利用生物技术向小麦导入冰草优异基因的研究 摘 要 小麦是世界上种植面积最大和最重要的粮食作物。利用生物技术向栽培作物转移向外源优异基因来拓宽小麦育种的遗传基础，是现代作物遗传育种学科中的一个非常重要的研究领域。 栽培小麦(Triticum aestivum L., 2n=4x-42, AABBDD)与冰草属(Agropyron Gaertn., P genome )(这儿所说的冰草属是现代小麦族植物分类学上的概念，而非传统的广义冰草属概念，即与一些小麦遗传育种学家将长穗偃麦草(Elytrigia elongata)和中间偃麦草(Elytrigia intermedia)等偃麦草属种也称为冰草的传统概念截然不同)植物间的杂交，可追溯到本世纪30年代(White, 1940; Smith, 1942; Dewey , 1984), 但直到90年代一些学者才先后报道了小麦与冰草属植物间的成功杂交(李立会等， 1990， 1995；Li & Dong, 1991; Chen et al., 1990; Limin & Fowler, 1990; Ahmad & Comeau , 1992; Jauhar, 1992)。 尽管这儿所列出的国外一些科学家也曾获得了小麦与冰草属植物间的杂种，但由于外源种选择的盲然性，即对要从冰草属植物向小麦转移哪些基因不明确(Chen et al., 1990; Jauhar, 1992), 或杂种F1的高度不育性(Ahmad & Comear, 1992), 或要转移的目标基因难以在小麦背景下表达(Limin & Fowler, 1990 )等原因，未见进一步的报道，而是基本上放弃了该领域的研究工作(私人通信，1997)。 在本项研究工作中，我们以普通小麦品种Fukuho (春性，具3对可杂交性基因，农艺性状良好，原产于日本)为母本，以分别采自新疆和内蒙古的3份冰草(Agropyron cristatum Gaertn., 2n=4x=28, PPPP)为父本进行杂交，并对杂种后代进行了研究。主要结果包括： 1、科学选择远缘杂交亲本，为杂交和外源优异基因转移的成功奠定了坚实的基础。在选择外源供体种的过程中，本项研究首先由中国农业科学院作物品种资源研究所和植物保护研究所、澳大利亚Division of Plant Industry, CSIRO, 加拿大Cytogenetics Section, Ottawa Research Station, Agriculture Canada 等单位对上千份小麦野生近缘植物的农艺性状、抗逆性和抗病性进行了联合鉴定，然后根据综合鉴定结果才精选出本项研究所利用的3份冰草－最佳外源供体种。因为这3份冰草不仅具有其它外源种难以比拟的众多优异基因（性状），包括小麦超高产育种所需的合理株型结构(株高小于60cm且穗下茎长度约占株高的2/3、有效分蘖>50、叶片窄短上挺)、大穗多粒（每穗结实在150粒以上）、黑粒且蛋白质含量极高、极强的抗旱和抗寒性、适度的耐盐性、对三种锈病、白粉病和黄矮病免疫、高抗赤霉病等，而且更为重要的是上述优异基因都是当前小麦育种迫切需要的。 2、利用现代远缘杂交方法和幼胚拯救技术，在国际上首次获得了具部分自交可育性的普通小麦与冰草间的杂种，并发现以不同来源的冰草为父本，不仅杂交结实率不同，而且杂种F1的表现型亦不同。这一结果，一方面突破了前人(Dewey, 1984)所认为的“冰草属P染色体组在小麦族中具有独立的遗传地位，与小麦之间不可能杂交”的论断；另一方面杂种F1具部分自交可育性，为实现外源基因的成功转移 奠定了坚实的物质基础。 3、以杂种F1幼穗为外植体，通过诱导愈伤组织产生体细胞无性系变异，首次发现杂种F1在无染色体数量变异情况下，其自交可育性或从无到有(0Õ ),或显著提高(10倍)。这一发现之所以重要，是因为它向人们展示了通过这一技术有可能在杂种F1就实现外源基因转移的美好前景。 4、通过精细分析，首次阐明了一些遗传学机理：一是杂种F1自交可育性是由于2条P染色体含有控制减数分裂过程中染色体分离的基因，从而能够形成有功能的近等2n或未减数配子；二是另外1条P染色体上具有抑制小麦Ph基因的遗传因子，能够诱发冰草P染色体组和小麦A、B、D染色体组间的染色体相互配对；三是证实了通过染色体间的自发易位可实现小麦－冰草间的基因交流。这些发现，一方面彻底突破了国际权威所认为的“小麦－冰草间不可能进行基因交流”的论断，另一方面为更加有目的、更加高效率地转移冰草优异基因提供了重要的理论指导。 5、利用回交、选择和形态学、细胞学、等位酶以及基因组原位杂交检测等综合技术，首次育成了11个遗传稳定的小麦－冰草异源二体附加系，并提出了有效产生异源二体附加系（列）的可行做法。异源二体附加系的产生，是研究每条P染色体上的基因在小麦背景下的遗传效应及其有效利用的重要工具。 6、首次创造了一批携带冰草优异基因、遗传稳定(2n=42, 21II ,异源易位系或代换系)且能为育种和生产利用的新种质。其普遍的特点是：有效分蘖多(15~82穗/株)；株高70～95cm且穗下茎约占株高的1/2；旗叶上挺；大穗(55～112粒/穗)；籽粒外观白色或黑色、蛋白质含量高(); 千粒重>38g; 综合抗病性（白粉病、条锈病、黄矮病和赤霉病)、抗寒性和抗旱性良好，特别是一些新种质具超高产潜力(理论产量高于600 kg/亩)。目前，这批新种质已为我国小麦主产区的9省15家育种单位利用，其中在陕西、山西有5个新种质正参加产量比较试验。 7、对获得的遗传稳定(2n=42, 21II ,异源易位系)的黑色籽粒和对白粉病免疫新种质中的黑色籽粒基因和抗白粉病基因(均来自冰草)进行了初步遗传分析，证明二者皆为显性单基因遗传。关键词：普通小麦(Triticum aestivum, L., 2n=6x=42, AABBDD); 冰草(Agropyron cristatum Gaertn., 2n=4x=28, PPPP)；属间杂种；自交可育性；异源二体附加系；遗传分析；新种质Introduction of Desirable Genes from Agropyron cristatum (L.) Gaertn. to Triticum aestivum L. Using BiotechnologyDoctoral student : Li-Hui LiSupervisor: Professor Yu-Shen Dong (Institute of Crop Germplasm Resources, Chinese Academy of Agriculture Sciences, Beijing, 100081)AbstractAs in most other crops, the genetic variation of cultivated wheat has been greatly eroded under modern agricultural systems. Genetic erosion not only limits the further improvement of yield and quality but also makes