Microspore Culture Of Non-Heading Chinese Cabbage And Its Mechanism Of Embryogenesis

Non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) is one of the most important vegetable crops in the Brassica genus. Non-heading Chinese cabbage, known as Pak Choi, originated in China, is among the most popular vegetables crops in eastern Asia like China, Korea and Japan and is now common in Europe and America. Like the other Brassicas, Non-heading Chinese cabbage is a good source of nutrients. Non-heading Chinese cabbage obtained the character of heterosis, so it’s very important to study on the embryogenesis mechanism and optimize the isolated microspore culture technology for the crop breeding. The results are as follows:1. Study on the isolated microspore culture technology of non-heading Chinese cabbageFactors affected on microspore culture were studied on four early bolting cultivars of non-heading Chinese cabbage. Established a stable and reproducible embryogenesis system of microspore culture technology, then applied this program to18materials. The results showed that kept picking small inflorescence from the early flowering season can remained on the fresh inflorescence for a long time, and help reduce or overcome contaminate which came from the crack bud. There were some factors that could improve the induction rate of microspore-derived embryos significantly, such as pre-culture under32.8℃for2-3days; with1%of active charcoal in the NLN culture medium; the value of pH were5.8and add6-BA with0.05mg·L-1both for NLN culture medium and B5.The response frequency of genotype was88.9%, and the embryogenesis frequency of almost cultivars can reached or exceeded ten embryos per dish with the optimized program.2. Plant regeneration and transplanting in non-heading Chinese cabbageThe microspore-derived embryos we had obtained were used as materials for research the effect on the development of the embryoids and plantlet formation. Results showed that: heat treatment time would affect the survival rate of plantlet, the rate of heat treatment for2days and3days was higher than survival rate for1day in’qinggeng’; After cultured for25days, the percentage of embryos germination was highest in the solid medium; Seedling rate of embryonic roots into the medium was higher than keeping flat on the medium, and cold treatment didn’t help improve the survival number; Improved B5added with1.2%agar,0.4g-L"1AC and0.05m g-L-16-BA could promote the development of the embryoids and plantlet formation. The survival rate of the ’qinggeng’ plantlets was93.51%after being transplanted into soil. The average survival rate was more than74%, when the plantlets were induced roots and transferred under wet environment. Most of the plantlets after transplanted could grow to flower.3. Studies on the determination of ploidy level of microspore-derived plantsThe relationship between the ploidy level of microspore-derived plants and chloroplast number in stomatal guard cells was studied in non-heading Chinese cabbage. We had determined123plants by the FCM (Flow Cytometry) and113plants by chloroplast counting, then morphology identifying and chromosome counting through meiosis were used to test accuracy. The results showed there were haploid, diploid, triploid, tetraploid and chimera for the plantlets. The average rate of spontaneously doubled haploid was55.3%. Different cultivars had different rate, as follow,57.5%of huajing,73.1%of huaguan,80.0%of xialv No.2and the highest rate was qinggeng (94%). The accuracy of the two methods for identification of different ploidy plants was92.8%. The chloroplast average number in stomatal guard cells varied significantly among the different ploidy stoma in the same variety. A correlation has been established between ploidy and chloroplast number in the stomatal guard cells. In every single stoma of microspore-derived plants, the chloroplast number for a haploid should not be more than9, that for diploids should be10to16, and polyploids should be more than16. The result of this method was the same with the morphology identifying method, and was simpler than by the FCM. Furthermore, the accuracy of this method was reliable and did not vary with the plants growth conditions (seedling or bolting stage). The multiploids plants grew faster and the petal was larger.10univalents were observed in the haploid cells at metaphase I stage, and they symmetrically divide at metaphase Ⅱ stage. The shape of pollen from haploid was irregular; the germinated groove was deeper than diploids and there was little fluorescence area (no DNA); For the normal form of diploids,10bivalents could be observed at diakinesis and metaphase Ⅰ stage; But more than10chromosomes could be observed in the tetraploid cells at metaphase Ⅱ stage and so on. For the normal form of tetraploid,20bivalents could be observed.4. Cytological studies on pollen mother cell meiosis, embryogenesis and developmentChromosomal meiotic behaviors and male gametophytes development of non-heading Chinese cabbage were studied using chromosome preparation and fluorescence staining technique. The research showed that:the cytokinesis and chromosomes meiosis of pollen mother cells of non-heading Chinese cabbage was simultaneous type; diakinesis bivalents was mostly rod and ring. A few lagging bivalents appeared in anaphase Ⅰ and the spindle arranged in vertical, parallel and an acute angle in metaphase Ⅱ. The microspores in tetrad stage showed the arrangement of decussate type, isobilateral type and tetrahedral type. Diakinesis, metaphase Ⅰ and metahase Ⅱ was the best period for identification of chromosome numbers. The length of buds which were from1.5to2.49mm was usually at the stage of uniuclaete microsore in center. Uniucleate microsore at periphery and early2-celled pollen existed in buds long from2.5to3.0mm.2.0～3.0mm long flower buds were adapt for anther or microspore culture. Most of microspore embryos development was same to the zygotic embryos, and the development process was very fast. The embryos grow to globular stages after6-9days of culture, and to cotyledonary stages after13days. There were two kinds of male gametophyte development, one was asymmetrical division (names A pathway), other was B pathway. Both A and B pathway were observed in our study. After the first asymmetrical division, we found that both the vegetative and germ cells could be inducted to embryos, or only the vegetative cells could be inducted, but not the germ ones.