In vitro micropropagation of the orchid (dendrobium crystallinum var. alba)

AGU International Journal of Sciences – 2019, Vol 7 (3), 1 – 8<br /> <br /> <br /> <br /> <br /> IN VITRO MICROPROPAGATION OF THE ORCHID (DENDROBIUM CRYSTALLINUM<br /> VAR. ALBA)<br /> <br /> Tran Thi Ngoc Lan1<br /> 1<br /> South Central and Highland Institute of Science<br /> <br /> Information: ABSTRACT<br /> Received: 07/08/2018<br /> Studies of micropropagation of Dendrobium crystallinum var. alba. were<br /> Accepted: 17/10/2018<br /> conducted in order to conserve and develop this precious orchid species. The<br /> Published: 11/2019<br /> results showed that protocorms were formed from seeds culture on the ½ MS<br /> Keywords: medium supplemented with 10% potatoes extract within 8 weeks. Protocorms<br /> BA, Dendrobium crystallinum which were cultured on the ½ MS medium containing 30 g/l sucrose, 0.5 g/l AC,<br /> 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<br /> protocorm. PLBs/sample) after 8 weeks culture. Protocorms converted into normal plants<br /> with well-developed shoots and roots on the ½ MS medium supplemented with 20<br /> g/l sucrose, 10% potatoes extract, 0.5 g/l AC, and 7 g/l agar after about 90 days.<br /> PLBs converted into normal plants on the same medium supplemented with 0.5<br /> mg/l NAA and 0.5 mg/l BA, too. No abnormal morphological changes were found<br /> in these Dendrobium seedlings.<br /> <br /> <br /> <br /> 1. INTRODUCTION Micropropagation provided an important method<br /> for mass propagation of many orchid species.<br /> Vietnam has a tropical monsoon climate that is<br /> There were perusals of Dendrobium<br /> appropriate for orchid growth such as the<br /> micropropagation on the world and Vietnam.<br /> Dendrobiums. Dendrobiums are not a beautiful<br /> Sunitibala & Rajkumar (2009) cultured seeds of<br /> species although they are highly valued in the<br /> Dendrobium transparens L. on the ½ Murashige<br /> flower industry as potted plants and cut flowers.<br /> and Skoog (½ MS) suplemented with 1 mg/l NAA<br /> Even though these wild orchids are diverse, they<br /> and 2 mg/l BA. Nguyễn Văn Song (2011) cultured<br /> have been exploited almost to extinction. The<br /> seeds of Dendrobium chrysotosum Lindl on MS<br /> Dendrobium crystallinum var. alba is a very<br /> medium. Nguyễn Thị Sơn et al. (2014) propagated<br /> graceful wild orchid of Vietnam and is a rare<br /> Dendrobium officinale Kimura et Migo with<br /> species. It is a epiphytic, sympodial orchid which<br /> sowing seeds on the Vacin Went and cultured<br /> also grows in Hymalayan Myanmar, Thailand and<br /> nodal segment on the MS medium. Vũ Kim Dung<br /> Vietnam at altitudes of 700 – 1700 m. It has<br /> et al. (2016) propagated Dendrobium<br /> beautiful, long-lasting flowers, noble aroma and<br /> gratiosissimum Rchb.f. Nguyễn Văn Việt (2017)<br /> blooms in April- May every year. It is classified as<br /> cultured the Dendrobium lituiflorum Lindley<br /> a group of rare orchids and an endangered species<br /> seeds. Rattana & Sangchanjirade (2017)<br /> (EN, IUCN, Averyanov, 2005).<br /> propagated Dendrobium signatum Rchb.f.<br /> However, there is no article refering to clonal<br /> <br /> <br /> <br /> 1<br /> AGU International Journal of Sciences – 2019, Vol 7 (3), 1 – 8<br /> <br /> propagation of Dendrobium crystallinum var. alba. 2.1 Materials<br /> This study reports micropropagation of Research subject: Den. crystallinum var.alba plant<br /> Dendrobium crystallinum var. alba in order to with fruits were collected in the family garden.