Acclimatization of in Vitro-derived Dendrobium

May 2017. Horticultural Plant Journal, 3 (3): 110–124.

Horticultural Plant Journal Available online at www.sciencedirect.com The journal’s homepage: http://www.journals.elsevier.com/horticultural-plant-journal

Acclimatization of in Vitro-derived Dendrobium Jaime A. Teixeira da Silva a,*, Mohammad Musharof Hossain b,*, Madhu Sharma c,*, Judit Dobránszki d,*, Jean Carlos Cardoso e,*, and ZENG Songjun f,* a

P.O. Box 7, Miki-cho post office, Ikenobe 3011-2, Kagawa-ken 7610799, Japan Department of Botany, University of Chittagong, Chittagong 4331, Bangladesh c Division of Biotechnology, CSIR—Institute of Himalayan Bioresource Technology, Palampur, 176061 Himachal Pradesh, India d Research Institute of Nyíregyháza, University of Debrecen, Nyíregyháza, P.O. Box 12, H-4400, Hungary e Centro de Ciências Agrárias, UFSCar, Via Anhanguera, km 174, CP 153, CEP 13.600-970, Araras, Brazil f Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China b

Received 12 October 2016; Received in revised form 20 December 2016; Accepted 28 April 2017 Available online 5 July 2017

A B S T R A C T The successful ex vitro establishment of Dendrobium plantlets raised in vitro determines the quality of the end product (cut flowers or potted plants) in commercial production for economic gain. When in vitro Dendrobium plantlets are transplanted from the culture room to greenhouse conditions, they may desiccate or wilt rapidly and can die as a result of changes in the environment, unless substantial precautions are taken to adapt plantlets to a new environment. The acclimatization of in vitro-grown Dendrobium plantlets to an ex vitro environment by gradually weaning them towards ambient relative humidity and light levels facilitates better survival of young and physiologically sensitive plantlets. Dendrobium plantlets raised in vitro must thus undergo a period of acclimatization or transitional development to correct anatomical abnormalities and to enhance their physiological performance to ensure survival under ex vitro conditions. The most common approach to improve the survival of Dendrobium plantlets upon transfer to an ex vitro environment is their gradual adaptation to that environment. Under such conditions, plants convert rapidly from a heterotrophic or photomixotrophic state to an autotrophic growth, develop a fully functional root system, and better control their stomatal and cuticular transpiration. Gradual adaptation is carried out in a greenhouse by decreasing relative humidity using fog or mist chambers and by increasing light intensity using shading techniques. This review details the acclimatization and ex vitro survival of Dendrobium plants produced in vitro. This advice is also useful for other orchids. Keywords: Orchidaceae; acclimation; hardening; autotrophic; light intensity; medicinal orchid; relative humidity

1. Introduction Dendrobium is the second-largest genus in the Orchidaceae, consisting of more than 1 100 natural species and a large number of hybrids (Pridgeon and Morrison, 2006; Wood, 2006; Wu et al., 2009). Many Dendrobium hybrids produce flowers several times a year and the myriad of shapes, vibrant colours, shapes and textures of Dendrobium flowers have a high demand in cut-flower and pot plant markets (Zeng and Cheng, 1996; Zeng and Hu, 2004; Limpanavech et al., 2008). Dendrobium is conventionally propagated by separat-

ing back bulbs and keikies, or by vegetative cuttings, but these are very slow and laborious methods that result in the regeneration of only a few propagules in a year (Venturieri and Pickscius, 2013). Tissue culture, on the other hand, provides an alternative solution for producing a large number of genetically similar, phytosanitarily and physiologically high quality plantlets within a limited time period. In this process, a high survival percentage, associated with a high standard of acclimatized plantlets, is desirable in commercial labs and companies involved with orchid

* Corresponding author. Tel.: +86 20 37252993 E-mail addresses: [email protected]; [email protected]; [email protected]; [email protected]; jeancardosoctv@ gmail.com; [email protected] Peer review under responsibility of Chinese Society for Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS) http://dx.doi.org/10.1016/j.hpj.2017.07.009 2468-0141/© 2017 Chinese Society for Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Acclimatization of in Vitro-derived Dendrobium

Fig. 1 The successfully propagated in vitro Dendrobium officinale transplanted in a greenhouse for 6 months (A) and 30 months (B) in South China Botanical Garden, the Chinese Academy of Sciences micropropagation (Fig. 1). Tissue culture is also used to obtain new Dendrobium cultivars (Cardoso, 2012; Teixeira da Silva et al., 2015a) while the in vitro environment allows for the production of seedlings derived from symbiotic (Teixeira da Silva et al., 2015b) or asymbiotic (Teixeira da Silva et al., 2015c) germination within a sanitized environment following the appropriate disinfection procedures (Teixeira da Silva et al., 2016a). Tissue culture–raised plants often require extensive hardening treatments to prevent high mortality after transfer to ex vitro conditions (Pospišilová et al., 1999; Ziv and Chen, 2008). Plantlets produced in vitro are unable to develop resistance against minor and major microbial pathogens or other biotic and abiotic stresses caused by in vitro controlled conditions, which are characterized by an aseptic environment with small variations in temperature, high relative air humidity, high availability of nutrients, low light intensity and a low carbon dioxide (CO2) concentration (Teixeira da Silva et al., 2015b). These conditions result in photomixotrophic development and the need for a carbohydrate source in the culture medium. Their subsequent survival, after transfer to greenhouse or field conditions, is adversely affected by physiological and anatomical deficiencies. There are no studies that directly assess the response of Dendrobium to biotic stresses. However, slugs, snails, Dendrobium beetles, thrips, mealybugs, and other pests have been observed to feed on Dendrobium plants during acclimatization (MM Hossain, personal observation) while fungus gnats or sciara (Family Sciaridae; Order Diptera) at the larval stage affect and kill many Dendrobium plantlets at the acclimatization stage in Brazil (JC Cardoso, personal observation). The acclimatization of plantlets by weaning towards an ambient environment enhances the survival and growth of in vitro plantlets after transfer to ex vitro conditions. The survival of asymbiotically raised orchid seedlings transferred directly to natural habitats is unsuccessful unless they develop mycorrhizal associations (Hou and Guo, 2009; Dan et al., 2012a, 2012b; McCormick et al., 2012; Wang et al., 2013; Teixeira da Silva et al., 2015b). Therefore, the acclimatization of in vitro raised plantlets plays an important role in the mass propagation of different orchid species. Acclimatization can be sped up by hardening plantlets in vitro (Cardoso et al., 2013) or after transplantation by decreasing the transpiration rate by antitranspirants, including abscisic acid (ABA), or by increasing the photosynthetic rate by creating an ambient with an elevated CO2 concentration (Pospišilová et al., 1999; Kadlecˇek et al., 2001; Hronková et al., 2003).

