Efficient Transformation of Somatic Embryos and Regeneration of Cork Oak Plantlets with a Gene (CsTL1) Encoding a Chestnut Thaumatin-Like Protein

Abstract

We present a reproducible procedure for transforming somatic embryos of cork oak with the <i>CsTL1</i> gene that codes for a thaumatin-like protein, in order to confer tolerance to <i>Phytophthora cinnamomi</i>. Different concentrations/combinations of the antibiotics carbenicillin and cefotaxime, as bacteriostatic agents, and kanamycin, as a selective agent, were tested. A lethal dose of 125 mg/L kanamycin was employed to select transgenic somatic embryos, and carbenicillin was used as a bacteriostatic agent at a concentration of 300 mg/L, which does not inhibit somatic embryo proliferation. The transformation efficiency was clearly genotype-dependent and was higher for the TGR3 genotype (17%) than for ALM80 (4.5%) and ALM6 (2%). Insertion of the transgenes in genomic DNA was confirmed by PCR analysis, whereas expression of the <i>CsTL1</i> gene was evaluated by semi-quantitative real-time PCR (qPCR) analysis. A vitrification treatment successfully cryopreserved the transgenic lines generated. The antifungal activity of the thaumatin-like protein expressed by the gene <i>CsTL1</i> was evaluated in an in vitro bioassay with the oomycete <i>P. cinnamomi</i>. Of the eight transgenic lines analyzed, seven survived for between one or two times longer than non-transgenic plantlets. Expression of the <i>CsTL1</i> gene and plantlet survival days were correlated, and survival was generally greater in plantlets that strongly expressed the <i>CsTL1</i> gene.