The maize callus was incubated in a saturated Agrobacterium culture for 1 h, then cultivated on the MS medium containing cefotaxime to eliminate its contamination by the bacteria. After the formation of shoots, the latter were transferred to the medium supplemented with kanamycin (Kn) to select the plants resistant to this antibiotic. The frequency of producing the Kn-resistant plants by such a method of plant transformation was found to amount to 3.3 and 14.4% for A188x91 and 91 line, respectively. However, our attempts to select the Kn-resistant plants of A188 appeared to be unsuccessful.

In order to accelerate infection of the explants, the callus cultivated with Agrobacterium was vortexed for 30 sec or subjected to vacuum infiltration. According to the data reported by Ishida et al. (Nature Biotechnology 14:745-750, 1996), which used immature maize embryos as explants, the vortex treatment of the explants enhanced an efficiency of their genetic transformation and did not bring about any embryo injury. However, the embryonic callus used by us appeared to be more sensitive to mechanic injuries: the vortexed callus inocula grew slowly, and their regeneration capacity was found to significantly decrease. Among the few plants recovered from the use of this procedure, Kn-resistant ones appeared to be absent. The use of vacuum infiltration allowed an increase in the yield of A188x91 green plants of about three-fold, but was found to be ineffective in the case of the A188 line that was more sensitive to such a treatment.

To enhance virulence of Agrobacterium, the overnight culture used for co-cultivation with maize callus was supplemented with acetosyringone or tobacco leaf exudate. The application of acetosyringone resulted in the increase of about three-fold of the frequency of the regeneration of the Kn-resistant A188x91 plants, but had only a slight effect on the transformation efficiency of A188 and 91 lines. The exudate-induced activation of Agrobacterium was more effective, because in this case the amount of the Kn-resistant plants of 91 line and A188x91 appeared to be 2.4 and 5.4 times, respectively, higher as compared to that of the plants obtained by using inactivated Agrobacterium. This may be due to the fact that the tobacco leaf exudate, unlike acetosyringone, contains a wider spectrum of phenols and other compounds capable of affecting the gene transfection.

We have found that incubation of maize callus in a suspension of Agrobacterium results in a significant decrease of its morphogenetic capacity. This effect was expressed in a marked decrease in a probability of selection of the transformed plants. For this reason the duration of the co-cultivation was confined, as a rule, to 1 h. Using these findings as the base, we offer a new method of infection of explants that involves cultivation of callus inocula for 15-20 days on the lawn of the Agrobacterium activated by tobacco leaf exudate. The advantage of the suggested method is prolonged contact of growing plant tissues with the activated Agrobacterium, which provides a high enough probability of plant infection. In addition, it should be noted that in the Agrobacterium-containing cultivation medium, both an active growth of the embryogenic callus and regenerative capacity of the latter are still retained.

The above described method of plant transformation was found to be effective upon infection of both immature maize embryos and embryogenic callus. According to our results, the frequency of producing the Kn-resistant plants of A188, 91 and A188x91 was estimated as 17.8, 40.6 and 21.1%, respectively. With the use of fluorometric and histochemical methods, an expression of the inserted reporter GUS gene in tissues of the plants obtained was demonstrated. The resistance to Kn was inherited to the T1 generation.

The designed method of genetic transformation of plants was applied for insertion in embryonic maize callus of plasmid pK22rs containing the genes nptII and rs from radish, encoding defensin. This transformation was used for the purpose of enhancing plant resistance to some phytopathogenic fungi. The transfer of the plasmid into plant cells was performed with the use of strain LBA 4404 of A. tumefaciens. In the transgenic plants thus obtained the insertions of genes nptII and rs were confirmed by both PCR and Southern blot hybridization.
 
 

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