In vitro physiology of recalcitrant tissue cultured plants in the nymphaeaceae, alismataceae, and orchidaceae

Comparative effects of two fungal strains, Dlin-379 or Dlin-394, on Ghost orchid seedling development stage during symbiotic culture on oatmeal agar medium OM or asymbiotic culture on P

IN VITRO PHYSIOLOGY OF RECALCITRANT TISSUE CULTURED PLANTS IN THE

NYMPHAEACEAE, ALISMATACEAE, AND ORCHIDACEAE

By HOANG NGUYEN

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA

2016

© 2016 Hoang H Nguyen

To Huong, Mom, Dad, Huy, Dale and Dr Kane

ACKNOWLEDGMENTS

“I once dreamed that I could walk through the trees that are full of blooming ghost orchids” said Larry Richardson of the Florida Panther National Wildlife Refuge

Dear Larry, your dream is coming true And so is mine

My dream, reflected through this study and this dissertation, could not be

completed without the continuing support from friends, colleagues, and instructors I deeply thank my major advisor, Dr Michael Kane, who has guided me from the first days in the US He is not only my teacher, my mentor, but he is also my cheerleader Thank you for letting me do what I love, and giving me room to grow

I would like to thank my committee members, Drs Charles Guy, Dennis Gray, Gregory Macdonald, and Hector Perez for their guidance I especially appreciate Drs Sandra Wilson, Hector Perez, Stuart McDaniel, and Thomas Colquhoun for allowing me

to use their research equipment and spending their time to discuss my research with

me I thank Drs Thomas Sheehan, Harold Koopowitz, Carlos Fighetti, and Mr Frank Smith of American Orchid Society, who inspired me tremendously with their humility, knowledge, and true love of orchids

This study is proof of the strong collaboration between the University of Florida, the Florida Panther National Wildlife Refuge (Larry Richardson, Mark Danaher, Ben Nottingham, Kevin Godsea), and Illinois College (Dr Lawrence Zettler) The orchid research was financially supported by funding from U.S Fish & Wildlife Service under contract F12AC01245 The mycorrhizal fungus strains in this study were isolated by Ellen Radcliffe, Dr Zettler’s student

I am grateful for the friendship and support of of Tanh Nguyen, Nguyet Doan, Addison Nguyen, Oren Ehrlich, Cong Dang, Trang Nguyen, Yen Nguyen, Kien

Nguyen,Tu Nguyen, Manh Tran, Du Huynh, Lien Dieu, Phu Nguyen, Dung Phan, Long Kap, Brian Owens, John Ingram, Marian Sheehan, John Bourret, JJ Sadler, Nancy Philman, Tim Johnson, Jonathan Jasinski, Phil Kauth, Rinnie Rodenius, Ben Hughes, Paulina Quijia, Jameson Coopman, Wendy Vidor, Adrienne Smith, Candace Prince, Nick Genna, Tia Tyler, Leah Cobb, Ryan Dickson, Gabriel Campbell, and Kim Backer-Kelley I thank you all for your friendship and for the years we have spent together

I am blessed to know Stacey and Anthony Barber, Dale and Karen, Haskell, Rose Steeves, Fe Almira, Cuong Nguyen, Tuan Nguyen, Anh Lac, Qui Lac, Lien Duong, Ly Lam, Meredith Course, Atossa Shaltouki, Sally Kim, Bart Schutzman, Edritz Javelosa, Sandy Dang, Anh Lan, Moo Prasert, and their family members Their hearts are among the biggest and their minds are the most beautiful

With the unconditional love of my wife, mom, dad, and my little brother, I am the richest man in the world

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS 4

LIST OF TABLES 10

LIST OF FIGURES 11

LIST OF ABBREVIATIONS 14

ABSTRACT 15

CHAPTER 1 LITERATURE REVIEW 17

Plant Propagation 17

Plant Micropropagation 18

Aquatic Plant Micropropagation 20

Orchid Micropropagation 23

Orchid Propagation from Seeds 26

Plant Tissue Culture Applications in Conservation 28

Wetland loss 29

Orchid conservation 30

Micropropagation Industry 32

Tissue-culture Recalcitrant Plants 34

Recalcitrance during Culture Initiation 35

In vitro Culture Initiation 35

Aquatic Plant Culture Initiation 36

Recalcitrance during Shoot Multiplication 38

Carry-over Effects 38

Carry-over Effects and the Reliability of Plant Tissue Culture Research 40

Recalcitrance during Ex vitro Acclimatization 41

Species of Study 44

Nymphaeaceae Waterlilies 44

Traditional waterlily propagation 46

Waterlily in vitro propagation 47

Sagittaria latifolia 48

Ghost Orchid (Dendrophylax lindenii) 50

Project Description 54

Project Objectives 54

Rationale and Significance 55

2 IN VITRO CULTURE ESTABLISHMENT AND GERMINATION OF

NYMPHAEACEAE WATERLILIES 57

Introduction 57

Materials and Methods 60

Explant Sources 60

Ovule Development in Nymphaea × ‘Madame Ganna Walska’ 61

Experiment 1: Effects of sterilization method on sterility of N × ‘Madame Ganna Walska’ ovule tissue 61

Experiment 2: Effects of surface sterilization and seed density on in vitro seed germination of Nymphaea × ‘Madame Ganna Walska’ 62

Experiment 3: Effects of light and temperature on germination of Nymphaea × ‘Madame Ganna Walska’ 63

Experiment 4: Effects of temperature and seed coat removal on germination of Nymphaea ‘Mayla’ 64

Experiment 5: Effects of seed coat inhibitors on Nymphaea ‘Mayla’ excised embryo development 64

Experiment 6: Effects of GA3 on seed germination of Nymphaea ‘Mayla’ 65

Experiment 7: Effect of sterilization methods on sterility and germination of Victoria embryos 66

Statistical analysis 67

Results and Discussion 67

Ovule development in Nymphaea × ‘Madame Ganna Walska’ 68

Effects of Sterilization Methods on Sterility of Nymphaea × ‘Madame Ganna Walska’ Ovary 69

Effects of Seed Surface Sterilization and Density on Germination 70

Effects of Light and Temperature on Germination of Nymphaea × ‘Madame Ganna Walska’ 71

Effects of Temperature on N ‘Mayla’ Seed and Excised Embryo in vitro Germination 72

Effects of Seed Coat Inhibitors on Excised Embryo Development 73

Effects of GA3 on Seed Germination of Nymphaea ‘Mayla’ 74

Effect of Surface Sterilization Methods on Sterility of Victoria Seeds and Embryos 75

3 IMPORTANCE OF EVALUATING SHOOT PRODUCTION RATIOS OVER REPEATED SUBCULTURES DURING MEDIUM OPTIMIZATION: A CASE STUDY USING Sagittaria latifolia L 89

