怎样移栽小麦单倍体试管苗

由小麦花药离体培养而产生的小麦单倍体试管苗,一般在高温潮湿的夏季长期生长在试管里,其单倍体植株茎秆纤细嫩弱、分蘖少、叶色淡绿、根系生长不好,即使秋季移栽到大田里仍很难成活。为此,我们做了两季壮苗越夏的试验,结果如下: 一、促壮苗生根我们将小麦单倍体植株转移在附加NAA或IAA的壮苗培养基以后,有的单倍体植株不生根     

培养基和基因型对小麦x玉米诱导单倍体效率的影响

当培养条件、胚龄、胚发育期间的积温一致时,培养基就成为胚萌发和成苗的关键性影响因素。选用4种培养基（B5、B5 0.1 mg L-1 BAP 0.1 mg L-1 NAA、 1/2MS、1/2MS 0.1 mg L-1 BAP 0.1 mg L-1 NAA）培养3个小麦杂交组合F2与玉米杂交获得的单倍体胚,以比较不同培养基对单倍体胚的萌发和成苗的影响。结果显示不同培养基单倍体胚的平均萌发率和平均成苗率存在极显著差异,胚萌发率表现为1/2MS（75.24%）＞1/2MS 0.1 mg L-1 BAP 0.1 mg L-1 NAA（68.68%）＞B5 0.1 mg L-1 BAP 0.1 mg L-1 NAA（67.00%）＞B5（66.67%）;胚成苗率表现为： 1/2MS（63.11）＞B5（50.67%）＞B5 0.1 mg L-1 BAP 0.1 mg L-1 NAA（4.00%）＞1/2MS 0.1 mg L-1 BAP 0.1 mg L-1 NAA（3.30%）。无激素的1/2MS上单倍体胚的平均萌发率(75.24%)和平均成苗率(63.11%)都最高,能稳定地一次成苗。不同杂交组合后代的单倍体胚的平均萌发率和平均成苗率也存在极显著差异。表明小麦基因型是影响单倍体胚萌发和成苗的重要因素,无激素的1/2MS最适合于小麦单倍体胚的萌发和一次成苗。     

不同小麦组合与不同玉米基因型远缘杂交诱导小麦单倍体的研究

选用21个小麦F1杂交组合与4个玉米基因型远缘杂交诱导小麦单倍体。结果表明,不同小麦F1杂交组合的得胚率存在明显差异,最高组合D026/D035为11.6%,最低组合Ta01/C98仅为1.2%;二者相差10%以上;不同玉米基因型的得胚率趋势表现为:甜玉米(35.7%)>普通玉米(11.6%)>爆裂玉米(8.7%)>糯玉米(7.3%);玉米扬花期间,不同阶段授粉对得胚率具有显著影响,以开花当日授粉最佳,越往后得胚率越低。     

培养基对小麦单倍体胚成苗率的影响

为筛选出适宜小麦单倍体幼胚生长的培养基,提高小麦单倍体胚成苗率,将小麦×玉米远缘杂交得到的小麦单倍体幼胚接种在8种培养基上,每种培养基上接种2个材料,平均值用于数据分析。8种培养基中幼苗平均产生率分别为：46.07%、45.90%、34.11%、41.15%、37.99%、36.91%、37.70%和52.51%。结果表明1/2MS +1.0 mg/L KT+3.0 mg/L多效唑+100 mg/L肌醇+5%蔗糖+0.6%琼脂,pH 5.8培养的幼胚成苗率最高,白化苗率低,且单倍体苗的长势较好,有利于培育壮苗。以1/2MS为基本培养基,加入微量的多效唑、KT、肌醇以及较高浓度的蔗糖可以有效促进幼胚生长。     

不同基因型玉米材料的单倍体诱导和加倍研究

利用12份不同基因型F1进行单倍体杂交诱导与自然加倍研究,分析不同材料单倍体诱导率和加倍结实率的差异性。结果表明:12份不同基因型F1的单倍体诱导率和加倍结实率都存在显著的差异,诱导率最高达11.03%,最低为3.34%;加倍结实率最高达13.20%,最低为2.61%。2组正反交组合的单倍体诱导率差异较小,而其中1组正反交组合的加倍结实率存在显著差异,最高相差3.34%。单倍体的雄穗加倍率与加倍结实率存在极显著的相关性,且雄穗正常散粉植株加倍获得稳定纯系的比例较高,达89.52%。     

小麦单倍体育种中培养基的研究

小麦单倍体育种中培养基直接影响幼苗产生率,为了找到一种适宜小麦幼胚生长的培养基,对16份F1单交组合幼胚在8种培养基上的生长发育状况进行了研究,8种培养基的幼苗平均产生率分别为32.71%、41.48%、47.15%、53.59%、57.98%、62.05%、53.26%和42.21%。培养基①、②、③、⑧的幼苗平均产生率较低,培养基④、⑤、⑥、⑦的较高,其中培养基⑥(1/2MS+0.2 mg/L IAA+0.2 mg/L 6-BAP+2%蔗糖+7.5g/L琼脂 PH=5.8)的幼苗产生率为62.05%,且幼芽分化明显,根系弯曲至瓶底,叶色深绿,较其他7种培养基培养效果最好。     

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.     

