枸杞体细胞胚发生中外源Ca2+的作用

脱分化的枸杞叶片外植体愈伤组织转入含有2,4-D的MS培养基上分化培养后有大量胚性细胞的分化和体细胞胚发生;加入一定量的外源Ca2 或45Ca2 ,明显地提高了胚性愈伤组织中体细胞胚发生的频率;加入Ca2 的鳌合剂EGTA则显著降低了体细胞胚发生频率;胚性愈伤组织中CaM的水平在多细胞原胚期和球形胚期显著升高,加入外源Ca2 后CaM含量几乎成倍增加;胚性愈伤组织中蛋白质组分与活性都远远多于或高于非胚性愈伤组织,加Ca2 后蛋白质组分种类也增加.

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 (45)Ca(2+) and EGTA cultured, and microscopic examination of tissue sliced, gamma-ray energy spectrum analysis, ELISA and two-dimensional polyacrylamide-gel electrophoresis were used to study the relation between changes in Ca(2+) 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; 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). (2) SE frequency of EC was rised with exogenous Ca(2+) concentration was going up, and adding peak value (70.5% to 74.5%) when Ca(2+)concentration was from 0.8 to 1.6 mmol/L, then SE frequency was dropped markedly with Ca(2+) concentration was farther increasing. Formation of meristematic cell aggregates of NEC was also enhanced when exogenous Ca(2+) concentration was from 0.8 to 1.6 mmol/L. (3) After adding EGTA, which was Ca(2+) 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 micromol/L, SE frequency dropped to 5%, and the formation of meristematic cell aggregates on NEC was inhibited. (4) When 2 microCi (45)Ca(2+)/mL was added, the uptake of (45)Ca(2+) by EC and NEC was different, two uptake peaks of (45)Ca(2+)appeared in EC at the embryogenic cell differentiation of stage, and the uptake of (45)Ca(2+) of EC was 4-5 times higher than that of NEC, and the uptake frequency of (45)Ca(2+) improved from 54.1% to 74.5%. The uptake of (45)Ca(2+) by NEC during development not only was lower than that by EC but also there were no such marked peak as those with EC. (5) The CaM content examined by ELISA was increased markedly at multi-cellular embryo and globular embryoid stage of EC. After adding Ca(2+), the CaM content increased significantly, the CaM content of EC was 2-3 times that of NEC. (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 Ca(2+) 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.