利用小麦×玉米诱导小麦单倍体形成的研究

利用小麦（Triticum aestivum L.）与玉米（Zea mays L.）进行远缘杂交,是获得小麦单倍体的一条重要途径,也是获得双单倍体（Double haploid,DH）植株、构建小麦遗传群体的重要途径之一。为创建小麦抗吸浆虫DH群体,在温室条件下对5个小麦抗、感吸浆虫杂交组合进行了单倍体诱导试验。结果表明：小麦不同基因型对诱导单倍体胚形成有较大影响;小麦授粉后适宜的2,4-D点药时间为6-36h;适宜的2,4-D药液为10-30mg/L;授粉后适宜的剥胚培养时间为14-16天,授粉后茎秆离体培养有较好的诱导效果,培养温度以20~25℃为宜。提出了在温室条件下高效诱导小麦单倍体植株的方法及条件。     

苦瓜未授粉子房的胚状体诱导

苦瓜常规育种获得纯合株系耗时长、成本高,通过离体雌核培养途径获得单倍体可加速苦瓜育种进程。为建立苦瓜未授粉子房离体培养再生体系,本研究利用苦瓜未授粉子房为外植体,研究基因型、接种方式、激素配比、胚囊发育时期、预培养处理对胚状体诱导效果的影响。结果表明:MS+6-BA 0.5 mg/L+NAA1.00 mg/L+2,4-D 1.0 mg/L+TDZ 0.04 mg/L培养基胚状体的诱导率最高,为19.60%;在8个苦瓜品种中,2个植株生长势强的品种‘桂农科3号’和‘泰国山’苦瓜胚状体诱导率较高,适合作为离体雌核诱导的实验材料;开花当天的子房去除表皮后横切成2 mm的薄片可有效降低愈伤组织的形成,提高胚状体的诱导率;在黑暗和33℃条件下热激3 d有利于子房的转绿和出胚。本研究对苦瓜未授粉子房的胚状体诱导关键因子进行优化,为创制苦瓜单倍体育种材料提供帮助。     

小麦×玉米远缘杂交技术以及在单倍体育种上的应用评价

为了提高小麦作物育种选择的精确性和效率,应用小麦亲本杂交后代F1(或F2)代与玉米进行远缘杂交,经激素诱导和组织培养形成单倍体胚及植株,加倍单倍体(DH)育种结合分子标记和性状表达,达到了单代纯化培育小麦新品系的生产能力;本文阐述了小麦×玉米远缘杂交发展进程,分析了小麦×玉米远缘杂交较孤雄生殖、球茎大麦技术等具备的优势...     

小麦与玉米杂交产生小麦单倍体与双单倍体的稳定性

小麦与玉米杂交是诱导小麦单倍体最有效的途径之一, 但单倍体和双单倍体产生频率不稳定影响了该技术的应用。选用13个小麦杂种F₁代单交组合与玉米杂交, 研究了不同小麦生长环境、生长素处理、培养基和壮苗处理对单倍体及双单倍体产生频率的影响。小麦生长在大田, 去雄后割穗培养与玉米杂交平均得胚率为23.9%, 每个杂交穗平均得胚数6.8个, 均是返青后从大田移回冷温室盆栽的3倍以上;不同小麦杂交组合间胚产生频率存在明显差异。生长素Dicamba蘸穗处理平均得胚率是21.5%, 与2,4-D处理得胚率(21.1%)无显著差异, 但不同杂交组合间差异显著。B5培养基幼胚萌发率为70.9%~88.3%, 平均82.0%;1/2 MS培养基胚萌发率为70.0%~86.0%, 平均76.6%;两种培养基平均胚萌发率无显著差异。试管苗经壮苗培养基壮苗处理与试管苗经移栽壮苗处理后加倍效率分别是67.6%和8.6%。移栽壮苗处理的苗分蘖少, 生长较弱, 加倍处理后存活率低和加倍率低是其单倍体加倍效率低的原因。     