<br /> conserve and develop this precious orchid species. Fruit was harvested 180 days after pollination in<br /> 2. MATERIALS AND METHODS October, 2017. (fig. 1).<br /> <br /> <br /> <br /> <br /> Figure 1. Dendrobium crystallinum var.alba. a. Plants with their flowers. b. Plants with their fruits (arrow).<br /> <br /> 2.2 Experiment conditions consisting of MS, ½ MS, supplemented with<br /> different organic compound (15% coconut water<br /> All tests were kept at 25±1οC under a photoperiod<br /> (CW) – Thickness of coconut pulp = 3 – 4 mm;<br /> of 16 h light/8 h dark, light intensity of 2000 lux<br /> 10% potatoes extract – 100g potatoes were boiled,<br /> and 70% relative humidity.<br /> extract to 1 l solution; 5% mashed banana), 30 g/l<br /> Basal cultured medium: MS, ½ MS, at pH 5.7, 121<br /> sucrose, 0.5 g/l AC, 7 g/l agar. Seeds were cultured<br /> οC, 1 atm, autoclaved for 20 minutes.<br /> in the bottles (volume = 500 ml with 70 ml<br /> 2.3 Methods medium/bottle). Each treatment had 5 bottles. The<br /> 2.3.1 Effect of different media and organic percentage of orchid seed germination was<br /> supplements on germination of Den. obtained by estimating the surface area of seed<br /> crystallinum var.alba seeds germination in the tissue culture bottle with a<br /> The mature capsules of Den. crystallinum var.alba diameter of 6.5 cm. The total surface area of the<br /> were collected six months after pollination, soaked tissue culture bottle was defined as 100%. After<br /> in aqueous solution of commercial detergent cultivation for eight weeks, the percentage of seed<br /> (Sunlight, Vietnam) for 10 min, then rinsed germination was recorded. Observations on the<br /> thoroughly three times with sterile distilled water, percentage germination of seeds, protocrom<br /> followed by dipping them in 70% ethanol for 20 formation rate, shoot formation rate were recorded<br /> sec. Capsules were then surface sterilised by 8 weeks after culture.<br /> dipping in 70% ethyl alcohol and flamed 2.3.2 Effect of NAA and BA on protocorm<br /> immediately four to five times in laminar air flow. proliferation of Den. crystallinum var.alba<br /> The capsules were then cut longitudinally in a Protocorms from the above study were used as<br /> sterilised petri dish. Seeds were scraped from the culture material (the average weight of 20 ± 5 mg)<br /> capsules, mixed with 100 ml sterile distilled water were proliferated on ½ MS supplemented with 30<br /> and pipetted into 1ml tubes and then cultured on g/l sucrose, 10% potatoes extract, NAA (0; 0.1; 0.2<br /> 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.<br /> media were used in the whole experiment Protocorms were cultured in the bottles (volume =<br /> 2<br /> AGU International Journal of Sciences – 2019, Vol 7 (3), 1 – 8<br /> <br /> 500 ml with 70 ml medium/bottle) with 20 days after culture) on the optical microscope<br /> explants/bottle. Each treatment had 5 bottles. (Olympus CX21, Japan) with degrees<br /> Observations on the protocorm-like body (PLB) magnification of 40 - 100 times.<br /> formation rate, number of PLB/explants, figure of 2.3.5 Statistical analysis<br /> PLBs were recorded 8 weeks after culture.<br /> All the experiments were set up in completely<br /> 2.3.3 Effect of plant growth regulators (PGRs) on randomised design (CRD). Each treatment<br /> development of protocorms and PLBs Den. consisted of 3 replicates. The difference among the<br /> crystallinum var.alba treatment means was compared based on Duncan’s<br /> Protocorms from the study (2.3.1) and PLBs from multiple range test (DMRT) analysis (with a level<br /> the study (2.3.2) with being used as explants (the of significance of P