番茄游离小孢子培养研究进展

雄核发育是小孢子或未成熟花粉细胞沿孢子体发育途径形成植株的过程，也是创制育种材料的有效途径之一。为建立重复性好、再生频率高的番茄小孢子诱导方法，文章通过回顾国内外研究进展，总结了番茄雄核番茄雄核发育各阶段（胚胎发生能力的获得、诱导小孢子分化形成胚状体以及胚状体再生形成单倍体植株）的主要影响因素，并简要归纳了现有的游离小孢子分离培养技术。分析表明，基因型是制约番茄小孢子诱导体系的关键因素，且需与发育时期、培养条件等多个影响因素同期发生，才能使雄核发育偏离配子体途径。最后，展望了番茄小孢子培养未来的研究方向与发展趋势，以期为番茄双单倍体技术的深入研究提供参考。     

甘蓝型油菜小孢子培养、染色体加倍、试管苗继代越夏和田间移栽配套技术的研究及其在油菜育种中的应用

鉴于国内油菜育种单位一般没有大型气候室，供试材料只能种在田间，使小孢子培养技术在油菜育种中应用难度大，成本高效率低的状况，自1992年以来我们系统地研究了生长于田间的油菜的小孢子培养，染色体加倍，试管苗继代越夏和田间移栽等问题，建立了油菜双单倍体育种技术体系，利用该技术选育出甘蓝型双低油菜品系31个和优质高产品种华双3号（原系号91806），该品种的推广面积已达2000多万亩。     

油菜小孢子培养影响因素及黄籽油菜双单倍体群体的构建

针对我国油菜小孢子培养供体材料一般种植在大田,受外界环境影响较大,出胚率较低的现状,对大田环境条件下小孢子培养的主要影响因素进行了研究。同时,为了构建黄籽油菜双单倍体群体,研究了黄籽油菜的出胚情况。结果表明,不同取样时间对小孢子胚状体再生频率的影响很大;秋水仙素处理游离小孢子对胚状体的诱导有一定的负面影响,浓度越高,影响越大;黄籽油菜的出胚率较低(0.56枚/皿),同样条件下,不到黑籽油菜的1/8。不同黄籽单株间出胚率差异较大,变异幅度在0.04~1.97枚/皿。采用小孢子苗直接移栽大田技术,移栽成活率达89.0%,经染色体加倍后,最终构建了含有127个株系的黄籽油菜双单倍体群体。     

甘蓝型油菜小孢子培养技术及其单倍体应用研究进展

油菜小孢子培养已经成为油菜育种和生物技术的一项重要内容.由于影响小孢子成胚和成苗的因素较多,低诱导频率及成苗率严重限制了该技术在育种中的应用.为了全面了解小孢子胚状体发生能力及影响再生体系的各项因素,掌握该技术的研究动态和发展方向,总结了近20年来国内外影响小孢子培养的主要因素:供体植株基因型、供体植株生长条件及生理状态、小孢子发育时期、培养基组成、培养条件,及其在双单倍体育种、诱变育种、转基因育种等领域中的应用.     

通过花粉照射技术获得南瓜单倍体胚胎和植株

对育种者和遗传学家而言，单倍体化很容易在单一世代用杂合性植株生产纯系，并且有显著的优点。单倍体植株也可以自然获得，但这种事件的频率极低。目前采用花药小孢子培养和胚珠一子房培养或授与不完全的花粉(用化学药剂处理和照射等)来生产单倍体植     

芜菁小孢子培养中单倍体植株的高频率自然加倍

小孢子培养技术在培养单倍体方面，技术相对简单、效率较高，所以它在植物遗传研究和育种中具有重要的应用价值。芜菁是一种重要的蔬菜作物，但是其小孢子培养难度较大。对B．rapa ssp．chinensis两个变种的各—个基因型进行小孢子培养，共得到3．8～42．4个胚，芽。通过改良的方法，包括诱导48小时后更换培养基和降低蔗糖浓度，在未经继代培养的条件，直接从小孢子诱导胚获得了高频率的再生植株。其中，     

青麻叶大白菜小孢子培养及新品种选育

对影响青麻叶大白菜游离小孢子培养的因素进行了研究。结果表明：供体植株的基因型与小孢子胚胎发生密切相关;33℃,24h高温处理有助于小孢子转化成胚状体,每蕾成胚数比25℃恒温培养提高4．5倍;在培养基中加入外源激素和多种氨基酸可以提高胚状体的诱导效率,平均比对照增加59．64％;培养基的琼脂含量增加到12g／L能显著提高小孢子胚的成苗率,比含琼脂8g／L的高出40．5个百分点。通过对青麻叶大白菜小孢子胚的培养,获得二倍性的双单倍体植株,从中选出性状优良的纯系进行杂交组合的选配,培育出青麻叶大白菜的新品系。     

