while Camouflage exhibited the highest shoot formation (6.7 shoots/callus).

Variation in callus morphology and callus forming ability of different genotypes has been

previously reported in other species (Thao et al., 2003; Landi and Mezzetti, 2005). Our results

were consistent with these reports. Kallak et al (1997) concluded that callogenesis was not only a









result of dedifferentiation of explant tissues, but also an essential preparatory stage for in vitro

morphogenesis and callus type may be an indicator for shoot organogenic potential. In their

analysis of genetic relatedness of Dieffenbachia cultivars using AFLP, Chen et al (2004) found

that cv. Camouflage significantly differed from cvs. Camille, Octopus and Star Bright as it was

positioned in one genetic cluster and the other three shared another cluster. This might partially

explain why cv. Camouflage performed differently in terms of the frequency of callus formation

and shoots/callus compared to the other 3 cultivars. Phillips (2004) reported that specific genes

involved in each stage of shoot organogenesis dedifferentiationn, induction and differentiation).

In some genotypes genes involved in shoot organogenesis may be suppressed due to

inappropriate culture condition.

PGR Effects

The type, concentration and combination of PGRs in media are another key factor

regulating shoot organogenesis. The present study showed that PGRs have significant effects on

the induction of calli. 2,4-D has been shown to be the most effective for callus induction in a

variety of species (Ma and Xu, 2002; Thao et al., 2003). In contrast, we observed that 2,4-D was

not required for callus initiation in 3 of 4 cultivars. Among PGRs screened, TDZ was most

effective in stimulating callus formation. TDZ has also been found effective in in vitro

regeneration in a variety of species, such as pelargonium (Haensch, 2004b), African violet

(Mithila et al., 2003), geranium (Robichon et al., 1997). Hutchinson et al (1996) reported that

TDZ treatment could result in an increase in endogenous auxin levels. This may explain why

TDZ was most effective in callus induction in present study because auxins usually stimulate

callus formation.

TDZ also has cytokinin-like activity (Zhang et al., 2001; Landi and Mezzetti, 2006).

Induction of indirect shoot organogenesis in Dieffenbachia cultures supports this claim. It was









noted that many small shoot meristems formed on the surface of calli on media with TDZ, but

these shoots did not elongate. Attempt to include GA3 in media to elongate these stunted shoots

failed. Bacchetta et al (2003) reported that TDZ induced multiple shoots with stunted growth in

Lilium. Orlikowska et al (1995) noted 4.5 [iM TDZ in combination with 5.4 [iM NAA induced

direct shoot organogenesis on petiole explants in an unidentified Dieffenbachia cultivar.

Carry-Over Effect of PGRs

Depending on the type and concentration, PGRs in the callus induction media may have

had either a negative or positive carry-over effect on subsequent shoot formation (Vardja and

Vardja, 2001). In our study, we observed that shoot forming ability was higher from calli

cultured on media containing TDZ alone. Similar result was found in indirect shoot

organogenesis in Duboisia myoporoides in which the combination of cytokinin/auxin used for

callus induction had effect on subsequent shoot induction (Khanam et al., 2000). Calli derived

from TDZ-supplemented media may have accumulated TDZ or a metabolite in their cells during

the period of culture. When these calli were transferred to shoot induction media, high TDZ

levels in callus cells may have stimulated shoot proliferation.

Explant Effects

The effect of explant sources on indirect shoot organogenesis has been reported by other

authors (Orlikowska et al., 1995; Kallak et al., 1997; Berrios et al., 1999; Mithila et al., 2003).

This study showed that only leaf, not root explants, exhibited the capacity for indirect shoot

organogenesis. Root explants failed to indirect shoot organogenesis. An explanation for this

difference would be that cells from root explants were organogenically less competent than those

of leaf explants. Another possible reason could be that PGR combinations and/or their

concentrations screened were inappropriate for callus induction on root explants. Orlikowsika et

al (1995) also reported that Dieffenbachia root explants were non-regenerative in vitro.









