Asymbiotic germination and seed storage of Paphiopedilum insigne, an endangered lady's slipper orchid

South African Journal of Botany 112 (2017) 215–224

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Asymbiotic germination and seed storage of Paphiopedilum insigne, an endangered lady's slipper orchid Reema Vareen Diengdoh a, Suman Kumaria a, Pramod Tandon b, Meera Chettri Das a,⁎ a b

Plant Biotechnology Laboratory, Department of Botany, Centre for Advanced Studies, North-Eastern Hill University, Shillong 793022, Meghalaya, India Biotech Park, Lucknow, India

a r t i c l e

i n f o

Article history: Received 5 April 2017 Received in revised form 9 May 2017 Accepted 22 May 2017 Available online xxxx Edited by J Van Staden Keywords: Endangered – orchids High frequency germination Propagation Mature seeds- storage studies SEM

a b s t r a c t The present scenario of urbanization and commercialization has adversely affected orchid's population; as a consequence they are diminishing from the nature very rapidly. Paphiopedilum insigne (lady's slipper orchid) is one such orchid of horticultural importance. Apart from being listed as endangered in IUCN red data list; it finds a place in Appendix I of CITES in the global platform. In the present study, capsules b180 days after pollination (DAP) were found to be immature and 180 DAP was found optimal for seed germination. Capsules N 240 DAP were found to have mature seeds that were treated prior to germination and TTC tested for viability. Seeds derived from 180 DAP capsules showed the highest germination of 88.5% in modified Burgeff medium (BG1) with initiation in 26 days. Incorporation of plant growth regulators like 5 μM kinetin (KN) + 10 μM indole-3acetic acid (IAA) in 1/2 MS medium influenced the stage-wise development of the seedlings in a short duration. Mature seeds stored at −196 °C for 360 days followed by pre-treatment with 3% NaOCl showed viability of 70.5% and recorded germination of 79.2%. Sixty-nine percent of the plantlets were successfully hardened and acclimatized in the green house. © 2017 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction There is an unmatched fascination that we the people have with orchids and their unique beautiful flowers. However, there is an undying fact that their very existences in nature today, is deteriorating globally. Paphiopedilum insigne (known as lady slippers), is one such beautiful terrestrial orchid. It is distributed in small pockets of Northeast India (namely, Nagaland, Manipur, Mizoram and Meghalaya which is part of the Indo-Burma mega biodiversity hotspot of India) and other Southeast Asian countries like China; Myanmar; Thailand; Laos and Vietnam (Chowdhery, 2004; Tandon and Kumaria, 2011; http://www. iucnredlist.org/). This species is marketed as an attractive potted plant due to long shelf-life (60–90 days) of the flowers which are used as parents in producing hybrids in breeding programmes. There are a few reports on ethno-botanical utilization of P. insigne as a medicine in curing stomach ailments (Friesen and Friesen, 2012). Poor germination and slow plantlet growth have added to it being rare in the wild (Yam and Arditti, 2009; Zeng et al., 2015). In the present scenario, the orchids are experiencing a steady decline from their natural habitats. With concerns on prioritizing for conservation, P. insigne has not only been included under the latest IUCN red list as endangered plant (Rankou ⁎ Corresponding author at: Department of Botany, Centre for Advanced Studies, NorthEastern Hill University, Shillong 793022, Meghalaya, India. E-mail address: [email protected] (M.C. Das).

http://dx.doi.org/10.1016/j.sajb.2017.05.028 0254-6299/© 2017 SAAB. Published by Elsevier B.V. All rights reserved.

and Kumar, 2015; http://www.iucnredlist.org/) but, also enlisted in Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Although orchid seeds are the best source to conserve their genetic diversity still, they are under-rated, hence rarely collected, sowed or stored. The heterogeneous nature of seeds makes them suitable for conserving genetic diversity of plant populations in nature. Orchid seeds too exhibit this nature in conjunction with other features like minute seed size and ample availability per capsule. Despite of several attempts on preserving orchid population in the wild yet, they are still abating at an alarming rate. There is a need to strategize in-situ and ex-situ conservation methods to help preserve this orchid. Ideally, approach of conservation is in-situ (Tandon, 2004), however this practice is unsafe due to many anthropogenic activities. Alternatively, ex-situ conservation offers not only safer security backup system for conservation (Chugh et al., 2009; Engelmann, 2010) but also allows accessibility for research work evaluation. In-vitro technologies like asymbiotic seed germination, mass propagation, short-long term storage etc. based on ex-situ conservation have been used to conserve rare, endangered and threatened plants especially orchids (Arditti, 1994; Kumaria and Tandon, 2001; Decruse et al., 2003; Dutra et al., 2008; Yam and Arditti, 2009; Mohanty et al., 2012; Bhattacharyya et al., 2017). Long-term preservation of orthodox seeds via. cryopreservation in liquid nitrogen (LN; − 196 °C) helps in maintaining the plant germplasm in stasis for years and is also applied for rescue of rare and endangered plant species (Engelmann,

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2004; Nikishina et al., 2007; Thammasiri and Soamkul, 2007; Hirano et al., 2009; Kaczmarczyk et al., 2011). Terrestrial orchids unlike their epiphytic counterparts, are difficult to germinate in-vitro and fail to establish in soil on a large scale (Batty et al., 2001; Stewart and Kane, 2006; Swarts and Dixon, 2009). There are a number of reports on asymbiotic germination of immature seeds of this genus which varies from species to species (Arditti and Ernst, 1984; Kauth et al., 2006; Zeng et al., 2013). Mature orchid seeds may have a greater potential for propagation and storage because of fully developed testa and lower water content (Miyoshi and Mii, 1998; Fu et al., 2016). Yet, for many species, prospects for this kind of conservation are hampered by poor storage conditions and regeneration protocols need to be standardized (Stimart and Ascher, 1981; Long et al., 2010; Merritt et al., 2014; Zeng et al., 2015). Therefore, in this present study, we report an effective protocol for ex-situ conservation of P insigne with high frequency seed germination, storage of mature seeds and seedling growth comparatively in a shorter period. 2. Materials and methods 2.1. Capsules collection and sterilization The plants of P. insigne were collected from the nurseries of Upper Shillong, Cherrapunjee, Mawsynram and maintained in the greenhouse of Plant Biotechnology Laboratory, Department of Botany, NorthEastern Hill University, Shillong, India. The collection is in compliance with the fulfilment of legal requirements. During the flowering season 70 flowers were hand pollinated by dusting pollens onto the stigma of the same flower and covered with polythene bags. From the time of hand pollination, the immature capsules and mature seeds were harvested at interval of 30 days. The capsules b240 days after pollination (DAP) were washed thoroughly with tween 20 and rinsed under running tap water 3–4 times. These capsules were then treated with 70% alcohol for 30s followed by flame sterilization. Mature seeds N240 DAP were collected from the polythene bags and subjected to various pretreatments like pre-soaking in water for 30 min (control), 1%, 3%, 5% and 7% of NaOCl (4% available chlorine; Himedia) for 30 min, After every pre-treatment with NaOCl, the seeds were rinsed with sterile distilled water 6–8 times. 2.2. Asymbiotic seed germination of immature and mature seeds Immature seeds were scooped out of the capsule and inoculated on different media namely, Murashige and Skoog (MS), BM Van Waes (BM), modified Burgeff (BG1), 1/2 MS (Table 1) contained in 25 × 150 mm glass test tubes each containing 10 ml of medium. The cultures were incubated in the dark for 10 days, and to16 h photoperiod at 50 μmol m2 s−1 light intensity. Initiation of seed germination was observed after every 10 days but percentage germination was recorded 60 days after inoculation (DAI). Ten replicates were maintained and the experiment was repeated thrice. Similarly, pre-treated mature seeds were inoculated for seed germination while the seed morphology for mature seeds after every treatment was observed under Scanning Electron Microscopy (SEM). For SEM studies, seed samples were air dried in laminar air flow for 1 h followed by drying with a JFD-310 freeze dryer and affixed to aluminium stubs and coated with gold in a JFC-1100 (JEOL) ion sputter coater and observed using a JEOL, JSM-6360 SEM at 20 kV (SAIF, NEHU, India). The proliferation of protocorms into seedlings at different developmental stages of protocorms was assessed (Table 2). Thirty days old protocorms were subjected to different concentrations of plant growth regulators namely, 6-benzylaminopurine (BAP), kinetin (KN), α-naphthalene-acetic acid(NAA) and indole-3-acetic acid (IAA) ranging from 0 to 25 μM concentrations singly and in combination, were

