Regeneration of Cytologically Stable Plants Through Dedifferentiation, Redifferentiation, and Artificial Seeds in Spathoglottis plicata Blume. (Orchidaceae)

Abstract

Spathoglottis plicata Blume. is a horticulturally important vulnerable ground orchid with beautiful flowers blooming round the year. High-frequency protocorm-like body (PLB) formation was established via callus culture from vegetative tissues of in vitro germinated seedlings of S. plicata. Media containing MS salts and Gamborg's B5 vitamins supplemented with 1.0 mg⋅L−1 2,4-dichlorophenoxyacetic acid (2,4-D), 3.0 mg⋅L−1α-naphthaleneacetic acid (NAA), 1.0 mg⋅L−1 kinetin (KIN), and 10% (v/v) ‘Aloe vera gel’ (AvG) were effective in fragile calli induction. A maximum of (22.3 ± 0.52) PLBs were induced from about 250 mg callus within 45–55 days in the presence of 2.0 mg⋅L−1 NAA and 3.0 mg⋅L−1 6-benzylaminopurine (BAP). Briefly, 3.0% sodium alginate was found to be most suitable for the formation of an appropriate shape and good germination rates (86.7%) of artificial seeds. Out of three different temperatures (4, 15, and 24 °C), the best result was achieved at 4 °C with 66.7% germinability even after 90 days of storage. Plantlets were acclimatized with 86.6% survival rate and 76.3% of these plants produced flowers within 12–15 months of field transfer. Chromosomal studies revealed cytological stability of all regenerants containing 2n = 40 chromosomes as in the parental plants. The present protocol can be applied reliably for the purposes of large-scale commercial propagation and short-term conservation of this orchid.