Acclimatization

In contrast to their recalcitrant nature for callus induction and shoot regeneration,

microcuttings of Dieffenbachia cultivars were easy-to-root and 100% ex vitro survival of shoots

was achieved in all the 3 cultivars exhibiting the capacity for indirect shoot organogenesis. Root

formation occurred concurrently with shoot formation on shoot induction medium. It was

conceivable that endogenous auxin levels in Dieffenbachia microcuttings were sufficient for root

formation. Variations in leaf variegation and color (in cv. Camouflage) and leaf morphology (in

cv. Camille) were observed among acclimatized plants. The evaluation of this regeneration

protocol for potential isolation of somaclonal variants will be described in Chapter 4.












































Figure 3-1 .Characterization of calli cultured on BM supplemented with 5 iM TDZ and 1 iM
2,4-D after 8 weeks culture in dark for cvs. Camouflage, Camille and Octopus (12
weeks for cv. Star Bright) and 4 weeks culture in a 16 h light photoperiod. A) Green
nodular calli of cv. Camouflage. Bar = 1 mm. B) brown nodular calli of cv. Camille.
Bar = 1 mm. C) Yellow, friable calli of cv. Octopus. Bar = 5 mm. D) green compact
calli of cv. Star Bright. Bar = 5 mm.











Table 3-1. Effects of TDZ and 2,4-D on the frequency of callus formation on leaf explants and shoot number per callus of
Dieffenbachia 4 cultivars Camouflage, Camille, Octopus and Star Bright.
Camouflage Camille Octopus Star Bright
Callus Callus Callus Callus
formation formation formation formation
TDZ 2,4-D frequency Shoots/ frequency Shoots/ frequency Shoots/ frequency Shoots/
(GM) (GM) (%) + SE callus + SE (%) + SE callus SE (%) + SE callus + SE (%) + SE callus + SE
0 0 Oa Oa Oa Oa Oa 0 Oa 0
0 0.5 0a 0a 0a 0a 2+0.2a 0 0a 0
0 1.0 Oa Oa Oa Oa Oa 0 Oa 0
1 0 4+0.1a Oa Oa Oa 6+0.3a 0 Oa 0
1 0.5 46 0.3 b 3.7 0.5 b 8 0.2 a 0 a 30+ 0.4 bc 0 0 a 0
1 1.0 66 0.2c 2.7 0.5 c 28 0.2 bd Oa 30 +0.3 bc 0 4 0.1 a 0
5 0 10 +0.3 a 0 a Oa Oa 14 0.4 ab 0 Oa 0
5 0.5 60 +0.3 c 4.4 0.5 b 22 0.2 b 0a 36 0.4c 0 4 0.1 a 0
5 1.0 96 0.1 d 3.5 0.5bc 62 0.3 c 3.7 0.5 b 66 0.3 d 0 52 0.3 c 3.5 1.6
10 0 26 0.3 e 6.7 1.3 d Oa Oa 24 0.3 b 0 4 0.1 a 0
10 0.5 76 0.2 f 5.3 0.7 e 36 0.3 d 0 a 54 0.3 de 0 28 0.2 b 0
10 1.0 78 0.2 f 3.8 0.4 b 56 0.3 c 4.4 0.5 c 40 + 0.2 ce 0 32 0.1 b 0

Callus formation frequency was evaluated after 8 weeks for cv. Camouflage, Camille and Octopus (12 weeks for cv. Star Bright) in
dark and 4 weeks in a 16 h light photoperiod on BM supplemented with different combination of TDZ and 2,4-D.
Shoots per callus were scored after 8 weeks in a 16 h light photoperiod on BM supplemented with 80 iM 2iP and 2 iM IAA.












































Figure 3-2. Indirect shoot organogenesis in Dieffenbachia cv. Camille. A) Induction of calli on
leaf explants on BM supplemented with 5 [iM TDZ and 1 [iM 2,4-D after 8 weeks
culture in dark and 4 weeks culture in a 16 h photoperiod. Bar = 500 Lim. B) Small
green meristems formed on the surface of calli on BM containing 80 [iM 2iP and 2
[iM IAA within 4 weeks. Bar = Icm. C) Shoot buds formed from these meristems by
the 6th week. Bar = 1cm. D) Leaf formation and shoot elongation occurred. Bar =
Icm. E) Well-developed shoots with roots within 8 weeks of culture. Bar = 1 cm.
F) Acclimatized plants in the greenhouse. Bar = 1 cm.


