Role of calcium-calmodulin in auxin-induced somatic embryogenesis in leaf base cultures of wheat (Triticum aestivum var. HD 2329)

Wheat leaf bases cultured for 1 day on 2,4-D (10 mu M) display the induction of somatic embryogenesis. The induction of somatic embryogenesis by 2,4-D appears to be calcium-mediated as treatment of leaf bases with the calcium chelator, EGTA, prior to 2,4-D treatment, inhibited the induction of somatic embryogenesis. This sensitivity of auxin to reduced calcium levels can be reversed by calcium ions alone and not any other divalent cation like magnesium or zinc. Additionally, the expression of the three calcium-regulated genes, Triticum aestivum calmodulin binding protein kinase, calcium-dependent protein kinase, and putative calcium binding protein was analyzed in wheat leaf bases which suggest a specific role for Ca2+ in somatic embryogenesis. Application of the calcium ionophore, A23187, either alone or along with 2,4-D, induced somatic embryogenesis. This specificity for calcium was verified both by treatment with the calcium antagonist TMB8, and the elimination of calcium from the medium, resulting in reduction of somatic embryogenesis by 80%. Treatment with calcium channel blockers like verapamil and nifedipine, calcium antagonist, lanthanum, and calmodulin inhibitors chlorpromazine and fluphenazine, prior to the 2,4-D treatment, inhibited induction of somatic embryogenesis. The present study thus provides evidence for the involvement of calcium-calmodulin in the stimulus-response coupling of auxin-induced somatic embryogenesis in wheat leaf base system.