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In Vitro Application of Colchicine to Induce Tetraploids in Dieffenbachia 'Star Bright M-1'

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In Vitro Application of Colchicine to Induce Tetraploids in Dieffenbachia 'Star Bright M-1'
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HOLM, JAMES ROBERT
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2008

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Cytometry ( jstor )
Diploidy ( jstor )
In vitro fertilization ( jstor )
Leaves ( jstor )
Plant morphology ( jstor )
Plants ( jstor )
Ploidies ( jstor )
Polyploidy ( jstor )
Stomata ( jstor )
Tetraploidy ( jstor )
City of Mount Dora ( local )

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University of Florida
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University of Florida
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Copyright James Robert Holm. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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5/31/2008
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659898667 ( OCLC )

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1 IN VITRO APPLICATION OF COLCHICINE TO INDUCE TETRAPLOIDS IN Dieffenbachia x ‘Star Bright M-1’ By JAMES ROBERT HOLM A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

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2 2007 James Robert Holm

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3 ACKNOWLEDGMENTS I thank Dr. R. J. Henny, Dr. S. M. Scheiber, Dr . J. Chen and Dr. D. J. Norman for their support and mentoring, and the faculty and staff of the Mid-Florida Research and Education Center for making my graduate education possible. I want to especially thank Terri Mellich and Mary Brennan for their laboratory expertise and a dvice. In addition, I thank Diane Mealo for her help and assistance in coordinating my Gradua te studies and my good friend Chris Davern for her encouragement and support. I want to express my apprecia tion to Agri-Starts I, Inc., fo r their financial assistance and also for providing the equipment and materials to start my project . Without their support, this work would not have been possible. In addi tion, I thank the Florida Nursery, Growers, and Landscape Association – Action Chapter for their generous financial contribution.

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4 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................3 LIST OF TABLES................................................................................................................. ..........6 LIST OF FIGURES................................................................................................................ .........7 ABSTRACT....................................................................................................................... ..............9 CHAPTER 1 LITERATURE REVIEW.......................................................................................................11 Overview....................................................................................................................... ..........11 Reports of Polyploidization....................................................................................................11 Mutagens Used to Induce Polyploidy.....................................................................................12 Colchicine..................................................................................................................... ...12 Other Mutagens Used to Induce Polyploidization...........................................................17 Determination of Polyploids...................................................................................................18 Morpholgy...................................................................................................................... .18 Stomata Length................................................................................................................19 Flow Cytometry...............................................................................................................20 2 INDUCTION OF TETRAPLOIDY IN Dieffenbachia x ‘Star Bright M-1’..........................21 Introduction................................................................................................................... ..........21 Materials and Methods.......................................................................................................... .22 In Vitro Propagation........................................................................................................22 Colchicine Application....................................................................................................24 Greenhouse Conditions...................................................................................................25 Data Collected.................................................................................................................25 Survival....................................................................................................................25 Morphological characteristics..................................................................................26 Stomata length..........................................................................................................27 Flow cytometry........................................................................................................27 Statistical analysis....................................................................................................27 Results and Discussion......................................................................................................... ..28 In Vitro Survival..............................................................................................................28 Ex Vitro Survival.............................................................................................................29 Morphology.....................................................................................................................29 Morphological assessment of 422 surviving explants..............................................29 Morphological assessment of 63 potential polyploids.............................................31 Stomata Analysis of 63 Potential Polyploids..................................................................32 Flow Cytometry Analysis of 63 Potential Polyploids.....................................................32 Conclusion..................................................................................................................... .........34

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5 3 MORPHOLOGICAL COMPARISONS OF DIPLOID AND TETRAPLOID Dieffenbachia x ‘Star Bright M-1’.........................................................................................62 Materials and Methods.......................................................................................................... .62 Results and Discussion......................................................................................................... ..63 Conclusion..................................................................................................................... .........64 LIST OF REFERENCES............................................................................................................. ..71 BIOGRAPHICAL SKETCH.........................................................................................................75

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6 LIST OF TABLES Table page 2-1 In vitro percent survival of tissue cultured Dieffenbachia x ‘Star Bright M-1’ shoot clumps 12 weeks after treatment w ith four rates of colchicine in vitro .............................36 2-2 Number of tissue cultured Dieffenbachia x ‘Star Bright M-1’shoots harvested and mean percent survival of plants.........................................................................................37 2-3 Morphological characteristics of 422 Dieffenbachia x ‘Star Bright M-1’ plants 18 months after treatment with four rates of colchicine in vitro and 12 months after transfer to the greenhouse..................................................................................................38 2-4 Morphological characteristics of Dieffenbachia x ‘Star Bright M-1’ selected based on visual indicators of polypl oidy 18 months after treatm ent with four rates of colchicine in vitro and 12 months after tran sfer to the greenhouse...................................39 2-5 Mean stomata length of leaves from Dieffenbachia x ‘Star Bright M-1’ selected based on visual indicators of polyploidy 20 mont hs after treatment with four rates of colchicine in vitro and 14 months after tran sfer to the greenhouse...................................40 2-6 Ploidy level of 63 Dieffenbachia x ‘Star Bright M-1’ plants initially selected as potential polyploids based on morphological traits 20 mont hs after treatment with four rates of colchicine in vitro ..........................................................................................41 2-7 Comparison of percent tetraploids from tota l number of plants available for study to percent tetraploids from those selected for flow cytometry based on morphological markers and stomata analysis.............................................................................................42 3-1 Comparison of ploidy leve l with stomata lengths of Dieffenbachia x ‘Star Bright M-1’ 20 months after treatme nt with colchicine in vitro and 14 months after transfer to the greenhouse..................................................................................................................... ....66 3-2 Morphological comparison of diploids to tetraploids of Dieffenbachia x ‘Star Bright M-1’ 20 months after treatment w ith four rates of colchicine in vitro and 14 months after transfer to the greenhouse..........................................................................................67

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7 LIST OF FIGURES Figure page 2-1 Tissue cultured Dieffenbachia x ‘Star Bright M-1’ shoot clumps prior to treatment with 0, 250, 500 or 1000 mgL-1 colchicine in vitro ..........................................................43 2-2 In vitro Dieffenbachia x ‘Star Bright M-1’ shoot cl umps 12 weeks after treatment with four rates of colchicine in vitro ................................................................................. 44 2-3 In vitro Dieffenbachia x ‘Star Bright M-1’ shoot clum ps before second transfer to fresh media 12 weeks after treatment with four rates of colchicine in vitro ......................45 2-4 In vitro percent survival of tissue cultured Dieffenbachia x ‘Star Bright M-1’ shoot clumps......................................................................................................................... .......46 2-5 Dieffenbachia x ‘Star Bright M-1’ shoots ready to plant to greenhous e 26 weeks after treatment with liquid colchicine for 24 hours in vitro .......................................................47 2-6 Ex vitro percent survival of tissue cultured Dieffenbachia x ‘Star Bright M-1’plants......48 2-7 Crown height vs. treatment rate of plants from tissue cultured Dieffenbachia x ‘Star Bright M-1’.................................................................................................................... ....49 2-8 Height with leaves held up vs. trea tment rate of plants from tissue cultured Dieffenbachia x ‘Star Bright M-1’.....................................................................................50 2-9 Canopy height vs. treatment rate of plants from tissue cultured Dieffenbachia x ‘Star Bright M-1’.................................................................................................................... ....51 2-10 Canopy width vs. treatment rate of plants from tissue cultured Dieffenbachia x ‘Star Bright M-1’.................................................................................................................... ....52 2-11 Growth index vs. treatment rate of plants from tissue cultured Dieffenbachia x ‘Star Bright M-1’.................................................................................................................... ....53 2-12 Largest leaf length vs. treatment rate of plants from tissue cultured Dieffenbachia x ‘Star Bright M-1’.............................................................................................................. .54 2-13 Largest leaf width vs. treatment rate of plants from tissue cultured Dieffenbachia x ‘Star Bright M-1’.............................................................................................................. .55 2-14 Flow cytometry confirmed tetraploid from tissue cultured Dieffenbachia x ‘Star Bright M-1’ after treatment with 250 mgL-1 colchicine in vitro .......................................56 2-15 Flow cytometry confirmed tetraploid from tissue cultured Dieffenbachia x ‘Star Bright M-1’ after treatment with 500 mgL-1 colchicine in vitro .......................................57

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8 2-16 Flow cytometry confirmed tetraploid from tissue cultured Dieffenbachia x ‘Star Bright M-1’ after treatment with 1000 mgL-1 colchicine in vitro .....................................58 2-17 Flow cytometric histogram of a diploid plant from tissue cultured Dieffenbachia x ‘Star Bright M-1’.............................................................................................................. .59 2-18 Flow cytometric histogram of a tetraploid plant from tissue cultured Dieffenbachia x ‘Star Bright M-1’.............................................................................................................. .60 2-19 Flow cytometric histogram of a mixoploid plant fr om tissue cultured Dieffenbachia x ‘Star Bright M-1’.............................................................................................................. .61 3-1 Stomata imprints of Dieffenbachia x ‘Star Bright M-1’....................................................68 3-2 A 20-month-old diploid (left) and tetraploid (right) Dieffenbachia x ‘Star Bright M-1’.....69 3-3 Typical diploid leaf (left) and 3 typical tetraploid leaves (right) of Dieffenbachia x ‘Star Bright M-1’ taken fr om 20-month-old plants...........................................................70

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9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science IN VITRO APPLICATION OF COLCHICINE TO INDUCE TETRAPLOIDS IN Dieffenbachia ‘Star Bright M-1’ By James Robert Holm May 2007 Chair: R. J. Henny Major: Horticultural Science Treating in vitro diploi d Dieffenbachia x ‘Star Bright M-1’ with colchicine successfully induced tetraploids. Colchicine rates of 0, 250, 500 or 1000 mgL-1 were added to liquid MS media to treat rapidly growing shoot clumps for 24 hours. In vitro survival of shoot clumps significantly decreased as colchi cine concentration increased. Morphological assessment of 422 plants that survived colchicine treatment led to th e selection of 63 plants with visible traits that suggested a polyploid condition. These plants were subjected to flow cy tometry which confirmed the presence of 21 diploids, 13 tetraploids and 29 mixoploids among 63 selections. A treatment rate of 500 mgL-1 colchicine produced more tetraploids (40.0%) than did 250 (18.8%) or 1000 mgL-1 (6.3%). The 1000 mgL-1 treatment produced the most mixoploids (75.0%) compared to 40.6% and 26.7% at 250 and 500 mgL-1, respectively. Comparing diploid and tetraploid morphology showed significant differences in the shape, size, thickness, and weight of leaves and in stomata size. Tetraploid leaf area averaged almost 50% smaller th an diploid but tetraploid leaf width to length ratio was greater. Tetraploid leaf blades were thicker than diploid but there was no significant difference in leaf midrib thickness. Tetraploid leaf specific weight was significantly greater than

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10 diploid. Results suggest morphol ogical data alone is insufficient to confirm the presence of polyploids, but it is a valuable tool in selecting candidates for flow cytometry analysis.

