In vitro flowering of orchids

http://informahealthcare.com/bty ISSN: 0738-8551 (print), 1549-7801 (electronic) Crit Rev Biotechnol, 2014; 34(1): 56–76 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2013.807219

REVIEW ARTICLE

In vitro flowering of orchids Jaime A. Teixeira da Silva1, Gilberto B. Kerbauy2, Songjun Zeng3, Zhilin Chen4, and Jun Duan5 Bioresource Production, Department of Horticulture, Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Ikenobe, Kagawa, Japan, 2P.O. Box 11461, Department of Botany, Institute of Biosciences, University of Saõ Paulo, Brazil, 3Key Laboratory of South China Agricultural Plant Genetics and Breeding, South China Botanical Garden, Chinese Academy of Sciences, China, 4Horticultural Research Institute of Guizhou Province, Guiyang, China, and 5Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, China Abstract

Keywords

Flowering is the most elusive and fascinating of all plant developmental processes. The ability to induce flowering in vitro in orchids would reduce the relatively long juvenile phase and provide deeper insight into the physiological, genetic and molecular aspects of flowering. This review synthesizes all available studies that have been conducted on in vitro flowering of orchids with the objective of providing valuable clues as to the mechanism(s) that is possibly taking place.

ABC model, Cymbidium, Dendrobium, in vitro flowering, orchids

In vitro flowering within the broader context of flowering Orchids, one of the largest flowering families, are well known for their unique flower shapes and attractive colors. Generally, orchids have a long juvenile phase that requires several years of growth before they can flower (Duan & Yazawa, 1994a; Kostenyuk et al., 1999). The ability of inducing orchids to flower in vitro is capable of greatly reducing the time required (from years to months) for reaching the stage of maturity necessary for flowering in in planta or ex vitro plants. For example, in vitro flowering of Oncidium varicosum was observed after eight to nine months in comparison to 3 years to flower in vivo (Kerbauy, 1984), that Dendrobium candidum in vitro flowers were induced within three to six months versus 3 years to flower in vivo (Wang et al., 1997), while those of Cymbidium niveomarginatum were observed within three months in comparison to 4–7 years for in vivo flowering (Kostenyuk et al., 1999). Sim et al. (2007) reported that seed germination to in vitro flowering of Dendrobium Madame Thong-In took about 5–6 months and that the juvenile period was shortened to one fifth of the normal in vivo vegetative growth period. In addition, these seedlings were from self-pollinated seedpods and segregation (but not in a Mendelian ratio) of

Address for correspondence: Jaime A. Teixeira da Silva, P. O. Box 7, Miki-cho post office, Ikenobe 3011-2, Kagawa-ken, Japan. E-mail: [email protected] Songjun Zeng, Key Laboratory of South China Agricultural Plant Genetics and Breeding, South China Botanical Garden, Chinese Academy of Sciences, 510650, China. E-mail: [email protected]

History Received 22 September 2012 Revised 21 April 2013 Accepted 7 May 2013 Published online 23 July 2013

colors was observed. Therefore, early assessment of flower colors is possible within 6 months using in vitro flower induction, which shortens the time required for normal evaluation (at least 2 years), reduces the labor costs and optimizes the space required for normal orchid breeding. This system will be highly beneficial for orchid breeders. Since environmental signals affect flower initiation and development in many orchids, and indeed plant species, a good approach to flowering studies, is in vitro propagation or tissue culture since this allows for better control of growth and development than in vivo studies (Teixeira da Silva et al., 2007; Vaz et al., 2004). The ability to control flowering in vitro would be important for molecular and genetic studies aimed at elucidating the mechanisms of flower induction and for advancing orchid breeding programs. In vitro flowering is most likely the most elusive and most fascinating of all the in vitro plant developmental processes. However, like it’s ex vitro flowering counterparts in the greenhouse, the process, especially at the genetic and molecular levels, remains fairly rudimentarily understood, although great strides have already been made in orchids (Hsiao et al., 2011). Orchids exhibit exotic floral patterning with distinct structures and organ identity, making them unique amongst the angiosperms. Moreover, their exquisite reproductive biology, with a column in which the stamens and styles are fused, the co-evolution of pollinators, different maturation times of pollen grains and ovules, and synchronized micro- and mega-gametogenesis, makes for effective pollination (Yu & Goh, 2001). During 2006, a landmark year the term ‘‘in vitro bouquets’’ was coined (Sudhakaran et al., 2006) to refer to the ability not only to induce and control flowering in vitro,

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DOI: 10.3109/07388551.2013.807219

but to commercialize the concept, especially for orchid species which have a long juvenile period (Vaz & Kerbauy, 2008a) and which are capable of flowering for long periods of time making ‘‘in vitro bouquets’’ a potential high-end market product. Depending on the genotype, it can take anything from 1–13 years to produce a flowering plant from seeds in orchids, and this transition from the juvenile period to flowering also depends on environmental conditions (Bhadra & Hossain, 2003; Chia et al., 1999; Ziv & Naor, 2006). Moreover, it usually takes breeders 3–5 years to assess and select desired flower characteristics by conventional breeding. Since flowering is an extremely slow process, and in some cases being a rare event, orchid breeding programs are also very slow with clearly defined flowering seasons (Chang & Chang, 2003; Duan & Yazawa, 1995a; Kostenyuk et al., 1999). Consequently, in vitro biotechnology (more specifically tissue culture and micropropagation) would provide an effective tool not only to clonally propagate a genotype of interest or commercial value, but also serve as a way to allow the coordinated development such that greenhouse-acclimatized orchid plants all flower simultaneously, essential for market predictions (Teixeira da Silva, 2013a). In vitro conditions also have the ability of significantly shortening the juvenile period, and, depending on the explant used, the orchid species being discussed, and the set of biotic and abiotic factors, all which play their weighted role, it is possible, on some occasions, to induce in vitro flowers in orchids (Blanchard & Runkle, 2008; Goh & Yang, 1978; Hew & Clifford, 1993; Hsiao et al., 2011; Taylor & Van Staden, 2006). These factors are the subject of closer scrutiny later on in this review. Ovule development in orchids is triggered by pollination in contrast to most flowering plants where ovules are mature and egg cells are ready to be fertilized at anthesis. Studies on molecular aspects of ovule development in Phalaenopsis spp. led to the isolation and characterization of a series of orchid genes associated with ovule development (Yu & Goh, 2001). Orchidologists and horticulturists view in vitro flowering as a keen focus of interest because it provides a system for precocious flowering, the so-called ‘‘in vitro bouquet’’ (Sudhakaran et al., 2006), while providing a model system for studying flower induction as well as inflorescence and flower morphogenesis, much like the thin cell layer (TCL) system is highly advantageous in studying the fine-scale requirements for tissue organogenesis across a wide range of plant species (Teixeira da Silva et al., 2007; Teixeira da Silva & Tanaka, 2011; Teixeira da Silva, 2013b; Teixeira da Silva & Dobrańszki, 2013). Since the media components, the level of plant growth regulators (PGRs) applied, the light conditions, the genetic make-up of the species and the culture conditions are strongly correlated with the initiation and development of floral organs from buds or callus tissue, establishing a reliable in vitro protocol to induce early flowering in orchids is a vital tool for the study of molecular and genetic mechanisms of flower induction and for assisting orchid breeding programs, hand-in-hand with genetic transformation (Teixeira da Silva et al., 2011). Orchid breeders can exploit the short flowering period in order to develop new hybrids much earlier than conventional methods while in vitro flowering can also be applied to adjust commercial production of flowers and specific compounds from floral organs

In vitro flowering of orchids

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(Hsiao et al., 2008). There appear to be a number of inherent and imposed factors that determine the success of flowering both in vitro and ex vitro, and the concept of the florigen (flowering chemical stimulus) has been strengthened by a complex hierarchy of flowering time and meristem identity genes (e.g. Zeevaart, 2006). However, this review does not pretend to identify the molecular and genetic reasons for the success or the ability of a shoot apical meristem to transition from the vegetative to the flowering stage. Issues such as MADS-box genes and the ABCD(E) model of flowering are extremely well explored elsewhere (e.g. Hsiao et al., 2011; Pinyopich et al., 2003; Teixeira da Silva & Nhut, 2003a; Xu et al., 2006). The objective of this review is to highlight the successes that have so far been achieved in in vitro flowering in orchids, even though in vitro flowering has been reported widely in numerous plant families (Taylor & Van Staden, 2006). Chia et al. (1999) summarized almost 15 years ago the only 40 studies that had been reported in the literature at that time, but most of those studies were purely descriptive in which in vitro flowering was spontaneously observed, not induced, and with almost no understanding of the mechanisms as to why these events occurred in vitro. Nonetheless, the reader should always keep in mind that flower and inflorescence reversion involves a switch from floral development back to vegetative development, thus rendering flowering a phase in an ongoing growth pattern rather than a terminal act of the apical meristem (Tooke et al., 2005). Independent of whether flowering takes place in vitro or in vivo, the transitional phase in a competent shoot meristem can be divided into three stages: the shift from vegetative to the reproductive meristem (floral induction), maintenance of the floral meristem (floral determination), and the differentiation of the floral meristem (floral organ development). However, in vitro techniques are advantageous since they allow for better control of the environment and media components than the ex vitro milieu, allowing flower induction, initiation and development to be controlled and manipulated, step by step. A glance at the complex mixture of factors in Table 1 shows that no evident trend exists, although the information that has been compacted into this review will allow determined orchid tissue culture scientists to induce in vitro flowers for the study of physiological, biochemical and genetic mechanisms underlying developmental processes in their orchid of interest or closely-related genus, or to go one step further to commercialize the concept of the ‘‘in vitro bouquet’’.

In vitro flowering in orchids: timely (r)evolution Although there is an abundance of literature that reports on in vitro flowering in orchids, they only belong to nine genera focusing almost exclusively on Cymbidium and Dendrobium. Within these 9 genera, Cymbidium, Oncidium and Psygmorchis belong to the sub-family Epidendroideae, tribe Cymbidieae; Phalaenopsis, Doritis and Polystachya belong to the sub-family Epidendroideae, tribe Vandeae; Dendrobium belongs to the sub-family Epidendroideae, tribe Dendrobieae; Geodorum and Eulophia belong to the subfamily Orchidoideae, tribe Epidendreae. Since no clear-cut trend exists for in vitro flowering in orchids, this review will examine studies in a chronological order (Table 1).

Adventitious shoots cultured under different day/night temperatures.

Seed-derived protocorms. Flowering induced within 90 to 150 days. Cultures were maintained in 12-h photoperiod at 1000–1500 lux light at 25–27 C. Details not available.

Phalaenopsis Pink Leopard (‘‘Petra’’)

Dendrobium candidum Wall. Ex Lindl.

Aranda, Dendrobium, Aranthera and Oncidium Dendrobium candidum Wall. Ex Lindl.

Doritis pulcherrima Kingiella philippinensis

Cymbidium goeringii

Oncidium varicosum ‘‘Baldin’’

MS medium supplemented with BAP, NAA, ABA at different concentrations

Details not available.

MS medium supplemented with BA, NAA, ABA at different concentrations

VW þ 22 mM BA

VW medium and Hyponex medium BAP, KN, 2ip, CW and different N content

Knudson (1946) inorganic medium, hormone-free, 60 gl1 banana pulp, 1,0 gl1 activated charcoal MS supplemented with CW or BA combination NAA

Modified VW, Modified W and MS media supplemented with BA, ZT, CM, NAA, 2,4-D MS

MS medium

Culture medium

Wang et al., 1988; Wang, 1988

The plantlets from protocorms were cultured on MS most plantlets formed floral bud and bloomed.

