Direct regeneration and genetic transformation studies in Hemidesmus indicus (L.) R. Br. (Indian Sarsaparilla)

Abstract

Hemidesmus indicus L. R. Br. is a rare and endangered plant exhibits potent medicinal properties explored for the presence of flavoring compound in the roots namely 2-hydroxy-4- methoxybenzaldehyde (HMB) having applications in pharmaceutical and nutraceutical industries. The objective of the study is to improve the regeneration potential and optimize the genetic transformation in Hemidesmus indicus. Present study exhibits direct regeneration of Hemidesmus indicus through leaf and nodal explants with subsequent plant regeneration using Murashige and Skoog (MS) medium supplemented with various plant growth regulators (auxins, cytokinins, and gibberellic acid) TRIA and with adjuvant adenine sulphate,. The caulogenic response of 78.8%, 73.3% and 71.4% was observed when the leaf explant was inoculated on MS media containing 2.3 mgL^− 1 BAP + 0.2 mgL^− 1 2,4-D, 0.02 mgL^− 1 TRIA + 2 mgL^− 1 BAP and 1 mgL^− 1 KIN + 1 mgL^− 1 NAA respectively with creamish yellow nodular friable callus by 4 weeks. The initiation of shoot bud was observed within three days after inoculation of nodal explant on media supplemented with 1 mgL^− 1 BAP + 0.1 mgL^− 1 NAA, 1 mgL^− 1 BAP + 0.1 mgL^− 1 NAA + 40 mgL^− 1 AgNO₃, 1 mgL^− 1 BAP + 0.1 mgL^− 1 NAA + 40 mgL^− 1 AgNO₃ + 40 mgL^− 1 adenine sulphate respectively and incubated in the dark for 2 weeks. Shoot regeneration from the leaf explants was also observed within 4 weeks after inoculation in MS medium with 1 mgL^− 1 BAP + 0.1 mgL^− 1 NAA. In the present study, we also report the development of an effective and reproducible Agrobacterium tumefaciens mediated genetic transformation system in Hemidesmus indicus. Genetic transformation was achieved by inoculating in vitro calli of Hemidesmus indicus with the A. tumefaciens strain EHA105 harboring pCAMBIA1301 with 35 S CAMV promoter driving the expression of reporter β-glucuronidase gene (GUS) and hygromycin selection marker hptII. After co-cultivation, the calli were thoroughly washed and inoculated on MS basal medium with (2.3mgL^− 1 BAP + 0.2 mgL^− 1 2,4-D) along with cefotaxime (250 mgL^− 1) and hygromycin (30 mgL^− 1) and the co-cultivated callus cultures were maintained in dark at 25 ̊C ± 2 ̊C for 4 weeks. After two rounds of subculture, the putative transformed calli were assessed by performing histochemical GUS staining assay and genomic DNA PCR to detect the exogenous GUS gene sequence. The transformation efficiency was 26% with transformed calli tolerant to hygromycin (30 mg L⁻¹) and were positive to histochemical GUS staining assay and PCR. The results indicated a successful establishment of a reliable and efficient A. tumefaciens mediated genetic transformation system. The in-vitro micropropagation is a well-known technique for the mass production of the plant and thus aid in conservation of H. indicus. Through transformation, the regulatory and the biosynthetic genes can be manipulated for enhancement of nutraceutical, pharmaceutical bioactive compounds present in H. indicus and can also be applied for generating transgenic plants with desirable traits.