2,4-D浓度对玉米花粉诱导小麦产生单倍体幼胚得胚率的影响

在玉米花粉诱导普通小麦产生单倍体技术中,2,4-D浓度是影响幼胚得胚率的重要因素之一.为了确定2,4-D的适宜浓度,利用9个玉米材料和16个小麦材料进行了研究.在小麦散粉前和散粉后,授玉米花粉后12h和36 h分别用150、200、250、300、350 mg/L的2,4-D蘸穗2次,调查数据显示,散粉前处理得胚率平均值分别为6.74%、7.19%、8.44%、7.53%和6.61%;散粉后分别为2.53%、3.11%、4.70%、2.81%和2.67%.结果表明,小麦散粉前授玉米花粉后用250 mg/L的2,4-D处理得胚率最高.     

农杆菌转化小麦幼胚获得转bar基因再生植株

用农杆菌转化小麦授粉10d后的幼胚，经5‰PPT筛选获得大量正常再生植株。PCR及PCR－Southern杂交检测证明其中23％（18株）再生苗为转化bar基因植株，这些植株可明显提高对basta的抗性。还总结了农杆菌转化小麦幼胚高效获得转基因再生植株的处理程序。     

不同二倍体和四倍体小麦成熟胚再生体系研究

为了研究不同基因型和培养条件对小麦成熟胚愈伤组织诱导和分化的影响,以硬粒小麦、墨西里卡、野生一粒麦Tu和野生一粒麦Tb为实验材料,对其成熟胚在不同激素配比下愈伤组织的诱导和植株分化进行研究。结果表明,不同基因型小麦成熟胚愈伤组织诱导、分化及再生均存在很大差异。其中硬粒小麦的成熟胚培养效果最佳,其愈伤组织在不同2,4-D浓度（1.0~4.0 mg/L）下诱导率均在93%以上。不同激素配比对成熟胚愈伤组织绿点率、再生率均有显著影响。硬粒小麦、墨西里卡、野生一粒麦Tb在激素配比为KT 1.5 mg/L+NAA 0.5 mg/L的分化培养基中培养效果最好,其绿点率分别为85.22%,61.67%,8.50%,成苗率分别为40.40%,32.06%,1.72%;而野生一粒麦Tu的最适分化培养基激素配比为KT 1.0 mg/L +NAA 1.0 mg/L,其绿点率和再生率分别为18.64%和8.47%。研究表明,基因型是影响二倍体和四倍体小麦成熟胚培养的主要因素,愈伤组织的诱导和植株的再生是相互独立的。     

色素万寿菊杂交母本未授粉子房培养

以色素万寿菊雄性不育株未授粉子房为外植体,比较研究子房发育期、高低温处理和生长调节剂组合对愈伤组织诱导和分化的影响。结果显示:发育6~10 d,花丝刚好露出花萼,顶部呈圆柱状的花蕾愈伤组织诱导效果最好,为79.2%;低温预处理3 d,高温培养5 d有利于愈伤组织诱导;MS+2,4-D 0.5 mg/L+NAA0.5 mg/L+6-BA 0.5 mg/L+KT 0.5 mg/L为愈伤组织诱导最佳培养基,诱导率高达85.8%;NAA 1.0 mg/L+6-BA3.0 mg/L为不定芽分化适宜培养基,分化率达77.6%;1/2MS+NAA 0.2 mg/L为生根适宜培养基,生根率93.5%。本研究可为建立高效单倍体培养体系以及单倍体育种技术提供基础性资料。     

利用远缘杂交和未减数配子基因创制小麦加倍单倍体

未减数配子的结合实现染色体自动加倍,是多倍体物种起源的重要途径,也是提高作物单倍体育种效率的重要手段。我们前期从四倍体小麦发掘出控制未减数配子形成的强效QTL位点QTug.sau-3B,并通过人工合成小麦为“桥梁”,将其导入到综合农艺性状优良的小麦新品系中。本实验使用5份含未减数配子基因的优良小麦新品系与不含未减数配子基因的小麦推广品种的F1杂种作母本与3份白茅(Imperata cylindrica)进行远缘杂交,共授粉4610朵小花,结实1965粒,经幼胚拯救获得244个幼胚,其中50个幼胚发育正常生长为50个小麦单倍体植株。由于小麦单倍体植株未减数配子基因的表达易受环境影响,因此,对单倍体植株在相同光周期(18 h光照/6 h黑暗)下进行了不同温度25℃/18℃、25℃/15℃和25℃/10℃处理,结果表明, 25℃/18℃和25℃/10℃条件下编号为H31单倍体植株能够结实,自交结实率分别为4.35%和2.41%。该研究结果为建立“基于小麦-白茅杂交实现染色体消除和未减数配子基因实现染色体自动加倍”的小麦单倍体育种技术提供了参考。     

