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.     

原生质体融合选育高产脂肽LI-F多粘类芽胞杆菌及融合菌株表达差异分析

本研究以多粘类芽胞杆菌JSa-9前期诱变获得的两株带有营养缺陷型标记的菌株N1-37(Phe–)和N2-27(His–)作为亲本菌株,采用聚乙二醇作为促融剂,进行原生质体融合,筛选出高产LI-F类抗菌脂肽的融合菌株。通过HPLC对融合菌株和原始菌株产LI-F类抗菌脂肽进行定量检测,并利用实时荧光定量PCR对菌株JSa-9和融合菌株中LI-F类抗菌脂肽合成酶的关键基因fus A1、fus A2、fus C1和fus C2的差异性表达进行分析。结果表明,经过原生质融合获得一株LI-F类抗菌脂肽高产菌株F5-15。菌株F5-15的LI-F类抗菌脂肽产量为原始菌株产量的3.1倍,LI-F类抗菌脂肽合成酶的4个关键基因在融合菌株F5-15中的表达量分别是其在原始菌株JSa-9中的10.48、2.48、2.1和11.8倍。     

激光微束在遗传操作上的应用

本文概述了近年来激光微束在染色体微切割和微分离、分子细胞生物学、去除细胞壁、诱导原生质体融合等方面的应用,并对其今后的应用前景作了展望。     

Phospholipids of membranes of cultured cells and the products of protoplast fusion

The variety and distribution of phospholipids in the cell membranes of cultured cells and their fatty acid composition were analysed. Membranes of suspension cultured cells of Rauwolfia serpentina var. Bentham, Nicotiana tabacum var. Samsun, Atropa belladonna and Bouvardia ternifolia had almost the same composition of phosphatidylcholine, PC (ca 50%); phosphatidylethanolamine, PE (ca 25%); phosphatidylinositol, PI (ca 10%); phosphatidylglycerol, PG (several %); and phosphatidic acid, PA (several %). We determined the distribution of the molecular species of the three major phospholipid fractions (PC, PE and PI) in R.serpentina and N.tabacum. Membranes of both cell-types contained basically similar molecular species, 1–16:0/2–18:2 the main type, particularly in the PC- and PE- fractions; 1–18:2/2–18:2 and 1–18:0/2–18:2 for R. serpentina; and 1–16:0/2–18:3, 1-18:0/2-18:3 and 1-18:2/2-18:2 for N.tabacum. The influence of membrane fluidity on protoplast fusion as effected by the phase transition of the phospholipids, is discussed.     

Application of protoplast fusion technique to genetic recombination of Micromonospora rosaria

The optimum conditions for efficient formation and regeneration of Micromonospora rosaria protoplasts have been determined. The state of inoculum culture and stage of growth in a medium containing partially growth-inhibiting concentrations of glycine had significant effects on protoplasting. A high frequency of regeneration was accomplished with a hypertonic regeneration agar medium. A slight difference was found in the optimum culture age for formation and regeneration of protoplasts. Protoplast fusion was carried out using these optimum conditions. The recombinant frequency varied from 0.7 to 5.9% in the intraspecific crosses employing single and multiple auxotrophic markers. Electron microscopy showed stable and intact protoplasts when they were prepared with a hypertonic buffer. However, many protoplasts were shown to be damaged and many membraneous vesicles were observed when prepared in buffer without sucrose. The fusion process of protoplasts of Micromonospora was observed with the aid of electron microscopy.     