Introduction 89

Material and Methods 92

Explant Source 92

Stage II Medium Optimization 93

Greenhouse Acclimatization 94

Results 95

Media Optimization 95

Single Culture Cycle Medium Optimization 95

Multiple-subculture medium optimization 96

Greenhouse acclimatization 97

Discussion 98

Carry-over Effects 98

Stage II Media Optimization Methods 100

Greenhouse Acclimatization 102

4 COMPARATIVE SEED GERMINATION AND SEEDLING DEVELOPMENT OF THE GHOST ORCHID, Dendrophylax lindenii (ORCHIDACEAE), AND MOLECULAR IDENTIFICATION OF ITS MYCORRHIZAL FUNGUS FROM SOUTH FLORIDA 114

Introduction 114

Material and Methods 116

Study Site 116

Orchid Seed Source 117

Fungal Isolation, Preliminary Identification and Storage 118

Molecular Identification of Fungi 119

Asymbiotic Seed Germination for Seedling Developmental Stage Description 120

Comparative Asymbiotic and Symbiotic Seed Germination 121

Statistical Analysis 122

Results 123

Fungal Isolation and Identification 123

Ghost Orchid Germination and Seedling Development 124

Comparative Symbiotic and Asymbiotic Seed Germination 125

Discussion 127

Fungus Isolation and Identification 127

Comparative Symbiotic Seed Germination 134

5 GHOST ORCHID (Dendrophylax lindenii) IN VITRO CLONAL PROPAGATION FROM SEEDLING EXPLANTS 150

Introduction 150

Materials and Methods 152

Orchid Seed Source and Sterilization 152

Initial PLB Formation, Multiplication, and Cleansing Processes 153

Protocorm-like Body Regeneration 154

Greenhouse Acclimatization 155

Statistical Analysis 157

Results 158

Protocorm-like Body Regeneration 158

Greenhouse Acclimatization 159

Discussion 159

PLB Induction and Multiplication 160

Greenhouse Acclimatization of PLB-derived Ghost Orchids 162

6 ACCLIMATIZATION AND PRELIMINARY REINTRODUCTION OF THE

ENDANGERED FLORIDA GHOST ORCHID, Dendrophylax lindenii

(ORCHIDACEAE) 176

Introduction 176

Materials and Methods 178

Seed Collection, Symbiotic Germination, and Seedling Culture 178

Greenhouse Acclimatization 179

Pre-reintroduction Seedling Cultivation 181

Seedling Reintroduction Site 182

Seedling Reintroduction Experiment 183

Statistical Analysis 184

Results 184

Seedling Greenhouse Acclimatization 184

Seedling Reintroduction 185

Discussion 185

Seedling Acclimatization 186

Seedling Reintroduction 189

7 SUMMARY 199

LIST OF REFERENCES 202

BIOGRAPHICAL SKETCH 233

LIST OF TABLES

3-1 Greenhouse performance of ex vitro shoots t 113 4-1 Ghost orchid seedling development stages 138 5-1 Effects of PGRs on protocorm-like body (PLB) regeneration and growth in

Ghost orchid 169 5-2 Effects of two greenhouse acclimatization methods on Ghost orchid PLB-

derived plantlets 170 6-1 Effects of acclimatization method on greenhouse performance of Ghost

orchid seedlings 194 6-2 Effects of orchid mounting method on field performance of Ghost orchid

seedlings 195

LIST OF FIGURES

2-1 Nymphaea × ‘Madame Ganna Walska’ ovule and embryo development

before and after anthesis 77

2-2 Sterility of Nymphaea × ‘Madame Ganna Walska’ ovule tissue following different sterilization methods after three-week incubation 78

2-3 Nymphaea × ‘Madame Ganna Walska’ ovule tissue 18 days after being inoculated 79

2-4 Effects of surface sterilization and seed density on Nymphaea × ‘Madame Ganna Walska’ seed germination 80

2-5 Effects of light and temperature on germination of non-sterile Nymphaea × ‘Madame Ganna Walska’ 81

2-6 Nymphaea ‘Mayla’ fresh seed with enclosing aril, intact seed, and excised embryo 82

2-7 Effects of temperature on in vitro germination of Nymphaea ‘Mayla’ seeds and excised embryos 83

2-8 Effects of medium volume and seed coat/endosperm addition on excised embryo germination 84

2-9 Effects of medium volume and seed coat/endosperm addition on excised embryo germination 85

2-10 Effects of GA3 on germination of non-sterile Nymphaea ‘Mayla’ seed germination 86

2-11 Effect of surface sterilization methods on contamination and germination percentage of Victoria excised embryos 87

2-12 In vitro Victoria seedlings 88

3-1 In vitro Sagittaria latifolia culture 105

3-2 Stage II media optimization using two different methods 106

3-3 Comparative rhizome production, shoots per explant, and shoot height after a single four-week culture cycle 107

3-4 Comparative Sagittaria latifolia in vitro production during four subculture cycles 108

3-5 Comparative corm formation over four subculture cycles 109

3-6 Comparative root production per explant over four subculture cycles 110

3-7 Greenhouse acclimatized plants from first culture cycle after six weeks (bar = 4 cm) 111

3-8 Comparative height of plantlets produced on four media (2 µM BA, 4 µM BA, 1 µM mT, and 3 µM mT) 112

4-1 Florida Ghost orchid (Dendrophilax lindenii (Lindl.) Benth ex Rolfe) plant and flower 139

4-2 Initial and aged cultures of Ceratobasidium strain Dlin-394 on potato dextrose agar (PDA) plate 140

4-3 Ghost orchid asymbiotic embryo and seedling development from 0-80 days after germination 141

4-4 Ghost orchid embryo and seedling development from 0 to 60 days after germination (DAG) under SEM 142

4-5 Histological analysis of Ghost orchid embryo and seedling anatomy from 0-80 days after germination 143

4-6 Ghost orchid shoot meristem and floral stalk node 144

4-7 Comparative asymbiotic and symbiotic Ghost orchid seed germination 145

4-8 Comparative effects of two fungal strains, Dlin-379 or Dlin-394, on weekly Ghost orchid seed germination percentage during symbiotic culture on oatmeal agar medium (OM) or asymbiotic culture on P723 medium 146

4-9 Comparative effects of two fungal strains, Dlin-379 or Dlin-394, on Ghost orchid seedling development stage during symbiotic culture on oatmeal agar medium (OM) or asymbiotic culture on P723 medium 147

4-10 Ghost orchid embryo and seedling development stages 148

4-11 Ghost orchid in situ and in the greenhouse 149

5-1 Flowering Ghost orchid in situ 171

5-2 Ghost orchid protocorm-like bodies (PLBs) 172

5-3 Substrate evaluation using different fabric substrates mounted on plastic mesh 173

