用花药培养创建小麦加倍单倍体作图群体

用花药培养创建加倍单倍体作图群体是构建数量性状基因作图群体的有效途径,但通过花药培养生产小麦加倍单倍体的成功率较低。该文考查了小麦幼穗发育时期、低温处理幼穗及高温处理接种花药对花粉愈伤诱导率的影响。采用春播小麦做为花药供体,推迟花药接种时间,避免试管苗在低温条件下越夏,有利于早移栽,培育冬前壮苗,同时加强试管苗移栽后的田间管理,保证安全越冬,有效地提高了花药培养生产加倍单倍体的成功率。通过花药培养创建了一个具有１９１个个体的小麦加倍单倍体作图群体,该群体将用于小麦有关抗旱性状及产量性状基因的遗传作图     

水稻花药培养力的遗传分析及基因定位

在栽培稻的籼粳亚种间,花药培养力存在显著差异,这一差异主要是由遗传因素引起的。以适合籼粳稻杂种花药培养的SK_3培养基,经花药培养获得了一个籼粳交F_1代的加倍单倍体(DH)群体,对该群体的110个株系用同一种培养基进行花药培养,利用该群体构建的分子图谱进行有关水稻花药培养力的数量性状基因座位(QTLs)的分析。结果表明,与水稻花药培养力有关的4个性状在DH群体中均表现为连续分布,愈伤组织诱导率与绿苗分化率之间不存在相关性,而绿苗产率与愈伤组织诱导率和绿苗分化率均显著相关。在第6、7、8、10和12 5条染色体上分别检测到与愈伤组织诱导率有关的5个QTLs,其加性效应均为正。在第1和第9染色体上检测到与绿苗分化率有关的2个QTLs,这两个性状间的QTs不存在连锁。在第9染色体上有一个主效基因与白苗分化率有关,对绿苗产率则没有检测到特有的QTL。     

大麦花药培养技术研究进展

花药培养的原理是将雄配子由正常发育转向一个异常发育途径,促进愈伤组织或胚状体的形成,最终再生绿色小植株。花药培养再生的小植株往往是单倍体,经染色体加倍可形成可育的纯合二倍体。因此,花药培养提供了一个对育种很有价值的快速诱导纯系的方法(Lrz等1988)。通过花药培养诱导产生大麦单倍体植株的成功报道最早见于七十年代初期。一直以来,特别是近几年来,一些学者已就影响大麦花药培养技术的基因型、预处理、培养基以及培养方法等诸因素作了许多探索性研究,使大麦花药培养技术得到了明显的改进。目前,已有人能从任何一个品系或品种(包括几年前仅产生白化苗的品种“Bonanza”)得到绿色小孢子植株,并且获得了每100个被培养花药生产100株绿色植株的产量(Kao等1991)。     

禾木科植物染色体消除型远缘杂交的研究进展

植物远缘杂交是作物育种中广泛应用的技术。除了核型稳定的种间杂交可以获得杂种以外,还可以利用核型不稳定的种间杂交后父本染色体消除的现象,通过胚培养和染色体加倍处理获得加倍单倍体(DH)植株。然而从小麦×玉米杂交获得的DH后代与其理论上应完全同质的遗传表现却不相符,总有2～5％的DH植株发生了形态学变异。最近的研究证明。通过小麦×玉米的受精作用,一些玉米特异DNA可以被转移到小麦DH后代的基因组中。     

大花蕙兰多倍体的高效诱导

以大花蕙兰‘红瀑布’无菌苗丛芽为材料、秋水仙素为诱变剂,采用不同的处理浓度、时间诱导大花蕙兰体细胞加倍。通过形态学和细胞学观察、统计等方法对其进行倍性鉴定。结果表明：秋水仙素浓度0.05%,处理时间24 h的条件下,诱导率高达28.2%;多倍体苗外部形态、叶绿体数目、气孔数目和大小与二倍体差异大,加倍后的细胞核明显变大,染色体倍数增加。     