平菇种内原生质体分离与融合杂交育种技术的研究

将16个平菇资源品种的菌丝体,在25℃、pH5．8-6．0环境下,分别用1．5％溶菌酶加0．6mol／L KCl稳渗剂溶解其细胞壁2h,原生质体数得量最高。将获得的原生质体两两配对成39个组合,在30℃、pH9、0．6mol／L KCl稳渗剂、不加融合促进剂CaCl2和PEG6000的融合条件下,得到种内杂交融合子5株。其中4株融合子再生出菌丝体和子实体;同皿接种实验显示,新菌株与其双亲具明显的拮抗线。经栽培试验与生产示范,新育的“优生1号”品系表现适应性强,优质、丰产、抗杂、耐热。     

Electrical fusion for optimal formation of protoplast heterokaryons in Nicotiana

The electrical fusion of protoplasts has been studied in order to maximize the formation of heterokaryons for culture. Heterokaryons of Nicotiana tabacum L. mesophyll protoplasts and N. plumbaginifolia Viviani supension-cell protoplasts were identified in fixed and stained as well as living material; a quantitative fusion index was thereby developed. With this index the efficiencies of various electric fields and fusion-chamber designs have been determined. Optimal fusion was obtained with an alternating-current (AC) field of 150 V/cm and direct-current (DC) square-wave pulses of 1000 V/cm. A new, simple-to-use, largescale fusion chamber is described in which batches of up to 5·10⁵ protoplasts (0.5 ml of cells at 10⁶/ml) can be fused in 5–7 min with efficiencies approaching 40%. Half of the fusion products are heterokaryons, thus fusion is random. Of the fusion products, 60% are bi- or trinucleate. Using fusion procedures similar to those described here Bates and C. Hasenkampf (1985, Theor. Appl. Genet., in press) have recovered viable somatic hybrids which have been regenerated.Abbreviations AC alternating current - DC direct current - PEG polyethylene glycol     

Strain improvement ofArthrobacter simplex by protoplast fusion

Anl-tryptophan auxotroph and milky mutants were derived from an inducible cholesterol oxidase-producing bacterium,Arthrobacter simplex USA18, via UV-mutagenesis. Protoplasts of these mutants and a constitutive cholesterol oxidase producer, strain US3011, were prepared by growing cells in the presence of ampicillin (20g ml^–1) followed by digestion with lysozyme. Protoplast fusion between tested strains with complementary characteristics was achieved in the presence of 20–40% polyethylene glycol 6000. The fusion frequency was about 1.5–1.7×10^–3. The cholesterol oxidase activity of four fusants in a cholesterol-containing medium was 20–60% higher than that of parental strains. This study demonstrated that protoplast fusion is applicable to strain improvement ofArthrobacter strains for enzyme production.     

Interspecific hybridization between Volvariella volvacea and V. bombycina by PEG-induced protoplast fusion

Interspecific hybridization between Volvariella volvacea and V. bombycina was studied using the protoplast fusion technique. The fusion frequency was found to be in the range of 0.032 to 0.333%. Protoplasts from various hybrids were released and regenerated to determine whether they were heterokaryons. In all regenerated colonies, both parental types could not be recovered at the same time. The nuclear DNA contents of hybrids were compared with their parents, and no diploid (parent 1 genome plus parent 2 genome) was found. Some hybrids revealed novel fragments in mitochondrial rDNA PCR profiles, which indicated that rearrangement of mtDNA could have occurred after fusion. Results from arbitrarily-primed polymerase chain reaction (AP-PCR) fingerprints also revealed that the majority of hybrids were similar to one parental type, but heterologous fragments were found in some hybrids.     

原生质体融合构建直接利用淀粉产衣康酸菌株

采用衣康酸高产菌株栖土曲霉T-730的原生质体,与葡萄糖淀粉酶产生菌黑曲霉Ni-5k的原生质体进行融合处理.在以30%PEG6000为促融剂,30℃保温20min的融合条件下进行融合,所得异核体经诱导获得3株稳定的融合株(F3,F9,F11).以生淀粉为唯一碳源,对F3,F9,F11的发酵性能测定表明,F3在发酵过程中积累葡萄糖淀粉酶和衣康酸;对F3进行发酵条件初步探讨的结果表明,以10%生淀粉为碳源,连续发酵6d,F3的衣康酸产酸率达40.9mg/mL,对供给淀粉的转化率为40.9%.     

