An improved method for Agrobacterium-mediated genetic transformation from cotyledon explants of Brassica juncea
| Autor: | Kwan Sam Choi, Yong Pyo Lim, Won Yong Song, Youngsook Lee, Jang R. Liu, Won Joong Jeong, Sayeda Sultana, Sung Ran Min, Mohammed Shafi Ullah Bhuiyan |
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| Rok vydání: | 2011 |
| Předmět: |
Acetosyringone
food.ingredient Agrobacterium food and beverages Plant Science Genetically modified crops Biology biology.organism_classification Transformation (genetics) Horticulture chemistry.chemical_compound food Murashige and Skoog medium chemistry Botany Agronomy and Crop Science Cotyledon Selectable marker Biotechnology Transformation efficiency |
| Zdroj: | Plant Biotechnology. 28:17-23 |
| ISSN: | 1347-6114 1342-4580 |
| DOI: | 10.5511/plantbiotechnology.10.0921a |
| Popis: | An efficient Agrobacterium-mediated genetic transformation method was established for Brassica juncea by investigating several factors responsible for successful gene transfer. Four-day-old cotyledon explants from in vitro grown seedlings were co-cultivated with Agrobacterium strain GV3101 harboring the binary vector EnPCAMBIA1302-YCF1, which contained the hygromycin phosphotransferase (HPT) gene as a selectable marker and the yeast cadmium factor 1 (YCF1) gene. Two days co-cultivation period on shoot induction medium (MS medium supplemented with 0.1 mg l−1 α-naphthaleneacetic acid, 1.0 mg l−1 6-benzyladenine, and 2.0 mg l−1 silver nitrate) containing 20 mg l−1 acetosyringone and five days delaying exposure of explants to selective agent enhanced transformation efficiency significantly. A three-step selection strategy was developed to select hygromycin resistant shoots. Hygromycin-resistant shoots were subsequently rooted on root induction medium. Rooted plantlets were transferred to pot-soil, hardened, and grown in a greenhouse until maturity. Using the optimized transformation procedure, transformation efficiency reached at 16.2% in this study. Southern blot analysis was performed to confirm that transgenes (HPT and YCF1) were stably integrated into the plant genome. All transgenic plants showed single-copy of transgene integration in the host genome. Segregation analysis of T1 progeny showed that the transgenes were stably integrated and transmitted to the progeny in a Mendelian fashion. |
| Databáze: | OpenAIRE |
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