5-4 Greenhouse acclimatization of protocorm-like body-derived Ghost orchid

plantlets using two procedures 174 5-5 Protocorm-like body derived Ghost orchid plantlets 175 6-1 A Ghost orchid flower produced on an acclimatized seedling (three-year old)

blooming under greenhouse conditions 196 6-2 Effect of in vitro pre-acclimatization method on two-year old seedling

acclimation and growth ex vitro 197 6-3 Acclimatized Ghost orchid seedlings in the greenhouse and in situ 198

LIST OF ABBREVIATIONS 2,4-D

ABA

BA

2,4-Dichlorophenoxyacetic acid Abscisic acid

Benzylaminopurine or benzyladenine DAG Day(s) after germination

Internal-transribed spacer Gibberellic acid

Leifert and Waites (medium)

NAA 1-Naphthaleneacetic acid

Photosynthetic Photon Flux Density Plant preservative mixture

PGR Plant growth regulator

SEM Scanning electron microscope

Abstract of Dissertation Presented to the Graduate School

of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

IN VITRO PHYSIOLOGY OF RECALCITRANT TISSUE CULTURED PLANTS IN

NYMPHAEACEAE, ALISMATACEAE, AND ORCHIDACEAE

By Hoang Nguyen May 2016 Chair: Michael Kane Ph.D

Major: Horticultural Sciences

As the demand for efficient plant propagation techniques rises, in vitro

propagation becomes a powerful tool that is commercially applied to plant production, research, and conservation Unfortunately, there are plants that are difficult to

manipulate in vitro under culture conditions (tissue culture recalcitrant plants) However, little is known about their in vitro biology and methods to overcome this recalcitrance Some recalcitrant species are endangered and have high ornamental value

Nymphaeaceae waterlilies are examples of plants that have high ornamental value, but are very difficult to grow in in vitro culture Experiments on in vitro

establishment using seeds and ovule explants were conducted Nymphaea ovule

developmental stages were described in detail Ovule and seed explants were

established in vitro, but their regeneration was poor Chemical inhibitors in the seed

coats may inhibit hardy waterlily embryo germination in liquid medium Victoria

seedlings were successfully established by culturing isolated embryos

Several studies indicated that cytokinins, especially BA, produce negative over effects that could be associated with low shoot multiplication A new experimental

carry-design using Sagittaria latifolia, a wetland plant species that is very sensitive to

benzyladenine (BA), was used to illustrate BA carry-over effects Our results showed that the single culture cycle method generates insufficient information to select an

optimal medium that is reliable for commercial production, which relies upon multiple culture cycles However, multiple-cycle subculture method resembling commercial micropropagation conditions, could generate more reliable information regarding

medium selection

Some recalcitrant species can be established and propagated in vitro, but are

extremely difficult to acclimatize, such as is observed in the Ghost orchid (Dendrophylax

lindenii) While the species has become endangered in its shrinking habitat, attempts to

acclimatize this species to greenhouse conditions have not been successful

Unfortunately, very little scientific information is available An asymbiotic seed

germination procedure was developed and seedling development stages were

described based on morphological and anatomical features The effectiveness of both types of germination procedure, symbiotic (using mycorrhizal fungus strains Dlin379 and Dlin394, isolated from mature Ghost orchid roots) and asymbiotic, were compared Ghost orchid PLB regeneration was investigated Clonally propagated plantlets were used to develop an acclimatization procedure Ghost orchid seedlings without in vitro hardening were acclimatized under greenhouse intermittent mist Two orchid mounting methods were tested during a reintroduction experiment at Florida Panther National Wildlife Refuge (Naples, FL) There was no difference between two orchid mounting methods (with and without a layer of sphagnum moss) For the first time, Ghost orchid seedlings were successfully out-planted into their natural environment with 87% survival after 16 weeks

CHAPTER 1 LITERATURE REVIEW

Plant Propagation

The development of plant propagation methods paralleled increases in

agriculture production beginning 10,000 years ago (Beyl and Trigiano, 2014) Today, our growing human population (United Nations estimate of 7.3 billion as of July 2015) is heavily dependent on effective propagation methods Plant propagation not only serves the food production industry, but also plant production for medical, industrial, research, and conservation purposes Plants are propagated using various sexual or clonal

techniques depending on their reproductive biology, final uses, and most importantly, their responses to these methods (Beyl and Trigiano, 2014) Some crops respond poorly to certain propagation techniques, while other methods, if available, could allow these crops to be efficiently propagated

Many crops, such as rice, wheat, soy, or corn, are sexually propagated from seeds Sexual reproduction produces seedlings with high genetic variation This method includes traditional seed germination, in vitro seed germination, zygotic embryo culture, and zygotic embryo rescue techniques In vitro seed and embryo culture improve the production of many difficult to propagate plants, such as orchids (Arditti, 2009) The first reports of seed and zygotic embryo germination in vitro were by Thilo Irmisch in 1858 and Julius Sachs in 1859 (reviewed by Höxtermann, 1997) Seeds of many crops

germinate easily, while others have different germination requirements such as fungal colonization, cold stratification, warm stratification, scarification, heat treatment, etc (Baskin and Baskin, 2014) For conservation and habitat restoration, the high genetic

variation between seedlings generated from sexual propagation is required to preserve genetic diversity of any plant population (Huenneke, 1991)

Vegetative propagation, also called clonal propagation, preserves genetic

material of individual elite stock plants, allowing farmers to propagate and maintain the characteristics of their high-quality crops Traditional methods, such as cuttings (stem, leaf, or internode), grafting, or air layering, are widely used for their simplicity and cost efficiency For many recalcitrant and slow-growing plant species, propagation ratios are often low when these methods are used Cutting or division propagation may also accelerate disease transmission in unsterile environments, especially viral diseases (Hull, 2013) In vitro clonal propagation, typically called micropropagation, involves rapid production of propagules cultured on defined tissue culture media in culture vessels under highly control environments (Kane et al., 2015) The application of tissue culture techniques allows millions of high quality clonal plants of many families to be produced globally (Winkelmann et al., 2006) Given that plant tissue culture techniques are being used worldwide, the purpose of this review is to outline the in vitro biological

characteristics of diverse recalcitrant plant species and hybrids, such as Nymphaea hybrids, Sagittaria latifolia and Dendrophylax lindenii These species and hybrids, with

high ornamental and conservation value, are recalcitrant in different tissue culture

stages that affect their sexual and/or vegetative propagation capacity

Plant Micropropagation

Micropropagation is defined as “the true-to-type propagation of selected

genotypes using in-vitro culture techniques” (Kane, 2011) The term micropropagation was first described by Murashige in the mid-70s, referring to the application of tissue

culture techniques for the large-scale production of genetically uniform plants The technique was developed with orchids first by Morel (1964) and then applied to many other crops, resulting in the production of millions of plants each year North America commercial laboratory production capacity was estimated to be 120M-150M plants per year in 1997 (Hartman and Zimmerman, 1999, Zimmerman, 1997) There is no recent estimate of tissue culture plant production in the US