预处理和外源激素对黄瓜未授粉子房的胚状体诱导的影响

用正交设计法研究了预处理和4种外源激素对诱导黄瓜未授粉子房胚状体发育的影响。结果显示,诱导黄瓜大孢子发育的最佳培养条件是:36℃3 d 0.5 mg/L NAA 2.0 mg/L KT 1.5 mg/L BA,高温36℃预处理对再生植株的诱导率明显高于低温的预处理,且2,4-D,NAA和BA对胚状体的诱导有促进作用。     

Inheritance of resistance to wheat midge, Sitodiplosis mosellana, in spring wheat

Inheritance of resistance to a wheat midge, Sitodiplosis mosellana (Géhin), was investigated in spring wheats derived from nine resistant winter wheat cultivars. F₁ hybrids were obtained from crosses between resistant winter wheats and susceptible spring wheats, and used to generate doubled haploid populations. These populations segregated in a ratio of 1:1 resistant to susceptible, indicating that a single gene confers the resistance. The F₂ progeny from an intercross among spring wheats derived from the nine resistance sources did not segregate for resistance. Therefore, the same gene confers resistance in all nine sources of resistance, although other genes probably affect expression because the level of resistance varied among lines. Heterozygous plants from five crosses between diverse susceptible and resistant spring wheat parents all showed intermediate levels of response, indicating that resistance is partly dominant. Susceptible plants were reliably discriminated from heterozygous or homozygous resistant ones in laboratory tests, based on the survival and development of wheat midge larvae on one or two spikes. This powerful resistance gene, designated Sm1, is simply inherited and can be incorporated readily into breeding programmes for spring or winter wheat. However, the use of this gene by itself may lead to the evolution of a virulent population, once a resistant cultivar is widely grown.     

Genetic Variability for Haploid Production in Crosses Between Tetraploid and Hexaploid Wheats with Maize

Crosses were made between 14 wheat genotypes (11 tetraploid, 3 hexaploid) and a single Fl hybrid of maize that was used as the male parent. The experimental design consisted of randomized blocks with three replications. Plants were grown under controlled greenhouse conditions (day length 16 h and temperature 25 °C/15 °C, day/night). To enhance embryo survival, 2, 4-D treatment (10 mg/1) was applied to spikes 24 h after pollination with maize. Embryos were recovered from all tetraploid and hexaploid wheats at a rate of 2.09 to 26.76 per 100 pollinated florets. Haploid and doubled haploid plants were obtained from all hexaploid genotypes (T. aestivum) and from 5 of 11 tetraploid genotypes (T. turgidum var.). The most important point of these experiments was the ability to produce haploid plants from tetraploid wheat for two reasons: firstly, anther culture cannot be applied in tetraploid wheat (T. turgidum var.) due to the inefficiency of embryo formation and the high proportion of albino plants. Secondly, to date, crosses between tetraploid wheat and maize have resulted in embryo formation, but not in haploid plants.     

Wheat embryogenesis and haploid production in wheat × maize hybrids

Summary Embryogenesis was analyzed in wheat × maize hybrids using paraffin sectioning. Embryogenesis in wheat × maize hybrids is different from that in self-pollinated wheat plants. Development of the embryo is not accompanied by the formation of an endosperm. The endosperm nuclei remain free in the cytoplasm, fail to advance into the cellular stage, and degenerate at a later time. The antipodal cells quickly degenerate in the fertilized ovaries of wheat × maize hybrids similar to self-pollinated ovaries. The antipodal cells remain normal in unpollinated ovaries. The pre-embryo will abort if it is allowed to develop on the plant, because of a nutritional shortage in the absence of an endosperm. Therefore, embryo rescue is necessary for haploid production from a wheat × maize hybrids. Haploid polyembryos were obtained from spikelet culture of wheat × maize hybrids. The formation of polyembryos is due to the cleavage of the pre-embryo and the effect of 2,4-D. The frequency of haploid embryo production and plant regeneration is affected significantly by maize genotypes, but not by wheat genotypes. The concentration of 2,4-D affects only the size of the embryo.     