Plant hormones and polyamines played an important role in flower initiation. The addition of 20 mmol l1 spermidine, or 2.0 mg l1 BA, or the combination of 0.5 mg l1 NAA and BA in the culture medium induced protocorms or shoots to flower within 3–6 months with a frequency of 31.6–45.8%. The flowering frequency was further increased to 82.8% on average by pre-treatment of protocorms in a 0.5 mg l1 ABA-containing medium followed by transfer onto medium with 2.0 mg l1 BA. The induction of precocious flowering depends on the developmental stage of the experimental materials.

The in vitro plantlets induced flowering in the presence of BA under certain nutritional conditions, including appropriate sucrose content and nitrogen in the culture media. Use of KN, 2iP or CW was ineffective for floral bud induction. No floral buds formed in Hyponex medium, even with BA and sucrose. High N in the medium decreased or discouraged floral bud formation while low N content improved the formation of floral buds. Floral buds were not formed on the adventitious shoots cultured on VW medium lacking BA. Low nitrogen supply and cytokinin. Vernalization resulted in floral induction, but anthesis was not observed, possibly because of ethylene accumulation in flasks. Floral buds were induced in 2.0 mg l1 BA and 0.5 mg l1 NAA at a frequency of 27%. When protocorms were cultured in 0.5 mg l1 ABA for 15 days and then transferred to 2.0 mg l1 BAsupplemented medium the frequency of flowering increased up to 84.0%. No flower buds were observed when the protocorms were cultured on ABA-supplemented medium for long periods. BA important

90% of in vitro-grown seedlings formed floral buds.

Wang et al., 1995, 1997

Hew & Yong, 1997

Wang et al., 1995

Duan & Yazawa, 1994, 1995a

Duan & Yazawa, 1994, 1995b

Zhang et al., 1993

Kerbauy, 1984

Wang, 1984

Modified W medium supplemented with 1.0 mg l1 BA and 0.1 mg l1 NAA was suitable for floral bud induction of the in vitro plantlets.

The first study on in vitro flowering employing a cloned orchid plant. In vitro flowering of Oncidium varicosum was observed after eight to nine months in comparison to 3 years for in vivo flowering.

Wang et al., 1981

Reference(s)

Flower organ formed from plantlets or the flower bud directly grew from rosette protocorm.

Major observations, successes and failures

J. A. Teixeira da Silva et al.

Protocorms or shoots. Flowering induced within 3–6 months of culture. Cultures were maintained in a 12-h photoperiod at 1000–1500 lux at 25–27 C.

Shoot meristems and axillary. Cultures were maintained in 8-h photoperiod. Shoot apical meristem, 16-h photoperiod at 25 C, Gro-Lux tubes, flowering induced 8–9 months after transferring PLBs. In vitro-grown seedlings. Cultures were maintained in 8- or 12-h photoperiod at 25 2 C. In vitro plantlets. Flowering occurred within 7 months of culture. Cultures were maintained in 12-h photoperiod at 25 2 C.

Cymbidium ensifolium cv. Susin

Cymbidium ensifolium cv. Qiulan

Shoot meristems and axillary bud. Cultures were maintained in 8–9-h photoperiod at 25 C. Shoot meristems and axillary buds. Cultures were maintained in 8–9-h photoperiod at 25 C.

Explant, Culture conditions (temperature, light, photoperiod), duration of flowering

Cymbidium ensifolium cv. Qiulan

Species*

Table 1. In vitro flowering of orchids.


58 Crit Rev Biotechnol, 2014; 34(1): 56–76

In vitro grown seedlings at different size cultures were incubated at 25 2 C under a 16-h photoperiod of 30 mmol m2 s1.

Callus-derived rhizomes. Flowering occurred within 100 d of culture. The cultures were maintained for 100 d at 25 1 C and exposed to artificial light (daylight fluorescent tubes FL-30D/29, 40 W) of 10 mmol m2 s1 with a 16-h photoperiod. Two-three months old in vitro grown seedlings having 3–4 leaves. Flowering occurred within 90–100 days. Cultures were maintained at 25 2 C under a 16-h photoperiod of 1000 lux fluorescent light. Asymbiotically germination of immature seeds. Darkness, 6, 8, 12, 16, 20, 22 and 24 h

Psygmorchis pusilla

Cymbidium ensifolium var. misericors

Psygmorchis pusilla

Geodorum densiflorum (Lam.) Schltr.

Cymbidium ensifolium

Dendrobium huoshanase

Rhizomes (3–4 months old) and in vitro plants 3–5 months old. Cultures were maintained at 25–26 C, 50 mmol m2 s1, and a 16-h photoperiod. To stimulate short-day condition, an 8-h photoperiod was employed at low (4–6 C) and high (30–32 C) temperature. Nearly 100% of plants flowered within 90 days. In vitro-grown seedlings. Cultures were maintained in a 12-h photoperiod at 2000 lux at 25 2 C. Internode of flower branch. Cultures were maintained in 0or 24-h photoperiod at 25 2 C.

Cymbidium niveo-marginatum Mak

Solid VW with MS micronutrients, 6% (w/v) ripe banana pulp, 0.1% (w/v) activated charcoal and 2%

MS medium supplemented with BA, IAA and AC at different concentrations

MS containing NAA, TDZ, 2iP or BA at different concentrations

VW containing different concentrations total mineral salt, inorganic N (NHþ 4 and NO 3 ), P, K and Ca.

MS supplemented with BA, NAA and GA3.

MS containing 2,4-D and Zea at different concentrations

Modified MS medium supplemented with BA and restricted nitrogen supply with phosphorus enrichment

Wen et al., 1999

4-mon. plantlets were culture on MS medium containing 0.1 mg l1 2,4-D and 1.0 mg l1 Zea for flower induction, and MS medium containing 10 g l1 banana juice were the effective for achieving flower. Latent bud of internode cutting part of flower bud differentiated flower bud on the MS supplemented with 3-4 mg l1 BA and 0.1 mg l1 NAA and 1.0 mg l1 GA, the frequency of flowering were 75%. Flower and nutrient buds can all be induced secondprotocorm and pseudo-root stem. A low concentration of N stimulated flower formation and this effect was more pronounced than that observed for the lowest total salt level used. The presence of NHþ 4 in the medium was essential for plant growth and development, while the absence of NO 3 depressed flowering. Flower formation was also enhanced by half the ionic concentration of P and K while the highest Ca concentration showed some floral stimulation. Plants grown under nutrient-deficient conditions were understandably more yellow than those cultivated on full-strength medium. ABA and TDZ inhibited reproductive development. Precocious flowering occurred on a defined MS medium containing NAA with TDZ, 2iP or BA within 100 d of culture. Among eight cytokinins tested, TDZ at 3.3–10 mM or 2iP at 10–33 mM combined with 1.5 mM NAA were the most effective combinations for achieving flower induction. TDZ had a stronger inductive effect than BA on the flowering.

Vaz, 2002; Vaz & Kerbauy, 2000; Vaz et al., 2004,

Photoperiod and temperature effects were evaluated. Endogenous carbohydrate and pigment contents were measured. Inorganic N (NHþ 4 and NO3 ), P, K, Ca and total mineral salt concentrations effects on floral spike development were investigated. Effects of

In vitro flowering of orchids (continued )

Bhadra & Hossain, 2003

In vitro-grown seedlings flowered only on MS þ 2.0 mg l1 BA þ 1.0 mg l1 IAA and on MS þ 2.5 mg l1 BA þ 0.1% (w/v) AC within 3–4 months of culture. BA had a strong effect on in vitro flowering.

Chang & Chang, 2003

Vaz & Kerbauy, 2000, 2008b

Jia et al., 2000

Kostenyuk et al., 1999

A combined treatment of BA, restricted nitrogen supply with phosphorus enrichment, and root excision (pruning) resulted in flowering. When root excision and/or BA were omitted from the combined treatments, flower induction was significantly reduced. Root-excised plantlets cultured in medium containing BA with high P and low N content achieved about 2.5-fold higher number of in vitro flowers than cultures grown in MS medium containing BA without a modified P/N ratio. Different exogenous concentrations of NAA, BAP, TIBA, GA3 and PBZ were evaluated during plant rhizome propagation, plant regeneration and flowering induction.


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In vitro-grown seedlings. Cultures were kept at a temperature of 25 2 C and 12-h photoperiod with irradiance of 50–60 mmol m2 s1.

Cultures were kept in a growth room at 26 1 C with a 16-h photoperiod at 35–45 or 50–60 mmol m2 s1.

In vitro plants without roots. Flowering occurred within 40–120 days. Cultures incubated at 25 2 C with a 16-h photoperiod of 35–40 mmol

Dendrobium Second Love

Phalaenopsis Cygnus ‘Silky Moon’

photoperiods tested. 30 mmol m2 s1.


Dendrobium moniliforme

Species*

Table 1. Continued

medium composition (sugars, mineral nutrients, hormones, pH), photoperiodism and temperature on flower formation were investigated, also using SEM. Endogenous levels of carbohydrates, pigments, cytokinins, auxin and ABA were measured. IAA, ABA, Z, ZR, iP and [9R]iP quantification were based on indirect ELISA after purification in reverse-phase HPLC. Floral buds were induced in MS containing 0.05 mg l1 TDZ or MS containing 0.04 mg l1 PBZ and 1.0 mg l1 ABA for 90 d, then transferred to MS medium supplemented with 1.0 mg l1 PBZ þ 0.1 mg l1 TDZ for flowering, the frequency of flowering were all 93.3%, but the normal flower frequencies were 45.4% and 80.0%, respectively. The endogenous levels of cytokinins, IAA, soluble sugars and amino acids were measured during flowering of isolated shoots. The effects of different culture media, cytokinins and sucrose concentrations were also evaluated. Authors promoted precocious flowering in micropropagated rootless shoots as well as in seedlings of different crossings involving Dendrobium plants of Nobile group, by adding TDZ to the culture medium. TDZ was substantially involved with the floral transition in vitro, presumably by means of an endogenous increase in the levels of cytokinins and IAA. 1.8 mM TDZ had a profound effect on the amounts of these hormones compared to explants grown on TDZ-free medium. Among the studied cytokinins, [9R]iP and ZR were significantly predominant during the first peak at the 5th incubation day. Moreover, the levels of some sugars – mainly sucrose and glucose –, and amino acids – mainly asparagine, glutamine, tyrosine and ornithine – were markedly influenced by TDZ concentration, but a possible direct effect of TDZ on flower induction can not be completely discarded. Changes in hormone levels induced by thermoperiodic treatments were observed in lateral buds and mature leaves of adult plants cultivated in vivo. Z and ZR were the most prominent cytokinins found at the 15th day in cold-treated plants. However, the observed increases in total cytokinins, as well as in IAA levels, and the significant decrease in ABA content, do not mean that they are direct and final inductive factors. According to the authors, these hormones would be necessary, but not sufficient for flower induction. The highest number of plants (38–43%) induced flower buds after preculture in VW medium supplemented with 30 or 30–40 g l1 sucrose before transfer onto standard or modified VW (i.e., MVW) medium supplemented with 66.6 mM of BA for 40–120 days. BA and N concentration had major effects on flower bud formation.


