Satish, L. and Ceasar, S.A. and Ramesh, M. (2017) Improved Agrobacterium-mediated transformation and direct plant regeneration in four cultivars of finger millet (Eleusine coracana (L.) Gaertn.). Plant Cell Tissue Organ Culture, 131 (3). pp. 547-565.
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Abstract
We have developed an improved Agrobacteriummediated transformation and rapid regeneration system for four cultivars (‘CO(Ra)-14’, ‘PR-202’, ‘Try-1’ and ‘Paiyur- 2’) of finger millet using optimized transformation and direct plant regeneration conditions. The shoot apical meristems (SAMs) were used as explants in this study. Agrobacterium strain EHA105 carrying binary vector pCAMBIA1301 was used to optimize the transformation conditions. Concentrationwas achieved in cultivar ‘CO(Ra)-14’. The entire transformation procedure, from initiating SAMs to planting putative transgenic plantlets in the greenhouse, was completed within 45 days with the highest stable transformation frequency of 11.8% for ‘CO(Ra)-14’. PCR, gus staining and Southern blot analyses were performed in T0 and T1 generations to confirm the gene integration. Six events from T0 had a single copy of the transgene and showed a normal Mendelian pattern of segregation. To our knowledge, this is the first report on the high frequency transformation of finger millet by Agrobacterium and subsequent recovery of transgenic plants via direct plant regeneration without a callus phase, in short duration (45 days). The proposed protocol could be supportive in breaking through the bottleneck in transformation and regeneration of finger millet cultivars. of hygromycin, the optical density of the culture, infection time, age of the explants, co-cultivation period, the concentrations of acetosyringone and antibiotics were optimized to improve the transformation frequency. The highest frequency of mean transient gus expression (85.1%)was achieved in cultivar ‘CO(Ra)-14’. The entire transformation procedure, from initiating SAMs to planting putative transgenic plantlets in the greenhouse, was completed within 45 days with the highest stable transformation frequency of 11.8% for ‘CO(Ra)-14’. PCR, gus staining and Southern blot analyses were performed in T0 and T1 generations to confirm the gene integration. Six events from T0 had a single copy of the transgene and showed a normal Mendelian pattern of segregation. To our knowledge, this is the first report on the high frequency transformation of finger millet by Agrobacterium and subsequent recovery of transgenic plants via direct plant regeneration without a callus phase, in short duration (45 days). The proposed protocol could be supportive in breaking through the bottleneck in transformation and regeneration of finger millet cultivars.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | Agrobacterium · Direct plant regeneration · Finger millet, Shoot apical meristems,Southern blotting, Transgenic plants |
| Author Affiliation: | Department of Biotechnology, Alagappa University, Science Campus, Karaikudi, Tamil Nadu 630 004, India |
| Subjects: | Crop Improvement |
| Divisions: | Millet |
| Depositing User: | Mr T L Gautham |
| Date Deposited: | 31 May 2018 10:41 |
| Last Modified: | 31 May 2018 10:41 |
| URI: | http://eprints.icrisat.ac.in/id/eprint/15438 |
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