Primary Studies On Friable Embryogenic Calli Induction, Long-term Subculture And Plant Regeneration In Hevea Brasiliensis

Embryogenic calli induced successfully from explants of Hevea brasiliensis can provide sufficient and excellent materials for establishment of cell suspensions and protoplast culture. Juvenile self-root clones obtained from somatic embryogenesis are uniform and can be used as rootstocks. An efficient plant regeneration system of rubber tree is of great significance for the establishment of genetic transformation system and production of juvenile self-root clone and rootstock clone in large-scale. In this study, the tissue culture system of rubber tree was systematically studied, embryogenic callus was induced, embryogenic cell suspensions and plant regeneration system were established. Based on them, cryopreservation and plant regeneration from post-thaw embryogenic callus were studied. In addition, the different stages of somatic embryo induced from friable embryogenic callus were observed by histological paraffin section, and changes in morphological structure and characteristics were examined microscopically. The purpose of this study is to provide the theoretically and practically scientific data for cell engineering research and genetic transformation of rubber trees, and provide new ways and conditions for creating new excellent germ plasm resources. The main results are the following:Induction of friable embryogenic callus:The anthers of the rubber tree clone Reyan 8-79 at uninulear stage were placed on 100×20 mm-Petri dishes containing M1 medium in the dark at 26±2℃for inducing callus. Ml medium consisted of modified MS basal medium, 2.0 mg/L 2,4-D (2,4-Dichlorophenoxy acetic acid),1.0 mg/L NAA (a-naphthalene acetic acid),1.0 mg/L KT (kinetin),0.1 g/L inositol,70 g/L sucrose,5%(v/v) CW(coconut water) and 2.0 g/L phytagel. After 50 days, the primary calli with good appearance and bright-yellow color induced from anther were transferred onto fresh M1 medium and subcultured every 10 days, after several subcultures of selection, embryogenic calli were obtained. The inner integuments from young fruit (45～75 days after flowering) of the rubber tree clone Reyan 88-13 were placed on 100×20 mm-Petri dishes containing M2 medium in the dark at 26±2℃for inducing callus. M2 medium consisted of modified MS basal medium,1.0 mg/L 2, 4-D,1.0 mg/L BAP (6-benzyl amino purine),0.1 g/L inositol,70 g/L sucrose,5%(v/v) CW and 2.0 g/L phytagel. After 50 days, the primary calli with good appearance and bright-yellow color induced from inner integument were transferred onto fresh M2 medium and subcultured every 10 days, after several subcultures of selection, embryogenic calli were obtained.Establishment of embryogenic cell suspension:about 2 g embryogenic calli of the rubber tree clone Reyan 8-79 were transferred into a 100 mL Erlenmeyer flask containing 20 mL liquid M3 medium which consisted of modified MS basal medium, 1.0mg/L 2,4-D,1.0 mg/L NAA,0.5 mg/L BAP,0.1 g/L inositol,50 g/L sucrose and 5%(v/v) CW. Cultures were maintained on a rotary shaker (100 rpm) in the dark at 26±2℃and subcultured every 5-7 days. Only newly formed small cell aggregates were selected and transferred into new M3 medium at a dilution of 1:6 (inoculum:fresh medium) until a homogeneous and stable embryogenic cell suspension was established.Optimization of embryogenic cell suspension cultures:The effects of inoculum density, rotation velocity, sucrose concentration, liquid volume in flask and different concentrations of plant growth regulators had been studied. The optimal conditions for the growth of embryogenic cell suspension of the rubber tree clone Reyan 8-79 were the following:25 g/L cell FW,100 rpm,50 g/L sucrose,150 mL Erlenmeyer flasks with the liquid volume of 40mL, optimized medium for suspension culture was M4 medium which contained modified MS basal medium,1.0 mg/L 2,4-D,0.5 mg/L NAA,1.5 mg/L KT,0.1 g/L inositol and 5%(v/v) CW.Induction of friable embryogenic callus from the embryogenic cell suspension:The embryogenic cell suspension from the anther calli of the rubber tree clone Reyan 8-79 had been established. The friable embryogenic callus inducing percentage was 100% when suspension cell cultures were inoculated on M5 medium [modified MS medium supplemented with 1.5mg/L 2,4-D,1.0 mg/L NAA,0.5 mg/L BAP,0.1 g/L inositol,50 g/L sucrose,5%(v/v) CW and 2.0 g/L phytagel].Maintenance of embryogenic cell suspension cultures and friable embryogenic calli: Long-term subculture can be achieved by combined solid and liquid medium culture alternately, embryogenic cell suspensions and friable embryogenic calli of the rubber tree clone Reyan 8-79 were improved. Embryogenic cell suspensions and friable embryogenic callus lines of the rubber tree clone Reyan 8-79 were subculture for 2 years by this method, which grew well and maintained the embryogenic character.Induction and maturation of somatic embryos:The friable embryogenic calli of the rubber tree clone Reyan 8-79 were transferred onto optimized medium for somatic embryogenesis (M6) [modified MS medium supplemented with 3.0 mg/L KT,0.01 mg/L NAA,0.97 mg/L BAP,0.1 mg/L GA (gibberellic acid),0.1 g/L inositol,5%(v/v) CW and 2.0 g/L phytagel], high frequency of somatic embryogenesis was observed. After 1-2 times of subculture, somatic embryos developed to be mature.Germination and plant regeneration of somatic embryo:The mature somatic embryos of the rubber tree clone Reyan 8-79 were transferred onto medium for regeneration (M7) [modified MS medium supplemented with KT 0.5mg/L, IAA 0.2 mg/L, GA 0.5mg/L,50 g/L sucrose,5%(v/v) CW,2.0 g/L phytagel], an effective system for conversion of somatic embryos to regenerated plantlets was established. The rate of plant regeneration was 46.8%.Cryopreservation of friable embryogenic calli:The optimized cryopreservation procedure:the friable embryogenic calli of the rubber tree clone Reyan 88-13 after 15 days of subculture were transferred onto medium for preculture [M8 supplemented with 90 g/L sucrose and 5%DMSO (dimethylsulfoxide)] for 3 days at 4℃. Then they were pretreated with 60% PVS2 for 30 minutes at room temperature and dehydrated with PVS2 for 40 minutes at 0℃. Afterward the friable embryogenic calli were plunged into liquid nitrogen directly and conserved for 3 days. Then they were taken out from the liquid nitrogen rapidly and were thawed in a water bath at 40℃. They were washed three times with M5 containing 90 g/L sucrose(without phytagel). TTC analysis and recovery growth were employed. Friable embryogenic calli could be proliferated from the post-thaw calli. Then Somatic embryos were induced. And now plant regeneration is being induced.Histological paraffin section analysis of rubber tree:Friable embryogenic callus, non-embryogenic callus and several major developmental stages of somatic embryogenesis of rubber tree were analyzed and observed microscopically by paraffin sections, such as globular embryo, heart-shaped embryo, torpedo-shaped embryo and cotyledon embryo.