"


METRIC i
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METRIC 1
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CHAPTER 4
ASSESSMENT OF SOMACLONAL VARIATION IN Dieffenbachia PLANTS
REGENERATED FROM INDIRECT SHOOT ORGANOGENESIS AT PHENOTYPIC LEVEL

Introduction

Phenotypic variation observed among plants regenerated from tissue culture is referred to

as somaclonal variation (Larkin and Scowcroft, 1981). Somaclonal variation can be assessed by

analysis of phenotype, chromosome number and structure, proteins, or direct DNA evaluation of

plants (Bouman and De Klerk, 1997). The extent of phenotypic variation is usually determined

as the percentage of plants showing aberrations from the parental plant for one or more defined

characteristics. These characteristics may include change in foliar variegation pattern,

modification of flower color and size, alteration of leaf shape, increase in lateral shoots, and

change in overall plant form (Griesbach et al., 1988; Khalid et al., 1989). Plants with the deviant

phenotypes are known as somaclones or somaclonal variants. Commercially, visual phenotypic

evaluation is paramount since the value of the plants, especially floriculture crops, lies in

appearance. Chen and Henny (2006) documented the occurrence of somaclonal variation in 58

genera across 33 families of floriculture crops and proposed that somaclonal variation is an

important source for cultivar development of floriculture crops.

Many factors, including genotype, growth regulators, and tissue source are involved in

somaclonal variation (Karp, 1991; Chen and Henny, 2006). Among these factors, plant genotype

is probably the most important determinant of variation. Some cultivars show higher variation

rate while others are highly stable (Najaran and Walton, 1987; Skirvin et al., 1994; Bouman and

de Klerk, 1997). In addition, the duration of tissue culture also affects somaclonal variation.

Somaclonal variation generally increases with the time that a culture has been maintained in

vitro, especially for callus culture; the longer a culture remains in vitro, the greater somaclonal

variation (Orton, 1985; Rodrigues et al., 1998).









Dieffenbachia belongs to the family Araceae and consistently ranks among the top five

most popular ornamental foliage plant genera. Due to its naturally occurring dichogamy, limited

seed production, development of a new Dieffenbachia cultivar through traditional breeding

usually requires 7 to 10 years (Henny and Chen, 2003). With the establishment of in vitro culture

techniques for Dieffenbachia propagation in the 1980s, new cultivars have been selected from

somaclonal variants and released in the foliage plant industry (Chen and Henny, 2006).

However, there is no report on the evaluation of somaclonal variation among Dieffenbachia

plants regenerated from indirect shoot organogenesis.

Our previous studies established methods for regenerating the 3 Dieffenbachia cultivars

Camouflage, Camille, and Star Bright through indirect shoot organogenesis (Chapter 3). The

objectives of the present study were to phenotypically assess somaclonal variation among the

regenerants of the 3 Dieffenbachia cultivars and determine the effects of genotype and the

duration of callus culture on shoot regeneration and somaclonal variation.

