Susceptibility of Maize Mesocotyl Culture to Agrobacterium Transformation and Its in vitro Regeneration

The capacity of mesocotyls to agrobacterially transform four maize lines 4766, RB-179, L-1, L-2 and the conditions for their in vitro regeneration have been studied. Regeneration of maize mesocotyls was observed in media containing various auxins and cytokinins combinations. The efficiency of the process depended on the qualitative and quantitative compositions of growth regulators. The frequency of regenerated explants and the average number of shoots that form on one explant increased with increased cytokinin 6‑BAP and defoliant Dropp (thidiazuron) concentrations in the medium. 6-BAP (3–5 mg/L) and Dropp (3 mg/L) in combination with auxins (IAA, 0.5 mg/L and 2,4-D, 2 mg/L) were shown to be most effective for the mesocotyl regeneration. The number of regenerated explants reached 59.0%, whereas the average number of shoots per explant was 7.5 after 6 weeks of cultivation. The rooting and growing of regenerated shoots was successfully carried out on MS medium supplemented with 0.2 mg/L IBA. The capacity of the mesocotyl was determined by Agrobacterium tumefaciens–mediated transformation (ATMT) with an AGL0 strain containing the pVecActI-GUS genetic construction encoding the reporter gene uidA under the control of the promoter of rice actin ActI gene and the selective hptII hygromycin resistance gene. PCR analysis demonstrated that few regenerants with resistance to the selective agent contained the insertion of the hptII antibiotic-resistance gene in their genomes. Thus, it was shown that maize mesocotyls can be used as explants for agrobacterial transformation.