Literature reports on the commercial propagation of Dendrobium are scarce since plantlets produced in commonly used tissue culture media do not grow fast enough and do not produce roots strong enough to withstand acclimatization conditions (Puchooa, 2004). Moreover, hardening of tissue culture–raised plantlets, post transplantation conditions for growth of plantlets such as the potting mixture used, light intensity and moisture of the greenhouse, as well as genotype are important factors for the successful acclimatization of Dendrobium plants (Fig. 1), as described in detail in Table 1. There are approximately 100 studies in the mainstream Dendrobium literature dedicated to the tissue culture and in vitro propagation of Dendrobium (Teixeira da Silva et al., 2015a). However, not all of these studies have reported on the post-in vitro stage, i.e., on the hardening and acclimatization of in vitro-derived plantlets to ex vitro conditions. In fact, Table 1 reveals that approximately only 40% of all in vitro studies of this orchid genus have also examined the acclimatization of plantlets, with the earliest detectable evidence being in a study by Sagawa and Shoji (1967), which reports on the acclimatization and successful flowering of twoyear-old acclimatized D. ‘Jacquelyn Thomas’, although absolutely no protocols are provided to explain the methodology for achieving successful acclimatization. It is for this reason that only three studies in Table 1 are dated prior to 2002. Zeng Songjun and coworkers (unpublished data) were able to aseptically sow or tissue culture more than 50 Dendrobium species, and the plantlets of these species were successfully transplanted in a greenhouse, and flowered (Fig. 2).

2. Environmental abiotic acclimatization of Dendrobium

factors

affecting

2.1. Induction of a functional root system for acclimatization The formation of a functional root system in tissuecultured plantlets tends to be an essential step for them to survive after transplantation. Despite this, no study of Dendrobium tissue culture and subsequent acclimatization has examined the importance or need of roots for effective acclimatization and plantlet survival. In vitro roots remain functional and continue to grow during ex vitro acclimatization (Nowak and Pruski, 2004). Therefore, a strong and robust root system should be induced from in vitro plantlets before they are transferred to the outside environment.

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Jaime A. Teixeira da Silva et al. Table 1 Optimal conditions for the hardening and acclimatization of Dendrobium

Species and/or cultivar

Plantlet description

In vitro temperature and lighta conditions

Acclimatization environment Substrate

Environmenta

D. aggregatum

Well-rooted shoots

(25 ± 2) °C, 14-h PP, CWFTs

Well-rooted seedlings and plantlets

(25 ± 2) °C, 14-h PP, 60 µmol·m–2·s–1, 60% RH

D. aphylum

180-day-old plantlets

D. aqueum

Well-rooted plantlets

(24 ± 2) °C, 16-h PP, 150 µmol·m−2·s−1, 70%–80% RH (23 ± 2) °C, 30 µmol·m–2·s–1

Small charcoal pieces:brick pieces (1:1) Sterilized small brick:charcoal pieces:peat moss (1:1:0.5) Sand:vermiculite:chopped dry leaves (1:1:1)


(23 ± 2) °C, 40 µmol·m–2·s–1

Plantlets with 3–4 roots

(25 ± 1) °C, 12-h PP, 35 µmol·m–2·s–1


(25 ± 2) °C, 12-h PP, 1 000– 2 000 lx

Volcanite, pine bark, peat, perlite, moss, ceramsite or the combination of them


NR

Seedlings with 3–4 leaves and 3–5 roots hardened and acclimatized Plantlets

(25 ± 1) °C, 200–3 000 lx

5 substrates were tested: moss; pine needle:peat:perlite = 3:1:1; sawdust:perlite = 3:1; moss:peat:perlite = 3:1:1; coco coir:peat:perlite = 3:1:1 Brick:charcoal: tree fern: bark pieces:leaf mound:dry Sphagnum (1:1:1:1:1:2) with green Sphagnum topping Brick:charcoal:decaying litter (1:1:1)

D. candidum

D. chrysanthum

(25 ± 1) °C, 12-h PP, 40 µmol·m−2·s−1


(24 ± 2) °C, 14-h PP, 80% RH, 50 µmol·m–2·s–1


(25 ± 2) °C, 10-h PP, CWFTs, 37.5 µmol·m−2·s−1

Well-grown seedlings with leaves and roots

D. densiflorum

Well-rooted shoots

D. draconis D. fimbriatum

Rooted shoots 4–5 cm long Plantlets 5–8 cm in height Well-rooted plantlets

D. hookerianum

Seedlings 4–5 cm tall

(25 ± 2) °C ,16-h PP, CWFTs, 30 µmol·m−2·s−1 (25 ± 2) °C, 16-h PP, 350– 500 lx (25 ± 2) °C, 12-h PP, CWFTs, 40 µmol·m–2·s–1 (25 ± 2) °C, 16-h PP, CWFTs, 2 000–3 000 lx

(25 ± 2) °C, darkness for 2 weeks, then 12-h PP, 60 µmol·m–2·s–1, 70%–75% RH

Survival/%

Reference

75% shading

95

Greenhouse; 25–30 °C; 60%– 70% RH

80

Vijayakumar et al. (2012) Hossain (2013)

90%–100% AH

70–80

Das et al. (2008)

Greenhouse; well acclimatized plantlets were reintroduced to their habitat. Moss was tied to the bark to hold the roots intact. Greenhouse condition

56

Parthibhan et al. (2012)

96

Parthibhan et al. (2015)

(20 ± 2) °C; 14-h PP; 50 µmol·m–2·s–1; 65%–75% AH The most suitable medium was matrix including volcanite, pine bark (1:1, v/v), covered moss on surface of planting pot The optimum transplant medium was matrix including moss:peat:perlite = 3:1:1

95

Zhao et al. (2008)

95% after transplanting for 7 days and 90% for 30 days

Han et al. (2013)

95% in spring from trained plantlets

Xiao and Zhang (2013)

Chamber at (25 ± 1) °C, 200–3 000 lx for 2 weeks then transfer to net house

100

Sharma and Chauhan (1995)

Layer of moss on the top of the compost; plantlets were covered with holed polythene bags for 2–3 weeks; glasshouse at (18–25) °C and 70%–80% RH Greenhouse; (25 ± 2) °C; 80% RH

71

Hajong et al. (2010)

88

Rao and Barman (2014)

Subdued light

80

Roy et al. (2007)

Glasshouse 6–8 weeks

Successfully acclimatized

Nongdam and Tikendra (2014)

Greenhouse

100

Luo et al. (2008)

Cocopeat:litter:clay (2:1:1)

Gradual exposure to light

85

Coconut husk peat:perlite (1:1) NR

Plastic film

92

After a two-week period of acclimatization in the growth chamber with high humidity, plantlets adapted to greenhouse conditions (18–25) °C; 70%–80% AH; plastic film

83.6

Pradhan et al. (2013) Rangsayatorn, 2009 Kabir et al. (2013)

Compost, coir pith, bricks and charcoal pieces

Bricks and charcoal pieces (1:1). Moss was laid over the potting mixture to maintain moisture. Polythene bags were used to cover the pot to maintain high humidity. Vermiculite

Mixture of coarsely crushed sterile brick, charcoal and vermicompost Dried coconut husk:small pieces of brick:charcoal (1:1:1) Small plastic/clay pots with brick pieces:pine bark:charcoal pieces:moss (1:1:1:1) Vermiculite:bark:soil (1:2:2)

Broken brick:charcoal (1:1) + layer of moss

90.0 ± 4.1

Paul et al. (2012)

(continued on next page)

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Acclimatization of in Vitro-derived Dendrobium Table 1 (continued) Species and/or cultivar