Rojanawong et al., 2006

Ferreira, 2003; Campos & Kerbauy, 2004; Ferreira et al., 2006

Wang et al., 2006

Vaz & Kerbauy, 2008b

Reference(s)


VW medium supplemented with BA different concentrations of N supply

Liquid and solid VW and MS, TDZ, BAP, [9R]iP; Semisolid VW, TDZ, sucrose

MS containing TDZ or PBZ þ ABA for floral induce, Modified MS medium supplemented with PBZ þ TDZ for flowering

(w/v) sucrose, and solidified with 0.6% (w/v) ‘‘Oxoid’’ agar.

Culture medium



In vitro-grown seedlings. Flowering occurred within 60 d of culture. Cultures were maintained at 25 2 C and a 16-h photoperiod of 40 mmol m2 s1 from daylight fluorescent lamps.

In vitro seedlings with 2–3 leaflets. More than 60% of the flowers bloomed by the 6th week and 90% by the 9th week. Cultures were incubated at 26 2 C under a 16-h photoperiod of 35 mmol m2 s1 from daylight fluorescent lamps.

In vitro grown seedlings at different size cultures were incubated at 26 2 C under a 16-h photoperiod of 35 mmol m2 s1 from daylight fluorescent lamps.

Three-leaf stage plantlets. Cultures were kept at a temperature of 25 2 C and 16-h photoperiod

Dendrobium Chao Praya Smile

Dendrobium Madame Thong-In

Dendrobium Madame Thong-In

Dendrobium Sonia 17

Hybrid of Cymbidium goeringii and C. hybridium

m2 s1 provided by cool white fluorescent lights. In vitro-grown protocorms and seedlings. Cultures were maintained at 25 C

Modified MS medium supplemented with BA and different N/P concentrations

Modified KC

Modified KC supplemented with BA, CW and AC at different concentrations

Modified (two-layered) KC medium supplemented with BA and CW at different concentrations

MS medium supplemented with BA, NAA at different concentrations

Seedlings 1–2 cm, 2–4 cm, or 4–6 cm in height produced 19% 0% and 12% floral buds, respectively on MS medium containing 1.0 mg l1 BA and 0.1 mg l1 NAA within 80 d of culture. The frequency of floral bud formation did not significantly increased after pretreatment on MS supplemented with 0.5 mg l1 PBZ þ 0.5 mg l1 ABA for 35-d. The frequency of floral bud formation of protocorms was only 0–1.8% on all treatments. BA at 11.1 mM induced the highest percent of flowering (45%) within 6 months from germination. The percentage of inflorescence induction was increased to 72% in morphologically normal seedlings. Plantlets in culture produced both complete and incomplete flowers in a two-layer culture system. Pollen and female reproductive organs of in vitro developed complete flowers were morphologically and anatomically similar to flowers of fieldgrown plants. In addition, 65% of the pollen grains derived from in vitro developed flowers showed regular meiosis during microsporogenesis. Even so, a lower percentage of germination of the pollen grains derived from in vitro developed flowers could be self-pollinated and induced seed pods with viable seeds, comparable to field-grown plants. CW promoted vegetative growth but did not support the formation of flower buds. CW was essential to trigger the transition of the shoot apical meristem from the vegetative to floral state and BA enhanced inflorescence initiation and flower bud formation. Inflorescence stalks became visible after 3 weeks of culture upon transfer to two-layered medium. By the 9th week, about 40–55% of the protocorms produced inflorescence stalks. Both liquid and Gelrite-solidified medium with 22.2 mM BA and 0.03% AC produced flowers. Flower development was deformed in liquid medium but developed fully upon transferring to two-layered (liquid over Gelrite-solidified) medium. Healthy seedlings produced 100% flower buds, 75% of which resulted in abnormal flowers. Segregation of flower colors was observed and seed pods formed upon artificial pollination of the in vitro flowers. Seedling growth stage and endogenous level of cytokinin was important for vegetative growth and flower-inductive response. Cytokinin content varied in different growth stages of seedlings. Seedlings 1.0–1.5 cm in size cultured in flowering-inductive medium [liquid KC medium containing 4.4 mM BA and 15% CW)] showed up to 200 and 133 pmol g1 FW of iP and iPA, respectively. These levels were significantly higher than all other cytokinins analyzed in seedlings of the same stage and were about 80 - to 150fold higher than seedlings cultured in non-inductive medium. During the transitional (vegetative to reproductive) stage, the endogenous levels of iP (178 pmol g1 FW) and iPA (63 pmol g1 FW) were also significantly higher than cytokinins in the Zea and dihydrozeatin (DZ) families in the same seedlings. This report provided strong evidence that cytokinins, especially the iP family, play an important role in early in vitro flowering in this orchid. Medium enriched with high P and low N was effective for inducing inflorescences while low P and high N content could only effectively promote shoot formation. In 52% of plantlets that


In vitro flowering of orchids (continued )

Tee et al., 2008

Sim et al., 2008

Sim et al., 2007

Hee et al., 2007

Zheng & Pang, 2006

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In vitro seedlings. Flowering was observed about 4–6 months after seeds were sown. The cultures were maintained in a culture room kept at 25 C with a 12-h photoperiod provided by white fluorescent light at 60 mmol m2 s1.

In vitro seedlings. Cultures were maintained in a 12-h photoperiod at 2000 lux at 22–24 C. In vitro seed-derived rhizomes. All cultures were exposed to artificial light of 1000 lux with a 16h photoperiod at 25 1 C.

Dendrobium nobile Lindl.

Dendrobium denndanum

Polystachya sp.

In vitro shoots. Cultures were maintained under fluorescent

In vitro individual shoots 2–3 cm long consisted of 3–4 nodes. After 3 months of culture floral buds were induced. Cultures were maintained at 26 2 C under 1600 lux illumination for 14 h.

Friederick’s Dendrobium orchid

MS and VW medium

MS containing BA, NAA, ABA, and 2,4-D at different concentrations ¼ MS modified with coconut milk, potato powder, peptone and AC

Modified MS medium supplemented with PBZ and TDZ at different concentrations

MS medium supplemented with PBZ at different concentrations

B5 supplemented with BA and NAA at different concentrations

Culture medium

In vitro seed-derived rhizomes spontaneously sprouted flower stems followed by flowering and subsequent fruition to complete a full life cycle. About 44–61% of rhizomes spontaneously produced flower stems from the apex. Among the flowers, 50% showed normal floral organs, 40% lacked both left and right sepals and 10% lacked the right petal only. 18% of the flowers showed autogamous mating and developed fruits. The seeds were harvested and sown further, showing a 59.6% average germination percentage. In vitro flowering and fruit setting were mentioned but no specific reasons were provided to explain the phenomena.

formed an inflorescence in BA and high P- and low N-containing medium while only 20% of plantlets formed inflorescences in MS medium containing BA without any modification of the P/N ratio after 4 months. A repeatable method for in vitro inflorescence induction was found when the medium contained 20 mM BA. Different morphologies of in vitro flowers such as incomplete flower structures, abnormal and unresupinated in vitro flowers were observed. In vitro-grown seedlings were cultured in B5 medium supplemented with 2.0 mg l1 BA and 0.2 mg l1 NAA, the frequency of flowering were 78.2% with respect to 46.3% cultured in B5 supplemented with 1.0 mg l1 BA and 0.2 mg l1 NAA The mortality rate of flowers on the former was 12%, and the latter was 75%. Without supplementation of PBZ in culture medium no floral buds were induced. A low concentration of PBZ (0.025 mg l1) promoted a small number of floral buds (10%). PBZ at 0.05 mg l1 gave the highest percentage of floral bud induction (29%) and highest concentration of PBZ (0.1 mg l1) provided the lowest result (6.95%) of floral bud induction. Flowers obtained from all PBZ-containing media, i.e. at all concentrations of PBZ, showed normal morphology. Seedlings produced floral buds (33.3–34.8%) precociously on a defined MS medium containing PBZ at 0.5 mg l1. In TDZ at 0.1 mg l1 flowering occurred within 4 months of culture. The frequency of floral bud formation increased to 95.6% by growing seedlings in 0.3 mgl1 PBZ þ 0.5 mgl1 NAA followed by PP333 and TDZ. TDZ (0.05–0.1 mg l1) and PBZ (0.5–1.0 mg l1) were more effective for inducing floral buds of D. nobile plantlets than BA. Flowers that developed were deformed at 25 C but they developed fully when grown at a lower temperature regime (23 C/ 18 C, light/dark) for 45 days. In vitro seedlings without root were cultured on MS containing 2.0 mg l1 BA, the frequency of floral bud induction was 10%.


Sotthikul et al., 2010

Chang et al., 2010

Guan & Shi, 2009

Wang et al., 2009

Te-chato et al., 2009

Zhu et al., 2008

Reference(s)


Eulophia graminea Lindl.

In vitro-grown seedlings. Cultures were kept at a temperature of 25 2 C and 12-h photoperiod with irradiance of 30–40 mmol m2 s1.

with irradiance of 25 mmol m2 s1.


Cymbidium kanran

Species*

Table 1. Continued



In vitro grown seedlings at different size cultures were incubated at 25 3 C under a 12-h photoperiod of 20 mmol m2 s1.

Dendrobidium strongylanthum MS medium supplemented with single-factor PGR and multi-factor PGR treatments.

MS medium supplemented with combinations of PGRs including auxins (2,4-D, dicamba, NAA, IBA), cytokinins (2iP, BA, kinetin, TDZ) and GA3.

MS medium supplemented with single-factor hormone and multi-factor hormone treatments.

Among the single-factor hormone (PBZ or TDZ) treatments, a suitable concentration and the level of flower bud formation with the PBZ treatment was 0.2 mg l1 with 8.5%, respectively while that with the TDZ treatment was 0. 06 mg l1 with 15.5%, respectively. The effects of multi-factor hormone treatments on flowering induction were ranked as follows: (PBZ þ BA þ NAA þ TDZ)4(PBZ þ BA þ NAA)4(PBZ þ BA) and (PBZ þ NAA). The most suitable treatment was 0.3 mg l1 PBZ þ 0.5 mg l1 BA þ 0.5 mg l1 NAA þ 0.06 mg l1 TDZ. The level of flower bud formation and the level of flowers that blossomed reached 80.4% and 90.3%, respectively. When one-year-old callus, which was induced and subcultured in the presence of 3 mg l1 TDZ and 5 mg l1 dicamba (line 13 callus), was transferred onto MS medium supplemented with 0.1 mg l1 NAA and 3 mg l1 TDZ, it gave the highest number of petalbearing embryos. Flowering characteristics of plantlets from normal embryos and petal-bearing embryos were all not significantly different. The induction rate of flower buds in MS þ BA 2 mg l1 þ NAA 0.2 mg l1 with 3% sucrose for 70 d was over 96%. HyponexÕ medium supplemented with 0.2 mg l1 NAA and 0.5 g l1 AC was suitable for subsequently inducing flowering.

