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Asymbiotic Seed Germination of Vanda

Permanent Link: http://ufdc.ufl.edu/UFE0021379/00001

Material Information

Title: Asymbiotic Seed Germination of Vanda In Vitro Germination and Development of Three Hybrids
Physical Description: 1 online resource (63 p.)
Language: english
Creator: Johnson, Timothy R
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: asymbiotic, development, electron, germination, light, microscopy, orchid, propagation, scanning, seed, sem, vanda
Environmental Horticulture -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The majority of potted flowering orchids sold in the United States are Phalaenopsis hybrids. However, as consumers become accustomed to growing orchids in the home, demand for other genera will undoubtedly increase. The major objective of this study was to compare the germination and development of three Vanda hybrids, a genus with mass market potential. Specifically, I evaluated whether hybrids with different pedigrees had significantly different culture requirements. After 12 weeks culture, differences in germination and development were found between the three hybrids screened. Significantly more seed of Vanda Motes Primrose ? Ascocenda Tavivat (hybrid S014) germinated (82.0?95.5%) than seeds of Vanda Paki ? (Vanda tessellata ? Vanda cristata; hybrid S005) or (Vanda Joan Warne ? Vanda Paki) ? Vanda Loke (hybrid S013) on Knudson C (KC), half-strength Murashige & Skoog (?MS), or PhytoTechnology Orchid Seed Sowing Medium (P723) under three photoperiods (8/16 h, 12/12 h, 16/12 h light/dark). During 12 weeks of observation, protocorms of S005 and S013 did not develop past Stage 3 (first leaf present), while S014 seeds developed to Stage 4 (one leaf and more than one root present; 0.5?0.6%) and Stage 5 (two leaves and more than one root present; 2.7?5.6%) when cultured on P723 under all photoperiods tested. All hybrids appeared to undergo similar early development (Stage 0?3). Limited advanced development of hybrids S005 and S013 may be due either to inadequate culture conditions or low seed vigor. These data indicate that commercial production of some Vanda hybrids may be limited by slow growth and development. Efforts to breed hybrids which germinate and develop rapidly may be required to improve the mass market potential of Vanda hybrids. Attempts to initiate Vanda tissue cultures using explants from flowering size plants and seedlings were largely unsuccessful. Information on the protocols tested is included in the appendix.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Timothy R Johnson.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Kane, Michael E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021379:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021379/00001

Material Information

Title: Asymbiotic Seed Germination of Vanda In Vitro Germination and Development of Three Hybrids
Physical Description: 1 online resource (63 p.)
Language: english
Creator: Johnson, Timothy R
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: asymbiotic, development, electron, germination, light, microscopy, orchid, propagation, scanning, seed, sem, vanda
Environmental Horticulture -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The majority of potted flowering orchids sold in the United States are Phalaenopsis hybrids. However, as consumers become accustomed to growing orchids in the home, demand for other genera will undoubtedly increase. The major objective of this study was to compare the germination and development of three Vanda hybrids, a genus with mass market potential. Specifically, I evaluated whether hybrids with different pedigrees had significantly different culture requirements. After 12 weeks culture, differences in germination and development were found between the three hybrids screened. Significantly more seed of Vanda Motes Primrose ? Ascocenda Tavivat (hybrid S014) germinated (82.0?95.5%) than seeds of Vanda Paki ? (Vanda tessellata ? Vanda cristata; hybrid S005) or (Vanda Joan Warne ? Vanda Paki) ? Vanda Loke (hybrid S013) on Knudson C (KC), half-strength Murashige & Skoog (?MS), or PhytoTechnology Orchid Seed Sowing Medium (P723) under three photoperiods (8/16 h, 12/12 h, 16/12 h light/dark). During 12 weeks of observation, protocorms of S005 and S013 did not develop past Stage 3 (first leaf present), while S014 seeds developed to Stage 4 (one leaf and more than one root present; 0.5?0.6%) and Stage 5 (two leaves and more than one root present; 2.7?5.6%) when cultured on P723 under all photoperiods tested. All hybrids appeared to undergo similar early development (Stage 0?3). Limited advanced development of hybrids S005 and S013 may be due either to inadequate culture conditions or low seed vigor. These data indicate that commercial production of some Vanda hybrids may be limited by slow growth and development. Efforts to breed hybrids which germinate and develop rapidly may be required to improve the mass market potential of Vanda hybrids. Attempts to initiate Vanda tissue cultures using explants from flowering size plants and seedlings were largely unsuccessful. Information on the protocols tested is included in the appendix.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Timothy R Johnson.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Kane, Michael E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021379:00001


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1 ASYMBIOTIC SEED GERMINATION OF Vanda : IN VITRO GERMINATION AND DEVELOPMENT OF THREE HYBRIDS By TIMOTHY R. JOHNSON 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

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2 2007 Timothy R. Johnson

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3 To my wife, of course

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4 ACKNOWLEDGMENTS I would like to thank the me mbers of my committee, Dr. Tom Sheehan and Dr. Dennis Gray, for helpful comments on experimental designs as well as for their editorial feedback. I would like to thank my major professor, Dr. Mi chael Kane, for his con tinual dedication to all aspects of this project, as well as his dedication to my professional development. I also wish to acknowledge Daniela Dutra, Philip Kauth, Nanc y Philman, and Scott L. Stewart for providing the type of insight that only friends and lab-mate s can provide. Thanks also go out to Dr. Martin Motes and the Redland Professi onal Orchid Growers (Homestead, FL) for partial financial support of this project. Last but not least, I th ank my wife, Danielle, for putting up with so many late nights and long trips, as well as my generall y distracted demeanor ove r the past two years. We did it.

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5 TABLE OF CONTENTS ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ..............9 CHAPTER 1 LITERATURE REVIEW.......................................................................................................11 Introduction to the Family Orchidaceae.................................................................................11 The Wholesale Orchid Market................................................................................................13 Classification of Tribe Vandeae.............................................................................................14 Seed Propagation of Vanda ....................................................................................................15 Micropropagation of Vanda ....................................................................................................16 Initiation of Tissue Cultures............................................................................................18 Shoot tip explants.....................................................................................................18 Inflorescence and flower bud explants.....................................................................20 Leaf explants............................................................................................................20 Development and Rooting of Protocorm Like Bodies....................................................22 Summary........................................................................................................................ .........23 Objectives..................................................................................................................... ..........24 2 ASYMBIOTIC SEED GERMINATION OF Vanda HYBRIDS...........................................26 Introduction................................................................................................................... ..........26 Materials and Methods.......................................................................................................... .27 Results........................................................................................................................ .............29 Discussion..................................................................................................................... ..........33 APPENDIX ATTEMPTS TO INITIATE Vanda TISSUE CULTURES....................................................47 Introduction................................................................................................................... ..........47 Initiation of Vanda Tissue Culture Lines Using Newly Emerged Leaves of Flowering Size Plants.................................................................................................................... .......48 Materials and Methods....................................................................................................48 Results and Discussion....................................................................................................49 Initiation of Vanda Tissue Culture Lines Using Seedlings, Seedling Leaves, and Cut Protocorms in Liquid Culture..............................................................................................50 Methods........................................................................................................................ ...50 Results and Discussion....................................................................................................51

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6 Initiation of Vanda Tissue Culture Lines Using Seedling Leaf Sections...............................52 Methods........................................................................................................................ ...52 Results and Discussion....................................................................................................53 LIST OF REFERENCES............................................................................................................. ..56 BIOGRAPHICAL SKETCH.........................................................................................................63

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7 LIST OF TABLES Table page 1-1 List of interspe cific and intergeneric Vanda hybrids referenced in text.................................25 2-1 Parentage of Vanda hybrids used for experimentation...........................................................37 2-2 Composition of asymbiotic media used to test the effect of photoperiod on germination and development of Vanda hybrids S005, S013, and S014...............................................38 2-3 Developmental stages of Vanda hybrids................................................................................39

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8 LIST OF FIGURES Figure page 2-1 Germination of Vanda hybrids S005, S013, and S014............................................................40 2-2 Protocorm development of Vanda hybrid S005......................................................................41 2-3 Protocorm development of Vanda hybrid S013......................................................................42 2-4 Protocorm development of Vanda hybrid S014......................................................................43 2-5 Scanning electron micrographs of Vanda hybrid S014...........................................................44 2-6 Thin section light micrographs of Vanda hybrid S014............................................................45 2-7 Twelve month old cultures of Vanda hybrid S014..................................................................46 A-1 Tissue culture of Vanda tessellata Vanda Arjuna...............................................................55

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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 ASYMBIOTIC SEED GERMINATION OF Vanda : IN VITRO GERMINATION AND DEVELOPMENT OF THREE HYBRIDS By Timothy R. Johnson August 2007 Chair: Michael Kane Major: Horticultural Science The majority of potted flowering orchids sold in the United States are Phalaenopsis hybrids. However, as consumers become accust omed to growing orchids in the home, demand for other genera will undoubtedly increase. The major objective of this study was to compare the germination and development of three Vanda hybrids, a genus with mass market potential. Specifically, I evaluated whether hybrids with di fferent pedigrees had si gnificantly different culture requirements. After 12 weeks culture, di fferences in germination and development were found between the three hybr ids screened. Significantly more seed of Vanda Motes Primrose Ascocenda Tavivat (hybrid S014) germin ated (82.0.5%) than seeds of Vanda Paki ( Vanda tessellata Vanda cristata ; hybrid S005) or ( Vanda Joan Warne Vanda Paki) Vanda Loke (hybrid S013) on Knudson C (KC), half-s trength Murashige & Skoog (MS), or Phyto Technology Orchid Seed Sowing Medium (P 723) under three photoperiods (8/16 h, 12/12 h, 16/12 h light/dark). During 12 weeks of obser vation, protocorms of S005 and S013 did not develop past Stage 3 (first leaf present), whil e S014 seeds developed to Stage 4 (one leaf and more than one root present; 0.5.6%) and Stage 5 (two leaves and more than one root present; 2.7.6%) when cultured on P723 under all photoperi ods tested. All hybrids appeared to undergo similar early development (Stage 0). Limited advanced development of hybrids S005

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10 and S013 may be due either to inadequate cultu re conditions or low s eed vigor. These data indicate that commerc ial production of some Vanda hybrids may be limited by slow growth and development. Efforts to breed hybrids which ge rminate and develop rapidly may be required to improve the mass market potential of Vanda hybrids. Attempts to initiate Vanda tissue cultures using explants from flowering size plants and seedlings were largely unsuccessful. Informati on on the protocols tested is included in the appendix.

