Agrobacterium-Mediated Transfоrmation of Maize with Antisense Suppression of the Proline Dehydrogenase Gene by an In Planta Method

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.