6个烟草杂交组合花药再生苗的培养和DH群体的构建

以6个烟草(Nicotiana tabacum L.)杂交组合的花药为实验材料,对各杂交组合花药再生苗出苗状况及培养过程中添加H液体基本培养基对花药出苗状况的影响进行了比较分析;在此基础上,采用质量体积分数0.4%秋水仙素浸苗法构建加倍单倍体(DH)群体,对加倍处理后不同杂交组合再生苗的成苗率、大田移栽成活率及染色体加倍率等进行分析,并对杂交组合DH1单倍体和加倍单倍体植株叶片气孔保卫细胞叶绿体数的差异进行了研究。结果表明:不同杂交组合每枚花药的出苗数、加倍处理后再生苗的成苗率、大田移栽成活率及染色体加倍率有较大差异。其中,每枚花药出苗数为1.57～3.30,杂交组合DH5每枚花药的出苗数最多,达到3.30株,显著高于其他5个杂交组合(P<0.05);加倍处理后再生苗的成苗率为21.12%～33.42%,大田移栽成活率为91.87%～98.86%,杂交组合DH6的成苗率和大田移栽成活率最高;染色体加倍率为6.64%～10.78%,杂交组合DH4的染色体加倍率最高。花药培养约15 d后,添加H液体基本培养基能够显著促进杂交组合DH1和DH2花药出苗。杂交组合DH1单倍体苗和加倍单倍体苗叶片气孔保卫细胞的平均叶绿体数分别为9.27和17.46,二者比值接近1∶2,差异显著。通过花药培养和染色体加倍处理,分别获得了6个烟草杂交组合的DH群体。     

小麦花药培养后代和杂交后代中高分子量麦谷蛋白亚基变异分析

通过对Alondra、Orofen等5个小麦品种进行花药培养,同时以新春9号、京771、CB037等9个高分子量麦谷蛋白亚基组成不同的小麦品种相互间配制24个正、反交组合,分析小麦加倍单倍体无性系和品种间杂交后代中高分子量麦谷蛋白亚基变异,探讨利用花药培养和杂交手段改良HMW-GS组成的可能性。SDS-PAGE电泳分析发现,小麦加倍单倍体无性系中HMW-GS发生了频繁变异,Alondra加倍单倍体中变异率最高(61.8%),Verry加倍单倍体次之(16.7%),均出现了原始材料中所不具备的亚基类型;HMW-GS在部分F1杂种中呈现不完全共显性、亚基表达沉默和正、反交亚基表达不一致现象,宁春4号/CB037、京771/宁春4号2个组合中出现了双亲所不含有的亚基;通过连续自交和对新出现亚基的跟踪选择,获得了表达新亚基的高代株系。研究结果对于改良小麦加工品质,加深了解小麦HMW-GS编码基因的遗传特性、结构特性等具有一定理论意义和实践价值。     

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     

影响水稻花药培养力的数量性状基因座位间的互作

控制数量性状的多个基因间不仅存在加性效应,还存在非等位基因间的互作。对一个籼粳交后代的加倍单倍体群体花药培养,通过Ｅｐｉｓｔａｔ软件分析影响水稻花药 数量性状基因座位间的互作。结果表明,愈伤组织诱导率主要受两个单基因的影响,不存在双基因条件型互作,但有２对互适型互作与其有关。     

低温处理对水稻花药愈伤组织的诱导、分化的影响

近年来,花药培养工作在我.国广泛开展并取得一定的成果。但总的说来,培养水稻花粉植株的成功率还不高,这就直接影响花药培养单倍育种工作的效率,因此需进一步提高水稻绿苗分化率。目前一些单位采用低温处理各种作物的花药,以提高花药愈伤组织的诱导率。但具体做法和效果并不一致。胡忠等认为在10℃低温下(48—62小时)存放稻穗是暂时保存材料的有效方法,但并不能显著提高愈伤组织诱导率。钟华鑫等认为4～5℃低温对提高小麦花药愈伤组     

小麦单倍体叶片的离体培养

采用禾本科植物叶片进行组织培养,诱导 再生植株,目前国内已有成功的报道〔主,”。我们 于1980-1983年对小麦单倍体试管绿苗的叶 片进行离体培养,诱导出愈伤组织,并分化出绿 苗。经移栽加倍,获得结实单株。对其后代经 多代种植观察,表现整齐一致,性状稳定。     