In order to facilitate research and the application of micropropagation, Murashige elaborated the general micropropagation process into three sequential stages:

Establishment of the aseptic culture (Stage I); Multiplication of propagula (Stage II); and Preparation for establishment of ex vitro plants in soil (Stage III) (Miller and Murashige, 1976b, Murashige, 1974) During the commercialization of micropropagation (1970s-1980s), the definition of these stages standardized the key steps of a micropropagation project With maturation of the commercial micropropagation industry, five stages are now accepted globally (Kane et al., 2015)

maintaining them in clean conditions

meristems tissue) from stock plants, disinfecting and inoculating on

establishment media until stabilized, and then indexing for cultivable

contaminants using Leifert and Waites (LW) medium (Leifert et al., 1989)

shoots from stabilized cultures These shoots can either be used as material for the next cycle of shoot multiplication, transferring to rooting stage, or used as microcuttings to be rooted in the greenhouses

Stage II Some crops can skip this stage because they can produce roots during stage II or V under greenhouse conditions

acclimatization of propagules to greenhouse conditions

Using these five stages as a template, there have been large numbers of tissue culture and micropropagation procedures developed for diverse groups of plants,

including difficult ones, such as many woody plants or aquatic plants Investigation of these recalcitrant species under in vitro conditions provides critical information to

develop more efficient micropropagation procedures This review focuses on the use of

in vitro tissue culture biology to study the nature of recalcitrance in aquatic species (Nymphaeaceae and Alismataceae) and orchids (Orchidaceae)

Aquatic Plant Micropropagation

Aquatic plants are an economically important plant group In 2014, there were 84 nurseries in USA specializing in aquatic plant production, contributing $17.7M dollars in sales With 21 nurseries having a market value of $8.9M, Florida is the leading state in aquatic plant production, followed by New Mexico, with seven farms (USDA 2014

Census of Agriculture) These nurseries play a very important role in producing plants for all three categories of aquatic plant markets: native wetland habitat restoration and creation, aquaria, and water gardens Developing effective methods for aquatic and wetland plant production is always critical for profitability of aquatic and wetland plant nurseries

Aquatic plants are difficult to propagate under tissue culture conditions due to high contamination ratios encountered during initial establishment (Koch and Durako,

1991, Swindells, 1990) Several aquatic plants also respond poorly on tissue culture

media, especially Nymphaea waterlilies (Swindells, 1990, Lakshmanan, 1994,

Arunyanart et al., 2008, Bodhipadma et al., 2011) While micropropagation procedures have been developed for numerous aquatic plants, difficult aquatic plants are still

propagated by traditional methods in ponds In fact, aquatic plant production in the United States occurs mostly in open ponds (658 acres), in comparison to 26 acres under protection (USDA 2014 Census of Agriculture)

Even though aquatic plants are challenging to initiate in vitro, many species have been successfully micropropagated In most cases, in vitro germinated seedlings have

been used as the explant source Examples include Nymphaea alba L. (Sumlu et al

2010), Nymphaea tetragona Georgi (Tandon et al., 2010), Nymphoides coreana

(H.Lév.)Hara (Oh et al., 2010), Ceratophyllum demersum L. (Wyman and Francko, 1986),

Utricularia inflexa Forssk. (Ram and Swamy, 1966), Nelumbo lutea Willd. (Francko, 1986,

Kane et al., 1988), Nelumbo nucifera Gaertn. (Arunyanart and Chaitrayagun, 2005),

Polypleurum stylosum (Wight)J.B.Hall (Sehgal et al., 1993) Additional studies of in vitro seed germination and seedling growth could generate fundamental knowledge for further micropropagation research

There are successful results using submerged vegetative explants such as leaves (Kane and Albert, 1989b, Kane et al., 1991), leaf petioles (Jenks et al 2000), stem segments (Kane et al 1991), shoots (Huang et al 1994), and rhizomes (Ruiz-Carrera and Sánchez, 2008) of various aquatic species Explants from submerged donor plant tissues may require a series of specific treatments with media, antibiotic, and sterilization agents (Koch and Durako, 1991, Subhashini et al., 2014) Inducing adventitious regeneration of vegetative material is generally more difficult than

germinating seeds but micropropagated plants are genetically more uniform than seed propagated plants Production of large numbers of micropropagated aquatic plants also reduced the pressure of collecting from wild plant populations (Stanly et al., 2011)

Consequently, over the past 15 years, in vitro aquatic plant micropropagation has become commercialized in North America and Europe to meet retail market demands There is currently no published data on the number of in vitro aquatic plant products being produced and sold for this niche market

Tissue culture protocols have been developed as a rapid propagation technique

for many water garden plants, including lotus (Nelumbo sp.) (Arunyanart and

Chaitrayagun, 2005, Shou et al., 2008, Kane et al., 1988), but not yet for the

Nymphaeaceae waterlilies Although lotus and waterlilies are morphologically similar, they belong to two distantly related plant orders (Proteales and Nymphaeales)

Therefore, published studies on lotus are not applicable to Nymphaea waterlilies There are very few published papers on Nymphaea waterlily tissue culture due to poor

regeneration and high contamination ratios (Swindells, 1990, Lakshmanan, 1994, Arunyanart et al., 2008, Bodhipadma et al., 2011) Producing sterile explants for tissue culture research has been a major problem with waterlilies (Swindells, 1990)

Nymphaea rhizome tips are extremely difficult to surface sterilize, and they respond

poorly to plant growth regulator (PGR) treatments Even when established in vitro, they exhibit very limited propagule production (Swindells, 1990) Although mercury(II)

chloride (HgCl2) is prohibited due to its hazardous properties, it has been still used in

several studies for the in vitro establishment of Nymphaea meristem explants

(Swindells, 1990, Bodhipadma et al., 2011) This barrier has prevented further in vitro investigations, such as media optimization, which is important to understand the in vitro

physiology of Nymphaea

To investigate in vitro biology of recalcitrant species, such as Nymphaea

waterlilies, establishment of explant sources other than rhizome tips should be explored

as a possible solution Jenks et al (1990) used epiphyllous plantlets from leaves of the

viviparous waterlily Nymphaea 'Daubeniana' as explants for micropropagation

However, this study was limited to viviparous tropical waterlilies, while many waterlily cultivars, such as hardy waterlilies, do not produce epiphyllous plantlets Waterlily seeds and floral buds are available as explant sources due to their abundant number and limited contact with external surface contaminates In other non-aquatic species,

different flower parts have successfully been used as tissue explants such as ovules (Dunwell, 2013) or petals (Teixeira da Silva et al., 2015a) for adventitious shoot

regeneration However, in vitro waterlily seed germination biology and floral tissue regeneration for in vitro culture has not been investigated