Table 1 Composition of organic and inorganic nutrients used in various media. Constituents

MS

1/2 MS

BG1

BM

Inorganic nutrients mg/ml) NH4NO3 KNO3 KH2PO3 Ca(NO3)2·4H2O MgSO4·7H2O (NH4)2SO4 MgCl2·6H20 KCL KH2PO4·3H2O KI H3BO3 MnSO4·4H2O ZnSO4·7H2O Na2MoO4·2H2O CuSO4·5H2O CoCl2·6H2O FeSO4·7H2O Na2EDTA·2H2O CaCl2·2H2O CO(NO3)2·6H2O NaH2PO4·2H2O MnCl2 H3BO3

1650 1900 170 – 370 – – – – 0.83 6.2 22.3 0.25 0.25 0.25 0.025 27.8 37.3 440 – – –

825 950 85 – 185 – – – – 0.41 3.1 11.15 0.12 0.12 0.12 0.012 13.9 18.65 220 0.05 250 –

– – – 1000 250 250 – 250 250 – – – – – – – 20 – – – – –

– – – – 100 – – – 300 80 6.2 100 10 0.25 0.025 0.025 27.3 37.3 – – – 3.9 10

0.1 0.5 0.5 2 – – –

0.1 0.5 0.5 2 – – –

– – – – 90 – –

0.5 5 0.5 2 – 0.05 2

– 100 – 30,000 2000 8 5.8

– 100 – 30,000 2000 8 5.8

– – 20,000 – 2000 11 5.3

500 –

Organic nutrients (mg/ml) Thiamine HCl Nicotinic acid Pyridoxine HCl Glycine Citric acid Biotin glutamine Casein hydrolysate Meso Inositol Glucose Sucrose Activated charcoal Agar pH L

20,000 2000 12 5.2

incorporated in the optimal medium for seedling growth assessment. Percentage response of protocorms at each stage and cumulative developmental stages of the protocorms/seedlings were recorded after 30 DAI. Growth parameters like shoot number and length and root number and length were further evaluated separately. The data were recorded at 30 DAI. Twenty protocorms were taken for each treatment and all experiments were repeated thrice. 2.3. Mature seed storage studies Approximately, 200 mg mature seeds (N240 DAP) were placed per 2-ml sterile cryovials (polypropylene, Tarsons Pvt. Ltd., India.) followed by fixation in cryocane (Tarsons Pvt. Ltd., India) and stored at different temperatures for 280 days. After every 30 days of storage, the seeds were sterilized according to the protocol standardized for mature seeds and inoculated on the optimized regeneration medium. In case of seeds stored directly in LN, thawing was done by dipping the cryovials in distilled water at 45 °C for 2 min followed by the same

Table 2 Developmental stages of protocorm to seedlings. (Modified from Stewart et al., 2003) Stages

Description

I II III IV

Embryo enlarged, testa ruptured (=Germination) Appearance of protomeristem Emergence of two-first leaf primordia & elongation of shoot Root development

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procedure for sterilization before inoculation. Simultaneously, after every storage interval, seeds were subjected to viability test using 1% TTC where seeds showing some degree of pink or red were considered

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viable and scored accordingly (Vujanovic et al., 2000). For each treatment 6 replicates were maintained and the experiment was repeated thrice.

Fig. 1. In vitro propagation of P. insigne (a) flowering plant in natural habitat (b) 180 DAP seedpods (c)seedpods N240 DAP (d) mature seeds stored in cryovials (e) asymbiotic seed germination in modified BG1 medium 60 DAI (f)–(h) different developmental stages of protocorms in 1/2 MS medium (i)1% TTC tested staining mature seeds after storage in −196 °C liquid nitrogen for 180 days (j) seedling growth in 1/2 MS medium supplemented with 5 μM KN + 10 μM IAA (k) Subcultured plants after intervals of 35 days (l) primary hardened plantlets in the optimized compost mix after 60 days (m) secondary hardened plantlets after 90 days (n) hardened plantlets transferred to earth pots after 90 days survival compost mix. 1 bar = 10 mm.

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2.4. Acclimatization Plantlets with well-developed shoots (5–8 cm), leaf breadth of about 1–2 cm wide and roots (5–7 cm) were taken for hardening procedures. The plants were washed thoroughly under running tap water to remove traces of medium. These plantlets were transferred to thermocol pots containing different compost mixtures in varied ratios viz., (i)charcoal + soil + layer of moss litter (1:1), (ii)soil + decay litter + coconut husk (1:1:1), (iii)cocopeat + charcoal + decaying litter (1:2:1) with a top layer of moss and maintained in the green house. The thermocol pots were covered with perforated polythene bags for about 2 weeks to avoid direct exposure to sunlight, as transfer to direct sunlight of the in-vitro plantlets has been reported to dry up the leaves (Hiren et al., 2004; Lavanya et al., 2009; Gogoi et al., 2012). The plants were watered thoroughly after every two days. The survival percentage and shoot length were recorded after 60 days. The plantlets were transferred to earthen pots with the optimized pot mix after 90 days. 2.5. Culture conditions pH of the medium was adjusted to 5.8 using 1 N NaOH prior to autoclaving at 15 psi, 121 °C for 15 min. All the cultures were incubated at 25 ± 2 °C and 16 h photoperiod at 50 μmol m−2 s−1 light intensity. 2.6. Statistical analysis All data were subjected to analysis of variance (one way ANOVA) and significance (P b 0.05) was determined with Duncan's multiple range test. Statistical tests were performed with the help of SPSS statistical package version 15.0 (SPSS Inc., Chicago, USA). 3. Results & discussion 3.1. Capsules collection & sterilization Of the 70 hand-pollinated flowers, seed set was recorded only in about 50%. (Fig. 1a). Sterilization protocol was applicable to capsules b210DAP (Fig. 1b) however, mature seeds were subjected to pretreatments with NaOCl. Bursted seeds N240 DAP from desiccated capsules as shown in Fig. 1(c), were collected from the polythene bags. The seed set and seed viability from self-pollination were not significantly different to cross-pollination yet, the frequency of seed set was observed to be low and limited to just 50% as also reported by Zeng et al. (2012). 3.2. Asymbiotic seed germination of immature and mature seeds and plantlet development Capsule maturity plays an important role to decide the correct time capsule collection which affects germination and it diverges from orchid

