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Regeneration of Philodendron Micans K.koch Through Protocorm-Like Bodies and Improvement of Plant Form Using Growth Regu...

Permanent Link: http://ufdc.ufl.edu/UFE0041258/00001

Material Information

Title: Regeneration of Philodendron Micans K.koch Through Protocorm-Like Bodies and Improvement of Plant Form Using Growth Regulators
Physical Description: 1 online resource (75 p.)
Language: english
Creator: Xiong, Zhujun
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: mixploid, philodendron, plb, regeneration, somatic, variation
Environmental Horticulture -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Philodendron micans K. Koch, commonly known as velvet philodendron, has both a soft foliar texture and attractive colors that could make it an important genetic resource for breeding new ornamental traits and introducing improvements to the genus Philodendron. P. micans rarely flowers in nature and thus far no breeding program has utilized this plant for interspecific hybridization. However, in addition to traditional breeding, new cultivars could be developed using somaclonal variation produced during in vitro propagation. As a step toward this direction, bud, leaf, petiole, and internode explants were cultured on Murashige and Skoog (MS) medium supplemented with BA (N6-benzyladenine), CPPU N-(2-chloro-4-pyridyl)-N- phenylurea, or TDZ (N-phenyl-N? -1,2,3- thiadiazol-5-ylurea) with NAA (1-naphthaleneacetic acid). Bud explants showed the greatest regenerative potential by producing both callus and globular structures. Callus was friable and unable to produce adventitious shoots; while globular structures differentiated and produced shoots and roots. Histological analysis suggested that the globular structures were protocorm-like bodies (PLBs), a novel pathway for plant regeneration. Further experiments showed that 75% bud explants of P. micans cultured on MS medium supplemented with 1.0 mg L-1 TDZ and 0.5 mg L-1 NAA produced PLBs within 8 weeks. Each explant produced more than 50 shoots that subsequently rooted. The survival rate of plantlets regenerated through this protocol was 98%. Analysis of 20 randomly selected plantlets using DNA flow cytometry showed that two were mixoploid. Such a high frequency of ploidy change may suggest the occurrence of somaclonal variation. However, regenerated plants phenotypically resembled mother plants. This was the first in vitro study of P. micans, and a new and reliable protocol has been developed for efficient regeneration of this species. Growth regulators to improve appearance and marketability of P. micans were also examined. Four growth regulators, Bonziregistered trademark {?-(4-chlorophenyl)methyl-?-(1,1-dimethylethyl)-1H- 1,2,4-triazole-1-ethanol}, B-Nineregistered trademark (butanedioic acid mono (2,2-dimethylhydrazide), Cycocelregistered trademark (2-chlorethy) trimethylethanaminium chloride, and Florelregistered trademark (2-chloroethyl) phosphonic acid were applied as foliar sprays or soil drenches in attempt to shorten internode length and reduce a leggy appearance. Bonziregistered trademark application was the most effective treatment and resulted in plants with a more compact growth form.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Zhujun Xiong.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Chen, Jianjun.
Local: Co-adviser: Henny, Richard J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041258:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041258/00001

Material Information

Title: Regeneration of Philodendron Micans K.koch Through Protocorm-Like Bodies and Improvement of Plant Form Using Growth Regulators
Physical Description: 1 online resource (75 p.)
Language: english
Creator: Xiong, Zhujun
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: mixploid, philodendron, plb, regeneration, somatic, variation
Environmental Horticulture -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Philodendron micans K. Koch, commonly known as velvet philodendron, has both a soft foliar texture and attractive colors that could make it an important genetic resource for breeding new ornamental traits and introducing improvements to the genus Philodendron. P. micans rarely flowers in nature and thus far no breeding program has utilized this plant for interspecific hybridization. However, in addition to traditional breeding, new cultivars could be developed using somaclonal variation produced during in vitro propagation. As a step toward this direction, bud, leaf, petiole, and internode explants were cultured on Murashige and Skoog (MS) medium supplemented with BA (N6-benzyladenine), CPPU N-(2-chloro-4-pyridyl)-N- phenylurea, or TDZ (N-phenyl-N? -1,2,3- thiadiazol-5-ylurea) with NAA (1-naphthaleneacetic acid). Bud explants showed the greatest regenerative potential by producing both callus and globular structures. Callus was friable and unable to produce adventitious shoots; while globular structures differentiated and produced shoots and roots. Histological analysis suggested that the globular structures were protocorm-like bodies (PLBs), a novel pathway for plant regeneration. Further experiments showed that 75% bud explants of P. micans cultured on MS medium supplemented with 1.0 mg L-1 TDZ and 0.5 mg L-1 NAA produced PLBs within 8 weeks. Each explant produced more than 50 shoots that subsequently rooted. The survival rate of plantlets regenerated through this protocol was 98%. Analysis of 20 randomly selected plantlets using DNA flow cytometry showed that two were mixoploid. Such a high frequency of ploidy change may suggest the occurrence of somaclonal variation. However, regenerated plants phenotypically resembled mother plants. This was the first in vitro study of P. micans, and a new and reliable protocol has been developed for efficient regeneration of this species. Growth regulators to improve appearance and marketability of P. micans were also examined. Four growth regulators, Bonziregistered trademark {?-(4-chlorophenyl)methyl-?-(1,1-dimethylethyl)-1H- 1,2,4-triazole-1-ethanol}, B-Nineregistered trademark (butanedioic acid mono (2,2-dimethylhydrazide), Cycocelregistered trademark (2-chlorethy) trimethylethanaminium chloride, and Florelregistered trademark (2-chloroethyl) phosphonic acid were applied as foliar sprays or soil drenches in attempt to shorten internode length and reduce a leggy appearance. Bonziregistered trademark application was the most effective treatment and resulted in plants with a more compact growth form.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Zhujun Xiong.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Chen, Jianjun.
Local: Co-adviser: Henny, Richard J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041258:00001


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REGENERATION OF PHILODENDRON MICANS K. KOCH THROUGH PROTOCORM-LIKE BODIES AND IMPROVEMENT OF PLANT FORM USING GROWTH REGULATORS By ZHUJUN XIONG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009 1

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2009 Zhujun Xiong 2

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To my parents 3

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ACKNOWLEDGMENTS Before you read my thesis, I would like to let you know how fortunate I feel to have such a great supervisory committee. Without t heir help, it would be impossible for me to have completed the research and thesis. Firs t of all, I would like to thank Dr. Jianjun Chen for his great mental guidance and full s upport on every aspect of my studies. I will always remember that he spent weekends, which should be casual and relaxed, on reviewing my thesis. I learned more than hor ticultural knowledge from him. His wisdom and sincere advice inspired me to overcome many difficulties in my work and life. I would also like to show my greatest respec t and thanks to Dr. Richard J. Henny for all the thoughtful help, suggestions and use of hi s facilities. I could not have finished my research without his tremendous support. At the same time, I w ant to thank Dr. David J. Norman and Dr. Gary L. Leibee for comment s and suggestions on my proposal and thesis. Their encouragement and c oncern were always there. I wish to thank Dr. Feixiong Liao, Russell Caldwell, Min Deng, Qiansheng Li, and Luning Cui in Dr. Chens laboratory for their help on my improvement of research skills and their friendship. I appreciate the help from Ms. Terri Mellich in Dr. Hennys laboratory for the DNA flow cytometry anal ysis. Acknowledgement also goes to the College of Agricultural and Life Sciences, Mi d-Florida Research an d Education Center, and Dr. Chens program for providing matching assistantship to support this research. Lastly, I would like to thank my lov ed parents, Qiugu Xiong and Ruohong Gao, for their unselfish and endless love. I also extend my great appreciation to my friend Xue Zhang for his encouragement and kind help. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ..................................................................................................4 LIST OF TABLES ............................................................................................................7 LIST OF FIGURES ..........................................................................................................8 LIST OF ABBREVIATIONS .............................................................................................9 ABSTRACT ...................................................................................................................10 CHAPTER 1 LITERA TURE RE VIEW..........................................................................................12 The Genus Philodendron ........................................................................................12 Ornamental Value of P. micans and Potential for Improvement .............................13 Plant Regeneration Pathways .................................................................................14 Shoot Organogenesis .......................................................................................14 Somatic Embryogenesis ...................................................................................16 Protocorm-like Body Formation ........................................................................18 Histological Analysis of Regeneration Processes ...................................................19 Somaclonal Variation ..............................................................................................20 2 FACTORS INFLUE NCING REGENERATION OF PHILODENDRON MICANS .....22 Introduction .............................................................................................................22 Materials and Methods ............................................................................................25 Plant Materials and Explants ............................................................................25 Culture Medium ................................................................................................26 Experiments and Cultural Conditions ...............................................................26 Data Collection and Analysis ............................................................................27 Results ....................................................................................................................27 Bud Explants ....................................................................................................27 Internode Explants ...........................................................................................30 Leaf and Petiole Explants .................................................................................30 Bud Explants Cultured in the Dark ...................................................................37 Discussion ..............................................................................................................37 Effects of Explants ............................................................................................37 Effects of Growth Regulators ............................................................................38 Effects of Culture in the Dark ............................................................................38 3 ESTABLISHMENT OF AN EFFE CTIVE PROTOCOL FOR REGENERATION OF PHILODENDRON MICANS ..............................................................................40 5

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Introduction .............................................................................................................40 Materials and Methods ............................................................................................41 Plant Materials and Explants ............................................................................41 Culture Medium and Conditions .......................................................................41 Root Development and Ex Vitro Plantlet Establishment. ..................................42 Histological Analysis .........................................................................................43 Experimental Design and Data Analysis ..........................................................43 Results ...................................................................................................................44 Induction of Globular Structures .......................................................................44 A Model for Predicting Globul ar Structure Formation .......................................48 Histological Analysis .........................................................................................48 Shoot and Root Formation and Ex Vitro Transplanting ....................................48 Discussion ..............................................................................................................49 PLB Occurrence ...............................................................................................49 PLB Induction by TDZ ......................................................................................49 The Significance of the Established Regeneration System ..............................50 4 ANALYSIS OF REGENERATED P. MICANS USING DNA FLOW CYTOMETR Y.........................................................................................................52 Introduction .............................................................................................................52 Materials and Methods ............................................................................................53 Results and Discussion ...........................................................................................53 5 APPLICATION OF PLANT GR OWTH REGULATORS FOR IMPROVING GROWTH FORM OF P. MICANS ...........................................................................56 Introduction .............................................................................................................56 Materials and Methods ............................................................................................57 Plant Material and Plan t Growth Condition .......................................................57 Growth Regulators and Their Application .........................................................57 Results ....................................................................................................................59 Effects of Growth Regulator Foliar Spray .........................................................59 Effects of Growth Regulator Soil Drenching .....................................................59 Discussion ..............................................................................................................64 APPENDIX LIST OF RE FERENCES...............................................................................................66 BIOGRAPHICAL SKETCH ............................................................................................75 6