人工合成冬性六倍体小麦的研究

以冬性四倍体硬粒小麦(Triticum durum,2n=28,AABB)为母本与粗山羊草(Aegilops tauschii,2n=14,DD)杂交,得到的单倍体幼胚(n=21,ABD)经组织培养拯救,获得的幼苗经染色体加倍而成为合成小麦(AABBDD)。从中鉴定、筛选出冬性的合成小麦。幼胚仅在1/2 MS培养基上培养,成苗率为75.81%;根据幼胚的发育状态,将发育较完善的幼胚直接接种在1/2 MS培养基上,将发育不良的幼胚先接种于1/2 MS+2 mg/L 2,4-D培养基上进一步养育幼胚,之后视幼胚发育状况再将其转入1/2 MS培养基中培养成苗,此方法的成苗率为92.44%,较前者的成苗率提高了16.63%。染色体加倍在冬季塑膜拱棚内用0.05%秋水仙素进行半根法处理,较容易获得健壮苗,并且分蘖多。     

提高玉米花粉植株移栽成活率的研究

玉米花药培养的成功,为大量地产生单倍体提供了新的途径,所以引起了国内外遗传育种工作者的关注。但是花粉植株移栽成活率低,是当前存在的突出问题之一。据估计,国内有80％以上的花粉植株死于移栽;Green(1978)报告用玉米盾片培养再生的植株成活率也只有10～15％。看来,通过组织培养获得的玉米植株成活率普遍较低。提高试管苗移栽成活率的研究是极有意义的课题之一。     

黄瓜子叶节高频再生体系建立及再生植株倍性观察

以‘农城3号’黄瓜子叶节为试材,研究不同苗龄、不同激素对黄瓜离体植株再生频率及再生不定芽的影响,并对再生植株进行了根尖染色体计数和叶片气孔保卫细胞叶绿体数目的鉴定。结果表明,(1)6-BA对黄瓜不定芽诱导起关键作用,IBA不利于黄瓜不定芽的诱导。(2)MS 2.0mg/L6-BA培养基是‘农城3号’黄瓜通过子叶节进行不定芽诱导再生植株的最佳培养基。(3)正常发育的无菌苗4~5d左右子叶不定芽再生频率较高,苗龄超过5d,不定芽再生频率明显下降。黄瓜子叶再生植株与实生苗的根尖染色体数目均为2n=14,再生植株与实生苗的气孔保卫细胞叶绿体数均分布在6~10的范围内,说明再生植株的倍性没有发生改变。     

小麦花培苗染色体加倍技术研究

为了提高冬小麦花培苗染色体加倍效率,分别用不同浓度的秋水仙碱对参试的冬小麦材料的花药愈伤组织、再生植株根系和花培苗分蘖节进行了加倍处理。结果表明,用0.02‰和0.05‰秋水仙碱浓度处理的愈伤组织再生植株结实率达33.3%～61.5%。用0.2％的秋水仙碱浸根处理5 h,结实株率平均高达37.5％。用0.04％的秋水仙碱1%的二甲亚砜溶液浸泡分蘖节的时间应在5～10 h之间较为适宜,结实株率平均可达50％以上。     

利用花药培养快速创制小麦条锈病和黄矮病抗性新种质

利用中间偃麦草与普通小麦杂交育成的部分双二倍体－中５为外源抗性基因供体,通过花药培养途径,在它与几个冬小麦品种的５个杂交组合中,诱导出试管花粉绿苗１４４株,移栽加倍后,形成了４９个加倍单倍体株系。通过小麦条锈病和黄矮病抗性鉴定获得了几个高抗黄矮病或者对条锈病近免疫的新材料。其中ＤＨ７２８对条锈病菌近免疫,２ｎ＝４４,减数分裂构型为０．７０１Ｖ＋０．０３５ＩＩＩ＋２１．５７９ＩＩ＋０．４５６Ｉ,为双     