species to species (Deb and Pongener, 2013). In the present study, capsules b150 DAP did not show any germination and turned brown after 15–18 DAI, while 150 DAP capsules showed initiation of germination i.e., stage 1 within 45 DAI as shown in Table 3. Several reports on Paphiopedilum spp. suggest that immature seeds b 150 DAP either fail to germinate or take more time to respond compared to older capsules which may be due to under developed embryo that failed to recognize stimulating effect of medium composition to germinate (Nhat and Dung, 2006; Long et al., 2010; Ng et al., 2010; Zeng et al., 2012, 2015). The optimal age of the seeds initiating germination as early as 26 days was observed from 180 DAP capsules as compared to more mature capsules (Fig. 1e, Table 3). This initiation of seed germination from 180 DAP capsules may be due to precise age of the capsules for efficient protein mobilization during rehydration and embryonic unlignified testa allowing the permeability to nutrients (Rasmussen, 1995; Lee et al., 2006; Zeng et al., 2012, 2015). On the other hand, poor or no response to germination of seeds derived from mature capsules N 180 (Table 3).this is in conformity with the other reports (Ding et al., 2004; Lee et al., 2005; Nikishina et al., 2007; Long et al., 2010; Zeng et al., 2013). Composition of the medium plays a crucial role in influencing in-vitro seed germination. Amongst the medium types used, the earliest and highest germination of 88.5% was observed from seeds inoculated on BG1 medium recorded 60 DAI from 180 DAP capsules (Fig. 1e), while in other media significantly lower percentage of germination was seen (Table 3). Similarly, the time taken for initiation of stage 1 of germination (Tables 2, 3) was recorded earliest in modified BG1 medium irrespective of capsules maturity. Morphologically, protocorm colour in modified BG1 medium was light green to whitish as seen in Fig. 1(e) while, in 1/2 MS medium and BM medium it was whitish. The seed germination for same species on the different medium varies as also reported by Lo et al. (2004) and Chen et al. (2015). There are several reports on certain orchid seeds requiring higher salt content medium for germination (Dohling et al., 2008; Paul et al., 2011). On analysing BG1 medium (Table 1), it was found that it had low salts contents in its composition thereby making it more suitable for the seeds of P. insigne seeds to germinate. This is also supported by reports (Pierik et al., 1988; Nikabadi et al., 2014; Zeng et al., 2015) that suggest in few selected orchids the seeds require even less than half of both micro and macronutrients for initiating germination in seeds. Another key factor in the composition of BG1 which varied was in its source of carbohydrates as glucose. Glucose being the simplest form of carbohydrates may have adhered to the easy assimilation by the seeds to germination (Nikabadi et al., 2014). The beneficial effects of glucose for early germination has been reported by Traore and Guiltinan (2006) and Long et al. (2010). In contrary to earlier reports (Tay et al., 1988; Zeng et al., 2012; Chen et al., 2015) the results cater to the need of reducing the time taken for germination as well as adding to enhanced effect on seedling development. Our present study suggests that BG1 medium with added glucose resulted in high frequency of germination

Table 3 Seed germination of P. insigne on different media. Days after pollination

60 90 120 150 180 210 240 270 300

MS

1/2 MS

BG1

BM

G%

Time taken (days)

G%

Time taken (days)

G%

Time taken (days)

G%

Time taken (days)

– – – – 9.8 ± 4.8b 16.1 ± 0.9a – – –

– – – – 78 129 – – –

– – – – 41.5 ± 4.5a 38.4 ± 4.1a – – –

– – – – 51 80 – – –

– – 0c 48.1 ± 4.5b 88.5 ± 2.1a 58.1 ± 5.1b 4.8 ± 0.8c – –

– – – 45 26 41 86 – –

– – – – 32.0 ± 5.1a 35.9 ± 4.9a – – –

– – – – 47 74 – – –

Germination percentage (G%) after 60 DAI. Data recorded after every 10 days of inoculation (DOI). Mean values within a column followed by the same letter are not significantly different by Duncan's multiple range test (P C 0.05). Values correspond to means (±SE) of three independent experiments. 10 replicates were used for each experiment. Note: no response for germination.

R.V. Diengdoh et al. / South African Journal of Botany 112 (2017) 215–224 Table 4 Growth and development of P. insigne protocorms to seedlings after 60 DAI in different media. Medium

Shoot number

Shoot length (cm)

Root number

Root length (cm)

BG BM 1/2 MS

2.0 ± 0.1b 2.2 ± 0.1bc 3.4 ± 0.1a

1.8 ± 0.2b 2.1 ± 0.1bc 3.1 ± 0.3a

2.1 ± 0.2a 2.3 ± 0.2a 2.5 ± 0.2a

1.1 ± 0.2b 1.4 ± 0.2b 2.7 ± 0.3a

# Means followed by the same letter are not significantly different according to Duncan's multiple range test (p = 0.05) [Values are mean ± SEM of three experiments with ten replicates/experiment]. ANOVA test shows that seed germination is highly significant at 5% level.

in a short period of time. Results obtained in the present study showed that seed maturity and culture media have a distinct influence on the germination pattern of this species. The ideal medium for the seed germination of Paphiopedilum species is species-specific (Zeng et al., 2012; Chen et al., 2015). Protocorm development into seedling is a slow sequential process (Table 2) as also reported by Robinson et al. (2009). On assessing the trend of seedling development from protocorm and time taken for each stage to develop; the highest average shoot length (3.1 cm) and root length (2.7 cm) were observed in 1/2 MS medium which was significantly higher than that recorded in BG1 and BM medium at 35 DAI (Table 4). Therefore, 1/2 MS medium was regarded optimal medium for seedling growth. This also shows that the carbohydrate requirement of the protocorm shifts from glucose to sucrose. This implies that the nutrient requirement of the protocorm varies during the different stages of development of shooting and rooting also suggested in Cypripedium

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(Bae et al., 2010). The effect of auxins and cytokinins when incorporated singly in 1/2 MS fastened the protocorm development as recorded after 45 days of DAI (Table 5). The total cumulative response of 79.7% of protocorms to 1/2 MS incorporated with KN at 10 μM enhancing stage III (61.5%) in shorter time period which is significantly higher as compared to the control (total cumulative response of 65.7%, stage III of 10.6% as shown in Fig. 1 f–h). While, in case of the 1/2 MS medium incorporated with BAP at 15 μM gave a total response of 72.8% but was not significantly different from the control. Amongst the auxins; 1/2 MS incorporated with IAA at 10 μM a cumulative total response of 75.8% (stage IV − 26.5%) which was significantly higher than control. Incorporation of 2,4-D and NAA in 1/2 MS medium showed inhibitory effect on the seedling growth similar reports by Hossain (2008). Of the different combinations of KN with either IAA or NAA incorporated in 1/2 MS medium, it was found that protocorms cultured in medium containing 5 μM KN + 10 μM IAA and 20 μM KN + 15 μM IAA resulted in high cumulative response of 86.5% and 80.8% respectively with enhanced effect on stages III & IV and with no significant difference between them (Table 5). However, on further analysis on the growth parameters like shoot number, length, root number and length, significant differences were found between the two concentrations (Fig. 3). The best growth and development of the seedlings was recorded in 1/2 MS medium supplemented with 5 μM KN + 10 μM IAA, with maximum average number of shoots (4.5 cm), shoot length (4.2 cm), average root number (4.0 cm) and root length (3.6 cm) recorded after 30 DOI (Fig. 3). This shows that the nutrient requirement for different stages of development varies (Nadarajan et al., 2011; Zeng et al., 2013). In the present study, incorporation of KN and IAA was found

