Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: A non-tissue culture approach

Germinating seeds of Arabidopsis thaliana were cocultivated with an Agrobacterium tumefaciens strain (C58Clrif) carrying the pGV3850:pAK1003 Ti plasmid. This Ti plasmid contains the neomycin phosphotransferase II gene (NPT II) which confers resistance to kanamycin and G418. Seeds (T1 generation) imbibed for 12 h before a 24 h exposure to Agrobacterium gave rise to the highest number of transformed progeny (T2 generation). Over 200 kanamycin-resistant T2 seedlings were isolated. Some of the T2 seedlings and T3 families were characterized for genetic segregation of functional NPT II gene(s), NPT II activity, and the presence of T-DNA inserts (Southern analysis). Ninety percent of the T2 individuals transmitted the resistance factor to the T3 families in a Mendelian fashion. Of the T3 families segregating in a Mendelian fashion (n=111), 62% segregated for one functional insert, 29% for two unlinked or linked functional inserts, 5% for three unlinked inserts, 1% for four unlinked inserts, whereas 3% appeared to be homozygous for the insert(s). The 13 families that did not exhibit Mendelian segregation ratios fell into 2 classes, both of which had a deficiency of kanamycin-resistant seedlings. In the Group I T3 families (n=6) only 0%-2% of the seedlings were resistant to kanamycin (100 mg/l), whereas in the Group II families (n=7) 8%-63% of the seedlings were resistant. All of the kanamycin-resistant plants that were tested were found to possess NPT II activity. Southern analysis revealed that all of the resistant plants contained at least one copy of the T-DNA and that the majority of the plants had multiple inserts. Explants from kanamycin-resistant plants survived and formed callus when cultured on callus-inducing medium containg G418.