单倍体小麦染色体加倍的研究

从七十年代利用花药培养技术获得单倍体植株以来,单倍体植物在育种应用上的潜力日益显示出来。但是这种潜力只有使单倍体植物加倍成为纯合二倍体才有可能发挥。因此,对于单倍体植物的染色体加倍,许多植物育种工作者进行过多方面的试验研究,提出过多种加倍方法,其中秋水仙碱法应用最为广泛,但加倍成功率并不理想,植株死亡率也较高。近年来有人用秋水仙碱和DMSO结合处理单倍体植株,加倍效果显著提高。就小麦来说,最高成功率有达9.0％     

在低温预处理及花药培养期间高氯酸汞处理对大麦花药生产力的影响

在春大麦(sabarlis品种)幼穗低温预处理过程中,使用乙烯的吸收剂高氯酸汞溶液处理,明显提高花药培养中花药的诱导率,以及每个花药形成的愈伤组织数目。在花药培养中,此种处理仅增加花粉愈伤组织的形成,而对花药的诱导率影响不明显。     

花粉单倍体的遗传品质——花药培养是否降低了育种材料的农艺价值

自S.Guha和S.C.Maheshwari 1964年以曼陀罗(Datura)花药为材料,首次成功地诱导出离体幼胚以来,人们经过不断的发展和完善使之形成了一套完整的花药培养技术,即通过植物花药(或小孢子)的培养,大批创制单倍体并经染色体加倍进而获得双单倍体(Doubled haploid)植株。该技术被迅速而广泛地应用于作物育种程序之中。目前已有不少的栽培作物成功地获得了花粉植株,如:烟草、小麦、大麦、水稻、黑麦、小黑麦、玉米、甘     

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。     

Anther culture as an effective tool in winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) breeding

The aim of this study was to determine the effect of genotype and induction medium in anther culture of wheat (Triticum aestivum L.). Ten F1 winter wheat genotypes were tested in anther culture (AC) to compare the two most frequently applied induction media (W14mf and P4mf). Androgenesis was induced during the treatment of each tested genotypes and green plants were produced from them using both media. Based on statistical analysis, the genotypes significantly influenced (at the 0.001 probability level) the efficiency of AC (embryo-like structures (ELS), albinos, green plantlets and transplanted plantlets) and the media also had a significant effect on the number of ELS and albino plantlets. Both media can be used for AC in wheat doubled haploid (DH) plant production. The production of ELS and green plantlets was higher in P4mf medium (48.84 ELS/100 anthers, 4.82 green plantlets/100 anthers) than in W14mf medium (28.14 ELS/100 anthers, 4.59 green plantlets/100 anthers). However, the green plant regeneration efficiency of the microspore-derived structures was 16.9% when using W14mf medium, while this value was 9.6% in the case of ELS induced with P4mf medium. The application of W14mf medium thus proved to be time- and labour-saving medium in the large-scale production of DH wheat plants. In our experiments, 267 DH plants were produced for our winter wheat breeding program. The spontaneous rediploidization rate was 32.72%.     

Effect of the 1B/1R translocation on anther culture ability in wheat (Triticum aestivum L.)

Using two varieties, their reciprocal hybrids, F₈ lines and doubled haploids, results confirmed that three genetic components are involved in wheat anther culture ability, viz embryo induction frequency, regeneration ability and the frequency of albinism. In these experiments, no significant maternal effects were noticed. For embryo yields, transgressive lines were obtained from hybrids between distant genotypes. Regeneration of green plants depended upon two independent traits: regeneration ability and the frequency of albinos. F₈ lines and two doubled haploids equaled the 50% regeneration rate of the hybrids, but they only regenerated green plants. Based upon cytological examination and gliadin patterns, it is suggested that genes favoring regeneration ability could be linked to the 1BL-1RS translocated chromosome from Aurora.Abbreviations DH doubled haploids - MS Murashige and Skoog - MPG multicellular pollen grains     