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11 CHAPTER 1 LITERATURE REVIEW Introduction to the Family Orchidaceae The family Orchidaceae is estimated to ha ve about 25,000 species and 800 genera, which Dressler placed into five subfamilies (1993). Members of the family are highly variable in habitat and form. The vast majority of orch id species are epiphytic, but 4,000 known species are primarily terrestrial (Dressler, 1981 ). Orchids exhibit two distinct growth habits: sympodial and monopodial. The sympodial growth habit involve s determinate stem growth and subsequent spreading by rhizomes while the more evolutiona rily derived monopodial growth form is marked by continual apical growth with little or no axi llary sprouting. Many mo difications to these two general growth habits can be found, however mo st orchid species can be placed into one category or the other (Arditti and Ernst, 1992; Dressler, 1993). The flowers of orchid species are as variable as the grow th forms. Dressler (1981) hypothesized that the Orchidaceae has not yet undergone the degr ee of natural selection that older angiosperm families have undergone; [M]any of the evolutionary stages of the [O rchidaceae] are still found in living members. If we had only the most primitive living representations of the family, they might be dismissed as rather peculiar lilies. But th e vast array of not-missing links show these primitive orchids to be the first stages in the evolution of this distinctive family. Orchid flowers are typically bilaterally symm etrical and trimerous with inferior ovaries (Dressler, 1993). Typically one petal is larger or more ornate than the others and is termed the lip or labellum (Arditti and Ernst, 1992; Dressler, 1993). Orchid flowers usually display resupination, twisting or bending of the pedicel, during developm ent. Because of this, the labellum, which is adaxial in the flower bud, co mes to rest in an ab axial position (Arditti and Ernst, 1992; Dressler, 1993). Anot her defining characterist ic of orchids is th at their stamens are all located on one side of the flower rather th an being radially dispersed like those of other

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12 angiosperm orders (Dressler, 1981). In additi on, the style and filaments of orchid flowers are fused to form a column or gynostemium (Arditti and Ernst, 1992; Dressler, 1993). The column is marked by the presence of the rostellum, a modification to the column that separates the stigmatic surface from the anthers (Arditti and Er nst, 1992; Dressler, 1981), which are located at the end of the column (Arditti and Ernst, 1992; Dressler, 1981; 1993). While primitive orchids still exhibit powdery pollen, the po llen of more derived orchids is contained in discrete, hard packets or pollinia that are attached to a stic ky disc termed the viscidium. The viscidium attaches to potential pollinators, resulting in removal of pollinia and (possibly) subsequent pollination (Arditti and Ernst, 1992; Dressler, 1993). Darwin (1892) wrote extensively about the mechanisms by which orchid pollination occurs. While he studied and presented examples of specific pollinators to illustrate his theories about co-evolution, these may be the exceptions rather than the rule s in the Orchidaceae (Dressler, 1993). Most orchids appear to be adapted for insect-mediated pollination (Dressler, 1981) and often exhibit deceptive pollinator attr action strategies (Arditti and Ernst, 1992; Cozzolino and Widmer, 2005; Dressler, 1981). As many as 400 species of orchids are sexually deceptive, offering some resemblance to the sexu al partners of pollinator species, whereas onethird of all orchids are food deceptive (Co zzolino and Widmer, 2005). Deception takes many other forms in the Orchidaceae including simula tion of prey, simulation of substrata and resemblance to antagonists (Arditti and Erns t, 1992). Attraction without reward (i.e. deception) is believed to be evolutionarily primitiv e in orchids, as it is a prevalent characteristic and may be a long-term adaptive strategy that limits inbreeding depression and excessive fruit set, both of which may reduce fitn ess (Cozzolino and Widmer, 2005).

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13 The Wholesale Orchid Market Since the USDA began keeping statistics on orchid production, sales of orchid cut flowers and potted plants have increased significantly in th e United States; since 1996 there has been a 29% increase in the number of orchid growers (J erardo, 2006; Nash, 2003). Wholesale values of potted flowering orchids reached $100 million in 2001 with cut orchid flower sales adding an additional $8.6 million. While cut flower sales in the United States have been declining for the last five years (approximately $6.9 million in w holesale value during 2005), wholesales of potted flowering orchids rose to an estimated $144 milli on in 2005, second only to that of poinsettias. Florida wholesalers alone made an estimated $47 million net sales on orchid potted plants in 2005 (Jerardo, 2006). The resurgence of orchids as popular house plants can be largely attributed to one genus, Phalaenopsis (Griesbach, 2002; 2003; Nash, 2003). In 2003, Phalaenopsis species and hybrids accounted for 85% of all orchids so ld (Nash, 2003). At present, Phalaenopsis are being prominently featured in design and fashion ma gazines, business and hotel lobbies, and TV series sets. Phalaenopsis hybrids (which are affec tionately called beginne rs orchids for their relative ease of culture and willingness to rebloom) are new symbols of elegance and sophistication though the ease with which they are produced and grown has not always been feasible. Cattleya Cymbidium and Paphiopedilium were favored during the parlor plant craze of Victorian times. At that time, Phalaenopsis were considered difficult to culture since they were not suitable for the very hot rear parlor or very cool front parlor of Victorian style houses (Griesbach, 2002; 2003). In addition, Phalaenopsis plants proved difficult to ship during this era, as they do not have pseudobul bs to store water, and their so ft leaves are susceptible to bacterial and fungal infecti on under humid, stagnant condi tions (Griesbach, 2003).

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14 Commercial orchid production has not yet met demand (Griesbach, 2002) as evidenced by the steadily increasing number of orchids sold annually (Jerardo, 2006). As consumers become knowledgeable about growing Phalaenopsis they will undoubtedly become interested in growing other orchid genera. One genus that ma y be well suited for the growing orchid market is Vanda Modern breeding programs are changing the archetype of Vanda hybrids being difficult to grow and slow to bl oom (Grove, 1995; Motes, 1997). Vanda hybrids often exhibit large flowers, a floriferous habit, virtually pe st free culture and a wide color pallet (Motes, 1997). Ascocenda hybrids (Table 1-1 contains a list of pa rentages for hybrids that are listed in this chapter), for example, are t ypically compact in stature, form numerous large flowers and are free blooming (Fitch, 2005). Ot her bigeneric hybrids like Vandafinetia offer an even more compact habit with unusual flowers. Such plan ts may be marketable for windowsill cultivation (Grove, 1995). Although Vanda hybrids have many qualities that make them highly marketable, the production of Vanda hybrids is currently confined to small-scale growing operations (personal observation). Classification of Tribe Vandeae Classification of the vandaceous orchids has pr oven difficult and has lead to conflicting taxonomic visions of the group (Arditti and Er nst, 1992). Dressler (1981; 1993) placed Vanda and closely allied species into the largest of the Orchidaceae subfamilies, Epidendroideae, which includes Calypso Coelogyne Cymbidium Dendrobium Epidendrum and Maxillaria The common characteristics of this hi ghly variable subfamily include epiphytic habit, pseudobulbs or corms, fleshy leaves with distichous arrangemen t (often caduceus), lateral inflorescence, and hard pollinia with caduceus anthers (Dressler, 1993). Such a broad description of this subfamily warrants further taxonomic splitting, and so Dressler placed Vanda into the phylad Epidendroid (eight pollinia, often reduced to six, four, or

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15 two), and the subclad Dendrobiod (1993). Tr ibes of the Dendrobiod subclad (including Dendrobieae, Podochileae, and Vandeae) have distin ctive spherical silica bodies, or stegmata, present in their cells and typica lly lack pseudobulbs (Dressler, 1993) Interestingly, this subclad contains both sympodial and monop odial genera. Dressler (1993) wrote that these differences are minor, as [o]nly continued apical growth and rooting at the nodes is needed to convert [sympodial growth] to monopodial growth. Among the more recognized genera of the tribe Vandeae are Aerides Phalaenopsis and Vanda The Vandeae can be further divided into the subtribes Aeridinae, Angraecinae and Aerangidinae (Dressler, 1993). Th e Aeridinae consist of 103 gene ra of monopodial orchids with a Vanda -type velamen (multilayered with larger epivelamen cells marked by helical thickenings), two or four pollinia, one or more stipes and a viscidium (Arditti and Ernst, 1992; Dressler, 1993). Flower struct ures in the subtribe are vari able in both size and shape. Seed propagation of Vanda In the 1920s, Knudson (1922) demonstrated that orchid seed could be germinated without mycorrhyzal fungi by growing seed in vitro on medium containing mineral nutrients and sugar. Prior to this, Noel Bernard and Hans Burgeff had presente d evidence which supported the conclusion that orchid seeds could only be germinated symbiotically (discussed in Knudson, 1922). Seed germination studies have been conducted for several Vanda species and hybrids. Roy and Banerjee (2002) found that after th ree months culture, over 70% of immature Vanda tessellata seeds germinated on a variety of media. Bhaskar and Rajeevan (1996) reported fewer than 20% of immature Vanda John Club seeds germinated when sown on full-strength Murashige and Skoog (MS; Murashige and Sk oog, 1962) and Knudson C (KC; Knudson, 1946) media, and 80% germination of seeds cultured on half-strength Mura shige and Skoog medium (MS). Devi et al. (1998) we re able to germinate 90% of Vanda coerulea seeds using a wicking

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16 system with liquid Vacin and Went medium (VW; Vacin and Went, 1949) supplemented with Murashige and Skoog vitamins (Murashige a nd Skoog, 1962). Recently, Kishor et al. (2006) reported that seed of Ascocenda Kangla germinated readily when sown on VW containing 2.3 M kinetin and 0.5 M NAA (90% germination). The trend in Vanda hybridization programs is to breed increasingly more complex hybrids in an attempt to generate plants with superior flower form and color. Researchers have not yet compared the effect of media and photoperiod on seed germination of complex hybrids (those hybrids with more than two species in their heritage; i.e. greater th an F1 hybrids) in side-by-side experiments. For commercial growers of Vanda hybrids, understanding the influence of photoperiod and germination media on the growth and development of complex Vanda hybrids is important. If problems with growth and de velopment of complex hybrids are detected, the goals of breeders may need to be altered from fo cusing exclusively on flower characteristics. In order to make Vanda a commercially profitable crop, production of Vanda must be competitive with that of Phalaenopsis Breeders may need to focus on char acteristics such as vigor, time to first bloom, flower longevity, and floriferousness in order to mini mize the cost of growing plants and capitalize on mass market cons umption of flowering orchids. Micropropagation of Vanda While seed culture can be an effective means fo r producing plants in mass, it does not allow for the cloning of unique or highly prized specime ns. In 1960, Morel published a procedure for cloning Cymbidium via tissue culture. Th e tissue culture method, whic h was more completely described by Wimber (1963), invol ved isolating shoot tip s and culturing them aseptically in the presence of hormones and chemical nutrients un til they developed into a full plant (Morel, 1960). The first successful cloning of Cymbidium was recognized as a means to produce virusfree clones from prized plants that were entir ely contaminated with virus. Morel (1960;

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17 1964) and Wimber (1963) noted the inherent potential of this te chnique for the mass production of desirable plants for commercial sales. Since this early work, orchids have been su ccessfully propagated using roots, leaves, stems, flower buds and inflorescences as explan t materials (Arditti, 1993 ; Arditti and Ernst, 1992). Vanda has been notoriously difficult genus to micropropagate (Arditti, 1993; Morel, 1964), though successful tissue culture protocols have been published. Micropropagation has been used extensively with intergeneric Vanda crosses such as Aranda and Mokara both of which are grown extensively in the Indonesian fl ower markets (Arditti an d Ernst, 1992; Lee et al., 1996). Seed culture of Vanda is a productive way to mass produce plants, as previously discussed (Bhaskar and Rajeevan, 1996; De vi et al., 1998; Sharma, 1998), and seed derived protocorms can be used as explants to establish tissue culture lines (Mathews and Rao, 1979; Roy and Banerjee, 2002). However, the marketable quality of orchid seedlings is variable, making clonal propagation of awarded plants a mo re desirable method of multiplicat ion, if possible. The vast majority of the published work on the micropropagation of Vanda species, interspecific Vanda hybrids, intergeneric Vanda hybrids and Vanda allies has used shoot tips as explants (Cheah and Sagawa, 1978; Ghani et al., 1992; Kanika and Vij, 2004; Kunisaki et al., 1972; Lakshmanan et al., 1995; Malabadi et al., 2004; Seeni and Lath a, 1992; Teo et al., 1973; Van Le et al., 1999). When harvesting explants from sympodial orchids, sacrificing a shoot tip re sults in the loss of a single new growth, however, harvesting the api cal meristem from monopodial orchids like Vanda may result in plant death (Goh and W ong, 1990; Intuwong and Sagawa, 1973; Sharma and Vij, 1997; Vij et al., 1986). In addition, it can be difficult to excise and sterilize such large explants.