D. huoshanense

Well-developed roots




Environmenta

Cold pre-treatment: (5 ± 1) °C or (10 ± 1) °C for 1–10 weeks (25 ± 1) °C, 16-h PP, CWFTs, 50 µmol·m−2·s−1 (25 ± 2) °C ,12-h PP, 40 µmol·m–2·s–1

Vermiculite:bark:soil (1:2:2)

Well-developed plantlets Well-developed plantlets

(25 ± 2) °C, 12-h PP, CWFTs, 30 µmol·m−2·s−1 (25 ± 2) °C, 12-h PP, 50 µmol·m–2·s–1

D. microbulbon


(25 ± 2) °C, 16-h PP, CWFTs, 50 µmol·m−2·s−1

D. nanum

Well-rooted shoots

D. nobile

Plantlets with 7-months of in vitro culture and (1.0 ± 0.3) cm tall

26 °C, 14-h PP, CWFTs, 1 000–2 000 lx NR

D. lituiflorum D. longicornu

Survival/%

Reference

Plant growth chamber

65

Luo et al. (2009)

6 substrates were tested: bark; perlite; calcicalathina; sawdust; bark:sawdust = 3:7; ceramsite:sawdust = 3:7 Luffa sponge or cocopeat:perlite (9:1) Crushed brick:charcoal:shredded bark (1:1:1) + layer of moss Sand:soil:brick pieces:charcoal pieces (1:1:4:4) Vermiculite:sawdust (1:1); humus:sawdust (1:1) Different treatments with vermiculite + carbonized rice husk showed best results for percentage survival of plantlets and plantlet development 180 d after acclimatization Xaxim coir

The optimum transplant matrix was mixed with 3/10 bark in bottom and 7/10 sawdust on upper layer (25 ± 2) °C; 85%–90% RH

78

Qian et al. (2013)

NR

(24 ± 2) °C

68

Vyas et al. (2011) Dohling et al. (2012)

(28 ± 2) °C; 80%–90% RH

60

Sharma et al. (2007)

Greenhouse; 25 °C; low natural light; 95% RH Greenhouse; 50% shading; irrigated three times a week and foliar application of 2 g·L−1 of N-P-K (10-30-20) fertilizer each 15 d

85

Maridass et al. (2010) de Moraes et al. (2002)

(25–28) °C; 50% shading

NR

Greenhouse

92

Greenhouse

25–92

77.8–95.2

Plantlets

(25 ± 3) °C, 16-h PP, 22 °C

Well-rooted shoots

CWFTs, 100 µmol·m−2·s−1

Plantlets

25 °C, 2 000 lx, 16-h PP

Charcoal chips:coconut husk:broken tiles (2:2:1) Pine bark:styrofoam (3:1)

Seedlings

NR

Sphagnum

50% shading

NR

Plantlets

(25 ± 2) °C, 16-h PP, CWFTs

Sphagnum 60 d

NR

D. officinale

Seedlings

(22 ± 2) °C, 16-h PP, CWFTs, 36 µmol·m–2·s–1

D. phalaenopsis

Seedlings 180 d old; (1.0 ± 0.3) cm long

(25 ± 2) °C, 12-h PP, CWFTs, 18,9 µmol·m−2·s−1

20-cm pot diameter with bark:pebbles:coarse humus (3:1:1) Polypropylene 50 cm3 containing Sphagnum and coconut fibres (1:1, v/v)

Seedlings 180-d old; 1.2 cm long

(25 ± 2) °C, 12-h PP, CWFTs, 18,9 µmol·m−2·s−1

Polypropylene 50 cm3 (5.0 cm diameter; 4.0 cm tall) containing Sphagnum and coconut fibres (1:1, v/v)

Rooted plantlets

(25 ± 2) °C ,16-h PP, CWFTs

Cocopeat:Sphagnum moss (2:1)

26 °C, 14-h PP, 1 000– 2 000 lx (25 ± 1) °C, 16-h PP, CWFTs, 40 µmol·m−2·s−1

Humus:sawdust (1:1)

Greenhouse, 60% shading using black agro shade net Growth chamber; (25 ± 2) °C; 70%–80% AH; 16-h PP; 400–1 000 lx 1) Growth chamber; (25 ± 2) °C; 12-h PP, 9.45 µmol·m−2·s−1, CRFT for 30-d 2) Greenhouse; 162 µmol·m– –1 2·s (for 9 months) 3) Irrigation of 3 mm per day with weekly foliar fertilization 1) Growth chamber; (25 ± 2) °C; 12-h PP, CWFT + CRFT (for 30-d) 2) Greenhouse; 162 µmol·m– –1 2·s ; (22.6 ± 5) °C; 73.9% ± 10% AH (for 9 months) 3) Irrigation of 3 mm per day with weekly foliar fertilization 0.1% (w/v) Bavistin; plants were covered with plastic bags (for 30 days) then opening the bags gradually up to the 50th day; greenhouse 25 °C

Moss or tree fern or moss + tree fern

Greenhouse; 90% AH; irrigated twice a week

D. primulinum

D. strongylanthum Well-rooted shoots D. tosaense

Plantlets

NR

Faria et al. (2004) Malabadi et al. (2005) Lone et al. (2008) Faria et al. (2011) Faria et al. (2013b) Zhao et al. (2013)

87.5

Sorgato et al. (2015a)

90

Sorgato et al. (2016)

70

Pant and Thapa (2012)

>98

Kong et al. (2007) Lo et al. (2004)

80–97

(continued on next page)

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Jaime A. Teixeira da Silva et al.

Table 1 (continued) Species and/or cultivar




Environmenta

D. transparens

Plantlets

(25 ± 2) °C, 16-h PP

Brick:charcoal (2:1)

D. wilsonii

Seedlings

(26 ± 1) °C, 15–16 h PP, 1 000–1 500 lx

Dendrobium hybrids Dwarf hybrid Seedlings

D. ‘Serdang Beauty’ D. ‘Sonia’

Plantlets Well-rooted plantlets

Survival/%

Reference

50% shading

>90

Sphagnum moss

Seedlings were transplanted after immersed in millesimal thiophanate methyl for 5 min, then sprayed with millesimal carbendazim after transplanting for 3 days

100

Sunitibala and Kishor (2009) Zhan et al. (2010)

(25 ± 1) °C, 16-h PP, CWFTs, 1 960 lx

Coconut husk

Greenhouse

NR

(28 ± 1) °C, 16-h PP, 700– 1 000 lx, , 60% ± 10% RH (26 ± 2) °C, 16-h PP, CWFTs, 50 µmol·m−2·s−1 (26 ± 2) °C, 15-h PP, CWFTs, 3 000 lx

Charcoal:broken rock (1:1)

(28 ± 1) °C; 13.5 µmol·m–2·s– 16-h PP (26 ± 2) °C; 16-h PP CWFTs 500 µmol·m–2·s–1 Rooted plantlets transferred to room temperature and kept near window to get diffused sunlight for 2 weeks Biotron LPH200, (25 ± 2) °C, 12-h PP (25 µmol·m–2·s–1) and (85 ± 5)% RH for 30-d after which plantlets were transferred to net house (25 ± 8) °C; 70% shading

100

1;

Wood charcoal

D. Sonia ‘Earsakul.’


D. ‘Sonia 17’, ‘Sonia 28’

Plantlets

(25 ± 2) °C ,16-h PP, 25 µmol·m−2·s−1

Sand:brick:charcoal pieces:coir fibre (1:4:4:2)

D. ‘Tong Chai Gold’ × ‘Black Jack’ D. ‘Zahra FR 62’, D. ‘Gradita 31’

Seedlings

NR

Coconut fibre

25 °C, 12-h PP, 13 µmol·m– 60.6% RH

First into Cycas rumphii bulk for 2 months. Then wood charcoal:C. rumphii bulk (1:1)

Well-rooted plantlets (90 days old) with 3–5 leaves and 2–4 roots. Plantlets immersed in 1% pesticide solution of 50% benomyl and 20% streptomycin sulphate for 3 min to reduce root rot caused by Erwinia sp.