Table 5 Developmental stages of protocorms of P. insigne incorporated with growth regulators in the 1/2 MS medium recorded at 30 DAI. PGRS & Conc. (μM)

Percentage of stages of development after germination

NAA

IAA

2,4-D

KN

BAP

I

II

III

IV

Total %

0 5 10 15 20 25 – – – – – – – – – – – – – – – – – – – – 5 10 15 20 25 – – – – –

0 – – – – – 5 10 15 20 25 – – – – – – – – – – – – – – – – – – – – 5 10 15 20 25

0 – – – – – – – – – – 5 10 15 20 25 – – – – – – – – – – – – – – – – – – – –

0 – – – – – – – – – – – – – – – 5 10 15 20 25 – – – – 10 5 15 10 5 5 5 20 10 10

0 – – – – – – – – – – – – – – – – – – – – 5 10 15 20 25 – – – – – – – – – –

40.6 ± 1.5a 38.6 ± 1.5a 4.4 ± 1.2d 5.8 ± 1.3c 6.1 ± 1.3bc 6.4 ± 1.1b 5.4 ± 0.6c 4.1 ± 2.7de 4.2 ± 2.7de 4.9 ± 1.5 cd 4.4 ± 0.9d 3.5 ± 0.5e 2.8 ± 0.6f 2.6 ± 1.5f 3.6 ± 0.5e 4.5 ± 0.6d 6.4 ± 1.1b 4.4 ± 0.3c 6.4 ± 1.1b 3.8 ± 0.2e 5.4 ± 1.6c 2.8 ± 0.6f 2.8 ± 0.6f 4.0 ± 0.3de 4.2 ± 0.7d 2.8 ± 0.6f 4.9 ± 1.6b 4.8 ± 0.4b 3.7 ± 0.8c 3.9 ± 1.6bc 4.5 ± 0.4b 3.7 ± 0.8c 3.7 ± 0.8b 4.2 ± 0.8a 3.7 ± 0.8b 3.7 ± 0.8b

14.0 ± 0.6b 28.0 ± 0.6 g 17.4 ± 1.2b 23.8 ± 1.4ab 20.8 ± 1.3b 27.2 ± 1.1ab 6.4 ± 0.6ef 6.1 ± 1.7f 5.1 ± 1.7f 5.0 ± 1.5 g 5.4 ± 0.9 fg 23.2 ± 1.1ab 19.6 ± 0.5b 30.4 ± 0.6a 21.4 ± 0.9ab 13.1 ± 2.7b 27.2 ± 1.1ab 5.4 ± 0.3e 27.2 ± 1.1ab 6.2 ± 0.2f 7.4 ± 1.6e 7.8 ± 0.6e 7.4 ± 1.2e 10.8 ± 0.3f 10.8 ± 1.4d 8.8 ± 1.3de 14.8 ± 0.7ab 4.8 ± 0.4b 4.2 ± 1.3b 3.6 ± 0.8c 16.2 ± 0.8a 4.2 ± 1.3b 4.2 ± 1.3d 10.5 ± 0.5c 41.9 ± 2.4b 46.2 ± 4.8a

10.6 ± 0.5d 2.6 ± 1.5 h 35.8 ± 1.7d 30.6 ± 2.0d 39.6 ± 2.0d 36.3 ± 1.3d 40.0 ± 0.9 cd 40.2 ± 2.0b 49.2 ± 2.0b 44.2 ± 2.0c 41.6 ± 1.8c 23.6 ± 1.2e 19.6 ± 1.5e 9.5 ± 0.5 g 9.2 ± 0.5f 9.5 ± 0.5ef 38.3 ± 1.3d 61.5 ± 1.9a 36.3 ± 1.3d 50.8 ± 1.2b 42.3 ± 1.2c 51.6 ± 2.5ab 47.2 ± 0.5c 41.2 ± 0.9a 37.4 ± 0.8d 36.2 ± 0.5d 42.8 ± 1.3b 47.4 ± 0.8ab 58.4 ± 0.5a 48.8 ± 1.0ab 37.2 ± 1.3c 58.4 ± 0.5a 53.4 ± 0.5a 51.2 ± 1.9b 8.6 ± 1.8c 4.9 ± 2.2d

2.1 ± 0.5c 4.2 ± 0.5c 4.4 ± 1.2f 7.8 ± 1.4de 6.8 ± 1.3 cd 6.2 ± 1.1d 5.4 ± 0.6ef 26.5 ± 0.9a 9.5 ± 0.9b 7.9 ± 1.5bc 6.4 ± 0.9d 3.5 ± 0.5 g 2.8 ± 0.6 g 2.6 ± 1.5 g 0g 0g 5.2 ± 1.1d 7.4 ± 0.3b 6.2 ± 1.1d 1.4 ± 1.6 h 9.8 ± 0.2b 2.8 ± 0.6 g 2.8 ± 0.6 g 5.8 ± 0.3de 4.2 ± 0.2f 2.8 ± 0.6 g 4.9 ± 1.6a 4.5 ± 0.4a 4.7 ± 0.8a 4.4 ± 0.3a 4.5 ± 0.3b 4.7 ± 0.8a 27.7 ± 0.8a 14.6 ± 0.4b 4.6 ± 0.4b 4.5 ± 0.8b

65.7 ± 2.1b 69.7 ± 2.2bc 62.0 ± 3.2d 70.0 ± 2.2bc 69.0 ± 2.1b 70.1 ± 2.2bc 62.2 ± 3.2d 75.8 ± 1.2a 70.8 ± 1.2bc 62.0 ± 3.1d 57.8 ± 4.2e 45.8 ± 1.2de 41.8 ± 4.2f 40.1 ± 3.6e 34.8 ± 4.3f 31.6 ± 4.8f 71.1 ± 2.2b 79.7 ± 1.9a 70.1 ± 2.2b 69.2 ± 2.5a 64.9 ± 2.3 cd 65.0 ± 1.3c 67.4 ± 2.1b 72.8 ± 2.1b 56.6 ± 3.1e 50.6 ± 3.8ef 67.4 ± 2.1b 61.2 ± 2.7b 71.0 ± 2.1a 60.7 ± 3.1bc 60.3 ± 3.2bc 71.0 ± 2.1b 86.5 ± 3.1a 80.8 ± 2.1a 60.1 ± 3.2c 59.3 ± 4.1c

Means followed by the same letter are not significantly different according to Duncan's multiple range test (p = 0.05) [Values are mean ± SEM of three experiments with ten replicates/ experiment] ANOVA test shows that seed germination is highly significant at 5% level.

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stimulatory for seedling growth of P. insigne which is in contrast to earlier reports (Long et al., 2010; Zeng et al., 2012; Chen et al., 2015) in which BAP and NAA in combination was effectively used for in-vitro propagation of several orchids. It was observed that with further increase in concentration of both the cytokinins and auxins, seedling growth of P. insigne was inhibited. Nagaraju et al. (2003) had also reported that higher concentrations of cytokinins and auxins do show inhibitory effect on growth of orchids. The concentration of exogenous cytokinins and auxins and its synergistic effects in combinations under balanced condition, varies between species to species (Hossain, 2008; Hossain et al., 2010; Long et al., 2010; Roy et al., 2011; Zeng et al., 2012). To date, there has been limited success in inducing callus in Paphiopedilum, due to the initial difficulty in the induction process itself, slow growth rate, low regeneration capacity, and eventual browning

of the callus (Lin et al., 2000; Long et al., 2010; Zeng et al., 2013). The optimized medium was further used for subculturing at interval of 35 days (Fig. 1k). In this study, it may be noted that organic additives like coconut water, banana homogenate etc., were not used in the medium composition, which is contradictory to other earlier reports where such organic additives were used. 3.3. Pretreatment of mature seeds (N 240 DAP) and seed germination It is common for orchid seeds to be collected when capsules are partly immature. This practice arises from difficulties in identifying the plant populations (particularly of those species that are rare, vulnerable, endangered) and is compounded by the risk of losing thousands of seeds upon bursting of the mature capsules as reported by Merritt

Fig. 2. Surface scarification observed using SEM analysis on the pre-treatment of bursted seeds(N210 DAP) of P. insigne (a)1% NaOCl at 190× (b) 1% NaOCl at 37× (c)3% NaOCl at 190× (d) 3% NaOCl at 37× (e) 5% NaOCl at 190× (f) 5% NaOCl at 37× (g) 3% NaOCl at 160× (h) 7% NaOCl at 37×.

