AGU International Journal of Sciences – 2019, Vol 7 (3), 1 – 8

IN VITRO MICROPROPAGATION OF THE ORCHID ( CRYSTALLINUM VAR. ALBA)

Tran Thi Ngoc Lan1 1Forest Science Institute of Central Highlands and South of Central

Information: ABSTRACT Received: 07/08/2018 Studies of micropropagation of Dendrobium crystallinum var. alba. were Accepted: 17/10/2018 conducted in order to conserve and develop this precious orchid species. The Published: 11/2019 results showed that protocorms were formed from seeds culture on the ½ MS Keywords: medium supplemented with 10% potatoes extract within 8 weeks. Protocorms BA, Dendrobium crystallinum which were cultured on the ½ MS medium containing 30 g/l sucrose, 0.5 g/l AC, var. alba., NAA, PLB, 7 g/l agar, 0.1 mg/l NAA and 1 mg/l BA were optimal for PLB formation (8.67 protocorm. PLBs/sample) after 8 weeks culture. Protocorms converted into normal with well-developed shoots and roots on the ½ MS medium supplemented with 20 g/l sucrose, 10% potatoes extract, 0.5 g/l AC, and 7 g/l agar after about 90 days. PLBs converted into normal plants on the same medium supplemented with 0.5 mg/l NAA and 0.5 mg/l BA, too. No abnormal morphological changes were found in these Dendrobium seedlings.

1. INTRODUCTION Micropropagation provided an important method for mass propagation of many orchid species. Vietnam has a tropical monsoon climate that is There were perusals of Dendrobium appropriate for orchid growth such as the micropropagation on the world and Vietnam. . Dendrobiums are not a beautiful Sunitibala & Rajkumar (2009) cultured seeds of species although they are highly valued in the Dendrobium transparens L. on the ½ Murashige flower industry as potted plants and cut flowers. and Skoog (½ MS) suplemented with 1 mg/l NAA Even though these wild orchids are diverse, they and 2 mg/l BA. Nguyễn Văn Song (2011) cultured have been exploited almost to extinction. The seeds of Dendrobium chrysotosum Lindl on MS Dendrobium crystallinum var. alba is a very medium. Nguyễn Thị Sơn et al. (2014) propagated graceful wild orchid of Vietnam and is a rare Dendrobium officinale Kimura et Migo with species. It is a epiphytic, sympodial orchid which sowing seeds on the Vacin Went and cultured also grows in Hymalayan , and nodal segment on the MS medium. Vũ Kim Dung Vietnam at altitudes of 700 – 1700 m. It has et al. (2016) propagated Dendrobium beautiful, long-lasting flowers, noble aroma and gratiosissimum Rchb.f. Nguyễn Văn Việt (2017) blooms in April- May every year. It is classified as cultured the Dendrobium lituiflorum Lindley a group of rare orchids and an endangered species seeds. Rattana & Sangchanjirade (2017) (EN, IUCN, Averyanov, 2005). propagated Dendrobium signatum Rchb.f. However, there is no article refering to clonal

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propagation of Dendrobium crystallinum var. alba. 2.1 Materials This study reports micropropagation of Research subject: Den. crystallinum var.alba Dendrobium crystallinum var. alba in order to with fruits were collected in the family garden. conserve and develop this precious orchid species. Fruit was harvested 180 days after pollination in 2. MATERIALS AND METHODS October, 2017. (fig. 1).

Figure 1. Dendrobium crystallinum var.alba. a. Plants with their flowers. b. Plants with their fruits (arrow).