–1 2·s ,

Charcoal pieces:brick pieces (1:1)

Reduced light intensity (100– 150 µmol·m–2·s–1) using 50% shading net. 85%–95% RH when plantlets in plastic pots were covered by transparent plastic and 70%–90% RH after removing the plastic

84

Sujjaritthurakarn and Kanchanapoom (2011) Khosravi et al. (2008) Puchooa (2004)

66.67

Kumari et al. (2013)

81% (D. ‘Sonia 17’) and 84% (D. ‘Sonia 28’)

Martin et al. (2005)

100

Cardoso (2012)

90–100

Winarto et al. (2013); Winarto and Rachmawati (2013); Winarto and Teixeira da Silva (2015)

Note: AH, air humidity; CRFT, cool red fluorescent tube (Gro-lux®); CWFTs, cool white fluorescent tubes; NR, not reported; PP, photoperiod; RH, relative humidity. a The original light intensity reported in each study has been represented since the conversion of lx to µmol·m–2·s–1 is different for different illumination (main ones represented): for fluorescent lamps, 1 µmol·m–2·s–1 = 80 lx; the sun, 1 µmol·m–2·s–1 = 55.6 lx; high voltage sodium lamp, 1 µmol·m–2·s–1 = 71.4 lx (Thimijan and Heins, 1983).

In many cases, an auxin is suitable for the induction of a well-developed root system. The concentration of auxin in the culture medium is also important (Van Staden et al., 2008). A high constitutes ‘high’ will most likely differ on the plant and genotype — often induces long and thin roots while the excessive application of auxins after initial rooting may inhibit root regeneration and elongation and impair the formation of a functional root system (Overvoorde et al., 2010; Zhao, 2011). A high number of roots per plantlet and longer roots were obtained using high concentrations of auxins, alone, or in combination with cytokinins: 0.5 mg·L−1 N6-benzyladenine (BA; a cytokinin) + 3.0 mg·L−1 indole-3-butyric acid (IBA) (13.2 ± 1.3 and 4.4 ± 1.7 cm, respectively), 4.5 mg·L−1 1-naphthaleneacetic acid (NAA) (11.1 ± 2.1 and 4.6 ± 0.4 cm, respectively) and 2.5 mg·L−1 kinetin (Kin; a cytokinin) + 2.5 mg·L−1 NAA (12.0 ± 1.2 and 2.6 ± 1.4 cm, respectively) (Nongdam and Tikendra, 2014). The use of activated charcoal (AC) with auxin(s) can enhance rooting and the development of a well-developed root system

(Pan and Van Staden, 1998). Martin et al. (2005) observed that the addition of 2.0 g·L−1 of AC resulted in highest number of roots per shoot in D. ‘Sonia 17’ (9.4) and D. ‘Sonia 28’ (9.2) compared to the control (no AC added: 5.3 and 4.9, respectively). Different Dendrobium genotypes may or may not need an auxin in the culture medium for inducing roots in vitro. Hossain et al. (2013) reported that D. aphyllum shoot buds produced a strong and well-developed root system (>4 roots per seedling) in half-strength Phytamax or Mitra et al. (1976) media when supplemented with 0.5 mg·L−1 indole-3-acetic acid (IAA), although they developed very thin and long roots in 1.0 mg·L−1 IAA in the same media. Similar results were also found for D. aggregatum when grown in half-strength Murashige and Skoog (MS; Murashige and Skoog, 1962) basal medium with 1.5% (w/v) sucrose and 0.5 mg·L−1 IAA (Hossain, 2013). A welldeveloped root system formed in D. macrostachyum (Pyati et al., 2002) and D. microbulbon (Sharma et al., 2007) even though the medium was not supplemented with plant growth


115

Fig. 2 Successfully propagated in vitro Dendrobium flowered in a greenhouse in the South China Botanical Garden, the Chinese Academy of Sciences (A) D. aphyllum; (B) D. crystallinum; (C) D. densiflorum; (D) D. devonianum; (E) D. gratiosissimum; (F) D. heterocarpum; (G) D. hookerianum; (H) D. loddigesii; (I) D. nobile; (J) D. shixingense; (K) D. thyrsiflorum; (L) D. tosaense; (M) D. cochliodes; (N) D. linawianum; (O) D. draconis; (P) D. chrysotoxum; (Q) D. crepidatum; (R) D. fimbriatum; (S) D. flexicaule; (T) D. officinale; (U) D. huoshanense; (V) D. lituiflorum; (W) D. phalaenopsis; (X) D. unicum; (Y) D. strongylanthum; (Z) D. pendulum; (AB) D. moschatum.

116 regulators (PGRs), most likely due to the active biosynthesis of IAA by plants (Zhang et al., 1995). Thus, most likely, the concentration of exogenous auxin required to induce rooting depends on the endogenous level of auxins. Therefore, the careful selection of an auxin, its concentration and its combination with other PGRs are recommended to fortify the chance of successful acclimatization. The use of other PGRs to improve acclimatization has been poorly studied. Sorgato et al. (2015b) immersed D. phalaenopsis ‘Deang Suree’ plantlets at the pre-acclimatization stage in a solution containing 30 mg·L−1 gibberellic acid (GA3), which resulted in reduced survival percentage, but an increase in the fresh weight of acclimatized plantlets. A reduction in the nitrogen (N) concentration in culture medium also resulted in better root development in another orchid (Cardoso and Ono, 2011). In fact, most protocols used half-strength MS medium for root induction in Dendrobium (Faria et al., 2004; Martin et al., 2005) and the main benefit to root production could be the reduced N concentration in MS basal medium. Winarto and Rachmawati (2013) observed that a reduced N concentration in the culture medium promoted rapid root initial formation (22.8 days), and increased the number of roots per shoot (2.4) and root length (0.76 cm) in D. ‘Gradita 31’. 2.2. Hardening and potting mixtures for acclimatization Hardening of in vitro-raised plantlets implies acquiring the ability to tolerate adverse environmental conditions and is a prerequisite for the acclimatization of in vitro plantlets to ex vitro conditions. The ability of propagules to withstand transplanting stress frequently determines the success or failure of tissue culture operations (Nowak and Pruski, 2004). The controlled biotic and abiotic conditions during in vitro culture commonly result in high and rapid vegetative development. However, structural and physiological disorders are common, resulting in plant loss during acclimatization, thus reducing the efficiency of micropropagation systems. The most frequent disorder is caused by low photosynthetic rates, malfunctioning of stomata and reduced epicuticular wax in leaves. Plantlet hardening can start when plants are still in vitro, contributing to better survival percentage at the acclimatization stage in greenhouse conditions. This can be achieved by increasing the sucrose concentration in the culture medium, inducing photoautotrophic conditions by reducing sucrose in the culture medium, decreasing relative humidity in culture vessels by using desiccants or by using culture vessels that allow air exchanges between the in vitro and external environment, as well as other techniques aimed at hardening plantlets for acclimatization (Hazarika, 2006). Different laboratories use different techniques to harden in vitro plantlets. Sharma and Chauhan (1995) reported that D. chrysanthum plantlets could not survive when transferred directly from the laboratory to a net house, but when plantlets were hardened for three weeks in Knudson ‘C’ (Knudson, 1946) or Thomale GD (Thomale, 1954) medium at quarter nutrient strength, lacking sucrose but having sterilized earthen pot pieces (1 cm thickness) at (25 ± 1) °C and under 2 000–3 000 lx light intensity, 85%–100% of plantlets survived.


