Efficient Transformation of Somatic Embryos and Regeneration of Cork Oak Plantlets with a Gene (<i>CsTL1</i>) Encoding a Chestnut Thaumatin-Like Protein
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 carbeni...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2021-02-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/22/4/1757 |
| _version_ | 1797411885129138176 |
|---|---|
| author | Vanesa Cano Mᵃ Teresa Martínez José Luis Couselo Elena Varas Francisco Javier Vieitez Elena Corredoira |
| author_facet | Vanesa Cano Mᵃ Teresa Martínez José Luis Couselo Elena Varas Francisco Javier Vieitez Elena Corredoira |
| author_sort | Vanesa Cano |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-09T04:52:43Z |
| format | Article |
| id | doaj.art-31d34e0ca0b74ccaa7c3880213b20f24 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T04:52:43Z |
| publishDate | 2021-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-31d34e0ca0b74ccaa7c3880213b20f242023-12-03T13:08:45ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-02-01224175710.3390/ijms22041757Efficient Transformation of Somatic Embryos and Regeneration of Cork Oak Plantlets with a Gene (<i>CsTL1</i>) Encoding a Chestnut Thaumatin-Like ProteinVanesa Cano0Mᵃ Teresa Martínez1José Luis Couselo2Elena Varas3Francisco Javier Vieitez4Elena Corredoira5Instituto de Investigaciones Agrobiológicas de Galicia (IIAG), Avd Vigo s/n, 15705 Santiago de Compostela, SpainInstituto de Investigaciones Agrobiológicas de Galicia (IIAG), Avd Vigo s/n, 15705 Santiago de Compostela, SpainEdificio de Ciencias Experimentais, Bloque A, Campus Universitario, Universidad de Vigo, 36310 Vigo, SpainEstación Fitopatolóxica do Areeiro (EFA), Subida a la Robleda s/n, 36153 Pontevedra, SpainInstituto de Investigaciones Agrobiológicas de Galicia (IIAG), Avd Vigo s/n, 15705 Santiago de Compostela, SpainInstituto de Investigaciones Agrobiológicas de Galicia (IIAG), Avd Vigo s/n, 15705 Santiago de Compostela, SpainWe 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.https://www.mdpi.com/1422-0067/22/4/1757<i>Agrobacterium tumefaciens</i>in vitro <i>Phytophthora cinnamomi</i> tolerance testoak decline<i>Quercus suber</i>pathogenesis related proteinsvitrification procedure |
| spellingShingle | Vanesa Cano Mᵃ Teresa Martínez José Luis Couselo Elena Varas Francisco Javier Vieitez Elena Corredoira Efficient Transformation of Somatic Embryos and Regeneration of Cork Oak Plantlets with a Gene (<i>CsTL1</i>) Encoding a Chestnut Thaumatin-Like Protein International Journal of Molecular Sciences <i>Agrobacterium tumefaciens</i> in vitro <i>Phytophthora cinnamomi</i> tolerance test oak decline <i>Quercus suber</i> pathogenesis related proteins vitrification procedure |
| title | Efficient Transformation of Somatic Embryos and Regeneration of Cork Oak Plantlets with a Gene (<i>CsTL1</i>) Encoding a Chestnut Thaumatin-Like Protein |
| title_full | Efficient Transformation of Somatic Embryos and Regeneration of Cork Oak Plantlets with a Gene (<i>CsTL1</i>) Encoding a Chestnut Thaumatin-Like Protein |
| title_fullStr | Efficient Transformation of Somatic Embryos and Regeneration of Cork Oak Plantlets with a Gene (<i>CsTL1</i>) Encoding a Chestnut Thaumatin-Like Protein |
| title_full_unstemmed | Efficient Transformation of Somatic Embryos and Regeneration of Cork Oak Plantlets with a Gene (<i>CsTL1</i>) Encoding a Chestnut Thaumatin-Like Protein |
| title_short | Efficient Transformation of Somatic Embryos and Regeneration of Cork Oak Plantlets with a Gene (<i>CsTL1</i>) Encoding a Chestnut Thaumatin-Like Protein |
| title_sort | efficient transformation of somatic embryos and regeneration of cork oak plantlets with a gene i cstl1 i encoding a chestnut thaumatin like protein |
| topic | <i>Agrobacterium tumefaciens</i> in vitro <i>Phytophthora cinnamomi</i> tolerance test oak decline <i>Quercus suber</i> pathogenesis related proteins vitrification procedure |
| url | https://www.mdpi.com/1422-0067/22/4/1757 |
| work_keys_str_mv | AT vanesacano efficienttransformationofsomaticembryosandregenerationofcorkoakplantletswithageneicstl1iencodingachestnutthaumatinlikeprotein AT materesamartinez efficienttransformationofsomaticembryosandregenerationofcorkoakplantletswithageneicstl1iencodingachestnutthaumatinlikeprotein AT joseluiscouselo efficienttransformationofsomaticembryosandregenerationofcorkoakplantletswithageneicstl1iencodingachestnutthaumatinlikeprotein AT elenavaras efficienttransformationofsomaticembryosandregenerationofcorkoakplantletswithageneicstl1iencodingachestnutthaumatinlikeprotein AT franciscojaviervieitez efficienttransformationofsomaticembryosandregenerationofcorkoakplantletswithageneicstl1iencodingachestnutthaumatinlikeprotein AT elenacorredoira efficienttransformationofsomaticembryosandregenerationofcorkoakplantletswithageneicstl1iencodingachestnutthaumatinlikeprotein |