KEYWORDS: agarwood callus induction Gyrinops leaf disc explant plant growth regulators seedlings

"Comparison of Callus Induction via in vitro Seedlings and Leaf Disc Explants of Gyrinops walla Gaertn",

Gyrinops is a genus of eight species belongs to family Thymelaeaceae. They are native to
South East Asia and the Indian subcontinent. The species Gyrinops walla is closely related to
Aquilaria, which may produce a resinous substance called agarwood. Agarwood has been
well recognized as a primary raw material in perfumery and therapeutic industries worldwide.
G. walla has been listed as a potentially threatened species by the Convention on International
Trade in Endangered Species of Wild Fauna and Flora (CITES) due to overexploitation of
natural population. Therefore, developing in vitro micropropagation techniques may be an
applicable unravelment to implement healthy planting material to produce stocks for
commercial scale plantations.
Optimized surface sterilization protocol was used to eliminate microbial contaminations.
Carbendazim, Clorox® and ethanol were used as sterilizing agents. Mature seeds were used to
obtain in vitro seedlings. Parts from in vitro seedlings and young tender leaves from natural
plants were used as explants for callus initiation. Sterilized explants were inoculated on
growth regulator free Murashige and Skoog (MS) medium. After two weeks, pathogen free
explants were transferred to MS medium supplemented with different combinations of
benzylaminopurine (BA) and naphthaleneacetic acid (NAA). Survival rate of explants, rate of
callus induction and nature of calli were observed after three weeks of incubation.
Surface sterilization of explants with, 0.2% Carbendazim for 10 minutes, 10% Clorox® for
10 minutes and 70% ethanol for 30 seconds each followed by three successive washings in
sterile distilled water found to be the best surface sterilization method. MS medium
supplemented with a combination of 1.0 mg/L BA and 3.0 mg/L NAA found to be ideal for
callus induction from seedlings and leaf disc explants. Explants obtained from in vitro
seedling parts showed 90% survival, while leaf disc explant showed less (40%) survival rate.
Callus initiation was observed within three weeks from seedling parts, however leaf disc
explants took longer period (seven weeks). Calli obtained from both explant types were
yellowish in colour, compact in nature and observed along the mid rib of leaves. From the
results obtained it could be suggested that callus induction via in vitro seedlings are more
feasible to obtain healthy and high quality calli within a short period of time. Calli obtained
could either be used for mass propagation, through indirect organogenesis or for the
establishment of plant cell culture to secondary metabolite synthesis.

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