2.2 Experiment conditions consisting of MS, ½ MS, supplemented with different organic compound (15% coconut water All tests were kept at 25±1οC under a photoperiod (CW) – Thickness of coconut pulp = 3 – 4 mm; of 16 h light/8 h dark, light intensity of 2000 lux 10% potatoes extract – 100g potatoes were boiled, and 70% relative humidity. extract to 1 l solution; 5% mashed banana), 30 g/l Basal cultured medium: MS, ½ MS, at pH 5.7, 121 sucrose, 0.5 g/l AC, 7 g/l agar. Seeds were cultured οC, 1 atm, autoclaved for 20 minutes. in the bottles (volume = 500 ml with 70 ml 2.3 Methods medium/bottle). Each treatment had 5 bottles. The 2.3.1 Effect of different media and organic percentage of orchid seed germination was supplements on germination of Den. obtained by estimating the surface area of seed crystallinum var.alba seeds germination in the tissue culture bottle with a The mature capsules of Den. crystallinum var.alba diameter of 6.5 cm. The total surface area of the were collected six months after pollination, soaked tissue culture bottle was defined as 100%. After in aqueous solution of commercial detergent cultivation for eight weeks, the percentage of seed (Sunlight, Vietnam) for 10 min, then rinsed germination was recorded. Observations on the thoroughly three times with sterile distilled water, percentage germination of seeds, protocrom followed by dipping them in 70% ethanol for 20 formation rate, shoot formation rate were recorded sec. Capsules were then surface sterilised by 8 weeks after culture. dipping in 70% ethyl alcohol and flamed 2.3.2 Effect of NAA and BA on protocorm immediately four to five times in laminar air flow. proliferation of Den. crystallinum var.alba The capsules were then cut longitudinally in a Protocorms from the above study were used as sterilised petri dish. Seeds were scraped from the culture material (the average weight of 20 ± 5 mg) capsules, mixed with 100 ml sterile distilled water were proliferated on ½ MS supplemented with 30 and pipetted into 1ml tubes and then cultured on g/l sucrose, 10% potatoes extract, NAA (0; 0.1; 0.2 the surface of the medium. Two different basal mg/l), BA (0, 0.5, 1 mg/l), 0.5 g/l AC and 7 g/l agar. media were used in the whole experiment Protocorms were cultured in the bottles (volume = 2 AGU International Journal of Sciences – 2019, Vol 7 (3), 1 – 8

500 ml with 70 ml medium/bottle) with 20 days after culture) on the optical microscope explants/bottle. Each treatment had 5 bottles. (Olympus CX21, Japan) with degrees Observations on the protocorm-like body (PLB) magnification of 40 - 100 times. formation rate, number of PLB/explants, figure of 2.3.5 Statistical analysis PLBs were recorded 8 weeks after culture. All the experiments were set up in completely 2.3.3 Effect of plant growth regulators (PGRs) on randomised design (CRD). Each treatment development of protocorms and PLBs Den. consisted of 3 replicates. The difference among the crystallinum var.alba treatment means was compared based on Duncan’s Protocorms from the study (2.3.1) and PLBs from multiple range test (DMRT) analysis (with a level the study (2.3.2) with being used as explants (the of significance of P<0.05). Excel 2010 and the average weight of 20 ± 5 mg) were cultured on the statistical package SPSS (Statistical Program ½ MS supplemented with 20 g/l sucrose, 10% Scientific System)16.0 was used for the analyses. potatoes extract, 0.5 g/l AC, 7 g/l agar, NAA (0; 3. RESULTS AND DISCUSSION 0.5; 1 mg/l), BA (0; 0.5 mg/l) in examnination the 3.1 Effect of different media and organic growth of these protocorms and PLBs. They were supplements on germination of Den. cultured in the bottles (volume = 500 ml with 70 crystallinum var.alba seeds ml medium/bottle) with 20 explants/bottle. Each Naturally, orchid seeds have a poor germination treatment had 5 bottles. Observations on figure of rate because of the small size of the seeds and the seedlings, the PLB formation rate, the seedling lack of cotyledons and endosperm. Seeds can only formation rate, height of shoot, number of leaves, germinate with isolated mycorrhizal fungi but number of roots and length of root were recorded seeds run the risk of being infected and die. 90 days after culture. Observation was conducted Consequently, the sample orchid seeds were on the developing stages of protocorms and PLBs propagated in asymbiotic seed germination. The (4 weeks, 8 weeks and 90 days) after culture. results of the study on germinated seeds of Den. 2.3.4 Histological observation crystallinum var.alba on the different medium were Observation of the germination of orchid seeds and observed at 8 weeks after culture (Table 1). their developing stages (4 weeks, 8 weeks and 90 Table 1. Effect of different media and organic supplements on germination of Den. crystallinum var.alba seeds.

