利用玉米黄绿苗标记单倍体诱导系选育早期加倍双单系（EDH）的研究

介绍了一种新型玉米单倍体诱导系,该诱导系兼具籽粒标记、幼苗标记和成株标记,可在幼苗期通过黄绿苗性状鉴别出杂合二倍体植株,可在早期加倍（EDH）、高自然加倍、单倍体回交和单倍体轮回选等技术方面应用,有明显的技术优势与应用前景。     

玉米不同基因型诱导系诱导效果研究及应用

研究旨在对玉米诱导系进行改良,对改良诱导系进行诱导率鉴定,以期在单倍体育种中更好利用。研究引进的2个常规诱导系及用其改良选育的5个诱导系对杂交种‘浚单509’的单倍体诱导能力的差异。结果表明:7个诱导系中单倍体诱导率最高的是QYG1,为3.36%;最低的是ZYG1,仅为0.4%;改良后诱导系QYG1、ZYG2诱导率稍有提高。7个诱导系在玉米穗部不同部位(上部、中部、下部)诱导的准单倍体诱导率不同,5个中部居多,下部次之,上部最少。QYG1田间植株表现植株高大、雄穗花粉量大且散粉期长、抗逆性强,可以考虑作为新的诱导系利用。研究结果为开展玉米诱导系选育及单倍体技术进行种质改良提供了综合的参考依据。     

孤雌生殖诱导玉米单倍体育种技术研究

玉米单倍体育种技术可以有效地缩短育种进程,快速获得大量纯合系。孤雌生殖诱导是获得玉米单倍体的主要方法之一,该项技术在国外各大育种公司已被广泛使用,但在国内仍处于研究探索阶段。本文从诱导系的选择与繁殖、基础材料的组配、杂交诱导、单倍体籽粒的挑选、单倍体植株的种植、加倍以及双单系的繁殖等方面综述了孤雌生殖诱导系（简称诱导系）产生玉米单倍体育种技术的基本流程,同时展望了单倍体育种的应用前景。     

玉米单倍体鉴定方法研究进展

玉米单倍体鉴定是玉米单倍体育种三大关键技术(杂交诱导、单倍体鉴定、染色体加倍)之一,准确、高效的单倍体鉴定方法不仅可以提高玉米单倍体育种的效率,同时对筛选高诱导率的单倍体诱导系也十分重要。综述了国内外玉米单倍体鉴定方法的演变过程,旨在为玉米单倍体育种工作者把握鉴定方法发展的脉络提供帮助。     

激素浓度对茄子花药愈伤再生植株的影响

以4个茄子杂交品种为试材,对诱导花药获得的愈伤进行再生植株诱导。结果表明,4份材料中,苏茄7号、苏茄8号诱导出极少不定芽;苏茄6号能够诱导少量不定芽,但是在后期生长中极易玻璃体化;苏茄5号能够诱导大量不定芽。诱导苏茄5号形成不定芽的的激素浓度最佳配比是1.0 mg/L 6-Benzylaminopurine(6-BA)+0.05 mg/L 1-Naphthylacetic acid(NAA),诱导率可达50%。最终获得27个再生植株,经过根尖染色体计数和SSR标记鉴定,均为双单倍体。以上结果表明,合适的激素组合以及合适的浓度可以大大提高再生植株的诱导率,同时也可以诱导愈伤组织加倍直接获得双单倍体。     

在采用和不采用玉米授粉条件下单性生殖对小麦胚形成和单倍体生产的影响

小麦加倍单倍体(DH)品系有助于育种者鉴定控制重要品质性状的分子标记。此外，由于所生产的DH植株在单一步骤就产生出完全纯合性，因此DH品系能促进理想基因型的选择，加速品种的释放。培育DH品系必不可少的步骤是单倍体的生产。采用许多不同的技术都能生产出小麦单倍体；然而，采用玉米授粉的远缘杂交和单倍体生产     

玉米花药培养及再生植株倍性鉴定

对影响玉米花药培养的几个因素及再生植株的倍性鉴定的结果表明、基因型是影响玉米花培效果的重要因素,杂交种的花培效果好于不稳定系,不稳定系又好于自交系.N_6与正_(14)两种培养基都适于花药培养,但正_(14)培养基优于N_6培养基.特别是液体培养基效果更好.花药接种的适宜时期为单核中期和后期.单核后期又稍好于单核中期,单核早期的花药不能诱导愈伤组织.单倍体与二倍体花粉植株之间叶片气孔保卫细胞长度差异极显著,长度小于29μm的为单倍体.大于29μm的为二倍体或多倍体.鉴定的准确性可达到95%.因此可利用测量气孔保卫细胞长度的方法鉴定花粉植株的倍性.     

