Protoplast Fusion of Cosmos bipinnatus and Cosmos sulphureus

[ Objective] The aim was to study appropriate fusion conditions of protoplasts from 2 species Cosmos bipinnatus and Cosmos sulphureus. [ Method ] The protoplasts were separated from the blossom petals of Cosmos bipinnatus and Cosmos sulphureus availed with the function of pectinase and cellulase, and the fusion of two pretoplasts was performed with PEG. [ Results] A great deal of free pretoplasts were obtained from Cosmos bipinnatus by using 6% mannitol ＋ 1.3% cellu- lase ＋ 1.2% pectinase, and from Cosmos sulphureus by using 10% mannitol ＋ 1.3% cellulase ＋ 1.2% pectinase. Five hours after of enzymolysis treatment, large amount of dissociated protoplasts were observed in the solution. Pretoplast fusion was be obtained by using 30% PEG fusion liquid and 0. lmol/1 CaC12 solution （pH value = 9.5）. [ Conclusion] Our study established an optimal separation and fusion system of protoplasts from different coreopsis species, laying foundation for the breeding of novel coreopsis varieties.

Research Progress of Algae Protoplast Fusion Technique

This paper discussed the technique of algal protoplast fusion,including the method of protoplast-induced fusion,the screening and identification of hybrid cells and so on.In this paper,the research progress of protoplast-induced fusion of microalgae was reviewed,and the problems existing in the process of protoplast-induced fusion were discussed in order to provide a theoretical basis for the development of algae genetics and breeding.

Regeneration of intergeneric somatic hybrids by protoplast fusion between Onobrychis viciaefolia and Medicago sativa

Protoplast fusion was induced between sainfoin and alfalfa by an improved polyethyleneglycol (PEG) method. The intergeneric somatic calluses were selected based on complementation of hydroxyproline-resistance of sainfoin and hormone autonomy growth of alfalfa transformation cell line. 17 somatic hybrid plantlets were regenerat-ed. PEG could induce the tight agglutination of protoplasts. During diluting and washing process, cyclization of the linked membrane and formation of vesicle-like structures were observed, resulting in protoplast fusion. 5%-10% glycerol supplemented in the fusion inducing solution markedly increased the frequency of heterogeneous fusion. Better fusion results were obtained when mixed protoplast suspension was dripped in petri dishes in which PEG solution was previously placed. Chromosome number of regenerated hybrid buds varied from 30 to 60. The genome of hybrids in-cluded the small chromosome from sainfoin and two chromosomes with two clear constrictions from alfalfa. The hybrid-ity of obtained hybrid calluses was confirmed by their isozyme banding patterns and their nopaline synthetase activity.

The protoplast two-way fusions and fusant characteristics of Antrodia cinnamomea and Cordyceps militaris

This study generated two fused protoplasts of Antrodia cinnamomea and Cordyceps militaris in two ways.The protoplasts of A.cinnamomea were inactivated by heat to inactivate biochemical processes and enzymatic activities in the cytoplasm,and the protoplasts of C.militaris were inactivated by UV radiation to invalidate their genome function,then they were fused under optimal conditions to get a fusion rate as(7.42±0.8)×10^(-6) fusants/mL;the new fusants were abbreviated as Ac-Cm.On the other hand,when A.cinnamomea and C.militaris were treated with heat and UV oppositely using similar experiments,the fusion rate was(9.70±0.68)×10^(-5) fusants/mL,and the new fusants were abbreviated as Cm-Ac.We selected each of two best-growing fused colonies Ac-Cm-1,Ac-Cm-2,Cm-Ac-1,and Cm-Ac-2,together with parental A.cinnamomea and C.militaris,and studied their morphology,growth antagonism tests,and genetic relationships by 18 S rRNA sequencing.In comparison with the initial cultures of 4 fusants,the yields of adenosine,biomass,cordycepic acid,cordycepin,total polysaccharide,and total triterpenoids were increased up 1.305-50.1563 times in the optimal medium conditions.For gene stability tests,those of the four fusants and their outputs were stabilized within 10 generations.

Enhancement and selective production of avermectin B by recombinants of Streptomyces avermitilis via intraspecific protoplast fusion

Among eight components of avermectin, B1 fractions have the most effective antiparasitic activities and the lowest level of toxic side-effects and are used widely in veterinary and agricultural fields. In-traspecific protoplast fusion between two strains of Streptomyces avermitilis, one an avermectin high producer (strain 76-05) and the other a genetically engineered strain containing the mutations aveDˉ and olmAˉ (strain 73-12) was performed for enhancement and selective production of avermectin B in the absence of oligomycin. Two recombinant strains (F23 and F29) were isolated and characterized with regards to the parental merits. F23 and F29 produced only the four avermectin B components with high yield and produced no oligomycin. The avermectin production of F23 and F29 was about 84.20% and 103.45% of the parental strain 76-05, respectively, and increased about 2.66-fold and 3.50-fold, re-spectively, compared to that of parental strain 73-12. F23 and F29 were genetically stable prototrophic recombinants and F29 was quite tolerant of fermentation conditions compared to avermectin high producer parental strain 76-05. The ability to produce avermectin B with high yield without the produc-tion of other avermectin components and oligomycin will make F23 and F29 useful strains for aver-mectin production. Strain F29's tolerance of fermentation conditions will also make it suitable for in-dustrial applications.