Materials and Methods

Regeneration of the 3 Dieffenbachia cultivars Camouflage, Camille, and Star Bright

through indirect shoot organogenesis was described in Chapter 3, which can be briefly outlined

as follows:

Callus Induction

Leaves obtained from in vitro shoot culture of the 3 cultivars were cut into 5 mm2 sections

containing mid-vein. A callus induction medium consisted of a basal medium (BM) containing

MS (Murashige and Skoog, 1962) mineral salts, 0.4 mg 1- thiamine, 2.0 mg 1- glycine, 100.0 mg

1- myo-inositol, 0.5 mg 1- pyridoxine, 0.5 mg 1- nicotinic acid, and 25 g 1-1 sucrose which was

supplemented with 5 [iM TDZ and 1 [iM 2,4-D. Medium was adjusted to pH 5.8 with 0.1 N

KOH prior to the addition of 6 g 1- TC agar (PhytoTechnology Laboratories, Shawnee Mission,









KS) and autoclaved at 1.2 kg cm-2 for 20 min. Leaf explants were cultured in 100 x 15 mm

sterile Petri plates containing 20 ml medium. There were 5 explants per plate inoculated with

abaxial surface in contact with the medium. The culture was initially maintained in the dark for 8

weeks for cvs. Camouflage and Camille, and 12 weeks for Star Bright (due to no responses by 8

weeks culture), and then transferred to a 16 h light photoperiod at 40 imol m-2 s-1 provided by

cool white fluorescent lamps (General Electric F20WT12CW) at 22 3C for another 4 weeks.

Sustained Callus Culture

Calli were excised from leaf explants at the end of callus induction, and cut into pieces

weighing approximately 150 mg (fresh weight). Half the calli were transferred to the same fresh

callus induction medium for sustained callus production. The other half was transferred to shoot

induction medium to determine effects of subculture number on the capacity for shoot

regeneration from calli. Calli were subcultured at 8 week intervals by this manner.

Shoot Induction

Five callus pieces, each weighing approximately 150 mg, were transferred into individual

baby food jar containing 40 ml shoot induction medium. The shoot induction medium was

composed of BM supplemented with 40 iM 2iP and 2 iM IAA and solidified with 6 g 1- TC

agar. Cultures were maintained under a 16 h light photoperiod with a light intensity of 40 imol

m-2 s-1 at 22 3C for 8 weeks. The number of shoots per callus piece was recorded for each

subculture.

Acclimatization

After 8 weeks on shoot induction medium, shoots, some with roots, were removed from

the vessels and excised individually from the callus clumps. Medium was carefully rinsed from

the shoots. Shoots longer than 20 mm with 2-3 leaves were planted individually in 60-cell plug

trays (cell dimensions: 4.5 x 4 x 5 cm3, REB Plastics Inc, Apopka, FL) containing a 2:1:1 (v/v)









soilless mixture of Canadian peat: vermiculite: perlite. All plantlets were maintained in a shaded

greenhouse under natural photoperiod (10 to 14.5 h light) with a maximum irradiance of 345

mrnol m-2 s-1 and a temperature range of 20 to 3 10C. Plugs were hand watered twice a week.

Peters 20N-10P-20K liquid fertilizer (200 mg/1 N; The Scotts Company, Marysville, OH) was

applied weekly following 2 weeks acclimatization.

Determination of Somaclonal Variation

Plants grown in plug trays were regularly checked for the presence of variant phenotypes.

Emphasis was primarily placed on foliar characteristics as Dieffenbachia is valued for its leaf

variegation. Novel variegation patterns were described, photographed, and grouped into different

types of somaclonal variant. Numbers of plants per variant type were counted, and percentage of

such type in relation to the total number of regenerated plants was calculated.

Parental and variant types of plants regenerated from Dieffenbachia cvs. Camouflage and

Camille were then transplanted from plug trays to 15-cm diameter plastic pots containing Vergo

Container Mix A (Verlite Co. Tampa, FL). The components of the substrate were 2:1:1 (v/v) of

Canadian peat: vermiculite: perlite. Five grams of an 18.0N-2.6P-10.0K controlled-released

fertilizer with microelements (Multicote 18-6-12, Haifa Chemicals Ltd., Haifa Bay, Israel) was

applied to the soil surface in each pot. Plants were grown on raised benches in a shaded and

evaporated pad cooled greenhouse under a maximum photosynthetically active radiation of 350

mrol m-2 s-1. Temperatures ranged from 20 to 300C and relative humidity from 50 to 100%.

