Efficient propagation with in vitro seed germination of Vanda falcata

Efficient propagation with in vitro seed germination of Vanda falcata 基于种子萌发的高效风兰( Vanda falcata )繁殖体系 1 2 first-author 2 2 2 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:lifang68@zju.edu.cn †E-mail:lifang68@zju.edu.cn lifang68@zju.edu.cn National Engineering Laboratory of Cold Chain Logistics Technology and Facility for Horticultural Produce, Zhejiang University, Hangzhou 310058, China National Engineering Laboratory of Cold Chain Logistics Technology and Facility for Horticultural Produce, Zhejiang University, Hangzhou 310058, China College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310058, China College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310058, China Vanda falcata (Thunb.) Beer (Orchidaceae), a famous native orchid of China, Japan, and Korea, is known as one of the most beautiful and charming orchid species in the world (Ohwi, 1965 1965 ; Lawler, 1984 1984 ; Arditti, 2008 2008 ). V. falcata is widely cultivated and delights the world with its compact plant shape, elegant white blooms, and sweet coconut-like scent. However, vegetative propagation by division has limited the development of V. falcata because of its inefficiency (Mitsukuri et al., 2009a 2009a , 2009b 2009b ). 目的 2 鉴于风兰( Vanda falcata (Thunb.) Beer)传统繁殖能力弱,本文拟利用植物组织培养技术建立一种高效的繁殖体系,以满足市场对风兰数量增多的需要. 创新点 2 建立了适用于风兰的高效组培繁殖体系,包括离体种子萌发、原球茎的形成,以及芽和根的生成. 方法 2 本研究首先以5年生母株自花授粉4~5个月后生成的种子为试材,依次经70%乙醇和10%次氯酸钠进行表面消毒后,分别接种于含不同成分的13种培养基中,统计分析种子萌发情况.进而将形成原球茎的试材,接种于含不同植物生长调节剂组合的培养基,统计分析芽和根的生长情况. 结论 2 评估的13种不同配方的培养基中,1/2 MS+维生素+肌醇+20 g/L蔗糖+3 g/L 活性炭+100 g/L椰肉组合可达到100%种子萌发率并形成较大原球茎;0.2 mg/L α-萘乙酸+0.5 mg/L 6-苄基腺嘌呤组合具有最高的芽诱导效率;1 mg/L α-萘乙酸+ 0.5 mg/L 6-苄基腺嘌呤+40 g/L蔗糖组合具有最高的根诱导效率.长势良好的植株经炼苗后可适应露天栽培. Vanda falcata Seed germination Protocorm Aseptic seeding Plant regeneration 风兰 种子萌发 原球茎 无菌播种 植株再生 Vanda falcata (Thunb.) Beer (Orchidaceae), a famous native orchid of China, Japan, and Korea, is known as one of the most beautiful and charming orchid species in the world (Ohwi, 1965 1965 ; Lawler, 1984 1984 ; Arditti, 2008 2008 ). V. falcata is widely cultivated and delights the world with its compact plant shape, elegant white blooms, and sweet coconut-like scent. However, vegetative propagation by division has limited the development of V. falcata because of its inefficiency (Mitsukuri et al., 2009a 2009a , 2009b 2009b ). Previous findings have shown that both in vitro micropropagation and asymbiotic germination of orchids could have the advantage of ensuring a large supply of clonal plantlets throughout the year (Teixeira da Silva et al., 2015 2015 ). Thus, several studies have been focused on the establishment of a micropropagation system for V. falcata . Different kinds of explants have been studied for their ability to establish aseptic cultures, such as shoot apexes, florets, new stocks, scapes, leaves, and root tips (Shimasaki, 1993 1993 ; Mitsukuri et al., 2009a 2009a ). These scientists found that shoot apexes of V. falcata were the most suitable explant materials, although they exuded browning compounds into the medium (Mitsukuri et al., 2009a 2009a ). Generally, two to three weeks of dark-preconditioning treatment or treatment with L -2-aminooxy-3-phenylpropionic acid (AOPP) must be carried out on shoot apexes or leaf tissue cultures to control browning (Mitsukuri et al., 2009a 2009a , 2009b 2009b , 2010 2010 ). Asymbiotic germination involves the in vitro inoculation and germination of seeds with the aid of a sucrose-containing culture medium, and represents the most efficient method of native epiphytic orchid propagation for conservation purposes (Arditti et al., 1981 1981 ; Cardoso et al., 2020 2020 ). These studies showed that in vitro germination rates could rise higher than 70%, while these rates barely