Orchid Micropropagation

As opposed to Nymphaea waterlilies, members of the Orchidaceae have been

intensely studied for many decades The family has a long cultural history and use as ornamental and medicinal plants (Liu et al., 2014) The Orchidaceae is one of the

largest plant families, comprised of 17,000-35,000 species (Dressler, 1993) as well as 162,000 registered hybrids (Julian Shaw, Royal Horticultural Society, personal

communication, August 2015) Interest in orchids can be traced back to the Chinese literature of 2500-3000 years ago (Reinikka, 1995) Orchids were cultivated as specialty plants in Asian culture long before they were investigated by Carl von Linné (1707-1778), Peter Osbeck (1723-1805), Robert Allen Rolfe (1855-1921) and other European scientists (Arditti, 1992) A majority of orchid species and hybrids are used for

ornamental purposes and were mostly collected from the wild As epiphytic orchid

species are considered slow growing (Zotz, 1995), traditional propagation methods such

as stem cutting or rhizome division produce limited number of propagules As a result, orchid prices were very high Wild orchid collection remained the primary plant source until tissue culture techniques and micropropagation protocols were developed in the early1960s (Morel, 1960, Morel, 1964, Wimber, 1963)

The first orchid micropropagation success was published in 1960, in which orchid clonal production via meristem culture was described (Morel, 1960) Morel also applied

the same method on other orchids, such as Cattleya, Miltonia, and Phaius (1964)

Morel’s publications described the first successful method to clone orchids under

aseptic conditions However, it was not until Donald Wimber’s detailed descriptions on

Cymbidium in vitro clonal propagation (1963) that the commercial orchid

micropropagation industry began to expand The Orchidaceae is the plant family that benefited most from the commercialization of in vitro culture procedures

Micropropagation has allowed large-scale production of elite orchid lines (Arditti, 2009) Successful application of this method significantly reduces the cost of many slow growing orchid species that are recalcitrant to traditional propagation methods For

example, a division of Phalaenopsis Golden Emperor ‘Sweet’ cost $100,000 in 1978,

but now can be purchased in many retail stores in USA for $5.48 (Dou, 2013) This decreasing price is no doubt a result of the application of micropropagation and

improved culture techniques Pot orchid wholesale value in US market surpassed

poinsettias from 2009 to 2014 and is still exponentially increasing (USDA Floriculture Crops 2009-2014 summary) Rapid orchid propagation via micropropagation supplies

the growing orchid market with high quality plants of many varieties (Arditti, 2009)

Phalaenopsis, together with Aranda, Dendrobium, Oncidium, Vanda, Miltonia,

Cymbidium, and Cattleya are among the orchid genera that are widely propagated

using micropropagation (Hew, 1994, Griesbach, 2002, Arditti, 2009)

Orchid in vitro multiplication via production of protocorm-like body (PLBs) is commonly utilized in many orchid propagation procedures (Chugh et al., 2009) PLBs can be induced directly from shoot tips (Tokuhara and Mii, 1993), flower stalk buds (Paek et al., 2011), root tips (Park et al., 2003), or leaf segments (Huei-Lan Kuo, 2005) Orchid PLB histology origin and function is still debatable Many authors believe orchid PLBs, in general, are somatic embryos (Steward and Mapes, 1971, Lee et al., 2013, Tokuhara and Mii, 2003, Begum et al., 1994b, Teixeira da Silva and Tanaka, 2006)

Orchid callus-derived PLBs are definitely somatic embryos in Phalaenopsis, Oncidium, and Cymbidium, due to their single-cell origin (Lee et al., 2013, Tokuhara and Mii, 2003,

Begum et al., 1994b) However, in many other publications, PLB formation from other types of explants, such as PLBs, leaves, or shoots, were described as organogenesis rather than embryogenesis (reviewed by Teixeira da Silva et al., 2015c)

There are also many recalcitrant orchid species in which PLB formation and

regeneration ratios are very poor, e.g., Paphiopedilum or Cypripedium spp These

orchids are slow growing under greenhouse conditions and also have high level of recalcitrance during in vitro establishment and multiplication (Huang et al., 2001) The Florida Ghost orchid, however, is available in tissue culture flasks as seedlings, but they are extremely difficult to acclimatize and grow under greenhouse conditions (Mirenda,

2013, Davis, 2009a) This bottleneck limits availability of plants to researchers and

growers who want to conduct research or grow them Ghost orchid micropropagation via PLB induction could produce large numbers of plantlets, not only for research, but also for ornamental purposes This could satisfy orchid collectors’ demand for this

species Successful in vitro propagation of this rare orchid could protect the wild orchid populations from poaching pressure (Cruz-Garcia et al., 2015)

Orchid Propagation from Seeds

Orchid seeds typically contain rudimentary embryos bearing little or no

carbohydrate reserves (Arditti and Ghani, 2000) In nature, these embryos depend on endophytic mycorrhizal fungi associations (mycobionts) to supply nutrients,

carbohydrates, and water, which promote germination and seedling development

(Arditti, 1967, Rasmussen, 1995) The critical nature of this plant/fungus association

was discovered by Noel Bernard (1899) He successfully germinated Cattleya × Laelia

seed asymbiotically using a concentrated solution of pulverized salep powder (from the

tuber of Orchis spp.) (reviewed by Arditti, 1992) These results later directed Knudson to

develop a simple and effective germination technique using a defined mediuim with mineral salts and sucrose (Knudson, 1922) For the first time, this technique allowed growers to produce orchid seedlings on a large scale, and to supply the expanding market without the need to conduct overseas orchid hunting trips After Knudson’s success, there were many orchids species and hybrids propagated using this in vitro asymbiotic seed germination (culture) method (Arditti, 2009)

In vitro seed germination is still widely used to propagate orchids for ornamental, breeding, and conservation purposes (Yam and Arditti, 2009, Seaton and Pritchard, 2003) Asymbiotic germination is very cost effective for commercial orchid production, in

comparison to symbiotic seed culture methods or clonal propagation methods (Hossain

et al., 2009) Seed-derived plantlets have a high level of genetic variation, which is critical for breeding or conservation purposes (Seaton and Pritchard, 2003) Seed germination on media with mineral nutrients and sugar does not require the isolation, identification and co-culture of mycorrhizal fungi, which can be both complicated and time consuming The simplicity and effectiveness of this method allows it to be used on

a wide range of orchids, and potentially could be applied to species that have highly

abbreviated structures, such as Dendrophylax lindenii Using plantlets of endangered

species derived from asymbiotic seed germination for morphological and anatomical studies could be a valuable tool to circumvent the use of in situ endangered plants that are unavailable for experimentation (Zhang et al., 2015)

