The Establishment Of Transformation System For Pyrethrum Cinerariifolium With GUS Gene Mediated By Agrobacterium Tumefaciens

Pyrethrum (Pyrethrum cinerariifolium Trev.) is a perennial herb of Compositae family. It is an ancient and natural insecticidal plant cultivated in the world. With the tissue culture technology,we can effectively preserve the fine varieties of pyrethrum to meet the factory production.In this paper, the regeneration system of ’No.39’genotype was further stabilized and optimized by studying the ratio of different concentrations of 6-BA and IBA, dark culture time, and the concentrations of AgNO3. Moreover, the transformation system of pyrethrum was initially established by studying the concentration of kanamycin, the concentration of cefotaxime, dark culture time, the pre-culture time and infection time. PCR and GUS staining were used to determined the resistant seedlings. The Main main results were are as follows:1. Optimizing the regeneration system of pyrethrum’No.39’genotypeFor further stabilizing and optimizing the regeneration with the leaves of ’No.39’genotype, we studied the effects of different concentrations of 6-BA and IBA, dark time and AgNO3 on the regeneration system of’No.39’genotype. The results showed that 6-BA and IBA were significantly influenced on the regeneration.When the concentrations of 6-BA and IBA were 2.0mg/Land 0.2 mg/L, the regeneration was 86.7% and the average number of buds/explant was 1.5. Low concentration of AgNO3 played a significant role in promoting regeneration of pyrethrum’No.39’genotype. When the concentration of AgNO3 was 2mg/L, the regeneration of leaves was the highest (up to 90%), the average number of buds/explant was 2.4. The concentrations of 6-BA and IBA and AgNO3 were basically consistent with previous studies, but the dark time was slightly different. When the dark culture time was 2 weeks, the regeneration of leaves was highest (up to 90%), the average number of buds/explant was 2.2. Therefore, we selected the best combination of of 6-BA2.0mg/L and IBA0.2mg/L, the concentration of AgNO3 was 2mg/L, the dark time culture was 2 weeks.2. Initial establish the transformation system of pyrethrumWe studied the effects of the concentration of kanamycin and cefotaxime, dark culture time, pre-culture time and infection time on the transformation system of pyrethrum using the leaves of’No.39’genotype. The results showed that leaves were very sensitive to kanamycin, and the concentration of 10mg/L almost completely inhibited the regeneration of non-transformed seedlings. The pre-culture time can significantly improve the conversion rate of pyrethrum, but the time could not be too long. The infection time can also influence the conversion rate of pyrethrum. Finally, the optimum conversion conditions were selected and they were kanamycin 10mg/L cefotaxime 20mg/L, the dark culture time 2 weeks, the pre-culture time 2 days, and the infection time 5 minutes.3. The PCR and GUS staining of the resistant seedlingsWe selected 8 resistant seedlings obtained from pyrethrum’No.39’genotype and extracted DNA by CTAB, then tested the DNA by PCR. The results showed that the 8 resistant plants were PCR positive plants. GUS staining was used to test the leaves of the resistant seedlings, we found that only one positive plant had the leaves of (the No.4) had blue reaction, others had no blue reaction. The we used the whole seedling for GUS staining, the roots, stems and leaves of the seedling were all dyed blue. Therefore, it indicated that the GUS gene had been steadily transferred to the pyrethrum’No.39’genotype.