Expression of gus and gfp genes in amphidiploid spelt wheat (Triticum spelta L.) after Agrobacterium-mediated transformation
Purpose. To study the expression of gus and gfp genes in callus explants of amphidiploid spelt wheat (Triticum spelta L.) after Agrobacterium-mediated genetic transformation. Methods. Winter spelt wheat of ‘Europa’ variety was chosen for transformation. Calli obtained from mature embryos were used...
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| Format: | Article |
| Language: | English |
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Ukrainian Institute for Plant Variety Examination
2020-04-01
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| Series: | Plant Varieties Studying and Protection |
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| Online Access: | http://journal.sops.gov.ua/article/view/201377 |
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| author | А. В. Кирієнко М. В. Кучук Н. Л. Щербак М. Ф. Парій Ю. В. Симоненко |
| author_facet | А. В. Кирієнко М. В. Кучук Н. Л. Щербак М. Ф. Парій Ю. В. Симоненко |
| author_sort | А. В. Кирієнко |
| collection | DOAJ |
| description | Purpose. To study the expression of gus and gfp genes in callus explants of amphidiploid spelt wheat (Triticum spelta L.) after Agrobacterium-mediated genetic transformation.
Methods. Winter spelt wheat of ‘Europa’ variety was chosen for transformation. Calli obtained from mature embryos were used as explants. Callus pre-cultivation was carried out on MS nutrient medium (Murashige–Skoog) supplemented with 2 mg/L 2.4-D (2.4-Dichlorophenoxyacetic acid) and 10 mg/L silver nitrate. For genetic transformation, Agrobacterium tumefaciens Conn., strain GV3101 and a genetic construct with reporter genes beta-glucuronidase (GUS) and green fluorescent protein (GFP) were used. Calli were transformed by inoculation with agrobacteria and vacuum infiltration. Then they were co-cultured on MS medium with 2 mg/L 2.4-D and 10 mg/L AgNO3, but without antibiotics. The expression of the gus gene was checked by histochemical and the gfp gene by visual analysis (fluorescence of the GFP protein in UV light). Gfp and gus gene expression levels were evaluated using ImajeJ software. The integration of the gfp and gus genes into the spelt genome was verified by PCR.
Results. Genetic transformation of spelt callus explants by inoculation in a nutrient medium with agrobacteria and vacuum infiltration occurred at different frequencies. The level of expression of the gus gene during vacuum infiltration was 4.66 ± 0.74%, with inoculation – 4.00 ± 0.91%; and the gfp gene with vacuum infiltration – 3.66 ± 0.74%, with inoculation – 4.66 ± 1.39%. The level of expression of the gfp gene was higher when using inoculation with agrobacteria, and the gus gene was higher during vacuum infiltration. Using PCR analysis, the integration of the gfp and gus genes into the callus of spelt genome was confirmed. The length of the PCR product with primers for the gus gene was 240 bp, and 717 bp for the gfp gene.
Conclusions. The use of vacuum infiltration and inoculation methods for spelt genetic transformation gave different results. The frequency of genetic transformation ranged from 3.66 to 4.66%. Agrobacterium-mediated genetic transformation of amphidiploid spelt wheat allows us to study the expression of gus and gfp reporter genes using callus explants derived from mature embryos |
| first_indexed | 2024-12-11T02:21:57Z |
| format | Article |
| id | doaj.art-583b0bbb2f2a409e9573b86a42bb4a22 |
| institution | Directory Open Access Journal |
| issn | 2518-1017 2518-7457 |
| language | English |
| last_indexed | 2024-12-11T02:21:57Z |
| publishDate | 2020-04-01 |
| publisher | Ukrainian Institute for Plant Variety Examination |
| record_format | Article |
| series | Plant Varieties Studying and Protection |