Hossain and coworkers (unpublished data) applied a successive phase of acclimatization to in vitro-grown orchid plantlets of Acampe ochracea, Phalaenopsis cornu-cervi, Sarcanthus lanatus, Vanda tessellata (Nandy, 2003), D. aphyllum and Rhynchostylis retusa (Bhattacharjee, 2003). To achieve this, the mouth of culture vessels was maintained open for one day in the culture room, placed outside the culture room for 12 h, and then returned to the culture room. On the following day, plantlets were placed outside the culture room for 24 h and then returned to the culture room for 12 h followed by one week outside the culture room. This option is viable where greenhouse conditions are not available. Finally, plantlets were removed from culture vessels and washed with running tap water to remove agar attached to roots. They were then transferred to small pots containing moistened coconut coir, coir dust and wood coal pieces (1:1:1) and grown in the culture room (since a greenhouse was not available) for 20–30 days. Finally, 70%–80% of plantlets that were transferred to wooden orchid pots filled with moistened coconut coir, coal and small brick pieces (2:1:2) survived. Hossain et al. (2013) found that this gradual acclimatization resulted in very low contamination and 80% survival of D. aphyllum plantlets. Deb and Imchen (2010) described another method to acclimatize the plantlets of three orchids [Arachnis labrosa (Lindl. ex Paxt.), Cleisostoma racemiferum (Lindl.) Garay and Malaxis khasiana Soland ex Schwartz], achieving over 95% survival. They reported that tissue-cultured seedlings of these orchids were acclimatized and hardened in vitro in 1/10-strength MS basal medium without a carbon source or any PGRs, then transferred to new culture vials containing a sterile matrix of charcoal pieces, small brick chips, moss and decayed wood/ forest litter in different ratios as an alternative substrate. They reported that a combination of charcoal pieces (5–7 mm in thickness), small brick chips and moss at a 1:1 ratio was suitable for epiphytic orchids (A. labrosa; C. racemiferum) and a 1:1 ratio of moss and decayed wood/forest litter, together with charcoal pieces and brick chips, was better for the terrestrial orchid (M. khasiana). Parthibhan et al. (2015) successfully used a similar in vitro hardening period for D. aqueum using halfstrength MS medium without sucrose for 5 weeks before rooted plantlets were acclimatized (Table 1). Sorgato et al. (2015a) proposed a system involving the intermediate acclimatization of D. phalaenopsis plantlets in a growth chamber for 30 days, which resulted in an increase in the percentage of plantlets that survived and a greater fresh weight of plantlets. Dendrobium orchids are mainly epiphytes. In nature, their exposed roots absorb moisture from humid air as well as from dew (Atwell et al., 2010). Thus, when grown in artificial conditions, aeration, capillary action, water and nutrient-holding capacities of the substrate should be taken into consideration (da Silva et al., 1993; Spomer et al., 1997; Di Benedetto, 2007). The stability and weight of medium components, easy availability as well as costs and consistency also need to be considered (Oliveira et al., 2005). Most substrates used for the acclimatization of in vitro-raised Dendrobium plantlets are made up of brick or charcoal pieces, broken tiles, pine bark, Cycas bark, cocopeat, coconut coir, sawdust, perlite, vermiculite, peat or Sphagnum moss (Table 1). To prepare a potting mixture to


acclimatize in vitro plants, one must add one or two materials such as perlite, Sphagnum moss, fern roots, or coconut husk that can absorb water. Pure Sphagnum moss is probably the best potting material and may be used singly for growing young plantlets, although some problems with fungus gnats may be encountered (Evans et al., 1998). Sphagnum moss has a high water absorption capacity, which increases the reproduction of fungus gnats, an important pest in plantlet production with the potential of disrupting commercial Dendrobium cultivation, mainly at the acclimatization stage (JC Cardoso, personal observation). Many orchid growers transplant plantlets from flasks into plug trays with Sphagnum moss as the sole potting medium. Moss has a low pH and absorbs large quantities of water and mineral nutrients (da Silva et al., 1993). Therefore, Sphagnum moss may be used for the acclimatization of Dendrobium plantlets (Zhan et al., 2010; Han et al., 2013), especially in more temperate climates where pots lose water rapidly. Cocopeat, coconut coir, or sawdust may be alternatives to peat moss where it is not available or where it is a costly item (Meerow, 1994; Noguera et al., 2000). A good potting mixture will provide a suitable ambient condition for good drainage, sufficient moisture retention and good aeration, which are essential for good orchid growth (Qian et al., 2013; Xiao and Zhang, 2013). The selection of suitable potting mixtures for acclimatization is another condition for improving plantlet survival. Potting mixtures can be problematic when wild plant species are overexploited to produce substrates for cultivated plants (Di Benedetto, 2007), including Dendrobium orchids. In Brazil, until 2000, xaxim fibre was the main substrate used for orchid cultivation. Xaxim fibre is obtained by shredding the stems and leaf-sheaths of ‘samambaiaçú-imperial’ (Dicksonia sellowiana), but this plant has been listed, since 1992, as a near-extinct species by the Environmental Ministry of Brazil (MMA, 1992, 2008). Recently, xaxim fibre has been replaced by coconut mesocarp, obtained from the coconut industry, and was successfully used for the rooting and vegetative development of D. nobile (Assis et al., 2005). In China, many potting substrates, including peat, vermiculite, pine bark, sawdust, coco coir, fern roots, perlite, calcicalathina, vesuvianite, ceramsite, brickbat, broken charcoal, Sphagnum moss and their combinations are used in the acclimatization of Dendrobium; however, recently, pine bark and sawdust and their combination have most commonly been used in mass commercial production (Table 1; Zeng et al., 1998; Duan and Duan, 2013; Qian et al., 2013; An et al., 2014). In Bangladesh, tree ferns are nearly extinct and Sphagnum moss is rare, but coconut coir or cocopeat, charcoal and sawdust are cheap, readily available across the country and are thus the main substrates used for the acclimatization of Dendrobium (MM Hossain, personal observation). In India, Sphagnum moss, chopped fern roots, leaf mould, coconut coir and charcoal pieces are available and frequently used to acclimatize Dendrobium (M Sharma, personal observation). For example, D. chrysanthum exhibited 100% survival on a potting mixture containing brick pieces, charcoal pieces, leaf mould, tree fern pieces (Cyathea spp.) and dry Sphagnum in a 1:1:1:1:1:2 ratio, with or without green Sphagnum on the surface of the potting medium (Sharma and Chauhan, 1995). Interestingly,