Chromosomal regions associated with the in vitro culture response of wheat (Triticum aestivum L.) microspores

Generation of doubled haploid plants is a powerful tool in breeding, as homozygous individuals will be obtained directly from hybrids. However, genotype variability in regeneration efficiency of most European wheat (Triticum aestivum L.) varieties has limited its use in wheat. This study intended to identify quantitative trait loci (QTLs) for green plantlet regeneration from wheat microspore cultures. A QTL analysis using DArT markers was conducted based on a bi‐parental F₃ population, derived from a cross between the varieties Svilena and Jensen, which displayed markedly different capacity for plantlet regeneration. Two QTLs on chromosome 1B and 7B explained 53% of the variation in green plantlet regeneration. Furthermore, a collection of 94 European wheat varieties was genotyped and phenotyped. The microspore response level was low among western and northern European wheat varieties, and the positive QTLs found in the bi‐parental population were rare in the variety collection. Identification of the two QTLs enables introduction of high regeneration efficiency into wheat germplasm. Moreover, our results proved that the efficient regeneration observed for one variety could be crossed into modern winter wheat.     

The effects of parthenogenesis on wheat embryo formation and haploid production with and without maize pollination

Doubled haploid (DH) lines are important in wheat (Triticum aestivum L.) breeding, and haploids produced via maize pollination precede DH line development. Although maize pollination has proven reliable and broadly applicable to wheat, its success is determined by the wheat and maize genotypes employed. A wheat genotype consisting of nuclear and cytoplasm components predisposing it to parthenogenesis was compared with three other genotypes, each possessing only one or neither component necessary for parthenogenesis. In a glasshouse experiment, each genotype was pollinated with maize and subsequently treated with a2,4-Dichlorophenoxyacetic Acid (2,4-D) solution to determine if parthenogenesis affected embryo formation frequency (EFF)and haploid formation efficiency (HFE). Wheat genotypes were also treated with the2,4-D solution alone to determine if embryos and haploid plants could be produced in vivo without maize pollination. ‘Salmon(K)’, a parthenogenetic genotype consisting of a Salmon 1BL.1RS nucleus in a Ae. kotschyii cytoplasm, had a mean EFF of 32%; whereas, the non-parthenogenetic genotypes had mean EFF calculations ranging from 7 to 21%. Mean HFE for Salmon(K) was not significantly different than the mean HFE for non-parthenogenetic Salmon; however, EFF and HFE calculations for Salmon(K) and Salmon, each with a 1BL.1RS translocation, were generally higher than calculations for genotypes without the translocation. Salmon(K) was the only genotype to produce a 3% or higher EFF and HFE after treatment with 2,4-D alone. Parthenogenesis significantly affected the frequency at which embryos were produced after pollination with maize and the frequency at which embryos and haploid plants were produced after treatment with 2,4-D alone. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of Doubled Haploids by Anther Culture and Wheat X Maize Method in a Wheat Breeding Programme

Utilization of the doubled haploid method of breeding usually shortens the time to cultivar release, and methods of haploid production need evaluation in a breeding programme. Thirty-eight different three-way crosses were tested for anther culture response. On average 5.8 percent of the anthers cultured produced calli. Three crosses were found recalcitrant for callus induction. Overall, the anther culture method produced 0.6 plantlet per 100 anthers cultured. Five crosses with an average of 5.8 and 2.8 percent of anthers producing calli and plantlets, respectively, were compared using anther culture and wheat × maize crosses. Non-responsive genotypes for callus induction and plantlet formation in the anther culture method proved to be good parental material in wheat × maize crosses. The average percentages of embryo formation and plantlet production in wheat × maize crosses were 10.3 and 4.7, respectively. Anther-derived plants were cytologically unstable, whereas all the plants regenerated from wheat × maize crosses were haploids (n = 21 chromosomes). The chromosome numbers of the polyhaploids were doubled with a colchicine treatment. Improvement of the two haploid production methods to facilitate their efficient use in a breeding programme is discussed.     

