香石竹原生质体再生的研究

香石竹(Dianthus caryophyllus L.)是一种受到世界性欢迎的观赏植物。香石竹的快速繁殖已有不少研究报告。为了寻找新颖的育种手段,培育新的品种,我们进行了该植物的原生质体培养。米益(Mii, et al., 1682)已对香石竹的原生质体培养作过报道,但他们所用的材料为叶肉原生质体。为期望得到更多变异的再生植株,我们选用愈伤组织做为游离原生质体的材料。     

黑曲霉UB4和NB3的原生质体制备及其再生条件的研究

以两株黑曲霉营养缺陷型突变株ＵＢ４和ＮＢ３为材料,对于其原生质体制备及其原生质体再生条件作了详细的研究,从中找到了合适的破细胞壁方法和培养条件,为尔后的黑曲霉原生质体的利用如融合或诱变育种等方面的研究奠定了基础。     

原生质体融合技术在香菇育种中的应用

对香菇原生质体融合技术以及其融合育种研究作了简要介绍,并初步阐明原生质体融合育种的理论与实际意义。     

茶树菇原生质体再生及单核体菌株的特性

【背景】茶树菇遗传育种工作是茶树菇产业持续发展的保障和关键,原生质体的制备及单核体菌株的获得可为茶树菇遗传育种工作的开展提供技术支持。【目的】获得茶树菇原生质体的再生特性、单核化特性及其交配型,为开展茶树菇的杂交育种、融合育种、诱变育种、遗传转化和功能基因挖掘等奠定基础。【方法】以茶树菇保藏菌种Aa11的菌丝为材料,采用甘露醇溶液和溶壁酶溶液直接处理平板菌丝制备茶树菇原生质体,而后对原生质体进行分离和再生培养。通过原生质体单核菌丝体两两单单对峙培养,观察对峙培养过程中的菌落形态变化。【结果】当接种块数量为7、酶解温度为33-34℃、酶解时间为60-80 min时,原生质体数量为107个/mL。茶树菇原生质体在涂布平板7 d后肉眼才可见明显的再生菌落形成,在再生培养基上再生率为0.71%,单核化率为41.1%;再生异核体和再生单核体在形成再生菌落时有时间差,从第7天开始往后连续3 d的再生菌落均为异核体菌株,往后第4天开始陆续出现单核体菌落,之后时间内的菌落均为单核体菌株。试验共得到290个原生质体单核体,分为A1B1和A2B2两种亲本交配型,A1B1和A2B2二者的比例为138:152...     

烟草花粉原生质体与叶肉原生质体的融合及培养早期变化

用ＰＥＧ—高Ｃａ高ＰＨ法诱导抗卡那霉素的烟草（Ｎｉｃｏｔｉａｎａｔａｂａｃｕｍ）品系Ｎ３６４＋Ｋｍ＋花粉原生质体和黄花烟草（Ｎｉｃｏｔｉａｒｕｓｔｉｃａ）叶肉原生质体融合。幼嫩花粉原生质体和叶肉原生质体之间的融合体培养启动胚胎发生分裂,经卡那霉素筛选后,少数多细胞团存活并形成小愈伤组织。成熟花粉原生质体与叶肉原生质体之间的融合体则仅产生管状结构。这一结果表明,作为融合一方的花粉原生质体的发育时期对融合产物的发育途径有重要影响。     

杏鲍菇原生质体制备与再生条件初探

研究酶浓度、酶解时间、酶解温度、培养时间、稳渗剂种类、pH值和几种再生培养基对杏鲍菇原生质体制备与再生的影响。最适条件为 :在 30℃、pH5 .5、1.5 %溶壁酶条件下 ,以 0 .6mol L甘露醇作为稳渗剂 ,酶解 2 .5h ,原生质体产量达到 2 .90× 10 7个 mL。将所得原生质体过滤、纯化、稀释后涂布再生培养基 ,再生率为 0 .18%。为利用原生质体技术进行杏鲍菇育种奠定了基础。     

草菇(Volvariella volvacea)、银丝草菇(V.bombycina)原生质体分离研究

本文对草菇、银丝草菇菌丝原生质体制备的最佳条件作了探讨。结果表明,培养两天的草菇菌丝,以0.5MKCI或0.5MMgSO_(4.7)H_2O作渗透压稳定剂,1.5％Lywallzyme(v/m),35℃下酶解1.5—2小时,原生质体产量可达1.5×10~6个/ml以上,培养3天的银丝草菇菌丝,以0.5MKCl作渗透压稳定剂,1.5％Lywallzyme,28℃酶解2—3小时,原生质体产量可达2.8×10~6个/ml以上。此研究对以后的原生质体融合育种打下了基础。     

