| Doubled haploid (DH) plants are valuable in genetics and cytogenesis study, physiological research and breeding work, because the recessive genes are not covered by their dominant alleles. The isolated microspore culture is one way of getting doubled haploid.Five different pepper cultivars were used in this study, including 75-7-3-1, 77013, Haihua No.3, 83-58 and Milord. The effects of genotype, anther development period, pretreatment and culture condition on microspore culture were studied. At the same time, the development of microspore after isolated and the growing of callus and embryo were observed.The relationship between characters of bud and stages of microspore was studied. When most microspores at late uninucleate and early bincleate stage, the bud has white or light green petals, the sepal length same to the petals, anther is white or light yellow and tip of anther is light purple. The length of bud at this stage in most cultivars was above 4 mm, but the size of buds was different among different cultivars.The pretreatment effect on survival rate of microspore was studied by 5 kinds of pretreatment including 4 C, 36 C, no sugar treatment, Mannitol treatment and colchicine treatment. At the same time, the efficient pretreatment that elevating and maintaining survive rate of microspore was chosen for condition test. The result showed that different cultivars have different feedback after different pretreatment, 4 C and no sugar treatment have better effect on survive rate for 5 cultivars then control.The effects of 13 culture factors on isolated microspore culture are checked in this test through orthogonal tests. 13 factors include type of medium, sugar, time of sugar treatment, active charcoal, sliver nitrate, Vb1, lactabumin hydrolysate, glutamine, 2,4-D, KT, BA, IAA and NAA. Directly review analysis with Excel and variance analysis with SAS show that the sugar, sliver nitrate, lactabumin hydrolysate, Vb1 and glutamine are most important culture factors for the forming of callus and embryo. The mediums that benefit for the microspore development are found by Dun Can's new multiple rang test analysis, but the optimum culture medium varied with genotype. The different of callus and embryo rate between genotype were evaluated by T test.The development of microspore, callus and embryo are observed during culturing. The B division way is discovered in Haihua No.3 and 83-58, A division way is only discovered in 83-58. The course from ball stage of embryo to plantlet is also observed. The abnormal embryo development are summarized, some embryo developed into abnormal cotyledon, some stopped growing after formed 2 cotyledons because the plantlet lose growing point in the earlier stage, and others stop developing on the stage of embryo.In this study two different cultivation ways are checked for microspore to plantlet. One is that microspore develop directly into plantlet through embryo, the second is that microspore induce callus forming new growing point. The result shows that the successes rate of callus cultivation that from embryo directly to plantlet are higher than the other, and the V3 medium is better than improved MS medium for embryo cultivation. Finally the chromosome number of plantlet is identified. Colchicine is used to double the chromosome number, but all haploid plant are not doubled. The success rate of haploid and chromosome doubling for plantlet from embryo is higher than the plantle from callus. |
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