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7 6 5 4 3 2 1 0

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Means followed by the same letter are not significantly different according to Duncan’s multiple range test (p=0.05)[Values are mean±SEM of three experiments with ten replicates/experiment] ANOVA test shows that seed germination is highly significant Fig. 3. Seedling growth of P. insigne on 1/2 MS medium incorporated with growth hormones. Data recorded after 30 DAI.

percentage %

et al. (2014). There have also been several reports stating difficulties of in-vitro germination for Paphiopedilum seeds older than 190 days (Lee and Lee, 1999; Chen et al., 2004; Ding et al., 2004; Lee et al., 2006; Liao and Chen, 2006; Zeng et al., 2006, 2010, 2012, 2013). The factors affecting low germination percentage of mature seeds in orchids could be due to impermeable testa, or the presence of chemical inhibitors such as abscisic acid (ABA), or the lack of certain germinationpromoting hormones (Zeng et al., 2012). In the present study, of all the pretreatments for seeds N240 DAP, 3% NaOCl treatment for 30 min resulted in the highest germination percentage of 81.2% recorded 30 days DAI (Fig. 4). This treatment proved to be beneficial in combating the contamination issue, acting as a surface sterilant as the mature seeds have bursted from the protective capsules. The outcome of pre-soaking in water (Fig. 4) indicated that contamination and seed dormancy are factors that hinder the mature seeds from germination. From the SEM studies of the seeds it was revealed that various concentrations of NaOCl had varied effects on the outer covering of the orchid seed i.e., the testa. The control and 1% NaOCl showed similar effect (Fig. 2a,b) with no visible scarification on the testa but with maximum contamination and low germination (Fig. 4). Pre-treatment with 3% NaOCl showed an adequate amount of scarification on testa (Fig. 2c,d) which clearly showed small perforations of the testa (Fig. 2) that affected its rigidity and scarified the testa enough to break the dormancy and softened the walls allowing more nutrient permeability to the embryo of the mature seeds which is reflected with highest germination percentage (Fig. 4). This was beneficial in combating the contamination issue by acting as a surface sterilant. However, higher concentrations of 5%

100 90 80 70 60 50 40 30 20 10 0

NaOCl and 7% NaOCl showed heavy scarification and resulted in significant injury like shrinkage, scalded, deformed seeds (Fig. 2e–h) thereby, explaining the reduction in germination rate. This could be due to the toxic effect as well as excessive mechanical damage caused by NaOCl at higher concentration (Zeng et al., 2013). Furthermore, stimulatory effects of NaOCl or calcium hypochlorite pre-treatment have been reported in some orchid species (Miyoshi and Mii, 1995; Lee et al., 2005; Yamazaki and Miyoshi, 2006; Mweetwa et al., 2008; Zeng et al., 2014; Chen et al., 2015) which may have worked also on this species after optimization. 3.4. Mature seed storage studies and germination Two important characteristics of orchid seeds namely, i.e. (i) Mature seeds - propagation and storage, (ii) seeds - regarded as possessing orthodox storage behaviour (Miyoshi and Mii, 1995; Li and Pritchard, 2009; Nadarajan et al., 2011) were addressed in the present study. Immature seeds have the zygotic embryo which is not fully developed and the testa might not be lignified, allowing it to be permeable to water and nutrient, but in case of mature seeds a fully formed testa and low water content may show superior potential for propagation and storage as reported earlier (Miyoshi and Mii, 1998; Zhang et al., 2015; Fu et al., 2016). In the present findings, mature seeds collected in cryovials as shown in Fig. 1d; when stored at − 196 °C were found to be most appropriate for long term storage with 77.2% to 82.6% germination rate (Fig. 5), without significant variation, over a span of 360 days storage. This was also supported by TTC test which showed 80% (Fig. 5)

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Means followed by the same letter are not significantly different according to Duncan’s multiple range test (p=0.05)[Values are mean ± SEM of three experiments with ten replicates/experiment] ANOVA test shows that seed germination is highly significant

Fig. 4. Pre-treatment of mature seeds (N210 DAP) of P. insigne and in vitro germination on modified Burgeff medium.

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# Means followed by the same letter are not significantly different according to Duncan’s multiple range test (p=0.05)[Values are mean ± SEM of three experiments with ten replicates/experiment] ANOVA test shows that seed germination is highly significant

Fig. 5. Germination and TTC viability percentage of the stored seeds (N240DAP) of P. insigne in various temperatures recorded at 30 DAI.

seeds viability in 360 days stored seed (Fig. 1i). Storage at 0 °C resulted in seed germination from 60% to 80% with significantly reduced germination with long duration of storage. While, seeds stored at 25 °C, showed 79% to 81% germination on storage up to 180 days, however, the germination percentage reduced on further storage after 360 days (Fig. 5). The percentage of viability does not tally with in-vitro germination of the stored seeds as shown in Fig. 5. In-vitro germination of stored seeds showed higher percentage response than TTC seed viability test as this test might not give the accurate viability since it introduced a bias with respect to seed testa colour (Lemay et al., 2015). There are reports of seed viability being maintained in a number of orchid species stored for up to 12 months (Li and Pritchard, 2009). Seeds or shoot tips have been successfully stored in liquid nitrogen (LN) providing protection with the ability for revival and plant regeneration when needed in future. Cryopreservation has been used for seed and shoot-tip conservation, providing long-term storage (Engelmann, 2004; Li and Pritchard, 2009; Pritchard and Nadarajan, 2009; Engelmann, 2010; Reed et al., 2011). Poor response was observed in seeds stored at 25 °C, both using TTC test as well as in-vitro germination suggesting at higher temperature the seeds are subjected to excessive dehydration stress leading to imbibitional injury inducing rapid rehydration in free water as reported by Hirano et al. (2011). Depending on the duration method and temperature adopted, drying and long-term storage may lead to considerable reduction in germination or to eventual death of the

seeds. Thus, in the present findings, storage in (LN; − 196 °C) proved to be most effective in not only storing and retaining the seed viability but also in protecting the seeds against pathogen attack (Mweetwa et al., 2008). 3.5. Acclimatization Plantlets raised under in-vitro require stepwise and careful procedure for transfer to ex-vitro conditions. Plantlets transferred to the green house depends on the suitable size of seedlings and the compost used. Of all the compost mixtures used to check the survivability of in-vitro raised plantlets, the highest response of 69.2% with average shoot length of 12.1 cm (Fig. 6) was observed in a compost mix of cocopeat + charcoal + decaying litter in a ratio 2: 1: 1 with a layer of moss after 60 days of hardening (Fig. 1l). Similarly, compost mixture comprising soil + decay litter + coconut husk in the ratio 1:1:1 but without a layer of moss was found to have good survival of 53.4% with average shoot length 7.2 cm. On the other hand, the compost containing charcoal + soil in a ratio 1:1 with a layer of moss did not support survival of the transferred plantlets of P. insigne with only 30.9% survival and average shoot length of 6.5 cm. Complete established plantlets were obtained after 90 days (Fig. 1m). These plantlets were later transferred to earthen pot with standardized compost mix (Fig. 1n). In the present study, healthy and vigorous growing plantlets

80 a

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# Means followed by the same letter are not significantly different according to Duncan’s multiple range test (p=0.05)[Values are mean ± SEM of three experiments with ten replicates/experiment] ANOVA test shows that seed germination is highly significant

Fig. 6. Acclimitization of in-vitro raised seedlings of P. insigne in different compost mixture recorded at 65 days.

