Studies on Nanoparticles-Assisted Genetic Transformation of Stevia rebaudiana Bertoni

Abstract

Stevia rebaudiana Bertoni is a medicinally important plant, which is well known for its non-caloric sweetening properties. Because of its natural properties it is in huge demand but the rate of seed germination is very low and seed propagation does not lead to the production of homogenous populations. There are many limitations with the traditional breeding technique. Thus, biotechnological tools such as genetic engineering and genetic transformation are seen as important alternatives. The present report is focused on the use of silver nanoparticles for the improvement of genetic transformation efficiency of Stevia rebaudiana Bertoni. Effect of different combinations and concentration of 2,4-D, NAA and BA on shoot organogenesis and somatic embryogenesis was investigated. In MS-1 medium supplemented with 5 μM BA (92%) of explants resulted in shoot organogenesis. MS-1 Medium supplemented with 1 μM 2,4-D (95%) and 1 μM NAA (96%) showed maximum explant differentiating into embryo like structures.

The present study is aimed at the development of an efficient method genetic transformation in Stevia rebaudiana Bertoni through Agrobacterium tumefaciens and particles. In Agrobacterium tumefacien mediated genetic transformation explants pre-cultured (2 days) before bacterial infection (O.D600 – 0.6) showed maximum transient GUS expression (80%). The genetic transformation through chitosan stabilized silver nanoparticles coated with pBI121 plasmid was attempted while the effect of pristine silver nanoparticles in the presence of chitosan as a stabilizer was also studied. The explants injured silver NPs and transformed with Agrobacterium tumefacien showed (70%) of transient GUS expression while there was decline in transformation frequency in explants injured with chitosan stabilized silver NPs and transformed with Agrobacterium tumefacien showed (30%). GUS activity and molecular studies confirm stable transformation in regenerated shoots. To optimize the use of nanoparticles in genetic transformation further studies need to be conducted.