The Regeneration System Establishment And The Genetic Transformation Study Of The Calendula Officinalis L.

Calendula officinalis L. is belong to the Calendula of Compositae, a kind of annual or biennial herbaceous plant. As an early spring leading flower, Calendula officinalis L. originated in the Europe for a relatively long history and widely cultivated as an ornamental flower in the garden and along both sides of the street in countries. In our country, Calendula officinalis L. introduced from abroad since the 18th century and then widely used as an ornamental plant in landscape. With the introducing of larg size, usuallay double and low-growing herb, Calendula officinalis L. has become an important herbaceous plant in our country Since the 1980s and then takes on a new look. Besides as a type of ornamental flower, Calendula officinalis L. also can be used as a nice type of economical plants such as cosmetic and medicine and it brings good prospect for the future.But Calendula officinalis L. is apt to suffer from insect pests, especially when it blooms early in the spring. So Calendula officinalis L. was limited in farther developing. Therefore, the objective of this study is to transfer the kunitz trypsin inhibitor Cpkti gene to Calendula officinalis L. and to obtain new types of Calendula officinalis L. with characteristic of insect-resistant through the study of regeneration system and transformation system mediated by agrobacterium tumefaciens of Calendula officinalis L., and established a foundation of wide application of improving Calendula officinalis L. cultivars applying transgenic method. Furthermore, the transgenic Calendula officinalis L. can be widely applied into use around the country and serve the gardens.This study use the leaves of Calendula officinalis L. as the explant to probe into the system of regeneration and genetic transformation of Calendula officinalis L.. The main research work and results were as follows:1 The regeneration system establishment of Calendula officinalis L.With infancy leaves of Calendula officinalis L. as explants, MS was used as the culture medium and it was upplemented with 6-BA and NAA at different concentrations proportion for designing different cultivars media in order to study the optmium differentiation conditions of leaf discs of Calendula officinalis L.. Consequently, the efficient regeneration system of Calendula officinalis L. was established.The surface sterilization measure of infancy leaves of Calendula officinalis L. was in 75% alcohol solution 30 second and then in 0.1% mercuric chloride solution 10 minutes. The best callus inducing medium was the MS medium with 6-BA 1.0 mg/L and NAA 0.1 mg/L,3% cane sugar and 7g/L agar. Under such conditions, the callus inducing rate was 100%. The optmium differentiation condition was the MS medium with 6-BA 0.5 mg/L and NAA 0.1 mg/L,3% cane sugar and 7g/L agar. Under such conditions, the differentiation rate was 90% and the propagation coefficient was 4.5. The best root development medium was the 1/2MS medium with NAA 0.1 mg/L,3% cane sugar and 7g/L agar. After about 12 days, the rooting rate was 100%.2 The genetic transformation system study of Calendula officinalis L.Susceptibility to antibiotics of Calendula officinalis L. had been studied based on it's efficient regeneration system. The results showed that the regeneration system of Calendula officinalis L. was sensitive to kanamycin, in which, 10.0mg/L kanamycin could inhibit the differentiation of leaf discs, and 10.0mg/L Kanamycin inhibited rooting of seedlings. Consequently, 10.0mg/L Kanamycin can be used in the selecting culture medium. The experiments indicated that, as a kind of antibiotic,500-300mg/L carbenicillin had little distinct influence on the regeneration frequency and quantity of Calendula officinalis L.,but it was able to efficient restrain the growth of agrobacterium tumefaciens.Genetic transformation was preliminarily studied by using leaf discs by co-cultivation with Agrobacterium tumefaciens. The study probed into several factors effect on the genetic transformation rate of Calendula officinalis L., such as the time of pre-culturation,OD600 of agrobacterium,the time of infecting and co-cultivation. The results showed that these factors were critical on the genetic transformation rate of Calendula officinalis L.. The optimal method of transformation system was as follows:After the bacterial liquid whose OD600 was 1.0 by the second activation was centrifuged, thalli were collected. The OD600 of bacterial liquid should be about 0.4 after thalli was resuspended in liquid MS and AS100μm/L and MES 0.1%(PH=7.0) Then the leaf discs that had been pre-cultured for 2 days were put into the resuspended bacterial liquid to be infected for about 15 minutes in the shaking bed. These leaf discs were transferred into the medium MS and 6-BA 1.0mg/L and NAA 0.1mg/L in the darkness co-cultivated for 2 days. And then, it was the time to wash bacteria off on the surface of leaf discs in the liquid MS+Cef 500 mg/L for 30 minites. After infection, delayed cultivation for 2 days were suitable for transformation. It was important to inerease antibiotic from 5mg/L Kan to 10mg/L Kan step by step. And 500mg/L Carbenicillin was suitable at the beginning then decreased to 300mg/L. About one centimeter transgenic seedlings was transformed into 1/2MS with 10mg/L Kan for regenerating roots. Integration of Cpkti gene into Calendula officinalis L. was identified by detecting the transient expression of GUS gene and using PCR amplification. The transgenetic seedlings were obtained.