Agrobacterium tumefaciens-mediated transformation of Allium cepa L.: the production of transgenic onions and shallots

This paper describes the development of a reliable transformation protocol for onion and shallot (Allium cepa L.) which can be used year-round. It is based on Agrobacterium tumefaciens as a vector, with three-week old callus, induced from mature zygotic embryos, as target tissue. For the development of the protocol a large number of parameters were studied. The expression of the uidA gene coding for beta -glucuronidase was used as an indicator in the optimization of the protocol. Subspecies (onion and shallot) and culti var were important factors for a successful transformation: shallot was bet ter than onion and for shallot cv. Kuning the best results were obtained. A lso, it was found that constantly reducing the size of the calli during sub culturing and selection by chopping, thus enhancing exposure to the selecti ve agent hygromycin, improved the selection efficiency significantly. Furth ermore, callus induction medium and co-cultivation period showed a signific ant effect on successful stable transformation. The usage of different Agro bacterium strains, callus ages, callus sources and osmotic treatments durin g co-cultivation did not influence transformation efficiency. The highest t ransformation frequency (1.95%), was obtained with shallot cv. Kuning. A to tal of 11 independent transformed callus lines derived from zygotic embryos were produced: seven lines from shallot and four lines from onion. Large d ifferences in plantlet production were observed among these lines. The best line produced over 90 plantlets. Via PCR the presence of the uidA and hpt (hygromycin phosphotransferase) genes could be demonstrated in these putati ve transformed plants. Southern hybridization showed that most lines origin ated from one transformation event. However, in one line plants were obtain ed indicating the occurrence and rescue of at least three independent trans formation events. This suggested that T-DNA integration occurred in differe nt cells within the callus. Most transgenic plants only had one copy of T-D NA integrated into their genomes. FISH performed on 12 plants from two diff erent lines representing two integration events showed that original T-DNA integration had taken place on the distal end of chromosomes 1 or 5. A tota l of 83 transgenic plants were transferred to the greenhouse and these plan ts appeared to be diploid and normal in morphology.