Transformation experiments by pipetting Agrobacterium into the spikelets of wheat ( Triticum aestivum L.)

Abstract

We report the transfer of kanamycin resistance of bacterial origin into wheat via pipetting Agrobacterium suspension into wheat spikelets. Grain progenies obtained were screened for kanamycin resistance in a two-step procedure allowing the elimination of infected grains. As shown by Southern blot analysis and NPTII-activity assays the gene conferring kanamycin resistance had been transferred into the first and second sexual generation. The transformants obtained were fully fertile and showed no morphological abnormalities. Taking all experiments together, including some unsuccessful ones, transformation frequency was 1%, the highest transformation frequency in a single experiment being 2.6%. All transformants showed alterations concerning the transferred DNA sequences. In the selfed progenies of some first generation transformants the foreign gene had been lost. NPTII-activity was compartively low. Several tests including probing with vir probes prove that our transformants are not contaminated with Agrobacterium. Using the model plant Petunia hybrida, Agrobacterium-mediated gene transfer via pollen had been proven previously. As Petunia pollen, wheat pollen contain diffusible factors identified as flavonol glycosides which induce the plasmidial vir region. By pipetting Agrobacterium suspension into wheat spikelets, bacteria and pollen are brought in intimate contact. Therefore, pollen-mediated gene transfer seems the best explanation for the results obtained. The (pollen) system used in our experiments could be easily adapted to other plant species. It represents an alternative to protoplast-based transformation and leaf disk techniques by avoiding regeneration problems and somaclonal variation, introducing fertile plants. Our procedure is further characterized by its high efficiency and simplicity.