【作者】 孙玉强；

【导师】 张献龙；

【作者基本信息】 华中农业大学 ， 植物遗传育种， 2005， 博士

【摘要】 棉花是世界上最重要的天然纤维作物,也是世界上产量第二位的油料作物;同时是世界上附加值最高的作物。大约棉花价值有90%附加于棉纤维,然而,棉花产量和纤维品质正在逐步下降,尤其是过去的十五年。这种下降趋势归因于棉花品种资源遗传多样性的全面流失,作物对环境胁迫脆弱性的增加。根据增加棉花基因库多样性的迫切需要,棉花改良计划正在转向于多种分子育种技术的应用和资源的利用。丰富的棉花野生种（Gossypium spp.）是栽培棉遗传改良重要的种质资源和更新资源,并成为宝贵的遗传资源库,对棉花的遗传改良有着重大的现实和理论意义及潜在的应用价值。由于野生种和栽培种亲缘关系较远,杂交往往很难成功,或者F₁代没有育性,或育性很低。寻求把野生棉的有益基因转入到栽培种中去的途径对于栽培棉种的遗传改良具有重要意义。利用生物技术进行棉花的种质资源创新是一条有效途径,我们拟通过体细胞培养、原生质体培养和原生质体融合创造新的棉花种质。本研究内容主要涉及到野生棉的体细胞胚胎发生和植株再生;陆地棉和野生棉的原生质体培养及植株再生;栽培棉种和野生棉的原生质体对称融合及杂种植株再生。本研究取得的主要结果如下: 1.从9个野生棉种中诱导出愈伤组织,其中从G. davidsonii, G. klotzschianum, G. raimondii和G. stocksii愈伤组织通过体细胞胚胎发生得到正常的再生植株。从G. aridum也可以得到形态不正常的再生植株。G. anomalum, G. africanum, G. thurberi和G. bickii的愈伤组织仍处于非胚性状态。详细地研究了胚性愈伤组织的诱导和保存、促进体细胞胚成熟和植株再生与移栽的方法和途径,基本建立起一套适合野生棉体细胞培养操作程序。其中2,4-D/KT对所有供试棉种愈伤组织诱导都很有效;不同激素组合、糖源,悬浮培养,环境胁迫等能够不同程度地有效地促进胚性愈伤组织形成、体细胞胚成熟、萌发和植株再生;野生棉的胚性愈伤组织在含有IBA 0.984μM,KT 0.232μM的MSB固体培养基上保存4年多,仍然具有分化能力,这样就可以提供大量的实验材料。 这是首例报道从多个野生棉通过体细胞胚胎发生得到再生植株。更多还原

