Overexpression of the chestnut CsTL1 gene coding for a thaumatin-like protein in somatic embryos of Quercus robur

Abstract

Somatic embryos of Quercus robur derived from mature and juvenile trees were transformed with the CsTL1 gene coding for an antifungal protein. Clumps of somatic embryos were inoculated with Agrobacterium tumefaciens strain EHA105 harbouring a binary vector (pKWG2D-TAU) that included the green fluorescence protein (GFP) reporter gene. After 24 weeks of culture on selective medium, significantly higher transformation frequencies were obtained in a temporary immersion system (TIS) than in semi-solid medium (ranging from 1.4 to 9.6 % for the four genotypes). The first transformants appeared after 10 weeks and GFP activity was evaluated in all kan-resistant embryogenic lines. The transgenic nature of lines was confirmed by PCR and Southern blotting. CsTL1 transcripts were detected in all transgenic lines (TL) analyzed by quantitative real-time PCR, and levels of expression were significantly higher than in the wild type (wt), reaching up to 24 times higher in some genotypes. Sixty-two stable TL were obtained (38 derived from centenarian trees). Plant conversion rates of transformed somatic embryos were similar to those of the wt and were increased by use of the TIS. GFP fluorescence was detected in roots and shoots of transgenic plants, which confirmed stable transformation of regenerants.