Plants were overhead irrigated through sprinklers one to two times a week. Plant height, canopy

height and width, length and width of the largest leaf, number of basal shoots were recorded 8

months after growing in the shaded greenhouse.

Both shoot and cane cuttings were then made from parental plants and identified variants

and rooted in Vergo Container Mix A in the aforementioned shaded greenhouse. Morphological









characteristics of cutting-propagated progenies were compared to respective parents for

determining if the variant phenotypes were stable.

Examination of the Duration of Callus Culture Affecting Somaclonal Variation

Two populations of regenerated plants of Dieffenbachia cv. Camouflage were used to

examine the duration of callus culture phase influencing somaclonal variation. One population

was derived from 8 months callus culture in vitro. The other population was derived from 16

months callus culture in vitro. Plants with novel foliar variegation patterns were grouped as

mentioned above. Numbers of plants per variant type were counted and calculated in relation to

the total number of regenerated plants in each population.

Experimental Design and Statistical Analysis

All experiments in this study were established in a completely randomized design. Data for

plant growth were subject to analysis of variance using SAS (SAS institute Inc., 1999). Least-

squares mean test was used to compare means at the 95% level of probability.

Results

Somaclonal Variant Identification

A total of 2,248 plants from cv. Camouflage, 384 of cv. Camille and 62 of cv. Star Bright

were regenerated from indirect shoot organogenesis. Novel phenotypes were observed from

regenerants of cvs. Camouflage and Camille, but not from cv. Star Bright. Three types of

variants, called SV1, SV2, and SV3, were identified among regenerated Camouflage plants; each

had distinct phenotype (Figure 4-1). The parental plant had camouflaged leaves with random

green batches. Leaves of SV1, however, were green with whitish variegation along the midvein.

SV2 had light green leaves with many yellowish spots, and connections among the spots resulted

in large yellowish blotches. Leaf color of SV3 was similar to SV2 but had fewer yellowish spots.

The other differences between SV2 and SV3 were that SV2 had some white spots on leaves and









sparkled petioles. The leaf shape of the 3 variants was comparable to that of their parent. In

addition to these morphological differences, analysis of variance for plant growth parameters

showed that SV1 significantly differed in plant height, canopy height and width, leaf length from

the parental plant and SV2. SV1 was taller with larger canopy and longer leaves than parental

plants and SV2. SV2 and SV3 had no basal shoots (single stem) but basal shoot numbers

between SV1 and parental plants were similar ranging from 3 to 4 (Table 4-1). No statistical

analysis could be done with SV3 because only 1 plant was available.

One variant type was identified among the regenerants of cv. Camille. This variant had the

same foliar variegation pattern as its parent but possessed lanceolate leaves compared to the

oblong leaves of the parent (Figure 4-2). The ratio of leaf length and leaf width was 3.4 for the

variants compared to 1.5 for parental plants. The variant also exhibited significant difference

from the parent in growth parameters measured except for basal shoot number. Variants grew

taller with larger canopy and had longer, narrower leaves than the parent (Table 4-2). The

morphological characteristics of all variants, 3 types from cv. Camouflage and 1 from cv.

Camille, were stable in shoot and cane cutting propagated population.

Genotype Differences

Among the 2,248 regenerated plants of cv. Camouflage, a total of 908 variant plants were

isolated, of which 904 plants belonged to the SV1 type, 3 plants belonged to the SV2 type, and

one being the SV3 type. Correspondingly, the occurrence of SV1, SV2, and SV3 was at rates of

40.2%, 0.13%, and 0.04%, respectively. Thus, somaclonal variation rate at the phenotypic level

for regenerated cv. Camouflage plants was 40.4% (Table 4-3). Of the 384 regenerated plants of

cv. Camille, 10 variants had lanceolate leaves resulting in a somaclonal variation rate of 2.6%

(Table 3). No variation was observed among the 62 regenerated plants of cv. Star Bright.









