Studies On System Of Rapid Propagation And Shoot Regeneration Of Leaves In Raspberry In Vitro

Four varieties of raspberry such as 'Chilliwack','Dinkum','Kotata' and 'Shawnee' whose shoot tips and stem segments were used as material to set up the non-bacillus propagation system through initial induction, subculture proliferation and rooting cultured. By means of the orthogonal experiment design, analysis of variance and multiple comparisons, the factors including genotypes, basic media, plant growth regulators, dark periods and AgNO₃ concentrations were analyzed and the ideal regeneration system of raspberry by leaf explants was established. The results were as follows:(1)The stage of explants building: 'Kotata' whose rate of shoot survival was 48.29% was the highest rate shoot regeneration of breed among the four varieties, 'Shawnee' followed(41.73%). MS supplemented with 1.0mg/L BA, 0.2mg/L NAA and 0.5mg/L GA3 was the best combination to both 'Kotata' and 'Shawnee' .The highest rates of shoot survival of 'Chilliwack' and 'Dinkum' were 31.1% and 21.53% each and the best combination for the two varieties was MS supplemented with 1.0mg/L BA and 0.1mg/L NAA. It could get the conclusion that MS supplemented with 1.0～2.0mg/L BA and 0.1mg/L NAA was better to set up raspberry non-bacillus propagation system.(2) The stage of proliferation: for 'Chilliwack', shoot proliferation best on MS supplemented with BA1.0mg/L and NAA0.1mg/L. For 'Dinkum', shoot proliferation best on 1/2MS supplemented with BA1.0mg/L and IBA0.4mg/L. For 'Kotata' and 'Shawnee', MS supplemented with BA2.0mg/L and IBA0.2mg/L was the best combination.(3)The stage of rooting: the effect of rooting of 'Kotata' was better than the other three varieties. 1/4MS was the best among the four rooting basic mediums. Sugar was necessary for rooting. Auxin and AC were also necessary in the earlier stage of rooting, but not necessary in the later.(4)According to the observation of the shoot regeneration of leaves in four varieties of raspberry, 'Chilliwack' showed highest rate of regeneration, 'Kotata' followed, but there was on buds regenerated in 'Dinkum' and 'Shawnee'. During the 32 combinations, NN69 supplemented with BA1.0mg/L and NAA0.2mg/L was best for 'Chilliwack' with regeneration rate of 49.67%. For 'Kotata', shoot regeneration on NN69 supplemented with BA2.0mg/L and IBA0.2mg/L with12.13%.(5)According to the analysis of variance and multiple comparisons, two kinds of cytokines such as TDZ and BA had different effects in shoot regeneration of leaves in raspberry. The result showed that BA is better than TDZ when it referred to leaves culture.(6) Compared with the effect of four basic mediums to buds regeneration of leaves, it found that MS was better to bud regeneration for its higher concentration of salt and N ( the rate of bud regeneration of 'Chilliwack was 64.13% and the rate of bud regeneration of 'Kotata' was 23.21%). However, the other three basic mediums all lead to rate of bud regeneration of 'Chilliwack' and 'Kotata' descend.(7) Dark culture could improve the rate of shoot regeneration of leaves in raspberry. The rate of shoot regeneration of leaves in 'Chilliwack' rose from 62.00% to 83.32% when deal with dark culture 14 days, so does 'Kotata'. But the rate of regeneration of leaves would descend with the period of dark culture extending.(8) Adding 0.5mg/L～2.0mg/LAgNO₃ to basic medium can cause rate of shoot regeneration of leaves in 'Chilliwack' and 'Kotata' decrease and the higher concentration added, the more negative effect indicated. When the concentration of AgNO₃ reached 2.0mg/L, the rate of shoot regeneration of leaves in Chilliwack' was only 9.98% which descended 51.19 percents compared with the check. Similarly, the rate of shoot regeneration of leaves in Chilliwack' decrease from 22.10% to 7.42%. At the same time, AgNO₃ could change the method of regeneration from callus bringing buds to generating buds directly.