种间双亲灭活原生质体融合选育高产Monacolin K红曲菌

为获得高产MonacolinK的红曲菌菌株,将经农杆菌介导转化获得的携带潮霉素抗性基因且以甘油为原料液态发酵高产MonacolinK的发白红曲菌H2和以大米为原料固态发酵产Monaco-1inK的烟色红曲菌9908作为亲本,对其原生质体分别进行热灭活及紫外灭活,然后对灭活双亲用PEG作融合剂进行原生质体融合。从融合子中选出有潮霉素抗性的突变株,通过发酵与亲本对比,筛选得到一株以大米为原料固态发酵高产MonacolinK的融合株F12．11,其MonacolinK产量达到8．73mg／g;较发白红曲菌H2与烟色红曲菌9908分别提高了100．23％和48．98％;一株以甘油为原料液态发酵高产MonacolinK的融合株F13-2,其MonacolinK的产量达到1752．46mg／L,较发白红曲菌H2与烟色红曲菌9908分别提高了32．98％和1979．33％。     

跨界原生质体融合菌Xhhh基因组分子鉴定

Xhhh是由真核、原核两界生物3个亲株PC、SC、XZ的原生质体跨界融合而成的基因工程茵。本研究以Xhhh及其三亲株（PC、SC、XZ）的基因组DNA为扩增模板,筛选出的38条引物,应用随机扩增多态性DNA（R虹,D）技术扩增出739条清晰条带,平均每条引物扩增出了7条清晰可重复的条带。通过聚类分析,结果显示Xhhh与PC、SC与XZ的遗传相似指数S1分别为36．21％、37．73％和37．48％,提示Xhhh与三亲株间存在明显的亲缘关系。研究同时根据已公布的mnp、zip以及FLO1核酸序列设计了相应的功能基因引物,并从Xhhh及其亲株中扩增出了mnp、lip以及FL01功能基因片段,表明RAPD结合功能基因PCR扩增技术可以快速、经济、准确的用于跨界原生质体融合茵株的分子鉴定,以及其它融合子的鉴定。     

Interspecies protoplast fusion inLarix: Comparison of electric and chemical methods

Summary Larix was chosen for the study on interspecies protoplast fusion due to its ability to regenerate plants from protoplasts derived from embryogenic cultures.L. laricina line L2 was used in fusion experiments with eitherL. × eurolepis line L6 orL. × leptoeuropaea line L5. A method of unambiguous labeling of parental protoplasts prior to fusion was developed using vital fluorescent dyes. Of a number of dyes tested, only rhodamine B hexyl ester chloride (R6) and 3,3′-dihexylox-carbocyanine iodide (DiOC₆) stained the protoplasts in a consistent and uniform fashion. The fusion of mixed parental protoplasts that were internally labeled was carried out either in the presence of a 20% polyethylene glycol (PEG) solution or in an electric field. The progress of fusion was readily observed, taking only minutes under the experimental conditions. The fusion products could be identified by dual fluorescence several h after the onset of fusion. Heterofusion frequencies of approximately 18% and 6% in the presence of PEG and an electric field, respectively, were attained. Postfusion cultures betweenL. × laricina protoplasts and protoplasts ofL. × leptoeuropaea gave rise to cell colonies and betweenL. laricina andL. × eurolepis, to mature somatic embryos.     

Plant molecular and cellular laser microsurgery

The u.v. laser microbeam has facilitated new approaches to the genetic manipulation of plant cells. Under visual control, a focused laser microbeam is able to perforate a plant cell wall, thus facilitating the uptake of genes into target cells, induce protoplast fusion selectively and precisely destroy cells or specific subcellular structures in a single living cell. In addition, by expanding these applications to micro-dissection of chromosomes, microsurgery combined with an optical trap inside cells and patch-clamp studies, will open new genetic, biophysical and cell-specific wall development aspects in cell and molecular biology.     

