An improved method for transformation of regal pelargonium (Pelargonium X domesticum Dubonnet) by Agrobacterium tumefaciens.

An improved method for genetic transformation of the regal pelargonium cultivar, Pelargonium Xdomesticum Dubonnet, by Agrobacterium tumefaciens has been developed. One of the main modifications to the transformation protocol published previously was the use of 6-week-old in vitro grown plantlets as a source of leaf-lamina explants, rather than glasshouse grown plants. A test of three shoot regeneration media containing differing concentrations of the phytohormones, naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) revealed that shoot regeneration efficiency from in vitro leaf-lamina explants was more than doubled when the auxin and cytokinin concentrations in :he medium were twice those previously reported for glasshouse derived explants. The second modification to improve the transformation protocol was the use of the plant phenolic, acetosyringone to induce vir genes on the A. tumefaciens Ti plasmid. The disarmed A. tumefaciens strain LBA4404 used for inoculation was cultured in a three step process in which acetosyringone was added to the final culture. During both preculture and co-cultivation periods, the leaf-lamina explants were plated on regeneration medium containing 100 mu M of acetosyringone. The binary vector used in the experiment reported here was pLN54, which contains the phytochrome A (phyA) cDNA from oat (Avena sativa) along with the kanamycin resistance gene, nptII. Transgenic shoots were regenerated on selection medium containing lower concentrations of kanamycin monosulphate than reported previously (50 instead of 300 mg l(-1)), because the earlier work suggested that in vitro material was more sensitive to kanamycin than glasshouse material. Modifications to the rooting medium for transgenic shoots also included a reduction in the kanamycin concentrations, rom 300 to 200 mg l(-1), and the addition of 0.1 mg l(-1) NAA to promote rooting. Based on the rooting test in the presence of kanamycin, the transformation efficiency of this revised transformation method was 19.3%, with the production of 29 independent pLN54 transformants. Stable integration of the pLN54 T-DNA and expression of phyA in 24 of the transformants was confirmed by Southern and Northern analyses. This revised method has resulted in a more reliable and productive transformation system that can be used successfully throughout the year. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.