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LIST OF TABLES Table page 2-1 Bud explants of Philodendron micans responding to in vitro culture on MS medium supplemented with TDZ, BA, or CPPU with NAA for 8 weeks under a 16 h light photoperiod at 40 mol m-2 s-1.........................................................29 2-2 Responses of internode explants of Philodendron micans to in vitro culture on MS medium supplemented with TDZ or BA with NAA for 8 weeks under a 16 h light photoperiod at 40 mol m-2 s-1..........................................................30 2-3 Responses of leaf and petiole explants of Philodendron micans to in vitro culture on MS medium supplemented wit h TDZ with NAA for 8 weeks under a 16 h light photoperiod at 40 mol m-2 s-1.........................................................32 2-4 The frequency of swollen explants, necrotic callus, and formation of globular structures of bud, internode, leaf, and petiole expalnts cultured on MS medium regardless of growth regula tor combinations or concentrations evaluat ed............................................................................................................34 2-5 The frequency of swollen explants, necrotic callus and formation of globular structures produced by cytokinins of TDZ, BA, and CPPU regardless of the type of explant s tested........................................................................................35 3-1 Effect TDZ and NAA at differ ent concentrations on explant survival, production of necrotic callus, induction of protocorm-like bodies (PLBs) and shoots from Philodendron micans buds cultured on MS medium for 8 weeks in a 16 h light photoperiod at 40 mol m-2 s-1......................................................46 5-1 Average internode length, largest leaf length and leaf wid th of Philodendron micans after foliar spraying with B-nine Bonzi, Cycocel, and Florel for one mont h..........................................................................................................60 5-2 Average internode l ength(AIL), the largest leaf l ength (LLL) and width wildth (LLW) of Philodendron micans after soil drenching of B-nine, Bonzi, Cycocel, and Florel........................................................................................61 7

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LIST OF FIGURES Figure page 2-1 Different Philo dendron micans explants were necrotic after 8 weeks of cultur e.................................................................................................................33 2-2 The frequency of globular structur es formed under dark or 16 h light culture for 2, 4 and 8 weeks...........................................................................................36 3-1 Dark green and globular structures were initiated at the basal of bud explants.45 3-2 Histological analysis of the formation of PL Bs....................................................47 4-1 Histograms of relative fluorescence intensity obtained through PARTEC PA Flow Cytome ter..................................................................................................55 5-1 The largest leaf size for P. micans one month after one-time spraying with water, B-Nine at 5000 mg L-1, Bonzi at 100 mg L-1, Cycocel at 2000 mg L-1, and Florel at 1000 mg L-1...........................................................................62 5-2 The largest leaf size for P. micans one month after one-time drenching with water, B-Nine at 2500 mg L-1, Bonzi at 50 mg L-1, Cycocel at 1000 mg L1, and Florel at 500 mg L-1................................................................................63 8

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LIST OF ABBREVIATIONS B-Nine butanedioic acid mono (2,2-dimethylhydrazide) BA 6-benzyladenine BM basal medium Bonzi -[(4-chlorophenyl)methyl]-(1,1-dimethylethyl)-1 H 1,2,4-triazole1-ethanol CPPU N-(2-c hloro-4-pyridyl)-N-phenylurea Cycocel (2-chlorethy) tr imethylethanaminium chloride Florel (2-chloroethyl) phosphonic acid NAA 1naphthalene acetic acid PGR plant growth regulator PLBs protocorm-like bodies TDZ N -phenylN -1,2,3thiadiazol-5-ylurea 9

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Abstract of Thesis Pres ented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Master of Science REGENERATION OF Philodendron micans K. Koch THROUGH PROTOCORM-LIKE BODIES AND IMPROVEMENT OF PLANT FORM USING GROWTH REGULATORS By Zhujun Xiong December 2009 Chair: Jianjun Chen Cochair: Richard J. Henny Major: Horticultural Science Philodendron micans K. Koch, commonly known as velvet philodendron, has both a soft foliar texture and attractive colors that could make it an important genetic resource for breeding new ornamental traits and introducing improvements to the genus Philodendron. P. micans rarely flowers in nature and thus far no breeding program has utilized this plant for interspecific hybridiz ation. However, in addition to traditional breeding, new cultivars could be developed using somaclonal variation produced during in vitro propagation. As a step toward this direction, bud, leaf, petiole, and internode explants were cultured on Murashige and Skoog (MS) medium supplemented with BA (N 6 -benzyladenine), CPPU [N-(2-chloro-4-pyridyl)-Nphenylurea], or TDZ ( N -phenylN 1,2,3thiadiazol-5-ylurea) with NAA (1-naphthaleneacetic acid). Bud explants showed the greatest regenerative potential by producing both callus and globular structures. Callus was friable and unable to produce adventit ious shoots; while globular structures differentiated and produced shoots and roots. Histological analysis suggested that the globular structures were protocorm-like bodies (PLBs), a novel pathway for plant regeneration. Further ex periments showed that 75% bud explants of P. micans cultured 10

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on MS medium supplemented with 1.0 mg L -1 TDZ and 0.5 mg L -1 NAA produced PLBs within 8 weeks. Each explant produced more than 50 shoots that subsequently rooted. The survival rate of plantlets regenerated thr ough this protocol was 98%. Analysis of 20 randomly selected plantlets using DNA flow cyt ometry showed that tw o were mixoploid. Such a high frequency of ploidy change may suggest the occurrence of somaclonal variation. However, regenerated plants phenotypically resemb led mother plants. This was the first in vitro study of P. micans and a new and reliable protocol has been developed for efficient regeneration of this species. Growth regulators to improve appearance and marketability of P. micans were also examined. F our growth regulators, Bonzi { -[(4-chlorophenyl)methyl]-(1,1-dimethylethyl)-1 H 1,2,4-triazole-1-ethanol}, B-Nine (butanedioic acid mono (2,2-dimethy lhydrazide), Cycocel [(2-chlorethy) trimethylethanaminium chlori de], and Florel [(2-chloroethyl) phosphonic acid] were applied as foliar sprays or soil drenches in attempt to shorten internode length and reduce a leggy appearance. Bonzi applicati on was the most effective treatment and resulted in plants with a more compact growth form. 11

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CHAPTER 1 LITERATURE REVIEW The Genus Philodendron The genus Philodendron contains approximately 700 species, making it the second largest genus in the family Araceae (Croat 1997). Philoden drons are native to tropical America and comprise a conspicuous component of the native flora because of their abundance, different growth styles, and attractive, durable leaves. Based on their growth habits, philodendrons were divided into three groups by McColley and Miller (1965). The first group is the vi ning or scandent type, such as P. scandens K. Koch & Sello (heartleaf philodendr on). The second group has a self-heading and upright growing style represented by P. wendlandii Schott. The third group is the erectarborescent or tree type, such as P. bipinnatifidum Endl., which appear self-heading when they are young, but assume a more woody and treelike shape as they are mature. Philodendrons are popular or namental foliage plants, par ticularly the vining types, which are grown as either hanging baskets or potted plants where vines are supported by totem poles (Chen et al. 2005). Because of its ease in production, low maintenance and great durability indoors, P. scandens dominated all othe r genera of tropical ornamental foliage plant production, accounting for 50% of the national wholesale value of foliage plants in the U. S. in 1950 and 36% in 1967 (McConnell et al. 1989; Chen and Henny 2008). Recently, another vining type philodendron, Philodendron micans commonly known in the trade as velv et philodendron or red philode ndron, has been re-introduced to the foliage plant indus try and has claimed a significant market share. P. micans is easily recognized by cordate leaves that are velvet bronze on the upper surface and 12

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reddish to bronze on the lower surface. Taxonomically, it is unclear if P. micans is a distinct species. It has been documented as P. micans K. Koch (Marchan 1970), P. hederaceum (Jacq.) Schott (Croat 1997), and P. scandens forma micans (K. Koch) G. S. Bunting (Huxley 1994). Nevertheless, P. micans is closely related to P. scandens as both exhibit a vining growth style and have small chromosomes with 2 n = 32 (Marchan 1970). Ornamental Value of P. micans and Potential for Improvement Due to its unique foliar colors ranging from burgundy to bronze and copper, velvet philodendron is gaining popularity and offers cons umers variety in foliage assortments as small containers or as climbers supported by totem poles, or as hanging baskets. The growth form of P. micans is often regarded as having a leggy appearance due to long internodes. This could be improv ed by introducing new cultivars with a more compact growth style. P. micans could be an important genetic resource for plant breeders seeking to add color and foliage texture into lines of vining Philodendron types. However, P. micans similar to heartleaf philodendron, ra rely flowers in nature. To date, no plant breeding program has target ed this plant for hybridization, New cultivars can also be developed from somaclonal variation which is referred to as the phenotypic variation obse rved among plants regenerated after passage through a tissue culture stage (Larkin and Sc owcroft 1981). During the tissue culture process, there are two main pathwa ys of plant regeneration: organogenesis and somatic embryogenesis. Somaclonal variation may occur in plants regenerated from either pathway, and selected variants can be evaluated for cultivar development in floriculture crops. More than 82 cultivars of floricultural crops currently in the marketplace were developed through the sele ction of somaclonal variants (Chen and 13

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Henny 2008). Another approach for modifying pl ant growth form is the use of plant growth regulators. There are many grow th regulators commonly used by ornamental foliage plant growers to shorten internodes or improve branching of many foliage plant genera (Chen et al. 2005). The leggy growth habit of P. micans could be improved by growth regulator application. In the foliage plant industry, P. micans is propagated exclusively by single eye cuttings; an economically feasible micropropagat ion system using tissue culture has not yet been established. Chen and Henny (2006) proposed that the continuous vegetative propagation of asexual-propagated plants ma y have allowed an accumulation of unexpressed somatic mutations not eliminated by the meiotic sieve. Thus, in vitro culture of P. micans may provide an opportunity for mu tated cells to become expressed if plants arise from single cells that c ontain mutations. In addi tion to mutated cell expression, the process of in vitro culture itself can also induce mutations (Larkin and Scowcroft 1981; Lee and Phillips 1988; Chen and Henny 2008). Selection, characterization, and evaluation of the subsequent variants could result in new P. micans cultivars. Plant Regeneration Pathways Shoot Organogenesis Shoot organogenesis is the regeneration of adventitious shoot s from explants without pre-existing meristems and subseq uent rooting of the shoots (Schwarz and Beaty 2000). There are direct and indirect shoot organogenesis depending on the origin of shoots. Direct shoot organogenes is is the production of s hoots from single explants without any callus phase, while indirect organogenes is refers to the formation of shoots from an intermediary callus stage instead of directly from explants (Hicks 1980, Kane et 14

