In vitro propagation and field establishment of Eulophia cullenii (Wight) Bl., a critically endangered orchid of Western Ghats, India through culture of seeds and axenic seedling-derived rhizomes

An efficient protocol has been devised for the propagation and field establishment of Eulophia cullenii (Wight) Bl., a terrestrial orchid having ornamental potentialities, and is critically endangered in Western Ghats, India. Seeds extracted from 60-90-d-old capsules germinated in A1/2 MS, A1/4 MS, Knudson C, or Mitra liquid medium developed into 1.4-2.5-mm-diameter protocorms in 60 d. Supplementation of organic additives like coconut water, peptone, yeast extract, and casein acid hydrolysate (CH) significantly enhanced protocorm growth. Upon subculture onto agar-gelled Mitra medium fortified with 0.05% CH, 56% of protocorms regenerated into shoots through the formation of linear mini-rhizomes. The regenerated shoots grew vigorously in A1/2 MS, producing new rhizomes. Mature rhizomes from axenic seedlings produced maximum (13 A +/- 1.4) shoots/whole rhizome in A1/2 MS fortified with 44.4 mu M 6-benzylaminopurine (BAP), in 120-150 d. Horizontal and longitudinal halves of the rhizome also gave multiple shoots (6-8.5) in the presence of 44.4 mu M BAP. Shoots or shoot clumps sub-cultured onto A1/2 MS basal medium produced roots followed by rhizomes in 60-150 d. Seedlings with mature rhizomes showed 70% establishment in the nursery and added a new rhizome at the end of one growth cycle. An average of 70.6% of the rhizomes originating from seedlings during the second growth cycle sprouted to produce new shoots, when planted in the native localities. Asymbiotic germination and cloning through rhizomes thus can provide a large number of vigorous plants of E. cullenii for ornamental exploitation as well as eco-restoration, if rhizome as storage organ is ensured in the propagule.