exceed 5% in ex vitro (natural environmental) conditions (Rao, 1997 1997 ; Kunakhonnuruk et al., 2018 2018 ). However, no efficient in vitro propagation method has been reported for V . falcata . Here, we describe a simple and feasible protocol for propagation using immature seeds. Mature capsules were collected from five-year pot-grown mother plants in the Orchid Garden in Hangzhou Flower Garden, Hangzhou, China, after self-pollination for 4–5 months. First, the capsules were washed with tap water for 1 h and immersed in 70% alcohol for 30 s. Subsequently, the capsules were soaked in a solution of 10% sodium hypochlorite and agitated for 10 min. Finally, they were washed thoroughly in sterile water for five times after pouring the disinfectant out. The sterilized capsules were cut longitudinally using a sharp sterilized surgical blade to facilitate seed extraction. Seeds from these capsules were sown on modified MS (Murashige and Skoog, 1962 1962 ) basal medium containing half-strength macro-and micro-elements (Figs. 1a and 1b 1a and 1b ). To test the effect of some plant growth regulators (PGRs) and additives, which are commonly used in orchid tissue culture, we added the following to the basal MS medium, either individually or in combination: sucrose (30 g/L), activated charcoal (3 g/L), coconut water (100 mL/L), coconut meat (100 g/L), peptone (PEP, 3 g/L), lactoalbumin hydrolysate (3 g/L), α-naphthalene acetic acid (NAA; 0.1, 1.0, 3.0, or 5.0 mg/L), 6-benzyladenine (BA; 0.1, 1.0, 3.0, or 5.0 mg/L), and/or kinetin (KT; 1 mg/L); details are in Table 1 1 . The coconut water (liquid endosperm) and coconut meat (solid endosperm) were obtained from commercial coconuts. Our objective was not to test all permutations exhaustively, which would have resulted in hundreds of different treatments, but to test whether several key factors that were previously found to be effective in orchid seed germination (Kauth et al., 2008 2008 ) would also be successful for V. falcata seed germination. Each treatment was repeated twice with six replicates. All media were solidified with a 0.7% agar strip (Sigma, America). Media were adjusted to pH 5.6–5.8 with 1 mol/L NaOH or HCl before autoclaving at 121 °C 105 kPa for 15 min. The average number of seeds per capsule inoculated was more than 1000. The germination status was recorded after four weeks of culture. (a) Mature seeds (×100); (b) Aseptic seedling; (c) Protocorm multiplying; (d) Development of the protocorms; (e) Initiation of multiple shoots; (f) Development of multiple shoots; (g) Development of a stout root system in a plantlet; (h) A hardened plant growing in a pot in the greenhouse AC, activated charcoal; BA, 6-benzyladenine; CM, coconut meat; CW, coconut water; KT, kinetin; LH, lactoalbumin hydrolysate; MS, Murashige and Skoog; NAA, α-naphthalene acetic acid; PEP, peptone; S, sucrose Code name Medium composition  CW 1/2 MS+S 20 g/L+AC 3 g/L+CW 100 mL/L  CM 1/2 MS+S 20 g/L+AC 3 g/L+CM 100 g/L  PEP 1/2 MS+S 20 g/L+AC 3 g/L+PEP 3 g/L  N-1 1/2 MS+NAA 1.0 mg/L+BA 0.1 mg/L+AC 3 g/L+CW 100 mL/L  N-3 1/2 MS+NAA 3.0 mg/L+BA 0.1 mg/L+AC 3 g/L+CW 100 mL/L  N-5 1/2 MS+NAA 5.0 mg/L+BA 0.1 mg/L+AC 3 g/L+CW 100 mL/L  NP-3 1/2 MS+NAA 3.0 mg/L+BA 0.1 mg/L+AC 3 g/L+PEP 3 g/L  B-1 1/2 MS+NAA 0.1 mg/L+BA 1.0 mg/L+AC 3 g/L+CW 100 mL/L  B-3 1/2 MS+NAA 0.1 mg/L+BA 3.0 mg/L+AC 3 g/L+CW 100 mL/L  B-5 1/2 MS+NAA 0.1 mg/L+BA 5.0 mg/L+AC 3 g/L+CW 100 mL/L  BP-1 1/2 MS+NAA 0.1 mg/L+BA 1.0 mg/L+AC 3 g/L+PEP 3 g/L  K-1 1/2 MS+NAA 0.1 mg/L+KT 1.0 mg/L+AC 3 g/L+CW 100 mL/L  LH 1/2 MS+S 20 g/L+AC 3 g/L+LH 3 g/L We used the orthogonal matrix L9(34) method to measure the effects of the PGRs on multiple-shoot induction in MS medium. After a preliminary experiment to test the effects of various PGRs on multiple-shoot induction by one-factor-at-a-time, we selected