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11 CHAPTER 1 LITERATURE REVIEW Overview Polyploidization occurs due to duplication of chromosomes and can occur naturally or may be induced artificially. Since polyploidiza tion also results in the duplication of gene products, phenotypic variants can be observed, such as thicker leav es and stems, deeper color, and more compact growth habit (Gao et al. 1996 ). Two basic types of polyploids can exist: autopolyploids, which contain more than two ge netically identical genomes; and allopolyploids, which contain genomes from more than one spec ies (Leitch and Bennett 1997). One of the most important uses of polyploids in plant breeding is to overcome hybrid sterility that arises from chromosomal structural differences which reduc e the degree of homology and consequently pairing at meiosis (Lu and Bridge n 1997). The induction of a tetraplo id from a sterile diploid (or even a hexaploid from a triploid) insures that each chromosome has a homologous partner during meiosis. Fertility is restored with the production of viable gametes. Polyploidization has been used in many facets of plant breeding, crop improvement, and genetic studies. The focus of this study is the polyploidiza tion of a mutant of Dieffenbachia x ‘Star Bright' (United States patent #9051, 1995), a cultivar whose genus is economically important in the ornamental tropical foliage industry. Reports of Polyploidization The first reports of artificial induction of polyploids in plan ts come from Blakeslee (1937). Thereafter, polyploidization has be en reported in many crop species and ornamentals. Literature reports on polyploidization of vegetable species include: Beta vulgaris (Hansen A et al. 1998 & Hansen A et al. 2000); Zingiber officinale (Adaniya and Shirai 2001); Solanum tuberosum (Jacobsen 1981); Brassica napus (Weber et al. 2005, Zhao and Simmonds 1995); Zea mays

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12 (Kato 2002) and Allium spp. (Song et al. 1997). Fruit crops for which polyploidization has been reported include: Citrus spp (Wu and Mooney 2002); Musa spp. (Ganga and Chezhiyan 2002); Punica granatum (Shao et al. 2003); Pyrus pyrifolia (Kadota and Niimi 2002) and Zizyphus jujube (Gu et al. 2005). Other pl ant species in whic h polyploidization ha s been described include: Triticum aestivum (Redha A et al 1998, Hansen N and Anderson 1998, Zamani et al. 2000); Morus spp. (Chakraborti et al. 1998, Thomas et al. 2000); Lolium spp. (Paakinskien 2000); Scutelaria baicalensis (Gao et al. 2002) and Humulus lupulus (Koutoulis et al. 2005). In ornamentals, reports include: Agapanthus praecox (Nakano et al. 2003); Alstroemeria spp. (Lu and Bridgen 1997 and Ishikawa et al. 1999); Buddleia globosa (Rose et al. (2000a); Cyclamen persicum (Takamura and Miyajima 1996); Doritaenopsis sp. (Mishiba et al. 2001); Rhododendron hybrids (Vainola 2000); Rosa chinensis (Zlesak et al. 2005) and Syringa vulgaris x S. pinnatifolia (Rose et al. 2000b). In addition, reports include species and cultivars in the family Araceae: Spathiphyllum wallisii , (Eeckhaut et al. 2004); Alocasia micholitziana (Thao et al. 2003); Colocasia (Miyazaki et al. 1985); Xanthosoma sagittifolium (Tambong 1998) and Zantedeschia (Cohen and Yao 1996). Mutagens Used to Induce Polyploidy Colchicine Colchicine, an alkaloid derived fr om the bulb of the autumn crocus Colchicum autamnale , has been used successfully to induce polyploids in many different crops (Thao et al 2003). Chromosome doubling effects of colc hicine are attributed to its a ffinity for the spindle protein tubulin during both cell and nuclear division. These antimitotic e ffects inhibit sp indle function resulting in the prevention of nuclear division at the ana phase stage (Hancock 1997 and Tambong et al. 1998). During each mitotic cycle, the number of chromosomes per cell will be

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13 doubled as the chromosomes reproduce and divide to form separate daughter chromosomes (Ganga and Chezhiyan 2002). Reports of polyploidization of ornamental species using colchicine are numerous. Takamura and Miyajima (1996) treated in vitro Cyclamen persicum tubers on solid MS medium with 0, 20, 100, or 500 mgL-1 colchicine for 1, 2, 4, or 7 days. The study’s aim was to develop cultivars with increased flower size and d eeper color. Two tetraploids with 100 mgL-1 colchicine and 4 days treatment were reported after 8 weeks of culture on colchicine free medium. Stomata length and chromosome counts were used to assess ploidy. The authors acknowledged that the efficiency of polyploi dization in this study was low at < 1%. In vitro induction of tetraploidy with colchi cine has also been reported in Buddleia globosa (Rose et al. 2000a). The goal was to back cross B. globosa (2n = 38) with tetraploid B. davidii (2n = 76) to facilitate introgression of yellow flow er color. Nodal sections from in vitro grown plant material were treated with 0.01%, 0.05% or 0.10% w/v colc hicine for 1, 2, or 3 days. Treatments took place in liquid medium and on shaker set at 230 rpm. Ploidy was determined by chromosome counts. Six of the nineteen te traploids (32%) produced over the various treatments and durations were recovered from treatment with 0.10 % colc hicine and 3 days exposure. In another experiment, colchicine induced polyploids in three different interspecific selections of the lilac Syringa vulgaris x S. pinnatifolia (Rose et al. 2000b). Explants of sub-apical nodal sections were exposed to a range of 0 to 2.5 mM colchicine fo r 1, 2 or 3 days in liquid and on shaker (ca. 230 rpm). Twenty-two weeks after treatment, 11 te traploids and 17 mixoploids were recovered as confirmed by flow cytometry and by chromosome counts. The highest e fficiency of conversion to tetraploidy was 20% with 0.05 mM of colchicine and 2 day exposure. Furthermore, colchicine was used to induce polyploidization of ovules in the interspecific hybrid Alstroemeria ligtu L. x

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14 A. pelegrina L. var rosea (Ishikawa et al. 1999). Treat ments in this experiment consisted of a 0.05% liquid colchicine for 1, 2, 4, or 7 day duration. Recovery of tetraploids was best at 4 days exposure to colchicine. Two plan ts were recovered out of 246 ovul es cultured; these two plants were later determined to be tetraploid by chromosome counts. Colchicine has been effective in generating polyploids in several agronomic crops. In Brassica napus , colchicine was applied to haploid micros pore cultures in order to recover fertile doubled haploid homozygous plants (Weber et al. 2005). Microspores were exposed to 50 mgL-1 colchicine for 24 hours following th eir culture and isol ation. Frequency of recovered doubled haploids was very high (as high as 95.0% in one genotype). Spontaneous diploidization in the control (no colchicine) occurred at a recovery ra te of 18.8%. No data was given on the fertility of the recovered plants. In another agronomic crop, Allium fistulosum x A. cepa , colchicine was used at concentrations of 0.05%, 0.1% and 0.2% for 36, 48, and 72 hours (Song et al. 1997). The number of surviving calluses was more dependent on concentration used rather than exposure time. As the concentration of colchicine incr eased, the number of surviv ing calluses decreased. The highest number of tetraploid plantlets recovered (approximat ely 50%) had been treated with 0.1% or 0.2% colchicine for 48 or 72 hours. Finally, in ginger ( Zingiber officinal Roscoe) shoot tip cultures were exposed to 0.2% colchicine fo r 4, 8, 12, or 14 days in an attempt to restore fertility (Adaniya and Shirai 2001). In this experiment, optimum exposure time to generate tetraploids was 8 days with a re covery of 9% on a solid MS culture medium and 3% on a liquid MS culture medium. There are several reports of successful polyploidization of fruit species using colchicine. In jujube ( Zizyphus jujuba Mill.), colchicine at concentrations of 0.01, 0.03, 0.05, 0.1, and 0.3% in liquid MS was used to induce tetraploid plan ts (Gu et al. 2005). Exposure times of 24, 48, 72

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15 or 96 hours were also evaluate d. Recovery of tetraploids was greater than 3% at 0.05% colchicine for 48 or 72 hours exposur e. In another fruit species ( Pyrus pyrifolia N. cv. Hosui), tetraploids were not initially recovered using co lchicine concentrations of 0.01% and 0.1% for 1, 2, 4, or 8 days exposure (Kadota and Niimi 2002). However, 4 mixoploid species (2n = 2x + 4x) were recovered as determined by flow cytome try. Subsequently, these mixoploid individuals were further proliferated in vitro and 5 tetraploid shoots we re produced from them. In Citrus reticulate Blanco x C. sinensis [L.] Osb., embryogenic callus lines were treated with colchicine at concentrations of 0.05% and 0.1% (W u and Mooney 2002). Three non-chimeric autotetraploids were recovered fr om 0.05% colchicine and one from 0.1% colchicine. Finally, in Punica granatum , tetraploids were induced from s hoot cultures on solid MS medium supplemented with colchicine at 10 mgL-1 for 30 days or 5000 mgL-1 colchicine for 96 or 114 hours (Shao et al. 2003). Tetrap loids were recovered at a ra te of 20 % on medium with 10 mgL-1 colchicine as confirmed by flow cytometry. Recovery of tetraplo ids on medium with 5000 mgL-1 colchicine produced 3 mixoploids th at later separated into diploi ds or mixoploids after further subculture without colchicine. Reports on colchicine’s efficiency to indu ce polyploidization in the genus Araceae are varied in the literature. For example, in Xanthosoma sagittifolium (Tambong 1998), polyploids were successfully obtained when in vitro grown plants were treated with 1.25 mM or 2.5 mM of colchicine. Thirty shoots per treatment were exposed to various concentrations of liquid colchicine added to Gamborg B5 medium (Gambor g et al. 1968) for 2 days. An increase in stomata length correlated to an increase in ploidy. Ploidy level was confirmed by flow cytometry. The researchers found that 1.25 mM a nd 2.5 mM of colchicine produced tetraploids at a frequency of 83.3% and 80.0%, respect ively, and mixoploids at 16.0% and 20.0%,

