Studies On The System Establishment Of Tissue Culture And Transformation For Pyrethrum Cinerariifolium Trev.

Pyrethrum （Pyrethrum cinerariifolium Trev.） is one kind of beautiful flowers with high ornamental value, and also it is an ancient natural insecticide plant. For the germplasm conservation and the pyrethrum industrial production, the syetem of pyrethrum tissue culture had been set, using the orthogonal test on the propagation of two pyrethrum genotypes. Screened on genotype, hormones and other conditions, we established an efficient regeneration pyrethrum system and studied on planta transformation of different conditions. Main results were as follows:1. The establishment of rapid propagation system for pyrethrumIt was studied on the impact of 6-BA, NAA, sucrose concentration on the rapid propagation of pyrethrum genotypes CK-30 and 3-22, using the stems with buds as explants. The results showed that the best medium for the rapid propagation of two pyrethrum genotypes was MS + 6- BA 4 mg / L + NAA 0.3 mg / L + sucrose 3%. The genotype not only had the highest propagation rate but also had no vitrification. The best medium for rooting was 1/2 MS. During the transplant, sterile sand before transplanting into the soil can help to increase the survival rate reached to 100%.2. The establishment of regeneration system for pyrethrumThe leaves and petioles as explants for pyrethrum genotypes of TCl1-9, No.34, No.39, 3-22 and 3-32 were used for the direct and indirect regeneration. The different genotypes and different appendages were compared on the impact of pyrethrum regeneration. The results showed that there existed differences on the pyrethrum direct regeneration among different genotypes. The shoots were induced 15 days after inoculation and the regeneration rate was 96% from genotype No.39; The shoots induced from genotype No.34 appeared about 30 d and the regeneration rate was 30%. The regeneration rate of genotype 3-32 was 12% and genotype 3-22, TCL1-9 did not get any shoot. Thus, pyrethrum genotype was one of the most important factors for the regeneration. In addition, AgNO₃ also had influence of direct regeneration. The regeneration rate from genotype No.34 was 34% under the concentration of AgNO₃ was 2 mg/L, and significantly higher than the medium without AgNO₃. For pyrethrum indirect regeneration, callus induction was easy to reach 95% above, but in the medium with different growth regulator, no shoot was induced ultimately.3. Studies on the planta transformation of pyrethrumOne cotyledon from each germinated seed was removed by excision near the inter-cotyledonary region, thereby producing a clear wound site adjacent to the plumule, cotyledonary node and the hypocotyl for inoculation. Seedlings with one cotyledon emoved were submerged in the Agrobacterium suspension for several minutes, blot-dried and co-cultured. After co-culture, the seedlings were washed by sterile distilled water and then transferred upright in MS medium containing cefotaxime at 200 mg/L. After 4 pieces of leaves were grown, the seedlings were used for Histochemical analysis of GUS expression.Analyzed of transient expression of GUS gene, the factors on planta transformation of pyrethrum were discussed, the results showed that the infection time, co-culture time, and the inoculating ways all have certain impact on the transfomation efficiency. The transient expression activity of Gus was the highest and reached to about 100% with infected 20 min and co-cultured 3 d and inoculated transverse.