117 no plantlets survived in leaf mould, perlite, vermiculite and dry Sphagnum at a 1:1:1:2 ratio. Hazarika and Sarma (1995) achieved 90%–95% survival of D. transparens plantlets by dipping them in 2% dithane M-45 (a fungicide) for a few minutes, then transplanting them to a potting medium consisting of farmyard manure, chopped fern roots, leaf mould and charcoal pieces (1:2:1:2). This was accompanied by regular irrigation with Hoagland and Arnon’s nutrient solution (Hoagland and Arnon, 1950) while maintaining plantlets at ambient room temperature for two months. Rani et al. (2006) assessed the effects of different potting media on the survival and post-transplantation growth of Dendrobium hybrid plantlets for up to six months from deflasking. They showed that 66%–92% of plantlets survived, depending on the potting mixture. A combination of broken tiles, charcoal pieces and soilrite (2:2:1) and broken tiles, charcoal pieces and tree fern roots (2:2:1) were most suitable substrates for achieving highest survival (92%) and subsequent performance of plantlets with respect to the number and size of shoots, leaves and roots. Coconut coir and coconut fibre, mixed with brick and charcoal pieces, are also frequently used in the acclimatization of Dendrobium plantlets. Roy and Banerjee (2003) achieved 65% survival of D. fimbriatum plantlets when transferred to clay pots containing a mixture of coconut husk, small brick pieces and leaf mould (1:1:1) and maintained in a moist, shady place for acclimatization. Martin et al. (2005) showed 81% and 84% survival of Dendrobium hybrid ‘Sonia 17’ and ‘Sonia 28’ plantlets, respectively, in sand brick or tile, charcoal pieces and coir fibre (1:4:4:2) after acclimatization in a Biotron LPH 200 (Japan). Sunitibala and Kishor (2009) showed over 90% survival of D. transparens plantlets only in a mixture of brick and charcoal pieces (2:1) after acclimatization in a growth chamber. Winarto and Rachmawati (2013) reported 100% survival of D. ‘Gradita 31’ plantlets when acclimatized for two months in plastic pots containing Cycas rumphii bulk covered with transparent plastic, and placed under reduced photosynthetically active radiation (PAR) (37–74 µmol·m−2·s−1) in a glasshouse and finally transferred to bigger plastic pots containing wood charcoal and C. rumphii bulk (1:1). Sharma and Tandon (1992) observed 65% survival in a substrate made up of brick and charcoal pieces, and coconut fibre, under glasshouse conditions. Indhumathi et al. (2003) found that D. ‘Sonia-17’ plantlets hardened best in a 1:1:1 ratio of charcoal pieces, cocopeat and brick pieces. Hossain et al. (2013) reported that a potting mixture containing charcoal pieces, cocopeat, brick pieces and vermiculite (2:2:1:1) resulted in 80% survival of D. aphyllum plantlets, and gradual adjustment of in vitro plantlets to the ex vitro environment resulted in better survival after transplantation. Gangaprasad (1996) obtained the best survival of Dendrobium seedlings in only charcoal pieces and brick pieces (1:1). Vijayakumar et al. (2012) also reported similar findings for D. aggregatum. High survival (95%) of D. nobile plantlets was observed when they were transferred to small plastic pots containing peat moss, wood charcoal, and brick pieces (1:1:1) (Nayak et al., 2002). A 92% survival was reported in the same species when transferred to a mixture of charcoal chips, coconut husk and broken tiles (2:2:1) (Malabadi et al., 2005). In D. candidum, 95% survival of plantlets was possible on vermiculite alone (Zhao et al., 2008). Survival of D. ‘Sonia’ was

118 80% in a mixture of sand, brick or tile, charcoal pieces and coir fibre (1:4:4:2) (Martin and Madassery, 2006) while D. ‘Serdang Beauty’ plantlets showed 80%–100% survival only in charcoal (Khosravi et al., 2008). Vyas et al. (2011) reported that banana extract could help to synchronize plantlet development in vitro and Luffa sponge or cocopeat:perlite (9:1) was suitable as the supporting matrix in half-strength Knudson’s ‘C’ liquid medium for in vitro acclimatization of D. lituiflorum, but the survival percentage of plantlets after ex vitro transfer was not specified. Qian et al. (2013) measured the physical and chemical characters (gross porosity, porosity, water-hold capacity, conductivity and pH) of five transplanting substrates including ceramsite, bark, perlite, calcicalathina and sawdust. Then they used six substrate combinations for transplanting D. huoshanense, including (1) bark, (2) perlite, (3) calcicalathina, (4) sawdust, (5) bark (bottom):sawdust (upper layer) = 3:7, and (6) ceramsite (bottom):sawdust (upper layer) = 3:7; optimum transplant matrix was mixed with 3/10 bark at the bottom and 7/10 sawdust as the upper layer; survival reached 78% and the number of new buds was 2.7 per clump, which may be related to its good water retention and air permeability. de Moraes et al. (2002) tested different organic and mineral substrates to improve the acclimatization of D. nobile plantlets (1.0 ± 0.3 cm in height) and observed a percentage of survival ranging from 77.8% [vermiculite and Plantmax® (commercial substrate composed of ground Pinus bark), 2:1] to 95.2% (vermiculite and carbonized rice husk, 1:1). The 1:1 mixture of vermiculite and carbonized rice husk showed the best development of plantlets 180 days after transplantation. Mixtures of substrates are not practical in large-scale biofactories and result in additional costs for growers because additional resources are needed to prepare new substrate mixtures (Duan and Duan, 2013; An et al., 2014). Another problem is that a correct mixture and homogeneity depends on the density and granulation (i.e., porosity) of the substrate, and can result in different layers of substrate after mixing. Thus, only substantial increases in the percentage of survival of acclimatized plants will justify the use of mixed substrates in a commercial setting, although academics in scientific papers have a different objective. Since most Dendrobium species are epiphytic and seedling viability is low, the most suitable potting mixture for acclimatization should have strong permeability and retain high humidity (Qian et al., 2013). 2.3. Relative humidity and light intensity/PAR for acclimatization The retarded development of the cuticle, epicuticular waxes and functional stomatal apparatus during in vitro culture can cause high stomatal and cuticular transpiration of leaves in plantlets when removed from culture vessels. To avoid this, plantlets should be transferred slowly from high to low humidity, which allows stomatal and cuticular transpiration rates to gradually decrease because stomatal regulation of water loss is more effective and cuticle and epicuticular waxes develop (Pospišilová et al., 1999). The relative air humidity surrounding plantlets upon transplantation is usually 70%–80% (Martin et al., 2005; Sharma et al., 2007; Hajong et al., 2010; Vyas et al., 2011; Paul et al., 2012; Winarto and Teixeira da Silva, 2015), but occasionally 90% or higher (Lo et al., 2004; Das et al., 2008;