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18 Successful micropropagation of Vanda species and hybrids has been attempted using adult leaves (Lay, 1979; Sharma and Vij, 1997), axenic seedling leaves (Vij et al., 1986), undifferentiated inflorescence buds (Goh a nd Wong, 1990; Intuwong and Sagawa, 1973), differentiated flower buds (Valmayor et al., 1986), and inflorescence nodes (Decruse et al., 2003). Even with many reports of successful tissue culture of Vanda species and hybrids, it remains to be demonstrated that any one method can be applied to a wide range of Vanda The numerous methods by which Vanda have been tissue cultured may reflect a problem that must be assessed species-by-species or hybrid-by-hybri d. Additionally, incidences of somaclonal variation and potential alte rations to flower quality have not been studied in Vanda Random mutation has been reported in woody plants (Hashmi et al., 1997; Pogany and Lineburger, 1990; Tremblay et al., 1999; Vendrame et al., 1999) as well as herbaceous plants (Al-Zahim et al., 1999; Paek and Hahn, 1999; Thomas et al., 2006). Researchers have also found somaclonal variation in Phalaenopsis hybrids (Chen et al., 1998; Tokuhara and Mii, 2001), indicating that unwanted mutation could be a problem in the tissue culture of Vanda Initiation of Tissue Cultures Shoot tip explants Modified VW medium supplemented with 15% (v /v) coconut water (CW) has been used to produce protocorm like bodies (PLBs) from cultured shoot tips of Aranthera James Storei (Cheah and Sagawa, 1978), Vanda insignis Vanda tessellate (Teo et al., 1973), and Papilionanthe Miss Joaquim (synonym = Vanda Miss Joaquim; Kunisaki et al., 1972). While this may be an effective and highly appli cable method for mass clonal propagation of Vanda hybrids, none of these studies compared VW me dium supplemented with CW to other media. The medium of Mitra et al. (MM; Mitra et al ., 1976) has also been used for establishment of Vanda species and hybrids in culture. Kanika and V ij (2004) studied the effect of MM

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19 supplemented with auxins and cytokinins. They found that medium supplemented with 2.3 M 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 M 6-benzyladenine (BA), and 20% CW resulted in the formation of two to three PLBs in 100% of Vanda coerulea shoot tip explants. However, they did not examine the effects of other concen trations of 2,4-D and/ or BA on initiation of regeneration. Seeni and La the (2000) used MM supplemented with 10% CW, 500 mg L-1 peptone, 30 g L-1 sucrose, 8.8 M BA, and 4.1 M NAA to compare the response of shoot tips derived from mature plants and in vitro plantlets of V coerulea Testing both liquid and solid (7 g L-1 agar) media, they found that explants obtained from in vitro plantlets were 15% more responsive than explants harvested from flowering size plants. However, a shortcoming of this study was the small sample size (only five explants were tested for each media). Malabadi et al. (2004) studied the effects of th idiazuron (TDZ) on PLB production from shoot tips of V coerulea cultured on VW basal medium supplemented with 7 g L-1 agar, 2.0 g L-1 casein hydrolysate, 0.5 g L-1 L-glutamine, 1.0g L-1 meso-Inositol, 30 g L-1 sucrose, and 250 mg L-1 peptone. It was reported that the addition of 11.35 M TDZ resulted in optimum PLB production (96% of 30 cultures responded). TDZ has also been proven effective for production of PLBs on thin sectioned leaf explants of Doritaenopsis (Park et al., 2002). Lakshmanan et al. (1995) re ported PLB formation from Aranda Deborah shoot tips and thin sectioned shoot tips. They noted that addi tion of BA reduced the number of PLBs produced on explants cultured on VW, KC, and MS medi a supplemented with various amounts of CW. Valmayor et al. (1986) conducted a very exte nsive comparison of the effects of tissue culture media on regeneration from terminal flower buds of an Ascocenda hybrid, Vanda lamellata Ascocenda Tropicana. They screened 55 modified formulae of liquid KC, MS, VW media containing combinations of 2,4-D, BA, CW and kinetin (KN) for regenerative effects on

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20 terminal flower buds. Of all the explants test ed, only the youngest flow er buds initiated PLBs, and only when grown on KC with 15% CW and 4.65 M kinetin. Inflorescence and flower bud explants Intuwong and Sagawa (1973) presented a process for propagating newly emerged inflorescences of Ascofinetia Cherry Blossom in liquid VW with 15% CW and 20 g L-1. While they reported that other hybrids were also cult ured, a single inflorescence of both Vascostylis Blue Fairy and Neostylis Lou Sneary were cultured, but failed to produce PLBs. No other media formulae were tested for initiation of PLBs. Goh and Wong (1990) compared the effect of 0.2 M BA and 4.7 M KN alone and in combination on culture initiation using inflorescence tips of Aranda Deborah in modified formulae of KC and VW both with 15% CW and 20 g L-1 sucrose. They reported that none of the modified VW formulae were satisfactory for the production of PLBs or shoot induction. Explants cultured on KC contai ning CW, KN, and sucrose produced PLBs (32% of explants) or shoots (68% of explants). Explants cultured on KC supplemented with BA alone or with KN regenerated PLBs or shoots. Leaf explants Sharma and Vij (1997) reporte d PLB production using adult Vanda leaves. Newly emerged leaf explants of Vanda cristata cultured on MM containing 44.4 M BA and 26.9 M napthalaleneacetic acid (NAA) or 44.4 M BA and 28.6 M indole-3-acetic acid (IAA) produced PLBs only on media containing add itional copper II ions (2.2 mg L-1). Explants from leaves longer than 3 cm failed to regenerate. Only e xplants taken from the proximal one-third of each leaf formed PLBs. Seeni and Latha (1992) used solid MM supplemented with 20 g L-1 peptone and 20 g L-1 sucrose to test the response of newly emerged whole leaf explants of Renanthera imschootiana to NAA and BA alone and in combinati on for the production of PLBs. Inclusion

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21 of 44.4 M BA and 5.4 M NAA in the medium promoted initi ation of shoots in 80% of whole leaf explants. Seeni and Latha (2000) tested th e response of seedling leaf explants on MM containing 7 g L-1 agar, 10% CW, 500 mg L-1 peptone, 30 g L-1 sucrose, and various combinations of BA and NAA. They also tested the e ffect of leaf position on responsiv eness. After 12 weeks culture, 96% of second position leaves produced an average 13.6 PLBs when cultured on media containing 8.8 M BA and 4.1 M NAA. Leaves at the first, third, forth, and fifth positions were less responsive with respect to PLB produc tion. Seeni and Lathas attempts to initiate PLBs on adult leaves resulted only in ca llus formation after 12 weeks culture. In vitro derived plantlets have been used as leaf explant sources. Lay (1979) reported leaf bases of Aranda Noorah Alsagoff regenerated shoots wh en cultured on VW medium containing 50% CW and 20 g L-1 sucrose, or MS containing 15% CW, 1 mg L-1 glycine, 100 mg L-1 myoInositol, 0.25 mg L-1 nicotinic acid, 0.25 mg L-1 pyridoxine, 30 g L-1 sucrose, 1 mg L-1 thiamine, 9.05 M 2,4-D, and 8.9 M BA. Vij et al. (1986) induced initiation of Vanda testacea by culturing 0.5 cm thick leaf cr oss sections on MM, 2 mg L-1 peptone and either 2.9 M gibberellic acid or 4.4 M BA and 5.4 M NAA. Sheelavanthmath et al. (2005) reported on the effects of BA, KN, and TDZ on Aerides crispum leaf cross sections in MS medium with 10 g L-1 agar and 20 g L-1 sucrose. They were able to successfully initiate PLBs on 100% of leaf cross sections 3 mm thick on media containing 2 M BA. Explants formed an aver age of 22 PLBs after ei ght weeks of culture. Explants grown in the presence of 0.5 and 1 M TDZ produced PLBs on 83% of explants tested and an average 7.1 and 8.0 PLBs/explant, respectively.

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22 Development and Rooting of Protocorm Like Bodies Research on development and rooting of tissue cultured Vanda has focused on the effects of growth regulators and complex, undefined additi ves on shoot differentiation. While often not explicitly defined, shoot differen tiation probably refers to successful conversion of PLBs into leaf and root bearing seedlings, or direct organogenesis of shoots. Complex nutrients like banana powder and CW contain inorganic salts, amino acid s, plant hormones, sugars, and vitamins that effect plant development (Arditti and Ernst, 1992). Kunisaki et al. (1972) studied the effects of sucrose concentration and CW on Papilionanthe Miss Joaquim shoot tips cultured in solid VW medium and reported better differe ntiation when CW and 10 or 20 g L-1 sucrose were present, but did not support their findings wi th any measurements of success. Teo et al. (1973) reported that Vanda insignis Vanda tessellata PLBs failed to differentiate and perished on solid VW supplemen ted with 15% CW and sucrose, but developed shoots on solid sugar-free VW with CW, and produced both shoots and roots on solid VW containing sucrose. Seeni and Latha (2000) modi fied their initiation medium (modified MM) for Vanda coerulea shoot tips with 35 g L-1 ripe banana pulp, 30% CW and 1.08 M NAA, but did not report whether these additives lead to increased shoot differentiation. Decruse et al. (2003), studying Vanda spathulata reported on the effects of solid (6 g L-1 agar) MM containing different concentrations of BA and IAA alone and in combination on initiation of direct shoots. They reported that after three months cultu re, explants cultured on medium supplemented with 4.4.2 M BA produced one shoot. Hi gher BA concentrations (44.4.8 M) resulted in the production of less th an three shoots per explant. Explants cultured on medium containing 44.4 M BA with 17.1 M IAA and 66.6 M BA with 28.5 M IAA produced 12.6 and 12.1 shoots/explant, respectivel y. This indicates that the inclusion of both cytokinins and auxins may improve multiplication rates of Vanda cultures.

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23 Valmayor et al. (1986) conducted the most exte nsive research on shoot differentiation from Vanda lamellata Ascocenda Tropicana flower bud explants by testing the effects of three media (KC, MS, and VW) supplemented with va rious concentrations of complex, undefined nutrients, plant growth regulators (PGRs), and su crose. Their results indicate that many media were effective at inducing shoots: VC with 20 g L-1 sucrose, MS with 15% CW, KC with 15% CW, and KC amended with both 15% CW and 8.9 M BA. Malabadi et al. (2004) pub lished one of the few studies on root differentiation of Vanda in which the effects of various auxins (IAA, i ndolebutyric acid [IBA], and NAA) on rooting of in vitro derived shoots of Vanda coerulea were compared. Optimal results (87% rooting) were observed on half-strength VW containing 11.42 M IAA. VW basal medium with BA and IAA concentrations less than 26.9 M and 24.5 M, respectively, failed to induce root production. Seeni and Latha (1992) reported successful in vitro rooting of Renanthera imschootiana shoots in the presence of 5.4 M NAA and 1% activated charcoal (the exact medium used is unclear, but presumed to be MM with 20 g L-1 sucrose and 20 g L-1 peptone, as was used for initiation). Summary To date, no studies have conclusi vely assessed the feasibility for using any one micropropagation protocol for the tissue culture of interspecific and intergeneric Vanda hybrids. Given the number of potential species involved in any one breeding program, as well as the various ways in which hybrids are crossed and backcrossed, it seems unlik ely that any one protocol will work for all hybrids. Due to the limited br eadth of any one particular Vanda micropropagation protocol and the seemingly hybrid-specific requirements of all Vanda tested to date, seed propagation may be a more reliable means of producing large quantitie s of plants with minimal input. If so, the question then becomes whether Vanda seeds from unique and diverse lineages will germinate and grow under common in vitro conditions?

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24 Objectives OBJECTIVE I. Assess the effects of photoperiod and culture media on the germination and seedling development of Vanda hybrids of diverse breeding histories. OBJECTIVE II. Compare the seedling gr owth and development of Vanda hybrids using scanning electron microscopy and light microscopy. OBJECTIVE III. Interpret results in the cont ext of a growing market for potted flowering orchids and assess the feasibility of using seed propagation for the mass market production of Vanda hybrids.