Effects of colchicine on anther and microspore culture of bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The aim of this work was to study the effects of colchicine application on chromosome doubling and androgenic response in anther and microspore culture of different bread wheat genotypes. Colchicine was applied during a mannitol stress pretreatment or during the first 48 h of culture at concentrations of 0, 150 and 300 mg l⁻¹. When colchicine was applied during stress pretreatment, the percentage of doubling depended on genotype and concentration. A significant increase in doubling was observed with 300 mg l⁻¹ in the low androgenic responding cv. Caramba. Colchicine incorporation during the first hours of culture improved percentage of doubling in all genotypes, in both anther and microspore culture. Application of 300 mg l⁻¹ colchicine improved the percentage of doubling in the two low responding genotypes, to 118% of control in DH24033, and 75% in Caramba in microspore and anther culture, respectively. Concerning the androgenic response, the effect of colchicine on embryo formation and percentage of green plants depended on the genotype and on the culture method. In cv. Pavon, a 2- and a 3-fold increase in percentage of embryogenesis and green plants, respectively, were obtained with 300 mg l⁻¹ colchicine in microspore culture. However, no significant differences in these two variables were observed in anther culture. The number of green doubled haploid (DH) plants reflects the index of success of the procedure. Regardless of the culture method, when colchicine was incorporated during the first hours of culture, the number of green DH plants increased significantly in three of four genotypes. These results confirm the usefulness of colchicine application during the first hours of culture in wheat breeding programs.     

Improvement of green plant regeneration by manipulation of anther culture induction medium of hexaploid wheat

Anthers of three hexaploid wheat (Triticum aestivum L.) genotypes with high frequencies of albino regenerants in anther culture were compared to DH after inoculation on medium supplemented with ficoll, colchicine or maltose separately, pair-wise or combined, in an attempt to increase green plant regeneration. Maltose treatment produced more green regenerated plants than sucrose for all of the genotypes. The three chemicals combined in anther medium either reduced green plant regeneration or did not yield significantly different numbers of green regenerated plants compared to the maltose treatment. With DH fewer embryo-like structures (ELS) were obtained per 100 cultured anthers on all medium containing colchicine but greater frequencies of green plants per 100 ELS were obtained. It appeared that the increase in green regenerated plants per 100 ELS was due to a better quality of embryos that were capable of regenerating into green rather than albino plantlets. Smaller increases in green plants per 100 ELS were observed in ICR 4 and V-15 on colchicine containing medium compared to DH. Genotypic differences in anther culture response were observed for ELS per 100 cultured anthers (increased for V-37, decreased for DH and approx. the same for ICR 4 and V-15 in medium with all three chemicals compared to the sucrose control).     

Analysis of quantitative trait loci which contribute to anther culturability in rice (Oryza sativa L.)

Anther culturability of rice is significantly different between indica and japonica varieties. A doubled haploid (DH) population was established via anther culture of an indica/japonica hybrid on SK₃ medium, which had been shown particularly suitable for anther culture of indica/japonica hybrids. For analyzing the quantitative trait loci (QTLs) responsible for anther culturability, anthers of the DH lines were again cultured with SK₃ medium and parameters for four traits representing the anther culturability were surveyed and analyzed with the molecular map constructed from the same DH population. The parameters for four major traits were as follows: callus induction frequency (CI), green plantlet differentiation frequency (GPD), albino plantlet differentiation frequency (APD), and green plantlet yield frequency (GPY). All four traits displayed continuous distributions among the DH lines. The correlation coefficients between these traits were also tested and showed that there was no relationship between callus induction and green plantlet differentiation frequencies, but both showed strong positive correlation with the frequency of green plantlet yield. For callus induction frequency, five QTLs were identified on chromosomes 6, 7, 8, 10 and 12. Two QTLs for green plantlet differentiation frequency were located on chromosomes 1 and 9. There was a major QTL for albino plantlet differentiation frequency on chromosome 9. No independent QTL was found for green plantlet yield frequency. The results may be useful in the selection of parents with high response to anther culture for rice haploid breeding and in the establishment of permanent DH populations for molecular mapping.     

Effect of genome, induction medium and temperature pretreatment on green plant production in durum ( Triticum turgidum var. durum ) x bread wheat ( Triticum aestivum L. em Thell) crosses

The recalcitrancy of durum wheat (Triticum turgidum var. durum) to anther culture, was attempted to be overcome by transferring the responsible genes form bread wheat B-genome to the respective on durum wheat, determining an appropriate induction medium and clarifying the necessity of cold pretreatment. For this, three durum wheat cultivars were crossed to two bread wheat (Triticum aestivum L. em Thell) cultivars. The resulting F₁ plants and their original cultivars were grown in the field and anthers at the appropriate microspore stage were cultured on potato-2 and W₁₄ media with and without low temperature pretreatment. No green plants were produced from the parental durum wheat cultivars. In contrast, green plants were produced from the F₁ plants. The best results in three of the four F₁ hybrids were recorded when potato-2 was used as induction medium. A more variable response of the examined genotypes was noticed with respect to temperature pretreatment. Regarding green plant production, a negative effect of cold pretreatment was observed in two of the F₁ hybrids when they were cultured on potato-2. Chromosome counts on root tips from the resulting green plants revealed that they all carried D-genome chromosomes. The last observation could suggest that D-genome chromosomes are necessary for anther culture response in wheat. Yet, the production of one green plant with 15 chromosomes may indicate that the development of extracted durum genotypes from bread wheat genotypes with good response to in vitro anther culture might be possible. Further work however, is needed for this to be verified.     