four PGRs (NAA, BA, indole-3-butyric acid (IBA), and KT) with different concentrations. Three different combinations were used, including 0.2 mg/L NAA+0.5 mg/L BA, 0.5 mg/L NAA+1.0 mg/L BA+0.5 mg/L IBA+0.5 mg/L KT, and 1.0 mg/L NAA+2.0 mg/L BA+1.0 mg/L IBA+1.0 mg/L KT. The number of shoots (length of ≥2 mm) was investigated after 30 d of culture. Four influential factors in rooting, namely NAA, BA, sucrose, and PEP to induce roots, were optimized by orthogonal experiment. The combinations included 0.2 mg/L NAA+0.2 mg/L BA+10 g/L sucrose, 0.5 mg/L NAA+0.5 mg/L BA+20 g/L sucrose+1 g/L PEP, and 1.0 mg/L NAA+1.0 mg/L BA+40 g/L sucrose+3 g/L PEP. Each treatment was repeated twice with four replicates. We found that the best medium for rooting was the combination of solid MS supplemented with 1.0 mg/L NAA, 0.5 mg/L BA, and 40 g/L sucrose. We counted the number of roots (length of ≥2 mm) of eight-week-old plantlets. The 4–5-cm-high rooted seedlings were then transferred to plastic pots (12 cm diameter×18 cm height) containing sphagnum moss, wood charcoal, and gravel (1:1:1, volume ratio), and covered with polythene bags to maintain a high level of humidity. Plantlets were acclimated in the culture room for 3–4 weeks and then transferred to the greenhouse, where the climate conditions were the same (temperature range 20–25 °C, photoperiod 13 h/11 h (light/dark), and relative humidity 60%–70%). To evaluate the effectiveness of the culture medium on germination, we used Ridit analysis, which is designed to facilitate the data analysis of variables that are more than dichotomous classifications. These variables are ordered but do not reach the standards of refined measurement systems, such as those meeting the criteria for equal-interval or ratio scales (Bradburn, 1969 1969 ). The multiple-shoot induction and rooting data obtained using the orthogonal test were analyzed using SPSS software (Version 20, IBM, USA), and range analysis was applied to determine the impact of various factors on the experimental index. Statistical significance was determined using the least-significant-difference test. This statistical analysis used P =0.05. This is the first complete protocol that has been developed for mass propagation of V. falcata , an epiphytic orchid species. The main morphogenetic pathway is summarized in Fig. 1 1 . Although seeds germinated on all the media tested, the germination rate varied among them. On CM medium (code names of media are listed in Table 1 1 , the same for the following media), the embryos enlarged to occupy the entire seed coat (Figs. 1c and 1d 1c and 1d ) and developed into the protocorm stage, whereas the embryos remained swollen on other media. The highest seed germination rate ( R =0.9487) was observed on CM medium, followed by CW ( R =0.6538), B-3 ( R =0.6538), and B-5 ( R =0.6729) media (Fig. 2 2 ). LH, NP-3, and BP-1 media were not as effective as CM, CW, PEP, N-1, N-3, B-1, B-3, B-5, and K-1 media for seed germination. However, when PEP was applied in combination with NAA or BA, seed germination was poor. Code names of media are listed in Table 1 1 . Data are presented as the means of three replicates, and error bars indicate the standard deviation. The differences (with different lowercase letters) were tested at 0.05 levels of significance According to the average value in Table 2 2 , NAA exerted the most significant effect in multiplying shoots (Figs. 1e and 1f 1e and 1f ), and the order of importance that influenced multiplication was found to be NAA > KT > IBA > BA. NAA showed the most significant effects on rooting (Fig. 1g 1g ), followed by PEP ( P < 0.05, Table 2 2 ). The best medium for rooting was the combination of solid MS supplemented with 1.0 mg/L NAA, 0.5 mg/L BA, and 40 g/L sucrose. Each rooted seedling survived successfully