Maridass et al., 2010). However, it is difficult to control the relative air humidity of an environment and very few reports are available that specifically deal with humidity during Dendrobium plantlet transplantation. The in vitro environment, which tends to have a low light intensity/PAR, can be enriched by CO2 and macro- and micronutrients that are added to the culture medium, allowing for the large-scale production of plantlets. However, since plantlets suffer high mortality after transferring from in vitro to ex vitro conditions, there is a need for an effective acclimatization procedure to produce healthy plantlets that would allow their rapid adaptation to the ex vitro environment (Jeon et al., 2005, 2006; Yoon et al., 2009; Cha-um et al., 2010). In vitro plantlets are generally grown under low and well controlled light intensity (1 000–3 000 lx) and appropriate temperature (25 ± 2 °C), hence direct transfer to broad spectrum sunlight (4 000–12 000 lx) and temperature (26–36 °C) might cause charring of leaves and wilting of plantlets (de Moraes et al., 2002; Zhao et al., 2008; Maridass et al., 2010; Cardoso, 2012; Kumari et al., 2013; Winarto and Rachmawati, 2013; Winarto et al., 2013; Winarto and Rachmawati, 2013; Zhao et al., 2013; Winarto and Teixeira da Silva, 2015; Table 1). Winarto and Rachmawati (2013) reported that D. ‘Gradita 31’ plantlets showed 100% survival when acclimatized for two months under reduced light intensity/PAR (2 700–5 500 lx or 37–74 µmol·m−2·s−1) in a glasshouse. Nayak et al. (2002) reported that the acclimatization of D. nobile plantlets in a growth chamber for 8–10 weeks before transfer to a field at 35 µmol·m−2·s−1 (2 590 lx) and 80% relative humidity enhanced survival. Martin and Madassery (2006) achieved over 80% ex vitro establishment of D. ‘Sonia’ plantlets when acclimatized for 30 days at (25 ± 2) °C, 85% ± 5% relative humidity and a 12 h photoperiod at 25 µmol·m−2·s−1 (1 850 lx). Besides light intensity, wavelength can affect plantlet survival after acclimatization. Sorgato et al. (2015a) reported that insertion of a 30-day-long intermediate phase before acclimatization in a growth room using red fluorescent lighting (GRO-LUX®, 9.45 µmol·m–2·s–1) was able to increase survival after acclimatization from 60% (control without intermediate phase before acclimatization) or 75% [intermediate phase using white (18.9 µmol·m–2·s–1) or mixed lighting of red and white fluorescent light] to 87.5%. All photoconversions in the text and Table 1 are based on equations provided in Thimijan and Heins (1983).

3. Endogenous and biotic factors affecting the acclimatization of Dendrobium 3.1. Genotype and physiology of in vitro plantlets, and effect on acclimatization Genotype is an important factor that influences the effectiveness of acclimatization. In several crosses of D. phalaenopsis, Lone et al. (2008) observed large differences in survival percentage after acclimatization, varying from 25% to 94.8% depending on the genotype used for crossing, although the exact cultivars were not described (only the cross parents) as they were dealing with genetically heterogeneous seedlings. Although there are no reports specifically showing that genotype is an important factor influencing the acclimatization of Dendrobium, Zeng Songjun and coworkers (unpublished data)


found that the survival rates of in vitro D. candidum (syn. D. officinale) plantlets from different genotypes were significantly different. In other ornamental plants, Rout et al. (1989a, 1989b) reported the response of different rose genotypes to acclimatization with a range of survival percentages (92%– 98%) when transferred to the greenhouse, while Radojevic et al. (1990) also observed variation among carnation genotypes after transplant to ex vitro conditions. Few studies exist exclusively on Dendrobium to explain the physiology of in vitro plantlets during the acclimatization step and thus knowledge has been drawn from other plant literature. There are morphogenic differences between in vitro plantlets and ex vitro plants, mainly in the structure of roots, leaves and stems. The leaves of in vitro grown plants are generally thinner (weak vasculature) and have a poorly developed palisade layer with a reduced amount of mesophyll air space compared to ex vitro grown plants while in vitro plantlets are unable to close their stomata when first removed from culture; the malfunctioning of stomata can also contribute to excessive water loss (Kumar and Rao, 2012). The roots of in vitro plantlets are often smaller in diameter and have a uniseriate epidermis and limited periderm, the vascular bundles in roots are immature, the cambial activity is often limited, xylem and phloem are poorly developed and there are no or only few fine root hairs (Ziv and Chen, 2008). Plantlets cultured in vitro often show a low rate of photosynthesis and incomplete autotrophy, and these may be the reasons for low survival rates of plants during the acclimatization stage (Grout and Millam, 1985; Faria et al., 2004). Thus the ability to photosynthesize, manage water and respond to stress during and after in vitro culture determines the final performance of regenerants. Tissue-cultured propagules are produced in small vessels in a controlled environment under a preset temperature, low PAR and poor air exchange between containers and the external atmosphere, and this can lead to high humidity and the accumulation of ethylene (Jeong et al., 1995). Moreover, nutrients in growing media such as mineral salts, sucrose, vitamins and PGRs cause greater developmental distortions and repress or modulate several metabolic pathways than soil conditions. As a result, in vitro-grown plantlets develop small juvenile leaves with a poor cuticle layer, malfunctioning stomata and reduced photosynthetic capacity, while roots lack hairs or only have a few hairs (Ziv, 1986). The stomata of plants grown in vitro show a characteristic inability to close with poor epicuticular wax formation when first removed from culture which may cause excessive evapotranspiration after transplantation to ex vivo conditions (Drew et al., 1992). As a result, plantlets may rapidly wilt and die. To promote ex vitro survival and physiological competence, especially to guard against water loss and encourage autotrophy, a transitional environment is usually supplied for an acclimatization interval from one to several weeks (Grout and Millam, 1985). In this transitional environment, relative humidity is kept in a range of 70%–100% by misting or fogging, and PAR should not be much greater than it was in culture. Jeon et al. (2005, 2006) studied the morphology, photosynthesis and growth parameters of another orchid, Doritaenopsis for four months after acclimatization. Length, area, fresh and dry weight of leaves and their chlorophyll a/b ratio were highest

