Synthesis of nonembryonic synseeds in Hemidesmus indicus R. Br.: short term conservation, evaluation of phytochemicals and genetic fidelity of the regenerants

Abstract

Non-embryogenic, synthetic seeds were formed by encapsulating the nodal segments (NS) of Hemidesmus indicus R. Br. in calcium alginate hydrogel comprising of MS basal medium for short-term conservation. A 3% sodium aliginate (SA) with 100 mM CaCl2 was found most suitable for the preparation of isodiametrical beads. Highest shoot regrowth (84.50 ± 0.35%) was recorded when the gelling matrix, i.e. 3% SA in Murashige and Skoog (MS) basal medium was supplemented with 5.0 µM 6-benzyladenine (BA) + 0.5 µM indole-3-butyric acid (IBA) and inoculated onto the nutrient medium comprised of MS + 5.0 µM BA gave 5.53 ± 0.096 shoots/encapsulated NS. Rooting in microshoots was obtained on the half strength liquid MS + 0.1 µM IBA. Plantlets achieved from preserved synthetic seeds were acclimatized and relocated in the natural condition successfully with the immortality rate of 89.3%. During the days of acclimatization, the obtained plants were subjected for the assessment of their pigment content and biochemical assay. The results obtained clearly exhibited the enhancement in the pigment content as the acclimatization period increases. A significant rise and fall in Superoxide dismutase (SOD), Catalase (CAT), Glutathione reductase (GR) and Ascorbate peroxidase (APX) were evident, suggesting their preventive role against the several environmental stresses during the acclimatization of the plant. Further gas chromatography–mass spectrometry (GC–MS) analysis of both mother and in vitro raised plants showed the occurrence of more than 60 diverse phyto-chemicals in the methanolic root extract. HPLC analysis of the root extract revealed the presence of 2-hydroxy-4-methoxybenzaldehyde (2H4MB), a potential phytoactive compound and flavouring agent. 2H4MB content quantified by high-performance liquid chromatography (HPLC) in methanol extract was 17.94 μg/ml. The genetic stability of the regenerated plantlets with mother plant was confirmed by the presence of monomorphic bands generated through inter simple sequence repeat (ISSR) markers. A protocol with improved results has been developed for synthetic seed production and regeneration in H. indicus using nodal explants.