Culture Duration Effects on Somaclonal Variation

Duration of callus culture phase had no effect on somaclonal variation of cv.Camouflage.

The somaclonal variation rates for plants regenerated from 8 and 16 months callus culture were

41.2% and 39.5% respectively (Table 4-4).

Effects of Callus Subculture Number on Shoot Regeneration

The 3 Dieffenbachia cultivars displayed significant differences in sustained capacity for

shoot regeneration in long-term callus culture (Figure 4-3). Cultivar Camouflage showed a

greater capacity for shoot regeneration than cvs. Camille and Star Bright. For cv. Camouflage,

the number of shoots per callus increased from 4.7 at the first subculture to 6.3 by the sixth

subculture, and then gradually decreased to 2.9 by the twelfth subculture. For cv. Camille, the

number of regenerated shoots per callus increased from 2.6 at the first subculture to 4.0 by the

fifth subculture, then decreased to 2.8 at seventh subculture, and finally decreasing to no

regeneration by the eighth subculture (Figure 4-3). Cultivar Star Bright exhibited no capacity for

sustained callus culture as calli lost shoot regeneration ability after the first subculture. Calli of

cv. Camille maintained the capacity for regeneration in vitro up to 16 months while cv.

Camouflage calli retained regenerative after 24 months culture in vitro.

Discussion

Somaclonal Variation and Genotypes

The present study demonstrated that somaclonal variation occurred among Dieffenbachia

plants regenerated through indirect shoot organogenesis from leaf-derived calli. The variants

showed distinct phenotypes from their parents. These variants, particularly those isolated from

regenerated cv. Camouflage, could potentially be new commercial cultivars as their phenotypes

are novel and stable as demonstrated by their progenies after cutting propagation. Additionally,

the growth rates of these variants were either higher than or comparative to those of their









parental plants in our shaded greenhouse evaluation. Reserach thus far has primarily emphasized

phenotypic evaluation. This is because in the absence of reliable genetic markers of somaclonal

variation and considering the fact that Dieffenbachia is primarily propagated through asexual

means, phenotype represents the fastest and the most convenient way to identify somaclonal

variants. In addition, ornamental plants are prized for their visual phenotypic appearance. As

long as a novel phenotype has ornamental value and is stable, it could potentially become a new

cultivar.

The rate of somaclonal variation differed greatly, ranging from none for cv. Star Bright and

2.6% for cv. Camille to 40.4% for cv. Camouflage. The low number of regenerated plants, 62

and 384 for cvs. Star Bright and Camille respectively, may be a factor for the low rates of

variation observed. It is believed, however, that the genetic makeup of the cultivars plays an

important role. First, cvs. Camouflage, Camille, and Star Brigh differed in callus formation

frequencies, 96%, 62%, and 52% respectively (Table 3-1). Second, the cultivars also varied with

respect to in vitro shoot formation (Figure 4-3). Calli of cv. Star Bright had no capacity for

subculture, and calli of cv. Camille gradually lost the ability to form shoots at approximately 16

months subculture. In contrast, calli of cv. Camouflage maintained their capability to form shoots

for at least 24 months after being subcultured. Cultivar differences in the frequency of callus and

shoot formation frequencies resulted in low numbers of regenerated plants for cvs. Camille and

Star Bright. Thus, it is possible that a prerequisite for producing somaclonal variants through

indirect shoot organogenesis is that calli maintain a high and prolonged capacity for shoot

organogenesis. Chen et al (2004) reported that cv. Camouflage significantly differed from cvs.

Camille and Star Bright genetically as the former was positioned in one genetic cluster while the

latter two shared a common cluster. This might partially explain why cv. Camouflage exhibited









greater ability to form calli and shoots in vitro than cvs. Camille and Star Bright. High

multiplication rate is associated with rapid mitosis, thus more error may have occurred.

Somaclonal variation rates varying among species and cultivars have been widely

documented. Skirvin et al (1994) stated that the somaclonal variation rate expected in vitro was

probably about 1% to 3%. Our result with cv. Camille was similar to this rate since 2.6%

somaclonal variation occurred in this cultivar. The rate of somaclonal variation in cv.