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al. 1994). In practice, it is not always possi ble to distinguish between the two methods. Directly-formed meristems may proliferate to form a regenerative tissue similar in appearance to callus and may also become surrounded by callus so that it is difficult to ascertain its origin (George et al. 2008). In general, three phases of organogenesis ar e recognizable, namely competence, determination, and finally adventitious shoots (Sugiyama 1999). Competence is defined as a state of cells which have retained t he capacity for a particular kind of cellular differentiation. This includes the capability to respond to extra-cellular signals under stimulus that could be plant growth regulat ors or other alternative changes in culture environment. Determination is the state of pr eviously competent cells committing to a differentiation pathway of shoot organogenesis after induction. Adventitious shoots form and develop into buds after the differentiati on stage. The dedifferentiation phase refers to the span of time that cells are becoming competent and becoming able to response to growth regulating chemicals (Sugiyama 1999). The nature of the internal factors gover ning cell competence is largely unknown. However, the study on Nicotiana tabacum by Attfield and Evans (1991) showed that a 1-2 day exposure to basal medium without plant growth regulator first before placing explants on IBM would maximize the rate of shoot formation, which indicates that 2 days was the time explants required to gai n competence and growth regulators were not necessary in this period. During the indu ction phase, a competent cell or a group of competent cells become committed to a unique developmental fate on the stimulus of an inducing signal. At the end of the induction phase, cells become fully determined and 15

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capable of shoot organogenesis ev en with the removal of the inducing signals (Schwarz and Beaty 2000). Hu et al. (2005) studied morphogenesis in petiole derived callus of Amorphophallus rivieri Durieu and observed that compact callus consisted of 3 components: epidermis, subepidermis, and inner parenchyma cells. Cells in the subepidermis started to divide to give rise to the formation of a long and narrow meristematic zone after 1 week of cultur e on differentiation medium. During direct organogenesis, adventitious buds may not deriv ed immediately from explants. There is evidence that a single epidermal cell may sometimes give rise to meristematic centers from one of which up to 22 identical shoots may arise (Broertjes et al. 1976; Shen 2007). Similarly, shoots on cultured explants of Nicotiana tabacum leaves were found to arise indirectly from nodules at the edges of t he explants, which were mainly formed by divisions of palisade mesophyll cells ar ound the edge of the explants (Attifield and Evans 1991). Somatic Embryogenesis Somatic embryogenesis or non-zygotic embryogenesis is a process whereby somatic cells differentiate into embryos, and somatic embryo germination, like zygotic embryos, produces seedlings (Gray 2000; Merkle et al. 1990). Since the first report of somatic embryogenesis in carrot cell cultur e in 1958 (Steward et al. 1958), in vitro somatic embryogenesis has been reported in more than 100 species (Krishnaraj and Vasil 1995). Somatic embryos can be either induced from the explant without any intervening callus phase, called direct somati c embyrogenesis or indirectly after a callus phase, commonly known as indirect soma tic embryogenesis (Willams and Maheswaran, 1986). 16

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There are three developmental stages in somatic embryogenesis in vitro: induction, development, and matu ration (Zimmerman 1993). Ex plants are composed of heterogeneous cell types. During the induction stage, undifferentiated cells from explants can be induced to obtain an embryogenic determination and somatic embryos can be obtained directly from these cells via direct embryogenesis (Merkle 1997). Differentiated cells from explants can undergo dedifferent iation, meristemation, and redifferentiation into embryogenic cells. In this case, calli are usually formed on the explant tissue, and somatic embr yos form on the callus, a process which is referred to as indirect somatic embryogenesis (Kohlenbach 1985). Somatic embryogenic cells can be distinguished from non-em bryogenic cells because embr yogenic cells usually contain dense cytoplasm, promi nent nuclei, higher starch contents, thickened cell walls, and are less vacuolated (Mooney and van Staden 1987). During the development of somatic emby ros, a single embryogenic cell undergoes a series of transverse and longitudinal di visions, passing through globular, torpedo, and cotyledonary stages for dicots or globular, scut ellar and coleoptilar stages for monocots. Finally, it forms a bipolar st ructure with a root and a shoot on opposite ends of the same axis with the capacity to reproduce entire plants (Arnold et al. 2002). During the maturation stage, somatic embryos accumulate a reserve and achieve desiccation tolerance, which is very important for successful germination and growth (Ammirato 1974). The maturation stage in somatic em bryogenesis has just been recognized recently due to observations that the rate of germination and regeneration is often very poor even with well-developed somatic embryo s (Bhojwani and Razdan 1996). A period of reversible arrested growth is necessary for proper embryo germination; without such 17

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developmental stages, somatic embryos would germinate precociously and finally die (Gray and Purohit 1991). Protocorm-like Body Formation Term of protocorm-like bodies (PLBs) was first coined by Morel (1960) when the shoot apex of Cymbidium was cultured for producing vi rus-free plants, during which protocorms were formed from apical meristems rather than from seeds. A protocorm is the tuberlike swollen part of an orchid seed, which appears during the early stage of germination. Protocorm-like bodi es are composed of many meristematic centers that are able to differentiate into shoots and roots (da Silva et al. 2000), which resemble somatic embryos. However, the shoots and r oots of regenerated plantlets from PLBs are not on the same axis. Thus, there are two schools of thought regarding the identity of PLBs: one considers that PLBs are somatic embryos (Steward and Mapes 1971; Begum et al. 1994; Ishii et al. 1998; Chen and Chang 2000) and the other believes PLBs differ from somatic embryos (Norstog 1 979; da Silva et al. 2000; Cui et al. 2008; Tian et al. 2008). The claim favoring the soma tic embryo scenario is largely based on the evidence derived from in vi tro regeneration of or chids. In fact, usage of the term PLB had been initially restrict ed to orchids (Ishii et al. 1998 ). However, PLBs have been identified in a wide range of other plant genera including Anthurium (Yu et al. 2009), Colocasia (Abo El-Nil and Zettler 1976), Heliconia (Goh et al. 1995), Lilium ( Nhut et al. 2001), Musa (Venkatachalam et al. 2006), Pinellia (Liu et al. 2009) Rosa (Tian et al. 2008), and Syngonium (Cui et al. 2008). This research hypothesis assumes that PLBs could be an independent pathway in pl ant regeneration. PLBs are distinguished from somatic embryos by the lack of a si ngle embryonic axis (Norstog 1979) and also different from shoot organogenesis by the di rect formation of abundant PLBs that are 18

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able to produce plantlets with both shoots and roots resembling the characteristics of somatic embryogenesis. Histological Analysis of Regeneration Processes Histological analysis is critically import ant for identifying regeneration pathways. In shoot organogenesis, shoots are usually formed first. There is a well-developed vascular connection between shoots and maternal explants. Roots are then induced from the base of shoots, which result in plantlets with shoots and roots not in the same axis. In somatic embryogenesis, a somatic embr yo is an independent entity; there is no vascular connection between somatic embryos and parental explants (Chengalrayan 2001). As mentioned above, the development of a single em bryogenic cell undergoes a series of transverse and longitudinal divi sions, passing through globular, torpedo, and cotyledonary stages for dicots or globular, scut ellar and coleoptilar stages for monocots. Somatic embryo germination, like seed germi nation, results in shoots and roots on a bipolar structure. PLBs rese mble somatic embryos morphologically, but PLBs have a vascular connection with the maternal expl ants and lack the bipolar structure. Depending on plant species and explant types, many PLBs can be produced per explant, and PLBs can produce shoots and roots di rectly (Cui et al. 2008; Tian et al. 2008). In theory, plantlets can be regenerated fr om each of the three pathways, but different pathways result in different number s of regenerated plantlet s and the degree of somaclonal variation observed (Chen and Henny 2006). Additionally, for genetic transformation or germplasm conservation, one pathway may have more advantages than another. Thus, histological analysis is essential for ascertaining the plant regeneration pathway em ployed on a case by case basis. 19

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Somaclonal Variation Somaclonal variation refe rs to the heritable genet ic variation in plants regenerated from tissue culture (Larkin and Scowcroft 1981). Among factors affecting somaclonal variation, plant genotype is probably the most important. Some cultivars show high variation rates while other s are highly stable (N ajaran and Walton 1987; Bouman and de Klerk 1997). Reg eneration pathways also make significant differences in the somaclone occurrence. Plants regenerat ed from a pathway with callus phase (i.e. indirect shoot organogenesis or indirect somatic embryogenesis) are often high in somalconal variation (Merkle 1997). Plants regenerated from direct shoot organogenesis or direct somatic embry ogenesis generally have a low rate of somaclonal variation because these proce sses have no callus phase (Merkle 1997). Regeneration through PLBs without the occurrence of callus in theory is also low in somaclonal variation. Plants produced from pre-existing meri stematic cells are usually true-to-type. In addition, soma clonal variation generally incr eases with the time that a culture has been maintained in vitro, especia lly callus culture (Skirvin et al. 1994: Bouman and de Klerk 1997). Somaclonal variation could be derived fr om pre-existing genetic variation in explants being cultured or induced during t he tissue culture process (Evans 1989). To distinguish the source of variation has been experimentally difficult because the appearance of somaclonal variants relies on in vitro culture. However, with the advance of molecular genetics, it has become clear that somaclonal variation is not the result of a single genetic mechanism (Kaeppler et al. 2000). Changes at the chromosome level, variation of DNA sequences including activation of transposable elements, and 20

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epigenetic effects have been shown to be the underlying mechanisms of somaclonal variation ( Lee and Phillips 1988; Chen and Henny 2006). Somalconal variants can be identified through morphological, cytological, and molecular means. Morphological characterist ics such as plant form (height, width, internode length, leaf number, l eaf size), foliar variegation pa tterns, leaf colors, flower color if any, and petiole color are compar ed between the variants and parental plants (Chen and Henny 2006). Only those variants that show distinct differences in one or more characteristics from parental plants are then selected and maintained for further evaluation. For research purposes, further evaluation may include molecular marker and cytological analyses. Cytological assa y includes chromosome counts and DNA flow cytometry analysis. Among the available molecular marker techniques, amplified fragment length polymorphisms (AFLP) has been widely used for somaclonal and naturally occurring sport evaluation (Chao et al. 2005; Chen et al. 2004). AFLP can be used to detect variation on the DNA level an d has proven to be extremely effective in distinguishing closely relat ed genotypes (Chen et al. 2006). 21

