导　　师： 叶兴国

学科专业： 090102

授予学位： 硕士

作　　者： ;

机构地区： 中国农业科学院

摘　　要： 小麦离体培养高频率植株再生是开展细胞工程育种和转基因育种的重要环节之一，但培养效率受到多种因素的影响，其中，基因型的作用尤为显著。因此，选择具有高再生力的基因型，尤其是以农艺性状优良的基因型作为小麦组织培养的起始材料，将推动小麦转基因育种和单倍体育种的有效开展。本研究以近年来全国大面积推广种植的24个优良品种和优良新品系CB037为材料，从幼胚、成熟胚、花药三个方面评价了组织培养再生性能，并对幼胚、成熟胚的培养体系进行优化，同时探讨了小麦-玉米杂交和小孢子培养诱导小麦单倍体技术，旨在进一步提高小麦组织培养和单倍体植株诱导效率。取得了如下主要结果： 1．25个小麦优良品种（系）幼胚培养结果表明，愈伤组织分化率1.12/%-74.60/%，植株再生率2.25/%-531.92/%，基因型间存在极显著差异；其中，CB037、轮选987、扬麦16、内麦836、科农199、新春6号、郑麦366、郑麦9023、新冬20、烟农19和川麦42幼胚培养植株再生能力较强，植株再生率分别为531.92/%、370.16/%、363.03/%、328.32/%、296.82/%、210.94/%、131.93/%、109.35/%、104.19/%、89.37/%和81.44/%。成熟胚培养结果表明，愈伤组织分化率5.03/%-58.48/%，植株再生率3.24/%-84.34/%，基因型间差异显著；其中，CB037、新春6号、京冬8号、石麦4185、科农199和轮选987成熟胚培养植株再生率较高，分别为84.34/%、68.78/%、61.95/%、59.00/%、52.34/%和52.19/%。花药培养结果表明，愈伤组织诱导率0.38/%-64.19/%，愈伤组织分化率0/%-11.9/%，绿芽诱导率0-41.75/%，基因型间差异显著；其中，CB037绿芽诱导率最高（41.75/%），其次是石麦4185（39.6/%）和邯6172（15.76/%），其它基因型花药培养植株再生率均小于5.2/%。 2．以幼胚再生率较低的京冬8号、济麦22和邯6172为材料，研究不同浓度阿拉伯半乳聚糖蛋白（Arabinogalactan proteins, AGPs）和H2O2对其幼胚组织培养再生的影响。结果表明，愈伤组织诱导培养基中添加200mg//LAGP显著提高了京冬8号和济麦22幼胚植株再生率，分别达到374.51/%和84.29/%；添加10mg//LAGP时，邯6172植株再生率达到131.41/%。添加0.005‰H2O2时，邯6172和济麦22幼胚培养植株再生率比对照有显著增加，分别为343.89/%和47.72/%；H2O2为0.02‰时京冬8号反应最为显著，绿芽诱导率达到了218.88/%。说明AGP和H2O2促进植株再生的作用在基因型间存在差异。 3．为了延长田间小麦幼胚可利用的时间，以石麦366、矮抗58、科农199、新春9号和CB037较大幼胚（2.0-3.0mm）为材料，研究纤维素酶+果胶酶和1-甲基环丙烯（1-Methylcyclopropene,1-MCP）对大龄幼胚培养再生植株的影响。结果发现，2mg//L1-MCP显著提高了石麦366、矮抗58和科农199绿芽诱导率，分别为40.21/%、120.17/%和138.79/%。100mg//L纤维素酶+20mg//L果胶酶处理显著提高了矮抗58绿芽诱导率（75.12/%），但对石麦366、科农199和新春9号大龄幼胚再生无显著作用，反应了纤维素酶、果胶酶对不同基因型的作用效果不同。1-MCP处理对CB037较大幼胚再生率的影响与对照相比无显著差异，而用纤维素酶+果胶酶处理却降低了植株再生率。 4．以科农199和轮选987为材料，研究不同浓度AGP和H2O2对其成熟胚组织培养再生的影响。结果表明，愈伤组织诱导培养基中添加100mg//L AGP轮选987和科农199绿芽再生率分别为72.46/%和64.27/%。但添加不同浓度H2O2，成熟胚再生率都低于对照，表明培养基中添加H2O2在一定程度上抑制了成熟胚的再生。 5．以矮败小麦为材料，比较了玉米诱导系和普通自交系诱导小麦单倍体的影响。结果表明，两种玉米基因型得胚率没有显著差异。对获得的矮败小麦单倍体进行了细胞学、植物学和分子标记鉴定，证实获得了Ms2//Rht10矮败小麦单倍体植株，且矮败小麦的二种雌配子ms2//rht10和Ms2//Rht10经玉米花粉诱导后都具有发育为单倍体植株的潜力。进一步以扬麦16为材料，研究了不同浓度AGP处理对小麦-玉米杂交后小麦单倍体得胚率、萌发率和成苗率的影响，结果发现，2g//L AGP处理在一定程度上促进了小麦-玉米杂交后小麦单倍体胚的发育。 6．对小孢子培养技术进行了有意义的探索，发现在4℃条件下低温饥饿处理麦穗25天，并增加液体培养基中小孢子密度，对小麦小孢子培养具有至关重要的效果，并从小孢子培养中诱导形成了胚状体，获得了再生植株，为进一步建立小麦小孢子培养技术体系奠定了基础。 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.

关 键 词： 小麦 组织培养 远缘杂交 单倍体

分 类 号： [S512.11]

领　　域： [农业科学]