甜菊不同杂交组合结实率及其F1代萌发和生长及对NaCl耐性的比较

以甜菊(Stevia rebaudiana Bertoni)的耐盐性较强品种‘中山3号’和‘守田2号’及R-A高含量品种‘中山4号’和‘守田3号’为亲本配置7个杂交组合并获得杂交种子,对种子结实率和发芽率及F1代幼苗的存活率进行统计分析,在此基础上采用砂培和水培方法比较了亲本及F1代扦插苗对NaCl胁迫的耐性.结果表明:品种间杂交组合的结实率均显著高于同系列品种间杂交及自交组合,其中‘守田2号’ב中山3号’杂交组合的结实率最高,为74.9％;7个杂交组合F1代的种子发芽率为63.8％ ～ 89.0％,差异明显;‘守田2号’ב中山4号’杂交组合F1代幼苗存活率相对较低(79.80％),其他杂交组合F1代幼苗存活率均在93％以上.砂培条件下,用100 mmol·L-1NaCl胁迫7d,各杂交组合F1代扦插苗的存活率差异不显著;随NaCl胁迫时间的延长各杂交组合F1代扦插苗的存活率均明显下降;胁迫28 d,‘守田2号’ב守田 3号’杂交组合以及‘中山3号’自交组合F1代扦插苗的存活率显著高于其他杂交组合.水培条件下,用100、150、200和250 mmol·L-1 NaCl胁迫14 d,‘守田2号’ב中山3号’、‘中山3号’ב守田2号’和‘中山3号’ב守田3号’3个杂交组合F1代扦插苗的存活率均显著高于其耐盐亲本及其他杂交组合.研究结果说明:通过杂交提高甜菊耐盐能力是可行的,而亲本的耐盐能力及亲本配置对杂交后代目标性状有较大影响;‘中山3号’ב守田2号’、‘守田2号’ב中山3号’和‘中山3号’ב守田3号’是耐盐性较强的甜菊优良杂交组合.     

普通小麦与粗山羊草正,反交的育性研究

普通小麦与粗山羊草杂交需借助幼胚培养方可获得杂种,取授粉１２～１６天的幼胚进行拯拯救,成苗率较高,效果较好。而不同杂交方式对共杂种成胚率、成苗率及思胚拯救率影响很大。以普通小麦为母本,粗山羊草为父杂交时,１５个杂交组合平均杂种成胚率,成苗率及幼胚拯率分别为５．３５％和５８．７３％,而在１２个反交组合中,其平均成胚率,成苗率及幼胚拯救率分别为９８．８８％、１７．１０％和２４．４４％。由此可见,普通小     

Pollen plant formation from anther cultures of Digitalis obscura L.

Factors favouring pollen callus proliferation, induction of embryogenesis and plant regeneration from cultured anthers of Digitalis obscura L. were determined. The presence of auxins was essential for cell proliferation and morphogenesis, and incubation in darkness singificantlyincreased these responses. Callus proliferation usually preceded embryo development, although sometimes direct embryogenesis was observed. On the other hand, bud differentiation was achieved only when callus was transferred to media containing cytokinin or several auxin/cytokinin combinations. Different ploidy levels] were observed in the regenerated plants, with approximately 50% being haploid.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis and antioxidant enzymes activity in <Emphasis Type="Italic">Acanthophyllum sordidum</Emphasis>

The effect of various hormonal combinations on callus formation and regeneration of shoot and root from leaf derived callus of Acanthophyllum sordidum Bunge ex Boiss. has been studied. Proteins and activity of antioxidant enzymes were also evaluated during shoot and root organogenesis from callus. Calli were induced from leaf explants excised from 30-d-old seedlings grown on Murashige and Skoog medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid + 4.65 μM kinetin. Maximum growth of calli and the most efficient regeneration of shoots and roots occurred with 2.69 μM 1-naphthalene acetic acid (NAA), 2.69 μM NAA + 4.54 μM thidiazuron and 2.46 μM indole-3-butyric acid. Protein content decreased in calli and increased significantly during regeneration of shoots from callus. Superoxide dismutase activity decreased in calli comparing to that of seedlings, then increased in regenerated shoots and roots. High catalase activity was detected in seedlings and regenerated shoots, whereas high peroxidase activity was observed in calli and regenerated roots.     

Genotypic variation of quantitative trait loci controlling in vitro androgenesis in maize.