Nitrogen addition has a stronger effect on stoichiometries of non-structural carbohydrates,nitrogen and phosphorus in <Emphasis Type="Italic">Bothriochloa ischaemum</Emphasis> than elevated CO<Subscript>2</Subscript>

The current study attempted to obtain candidate doubled haploid (DH) wheat lines by serially combining two approaches: conventional chemical mutagenesis and anther culture. Additionally, the salt tolerance levels were examined between stress-treated (100 mM NaCl) and non-treated DH groups. For the molecular analysis, IRAP markers were used to characterize retrotransposon insertion polymorphisms induced by haploidization, chromosome doubling, and/or mutagenesis in the DH lines. Various sodium azide (NaN₃) concentrations (from 0 to 5 mM) were applied to seeds of the Pehlivan wheat cultivar to obtain an M₁ generation mutant population. Anther culture was set up from the M₁ mutant population. Green plant regeneration, the frequency of selected candidate mutants within the DH form and the levels of salt tolerance between samples were screened. A total of eight thousand anthers were cultured, and sixteen candidate salt-tolerant DH mutant lines, twenty-seven candidate DH mutant lines with different characteristics and one hundred and two candidate DH lines with morphologically normal appearances were obtained from the NaN₃-mutagenized population. The IRAP patterns were quite similar between the control DH lines, and the genetic differences between the controls and DHs originating from possible mutants showed close relatedness. According to previous studies, chemical mutagenesis and anther culture were combined for the first time to detect candidate salt tolerant genotypes at the DH stage. This approach might also be useful for determining the threshold dose and efficiency of wheat mutagens.     

Nuclear genes affecting percentage of green plants in barley (Hordeum vulgare L.) anther culture

Summary The genetics behind response in barley anther culture was studied with 22 reciprocal and one single: cross between three varieties with high and four varieties with low capacity for green plant formation. Effects of genotypes dominated embryo formation and percentages of green plants, accounting for 62 and 76% of total variation, respectively, with almost no genetic effect on the ability to regenerate plants from pollen embryos. Nuclear genes could explain all genotype effects in this plant material, since no reciprocal effects were indicated. The three parents with high and the four parents with low capacity for green plant formation formed two phenotypically homogeneous groups, producing 27–52% and 0–7% green plants, respectively. Genetic variation within hybrids for both embryo and green plant formation could be explained completely by general combining ability (GCA). The results are discussed with respect to a previous similar study in hexaploid wheat and the reported existence of DNA deletions in the plastid genomes in albino plants from anther culture of wheat and barley.     

Increased induction and chromosome doubling of androgenetic haploid rye

Summary Further progress of studies aimed at increasing production of androgenetic Secale cereale plants via the culture of anthers is described. Two culture media initially developed for rice and wheat anther culture have been shown to have pronounced influence on rye. It has been possible to increase the average percentages of responsive anthers (i.e. those producing embryoids or calluses) from 0.26% to 10% with a maximum in certain experiments of over 40 %. Of nearly 400 plants produced in 1976, 1/4 are green and can be grown further by transfer to potting compost; 3/4 are albino. Stable green haploid lines were present amongst the plants, and after vegetative propagation of the lines representative samples have been treated with colchicine resulting in diploid, triploid and tetraploid plants. The influence of the genetic background of the donor plants on the success rate of anther culture and on the percentage of albino formation is discussed.     

Methodical improvements in rye anther culture

Summary The crucial problem in anther culture of rye (Secale cereale L.) is the very low regeneration capacity. Our study was conducted to overcome this restriction. The plant material used included a doubled haploid line (DH), two single crosses between DH Unes, and a tetraploid Secale cereale L. population. The factors carbohydrate source, post-plating temperature treatment, and gelling agent were investigated. Substantial progress was achieved by substituting maltose for sucrose. Top rates of 49 % responding anthers and 20 % green plants were obtained from one of the single crosses after a post-plating cold treatment on geirrte solidified medium. We consider our results a methodical step forward in rye anther culture.