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CHAPTER 2 FACTORS INFLUENCING REGENERATION OF PHILODENDRON MICANS Introduction Although different species of Philodendron have been used as either landscape or potted ornamental plants, information regar ding micropropagation of philodendrons is limited. Vardja and Vardja (2001) reported that shoots were produced from P. tuxlanum and P. erubescens cultured on MS (Murashige and Sk oog 1962) medium containing N6-benzyladenine (BA) at concen trations from 2 to 4 mg L -1 with indole-3acetic acid (IAA) at 0.1 mg L -1 Blanco and Valverde (2004) micropropagated P. corcovadense using MS medium containing BA, N6-( 2-isopentenyl) adenine (2 iP), IAA, and naphthaleneacetic acid (NAA) in 53 comb inations. The highest rate of shoot multiplication, 3 shoots every four weeks, was achieved on MS medium containing 7 mg L -1 BA, and 3 mg L -1 kinetin. A shoot culture met hod was established by Gangopadhyay et al. (2004) for micropropagation of P. Xanadu on MS medium supplemented with 5.0 mg L -1 BA and 0.5 mg L -1 IAA. Thus far, all reports on in vitro culture of philodendrons were via shoot culture and BA was identified to be the most effective cytokinin for micropropagation. However, there were no reports on in vitro regeneration of P. micans. It has been well documented that species di ffer in regeneration capacity. Sears (1982) reported that variability occurred in different wheat genotypes for callus induction, regenerable callus formation, response to s ubculture, and plant regeneration potential. Similar results were shown by Mathias (1986) in wheat where t he initiation of callus from immature embryos was different among eight breeding lines. Somatic embryogenesis is also strictly genetically controlled among species or closely related varieties levels. In an evaluation of so matic embryogenesis from leaves of 22

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Dendranthema grandiflora 12 out of 23 cultivars tested produced somatic embryos, but only five regenerated plants. Likewise, vari ation in somatic embryogenic capacity ranging from 4.8 % to 72.7% was found among 15 genotypes of coffee ( Coffea arabica) (Molina et al. 2002). Explant source including petals, sepals, anthers, carpels, ovules, placenta, endosperm, microspores, shoot apices, axiliary buds, hypocotyls, mesocotyls, roots, leaves, and stems has been shown to affect somatic embryogenesis (Merkle et al. 1990). Somatic embryogenesis in the Nabali olive ( Olea europea L.) was shown to be 46% for root, 30% for cotyledon, 26% for hypocotyle, 20% for petiole, and 10% for leaf explants (Shibli et al. 2001). The ability to induce somatic embryogenesis decreases with the ageing of explants and they can mature to a point where they completely lose their ability to induce somatic embryogenesis (P arrott et al. 1995). In general, explants from less differentiated, imma ture or meristematic tissues such as immature zygotic embryos, cotyledons, and apical meristems, are more easily induced and established via somatic embryogenesis or organogenesis in vitro. Callus only initiates from young leaves, seed embryos or nodes, but never from mature leaves or stems in the Poaceae (George et al. 2008). Direct embr yogenesis usually takes plac e on explants taken from cotyledon sections and embryonic axes (P lata and Vieitez 1990), whereas indirect somatic embryogenesis is derived from well-differentiated tissues and might require higher growth regulator concentrations in the medium (Hartmann and Kester 1983). In addition to plant genotypes and explant s types, growth regulators used for induction are also important for regeneratio n. Induction of somatic embryogenesis by cytokinins alone is relatively rare; how ever, adventitious shoots can be induced on 23

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media containing only BA ( Simmonds 1984 ). Although an auxin is necessary for the induction of somatic embryogenesis, it has an inhibitory effect on subsequent differentiation and development of somatic embryos (Terzi and LoSchiavo 1990). The ratio of cytokinins to auxins is critical in determining shoot versus root formation. A high cytokinin to auxin ratio promotes shoot meristem formation, wh ile callus or root meristems are formed when the cyt okinin to auxin ratio is low. It has been reported that different combination of auxins and cytokinins at 0, 1, 2 mg L -1 levels resulted in either direct adventitious shoot formation, callus formation, or indirect adventitious shoot formation (Makunga et al. 2005). Dark treatment for newly inoculat ed explants can enhance morphogenesis. For example, keeping segments of apple leaves in the dark for the first three weeks of culture enhanced the subsequent regeneration of adventitious shoots and embryo-like structures (Welander 1988). Simi larly, an initial period of dark culture increased somatic embryogenesis in anthers or anther-der ived callus (Nair 1983). The highest frequency of white embryogenic tissue formation and the most normal embryoids of Triticum aestivum were obtained in the dark on MS medi um, compared to the light treatment when other factors were same (Ozias -Akins 1983).The optim un shoot regeneration could be achieved by germinating embryos in darkness before preparing cotyledon explants of watermelon w as reported by Compton (1999). Numerous report exist in the literature where the pr etreatment of plant tissue in darkness improves adventitious shoot regeneration from cytoledons, nodal tissue, leaf cells, petioles (Punja et al. 1990; Leblay et al. 1991; Mohamed et al. 1992). The exact mechanism of how dark pretreatments stimulate s ubsequent organogenesis under li ght is not completely 24

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understood. In some reports, the dark tr eatment was considered to increase endogenous auxin in the explants, which woul d promote callus formation or rooting (Huxter 1981). Except for the endogenous hormone regulation, dark-grown tissue produces much more ethylene than light-grown (Huxter 1981). The instances that ethylene would enhance or ganogenesis and organ development spread in a variety of culture systems (Kumar 1998). Because there has been no previous knowledge on regeneration of P. mican t he first portion of this project was to evaluate responses of different types of explants to selected combinations and concentrations of gr owth regulators as well as to compare dark versus light culture on regeneration of P. micans Materials and Methods Plant Materials and Explants P. micans stock plants were grown in 15-cm diameter pots in a shaded greenhouse under a maximum photosynthetic photon flux density of 300 mol m -2 s -1 at the University of Floridas Mid-Florida Rese arch and Education Center in Apopka, FL. Nodes, 10 mm in length containing buds, petioles, leaves, and the internode sections were collected from vines and immersed in 70% ethanol for 1 min by agitation. Explants of nodes, petiole and internode sections (10 mm in length), and leaf sections (10 mm 2 ) were cut in sterile Petri dishes and placed in sterilized bottles containing 100 ml solution of 1.0% sodium hypochlor ite (16%, v/v) and 1 to 2 drops of Tween-20 added as a surfactant. The bottles with explants and Clorox solution were agitated on a rotary shaker for 30 min at 120 rpm. To rinse the explants of the Clorox solution, the explants were immersed in 100 ml sterile distilled wa ter and agitated for 5 min on the shaker at 120 rpm. After the rinsing two mo re times, the expl ants were ready for culture initiation. 25

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Culture Medium Murashige and Skoog (MS) mineral salts and vitamins (Murashige and Skoog 1962) with 2.8% (w/v) sucrose and 0.66% (w/v ) agar (PhytoTechnology Laboratories, Shawnee Mission, KS) were used as a basal medium. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving at 121oC for 25 min. Plant growth regulator solutions of BA, CPPU [N-(2 -chloro-4-pyridyl)-Nphenylurea], TDZ (thidiazuron or N-phenyl-N -1,2,3thiadiazol-5-ylurea), and NAA were filter-sterilized and added to autoclaved basal medium based on t he requirements of the following four experiments when the temperature dropped to 50oC. The medium was then poured in 100 x 15 mm sterile Petri dishes (Fisher Sci entific, Pittsburgh, PA) with 20 mL each. Experiments and Cultural Conditions Four experiments were conducted with different explants. The fi rst experiment was the culture of node explants on MS basal medium for three weeks. Buds were excised and cultured on MS medium supplem ented with 1.0, 2.0, and 3.0 mg L -1 TDZ with 0.5 g L -1 NAA, respectively; 2.0 or 4.0 g L -1 BA with 0.5 g L -1 NAA; and 2.0 g L -1 CPPU with 0.5 g L -1 NAA. Each Petri dish was inoculated with four buds, six dishes per treatment. The bud explants were cultured under 16-h photoperiod provided by cool-white fluorescent tubes at a photon flux density of 8 mol m -2 s -1 The second experiment was internode expl ants cultured on MS basal medium supplemented with 1.0, 2.0, and 3.0 mg L -1 TDZ with 0.5 mg L -1 NAA, respectively and 4.0 g L -1 BA with 0.5 mg L -1 NAA. The explants were placed horizontally, four each on each medium-filled Petri dish with six dishes per treatment, which were cultured under 16-h photoperiod with a photon fl ux density of 8 mol m -2 s -1 26

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The third experiment was the culture of leaf and petiole expl ants on MS medium containing 1.0 and 2.0 mg L -1 TDZ with 0.5 mg L -1 NAA, respectively. Sterilized leaf explants were placed with the adaxial surfac e up, and petiole explants were placed horizontally. There were four explants per Petri dish with si x dishes per treatment for both leaf and petiole explant s. Leaf and petiole explants were cultured under 16-h photoperiod with a photon flux density of 8 mol m -2 s -1 The fourth experiment was the cultur e of bud explants on MS basal medium supplemented with 1.0, 2.0, and 3.0 mg L -1 TDZ with 0.5 mg L -1 NAA, respectively in the dark for 8 weeks. Again, there were four buds per dish with six dishes per treatment. Data Collection and Analysis The four experiments were arranged in a completely random design. Each Petri dish was considered an experimental uni t, and each treatment had six replications. Explants that showed a response, such as sw elling, formation of callus and/or globular structures, were recorded in each Petri di sh, and frequencies of the responses were calculated. Data were subjected to analysis of variance (SAS GLM, SAS Institute, Cary, N.C.), and means separations were determined using Tukey's honest significant difference (HSD) at the 5% levels. Results Bud Explants Fungal and bacterial contaminations were common to explants collected from the greenhouse-grown P. micans Before the outlined four experiments, several tests had shown an intensive chemical st erilization, i.e. 1.0% sodium hypochlorite (16%, v/v) with 1 to 2 drops of Tween-20 and agitation on a rota ry shaker for 30 min at 120 rpm, was required to minimize contamination. However, such sterilization often caused the 27

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bleaching of young explants. Further tests s howed that internodes and the node at or below the third leaf (counting back from th e shoot tip) were more durable for the chemical sterilization. The six combinations of growth regulat ors induced the expansion of lateral buds and production of callus and globul ar structures (Table 2-1) However, the frequencies in inducing bud expansion, callus, and globula r structures varied significantly among treatments. The highest frequencies in producing swollen buds, callus, and globular structure were those explants induced by TDZ at 1.0, 2.0, and 3.0 mg L -1 with NAA at 0.5 mg L -1 NAA, respectively. Calli were formed fr om one side of bud explants (Figure 21A), and were yellowish and in friable form (Figure 2-1B) and eventually died. Globular structures were formed from the other side (Figure 2-1B). On the other hand, globular structures were green, in solid and nodular form, and were able to differentiate to produce shoots with roots (data not shown). 28

