Induced ectopic expression of At-CBF1 in marker-free transgenic tomatoes confers enhanced chilling tolerance

In an attempt to improve chilling stress tolerance, an Arabidopsis C-repeat binding factor 1 (At-CBF1) gene driven by the inducible promoter RD29A was co-transferred into tomato var. Shalimar. Marker (NPTII)-free transgenic were obtained in T(1) generation because of unlinked integration of CBF1 and NPTII genes. Reverse transcription-polymerase chain reaction confirmed the expression of CBF1 in T(1) transgenic lines. Study of expression pattern in T(1) transgenic line showed a gradual increase with increasing chilling stress period and also confirmed the reversibility of expression on removal of stress. The transgenic plants exhibited no morphological and agronomical differences as compared to non-transformed plants. When young transgenic plants were exposed to chilling stress (4°C) for 3 days, increased survival (50%) was observed in transgenic lines than non-transformed plants (10%). Transgenic plants subjected to the chilling stress showed a significant decrease in membrane injury index and lipid peroxidation and also increased significantly free proline content in the leaf tissues as compared to non-transformed plants. Thus, these findings indicate that marker-free transgenic tomato plants expressing Arabidopsis CBF1 gene provided protection and conferred cold tolerance to transgenic tomato without any phenotypic variation.