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with well-developed roots in culture were used for transferring to the pots. Direct transfer to the outside environment leads to an increased mortality of the plantlets. Hence, in-vitro raised plantlets need to be acclimatized before being transferred to nature. For the transferred plantlets, humidity and temperature are important so as to acclimatize to the outside environment. The plastic pots were covered with perforated polythene bags for about 2–3 weeks to avoid direct exposure to sunlight, as transfer to direct sunlight of the cultured plantlets let to drying of the leaves (Lavanya et al., 2009). Seedlings were successfully acclimatized to greenhouse conditions and could be used for ornamental, eco-rehabilitation and conservation purposes (Gogoi et al., 2012; Bhattacharyya et al., 2016).

3.6. Conclusion The objective of the study was to establish high frequency in-vitro propagation and storage protocol of P. insigne as a priority for its conservation. In this aspect, we have standardized an efficient asymbiotic seed germination procedure which not only catered to high frequency of seed germination with reduced time for germination but is more effective than earlier reports. Further growth and development on optimal medium was recorded with use of lesser and balanced concentrations of auxins and cytokinins. The variation in the modified Burgeff medium was made with incorporation of glucose that resulted in significant increase in the initial seed germination compared to other media-types. The seedling development did not need a separate rooting experiment as the synergic effect of auxins and cytokinins resulted in well-developed shoots and roots. Acclimatization was also successful in the green house condition while eco-restoration is still under observation in the natural conditions. Another important aspect which was dealt with was the effective utilization of the mature seeds not only for in-vitro germination which is considered difficult to germinate but also for resourceful storage protocol for cryoconservation of these seeds. To the best of our knowledge, it is the first report on the in-vitro propagation and storage studies of P. insigne. Furthermore, the results showed that orchid seeds based on the degree of maturity can be successfully utilized not only to cater to the need of its commercial demands but also its germplasm can be conserved for sustainable utilization.

Acknowledgement Financial supports received from University Grants Commission {(UGC) major research project grant (F No. 42-984/2013(SR)}. The authors would like to thank staff of Sophisticated Analytical Instrumentation Facility (SAIF) for technical assistance in SEM and other resources from Centre for Advanced Studies (CAS) in Botany, (UGC), India. References Arditti, J., 1994. Micropropagation of orchids. Biological Conservation. http://dx.doi.org/ 10.1016/0006-3207(94)90551-7. Arditti, J., Ernst, R., 1984. Physiology of germinating orchid seeds. In: Arditti, J (Ed.), Orchid biology reviews and perspectives III. Cornell University Press, New York, pp. 178–222. Bae, kee h., Kim, chul hwan, Sun, B.Y., Choi, yong e., 2010. Structural changes of seed coats and stimulation of in-vitro germination. Propagation of Ornamental Plants 10, 2–3. Batty, A.L., Dixon, K.W., Brundrett, M., Sivasithamparam, K., 2001. Constraints to symbiotic germination of terrestrial orchid seed in a Mediterranean bushland. New Phytologist 152:511–520. http://dx.doi.org/10.1046/j.0028-646X.2001.00277.x. Bhattacharyya, P., Kumaria, S., Tandon, P., 2016. High frequency regeneration protocol for Dendrobium nobile: a model tissue culture approach for propagation of medicinally important orchid species. South African Journal of Botany 104:232–243. http://dx.doi.org/10.1016/j.sajb.2015.11.013. Bhattacharyya, P., Kumar, V., Van Staden, J., 2017. Assessment of genetic stability amongst micropropagated Ansellia africana, a vulnerable medicinal orchid species of Africa using SCoT markers. South African Journal of Botany 108:294–302. http://dx.doi.org/10.1016/j.sajb.2016.11.007.