CENH3介导的单倍体诱导技术研究进展

传统育种方法选育纯合自交系需要7~9个世代,含单个亲本染色体的单倍体经过染色体加倍一个世代就可以获得双单倍体(doubled haploid,DH),加快育种进程。许多单倍体获得和加倍的方法受物种或基因型限制,不能广泛应用。在拟南芥中通过遗传工程操控着丝粒特异组蛋白CENH3(centromere-specific histone 3 variant)可产生单倍体。CENH3是细胞分裂时染色体分离所必需的,其决定着丝点的定位。序列替换或点突变的CENH3以及异源CENH3可补偿cenh3的突变,形成单倍体诱导系,同野生型植株杂交诱导单倍体的产生。本研究论述了CENH3介导的单倍体诱导技术的研究进展。CENH3在植物中广泛存在,CENH3介导染色体消失诱导单倍体技术具有广阔的应用前景。     

甘蓝未受精子房离体培养及再生植株倍性的鉴定

甘蓝通过未受精子房离体培养诱导获得的再生植株,对再生植株的倍性进行有效的鉴定是将其进一步应用于优良品种选育的基础。本研究利用3种基因型的甘蓝材料(PMQM、QMF、RMQM)培育再生植株,优化甘蓝未受精子房离体培养体系,并通过形态学鉴定法、根尖染色体计数法、流式细胞仪鉴定法对组培植株进行倍性鉴定。结果表明:在0.4 mg/L ZT的分化培养基中,3种基因型材料的愈伤组织分化率明显高于1.0 mg/L 6-BA培养基中的组培苗,其中基因型RMQM的分化效果最好;最终确定诱导愈伤组织分化不定芽的最适培养基配方为MS+0.4 mg/L ZT+2.0 mg/L 2,4-D+0.1 mg/L NAA,且通过3种鉴定方法,得出再生植株倍性:单倍体3.4%,双倍体49.8%,四倍体15.9%,嵌合体35.3%。     

利用小麦ⅹ玉米诱导小麦单倍体形成的研究

摘 要：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。本研究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2,4-D适宜的点药时间为6－36小时;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20-250C为宜。本文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。     

Doubled Haploids of Cocoa (Theobroma cacao L.) 1. Observations of Fertility

Ovule and pollen fertility have been studied in doubled haploids of cocoa derived from spontaneous haploids, doubled by colchicine treatment. The two measures of fertility were very variable; occasionally, the levels were as high as those of the parent plant, but most were lower. The low fertility was not improved either by changing pollinators or after grafting. Pollen fertility also varied seasonally more in doubled haploids than in their parents. The lack of differentiation of embryo sacs seemed to be the cause of low values of ovule fertility. Both of these unfavourable characteristics were not observed in the progeny of doubled haploids, The low fertility is described in terms of homozygote depression, forced, on normally allogamous plants, its consequences for the use of doubled haploids in the production of hybrid seeds are discussed.     

Effectiveness of combining resistance to Thielaviopsis basicola and Tomato spotted wilt virus in haploid tobacco genotypes

Black root rot (BRR) caused by Thielaviopsis basicola as well as Tomato spotted wilt virus (TSWV) are the most serious problems in tobacco growing regions. We crossed the breeding line WGL 3 carrying BRR resistance derived from N.glauca with the line PW-834 the resistance of which to TSWV was transferred from cultivar Polalta. Anthers obtained from F₁ hybrid plants were cultured to induce haploids combining resistance to Th. basicola and TSWV. Flow cytometry analysis revealed 242 haploids and 2 spontaneous doubled haploids among regenerants. All haploids were cloned and then evaluated for BRR as well as TSWV resistance. The presence of pathogens was detected by microscopic evaluation of roots or using DAS-ELISA test. Microscopic assessment showed that, 132 haploids had no symptoms of Th. basicola which, together with the absence of symptoms in the F₁ hybrids, indicated a dominant monogenic mode of inheritance. At the same time only 30 haploids demonstrated resistance to TSWV. SCAR markers associated with TSWV resistance gene detection was applied. The results indicate that small proportion of TSWV-resistant haploids is probably due to the influence of deleterious genes flanking the resistance factor that reduced vitality of gametophytes. Altogether, 24 haploids showed multiple resistance to Th. basicola and TSWV.     

