Establishment Of Micropropagation Technique System Of Rosa Rugosa

Rosa rugosa stem with axillary bud and tender leaf was used as explant, tissue culture of Rosa rugosa was studied systematically in the article in order to set up the technique for commercial production of Rosa rugosa and provid technique support for molecular breeding and correlation study of Rosa rugosa.Ⅰ. Rosa rugosa stem with axillary bud was used as explant during the establishment of micropropagation technique system. The drawing material time of explant, the degerming results using different germicidal treatment programs, the browning phenomenon of explant and the control of browning method in different culture stages, the effect of culture conditions on plantlets, the effect of polyphenol content and polyphenol oxidase activity on explant browning and in vitro rooting, the effect of acclimatization disposal and matrix on survival rate, the optimal primary medium, the optimal proliferation medium and the optimal rooting medium was studied systematically. The results indicate that:1) The optimal drawing material time for Rosa rugosa tissue culture is April. In this period, the contamination rate of explant is low and the priming rate is high.2) Drawing Rosa rugosa explant in April, the optimal germicidal treatment program is T5 treatment, that is: 75% C2H5OH 1min+0.1% Hgcl2 (add tween-20) 10min.3) There is the identical law for the effect of plant growth regulating substance on the priming rate and the average growth of different Rosa rugosa cultivars. The optimal primary medium for primary culture of Rosa rugosa is MS+6-BA 0.5 mg/L+NAA 0.1 mg/L+GA3 0.4 mg/L. 4) The browning degree of different Rosa rugosa cultivars and the different bud locations of one Rosa rugosa cultivar is different, but there are"V"type change in the browning degree with the bud location of different Rosa rugosa cultivars and all the peak amplitude is the fourth bud. The optimal browning inhibitor in primary culture and rooting culture is 1.5 g/L active carbon (AC). The optimal browning inhibitor in proliferation culture is 0.2g/L ascorbic acid (Vc). Once the medium added AC, the plantlets leaves become more and more greener and glosser, and lignification degree of the plantlets is higher, so AC have the positive effect on primary culture and rooting culture of plantlets.5) There are same effect tendency of plant growth regulating substance on the height and the proliferation of different Rosa rugosa plantlets. There are prominent promotion effect of 6-BA with suitable density to the height and the proliferation coefficient, 6-BA is the dominating effect factor to proliferation of plantlets, but NAA is not the dominating effect factor, and GA3 is the important effect factor to proliferation of plantlets. The optimal proliferation medium of Rosa rugosa is MS+6-BA 1.0 mg/L+NAA 0.05 mg/L+GA3 0.8 mg/L.6) Adequate intensity of illumination is the essential condition for normal growth of plantlets. The optimal intensity of illumination scope for tissue culture of Rosa rugosa plantlets is 2000～3000LUX. The minimum intensity of illumination for the normal growth of plantlets is 2000LUX. The maximum intensity of illumination which ensure plantlets with relative higher proliferation coefficient is 3000LUX.7) The proliferation coefficient of plantlets begin to decline from the sixth proliferation culture, the proliferation coefficient of plantlets is continue to decline with the increase of proliferation culture frequency. The plantlets should be renewed regularly during commercial production.8) The effect of browning degree on plantlets rooting index is significantly negative. Polyphenol content, polyphenol oxidase activity and rooting index of different cultivars were different significantly. Polyphenol content, polyphenol oxidase activity was correlated negatively to rooting index respectively, and polyphenol oxidase activity has more influence to rooting index than polyphenol content. Both polyphenol oxidase activity and rooting index had significant correlation with pH, and the optimal pH of PPO was 6.5, while the optimal pH of in vitro rooting was 5.5.9) The effect of NAA on rootage of different Rosa rugosa plantlets is significant. IBA has smaller effect than NAA on rootage of plantlets. The optimal rooting medium of Rosa rugosa plantlets is 1/2MS+NAA 0.2 mg/L+IBA 0.5mg/L+AC1.5 g/L.10) Acclimation treatment can improve the survival rate significantly in the transplant of plantlets and it is the indispensable process before transplant of Rosa rugosa plantlets. The optimal matrix for the transplant of Rosa rugosa plantlets is pearlite: vermiculite: grass peat=1:2:1.Ⅱ. Rosa rugosa tender leaf was used as explant during the establishment of the callus inducement and plant regeneration system. The dynamic process of Rosa rugosa leaf callus was described in detail in the article. The effect of dark-induced, the inoculation location and the inoculation direction of the leaf,and plant growth regulating substance on the leaf callus inducement was studied in detail. The effect of dark-induced and plant growth regulating substance on plant regeneration was studied in detail. The results indicate that:1) After 20 days culture,there are some flavo-green callus at the leaf lump with leafstalk, and then the callus differentiate into three different kinds gradually in the following culture, one callus which is flavo-green, compact and granular can been induced adventitious bud in the adventitious bud inducement medium without secondary culture.2) Dark-induced has great effect on the callus inducement of the inoculated leaf. Under the experiment conditions, the optimal dark-induced time of is 3 weeks.3) Callus regeneration ability is different in different parts of the same leaf. The sequence of regeneration ability is: leaf base>leaf midst> leaf apex.4) The inoculation direction of the leaf has no distinct effect on the callus inducement.5) 2, 4-D is the dominating effect factor to the callus inducement rate and the callus growth ; 6-BA is the dominating effect factor to the callus quality. The optimal inducement medium of Rosa rugosa leaf callus is MS+2, 4-D1.0mg/L+6-BA0.5 mg/L.6) Dark-induced is not the necessary factor in the regeneration of Rosa rugosa leaf callus, but adequate dark-induced is benefit to the adventitious bud regeneration of Rosa rugosa callus.7) Plant growth regulating substance have great effect on the adventitious bud regeneration of Rosa rugosa callus, the necessary factor in the adventitious bud regeneration of Rosa rugosa leaf callus is TDZ. The optimal inducemrnt medium for the adventitious bud regeneration of Rosa rugosa callus is TDZ2.0mg/L+IBA0.1mg/L.