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25 Table 1-1. List of interspecific and intergeneric Vanda hybrids refe renced in text. Parentage of registered hybrids was found on the International Orchid Register ( http://www.rhs.org.uk/plants /registration_orchids.asp ). Intergeneric hybrid names Parentage Aranda Arachnis Vanda Ascocenda Ascocentrum Vanda Doritaenopsis Doritis Phalaenopsis Mokara Arachnis Vanda Ascocentrum Intergeneric hybrids Parentage (seed parent pollen parent) Ascofinetia Cherry Blossom Neofinetia falcata Ascocentrum ampullaceum Aranda Deborah Arachnis hookeriana Vanda lamellata Aranda Noorah Alsagoff Arachnis hookeriana Vanda Dawn Nisimura Aranthera James Storei Arachnis hookeriana Renanthera storei Ascocenda Kangla Vanda coerulea Ascocentrum auranticum Ascocenda Tropicana Ascocentrum curvifolium Vanda Betsy Sumner Neostylis Lou Sneary Neofinetia falcata Rhyncostylis coelestis Vascostylis Blue Fairy Ascocenda Meda Arnold Rhynchostylis coelestis Interspecific hybrids Parentage (seed parent pollen parent) Papilionanthe Miss Joaquim Papilionanthe hookeriana Papilionanthe teres

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26 CHAPTER 2 ASYMBIOTIC SEED GERMINATION OF Vanda HYBRIDS Introduction Sales of potted flowering orchids in the United Stat es have risen since data was first collected by the USDA in 1996. Wholesales of potted orchids reached $100 million in 2001, and an estimated $144 million in 2005 (Jerardo, 2006). It has been estimated that as many as 90% of all orchids sold are Phalaenopsis species and hybrids (Nash, 2003) However, as consumers become accustomed to growing and displaying or chids in residential and commercial settings, the demand for other orchid genera may also increase. Extensive research on the physiology of Phalaenopsis flower induction (Blanchard and Runkle, 2006; Li et al., 2006; Su et al., 2001; Wang et al., 2002) has undoubtedly contributed to the commercial success of this genus by decreasing time to flowering, thus lowering per unit production costs and retail sale prices. Vanda hybrids may also have a profitable place in the expanding orchid market, though their production in the United States is at this time limited by small-scale production and the long time to flowering. The result is a relatively high production cost for Vanda hybrids compared to that of Phalaenopsis Vanda hybrids have many characteristics that ar e amenable to mass consumption including a variable color pallet, long lasting flowers, fr ee-blooming habit, fragrant flowers and multiple inflorescences. In addition, some hybrids exhibit a compact stature and cold tolerance. Research on the growth and development of Vanda could lead to more efficient methods to produce salable (i.e. flowering) plants, thus making Vanda both profitable for larg e-scale production and affordable for consumers in the mass market. While germination studies of Vanda species and hybrids have been conducted (Bhaskar & Rajeevan, 1996; Devi et al., 1998; Kishor et al., 2006; Roy and Banerjee, 2002), limited

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27 information regarding protocorm and seedling development has been published. Asymbiotic seed germination studies of terrestrial orchids ha ve revealed that germination alone may not be a reliable indicator of subsequent plant growth and development (H oshi et al., 1994; Kauth et al., 2006; Stenberg and Kane, 1998). The use of dis tinct morphological stag es (= developmental stages), can be used to more precisely compar e growth and development of different orchid species, hybrids, and/or cultivars. The objective of this study was to de scribe and compare the growth and development of three complex Vanda hybrids asymbiotically cultured on a range of in vitro media and under different photoperiodic regimes. Materials and methods Seed source. Seeds of three Vanda hybrids (Table 2-1) were provided by Motes Orchids (Homestead, FL). Seeds were removed from mature, undehisced capsules on 2 February 2006, and stored at 5% relative humidity and 20 5C. Seeds were removed from dry capsules five weeks after capsules were harvested, then tr ansferred to filter pape r packets (Whatman No. 2) and stored over desiccant for four weeks prio r to experimentation. A small sample of seeds (approximately 300) from each capsule was subjected to a tetrazolium (TZ) viability test (Lakon, 1949). Seeds were pretreated for 15 min in 5.0% Ca(OCl)2 before being soaked for 24 h in distilled deionized (DD) water at 25C, followed by a 24 h soak in 1.0% TZ in darkness at 30 C. Seeds were observed with a dissec ting microscope and scored as vi able (pink or red embryo) or nonviable (white embryo). Percent viability wa s calculated by dividing the number of viable embryos by the total numb er of embryos scored. Effect of asymbiotic media and photoperiod. Seed germination and subsequent protocorm development of Vanda hybrids were compared on th ree media (Table 2-2): Knudson C (KC), Phyto Technology Orchid Seed Sowing Medium (P723) and half-strength Murashige & Skoog (MS). All media were commercially prepared by Phyto Technology Laboratories

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28 (Shawnee Mission, KS). To standa rdize the concentrati on of activated charcoal (AC), agar, and sucrose in the screened media, the fo llowing modifications were made: 8 g L-1 TC agar ( Phyto Technology Laboratories) and 1 g L-1 AC were added to KC; 8 g L-1 TC agar, 1 g L-1 AC and 20 g L-1 sucrose were added to MS. All media were adjusted to pH 5.7 with 0.1 M KOH before autoclaving at 121C and 117.67 kPa. Medi um was then dispensed as 30 mL aliquots into 9 cm diameter Petri plates. Seeds were surface sterilized in a solution of ethanol:NaOCl (Clorox):sterile distilled deionized (DD) water (5:5:90) fo r two min followed by three rinses in sterile DD water. Surface sterilized seeds were placed back in sterile DD water, agitated w ith a vortex shaker to keep seeds suspended, and the volume of water adjusted until a 30 L aliquot consistently yielded 25 seeds (average 30.9). Petri plates we re then inoculated with three 30 L aliquots of seed suspension from one of three Vanda hybrids and sealed with a singl e layer of NescoFilm (Karlan Research Products Corporation, Cottonwood, AZ). Each aliquot was treated as a subreplicate. Petri plates containing one of three media and seeds from one of three Vanda hybrids were maintained at 23 2C under a 8/16 h, 12/12 h, or 16/8 h light/dark (L/D) photoperiod (3 factorial) provided by cool white fluorescent lights (General El ectric F96T12, Fairfield, CT) at 50 M m-2 s-1 for 12 weeks. Plates were observed ever y two weeks for signs of germination and subsequent protocorm development. Developmenta l stages (Table 3) were adapted from Stewart and Zettler (2002). Five replicates of each treatment were performed. Percentage of seed/protocorms in each stag e was calculated for each replicate by dividing the number of seeds/protocorms in each stage by the total number of seeds in each plate. Data on week 12 individual stage percenta ges was arcsine transformed to normalize data. Data was then analyzed using general linear modeli ng and least square mean separation ( = 0.05). Time

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29 course data was analyzed using general lineal modeling, standard error, and least square mean separation ( = 0.05). Statistical analysis was comple ted with SAS v 9.1 (SAS Institute Inc., Cary, NC). Scanning electron microscopy. S014 seeds and protocorms were used for examination by scanning electron microscopy (SEM) since othe r hybrids did not develop beyond Stage 3. All samples were fixed in 10% FAA under vacuum pre ssure for at least 24 h before dehydration in a graded ethanol series and subsequently critical point dried (CPD). Stage 1 protocorms were processed in methanol since samples dehydrated in ethanol tended to collapse during CPD. Samples were gold sputter coated for 30 s at 50 mA. Samples were then observed and digital images captured with a Hitachi S4000 scanning electron microscope. Light microscopy. Stage 4 and 5 seedlings from S014 were fixed in FAA as previously described before being embedded in paraffin wax and sectioned at 4 m at the Molecular Pathology and Immunology Core Lab (University of Florida, Gainesville). Staining procedures were modified from Sakai (1973). Paraffin embe dded longitudinal cross-sections were stained with 0.05% toluidine blue O for 15 min, rinsed wi th water, and air dried. Paraffin was then removed with two 60 sec rinses in Hemo-De (Sci entific Safety Solvents; Keller, TX), a xylene substitute, before cover slips were mounted with Pro-Texx (American Scientific Products; McGaw Park, IL). Slides were observed with a Nikon Labophot-2 microscope. Digital images were captured with a Nikon Coolpix 990. Results Effects of asymbiotic media and photoperiod. After two weeks culture, seeds of all three Vanda hybrids had begun to germinate under all c onditions tested (Fig. 2-1). Cumulative germination of S005 (34.1.5%) under all cultu re conditions of media and photoperiod was significantly lower than S013 (50.8.7%) and S014 (82.0.3%). S005 seeds cultured under

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30 8/16 h L/D photoperiod initially germinated to a higher percentage th an S005 seeds cultured under 12/12 h and 16/8 h L/D photope riods, but cumulative differen ces in germination after 12 weeks were not significantly different betw een treatments (Fig. 2-1 A-C). Cumulative germination of S013 was similar under all cult ure conditions (Fig. 21 D-F). Cumulative germination of S014 on P723 under 8/16 h ( 95.5%) and 16/8 h (95.3%) L/D photoperiods was significantly higher than seeds cu ltured on KC and MS under all photoperiods (Fig. 2-1 G-H). Cumulative germination (93.7%) of S014 cultu red on P723 under a 12/12 h L/D photoperiod was significantly greater that of seeds cultured on MS under al l photoperiods and seeds cultured on KC under 12/12 h and 16/8 h L/D photoperiods. In addition, maximum germination of S014 seeds cultured on P723 occurred by week eight, whereas seeds cultured on KC or MS did not reach maximum observed germination until 1012 weeks of culture. TZ testing indicated that seeds were 53.5% (S005), 75.4% (S 013) and 92.0% (S014) viable. Embryos of S005 cultured on KC under 12/ 12 h or 16/8 h L/D photoperiods did not develop beyond Stage 1 (34.1% a nd 36.9%, respectively) in the 12 weeks they were observed (Fig. 2-2 A-C). Few Stage 2 protocorms ( 0.3%) developed under 8/16 h L/D photoperiod. S005 embryos developed to Stage 2 when culture d on MS (0.7.6%; Fig. 2-2 D-F) while seeds cultured on P723 (Fig. 2-2 G-I) developed to Stage 3 (0.7.7%) under all photoperiods screened, and Stage 5 under 12/12 h (0.2%) and 16/8 h (0.2 %) L/D photoperiods. Stage 4 protocorms were not observed. S013 embryos did not develop beyond Stag e 2 when cultured on KC (0.4.8%; Fig. 2-3 A-C) and MS (6.1.1%; Fig. 23 D-F). However, seeds cultured on P723 developed significantly more Stage 2 protocorms ( 36.8.2%) under all photoperiods. For S013 seeds cultured on P723 (Fig. 2-2 G-I), Stage 1 protoc orms were most abundant at week two (34.1

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31 45.1%). However, as Stage 1 protocorms continued to develop to more advanced stages, the percentage of Stage 1 protocorms decreased to 12.8% (8/16 h L/D), 15.2% (12/12 h L/D), and 16.1% (16/8 h L/D) by week 12. Stage 3 prot ocorms of hybrid S013 were observed on P723 under 8/16 h L/D (0.8%) photoperiod a nd were not significantly different than 0% when cultured on P723 under 16/8 h L/D (0.3%) photoperiod. When cultured on KC (Fig. 2-4 A-C) and M S (Fig. 2-4 D-F), seeds of S014 did not develop beyond Stage 2 regardle ss of photoperiod. However, seeds cultured on P723 developed to Stage 5 (Fig. 2-4 G-I) under a ll photoperiods tested. Stage 1 protocorms (Fig. 2-5 B) were most abundant after two week s culture on P723 under all phot operiods (8/16 h L/D, 51.5%; 12/12 h L/D, 54.2%; 16/8 h L/D, 44.7%). A majority of seeds cultured on P723 had developed to Stage 2 protocorms (Fig. 2-5 C) by week four with the highest percentage of Stage 2 seedlings observed during week six (8/16 h L/D, 60.7%; 12/12 h L/D, 61.9%; 16/8 h L/D, 68.2%). By week 10, S014 seeds had developed to Stages 3 (Fig. 2-5 D-F) on P723 under all three photoperiods tested. Final percentages of Stage 3 protocorms were not si gnificantly different for seeds cultured on P723 under different photope riods (8/16 h L/D, 13.1%; 12/12 h L/D, 14.2%; 16/8 h L/D, 14.2%). Significantly more Stage 4 protocorms were observed on P723 treatments under 12/12 h (5.0%) and 16/8 h (4.8%) than under 8/16 h L/D (0.3%) photoperiod. Significantly more Stage 5 seedlings were obs erved on P723 replicates cultured under 16/8 h L/D (7.9%) than those cultured under 8/16 h (2.7%) and 12/12 h L/D photoperiod. Visible contamination rate over all treatments was 8.9%. Developmental sequence. Unimbibed seeds of the three Vanda hybrids (Fig. 2-5 A) used in this study differed greatly in size (approximate lengths: S005 132 m; S013 172 m; S014 190 m). Some embryos of all hybrids imbi bed to the point of testa rupture by

