Optimization Of Wheat Tissue Culture Regeneration System And Haploid Induction Technology

High-frequency regeneration of wheat plant in vitro is essential for cell engineering breeding andtransgenic breeding of this species. But tissue culture frequency is influenced by various elements,among them, the effect of genotype is particularly significant. So Selection genotypes with high plantregenerations, especially with excellent agronomic traits as starting materials for wheat tissue culture,will effectively promote the development of wheat genetic engineering breeding and haploid breeding.This research evaluated the tissue culture performance of immature embryos, mature embryos andanther from24excellent wheat varieties popularized widely in recent years and a good line CB037, andoptimized in vitro culture system of immature embryos and mature embryos, meanwhile discussedwheat haploid induction technology of wheat×maize crossing and microspore culture, in order toimprove the efficiency of wheat tissue culture and induction frequency of haploid plantlets. The mainresults as follow:1. The result of immature embryo culture of25wheat good varieties (or line) showed that callusdifferentiation frequency (1.12%to74.60%) and plantlet regeneration frequency (2.25%to531.92%)were highly significantly different between genotypes. Higher plantlet regeneration frequencies werefound in CB037, Lunxun987, Yangmai16, Neimai836, Kenong199, Xinchun6, Zhengmai366,Zhengmai9023, Xindong20, Yannong19and Chuanmai42, with531.92%,370.16%,363.03%,328.32%,296.82%,210.94%,131.93%,109.35%,104.19%,89.37%,81.44%, respectively. And the Matureembryo culture result indicated that the callus differentiation rates were from5.03%to58.48%and theplantlet regeneration rates were from3.24%to84.34%. There was significant difference betweengenotypes. Among them, CB037, Xinchun6, Jingdong8, Shimai4185, Kenong199and Lunxuan987played better in plantlet regeneration frequency, with81.34%,68.78%,61.95%,59.00%,52.34%and52.19%, respectively. Moreover, the anther culture results demonstrated that the rates of callus induction,differentiation and green shoots were from0.38%to64.19%,0%to11.9%and0%to41.75%,respectively, showing significant difference in genotypes. Among them, the CB037had the highestgreen shoots induction rate (41.75%), followed by shimai4185(39.6%) and Han6172(15.76%), whileother genotypes were less than5.2%.2. By using low-frequency regeneration wheat cultivars Jimai22, Jingdong8and Han6172,different levels of AGPs and H2O2were tested for the effect of plant regeneration of wheat immatureembryos. Results indicated that adding200mg/L AGPs into callus induction medium (SD2) led to asignificant increase of shoot regeneration rates in immature embryos of Jingdong8and Jimai22, up to374.51%and84.29%respectively. Whereas adding10mg/L AGPs Han6172(131.41%) was moreresponsive. Moreover at0.005‰level of H2O2, Han6172and Jimai22had the significant increase ofregeneration rates with343.89%and47.72%, respectively, and Jingdong8(218.88%) was moreresponsive to0.02‰of H2O2. The efficacy of AGPs and H2O2were influenced by wheat genotypes.3. In order to prolong the utilization time of the wheat immature embryo, using large immature embryos (2.0-3.0mm) of Shimai366, Aikang58, KeNong199, Xinchun9and CB037as materials, theinfluence of1-MCP、cellulase and pectolyase on plantlet regeneration of large immature embryos inwheat tissue culture was studied. The results showed that2mg/L1-MCP significantly enhancedregeneration rates of Shimai366, Aikang58, KeNong199, up to40.21%,120.17%and138.79%,respectively. While100mg/L cellulase+20mg/L pectolyase significantly improved regeneration rate ofAikang58(75.12%), but had no effect on Shimai366, KeNong199and Xinchun9. The cellulase andpectolyase played different acts on genotypes. Additional different concentrations of1-MCP had nosignificant differences in regeneration rates of large embryos from CB037, while cellulase andpectolyase decreased the plant regeneration rates.4. Selection Kenong199and Lunxuan987as material, the impact of different levels of AGPs andH2O2on shoots plant regeneration of wheat mature embryos was investigated. Statistical analysisshowed that adding100mg/L AGPs to callus induction medium, Kenong199and Lunxuan987obtainedthe highest regeneration rates with72.46%and64.27%, respectively. However adding different levelsof H2O2the regeneration rates of mature embryos from Kenong199and Lunxuan987were lower thancontrols, but the difference was not significant, indicating that adding H2O2to callus induction medium,to a certain extent, inhibited the regeneration of mature embryo.5. Take Dwarf male sterile wheat as material, the wheat haploid induction efficiency of corninducer and normal inbred corn line was compared. It turned out that the efficiency for wheat embryosformation induced by the two corn lines were not different obviously. Then the obtained haploids wereidentified by cytology, botanical traits and molecular marker. The results conformed that Ms2/Rht10haploids were successfully obtained in this study. Once induced by maize pollens, the Ms2/Rht10andms2/rht10female gametes from Dwarf male sterile wheat had the same chance to develop into embryos.Next use Yangmai16as material, the impact of different AGPs concentration on embryos formation rate,germination rate and wheat haploid production rate were discussed. The results showed that2g/L AGPsto a certain extent, promoted the haploid embryo development.6. Exploration of microspore culture method, it was found that the condition at4℃with hungertreatment and the increased microspore culture density would play crucial effects on wheat microsporeculture, and callus as well as wheat haploids were successfully induced from wheat microspore culturein this study. This laid a foundation for further establishing wheat microspore culture technologysystem.