Laser-induced tobacco protoplast fusion

Laser tweezers can manipulate small particles, such as cells and organelles. When coupling them with laser microbeam selective fusion of two tobacco protoplasts containing some chloroplast was achieved. Physical and biological variables that affect laser trapping and laser-induced fusion were also discussed. The results show that the effect of chloroplast content and distribution on the yield of cell fusion is remarkable.

Protoplast Isolation and Fusion of Epipremnum aureum

[Objectives] This study was conducted to improve the genetic character of Epipremnum aureum and provide a new way for genetic breeding of E. aureum. [Methods] In the paper,protoplasts were isolated from tender leaves of E. aureum and Scindapsus aureus var. wilcoxii by using 1% cellulase and 1% peetinase. Then the protoplasts were purified and dynamically identified,and fusion of protoplasts of E. aureum and S. aureus was induced by PEG. [Results]A large quantity of protoplasts were obtained from tender leaves of E. aureum and S. aureus successfully,and the activity of protoplasts was over 90%. Fusion product was obtained by PEG induction,and the fusion rate was 16. 3%. [Conclusions]Protoplasts were isolated from tender leaves of E. aureum and S. aureus successfully,and a stable fusion system was built by PEG induction.

Protoplast Culture and Its Application in Fruit Breeding

The progress in protoplast technology and its application in fruit breeding were summarized,and the existing problems and application prospect were also discussed.

体细胞杂交在蔬菜作物上的研究与前景

体细胞杂交是一种将植物不同种、属,甚至科间的原生质体诱导融合,然后离体培养获得再生植株的技术。原生质体融合能克服传统育种的杂交生理障碍问题,实现不同材料间核质基因的重组,传递优异基因,创制并获得含目标性状的新种质,拓宽育种材料的遗传背景,同时能快速创制雄性不育材料,极大地缩短育种年限,是作物进行突破育种的有效手段和强大的性状改良工具。本文对该技术在蔬菜上的发展历程、研究和应用领域进行综述,重点总结了十字花科、茄科、伞形科蔬菜作物原生质体分离再生和体细胞杂交的研究进展,展望了体细胞杂交技术在蔬菜作物中的应用前景,讨论了该技术在应用过程中面临的问题与对策,为体细胞杂交在蔬菜上的广泛应用提供参考。

兰州百合原生质体的分离纯化和应用

该研究旨在建立兰州百合(Lilium davidii var.unicolor)原生质体分离纯化体系,以促进其基因功能研究和体细胞杂交育种。通过研究材料选择、渗透压调节剂浓度、酶添加量、酶解时间和纯化条件等关键因素,确定了酶解法提取兰州百合原生质体的最优参数。结果表明:继代30 d左右的兰州百合无菌苗叶片,在0.4 mol·L^(-1)的甘露醇环境下,使用含15 g·L^(-1)的纤维素酶和5 g·L^(-1)的离析酶的酶解液酶解3 h,即能分离完整均一的兰州百合叶肉原生质体,平均产量超过4×10^(6)个·g-1,且细胞活力超过90%。在0.4 g·mL^(-1)PEG-4000介导下转染35S::GFP质粒15 min,成功获得阳性转化子。同时,在0.5 g·mL^(-1)PEG-4000与5 mmol·L^(-1)CaCl_(2)(pH=10)作用下,原生质体发生融合,形成双核细胞。该体系的建立为兰州百合体细胞杂交育种提供了高质量的细胞材料,也为其基因功能研究搭建了平台。

应用原生质体融合技术获得茄子种间体细胞杂种

应用原生质体电融合技术 ,获得了茄子近缘野生种Solanumtorvum、S aethiopicum与栽培种S melongena (六叶茄 ,Dourga)的种间体细胞融合四倍体再生植株。染色体检测表明该植株为四倍体 (2n =4x =4 8) ,荧光免疫检测证明为种间体细胞杂种 ,青枯病抗性鉴定表明带有抗病基因 。

利用原生质体融合技术选育微生物菌种

对原生质体融合技术的遗传标记筛选、原生质体的制备与再生、原生质体融合等的方法 ,以及原生质体融合技术在提高抗生素生产效价、酵母菌工程菌株的构建、多功能菌株的选育以及污水处理工程菌的构建等方面的研究情况进行了综述。就原生质体融合技术在微生物育种技术上的应用前景进行了分析。

