IN VITRO PROPAGATION AND SECONDARY METABOLITE PRODUCTION OF Angelica glauca EDGEW. - AN ENDANGERED MEDICINAL PLANT OF HIMALAYAS
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Date
2024-03-26
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UHF Nauni
Abstract
The present study reports an optimized protocol for high frequency in vitro propagation through seeds
and rhizome buds, secondary metabolite production and molecular analysis in Angelica glauca. Seeds were
subjected to different pre-treatments prior to surface sterilization for breaking recalcitrance. Maximum percent
survival of seeds (85.19%) was achieved on treatment with 0.1% HgCl2 for 3 minutes with maximum in vitro
seed germination and culture establishment (87%) during spring, followed by winter season (65%). The
proliferated shoots showed highest multiplication (1:12) with rooting on half strength MS medium fortified with
1.0 mg/l BA + 0.2 mg/l NAA. In rhizome buds highest percent survival of 91.67 and 87.50% was achieved in
0.3-0.5 cm and 1.0-2.0 cm sized buds after surface sterilization with 0.1% HgCl2, for 2 and 3 minutes,
respectively. Maximum in vitro establishment (94.44%) was achieved on MS medium fortified with 0.3 mg/l
BA and 0.1 mg/l GA3 with 90% success during spring, followed by 78% in winter season. Highest
multiplication rate (1:15) with rooting was achieved on MS medium fortified with 1.0 mg/l BA +0.2 mg/l NAA.
Endophytic bacterial contamination could be observed in some cultures during multiplication stage after seven
to eight months which was uncontrollable and led to the mortality of cultures. The bacterium responsible for it
was identified as Agrobacterium pusense AG1, through morphological, biochemical and molecular
characterization. For callus induction in vivo leaves and roots were used as explants wherein, highest callus
induction in in vivo leaves (88.89%) and roots (80.59%) was observed under dark incubation. During secondary
metabolite production, GC-MS analysis revealed maximum (8.79%) production of 1-Monolinoleoylglycerol
trimethylsilyl ether from root callus and 6.72% from leaf callus. For enhancing the in vitro yield of bioactive
compounds, callus induced from in vivo leaves and roots were subjected to elicitation by incorporating different
concentrations of sucrose (1,3 and 5%), methyl jasmonate (0.5-1.5 mM), 2,4D (2.0 mg/l) and Kin (0.5 mg/l) in
production medium. Elicitation with 1% sucrose showed maximum Stigmasterol (37.33%) from leaf calli
whereas, maximum Oleic acid (34.33%) was monitored through root calli. Sucrose (3%) resulted in production
of Stigmasterol (55.85%) from leaf and Erucic acid (21.03%) from root callus, and at 5% concentration 17-
Pentatriacontene (25.71%) in leaf callus and 2-Pentanone, 4-hydroxy-4-methyl- (15.09%) in root callus were
identified. Addition of methyl jasmonate (0.5 mM) resulted in production of Furfural (15.54%) from leaf callus
and trans-13-Octadecenoic acid (31.88%) from root callus, and at 1.0 mM concentration 2-
Furancarboxaldehyde, 5-methyl- (22.53%) and trans-13-Octadecenoic acid (33.39%) were monitored from leaf
and root callus respectively. On increasing the concentration of methyl jasmonate to 1.5 mM, n-Hexadecanoic
acid (16.31%) and cis-13-Octadecenoic acid (27.82%) were identified from leaf and root callus. Addition of
2,4D in the medium resulted in production of 2-Furancarboxaldehyde, 5-methyl- (5.74%) and Furfural (10.62%)
from leaf and root calli, respectively. Whereas, addition of kinetin led to the accumulation of 1-
Monolinoleoylglycerol trimethylsilyl ether (16.93%) from leaf and (33.51%) from root callus in production
medium. Molecular analysis of samples from different altitudes through SCoT and CBDP markers revealed 75%
and 80.57% polymorphism, respectively suggesting genetic variations in samples.