Camouflage was high (40.4%) but not unique. High rates have been reported in other plants as

well. Legkobit and Khadeeva (2004) noted 50% to 80% somaclonal variation from Stachys

(betony) species. Bairu et al (2006) reported somaclonal variation in Cavendish banana cultivars

was high as 72%. The high somaclonal variation rate may also be due to the multiplication of

variant cells already produced in the previous cycle since regenerated plants were derived from

calli subcultured up to 8 cycles at 8 weeks interval for cv. Camouflage. Xie et al (1995) reported

that a particular labile portion of the rice genome was susceptible to stress (in vitro culture

condition) and showed higher rearrangement and mutation rates than other portion during in

vitro culture. Further research is warranted to determine if the deviant phenotypes resulted from

genetic or epigenetic changes and why such a high rate occurred in cv. Camouflage.

Callus Culture Duration

It is generally believed that the rate of somaclonal variation increases with the time a

culture has been maintained in vitro. The longer a culture is maintained in vitro, the greater the

somaclonal variation (Rodrigues et al., 1998; Kuznetsova et al., 2006). However, the present

study showed that for Dieffenbachia cv. Camouflage there was no difference in the rate of

somaclonal variation between plants regenerated from 8 and 16 months callus culture. It is not

known at this time if shortening the duration of each subculture (less than an 8 week interval)









and increasing time in maintenance or changing growth regulator combinations in media could

result in any difference in somaclonal variation rates of cv. Camouflage.

Both the type and the rate of somalconal variation are crucial factors for determining the

feasibility of using somaclonal variation for new cultivar development. In this study, wide

variation in leaf variegation, color, shape and plant forms were observed, and these traits are

novel and stable demonstrated by their progenies after cutting propagation. These results showed

that selection of somaclonal variants has great potential for new cultivar development in

Dieffenbachia. A future study will focus on analysis of genetic changes of the somaclonal

variants using molecular marker techniques.



















































Figure 4-1. Plants ofDieffenbachia cv. Camouflage regenerated by indirect shoot organogenesis
showing variation in leaf variegation and color. A) Parental plant: creamy,
camouflaged leaves with random green batches of different size. Bar = 10 cm. B)
SV1: solid dark green leaves with whitish variegation along the midvein. Bar = 10
cm. C) SV2: light green leaves with many yellowish spots, and connections among
spots resulted in large yellowish blotches. Bar = 10 cm. D) SV3: green leaves with
few scattered yellowish spots. Bar = 10 cm.




70

















Table 4-1. Quantitative evaluation of somaclonal variants of Dieffenbachia cv. Camouflage regenerated by indirect shoot
organogenesis and grown in the greenhouse for 8 months.

No. of Plant height Canopy height Canopy width Largest leaf Largest leaf Basal shoot
plants (cm) SE (cm) SE (cm) SE length (cm) SE width (cm) SE number SE

Parental type 114 22 0.9 a 39 1.0 a 47 1.0 a 30+ 0.4 a 11 0.2 a 3.7 0.1a

SV1 34 30 1.4 b 44 1.6 b 53 1.6 b 33 0.6b 12 0.3 a 3.9 0.2 a

SV2 3 15 4.7 a 35 5.3 a 41 5.2 a 26 2.0 c 12 1.0 a 1.0+ 0.0 b

SV3 1 26 17 24 22 8 1
Means followed by the same letter in each column are not significant at 0.05 level.
Parental type: regenerated plants exhibited the same leaf variegation as the parent.
SV1, SV2, SV3: three types of somaclonal variation observed in cv. Camouflage.














































Figure 4-2. Plants ofDieffenbachia cv. Camille regenerated by indirect shoot organogenesis
showing variation in leaf shape. A) Parental type plants with oblong shaped leaves.
Bar = 1 cm. B) Somaclonal variants with lanceolate leaves. Bar = 1 cm.














