Study On Callus Inducement And Protoplast Culture Of Rose(Floribunda Rose)

Floribunda Roses(Rosa L.) are one kind of perennial woody ornamentals, which are significant for city virescence. Because the temperature is low in the north of china, there are few cultivars of Floribunda Roses can live through the winter.Modern roses have a narrow genetic base, and traditional breeding methods need to cost a long term, which delays the development of rose breeding. Bioengineering offers a new approach for breeding work. By transferring new good genes of other sources into our plants, we can improve plants species in little time.This study discussed the factors that affected callus inducement of Floribunda Rose and acquired embryogenic callus. The cell suspension culture was established using embryogenic callus. On that base protoplasts were isolated from embryogenic callus, cell suspensions, and leaves. The fators that affected protoplast isolation were discussed. We were aimed to acquire regenerated plants from protoplasts, and provide base for cold-resistant species improvement and somatic hybridization breeding work. This study included :1. Establishment of micropropagation of Floribunda rose: The best medium for the germination of axillary bud and shoot multiplication was MS+6-BA2.0mg/l+NAA0.05mg/l, which was fit for shoot multiplication of 4 species. Shoot multiplication times were larger than 3.8. The best rooting medium was 1/2MS+ NAA0.05mg/l.2. Immature leaves , stem segments, immature seeds and immature embryos were used as explants to induce callus. Conclusions were : For Floribunda rose 8, the best callus inducement medium for leaf was MS+2,4-D2.0mg/l+TDZ0.5mg/l+ L-proline 300mg/l+sucrose 30g/l, and in the dark, the best subculturing medium for callus proliferation was MS+2,4-D1.0mg/l+TDZ 0.5mg/l,after 2 to 3 times subculturing, embryogenic callus formed. Immature stem segments were not fit for callus inducement. For Floribunda rose 1, the best callus inducement medium for immature seeds was MS+2,4-D3.0mg/l, subcultured on medium MS+2,4-D 0.5mg/l, cotyledon-like embryogenic structure formed, transferd to medium MS+6-BA1.0mg/l+ IBA0.1mg/l , adventitious shoots regenerated. Callus induced from immature embryos was bad. There were significant differences of callus inducement among different genotypes.3. Cell suspension culture was established: Subculturing must be performed at an interval of 4 to 5 days at first. Later the stable cell suspension culture had to be subcultured at an interval of 8 to 10 days. The best inoculum quantity was 1ml PCV(Packed cell volume) cell suspensions per 40ml culture fluid. There were no significant differences of growth characteristics between different genotypes.4. Protoplasts were isolated from immature leaves, embryogetic callus and cell suspensions. The best isolation condition for leaves was 2.0%Cellulase Onozuka R-10+1.0%Hemicellulase +0.05% Pectolyase Y-23+0.1%Macerozyme R-10+ 0.7mol/l mannitol, isolation for 14h; The best isolation condition for embryogetic callus was 1.0%～2.0% Cellulase Onozuka R-10+1.0% Hemicellulase +0.1% Pectolyase Y-23 +0.05% Macerozyme R-10+0.6 mol/l mannitol, isolation for 16h; The best isolation condition for cell suspensions was 2.0% Cellulase Onozuka R-10+1.0% Hemicellulase+0.1%～0.3%Pectolyase Y-23+0.1%Macerozyme R-10+0.6mol/l mannitol, isolation for 16h.5. Physiological character of material and pretreatment had effects on protoplast isolation: Protoplast yield of immature leaves was higher than mature leaves; culture days after subculturing and subculturing times were related to protoplast isolation. Pretreatment in osmoticum solution was propitious to protoplast isolation; darkness pretreatment was no effects; with coldness pretreatment protoplast yield decreased.6. Protoplasts culture was conducted: When solid-liquid two layer- midium culture method was used, culture density was 1×105 protoplasts ml-1 , and on medium KM8P +2,4-D0.5mg/l, protoplasts regenerated cell wall, but the cell failed to divide.