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Table 2-1. Bud explants of Philodendron micans responding to in vitro culture on MS medium supplemented with TDZ, BA, or CPPU with NAA for 8 weeks under a 16 h light photoperiod at 40 mol m -2 s -1 TDZ NAA BA CPPU Swollen explants (%) Explants producing necrotic callus (%) Explants producing globular structures (%) 1.0 0.5 0 0 83.3ab 33.3b 75.6a 2.0 0.5 0 0 92.3a 75.9a 54.8ab 3.0 0.5 0 0 96.0a 83.3a 42.3ab 0 0.5 4.0 0 47.1b 10.0b 42.0ab 0 0.5 2.0 0 41.0b 6.3.2b 36.0b 0 0.5 0 2.0 65.3b 55.8b 35.8b Different letters within a column represent significant difference among treatments tested by Tukey's honest significant difference (HSD) at the 5% level. 29

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Internode Explants Table 2-2 show the response of internode explants to the three concentrations of TDZ with 0.5 mg L -1 NAA as well as 4.0 mg L -1 BA with 0.5 mg L -1 NAA. Many internodes failed to respond the induction (F igure 2-1A) or died (Figure 2-1B). The highest frequencies in explant expansion an d callus formation occurred in those cultured on MS medium supplemented with 2.0 mg L -1 TDZ with 0.5 mg L -1 NAA and 3.0 mg L -1 TDZ with 0.5 g L -1 NAA, respectively. The calli, however, quickly became yellow and died (Figure 2-1C). There was no occurrence of globular structures in all the treatments except for a low frequency among explants treated by 3.0 mg L -1 TDZ with 0.5 g L -1 NAA. Leaf and Petiole Explants Leaf and petiole explants were cultured on MS medium containing 1.0 and 2.0 mg L -1 TDZ with 0.5 mg L -1 NAA. About 70% of cultured leaf and petiole explants were swollen (Figure 2-1 F, G, and I), but no leaf explants produced callus on medium supplemented with 1.0 mg L -1 TDZ with 0.5 mg L -1 NAA and only 4% of leaf explants produced callus on medium containing 2.0 mg L -1 TDZ with 0.5 mg L -1 NAA (Table 2-3; Figure 2-1H). Additionally, glob ular structures were not form ed from leaf explants. There were 46% and 26% of petiole explants that formed callus when cultured on medium containing 1.0 or 2.0 mg L -1 TDZ with 0.5 mg L -1 NAA (Figure 2-1J and K). However, the frequency for globular structures was only 8% from explants induced by 2.0 mg L -1 TDZ with 0.5 mg L -1 NAA (Table 2-3). 30

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Table 2-2. Responses of internode explants of Philodendron micans to in vitro culture on MS medium supplemented with TDZ or BA with NAA for 8 weeks under a 16 h light photoperiod at 40 mol m -2 s -1 TDZ NAA BA Swollen explants (%) Explants producing necrotic callus (%) Explants producing globular structures (%) 1.0 0.5 0 43.3ab 14.5b 0a 2.0 0.5 0 77a 15.5b 0a 3.0 0.5 0 31.8b 68.1a 5.9a 0 0.5 4.0 6.3.3c 10.3b 0a Different letters within a column represent significant difference among treatments tested by Tukey's honest significant difference (HSD) at the 5% level. 31

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Table 2-3. Responses of leaf and petiole ex plants of Philodendron micans to in vitro culture on MS medium supplemented wit h TDZ with NAA for 8 weeks under a 16 h light photoperiod at 40 mol m -2 s -1 Explant TDZ NAA Swollen explant (%) Explant producing necrotic callus (%) Explant producing globular structures (%) Leaf 1.0 0.5 72.5a 0a 0a 2.0 0.5 74.1a 4.9a 0a Petiole 1.0 0.5 73.5a 46.5a 0a 2.0 0.5 71.a 26.0a 8.2a Explant significance NS ** NS Treatment significance NS NS NS NS = No significant difference between expl ant types or treatments at P <0.05; ** = Significant difference at P< 0.01. Different letters within a column represent significant difference among treatments tested by Tukey's honest significant difference (HSD) at the 5% level. 32

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Figure 2-1. Different Philodendron micans explants were necrotic after 8 weeks of culture. Bud explants produced calli t hat were yellowish and friable (A) and one side of a bud explant produced calli that died and globular structures produced on the other side were alive (B ). Internode explants did not respond to induction (C), died (D), and produc ed calli but died (E). Leaf explants became swollen (F and G) or produce calli (H), but died later. Petiole explants were swollen (I) and produced calli (J and K); the calli died after about 10 weeks. 33

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Table 2-4. The frequency of swollen explants, necrotic callus, and formation of globular structures of bud, internode, leaf, and petiole expalnts cultured on MS medium regardless of growth regula tor combinations or concentrations evaluated. Explant type Swollen explants (%) Explants producing necrotic callus (%) Explants producing globular structures (%) Bud 90a 64a 75a Petiole 71b 35bc 5b Internode 49c 34c 2b Leaf 73ab 2d 0b Significance ** ** ** ** = Significant difference at P < 0.01. Different letters within a column represent significant difference among treatments tested by Tukey's honest significant di fference (HSD) at the 5% level. 34

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Table 2-5. The frequency of swollen explants, necrotic callus and formation of globular structures produced by cytokinins of TDZ, BA, and CPPU regardless of the type of explants tested. Cytokinin Type Swollen explant (%) Explant producing necrosis callus (%) Explant producing globular structures (%) TDZ 90a 64a 57a BA 44c 8.15b 39a CPPU 65bc 55ab 35b Significance ** ** NS NS = No significant difference between expl ant types or treatments at P <0.05; ** = Significant difference at P< 0.01. Different letters within a column represent significant difference among treatments tested by Tukey's honest significant difference (HSD) at the 5% level. 35

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36 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 2week4week8week% of explan t 1.0TDZ 0.5 NAA Dark 2.0TDZ 0.5NAA Dark 1.0TDZ 0.5NAA Light 2.0TDZ 0.5NAA Light Figure 2-2. The frequency of globular structur es formed under dark or 16 h light culture for 2, 4 and 8 weeks. There were significant differences between dark and light treatment with P < 0.05.

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Bud Explants Cultured in the Dark Bud explants cultured on MS medium containing 1.0 or 2.0 mg L -1 TDZ with 0.5 mg L -1 NAA and held in the dark had significantly higher frequencies of globular structures 2 and 4 weeks after inoculation compared to those cultured under 16-h photoperiod with a photon flux density of 8 mol m -2 s -1 (Figure 2-2). However, t he frequencies for globular structure formation were similar between dark and light cultures for explants inoculated 8 weeks on the same growth r egulator treatments. Additionally the dark culture, like the light culture, produced both callu s and globular structures. Discussion Initial investigation of P. micans showed that bud, internode, leaf, and petiole explants differed significantly in response to in vitro culture. Growth regulator combinations and concentrations resulted in different frequencies in callus and globular structure formation. In addition, the dark culture of bud explants greatly promoted the formation of globular structures at 2 and 4 weeks after culture. Effects of Explants Bud explants, regardless of growth regulator combinations or concentrations, exhibited the highest frequencies in explant expansion (90%), callus formation (64%), and production of globular structures (75%) in this study (Table 2-4). Internode and petiole explants had significantly lower fr equencies in callus and globular structure formation. Leaf explants were in ferior in formation of both callus and globular structures. The explant differences in response to in vi tro culture could suggest that cells from internode, leaf, and petiole explants were less responsive to growth regulator induction. Another possibility could be that growth regulator combinations and/or their 37

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concentrations screened were not optimal for induction of internode, leaf, and petiole explants. Effects of Growth Regulators With the NAA concentration used in the first three experiment s set at 0.5 mg L -1 TDZ was found to be a more effective cyt okinin at inducing callus and globular structures, whereas BA produc ed globular structures and CPPU stimulated the most callus formation (Table 2-5). In addition there were two trends for bud explants: with an increase of TDZ concentration, callus fo rmation frequency increased; meanwhile, the production frequency for globular structures decreased. TDZ has also been found to induce callus in grape (Lin et al. 1989; Kartonmysheva et al. 1983), peanut (Gill 1999), orchid (Huan 2004), and several woody species (Huetteman 1993). However, there is little information concerning what type of callus was induced by TDZ. In the present study, the callus initiated was friable and loosely adhered on the surface of explants. The callus died a fter being transferred to a fresh medium. Continuing culture of explants with the callus, however, resulted in the death of explants. Therefore callus had to be re moved from explants to secure survival of globular structures. Since the increased concentrations of TDZ enhanced callus formation, TDZ concentrations at or above 3.0 mg L -1 should be avoided in regeneration of P. micans Effects of Culture in the Dark Maintaining cultures of explants in dar kness enhanced the formation of globular structures during the first two to four weeks. After 2 wee ks, most bud explants in the dark culture environment were swollen at t he base of buds, which was followed by the appearance of light yellow or wh ite globular structures (F ig 2-2). However, this 38

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enhancement diminished once cultures approached 8 weeks in the dark (Figure 2-2). It is unknown if an initial 4-week dark cultur e followed by culture under lighted conditions would accelerate the occurrence of globular structures. Future research to determine the effect of this proposed practice is warranted. Although the exact mechanism underlyi ng dark-culture mediated organogenesis is not completely understood, dark treatments were reported to promote adventitious shoots and embryo-like stru cture (Welander 1988), somati c embryogenesis (OziasAkins 1983), as well as an increase in auxin and ethylene in the explants (Huxter 1981). Microscopic examination compar ing dark and light cultured bean ( Phaseolus vulgaris ) showed more parenchyma cells and a higher proportion of less differentiated tissue in plantlets grown in the dark compared to the light (Herman and Hess 1963). In this study, where the globular structures observed cons isted of parenchyma cells in the center area of bud explants, the dark conditions could stimulate the r egeneration of those parenchyma cells and accelerate the formati on and enlargement of gl obular structures. 39

