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.

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.

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

Laser-induced tobacco protoplast fusion

Laser tweezers can manipulate small particles, such as cells and organdies. 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.

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。

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.

巨大口蘑与双孢蘑菇原生质体融合育种

为了培育出生长速度快、生长周期短的巨大口蘑优良新菌株,以巨大口蘑和双孢蘑菇2796为亲本菌株,建立原生质体聚乙二醇(PEG)融合体系。将分别经过紫外灭活的巨大口蘑和热灭活的双孢蘑菇原生质体按照体积比1∶1混合,PEG(分子量6000)质量分数为30%,Tris浓度为0.016 mol·L-1,Ca2+浓度为0.014 mol·L-1,30℃水浴20 min,该最佳融合条件下融合率达0.0387%。利用灭活原生质体作为标记筛选出融合子,再生培养后通过菌落形态、细胞学观察、拮抗试验、出菇试验验证再生菌株为融合菌株,最终从7个再生菌株中筛选出3个融合菌株,进行出菇试验。试验表明,3个融合菌株的母种和栽培种生长速度明显快于母本菌株巨大口蘑,栽培周期明显缩短。

原生质体融合结合基因编辑技术显著提高酿酒酵母2-苯乙醇产量

酿酒酵母(Saccharomyces cerevisiae)已用于生产天然2-苯乙醇(2-phenylethanol,2-PE),但其产量低,耐受性差。该研究筛选出3株具有不同优良性状的酿酒酵母菌株,其中菌株LSC-1的2-PE产量为3.41 g/L,菌株NGER对2-PE的耐受性达3.60 g/L,菌株S.C-1耐热性好并能在41℃下生长。以这3株菌为亲本,通过2轮原生质体融合获得融合子菌株RH2-16,对2-PE的耐受性提高了20%。以L-苯丙氨酸为底物,发酵转化36 h,2-PE产量达到4.31 g/L,与亲本菌株LSC-1和2-PE工业生产菌株CWY132相比,分别提高26.4%和38.1%。利用CRISPR/Cas9系统,在RH2-16中构建了含突变的Ras特异性鸟嘌呤核苷酸交换因子CDC25(W1416C)菌株,2-PE的产量提高了8%。在RH2-16中过表达艾氏途径合成2-PE关键基因ARO8,ARO10和ADH2未能提高2-PE产量。研究为筛选合成2-PE等天然产物的新型酿酒酵母菌株及其育种提供了一种有效策略。

益生菌遗传育种方法研究进展

益生菌的多样化益生作用使其在人类健康领域发挥着重要作用,基于益生菌生产的药品、食品和膳食补充剂也受到越来越多的关注。目前,研究人员致力于为药品和食品行业选育新型益生菌,以期为食品药品行业的发展提供新的思路。遗传育种为选育具有有益特性的益生菌菌株提供了一个很好的平台,这对提升益生菌的市场价值和人类健康具有重要意义。为了今后更好的开展益生菌育种工作,该文综述了原生质体融合、基因组重排、常压室温等离子体诱变及基因编辑技术[成簇规律间隔的短回文重复序列相关蛋白(clustered regularly interspaced short palindromic repeats-associated,CRISPR-Cas)技术和碱基编辑技术]等遗传育种技术的研究现状及在益生菌中的应用,并讨论了遗传育种的安全性,旨在为优良益生菌的选育提供参考,促进益生菌资源的开发和利用。

远缘原生质体融合菌株的构建及生防作用研究

研究基于沼泽红假单胞菌PSB-06促生和增强寄主抗病性,苏云金芽孢杆菌KY-1抗逆性强等优点,采用原生质体融合技术获得这2种菌株的融合菌株,测定了融合菌株对生姜根结线虫的防治效果,以期得到综合亲本优良性状的新型高效生防菌株。结果表明:融合菌株显著降低了土壤根结线虫的数量,收获期相对防效达73.68%;融合菌株处理显著提高了生姜的农艺性状、产量和品质,单株姜球数(13.80个)、姜球宽度(33.47 cm)和单株产量(1.29 kg)均优于空白对照;同时提高了生姜收获期叶片的总叶绿素含量、POD和SOD活性。综合来看,沼泽红假单胞菌和苏云金芽孢杆菌融合菌株的促生及生防潜力较亲本沼泽红假单胞菌有所提高,为微生物菌种改良提供了依据。

基因组重排技术在食用菌资源创新中的应用

基因组重排技术,标志着育种技术的研究从单一自然表型定向进化至理想全基因型的遗传改造阶段,突破了现有育种技术的范式,实现了种质跨界育种的资源创新。对基因组重排技术在食用菌组学领域的研究现状,及以多亲本原生质体融合技术为核心的基本技术进展进行了综述,介绍了基因组重排实现部分食用菌耐受性的提高,以及在高产新品种菌株育种中的应用,并对基因组重排技术促进食用菌产业发展进行了展望。

一株橘绿木霉TR673原生质体的制备、再生及融合研究

从齐齐哈尔市大豆根围土壤中分离得到1株木霉菌株TR673,经分子生物学及形态学鉴定,确定其为橘绿木霉(Trichoderma citrinoviride)。采用崩溃酶对TR673的菌丝体进行裂解制备原生质体,分别采用50℃水浴5 min和紫外线照射(20 W、40 cm)90 s对原生质体进行灭活,再将灭活的原生质体用聚乙二醇融合法进行融合,并对TR673原生质体的制备和再生条件进行了优化。结果表明,用20 g·L^(-1)的崩溃酶对培养16 h的TR673菌丝体酶解(裂解)5 h时,原生质体产量最大,达到1.2×10^(6)个·mL^(-1)。用ZS1培养基对原生质体进行再生培养,其再生率可达12.3%。融合菌株TR673-3的哌珀霉素产量达1282.67 mg·L^(-1),较原始菌株TR673的哌珀霉素产量提高了18.11%。本研究为后续的橘绿木霉基因组的编辑、转化以及其它木霉菌原生质体的制备、再生与融合等实验提供了参考依据。

原生质体形成及融合因素分析

本实验以经过青霉素筛选后的两株絮凝菌为对象,分别测定其溶菌酶处理前总菌数、溶菌酶处理后总菌数和原生质体再生菌数,计算原生质体形成率和再生率,并对PEG的作用浓度和时间进行优化。实验结果:F2原生质体形成率为99.97%,F6原生质体形成率为99.96%,F2原生质体再生率为0.4225%,F6原生质体再生率为0.5423%,初步判断溶菌酶处理后得到的原生质体在质量和数量上满足细胞融合需求,确定了原生质体融合PEG最佳作用浓度为30%,最佳作用时间为10 min,以保证原生质体融合的完成。

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。