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32 week two (Fig. 2-1; Fig. 2-5 B). Stage 1 protoc orms were polarized with the suspensor end of the embryo comprised of much smaller cells than t hose in the apical regio n. By week four, some protocorms had developed to Stag e 2 (Fig. 2-5 C) with slightly elongated apical regions defining the protomeristem. Rhizoids were occasionally present on Stage 2 protocorms. With further development, the protomeristem developed an a ngular opening from which the first true leaf emerged (Stage 3, Fig. 2-5 D). At least one true root typically em erged from protocorms at this time (Stage 4, Fig. 2-5 E) and prior to the emerge nce and elongation of a second true leaf (Fig. 25 F). Thick sections of Stage 5 seedlings reve aled a collar through whic h leaves emerged (Fig. 2-6 A), which constituted a larg e portion of the protocorm body. The apical meristem (Fig. 2-6 B) was spade-shaped and located near the center of the protocorm. Lateral meristems (Fig. 2-6 C) were found along leaf axes in Stag e 4 protocorms and Stage 5 seedlings. Observations on subculture of Vanda hybrids. Three month old S014 seedlings were transferred to newly prepared P723 in sterile Phyto Tech Culture Boxes ( Phyto Technology Laboratories; product # P700) and sealed with a single layer of NescoFilm. Approximately three months after transfer, a proportion of seedlings began to exhibit symptoms of decline (loss of chlorophyll, tissue translucency, ce ssation of growth; Fig. 2-7 A). Believing this to be the result of media depletion or ethylen e accumulation, six month old s eedlings that did not exhibit symptoms were transferred to fresh Phyto Tech Culture Boxes containing P723. These vessels were not sealed with NescoFilm. Seedlings ag ain displayed symptoms of decline following the second transfer. Culture indexing with liquid Leif ert and Waites Sterility Test Medium (Leifert et al., 1989) indicated that tested seedlings did not harbor pa thogens. Twelve months after seeds were sown, nearly all seedlings lost pigmenta tion, became transparent, and were considered

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33 dead. A small number of seedlings formed callus (Fig. 2-7 B), some of which regenerated shoots and/or somatic embryos (Fig. 2-7 C). Discussion Few studies on germination and seedling production of Vanda species and hybrids have been conducted (Bhaskar and Rajeevan, 1996; Roy a nd Banerjee, 2002). Anatomical studies of Vanda seeds and protocorms have focused on embryo development (Swamy, 1942) and early protocorm ultrastructure (Ri cardo and Alvarez, 1971). Ricar do and Alvarez (1971) included a diagram of a Vanda protocorm with emerging leaves, howev er this figure did not depict the presence of an apical dome or protocorm collar as illustrated herein. This report constitutes the first comparison of germination and seedling development of Vanda hybrids under several different asymbiotic culture conditions. In this study, S014 seeds cultured on KC exhibited significan tly greater percentage germination compared to S014 seeds cultured on MS. These results differ from t hose reported by Bhaskar and Rajeevan (1996) who reported that the hybrid Vanda John Club, germinated better on M S (80%) than on KC (20%). In addition, germination of Vanda tessellata seeds cultured on various modified formulations of MS and KC did not differ in final percent ge rmination (Roy and Banerjee, 2002). Similar to hybrids S005 and S013, Vanda tessellata grew slowly and produced few advanced stage seedlings, even after three months culture. TZ based estimates of viability for hybrids S005 (53.5%) and S013 (75.4%) were considerably greater than observed maximum germination (43.5% and 61.7%, respectively). Viability estimat es for hybrid S014 (92.0%) were comparable to observed germination on P723 (93.7.3%). It is possible that the sterili zation protocol used in this study impaired the germination of hybrids S005 and S013 by damaging the embryos, however this hypothesis does not account for the concurrence between observed germination and estimated viability of hybrid S014. However, orchid seed viability estimates do not appear to be

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34 good indicators for germinability (Lauzer et al., 1994; Shoushtari et al., 1994; Vujanovic et al., 2000). Therefore, germinability should be tested as well as viability. Protocorm development of Vanda hybrid seeds cultured on MS was consistently more advanced than that of seeds cultured on KC. Th is may be due to greate r total nitrogen or a higher NO3:NH4 ratio in MS compared to KC. Cur tis and Spoerl (1948) reported that Vanda tricolor seeds cultured on media containing NO3 developed more rapidly then seeds cultured in the presence of NH4 when nitrogen concentrations were kept constant. This is in contrast to reports of the nitrogen preference of Calopogon tuberosus, Cattleya and Cymbidium These orchids were better able to utilize NH4 than NO3 during germination and early growth (Curtis and Spoerl, 1948; Kauth et al., 2006 ; Raghavan and Torrey, 1964). In these species the ability to utilize nitrate may be delayed until nitrate redu ctase is synthesized (approximately 60 days after imbibition for Cattleya ; Raghavan and Torrey, 1964). Nitrate reductase in Vanda may be synthesized or activated sooner after imbibition than other studied species. Protocorm development of all three Vanda hybrids was significantly greater on P723. This medium has also been shown to support high germination percentage and development of C. tuberosus (Kauth et al., 2006). P723 contains less total ammonium and nitrogen than either MS or KC, but contains additi onal organic nitrogen in the form of peptone. Peptone has also been shown to promote germination and development of V. tricolor Paphiopedilum species, Prosthecia cochleata and Spathoglottis plicata (Curtis, 1947). Clear trends in the effect of photoperiod were not obser ved for hybrids S005 and S013, while advanced development of S014 was sli ghtly greater for seeds cultured on P723 under longer photoperiods. S014 seeds cultured on P723 developed signifi cantly more Stage 4 protocorms under 12/12 h and 16/8 h L/D photoperiods, as well as more Stage 5 seedlings under

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35 a 16/8 h L/D photoperiod. Preliminary data indicated that Vanda hybrid seeds cultured in dark (0/24 h L/D) germinated poorly and embryos did not develop as rapidly as those cultured in lighted conditions. Although further study is neede d, this may indicate that culturing seeds under longer photoperiods results in more rapid protocorm development. It is unclear why subcultured S014 seedlings lost pigmentation and died following transfer to fresh media. Since Phyto Tech culture boxes seal tightly, a possible cause of seedling decline is accumulation of ethylene in the head space. If accumulation of gaseous respiratory byproducts is the problem, in vitro culture of Vanda may be improved by aerating cu lture vessels (Lai et al., 1998), using photoautotrophic culture (Nguyen et al ., 2001; Xiao et al., 2003) or incorporating ethylene inhibitors in the medium (Brar et al., 1999). Another possible explanation for seedling death is that transferred seedlings were not able to adjust to the sudden cultural change from depleted media to fresh media with relatively hi gher mineral salt concentr ations. If the observed seedling death is linked to subcu lture, seedlings may benefit from being sown in large culture vessels containing enough media to support long-term growth. Alte rnatively, media that support germination and early development may not meet the nutritional requiremen ts of older seedlings. Callus formation from germinating seeds has been documented with V. tessellata (= Vanda roxburghii ; Bose and Mukerjee, 1974) and Vanda coerulea (Devi et al., 1998). Screening for genotypes that produce callus was not an objective of this stu dy. However, callus-forming genotypes may be useful for studying both the factors that induce callus formation of Vanda and the factors that promote shoot organ ogenesis, embryogenesis, and conversion. When studying the growth and development of a genus as large and interbred as Vanda comparing several genotypes, species, or hybrid s can be valuable. Had hybrid S014 been excluded in this study, the mistaken conclusion c ould have been drawn that the media and/or

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36 photoperiods tested in this study were inad equate for supporting advanced growth of Vanda hybrids since hybrids S005 and S013 did not devel op to advanced stages under most conditions tested. However, since S014 seeds germinated ra pidly and developed to advanced stages, it can not be ruled out that S005 and S013 have low seed vigor or that protocorms of these two hybrids exhibit symptoms of inbreeding depression. Such problems may contribute to slow growth and a long time to flowering of certain Vanda This may be the result of Vanda breeders tendency to breed for improved flower form, size, and colo r (features that are r ecognized and awarded by orchid societies) while disregarding the impor tance of rapid growth and development in a commercial setting. The current breeding trends which cater to a hobbyist niche market, could be limiting the potential success of Vanda hybrids in the expanding orch id market. In addition, commonly employed breeding practices such as sibling mating and backcrossing (two techniques used to produce plants that are homozygous for r ecessive flower alleles) may lead to the buildup of deleterious alleles. Such practices could contribute to poo r seedling germination and vigor.

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37 Table 2-1. Parentage of Vanda hybrids used for experimentation (seed parent po llen parent). Parentheses are used when a pare nt is itself an unnamed hybrid. ID Hybrid S005 Vanda Paki ( Vanda tessellata Vanda cristata ) S013 ( Vanda Joan Warne Vanda Paki) Vanda Loke S014 Vanda Motes Primrose Ascocenda Tavivat

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38 Table 2-2. Composition of asymbiotic media used to test the effects of photoperiod on germination and development of Vanda hybrids S005, S013, and S014. S005 Vanda Paki ( Vanda tessellata Vanda cristata ), S013 ( Vanda Joan Warne V. Paki) Vanda Loke, S014 Vanda Motes Primrose Ascocenda Tavivat, KC Knudson C, MS half-strength Murashige & Skoog, P723Phyto Technology Orchid Seed Sowing Media. KC P723 MS Macronutrients (mM) Ammonium 13.8 5.15 10.31 Calcium 2.12 0.75 1.5 Chlorine 3.35 1.5 1.5 Magnesium 1.01 0.62 0.75 Nitrate 10.499.85 19.7 Potassium 5.19 5.01 10.02 Phosphate 1.84 0.31 0.63 Sulfate 4.91 0.71 0.86 Sodium 0.10 1.51 Micronutrients ( M) Boron 30 50 Cobalt 0.03 0.11 Copper 0.03 0.10 Iron 90 50 50 Iodine 1.20 2.50 Manganese 30 30 37.9 Molybdenum 26 0.52 Zinc 9.2 30 Vitamins (mg L-1) Myo-Inositol 100 Nicotinic acid 1 Peptone 2000 Pyridoxine 1 Thiamine 10 Total N (mM) 24.31unknown30.01 NH4:NO3 1.32 0.52 0.52

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39 Table 2-3. Developmental stages of Vanda hybrids. Stage Description 0 Ungerminated seed with embryo 1 Enlarged embryo, testa ruptured (= germination) 2 Appearance of protomeristem and/or rhizoids 3 Emergence and elonga tion of first leaf 4 One leaf and one or more roots present 5 Presence of two or more leav es, roots present (= seedling)

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40 GTime (wks) 024681012 0 20 40 60 80 100 H 024681012 2D Graph 2 024681012 KC MS P723S005 S013S014 D Germination (%) 0 20 40 60 80 100 E F A 0 20 40 60 80 100 B 8/16 h L/D 12/12 h L/D 16/8 h L/D C I MediumHybrid Figure 2-1. Germination of Vanda hybrids cultured on three di fferent media under an 8/16 h, 12/12 h, or 16/8 h light/dark photoperiod. Re d lines represent es timated viability based on tetrazolium staining. A) V anda Paki ( Vanda tessellata Vanda cristata ) (S005) cultured on Knudson C Medium (KC) B) S005 cultured on half-strength Murashige and Skoog Medium (MS). C) S005 cultured on Phyto Technology Orchid Seed Sowing Medium (P723). D) ( Vanda Joan Warne V Paki) Vanda Loke (S013) cultured on KC. E) S013 cultu red on MS. F) S013 cultured on P723. G) Vanda Motes Primrose Ascocenda Tavivat (S014) cultured on KC. H) S014 cultured on MS. I) S014 cultured on P723.

