Study On Anther & Microspore Culture And Mechanism Of Embryogenesis In Pepper

Aimed at the problems of haploid culture of pepper, the protocols included interrelation of cytological development period of microspore and the morphology of flower organ in pepper, influencing factors to microspore embryogenesis, the methods of increasing microspore activity, transplant of microspore plantlet, the ploidy of microspore plantlet and ploidy identification method were carried out. At the same time, the development of microspore and the growing of embryo were observed at both light and electron microscopy levels. The related gene with embryogenesis was also cloned. The aim of the research was to set up high-efficient culture technique system of the anther and microspore culture of pepper, and to probe into the mechanism of androgenesis, in the end, to manipulate haploid culture .The results were as follows:1. Microspore development showed correlativity with the morphological characteristics of buds and the color of anthers. The buds 4.253~5.074mm in height and 4.191~5.367mm in diameter, and the anthers 2.020~2.565mm in length and 0.982~1.417mm in width were determined to contain microspores at the uninucleate. At this stage, the length of corolla was about the same as or slightly greater than that of the calyx. Pepper was characterized by an asynchrony of pollen development within a single anther. Therefore, the cytological development period of microspore can be decided by size, color and shape of the flower organ.2. We assayed the content of endogenous hormones IAA and ABA of pepper anther in the process of the pretreatment using ELISA method. On the basis of results of our test, We presume that low temperature pretreatment changes content of endogenous IAA of anther, thus interrupts previous developmental direction of pollen and induces microspores develop from a gametophytic way to a sporophytic way. ABA content in anthers is not the only factor for obtaining embryos from the anther culture of peppers.3. Haploid plants of pepper have been regenerated from in vitro anther cultures by a heat treatment at 35℃for 8 days. Haploid plants of pepper have been regenerated from in vitro anther cultures by a heat treatment at 35℃for 8 days. Genotype was the most important factor affecting anther culture. Hormone was another significant factor. On the different media, the effect of anther culture of various genotypes pepper were different. The most suitable hormone and perfect concentration varied from genotype to another. The time of inoculation affected inducing rate of embryo. Commonly, from flowering to 1~4 weeks, there were a good result for anther culture. Embryoids by inducing anther were characters of asynchronism。4. We created a method of embryoid obtainment from induced microspore of pepper, and patented it. The method of natural released microspore from pepper anther was simple and efficient. The solid medium with 0.5%~1.0% active charcoal could improved induced rate of embryo. The flower bud pretraeted at 4℃low temperature had a positive effect on the total embryos number, and had a more evidence influence of the number of normal embryo. Although the difference was unmarkable, two days pretreatment at 4℃low temperature had a good result.It was a effective method to isolate microspore by crushing flower bud removed sepal or dissected anther. To get clean and active microspores, low speed centrifuge was recommended during microspore collection and purification. In microspore culture, activity of microspore was assayed according to different age of donor plants and different genotypes. It varied among different genotypes and various age of the donor plants in the same genotype, the more activity the microspores had, the higher embryogenesis frequency the genotype had. It was recommended that microspores first be cultured at 35℃or 32℃less than 24 hours was advantage of the dedifferentiation of microspore. Low temperature pretreatment favored the maintaining of microspore activity, but the effect on the microspore dedifferentiation was not obvious.5. Pepper was characterized by a strong asynchrony of pollen development within a single anther. With the culture period changed and the proportion of dead pollen increased drastically from day 2 after culture. Microspores that were cultured at the late-uninucleate stage followed one of two developmental pathways. In the more common route, the first sporophytic division was asymmetric and produced what appeared to be typical bicellular pollen. Embryogenic pollen was formed by repeated divisions of the vegetative nucleus. In the second pathway, which occurred in fewer microspores, the first division was symmetric and both nuclei divided repeatedly to form embryogenic pollen. However, division of the generative nucleus alone to form the embryo was never observed.An exine with its specific pattern had already been formed, when microspores were released from tetrads. During subsequent pollen development, microspores increased in size and continued to strengthen the exine. After 24 h in culture, the microspores had increased in size. Thereafter, embryogenesis was indicated in some microspores by two different morphological changes. One featured an expansion in volume of the cell cluster around the germination aperture, the other showed cell cluster volume expansion over the entire microspore surface.Morphogenesis of microspore-derived embryos has been analysed, at both light and electron microscopy levels. The changes in cell organization after embryogenesis induction, and the characterization of the time sequence of a set of structural events, had been also explained. These changes mainly affected the plastids, the vacuolar compartment, the cell wall and the nucleus. Further differentiation processes mimicked that of the zygotic development. The occurrence of defined subcellular rearrangements has been investigated during early microspore embryogenesis in pepper, in relation to proliferation and differentiation events. They have increased the understanding of the mechanisms controlling the switch and progression of the microspore embryogenesis, which could help to improve its efficiency and to direct strategies.6. The RT-PCR method was applied to analyse differential gene expression in the process of microspore-derived embryogenesis in pepper(Capsicum annuum L). The results showed that two sequences were proved to be expressed at the embryoids. Blast results in GeneBank showed that PELTP was highly homologous to LTP gene of pepper and PEGST to GST gene of pepper(98% and 99%, respectively).Therefore, we presumed that the two genes may play an important role in the early stages of pepper microspore embryogenesis.7. The procedures of domestication transplant of anther culture plantlets were established in pepper .The transplant technology is as follows: The transplanting surviral rate of plant1ets could be increased to 90% through the procedure of hardening plantlets for 24～48h,growing with medium supplemented with equal volume fine river grit or perlite and peat.The survived plants with deep grown color-leaves and vigor roots could grow strongly in the field.8. The discrepancy of the average chloroplast number was extremely prominent. Chloroplast number of the haploid was below 13, and that of the diploid was equal to or more than 13. The number of stomatal chloroplasts increased with the increase in ploidy levels. The reliability of ploidy identification using count stoma guard cell chloroplast number was higher, with the average accurate rate of 92.68%, showing that measurement of the number of stoma chloroplast in guard cell could be considered as a fast and accurate method to determine haploid, diploid of pepper in the seedling stage.The difference between them reached the highly significant level. These plants with stoma guard cell circumferences shorter than 103.3μm were haploids ones, and those with stoma guard cell circumferences longer 103.3μm were diploid ones. The stoma guard cell circumferences in the fifth leaf among these plantlets were relatively steady. So the stoma guard cell circumferences of plantlets could be used as a subsidiary index to estimate the ploidy level of Capsicum annuum L.