Silver nanoparticles for agrobacterium mediated genetic transformation

Abstract

The study entitled “Silver nanoparticles for Agrobacterium mediated transformation” was carried out during 2018 - 2020 in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The objective of the study was to evaluate the efficacy of silver nanoparticles in improving the transformation efficiency of Agrobacterium tumefaciens. Growth kinetics of Agrobacterium tumefaciens strain EHA105 was determined by plotting Absorbance (OD600) vs Time. The exponential phase started 12 h after inoculation. Toxicity assay of silver nanoparticles (100 nm) on Agrobacterium cells was carried out in the exponential phase by exposing to different concentrations of silver nanoparticles viz., 0.01, 1, 5, 10 and 20 mgL-1. Maximum toxicity (91%) was recorded at a concentration of 20 mg L-1 and minimum (3.3%) at a concentration of 0.01 mgL-1. The half maximal effective concentration (EC50) was estimated as 8.707 mgL-1 by carrying out Probit analysis using the SPSS software. Efficacy of different concentrations of silver nanoparticles (below EC50 value) for induction of competency and transformation of Agrobacterium tumefaciens with pART27 (11.6 kb) plasmid vector was assessed and compared with conventional calcium chloride (20 mM) freeze-thaw technique. The different techniques tried were silver nanoparticles alone, silver nanoparticles with calcium chloride, silver nanoparticles with freeze-thaw and silver nanoparticles with calcium chloride along with freeze-thaw. For the treatments involving silver nanoparticles alone, six different concentrations (0.01, 0.5, 1, 2, 4, 6 mgL-1) were tried. The four concentrations (0.01, 0.5, 1, 2 mgL-1) that recorded better transformation efficiency were selected for inclusion in combinations with calcium chloride and freeze-thaw technique. All the treatments were carried out with cultures at an early exponential phase and the transformants were selected on Luria Bertani agar supplemented with rifampicin (100 μg/mL) and spectinomycin (100 μg/mL). Transformation efficiency of different techniques was assessed. Colonies started appearing in the plates after 48 h in conventional technique and treatments involving silver nanoparticles with calcium chloride, whereas, in all other treatments involving silver nanoparticles, colonies were observed after 60 h of plating. Size of the colonies that emerged from cells involving treatments with silver nanoparticles were smaller compared to that of conventional technique. However, all treatments involving silver nanoparticles showed significantly higher or comparable transformation efficiency with conventional technique (2.31 log cfu/μg of DNA) except two treatments (1 mgL-1 or 2 mgL-1 of silver nanoparticles with freeze-thaw). Maximum transformation efficiency (3.34 log cfu/μg of DNA) was recorded in the treatment with combination of 0.5 mgL-1 silver nanoparticles along with calcium chloride (20 mM) which was on par with 0.01 mgL-1 silver nanoparticles along with calcium chloride (3.33 log cfu/ μg of DNA). Transformation was confirmed by the re-isolation of plasmid from the transformed colonies. Colony PCR of the transformed colonies using nptII gene specific primer exhibited an amplicon of predicted size (475 bp) confirming transformation. Results of the present study indicated that silver nanoparticles can improve transformation efficiency in Agrobacterium tumefaciens. Silver nanoparticles of size 100 nm at a concentration of 0.01 mgL-1 with calcium chloride (20 mM) showed ten-fold increase in the transformation efficiency (3.3 3 log cfu/μg of DNA) of Agrobacterium tumefaciens compared to conventional calcium chloride freeze-thaw technique. From the study it was inferred that induction of competency using silver nanoparticles is economically feasible.