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41 G 024681012 0 10 20 30 40 50 D % Seedlings in Stage 0 10 20 30 40 50 A 0 10 20 30 40 50 HTime (wks) 024681012 I 024681012 E f 8/16 h L/D12/12 h L/D16/8 h L/DKCMSP723 B C Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 PhotoperiodMediumF Figure 2-2. Protocorm development (see Table 2-3 for stage definitions) of Vanda Paki ( Vanda tessellata Vanda cristata ) (S005), cultured for 12 weeks on Knudson C Medium (KC), half-strength Murash ige & Skoog Medium (MS), or Phyto Technology Orchid Seed Sowing Medi um (P723) under an 8/16 h, 12/12 h, or 16/8 h light/dark (L/D) photoperiod. A) KC, 8/16 h L/D. B) KC, 12/12 h L/D. C) KC, 16/8 h L/D. D) MS, 8/16 h L/D. E) MS, 12/12 h L/D. F) MS, 16/8 h L/D. G) P723, 8/16 L/D. H) P723, 12/12 h L/D. I) P723, 16/8 h L/D.

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42 G 024681012 0 10 20 30 40 50 60 HTime (wks) 024681012 I 024681012 8/16 h L/D12/12 h L/D16/8 h L/DKCMSP723 D % Seedlings in Stage 0 10 20 30 40 50 60 E A 0 10 20 30 40 50 60 B C Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 FPhotoperiodMedium Figure 2-3. Protocorm development (see Table 2-3 for stage definitions) of ( Vanda Joan Warne Vanda Paki) Vanda Loke (S013), cultured for 12 weeks on Knudson C Medium (KC), half-strength Murashige & Skoog Medium (MS), or Phyto Technology Orchid Seed Sowing Medium (P723) under an 8/16 h, 12/12 h, or 16/8 h light/dark (L/D) photoperiod. A) KC, 8/16 h L/D. B) KC, 12/12 h L/D. C) KC, 16/8 h L/D. D) MS, 8/16 h L/D. E) MS, 12/12 h L/D. F) MS, 16/8 h L/D. G) P723, 8/16 L/D. H) P723, 12/12 h L/D. I) P723, 16/8 h L/D.

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43 G 024681012 0 20 40 60 80 100 HTime (wks) 024681012 I 024681012 8/16 h L/D12/12 h L/D16/8 h L/DKCMS P723 D % Seedlings in Stage 0 20 40 60 80 100 A 0 20 40 60 80 100 Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 E F B C PhotoperiodMedium Figure 2-4. Protocorm development (see Table 2-3 for stage definitions) of Vanda Motes Primrose Ascocenda Tavivat (S014), cultured fo r 12 weeks on Knudson C Medium (KC), half-strength Murashige & Skoog Medium (MS), or Phyto Technology Orchid Seed Sowing Medium (P723) under an 8/16 h, 12/12 h, or 16/8 h light/dark (L/D) photoperiod. A) KC, 8/16 h L/D. B) KC, 12/12 h L/D. C) KC, 16/8 h L/D. D) MS, 8/16 h L/D. E) MS, 12/12 h L/D. F) MS, 16/8 h L/D. G) P723, 8/16 L/D. H) P723, 12/12 h L/D. I) P723, 16/8 h L/D.

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44 Figure 2-5. Scanning electron micrographs of th e germination and early development of hybrid S014 ( Vanda Motes Primrose Ascocenda Tavivat) (see Table 2-3 for stage definitions). A) Stage 0 seed. Scale bar = 50 m. B) Stage 1 protocorm with ruptured testa (T). Scale bar = 100 m. C) Stage 2 protocorm with developing protomeristem (PM) and rhizoids (RZ). Scale bar = 0.5 mm. D) Stage 3 protocorm with emerging first leaf (FL). Scale ba r = 1.0 mm. E) Stage 4 protocorm with emerging root (RT). Scale bar = 1.0mm. F) Stage 5 seedling with elongating second leaf (SL). Scale bar = 1.0 mm.

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45 Figure 2-6. Thin section light micrographs of hybrid S014 ( Vanda Motes Primrose Ascocenda Tavivat). A) 4 m thin section of Stage 5 seedling with first leaf (FL), second leaf (SL), third leaf (TL), and root (RT) present. Scale bar = 0.5 mm. B) High magnification image of a seedling apical meristem (AM). Scale bar = 200 m. C) Thin section through the apical dome of a seedling containing a lateral meristem (LM). Scale bar = 100 m.

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46 Figure 2-7. Twelve month ol d cultures of hybrid S014 ( Vanda Motes Primrose Ascocenda Tavivat) cultured on Phyto Technology Orchid Seed Sowing Medium (P723). A) Typical culture of dead seedli ngs that have lost pigmentatio n and become transparent. Scale bar = 2 cm. B) Mass of tissue with globular masses, which appear to be protocorm like bodies. Scale bar = 2.5 mm. C) Embryogenic and/or organogenic callus with differentiating s hoots. Scale bar = 2.5 mm.

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47 APPENDIX ATTEMPTS TO INITIATE Vanda TISSUE CULTURES Introduction Propagation of orchids from seed can be an efficient method of commercial production. However, the quality of individua l seeds resulting from any singl e breeding event can be highly variable in both growth rates and flower characte ristics due to recombinat ion. It is only through clonal propagation that the unique genetics of an awarded specimen can be retained. Clones of unique and desirable plants are often in demand, but developing a salable crop of plants through traditional methods of plant division is a slow process. In such cases, growers and producers may be able to employ micropropagation techniqu es in order to rapidly and reliable produce plants of identical or nearly identical quality. As discussed in Chapter 1, micr opropagation of flowering size Vanda species and hybrids has been documented by many different research ers (Cheah and Sagawa, 1978; Decruse et al., 2003; Ghani et al., 1992; Goh and Wong, 1990; In tuwong and Sagawa, 1973; Kanika and Vij, 2004; Kunisaki et al., 1972; Lakshmanan et al., 1995; Lay, 1979; Le et al., 1999; Malabadi et al., 2004; Seeni and Latha, 1992; Sharma and Vij, 1997; Teo et al., 1973; Valmayor et al., 1986). Taken as a whole, the body of literature on Vanda micropropagation indicates that there are many suitable explants for initiati ng cultures, but the effectiveness of any one protocol appears to be species, hybrid, or genotype specific. During the course of this research, several attempts were made to initiate cultures of various Vanda hybrids from both flowering size plants and seedlings. Attempts to initiate Vanda cultures from flowering size plants and seedling explants were conducted, but not in the context of a scientific experiment. An experiment on the effect of various plant growth regulatos (PGRs) on PLB formation from seedling leaf explants was conducted and is also included in this

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48 appendix. Attempts to establish cultures were largely unsuccessful, alth ough a single culture of ( Vanda Motes Honeybun Vanda Rasri Gold) Vanda Rasri Gold was established (Fig. A-1). The purpose of this appendix is to provide info rmation on the initiation protocols that were attempted so as to provide information to future researchers. Initiation of Vanda Tissue Culture Lines Using Newly Emerged Leaves of Flowering Size Plants Explants from newly emerged leaves of Vanda cristata less than 3 cm in length cultured on modified MM regenerated PLBs when cultured in the presence of high conc entrations of BA and NAA combined (44.4 M and 26.9 M, respectively), and BA and IAA combined (44.4 M and 28.6 M, respectively; Sharma and Vij, 1997). When culturing monopodial orchids such as Vanda initiating tissue cultures from leaf explants is an attractive option since harvesting shoottips may result in plant death (Goh and Wong, 1990; Sharma and Vij, 1997; Vij et al. 1986) and can be difficult to steril ize (personal observation). Materials and Methods Plant material. Newly emerged leaves less than 4 cm in length were removed from flowering sized plants of Vanda tessellata Vanda Arjuna, and Ascocenda Motes Burning Sands. Leaves were washed in running tap water for 15 min, rinsed in 70% ethanol for 30 s and surface sterilized in a solution of 1.2% NaOCl (v:v; 1:5; Clorox bleach: sterile di stilled deionized [DD] water) for 12 min, then rinsed three times in st erile DD water for 60 s per rinse. Each surface sterilized leaf was cut in half along the midve in and cut into two or three sections, each approximately 1 cm2. Each leaf half was treated as an e xplant and inoculated into a single Petri plate containing one of five media. Culture media and culture conditions. Explants were cultured on one of five solid media: Knudson C Medium (KC) with 15% (v/v ) coconut water (CW), Vacin and Went medium

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49 (VW) with 15% CW, Mitra et al. medium (MM) with 15% CW, MM containing 22.5 M 6benzyladenine (BA) and 13.5 M -napthalaleneacetic acid ( NAA), and MM containing 45 M BA and 27 M NAA. Basal media were ad justed to pH 5.7 with 0.1 M NaOH before autoclaving for 40 minutes at 121C and 117.67 kPa. PGRs were filter steri lized and added after media was autoclaved and cooled. Autoclaved me dia were dispensed as 30 mL aliquots into 9 cm diameter Petri plates and allowed to harden. After inoculation, plates were sealed with a single layer of NescoFilm and cu ltured at 23 2C under a 16/8 h light/dark (L/D) photoperiod provided by cool white fluorescent lights at 40 M m-2 s-1. Results and Discussion Leaf explants from the three Vanda hybrids screened did not respond to any of the media tested. Contamination rates were high: approximately 75% of Ascocenda Motes Burning Sands cultures, 60% of Vanda Arjuna cultures and 10% of Vanda tessellata leaf explants were visibly contaminated. This may indicate that the su rface sterilization proc edure employed was not adequate. The fungal and/or bacterial load on Vanda explants may need to be reduced prior to explant harvest. Sharma and Vij (1997) soaked leaves of wild collected Vanda cristata in 0.1% streptomycin for 20 min followed by 15 min in 0.1% HgCl2, but did not report contamination rates. In addition, stage 1 contamination may be reduced by selecting pathogen-free donor plants (Leifert and Cassells, 2001), sele cting plants with lower bacter ial/fungal loads, or by treating donor plants with antibiotics or fungicides prior to explan t harvest (Nagy et al., 2005). Within two months of culture initiation, all e xplants from the three hybrids screened turned black and were presumed dead. The screened cu lture media or culture conditions may not have been conducive to regeneration. Explan ts from newly emerged leaves of V. cristata cultured on modified MM regenerated PLBs when cultured in the presence of high c oncentrations of BA (44.4 M), NAA (26.9 M) and a combination of BA and indole-3-acetic acid (44.4 M and

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50 28.6 M, respectively; Sharma and Vij, 1997). Leaves from adult Renanthera imshootiana plants also required a relativel y high concentration of BA co mbined with NAA (44.4 M and 4.1 M, respectively) for maximum shoot regeneratio n (80% response; 38 shoo ts/explant; Seeni and Latha, 1992). It is unknown whether similar protocols can be used for Vanda hybrids. Initiation of Vanda Tissue Culture Lines Using Seed lings, Seedling Leaves, and Cut Protocorms in Liquid Culture The establishment of orchid tissue cultures from seedling explants is well documented (see Chen and Chang, 2006; Hoppe and Hoppe, 1988; Kuo et al., 2005; Lu, 2004; Park et al., 2002; Sheelavantmath et el., 2000; Vaz et al., 2004; Ya n, 2005). Sterile seedlings of many orchid genera can be easily produced using asymbiotic germination. Establishing cultures this way may bypass difficulties associated with sterilizing seasoned plant mate rial. In addition, young plant material has been shown more responsive than more mature explants in many non-orchid species (Becerra et al, 2004; Hoque and Mansfield, 2004; Molina et al., 2002). However, the genotype and plant quality of cultures init iated from seedlings is unknown. The objective of this protocol was to assess the regenerative potential of various explants from hybrid Vanda seedlings. Materials and Methods Plant material. Four month old seedlings of Vanda tessellata V anda Arjuna served as explants. Seeds were sterilized as described in Chapter 2 and sown onto Phyto Technology Orchid Seed Sowing Medium (P723). Seeds were first cultured under 8/16 h, 12/12 h, or 16/8 h L/D photoperiod for four weeks, then transferred to a common growth chamber under a 16/8 L/D hour photoperiod for six months. F our different explants were us ed in this protocol: intact protocorms, bisected protocorm halves, excise d intact leaves, and bi sected leaf halves. Culture media and culture conditions. Two different media were used for this investigation: KC with 15% CW (v/v) and Orchid Multiplication Medium ( Phyto Technology cat.