利用小孢子培养技术创制红菜薹早熟DH系

为获得早熟型的双单倍体(DH)植株,加快早熟型红菜薹育种进程,本研究以15个早熟型红菜薹品种为试材,进行小孢子培养,观察小孢子发育过程并对再生植株进行倍性鉴定。结果表明,当花瓣/花药(P/A)长度比值为0.6～0.8时,红菜薹小孢子72%～78%处于单核靠边期;共有6个基因型红菜薹出胚,其中出胚率最大的为gy21-55,平均出胚率为14.3胚/蕾;选取子叶形胚进行植株再生,成苗率在90%以上;利用流式细胞仪对再生植株进行倍性鉴定,平均自然加倍率达76.8%。gy21-40、gy21-55表现出早熟特性,与对照植株相比获得的DH植株可分别提早5 d和8 d抽薹。     

中国白菜小孢子离体培养和双单倍体植株再生

摘 要: 采用略加修改的NLN培养基（无机大量元素1/2）和甘蓝型油菜小孢子的胚状体诱导方法,培养了5个中国菜用小白菜品种和1个中国大白菜品种的小孢子,进行了双单倍体的诱导。结果表明,不同品种的小孢子产胚量差异很大,在接种的6个基因型中,有5个诱导出胚,诱导成功率83.33％ 。其中的中萁青抗热605产胚量最高,是8.676个胚／10蕾。这些胚在添加了NAA和PP333（多效唑）的B5培养基发育成健壮的小植株。关于6个基因型的小孢子胚胎发生能力,还有待进一步研究。     

油菜小孢子培养技术体系研究

小孢子培养在油菜的基础研究和应用研究中均具有十分重要的意义。自1982年Lichter首次在甘蓝型油菜中进行小孢子培养获得成功以来,国内外在油菜小孢子培养技术方面已取得大量研究成果,包括油菜小孢子胚状体发生的影响因素,小孢子植株的再生、成苗、大田移栽、染色体加倍等,近年来又对一些关键技术环节加以了改进,笔者在对这些研究成果进行总结的基础上针对中国国情建立了大田条件下油菜高效小孢子培养技术体系。用该体系对甘蓝型油菜和新疆野生油菜的体细胞杂种后代进行小孢子培养的出胚率达到300枚/皿以上,采用小孢子苗直接移栽大田技术,成活率达到89.0%。此外还成功构建了含127个DH系的黄籽油菜DH群体及含115个DH系的粒重分离群体。     

RAPD markers linked to a clubroot-resistance locus in Brassica rapa L.

Linkage of random amplified polymorphic DNA (RAPD) markers with resistance genes to clubroot (Plasmodiophora brassicae Wor.) in Brassica rapa L. was studied in a doubled haploid (DH population obtained by microspore culture. Thirty-six DH lines were obtained from F₁ plants from a cross between susceptible ‘Homei P09’ and resistant ‘Siloga S2’ plants. ‘Homei P09’ was a DH line obtained by microspore culture of the Chinese cabbage variety ‘Homei’, which is highly responsive in microspore culture. The resistant line ‘Siloga S2’ was obtained by two rounds of selfing of the fodder turnip ‘Siloga’. Three RAPD markers, RA12-75A, WE22B and WE49B, were found to be linked to a clubroot-resistance locus. These three markers were linked in the DH lines and an F₂ population and should be useful for marker-assisted selection in breeding programs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Segregation of 12 isozyme genes among doubled haploid lines derived from a japonica x indica cross of rice (Oryza sativa L.)

Summary The segregation of 12 heterozygous isozyme markers was analyzed among F₂ plants and 51 anther culture (AC)-derived lines obtained from the japonica × indica cross of rice, IRAT 177 × Apura. All the lines except two were homozygous products of recombination of the two parental phenotypes. Doubled haploid (DH) lines derived from plants regenerated from the same callus were identical, confirming previously obtained results in rice. Surprisingly, some lines derived from different calli were also identical, suggesting a phenomenon of early callus fragmentation. All these observations at the isozyme level were confirmed by field evaluation. Deviations of segregations from the expected 1 : 1 ratio were observed at 4 loci among the DH lines. Among these, two were also noted among the F₂ plants. The two other distortions, both in favor of the japonica allele, were observed specifically in the AC-derived materials.Although this concerns a small proportion of the genes under study, it suggests that the embryogenic microsporal population does not represent a random gametic array. On the other hand, evaluation of recombination between isozyme genes located on chromosome 6 appears consistent with F₂ data and data previously recorded on the other japonica × indica crosses. The potential use of isozymes in breeding doubled haploids derived from remote crosses in rice is discussed.Abbreviations MCPA = 2-methyl-4-chlorophenoxyacetic acid - IAA = indolacetic acid - AC plant or line = anther culture-derived plant or line - DH line = doubled haploid line     