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CHAPTER 3 ESTABLISHMENT OF AN EFFECTIVE PROTOCOL FOR REGENERATION OF PHILODENDRON MICANS Introduction The previous study in Chapter 2 demonstrat ed that TDZ was effective in induction of globular structures in P. micans but it was uncertain whether the globular structures were somatic embryos or PLBs. The use of TDZ in regeneration of dicot plants has been widely reported (Huettem an and Preece 1993; Lu 1993; Murthy et al. 1998). The success in regeneration of P. micans was still one of a few examples of globular structures induced by TDZ in monocot pl ants. Other regenerated monocots by TDZ or TDZ with auxins include wheat, barley, Epipremnum (Qu et al. 2002; Zhang et al. 2005), Phalaenopsis (Shan et al. 2000; Chen et al. 2000), and Syngonium (Zhang et al. 2006). TDZ is a plant growth regul ator with cytokinin-like activity (Zhang et al., 2001; Landi and Mezzetti 2006), but Victor (1999) claimed that changes induced via TDZ undergo a different morphological route of development t han that those induced by purine-cytokinins. TDZ has been shown to prov ide sufficient stimulus for induction of somatic embryos or can substi tute for the combined auxin and cytokinin requirements in a variety of plant species, including peanut, tobacco, and geranium (Gill and Saxena 1992, 1993; Visser et al. 1992). Additionally TDZ can promote the accumulation of purine cytokinins (Thomas and Katterman 1 986) and/or the inhibition of cytokine oxidase activity (Kaminek and Armstrong 1990), which may also enhance its effectiveness in somatic embryogenesis. Preece et al. (1991) showed that TDZ was effective at concentrations as low as 10 pM and stimulated regeneration under a relatively short period exposure (Visser et al. 1992; Hutchinson and Saxena 1996). In 40

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the present study, a lower conc entration of TDZ (1 mg L -1 ) was better on inducing globular structures of P. micans compared with other cytokinins studied. The present study was intended to further ev aluate TDZ effects on in vitro culture of bud explants of P. micans The objectives were to identify the optimal concentration of TDZ and NAA in producing globular stru ctures, to investi gate the capability of globular structures for plantle t production, and also to perfo rm histological analysis to determine whether the globular structures were PLBs or somatic embryos. Materials and Methods Plant Materials and Explants Stems, 3rd lateral bud and below and 10-15 cm in length with lateral buds, were cut from the stock plants of P. micans grown in the shaded greenhouse. Leaves and roots around the nodes were removed. The st ems were immersed in 70% ethanol for 1 min by agitation. Nodal explants, 10 mm in lengt h, were cut in sterile Petri dishes and placed in a sterilized bottle containing 100 ml solution of 1.0% sodium hypochlorite (16%, v/v) and 1 to 2 drops of Tween-20 added as a surfact ant. The bottle with explants and Clorox solution was agitated on a rotary shaker table for 30 min at 120 rpm. To rinse the Clorox solution, the explants were immersed in 100 ml sterile distilled water and agitated for 5 min on the shaker tabl e at 120 rpm. Rinsing was repeated twice. Culture Medium and Conditions MS mineral salts and vitamins with 2.8% (w/v) sucrose and 0.66% (w/v) agar (PhytoTechnology Laboratories, Shawnee Mi ssion, KS) were used as a basal medium. The pH of the medium was adjusted to 5. 8 with 1 M NaOH before autoclaving at 121oC for 25 min. TDZ and NAA stock solutions were filter-sterilized and added to autoclaved basal medium when the tem perature dropped to 50oC. The medium was then poured in 41

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100 x 15 mm sterile Petri dishes (Fisher Scientific, Inc., Pitts burgh, PA) with 20 mL each, which was used as basal medium for inducing lateral bud growth. Another batch of MS medium supplemented with TDZ at 0.5, 1.0, and 2 mg L -1 with NAA at 0.0, 0.5, and 1.0 mg L -1 respectively were used for culture of bud explants for globular structure induction. Additional treatments included MS basal medium without growth regulator or containing 3.0 mg L -1 TDZ with 0.5 mg L -1 NAA for culture of bud explants. Thus, there was a total of 11 treatments for inducing gl obular structure using bud explants. Nodal explants were cult ured under 16-h photoperiod provided by cool-white fluorescent tubes at a photon flux density of 8 mol m -2 s -1 with a temperature of 25 2 C. Three weeks after culture, lateral buds h ad grown to approximately 0.5 mm in length and were excised from nodes. After cutting bud tips off, the bud explants were cultured on the same MS medium with the respecti ve 11 treatments. T here were four bud explants per Petri dish and six dishes per treatments. Four weeks later, the bud explants were subcultured on the same me dium with respective treatments. Data including survival rate, callus formation, and globular structur es, and shoots numbers were recorded 8 weeks after bud culture. Root Development and Ex Vitro Plantlet Establishment. Shoots produced from globular structures were able to produce roots in MS medium supplemented with TDZ and NAA. For be tter shoot growth and rooting, globular structures with shoots were tr ansferred to baby food jars c ontaining MS basal medium without growth regulator s or MS medium suppl emented with 0.5 mg L -1 IBA. Regenerated plantlets were separated, wa shed free of agar us ing tap water, and transplanted into a soilless medium with sphagnum peat, vermiculate, and perlite at a 2:1:1 ration based on volume. Potted plants were grown in a shaded greenhouse under 42

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a maximum photosynthetic photon flux density of 200 mol m -2 s -1 temperature range of 20 to 28 o C, and ambient relative humidity of 70 to 90%. Survival rates of plantlets in the shaded greenhouse were recorded tw o months after transplanting. Histological Analysis To determine the identity of the globular structures, bud culture samples were collected at different growth periods for hi stological examination. Globular structures, varying in size, were fixed in FAA solution (formalin: glacial acetic acid: 70% ethanol at 5:5:90 by volume) for 3 days. Samples were dehydrated in a series of ascending aqueous ethanol solutions at 70%, 80%, 95% fo r 2 hours each, and at 100% for 1 hour twice. To increase the transpar ency, samples were treated with a xylene:ethanol (1:1 by volume) solution for 2 hours, followed by pur e xylene for 1 hour. The specimens were infiltrated by moving them into a vessel t hat contained paraffin wax maintained in 40-56 C until complete saturation. Finally, the specimens were embedded in the paraffin wax for at least 24 h. Sections (10 m) were cut using a rotary microtome, and mounted on glass slides. The sections were de-waxed in xylene, xyl ene:ethanol (1:1 by volume), 100%, 95%, 85%, 70% ethanol solution, stai ned with Heindenhains haematoxylin and then covered by the cover slips with a dr op of neutral balsam bef ore examination under microscope. All sections were observ ed under a Nikon OPTIPHOT microscope and photographed using a Canon S3 IS digital camera. Experimental Design and Data Analysis All experiments were established in a completely randomized design with six replications. The frequency of bud explants in response to different concentrations of TDZ and NAA in the formation of globular structures was analyzed by SAS (SAS Inc. 1999). Mean separation was achieved by least si gnificant difference (LSD) test at 95% 43

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level. Additionally, a multiple linear regression model was developed for prediction of the frequency of globular st ructure formation from P. micans when TDZ and NAA were used for induction of bud explants. Results Induction of Globular Structures Buds sprouted from nodal explants and reached a length about 0.5 cm three weeks after culture on MS basal medium. Eight weeks after the buds were cultured on MS medium supplemented with TDZ and NAA under 16 h lighting, dark green and globular structures were initiated at the basal of bud explants (F igure 3-1A). Those globular structures had a solid surface t hat differed from callus and more closely resembled somatic embryos as they could be removed as individuals from explants (Figure 3-1B). PLBs differentiated to form shoots (Figure 3-1C-F). TDZ was necessary for formation of globular structures. TDZ at 0.5, 1.0, and 2.0 without NAA resulted in 37, 58, and 46% of bud explants producing globular structures, respectively (Table 3-1). However, the highest frequency in globula r structure formation was 75%, which occurred in MS medium supplemented with 1.0 mg L -1 TDZ with 0.5 mg L -1 NAA. A frequency of 67 and 62% occurred in MS medium containing 2.0 and 1.0 mg L -1 TDZ with 1.0 mg L -1 NAA, respectively. Similar to t he results in Chapter 2, bud explants showed increased frequencies in callus fo rmation when cultured with elevated concentration of TDZ with 0.5 mg L -1 NAA. The calli had to be removed; otherwise, the browning of calli caused death of globular structures. 44

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Figure 3-1. Dark green and globu lar structures were initia ted at the basal of bud explants (A); PLBs were removable as individuals from explants (B); differentiation of PLBs produced shoots (C-F), shoots produced roots but the shoot and root were not in the same axis (G); and regenerated plantlets grown in soilless potting medium in a shaded greenhouse with a survival rate at 98%(H). Bar=2mm 45

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Table 3-1. Effect TDZ and NAA at differ ent concentrations on explant survival, production of necrotic callus, induction of protocorm-like bodies (PLBs) and shoots from Philodendron micans buds cultured on MS medium for 8 weeks in a 16 h light photoperiod at 40 mol m -2 s -1 TDZ NAA Explants survived (%) Explants producing necrosis callus (%) Explant producing PLBs (%) Shoot numbers per explant 0.0 0.0 95.9 0f 0c 0.5 0.0 79.2 13.5ef 37.6b + 0.5 0.5 88.7 17.3ef 54.7ab ++ 0.5 1.0 92.3 50.5bcd 50.5ab ++ 1.0 0 83.2 20.2def 58.3ab ++ 1.0 0.5 92.3 33.3cde 75.1a +++ 1.0 1.0 83.2 87.6a 62.5ab +++ 2.0 0 79.8 33.3cde 46.7ab +++ 2.0 0.5 75.0 75.1ab 54.7ab +++ 2.0 1.0 96.1 63 10.7abc 67.3ab +++ 3.0 0.5 67.3 83.2a 42.3b +++ TDZ NS ** ** ** NAA NS ** ** TDZ*NAA NS ** NS NS NS = No significant difference between expl ant types or treatment s at P < 0.05; = significance at P < 0.05 level; and ** = significant difference at P < 0.01. Different letters within a column represent significant difference among treatments tested by Tukey's honest significant di fference (HSD) at the 5% level. + indicates <20 plantlets, ++ 20-50 plantlet, +++ >50 plantlet per explant 46

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Figure 3-2. Histological analysis of the formation of PLBs. Cell differentiation (A) and formed globular structures (B); the globular structure resembled somatic embryos (C), but there was vascular connection between explants and the globular structure (D); development of PLB produced multiple shoot meristems (E-I). Bar=100 m. 47