223

Chen, T.Y., Chen, J.T., Chang, W.C., 2004. Plant re- generation through direct shoot bud formation from leaf culture of Paphiopedilum orchids. Plant Cell, Tissue and Organ Culture. 76, 11–15. Chen, Y., Goodale, U.M., Fan, X.L., Gao, J.Y., 2015. Asymbiotic seed germination and in-vitro seedling development of Paphiopedilum spicerianum: an orchid with an extremely small population in China. Global Ecology and Conservation 3:367–378. http://dx.doi.org/10.1016/j.gecco.2015.01.002. Chowdhery, H.J., 2004. Lady's slipper orchids in India. In: Kumar, C.S. (Ed.), Orchid Memories: A Tribute to Seidenfaden. Mentor Boosk, Calicut. Government, pp. 35–48. Chugh, S., Guha, S., Rao, I.U., 2009. Micropropagation of orchids: a review on the potential of different explants. Scientia Horticulturae 122:507–520. http://dx.doi.org/10.1016/ j.scienta.2009.07.016. Deb, C.R., Pongener, A., 2013. A study on the use of low cost substrata against agar for non-symbiotic seed culture of Cymbidium iridioides D. Don. Australian Journal of Crop Science 7, 642–649. Decruse, S.W., Gangaprasad, A., Seeni, S., Menon, V.S., 2003. Micropropagation and ecorestoration of Vanda spathulata, an exquisite orchid. Plant Cell, Tissue and Organ Culture 3937, 199–202. Ding, C.C., Wu, H., Liu, F.Y., 2004. Factors affecting the germination of Paphiopedilum armeniacum. Acta Botanica Yunnanica 26, 673–677. Dohling, S., Kumaria, S., Tandon, P., 2008. Optimization of nutrient requirements for asymbiotic seed germination of Dendrobium longicornu Lindl. and D. formosum Roxb. Proceedings of the Indian National Science Academy 4, 167–171. Dutra, D., Johnson, T.R., Kauth, P.J., Stewart, S.L., Kane, M.E., Richardson, L., 2008. Asymbiotic seed germination, in vitro seedling development, and greenhouse acclimatization of the threatened terrestrial orchid Bletia purpurea. Plant Cell, Tissue and Organ Culture 94:11–21. http://dx.doi.org/10.1007/s11240-008-9382-0. Engelmann, F., 2004. Plant cryopreservation: progress and prospects. In Vitro Cellular & Developmental Biology: Plant 40:427–433. http://dx.doi.org/10.1079/IVP2004541. Engelmann, F., 2010. Use of Biotechnologies for the Conservation of Plant Biodiversity. http://dx.doi.org/10.1007/s11627-010-9327-2. Friesen, A., Friesen, B., 2012. The Herbal Power of Orchids. chnell und portofrei erhältlich bei beck-shop.de Die Fachbuchhandlung Themat. Verlag C.H. Beck (im Internet: www.beck.de. ISBN 978 3 86371 051 4). Fu, Y.Y., Jiang, N., Wu, K.L., Zhang, J.X., Duan, J., Liu, H.T., Zeng, S.J., Improvement, G., Garden, B., 2016. Stimulatory effects of sodium hypochlorite and ultrasonic treatments on tetrazolium staining and seed germination in vitro of Paphiopedilum SCBG Red Jewel. Seed Science and Technology 44, 77–90. Gogoi, K., Kumaria, S., Tandon, P., 2012. Ex situ conservation of Cymbidium eburneum Lindl.: a threatened and vulnerable orchid , by asymbiotic seed germination. 3 Biotech. http://dx.doi.org/10.1007/s13205-012-0062-8. Hirano, T., Godo, T., Miyoshi, K., Ishhikawa, K., Mii, M., 2009. Cryopreservation and lowtemperature storage of seeds of Phaius tankervilleae. Plant Biotechnology Reports 3: 103–109. http://dx.doi.org/10.1007/s11816-008-0080-5. Hirano, T., Yukawa, T., Miyoshi, K., Mii, M., 2011. Wide applicability of cryopreservation with vitrification method for seeds of some Cymbidium species. Plant Biotechnology 102:99–102. http://dx.doi.org/10.5511/plantbiotechnology.10.1115a. Hiren, A.P., Saurabh, R., Subramanian, M., 2004. In vitro regeneration in Curculigo orchioides Gaertn. An endangered medicinal herb. Phytomorphology 54, 85–95. Hossain, M.M., 2008. Asymbiotic seed germination and in vitro seedling development of Epidendrumibaguense Kunth. (Orchidaceae). African Journal of Biotechnology 7, 3614–3619. Hossain, M.M., Sharma, M., Teixeira da Silva, J.A., Pathak, P., 2010. Seed germination and tissue culture of Cymbidium giganteum Wall. ex Lindl. Scientia Horticulturae 123, 479–487. Kaczmarczyk, A., Turner, S.R., Bunn, E., Mancera, R.L., Dixon, K.W., 2011. Cryopreservation of threatened native Australian species-what have we learned and where to from here? In Vitro Cellular & Developmental Biology: Plant 47:17–25. http://dx.doi.org/ 10.1007/s11627-010-9318-3. Kauth, P.J., Vendrame, W.A., Kane, M.E., 2006. In vitro seed culture and seedling development of Calopogon tuberosus. Plant Cell, Tissue and Organ Culture:91–102. http://dx.doi.org/10.1007/s11240-005-9055-1. Kumaria, S., Tandon, P., 2001. In: Pathak, P., Sehgal, R.N., Shekhar, N., Sharma, M., Sood, A. (Eds.), Orchids: The World’s Most Wondrous Plants. In: Orchids: Science and Commerce. Bishen Singh Mahendra Pal Singh, India, pp. 17–28. Lavanya, M., Venkateshwarlu, B., Devi, B.P., 2009. Acclimatization of neem microshoots adaptable to semi- sterile conditions. Indian Journal Biotechnology 8, 218–222. Lee, N., Lee, Y.I., 1999. Effect of capsual maturity, medium composition and liquid culture on seed germination in vitro of Paphiopedilum spp. In Paphiopedilum in Taiwan II. Taiwan Paphiopedilum Society, pp. 10–11. Lee, Y., Lee, N., Yeung, E.C., 2005. Embryo development of Cypripedium formosanum in relation to seed germination. Journal of the American Society for Horticultural Science 130, 747–753. Lee, Y., Yeung, E.C., Lee, N., 2006. Embryo Development in the Lady's Slipper Orchid, Paphiopedilum delenatii, With Emphasis on the Ultrastructure of the Suspensor. 1311–1319. http://dx.doi.org/10.1093/aob/mcl222. Lemay, M.A., De Vriendt, L., Pellerin, S., Poulin, M., 2015. Ex situ germination as a method for seed viability assessment in a peatland orchid, Platanthera blephariglottis. American Journal of Botany 102:390–395. http://dx.doi.org/10.3732/ajb.1400441. Liao, Y.Z., Chen, J.J., 2006. Asymbiotic seed germination of Paphiopedilum. In: Paphiopedilum in Taiwan IV. Taiwan Paphiopedilum Society, pp. 11–14. Li, D.Z., Pritchard, H.W., 2009. The science and economics of ex situ plant conservation. Trends in Plant Science 14:614–621. http://dx.doi.org/10.1016/j.tplants.2009. 09.005. Lin, Y.H., Chang, C., Chang, W.C., 2000. Plant regeneration from callus culture of a Paphiopedilum hybrid. Plant Cell Tissue Organ Culture 62, 21–25.

224

R.V. Diengdoh et al. / South African Journal of Botany 112 (2017) 215–224

Lo, S., Nalawade, S.M., Kuo, C., Chen, C., 2004. Asymbiotic germination of immature seeds, plantlet development and ex vitro establishment of plants of Dendrobium tosaense makino – a medicinally important orchid. In Vitro Cellular & Developmental Biology: Plant:528–535. http://dx.doi.org/10.1079/IVP2004571. Long, B., Niemiera, A.X., Cheng, Z., Long, C., 2010. In vitro propagation of four threatened Paphiopedilum species ( Orchidaceae). Plant Cell, Tissue and Organ Culture: 151–162. http://dx.doi.org/10.1007/s11240-010-9672-1. Merritt, D.J., Martyn, A.J., Ainsley, P., Young, R.E., Seed, L.U., Thorpe, M., 2014. Storage Examining Seed, Plant, and Environmental Traits Associated With Longevity. 1081–1104. http://dx.doi.org/10.1007/s10531-014-0641-6. Miyoshi, K., Mii, M., 1995. Phytohormone pre-treatment for the enhancement of seed germination and protocorm formation by the terrestrial orchid, Calanthe discolor (Orchidaceae), in asymbiotic culture. Scientia Horticulturae 63, 263–267. Miyoshi, K., Mii, M., 1998. Stimulatory effects of sodium and calcium hypochlorite, prechilling and cytokinins on the germination of Cypripedium macranthos seed in vitro. Physiologia Plantarum 102, 481–486. Mohanty, P., Das, M.C., Kumaria, S., Tandon, P., 2012. High-efficiency Cryopreservation of the Medicinal Orchid Dendrobium nobile Lindl. 297–305. http://dx.doi.org/10. 1007/s11240-011-0095-4. Mweetwa, A.M., Welbaum, G.E., Tay, D., 2008. Effects of development, temperature, and calcium hypochlorite treatment on in vitro germinability of Phalaenopsis seeds. Scientia Horticulturae 117:257–262. http://dx.doi.org/10.1016/j.scienta.2008. 03.035. Nadarajan, J., Wood, S., Marks, T.R., Seaton, P.T., Pritchard, H.W., 2011. Nutritional requirements for in vitro seed germination of 12 terrestrial, lithophytic and epiphytic orchids. Journal of Tropical Forest Science 23, 204–212. Nagaraju, V., Das, S.P., Bhutia, P.C., Upadhyaya, R.C., 2003. Response of Cymbidium lunavian Atlas protocorms to media and benzyl aminopurine. Indian Journal of Horticulture 60, 98–103. Ng, C., Saleh, N.M., Zaman, F.Q., 2010. In vitro multiplication of the rare and endangered slipper orchid, Paphiopedilum rothschildianum (Orchidaceae). South African Journal of Botany 9, 2062–2068. Nhat, N.T.H., Dung, T.T., 2006. In vitro propagation of Dendrobium orchid through thin stem section culture. Proceedings of International Working Biotechnology and Agroculture, pp. 154–155. Nikabadi, S., Bunn, E., Stevens, J., Newman, B., Turner, S.R., Dixon, K.W., 2014. Germination Responses of four Native Terrestrial Orchids from South-west Western Australia to Temperature and Light Treatments. http://dx.doi.org/10.1007/ s11240-014-0507-3. Nikishina, T.V., Popova, E.V., Vakhrameeva, M.G., Varlygina, T.I., Kolomeitseva, G.L., Burov, A.V., Popovich, E.A., Shirokov, A.I., Popov, A.S., 2007. Cryopreservation of Seeds and Protocorms. 54:pp. 137–143. http://dx.doi.org/10.1134/S1021443707010189. Paul, S., Kumaria, S., Tandon, P., 2011. An effective nutrient medium for asymbiotic seed germination and large-scale in vitro regeneration of Dendrobium hookerianum, a threatened orchid of northeast India. AoB Plants 2012:plr032. http://dx.doi.org/ 10.1093/aobpla/plr032. Pierik, R.L.M., Sprenkels, P.A., Van Der Harst, B., Van Der Meys, Q.G., 1988. Seed germination and further development of plantlets of Paphiopedilum ciliolare Pfitz. in vitro. Scientia Horticulturae (Amsterdam) 34:139–153. http://dx.doi.org/10.1016/03044238(88)90084-2. Pritchard, H.W., Nadarajan, J., 2009. Fundamental aspects of plant cryopreservation. CryoLetters 30, 382–397. Rankou, H., Kumar, P., 2015. Paphiopedilum insigne,. IUCN Red List Threat. Species 2015, e.T43320499A43327884. http://dx.doi.org/10.2305/IUCN.UK.2015-2.RLTS. T43320499A43327884.en 8235. Rasmussen, H.N., 1995. Terrestrial Orchids: Terrestrial Orchids: From Seed to Mycotrophic Plant. 444. Cambridge University Press, pp. 708–709 (£45.00/$64.95 hardback. ISBN 0 521 45165 5). Reed, B.M., Sarasan, V., Kane, M., Bunn, E., Pence, V.C., 2011. Biodiversity conservation and conservation biotechnology tools. In Vitro Cellular & Developmental Biology: Plant 47:1–4. http://dx.doi.org/10.1007/s11627-010-9337-0.

