Ex vitro rescue, physiochemical evaluation, secondary metabolite production and assessment of genetic stability using DNA based molecular markers in regenerated plants of Decalepis salicifolia (Bedd. ex Hook.f.) Venter

Abstract

To ensure replenishment, a refine protocol for micropropagation of Decalepis salicifolia (Bedd. ex Hook.f.) Venter a critically endangered and endemic medicinal plant was developed using mature nodal explants. A high frequency shoot regeneration system was obtained on Murashige and Skoog (MS) medium comprised of 6- benzyladenine (BA) (5.0 µM) + α-naphthalene acetic acid (NAA) (0.5 µM) + adenine sulphate (ADS) (30.0 µM) corresponds to a highest mean number of 9.97 ± 0.01 shoots/explants with maximum shoot length of 6.46 ± 0.1 cm. Successful rooting in microshoots was achieved on half strength MS medium supplemented with indole-3-butyric acid (IBA) (2.5 µM). A maximum of 6.10 ± 0.07 roots/microshoot with average root length of 2.30 ± 0.06 cm was obtained. As much as 90% plantlets survived when Soilrite™ was used as planting substrate and finally established in soil without any casualty and morphological variation. Acclimatized plantlets were screened for pigment content, net photosynthetic rate (PN), stomatal conductance (Gs) and transpiration rate (E) during subsequent days of acclimatization as well as the changes in antioxidant was also evaluated. A steady rise was observed in the activity of superoxide dismutase (SOD) for initial 21 days and then after a decrease was found showing improved acclimatization efficiency of the plant. Similarly, the activities of catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) enzyme shows reliable increase as the days of acclimatization advanced which play their precautionary role against oxidative damage to the plant. The genetic fidelity of the in vitro raised plantlets with that of mother plant was further confirmed using random amplified polymorphic DNA (RAPD) and inters simple sequence repeats (ISSR) analysis. Additionally, the effect of acclimatization on the biosynthesis of 2-hydroxy-4-methoxybenzaldehyde (2H4MB) in the root system was also evaluated in relation to their biomass production. Maximum fresh weight (4.9 g/plant), dry weight (0.65 g/plant) of roots and 2H4MB content (6.8 µg ml−1 of root extract) was obtained after 10 weeks of acclimatization. Accelerated multiplication rate with the stability of genetic virtue, physiological and biochemical parameter assure the efficacy of the protocol developed for the propagation of this critically endangered medicinal plant.