Dicamba和2,4-D处理对小麦与玉米杂交得胚率的影响

设置生长调节剂Dicamba和2,4-D不同浓度混合液共6种处理,研究Dicamba和2,4-D对5个小麦基因型与玉米杂交得胚率的影响。结果表明,生长调节剂处理间、小麦基因型间以及二者之间互作均显著影响小麦与玉米杂交得胚率。单独使用100mg/L2,4-D的得胚率最高(26.2%,极显著高于25mg/L2,4-D+75mg/L Dicamba处理的得胚率(19.9%)和50mg/L2,4-D+50mg/L Dicamba混合处理的得胚率(20.0%)。其余3种混合型生长调节剂处理间得胚率差异不显著。襄麦56平均得胚率最高,显著高于扬麦158、郑麦9023和组合0173,其次为裹麦25。同一小麦基因型不同生长调节剂处理的得胚率及同一生长调节剂处理不同小麦品种间得胚率均存在差异。     

球茎大麦在大麦育种上的应用——Ⅰ.大麦单倍体的诱导与加倍单倍体的产生

以三种栽培大麦品种为母本,三种球茎大麦为父本进行杂交。诱导栽培大麦单倍体,结果杂交成胚率平均为47.9％,幼胚离体培养成苗率平均为54.0％。这些胚培苗的根尖细胞染色体数目分别含有7,14,21条,不同组合杂交后代含有的染色体数目不同,所有含有7条染色体的后代,其染色体C-带带型显示为父本栽培大麦的染色体,表明该后代为栽培大麦单倍体,用滴加0.04％秋水仙素的Knop溶液进行单倍体植株的染色体加倍,结果加倍率达90％以上,另外,我们还讨论了染色体消失法诱导单倍体的优越性及其机理。     

小麦单倍体幼胚离体培养研究

小麦单倍体幼胚由于胚乳发育异常不能提供营养物质,因此幼胚必须经过离体培养才能正常生长,而适宜的培养基是离体培养的关键。将小麦幼胚接种在8种培养基上,幼苗平均产生率分别为:30.95%、40.20%、42.20%、47.75%、45.00%、63.35%、50.00%和36.65%,结果表明1/2MS 0.2mg/LI-AA 0.2mg/L6-BAP 2%蔗糖 7.5g/L琼脂pH=5.8培养效果最好。     

Improved Techniques for Haploid Production in Wheat using Chromosome Elimination

Wheat × maize and wheat × pearl millet crosses have proved efficient for haploid production using various genotypes of wheat; 22 and 27 % of florets produced embryos. In favourable conditions 6—9 haploid plants per spike were produced. The following simplifications or improvements in technique are recommended: 1. Only a single treatment with an aqueous solution of dicamba or 2,4-D (50–100 ppm) for embryo stimulation in vivo; 2. Application by spraying or dipping the spikes; 3. Application time two to four days after pollination; 4. Embryo rescue 15 to 18 days after pollination; 5. Crosses without emasculation are possible if pollination occurs 1–2 days before anthesis. More than 450 haploids and some doubled haploid (DH) lines (after colchicine treatment in vitro) were produced using these methods. No hybrid plants, chromosome additions or substitutions were found.     

Effect of parental genotypes on haploid embryo and plantlet formation in wheat × maize crosses

Genotypic influence of both male and female parents on haploid production through interspecific crosses was studied using eight wheat and four maize genotypes. The average numbers of embryos and green haploid plantlets obtained per pollinated floret were 17.6% and 10.1%, respectively. Clear genotypic influence of the wheat genotype was detected, but heterozygosity of the wheat did not affect haploid production. Analogous response to anther culture and interspecific crossing was observed, still a wheat variety which did not respond to anther culture, produced 1.1 plantlets per pollinated spike upon maize pollination. This appears to be a major advantage of interspecific crossing compared to anther culture technique in wheat. Circumstantial evidence is presented for specific wheat × maize interaction on haploid plantlet formation. Rye chromatin enhanced haploid production but only in a complete 1B/1R substitution line. Ovaries with an embryo were found to be dispersed evenly all over the wheat spike, suggesting that within certain limits the developmental stage of ovaries and thus time of pollination within a spike are not as important as it was previously assumed. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