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51 No. P793). Media were adjusted to pH 5.7, dispen sed as five mL aliquots into 25 mL screw cap test tubes, and sterilized at 121C and 117.67 kPa for 10 min. Test tubes containing sterile media were capped and sealed with NescoFilm. Test tube s were placed in a roller drum (Bellco Glass, Inc., Vineland, NJ) at 10 rpm under 16/8 h L/D photoperiod. Illumination was provided by cool florescent lights at a maximum of 58 M m-2s-1. Protocol Design. All replicates contained three subreplicates. Seven replicates of all explant media combinations were complete d. Explants were observed for signs of regeneration after four weeks culture. Results and Discussion A few leaf explants and bisected protocorms cultured in KC with 15% CW showed signs of regeneration. Responding cultures were transfe rred to 250 mL Erlenmeyer flasks containing 50 mL of sterile KC with 15% CW (v/v) and seal ed with aluminum foil and NescoFilm. Cultures were placed on a G10 Gyratory Shaker (New Br unswick Scientific, Edison, NJ) and agitated at 60 rpm. Regenerating leaf explants became n ecrotic under these conditions and many were discarded. Three responding bi sected protocorm explants c ontinued to grow and produced between two and five shoots after approximately four weeks culture. Cultures began to show signs of necrosis after approxima tely four months culture and se veral transfers to fresh liquid medium. Explants were eventu ally transferred to KC with 15% CW solidified with 0.8% TC agar. One culture responded positively to solid media (Fig. A-1 A), while two cultures became necrotic. The proliferating cu lture produced numerous shoots and was subsequently subcultured (Fig. A-1 B). Liquid culture has been shown to be an e fficient method of propagating agronomic crops such as potato (Akita and Ohta, 1998; Jimnez et al., 1999; Piao et al., 2003), and various ornamental crops (Adelberg and Toler, 2004; Paek et al., 2001; Prasad and Gupta, 2006)

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52 including Phalaenopsis (Young et al., 2000). Such a method may also be effective in multiplication of Vanda cultures. Initiation of Vanda Tissue Culture Lines Using Seedling Leaf Sections Leaf explants have several advantages over s hoot tips as donor material for tissue culture. Leaves are easier to excise from plants than s hoot tips (especially when working with seedlings), and excision of leaves is less traumatic to donor pl ants. Finally, the surfac e area of a leaf offers the potential for very efficient and rapid rates of multiplication given the probable single cell origin of PLBs, provided that a suitable prot ocol can be developed. Leaf explants from terrestrial orchid sp ecies and several epi phytic genera including Vanda have been used to establish in vitro cultures (Chen et al., 1999; 2003; Chung et al., 2005; Churchill et al., 1973; Kuo et al., 2005; Park et al., 2002; Seeni and Latha, 2000; Yam and Weatherhead, 1991). Much tissue culture resear ch has been done with Phalaenopsis as it is the most important genus of orchids commercially available (Griesb ach, 2003). Researchers ha ve demonstrated that thidiazuron, in concentrations ranging from 3 M, is an effective PGR for inducing PLBs from Phalaenopsis seedling leaf explants (Chen and Chang, 2006), Doritaenopsis seedling leaf explants (Park et al, 2002), and 18 month old Phalaenopsis leaf explants (K uo et al, 2005). The objective of this experiment was to test the e ffects of cytokinin type and concentration on PLB production from Vanda hybrid leaf explants. Materials and Methods Plant material. Seeds of ( V anda Motes Honeybun Vanda Rasri Gold) V anda Rasri Gold were surface sterilized and cultured on P723 at 23 2C under a 16/8 h L/D photoperiod provided by cool white fluorescent lights at 40 M m-2 s-1. Leaves at least 0.5 cm in length were harvested from sterile four month old seedlings. For each replicat e, one whole leaf was cut in

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53 half, separating the base and tip, and both sections were inoculat ed into a single culture vessel containing one of 13 cytokinin treatments. Culture media and culture conditions. P723 was used as the basal medium in this experiment. Medium was modified with the addition of 1, 10, or 50 M of BA, 6, dimethylallyamino purine, kinetin, or TDZ. Basal medium without PGRs was used as the control. Media was adjusted to pH 5.7 with 0.1 M NaOH. Ten mL aliquots were then dispensed into 20 mL glass scintillation vials. Scintillatio n vials were autoclaving for 10 min at 121C and 117.67 kPa. Each replicate consisted of the tip and base sections of a single leaf (adaxial side in contact with media). Inoculated vessels were capped and sealed with a singl e layer of NescoFilm. Cultures were wrapped in two layers of alum inum foil to exclude light (0/24 h L/D) and incubated at 23 2 C for four weeks. At this time, cultures were examined for signs of tissue response and replicates were tran sferred to fresh media containing the same media treatment that they were originally cultured on. Cultures were again covered with aluminum foil and incubated at 23 2C for an additional four weeks. Experimental design. Five replicates of each treatment were used and the experiment was repeated twice. Treatment eff ects on explant response were obse rved after four and eight weeks culture. Results and Discussion Visible contamination for this experiment was 0%. Explants did not respond to any of the treatments after eight weeks cult ure, however, the majority of l eaf explants were still green by the end of eight weeks culture. Visible phenol e xudates were not observed in the medium of any treatments. In a study by Seeni and La tha (2000), it was fo und that 70% of Vanda coerulea seedling leaves cultured in the presence of 8.8 M BA combined with 4.1 M NAA produced

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54 PLBs. It is possible that leaf explants would respond to PGRs used in combination, though they did not respond to any single PGR te sted in this experiment.

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55 Figure A-1. Tissue culture of Vanda tessellata Vanda Arjuna initiated in liquid Knudson C with 15% coconut water (v/v). A) Regenerated shoots grow ing on original explant. Scale bar = 2 cm. B) Division and subsequent regeneration of cultures. Scale bars = 2 cm.

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56 LIST OF REFERENCES Adelberg J, Toler J (2004) Comparison of agar and an agitated, thin-f ilm, liquid system for micropropagation of ornamental elep hant ears. Hortscience 39:1088-1092 Akita M, Ohta Y (1998) A simple met hod for mass propagation of potato ( Solanum tuberosum L.) using a bioreactor without fo rced aeration. Plant Cell Rep 18:284-287 Al-Zahim MA, Ford-Lloyd BV, Newbury HJ (1999) Detection of somaclonal variation in garlic ( Allium sativum L.) using RAPD and cytological analysis. Plant Cell Rep 18:473-477 Arditti J (1993) Micropropagation of orchid s. John Wiley and Sons, Inc.: New York Arditti J, Ernst R (1992) Fundamentals of orch id biology. John Wiley and Sons, Inc.: New York Becerra DC, Forero AP, Gngora GA (2004) Age and physiological condition of donor plants affects in vitro morphogenesis in leaf explants of Passiflora edulis f. flavicarpa Plant Cell Tiss Org Cult 79:87-90 Bhaskar J, Rajeevan PK (1996) Embryo culture of Vanda John Club. S Indian Hort 44:36-38 Blanchard MG, Runkle ES (2006) Temperature duri ng the day, but not during the night, controls flowering of Phalaenopsis orchids. J Exp Bot 15:4043-4049 Bose TK, Mukherjee (1974) Effect of growth substances on seedling growth and differentiation from callus of Vanda in vitr o culture. Orchid Rev 82:148-149 Brar MS, Moore MJ, Al-Khayri JM, Morelock TE, Andersen EJ (1999) Ethylene inhibitors promote in vitro regeneration of cowpea ( Vigne unguiculata L.) In Vitro Cell Dev Biol Plant 35:222-225 Cheah KT, Sagawa Y (1978) In vitro propagation of Aranda Wendy Scott and Aranthera James Storei. Hortscience 13:661-662 Chen JT, Chang WC (2006) Di rect somatic embryogenesis and plant regeneration from leaf explants of Phalaenopsis amabilis Bio Plant 50:169-173 Chen JT, Chang C, Chang WC (1999) Direct somatic embryoge nesis on leaf explants of Oncidium Gower Ramsey and subsequent plant regeneration. Plant Cell Rep 19:143-149 Chen TY, Chen JT, Chen WC (2003) Plant rege neration through direct shoot bud formation from leaf cultures of Paphiopedilum orchids. Plant Cell Tiss Org Cul 76:11-15 Chen WH, Chen TM, Fu YM, Hsieh RM, Chen WS (1998) Studies on so maclonal variation in Phalaenopsis Plant Cell Rep 18:7-13 Chung HH, Chen JT, Chang WC (2 005) Cytokinins induce dir ect somatic embryogenesis of Dendrobium Cheingmai Pink and subsequent plant regeneration. In Vitro Cell Dev Biol 41:765-769

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57 Churchill ME, Ball EA, Arditti J (1973) Tissue cultu re of orchids: I. methods for leaf tips. New Phytol 72:161-166 Cozzolino S, Widmer A (2005) Orch id diversity: an evolutionary consequence of deception? Trends Ecol Evol 20:487-494 Curtis JT (1947) Studies on the nitrogen nutrition of orchid embryos I. complex nitrogen sources. Am Orchid Soc Bull 16:654-660 Curtis JT, Spoerl E (1948) Studies on the nitr ogen nutrition of orchid embryos II. comparative utilization of nitrate a nd ammonium nitrogen. Am Orchid Soc Bull 17:111-114 Darwin C (1892) The various contrivances by which orchids are fertilized by insects. 2nd ed. University of Chicago Press: Chicago Decruse SW,Gangaprasad A, Seeni S, Menon VS (2003) Micropropagation and ecorestoration of Vanda spathulata an exquisite orchid. Plan t Cell Org Tiss Cult 72:199-2003 Devi CG, Damayanti M, Sharma GJ (1998) Aseptic embryo culture of Vanda coerulea Griff.. J Orchid Soc India. 12:83-87 Dressler RL (1981) The orchids, natural history and classification. Harvard University Press: Cambridge, MA Dressler RL (1993) Phylogeny and classification of the orchid family. Cambridge University Press: Melbourne, AU Fitch CM (2005). Ascocentrums. Orchids. 74:20-25 Ghani AKBA, Haris H, Hajiujang NB (1992) Production of Renantanda plantlets from shoot tips in vitro Lindleyana. 7:3-6 Goh CJ, Wong PF (1990) Micropropaga tion of monopodial orchid hybrid Aranda Deborah using inflorescence explants. Sci Hort 44:315-321 Griesbach RJ (2002) Development of Phalaenopsis orchids for the mass-market. In Janick J and Whipsey A (eds) Trends in new crops a nd new uses. ASHS Press: Alexandria, VA Griesbach RJ (2003) Orchids emerge as ma jor world floral cro p. Chron Hort 43:6-9 Grove D L (1995) Vandas and Ascocendas and thei r combinations with other genera. Timber Press: Portland Hashmi G, Huettel R, Meyer R, Krusberg L, Hammerschlag F (1997) RAPD analysis of somaclonal variation derived from embryo ca llus cultures of peach. Plant Cell Rep 16:624627 Hoppe AG, Hoppe HJ (1988) Tissue culture of Ophrys apifera Lindleyana 3:190-194