Transmission genetics of Nicotiana hybrids produced by protoplast fusion

Somatic cell hybrids were produced by fusing protoplasts isolated from callus cells of a tobacco line transformed by Agrobacterium tumefaciens (octopine synthesizing strain B6S3), and mesophyll protoplasts from haploid plants of Nicotiana plumbaginifolia. Hybrids were selected by using differential medium (hormone-independent growth plus greening capacity), or by mechanical isolation and cloning of individual heterokaryocytes. The analysis of hybrid cell lines included the determination of lysopine dehydrogenase activity (encoded by the T-region of Agrobacterium tumefaciens plasmid), examination of isozymes of esterase, and study of chromosome number and morphology. All eight cell lines selected on the screening medium were identified as nuclear hybrids, while only three of the eight evaluated clones obtained by mechanical isolation and cloning were found to be nuclear hydrids; the rest of them were nuclear segregants of tobacco [1] or N. plumbaginifolia [4] type. These data give independent evidence for the occurrence of non-fusion and segregation of nuclei in fusion products, that can be revealed only by using nonselective methods for hybrid screening. In this paper we demonstrate the value of microisolation for the recovery of cytoplasmic hybrids.     

Genetic analysis of intraspecies recombinant formation by protoplast fusion with three species of Streptomyces

Abstract Protoplast fusion was shown to produce high frequencies of recombinant progeny in intraspecies crosses with auxotrophic mutants of Streptomyces canescens, Streptomyces griseus and Streptomyces limosus . The fused protoplasts were regenerated on non-selective media and the progeny spores subsequently analysed on selective media to allow detection of all possible genotypes. Prototrophic recombinants arose with frequencies of between 1% and 8%. All 4 possible genotypes were recovered in a series of 2-factor crosses and 6 of the 8 possible genotypes were detected in a 3-factor cross. In spite of attempts to equalise the ratios of parental protoplasts in the fusion mixture, there were noticeable deviations from unity in the ratios of parental genotypes in the progeny.     

Identification of protoplast fusion strain Fhhh by randomly amplified polymorphic DNA

A functional strain Fhhh was constructed through protoplast fusion of three parental strains (Phanerochaete chrysosporium, Saccharomyces cerevisiae and native bacterium YZ) to improve the degradation efficiency of purified terephthalic acid wastewater. Randomly amplified polymorphic DNA (RAPD) and scanning electron microscope (SEM) analysis were applied to identify and confirm the fusant Fhhh through phenotypic and genetic relationship. The result of SEM analysis demonstrated that the cell shape of fusant Fhhh differed from all three parental strains. RAPD analysis of 40 arbitrary primers generated a total of 1,135 bands. The genetic similarity indices between Fhhh and parental strains Phanerochaete chrysosporium (PC), Saccharomyces cerevisiae (SC) and native bacterium (YZ) were 34.01%, 33.16%, and 35.97%, respectively. The targeted-gene PCR results showed that Fhhh inherited the DNA fragments of mnp and lip genes from parental strain PC and FLO1 gene fragment from parental strain SC. Our results suggested protoplast fusion technique may be considered as a promising technique in environmental pollution control.     

Intraspecific protoplast fusion of amylase-producing strains of Candida fennica

The production of -amylase was increased by protoplast fusion of auxotrophic mutants of Candida fennica FTPT-8903. One prototrophic fusant was 90% and 32% more efficient in producing -amylase in semi-solid and liquid fermentation, respectively, than the parental strains. Protoplast fusion did not significantly stimulate the synthesis of glucoamylase in the fusants.     

Recombination after protoplast fusion in the yeast Candida tropicalis

Candida tropicalis protoplasts obtained by snail enzyme treatment were induced to fuse by the use of polyethylene-glycol. Heterokaryons formed by two auxotrophic strains were selected by complementation on minimal medium. These heterokaryons were unstable and readily dissociated into their nuclear components. Under appropriate conditions, the parental nuclei of an heterokaryon fused. The homokaryon so obtained was unstable and segregated into various types of auxotrophic and prototrophic recombinants.List of Abbreviations Used MM minimal medium - YEA yeast extract agar (complete medium) - YPGT yeast-peptone-glucosethiol (medium for protoplast preparation) - PTP medium for cell pretreatment (used before the action of snail enzyme) - PEG polyethylene glycol - p-FPA para-fluorophenylalanine - 5-FC 5-fluorocytosine