Survival and formation Survival and formation Culture medium rate of protocorms (%) rate of shoots (%)

MS 26.33f* 4.33b MS + 15% CW 55.67d 7.33a MS + 10% potatoes extract 80.67b 7.67a MS + 5% mashed banana 68.67c 6.67a ½ MS 37.33e 4.33b ½ MS + 15% CW 98.67a 1.33c ½ MS + 10% potatoes extract 99.33a 0.33c ½ MS + 5% mashed banana 70.67c 6.67a

*: Different alphabetical letters are significantly different according to Duncan’s multiple range tests at P < 0.05.

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It was observed that seeds had been green at 4 (2009) for sowing seeds Den. transparent L. with weeks after culture (Fig. 2c), germinated and using PGRs (1 mg/l NAA and 1mg/l BA), in this formed green protocorms. Protocorm had emerge experiment only used MS medium and organic from capsules and come on growing at 8 weeks compounds for Den. crystallinum var.alba seeds after culture (Fig. 2a, 2d). The most suitable media, gemination. providing seed germination were ½ MS medium Beside protocorm formation, there was the low supplemented with 15% CW and ½ MS medium shoot formation rate from orchid seeds (0,33 – supplemented with 10% potatoes extract 7,67%). It show that orchid embryos can develop (protocorm formation rate corresponding 98.67% to the different stages of seeds, accordance with và 99.33%). The less protocorm formation rate reports on Dendrobium culture (Arditti, 2008; Vũ gained 26.33% và 37.33% of seeds which were Ngọc Lan & Nguyen Thi Ly Anh, 2013, Vu Kim sowed on the MS medium and ½ MS medium. Dung và cs., 2016; Rattana & Sangchanjirade, Combinations of organic supplements in the media 2017). enhanced seed germination. It show that the 3.2 Effect of NAA và BA on protocorm importance of organic compounds such as coconut proliferation of Den. crystallinum var.alba water, potatoes extract… for orchid seeds The results of the study on effect of NAA và BA germination. Den. crystallinum var.alba is on protocorm proliferation of Den. crystallinum epiphytic orchid, growth rate is low, so, the high var.alba were observed at 8 weeks after culture concentration of N, P, K compounds were not (Table 2). suitable to culture orchid seeds. However, the The protocorm explants had showed the induction minerals derived from organic compounds such as of meristem, parenchyma with PGRs. Protocorm is coconut, potatoes extract were useful in culture the germinated stage of orchid embryos, along with orchid seeds. The reports on the world were translation of PGRs sush as NAA, BA to acknowledged this too (Arditti, 2008; Vũ Ngọc differentiate to PLBs. PLBs and protocorms are Lan & Nguyễn Thị Lý Anh, 2013; Parthibhan và potential in embryo proliferation. Subculture PLBs cs., 2015, Nguyễn Văn Việt, 2017). was conducted in micropropagation of orchid such The best medium for seed germination in treatment as Dendrobium transparent L.(Sunitibala & was 10% potatoes extract or 15% CW. Potatoes Rajkumar, 2009), Dendrobium aqueum Lindley extract consists of carbohydrates, amino acids, (Parthibhan et al., 2015) or Dendrobium lituiflorum important vitamins (C, B1, B6…) and mineral Lindley (Nguyen Van Viet, 2017). elements (potassium, iron, magnesium) (Molnár et al., 2011; Sandoval et al., 2014, Parisa et al., 2014). Adding to NAA, BA, potatoes extract was In the in vitro culture, potato had useful effects on effective in forming secondary PLBs. In fact, the some orchid species such as Phalaenopsis and composition of potatoes extract includes inorganic ions (e.g. phosphorus, potassium), nitrogenous Dendrobium (Rattana & Sangchanjirade, 2017). This study found that the nutrients of CW and compounds, amino acids, vitamins…). All these potato were suitable for orchid seeds. The suplements may be the reason for seed germination effciency of CW may depend on species, age and in orchids (Arditti, 2008; Rattana & Sangchanjirade, 2017). Using BA individually had thickness of coconut pulp. At this study, CW was undertaken of coconut fruits with the thickness of not much effective but the addition of BA in coconut pulp = 3 – 4 mm was suitable for orchid combination with NAA improved ability of cell seeds germination. However, CW was rather division of explants. The optimal concentrations expensive than potato. Potato was easy to look for, of NAA (0.1 mg/l ) and BA (1 mg/l) gave the so, potatoes extract was selected in the following highest PLB formation rate, the number of tests. Unlike the results of Sunitibala & Rajkumar PLBs/explant (corresponding 100%, 8.67

