Study On Microspore Culture And Differential Expression Of Genes Related To Early Microspore Embryogenesis In Cabbage (B. Oleracea L. Var.Capitata) And Broccoli (B. Oleracea L. Var. Italica)

Cabbage (B. oleracea L. var. capitata) and Broccoli (B. oleracea L. var. italica) are important cole crops getting greater breeding attention by seed companies due to the increasing production of these vegetables in recent years and citation of potential anticarcinogenic properties in some cultivars. Most of the commercial cultivars of cabbage and broccoli are F1 hybrids, which ensure high uniformity and yield as well as protection of plant breeder's rights and the markets of seed companies. The production of hybrid cultivars requires the use of homozygous parental lines. In cabbage and broccoli breeding programs, production of homozygous lines by recurrent selection and selfing (e.g., by hand bud pollination) is time-consuming (6 to 7 generations of selfing) and labour-intensive. Microspore culture is an effective alternative technique for the production of doubled-haploid (DH) lines that can also be parental inbreds for the production of hybrids in cabbage and broccoli. DH line development accelerates plant breeding process by saving labour and time.Successful microspore cultures of different genotypes have been reported in cabbage and broccoli. However, the practical application of microspore culture technique still involves limitations. One big problem is that embryo yield in many cabbage and broccoli genotypes is still very low, and certain genotypes do not appear amenable to the process at all. The second is that the frequency of plant regeneration from microspore-derived embryos in many cabbage and broccoli genotypes is still very low. Thirdly, an easy and reliable method on identifying pliody levels of plants in Brassica oleracea is absent. Another important consideration in the practical application of microspore culture technique involves chromosome doubling of microspore-derived plants.In the present paper, in order to test and incorporate recent advances in the technique to develop an efficient and reliable protocol for cabbage and broccoli microspore cultures, some effects on microspore embryogenesis and plant regeneration were studied. In order to improve efficiency on chromosome ploidy identification and chromosome doubling, the optimal method of identifying chromosome ploidy and doubling chromosome was also researched. Based on the effects of heat shock and cold pretreatment on microspore embryogenesis in cabbage and broccoli, one research was carried out to gain insight into the molecular events occurring in the very early stages of cabbage and broccoli microspore embryogenesis induced by different stress pretreatments. In this study, mRNA differential display PCR (eg. cDNA-AFLP) was used to isolated and identified cDNA clones corresponding to genes differentially expressed during the early stages of microspore culture. Main conclusions are listed as follows:1. The effect of genotype, heat shock, cold pretreatment and pH values on embryogenesis in microspore culture was investigated in cabbage and broccoli. There was a variation in the response of different genotypes to heat shock at 32.5°C for 1day. In cabbage,'Zhonggan 11'was the highly responsive genotype, producing an average of 22.30 embryos per bud; while'Zhonggan 8'was the only no-response genotype. In broccoli,'Lüxiu'was the only high-response genotype, producing an average of 9.93 embryos per bud; the low-response genotypes'TI-111','TI-089','Yuguan'and 'Lüyu'produced 0.07~1.33 embryos.For most cabbage genotypes, the heat shock at 32.5°C for 1 day was most optimal. In broccoli, the heat shock at 32.5°C for 1 day was most optimal for all genotypes in this experiment.In cabbage, cold pretreatment for 1, 2 or 3 days was not effective when applied alone in three cultivars; while cold pretreatment for 1, 2 or 3 days was effective when applied alone in three broccoli genotypes. In three cabbage genotypes, the combination of cold pretreatment (at 4°C) for 1, 2, 3 or 4 days and heat shock (at 32.5°C) for 1 day can not improve embryogenesis efficiency except'Xiwang'. Fortunately, in broccoli, the combination of cold pretreatment (at 4°C) for 1 or 2 days and heat shock (at 32.5°C) for 1 day significantly enhanced microspore embryogenesis efficiency, especially with'TI-111'for which it was increased by 63~72 fold. This is the first time that the effects of cold pretreatment and combination of cold pretreatment and heat shock on cabbage and broccoli microspore cultures have been reported.For all of three cabbage genotypes and three broccoli genotypes, 6.2 or 6.4 was the optimal pH value in NLN-13 and 1/2NLN-13, which significantly enhanced microspore embryogenesis efficiency by 2 fold compared to standard medium (NLN-13 and 1/2NLN-13) with pH 