Zhao et al., 2012

Chen, 2012

Cen et al., 2010

2,4-D, 2,4-dichlorophenoxyacetic acid; 2ip, N6-isopentenyladenine or 6-g,g-dimethylallylaminopurine; ABA, abscisic acid; AC, activated charcoal; BA, N6-benzyladenine (equiv. to BAP, benzylaminopurine; see Teixeira da Silva, 2012); CW, coconut water; IAA, indole-3-acetic acid, IBA, indole-3-butyric acid; iP, N6-(D2-isopentenyl)-adenine); iPA, N6-(D2-isopentenyl)-adenosine; KC, Knudson medium (1946); KN, kinetin; MS, Murashige and Skoog (1962) medium; NAA, -naphthaleneacetic acid; PBZ, paclobutrazol; PGR, plant growth regulator; PLB, protocorm-like body; PBZ, paclobutrazol; SEM, scanning electron microscopy; TDZ, thidiazuron (N-phenyl-N0 - 1,2,3-thiadiazol-5-ylurea); VW, Vacin and Went (1949) medium; Zea, zeatin; ZR, zeatin riboside *Note: Cymbidium, Oncidium and Psygmorchis belong to sub-family Epidendroideae, tribe Cymbidieae; Phalaenopsis, Doritis and Polystachya belong to sub-family Epidendroideae, tribe Vandeae; Dendrobium belongs to sub-family Epidendroideae, tribe Dendrobieae; Geodorum and Eulophia belong to sub-family Orchidoideae, tribe Epidendreae.

The petal-bearing embryos were derived from long-term root callus. All cultures were exposed to artificial light of 28–36 mmol m2 s1 with a 16-h photoperiod at 26 1 C.

Oncidium ‘Gower Ramsey’

Dendrobium officinale

light at an intensity of 30 mmol m2 s1, 16-h photoperiod. Non- root plantlets induced by stem explants. All cultures were exposed to artificial light of 1000–1800 lux with a 16-h photoperiod at 26 1 C.


DOI: 10.3109/07388551.2013.807219

In vitro flowering of orchids 63


64


Later, we will examine the factors that have led to successful or unsuccessful in vitro flowering. Historically, the first person to induce orchids (Laeliocattleya hybrid) to flower successfully in vitro was Lewis Knudson, from Cornell University, in 1928. At that time, his experiments were aimed at demonstrating that an orchid plant could grow and flower perfectly in the absence of mycorrhiza, i.e. asymbiotically, when, 8 years after sowing seeds, flowers developed (Arditti, 1967). Since then, a considerable number of orchid species and few nonorchidaceous plants have flowered under in vitro conditions. In the West, Kerbauy (from Brazil) in 1984 seems to have been the pioneer of modern times, stimulating in vitro flowers in Oncidium varicosum. Considering this, the history of orchid in vitro flowering, at least that reported in the literature, is extremely young – less than 30 years – relative to the history of plant tissue culture, possibly because it is extremely difficult to induce in vitro flowers. Only 10 years later was there a slight intensification of research by Duan & Yazawa (1994a, 1995a,b) in Doritis pulcherrima Kingiella philippinensis and Phalaenopsis sp. In vitro Doritis pulcherrima Kingiella philippinensis plantlets induced flowering in the presence of 6-benzyladenine (BA) when sucrose and nitrogen content in the culture media were at specific levels (Duan & Yazawa, 1994a, 1995b). Use of kinetin (KN), 6-g,g-dimethylallylaminopurine (2iP) or coconut water (CW) was ineffective for floral bud induction. No floral buds formed in HyponexÕ medium, even with BA and sucrose. A high nitrogen (N) [standard or 1/2 nitrogen content in MS medium (Murashige & Skoog, 1962) or double nitrogen in VW (Vacin & Went, 1949) medium] in the medium decreased or discouraged floral bud formation while low N content (1/10 nitrogen content in MS medium) improved the formation of floral buds (Duan & Yazawa 1994a, 1995b). Duan & Yazawa (1995a) reported that floral buds were not formed on the adventitious shoots cultured on VW medium lacking BA, low nitrogen supply and cytokinin resulted in floral induction. Historically, Wang in the 1980’s first induced in vitro flowering in orchids, and represents the efforts made by Eastern, in this case Chinese, scientists, focusing on Cymbidium ensifolium (Wang et al., 1981, 1988; Wang, 1988; Jia et al., 1998). They reported modified W medium supplemented with 1.0 mg l1 BA and 0.1 mg l1 a-naphthaleneacetic acid (NAA) was suitable for floral bud induction of the in vitro plantlets and flower organs were also formed from rosette protocorms with efforts later reinforced by Chang & Chang (2003). In this latter study, precocious flowering occurred on a defined half-strength MS medium containing NAA with thidiazuron (TDZ or N-phenyl-N0 -1,2,3thiadiazol-5-ylurea), 2iP or BA within 100 d of culture and among eight cytokinins (CKs) tested, TDZ at 3.3–10 mM or 2iP at 10–33 mM combined with 1.5 mM NAA were the most effective combinations for inducing flowers. TDZ had a stronger inductive effect on flowering than BA. Wang et al. (1995) induced floral buds in Dendrobium candidum with 2.0 mg l1 BA and 0.5 mg l1 NAA at a frequency of 27% and when protocorms were cultured in 0.5 mg l1 abscisic acid (ABA) for 15 days and then transferred to 2.0 mg l1 BA-supplemented medium, the frequency of flowering increased to 84.0%. No flower buds were observed when

Crit Rev Biotechnol, 2014; 34(1): 56–76

the protocorms were cultured on ABA-supplemented medium for long periods. Subsequent studies by the same group (Wang et al., 1997) indicated that PGRs and polyamines played an important role in flower initiation. The addition of spermidine, or BA, or the combination of NAA and BA in the culture medium induced protocorms or shoots to flower within 3–6 months with a frequency of 31.6–45.8%, further increased to 82.8% by pre-treatment of protocorms in an ABA-containing medium followed by transfer onto medium with BA. The induction of precocious flowering depended on the developmental stage of the initial experimental materials. Cymbidium niveo-marginatum (Kostenyuk et al., 1999) could be induced to flower in vitro with a combined treatment of BA, restricted N supply with phosphorus (P) enrichment, and root excision (pruning). When root excision and/or BA were omitted from the combined treatments, flower induction was significantly reduced. Root-excised plantlets cultured in MS medium containing BA with high P (five times P in MS medium) and low N (1/20 N in MS medium) content achieved about 2.5-fold more in vitro flowers than cultures grown in MS medium containing BA without a modified P/N ratio. Four-month-old Dendrobium huoshanase plantlets (Wen et al., 1999) could be induced to flower in vitro on MS medium containing 0.1 mg l1 2,4-D and 1.0 mgl1 zeatin (Zea) although MS medium containing 10 g l1 banana homogenate was effective for achieving in vitro flowers. The new millennium ushered in a considerable increase in in vitro flowering studies in orchids. Bhadra & Hossain (2003) showed that in vitro-grown Geodorum densiflorum seedlings flowered only on MS þ 2.0 mg l1 BA þ 1.0 mg l1 indole-3-acetic acid (IAA) and on MS þ 2.5 mg l1 BA þ 0.1% (w/v) activated charcoal (AC) within 3–4 months of culture. BA had a strong effect on in vitro flowering. Psygmorchis pusilla in vitro flowering has been well studied (Vaz, 2002; Vaz et al., 2004; Vaz & Kerbauy, 2000; Vaz & Kerbauy, 2008b). In these studies, several important observations were made: (a) flower induction was possible in light and darkness; (b) there was a negative correlation between mineral salt concentration and the number of floral spikes; (c) a low concentration of N stimulated flower formation; (d) the presence of NHþ 4 as the sole nitrogen source in the medium was essential for plant growth and development, while NO 3 absence depressed flowering; (e) flower formation was enhanced by K and Ca enrichment of medium; (f) in vitro flowering plants had higher endogenous levels of Ca and K than non-flowering plants but lower contents of Mg and P, while S level was similar in both plants; (g) floral spike formation and BA were positively correlated but a BA pulse for 48 h not sufficient to enhance floral spike formation after plants were transferred to hormone-free media while continuous exposure to BA in the medium was important for flowering process, 1 mM being the most efficient concentration and with higher concentrations not leading to a further increase in floral spike number; (h) higher levels of endogenous Zea, zeatin-riboside (ZR) and N6-(D2-isopentenyl)-adenine (iP) were observed during floral meristem development and floral organ differentiation while the isopentenyladenosine ([9R]iP) content was relatively lower than the other quantified CKs; (i) floral bud development was accompanied by glucose and fructose consumption;


DOI: 10.3109/07388551.2013.807219

(j) for the development of floral buds, growth of floral spikes and floral bud anthesis, plants require different media composition, temperatures and photoperiods, although more conspicuous flowering in these plants was dependent on the size and number of leaves, indicating that floral development is somewhat linked to vigour during vegetative growth. CKs might be involved in controlling the expression of floral meristem genes, thereby affecting later stages of flower development and floral organ differentiation, at least in Arabidopsis (Venglat & Sawhney, 1996). According to various authors, higher CK levels can negatively affect flower development in orchid plants cultivated in vitro (Duan & Yazawa, 1995a,b; Hee et al., 2007; Hew & Clifford, 1993; Hew & Yong, 1997). Matsumoto (2006), however, reported that GA3 alone (2.5 mM) was effective in promoting inflorescence emergence in Miltoniopsis. Most of the efforts during the past 10 years have focused on Dendrobium spp., probably because of the ease with which it can be propagated in vitro. Hee et al. (2007) induced the highest percent of in vitro flowering (45%) in Dendrobium Chao Praya Smile within 6 months from germination on a medium containing 11.1 mM BA. The percentage of inflorescence induction was increased to 72% in morphologically normal seedlings. Plantlets in culture produced both complete and incomplete flowers. Pollen and female reproductive organs in vitro developed complete flowers that were morphologically and anatomically similar to flowers of field-grown plants. In addition, 65% of the pollen grains derived from in vitro developed flowers showed regular meiosis during microsporogenesis. Even so, a lower percentage of germination of the pollen grains derived from in vitro developed flowers could be self-pollinated and induced seed pods with viable seeds. In their first study on Dendrobium Madame Thong-In, Sim et al. (2007) showed that CW promoted vegetative growth but did not support the formation of flower buds, although it was essential to trigger the transition of the shoot apical meristem from the vegetative to floral state. BA enhanced inflorescence initiation and flower bud formation. Inflorescence stalks became visible after 3 weeks of culture upon transfer to a two-layered medium. By the 9th week, about 40–55% of the protocorms produced inflorescence stalks. Both liquid and Gelrite-solidified medium with 22.2 mM BA and 0.03% AC produced flowers. Healthy seedlings produced 100% flower buds, 75% of which resulted in abnormal flowers. In their follow-up study (Sim et al., 2008), seedling growth stage and endogenous CK level were important factors determining vegetative growth and the flower-inductive response. CK content varied in different growth stages of seedlings. Seedlings 1.0–1.5 cm in size cultured in flowering-inductive medium [liquid KC medium containing 4.4 mM BA and 15% CW)] showed up to 200 and 133 pmol g1 fresh weight (FW) of iP and N6-(D2-isopentenyl)-adenosine (iPA), respectively. These levels were significantly higher than all other CKs analyzed in seedlings of the same stage and were about 80- to 150-fold higher than seedlings cultured in non-inductive medium. During the transitional (vegetative to reproductive) stage, the endogenous levels of iP (178 pmol g1 FW) and iPA (63 pmol g1 FW) were also significantly higher than CKs in the Z and dihydrozeatin (DZ) families in the same seedlings,


