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

Vanesa Cano,Elena Corredoira,Francisco Javier Vieitez,Elena Varas,José Luis Couselo,Mᵃ Teresa Martínez

doi:10.3390/ijms22041757

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.

Highlights

Cork oak (Quercus suber L.) forests have been exploited over the years for cork production, which is of great economic interest
Embryo Proliferation determine the lethal of kanamycin (Kan), cork oak somatic embryos isolated
Comparison of the three genotypes showed that non-transgenic plantlets (ALM6This is the and first TGR3-wt) report of the transformation of cork oakofwith gene days, in order imwt, ALM80-wt survived for a similar length timea

Summary

Introduction

Cork oak (Quercus suber L.) forests have been exploited over the years for cork production, which is of great economic interest. Cork is the outer bark of the tree and is harvested periodically, usually every 9–12 years, depending on the region [1]. This material is considered an elegant, high-quality product, due to its low weight, water resistance, elasticity and durability. Cork oak acorns are used to fatten the Iberian pig, from which high-quality cured ham is obtained This production system is at risk due to heavy tree mortality across the southwest of the Iberian Peninsula, due to a syndrome known as “La seca” in Spanish, “A secca” in Portuguese and “cork oak decline” in English

Objectives

Methods

Results

Discussion

Conclusion