Evaluation of the parameters affecting agrobacterium-mediated transformation process to produce cold stress-tolerant tomato by targeting Solanum lycopersicumC-repeat binding factor 2 gene

Abstract

C-repeat binding factors (CBFs) are a part of the AP2/ERF superfamily of transcription factors, which are known to regulate gene expression under different abiotic stress conditions, especially cold. In this study, when tomato cultivar Pusa Ruby (PR) was subjected to cold stress at 4 °C for 24 h, SlCBF2, along with SlCBF1, was found to be simultaneously induced at the early hours (3rd to 24th) of cold exposure. Similar simultaneous induction observed in another tomato variety, Arka Rakshak (AR), at the 3rd hour post-cold exposure. This brief yet strong simultaneous induction of both SlCBF1 and SlCBF2 provides a new insight into the initial induction of CBFs in cold-stressed tomato, which was not previously reported in PR or AR. To elucidate the SlCBF2-mediated cold response in tomato, we have developed stable overexpression lines that showed better cold tolerance than the wild-type PR. We have also evaluated and optimized two important parameters in the Agrobacterium-mediated transformation process and reported an improved transformation efficiency of 28.33% using 1 mg/L zeatin alone and 25% using a combination of 2 mg/L BAP and 0.1 mg/L IAA. Considering the varying degree of transformation efficiency in PR, this optimization will surely be of interest to future researchers to generate stable tomato transgenic lines. However, further works such as generating stable mutant lines targeting SlCBF2 and studying how it reacts to cold-stress, whether SlCBF2 directly regulates SlCBF1 or other downstream cold-responsive genes would provide a more definitive framework for establishing SlCBF2 as an important cold-stress-related transcription factor in tomato.