Orchid seeds can also germinate in vitro symbiotically in the presence of

germination mycobionts isolated from protocorms or roots (Rasmussen, 1995) Due to the complexity of fungal isolation and identification, symbiotic seed germination is limited mostly to conservation purposes and not commercial orchid production While the role and specificity of germination mycobionts in situ remain unclear, the use of specific orchid mycobionts during conservation may be required for a reintroduced population to successfully reproduce (Fay et al., 2015) Germination mycobionts may or may not be present and function in adult orchid roots, depending on the species

(Rasmussen et al., 2015) In the leafless orchid Dendrophylax lindenii, adult roots were observed to have digesting fungal pelotons (Chomicki et al., 2014) Dendrophylax

lindenii seedlings are also highly recalcitrant during greenhouse acclimatization

(Mirenda, 2013) These observations suggest the need of mycorrhizal infection for successful acclimatization of this recalcitrant species

To measure the effectiveness of orchid seed germination culture conditions on responses other than germination percentage, rate of seedling development is an

important parameter For example, in vitro seedling development stages have been

morphologically defined for various leafy terrestrial and epiphytic orchids: Calopogon

tuberosus (Kauth et al., 2011), Bletia purpurea (Johnson et al., 2011), Habenaria

macroceratis (Stewart and Kane, 2006), Spiranthes odorata (Zettler et al., 1995), or Cyrtopodium punctatum (Dutra et al., 2009) Quantifying orchid seedling development

ratios requires seedlings to be categorized into different stages (0-V) However, there are several genera in Orchidaceae, such as the leafless orchids, which have highly modified morphologies and growth habits, and for which seedling developmental stages having not been described This lack of information causes difficulty when comparing and developing effective seed germination procedures

Plant Tissue Culture Applications in Conservation

Urbanization and increasing human population (United Nations estimate of 7.3 billion as of July 2015) have created many negative impacts on natural habitats Vast acreages of forestland have been converted to monoculture agriculture production Demands for timber and non-timber products, agriculture land, irrigation water, as well

as anthropogenic climate change, are accelerating the sixth extinction (Canadell and Noble, 2001) Unlike animals, plants cannot move to escape from habitat disturbance 12.5% of global vascular plant species in 369 families face extinction (Walter and Gillett, 1998) In the USA, 31% of native plant species are considered at risk of extinction and

11% of them are protected under the Endangered Species Act (ESA) State and federal funding for plant conservation (investment per species) is lowest in comparison to other taxa (Negrón-Ortiz, 2014) California, Hawaii, and Florida are three endangered plant species hot spots in the USA (Dobson et al., 1997)

Tissue culture techniques have been widely used for rare plant conservation (Fay, 1992, Pence, 2010) In vitro propagation procedures can use seeds or

meristematic tissues as explant sources, depending on the availability and the

endangered status of the species (Fay, 1994) While there are some exceptions with recalcitrant types of seeds, germination percentage are usually enhanced under in vitro conditions (Fay, 1994) The effects of seed dormancy mechanisms, in many cases, could be controlled and minimized using different in vitro techniques, such as embryo culture or embryo rescue, with the supplement of PGRs (many examples were provided

by Sharma et al., 1996) Additionally, clonal propagation techniques from vegetative material could allow plantlet regeneration from minimum amount of vegetative tissue This method is critical during conservation of extremely rare species because the

number of individuals is limited and other traditional plant propagation methods are not suitable (Fay, 1992, Pence, 2010)

Wetland loss

From 1800 to the present, it is estimated that the world has lost 54-57% of its wetlands (Davidson, 2014) Human-related activity such as dredging, filling, draining, etc., are major causes of continuing wetland fractionation and damage (Johnston, 1994) Many wetland areas in the southern US, especially Florida, have 46 endangered orchid species in 32 genera (Weaver and Anderson, 2010a) Several endangered

orchid species, such as Dendrophylax lindenii or Habenaria distans, can only be found

in Florida wetlands (Coile and Garland, 1996) US government policies such as “no net loss” mandated wetland creation and restoration (Robertson, 2000), which

tremendously increased the demand for many wetland plant species Native plant

propagation for habitat restoration, therefore, has become much more important

Wetland plants used for restoration are usually collected from donor populations, which often results in negative consequences on vulnerable donor sites (Ticktin 2004) Seed propagation produces low-cost and genetically diverse seedlings that are ideal for restoration Seed or whole-plant collecting from natural wetlands is an economical practice for small nurseries, but overexploitation is always a risk without strict regulation Developing more effective wetland plant production methods is a solution to restore and protect current wetland areas

Orchid conservation

Among the plant families, members of the Orchidaceae depend highly on other organisms, which makes them very sensitive to habitat changes Habitat destruction affects wild orchid populations at three levels: partial habitat disturbance (e.g selective logging, forest fragmentation, genetic erosion, weed introduction, pesticide application, timber or non-timber product exploitation); habitat complete destruction (e.g fire, land clearing, mining, urbanization, desertification, or logging); and global disruptive patterns (e.g increased temperature, rising sea level, acid rain, or pollution) (Koopowitz et al., 2003)

In addition, each orchid population can be threatened by different factors, such

as phytophagous pests (Zettler et al., 2012), habitat destruction, lack of pollination, hydrology changes, or poaching/collecting (Koopowitz et al., 2003) Most orchids rely on

insect pollinators to produce capsules with millions of microscopic seeds, and in situ germination of these seeds is dependent on mycorrhizal infection (Rasmussen, 1995) Availability of pollinators and mycobionts that promote sustainable reproduction is critical to any orchid population (Rasmussen et al., 2015, Roberts, 2003) Investigations

of orchid conservation must take into account the pollinator and mycorrhizal

associations on establishment of future generation seedlings to avoid the formation of decreasing (senile) populations (Rasmussen et al., 2015)

In situ orchid populations, with specialized pollination systems, can be negatively impacted by pollinator rarity (Phillips et al., 2015) The dependence of orchids on

pollinators is the reason why orchid populations can be affected by insecticide

application (Brittain and Potts, 2011) Extreme dependence on pollinators was reported

for nine species of Australian Drakaea, pollinated by different thynnine wasp species

The wasps are parasitic on native scarab beetle larvae, which are heavily dependent on the roots of another native plant (Menz et al., 2013, Swarts and Dixon, 2009b) The frequency of insect pollination, interestingly, could be significantly improved with the presence of magnet plants that provide nectar (Johnson et al., 2003) Any human

impact on natural habitat, microbiome, or insect population could indirectly affect orchid life-cycle (Swarts and Dixon, 2009b)

Beside the orchid’s close relationship with mycorrhizae and insect pollinators, orchid seed germination and establishment is affected by many factors, such as

substrate chemical composition (Frei and Dodson, 1972), seed dormancy, interfering vegetation, interfering organisms, microbiomes, light, mineral nutrients, temperature, humidity, mutual seedling competition, and seed antagonists (Rasmussen et al., 2015)