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16 respectively. Colchicine has also been reported to induce tetraploidy in nine Zantedeschia cultivars (Cohen and Yao 1996). Rapidly multiplying in vitro shoot cultures were exposed to 0.05% (w/v) colchicine on solid MS media for 1, 2, or 4 days and thereafter subcultured on colchicine free MS media. Among the cultivars exposed to colchici ne, a recovery of tetraploids ranged from 12.9% to 41.8%. Stomata length wa s used to screen put ative tetraploids and chromosome counts verified their presence. An in crease in ploidy level co rrelated to an increase in stomata length. Other reports on the polyploidization of Araceae compare colchicine efficacy to other mutagens such as oryzalin and trifluralin. In Alocasia micholitziana ‘Green Velvet’, the ability of colchicine to induce tetraploids was compared to oryzalin (Thao et al . 2003). Shoot tips from in vitro grown plants were exposed to liquid colc hicine at concentrations of 0, 0.01, 0.05 or 0.1% or liquid oryzalin at concentrations of 0, 0.005, 0.01, or 0.05%. Each of the 4 concentrations of mutagens was tested at exposure times of 24, 48, or 72 hours. Initial ploidy determination was by stomata length and then later confirmed by flow cytometry. After 3 months of culture on MS media without colchicine and oryz alin, the researchers reported th at oryzalin at 0.01% and 24 or 48 hour exposure was more effective than colchici ne. Over the various treatments of oryzalin, 6.8% of surviving shoots were te traploid as compared to 4.5% in treatments with colchicine. Moreover, colchicine could be efficiently replaced by oryzalin or trifluralin at 10 M when applied to spadix derived secondary somatic embryos of Spathiphyllum wallisii Regal (Eeckhaut et al. 2004). Colchicine at 100 M was used as a ‘shock’ treatm ent and applied in liquid form for 1 to 2 days. Oryzalin and trifluralin at 10 M each were added to solid culture medium and remained throughout the duration of the experiment . Flow cytometry was used exclusively to

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17 determine ploidy. Oryzalin a nd trifluralin treatments yielde d approximately 5% polyploids, whereras colchicine yielded 2% polyploids. Other Mutagens Used to Induce Polyploidization Chemicals such as oryzalin, triflurali n, amiprophos-methyl, 2, 4-dichlorophenoxyacetic acid and nitrous oxide have been used to i nduce polyploidization . Three anti-microtubule herbicides, oryzalin, trifluralin and amiprophos-met hyl, have a mode of action similar to that of colchicine. One advantage of these compounds over colchicine is that thei r affinity to animal tubulin is quite low (Hansen et al. 1998). T hus, their use in the laboratory is safer than colchicine (Zhao and Simmonds 19 95). Oryzalin was effective in producing tetraploids at a concentration of 0.005% for 24 hours in shoot cultures of Rhododendron (Vainola 2000) where 18.2% of surviving plants were confirmed tetraplo id by flow cytometry. In another experiment, Ganga and Chezhiyan (2002) exposed four in vitro grown diploid banana cultivars to oryzalin at 10, 20, 30, or 40 M for 3 days and 10 or 20 M for 6 days. For one cultivar, recovery of tetraploids was the highest at 30 M of oryzalin for 3 days as confirmed by stomata analysis and chromosome counts. In Rosa chinensis minima (Sims) seedlings, tr ifluralin at 0.086% was effective at producing polyploids as compared to colchicine at 0.5% (Zlesak et al. 2005). Trifluralin was placed directly be tween the expanding cotyledons of ex vitro seedlings. After treatment with 0.086% trifluralin, 57% of surviving seedlings were determined by stomata length and pollen diameter. No polyploids were recovered from colchicine treatment. Trifluralin was also used successfully to convert in vitro grown Brassica napus haploid plantlets to doubled haploids (Zhao and Simmonds 1995) . Cultures treated with 1 or 10 M trifluralin for 18 hours recovered tetraploids at fre quencies approaching 60%. Fina lly, amiprophos-methyl (APM) was efficient at doubling chromosomes in Allium cepa L. embryos (Grzebeluse and Adamus 2004). APM at 50 M was found to be most efficient ov er oryzalin or triflu ralin because APM caused

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18 the least amount of vitrificati on in the developing embryos. Hansen et al. 1998 used APM to induce polyploidization in Beta vulgaris at an efficiency of conversion of 89%, and like Grzebelus and Adamus 2004, was found to be more advantageous over oryzalin and trifluralin. Induction of polyploidy by chemical s other than the micro-tubule inhibitors has also been tested. Exposure of various plant parts ( in vitro and ex vitro ) to nitrous oxide (N2O) has proven to be effective in converting diploid cells to tetr aploid. Several examples exist in the literature including Zea mays (Kato 2002), Trifolium pretense (Matsuura et al. 1974), and Sorghum bicolor (Tsvetova and Larin 1996). Th e compound 2, 4-dichlorophenoxyaceti c acid (2, 4-D) has been shown to induce pol yploidization in Doritaenopsis sp. (Mishiba et al . 2001). And, somatic hybridization has been show n to be effective in Agapanthus sp. (Nakano et al. 2003) and Solanum tuberosom (Mattheij et al. 1992). Determination of Polyploids Morpholgy Morpholoical traits, stomata length, and cytolo gical analyses can di stinguish polyploids from diploids. Polyploids usually have thicke r leaves and stems, a deeper green color, an increased width-to-length ratio of leaves and a more compact growth habit (Eeckhaut et al. 2002). In tetraploids of Alstroemeria , plant height, leaf area, and leaf specific weight were greater than that of the diploid coun terpart (Ishikawa et al. 1999). In Zizyphus jujuba , tetraploids had significantly rounder leaves a nd shorter, thicker s hoots than diploid plan ts (Gu et al. 2005). Polyploidization can also a ffect flower morphology. In Punica granatum , flower bud diameter was significantly greate r than diploids by 25% (Shao et al. 2003). However, flower length was significantly less than diploids by 10%. Polyploids may possess deeper color in flowers. In Cyclamen , depth of flower color of tetraploid s was greater than that of diploids (Takamura and Miyajima 1996). Similarly, roots of polyploids can often be darker, thicker, and

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19 shorter than diploids, such as in pomegranate (Shao et al. 2003) and mulberry (Chakraborti 1998). Chimeras can often result from attempts to induce polyploidization. Frequency of mixoploidy during the process of polyploidization can depend on th e species tested and also on the conditions in vitro . For example, Alocasia mixoploids of 2n = 2x + 4x nuclei had deformed or asymmetric leaves (Thao et al.2003). In Rhododendron , recovery of mixoploids was 2 to 3 times higher than tetraploids (Vainola 2000). However Chakraborti (1998), observed very few mixoploids in Morus and attributed the findings to cont rolled environment, temperature, and photoperiod promoting the synchronous division of meristematic cells in vitro . Stomata Length Stomata length can be an effective way to se lect between polyploids and diploids. The methods used to measure stomata length (e.g. epidermal peels) are simple, almost nondestructive, and do not require expensive equipment (Kadota and Niimi 2002). Research has reported that induced polyploids have stomates th at are significantly larger than the diploid counterparts. In Alocasia , stomata length was 49% greater in te traploids than in diploids (Thao et al. 2003). In Acacia mearnii , stomata length was 48% greater in tetraploids than in diploids (Beck et al. 2003); and in Alstromeria x caryophyllaea , stomata length wa s 43% greater in tetraploids than in diploids (Lu and Bridgen 1997) . Frequency of stomates is generally lower in tetraploids than in diploids as reported for Solanum villosum (Ojiewo et al. 2006 ), Chamomilla recutita (Seidler-Lozykoska 2003), Aegilops neglecta (Aryavand et al. 2003), and Bixa orellana (Carvalho et al. 2003). Although stomata size, stomata pore size, or guard cell length can provide a good general guide to ploidy level, as seen in Solanum villosum , they do not provide a comprehensive determination of ploidy (Ojiewo et al. 2006).

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20 Flow Cytometry The most accurate and reliable tool for ploidy de termination is cytological analysis by flow cytometry (Heller 1973). Flow cytometry utilizes the analysis of fluorescence and lightscattering properties of single pa rticles during their passage thr ough a narrow, precisely defined liquid stream (Eeckhaut 2004). Tens of thousands of nuclei can usually be analyzed within a few seconds. A typical flow cytometer will generate an on screen histogram in which fluorescent intensity is illustrated by peaks. These peaks correspond to the density of the nucleus, which is the primary indicator of the level of ploidy. Flow cytometry has been used extensively in plant genetic research and breeding (Eeckhaut 2004). Some ornamental cr ops in which flow cytometry has been used to confirm ploidy level include: Alocasia (Thao et al. 2003), Xanthosoma (Tambong 1998), Spathiphyllum (Eeckhaut et al. 2004), Rhododendron (Vainola 2000), Agapanthus (Nakano et al. 2003), and Syringa (Rose et al. 2000a).

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21 CHAPTER 2 INDUCTION OF TETRAPLOIDY IN Dieffenbachia x ‘Star Bright M-1’ Introduction In vitro induction of tetraploidy (polyploidizat ion) was first achieved in tobacco by Murashige and Nakano (1966). Thereafter, inducing polyploidy by way of chemical mutagenesis with substances such as colchicine has offered plant breeders a valuable tool for scientific investigations as well as overcoming sexual steril ity, particularly among interspecific hybrids. By definition, polyploids contain two or more genomes, where a genome is one basic set of chromosomes. A polyploid can contain two or more identical genomes as in an autopolyploid, or different genomes as in an allopolyploid (Leitch and Bennett 1997). From a crop improvement standpoint, polyploid plants are mo re robust, have thicker leaves, larger fruit, and a greater degree of drought and disease toleran ce (Eeckhaut et al. 2004). Triploids resulting from crosses between diploid and te traploid plants have been reporte d to be superior to diploids with respect to leaf nutrition, genetic adapta bility and resistance to environmental stress (Chakraborti et al. 1998). A ttempts at inducing polyploidiza tion may result in mixoploidy (i.e, plant tissue having cells of different ploidy levels such as 2n = 2x + 4x). The plant family Araceae (collectively referred to as ‘aroids’) includes many economically important plant genera, species, and cultivars. The most important ornamental aroid genera include Aglaonema , Anthurium , Dieffenbachia , Epipremnum , Spathiphyllum , and Syngonium . Historically, new ornamental ar oid cultivars originated by the introduction of plants collected from the wild or by mutations of estab lished cultivars (Henny 2000). The genus Dieffenbachia is valued for its colorful foliage, ease of production and durability in interiorscape environments. Since 1980, many commercial Dieffenbachia cultivars are the result of breeding programs that select for both aesthetics and disease tole rance (Henny 2000). To facilitate commercial