| spelling | doaj.art-583b0bbb2f2a409e9573b86a42bb4a222022-12-22T01:24:01ZengUkrainian Institute for Plant Variety ExaminationPlant Varieties Studying and Protection2518-10172518-74572020-04-0116110311310.21498/2518-1017.16.1.2020.201377201377Expression of gus and gfp genes in amphidiploid spelt wheat (Triticum spelta L.) after Agrobacterium-mediated transformationА. В. Кирієнко0М. В. Кучук1Н. Л. Щербак2М. Ф. Парій3Ю. В. Симоненко4Institute of Cell Biology and Genetic Engineering, NAS of Ukraine; Ukrainian Scientific Institute of Plant BreedingInstitute of Cell Biology and Genetic Engineering, NAS of UkraineInstitute of Cell Biology and Genetic Engineering, NAS of UkraineUkrainian Scientific Institute of Plant Breeding; National Universityof Life and Environmental Sciences of UkraineInstitute of Cell Biology and Genetic Engineering, NAS of Ukraine; Ukrainian Scientific Institute of Plant BreedingPurpose. To study the expression of gus and gfp genes in callus explants of amphidiploid spelt wheat (Triticum spelta L.) after Agrobacterium-mediated genetic transformation. Methods. Winter spelt wheat of ‘Europa’ variety was chosen for transformation. Calli obtained from mature embryos were used as explants. Callus pre-cultivation was carried out on MS nutrient medium (Murashige–Skoog) supplemented with 2 mg/L 2.4-D (2.4-Dichlorophenoxyacetic acid) and 10 mg/L silver nitrate. For genetic transformation, Agrobacterium tumefaciens Conn., strain GV3101 and a genetic construct with reporter genes beta-glucuronidase (GUS) and green fluorescent protein (GFP) were used. Calli were transformed by inoculation with agrobacteria and vacuum infiltration. Then they were co-cultured on MS medium with 2 mg/L 2.4-D and 10 mg/L AgNO3, but without antibiotics. The expression of the gus gene was checked by histochemical and the gfp gene by visual analysis (fluorescence of the GFP protein in UV light). Gfp and gus gene expression levels were evaluated using ImajeJ software. The integration of the gfp and gus genes into the spelt genome was verified by PCR. Results. Genetic transformation of spelt callus explants by inoculation in a nutrient medium with agrobacteria and vacuum infiltration occurred at different frequencies. The level of expression of the gus gene during vacuum infiltration was 4.66 ± 0.74%, with inoculation – 4.00 ± 0.91%; and the gfp gene with vacuum infiltration – 3.66 ± 0.74%, with inoculation – 4.66 ± 1.39%. The level of expression of the gfp gene was higher when using inoculation with agrobacteria, and the gus gene was higher during vacuum infiltration. Using PCR analysis, the integration of the gfp and gus genes into the callus of spelt genome was confirmed. The length of the PCR product with primers for the gus gene was 240 bp, and 717 bp for the gfp gene. Conclusions. The use of vacuum infiltration and inoculation methods for spelt genetic transformation gave different results. The frequency of genetic transformation ranged from 3.66 to 4.66%. Agrobacterium-mediated genetic transformation of amphidiploid spelt wheat allows us to study the expression of gus and gfp reporter genes using callus explants derived from mature embryoshttp://journal.sops.gov.ua/article/view/201377triticum spelta l.speltcallusogenesisgus genegfp genegenetic transformation |
| spellingShingle | А. В. Кирієнко М. В. Кучук Н. Л. Щербак М. Ф. Парій Ю. В. Симоненко Expression of gus and gfp genes in amphidiploid spelt wheat (Triticum spelta L.) after Agrobacterium-mediated transformation Plant Varieties Studying and Protection triticum spelta l. spelt callusogenesis gus gene gfp gene genetic transformation |
| title | Expression of gus and gfp genes in amphidiploid spelt wheat (Triticum spelta L.) after Agrobacterium-mediated transformation |
| title_full | Expression of gus and gfp genes in amphidiploid spelt wheat (Triticum spelta L.) after Agrobacterium-mediated transformation |
| title_fullStr | Expression of gus and gfp genes in amphidiploid spelt wheat (Triticum spelta L.) after Agrobacterium-mediated transformation |
| title_full_unstemmed | Expression of gus and gfp genes in amphidiploid spelt wheat (Triticum spelta L.) after Agrobacterium-mediated transformation |
| title_short | Expression of gus and gfp genes in amphidiploid spelt wheat (Triticum spelta L.) after Agrobacterium-mediated transformation |
| title_sort | expression of gus and gfp genes in amphidiploid spelt wheat triticum spelta l after agrobacterium mediated transformation |
| topic | triticum spelta l. spelt callusogenesis gus gene gfp gene genetic transformation |
| url | http://journal.sops.gov.ua/article/view/201377 |
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