65

providing strong evidence that CKs, especially the iP family, play an important role in early in vitro flowering in this orchid. For Dendrobium Sonia 17, Tee et al. (2008) found that a medium enriched with high P and low N (KH2PO4 was used at a concentration corresponding to 1.25 times full-strength MS medium while the nitrogen content [both KNO3 and (NH4)2NO3] was reduced to 0.25 times) was effective for inducing inflorescences while low P and high N content (KH2PO4 concentration corresponded to 0.25 times that of full-strength MS medium while the nitrogen content [both KNO3 and (NH4)2NO3] was reduced to 1.25 times) could only effectively promote shoot formation. In 52% of plantlets that formed an inflorescence in BA and high P- and low N-containing medium while only 20% of plantlets formed inflorescences in half-strength MS medium containing BA without any modification of the P/N ratio after 4 months. The method for inducing inflorescences in vitro was repeatable when the medium contained 20 mM BA, reinforcing the observations already made by Sudhakaran et al. (2006) for the same orchid. Different morphologies of in vitro flowers such as incomplete flower structures, abnormal and unresupinated in vitro flowers were observed. Without supplementing paclobutrazol (PBZ) in culture medium, no floral buds were induced in Friederick’s Dendrobium (Te-Chato et al., 2009). A low concentration of PBZ (0.025 mg l1) promoted a small number of floral buds (10%), PBZ at 0.05 mg l1 gave the highest percentage of floral bud induction (29%) and the highest concentration of PBZ (0.1 mg l1) provided the lowest level (6.95%) of floral bud induction. Flowers obtained from all PBZ-containing media, i.e. at all concentrations of PBZ, showed normal morphology. Dendrobium nobile (Wang et al., 2009) seedlings formed in vitro floral buds (33.3–34.8%) precociously on a defined MS medium containing 0.5 mg l1 PBZ (paclobutrazol, abbreviated as PP333 in the original source paper). Flowering occurred within 4 months of culture in the presence of 0.1 mg l1 TDZ, and the frequency of floral bud formation increased to 95.6% by growing seedlings in 0.3 mg l1 PBZ þ 0.5 mg l1 NAA followed by PBZ and TDZ. TDZ (0.05–0.1 mg l1) and PBZ (0.5–1.0 mg l1) were more effective for inducing floral buds than BA. Flowers that developed were deformed at 25 C but they developed fully when grown at a lower temperature regime (23 C/18 C, light/dark) for 45 days. In vitro seed-derived Eulophia graminea rhizomes spontaneously sprouted into flower stems followed by flowering and subsequent fruition to complete a full life cycle. About 44–61% of rhizomes spontaneously produced flower stems from the apex. Among the flowers, 50% showed normal floral organs, 40% lacked both left and right sepals, 10% lacked the right petal only while 18% of the flowers showed autogamous mating and developed fruits. The seeds were harvested and sown, with 59.6% germinating (Chang et al., 2010). Sotthikul et al. (2010) induced in vitro flowering and fruit setting in Polystachya sp. but no specific reasons were provided to explain the phenomena.

Factors inducing in vitro flowering of orchids The nature of the signal that induces flowering remains unclear although physiological studies of the floral


66



Figure 1. Orchid flowering and fruiting in vitro. (a) Flowering in vitro of Dendrobium officinale Kimura et Migo cultured on MS medium supplemented with 1.0 mg l1 BA, 0.2 mg l1 NAA, 3.0% sucrose and 0.1% activated carbon (AC); (b) Flowering in vitro of Dendrobium huoshanense C. Z. Tang et S. J. Cheng cultured on MS medium supplemented with 2.0 mg l1 BA, 0.2 mg l1 NAA, 3.0% sucrose and 0.1% AC; (c) Flowering in vitro of Dendrobium unicum Seidenf cultured on MS medium supplemented with 3.0 mg l1 BA, 0.5 mg l1 NAA, 3.0% sucrose and 0.1% AC; (d) Flowering in vitro of Cymbidium ensifolium (Linn.) Sw. cultured on MS medium supplemented with 0.5 mg l1 TDZ, 0.5 mg l1 NAA, 3.0% sucrose and 0.1% AC; (e) Fruiting in vitro of Dendrobium officinale culture on MS medium supplemented with 0.5 mg l1 NAA, 100 g l1 banana homogenate, 3.0% sucrose and 0.1% AC. Cultures were kept at a temperature of 25 2 C and 16-h photoperiod with irradiance of 30–40 mmol m2 s1. Scale bars: (a) and (b) 2 cm, (c) 1 cm, (d) 2 cm, (e) 1.5 cm. All figures unpublished.

transition led to the identification of several putative floral signals including CKs, sucrose, gibberellin and reduced N-compounds (Corbesier & Coupland 2006). To compound this complexity, many factors such as photoperiod, irradiance, temperature and hormonal control also affect the in vitro flowering of orchids (Chia et al. 1999) (Figure 1). Photoperiod Photoperiod has a significant effect on in vitro flowering of orchids. Long-day orchids need the least light period for flowering and short-day orchids cannot exceed a longest light critical point. Zhu (2006) reported that flower induction in vitro of Cymbidium kanran was significantly affected by photoperiod. The percentage of bud induction gradually decreased when the light period was prolonged from 8 h to 14 h while the highest flower bud induction percentage (41.67%) was achieved in an 8-h photoperiod. However, the percentage of bud induction was 28.26% in a 16-h photoperiod, which was higher than that observed in a 12- or 14-h photoperiod, but the induced flower buds did not develop into fully-opened blooms. Jia et al. (2000) noted that flower induction in vitro of Cymbidium ensifolium could be achieved only with continuous light and never in darkness. Vaz et al. (2004) noted a positive relationship between long days and floral spike formation of Psygmorchis pusilla. However, plants incubation under a 20-h photoperiod or longer showed reduced floral bud development in which anthesis was inhibited and flower longevity reduced.

In contrast, 12- and 16-h photoperiods resulted in perfectly opened flowers while flower buds that had been kept in darkness did not open even though a floral spike was induced. Two major flowering responses were verified, namely, when switching photoperiod from 6 to 8 h and from 22 h to continuous light. The increase in floral spike number under 8 h and continuous light may be related to carbohydrate accumulation observed just before 6- and 22-h photoperiods. This increase in carbohydrate content may have promoted the development of preexisting floral buds, which were kept dormant under shorter photoperiods. However, floral spike formation in darkness could have resulted from sugar absorption from the culture medium (Vaz et al. 2004). Temperature Although vernalization may induce or facilitate flowering in some orchid genera, its effects are not consistent in this plant family. Nevertheless, temperature is usually used to control flowering time in commercial cultures of some orchids such as Cymbidium, Dendrobium and Phalaenopsis (Chen et al., 1994; Goh & Arditti, 1985; Hew & Yong, 1997). However, similarities between temperature effects on plants cultivated in vitro and ex vitro is not a rule for all species. Spikes often did emerge when temperatures were over 28 C during the growth period; a low-temperature requirement (20 C or 25 C) for in vivo floral induction was documented for Phalaenopsis (Sakanishi et al., 1980) and the day/ night temperature (25 C/20 C or 25 C/15 C) was suitable



DOI: 10.3109/07388551.2013.807219

for flower bud induction (Wang et al., 2005). When Phalaenopsis amabilis was grown at high temperatures (30/25 C, day/night), flowering was blocked, and this could be reversed by gibberellin A3 or gibberellic acid (GA3) treatment (Wang et al., 2005). Similarly, GA3 and temperature influenced carbohydrate content and flowering in Phalaenopsis, although no specific details were provided by the authors (Chen et al., 1994). However, Duan & Yazawa (1995a) reported that low temperature treatments (using day/ night temperatures of 25/15 C and 25/10 C) did not promote the formation of floral buds in vitro and suggested that the conditions for the induction of floral buds in Phalaenopsis in vitro might be different from those in vivo. Vaz et al. (2004) reported great sensitivity to temperature variations by Psygmorchis pusilla, 27 C being the most adequate for growth, leaf and floral spike formation. Temperatures of 22 C and 32 C were not appropriate for in vitro development of P. pusilla. High temperatures might have increased respiration rates and lowered CO2 absorption, resulting in carbohydrate depletion, thereby inhibiting growth and delaying flowering. In addition, the considerable reduction in pigment content observed in P. pusilla incubated under 32 C may be deleterious to the plant photosystem. Su et al. (2001) suggested that flowering inhibition in high temperaturetreated Phalaenopsis plants was associated with too low levels of endogenous gibberellins. However, in P. pusilla, the application of GA3 could not enhance floral spike formation in vitro (Vaz et al., 2004). Wang et al. (2009) reported deformed flower development of Dendrobium nobile at 25 C but normal development at a lower temperature regime (23 C/18 C, light/dark) for 45 days. The temperature difference between day and night may have favored the accumulation of carbohydrates (Bernier et al., 1993). Nutrition In vitro flowering is influenced by the levels and ratios of the two major components, carbohydrates and minerals (reviewed by Scorza, 1982; Tee et al., 2008; Ziv & Naor, 2006). Sugars are considered to be necessary carbon sources in culture media for the viable induction and development of flowers. Vu et al. (2006) suggested that sucrose was the only factor needed in floral bud induction or initial development while other factor(s) are required to help them develop fully in later stages of in vitro floral morphogenesis, the most suitable sucrose concentration being 30 mg l1. In MS medium, high concentrations of N usually inhibited flowering and promoted vegetative growth while the use of half-strength MS mineral medium or a reduced nitrogen level enhanced in vitro flowering in Cymbidium (Kostenyuk et al., 1999), Doritis (Duan & Yazawa, 1994a), and Phalaenopsis (Duan & Yazawa, 1995a). Duan & Yazawa (1994a) reported the percentage of explants with floral buds on VW medium to be 93.3% but noted no floral bud formation on MS medium, although the presence of sucrose at 25 or 50 g l1 was indispensable. High P and low N in the medium stimulated flowering in Cymbidium (Duan & Yazawa, 1994a; Kostenyuk et al., 1999) and Dendrobium (Tee et al., 2008). Tee et al. (2008) reported

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52% plantlets cultured on the medium containing BA with high P and low N content formed inflorescence while only 20% of plantlets formed inflorescences in half-strength MS medium containing BA without any modification of the P/N ratio after four months. The percentage of adventitious shoots with floral buds decreased and the time required for increased buds to form. The lowest tested concentration of N (4.5 mM) in VW medium was the most effective for the formation of floral buds in Phalaenopsis (Duan & Yazawa, 1995a). Kostenyuk et al. (1999) reported that root-excised Cymbidium plantlets cultured in MS medium containing BA with high P and low N content achieved about 2.5-fold more in vitro flowers than cultures grown in half-strength MS medium containing BA without a modified P/N ratio. In addition, the NHþ 4 /NO3 ratio is an important factor for the in vitro flowering of plants (as it is for the development of other organs (Teixeira da Silva et al., 2005). Raising the ratio depressed in vitro flowering while reducing the NHþ 4 concentration promote in vitro flowering (Duan & Yazawa, 1994a; Kachonpadungkitti et al., 2001). Duan & Yazawa (1994a) also reported VW medium suited the formation of floral buds and the development of floral stalks when supplemented with BA, but it was not suitable for subsequently inducing flowering; inversely, HyponexÕ medium (with a very low NHþ 4 /NO3 ratio) did induce the formation or flowers nor did it permit them to develop, but it could induce flowering in flower buds formed from VW medium supplemented with BA. The same result was reported for Dendrobidium strongylanthum by Zhao et al. (2012). Vaz & Kerbauy (2000) reported a negative correlation between the total mineral salt concentration and the number of floral spikes of Psygmorchis pusilla. A low concentration of N stimulated flower formation and this effect was more pronounced than that observed for the lowest total salt level used. The presence of NHþ 4 in the medium was essential for plant growth and development, while the absence of NO 3 depressed flowering. Flower formation was also enhanced by half the ionic concentration of P and K while the highest Ca concentration showed some floral stimulation. Plants grown under nutrient-deficient conditions were understandably more yellow than those cultivated on full-strength medium. Plant growth regulators Auxins and cytokinins In most in vitro flowering studies of orchids, a single PGR such as BA, 2iP, TDZ, PBZ, ABA, KN, GA3, TIBA (2,3,5triiodobenzoic acid) or NAA, or combinations of other PGRs and nutrients were used to induce in vitro flowering (Chang & Chang, 2003; Duan & Yazawa, 1994a; Kostenyuk et al., 1999; Sim et al., 2007; Te-Chato et al., 2009; Wang et al., 2009). BA has been used for most in vitro flowering experiments of a number of orchids, including Dendrobium primulinum Lindl. (Deb & Sungkumlong, 2009), Doriella Tiny (Duan & Yazawa, 1994a), Dendrobium Chao Praya Smile (Hee et al., 2007), Cymbidium niveo-marginatum Mak (Kostenyuk et al., 1999), Cymbidium ensifolium var. misericors (Chang & Chang, 2003), Dendrobium Madame Thong-In (Sim et al., 2007), Dendrobium candidum (Wang et al., 1997), and Dendrobium nobile (Wang et al., 2009), Psygmorchis pusilla


