[Role of exogenous Ca2+ in the somatic embryogenesis of Lycium barbarum L].

In this study, Embryogenic and non-embryogenic calli were separately obtained by cultivation of leaf segments on MS medium containing and not containing 2,4-D 0.2 mg/L. The calli were transferred to an 2,4-D-free MS medium containing different concentrations of 45Ca2+ and EGTA cultured, and microscopic examination of tissue sliced, y-ray energy spectrum analysis, ELISA and two-dimensional polyacry-lamide-gel electrophoresis were used to study the relation between changes in Ca2+ concentration and protein composition changes during somatic embryogenesis. The results showed that: (1) Calli of dedifferentiation were obtained by cultivating in the inductive medium (MS+2,4-D 0.2 mg/ L) and then transferred to the 2,4-D-free (MS) differentiation medium. After cultivating, the large number of embryogenic cells divided and somatic embryogenic calli (EC) were formed (Fig.l); embryogenic cell differentiation and somatic embryo ware not formed when the dedifferentiation calli, which were cultivated in the inductive medium without 2,4-D, ware transferred to the cultivating of differentiation, so calli were called non-embryogenic calli (NEC) (Fig. 3). (2) SE frequency of EC was rised with exogenous Ca2+ concentration was going up, and adding peak value (70.5% to 74.5%) when Ca2+concentration was from 0.8 to 1.6 mmol/L, then SE frequency was dropped markedly with Ca2+ concentration was farther increasing, (Fig.2A). Formation of meristematic cell aggregates of NEC was also enhanced when exogenous Ca2+ concentration was from 0.8 to 1.6 mmol/L (Fig.3). (3) After adding EGTA, which was Ca2+ antagonist, SE frequency was dropped markedly, and SE frequency was fallen along with increased of EGTA concentration. When EGTA concentration went up to 1.2 μmol/L, SE frequency dropped to 5% (Fig.2B), and the formation of meristem-atic cell aggregates on NEC was inhibited. (4) When 2 μCi 45Ca2+/mL was added, the uptake of 45Ca2+ byEC and NEC was different, two uptake peaks of 45Ca2+appeared in EC at the embryo-genie cell differentiation of stage, and the uptake of 45Ca2+ of EC was 4-5 times higher than that of NEC, and the uptake frequency of 45Ca2+ improved from 54.1% to 74.5%. The uptake of 45Ca2+ by NEC during development not only was lower than that by EC but also there were no such marked peak as those with EC (Fig.4). (5) The CaM content examined by ELISA was increased markedly at multi-cellular embryo and globular embryoid stage of EC. After adding Ca2+, the CaM content increased significantly, the CaM content of EC was 2-3 times that of NEC (Fig.5). (6) The IEF/SDS-PAGE results showed that the numbers and amount of protein components were widely different between the two kinds of callus with different morphogenesis patterns, the number of proteins of EC had more components than those of NEC. The largest differences protein species presented with Ca2+ ware added, the more proteins presented on the range of molecular weight was from 43 kD to 66 kD and pI values was from 4.0 to 7.0 (Fig. 6, Table 1).