5.8.2. In order to improve plant generation frequency, several factors affecting plant regeneration were studied in a cabbage hybrid F1'Zhonggan 11'and a broccoli hybrid F1'TI-111'. Results showed that B5 medium with 1%~1.25% agar was optimal to germination and regeneration of embryos, and that frequency of plant regeneration from cotyledonary embryos was higher significantly than other embryos, especially the cotyledonary embryos obtained from the 25-day-cultured microspores. When the cotyledonary embryos obtained from the 25-day-cultured microspores in 8 genotypes of cabbage and broccoli were transferred to B5 medium with 1% agar, the frequency of plant regeneration was in the range of 77.8%~97.2%.3. The relationship between the ploidy level of microspore-derived plants and chloroplast number in stomatal guard cells was studied in cabbage, broccoli and Chinese kale. In the experiment, morphology identifying and chromosome counting were used to test accuracy of counting chloroplast number in stomatal guard cells. 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 than 10, diploids 11 to 15, and polyploids more than 15. The accuracy of this method for identification of different ploidy plants was 93.93%. Further more, the accuracy of this method was reliable and did not vary with the plants growth conditions. Therefore, the chromosome ploidy of plants derived from microspore culture in cabbage, broccoli and Chinese kale can be identified by simply counting the chloroplast number in stomatal guard cells.4. In the experiment, spontaneous chromosome doubling occurs randomly and is genotype dependent. In plant population derived from microspores, there were haploid and diploid, and even polyploid and Ploidy mixed were existed. In four cabbage genotypes, frequency of spontaneous chromosome douling was in the range of 46.19%~84.62%, and in six broccoli genotypes from 52.17% to 100%. In addition, the combined treatment (the combination of cold pretreatment at 4°C for 1d and heat shock at 32.5°C for 1d) resulted in higher diploids frequency of the regenerated population compared to traditional heat shock at 32.5°C for 1d in broccoli'TI-111'.5. With a haploid genotype plant from cabbage'Zhonggan 11'and broccoli'TI-111'separately, colchicine was used as chromosome-doubling agent in artificial method. In this experiment, the roots of plants were immersed in different solution concentration of colchicines with different treated time. In cabbage, a 0.2% working solution of colchicine for 9 or 12h, or a 0.4% working solution for 3 or 9h was appropriate, producing doubling rates from 58.33% to 86.42%, and meanwhile, the survival rate of plants was in the range of 73.33%~93.33%. In broccoli, a 0.05% working solution of colchicine for 6 or 12h was good, producing doubling rates from 54.55% to 75.00%, and the survival rate of plants was in the range of 57.14%~80.00%.6. Different microspores samples induced by different pretreatments (including no-pretreatment, heat shock at 32.5°C for 1d, cold pretreatment at 4°C for 1d, the combination of cold pretreatment at 4°C for 1d and heat shock at 32.5°C for 1d) and then cultured at 25°C for 0~3d were used to analyze the gene expression by means of cDNA-AFLP. In total, 256 pairs of primers were used for selective amplification, and 164 differentially expressed TDFs were got. Among of them, 45 TDFs expressed differentially in both cabbage and broccoli, and 2 TDF expressed differentially in broccoli microspores induced by cold pretreatment. So these 47 TDFs maybe were related to microspore embryogenesis.7. Some of these 47 TDFs were cloned and sequenced, and finally 26 TDFs sequences were obtained. The sequences of 26 TDF were submitted to dbEST, and GenBank asseccion numbers of them were GR301387~GR301412 in broccoli and GR301413~GR301436 in cabbage. Compared with the publicly available databases, 21 TDFs presented some significant similarity with known plant protein gene sequences, and function of them were involved in signal transduction, stress defense, gene regulation and transcription, Hormone modulation, synthesis and hydrolysis of protein, etc. Most of them were involved in the stress defense and signal transduction. In addition, these 21 TDF were from new genes in cabbage and broccoli.8. 3′end of full-length cDNA of 13 TDFs presenting well significant similarity with known plant protein gene sequences were cloned in broccoli, and sequences of cDNA 3′end and TDF were assembled. The 13 assembled sequences had been submitted to dbEST, and GenBank asseccion numbers were GR312927~GR312939.9. Among of 21 TDFs, expression pattern of 5 TDFs, e.g. TDF4,TDF8,TDF14,TDF16 and TDF20, in different'TI-111'microspore RNA samples were tested using real-time PCR method. Results of relative expression showed that these TDFs expression patterns accorded with that using cDNA-AFLP method.