Because of this complexity, orchids could be used as indicator species to measure vegetation condition (Newman et al., 2007) Unfortunately, this also means that any habitat disturbance could potentially result in damaging orchid populations

Micropropagation Industry

Commercial laboratories are important components of the propagation industry, where plants are clonally propagated on a large scale under in vitro conditions (Suttle, 2005) After Morel (1960) published the first micropropagation study, the first

commercial lab in the USA was established in 1965 (Hartman and Zimmerman, 1999) Size and number of commercial laboratories in US increased rapidly from 1965 to 1985, resulting in overproduction and more competition in subsequent years Increase in importation of low-priced tissue-cultured plants closed many laboratories and

consolidated many others (Hartman and Zimmerman, 1999)

Growth of the micropropagation industry in the USA is limited by high labor costs, lack of research information on recalcitrant plants, and many unsolved commercial production problems Commercial micropropagation laboratories require costly lab equipment and well-trained workers In fact, the most important challenge facing

micropropagation businesses in the USA and Europe is high labor cost, which can amount to 60%-65 of final product price (Hartman and Zimmerman, 1999, Chu, 1995, Van Huylenbroeck, 2010) In comparison $1.36 per hour in China (2012) for example, the high cost of technicians in the USA (minimum wage: $7.25, hourly labor cost:

$35.67 in 2012) contributed to the elevated cost of final products (US Bureau of Labor Statistics, International Labor comparisons, 2013) There have been many attempts to automate the micropropagation process (Aitken-Christie et al., 1995), such as the

Toshiba tissue culture robot developed by Fujita and Kinase (1991) However,

micropropagation always requires skillful manipulations during explant transfer, which limits the application of robotics to very few crops Automated systems are also

restricted by large scale contamination (Cassells, 1991) Thus, labor cost will remain a challenge for micropropagation businesses in the USA and other developing countries,

at least in the near future

Beside labor cost, lack of commercially reliable production information can also contribute to increasing costs of commercial research and development Commercial laboratories are heavily dependent on available micropropagation procedures

developed by different scientists at research institutes However, many procedures have very limited commercial application value, and other than a statement by Chu (1992) this issue has not been addressed scientifically in the literature Many published

procedures cannot be replicated due to differences in explant type, genotypic

differences, physiological conditions (Wojtania, 2010), excision methods (Chow et al., 1992), explant size (Voyiatzi and Voyiatzis, 1989), explant collection timing (Brand, 1993), culture conditions, media ingredient purity (Scholten and Pierik, 1998), or

research equipment Most importantly, many micropropagation studies, conducted on a small scale, are very difficult to scale up in commercial settings Micropropagation

media or protocol optimization is based on single-cycle subculture methods, whereas explants are multiplied repeatedly in commercial laboratories Addressing this issue could increase reliability of micropropagation studies and, therefore, facilitate

commercial applications

Tissue-culture Recalcitrant Plants

The general term “recalcitrant” is defined as difficult to manage or operate, or not responsive to treatment (Merriam-Webster Dictionary, 2015) Tissue culture recalcitrant plants are plants with cells, tissues, or organs that respond poorly in vitro, and therefore

are difficult to manipulate at any stage during the in vitro culture process (Benson, 2000) This definition covers plant species that are challenging to establish and multiply

in culture or acclimatize under greenhouse conditions Certain crops may be highly

recalcitrant, which makes them extremely difficult to propagate Holland and Polacco (1994) suggested that reports on recalcitrant crops are rarely published because poor results (e.g high contamination ratios or low regeneration) are usually discarded

Examples of recalcitrant crops with high economic or conservation value are listed here:

 Nymphaea waterlilies: difficult to sterilize during Stage I and poor regeneration

during Stage II (Arunyanart et al., 2008)

 Dioscorea spp (Yam): poor regeneration during Stage II (Malaurie et al., 1995)

 Fraxinus spp (Ash): poor regeneration during Stage II (Van Sambeek and

Preece, 2007)

 Quercus spp (Oak) low regeneration rate during Stage II (Vengadesan and Pijut,

2009)

 Pistacia vera (Pistachio) poor regeneration during Stage II (Onay, 2000)

 Juglans regia (Walnut) poor regeneration during Stage II and difficult to root

during Stage III (Leal et al., 2007)

 Ziziphus celata (Florida ziziphus): difficult to root during Stage III (Wiese and

Kane, 2007)

 Paphiopedilum spp (Lady slipper orchid): difficult to establish during Stage I and

poor regeneration during Stage II (Long et al., 2010)

 Dendrophylax lindenii (Ghost orchid): difficult to acclimatize under greenhouse

conditions (Mirenda, 2013)

Recalcitrance during Culture Initiation

In vitro Culture Initiation

Isolating plant cells and tissues from microbes under sterile conditions is crucial

to enhance plant development on nutrient media In vitro culture initiation refers to explant preparation (Stage 0) and surface sterilization (Stage I), the initial steps in any micropropagation protocol (Debergh and Maene, 1981) Culture initiation also includes the selection of viable and sterile explants that can be used for the next stages

Different sterilants and surface sterilization methods were developed to rid explants of bacterial and fungal contaminants

Surface sterilization has been successfully conducted on many different plant species using various sterilants, such as sodium hypochlorite (NaClO), calcium

hypochlorite (CaClO), sodium dichloroisocyanurate (Na-DCC), mercury(II) chloride (HgCl2), hydrogen peroxide (H2O2), ethanol (Pierik, 1987, Fay, 1994), silver nitrate (AgNO3), and bromine water (2% Br2 in H2O) (Beyl, 2014) Plant Preservative Mixture (PPMTM), antibiotics, bactericides and fungicides have been added directly into culture media to reduce microbial growth (Niedz and Bausher, 2002, Shields et al., 1984) Negative effects of PPM and the addition of antibiotics on in vitro plant development have also been reported (Leifert et al., 1992, Compton and Koch, 2001) The selection

of surface sterilants depends on the explant (Pierik, 1987) Surface sterilant treatment times and concentration are usually adjusted, depending on contamination level and physical structure of explants There are recalcitrant species, such as many aquatic plants, where traditional sterilization methods are not effective

Successful culture initiation, in many published studies, is mistakenly associated with generating visible-clean explants without using any indexing methods (Kane et al., 2011) Visible growth of bacteria and fungus could be temporarily inhibited on plant tissue culture media, causing large scale contamination issues later (Leifert et al., 1991) To index for cultivable contamination, Leifert and Waites Sterility Test Medium, a mixture of half-strength MS and microbial media (Leifert et al., 1989), is recommended for both research and production Culture indexing material and techniques were

described in detail by Kane et al (2011)