3个辣椒花药培养再生株群体主要农艺性状及苗期抗病性研究

对3个辣椒花药培养再生株DH-R2群体的主要农艺性状,包括株高、株幅、首花节位、叶色、单果质量、商品果横径、商品果纵径、果形指数、老熟果色等进行了较详细的测量和统计分析。结果表明:辣椒花药培养再生株群体的农艺性状存在基因型偏性;再生株群体的单果质量、商品果纵径、果形指数这3个性状都表现为低于供体,株高、株幅、首花节位、商品果横径这4个性状都表现为近似于供体;通过单倍体培养途径可以得到不同颜色层次的老熟果色的辣椒纯系,证明了该途径是多基因控制性状育种的有效途径;对耐TMV和CMV的亲本与感病亲本的杂种进行花药培养,获得了兼具TMV和CMV抗性的株系,证明了单倍体培养途径可以创造新的超亲抗病种质。     

Early diagnosis of ploidy status in doubled haploid production of maize by stomata length and flow cytometry measurements

Reliable discrimination of haploid (H), doubled haploid (DH) and crossing (C) plants in early growth stages could streamline DH production in maize. By detecting in early growth stages undesirable sterile H plants and undesirable heterozygous C plants, a large proportion of resources required for DH production could be saved. The goal of this study was to evaluate the effectiveness of early classification of plants in growth stages V3‐V4 as H, DH or C in the context of DH production using flow cytometry and stomata length. As the reference classification, we used a field score based on plant phenotype commonly applied in DH production and research. Our results show that identification of misclassified C seeds is possible because the overlap in distributions of stomata length between H&DH and C plants is small and the association between flow cytometry and the reference field score is high. In contrast, overlap between H and DH distributions is substantial. Consequently, the main application we see for these classification methods in early growth stages is the identification of C seedlings.     

矮秆紧凑型油菜细胞质雄性不育恢复系的选育和应用

为选育高油矮杆紧凑型油菜资源,通过对亲本‘矮油A05’（母本）和‘DH09-3048’（父本）的杂交后代F₁进行小孢子培养,获得DH分离群体,以植株矮秆、分枝特征、角果数量、粒数、成熟期、抗病性、抗倒性、含油量等主要性状以及较高的收获指数为选育目标,对DH分离群体进行不同生态区田间表型鉴定。选育出了具有高含油量、矮秆、株型紧凑、收获指数高等特性的细胞质雄性不育恢复系‘DH16-202’;在杂交组合配制中,‘DH16-202’表现出较强的配合力,群体株型中矮紧凑、角果性状优良、熟期集中、产量高,抗性好,符合当前机械化栽培油菜品种需求的特点,有较强的应用潜力。综上‘DH16-202’可用于机械化品种的选育,同时也为育种同行提供基础资源。     

A single dominant gene for downy mildew resistance in broccoli

Downy mildew, incited by Peronospora parasitica (Pers.: Fr.) Fr., is a destructive disease of broccoli (Brassica oleraceaL., Italica Group). Resistant cultivars represent a desirable control method to provide a practical, environmentally benign, and long-term means of limiting damage from this disease. Doubled-haploid (DH) lines developed by us exhibit a high level of downy mildew resistance at the cotyledon stage. To determine the mode of inheritance for this resistance, a resistant DH line was crossed to a susceptible DH line to make an F₁, from which F₂ and backcross (BC) populations were developed. All populations were evaluated for response to artificial inoculation with P. parasitica at the cotyledon stage. All F₁ plants (including reciprocals) were as resistant as the resistant parent, indicating no maternal effect for this trait. F₂ populations segregated approximately 3resistant to 1 susceptible, BC populations using the resistant parent as the recurrent parent contained all resistant plants, and the BC to the susceptible parent segregated 1 resistant to 1 susceptible. These results indicate that resistance is controlled by a single dominant gene. This gene should be easily incorporated into F₁ hybrids and used commercially to prevent downy mildew at the cotyledon stage. This revised version was published online in August 2006 with corrections to the Cover Date.     