72


















Table 4-2. Quantitative evaluation of somaclonal variants ofDieffenbachia cv. Camille regenerated by indirect shoot organogenesis
and grown in the greenhouse for 8 months.

Largest leaf Largest leaf Leaf length/ Basal shoot
No.of Plant height Canopy height Canopy width length (cm) width (cm) leaf width number
plants (cm) SE (cm) SE (cm) SE SE SE SE SE

Parental type 30 6.0 + 0.2 a 11 0.3 a 10 +0.2 a 5.1 0.2 a 3.4 0.1 a 1.5 0.1 a 3.6 0.2 a

Variant type 10 6.7 0.3 b 13 0.6 b 12 0.4 b 7.0 0.3 b 2.1 0.3 b 3.4 0.1 b 3.8 0.4 a
Means followed by the same letter in each column are not significant at 0.05 level.
Parental type: regenerated plants exhibited the same leaf shape as the parent.
Variant type: regenerated plants exhibited different leaf shape.


















Table 4-3. Effects of genotype on the number and rate of somaclonal variation among Dieffenbachia plants regenerated by indirect
shoot organogenesis and grown in the greenhouse for 8 months.

No. of somaclonal variants % of somaclonal variants
Cultivars No. of plants
regenerated Total SV1 SV2 SV3 Total SV1 SV2 SV3

Camouflage 2248 908 904 3 1 40.4 40.2 0.13 0.04

Camille 384 10 2.6

Star Bright 62 0 0
SV1, SV2, SV3: three types of somaclonal variation observed in cv. Camouflage.


















Table 4-4. Effects of the duration of callus culture on the number and rate of somaclonal variation of Dieffenbachia cv. Camouflage
regenerated by indirect shoot organogenesis and grown in the greenhouse for 8 months.

No. of plants
Duration (months) regenerated No. of variants Parental type SV1 SV2 SV3

8 1171 483 (41.2)* 688 (58.8) 479 (40.9) 3 (0.26) 1 (0.09)

16 1077 425 (39.5) 652 (60.5) 425 (39.4) 0 (0) 0 (0)
*Figures in parenthesis are the rate of somalconal variation.
Parental type: regenerated plants exhibited the same leaf variegation as the parent.
SV1, SV2, SV3: three types of somaclonal variation observed in cv. Camouflage.

























- cv Camouflage
--- cv Camille
A cv Star Bright


4


1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8


9 10 11 12


Subculture



Figure4- 3. Effects of subculture number on shoot regeneration (shoots/callus + SE) from calli
of the 3 Dieffenbachia cultivars. Calli were subcultured at 8 week intervals on the
callus induction medium under a 16 h light photoperiod. Shoots/callus were recorded
after 8 weeks culture on shoot induction medium under a 16 h light photoperiod.









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BIOGRAPHICAL SKETCH

Xiuli Shen was born in Harbin city, Heilongjiang province, China, on October 2, 1964.

She grew up in a happy family with loving parents and two younger brothers. From a very young

age, she showed her love for science and nature. She was an excellent student and did very well

in every subject in school. After graduating from high school in 1982, she moved to Changchun

and enrolled at Jinlin University and received a Bachelor of Science degree in July1987 with a

major in molecular biology. Then she moved back to her home city and worked in the

Department of Biotechnology in the Northeast Agricultural University as assistant professor

from 1987 to 1996, and as an associate professor from 1996 to 2000.

To fulfill her dream to look at the world outside of China and to be able to read a novel

written in a foreign language, she came to Saskatoon, Saskatchewan, Canada with her 9-year-old

son in May 2000. After 3 years of studying at the University of Saskatchewan, she earned her

Master of Science degree in plant science in 2003. She worked as intern in the Plant

Biotechnology Institute, National Research Council of Canada during summer 2003. In August

2003, she entered graduate school at the University of Florida to work on a doctoral program in

horticultural science. She expects to earn her Ph.D. in May 2007. She has one son, Hao Xu,

living with her in Gainesville.