Aquatic Plant Culture Initiation

Aquatic plants usually grow in the aqueous environment, resulting in high

numbers of potentially contaminating microbes Surface sterilization can be especially difficult because of the high density of microflora on plant surfaces Many aquatic plants have non-cutinized epidermal cells, which makes the explants more fragile and

sensitive to surface sterilants (Koch and Durako, 1991) There are few species that have been initiated and fully established under in vitro conditions These include:

Anubias barteri (Huang et al., 1994) Nymphaea alba (Sumlu et al 2010), Nymphaea

‘Daubeniana’ (Jenks et al., 1990), Nymphaea tetragona (Tandon et al 2010),

Nymphoides coreana (Oh et al 2010), Myriophyllum sp (Kane et al., 1991),

Proserpinaca sp (Kane and Albert, 1989a), Ceratophyllum demersum (Wyman and

Francko 1986), Utricularia inflexa (Ram and Swamy 1966), Nelumbo lutea (Francko 1986; Kane et al 1988), Nelumbo nucifera (Arunyanart and Chaitrayagun 2005),

Polypleurum stylosum (Sehgal et al 1993), and Vallisneria americana (Ruiz-Carrera

and Sánchez, 2008) Various types of explants, such as seeds, shoot tips, petioles, or

leaves, have served as explants Submerged explants may require a series of specific treatments with media, antibiotic and sterilization agents before successful

establishment is achieved (Koch and Durako, 1991, Subhashini et al., 2014)

Culture initiation for clonal propagation is still difficult in many other aquatic

plants, especially Nymphaea waterlilies Mercury chloride was frequently used to

sterilize Nymphaea rhizomes (Swindells, 1990, Bodhipadma et al., 2011), though this

sterilant is prohibited due to its toxicity to humans and the environment (Clarkson, 1997) Established shoot tips also respond poorly during shoot multiplication (Swindells,

1990, Bodhipadma et al., 2011) In this case, investigation of in vitro seed germination and seedling growth could generate explant sources for media optimization and

fundamental information for further micropropagation research

Many submerged angiosperms produce abundant floral tissue that could be

effectively surface sterilized Depending on regenerative capacity, floral tissue, such as

sepals, petals, ovules, stigmas, or filaments could be important sources of explants Ovules and anthers are enclosed inside the flower bud and could be surface sterilized more effectively While floral tissue has been studied in many land plants (for example, Doi et al., 2013, Li et al., 2013, Rotino, 2016), very few studies were conducted on

aquatic plants Anther explant from water chestnut (Trapa sp.) was reported as

responding poorly to media with different levels of BA, NAA, GA3 and 2,4-D (Hoque et al., 2007) However, callus induction and plant regeneration procedure was successfully

tested using ovule explants of Aponogeton madagascariensis (Carter and

Gunawardena, 2011) Pollinated ovules and embryos of Nelumbo nucifera in different

stages of development and were also developed into plantlets in liquid and solid MS

medium (Murashige and Skoog 1962) (Vasil'eva and Batygina, 1981) These results

suggested that floral explants from many recalcitrant aquatic plants, such as Nymphaea

waterlilies, should be further investigated as a substitute explant source

Recalcitrance during Shoot Multiplication

During Stage II, shoots are rapidly multiplied by inducing auxiliary shoot

proliferation from stabilized cultures by disruption of apical dominance These shoots can either be used as propagules for the next shoot multiplication cycle (Stage II), transferred to rooting stage (Stage III), or direct rooting under greenhouse conditions (Stage IV) (Kane et al., 2015) Auxiliary shoot proliferation is usually enhanced by the addition of cytokinins, mostly BA However, there are many recalcitrant crops that

respond poorly to Stage II media after being successfully initiated in vitro culture

(examples listed above) Shoot multiplication in recalcitrant species could be difficult due to genetic reasons (McCown, 2000), which requires further media specialization for each genotype The focus of this study is to study recalcitrance caused by negative carry-over effects of cytokinins supplemented in shoot multiplication media This is an issue that has not been scientifically addressed

Carry-over Effects

In vitro carry-over effects are changes in growth and development as a result of previous treatment conditions within the micropropagation stages (0-IV) In this broad sense, carry-over effects, even though they may not be directly stated by the authors, can be drawn from these examples:

 Changes in multiplication ratios of Rosaceae species during repeated subculture

on Stage II media containing BA (Norton and Norton, 1986)

 Differential rooting capacity of Capsicum plants in the greenhouse, due to Stage

III shoot elongation treatment with BA and IAA (Peddaboina et al., 2006)

 Differential survival ratios during greenhouse acclimatization of sea oat due to BA

and meta-Topolin (mT) treatments in stage II shoot multiplication

(Valero-Aracama et al., 2010)

 Reduction of greenhouse performance and survival rate of Rehmannia glutinosa

in the greenhouse due to Stage II sugar concentrations (30 gL-1) (Seon et al., 2000)

 Different endogenous cytokinin and phenolic profiles in one-year old ex vitro

Tulbaghia simmleri plants pre-treated with different cytokinin types (Aremu et al.,

Cytokinin carry-over effects could impact shoot multiplication ratios (Makara et al., 2012) and callus production ratios following subculture (Moncaleán et al., 2001), low greenhouse survival rate (Valero-Aracama et al., 2010), albino plant formation

(Werbrouck et al., 1995), and rooting inhibition (Bennett et al., 1994) Significant

cytokinin carry-over effects are usually found when BA is used It is the most stable, effective and frequently used cytokinin for Stage II shoot multiplication in both research

and commercial production (Bairu et al., 2007, Kleyn et al., 2013) BA carry-over effects have been explained by the accumulation of toxic and slowly-metabolized BA

conjugations, such as 9G BA (Werbrouck et al., 1995) BA toxicity during shoot

multiplication could be mitigated by replacing or reducing BA levels in the media

However, the latter option may not be economically viable for commercial production Detection of cytokinin carry-over effects allow solutions to be developed early before the protocol is applied in large-scale production However, most published media

optimization experiments are conducted using a single subculture cycle, and this

method does not allow cytokinin carry-overs effect to be detected

Carry-over Effects and the Reliability of Plant Tissue Culture Research

During the development of the micropropagation industry, refereed research publications were the primary information sources, providing commercial laboratories with information on various facets of micropropagation, from effects of explant excision methods, sterilization methods, media optimization, and culture conditions to rooting and Stage IV survival (Chu, 1992) Some of these published protocols have facilitated commercial production of millions of high quality clonal plants (Winkelmann et al., 2006, Murashige, 1978, Brown and Thorpe, 1995, Govil and Gupta, 1997) However, a

commercially viable micropropagation protocol must provide sufficient information for large scale and sustainable plant production (Suttle, 2005) Unfortunately, many

published protocols are either not reproducible or cannot be scaled up for commercial production (Kitto, 1997) These differences could partially be explained by variances in explant types, genotype differences, physiology conditions (Wojtania, 2010), excision methods (Chow et al., 1992), explant size (Voyiatzi and Voyiatzis, 1989), explant