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PLBs/explants). The mean weight of PLBs was cytokinins was observed in present study is in higher the mean weight of protocorm ( 32.4 mg and accordance with report on Dendrobium 25.4 mg) and had deep green (Fig.2b). Compared transparens L. (Sunitibala, 2009), Dendrobium to PLB proliferation (multiplication factor: 4.2 in signatum (Rattana K. & Sangchanjirade, 2017). protocorm proliferation of Dendrobium nobile Thus, the ½ MS medium suplemented with 0.1 Lindl; Vũ Ngọc Lan & Nguyễn Thị Lý Anh, 2013), mg/l NAA and 1 mg/l BA is optimal to form the the results of this study had higher multiplication secondary PLB. factor (8.67). The combined of auxin and

Table 2. Effect of NAA và BA on protocorm proliferation of Den. crystallinum var.alba after 8 weeks culture. NAA BA Formation rate Number of Figure of PLBs (mg/l) (mg/l) PLB (%) PLBs/explant 0 0 72.67c* 2.01d Small, light green 0.1 0 92.33b 2.69d big, light green 0 0.5 74.33c 4.45c Small, light green 0.1 0.5 91.67b 2.23d Big, dark green 0.1 1 100.00a 8.67a Big, dark green 0.2 0 91.33b 4.74c Big, light green 0.2 0.5 92.33b 6.33b Big, light green 0.2 1 73.33c 4.84c Big, light green

*: Different alphabetical letters are significantly different according to Duncan’s multiple range tests at P < 0.05. 3.3 Effect of plant growth regulators on development of Den. crystallinum var.alba protocorm and PLB after 90 days culture Effect of plant growth regulators (NAA and BA) on development of Den. crystallinum var.alba protocorm and PLB after 90 days culture showed in table 3 and table 4.

Table 3. Effect of NAA and BA on development of protocorm Den. crystallinum var.alba after 90 days culture. Average Average Average Medium supplement root of shoot Formation Formation number Average with length height rate PLB seedling of roots number (%) (%) (cm) (cm) of leaves NAA(mg/l) BA(mg/l)

0 0 17.67c* 82.33b 2.67b 0.92c 3.18b 2.47b 0.5 0 37.33b 62.67c 2.74b 1.93b 3.15b 2.43b 0.5 0.5 7.67d 92.33a 4.13a 3.61a 4.36a 4.87a 1.0 0.5 66.33a 33.67d 1.13c 0.20d 0.52c 2.05b

*: Different alphabetical letters are significantly different according to Duncan’s multiple range tests at P < 0.05.

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Table 4. Effect of NAA and BA on development of PLB, derived from protocorm Den. crystallinum var.alba after 90 days culture. Average Average Average Medium supplement root of shoot Formation Formation number Average with length height rate PLB seedling of roots number (%) (%) (cm) (cm) of leaves NAA(mg/l) BA(mg/l)

0 0 38.67b* 23.67c 1.57b 0.63c 0.76c 2.17c 0.5 0 39.33b 60.67b 1.79b 1.85b 2.07b 3.18b 0.5 0.5 9.67c 90.33a 4.22a 3.02a 4.19a 4.59a 1.0 0.5 69.33a 16.67d 0.53c 0.13d 0.55c 2.01c