119 when acclimatization occurred under intermediate light conditions (270 µmol·m−2·s−1) compared to acclimatization at light conditions of low (175 µmol·m−2·s−1) and high (450 µmol·m−2·s−1) PAR. There were no significant differences between light conditions for maximum chlorophyll fluorescence (Fv/Fm); however, net CO2 assimilation, transpiration and stomatal conductance were higher when plants were acclimatized at higher light conditions while low and intermediate PAR led to 90% and 89% survival compared to 73% in high PAR. Wax formation and a thick velamen layer of leaves, as indicators of adaptation to ex vitro conditions, were highest when plants were acclimatized at high PAR. A multilayered velamen in roots was observed four months after acclimatization at high PAR, and increased cell activity was detected in the roots of plants grown at high PAR for four months compared to in vitro roots. When observing the studies listed in Table 1, even though there is a wide range of in vitro and ex vitro conditions, and substrates used, no obvious trend between the state of roots in vitro and the success of acclimatization can be discerned for Dendrobium. Photoautotrophic culture of the final phases of micropropagation, namely rooting and elongation, could be an alternative to improve the quality of in vitro-derived plantlets to increase the level of plantlet survival at the acclimatization stage, and reducing or avoiding anatomical and physiological deficiencies caused by photomixotrophic culture, as was demonstrated for Spathiphyllum (Teixeira da Silva et al., 2006) and papaya (Teixeira da Silva, 2014), and could also be applied to Dendrobium. Using conventional photoheterotrophic culture, Faria et al. (2004) observed that the development (plant height, root length and number of shoots) of D. nobile plants was reduced when culture medium contained no sucrose and when plants were cultured under white fluorescent lamps. Unfortunately, they did not report the light intensity or PAR used for Dendrobium in vitro cultivation. In contrast, Mitra et al. (1998) tested the in vitro growth of an unspecified Dendrobium cultivar in a CO2-enriched environment on Vacin and Went (1949) medium, and at (25 ± 3) °C, 60%–70% relative humidity, a 16 h photoperiod and a PAR of 42–45 µmol·m−2·s−1. They observed that roots (2 roots per plantlet) were obtained only in sucrosefree medium using both 0.6 g·CO 2 ·m −3 (natural CO 2 concentration in air) or enrichment with 40 g·CO2·m−3. In contrast, in the presence of 2% sucrose, no roots formed after 4 weeks in culture. Photoautotrophic culture was tested by Nguyen et al. (2010) for Dendrobium ‘Burana White’ using halfstrength liquid MS medium without sucrose, vitamins or PGRs, and using perlite as the support substrate during culture. The authors observed that photoautotrophic plantlets submitted to a ventilation rate of 4.9 h·day−1 of ventilation under a PAR of 110 µmol·m−2·s−1 showed the highest increases in fresh and dry weights 65 days after cultivation under these conditions. 3.2. Mycorrhization of Dendrobium plantlets at the acclimatization stage Under natural conditions, Dendrobium orchids form strong associations with bacteria, especially cyanobacteria, as well as with mycorrhizal fungi to form complex interactions that provide and improve the growth and reproduction of all species

120 involved (Tsavkelova et al., 2003; Tsavkelova, 2011; Teixeira da Silva et al., 2015b). The benefits of the interactions with these microorganisms for Dendrobium are related to improved seed germination (Guo and Xu, 1990; Kolomeitseva et al., 2002; Tsavkelova et al., 2007) and plantlet development (He et al., 2010; Faria et al., 2013a), nutrient (N and phosphorus) and water availability and uptake, and protection of plants from pathogenic infections (Patten and Glick, 2002; Spaepen et al., 2007; Ahmad et al., 2008; Bhattacharyya and Jha, 2012). When Dendrobium plantlets are acclimatized, part of the stress is related to nutrient and water uptake and pathogenic infections that lead to the death of acclimatized plants. Under these conditions, the survival of in vitro-derived plantlets may be improved by inoculation with microorganisms at later stages of rooting and acclimatization when the root system is more developed since microorganisms tend to grow rapidly under highnutrient conditions in vitro at early stages of plantlet development and thus, possibly, negatively influence plantlet growth (Zhou et al., 2009; Wang et al., 2013). The effective and practical use of mycorrhization requires suitable microorganisms to be prospected, isolated in vitro and co-cultivated with Dendrobium seeds or plantlets. Wang et al. (2011), Dan et al. (2012a) and Wu et al. (2012) used this technique to obtain potential microorganisms to improve symbiotic seed germination of different Dendrobium species. These techniques could also be useful in asymbiotic in vitro plantlet production for hardening plantlets at the stage of in vitro rooting (pre-acclimatization) or even at the acclimatization stage, resulting in increased plant survival. For example, Zhang et al. (2011) used Mycena mycorrhizal fungus to inoculate acclimatized D. officinale plantlets and obtained gains compared to non-inoculated plantlets, evaluated after four months of cultivation: plant height (8.10 cm vs. 2.90 cm), number of new buds (5.0 vs. 2.0), fresh (61.4 mg vs. 36.7 mg) and dry weight (1.27 mg vs. 0.68 mg). Hossain et al. (2013) recommended the use of orchid mycorrhizal fungi to promote increases in in vitroderived plantlet survival, to enhance vegetative growth and to reduce disease infection rates. However, the optimal stage for inoculation of microorganisms on in vitro-derived plantlets needs to be studied because this technique involves changes and additional labour and costs in biofactories of in vitro production of Dendrobium plantlets.

Jaime A. Teixeira da Silva et al. light conditions, media components such as PGRs or carbohydrate content, can modify key components of successful acclimatization (Teixeira da Silva et al., 2015a), the development and function of the photosynthetic apparatus and stomatal regulation of transpiration, as was proved in other plant species (Pospišilová et al., 1999; Kadlecˇek et al., 2001; Dobránszki and Mendler-Drienyovszki, 2014a, 2014b). Ex vitro conditions, such as environmental factors and the substrate used for acclimatization, as applied to different Dendrobium species and hybrids, are listed in Table 1. The acclimatization environment in these studies appears random, but should be improved and optimized by monitoring the morphological, structural and physiological changes in the leaves, stems and roots of plants during acclimatization. More complete studies about the use of plain or mixed substrates are required to better understand the nutritional and rooting requirements of Dendrobium cultivars and their correlation with plant survival at the acclimatization stage. Mycorrhization could be an efficient alternative to improve the rooting and survival of Dendrobium. Photoautotrophic culture of Dendrobium is another unexplored area, but could be a way to improve the efficiency of acclimatization procedures. In this sense, pre-acclimatization in the greenhouse is used in some commercial biofactories to produce plantlets (Cardoso et al., 2013), mainly in South America and Asia, but is underexplored at a scientific level. This technique consists of inducing rooting in vitro (i.e., before acclimatization) and hardening plantlets while still in their flasks in the greenhouse environment by exposing plantlets to variations in temperature and increases in light intensity and quality (natural sunlight). This also reduces the costs associated with artificial illumination in the growth room. The molecular genetics of the Dendrobium genus is advancing very rapidly, allowing for the genetic dissection of this economically important species (Teixeira da Silva et al., 2016b). If genes of value, such as for increased abiotic or biotic stress tolerance, or even for novel flower colour or plant architecture, then such genes could be introduced through genetic engineering (Teixeira da Silva et al., 2016c). Transformed plants that are derived from the product of these molecular biology and genetic transformation trials also require a robust acclimatization protocol to bring their novel phenotypes to fruition.

Conflicts of interest 4. Conclusions and future perspectives Sudden environmental changes during transfer from in vitro to ex vitro conditions are a stress for plantlets. Plantlets have to repair structural and functional abnormalities caused by in vitro environments (Pospišilová et al., 1999; Ziv and Chen, 2008) and they have to adapt to a new environment. Chemical and physical factors of both the in vitro and the ex vitro environments may affect the success of this transition, i.e., the success of acclimatization. Therefore, there are two opportunities to increase the rate of survival and enhance the growth of plants during acclimatization: one is by harnessing and utilizing the long-term or post-effects of in vitro conditions (Teixeira da Silva et al., 2015a), and the other is the appropriate choice of conditions for acclimatization. In vitro conditions prior to acclimatization can modify the morphology and physiology of transplanted plantlets. In Dendrobium,

The authors declare no conflicts of interest.

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