Establishment of in planta transformation protocol of tomato (Solanum lycopersicum L.) through antiporter gene for improved salinity tolerance

Abstract

Tomato stands as the world’s third most consumed vegetable, but its production has been suffering due to climate vulnerability, notably for saline sensitivity. Despite its economic importance, developing salinity tolerant tomato has not been prioritized lately. Current study was aimed to establish a simple and efficient Agrobacterium-mediated in planta transformation protocol to transform Na+/H+ antiporter gene into 5 Bangladeshi tomato varieties, namely BARI tomato 2, BARI tomato 3, BINA tomato 2, BINA tomato 3 and Bahar, to improve their salt tolerance, through optimization of crucial transformation factors like optical density, infection time, co-cultivation period etc. Two vectors were constructed by cloning Na+/H+ antiporter gene from Arabidopsis (pK7WG2_AtNHX1_1.6) and Rice (pK7WG2_OsNHX1_1.6) individually to gateway vector pENTR/D-TOPO and electroporated to Agrobacterium while another vector pBI121 was used as control. Non-pricked seeds were found optimum for achieving more than 90% efficiency for GUS expression and germination percentages under conditions of OD600 1.1-1.4 with 30 min of infection time followed by 24 hrs co-cultivation period during transformation using the 3 vectors. Transformed plantlets were screened through resistance to Kanamycin 50 mg/l in germination medium while Cefotaxime 100 mg/l was applied to prevent Agrobacterium overgrowth during co-cultivation. Tolerance of 100 mM NaCl for 14 days has been observed in putative transformants in Leaf Disc Bioassay. No significant morphological changes were observed during the acclimatization of putatively transformed plantlets. This established protocol is novel and can efficiently produce genotype-independent transgenic tomato plants obviating intervening tissue culture. Hence, this study provides scope for climate-resilient crop improvement to ensure nutritional security.