Development of transgenic Brassica napus plants with AtATM3 gene to enhance cadmium and lead tolerance

Abstract

Four popular genotypes of Brassica napus, cv. BARI Sarisha-8, BARI Sarisha-13, Binasarisha-4, and Binasarisha-8 were subjected to cadmium (Cd) and lead (Pb) stress to identify a genotype with enhanced tolerance of Cd and Pb. The goal was to use the selected genotype for genetic engineering, thereby improving traits related to heavy metal tolerance and accumulation to remediate polluted agricultural soils. The genotype, BARI Sarisha-8 with superior tolerance to both Cd and Pb stresses, was chosen for genetic engineering with Arabidopsis thaliana ABC transporter of the mitochondrion 3 (AtATM3) gene. The transformation process involved coculturing the cotyledon explants with Agrobacterium strain GV3101 carrying a binary vector containing the hygromycin phosphotransferase (HPT) gene as a selectable marker and the AtATM3 gene. Transformation efficiency was significantly enhanced with a two-day co-cultivation period on shoot induction medium composed of MS medium, α-naphthaleneacetic acid (0.5 mg/L), and 6-benzyladenine (3 mg/L), supplemented with acetosyringone (20 mg/L), along with a four-day delay in exposing the explants to the selective agent hygromycin. Hygromycin-resistant shoots were obtained by employing a three-step selection process. The refined protocol resulted in a transformation efficiency of 15.38%. Polymerase chain reaction (PCR) analysis confirmed the integration of AtATM3 and HPT genes into the host genome in all recombinant plants. Transgenic B. napus plants expressing the AtATM3 gene exhibited notable improvements in tolerance, demonstrating a 1.4- to 1.7fold increase in Cd tolerance and a 1.3- to 1.5-fold increase in Pb tolerance compared to the wild type (nontransformed) under both Cd and Pb stress conditions.