原生质体技术在食用菌遗传育种中的应用

本文着重介绍了原生质体技术如单核和同核原生质体技术、诱变技术、原生质体融合技术、ＲＡＰＤ技术、ＲＦＬＰ技术等在食用菌遗传育种中的应用及研究进展概况。     

微生物原生质体技术

从影响原生质体制备、再生的因素、原生质体融合、原生质体诱为、原生质体再生育种、原生质体转化和原生质体固定化等几个方面,较为全面地介绍了目前原生质体技术的发展现状,对相关机制和技术环节作了简要概述。     

禾本科植物原生质体培养及研究进展

植物原生质体培养是高等植物细胞工程和基因工程的重要基础之一。许多遗传操作,如体细胞杂交、细胞质重组和直接的 DNA 摄入等,都依赖于原生质体的再生。这种再生在双子叶植物中已不是一件困难的事。然而,多年来禾本科植物原生质体培养一直被认为是个十     

灭活原生质体作黑木耳融合育种遗传标记研究

本文采用0.83％碘乙酰胺(CIA)化学灭活剂和热处理灭活方法,对黑木耳单株原生质体灭活,观察了化学灭活与不同温度热灭活的灭活效果。并通过对灭活原生质体与未经灭活处理的原生质体融合后再生产物分析、证明,不论化学灭活还是热灭活处理,只要筛选好灭活条件,作为黑木耳种内原生质体融合遗传标记是可行的。     

Protoplast Fusion in Banana (<Emphasis Type="Italic">Musa</Emphasis> spp.): Comparison of Chemical (PEG: Polyethylene Glycol) and Electrical Procedure

Optimization of protoplast fusion parameters is a prerequisite for the establishment of somatic fusion technology for banana breeding. In the present investigations, we compared the most frequently used fusion methods: the electrofusion technique and chemical procedure (polyethylene glycol). With regard to frequency of binary fusion, protoplast fusion with the fusogen polyethylene glycol was best. Conversely, electric fusion was found to be better with respect to mitotic activities, somatic embryogenesis and plantlet regeneration rate.     

食用菌原生质体技术的研究进展

综述了食用菌原生质体再生、诱变、融合技术以及原生质体技术与其他生物技术相结合的研究进展,提出了在现有研究中存在的问题,并展望了原生质体技术的发展趋势.     

斑玉蕈育种中漆酶转化体系建立的初步研究

漆酶具有降解木质素,氧化降解酚类物质,抑制杂菌,改善出菇品质等作用。采用酶学与原生质体融合相结合的技术,建立斑玉蕈育种中漆酶转化体系,将漆酶活性较高、生长速度较快的凤尾菇原生质体经高温灭活与斑玉蕈原生质体融合,通过RB-PDA平板显色技术筛选出具有漆酶活性较高的两融合菌株Ⅲ18C、Ⅲ2A,并对其进行了拮抗试验、RAPD分子标记、漆酶基因扩增等研究。结果表明,筛选出的两融合菌株与两亲本具有明显的拮抗线,随即引物扩增的条带与两亲本有明显的差异,并扩增出漆酶基因的一个片段。同时也表明利用漆酶转化体系筛选融合菌株具有目标明确、准确、快速的优点。     

Protoplast fusion: a tool for intergeneric gene transfer in bacteria

Protoplasts can be isolated from bacterial cells by digestion of the cell wall with the help of lysozyme in presence of osmotic stabilizers. Fusion of protoplasts can be induced by chemical fusogens like polyethylene glycol. The electrofusion technique has been reported in bacteria in which the fusion frequency is much higher than that obtained by PEG induced protoplast fusion. This technology allows recombination to take place not only between related species but also between unrelated genera and is of great potential in the breeding and improvement of industrial strains. This review includes the information and developments on the protoplast fusion in bacteria with special reference to genetic recombination by protoplast fusion between phylogenetically unrelated bacteria.     

Somatic hybridization in citrus: An effective tool to facilitate variety improvement

Summary Citrus somatic hybridization and cybridization via protoplast fusion has become an integral part of citrus variety improvement programs worldwide. Citrus somatic hybrid plants have been regenerated from more than 200 parental combinations, and several cybrid combinations have also been produced. Applications of somatic hybridization to citrus scion improvement include the production of quality tetraploid breeding parents that can be used in interploid crosses to generate seedless triploids, and the direct production of triploids by haploid + diploid fusion. Applications of somatic hybridization to citrus rootstock improvement include the production of allotetraploid hybrids that combine complementary diploid rootstocks, and to combine citrus with sexually incompatible or difficult to hybridize genera that possess traits of interest for germplasm expansion. A few somatic hybrid tetraploid breeding parents have flowered, are fertile, and are being used as pollen parents to generate triploids. Several allotetraploid somatic hybrid rootstocks are performing well in commercial field trials, and show great promise for tree size control. Seed trees of most of these somatic hybrid rootstocks are producing adequate nucellar seed for standard propagation. Somatic hybridization is expected to have a positive impact on citrus cultivar improvement efforts.     