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22 production, tissue culture methods ha ve been employed as a tool fo r fast and reliable propagation of Dieffenbachia . Dieffenbachia x ‘Star Bright M-1’ is a somaclonal variant of the commercial cultivar Dieffenbachia x ‘Star Bright'. The M-1 variant was se lected out of a population of tissue culture derived plants because its lower leaves pers ist longer under interior conditions, plus leaf variegation is more striking and l eaf coloration is not lost as the leaves mature. In addition, the internodal length is shorter giving the plant a mo re compact appearance. These traits make the plant desirable as a parent for breeding new lines of commercial cultivars of Dieffenbachia . However, like its parent ‘Star Bright’, ‘Star Brig ht M-1’ is sterile. This report describes an attempt to create a tetraploid fo rm of ‘Star Bright M-1’ that could later be used in breeding. The goal of this study was to induce polypl oidization by using co lchicine treatment in vitro and determine the optimum colchici ne concentration for efficient induction of tetraploidy. In addition, a putative identificati on of polyploids was based on an assessment of morphological indicators including stomata size, and flow cytometry was used as a tool to validate the occurrence of polyploids. Finally , leaf morphology of confirmed di ploid and tetraploid plants was compared. Materials and Methods In Vitro Propagation Plant material to establish tissue cultures of Dieffenbachia x ‘Star Bright M-1’ was selected out of a population being researched at the Univ ersity of Florida, Mid-Florida Research and Education Center in Apopka, Florida. Explants measuring 10 – 15 mm in length were dissected out of shoot tip apices. Ten explants we re used to initi ate cultures. The explants were placed in a mason jar with a screen fitted li d and flushed under tap water for 10 minutes to remove surface contamination. By gradually rem oving the outer most leaf primordials, shoot

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23 tips were trimmed down to about 5 – 7 mm in lengt h. Explants were placed in a 500 ml media bottle containing a 200 ml solution of 10% Clor ox bleach (1% sodium hypochlorite) and 1 to 2 drops of Joy detergent added as a surfactant. The media bottle with explants and Clorox solution was agitated on a rotary shaker table for 15 minutes at 100 rpm. To rinse the explants of the Clorox solution, the explants were first pour ed over a sieve to remove them from the sterilizing solution and then placed in another 500 ml media bottle cont aining 200 ml of sterile distilled water and agitated for 5 minutes on the shaker table at 100 rpm. Rinsing was repeated three times. Utilizing a binocul ar dissection scope placed inside a laminar flow hood, the explants were aseptically trimmed by carefully removing layers of leaf primordials. The final explants measured 2 to 3 mm in length and consis ted of 2 to 3 leaf primordials, meristematic tissue, and a small section of basal vascular tissue measuring approxima tely 1 mm in length. After dissection, the explants were placed asep tically on a culture medium consisting of the following: Murishige and Skoog salts (Mur ashige and Skoog 1962) supplemented with Gamborg B5 vitamins (Gamborg et al. 1968), 30 gL-1 sucrose, 10 mgL-1 6-( , Dimethylallylamino) Purine (2iP), 0.10 mgL-1 Indole-3-acetic acid (IAA), and 8 gL-1 of tissue culture grade agar. Before use, the pH was ad justed to 5.7 using 1N KOH. The medium was then dispensed into culture tubes measuring 25 x 150 mm, and autoclaved for 30 minutes at 15 PSI and 121 C. Each culture tube containe d approximately 20 ml of prepared medium. The explants were placed in a growth r oom equipped with cool white florescent lights (PAR 40 mol m-2 s-1), under a 16/8 hour photoperiod, and a c onstant temperature of 26 2C. The explants were subcultured every 6 weeks. At the fifth subculture, th e proliferating explant tissue was transferred to 177 ml baby food jar style culture vessel s fitted with Magenta B-Cap closures. The material was subc ultured every 6 weeks until a tota l of 40 jars was achieved (8

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24 subcultures or approximately 48 weeks from initia tion). Each jar contained 3 clumps of rapidly dividing shoot cultures with 6 to 8 potentia l shoots per clump along with callus tissue. Colchicine Application Colchicine was chosen over ot her possible mutagens since it is widely reported in the literature and its effects are well documented (Van Harten 1998). The 40 jars of shoot clumps were randomly divided into 4 groups of 10 jars e ach to receive specified levels of colchicine treatment. Prior to treatment, all shoots over 2 cm in length were excised off the callus and discarded. Small, undifferentiated shoot primordi al was preferred for colchicine treatment over large and differentiated shoots (Figure 2-1). Final colchicine treatments rates of 0, 250, 500, and 1000 mgL-1 were made from dilutions of an initial 10,000 mgL-1 colchicine stock solution that wa s prepared by disso lving 3 grams of powered colchicine into 300 ml of deionized wate r. Next, six liters of liquid MS medium were prepared. The medium contained full stre ngth MS salts, no hormones, and 30 gL-1 sucrose. To make a 250, 500, and 1000 mgL-1 colchicine/MS preparatio n, 37.5 ml, 75 ml and 150 ml, respectively, of the colchicine stock solution were combined with the MS medium to final volumes of 1.5 liters. One 1.5 lite r MS preparation was reserved without colchicine for control (0 mgL-1 colchicine). Forty empty baby food jars were filled with 50 ml of the various treatment preparations. The pr eparations were autoclaved fo r 30 minutes at 15 psi and 121 C and allowed to cool before use. Shoot clumps were aseptically tran sferred into the jars containing liquid colchicine/MS media treatments. The jars were then placed for 24 hours on a shaker table set at 80 rpm. Afterwards, shoot clumps were removed aseptically from the colchicine/MS treatments, rinsed in autoclaved deionized water and then placed back onto a colchicine free medium containing MS salts, 2 mgL-1 6-( , -Dimethylallylamino)Purine (2iP), 0.02 mgL-1 Indole-3-acetic acid (IAA), 30 gL-1 sucrose, Gamborg B5 vitamins, and 8 gL-1

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25 tissue culture grade agar. For th e next 26 weeks, the cultures de veloped differentiated shoots and at approximately 6 week in tervals were transferred to fresh medi a as stated above. At the time of the second and third transfers, all developed s hoots exceeding 2 cm in length were harvested, counted, removed from culture ve ssels and transferred to the gr eenhouse. At 26 weeks following colchicine treatment, all developed shoots were harvested, counted, and planted. Since the number of control plants exceeded a manag eable amount, 90 control plants were randomly selected for transfer to the greenhouse (3 plants from each of 10 controls for each of the 3 harvest dates). Greenhouse Conditions The plants were planted into 288 cell pack trays containing Vergro container Mix A which consisted of 50% Canadian peat, 25% perlite, and 25% vermiculite (Ver lite Co. Inc., Tampa, FL). The plants were acclimated for 2 weeks under a light intensity of 800 to 1000 foot candles (160 to 200 mol m-2 s-1). Plants were hand watered as needed. Once acclimated, the plants were moved to 1200 to 1500 foot candles of light (240 to 300 mol m-2 s-1). Thereafter, the plants were fertilized weekly with Peter’s 20:20:20 (T he Scotts Co., Marysville, Ohio) at 250 ppm nitrogen and hand watered as needed. After 17 weeks in the greenhouse, plants were repotted into 50 cell pack trays and allowed to grow. After 26 weeks in the greenhouse, they were stepped up to 5 inch round pots and allowed to gr ow for 26 more weeks. After 52 weeks in the greenhouse, the plants had developed 8 to 10 ma ture leaves and were determined ready for morphological analysis. Data Collected Survival In-vitro survival was determined 12 weeks after colchicine treat ment by counting the number of surviving shoot clumps per treatment. Cultures were maintained another 14 weeks.

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26 Ex-vitro survival was determined 26 weeks af ter transfer to the gr eenhouse by calculating the mean number of surviving plants per treatment and per replica tion. A total of 422 plants (332 colchicine-treated and 90 controls) survived ex vitro and were grown on for later analysis. Morphological characteristics In this study morphological markers were us ed to screen all 422 surviving plants (90 controls and 332 colchicine treated ) at approximately 12 months af ter transfer to the greenhouse. By this stage, plants had developed 8 to10 mature leaves and the following morphological characteristics were measured: crown height (cm), height with leaves held up (cm), canopy height (cm), canopy width (cm), growth index (m3), largest leaf length (cm), and largest leaf width (cm). Crown height was determined as the length of the primary stem from the base of the plant to the point at where the newly emerging leaves form an apex (crown) at the top of the plant. Height with leaves held up was determined by measuring from the base of the plant to the outer most tips of leaves as th e leaves are held up above the crow n of the plant. Canopy height was determined by an approximate measurement from the base of the plant to a point where the upper most leaves form an arch and begin to curve downward. Canopy wi dth was calculated as an average of two horizontal lengt hs (approximately 90 degrees from one another) from side to side across the canopy of the plants. Largest l eaf length was determined by measuring at the point of attachment of the petiole to the primary stem and outward to the tip of the leaf blade. Largest leaf width was determined by measuring from edge to edge the widest portion of the leaf blade. Growth index (m3) was calculated by the multiplying the product of 2 canopy widths by canopy height. Two separate morphological asse ssments were made: The first included all 422 surviving plants and the second included 63 colc hicine-treated plants that exhibited visual indicators of increased ploidy. Ten additional co ntrol plants were also included. All plants included in the second assessment were additiona lly evaluated by measuring stomata length and

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27 then were subjected to flow cytometric analys is. For both morphological assessments, statistical comparisons were made among the four colchicine treatment levels. Stomata length The stomata of 63 colchicine-treated plus 10 c ontrol plants were ‘fixed’ onto a glass slide by using an epidermal peel. One drop of Supe r Glue was placed on a 75 x 25 mm slide. The slide was pressed against the underside of a leaf , held for 30 seconds, and then removed. The result was a permanent impression of the epidermi s from the underside of the leaf. Stomata were observed under a light microscope at 400x ma gnification. Stomata were measured in micrometers (m) by placing the scale end to e nd along the lengths of th e guard cells in the longitudinal direction. Ten stomata per leaf were measured. Flow cytometry The same 73 plants that were used for stom ata measurements above were then examined for ploidy by flow cytometry. A Partec PA flow cytometer (Partec GmbH, Mnster, Germany) was used. Leaf squares measuring 5 x 5 mm we re cut from edges of newly matured leaves, placed in a 55 mm plastic Petri dish along w ith 0.4 ml nuclei extraction buffer (Cystain UV Precise), and chopped using a razor blade into approximately 100 pieces. The contents of the Petri dish were then filtered (pore size equals 50 m) into a 3.5 ml test tu be to which 1.6 ml of staining buffer (Cystain UV Precise ) was added. The flow cytometer’s gain value was set at 363.0 and speed was set at 0.40 ls-1. A minimum of 2000 nuclei was analyzed per sample. The standard peak of a known diploid Dieffenbachia was calibrated to appear at approximately 100 of fluorescence intensity (channel number). Statistical analysis Survival statistics were based on 4 colchi cine treatment levels and 10 baby food jar replications per treatment. Fo r morphological analysis, stomata analysis, and flow cytometry,