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(Vaz & Kerbauy, 2008b), Bulbophyllum auricomum (Than et al., 2009), Dendrobidium strongylanthum (Zhao et al., 2012). Nevertheless, the most suitable BA concentration showed a wide range depending on the orchid species. Duan & Yazawa (1994b) reported 94.7% of shoots forming floral buds in VW medium with 5 mg l1 BA in Doriella Tiny and floral buds formed on the VW medium supplemented with KN, Z and 2iP, except for Z (5 mg l1) where 13% of explants produced floral buds, but when the medium was supplemented with BA, subsequent flower induction was inhibited. Similarly, more than 70% of shoots formed floral buds in VW medium with 5 mg l1 BA in Phalaenopsis (Duan & Yazawa, 1995a). Than et al. (2009) reported in vitro flowering within six months after sowing Bulbophyllum auricomum seed, with the highest percentage (50 15.8%) of flowering observed in MS medium with 1.0 mg l1 BA þ 0.5 mg l1 NAA; Hee et al. (2007) reported 45% of shoots forming floral buds at with 11.1 mM (2.0 mg l1) BA, while Wang et al. (1997) reported that 82.8% of Dendrobium candidum shoots formed floral buds on MS medium with 0.01 mg l1 BA. Wang et al. (2009) reported that a high dose of BA (1.0 mg l1) was more effective for floral bud induction (20.0%) that lower doses (0–0.5 mg l1). Chang & Chang (2003) reported that 10–33 mM 2iP and 3.3–10 mM TDZ produced inflorescences more effectively than 0.1–33 mM BA. Sim et al. (2007) reported BA alone in the medium was not effective for floral induction but, in the presence of CW, 22.4 mM BA enhanced earlier formation of inflorescence stalks and promoted flower bud induction. Nadgauda et al. (1990) suggested that CKs might be involved in vitro flowering perhaps with CW supplying inositol and CK oxidase inhibitors, which promote CK responses. The role of CKs in floral evocation might be in controlling early mitotic activity, precocious initiation of axillary meristems, and increased rate of appendage production by the meristems (Scorza & Janick, 1980). Also, CK was cited as a probable component of a multi-factored flowering stimulus (Bernier, 1988). BA was also required for normal in vitro development of rose floral buds (Vu et al., 2006), which possibly regulated floral development through genes controlling shoot apical meristem activity (Lindsay et al., 2006). TDZ was more effective than BA for flower bud induction in vitro for Cymbidium niveo-marginatum Mak (Kostenyuk et al., 1999), Cymbidium ensifolium (Chang & Chang, 2003) and Dendrobium Second Love (Ferreira et al., 2006), but TDZ caused poor plant growth, and floral buds withered soon (Kostenyuk et al., 1999). Wang et al. (2009) also reported that TDZ promoted floral bud formation (31.9–34.8%) better than BA at low concentrations (0.05–0.1 mg l1), but floral bud (12.6–15.6%) induction was reduced at high concentrations (0.5–1.0 mg l1). TDZ is known to stimulate the synthesis of endogenous CKs and destroy CK oxidase (Murthy et al., 1998) and it can be considered responsible for the much higher increase in endogenous CKs. Also, there is some evidence that TDZ may influence the endogenous level of IAA (Ferreira et al., 2006; Murthy et al., 1995). Ferreira et al. (2006) reported that 1.8 mM TDZ induced an increase in the CK/IAA ratio for flower determination between 10 and 25 d of culture, with a sharp decrease in the development of flower buds after 25 d of culture. In addition,


Chen (2012) reported that when one-year-old callus of Oncidium ‘‘Gower Ramsey’’, which was induced and subcultured with 3 mg l1 TDZ and 5 mg l1 dicamba (line 13 callus), was transferred onto 1/2MS medium supplemented with 0.1 mg l1 NAA and 3 mg l1 TDZ, it gave the highest number of petal-bearing embryos. Flowering characteristics of plantlets from normal embryos and petal-bearing embryos were not all significantly different. Te-chato et al. (2009) reported PBZ at 0.05 mg l1 gave the highest percentage (29%) of floral bud induction of Friederick’s Dendrobium. Flowers obtained at all PBZ concentrations had a normal morphology. Wang et al. (2009) reported PP333 at high concentrations (0.5– 1.0 mg l1) promoted floral bud formation (28.9–33.3%) but dramatically reduced the percentage of floral buds (8.9– 17.0%) at low concentrations (0.05–0.1 mg l1). This result disagrees with the findings that PP333 totally blocked the inductive effects of CK (Kostenyuk et al., 1999). TDZ (0.05–0.1 mg l1) and PP333 (0.5–1.0 mg l1) were more effective for inducing floral buds of Dendrobium nobile plantlets than BA (Wang et al., 2009). Chang & Chang (2003) reported, among eight CKs tested, only TDZ, 2iP and BA inducted de novo flowering of rhizomes, with TDZ and 2iP having a stronger inductive effect than BA, respectively. The other five cytokinins [KN, DPU (1,3-diphenylurea), ADE (6-aminopurine), Zea, ZR] tested induced rhizome proliferation and shoot bud formation but not flowering. Cen et al. (2010) reported that among the single-factor hormone treatments, the most suitable concentration for flower bud formation was 0.2 mg l1 PBZ (8.5%) while 0.06 mg l1 TDZ resulted in 15.5% of shoots forming flower buds; a multi-factor PGR experiment ranked PGRs effects on flowering induction as follows: PBZ þ BA þ NAA þ TDZ4 PBZ þ BA þ NAA4PBZ þ BA and PBZ þ NAA; the most suitable treatment was 0.3 mg l1 PBZ þ 0.5 mg l1 BA þ 0. 5 mg l1 NAA þ 0.06 mg l1 TDZ. The percentage of flower bud formation and the percentage of flowers that blossomed reached 80.4% and 90.3%, respectively. PBZ and TDZ could both induce flowering in vitro of Dendrobium officinale Kimura et Migo although the effect of TDZ was stronger (i.e. induced more flower buds) than PBZ. Wang et al. (1997) reported that a flowering frequency of Dendrobium candidum was further increased to more than 80% by pretreatment of protocorms in a 0.5 mg l1 ABAcontaining medium followed by transfer onto an MS medium with 2.0 mg l1 BA. Wang et al. (2009) reported 1.0 mg l1 PBZ þ 0.1 mg l1 TDZ induced flowering in vitro over half of Dendrobium nobile shoots (62.2%) that were pre-cultured on half-strength MS medium supplemented with PA (0.5 mg l1 PBZ þ 0.5 mg l1 ABA); 15.4% of flowers were normal. However, 95.6% of shoots could be induced to form floral buds after pre-culture in PN (0.3 mg l1 PP333 þ 0.5 mg l1 NAA)-containing medium followed by transfer onto halfstrength MS medium with 1.0 mg l1 PBZ þ 0.1 mg l1 TDZ; in this case, 27.8% of flowers were normal. A similar pattern was observed in Dendrobium moniliforme (L.) SW. (Wang et al., 2006). Goh & Yang (1978) demonstrated that IAA could suppress the promotive effect of BA on flowering in two Dendrobium hybrids. Wang et al. (1997) reported there were a few rooted



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Dendrobium candidum plantlets that flowered in vitro, although the percentage of in vitro flowering was much lower than rootless plantlets. Wang et al. (1997) also reported that NAA alone suppressed flower formation, possibly because roots act as the major site of CK biosynthesis, thus plantlets with roots were less sensitive to exogenous CK than rootless ones (Wang et al., 1997). On the other hand, regenerative roots may be a sink for some substances that are required for floral initiation or for the release of some signals which inhibit flower induction, as occurs in tobacco (reviewed by McDaniel, 1996). The importance of iP family members and other types of CK in flowering has been observed by many authors. Sim et al. (2008) reported CKs, especially iP and iPA, to play an important role in early in vitro flowering of Dendrobium Madame Thong-In. Ferreira et al. (2006) found that after culturing Dendrobium Second Love shoots in medium containing TDZ inductive to in vitro flowering for 5 d, endogenous levels of iP9G [N6-(D2-isopentenyl)-adenine-9glucoside], zeatin-9-glucoside (Z9G) and Z increased, but the level of iP did not change greatly. Gibberellins A sufficient amount of GA is required if flowering is to occur, and the deficiency in the GA biosynthetic pathway increases the photoperiodic sensitivity. A high concentration of GA3 suppressed flower bud induction and blooming (Kostenyuk et al., 1999). Abscisic acid and ethylene Many preliminary observations suggest that the role of ABA is inhibitory to flowering in vitro (Vaz & Kerbauy, 2008b). Exposure to ethylene is capable of inducing flowering in some species. The flowering frequency was further increased to 82.8% on average by pre-treatment of protocorms in a 0.5 mg l1 ABA-containing medium followed by transfer onto medium with 2.0 mg l1 BA (Wang et al., 1997). Anthesis was favoured by removal of ethylene using a KMnO4-trap (Vaz & Kerbauy, 2000).

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inflorescence stalks increased as the CW concentration (50, 100 and 150 ml l1) increased. Similar results were achieved with Doriella Tiny (Duan & Yazawa, 1994b), but CW alone did not induce the formation of flower buds. CW contains sugars, vitamins, amino acids and PGRs (Tulecke et al., 1961; Raghavan, 1966). Ge et al. (2006) and Sim et al. (2008) found ZR to be one of the major CKs in CW. The addition of AC to Gelrite-solidified agar reduced the effective BA concentration for in vitro flowering (Sim et al., 2007) as AC is known to adsorb many types of molecules, including growth substances added to the culture medium (Weatherhead et al., 1978; Thomas, 2008). Explant status Sim et al. (2007) noted that the presence of tiny roots in young protocorms of Dendrobium Madame Thong-In did not affect their response towards a BA-enriched medium when compared to rootless young protocorms in terms of axillary shoot formation, root production, percentage of cultures with inflorescence stalks and flower buds. Culture methods Sim et al. (2007) reported the use of a two-layered medium (liquid over Gelrite-solidified) to promote the induction of inflorescence stalks, flower buds and flower development from protocorms of Dendrobium Madame Thong-In. The volume of liquid medium in this two-layered system also affected flower bud formation and flower development. The highest percentage of normal flowers produced was with 20 ml of KC medium (Knudson, 1946) supplemented with 22.2 mM BA (K5, 75%), followed by 43% in 30 ml of K5 and 26% in 5 ml of K5. This method was also successfully applied to induce in vitro flowering of another Dendrobium hybrid, Chao Praya Smile (Hee et al., 2007). However, in D. Second Love, shoots could be induced to produce flowers in vitro after culturing in a Phytagel-solidified medium with 1.8 mM TDZ (Ferreira et al., 2006). Therefore, the physical state of the medium i.e. liquid or Gelrite-solidified medium, and probably air exchanges (liquid medium) also played an important role in in vitro flowering.

