In vitro culture of immature embryos of Cinnamomum tamala Nees.— The role of different factors.

Seed characteristics and in vitro culture of C. tamala embryos were studied. Embryos desiccated below 50% (fresh weight) exhibited poor morphogenetic response in vitro and confirmed the recalcitrant nature of seeds. The immature embryos of various developmental ages (4-16 week after flowering, WAF) were cultured on different strengths of MS medium. Morphogenesis responses were recorded after 10 days of culture. The best culture responses were achieved from the immature embryos of 12 WAF on MS medium with sucrose (3%, w/v), polyvinyl pyrollidone (100 mg L-1) and benzyl adenine (12 µM). Under optimum condition ~60% explants responded; and ~7.3 shoots buds developed per explants after 35 days of culture initiation. The shoot buds could be converted into micro-shoots on MS medium with sucrose (3%) and kinetin (3 µM). About 5.3 micro-shoots/shoot buds sprouted per sub-culture. The micro-shoots were rooted by maintaining them on MS medium with α-naphthalene acetic acid (3 µM) where within 6-8 wk of culture ~8-10 roots developed. The rooted plantlets were acclimatized in vitro before they were transferred to community potting mix and maintained in the poly-shade ca 75% shading. The transplants registered ~70% survival after two months of transfer.

An efficient in vitro regeneration protocol for a natural dye yielding plant, Strobilanthes flaccidifolious Nees., from nodal explants.

Adventitious shoot buds formation from axillary buds of nodal segments of S. flaccidifolious was achieved on MS medium containing sucrose (3%, w/v), and α-naphthalene acetic acid (NAA; 3 µM) and benzyl adenine (3 µM) in combination. The nodal segments were primed on ‘Growtak Sieve’ for 48 h on MS medium containing sucrose (2%), polyvinyl pyrollidone (200 mgL-1) as antioxidant. About 80% of primed nodal segments responded positively and formed ~12 adventitious shoot buds per explants from explants collected during October-November months of every year. The shoot buds converted into plantlets on MS medium containing sucrose (3%) and kinetin (3 µM) where ~7 micro shoots developed per subculture after 8 weeks of culture. The regenerated micro shoots induced average 14 roots/ plant on medium containing NAA (3 µM). The regenerates were hardened for 6-7 weeks on medium with ½MS salt solution and sucrose (2%) under normal laboratory condition before transferring to potting mix. About 70% transplants survived after two months of transfer.

Effects of different factors on immature embryo culture, PLBs differentiation and rapid mass multiplication of Coelogyne suaveolens (Lindl.) Hook.

In vitro mass production of C. suaveolens (Lindl.) Hook, an endangered orchid with its snowy white flowers having horticultural potential was accomplished through immature seed culture, and subsequent plant regeneration. The developmental stage of the immature seeds and nutrient media significantly influenced the germination frequency. Seeds at 13 months after pollination cultured on 3% sucrose containing Murashige and Skoog (MS) medium with 9 microM alpha-naphthaleneacetic acid (NAA), and 15% coconut water exhibited 93% germination after 40 days of culture. Upon subculture, the germinated shoots on MS medium with 9 microM BA, 6 microM NAA, 3% casein hydrolysate and 0.1% activated charcoal (AC) yielded >12 shoots per shoot or bud. Addition of AC favoured the enlargement of pseudobulbs and better rooting. The plantlets transferred to community potting mix after in vitro hardening (8-10 wk) displayed 85% survival.

In vitro propagation of threatened terrestrial orchid, Malaxis khasiana Soland ex. Swartz through immature seed culture.

Rapid in vitro propagation of the terrestrial orchid, M. khasiana through immature seed culture was achieved. Immature seeds of 8-9 week after pollination (WAP) cultured on MS medium (2% sucrose) supplemented with 500 mgl(-1) casein-hydrolysate and 1 microM N6-benzyladenine (BA) exhibited germination of 75% seeds after 107 days of culture and subsequently supported the development of PLBs. Subsequent culture on MS medium enriched with 6 microM of indole-3-acetic acid (IAA), 18 microM each of BA and kinetin induced multiple shoots and plantlets. Transfer of PLBs to MS medium with 0.1% activated charcoal (AC) facilitated rapid proliferation of PLBs, while AC at 0.2% favored shoot bud induction and rhizome enlargement. The plantlets, developed on medium with IAA, BA and kinetin, after hardening in vitro for 8-10 weeks were planted in community pots and transferred to poly-house. The plantlets showed 65% survival under field conditions.

Regeneration of plantlets from in vitro raised leaf explants of Cleisostoma racimeferum Lindl.

Protocorm like bodies (PLBs), callus and shoot buds developed in culture from in vitro raised foliar explants of Cleisostoma racimeferum. Among the different basal media, better result was obtained on MS medium containing sucrose (3%) and BA (2 microM) with approximately 80% frequency after 40 days of culture. Young leaves (15 week old) produced better PLBs. Whole leaf placed vertically upside-up orientation can regenerate PLBs and shoot buds (80%). PLBs and shoot buds formed on entire surface of the leaves. Cultures on BA and NAA (2 and 2 microM respectively in combination) stimulated callus mediated regeneration (68%). The rooted plantlets regenerated within 8-10 week from PLBs and shoot buds on MS medium containing IAA and kinetin (2 microM each in combination). BA containing medium triggered multiple shoot bud formation, while NAA alone or in combination with other growth regulators was inhibitory. Incorporation of activated charcoal (0.01%) in the medium stimulated formation of repetitive PLBs and multiple shoot buds. Rooted plants were ready for harvest after 20-22 week of initiation of culture. About 65% of the potted plants survived after 3 months in the poly house.