Preliminary Characterization of the DREB Genes in Transgenic Wheat

Abstract

The DREB1A gene identified from Arabidopsis thaliana and involved in the plant response to several abiotic stresses was introduced in wheat. More than 20 independent transformation experiments were conducted using wheat cv. Bobwhite SH 98 26. For the preliminary screening of transformed lines, a solution of 0.3% Basta herbicide was sprayed at the 5- to 6-leaf stage. Molecular analyses for the presence of bar gene were carried out independently and 100% of the Basta-resistant plants exhibited bar, while only 113 of the 447 lines evaluated were positive to rd29DREBIA. Southern blot analysis of DNA from a T1 progeny using 2 probes consisting of coding regions of the DREBIA and bar genes, indicated that the 2 transgenes were present in their entirety and integrated into the genome of the transgenic plants. Among the 113 lines positive to DREB1A, 13 were selected based on their simple segregation patterns of the transgenes: 3:1 in progenies derived from selfed plants and 1:1 in progenies derived from transgenic plants crossed with the control plants. The expression of the 2 genes differed when determined by RT-PCR. The presence of mRNA for the bar genes, whose expression was driven by the maize ubiquitin promoter, was easily detected in the transgenic plants; transcripts of the DREB1a gene, driven by the RD29a promoter, were detected after 2 days of water stress. After sowing, a delay in germination was observed in the transgenic lines. The control plants started to germinate after 2-3 days, while the DREB plants showed nonuniform germination under both stressed and fully irrigated conditions. However, no differences related to the presence of the transgene were observed within the transgenic lines during the first days of water stress conditions. At this stage, no significant differences were observed between the development of the transgenic and the control lines. At the 4- to 5-leaf stage, the control and the transgenic plants were randomly distributed on the trays and subjected to water stress by simply withholding water. After 10 days without water, the transgenic lines started to show differences in drought tolerance, determined by the visible wilting of the leaves. The control plants began to show drought symptoms (loss of turgor and bleaching of the leaves) after 10 days of stress. Severe symptoms (death of all leaf tissue) were evident in the control samples after 15 days without water. The selected transgenic plants either did not show any symptoms or showed reduced leaf turgor after 10-12 days without water.