Establishment of an efficient in vitro regeneration and Agrobacterium rhizogenes-mediated genetic transformation protocol for safed musli (Chlorophytum borivilianum Santapau & R.R.Fern.)

Abstract

Safed musli (Chlorophytum borivilianum Santapau & R.R.Fern.) is a medicinal herb valued for its tuberous roots that contain several bioactive compounds. Conventionally, safed musli tubers are harvested for secondary metabolites 9 mo after planting. Alternatively, in vitro culture can be used for mass production and to shorten the time required to produce secondary metabolites. This study was designed to develop an efficient hairy root induction protocol for safed musli. Shoot bud explants of various lengths were tested on Murashige and Skoog medium with 3 mg L−1 6-benzylaminopurine. Shoot buds >4 cm resulted in 100% multiplication, but 2- to 4-cm shoot buds produced a high multiplication rate (92%), the highest number of multiple shoots (15.55 ± 0.37), and the longest shoots (6.83 ± 0.08 cm) after 30 d of culture. In vitro shoots were infected with one of five strains of Agrobacterium rhizogenes. Only strains MAFF106590, MAFF106591, and MAFF720002 induced the hairy root phenotype indicative of genetic transformation. MAFF720002 strain had a positive transformation response in all in vitro raised safed musli stem segments of different lengths. Stems 2.5 cm in length had the highest transformation percentage (76 to 81%), greatest number of hairy roots per explant (5.02 ± 0.06), and the longest hairy roots (23 ± 0.49 cm). PCR analysis detected the rolB gene in the transformed samples. The A. rhizogenes strain played a significant role in producing and promoting the growth of hairy roots in safed musli. This approach can potentially be utilized for the replacement of conventional field methods for secondary metabolite production from safed musli.