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28 individual plants harvested from the 4 treatment levels were evaluated as a single replication. The statistical program MINITAB Release 14 (2005) was used for mean separation by Fisher’s Protected least significant difference and for P earson correlation. Microsoft Excel (2003) was used to generate regression equations and plots. Results and Discussion In Vitro Survival Colchicine effects on clumps of shoot cultur es could be observed 12 weeks after treatment (Figures 2-2 and 2-3). Clumps not exposed to colchicine grew normally and rapidly and little necrosis was observed. Some clumps exposed to 250 mgL-1 colchicine showed growth retardation and a small amount of necrosis, while others grew normally similar to the control. Many clumps exposed to 500 and 1000 mgL-1 became necrotic, eventually died and were discarded. In vitro survival was significantly di fferent between control (0 mgL-1 colchicine) and treatment rates (250, 500, and 1000 mgL-1 colchicine), P < 0.05 (Table 2-1). Mean percent survival of clumps without colchicine was 93.3%, whereas mean percen t survival at 250, 500, and 1000 mgL-1 colchicine was 63.3%, 30.0%, a nd 23.3%, respectively. At 250 mgL-1 colchicine, in vitro survival was significantly greater than at 500 and 1000 mgL-1. In vitro survival was not significantly different between 500 and 1000 mgL-1, P > 0.05. In general, increasing the colchicine concentration reduced in vitro survival. Regression analysis showed a quadratic relationship between in vitro percent survival and co lchicine concentration, R2 = 0.99 and P < 0.05 (Figure 2-4). Colchicine proved to be lethal at > 500 mgL-1 due to its penetration into the apical cell layers and affect on cell division (Tha o et al. 2003). In future experimentation, improved success of colchici ne induced polyploidization and survival in vitro could be facilitated by more fre quent transfer of explants to fresh media, as observed in Buddleia

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29 globosa (Rose et al. 2000b), or shorter exposur e times and smaller dosages as seen in Spathiphyllum (Eeckhaut et al. 2004). Ex Vitro Survival Figure 2-5 shows typical shoots from all treatments that we re harvested from culture and ready for planting in the greenhouse 26 weeks after treatment with colchicine. All 90 controls (0 mgL-1 colchicine) survived ex vitro (Table 2-2). Survival of c ontrols was significantly higher than all other treatments P < 0.05. Total plan t survival among treatments at 250, 500, and 1000 mgL-1 colchicine was 204, 59, and 69, respectively 26 weeks after transfer to the greenhouse. Percent survival at 250, 500, and 1000 mgL-1 colchicine was 66.4%, 80.2%, and 80.4%, respectively. No significant difference in mean ex vitro percent survival was observed among colchicine treatments P > 0.05. Over all, survival appeared to be determined within the first few days after transfer to the gr eenhouse. Many of the shoots tr eated with colchicine were translucent and brittle. Shoots showing these sy mptoms did not survive. Non-treated control shoots were normal in appearance and acclimated quickly. Therefore, mean ex vitro percent survival was affected primarily by the presence of colchicine and not by concentration effects. This is confirmed by regression analysis whic h showed a weak relatio nship between percent survival of ex vitro shoots and concentration of colchicine, R2 = 0.55 and P > 0.05 (Figure 2-6). Although the significance of ex vitro survival affected by colchici ne concentration was low, any decrease in survival of colchicine treated shoot s over non-treated control is likely the result of a carry over effect of colchicine ex vitro as seen in Pyrus pyrifolia (Kadota and Niimi 2002). Morphology Morphological assessment of 422 surviving explants. A total of 422 surviving explants (332 from colchicine treatments and 90 controls) were examined morphologically after growing for one year in the greenhouse. In all morphological

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30 observations (Table 2-3), control plants (0 mgL-1) had significantly greater values than plants treated with 250, 500, or 1000 mgL-1 colchicine (P < 0.05). Crown height, height with leaves held up, and canopy height of plants exposed to 250 mgL-1 colchicine were significantly different from plants exposed to either 500 or 1000 mgL-1 colchicine. Crown height of control plants was the tallest (13.8 cm), whereas cr own height of plants exposed to 1000 mgL-1 colchicine was shortest (8.6 cm). Similarly, heig ht with leaves held up was greatest in control (36.2 cm) and the least in plants exposed to 1000 mgL-1 colchicine (23.9 cm). Control plants also yielded the greatest canopy he ight (30.6 cm), whereas 1000 mgL-1 colchicine produced the least canopy height (20.1 cm). No significant difference in th ese three observations was found between plants exposed to 500 or 1000 mgL-1 colchicine. Regression analysis showed crown height, [R2 = 0.99 (Figure 2-7A)], height with leaves held up [(R2 = 0.98 (Figure 2-8A)], and canopy height [R2 = 0.98 (Figure 2-9A)] decreased with an increase in colchicine concentration in a quadratic relationship. Canopy width, growth index, largest leaf length, a nd largest leaf width of plants exposed to 250 mgL-1 colchicine were significantly diffe rent from plants exposed to 1000 mgL-1 colchicine, but not significantly diffe rent from plants exposed to 500 mgL-1 colchicine. Canopy width was greatest in controls ( 30.0 cm) and the least in 1000 mgL-1 colchicine (21.0 cm). Similarly, controls produced the greatest valu es for observations of growth index (0.0278 m3), largest leaf length (22.4 cm), a nd largest leaf width (6.0 cm). Plants exposed to 1000 mgL-1 colchicine yielded the smallest values in growth index (0.0101 m3), largest leaf length (15.3 cm), and largest leaf width (4.6 cm). For these f our observations, no signi ficant differences were observed among plants exposed to 500 or 1000 mgL-1 colchicine. Regression analysis showed canopy width [R2 = 0.97 (Figure 2-10A)], growth index [R2 = 0.96 (Figure 2-11A)], largest leaf

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31 length [R2 =0.97 (Figure 2-12A)], and largest leaf width [R2 = 0.99 (Figure 2-13A)] decreased with an increase in colchicine concen tration in a quadrat ic relationship. Morphological assessment of 63 potential polyploids. The morphological examination of the 422 surviving plants led to the selection of 63 plants that displayed morphological tra its indicating strong lik elihood of being polyploid. Compared to control plants, these 63 treated plants selected we re shorter, had thicker and darker green leaves, and thicker stems. In addition, the leaves had a leathery feel and the fo liar variegation across the entire leaf blade was less defined and appeared diffuse rather than in a defined pattern. The morphological data for these 63 plants, plus 10 ra ndomly selected control plants was collected from the original data tables to allow a second statistical analysis. Assessment of these selected plants substantia ted the effects of colchicine concentration on morphology (Table 2-4). In all observations, control (0 mgL-1 colchicine) had significantly greater values than plants treated with 250, 500, or 1000 mgL-1 colchicine (P < 0.05). Crown height, height with leaves held up, canopy height, gr owth index, and largest le af length of plants exposed to 250 mgL-1 colchicine were significantly differe nt from plants exposed to 1000 mgL-1 colchicine, but not significan tly different from 500 mgL-1 colchicine. In these five observations, plants treated with 500 mgL-1 colchicine were not significantly di fferent from plants treated with 1000 mgL-1 colchicine. Crown height of control pl ants was tallest (13.4 cm), whereas crown height of plants exposed to 1000 mgL-1 colchicine was shortest (6.0 cm). Also, plants exposed to no colchicine had the highest values in observations of height with leaves held up (35.4 cm), canopy height (29.9 cm), growth index (0.0255 m3), and largest leaf length (22.1 cm). Likewise, plants exposed to 1000 mgL-1 colchicine had the lowest valu es in these four observations (17.5 cm, 14.7 cm, 0.0048 m3, and 11.6 cm, respectively). Regr ession analysis showed crown height [R2 = 0.96 (Figure 2-7B)], height with leaves held up [R2 = 0.95 (Figure 2-8B)], canopy

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32 height [R2 = 0.95 (Figure 2-9B)], growth index [R2 = 0.91 Figure 2-11B)] and largest leaf length [R2 = 0.94 (Figure 2-12B)] decreased with an increas e in colchicine concen tration in a quadratic relationship. Canopy width and larg est leaf width of control plants were greatest at 29.2 cm and 5.7 cm, respectively. Plants exposed to 1000 mgL-1 colchicine had the least values in canopy width and largest leaf width (17.5 cm and 3.8 cm , respectively). For these two morphological observations, 250, 500, or 1000 mgL-1 colchicine had no significant affect. Regression analysis showed canopy width [R2 = 0.95 (Figure 2-10B)] and largest leaf width [R2 = 0.89 (Figure 2-13B)] decreased with an increase in colchicine concentr ation in a quadratic rela tionship. Assessment of morphological traits on plants selected based on observations of poten tial polyploidy confirms that an increase in the con centration of colchicine has an effect on the morphology of Dieffenbachia x ‘Star Bright M-1’ and further indi cated a likelihood that polyploids were present. Stomata Analysis of 63 Potential Polyploids Comparing stomata length among colchicine tr eatment levels did not produce significant differences between the treatment means (Table 2-5). This sugge sts that increasing colchicine concentration does not have a pr oportional affect on stomate size (Pearson correlation coefficient = 0.134, P > 0.05). However, at least 21% of plants exposed to 250, 500, or 1000 mgL-1 colchicine had stomata lengths greater than 42.5 m . Since no observations of plants exposed to 0 mgL-1 colchicine had stomata lengths greater th an 42.5 m, early screening of stomata of Dieffenbachia x ‘Star Bright M-1’ coul d indicate polyploidy if stomat a are greater than 42.5 m. Flow Cytometry Analysis of 63 Potential Polyploids Flow cytometry results determined the ploidy of the 63 colchicine tr eated plants. Twentyone treated and 10 controls were confirmed diploi ds, 13 treated were tetraploids and 29 treated were mixoploids. The number and percentage per treatment of diploi ds, tetraploids, and