High Frequencies of Fertilization and Haploid Seedling Production in Crosses Between Commercial Hexaploid Wheat Varieties and Maize

Nineteen commercial hexaploid wheat varieties were crossed with the maize F₁ hybrid ‘Seneca 60’. Fertilization frequencies ranged from 32.1 % to 47.5 % of pollinated florets (mean 39.5 %) in the 14 winter wheat varieties and from 40.7 % to 51.4 % (mean 47.8 %) in the five spring wheat varieties. In some cases only an endosperm was formed and the frequencies of embryo formation were therefore slightly lower, being 28.2 % to 45.9 % (mean 36.4 %) for winter wheats and 39.8 % to 48.6 % (mean 45.1 %) for spring wheats. Mean values were significantly higher in the spring wheats but no significant variation was found between varieties within the spring or winter categories. In the five spring wheats the mean yield of embryos, and hence the potential yield of haploid plants, was 3.4-fold higher than with the tetraploid Hordeum bulbosum clone PB179. For the 14 winter wheats the figure was 10.9-fold higher. These differences were highly significant (p < 0.001) in all varieties. A single 2,4-D treatment given to spikes one day after pollination with maize enabled embryos to be recovered from all 19 varieties. A total of 311 embryos were recovered from 950 florets (an average of 7.3 embryos per spike) of which 191 germinated, giving an average yield of one haploid plant for every 5.0 florets pollinated (4.4 haploid plants per spike).     

Relative Efficiency of Anther Culture and Chromosome Elimination Techniques for Haploid Induction in Triticale × Wheat and Triticale × Triticale Hybrids

Summary The study was undertaken to evaluate the relative efficiency of anther culture and chromosome elimination (by crosses with maize) techniques of haploid induction in intergenotypic triticale and triticale × wheat hybrids. For this, 15 triticale × wheat and 8 triticale × triticale F₁ hybrids were subjected to anther culture and were also simultaneously crossed with the `Madgran Local' genotype of maize (Zea mays L.) to induce haploids through the chromosome elimination technique. The haploid embryo formation frequency through the chromosome elimination technique was significantly higher in both, triticale × wheat (20.4%) and triticale × triticale (17.0%) F₁ genotypes, as compared to the calli induction frequencies through anther culture (1.6 and 1.4%, respectively). Further, four triticale × wheat and three triticale × triticale F₁ genotypes failed to respond to anther culture, whereas, all the F₁ genotypes formed sufficient number of haploid embryos through the chromosome elimination technique with no recovery of albino plantlets. The haploid plantlet regeneration frequencies were also significantly higher through the latter technique in both triticale × wheat (42.7%) and triticale × triticale (49.4%) F₁s as compared to anther culture (8.2 and 4.0%, respectively), where the efficiency was drastically reduced by several constraints like, high genotypic specificity, low regeneration frequency and albinism. The overall success rates of obtaining doubled haploids per 100 pollinated florets/anthers cultured were also significantly higher through the chromosome elimination technique (1.1% in triticale × wheat and 1.5% in triticale × triticale hybrids), proving it to be a highly efficient and economically more viable technique of haploid induction as compared to anther culture, where the success rates were only 0.2% and 0.1%, respectively.     

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.     

Dicamba and growth condition effects on doubled haploid production in durum wheat crossed with maize

Doubled haploid plants are useful in genetic studies and plant breeding, but a consistent and satisfactory frequency of production has been difficult to achieve in durum wheat. Triticum turgidum L., using the maize pollen method. The objective of this study was to develop an objective method of producing doubled haploids in durum wheat. Plant growing and handling conditions, aspects of hormone treatments, wheat genotype and pollen source were considered. The number of caryopses, embryos, haploids, doubled plants and doubled plants that set seed were measured. Although growth conditions, pollen source, method of handling plants and wheat genotype are important considerations, the type of hormone was found to be most significant in the production of doubled haploid plants. When 50mg/l dicamba was substituted for 100 mg/l 2,4‐D the number of doubled haploids per spike increased from 0.2 for the best 2,4‐D treatment to 1.3 for the dicamba treatment. This increased frequency was largely attributed to an increase in the number of caryopses generated for each spike emasculated and from an increased frequency of germination of embryos to haploid plantlets. The best production of caryopses was 0.41 caryopses per florest with 2,4‐D. The best production of haploids per 100 florets was 12 with dicamba and 1.65 with 2,4‐D. The frequency of one doubled haploid per emasculated spike through the use of dicamba is a practical level for generating populations for genetic studies.