原生质体融合提高农抗武夷菌素的效价

从农抗武夷菌素产生菌不吸水链霉菌武夷变种菌株Co-N-31诱变获得两个突变株M35(Leu^-,孢子颜色灰色)和M46(ser^-.孢子颜色灰白色),并以此两突变株为直接亲本在25％ PEG1000诱导下进行种内原生质体融合。M35和M46原生质体再生率分别为3.72％和0.248％,重组频率为55.20％。采用间接法选择营养标记互补的稳定的原养型重组子,并从中获得一株高产菌株F31-24;其效价比原始亲本Co-N-31提高了82％。薄层层析结果表明,菌株F31-24和Co-N-31的发效产物在Rf值为0.50和0.26处均有斑点,但含量有异。测定斑点生物活性证明其均有抑菌活性。温室试验表明,菌株F31-24发酵产物对小麦白粉病的防治效果优于菌株Co-N-31。

属间体细胞杂交创建甘蓝型油菜细胞质雄性不育系及其鉴定

利用原生质体融合技,获得了甘蓝型油菜品种中双4号与新疆野生油菜野油18的对称性体细胞杂种。杂种当代建成的6株植株中有2株雄性不育株。用中双4号做轮回亲本与不育株回交,不育株率随回交代数增加而升高,至BC3代大部分株系的不育株率接近100%,到BC4代不育性稳定,群体中无育性分离现象。用波里马CMS的恢复系和保持系与其测交,6个甘蓝型油菜品系均可保持该不育系的不育性,表明该雄性不育胞质与Pol不育胞质不同。

原生质体融合选育L-亮氨酸高产菌的研究

以黄色短杆菌TQ5 35 1(Met- +Ile- + 2 TAr+SGr)和TQ5 35 6(Met- +Ile- +α ABr+ β HLr)为亲株 ,采用原生质体融合技术 ,定向选育出一株L 亮氨酸高产菌TQ980 6(Met- +IleL + 2 TAr+SGr+α ABr+ β HLr) ,该菌株经 5L罐分批发酵 64h产L 亮氨酸2 2 7g/L。

麦角固醇高产菌株的构建及其培养优化条件的研究

通过初筛、单倍体分离、诱变及酵母菌种间原生质体融合技术构建了２株麦角固醇产量明显提高的优良菌株ＹＥＦ２１和ＹＥＦ２９。并对ＹＥＦ２１的培养优化条件进行了研究。结果表明在优化的实验条件下ＹＥＦ２１的生物量及麦角固醇含量的综合值分别为原始亲株ＹＥ２２７和ＹＥ１８０的１５４和１５５倍。经遗传稳定性分析，没有发现标记基因的分离现象，从而证明获得的融合菌株是遗传稳定的，是具有一定实际应用价值的麦角固醇高产菌株。

细菌原生质体融合育种技术及其应用进展

原生质体融合技术是细菌遗传育种的有效方法之一,发展迅速,应用广泛。文中综述了亲本菌株选择性遗传标记方法、影响原生质体制备与再生因素、原生质体融合方法和条件。介绍了细菌原生质体融合技术在遗传育种中的应用,并展望了细菌原生质体融合技术的发展前景。

构建直接发酵淀粉产生酒精的酵母融合菌株的研究

以酒精酵母和热带假丝酵母为亲本 ,通过单亲灭活原生质体融合技术 ,获得了既具有糖化酶活性又能高产酒精的稳定的酵母融合株 ,并测定和比较了融合子的细胞大小、DNA含量以及比增长率 μ、淀粉利用能力、乙醇耐受性、α 淀粉酶和糖化酶活力等生产性能。属间原生质体融合率为 9 2× 10 -6。F 1和F 5两株融合子性状较好 ,酒精发酵产量可达 8 8%和 11 5 % ,具有良好的应用前景。

降解精对苯二甲酸生产废水特化菌株性能测定

将黄孢原毛平革真菌 (Phanerochaetechrysosporium ,PC)真核细胞与YZI土著细菌原核细胞的原生质体进行融合 ,构建而成Fhh融合子 ;再将Fhh与酵母 (Saccharomycescerevisiae ,Y99)真菌真核细胞的原生质体进行融合 ,构建而成Fhhh融合子 .Fhh与Fhhh是跨真核原核两界细胞的两株融合子 ,对制霉菌素和链霉素同时存在抗性 ,孢子和菌丝形态均不同于PC菌 .在精对苯二甲酸生产废水中降解反应 6h ,Fhhh的比增长率 μ和比降解率q均处最高水平 ,分别是 0 12 6 5h- 1 和 0 .3348h- 1 ,分别为PC、YZI、Y99三亲株菌的 5～ 10倍和 11～ 19倍 .研究结果对于提高PTA废水处理系统的处理效率 ,降低处理费用 ,有着重要的意义 .

香菇菌丝原生质体制备及融合条件的研究

讨论不同菌龄、酶解时间、酶解温度、渗透压稳定剂对香菇原生质体产率的影响。结果表明 ,香菇菌丝制备原生质体时最适菌龄为 6天 ,酶解时间为 3h ,酶解温度为 34℃ ,渗透压稳定剂用 0 .6mol/L甘露醇为最佳。在此基础上 ,以 35 %的PEG为融合诱导剂进行香菇B0 1、L2 6种间原生质体融合 。