Polyamines Polyamines played an important role in flower initiation in vitro of Dendrobium candidum. The addition of 20 mmol l1 spermidine, or 2.0 mg l1 BA, or the combination of 0.5 mg l1 NAA and BA in the culture medium induced protocorms or shoots to flower within 3–6 months with a frequency of 31.6–45.8% (Wang et al., 1997). Polyamines are believed to cooperate with CKs in the control of several processes, including cell division (Kuznetsov & Shevyakova, 2007; Pang et al., 2007). However, their exact function in the flowering process is so far unknown (Wang et al., 1997). Other conditions Additional media additives Sim et al. (2007) reported CW to be necessary for the growth of protocorms and for the transition of the vegetative apical shoot meristem of Dendrobium Madame Thong-In to an inflorescence meristem: the percentage of protocorms with

In vitro pollination and fruiting Orchid breeding involves pollination, seedpod maturation, protocorm development, in vitro growth of seedlings and subsequent ex vitro establishment of seedlings (Hossain et al., 2013). The entire breeding cycle is between 3 and 5 years in duration depending on the genotype involved (Hee et al., 2007; Kamemoto et al., 1999). Sim et al. (2007) reported that in vitro flowers were artificially self- or cross-pollinated using pollinia from in vitro flowers and that in vitro pollination was not successful although in vitro pollination was successful (fruiting achieved) if the anthers from in vivo-grown mother plants were harvested, surface-sterilized and the pollinia from such flowers used to pollinate the in vitro flowers. However, the density (number) of the seed of the seedpods pollinated in vitro was low compared to the seedpods pollinated in vivo, and most of the seeds in vitro appeared to be non-fertile (about 0.1% of the seeds were fertile) and only a few seeds germinated. However, Hee et al. (2007) reported, despite the


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low percentage of germination of pollen derived from in vitrodeveloped flowers, that pollination of in vitro-developed flowers and subsequent seedpod formation produced a large number of seeds sufficient for breeding purposes. The process from seed germination to production of the next generation of seeds in culture could be shortened from over 35 months to only about 11 months. The success of in vitro pollination and viable seed formation in Psygmorchis pusilla (Vaz & Kerbauy, 2008b), Dendrobium Chao Praya Smile (Hee et al., 2007) and Dendrobium Madame Thong-In (Sim et al., 2007) demonstrate that the gametes produced from in vitro flowers were functional. Thus, such a system could also be used for early determination of hybrid fertility. Chiu et al. (2011) reported that pollen tube growth and fertilization were successful after hand pollination in vitro. Zygotes were observed in embryo sacs 3–4 weeks after pollination (WAP). Fruits began to develop at 6 WAP, with seeds containing a minute embryo, but they were unable to germinate. The seed germination percentage was 7.5% at 8 WAP and 39.4–62.7% at 10–14 WAP. The fruits could generate more than 63 838 seeds capsule1 and more than 95% of the seeds contained an embryo; however, there were no follow-up germination experiments testing the viability of seeds derived from in vitro fruiting. In our study on Dendrobium candidum, from seed germination to production of the next generation of seeds in culture could be shortened from over 36 months to only about 8 months, and seed development needed only 60 d from pollination in comparison with 120–150 d in vivo (greenhouse), the germination percentage of seeds from in vitro fruiting was over 85%, which was similar to the seeds from in vivo (unpublished). Therefore, the whole breeding cycle could be considerably shortened. Chang et al. (2010) showed in vivo or in vitro seed-derived rhizomes of Eulophia graminea to spontaneously sprout flower stems which then flowered and subsequently fruited to complete a full life cycle without artificial pollination when Woody Plant Medium (WPM) (Lloyd & McCown, 1981) was supplemented with 1.0 mg l1 BA, 1.0 mg l1 NAA, 1 g l1 AC and 20 g l1 sucrose. A total of 4 generations were cultured over a 4-year period.

Possible mechanisms of in vitro flowering in orchids Some reports have indicated that flowering in orchids is accomplished by the combined synchronous effect of many endogenous substances, especially PGRs (Bhadra & Hossain, 2003; Hsiao et al., 2011; Kostenyuk et al., 1999; Wang, 1988). The proper manipulation of PGRs at appropriate doses to an appropriate explant can induce in vitro flowering, especially in orchids (Vaz & Kerbauy, 2008a). However, different experimental conditions and types of orchids may have various effects. For Phalaenopsis, CKs (e.g. BA) stimulate flowering, and auxin suppresses the effect of BA; GA3 is not effective when applied alone but when added in combination with BA it seems to accelerate the effect of BA slightly (Goh & Arditti, 1985; Goh & Yang, 1978; Hew & Clifford, 1993). For Dendrobium (Lee & Koay, 1986), Doritaenopsis and Phalaenopsis (Blanchard & Runkle, 2008), GA3 did not stimulate the effect of BA. The encouragement of flowering by the application of BA seems to suggest that CKs play a


part in regulating inflorescence initiation of Doritaenopsis and Phalaenopsis, although the promotion of flowering depends on certain conditions (Blanchard & Runkle, 2008). Gibberellins are thought to play an essential role in the vegetative and reproductive development of plants (Arditti, 1992; Cecich, 1985; Tymoszuk et al., 1979). Only 2.5 mM GA3 effectively promoted inflorescence emergence in Miltoniopsis (Matsumoto, 2006) by increasing flower spike length and size (Sakai et al., 2000). Dewir et al. (2007) reported that GA3 was mandatory for a shift from the vegetative to the reproductive stage in Spathiphyllum (Araceae), an important tropical ornamental. However, Chen et al. (1997) reported that flowering in hybrid Phalaenopsis was clearly induced by GA3 at 1 mg/shoot at higher temperatures (30 C day/25 C night), and that flower primordial elongation, even though promoted by GA3, was fully inhibited by BA treatments while a combined treatment with BA increased flower quality. In Cymbidium niveo-marginatum, a high concentration of GA3 (15 mg l1) delayed flowering and decreased the percentage of plants exhibiting flower induction. However, it did not totally block flower induction (Kostenyuk et al., 1999). The blockage of flower development under high temperatures can be rescued by applying GA3 exogenously, as observed in Phalaenopsis hybrida (Su et al., 2001). Molecular flowering-time control and their putative triggering in vitro and ex vitro The reproductive phase in angiosperms involves substantial changes in vegetative-shoot apical and/or shoot-lateral, meristems whereas annual herbaceous plants usually die after flower/seed bearing (monocarpy), perennials (trees and shrubs) produce flowers throughout their lifetime (polycarpy) (Ruan & Teixeira da Silva, 2012). Nonetheless, it is interesting to note that, despite their herbaceous architecture, all Orchidaceae species are polycarpic. Regardless of the flowering habit, reproductive success in angiosperms depends on the age of the plant, as well as environmental seasonal cues, such as daylight (photoperiodism) and wintertime (vernalization), the former sensed by mature leaves (phytochrome pigments) and the latter directly in the apical shoots or leaves (Figure 2). Some transient environmental conditions, such as nutrients and water fluctuation, can also influence the flowering time in some species. On considering nutrient availability, the in vitro flowering of isolated shoots of Dendrobium Second Love was improved during incubation in a more diluted VW medium, when compared to the more concentrated MS formula. The oligotrophic nutritional habit of this epiphytic orchid was postulated as a possible explanation for the above mentioned in vitro result (Ferreira, 2003). The transition from vegetative to reproductive growth occurs when competent meristematic cells respond to signals that evoke floral initiation. This developmental process, representing a complex and poorly understood event, involving multiple signaling control mechanisms (Bernier et al., 1993; Huijser & Schmid, 2011), will be discussed next. It has been demonstrated that environmental cues trigger substantial changes in metabolism and the hormonal status


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Figure 2. Simplified diagram depicting exogenous and endogenous signaling factors putatively involved in the transition of orchid shoot apical meristem (SAM) and shoot lateral meristem (SLM) into floral meristems through long and short distance signals. Figure adapted from A. thaliana as proposed by Turnbull (2011) and adopted by the authors for orchids.

in plants. The inductive effects of treatments with exogenous cytokinins and gibberellins on plant development have already been demonstrated for some mature orchid species (Goh et al., 1982; Matsumoto, 2006). The advantageous effects of cytokinins in in vitro orchid flowering have been extensively discussed throughout this review. Nonetheless, despite few studies having been addressed to the role of endogenous hormones in this developmental process in orchid species, the results obtained have corroborated those found through exogenous treatments. Endogenous hormonal levels and orchid flowering have been predominantly studied in thermoperiodic orchids (Campos & Kerbauy, 2004; Chou et al., 2000; Sakanishi et al., 1980). In this case, cold-treated plants of Phalaenopsis and Dendrobium (Nobile group) resulted in enhanced levels of endogenous cytokinins, marked by the abundance of zeatin and zeatin riboside types (Campos & Kerbauy, 2004; Chou et al., 2000). According to Wang et al. (2002), the decrease of endogenous ABA in Phalaenopsis shoots could possibly be associated with lateral reproductive-bud development. Furthermore, the absence of flowering in the control plants of Dendrobium Second Love treated with 200 mg l1 BA, indicated that, even though CKs could be a necessary signal, they are incapable of triggering floral bud differentiation (Campos & Kerbauy, 2004). Gibberellins represent another important group of plant growth regulators (PGRs), with conspicuous effects, not only in several short-day flowering plants, but also in some mature


