Study On Somatic Embryogenesis And Ploidy Germplasm Creation In Ziziphus Jujuba Mill.

Various culture factors suitable for the somatic embryogenesis of leaves from Ziziphus jujuba Mill. cv. Dongzao plantlets were studied using solid culture techniques. The mechanisms of morphogenesis of somatic embryogenesis was studied by cyto-histological observation. The pure polyploidies were induced using the established somatic embryogenesis culture system. In addition, ploidy germplasm creation were carried out by anther culture and polyploidy inducing by direct shoot regeneration system from leaves. The main results are as following:1 Somatic embryogenesis culture system of leaves was established for the first time. Callus was induced from leaves of 'Dongzao' plantlets on the solid medium of MS+maltose 20g/L+agar 3g/L+TDZ 10.0mg/L, which could not be subcultured even with adjusted osmotic pressure, hormone, light intensity and anti-brown reagents. Then, one-step method of somatic embroids differentiation from callus was set up successfully. It means that the calli were induced on the solid medium of MS+maltose 20g/L+agar 3g/L+TDZ 10.0mg/L+AgNO₃ 2.0mg/L, subsequently, embryoids differentiated from callus of leaves on the same medium with dark culture for about 30 days. The differentiation rate reached 84.4%. The embryoids germinated to plantlets on the solid medium of MS+sucrose 40g/L+agar 4g/L without any hormones and rooted on the solid medium of 1/2MS+sucrose 20g/L+agar 4g/L+IBA 2.0mg/L, then the integrate plantlets were obtained.2 By the paraffin-cut section method, the somatic embryoids induced indirectly from callus of leaves and originated from one single cell of embryogenic callus. The phenomenon of isolation that embryoids separated from ambient cells was observed. Moreover, developments of somatic embryoids were non-synchronous, different phase embryoids could be seen in the same time. In addition there are three fastigium of granulose accumulation during the course of simatic embryogenesis, they are embryogenic callus, globular embryoid and fish torpedo-shaped embryoid.3 The anther culture of Chinese jujube was carried out. The milk white callus was induced from anthers on the solid medium of MS+maltose 20g/L+agar 3g/L+2,4-D 1.0mg/L. But the milk white callus could not be subcultured. The weak yellow and grain-shaped callus were induced from anthers on the solid medium of MS+maltose 20g/L+agar 3g/L+TDZ 1.0mg/L. The embryoids differentiated from the weak yellow and grain-shaped callus of anthers on the solid medium of MS+maltose 20g/L+agar 3g/L+TDZ 1.0mg/L+AgNO₃ 0.5mg/L. The embryoids from anthers showed serious problem of vitrification, and only one normal plantlet of cultivar 'Zanhuangdazao' embryoids was obtained on the solid medium of MS+sucrose 40g/L+agar 4g/L. According to the identification of DNA content and chromosome number, diploid plantlet was firstly obtained from the diploid pollen of 'Zanhuangdazao'. By histological observation, the somatic embryogenesis of anthers were also originated from the single cell of embryogenic callus and induced indirectly from anther callus. The phenomenon of isolation and non-synchronous of somatic embroids were observed. The abnormal anther embroids were obsereved.4 The pure tetraploid was firstly induced using the established somatic embryogenesis culture system of leaves.The method of dipping in colchicine had great harm to leaves, somatic embryoids could not be induced, when colchicine mixed in medium, the regenerated embryoids from leaves were doubled on the medium supplemented 15mg/L colchicine. By identification of DNA content and chromosome number, the pure tetraploids were obtained with chromosome number 2n=4x=48.5 The polyploidy was also induced using the direct shoot regeneration culture system of leaves. The direct shoot regenerated from leaves on the solid medium of MS+maltose 20g/L+agar 3g/L +BA 1.0mg/L or TDZ 1.0mg/L+AgNO₃ 1.0mg/L. The shoot regeneration rate reached 97.3% and 90.1% respectively. The regenerated shoots were proliferated and rooted on the medium of MS+sucrose 40g/L+agar 4g/L+BA 1.0mg/L+ IBA 0.5mg/L and 1/2MS+sucrose 20g/L+agar 4g/L+IBA 1.0mg/L respectively. The ploidy of the regenerated shoots were doubled on the medium of direct shoot regeneration system supplemented with 15mg/L colchicine. By identification of DNA content, the doubled shoots were chimeras.6 The polyploidy was induced using the shoot regeneration from callus of branches in the field. The shoot regenerated from callus of branches with the treatment of TDZ 4.0mg/L+AgNO₃2.0mg/L. The callus inducing rate could reach 100%, and the shoot inducing rate reached 74.2%. But no regenerated shoots were doubled basing on the identification of DNA content.