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

The results showed that protocorms were formed from seeds culture on the ½ MS medium supplemented with 10% potatoes extract within 8 weeks. Protocorms which were cultured on the ½ MS medium containing 30 g/l sucrose, 0.5 g/l AC, 7 g/l agar, 0.1 mg/l NAA and 1 mg/l BA were optimal for PLB formation (8.67 PLBs/sample) after 8 weeks culture. Protocorms converted into normal plants 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.

AGU International Journal of Sciences – 2019, Vol (3), – IN VITRO MICROPROPAGATION OF THE ORCHID (DENDROBIUM CRYSTALLINUM VAR ALBA) Tran Thi Ngoc Lan1 South Central and Highland Institute of Science Information: Received: 07/08/2018 Accepted: 17/10/2018 Published: 11/2019 Keywords: BA, Dendrobium crystallinum var alba., NAA, PLB, protocorm ABSTRACT Studies of micropropagation of Dendrobium crystallinum var alba were conducted in order to conserve and develop this precious orchid species The results showed that protocorms were formed from seeds culture on the ½ MS medium supplemented with 10% potatoes extract within weeks Protocorms which were cultured on the ½ MS medium containing 30 g/l sucrose, 0.5 g/l AC, g/l agar, 0.1 mg/l NAA and mg/l BA were optimal for PLB formation (8.67 PLBs/sample) after weeks culture Protocorms converted into normal plants 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 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 Micropropagation provided an important method for mass propagation of many orchid species There were perusals of Dendrobium micropropagation on the world and Vietnam Sunitibala & Rajkumar (2009) cultured seeds of Dendrobium transparens L on the ½ Murashige and Skoog (½ MS) suplemented with mg/l NAA and mg/l BA Nguyễn Văn Song (2011) cultured seeds of Dendrobium chrysotosum Lindl on MS medium Nguyễn Thị Sơn et al (2014) propagated Dendrobium officinale Kimura et Migo with sowing seeds on the Vacin Went and cultured nodal segment on the MS medium Vũ Kim Dung et al (2016) propagated Dendrobium gratiosissimum Rchb.f Nguyễn Văn Việt (2017) cultured the Dendrobium lituiflorum Lindley seeds Rattana & Sangchanjirade (2017) propagated Dendrobium signatum Rchb.f However, there is no article refering to clonal INTRODUCTION Vietnam has a tropical monsoon climate that is appropriate for orchid growth such as the Dendrobiums Dendrobiums are not a beautiful species although they are highly valued in the flower industry as potted plants and cut flowers Even though these wild orchids are diverse, they have been exploited almost to extinction The Dendrobium crystallinum var alba is a very graceful wild orchid of Vietnam and is a rare species It is a epiphytic, sympodial orchid which also grows in Hymalayan Myanmar, Thailand and Vietnam at altitudes of 700 – 1700 m It has beautiful, long-lasting flowers, noble aroma and blooms in April- May every year It is classified as a group of rare orchids and an endangered species (EN, IUCN, Averyanov, 2005) AGU International Journal of Sciences – 2019, Vol (3), – propagation of Dendrobium crystallinum var alba This study reports micropropagation of Dendrobium crystallinum var alba in order to conserve and develop this precious orchid species 2.1 Materials Research subject: Den crystallinum var.alba plant with fruits were collected in the family garden Fruit was harvested 180 days after pollination in October, 2017 (fig 1) MATERIALS AND METHODS Figure Dendrobium crystallinum var.alba a Plants with their flowers b Plants with their fruits (arrow) consisting of MS, ½ MS, supplemented with different organic compound (15% coconut water (CW) – Thickness of coconut pulp = – mm; 10% potatoes extract – 100g potatoes were boiled, extract to l solution; 5% mashed banana), 30 g/l sucrose, 0.5 g/l AC, g/l agar Seeds were cultured in the bottles (volume = 500 ml with 70 ml medium/bottle) Each treatment had bottles The percentage of orchid seed germination was obtained by estimating the surface area of seed germination in the tissue culture bottle with a diameter of 6.5 cm The total surface area of the tissue culture bottle was defined as 100% After cultivation for eight weeks, the percentage of seed germination was recorded Observations on the percentage germination of seeds, protocrom formation rate, shoot formation rate were recorded weeks after culture 2.2 Experiment conditions All tests were kept at 25±1οC under a photoperiod of 16 h light/8 h dark, light intensity of 2000 lux and 70% relative humidity Basal cultured medium: MS, ½ MS, at pH 5.7, 121 οC, atm, autoclaved for 20 minutes 2.3 Methods 2.3.1 Effect of different media and organic supplements on germination of Den crystallinum var.alba seeds The mature capsules of Den crystallinum var.alba were collected six months after pollination, soaked in aqueous solution of commercial detergent (Sunlight, Vietnam) for 10 min, then rinsed thoroughly three times with sterile distilled water, followed by dipping them in 70% ethanol for 20 sec Capsules were then surface sterilised by dipping in 70% ethyl alcohol and flamed immediately four to five times in laminar air flow The capsules were then cut longitudinally in a sterilised petri dish Seeds were scraped from the capsules, mixed with 100 ml sterile distilled water and pipetted into 1ml tubes and then cultured on the surface of the medium Two different basal media were used in the whole experiment 2.3.2 Effect of NAA and BA on protocorm proliferation of Den crystallinum var.alba Protocorms from the above study were used as culture material (the average weight of 20 ± mg) were proliferated on ½ MS supplemented with 30 g/l sucrose, 10% potatoes extract, NAA (0; 0.1; 0.2 mg/l), BA (0, 0.5, mg/l), 0.5 g/l AC and g/l agar Protocorms were cultured in the bottles (volume = AGU International Journal of Sciences – 2019, Vol (3), – 500 ml with 70 ml medium/bottle) with 20 explants/bottle Each treatment had bottles Observations on the protocorm-like body (PLB) formation rate, number of PLB/explants, figure of PLBs were recorded weeks after culture days after culture) on the optical microscope (Olympus CX21, Japan) with degrees magnification of 40 - 100 times 2.3.5 Statistical analysis All the experiments were set up in completely randomised design (CRD) Each treatment consisted of replicates The difference among the treatment means was compared based on Duncan’s multiple range test (DMRT) analysis (with a level of significance of P