A quantitative trait loci (QTL) analysis for androgenetic capability has been conducted on three different crosses in maize, including very high and nonresponding lines for androgenesis. The doubled haploid lines derived by anther culture from the crosses DH5 x DH7, A188 x DH7, and R6 x DH99 showed a range of 0-70%, 0-40%, and 0-50% androgenetic responding anthers, respectively. The genotypic heritability of means for this trait is close to 0.90 for A188 x DH7 and 0.78 for R6 x DH99. The QTL analysis involved in each population the mapping of more than 100 loci covering a large part of the genome with reasonably spaced markers averaging 12 cM. Different measurements describing the androgenetic process were studied: AC, percentage of responding anthers; ELS, number of androgenetic embryos produced per 100 plated anthers; PLE, number of plantlets regenerated per 100 embryos; PLA, number of plantlets per 100 plated anthers. In each cross, three to four QTLs were found for AC, explaining 30-40% of the phenotypic variation. The QTL detected for PLA was also strong QTL for AC or ELS. This agrees with the observation that these last two traits are good predictors for final plantlet yield. The QTLs found were specific, although the same line DH7 was used in two crosses and DH99 derived from DH5 and DH7 in the third cross. These results suggest that the transfer of the androgenetic capabilities in elite germplasm will still involve a phenotypic evaluation of the androgenetic performances. A backcross-assisted selection based only on the genotype at the QTL is probably possible but only within the crosses used for this QTL analysis.     

Mapping of QTLs for androgenetic response based on a molecular genetic map of x Triticosecale Wittmack.

Quantitative trait loci (QTLs) for androgenetic response were mapped in a doubled haploid (DH) population derived from the F1 hybrid of 2 unrelated varieties of triticale, 'Torote' and 'Presto'. A molecular marker linkage map of this cross was previously constructed using 73 DH lines. This map contains 356 markers (18 random amplified 5 polymorphic DNA, 40 random amplified microsatellite polymorphics, 276 amplified fragment length polymorphisms, and 22 simple sequence repeats) and was used for QTL analysis. The genome was well covered, and of the markers analysed, 336 were located in 21 linkage groups (81.9%) identified using SSR markers. The map covered a total length of 2465.4 cM with an average of 1 marker for each 6.9 cM. The distribution of the markers was not homogeneous across the 3 genomes, with 50.7% detected in the R genome. Several QTLs were found for the following variables related to the androgenetic response: number of embryos/100 anthers; plants regenerated from 100 embryos; number of green plants/total number of plants; and number of green plants/1000 anthers. Two were detected on chromosome 6B and 4R, which together had a 30% total influence on the induction of embryos. Another was found on 6B and on the unidentified LG1; these influenced the production of total plants from haploid embryo cultures. One QTL on chromosome 3R determined the photosynthetic viability of the haploid plantlets regenerated from microspores. Other QTLs were found on chromosomes 1B, 1R, 4R, and 7R, which helped the control of the final androgenetic response (the number of plantlets obtained for every 1000 anthers cultured).     

欧石楠体细胞胚发生过程中的DNA甲基化

利用甲基化敏感扩增多态性（MSAP）方法,对欧石楠大田苗、胚性愈伤组织和再生苗的DNA甲基化进行了研究。从64对选扩增引物中筛选出19对,共扩增得到506条带,统计显示,大田苗、胚性愈伤组织和再生苗的全基因组DNA甲基化水平分别为31．42％、27．86％和29．05％,3种试材发生甲基化变异的有175条带,变异率为34．58％。体细胞胚诱导形成胚性愈伤组织过程中,甲基化水平降低,而在再生苗中有所恢复,与大田苗接近。在外侧胞嘧啶甲基化水平上,胚性愈伤组织的甲基化水平有所增加,且在再生苗中可部分维持。另外,在175条变异带中,再生苗恢复到大田苗DNA甲基化模式的有62条,占总变异条带的35．43％,而与胚性愈伤组织维持相同DNA甲基化模式的有59条,占33．71％。回收部分甲基化变异条带,最终得到8条有效的基因组DNA序列。BLASTnI：对分析表明,在欧石楠基因组中,包括抗性基因、蛋白激酶、质体基因等在内的多种DNA序列均存在DNA基化修饰现象。     

