Identification of thidiazuron-induced ESTs expressed differentially during callus differentiation of alfalfa (Medicago sativa)

Yellowish embryogenic callus, which had been induced from the petioles of alfalfa (Medicago sativa L. cv. Jinnan) and maintained on kinetin (KN)-containing B5h medium, turned non-embryogenic with the differentiation of tracheary elements when KN in the medium was replaced by thidiazuron (TDZ). To investigate the molecular mechanisms underlying the TDZ-induced morphological competence transition, we cloned the differentially expressed sequence tags from TDZ-treated alfalfa callus using suppression subtractive hybridization. The gene expression patterns induced by TDZ were found to change throughout the period of TDZ treatment. TDZ induced the expression of stress-related genes, including trehalose-6-phosphate phosphatase (TPP) gene, 1-aminocyclopropane-1- carboxylate synthase (ACS) gene and proline dehydrogenase gene, in both early days (day 2) and late days (day 28) of treatment, which are suggestive of the stress responses from the TDZ-treated callus. The TDZ-induced expression of TPP, which tends to lower the level of trehalose-6-phosphate, and ACS, the gene involved in ethylene production, appears to be the major cause for the termination of somatic embryogenesis.