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 CsTL1 gene that codes for a thaumatin-like protein, in order to confer tolerance to Phytophthora cinnamomi. 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 CsTL1 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 CsTL1 was evaluated in an in vitro bioassay with the oomycete P. cinnamomi. Of the eight transgenic lines analyzed, seven survived for between one or two times longer than non-transgenic plantlets. Expression of the CsTL1 gene and plantlet survival days were correlated, and survival was generally greater in plantlets that strongly expressed the CsTL1 gene.

Keywords: Agrobacterium tumefaciens; Quercus suber; in vitro Phytophthora cinnamomi tolerance test; oak decline; pathogenesis related proteins; somatic embryogenesis; thaumatin-like protein; vitrification procedure.

Figure 1

Effect of kanamycin concentration on percentage of explants with secondary somatic embryos (SSEs) and on the number of bipolar SSEs per Petri dish, in the genotypes ALM80 (a) and TGR3 (b). Vertical lines indicate the standard errors of mean values.

Figure 1

Effect of kanamycin concentration on percentage of explants with secondary somatic embryos (SSEs) and on the number of bipolar SSEs per Petri dish, in the genotypes ALM80 (a) and TGR3 (b). Vertical lines indicate the standard errors of mean values.

Figure 2

Effect of genotype on the percentage of kanamycin-resistant explants and transformation efficiency of cork oak somatic embryos transformed with strain EHA105pK7TAU. Ten Petri dishes were used for each genotype and each Petri dish contained 10 groups of somatic embryos. Vertical lines indicate the standard errors of mean values.

Figure 3

Different steps in the production of transgenic somatic embryos and transgenic cork oak plantlets. (a,b) Kan-resistant somatic embryos derived from genetic transformation of cork oak embryogenic genotypes ALM80 (a) and ALM6 (b) observed after 10 weeks in selective medium; (c,d) Transgenic cluster of somatic embryos visualized under white light (c) and green fluorescence protein (GFP) filter (d); (e,f) Plantlets obtained from germination of non-transgenic (e) and transgenic (f) somatic embryos after 6 weeks on germination medium; (g) GFP expression on a root of non-transgenic (left; arrow) and transgenic (right) plantlets, visualized in an epi-fluorescence stereomicroscope; (h) GFP expression on a leaf isolated from non-transgenic (left; arrow) and transgenic (right) plantlets visualized in an epi-fluorescence stereomicroscope.

Figure 4

PCR analysis of transgenic lines to detect the presence CsTL1 (in both transcriptional senses), NPTII and GFP genes. M: Molecular ladder, ALM6 (a), ALM80 (b) and TGR3 (c). P: plas-mid (positive control), wt: non-transgenic somatic embryos of cork oak (negative control) and lanes 1–12: putative cork oak transgenic lines.

Figure 5

Expression analysis of CsTL1 gene in somatic embryos of cork oak determined by qPCR. Total RNA was extracted from 18 independent transgenic lines and the corresponding non-transgenic lines (wt) of three cork oak embryogenic genotypes: ALM6 (a), ALM80 (b) and TGR3 (c). Values are mean ± standard error from at least three independent experiments, and vertical lines indicate the standard errors of mean values. Data analyzed by ANOVA I (p ≤ 0.05). Values in columns indicated with the same letter are not significantly different (p = 0.05, Duncan’s test).

Figure 6

Morphological appearance and progress of infection in non-transgenic plantlets (a,b) and transgenic plantlets (c,d) of genotype ALM80 immediately after inoculation (a,c) and 15 days later (b,d). Diameter of the tube: 20 mm.

Figure 7

Survival rate of transgenic and non-transgenic (wt) cork oak lines derived from genotypes ALM6 (a), ALM80 (b) and TGR3 (c) 31 days after infection with P. cinnamomi. A total of 18 plantlets were evaluated for each transgenic and wt line. Vertical lines indicate the standard errors of mean values. Data were analyzed by ANOVA I (p ≤ 0.05). Values in columns indicated with the same letter are not significantly different (p = 0.05, Duncan’s test).