Robinson, J.P., Balakrishnan, V., Britto, J., 2009. In vitro seed germination and protocorm development of Dendrobium aqueum lindl. A rare orchid species from eastern ghats of Tamil Nadu. Botany Research International 2, 99–102. Roy, A.R., Patel, R.S., Patel, V.V., Sajeev, S., Deka, B.C., 2011. Asymbiotic seed germination, mass propagation and seedling development of Vanda coerulea Griff ex-lindl. (Blue Vanda): An in vitro protocol for an endangered orchid. Scientia Horticuturae 128, 325–331. Stewart, S.L., Kane, Æ.M.E., 2006. Asymbiotic Seed Germination and In Vitro Seedling Development of Habenaria macroceratitis (Orchidaceae), a Rare Florida Terrestrial Orchid. :pp. 147–158. http://dx.doi.org/10.1007/s11240-006-9098-y. Swarts, N.D., Dixon, K.W., 2009. Terrestrial orchid conservation in the age of extinction. Annals of Botany 104:543–556. http://dx.doi.org/10.1093/aob/mcp025. Stewart, S.L., Zettler, L.W., Minso, J., Brown, P.M., 2003. Symbiotic germination and reintroduction of Spiranthes brevilabris Lindley, an endangered orchid native to Florida. Selbyana 26, 64–70. Stimart, D.P., Ascher, P.D., 1981. In vitro germination of Paphiopedilum seed on a completely defined medium. Scientia Horticulturae. 14, 165–170. Tandon, P., 2004. Conservation And Sustainable Development Of Plant Resources Of Northeast India. Man And Society 1 (1), 49–59. Tandon, P., Kumaria, S., 2011. Orchid resources of the northeast India and their sustainable utilization. In: Hasnain, S.E., Rashmi, B. Jha, Sharan, R.N. (Eds.), Biotechnology for Sustainable Development – Achievements and Challenges. Tata McGraw Hill Education Private Limited, New Delhi, India, pp. 183–191. Tay, L.J., Takeno, K., Hori, Y., 1988. Culture conditions suitable for in vitro seed germination and development of seedling in Paphiopedilum. Journal Japan Society Horticultural Science 57, 243–249. Thammasiri, K., Soamkul, L., 2007. Cryopreservation of Vanda coerulea Griff. ex Lindl. Seeds by Vitrification. 33:pp. 223–227. http://dx.doi.org/10.2306/scienceasia15131874.2007.33.223. Traore, A., Guiltinan, M.J., 2006. Effects of carbon source and explant type on somatic embryogenesis of four Cacao genotypes. Hortscience 41, 753–758. Vujanovic, V., St-arnund, Barabe, D., 2000. Viability testing of orchid seed and the promotion of colouration and germination. Annals of Botany 86:79–86. http://dx.doi.org/10. 1006/anbo.2000.1162. Yamazaki, J., Miyoshi, K., 2006. In vitro asymbiotic germination of immature seed and formation of protocorm by Cephalanthera falcata (Orchidaceae). Annals of Botany 98, 197–1206. Yam, T.W., Arditti, J., 2009. History of orchid propagation: a mirror of the history of biotechnology. Plant Biotechnology Reports 3:1–56. http://dx.doi.org/10.1007/ s11816-008-0066-3. Zeng, S.J., Chen, Z.L., Duan, J., 2006. Asepsis sowing and in vitro propagation of Paphiopedilum hirsutissimum Pfitz. Plant Physiology Communications 42 (2), 247. Zeng, S.J., Chen, Z.L., Wu, K.L., Zhang, J.X., Duan, J., 2010. In vitro propaga- tion of Paphiopedilum henryanum Bream. Plant Physiology Communications. 46 (5), 471–472. Zeng, S., Wu, K., Teixeira da Silva, J.A., Zhang, J., Chen, Z., Xia, N., 2012. Asymbiotic seed germination, seedling development and reintroduction of Paphiopedilum wardii Sumerh., an endangered terrestrial orchid. Scientia Horticulturae 138:198–209. http://dx.doi. org/10.1016/j.scienta.2012.02.026. Zeng, S., Wang, J., Wu, K., Teixeira da Silva, J.A., Zhang, J., Duan, J., 2013. In vitro propagation of Paphiopedilum hangianum Perner & Gruss. Scientia Horticulturae (Amsterdam) 151:147–156. http://dx.doi.org/10.1016/j.scienta.2012.10.032. Zeng, S., Zhang, Y., Teixeira da Silva, J.A., Wu, K., Zhang, J., Duan, J., 2014. Seed biology and in vitro seed germination of Cypripedium. Critical Reviews in Biotechnology 34: 358–371. http://dx.doi.org/10.3109/07388551.2013.841117. Zeng, S., Huang, W., Wu, K., Zhang, J., Teixeira da Silva, J.A., Duan, J., 2015. In vitro propagation of Paphiopedilum orchids. Critical Reviews in Biotechnology 0:1–14. http://dx. doi.org/10.3109/07388551.2014.993585. Zhang, Y., Wu, K., Zhang, J.-X., 2015. Embryo development in association with asymbiotic seed germination in vitro of Paphiopedilum armeniacum S. C. Chen et F. Y. Liu. Scientific Reports - Nature:1–15. http://dx.doi.org/10.1038/srep16356.