The effect of colchicine on triticale anther-derived plants: Microspore pre-treatment and haploid-plant treatment using a hydroponic recovery system

In cereals, chromosome doubling of microspore-derived haploid plants is a critical step in producing doubled haploid plants. This investigation was undertaken to study the effect of incorporation of colchicine in the induction medium for anther culture, and the effect of colchicine on anther culture-derived plants of triticale grown under controlled greenhouse conditions. In the latter case, chromosome doubling of adult sterile plants derived from anther culture of fourteen triticale populations was attempted, where androgenetic plants with non-dehiscent anthers were cloned and subjected to the colchicine treatment, and then grown with the aid of hydroponics. The hydroponic system provided optimal conditions for recovery of the affected haploids from the toxic effects of colchicine treatment and all colchicine-treated plants survived. A topcross-F₁ (TC₁F₁) population with timopheevii cytoplasm produced the highest percentage of plants with seed-set either due to chromosome doubling by colchicine (98%) or spontaneous doubling of chromosome number (15%). Colchicine-treated anthers performed inferior than control in both induction and regeneration phases. One of the key observation of this study was the reversal from reproductive stage back to the vegetative stage which in turn enabled further cloning of haploid plants under hydroponic conditions once they were identified as sterile. The one hundred percent survival rate of in vitro-derived plants, 100% survival rate of colchicine treated haploid plants and the high chromosome doubling success rate (X = 82.3) observed in this study imply that a temperature-controlled greenhouse with an hydroponic system provides an efficient environment for inducing chromosome doubling of haploid plants in cereals. This revised version was published online in August 2006 with corrections to the Cover Date.     

Optimization of culture conditions for improved plant regeneration efficiency from wheat microspore culture

The production of haploid plants through microspore culture is a very important tool for plant breeding. However, progress in microspore culture for many species has been hampered by a number of factors that have resulted in low recovery of regenerated green plants. In this study, a series of experiments were conducted to increase the regeneration of haploid green plants from isolated wheat microspores. The use of different basal media and variations in media components resulted in the increased recovery (approximately double) of regenerated haploid wheat plants. Our findings demonstrate that CHB medium, in combination with 2,4-d, was a better medium for embryoids induction and plant regeneration than medium MC17 with either 2,4-d or PAA growth hormones. Wheat microspores cultured without ovary co-cultivation did not respond. Furthermore, high efficiency of microspore derived embryoids (up to 296 MDEs per 100 anthers) and green plant regeneration (up to 71 green plants per 100 anthers) were achieved by the use of gelrite instead of agarose as a gelling agent, and by the addition of media additives such as spent medium or MET.     

Durum wheat × maize crosses for haploid wheat production: Influence of parental genotypes and various experimental factors

To improve haploid plant production in durum wheat, the haplomethod involving intergeneric crossing with maize followed by embryo rescue was used. The influence of parental genotypes and various experimental factors were studied. Ten cultivars of Triticum turgidum ssp. durum (female parent) were crossed with eight genotypes of Zea mays. After pollination, plant stems were either maintained in situ or cut near the base and kept in a 2,4‐dichlorophenoxyacetic acid (2,4‐D)‐sucrose solution. Ten to 18 days after pollination, embryos were excised from developed ovaries and cultured on one of MS, MS/2, or B5 media. Haploid embryos and plants were obtained (78 green haploid plants regenerated in 0 year). The wheat genotype was significant for ovary development, embryo and plant formation, whereas the maize genotype was significant only for embryo formation. Detailed results of all crosses showed the best crossing partner for each wheat genotype. Cutting the plant stems after pollination gave better results than maintaining them in situ. The optimal stage for embryo rescue was 14 days and B5 and MS/2 media were more efficient than MS for embryo culture.