| Botanically, Artemisia annua L., an annual herb, is one member of the genus of Artemisia, which belongs to the family of Asteraceae. It was recorded in many medical books as traditional Chinese medicine. Artemisinin, a sesquiterpene lactone endoperoxide derived from the acrial part of the plant A. annua, is the most effective anti-malarial drug. However, the low content of artemisinin in the plants leads to high cultivation cost, which hampers its cultivation and causes short supply of artemisinin in the commercial market. There are many countries aiming at the research of A. annua with high content of artemisinin and having already obtained some positive results. Nevertheless, because of the self-incompatibility of A. annua, it's difficult for A. annua to inherit the good traits into the following generations through traditional selfing means.Anther culture is a breeding means created by Indian scientists in the 1960s. Regenerated haploid plants can be obtained through in vitro culture of the isolated anthers. With the use of chromosome doubling agents such as colchicine, homozygous lines can be got and the progenies will maintain the good traits since the separation of the excellent genes would not occur. Therefore, anther culture is of great significance to the research of A. annua and will contribute to the breeding of homozygous lines with good traits including high yield, high content of artemisinin, etc. And also, anther culture will contribute to the development of artemisinin industry and the solution of malaria problem. However, there are few reports about anther culture system as well as haploid breeding of A. annua so far.The purpose of this study is to establish the anther culture system of A. annua and screen out the optimal conditions of this system. In this study, the developmental stages of the microspores of A. annua were identified and then anthers were isolated which were used in anther culture. Anther derived plants were regenerated and flow cytometry was used to test the ploidy level of the regenerated plants. The results are as follows:1) The developmental stages of the microspores of A. annua were identified through anatomical analysis.2) Callus induction experiments demonstrated the inducibility of androgenesis pathway of A. annua with the mononuclear microspores, meanwhile, the optimized callus induction medium (MS +1.0 mg / L 6-BA +0.3 mg / L NAA )was screened out.3) The optimized callus differentiation medium (MS+4.0 mg/L 6-BA+0.05 mg/L NAA)was screened out through callus differentiation experiments.4) The optimized rooting medium (1/2MS +0.5 mg / L NAA) was screened out through rooting experiments.5) The optimized dark incubation time (20-30d) and illumination time (7-10d) were determined through studying the incubation time gradient of darkness and illumination after inoculation of the anthers.6) The phenomenon was found that low temperature pretreatment to the inoculation material could not improve callus induction and differentiation rate.7) An optimized anther culture system of A. annua was established and verified through studying the combination of the screened optimized conditions.8) Flow cytometry was used to test the ploidy level of 23 regenerated plants, among which, the number of haploid, diploid, triploid, aneuploid was 3,11,3,6 and the proportion of them was 13.0%,47.9% ,13.0% and 26.1%, respectively.In this study, an anther culture system of A. annua was established and optimized. The present study provides a basis for haploid breeding of A. annua in the future. |
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