【Abstract】 Cotton is the world’s leading natural fiber and the second largest oilseed crop in production; cotton is the number one value-added crop. Approximately 90% of cotton’s value resides in the fiber, yet yield and fiber quality have declined, especially over the last 15 years. This downward trend has been attributed to general erosion in genetic diversity of cotton varieties, and an increased vulnerability of the crop to environmental stress. In light of the critical need to increase diversity in the gene pool, cotton improvement programs are increasingly turning to the application of molecular approaches to breeding and germplasm utilization. The abundant cotton wild species {Gossypium spp.) is an important renewable resource and has been the valuable genetic germplasm for cotton genenric improvement, and which has been significant in the reality and theory, potential in the application. It is very difficult to widecross between cultivars and wild species for the distant relationship, or no fertility of F₁ hybrids, or very low fertility. Application of biotechnology is an effective way for new germplasm development in cotton, and we wanted to develop new sources of cotton germplasm via somatic cell culture, protoplast culture and protoplast fusion. Our studies involved somatic embryogenesis and plant regeneration in wild cotton species, protoplast culture in Gossypium hirsutum L. and wild species, protoplast fusion between cultivars and wild species. The main results of this research were as fellows.1. Calluses were induced from 9 wild cotton species. Among them, the normal regenerated plants were obtained from G davidsonii, G. klotzschianum, G. raimondii and G stocksii via somatic embryogenesis, regenerated plants with abnormal morphology from G. aridum. Only non-embryogenic calluses were obtained from G. anomalum, G. africanum, G. thurberi and G. bickii. We studied the methods and factors for embryogenic callus induction and conservation, improving somatic embryos maturation andgermination, plant regeneration in detailed, and then a new and elementary protocol has been developed for somatic cell culture, mainly somatic embryogenesis and plant regeneration in wild cotton species. The combination of 2, 4-D/KT was very resultful for callus induction in all tested wild species. Different combinations of PGR, sugar sources, suspension culture and environmental stress et al improved embryogenic callus formation, somatic embryos maturation and germination, plant regeneration to some degree. Embryogenic calluses of wild species were subcultured and conserved on MSB semi-solid medium supplemented with IBA 0.984 (iM, KT 0.232 fiM for 4 years, they still have the capability of differentiation and then provide a mass of materials.This is the first report of regeneration of plants via somatic embryogenesis in many wild cotton species.2. Protoplasts were isolated from different explants of 2 species （Coker 201 and YZl） in Gossypium hirsutum L. （embryogenic cell suspension culture, embryogenic callus, immature somatic embryos, hypocotyls, young roots and leaves）. Plants regenerated from cultured protoplasts of 6 explants in Coker 201, but the plating frequencies of protoplasts from different explants varied significantly, the plating frequency of suspension culture-protoplast was 10%, that of embryogenic callus- and somatic embryo-protoplast was 6%, that of hypocotyl-, young root- and leaf-protoplast was not over 2%. Plants regenerated form protoplast cultures isolated from embryogenic suspension cultures, somatic embryos and embryogenic callus in YZl, the plating frequencies were lower （l%-2%） than those from same explants in Coker 201.Plants regenerated from protoplasts isolated from somatic embryos and embryogenic suspension cultures in wild cotton G klotzschianum with the plating frequencies ranging 6% to 8%. Analysis of RAPD demonstrated that the regenerated plants have genetic homogeneity.This study emphasized on enzyme combinations for protoplast isolation, the influences of culture density and PGR combinations et al for protoplasts sustained division, callus formation, and then a practical protocol for protoplast culture in cotton has been developed.3. In this research, symmetric fusion mediated by electricity was carried out,including 8 combinations. Plants regenerated from Coker 201+G klotzschianum, Coker 201+G davidsonii, Coker 201+G bickii, Coker 201+G stockii, which morphologically mediated fusion parents, apt to wild cotton parent. Cytological examinations and flow cytometric analysis showed that all of the tested plants were hexaploids with chromosomes （2n=2x=78）, or aneuploids nearing 78 chromosomes, sum of that of parents, and were somatic hybrids. Analysis of RAPD and other molecular markers demonstrated that the majority of regenerated plants had band patterns sum of two parents’ specific bands. Somatic hybrid plants of Coker 201+G klotzschianum, Coker 201+G davidsonii flowered and set bolls in green house, but difficultly flowered outside in the field. Somatic hybrid plants of Coker 201+G bickii, Coker 201+G stockii flowered and set bolls in the fall in the field and during the winter in the green house. The fertility of four somatic hybrids was comparatively high, perhaps related with photoperiod response. Somatic hybrid callus was obtained from G arboreum +G stockii, but this callus differentiated very difficultly.This is the first report on production of somatic hybrid plants between cultivars and wild species via protoplast fusion in cotton.4. In this research, we studied various factors in efforts to improve somatic embryogenesis and plant regeneration in wild cottons through 3 aspacts of somatic cell culture, protoplast culture in wild cotton species and regeneration of somatic hybrids between wild species with cultivars, in order to improve the efficiency of regeneration in wild cotton species and broard the range of wild species in which plant regenerated via somatic embryogenesis.更多还原