Agrobacterium-Mediated Transfоrmation of Maize with Antisense Suppression of the Proline Dehydrogenase Gene by an In Planta Method
Journal Title: Biotechnology Journal International - Year 2014, Vol 4, Issue 2
Abstract
Aims: The aim of this study was to estimate the efficiency of T-DNA transfer during Agrobacterium-mediated transformation of maize (Zea mays L.) at different temperatures. In addition, the way of T-DNA transfer was studied after application of an Agrobacterium suspension at maize pistil filaments. Study Design: Transgenic maize plants were obtained with an antisense suppressor of the proline dehydrogenase gene (ASPG) by using the binary vector pBi2E. Temperatures of 28-35ºC were used to establish suitable conditions for transformation in planta. Place and Duration of Study: Laboratory of Bioengineering, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences; between May 2008 and May 2013. Methodology: A. tumefaciens strain LBA4404 (pBi2E), containing the marker gene and the ASPG from Arabidopsis thaliana was used for maize transformation. The presence of T-DNA in the maize genome was detected by PCR. The proline concentration in transgenic hybrids of maize lines was determined colorimetrically. Results: T-DNA carrying the marker genes (nptII, gus) and the ASPG construct was detected in the maize genomes after Agrobacterium-mediated transformation. PCR analysis of total DNA isolated from 409 kanamycin-resistant diploid F1 seedlings revealed T-DNA insertions in the genomes of 30 plants. Expression of the ASPG in the maize genome led to a 4.5-fold increase (P=0.05) in free proline content in the transformed plants. Temperatures above 3ºC blocked the T-DNA transfer. Conclusion: The transfer of the ASPG by Agrobacterium T-DNA into the maize genome was achieved with a frequency of 0.3-2.3% at temperatures not higher than 31ºC. The PCR-positive maize plants had a statistically significant increase in the proline concentration in leaf tissues as compared with the non-transformed control. T-DNA may be transported into the maize egg cell by the growing pollen tube after the pistil filaments are inoculated with an Agrobacterium suspension.
Authors and Affiliations
M. I. Chumakov, Y. M. Moiseeva, V. A. Velikov, I. V. Volokhina, Yu. S. Gusev, O. S. Yakovleva
Keywords
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- EP ID EP237190
- DOI 10.9734/BBJ/2014/6504
- Views 126
- Downloads 0
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