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33 mixoploids confirmed among 10 controls and 63 pl ants selected based on morphological markers and stomata analysis are represented in Table 2-6. Tetraploid plants were isolated from all colchicine treatment levels excep t controls, and these tetraploids had similarities in plant height, plant width, and leaf shape (Figur es 2-14, 2-15, & 2-16). Flow cy tometry proved to be essential since selection based on morphology alone was not a definitive eval uation of ploidy. Histograms with excitation peaks of flores cent intensity at cha nnel 100 corresponded to diploids (Figure 2-17) and cha nnel 200 corresponded to tetraplo ids (Figure 2-18). Histograms with excitation peaks at florescent intensity of both channels 100 and 200 corresponded to mixoploids (Figure 2-19). All ten control plants were verified diploid. At treatment level 250 mgL-1 colchicine, 6 of 32 plants were confirmed tetraploid (18.8%) and 13 of 32 plants were confirmed mixoploid (40.6%). At treatment level 500 mgL-1 colchicine, 6 of 15 plants were confirmed tetraploid (40.0%) and 4 of 15 pl ants were confirmed mixoploid (26.7%). At treatment level 1000 mgL-1 colchicine, 1 of 16 plants was co nfirmed tetraploid (6.3%) and 12 of 16 plants were confirmed mixoploid (75.0%). All ot hers were confirmed di ploid. Colchicine at 500 mgL-1 produced more tetraploids by percent ( 40.0%) than all other treatment levels. Colchicine at 1000 mgL-1 produced more mixoploids by percen t (75.0%). These data suggest that colchicine concentrations of 500 mgL-1 is the optimum concentration under the conditions of this experiment for c onversion to tetraploidy in Dieffenbachia x ‘Star Bright M-1’. Similarly, high levels of colchicine te nded to produce mixoploids in Alocasia (Thao et al. 2003), Syringia vulgaris (Rose et al. 2000), Morus alba (Chakraborti et al. 1998), and Zizyphus jujube (Gu et al. 2005). In addition, these authors showed that lo w concentrations of colchicine decreased the efficiency to convert diploi d plants to tetraploids.

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34 Morphological screening and st omata analysis prior to flow cytometry increased the efficiency of polyploid iden tification in all colchicine treatment levels and therefore is useful in eliminating the need to perform flow cytometr y on a large number of plants. The use of morphological markers can be important for rese archers who do not have easy access to flow cytometry. Due to the expense of running larg e numbers of samples on a flow cytometer, reducing sample size based on morphology can signifi cantly lessen the cost of screening. Table 2-7 represents the number and pe rcentage of confirmed tetraplo ids calculated based on total plants available for study in each treatment level and also ca lculated based on number of morphologically selected plants in each treatment level, as show n in column 6 of Table 2-6. Comparison of percent tetraploid of total number of plants to percent tetraploid of selected plants showed that efficiency of tetraploid identifica tion increased when flow cytometry was limited to plants exhibiting morphological traits of increased ploidy. Conclusion This study showed that tetr aploids could be induced by treating rapidly growing in vitro shoot cultures of Dieffenbachia x ‘Star Bright M-1’ with colchi cine at concentrations of 250, 500 or 1000 mgL-1. Treatment of cultures at 500 mgL-1 colchicine produced more tetraploids by percent (40.0%) than did all other treatment levels. As seen in Alocasia (Thao et al. 2003), lower concentrations seemed to reduce the producti on of mixoploids and increase the production tetraploids. Flow cytometry confirmed the presence or absence of polyploidy. Colchicine concentrations at 500 mgL-1 or 1000 mgL-1 significantly reduced in vitro survival compared to 250 mgL-1. Colchicine reduced ex vitro survival at all treatment rates compared to untreated plants. Measuring morphological traits on 422 on e-year-old plants in the greenhouse led to selection of 63 plants that had significant variation in their ex vitro morphology. However, ploidy determination based on morphol ogical traits and stomata analysis alone could not

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35 precisely conclude the presence of polyploids. Flow cytometry was an essential tool in confirming ploidy level of the 63 plants selected based on morphological ma rkers. Fertility of these tetraploids can be determined by pollen anal ysis once plants are larg e enough to flower. In addition, this research can suggest an optimum treatment method and concentration range of colchicine for use in the pol yploidization of other sterile Dieffenbachia hybrids deemed valuable for breeding.

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36 Table 2-1. In vitro percent survival of tissue cultured Dieffenbachia x ‘Star Bright M-1’ shoot clumps 12 weeks after treatment w ith four rates of colchicine in vitro . Cultures were treated in liquid colchicine for 24 hours on shaker. Colchicine mgL-1 No. of jars No. of clumps per jar No. of jars with surviving shoots Total no of clumps survived Mean percent survival 0 10 3 10 28 93.3az 250 10 3 10 19 63.3b 500 10 3 6 9 30.0c 1000 10 3 5 7 23.3c zValues followed by the same letter are not si gnificantly different usi ng Fisher LSD at p=0.05.

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37 Table 2-2. Number of tissue cultured Dieffenbachia x ‘Star Bright M-1’shoots harvested and mean percent survival of plants 26 week s after transfer to the greenhouse and 52 weeks following treatment with four rates of colchicine in vitro . Colchicine mgL-1 Total no. of shoots harvested Total no. of shoots survived Mean percent survival 0 690 690z 100.0ay 250 306 204 66.4b 500 73 59 80.2b 1000 88 69 80.4b z90 controls were randomly selected for growi ng on out of 690 viable controls harvested. yValues followed by the same letter are not signi ficantly different using Fisher LSD at p = 0.05.

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38 Table 2-3. Morphological characteristics of 422 Dieffenbachia x ‘Star Bright M-1’ plants 18 months after treatment with four rates of colchicine in vitro and 12 months after transfer to the greenhouse. Colchcine mgL-1 Number of plants Crown height (cm)z Height with leaves held up (cm)y Canopy height (cm)x Canopy width average (cm)w Growth index (m3)v Largest leaf length (cm)u Largest leaf width (cm)t 0 90 13.8as 36.2a 30.6a 30.0a 0.0278a 22.4a 6.0a 250 204 10.5b 28.5b 24.0b 24.1b 0.0148b 18.0b 5.3b 500 59 9.2c 26.1c 21. 9c 22.6bc 0.0126bc 16.9bc 5.0bc 1000 69 8.6c 23.9c 20.1c 21.0c 0.0101c 15.3c 4.6c zLength of the primary stem from the base of th e plant to the point at where the newly emerging leaves form an apex (crown) at the top of the plant. yDistance from the base of the plant to the outer most tips of leaves as the leaves are held up above the crown of the plant. xApproximate length from the base of the plant to a point wher e the upper most leaves form an arch and begin to curve downward. wAverage of two horizontal lengths (approximately 90 degrees from one another) from side to side across the canopy of the plant. vProduct of 2 canopy widths x canopy height. uLength from the point of attachment of the pe tiole to the primary stem to the tip of the leaf blade. tLength from edge to edge of the widest portion of the leaf blade. sValues followed by the same letter are not significantly different using Fisher’s LSD at p = 0.05.

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39 Table 2-4. Morphological characteristics of Dieffenbachia x ‘Star Bright M-1’ selected based on visual indicators of polyploidy 18 months after treatment with four rates of colchicine in vitro and 12 months after tran sfer to the greenhouse. Colchcine mgL-1 Number of plants Crown height (cm)z Height with leaves held up (cm)y Canopy height (cm)x Canopy width average (cm)w Growth index (m3)v Largest leaf length (cm)u Largest leaf width (cm)t 0 10s 13.4ar 35.4a 29.9a 29.2a 0.0255a 22.1a 5.7a 250 32 7.5b 21.3b 17.9b 19.5b 0.0070b 13.8b 4.1b 500 15 6.5bc 19.3bc 16. 1bc 17.9b 0.0064bc 12.8bc 4.1b 1000 16 6.0c 17.5c 14.7c 17.5b 0.0048c 11.6c 3.8b zLength of the primary stem from the base of th e plant to the point at where the newly emerging leaves form an apex (crown) at the top of the plant. yDistance from the base of the plant to the outer most tips of leaves as the leaves are held up above the crown of the plant. xApproximate length from the base of the plant to a point wher e the upper most leaves form an arch and begin to curve downward. wAverage of two horizontal lengths (approximately 90 degrees from one another) from side to side across the canopy of the plant. vProduct of 2 canopy widths x canopy height. uLength from the point of attachment of the pe tiole to the primary stem to the tip of the leaf blade. tLength from edge to edge of the widest portion of the leaf blade. sFor control (0 mgL-1 colchicine), 10 plants were selected as average plants among 90 untreated controls. rValues followed by the same letter are not signi ficantly different using Fisher’s LSD at p = 0.05.

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40 Table 2-5. Mean stomata length of leaves from Dieffenbachia x ‘Star Bright M-1’ selected based on visual indicators of polypl oidy 20 months after treatm ent with four rates of colchicine in vitro and 14 months after tran sfer to the greenhouse. Colchicine mgL-1 No. of plants selected Stomata length (SL) (m)z Percent of plants with SL > 42.5 m 0 10 37.8y 0.0 250 32 38.3 21.2 500 15 42.5 33.3 1000 16 40.0 31.3 zAverage of 10 observations per plant. yNo significance based on Fi sher’s LSD at p = 0.05.

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41 Table 2-6. Ploidy level of 63 Dieffenbachia x ‘Star Bright M-1’ plants initially selected as potential polyploids based on morphological traits 20 mont hs after treatment with four rates of colchicine in vitro . Plants had grown for 14 m onths after transfer to the greenhouse. Final ploidy level determined by flow cytometry 2x 4x 2x + 4x Colchicine mgL-1 No. selected for flow cytometryz No. Percent No. Percent No. Percent 0 10y 10 100.0 0 0.0 0 0.0 250 32 13 40.6 6 18.8 13 40.6 500 15 5 33.3 6 40.0 4 26.7 1000 16 3 18.8 1 6.3 12 75.0 Total 73 31 13 29 zPlants were selected based on vi sual characteristics of polyploi dy, i.e. thicker leaves, deeper green color, and slower growth rate. yTen controls included for a tota l of 73 plants analyzed with flow cytometry.

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42 Table 2-7. Comparison of percent tetraploids from total number of plants available for study to percent tetraploids from those selected for flow cytometry based on morphological markers and stomata analysis. Colchicine mgL-1 Total no. of plants Percent 4x of total no. of plants No. selected for flow cytometry Percent 4x of no. selected for flow cytometry 250 204 2.9 32 18.8 500 59 10.2 15 40.0 1000 69 1.4 16 6.3

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43 Figure 2-1. Tissue cultured Dieffenbachia x ‘Star Bright M-1’ shoot clumps prior to treatment with 0, 250, 500 or 1000 mgL-1 colchicine in vitro . Arrow indicates one potentially harvestable shoot.

