Studies On Establishment Of Regeneration System And Co-transformed By Electroporation In Hevea Hybrid B1 Suspension Cells

Hevea Brasiliensis is the cross-pollination of perennial trees, because of the breeding cycle long, narrow genetic base, polygene heterozygous state, the traditional of cross-breeding and the utilization of heterosis is limited. Currently, the variety breeding is mainly used conventional breeding, which materials come from the weik han system of strain and its derived clones.Therefore, exploring other breeding methods to shorten the breeding cycle, improving breeding efficiency and overcoming the traditional breeding defects that is important significance. There are many advantages expected from cell suspension cultures system over the conventional cultivation of whole plants, such as faster growth rate, direct contact with the medium nutrients, high dispensability and uniformity, independence from various environmental and seasonal factors, cell contents enrichment, good experimental reproducibility and convenient harvest. Therefore, cell suspension cultures not only can be directly used for protoplast isolation, culture and hybridization, obtained secondary metabolites, large-scale clonal propagation, but also select mutants in the short term. This study establishs hybrid cell B1(Hevea brasiliensis×H.nitida) regeneration system and uses cells co-transformation by electroporation.Our objectives are to provide the theoretic and pratical scientific data for cell engineering research of Hevea rubber. The main results are as follows:1. Embryogenic cell suspension cultures subculture:Take 10mL of cell suspension cultures are inoculated into 100mL erlenmeyer flask and dry adsorbed medium, then add 20mL of fresh suspension culture medium(M1)[modified MS medium and add 2.0mg/L 2,4-D, 1.0mg/L NAA, 1.0mg/L KT,0.1 g/L inositol,70g/L sucrose,5%(V/V)coconut water]. Cells are maintained on a 7-9-day subculture. If change the inoculation volume and rotation speed, its can reduce the frequency of vaccination.2. Cell suspension induce embryogenic callus:suspension cells are inoculated into the callus medium(M2)[modified MS medium and add 1.5mg/L 2,4-D, 1.0mg/L NAA,0.5mg/L BAP,0.1 g/L inositol,70g/L sucrose,5%(V/V) coconut water, 2.2g/L phytagel]that can be induced callus. The induction rate up to 100%. Putting the callus into M2, they can grew well and embryogenic will not disappear when subculture and proliferative in a long-term.3. Induction and maturation of somatic embryos:The friable embryogenic calli of the rubber tree species B1 are inoculated into medium for somatic embryogenesis (M3)[modified MS medium containing 0.9mg/L KT,2mg/L BAP，0.01mg/L NAA,0.1 mg/L GA,0.1 mg/L inositol,70g/L sucrose,5%(Ⅴ/Ⅴ) coconut water, 2.2g/L phytagel], then can induce somatic embryogenesis. Following the generation of 1 or 2 times, somatic embryos develop to be mature.4. Plant regeneration:The mature somatic embryos of the rubber tree species B1 are inoculated into medium for regeneration(M4)[modified MS medium and add 0.5mg/L KT,0.2mg/L IAA,3.0mg/L GA3,70g/L sucrose,5%(Ⅴ/Ⅴ) coconut water,2.2g/L phygageI], then can induce plant regeneration. The plant regeneration rate up to 16.6%.5. Electroporation system optimization:Using the uniform design with electric field strength (X1) 18 levels, capacitor (X2) and sucrose concentration (X3) 2 factors in 6 levels, resistance (X4), growth state (X5), DNA concentration (X6), vacuum pressure (X7) 4 factors and 3 levels of each factor as investigation factors, with transient expression (Y1) and cell survival rate (Y2) as the dependent variable and compose of consolidated results(Y1+Y2), then optimization of electroporation system parameters. It obtaines the best system for electroporation:Under the electric shock conditions of lOuF,2Kn,1300v/cm, a final concentration of 90 ug/ml linear plasmid CaMV35S-EGFP-NOS in 0.4M sucrose electroporation solution and one minute of 600mbar vacuum-pumping, logarithmic phase suspended cells, cultured 3d, could reach an instant expression efficiency and survival rate in 0.6555% and 57.9262% respectively.6.Analysis on co-transformation by electroporation:Using the electric shock parameters and coconversing marker gene expression cassette (GFP or GUS): gene expression cassette (HbCBF1):gene expression cassette (Kana)-1:1:1 total concentration by electroporation,obtaining conversion of callus successfully. When 95 callus are extracted and PCR, they have 10 callus with positive. The primary conclusion that conversion efficiency up to 1.05%, but it do not get transgenic plants.