Holm oak populations have deteriorated drastically due to oak decline syndrome. The first objective of the present study was to investigate the use of axillary budding and somatic embryogenesis (SE) to propagate asymptomatic holm oak genotypes identified in disease hotspots in Spain. Axillary budding was achieved in two out of six tolerant genotypes from the south-western region and in two out of four genotypes from the Mediterranean region. Rooting of shoots cultured on medium supplemented with 3 mg L−1 of indole-3-acetic acid plus 0.1 mg L−1 α-naphthalene acetic acid was achieved, with rates ranging from 8 to 36%. Shoot cultures remained viable after cold storage for 9–12 months; this procedure is therefore suitable for medium-term conservation of holm oak germplasm. SE was induced in two out of the three genotypes tested, by using nodes and shoot tips cultured in medium without plant growth regulators. In vitro cloned progenies of the tolerant genotypes PL-T2 and VA5 inhibited growth of Phytophthora cinnamomi mycelia when exposed to the oomycete in vitro. Significant differences in total phenol contents and in the expression profiles of genes regulating phenylpropanoid biosynthesis were observed between in vitro cultured shoots derived from tolerant trees and cultures established from control genotypes.

Micropropagation, Characterization, and Conservation of Phytophthora cinnamomi-Tolerant Holm Oak Mature Trees

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.

/pdf/efficient-transformation-of-somatic-embryos-and-regeneration-3frl2ue1gi.pdf

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

Casuarina equisetifolia is an important tree of the forest, cultivated in tropical and subtropical regions, providing fuelwood, land reclamation, dune stabilization, paper production, and nitrogen fixation. We have developed a systematic in vitro propagation protocol in C. equisetifolia using nodal segments (NS). Murashige and Skoog (MS) medium augmented with BA (5.0 μM) and NAA (0.5 μM) gave rise to a maximum of 32.00 ± 0.31 shoots per explant (S/E) with shoot length (SL) of 3.94 ± 0.02 cm, and a maximum of 70% regeneration potential (RP) was recorded after 8 weeks of post inoculation. For root induction, in vitro derived shoots were transferred to the nutrient medium consisting of a half-strength (½) MS medium augmented with 2.5 μM NAA, which produced a maximum of 12.68 ± 0.33 roots/shoot (R/S) with 3.04 ± 0.50 cm root length (RL) in 60% of culture after 6 weeks. Micropropagated plants with healthy shoots and roots were successfully acclimatized in vermicompost + garden soil + sand (1:2:1) and a maximum survival percentage of 95.1% was recorded. NS was taken from a 6-weeks-old in vitro derived plant of C. equisetifolia for synthetic seed production, and it was reported that CaCl2 · 2H2O (100 mM) + Na2-alginate (4%) resulted in clear and uniform beads. Furthermore, the maximum conversion of synthetic seeds into plantlets occurred over a period of 4 weeks of storage at 4°C. Scanning Electron Microscopy (SEM) revealed the formation of direct shoot buds without any intermediate callus formation. In addition, the chlorophyll and carotenoid contents of the direct regenerated and mother plant were compared. Similarly, RAPD and ISSR primers were used for genetic homogeneity assessment of the direct regenerated plants, where a total of 18 and 19, respectively, clear and reproducible bands with 100% monomorphism were recorded. The developed micropropagation protocol can certainly be used for large-scale multiplication and germplasm preservation of C. equisetifolia. It will also help in meeting the growing demands of C. equisetifolia in the forest industry.

/pdf/micropropagation-encapsulation-physiological-and-genetic-1w2nxg8u.pdf

Micropropagation, encapsulation, physiological, and genetic homogeneity assessment in Casuarina equisetifolia

Somatic embryogenesis in Solanum betaceum (tamarillo) has proven to be an effective model system for studying morphogenesis, since optimized plant regeneration protocols are available, and embryogenic competent cell lines can be induced from different explants. Nevertheless, an efficient genetic transformation system for embryogenic callus (EC) has not yet been implemented for this species. Here, an optimized faster protocol of genetic transformation using Agrobacterium tumefaciens is described for EC. The sensitivity of EC to three antibiotics was determined, and kanamycin proved to be the best selective agent for tamarillo callus. Two Agrobacterium strains, EHA105 and LBA4404, both harboring the p35SGUSINT plasmid, carrying the reporter gene for β-glucuronidase (gus) and the marker gene neomycin phosphotransferase (nptII), were used to test the efficiency of the process. To increase the success of the genetic transformation, a cold-shock treatment, coconut water, polyvinylpyrrolidone and an appropriate selection schedule based on antibiotic resistance were employed. The genetic transformation was evaluated by GUS assay and PCR-based techniques, and a 100% efficiency rate was confirmed in the kanamycin-resistant EC clumps. Genetic transformation with the EHA105 strain resulted in higher values for gus insertion in the genome. The protocol presented provides a useful tool for functional gene analysis and biotechnology approaches.

/pdf/an-efficient-agrobacterium-mediated-genetic-transformation-1c0fh9xm.pdf

An Efficient Agrobacterium-Mediated Genetic Transformation Method for Solanum betaceum Cav. Embryogenic Callus

The CRISPR/Cas9 ribonucleoprotein (RNP)-mediated technology represents a fascinating tool for modifying gene expression or mutagenesis as this system allows for obtaining transgene-free plants, avoiding exogenous DNA integration. Holm oak (Quercus ilex) has an important social, economic, and ecological role in the Mediterranean climate zones of Western Europe and North Africa and is severely affected by oak decline syndrome. Here we report the first example of the application of the CRISPR/Cas9-RNP technology in holm oak. Firstly, we evaluated the protoplast isolation from both in vitro leaves and proembryogenic masses. Proembryogenic masses represented the best material to get high protoplast yield (11 x 106 protoplasts/ml) and viability. Secondly, the protoplast transfection ability was evaluated through a vector expressing green fluorescence protein as marker gene of transfection, reaching a transfection percentage of 62% after 24 hours. CRISPR/Cas9 RNPs were successfully delivered into protoplasts resulting in 5.6% ± 0.5% editing efficiency at phytoene desaturase (pds) target genomic region. Protoplasts were then cultured in semisolid media and, after 45 days in culture, developed embryogenic calli were observed in a Murashige and Skoog media with half concentration of NH4NO3 and KNO3 supplemented with 0.1 mg/L benzylaminopurine and 0.1 mg/L 2,4-dichlorophenoxyacetic acid.

Advances in Quercus ilex L. breeding: the CRISPR/Cas9 technology via ribonucleoproteins

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%.

Regeneration of transgenic plants by Agrobacterium-mediated transformation of Quercus ilex L. somatic embryos with the gene CsTL1

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Regeneration of transgenic plants by Agrobacterium-mediated transformation of Quercus ilex L. somatic embryos with the gene CsTL1