Establishment And Application Of The System For Isolated Microspore Culture In Kale (Brassica Oleracea L.var.acephala DC.)

In order to establish an efficient technique system of isolated microspore culture in kale and accelerate the speed of breeding materials purity, isolated microspore culture was performed using 10 F₁ hybrids of kale. The effects of media and culture conditions on embryoid induction, development and plantlet regeneration were studied. At the same time, the methods of ploidy identification for microspores-derived plants and doubling techniques for haploid plantlets were studied. Meanwhile, horticulture characters of double haploid (DH) plants were investigated and valued in the study. The results are as follows:1. The suitable sampled bud is different for different genotype hybrids. For most varieties, the range of buds with 3.0-4.5mm length and petal/anther with 4/5-7/6 are optimal for isolated microspore culture of kale. Choosing the buds with most rotundity microspores are proper while inoculation.2. Genotype is one of the key factors affecting the embryoid induction of kale. 7 genotypes among 10 get embryoid successfully in the research. The highest embryo yield is "Y009" with 0.88 embryo per bud.3. Plant growth regulators have great effects on embryoid induction and development. The results showed that 6-BA can promote embryoid induction and the suitable content is 0.2 mg·L^-1 NAA can not promote embryoid induction, but can increase the proportion of normal embryos; 2, 4-D has significant inhibition to embryogenesis. In addition, harmony action of 6-BA and NAA dramatically increase the frequency of microspore-derived embryos and enhance the rate of germinated and cotyledon-shaped embryos. The suitable content ration is 1 : 1 or 2 : 14. Agarose and active carbon can promote embryoid induction and development, especially for recalcitrant genotypes to get embryoids. The suitable content is 100μL per dish with the mix of agarose (0.5 g·L^-1) and active carbon (10 g·L^-1).5. 4℃ low-temperature pretreatment can increase the frequence of microspore-derived embryos. The effect isn't notable on embryoid induction for "Y009", but it can keep materials fresh for isolated microspore culture temporarily.6. 33℃ high-temperature pretreatment can change the developmental direction and make it from gametal developmental path to sporophyte developmental path. Meanwhile it can make microspores keep higher vitality cell frequency and promote cell division and embryoid induction. The optimal time treatment is 24 h.7. The sample buds in flourishing florescence get the highest embryo yield; primary...