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58 Hoque ME, Mansfield JW (2004) Effect of ge notype and explant age on callus induction and subsequent plant regeneration from root-derived callus of Indica rice genotypes. Plant Cell Tiss Org Cult 78:217-223 Hoshi Y, Kondo K, Hamatani S (1994) In vitro germination of four Asiatic taxa of Cypripedium and notes on the nodal micropropagation of American Cypripedium montanum Lindleyana 9:93-97 Intuwong O, Sagawa Y (1973) Clonal propagation of Sarcanthine orchids by aseptic culture of inflorescences. Am Orchid Soc Bull 42:209-215 Jerardo A (2006) Floriculture a nd nursery crop outlook. USDA FLO-05 Jimnez W, Prez N, de Feria M, Bardn R, Ca pote A, Chvez M, Quiala E, Prez JC (1999) Improved production of potato microtubers us ing a temporary immersion system. Plant Cell Tiss Org Cult 59:19-23 Kanika, Vij SJ (2004) Micropropagation of Vanda coerulea (Orchidaceae) through shoot-tip culture. Harayana J Hort Sci 33:227-228 Kauth PJ, Vendrame WA, Kane ME (2006) In vitro seed culture and se edling development of Calopogon tuberosus Plant Cell Tiss Org Cult 85:91-102 Kishor R, Sha Valli Khan PS, Sharma GJ (2006) Hybridization and in vitro culture of an orchid hybrid Ascocenda Kangla. Sci Hort 108:66-73 Knudson L (1922) Nonsymbiotic germinati on of orchid seeds. Bot Gaz 73:1-25 Knudson L (1946) A new nutrient solution for the ge rmination of orchid seed. Am Orchid Soc Bull 15:214-217 Kunisaki J, Kim K, Sagawa Y (1972) Shoot-tip culture of Vanda Am Orchid Soc Bull 41:435439 Kuo HL, Chen JT, Chang WC ( 2005) Efficient plant regenera tion through direct somatic embryogenesis from leaf explants of Phalaenopsis Little Steve. In Vitro Cell Dev Biol 41:453-456 Lai CC, Yu TA, Yeh SD, Tang JS (1998) Enhancem ent of in vitro growth of papaya multishoots by aeration. Plant Cell Tiss Org Cult 53:221-225 Lakon G (1949) The topographical tetrazolium me thod for determining the germination capacity of seeds. Plant Physiol 24:389-394 Lakshmanan P, Loh CS, Goh CJ (1995) An in vitro method for rapid regeneration of a monopodial orchid hybrid Aranda Deborah using thin section culture. Plant Cell Rep14:510-514

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60 Nagy JK, Sule S, Sampaio JP (2005) Apple tissue culture contamination by Rhodototula spp.: identification and prevention. In V itro Cell Dev Biol Plant 41:520-524 Nash N (2003) Phalaenopsis primer: A beginners guide to growing moth orchids. Orchids. 72:906-913 Nguyen QT, Kozai T, Heo J, Thai DX (2001) Photoautotrophic growth response of in vitro cultured coffee plantlets to ventilation methods and photos ynthetic photon fluxes under carbon dioxide enriched condition. Plant Cell Tiss Org Cult 66:217-225 Paek KY, Hahn EJ (1999) Variation in Afri can violet Crimson Frost micropropagated by homogenized leaf tissue culture. HortTech 9:625-627 Paek KY, Hahn EJ, Son SH (2001) Application of bioreactors for large-scale micropropagation systems of plants. In Vitro Cell Dev Biol Plant 37:149-157 Park SY, Yeung EC, Chakrabarty D, Paek KY (2002) An efficient direct induction of protocormlike bodies from leaf subepidermal cells of Doritaenopsis hybrid using thin-section culture. Plant Cell Rep 21:46-51 Piao XC, Chakrabarty D, Hahn EJ, Paek KY (2003) A simple method for mass production of potato microtubers using a biore actor system. Curr Sci 84:1129-1132 Pogany MF, Lineburger RD (1990) Phenotypic variation during micropropagation of the chimeral Rhododendron President Roosevelt. Plant Cell Tiss Org Cult 21: 201-209. Prasad VSS, Gupta SD (2006) In vitro shoot regeneration of gladiolus in semi-solid agar versus liquid cultures with support system s. Plant Cell Tiss Org Cult 87:263-271 Raghavan V, Torrey JG (1964) Inorganic nitroge n nutrition of the seedlings of the orchid, Cattleya Am J Bot 51:264-274 Ricardo Jr. MJ, Alvarez MR (1971) Ultrastructural ch anges associated with utilization of metabolite reserves and trichome differentiation in the protocorm of Vanda Amer J Bot 58:229-238 Roy J, Banerjee N (2002) Optimization of in vitro seed germination, protocorm growth and seedling proliferation of Vanda tessellata (Roxb.) Hook. Ex G. Don. Phytomorph 52:167178 Sakai WS (1973) Simple methods for differentia l staining of paraffin embedded plant material using toluidine blue O. Stain Tech 48:247-249 Seeni S, Latha PG (1992) Foliar regeneration of the endangered Red Vanda, Renanthera imschootiana Rolfe (Orchidaceae). Plant Cell Tiss Org Cult 29:167-172 Seeni S, Latha PG (2000) In vitro multiplication and ecorehabilitation of the endangered Blue Vanda. Plant Cell Tiss Org Cult 61:1-8

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61 Sharma J (1998) Studies on Vanda : effects of age of capsules (pods) on in vitro seed germination. J Orchid Soc India 12:43-45 Sharma V, Vij SP (1997) Effect of CuSO4.5H2O on in vitro regenerative capacity of foliar explants excised from mature Vanda cristata Lindl. plants. Phytomorph 47:203-208 Sheelavanthmath SS, Murphy HN, Hema BP, Hahn EJ, Paek KY (2005) High frequency of protocorm like bodies (PLBs) induction and pl ant regeneration from protocorm and leaf sections of Aerides crispum Sci Hort 106:395-401 Sheelavantmath SS, Murthy HN, Pyati AN, Kumar HGA, Ravishankar BV (2000) In vitro propagation of the endangered orchid, Geodorum densiflorum (Lam.) Schltr. through rhizome section culture. Pl ant Cell Tiss Org Cult 60:151-154 Shoushtari BD, Heydary R, Jogson GL, Arditti J (1994) Germination and viability staining of orchid seed following prolonged storage. Lindleyana 9:77-84 Stenberg ML, Kane ME (1998) In vi tro seed germination and greenhouse cultivation of Encyclia boothiana var. erythronioides an endangered Florida orchid. Lindleyana 13:101-112 Stewart SL, Zettler LW (2002) Symbiotic germ ination of three semi-aquatic rein orchids ( Habenaria repens H. quinquiseta H. macroceratitis ) from Florida. Aquat Bot 72:25-35 Su WR, Chen WS, Koshioka M, Mander LN, Hung LS, Chen WH, Fu YM, Huang KL (2001) Changes in gibberellin levels in the flowering shoot of Phalaenopsis hybrida under high temperature conditions when flower development is blocked. Plant Physiol Biochem 39:45-50 Swamy BGL (1942) Morphological st udies in three species of Vanda Curr Sci 11:285-286 Teo CKH, Kunisaki JT, Sagawa Y (1973) Clonal propagation of strap-leaved Vanda by shoot-tip culture. Am Orchid Soc Bull 42:403-405 Thomas J, Vijayan D, Joshi SD, Lopez SJ, Ku mar PR (2006) Genetic in tegrity of somaclonal variation in tea ( Camellia sinensis (L.) O Kuntze) as reveal ed by inter simple sequence repeats. J Biotech 123:149-154 Tokuhara K, Mii M (2001) Induction of embryog enic callus and cell su spension culture from shoot tips excised from flower stalk buds of Phalaenopsis (Orchidaceae). In Vitro Cell Dev Biol Plant 37:457-461 Tremblay L, Levasseur C, Trembl ay FM (1999) Frequenc y of somaclonal variation in plants of black spruce ( Picea mariana Pinaceae) and white spruce ( P. glauca Pinaceae) derived from somatic embryogenesis and identificati on of some factors involved in genetic stability. Am J Bot 86:01373-1381 Vacin E, Went F (1949) Some pH changes in nutrient solutio ns. Bot Gaz 110:605-613

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62 Valmayor HL, Pimentel ML, Martinez MT (1986) Callus formation and plantlet morphogenesis in Vanda Malayan Orchid Rev 20:22-30 Van Le B, Phuong NTH, Hong LTA, Van KTT (1999) High frequency shoot regeneration from Rhynchostylis gigantean (Orchidaceae) using thin cell layers. Plant Growth Reg 28:179185 Vaz APA, Figueiredo-Ribeiro RC, Kerbauy GB (2004) Photoperiod and temperature effects on in vitro growth and flowering of P. pusilla an epiphytic orchid. Plant Physiol Biochem 42:411-415 Vendrame WA, Kochert G, Wetzst ein HY (1999) AFLP analysis of variation in pecan somatic embryos. Plant Cell Rep 18:853-857 Vij SP, Sood A, Sharma M (1986) In vitro segment culture of Vanda testacea (Lindl) Reichb F (= V. parviflora Lindl) (Orchidaceae). Curr Sci 55:1100-1101 Vujanovic V, St-Arnaud M, Barab D, Thibeault G (2000) Viability testing of orchid seed and promotion of coloration and ge rmination. Ann Botany 86:79-86 Wang WY, Chen WS, Chen WH, Hung LS, Chang PS (2002) Influence of abscisic acid on flowering in Phalaenopsis hybrida Plant Physiol Biochem 40:97-100 Wimber DE (1963) Clonal multiplication of Cy mbidiums through tissue culture of the shoot meristem. Am Orchid Soc Bull 32:105-10 Yam TW, Weatherhead A (1991) L eaf-tip culture of several native orchids of Hong Kong. Lindleyana 6:147-150 Yan N, Hu H, Huang J, X K, Wang H, Zhou Z (2005) Micropropagation of Cypripedium flavum through multiple shoots of seedlings derived fr om mature seeds. Plant Cell Tiss Org Cult 84:114-118 Young PS, Murthy HN, Yoeup PK (2000) Mass mu ltiplication of protocorm-like-bodies using bioreactor system and subse quent plant regeneration in Phalaenopsis Plant Cell Tiss Org 63:67-72 Xiao YL, Lok YH, Kozai T (2003) Photoaut otrophic growth of sugarcane plantlets in vitro as affected by photosynthetic photon flux and vesse l air exchange. In Vitro Cell Dev Biol Plant 39:186-192

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63 BIOGRAPHICAL SKETCH Tim Johnsons research experience began as an undergraduate at the University of Wisconsin-Eau Claire (UWEC) where he worked with Dr. Wilson Taylor on the evolution of early land plants. Tim graduated from the UW EC in 2003 with a BS in biology and minor in anthropology. His experience as a student researcher and his intere st in orchids lead him to the Plant Propagation, Conservation and Biotechnology Lab at the University of Florida, where he worked on the propagation of commercial orchids, while cultivating an interested in the conservation of Floridas native orchids. Tim will begin working towards a Ph.D. in Environmental Horticulture at th e University of Florid a in the fall of 2007. He plans to study the seed physiology, seed ecology, and reintroduction scie nce of native orchids. He is dedicated to improving the scientific understand ing of orchid ecology and educat ing the public about the need for protecting wildland plant communities. In his spare time, Tim enjoys reading, traveling, gardening, running, being lazy with his family, a nd fishing (which he doesnt get to do nearly enough).