不同除草剂加倍玉米单倍体的效率

通过比较3种除草剂加倍玉米单倍体的效率,提出了利用除草剂加倍玉米单倍体的新方法。以先玉335、中农大4号和8607×8609三个基因型诱导的单倍体籽粒为材料,利用20、40、80和160 μmol L^-1浓度的甲基胺草磷、炔苯酰草胺和氟乐灵作为加倍药剂,在单倍体植株生长到三叶期和五叶期时,用滴心法处理幼苗,选择有花粉的单株自交,收获后调查果穗加倍率;采用细胞学方法观察单倍体的染色体数目和花粉的活性。结果表明,20~160 μmol L^-1的3种除草剂对玉米单倍体加倍均有效果,加倍率在3.42%~26.32%之间。甲基胺草磷、炔苯酰草胺和氟乐灵的加倍率分别为4.29%~26.32%、3.85%~20.81%和3.42%~17.61%;其中80 μmol L^-1甲基胺草磷的加倍效果最佳,使用80 μmol L^-1甲基胺草磷处理3个杂交种的单倍体,平均加倍率分别为25.02%、20.13%和14.99%。方差分析表明,3个基因型间的单倍体加倍率均呈极显著差异,可见使用甲基胺草磷、炔苯酰草胺、氟乐灵可以提高玉米单倍体的加倍频率,但不同基因型单倍体对除草剂的敏感性存在差异。     

Difference between USA and French isolates of tobacco etch virus and pepper mottle virus displayed by doubled haploid line analysis

Summary Doubled haploid peppers resistant in France to specific strains of tobacco etch virus (TEV) and pepper mottle virus (PeMV) were indexed for resistance to USA strains of TEV and PeMV. The doubled haploids were inoculated when four to six true leaves had developed. The peppers were indexed after an incubation period, and a disease index was calculated. Plants without viral symptoms were reinoculated. Plants challenged to TEV were inoculated four times. Doubled haploids, TEV-resistant in France, were susceptible to the USA strain by the fourth screening. All doubled haploid lines showed TEV symptoms after the fourth inoculation. Two doubled haploids lines, no. 4 and no. 8, had about 6% resistance to TEV after the fourth inoculation. Doubled haploid line no. 5 was 100% resistant to PeMV. The results confirm that the USA PeMV and the USA TEV isolates are different pathotypes than the French ones used originally to screen the doubled haploids. In addition, the use of doubled haploids to identify pathotypes of PeMV and TEV is demonstrated.     

玉米DH系的表现及其同工酶电泳分析

以孤雌生殖诱导系高诱1号诱导的单倍体植株为材料,由单倍体加倍后获得的DH系性状上表现高度一致,其中一些DH系的长势和果穗性状达到了育种上的要求.DH系的过氧化物酶和酯酶同工酶电泳表明,部分DH系具有母本谱带,部分具有父本谱带,由此说明,孤雌生殖诱导系诱导单倍体具有一定的随机性.     

Molecular identification of Pm12-carrying introgression lines in wheat using genomic and EST-SSR markers

The traditional process of obtaining maize hybrids involves the generation of inbred lines through successive generations of selfing and subsequent testcrosses in order to identify the best combining ability by allelic complementation. A fast alternative to obtain inbred lines is to induce the formation of haploids followed by chromosome doubling. However, even with the aid of haploid-inducing genetic sources, this strategy has not been widely used in maize breeding programs, partly due to difficulties inherent to haploid generation and identification. In order to evaluate the possibility of using dihaploids to generate homozygous maize tropical lines, we used the androgenetic haploid inducer line W23 as a female parent in crosses with the tropical single-cross hybrid BRS1010. Within the progeny of these crosses, 462 seeds were phenotypically selected as putative haploids by the purple-colored endosperm and colorless embryo conditioned by the R1-nj gene. Among these, only four individuals were confirmed as being haploids using SSR markers, chromosome counting and flow cytometry, showing that the phenotypic marker was not efficient in detecting haploids in the tropical maize genotype used. All four haploids as well as some diploid plants presented reduced size, corroborating the difficulties for haploid identification by phenotypic evaluation. Genetic diversity analysis revealed by SSR markers divided the haploids in two groups represented by flint and dent maize inbred lines, which could be helpful in identifying complementary dihaploid lines. The present article demonstrates that a combination of haploid production and SSR fingerprinting is a feasible strategy for maize hybrid development in tropical germplasm.     

