AN EFFICIENT IN VITRO PROPAGATION, ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF APHYLLORCHIS MONTANA (REICHENB.F.)

Abstract

Keywords: Antimicrobial activity, Antioxidant activity, Aphyllorchis montana, Asymbiotic seed ger- mination, In vitro propagation, Plant growth regulators, 2,2-Diphenyl-1- picrylhydrazyl. Abbreviations: IAA (Indol-3-acetic acid), BM-TM (BM-1Terrestrial orchid medium), IBA (Indole- 3-butyric acid), NAA (α- Naphthaleneacetic acid), TDZ (Thidiazuron), BA (6-Benzyl adenine), KIN (6-Furfurylaminopurine), 2-iP (2-Isopentenyladenine), GA3 (Gibberellic acid), Zt (Zeatin), DPPH (2, 2-diphenyl-1-picrylhydrazyl), ABTS (2, 2'azinobis (3-ethylbenzothiozoline-6-sulfonic acid) diammonium salt), TPTZ (2, 4, 6-tripyridyl-S-triazine), EDTA (Ethylenediamine tetraacetic acid). Abstract An in vitro plant regeneration protocol was successfully established in Aphyllorchis montana, a saprophytic achlorophyllous orchid by culturing immature seeds. Among the six basal media evaluated for seed germination, BM-TM medium was found to be the best followed by KC medium. After 40 days, all the media turned brown and the growths of the protocorms were arrested. Activated charcoal, 1 g/l in half strength BM-TM was found to be suitable for further development of protocorms. Half strength BM-TM medium was supplemented with different growth regulators either individually or in combinations for multiplication of shoots. Of the five cytokinins tested, TDZ at 6.8 μM was found to be most effective for multiple shoot induction yielding 17.24 ± 0.27 shoots after 10 weeks of culture. Addition of low concentration of NAA (1.3 μM) in MS medium supplemented with the cytokinin TDZ (6.8 μM) favoured shoot multiplication. A mean number of 27.56 ± 0.54 shoots with 3.92 ± 0.11 number of roots were produced per explant. The response of the seed derived protocorm to the different types of organic additives viz., peptone and yeast extract and coconut water was also evaluated. The addition of these organic additives to the medium containing TDZ enhanced the number of shoot regeneration. The plantlets were acclimatized in plastic pots containing sterilized vermiculite. The survival rate was 100 % when maintained in the culture room condition (25 ± 2 °C). Screening of the antibacterial, antioxidant activity and estimation of total phenolics and flavonoid content of methanolic extracts of micropropagated plants were also carried out and compared with that of the wild-grown plants. In all the tests, methanolic extract from wild- grown plants showed higher antioxidant, antimicrobial activity, total phenolics and flavonoid content than in vitro propagated plants.