Isolated Microspore Culture And Identification Of Markers Linked To Red Leaf Traits In Kale

Kale is an important ornamental plant and vegetable. The flower bud differentiation ofkale takes a long time and it takes two years to accomplish a cycle of sexual life as it is avernalization plant. Six to eight years needed to cultivate an inbred line using conventionalmeans of breeding. Breeding method of double haploid utilized can effectively shorten thebreeding period and raise the efficiency of breeding instead of breeding process for inbredlines. Microspore culture is a technique of cell breeding to cultivate double haploid efficiently.Thus, the optimization of system for culture technique in kale has practical significance.Color of leaves as the most important ornamental trait in kale, and exploiting the molecularmarker closely linked to color of leaves forms an important premise of exploring themechanism of color conformation and carrying out research of breeding assisted bymolecular marker.Microspore culture was conducted using16F1hybrids of kale as materials in this study.We studied the initiation and development of microspore embryos and the effect factors forregeneration of microspore embryo. The comparison of identifications for ploidy fromdifferent plant was made. The artificial induction for chromosome doubling method wasexplored. Identification of self-compatibility from DH lines and the horticultural traits forhybrid combinations from DH lines was taken. Hybrid combinations prepared using DH linesto build F2segregating populations to filter out SRAP markers on red leaf genes. The mainresults are as follows:1. Optimization for mbryogenesis system of microspore in kale was conducted. It wasrevealed that genotype played a key role in the conformation of embryoids from isolatedmicrospore culture of kale.15microspore embryos obtained in the16tested materials.While, the differences of embryo rate for varieties were great. The difference of embryo ratebetween the highest ‘Zhouyehongxin’ and the lowest ‘Baijiu’ was1.94embryos per bud.The highest proportion of medium and late uninucleate period for microspore was67.31%,when the range of bud length for kale was from3.01to3.50mm. Thus, the range of budlength should be3.01³.50mm when microspore culture of kale was taken, and contributingfor embryo induction. The ratio between petals and anthers was2/3⁴/5. The production ofembryo was enhanced by low temperature（4℃）pretreatment on bud and24hours of treatment was the best result. Counterproductive was occurred when processing time wastoo long. Microspore treated with heat shock of33℃for24h before cultured contributed forstarting the developmental pathway of embryo. Adding and replacing the medium forculture of isolated microspore significantly improved the production of embryos. Addingliquid medium culture contributed to increasing production of embryos. The response forvarious materials to hormone was different. The embryo rate for ‘Bolangyehongxin’ inmedium without hormone was the highest. In vitro culture of0.10mg·L^-1of6-BA mediumwhen ‘Hongou’ added was the best, and0.10mg·L^-1of6-BA+0.10mg·L^-1of NAA mediumwhen ‘Baijiu’ added was the best. Shaking culture contributed for the initiation anddevelopment of embryos. The embryo rate of microspore and initiation of cotyledonaryembryo were promoted, as well as the proportion of cotyledonary embryo. In addition, theincidence of deformed embryos was reduced by shaking culture.2. System for high-frequency regeneration of microspore-derived plant in kale was built.B5solid medium was most suitable for regeneration of microspore embryo in kale. Whenmicrospore embryo formed, and then transferred onto B5solid medium, after that, quicklygerminates and rapid differentiation of germ was found. It contributed for embryo developinginto regeneration plant. The regeneration frequency of cotyledonary embryo was significantlyhigher than other types of embryos. Torpedo-shaped embryos can develop into cotyledon,then into seedling. It also develops into callus, and then differentiates into plant. Only a smallfraction of heart-shaped embryo, globular embryo and embryonic malformations can developinto plant. The most appropriate time of embryo transfer was the25thday after microsporeculture. Higher regeneration frequency of plant observed when embryos stranded in NLNliquid medium was minimized. Microspore embryo was transferred into solid medium, andthen cultivated under low temperature（4℃）pretreatment for a certain period couldsignificantly increase the regeneration frequency of plant. Microspore embryo of ‘Baiou’ and‘Zhouyebaixin’ pretreated for5d was best, and the regeneration frequency of‘Zhouyehongxin’ pretreated for2d was the highest. However, the period for pretreatment oflow temperature should not be too long. Germination of embryos inoculated into the medium,which added activated carbon was faster, and the regeneration plant grew stronger andreached higher survival percentage. Howeve, addition of activated carbon did notsignificantly increase the percentage of plant regeneration. Appropriate concentration of AgNO₃was added to promote microspore embryo to develop into plant. The regenerationfrequency of ‘Hongou’ when3.0mg·L^-1of AgNO₃added was the highest, and when‘Zhouyehongxin’ added with5.0mg·L^-1of AgNO₃was the highest.3． Identification and doubling methods for microspore-derived ploidy in kale werestudied. The application of combination for directs identification of chromosome count andindirect identification of characteristics of morphological and stoma, size and vitality ofpollen grain, from outcrossing and flow cytometry to evaluate the microspore regenerationplant. Haploid, double haploid and tetraploid existed among plant groups of microsporeregeneration. However, natural doubling rate from different genotypes of regenerated plantmade a great difference. It is necessary to carry out artificial redouble for natural doublinglow genotypes. Small spores treated with colchicines among artificial chromosome doublingwere the best. Doubling efficiency was52.9%when treated with concentration of50mg· L^-1colchicine for36h and it was significantly higher than control. Cut root plants treated withcolchicines was the worst, and it is difficult for genetics and breeding research as a highmortality rate obtained. During baptist root test of plant, the redouble effect was44.8%and wasbest when the root immersed in1000mg·L^-1of colchicine for4h. While, this method was likely tocause root damage and there was a weak growth after transplanting field.4．Identification for DH lines population of kale was made. Two hundred and thirty nineof microspore-derived plntas were from10species of embryos in kale by optimization forsystem of isolated microspore culture. Eightteen excellent DH lines were identificatedaccording to horticultural traits. All DH lines fitted with standard of self-incompatibilitybased on the investigation of self-compatibility index. Seven hybrized combinations withexcellent comprehensive properties selected using DH lines to obtain the combination.5．SRAP marker linked to trait of red leaves closely was obtained by filtering. The lawwas found through genetic analysis indicated that a couple of dominant gene on quality traitcontrol the red and white leaf trait of kale. Marker analysis was carried out with BSA strategyand3SRAP markers linking to Re gene were developed with an F2population derived fromcrossing D05×D10. These markes, Me8Em4、Me8Em17and Me9Em11, were mapped to bothsides of Re with genetic distances of2.2cM,3.7cM and6.4cM, respectively.