Simulations and practical problems of applying multiple marker assisted selection and doubled haploids to wheat breeding programs

Marker assisted selection (MAS) and wheat doubled haploids (DH) are relatively new technologies, recently applied to wheat breeding programs. Simulations demonstrate that DHs increase the efficiency of MAS, and offer faster strategies for combining large numbers of genes with a minimum number of marker tests. When small numbers of marked loci (1-3) are selected simultaneously, selection of DH progeny is 5-6 times more efficient than selecting F₄ derived families. Combining 4-8 marked loci, screening of F₂ plants and using only those plants homozygous or segregating for all of the marked loci as parents for DH production (10-31% of F₂ plants) is 3-10 times as efficient as using F₁ plants. A number of protocols have been proposed involving sib-matings and selection to fix some genes, with further selection in the second generation to improve the proportion of useful DH lines. In one scheme (recombinant F₂ selection) all F₂ plants, either homozygous or heterozygous for the marked alleles, are intercrossed at random and the recurrent F₁ plants still having these alleles are used for DH production. An alternative strategy (recurrent DH selection) is to select from an initial DH population and intercross those lines having most favourable marked loci with a second cycle of DHs to fix all favourable marked loci. Combining more than 12 marked gene loci does not seem feasible, due to the very large numbers of F₂s (>2000) required. This has implications when using MAS for quantitative trait loci, where many minor gene loci would have to be combined. Direct selection for some multi-genic quantitative traits amongst the DH lines may be more efficient than using MAS where recurrent selection is used. At the Cereal Research Centre, the practical problems of using these protocols as part of the spring wheat breeding program are being evaluated. This revised version was published online in August 2006 with corrections to the Cover Date.     

种植密度对不同株高夏玉米品种茎秆性状与抗倒伏能力的影响

倒伏是影响夏玉米在密植条件下获得高产的重要限制因素之一,本研究旨在探讨种植密度对不同株高夏玉米品种茎秆性状与抗倒伏能力的影响。以矮秆品种登海661 (DH661)和高秆品种鲁单981 (LD981)为试验材料,通过设置4.50×10⁴株 hm^-2、6.75×10⁴株 hm^-2和9.00×10⁴株 hm^-2 3个种植密度,研究茎秆节间长度、茎秆穿刺强度、茎秆显微结构以及倒伏率等方面的变化。结果表明,随种植密度增加,夏玉米的基部第3茎节间和穗位节间变细,茎秆穿刺强度显著下降,较密度4.50万株 hm^-2,DH661和LD981 6.75万株 hm^-2、9.00万株 hm^-2地上第3节间茎秆穿刺强度分别降低了8.5%、22.6%和13.3%、29.6%;茎秆皮层和维管束内部厚壁细胞厚度及维管束数目均随种植密度的增加显著下降,倒伏风险增加,但矮秆品种的下降幅度小于高秆品种,而产量的增加幅度大于高秆品种,说明矮秆品种在高密度下能够保持较好的抗倒伏性能,有助于其在高密度种植条件下获得高产、稳产。     

Identification of Cr-tolerant lines in a rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>) DH population

Identification of Cr-tolerant lines in a rice DH population was conducted based on the modified weighted function analysis. The significant difference was found between the two parents and among DH population lines, with lines 117, 101 and parent ZYQ8 showing the high Cr tolerance, lines 41 and 49 showing Cr sensitivity. A dramatic difference also existed in Cr accumulation of plant tissues, with lines 19, 18 and 1, 5 having the minimum and maximum shoot Cr accumulation, respectively, and line 19, parent JX17 and lines 1, 56, ZYQ8 having the minimum and maximum root Cr accumulation, respectively. There was a significant difference in shoot/root ratio of Cr accumulation among the population, with line 18 and parent ZYQ8 ranking the tops and lines 109, 71, 19, parent JX17 ranking the bottoms. Zn uptake and accumulation was reduced when the plants were exposed to Cr stress. In addition, three QTLs were detected, which are, respectively, associated with Cr accumulation in shoot and root, and ratio of shoot to root.     

玉米Reid种质DH系遗传分析

为探讨玉米Reid种质遗传改良效果,本研究以Reid类群重要种质J1491、J1492、J1493、J1495、J1498与PH6WC构建基础群体,通过DH技术育成DH系,经鉴定从中选取5个代表DH系6DH6、6DH7、6DH9、6DH10、6DH12作母本,以Non-Reid群5个优良自交系作父本,按NCII设计组配25个杂交组合,对DH系单株产量及其构成因素的杂种优势、配合力和相关方面进行了研究。结果表明,单株产量杂种优势以组合6DH7×J1628最高为180.22%,组合6DH12×J1673最低为54.94%;单株产量一般配合力效应值最高的有6DH7和6DH10,穗长、穗粗亦为正效应,因此认为6DH7和6DH10更具应用潜力;特殊配合力效应值最高的组合是6DH7×J1673。各产量性状中穗长、穗粗和秃尖长,狭义遗传率高,主要以基因加性效应为主。本研究为鉴定有潜在价值的DH系及杂交种提供选择,为玉米遗传改良提供参考。