Genetic engineering of maize with the Arabidopsis DREB1A/CBF3 gene using split-seed explants

Transformation of maize (Zea mays L.) split-seed explants from inbred line R23 was performed following particle bombardment with a construct carrying the Arabidopsis transcriptional factor CBF3 under the control of the inducible promoter rd29A and the selectable marker hygromycin phosphotransferase. Overexpressing CBF3 has been shown to enhance cold, drought, and salt tolerance in Arabidopsis, tobacco (Nicotiana tabacum L.), and wheat (Triticum aestivum L.). The CBF3 gene was detected in 18 lines by polymerase chain reaction (PCR), and stable integration of multiple copies of CBF3 was confirmed by Southern blot analysis in three selected lines. Reverse transcription PCR detected expression of CBF3 in the transgenic lines under unstressed conditions despite the use of the stress-inducible rd29A promoter. This constitutive expression was associated with growth retardation and sterility in most of the transgenic lines. Transmission of the gene in a Mendelian fashion to T₁ and T ₂ generations was confirmed in one line by Southern blot analysis. Plants of this line showed stress-inducible expression of the CBF3 gene and hardly detectable expression under unstressed conditions along with significant tolerance to cold, drought, and salinity compared with wild-type plants. These results demonstrate that stress-inducible overexpression of CBF3 has the potential to enhance abiotic stress tolerance in corn.