Embryogenesis and haploid induction using anther culture in lovage (Levisticum officinale W.D.J. Koch)

An efficient and robust protocol to induce embryogenesis in lovage (Levisticum officinale W.D.J. Koch) has been developed. Immature anthers, with most of the microspores at the late uninucleate stage, were used as explants, and embryogenesis was induced in medium with combinations of plant growth regulators including α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 6-benzylaminopurine (BAP). The frequencies of in vitro embryogenesis ranged from 0.42 to 18.25% depending on the combinations of plant growth regulators in the induction medium. Induced globular embryos successfully developed into heart and torpedo-staged embryos. Fresh anther explants produced the highest embryo formation rate (17.75%). Anthers treated at 4?ºC for 3, 5, or 8 d, significantly reduced the embryogenic response (to 3.52–7.85%). More embryos were induced when the sucrose content in the medium was increased from 3 to 6% (w/v), but significantly fewer embryos were produced when sucrose was 8% or more. Nearly 20% of fresh anthers were able to produce embryogenic structures when cultured on Murashige and Skoog medium supplemented with 10.74 μM NAA, 8.80 μM BAP, 9.05 μM 2,4-D, and 6% sucrose. Furthermore, when silver nitrate was added to the embryo induction medium at 90 μM, the frequency of anther browning decreased by 30% and the embryo formation rate increased to 24.75% of anthers cultured. In total, 418 plants were regenerated and cytological analysis confirmed 11 haploid lines from 187 samples randomly selected.     

Haploid callus and regeneration of plants from anthers of Digitalis purpurea L.

Summary Production of callus from anthers of D. purpurea was obtained on several basal media supplemented with various amounts of auxins. Chromosome counts showed that the callus produced was haploid when the anthers 1) were of a dark-brown to black color, and 2) were cultured in the late tetrad stage of microspore development. Subsequent differentiation to plants at high frequencies was possible only 1) when the anthers had been cultured on the medium of Nitsch and Nitsch (Science 163, 85–87; 1969) supplemented with 5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 2) when the callus was transferred to the same medium but without 2,4-D, and 3) when it was cultured under continuous light from fluorescent lamps. Proliferation of the callus and regeneration of plants did not diminish through as many as 20 subcultures. The high frequency of regenerates permits the propagation of a distinct geno-type to a virtually unlimited number of plants. Diploid plants were obtained when the anthers had been cultured in the dark. Tetraploid plants were regenerated by callus from anthers which had been cultured in light. When the time of 2,4-D treatment was shortened a few haploid plants were produced which however did not survive transfer to soil. Cytological observations demonstrated that regeneration started from haploid callus, leading to intermediate degrees of ploidy and finally to diploid plants. Most of the regenerated plants were euploid and flowered and fruited normally under greenhouse and field conditions. If the anther-derived callus was cultured on the medium of Nitsch and Nitsch supplemented with 2.2 mg/l kinetin, plants regenerated only under photoperiodic conditions of 16 h light at 28° and 8 h dark at 20° but the survival was lowered to one third. These plants had a different leaf and flower morphology as compared to the control without kinetin and to the starting material, but their progeny was again essentially normal.     

Rice anther culture: callus initiation and androclonal variation in progenies of regenerated plants

Summary Anthers from rice (Oryza sativa L.) subspecies japonica initiated more callus than their indica or indica x japonica counterparts. A mild stress, either by slow desiccation or heat shock, prior to the plating of anthers enhanced the ability to initiate callus. Slow dessication of anthers enhanced the ability of the japonica anthers to initiate callus even in medium that was supplemented with NaCl. The ability to initiate callus by the anthers plated on NaCl-supplemented medium decreased as the NaCl level in the medium increased. Among the regenerated plants 2.5% were albino and another 2% were haploid. Androclonal variation for tiller numbers, shoot height, plant dry matter and flowering were noticed in the progenies of the regenerated plants.Abbreviations NAA -napthaleneacetic acid - KT Kinetin - 2,4-D 2, 4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - BAP N⁶ benzylaminopurine - NFM NaCl-free medium