after acclimatization (Fig. 1h 1h ). A value followed by the same letter within the same row (shoot multiplication or rooting) indicates insignificance at P =0.05. BA, 6-benzyladenine; IBA, indole-3-butyric acid; KT, kinetin; NAA, α-naphthalene acetic acid; PEP, peptone 2 Composition (average) 3 Number of shoots/roots Level 1 Level 2 Level 3 2 Shoot multiplication  NAA (7.92) 21.08 a 14.50 b 13.17 b  BA (6.58) 20.17 a 13.58 b 15.00 b  IBA (7.17) 19.92 a 12.75 b 16.08 ab  KT (7.25) 20.83 a 14.33 b 13.58 b Rooting  NAA (4.63) 3.61 b 2.91 b 7.54 a  BA (1.06) 4.17 a 5.22 a 4.67 a  Sucrose (0.91) 4.52 a 4.32 a 5.22 a  PEP (1.91) 5.37 a 3.46 b 5.22 a The effects of various PGRs or additives on seed germination and plant regeneration in V. falcata have been thoroughly studied. The simple and efficient method of regenerating a large number of plantlets from a single capsule containing coconut meat, NAA, and BA described here could be useful for large-scale propagation and ex situ conservation of the commercially important epiphytic orchid species. In vitro seed germination has been suggested as a suitable propagation method for conservation of orchids (Arditti et al., 1981 1981 ; Ballard, 1990 1990 ; Thornhill and Koopowitz, 1992 1992 ; Zettler and McInnis, 1993 1993 ; Kauth et al., 2006 2006 ; Stewart and Kane, 2006 2006 ; Mahendran and Narmatha Bai, 2009 2009 ; Hossain et al., 2010 2010 ). It has been documented that coconut water is a useful and potent germination enhancer and that it aids the germination of orchids in vitro (Withner, 1959 1959 ; Minea et al., 2004 2004 ). This finding is also supported by the results obtained in the present experiment, albeit at different concentrations of coconut water. However, coconut meat was shown to possess stronger stimulatory properties compared to those of coconut water, although the mechanism by which this occurs remains to be elucidated by further study. Initially, the protocorms were achlorophyllous and subsequently acquired chlorophyll, which agrees with the view of McKendrick ( 2000 2000 ). Much research has been focused on determining the nutritional and culture requirements for optimal germination and early seedling development in vitro. Beneficial modifications in the present study include reducing the concentration of inorganic salt (Fast, 1982 1982 ; Mitchell, 1989 1989 ; Anderson, 1991 1991 ) and adding half-strength macro-and micro-elements to the MS medium. The success of rapid and direct shoot and root regeneration from protocorm explants reveals another efficient way to mass-propagate V. falcata . Generally, auxins stimulate root formation, cytokinins enhance the development of shoots and cell division, and sugar stimulates the development of seedlings (Wotavová-Novotná et al., 2007 2007 ; Palama et al., 2010 2010 ). In the present study, protocorms developed multiple shoots and roots directly on medium supplemented with NAA and BA. In most orchids, including Dendrobium (Yasugi and Shinto, 1994 1994 ), Coelogyne stricta (Basker and Narmatha Bai, 2006 2006 ), Geodorum densiflorum (Sheelavantmath et al., 2000 2000 ), Oncidium varicosum (Kerbauy, 1984 1984 ), and Cymbidium pendulum (Nongdam et al., 2006 2006 ), the combination of NAA and BA (with a concentration range from 0.1 to 2.0 mg/L for NAA and 0.1 to 5.0 mg/L for BA) promoted optimal shoot proliferation and rooting. 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B2000505 Project supported by the Major Science and Technology Special Agricultural Projects in Zhejiang Province (No. 2007C12064) and the Zhejiang Province Breeding New Flower Varieties Major Science and Technology Key Projects (No. 2012C12909-10), China 2020-08-28 2020-11-05 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2020 2020 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 12 21