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orchids (Goh et al., 1982; Matsumoto, 2006). Cold-flowering induction and carbohydrate accumulation in mature Phalaenopsis plants were both substituted by gibberellin treatment (Chen et al., 1994; Su et al., 2001). The importance of sugars, as a signalizing factor in flowering, is shown in Figure 2. From the above observations, the question is: what leads to precocious in vitro attainment of the reproductive phase in some young orchid plants? A facile conclusion is that an answer to this question is notoriously difficult, especially when considering that this crucial phase in plant development, which is controlled by multiple physiological and molecular pathways, is little understood. Perhaps, some insight could be found, if multifactorial control of plant flowering were taken into consideration (Bernier et al., 1993). According to these authors, flowering induction would occur as a consequence of multifactorial controlling mechanisms, represented mainly by sugar and hormonal signaling pathways. Plants cultured in vitro are considered to have little photosynthetic ability to achieve a positive carbon balance (Hew & Yong, 1977). Culture media, through usually containing all the main factors involved in orchid flowering, such as PGR, sugar and nutrients, mimic the multifactorial hypothesis, as proposed by Bernier et al. (1993). In fact, added-sucrose/CK media are critical for both shoot multiplication (Ferreira & Kerbauy, 2002) and in vitro flowering stimulation of Dendrobium Second Love (Ferreira, 2003). Significant effects of sucrose on in vitro flowering have also been demonstrated in Doriella Tiny (Duan & Yazawa, 1994a). Thus, it is plausible to assume that under certain circumstances the media culture composition, together with certain environmental incubation conditions, could mimic this endogenous status (multifactorial), so necessary for flowering in mature orchid plants, by hastening conversion of the vegetative meristem into a reproductive one. In spite of high diversity and the economic importance of several genera of Orchidaceae, research on orchid developmental physiology is still relatively scarce. The available studies on this trait are either concentrated on the vegetative to reproductive meristem transition (flowering) stage, or the floral organ specification (sepal, petal, label, stamen and carpel). Furthermore, these studies are deeply anchored on studies carried out with the small mustard plant model, Arabidopsis thaliana (Brassicaceae). Apparently, one of the first cases of evidence related to genetic flowering transition control in orchid plants was presented by Yu & Goh (2000a) in Dendrobium Madame Thong-In, cultured in vitro. The isolated DOH1 gene shared great similarity with the KNOX gene expressed in the shoot apical meristem of A. thaliana. Molecular studies with A. thaliana have corroborated the former idea, that floral meristem transition might be controlled, either by a long-distance signaling process involving mobile leaf-formed signals (photoperiodism), or directly by meristem-formed signals (thermoperiodism/vernalization), both briefly shown in Figure 2. The main flowering signals found in A. thaliana, i.e. photoperiodism, vernalization and hormones, are the same as those described for orchid plants, both ex vitro and in vitro. Although the names of genes used in Figure 2 refers to A. thaliana, genes


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with equivalent functions (orthologous) have been described in other angiosperms, including orchids (Hou & Yang, 2009; Pan et al., 2011). Recent research on gene expression in Phalaenopsis aphrodite, revealed substantial differences between vegetative and reproductive organs; whereas 762 were expressed in the former, 2608 were found in the later phase (Su et al., 2011). Nowadays, FLOWERING LOCUS T (FT) can be considered as a well-established floral leave-integrator gene, playing an important role in flowering time (Huang et al., 2012). Its small FT protein is capable of acting as a phloem mobile florigen signal. ‘‘The collective evidence from several laboratories, mainly from research on the photoperiod, indicates that FT and its homologues (like TSF, for instance – Figure 2), are universal signaling molecules for flowering plants’’ (Turnbull, 2011). Functional analysis of the ortholog FT in Oncidium Gower Ramsey indicated its involvement in the photoperiodic control of floral transition (Hou & Yang, 2009). In A. thaliana photoperiodic-controlled flowering depends on the phytochrome family genes (PhY) involved in photoperiodism/ circadian clock systems. Furthermore, in this model plant, the protein receptor PhY appears to be involved with FT leaf expression through gibberellin activity (Figure 2). In cold-treated plants of Phalaenopsis hybrida, coincidence was observed between the enhanced amount of endogenous gibberellins and the onset of flowering. According to Turnbull (2011), cold perception could occur in the vicinity of, or outside the shoot apex meristem, i.e. in the leaves. In the mango, a typical tropical tree, flowering appears to be defined by a temperature-regulated florigenic promoter (FT), synthesized in leaves and translocated to the buds, via phloem (Ramirez et al., 2010). Cold treatments also enhanced the endogenous levels of cytokinins in whole plants of Phalaenopsis (Chou et al., 2000) and Dendrobium (Campos & Kerbauy, 2004). The involvement of light in flowering time control appears to also occur through sugar, especially sucrose, availability (photosynthesis) (King et al., 2008). A relationship between FT and sucrose synthase gene expression was recently proposed for A. thaliana (Seo et al., 2011). From the above, the proposal is that, regardless of the sites of FT expression – leaves or shoot apex – the product of this expression interacts directly with the FD element (FLOWERING LOCUS D), the resultant complex then inducing LEAFY (LFY) and APETALA1 (AP1) expression, both necessary and sufficient for transition from the vegetative to the reproductive stage (Figure 2). This figure also shows gibberellin’s putative involvement in LFY expression. Research with Oncidium has been significant in identifying several genes involved in flowering time regulation (Hsu et al., 2003). The OMADS1 gene and AGL6-like gene isolated from this orchid species were capable of up-regulating the expression of both flowering time FT genes and flower meristem identity genes LFY and AP1, in A. thaliana transgenic plants (Hsu et al., 2003). The presence of OnFT mRNA was detected in various organs (axillary buds, leaves, pseudobulbs and flowers) of photoperiodically induced Oncidium Gower Ramsey plants (Hou & Yang, 2009). Transformed Oncidium Gower Ramsey plants


overexpressing the OMADS1 gene (related to the ABCDE MADS box genes), flowered significantly earlier than non-transgenic ones (Hsu & Yang, 2002; Thiruvengadam et al., 2012). Such tissue-specific promoters are key to tissuetargeted genetic transformation of orchids (Teixeira da Silva et al., 2011). Xu et al. (2006) isolated candidates for A, B, C, D and E function genes from Dendrobium crumenatum, which included AP2-, PI/GLO-, AP3/DEF-, AG- and SEP-like genes. The expression profiles of these genes exhibited different patterns from their Arabidopsis orthologs in floral patterning. Functional studies showed that DcOPI and DcOAG1 could replace the function of PI and AG in Arabidopsis, respectively, DcOAP3A was found to be another putative B function gene. Moreover, yeast two-hybrid analysis demonstrated that DcOAP3A/B and DcOPI could form heterodimers, which could further interact with DcOSEP to form higher protein complexes. Once the shoot apical meristem has acquired floral commitment, expression starts in genes involved in flower organ identity (ABC model). Pan et al. (2011) characterized the orthologous orchid B-class homeotic genes APETALA (AP) and PISTILATA (PI) in 11 orchid species, from which, 24 were identified as AP3 like-genes and 11 as PI like-genes. In both groups, divergence was extensive and relationships complex, among the orchid species studied, thereby placing in evidence the impossibility of commonly and perfectly explaining floral development in orchids, by way of the zygomorphic morphogenesis ABC model (Pan et al., 2011). Dormant buds of Phalaenopsis contain a relatively high level of free ABA, whereas no detectable free or bound ABA was found in flowering shoots. A decrease in free ABA in buds may be associated with bud activation and the development of flowering shoots (Hsiao et al., 2011). Molecular techniques have been applied to study the mechanisms of flowering in orchids and more than 70 genes have been cloned from 7 orchid genera, some of these genes are related to flowering. The profile of gene expression during the transition to flowering has been established in Dendrobium spp. using an in vitro flowering system (Yu & Goh, 2000b). For example, four color-related genes in Oncidium, including OgCHS, OgCHI, OgANS and OgDFR, have been identified and are expressed during floral development (Chiou & Yeh, 2008). Among them, OgCHI and OgDFR showed especially low expression in yellow lip tissue but greater expression in the red part of Oncidium flowers (Hsiao et al., 2011). A UDPglucose anthocyanidin flavonoid glucosyltransferase (UFGT), which was isolated from P. equestris flower buds, showed high expression in red cultivars (Chen et al., 2011). Knockdown of the expression of PeUFGT3 by RNA interference resulted in various levels of fading color in Phalaenopsis. Consequently, PeUFGT3 may be associated with red color formation in Phalaenopsis. Chiou et al. (2008) identified OgCHRC and its promoter (Pchrc) in Oncidium that specifically expresses in flowers. Thiruvengadam et al. (2012) reported that overexpression of the Oncidium MADS box (OMADS1) gene promotes early flowering in transgenic Oncidium Gower Ramsey. Recently, some significant advances have been made in our understanding of the Cymbidium sinense transcriptome, which would allow the mechanisms responsible for floral development, and hence


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in vitro flowering, in this orchid to be better understood (Zhang et al., 2013). In that study, a total of 41 687 unique sequences were annotated, 23 092 of which were assigned to specific metabolic pathways while 120 flowering-associated unigenes, 73 MADS-box unigenes and 28 CONSTANS-LIKE (COL) unigenes were identified. Such tissue-specific promoters are key to tissue-targeted genetic transformation of orchids (Teixeira da Silva et al., 2011). Guo et al. (2006) cloned the pPI9 gene from Phalaenopsis but it only expressed in the reproductive organs, suggesting that it was probably involved in the regulation of floral morphogenesis. A more detailed review on the molecular basis underlying floral color and development in orchids has recently been reviewed by Hsiao et al. (2011), and thus details will not be covered here.

Future perspectives Physiological and molecular studies have contributed to great advances in our understanding about the flowering process. Besides these techniques, in vitro culture has certainly contributed to this research progress. So far, considering the relative facility that some young orchid plants flower under in vitro conditions, they could be seen as natural and noticeable models to study the yet unknown process of meristem conversion, particularly in plants with a long vegetative growth period. The conditions that induce floral bud formation in vivo may be different from the promotive cues under in vitro culture, characterized by some authors as an artificial environment. Independent of this, in both in vivo and in vitro systems, flowering is achieved when intact plants or isolated shoots reach maturity and are able to shift from the vegetative to reproductive stage (Teixeira da Silva & Nhut, 2003b). It is plausible to think that medium composition, PGRs, or even particular changes in internal vial atmospheric conditions can hasten growth rate, shortening the vegetative period and leading to precocious flowering. Despite the fact that many studies have already been performed on the in vitro flowering of orchids, further elucidation of the relationships among gene expression and the establishment of possible links between them and the activities of PGRs, carbohydrates, mineral nutrients and environmental cues is still necessary to unravel the complex mechanisms of floral transition and to assist orchid breeding programs. This means that many avenues of research are still available for exploration. In many commercial orchids, juvenile periods range between two and four years. Orchid breeders usually take a few years to grow the thousands of seedlings from each seedpod to maturity before flower quality can be evaluated. Therefore, the development of an early, in vitro flowering method will have a significant impact on the orchid industry. Such a system would allow earlier assessment of certain desired characteristics of the flowers such as size, shape, tones and variation in colors. Once the desired characteristics are selected, the clone could be mass propagated through tissue culture. In addition, such miniaturized orchid plantlets with flowers have potential commercial value as gifts or decoration, the ‘‘in vitro bouquets’’ (Sudhakaran et al., 2006). Furthermore, significant shortening of the juvenile phase can provide a model system for studying flower initiation and development (Sim et al., 2007)

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Tee et al. (2008) reported only 4% of inflorescence stalks Dendrobium Sonia 17 were able to flower and that the size of in vitro flowers induced was far smaller than the original flower size. Different types of incomplete floral structures were observed, for example, abnormal flowers with fewer petals or sepals, flowers without the lip structure, flowers with different colors and sizes, and resupinated and unresupinated flowers. Similar abnormalities were observed previously by others for Doriella (Duan & Yazawa, 1994a) and in rose (Zeng et al., 2013). Various abnormalities of in vitro flower buds formed indicate that different conditions might be required for the initiation and development of flowers. In ornamental biotechnology, however, except where clonal production is required, variation in such factors can actually be a beneficial factor by creating new phenotypes with new and unique ornamental value. In conclusion, an in vitro flowering system of orchids is considered to be a convenient tool to study the switch-on flowering mechanism of orchids. Establishing a reliable in vitro protocol to induce early flowering in orchids is important for advancing studies of molecular and genetic mechanisms of flower induction more rapidly and for assisting orchid breeding programs, which would also benefit the orchid industry.

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