*: Different alphabetical letters are significantly different according to Duncan’s multiple range tests at P < 0.05. At treatment devoid of PGRs, there was 82.33% of growth, lateral growth increases so it reduces protocorms converted into seedlings (Table 3, height of shoot as well as root formation. The Fig.2h) but only 23.67% of PLBs converted into combination of BA and auxin (NAA), the cells seedlings (Table 4). Formation rate PLB in this expand and differentiate, proliferate effectively treament was also rather low (corresponding protocorms. In the medium concentraton of NAA 17.67% and 38.67%). This show that the and BA (0.5 mg/l), an increase cell division and embryogenesis nature of protocorms. They can shoot and root morphogenesis took place with invert into seedlings that no using PGRs. However, organogenesis (Fig. 2i, 2j.). Thus, the treatment PLBs were not so. PLBs need assisting of NAA with culture medium suplemented with 0.5 mg/l and BA to develop and convert into seedings (the NAA and 0.5 mg/l BA was noted to differentiate highest formation seedling at treatment protocorms and PLBs into seedlings. supplemented with 0.5 mg/l NAA and 0.5 mg/l No morphological variations were observed in this BA, 90.33%). Similar to protocorm, the seedling study through micropropagation Den. crystallium formation rate was 92.33% in this result, too. Shoot var. alba with protocorms and PLBs. (Fig. 2i, 2j). height, number of roots, number of leaves, root 3.4 Histological observation length of the seedlings in this treatment were highest. The combination of NAA and BA made Protocorm is a earliest structure that is formed in the cells loosen, elongate dimension, improved embryogenesis of orchid seed and is united in all of ability of cell division, made the cell survive and (Batygina et al., 2003; Arditti, 2008). convert into seedling. The combination of NAA Observating on optical microscope showed that, and BA in micropropagation is uasually applied in the orchid seeds with green embryos after 4 weeks orchid embryo proliferation or in shoot culture (Fig. 2c), formed protocorms after 8 weeks proliferation with axenic pseudobulb segment culture (Fig. 2d), after that, formed shoots, roots, (Sunitibala & Rajkumar, 2009; Nguyễn Thị Sơn et converted into seedlings after 90 days culture (Fig. al., 2014; Parthibhan et al., 2015) or seedlings 2g). PLB derived from protocorm (Fig. 2b, 2e) formation of Dendrobium signatum Rchb.f. developed with the stages similar to protocorm (Rattana & Sangchanjirade, 2017). (Stage 1: PLB formed meristem; Stage 2: PLB formed leaflets, vascular tissue; Stage 3: formed At treatment devoid of PGRs, PLBs still converted seedling with shoot and root). That was confirmed into seedlings (23,67%). It show that PLBs can in this study (Fig. 2f, 2j). Thus, protocorms could develop to the different stages. At treatment directly convert into seedlings or proliferate, medium suplemented with 1 mg/l NAA and 0,5 forming PLBs and PLBs could proliferate and mg/l BA, PLB proliferation rate was high convert into seedlings, too. This is a effective (corresponding 66,33% and 69,33%) but formation orchid micropropagation. seedling rate was low (33,67% and 16,67%). Citokinin ( BA) uasually inhibits apical bud 6 AGU International Journal of Sciences – 2019, Vol 7 (3), 1 – 8

4. CONCLUSION AND PETITION Protocorms and PLBs converted into seedlings (corresponding 92.33% and 90.33%) on the ½ MS 4.1 Conclusion medium (supplemented with 10% potatoes extract, ½ MS medium (suplemented with 10% potatoes 20 g/l sucrose, 0.5 g/l AC, 0.5 mg/l NAA, 0.5 mg/l extract, 30 g/l sucrose, 0.5 g/l AC, 7 g/l agar) is the BA and 7 g/l agar) after 90 days culture. most suitable for Dendrobium crystallinum var. 4.2 Petition alba seed germination (the protocorm formation rate: 99.33%). PLBs were formed from protocorms The following studies are necessary to the growth on the medium suplemented with 0.5 mg/l NAA and development of these seedlings in the ex vitro and 1 mg/l BA (number of PLBs/explant: 8.67). condittion for mass production this valuable species.

Figure 2. Micropropagation Dendrobium crystallinum var.alba. a. Seeds germinated, formed protocorm after 8 weeks culture; b. PLBs formation from protocorms after 8 weeks culture; c. The seeds with embryos after 4 weeks culture; d. Protocorms formation from seeds after 8 weeks culture; e. PLB with shoot were formed from protocorms after 8 weeks culture; f. Seedling formation from PLB, derived from protocorm after 90 days culture; g. Seedlings formation from protocorm after 90 days culture; h. Seedlings formation on no PGRs medium; i. Seedlings formation derived from protocorm on the medium supplemented with 0.5 mg/l NAA and 0.5 mg/l BA; j. Seedlings formation derived from PLB on the medium supplemented with 0.5 mg/l NAA and 0.5 mg/l BA. Acknowledgements. The authors would like to thank College of Economics and Technical Lam Dong for supporting this study. REFERENCES Arditti, J. (2008). Micropropagation of orchids. Oxford: Blackwell Publishing.

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