wheat increasingly vulnerable to biological and environmental stresses. A large amount of genetic variation exists in the wild relatives of cultivated wheat. The introduction of genetic variation from alien species has been a valuable method for increasing the amount of genetic diversity available to wheat this experiment, intergeneric hybrids of Triticum aestivum cv Fukuho (2n=6x=42, AABBDD) with three accessions of tetraploid A. cristatum (2n=4x=28, PPPP) were synthesized through immature embryo rescued in artificial medium. These hybrids can be used to: (1) transfer the desirable traits from A. cristatum into common wheat; (2) identify the effects of the P genome on self-fertility in intergeneric hybrids; and (3) produce disomic addition lines of wheat-A. cristatum. Through study of the intergeneric hybrids and their derivatives, the following results were obtained:1. A. cristatum may be one of the best potential alien donors in the Triticeae for wheat improvement. Agropyron Gaertn. is a small genus of no more than ten species, which constitute what is known as the “crested wheatgrass complex” with the P genome, in accordance with the terminology of many modern botanists. A. cristatum (L.) Gaertn. is the type species of this genus. All species of the genus are very valuable; they are cultivated as predominantly pasture-fodder plants, distinguished by their high level of drought and cold tolerance; some species have be successfully used for fixing drifting sands. In addition, A. cristatum has also been found to possess the other desirable traits that are potentially valuable for wheat improvement through evaluation of all Triticeae collections from the northern part of China; these include shorter stem (usually less than 60 cm), more tillers and florets, immunity to wheat diseases such as rusts, powdery mildew, and barley yellow dwarf virus (BYDV) as well as resistance to wheat scab. 2. In the three hybridization combinations, seed set () and plant development were different. Each one plant obtained from Fukuho with A. cristatum accession No. Z540 and Z602 respective developed poorly. The former died before heading. Although the later produced two spikes, neither selfed nor backcross seed was obtained from these two spikes. The two plants obtained from the Fukuho×A. cristatum Z559 showed vigorous tillering. This result indicated that the three accessions of A. cristatum used in this experiment are different in crossability with the Fukuho. The root-tips of all hybrid seedlings were observed, and revealed that somatic chromosome number of each was 2n=35 as . A self-fertile intergeneric hybrids between Triticum aestivum cv Fukuho (2n=6x=42, AABBDD) and tetraploid A. cristatum (2n=4x=28, PPPP) were obtained for the first contrast with the reports that either no BC1 derivatives from wheat-Agropyron hybrids was obtained or BC1 derivatives obtained were very difficult, in the Fukuho×A. cristatum Z559 hybrids, however, they not only had a high seed set () of backcrossing with common wheat, but