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A Model for Predicting Gl obular Structure Formation A multiple linear regression model: Y = 0.284x 1 0.43x 2 0.18x 1 /x 2 + 0.91, was developed. Where Y was the fre quency of globular structure, x 1 was TDZ concentration, and x 2 was NAA concentration and should not be equal to 0. This model revealed the relationship between TDZ and NAA concentra tions influencing the frequency of the formation of globular structures with the significance at P < 0.10. Histological Analysis The globular structures we re first observed from bud explants on MS medium supplemented with TDZ and NAA after 28 days of culture. There were generally three types of cells observed under light microscopy: regenerative cells which were smaller in size and compact with more densely stai ned cytoplasm and more prominent nuclei; parenchyma cells which were larger with thin cells and less compact; non-regenerative smaller cells with thick cell walls and less stained cytoplasm (Figure 3-2A). Cell differentiation resulted in the formation of fo rmed globular structures (Figure 3-2B); the globular structure resembled somatic embryo s (Figure 3-2C), but there was vascular connection between explants and the globular st ructure (Figure 3-2D), suggesting they were PLBs. The meristematic mass, deriv ed from those regener ative epidermal cells, formed several more meristematic domes t hat represented apical me ristems after a few days (Figure 3-2E-I). Shoot and Root Formation and Ex Vitro Transplanting After 12 weeks of culture on MS m edium supplemented with TDZ and NAA, shoots appeared from PLBs (F igure 3-2). Shoot numbers per explant varied depending on TDZ and NAA concentrations (Table 3-1). Explants with more than 50 shoots were 48

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those cultured on MS medium at 1 mg L -1 TDZ with 0.5 or 1.0 mg L -1 NAA; 2 mg L -1 TDZ with 0, 0.5, or1.0 mg L -1 NAA, and 3.0 mg L -1 TDZ with 0.5 mg L -1 NAA. Shoots produced roots on MS medium with TDZ and NAA, also on MS medium with 0.5 mg L -1 IBA or without growth r egulators. The roots were initiated from the base of the shoots. However, the shoot and root were not on the same axis (Figure 3-2). Plantlets were easily acclimatized (Figure 3-2). The survival rate in soilless medium was 98%. Discussion PLB Occurrence The globular structures formed from bud explants we re initially thought to be somatic embryos. But histol ogical analysis showed that t he structures clearly had a vascular connection with explants Additionally, no polar stru ctures were identified. These results suggest the globular structures were not somatic embryos. The globular structures were also different from callus, as they were solid with a smooth surface and once individual globular structures removed from explants were able to produce shoots when cultured on MS basal medium without grow th regulators. On the other hand, PLBs are similar to somatic embryos but with vascular connection with explants (Norstog 1979; Cui et al., 2008; Tian et al. 2008). PL Bs were able to produce more than 50 plantlets, and the plantlets produced roots. Thus, histological analysis along with the characteristics in regeneration of plantlets suggested the globula r structures were actually PLBs formed from bud explants of P. micans PLB Induction by TDZ It is interesting to note that TDZ alone wa s able to induce PLBs at a frequency of 46% when bud explants were cultured on MS containing TDZ at 2.0 mg L -1 This could 49

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be additional evidence supporting that TDZ can substitute for the combined auxin and cytokinin requirements of morphogenesis. TD Z alone has been demonstrated to induce somatic embryogenesis in a variety of plant species, including peanut, tobacco, and geranium (Gill and Saxena 1992, 1993; Visser et al. 1992). The highest frequency in PLB formation in P. micans however, occurred in bud explants induced by TDZ in combination with NAA. The concentrations and the ratios of TDZ and NAA appeared to be significantly impor tant (Table 3-1). However, a simple increase or decrease of TDZ or NAA did not result in a linear increase of PLB formation frequency. Thus, a model was developed for describing their relationships. It reveals that PLB formation frequencies will increase wit h increased concentrations of TDZ but will be limited by NAA concentrations and TDZ and NAA ratios. Conversely, PLB formation frequencies will increase with decr eased concentrations of NAA but will be affected by TDZ concentrations and TDZ and NAA ratios. Based on the average value inequality, the frequency of PLBs would be maximized when 0.43 x TDZ concentration = 0.18 x TDZ/NAA ratio, which resu lted in the value of TDZ/NAA 2 is equal to 2.4. Although significance of the developed eq uation requires further testi ng, it suggests that for high frequency of PLB induction, it is essential to control the concentrations of TDZ and NAA and keep their ratio in appropriate ranges. The Significance of the Established Regeneration System As mentioned previously, effi cient regeneration systems for Philodendron have not been well established. This is the first established regeneration system for Philodendron and it is through PLBs. Nodal explants were cultured on MS basal medium to produce bud explants in three weeks; bud explants then cultured on MS medium supplemented with 1.0 mg L -1 TDZ with 0.5 mg L -1 NAA resulted in PLB formation in 4-8 weeks, and 50

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PLBs produced multiple shoot s in another 4 weeks. More than 50 plantlets were produced from single explants. The resultant plantlets were readily grown in soilless substrate with 98% survival rate. Thus, this established system can be used for effective micropropagation of P. micans Additionally, this protocol could also be readily used for stable genetic transformation (Chai et al. 2002), mass multiplication us ing bioreactors or synthetic seed production (Ara et al. 2000; Young et al. 2000), and for cryopreservation as described for orchids (Nik ishina et al. 2007). 51

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CHAPTER 4 ANALYSIS OF REGENERATED P. MICANS USING DNA FLOW CYTOMETRY Introduction P. micans after being established in soill ess media as potted plants were morphologically and phenotypical ly stable and were identical to the parent plant and exhibited elongated internodes with velvet foliage. This suggested that regeneration through PLBs, similar to the regeneration through direct somatic embryogenesis, can produce plants with genetic st ability. This stability is attribut ed in part to the fact that the formation of PLBs had no intervening callus phase As discussed previously, although callus was formed from bud explants, it was independent from PLB formation. The callus had no capability for organogenesis and had to be removed for PLB proliferation. As a result, regenerants through PLBs exhibited little somaclonal variation. Recent evidence, however, has shown t hat direct somatic embryogenesis can have a multi-cellular origin that may cause chromosomal alterations and ploidy changes (Endemann et al. 2001; Tremblay et al. 199 9; Wilhelm 2000). Although it was unclear whether the PLBs of P. micans had resulted from single cells or multiple-cellular origin, an attempt to examine the ploidy level was important. However, due to the small size of chromosomes in P. micans (Marchan 1970), chromosome c ounting could be difficult. Recently, DNA flow cytometry analysis has been shown to be a quick and effective method for determining ploidy levels (J ohnston et al. 1999; Dolezel and Bartos 2005) including regenerated plants (Cui et al. 2009). The objective of this study was to determine the ploidy levels of randomly selected regenerated P. micans plants using DNA flow cytometry. 52

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Materials and Methods A stock plants of P. micans grown in the shaded greenhouse and 20 regenerated young plants were collected. Young leaves of the 21 plants were chopped and analyzed using a PARTEC PA cytometer (Partec GmbH, Mnster, Germany) based on the procedures described below. How many of each plant Young leaves of each plant were chopped with a new razor blade in a Petri dish containing 0.5 ml ice-cold Partec CyStain UV Ploidy solution (Partec GmbH, Mnster, Germany) supplemented with 0.1% mercaptoethanol and 2.0% polyvinylpyrroli done. An additional 1.5 ml of the same solution was added to the Petri dish and incu bated on ice for 2 minutes. The suspension was filtered through a Partec 50 m CellTrics disposable filter and analyzed using the PARTEC PA cytometer for determining the m ean sample nuclei fluorescence intensity. The DNA histograms of nuclei from stock leaf tissue of P. micans were compared to those of the 20 regenerants to determine the possible occurrence of polyploidy. Results and Discussion P. micans has small chromosomes and a dipl oid number that has been reported as 2 n = 32 (Marchan 1970). Flow cyt ometry analysis showed stock plants and 18 out of the 20 regenerated plants had a single peak, i ndicating that there was no ploidy variation among the 18 plants. However, plants numbered 9 and 16 showed two peaks, which meant they were mixoploids. Since t he two mixoploid plants are still young, just potted in a soilless medium, their morphologi cal characteristics will be determined when they become mature. The histograms of the stock plants, and regenerated plants number 9, 16, and 22 are present ed in Figure 4-1. Flow cyt ometric analysis indicated that 10% of the regenerated plants had a ploidy change which is a high percentage of chromosome variation among the regenerated population. 53

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Such a high percentage of regenerated plan ts with chromosome alternation is surprising considering the fact the plant s were regenerated from PLBs that had no intervening callus stage. Somaclonal variatio n therefore should be theoretically lower compared to regeneration through indirect shoot organogenesis although prolonged time in culture has also been suggested to increase the incidence of polyploidization (Endemann et al. 2001). The duration in regeneration of P. micans from bud explants to plantlets only required 12 weeks. It is unlik ely that such a short duration could cause this high variation rate. Another likely possibility could be due to the pre-existing somatic variation. P. micans does not flower under production or in teriorscape settings and its commercial propagation is through eye cuttings. Contin uous vegetative pr opagation of highly heterozygous plants may allow an increase in variation through the accumulation of somatic mutations because of the missi ng meiotic sieve (Buss 1983). In vitro regeneration differs from traditi onal vegetative propagation by allowing single or a few cells from pieces of explants to differentiate in vitro and develop into plantlets, which provides a greater chance of uncovering mutated cells. This study demonstrated that chromosome alternation occurred in the regenerated population of P. micans Further evaluation of the regener anted plants may reveal that some may have a ploidy level such as te traploidy and hexaploidy. Polyploidization is often associated with morphological changes such as robust growth, larger and thicker leaves, and resistance to biotic and abiotic stresses (Levin 1983). These desired traits could expand genetic diversity and thus can be used for new cultivar development of P. micans. 54

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55 Figure 4-1. Histograms of re lative fluorescence intens ity obtained through PARTEC PA Flow Cytometer. The peak represents nuclei at G 1 phase of P. micans stock plant (A), regenerated plants No. 19 (B ), No. 9 (C), and No. 16 (D).