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44 Control = 0 mgL-1 250 mgL-1 500 mgL-1 1000 mgL-1 colchicine colchicine colchicine colchicine Figure 2-2. In vitro Dieffenbachia x ‘Star Bright M-1’ shoot cl umps 12 weeks after treatment with four rates of colchicine in vitro . Cultures were treated with liquid colchicine for 24 hours on shaker.

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45 A B C D Figure 2-3. In vitro Dieffenbachia x ‘Star Bright M-1’ shoot clum ps before second transfer to fresh media 12 weeks after treatment with four rates of colchicine in vitro . Cultures were treated with liquid colchicine fo r 24 hours on shaker. A) Control = 0 mgL-1 colchicine. B) 250 mgL-1 colchicine. C) 500 mgL-1 colchicine. D) 1000 mgL-1 colchicine.

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46 Figure 2-4. In vitro percent survival of tissue cultured Dieffenbachia x ‘Star Bright M-1’ shoot clumps 12 weeks after treatment w ith four rates of colchicine in vitro . y = 0.0001x 2 0.1732x + 95.242 R 2 = 0.99 0 10 20 30 40 50 60 70 80 90 100 0 250 500 750 1000 Colchicine ( mgL-1)In vitro % survival

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47 A B C D Figure 2-5. Dieffenbachia x ‘Star Bright M-1’ shoots rea dy to plant to greenhouse 26 weeks after treatment with liquid colchicine for 24 hours in vitro . A) Control = 0 mgL-1 colchicine. B) 250 mgL-1 colchicine. C) 500 mgL-1 colchicine. D) 1000 mgL-1 colchicine.

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48 Figure 2-6. Ex vitro percent survival of tissue cultured Dieffenbachia x ‘Star Bright M-1’plants 26 weeks after transfer to the greenhouse and 52 weeks following treatment with four rates of colchicine in vitro . y = 7E-05x 2 0.0843x + 95.416 R 2 = 0.55 0 10 20 30 40 50 60 70 80 90 100 0 250 500 750 1000 Colchicine ( mgL-1)Ex vitro % survival

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49 A B Figure 2-7. Crown height vs. treatment rate of plants from tissue cultured Dieffenbachia x ‘Star Bright M-1’ 18 months after treatment with four rate s of colchicine in vitro and 12 months after transfer to the greenhouse. A) 422 surviving explants. B) 63 potential polyploids. y = 9E-06x 2 0.0136x + 13.674 R 2 = 0.99 0 2 4 6 8 10 12 14 16 0 250 500 750 1000 colchicine ( mgL-1)Crown Height (cm) y = 1E-05x 2 0.0218x + 12.995 R 2 = 0.96 0 2 4 6 8 10 12 14 16 0 250 500 750 1000 colchicine ( mgL-1)Crown Height (cm)

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50 A B Figure 2-8. Height with leaves held up vs. treatment rate of plants from tissue cultured Dieffenbachia x ‘Star Bright M-1’ 18 months af ter treatment with four rates of colchicine in vitro and 12 months after transfer to the greenhouse. A) 422 surviving explants. B) 63 potential polyploids. y = 2E-05x 2 0.03x + 35.837 R 2 = 0.98 0 5 10 15 20 25 30 35 40 0 250 500 750 1000 Colchicine ( mgL-1)Height with leaves held up (cm) y = 3E-05x 2 0.0515x + 34.474 R 2 = 0.95 0 5 10 15 20 25 30 35 40 0 250 500 750 1000 Colchicine ( mgL-1)Height with leaves held up (cm)

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51 A B Figure 2-9. Canopy height vs. treatment ra te of plants from tissue cultured Dieffenbachia x ‘Star Bright M-1’ 18 months after treatment with four rate s of colchicine in vitro and 12 months after transfer to the greenhouse. A) 422 surviving explants. B) 63 potential polyploids. y = 2E-05x 2 0.0259x + 30.296 R 2 = 0.98 0 5 10 15 20 25 30 35 0 250 500 750 1000 Colchicine ( mgL-1)Canopy height (cm) y = 3E-05x 2 0.044x + 29.104 R 2 = 0.95 0 5 10 15 20 25 30 35 0 250 500 750 1000 Colchicine ( mgL-1)Canopy height (cm)

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52 A B Figure 2-10. Canopy width vs. treatment rate of plants from tissue cultured Dieffenbachia x ‘Star Bright M-1’ 18 months after trea tment with four rates of colchicine in vitro and 12 months after transfer to the greenhouse. A) 422 surviving explants. B) 63 potential polyploids. y = 1E-05x 2 0.0221x + 29.645 R 2 = 0.97 0 5 10 15 20 25 30 35 0 250 500 750 1000 Colchicine ( mgL-1)Canopy width (cm) y = 3E-05x 2 0.0367x + 28.623 R 2 = 0.95 0 5 10 15 20 25 30 35 0 250 500 750 1000 Colchicine ( mgL-1)Canopy width (cm)

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53 A B Figure 2-11. Growth index vs. treatmen t rate of plants from tissue cultured Dieffenbachia x ‘Star Bright M-1’ 18 months after treatment with four rates of colchicine in vitro and 12 months after transfer to the greenhouse. A) 422 surviving explants. B) 63 potential polyploids. y = 3E-08x 2 5E-05x + 0.027 R 2 = 0.96 0 0.005 0.01 0.015 0.02 0.025 0.03 0 250 500 750 1000 Colchicine ( mgL-1)Growth index (m3) y = 4E-08x 2 6E-05x + 0.0241 R 2 = 0.91 0 0.005 0.01 0.015 0.02 0.025 0.03 0 250 500 750 1000 Colchicine ( mgL-1)Growth index (m3)

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54 A B Figure 2-12. Largest leaf length vs. treat ment rate of plants from tissue cultured Dieffenbachia x ‘Star Bright M-1’ 18 months after trea tment with four rates of colchicine in vitro and 12 months after transfer to the greenhouse. A) 422 surviving explants. B) 63 potential polyploids. y = 1E-05x 2 0.0164x + 22.163 R 2 = 0.97 0 5 10 15 20 25 0 250 500 750 1000 Colchicine ( mgL-1)Largest leaf length (cm) y = 2E-05x 2 0.0297x + 21.479 R 2 = 0.94 0 5 10 15 20 25 0 250 500 750 1000 Colchicine ( mgL-1)Largest leaf length (cm)

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55 A B Figure 2-13. Largest leaf width vs. treatme nt rate of plants from tissue cultured Dieffenbachia x ‘Star Bright M-1’ 18 months after trea tment with four rates of colchicine in vitro and 12 months after transfer to the greenhouse. A) 422 surviving explants. B) 63 potential polyploids. y = 2E-06x 2 0.0029x + 6.0266 R 2 = 0.99 0 1 2 3 4 5 6 7 0 250 500 750 1000 Colchicine ( mgL-1)Largest leaf width (cm) y = 4E-06x 2 0.0055x + 5.5911 R 2 = 0.89 0 1 2 3 4 5 6 7 0 250 500 750 1000 Colchicine ( mgL-1)Largest leaf width (cm)

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56 Figure 2-14. Flow cytometry confir med tetraploid from tissue cultured Dieffenbachia x ‘Star Bright M-1’ after treatment with 250 mgL-1 colchicine in vitro . Photo was taken 20 months after colchicine treatment and 14 months after transfer to the greenhouse.

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57 Figure 2-15. Flow cytometry confir med tetraploid from tissue cultured Dieffenbachia x ‘Star Bright M-1’ after treatment with 500 mgL-1 colchicine in vitro . Photo was taken 20 months after colchicine treatment and 14 months after transfer to the greenhouse.

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58 Figure 2-16. Flow cytometry confir med tetraploid from tissue cultured Dieffenbachia x ‘Star Bright M-1’ after treatment with 1000 mgL-1 colchicine in vitro . Photo was taken 20 months after colchicine treatment and 14 months after transfer to the greenhouse.

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59 Florescence (channel number) Figure 2-17. Flow cytometric histogram of a diploid plant from tissue cultured Dieffenbachia x ‘Star Bright M-1’ 20 months after treatment with 0 mgL-1 colchicine in vitro and 14 months after transfer to the greenhouse. Control plant (2n = 2x)

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60 Florescence (channel number) Figure 2-18. Flow cytometric histogram of a tetraploid plant from tissue cultured Dieffenbachia x ‘Star Bright M-1’ 20 months after treatment with 500 mgL-1 colchicine in vitro and 14 months after transf er to the greenhouse. Tetraploid (2n = 4x)

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61 Florescence (channel number) Figure 2-19. Flow cytometric histogram of a mixoploid plant from tissue cultured Dieffenbachia x ‘Star Bright M-1’ 20 months after treatment with 1000 mgL-1 colchicine in vitro and 14 months after transfer to the greenhouse. Mixoploid (2n = 4x + 2x)

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62 CHAPTER 3 MORPHOLOGICAL COMPARISONS OF DIPLOID AND TETRAPLOID Dieffenbachia x ‘Star Bright M-1’ Materials and Methods The objective of this study wa s to compare stomata size and leaf morphology of diploid and colchicine induced tetraploid plants of Dieffenbachia x ‘Star Bright M-1’. First, stomata length was compared among 16 diploid (2n = 2x) and 13 tetraploid (2n = 4x) plants whose ploidy level had been previously confirmed by flow cytometry. Ten plants from untreated controls and two each from 250, 500, and 1000 mgL-1 colchicine treatments composed the diploid population. Among the 13 tetraploids, six came from the 250, six from the 500 and one from the 1000 mgL-1 colchicine treatments. Second, seve ral leaf morphological characteristics were compared among the same 29 plants. To obtain stomata measurements an epidermal p eel was used to ‘fix’ the stomata to a glass slide. A 75 x 25 mm microscope slide containing one drop of Super Glue was pressed against the underside of a leaf for 30 sec onds, then removed. This resulte d in a permanent impression of the epidermis from the underside of the leaf. Stomata were measured in micrometers (m) under a light microscope at 400x magni fication by placing the scale end to end along the lengths of the guard cells in the longitudinal di rection. Ten stomata per leaf we re measured. Stomata length measurements were obtained from the data pool in Chapter 2. Morphological observations included ratio of l eaf width to leaf length, leaf area, leaf thickness, mid-rib thickness, and leaf specific weight. A micrometer (Micromaster MM 2000, Brown and Sharp, Switzerland) was used to measur e leaf thickness and leaf mid-rib thickness. For each leaf thickness, three observations were recorded and averaged by measuring at points along the flat side of the leaf bl ade in the approximate center to the right or left of the mid-rib. The thickness of the mid-rib was measured by cutti ng the leaf at a 90 degree angle to the mid-rib