also were partially self-fertile. The mean configurations at meiotic metaphase I of the hybrids were I + rod Ⅱ + ring Ⅱ + Ⅲ + Ⅳ. Some of bivalents per cell were clearly heteromorphic on the basis of various chromosome size, indicating that these bivalents were heterogenetic pairing. At anaphase I, chromosome separation was mainly the most (16~30 chromosomes) of 35 chromosomes to assemble at one pole, resulting in that the bigger daughter cells receiving most of 35 chromosomes might develop the functional . In order to induce somaclonal variation, the immature inflorescences of the hybrids between Triticum aestivum cv Fukuho and A. cristatum Z559 were cultured. Although the regenerants did not exhibit variation in chromosome number, they did show a higher degree of meiotic instability than the initial hybrids. Especially, the selfed seed set could be increased greatly in the regenerated plants, being from to . As a result, a total of 61 selfed seeds were obtained. Obtaining of so more selfed seeds from the Fukuho×A. cristatum Z559 is rare in the intergeneric hybrids involving wheat, and makes a substantial foundation for transferring the desirable genes from A. cristatum into common wheat. 5. Using methods such as morphology, cytology, isozymes and genomic in situ hybridization, the selfed and backcross derivatives were analysed. The results showed that all plants with the alien characters carried the genetic materials of the P genome. Meanwhile, a total of 11 disomic alien addtion lines were obtained. 6. After all cytological data obtained from this experiment were summed up and analysed, some conclusions could be obtained. They were: (1) the A. cristatum Z559 used in this experiment carried a genetic system suppressing Ph activity, and this genetic system might be mainly involved one of the P genome; (2) the P genome contained genes controlling chromosomes segregation at meiotic anaphase, and the genes might be mainly involved two of the P chromosomes; and (3) the spontaneous wheat-A. cristatum translocations can occur in the selfed and backcross . In this experiment, the other very important result is that some new germplasm with the desirable alien genes were obtained. They showed more effective tillerings (15-82 spikes per plant), plant height ranged from 70 to 95 cm, 55-112 grains per spike, a higher content of protein (), resistance to wheat diseases such as powdery mildew, stripe rust, BYDV and wheat scab, and tolerance to drought and cold. So far, all new germplasm obtained from this experiment have been utilized by the 15 institutions for wheat breeding in . The new germplasm with black-grain in color and immunity to powdery mildew were analyzed. Genetic analysis revealed that these two characters were from A. cristatum Z559 and were controlled by a dominant gene respectively. Key Words: Triticum aestivum L., Agropyron cristatum, Intergeneric hybrids, Self-fertility, Alien addition and translocation lines, Genetic analysis.
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