Studies On Microspore Culture And Plant Regeneration And Innovation Of Tetraploidy Germplasm In Cruciferous Species

Ploidy breeding is very important in Cruciferous species breeding. The isolated microspore culture can obtain pure and stable materials in short time, accelerate breeding process, shorten breeding times and get recessive characters. It is useful in creating excellent inbred lines and promoting breeding efficient. The content of biological component and biomass yield in polyploidy plants is promoted than in diploid. It is very important in vegetables and other special useful crops.In the present paper, several Cruciferous species, such as cabbage, broccoli, kale and radish, were used to study the factors on microspore embryogenesis and plantlet regeneration. The tetraploidy radish, rich in red-pigment, was induced by colchicine treatment, and anthocyanin distribution and content in different parts were studied. Main results are listed as follows:1. The factors that affect embryogenesis and plant regeneration efficiency of several cultivars of Cruciferous species (cabbage, broccoli, kale and radish) were studied. The results showed that genotype was key factor that influenced microspore embryogenesis;4℃cold temperature pre-treated for1d combining32.5℃treated for1d could improve embryogenesis efficiency significantly. In solid differentiation medium,1-2%concentration of agar was suitable to microspore embryo germination and plantlet regeneration. The embryos cultured in NLN-13liquid medium about25d were best for germination.2. The floewer bud with microspore at late uninucleate stage was investigated by DAPI staining, and microspores of different B. oleracea species were co-cultured with that of B. napus (good responsive to microspore embryogenesis). The results showed that the size of buds containing late uninucleate microspores was different in various genotypes. The ratio of P/A (petal/anther) around1could be used as an index to select buds of B. oleracea species. Co-cultured with microspores of B. napus, cv. Zheshuang758, different genotypes of B. oleracea species exhibited improved frequency of embryo production and plant regeneration than those of non-co-culture controls. The efficiency of embryogenesis was different among different B. oleracea species. Microspore-derived plants of different cultivars of B. oleracea were discriminated from B. napus according to the morphological characters of plant. The results indicated that B. oleracea produced higher number of microspore-derived plants in co-culture treatments than controls. 3. DAPI staining was used to observe microspore developmental stage and found that good results were observed from microspore at late uninucleate to early binucleate stage, with the flower buds average length of3.0-5.0mm and P/A of0.8-1.2for cabbage;2.5-4.0mm and P/A of0.8-1.25for broccoli;3.0-5.0mm and P/A of0.8-1.2for kale, and3.0-4.0mm and P/A of0.9-1.2for radish. When microspores experienced heat shock, the volume began to expand, exine broke down gradually and many microspores entered into symmetric division. About15d after culture, macroscopic embryos showed up and developed into embryoids25d later which were suitable to transfer into solid differentiation medium. After heat shock, the vacuoles in microspore vanish, nucleus moved to center and microspores prepared for the first division. Simultaneously, lots of organelles gathered in cytoplasm and nucleus around abundant of starch grains. After first division, two similar cells were developed and the two nucleuses were light in color, and organelles spread in cytoplasm uniformly. The two cells closed each other but no cell wall envelope.4. In microspore-derived plant population, the ploidy level was mixed. Besides haploids, there were also diploids and other polyploidy plants. Flow cytometry analysis could be used to identify the ploidy level rapidly. Various concentrations of colchicine for different durations were used to treat haploid plants. The results indicated that the combination of200mg-L"1colchicine for20h was better than other treatments in term of plant survival and diploid inducement efficiency in three rested genotypes. The differences existed in stomatal size and morphologies, and guard cell chloroplasts in different ploidy level. The methods of FDA and I-KI staining could be used to observe the differences clearly. We found that stomatal length of haploid was approximately20μm, and25μm for diploid, while more than33μm for tetraploid. The number of guard cell chloroplasts in haploid was about7, and13for diploid and more than23for tetraploid. The difference of number of guard cell chloroplasts was significant in different ploidy levels.5. The apical tips of cotyledon-stage seedlings of a unique red-core radish cultivar "Yanzhi" were treated with various concentrations of colchicines (0.1,0.15,0.2g-L-1) for different durations (0,24,48,72h). The morphological characteristics and stomatal size were used to pre-screen putative polyploids one month later after treatment, and then ploidy level was confirmed by using the flow cytometry and chromosome counting. Morphologies and total anthocyanin content were compared between tetraploid and diploid plants of red-core radish. The results indicated that high concentration of colchicine treatments resulted in high mortality rate. The highest percent of variation plants was62.0%when treated with0.2g·L-1colchicine for72h, and the highest tetraploid induction efficiency was11.3%when seedlings treated with0.2g·L-1colchicine for48h where37plants of these putative tetraploids were indeed doubled. The tetraploid and diploid plantlets differed significantly in leaves and flower morphologies, plant vitality and also the stomatal and pollen grain sizes and guard cell chloroplasts. The anthocyanin content in tetraploid plants was173.6mg/100g FW,11.8%higher than that of diploid plants significantly.6、The synthesis of anthocyanin and their correlation to phenylalanine ammonia-lyase (PAL) activity, soluble sugar and chlorophyll contents were investigated in the present experiment. The results showed that the anthocyanin was synthesized during germination. The configuration order of anthocyanin was from top to bottom in hypocotyls and distributed in a non-uniform manner. The anthocyanin content in root peels was higher than that in flesh and was higher in aboveground parts as compared to underground parts. The contents of pigment, soluble sugar, and PAL activity were higher in light than in the dark. The changes of anthocyanin content of hypocotyls in roots under the light were raised. The trend of cotyledons in the light and cotyledons and hypocotyls in the dark increased initially and then reduced. Light influenced the trends of PAL activity and soluble sugar content during germination and growth in the field in a different mode. The chlorophyll content in leaves was fluctuated during growth period and the trend was on the rise, but almost unchanged in the petioles. In roots, the content of pigment in the peels was the highest (25.03mg/100g), followed by flesh and root apex, and least in petioles. The correlation of pigment to PAL activity and soluble sugar content was positive, but the correlation coefficient was different (at0.607and0.678, respectively). The relevance of chlorophyll to pigment was also positive with the correlation coefficient of0.496.