Silver nanoparticles for biolitics based gene delivery in plants

Abstract

The study entitled “Silver nanoparticles for biolistics based gene delivery in plants” was carried out at the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2018 to 2020. The objective of the study was to evaluate the efficacy of silver nanoparticles as carriers for gene delivery in Nicotiana tabacum using biolistic method. Standardisation of in vitro callus induction and regeneration medium of Nicotiana tabacum was carried out for screening of transformants. Leaf explants from in vitro grown seedlings were inoculated in basal Murashige and Skoog (MS) medium with varried concentrations of cytokinin and auxin for callus induction. MS medium with 1 mgL-1 NAA + 1 mgL-1 BA showed cent per cent callus induction. Calli exhibited cent per cent shoot induction on transfer to MS medium supplemented with 1 mgL-1 BA + 0.5 mgL-1 GA3 + 0.25 mgL-1 IAA and the shoots exhibited cent per cent rooting on transfer to half strength MS medium containing 0.1 mgL-1 IAA. For biolistic transformation, plasmid vector pBI121 (14.6 kb) was isolated from freshly cultured Agrobacterium tumefaciens EHA105 by alkaline lysis method and the quality of DNA was confirmed by double restriction digestion with EcoRI and HindIII producing bands of predicted size (12-13 kb and 3 kb). Biolistic bombardments were performed using PDS-1000/He apparatus (Bio- Rad) using gold microcarriers (size 0.6 micron at a concentration of 60 mgL-1) or silver nanoparticles (size 100 nm, varying concentrations viz., 0.01, 0.05, 0.5, 1, 2, 10, 20 and 100 mgL-1) by maintaining a vacuum pressure of 28 inch in the chamber. Helium pressures (450 psi, 650 psi, 900 psi, 1100 psi) and target distances (6 cm and 9 cm) were optimised to determine the maximum transformation efficiency of gold microcarriers and silver nanoparticles for comparison. Leaf discs of Nicotiana tabacum surface sterilized using 4% sodium hypochlorite for 2 min were used for bombardment. Transformation efficiency was determined by counting the number of blue spots on GUS assay. Helium pressure of 650 psi and target distance of 9 cm exhibited maximum transformation efficiency for Nicotiana tabacum using gold microcarriers. Among the different concentrations of silver nanoparticles tried as carriers for bombardment, 10 mgL-1 exhibited significantly higher transformation efficiency. Interaction effect of variation of helium pressure and target distance revealed that combination of 900 psi and 6 cm exhibited maximum transformation efficiency of silver nanoparticles. Comparison of gold microparticles and silver nanoparticles as carriers of DNA during biolistic bombardment indicated that transformation efficiency of silver nanoparticles was significantly higher than gold microparticles (81.0 vs 67.7). The bombarded leaf discs from the treatments of gold and silver carriers showing maximum transformation efficiency along with un-bombarded leaf discs were screened in MS medium supplemented with 100 mgL-1 selection agent kanamycin. All the un-bombarded leaf discs started drying within one week of transfer to selection medium whereas 65% of the leaf discs bombarded with DNA using gold microparticles and 70% of leaf discs bombarded with silver nanoparticles as carriers exhibited callus induction. The green calli were transferred to regeneration medium after two weeks for induction of shoots and further rooting and hardening. To conclude, the results of the study indicated that silver nanoparticles at a concentration of 10 mg L-1, He pressure of 900 psi and target distance of 6 cm exhibited maximum efficiency in leaves of Nicotiana tabacum without cytotoxic effect on callus induction. The maximum transformation efficiency of silver nanoparticles was sixteen per cent higher compared to maximum transformation efficiency of gold microcarriers and exhibited 37.5 fold cost reduction of consumables. This is the first report of efficient use for silver nanoparticles for biolistics based gene with delivery in plants.