Somatic embryogenesis, acclimatization and genetic homogeneity assessment of regenerated plantlets of Anoectochilus elatus Lindl., an endangered terrestrial jewel orchid

Abstract

An efficient in vitro plant regeneration system was established through somatic embryogenesis for Anoectochilus elatus Lindley, an endangered jewel orchid. Direct somatic embryogenesis was achieved from nodal explants (17.4 embryos per explant with 63.4% response) on Mitra medium supplemented with Morel vitamins, thidiazuron (4.54 µM) and ∞-naphthaleneacetic acid (2.69 µM). Simultaneously, a protocol was developed for indirect somatic embryogenesis from internodal explant, produced embryogenic calli and embryos (31.3 embryos with 76.4% response) on same medium amended with 50 mg/L peptone and 5% coconut water. Both types of embryogenic pathways, produced morphologically similar globular embryos in the form of protocorm like bodies and successfully germinated on hormone free Mitra medium supplemented with Morel vitamins. Morpho-histological investigation of the embryo revealed the initiation and developmental features of somatic embryos. In vitro regenerated plantlets were successfully established from heterotrophic to a photoautotrophic stage by reducing the nutrient content in culture media, adjusting temperature and humidity through three step method. During the process, no morphological and physiological abnormalities were observed. Hardened plantlets were successfully acclimatized at poly tunnel chamber with 95% of survival rate. Further, inter simple sequence repeats (ISSRs) molecular markers were used to analyse the genetic homogeneity of regenerated plants. Analysis with this method showed that the homogeneity is comparatively higher in direct somatic embryo regenerated plants (94.22%) as compared to plants elevated from an indirect somatic embryo (93.05%). The present study provides morpho-histological and genetically stable plants for germplasm conservation and further utility of this endangered jewel orchid.