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CHAPTER 5 APPLICATION OF PLANT GROWTH REGU LATORS FOR IMPROVING GROWTH FORM OF P. MICANS Introduction Tissue culture plantlets often have desirable growth habits when compared to plants propagated by standard methods. For example, Anthurium Dieffenbachia Spathiphyllum and Syngonium develop multiple basal shoots when grown from tissue culture, exhibit more compact growth and produce finished plants that are fuller than plants produced by other met hods (Conover 1985). However, P. micans plants regenerated through PLBs maintained their leggy growth style with elongated internodes after being produced in soilless potting medium in a shaded greenhouse, which suggests that additional breeding effort s, such as hybridization or creation of induced mutations, will be needed for improving the growth style of P. micans An alternative approach for reducing inte rnode length is to use antigibberelin growth regulators since gibberelli ns promote stem elongation. In the floriculture industry, plant growth retardants have been used as a foliar spray or soil drench (Davis 1987). Bonzi or Paclobutrazol [ -(4-chlorophenyl)methyl-(1,1-dimethylethyl)-1 H -1,2,4triazole-1-ethanol] has been shown to control the height of Caladium x hortulanum Bird., Codiaeum variegatum (L.) Blume, Schefflera actinophylla Endl., Euphorbia pulcherrima Wind., and Impatiens wallerana (L.) Hook. f. (Barrett et al. 1994) as well as Ficus. benjamina (Barrett and Nell 1983). B-Nine or daminozide ; [butanedioic acid mono (2,2-dimethylhydrazide] was used to reduce the height of Mussaenda L., a tropical ornamental shrub ( Cramer and Bridgen 1998). Plant Height suppression was reported in Canna lily ( Canna x generalis Florence Vaughan) by B-Nine application and Cycocel [(2-chlorethy) trimethylethanam inium chloride] (Bruner et al. 2001). 56

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Application of foliar spray of Florel [(2-chloroethyl) phosphonic acid] reduced internode elongation of Gynura aurantica (Blume) DC (Chen et al. 2002). The study in Chapter 5 was intended to determine if growth regulators Bonzi, BNine, Cycocel, and Florel applied to P. micans as a foliar spray or as a soil drench could suppress internode elongation and improve the growth form of P. micans. Materials and methods Plant Material and Plant Growth Condition Single node cuttings of P. micans 4 to 5 cm in length, were rooted in 15-cm diameter pots filled by Fafard 2 Mix (Conrad Fafard Inc., Apopka, FL) at 5 cuttings each. Cuttings were grown in a shaded greenhouse under a maximum light level of 284 mol m 2 s -1 a temperature range of 2028 C, and a relative humidity of 60-100%. Three weeks after rooting, each pot received 5 grams of a 15N-7P 2 O 5 -15K 2 O controlledrelease fertilizer, Multicote with an 8-mont h longevity at a temperature of 21 C ( Haifa Chemicals Ltd., Haifa Bay, Israel ). Plants were watered one or two times per week as needed. One month later, uniform 3 rooted cuttings were selected from the original 5 and the other 2 cuttings were pulled and disc arded. The vines of selected plants were 20-25 cm in length. Growth Regulators and Their Application A total of 250 potted P. micans were prepared. The plants were subjected to either foliar spray or drenching treatment on August 3, 2009. Solutions of daminozide (B-Nine SP, Uniroyal Chemical Co., Middl ebury, CT) at 1,250, 2, 500, and 5,000 mg L -1 ; paclobutrazol (Bonzi, Uniroyal Chem ical Co.) at 15, 50, and 100 mg L -1 ; chlormequat chloride (Cycocel, Olympic Horticultural Pr oducts, Mainland, PA) at 500, 1,000, and 2,000 mg L -1 ; and ethephon (Florel, Monterey Law n and Garden Products, Fresno, CA) 57

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at 250, 500, and 1,000 mg L -1 were made using deionized water. The solutions, along with a deionized water as a control, were immediately foliar sprayed until runoff, approximating 25 mL per plant. Another group of the plants were subjected drenching tr eatment on the same day. Solutions of daminozide (B-Nine) at 625, 1250, and 2500 mg L -1 ; paclobutrazol (Bonzi) at 7, 15, and 50 mg L -1 ; chlormequat chloride (C ycocel) at 250, 500, and 1000 mg L -1 ; and ethephon (Florel ) at 125, 250, and 500 mg L -1 were prepared using deionized water. The solutions were app lied as soil drenches at 125 mL per pot. After both foliar spray and soil drenching, a hole was punched in the youngest leaf of each vine. Four weeks later, the same treatments, spray and drenching, were repeated to the same plants. Immediately following the second treatment, the shoot length from the marked leaf to the tip, the nu mber of nodes from t he marked leaf to the tip, largest leaf length and width were measured and the average stem length for each vine were calculated. The experiment was arranged in a co mpletely randomized design with 10 replications for each treatment. Internode leng th, the largest leaf length and width were recorded one month after the tr eatments. Data were analyzed by analysis of variance (SAS Institute 1999), and means separ ations were determined using Tukey's honest significant difference (HSD) at the 5% level. Additional ly, data between growth regulator treated plants were compared to those of control plants using Tukey's Studentized Range at P < 0.05. 58

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Results Effects of Growth Re gulator Foliar Spray Internode lengths of plants sprayed by grow th regulators regardless of types and concentrations significantly varied from t he control plants (Table 5-1). However, among the growth regulator treated pl ants, there were no significant differences in internode length except for those sprayed by Florel at 500 and 1000 mg L -1 Internode length were 5.3 and 4.3 cm respectively for the two concentrations compared to 8.8 cm of the control plants. Foliar spray of Florel at concentrations of 500 and 1000 mg L -1 resulted in defoliation. Leaf length and width were signi ficantly reduced by the sprays of Florel at 250 mg L -1 Additionally, shoot tips of vines sprayed by Florel grew upright compared to the other all ot her treated and control plants which grew horizontally. In general, there was a slight reduction in leaf size for P. micans plants sprayed with growth regulators, but the reduction was not significant (Figure 5-1, Table 5-1). Effects of Growth Regulator Soil Drenching Similar to the results of foliar spray, internode lengths of P. micans plants drenched with growth regulators differed significantly from the control plants (Table 5-2). There was some variation in internode le ngth among all growth regulator drenched plants, however, the variation was non-significant except the plants drenched with 50 mg L -1 Bonzi whose internode lengths were 6. 4 compared to 8.8 cm of the control plants. Contrary to foliar spray, soil drenching of Floreldid not result in defoliation of plants; and soil drenched with Fl orel did not induce upright growth of shoot tips. Leaf length and width were decreased by soil drenching treatments, but the effects were not significant except for those tr eated by Cycocel at 1000 mg L -1 and Florel at the three concentrations (Figure 5-2, Table 5-2). 59

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Table 5-1. Average internode length, la rgest leaf length and leaf width of Philodendron micans after foliar spraying with B-nine, Bo nzi, Cycocel, and Florel for one month. Growth regulator Concentration (mg L -1 ) Internode length (cm) Largest leaf length (cm) Largest leaf width (cm) Control 0 8.8 9.6 5.0 B-nine 1250 7.1a* 8.8a 4.9 2500 7.0a* 8.7a 5.0 5000 6.4a* 8.9a 5.15 Bonzi 15 7.6a* 8.7a 4.6 50 7.4a* 9.2a 4.7 100 6.4a* 9.0a 4.7 Cycocel 500 7.7a* 8.5* 4.5 1000 7.2a* 9.1 4.8 2000 7.4a* 9.2 4.9 Florel 250 7.2a* 6.6* 3.3* 500 5.3b* 1000 4.3b* Indicates significant difference between growth regulator tr eated plants and control plant tested by Tukey's Student ized Range at P < 0.05; -, Different letters within a column represent significant difference among treatments tested by Tukey's honest significant di fference (HSD) at the 5% level. 60

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Table 5.2. Average internode length(AIL), th e largest leaf length (LLL) and width wildth (LLW) of Philodendron mican s after soil drenching of B-nine, Bonzi, Cycocel, and Florel. Growth regulator Concentrat ion (ppm) AIL(cm) LLL (cm) LLW(cm) Control 0 8.8 9.6 5.0 B-Nine 625 8.1a* 8.4* 4.3 1250 8.2a* 8.6 4.5 2500 8.3a* 8.6 4.3 Bonzi 7 7.5a* 9.4 4.8 25 6.8ab* 9.3 4.6 50 6.4b* 8.7 4.7 Cycocel 250 7.1a* 9.0 4.6 500 7.0a* 9.2 5.0 1000 6.7a* 8.5* 4.2* Florel 125 6.9a* 8.3* 4.3a* 250 6.9a* 8.0* 4.3a* 500 7.2a* 8.3* 4.2a* Indicates significant difference between growth regulator treated plants and control plant tested by Tukey's Studentized Range at P < 0.05; -, Different letters within a column represent significant difference among treatments tested by Tukey's honest significant difference (HSD) at the 5% level. 61

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Figure 5-1. The largest leaf size for P. micans one month after one-time spraying with water, B-Nine at 5000 mg L -1 Bonzi at 100 mg L -1 Cycocel at 2000 mg L -1 and Florel at 1000 mg L -1 62

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Figure 5-2. The largest leaf size for P. micans one month after one-time drenching with water, B-Nine at 2500 mg L -1 Bonzi at 50 mg L -1 Cycocel at 1000 mg L -1 and Florel at 500 mg L -1 63

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Discussion The results from both foliar spray and so il drenching experiments showed that BNine and Cycocel with the concentrations app lied were not effective in control of internode length (Table 5-1 and Table 5-2). Folia r spray of Florel significantly reduced internode length, but at the same time it caused leaf dr op, indicating that Florel cannot be used on P. micans as foliar spray. It is not surpri sing that foliar applic ation of Florel resulted in defoliation because Florel breaks down into ethy lene, a naturally occurring compound, which causes leaf drop and blossom abortion. However, Florel used as a soil drench did not cause defo liation or reduce internode length. However Florel did significantly reduce leaf size, and thus is not a suitable growth regulator to control internode length in P. micans Bonzi appears to have potential to reduce internode length in P. micans The internode of plants sprayed at 100 mg L -1 or drenched at 50 mg L -1 with Bonzi was 6.4 cm compared to 8.8 cm for the control. Bonz i applications did not significantly reduce leaf size. Bonzi is an effective inhibi tor that blocks gibberellin biosysnthesis by inhibiting kaurene oxidase, an enzyme conver ting kaurene to kaurenoic acid (Wang et al. 1986). When gibberellin biosysnthesis is blocked, cell division still occurs, but the new cells do not elongate, which results in shoots with the same numbers of leaves but compressed internodes ( Chaney 2003) Successful use of Bonzi application to reduce internode length has been reported in Plectranthus australis R. Br., Zebrina pendula Schnizl., and Ficus benjamina (Davis 1987) as well as Gynura aurantiaca (Blume) DC (Chen et al. 2002) and other floricultu re crops (Barrett et al. 1994). Although internode length was reduced by application of Bonzi, it appears that further tests with increased concentration in both foliar spray and soil drenching is 64

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needed to determine the optimum concentration ra tes for effective control of internode length and improve the growth form of P. micans 65

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BIOGRAPHICAL SKETCH Zhujun Xiong was born in Jiangxi province, P.R. China. She obtained her Bachelor of Science degree in biology from Fudan University in July 2007, Shanghai. At the same time, she was admitted to the graduate progr am in the Department of Environmental Horticulture, University of Florida. Her study was sponsored by Dr. Jianjun Chen with a research assistantship. She worked as a research assistant in t he plant ecology lab while she pursued her bachelors degree in Fudan University and studied the invasive pattern of Alternanthera philoxeroides, a alien species in China. At the University of Florida, she was employed as research assistant in a plant physiol ogy lab and helped with pl ant micropropagation. 75