IN VITRO REGENERATION AND PHYTOCHEMICAL STUDIES IN KAKOLI (Fritillaria roylei HOOK.) – HIGH VALUE CRITICALLY ENDANGERED HIMALAYAN HERB
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Date
2023-09-04
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UHF,NAUNI
Abstract
ABSTRACT
Fritillaria roylei Hook. (Liliaceae), commonly known as kakoli or janglilahsun, is a bulbous
perennial herb that thrives in the Indian Himalayan region at altitudes of 2800-4600 m above mean sea
level. The plant's bulbs are known for their abundant metabolite compounds with medicinal properties,
making them valuable for various therapeutic uses. However, natural propagation through seeds and bulbs
is slow, taking approximately 5-6 years for a plant to grow and mature. Due to the high demand for its
bulbs, overexploitation has become a concern. Therefore, biotechnological intervention is crucial for
conserving, restoring, and sustainably producing these valuable bioactive metabolites. In the present study,
in vitro cultures were established using bulb scales as explants. The highest survival percentage (88.77%)
was achieved by treating the explants with 0.5% bavistin for 20 minutes, followed by 70% ethanol for 45
seconds, and HgCl2 (0.1%) for 14 minutes. By using different combinations and concentrations of plant
growth regulators, a high-efficiency callus (100%) was obtained using 4.0 mg/L Picloram and 2.0 mg/L
TDZ at 25°C and 15°C, respectively, after 30 and 21 days. Transferring the callus from dark to light
conditions and utilizing a high sucrose concentration led to the formation of more bulblets. For bulblet
production, different plant growth regulators were tested, and the highest number of bulblets per explant
(10.07) with a regeneration response of 90.98% was achieved using NAA and KIN at 1.0 mg/L each after
25 days. Phytochemical analysis was performed using crude extracts from different in vivo (leaf, stem and
bulb) and in vitro (callus and bulblets) samples. Among the in vivo samples, the highest total phenol
content was observed in leaves (63.64 mg GAE/g), while in vitro callus exhibited the highest phenolic
content (54.31 mg GAE/g). Flavonoid content was found to be highest in leaves among in vivo samples
(133.0 mg RE/g) and in bulblets among in vitro samples (38.38 mg RE/g). Regarding antioxidant activity,
leaves showed the highest activity among the in vivo samples, while in vitro callus and bulblets displayed
antioxidant activity statistically at par in comparison to the in vivo samples. UHPLC-based quantification
was done for targeted steroidal alkaloids namely peimisine, sipeimine and peimine. Among in vivo sample
extracts, highest peimisine, sipeimine and peimine were quantified as 119.27 μg/g DW, 394.62 μg/g DW
and 25.46 μg/g DW respectively in bulbs. In contrast, in vitro, highest peimisine, sipeimine and peimine
were quantified as 26.46 μg/g DW, 0.60 μg/g DW and 13.92 μg/g DW, respectively in bulblets. Thus,
rapid in vitro regeneration protocol enables large-scale production of these targeted bioactive compounds
in a shorter period and throughout the year. Consequently, it offers a reliable and sustainable source for the
development of new drugs or natural remedies without the need to uproot the entire plant