Regeneration of transgenic plants by Agrobacterium -mediated transformation of Quercus ilex L. somatic embryos with the
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Regeneration of transgenic plants by Agrobacterium‑mediated transformation of Quercus ilex L. somatic embryos with the gene CsTL1 V. Cano1 · M. T. Martínez1 · M. C. San José1 · J. L. Couselo2,3 · E. Varas3 · L. Bouza‑Morcillo1 · M. Toribio4 · E. Corredoira1 Received: 28 February 2019 / Accepted: 14 January 2020 © Springer Nature B.V. 2020
Abstract Here we describe for the first time a protocol for the genetic transformation of the holm oak Quercus ilex. Holm oak populations are seriously affected by a disorder known as oak decline syndrome. However, tolerant plants can be obtained by overexpression of pathogenesis-related (PR) proteins. The aim of the present study was to define a protocol for transforming somatic embryos (SEs) of holm oak with the gene CsTL1 encoding a chestnut thaumatin-like protein (PR protein). Lethal concentrations of kanamycin (kan) were determined in previous studies, in which SE formation was found to be inhibited by kan concentrations of 100 mg/L and above. Genetic transformation was only achieved when target explants were pre-cultured for one or 2 weeks, with a transformation efficiency of 2%. Transformation was also affected by genotype and was only successful in 2 of the 3 embryogenic lines evaluated. A total of 11 transformed lines (10 corresponding to line Q8 and 1 to line Q10–16) were obtained, all of which were maintained by secondary embryogenesis on proliferation medium (Schenk and Hildebrandt medium lacking plant growth regulators). The transgenic embryogenic lines were successfully cryopreserved by a vitrification-based procedure, thus enabling the lines to be preserved while tolerance tests were performed. The presence of CsTL1 in genomic DNA was confirmed by PCR, and expression of the gene was determined by qPCR. CsTL1 expression was up to 5.93 times higher in transgenic lines than in the corresponding untransformed line. Transgenic plants were produced from transformed SEs, with embryo conversion rates ranging from 2.8 to 66.7%. Keywords Holm oak · Cryopreservation · GFP · Genetic transformation · Oak decline · Pathogenesis related proteins · Somatic embryogenesis · Thaumatin-like protein
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s1105 6-020-09771-9) contains supplementary material, which is available to authorized users. * E. Corredoira [email protected] Extended author information available on the last page of the article
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New Forests
Introduction Quercus ilex L. (holm oak) is one of the main constituents of the Spanish agrosylvopastoral systems known as “dehesas”, reported to the most widespread type of agroforestry system in Europe and covering a total area of 3.1 million hectares (Moreno and Pulido 2009). Holm oak acorns provide a rich source of nutrients for foraging animals, especially Iberian pigs (the meat of which is the basis of a high-quality food industry), as well as sheep, cattle, horses and fighting bulls (Cañellas et al. 2007). In addition, the ectomycorrhizal fungus Tu
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