Development of the components of a cytoplasmic male sterility hybrid system in rye through anther culture

Anther culture was applied as a method to develop the essential components of a cytoplasmic male sterility hybrid system in rye (Secale cereale L.). These components are the male sterile seed parent (A line), its isogenic maintainer counterpart (B line) and the restorer pollen parent (R line). Australian rye cultivars were crossed reciprocally to the cultivar ‘Luchs’ which carries the Pampa male sterile cytoplasm (cms-P). Anthers of the F1s in the cms-P cytoplasm (primary cross) and their reciprocals in the normal cytoplasm (reciprocal cross) were cultured in a modified C17 medium. Male sterile and male fertile doubled haploids were obtained from the anther culture of the F1s in the cms-P cytoplasm. Testcrosses indicated that the male sterile doubled haploids were A lines and the male fertile doubled haploids were R lines (restorers). The anther culture of genotypes in the normal cytoplasm (reciprocal cross) gave all male fertile doubled haploids. Testcrosses indicated that the male fertile doubled haploids were R lines (restorers) in the normal cytoplasm. The expected maintainer B lines were not identified because of the limited number of doubled haploids obtained from the anther culture of reciprocal crosses. Experimental single cross hybrids between male sterile and restorer male fertile doubled haploids showed high levels of heterosis. The results of this study have significant economic implications especially in the production of hybrids in several species. This revised version was published online in July 2006 with corrections to the Cover Date.     

AFLP analysis indicates no introgression of maize DNA in wheat x maize crosses

Amplified fragment length polymorphisms (AFLPs) were used to follow the possible introgression of maize DNA into haploids of wheat as a side‐effect of exploiting wheat x maize hybridization for haploid production. AFLPs were generated with 64 MseI/ EcoRI and 64 MseI/ PstI primer combinations, and the AFLP profiles of haploids were tested against those of maize and of the regular wheat varieties involved in the crosses. On average, 45.1 and 110.7 fragments were produced per assay with the MseI/EcoRI and MseI/PstI combinations, respectively. Different numbers of fragments were produced for wheat and maize: an average of 81 in the haploid, 80 in the wheat parent, and only 67.1 in maize. No evidence was found for introgression of maize into the wheat genome. Three unique AFLP fragments were detected in haploids, which were not present in the parental wheat genotypes. These ‘novel’ AFLP bands in the haploids could be caused by nucleo‐cytoplasmic interaction in the hybrid zygote. Such instability in the wheat genome is defined as temporal, as it was not detected in further generations when colchicine‐doubled progeny of the haploids was tested for the presence of polymorphic fragments.     

Variation among androgenic and gynogenic doubled haploids of tobacco (Nicotiana Tabacum L.)

Summary Agronomic field performance traits and leaf chemical characteristics of gynogenic doubled haploids (GDH) and androgenic double haploids (ADH) were compared with selfed lines of a burley tobacco cultivar to determine which type of doubled haploids was superior. Three sets of ADH and GDH lines performed similar to their selfed lines, but one set each of ADH and GDH lines had reduced yield, plant height, leaf number, stalk diameter, and leaf length in comparison to the respective selfed lines of KY 17.Contribution from the Department of Agronomy, University of Kentucky, Lexington, KY 40546. This paper (No. 87-3-287) is published with the approval of the Director of the Kentucky Agricultural Experiment Station.     

Properties of maternal haploid maize plants and potential application to maize breeding

Summary Presented are the results of a two-year study of haploid maize plants in the field. The haploids were produced with the aid of inducer line ZMS. In total, 604 and 1030 haploids were obtained and studied in the first and second years, respectively. Tassels of haploid plants were found to be almost completley sterile. Fertility of ears was studied by pollinating them with the pollen from diploid inbred lines, the cross resulting in almost all of the haploid ears carrying kernels. On average 27.4 kernels per ear of haploid plant were obtained in the first year of study and 26.3 in the second. These gave rise to normal diploid plants. This property allows genotypes selected at the level of haploid plants to be involved in breeding process. Unusual plants were found among haploids, phenotypically resembling homozygous lines. It was assumed that the plants had resulted from spontaneous chromosome doubling in haploids. The results of comparative studies of progenies of unusual plants and inbred lines derived from the same synthetic population are presented.