双亲灭活制备粘质沙雷氏菌和红曲霉的跨界产色素融合子

目的：采用双亲灭活原生质体技术制备粘质沙雷氏菌和红曲霉的跨界产色素融合子,并测定其抑菌活性。方法：经0.2%溶菌酶处理获得粘质沙雷氏菌的原生质体并热灭活;经混合酶（0.8%溶菌酶＋1.2%蜗牛酶＋1.6%纤维素酶）处理获得红曲霉的原生质体并紫外灭活;用含25%PEG的原生质体融合剂进行促融合与再生。观察融合子的菌落形态和色素合成能力,测定融合子色素提取物对金黄色葡萄球菌的抑制活性。结果：在优化条件下,粘质沙雷氏菌原生质体的形成率为92.58%,红曲霉原生质体形成数约为106个/mL,两菌原生质体灭活率均为100%。共获得13个融合子,9个能产红色素,融合率为1×10-5%。其中8个融合子的95%乙醇提取物对金黄色葡萄球菌表现出不同程度的抑制。结论：采用双亲灭活原生质体技术,能够制备具有抑菌活性的粘质沙雷氏菌和红曲霉的跨界产色素融合子。     

枯草芽胞杆菌基因组混组方法

基因组混组作为一种育种方法,通过循环原生质体融合等手段,使得不同菌株来源的基因组能够得到充分重组,增加将正向突变整合到同一重组子中的机会。使用4株带有4种不同标记的枯草芽胞杆菌亲本为初始菌株,通过循环转化、循环转导或循环原生质体融合的手段进行基因组混组,统计后代中非亲本类型占整个群体的比例,以衡量基因组混组的效果。分别经过5轮循环原生质体融合、循环转化或者循环转导,结果显示,重组程度较高者在后代群体中的比例较低,带有4种标记的后代未出现,带有3种标记的后代最高分别为4.53×10?4、1.64×10?4、4.47×10?3,明显低于文献报道的天蓝色链霉菌中同样实验的结果:带4种和3种标记的后代分别占2.5%、17%。对比上述实验的结果和文献报道的天蓝色链霉菌、乳杆菌基因组混组的结果,并结合计算机模拟循环融合过程,分析后认为:要达到较充分的基因组混组,需要有能够实现微生物细胞间高频重组的操作技术作为基础,重组频率应该不低于10?3～10?2数量级。     

Variety of Hybrid Characters among Recombinants Obtained by Interspecific Protoplast Fusion in Streptomycetes

To discover whether the protoplast fusion method is useful or not for interspecific breeding, some methods were devised, and the appearance of various hybrids with different characters and the change of antibiotic activities in the recombinants obtained by the protoplast fusion were investigated. The purification of protoplasts, the choice of parental natural characters as selection markers, and the adoption of a replica method for selecting all types of recombinants were devised and used for these experiments. Protoplast fusion was done between S. griseus KCC S-0644 and each strain of 5 species that were clearly different species from S. griseus, in addition to being streptomycin sensitive (SM^s) and capable of L-arabinose utilization for growth (Ara⁺). Recombinants (SM^r, Ara⁺) obtained by protoplast fusion displayed a great variety of hybrids in their taxonomic characters, e.g., 21 recombinant strains obtained by the cross between S. griseus and S. griseoruber consisted of 14 types of hybrids. Antibiotic productivity was examined in all recombinants obtained. Although both parental species produced their respective antibiotics, 60% of the recombinants did not produce any antibiotic and 24% produced different antibiotics from those of their parents. Among those recombinants, it was also found that the distribution of the productivity of each antibiotic among the recombinants was entirely different from that of the allelo-character in each taxonomic feature.     

双亲灭活制备粘质沙雷氏菌和红曲霉的跨界产色素融合子

目的:采用双亲灭活原生质体技术制备粘质沙雷氏菌和红曲霉的跨界产色素融合子,并测定其抑菌活性。方法:经0.2%溶菌酶处理获得粘质沙雷氏菌的原生质体并热灭活;经混合酶(0.8%溶菌酶+1.2%蜗牛酶+1.6%纤维素酶)处理获得红曲霉的原生质体并紫外灭活;用含25%PEG的原生质体融合剂进行促融合与再生。观察融合子的菌落形态和色素合成能力,测定融合子色素提取物对金黄色葡萄球菌的抑制活性。结果:在优化条件下,粘质沙雷氏菌原生质体的形成率为92.58%,红曲霉原生质体形成数约为106个/mL,两菌原生质体灭活率均为100%。共获得13个融合子,9个能产红色素,融合率为1×10-5%。其中8个融合子的95%乙醇提取物对金黄色葡萄球菌表现出不同程度的抑制。结论:采用双亲灭活原生质体技术,能够制备具有抑菌活性的粘质沙雷氏菌和红曲霉的跨界产色素融合子。