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63 and placing the micrometer at th e edge of the cut mid-rib. Le af length was measured as the distance from the point of attachme nt of the petiole to the primary stem to the tip of the leaf blade. Leaf width was measured as the distance fr om side to side in the widest portion of the leaf. A ratio of leaf width to leaf length wa s than calculated. To m easure leaf area, a Li-3100 area meter (Li-Cor, Inc., Lincoln, Nebraska) was used. Leaf specific weight was calculated as the fresh weight per unit area. The average of 3 leaves per plant was recorded for all observations. Mean stomata lengths and leaf morphological characteristics of diploid and tetraploid plants were compared using Fish er’s Protected least significan t difference. In addition, a Pearson correlation coefficient for mean st omata length to ploidy level was computed. Results and Discussion Table 3-1 compares the mean stomata lengths of diploids and tetraplo ids. Stomata lengths of tetraploids were sign ificantly larger than diploids (P < 0.05) by roughly 20% (Figure 3-1). Stomata lengths of tetraploids averaged 47.0 m, whereas stomata lengths of diploids averaged 37.5 m. In addition, a positive correlation existe d between stomata size and level of ploidy (Pearson correlation coefficien t = 0.741, P < 0.05). These findi ngs are supported by previous Dieffenbachia research which found stomata of tetrap loids to be roughly 20% larger than diploids1. Research in other plant species wh ere polyploid stomata were found to be significantly larger than in diploid stomata includes: Xanthosoma (Tambong et al. 1998), Alocasia (Thao et al. 2003), and Alstromeria (Lu and Bridgen 1997). All leaf morphological comparisons of tetraploids to diploids were sign ificantly different [(P < 0.05); Table 3-2], except mid-rib thickness (P > 0.05). Leav es of tetraploids were thick, dark green and leathery. Leaf 1 Henny RJ (2006) Personal communication. University of Florida, Mid-Florida Research and Education Center.

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64 thickness of tetraploids (0.637 mm) averaged 83% greater than diploids (0.348 mm). Leaves of tetraploid plants could be felt to have more bul k and sturdiness over leaves of diploids. Leaves of diploid plants tended to droop and arch more th an tetraploid leaves th at were more rigid and straight. Similar morphology was observed in Citrus polyploids (Wu and Mooney 2002). Figure 3-2 shows a comparison in the size and shape of a diploid plant versus a tetraploid plant of Dieffenbachia x ‘Star Bright M-1.’ Compared to the diploid, the tetraploid was less full (i.e. fewer basal shoots), had a coarse r texture, and the variegation was less pronounced. Tetraploid leaves tended to be broader than diploid leaves. Ratio of leaf wi dth to leaf length of tetraploids (0.305) averaged 22% greater than diploids (0.249). Leaf area of tetraploids (67.4 cm2) averaged 53% less than diploids (115.0 cm2). This confirms the observation that diploid leaves were more elongate and lance shaped, whereas those of te traploids tended to be shorter and more oval (Figure 3-3). Leaf specific we ight of tetraploids (56.3mg/cm2) averaged 53% greater than in diploids (36.7 mg/cm2). Similar results were observed in Alstromeria (Lu and Bridgen 1997) where leaf specific weight was found to be signifi cantly greater in tetraplo ids than in diploids. No significance in mid-rib thickness was found be tween tetraploids and diploids, 2.31 mm and 2.08 mm, respectively. Overall, these fi ndings showed that ploidy level in Dieffenbachia has a significant effect on leaf shape. An increase in ploidy has been shown to affect leaf shape in other aroids such as Alocasia (Thao et al. 2003) and Spathiphyllum (Eeckhaut et al. 2004). Conclusion This study showed that Dieffenbachia stomata length is greater at higher ploidy levels and that quantitative comparison leaf morphology of dipl oids and tetraploids al so yields significant differences. Tetraploid plants at 1 year from tissue culture had thicker and broader leaves. In addition, previous data (Table 2-4) showed tetr aploid plants were shorter and produced fewer

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65 basal shoots than diploids. In combination, the a bove traits can be useful in the identification of potential tetraploids in a large population of colchicine treated Dieffenbachia .

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66 Table 3-1. Comparison of ploidy level with stomata lengths of Dieffenbachia x ‘Star Bright M-1’ 20 months after treatme nt with colchicine in vitro and 14 months after transfer to the greenhouse. Plants examined were select ed based on flow cytometric results. Ploidy Number of plants Mean stomata length 2x 16 37.5bz 4x 13 47.0a zValues followed by the same letter are not signi ficantly different using Fisher LSD at p = 0.05.

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67 Table 3-2. Morphological comparison of diploids to tetraploids of Dieffenbachia x ‘Star Bright M-1’ 20 months after treatment w ith four rates of colchicine in vitro and 14 months after transfer to the greenhouse. Plants examined were selected based on flow cytometric results. Ploidy Number of plants Ratio leaf width: leaf lengthz Leaf area (cm2)y Leaf thickness (mm)x Mid-rib thickness (mm)w Leaf specific weight (mg/cm2)v 2x 16 0.249bu 115.0a 0.348b 2.08a 36.7b 4x 13 0.305a 67.4b 0.637a 2.31a 56.3a zRatio by dividing the average of three leaf leng ths by the average of three leaf widths. Length was measured from the point of attachment of th e petiole to the primary stem to the tip of the leaf blade. Leaf width was measured from edge to edge of the widest por tion of the leaf blade. yLeaf area was measured from the average of th ree leaves using a Li-3100 area meter (Li-Cor, Inc., Lincoln, Nebraska). xLeaf thickness was measured from the average of three leaves and three observations per leaf at point s along the flat side of the leaf blade in the approximate center to the right or left of the mid-rib. wMid-rib thickness was measured from the average of three leaves by the thickness of the mid-rib when a leaf is cut at a 90 degree an gle to the mid-rib and a micrometer is placed on the edge of the cut mid-rib. vLeaf specific weight was measured as the fresh weight per unit area. uValues followed by the same letter are not significantly different using Fisher LSD at p = 0.05.

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68 A B Figure 3-1. Stomata imprints of Dieffenbachia x ‘Star Bright M-1’ 20 months after treatment with colchicine in vitro and 14 months after transfer to the greenhouse. A) Diploid (2n = 2x). B) Tetraploid (2n = 4x). Bar = 40 m.

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69 Figure 3-2. A 20-monthold diploid (left) a nd tetraploid (right) Dieffenbachia x ‘Star Bright M-1.’

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70 Figure 3-3. Typical diploid l eaf (left) and 3 typical tetraploid leaves (right) of Dieffenbachia x ‘Star Bright M-1’ taken from 20-month-old plants.

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71 LIST OF REFERENCES Adaniya S, Shirai D (2001) In vitro induction of tetraploid ginger ( Zingiber officinale Roscoe) and its pollen fertility and germinabil ity. Scientia Horticulturae 88:277-287 Aryavand A, Ehdaie B, Tran B, Waines JG ( 2003) Stomatal frequenc y and size differentiate ploidy levels in Aegilops neglecta . Genetic Resources a nd Crop Evolution 50:175-182 Beck S, Dunlop R, Fossey A (2003) Stomatal le ngth and frequency as a measure of ploidy level in black wattle, Acacia mearnsii (de Wild). Botanical Journal of the Linnean Society 141:177-181 Blakeslee A, Avery A (1937) Methods of i nducing doubling of chromosomes in plants. Journal of Herdity 28:393-411 Carvalho JF, Carvalho CR, Otoni WC (2003) In vitro induction of polyploidy in annatto ( Bixa orellana ). Plant Cell, Tissue and Organ Culture 80:69-75 Chakraborti SP, Vijayan K, Roy BN, Qadri SMH (1998) In vitro induction of tetraploidy in mulberry ( Morus alba L.). Plant Cell Reports 17:799-803 Cohen D, Yao JL (1996) In vitro chromosome doubling of nine Zantedeschia cultivars. Plant, Cell, Tissue and Organ Culture 47:43-49 Eeckhaut T, Samyn G, van Bockstaele E (2002) In vitro polyploidy induction in Rhododendron simsii hybrids. Acta Ho rticulturae 572:43-49 Eeckhaut TGR, Werbrouck SPO, Leus LWH, Van Bockstaele EJ, Debergh PC (2004) Chemically induced polyploidization in Spathiphyllum wallisii Regal through somatic embryogenesis. Plant, Cell, Tissue and Organ Culture 78:241-246 Gamborg O, Miller R, Ojima K (1968) Nutrie nt requirements of suspension cultures of soybean root cells. Experimental Cell Research 50:151-158 Ganga M, Chezhiyan N (2002) Influence of the an timitotic agents colchici ne and oryzalin in in vitro doubling of diploid bananas ( Musa spp.). Journal of Horticultural Science & Biotechnology 77: 572-575 Gao SL, Chen BJ, Zhu DN (2002) In vitro production and identification of autotetraploid of Scutellaria baicalensis . Plant Cell, Tissue and Organ Culture 70:289-293 Gao SL, Zhu D, Cai Z, Xu D (1996) Autotetraplo id plants from colchici ne-treated bud culture of Salvia Miltiorrhiza Bge. Plant Cell, Tissue and Organ Culture 47: 73-77 Grzebeluse E, Adamus A (2004) Effect of an ti-mitotic agents on de velopment and genome doubling of gynogenic onion ( Allium cepa L.) embryos. Plant Science 2004 167(3):569574

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75 BIOGRAPHICAL SKETCH James Robert Holm was born May 4, 1971 in Jamestown, New York. The youngest of four children, he grew up in Mount Dora, Florid a, graduating from Mount Dora High School in 1990. He graduated from Lake-Sumter Community College in 1995 with an Associate of Arts degree. He earned his B.S. in biology from the University of Central Florida in 1997 and graduated with honors. Following graduation, Mr. Holm worked as a Biologist at the University of Florida Research and Education Center in Sanford, Florid a. In 1999 he began a position in Research and Development at Agri-Starts I, In c. in Apopka, Florida. He en tered the master’s program in environmental horticulture at the University of Florida in 2003. While working toward his M.S., he worked concurrently with the Florida Di vision of Plant Industry as an inspector. Upon graduation, Mr. Holm plans to remain in Mount Dora and work toward developing a commercial tissue culture company to bene fit the ornamental foliage industry.