A Comparison of Methods for Plasmid Delivery into Plant Protoplasts

Three methods of plasmid delivery to mesophyll protoplasts ofNicotiana tabacum cv. Xanthi have been evaluated. Specifically;a) chemically stimulated uptake of isolated plasmid, b) deliveryof plasmid encapsulated in liposomes, and c) fusion of plasmid-containingspheroplasts, were combined with divalent cation (Ca²⁺ and Mg²⁺)or polyalcohol [polyethylene glycol (PEG) and polyvinyl alcohol(PVA)] treatments. The quantity and quality of plasmid associatedwith intact protoplasts, was assessed by DNA-DNA blot hybridisationanalysis, following stringent washing to separate intact protoplastsfrom non-viable protoplasts and debris. Treatments which increasedassociation of plasmid with protoplasts decreased protoplastviability. Optimum association of plasmid with protoplasts,in the context of acceptable loss of viability, was achievedwhen protoplasts were interacted with either naked plasmid orliposomeencapsulated DNA in the presence of 15% w/v PEG 6000,or with Escherichia coli spheroplasts containing chloramphenicol-amplifiedplasmid in the presence of 25% w/v PEG 6000. Divalent cationsdid not stimulate significant plasmid delivery without unacceptableloss of protoplast viability. Strategies to further increasethe efficiency of plasmid delivery are discussed. (Received June 21, 1984; Accepted August 20, 1984)     

Transformation of Streptococcus lactis Protoplasts by Plasmid DNA

Polyethylene glycol-treated protoplasts prepared from Streptococcus lactis LM3302, a lactose-negative (Lac⁻) derivative of S. lactis ML3, were transformed to lactose-fermenting ability by a transductionally shortened plasmid (pLM2103) coding for lactose utilization.     

Polyethylene Glycol-induced Uptake of Bacteria into Yeast Protoplasts

Cells of the nitrogen-fixing bacterium Azotobacter vinelandii and the unicellular cyanobacterium Anacystis nidulans were introduced into protoplasts of Saccharomyces cerevisiae by the polyethylene glycol (PEG) method. Factors influencing the uptake frequency were examined, and experimental conditions were established for maximizing the uptake frequency. Under optimal conditions, each protoplast took-up a few bacterial cells. Electron-microscopic studies showed the localization of integrated bacterial cells in membrane-bound vesicles of the cytoplasm or large vacuoles. The protoplasts at the intermediate stages of uptake revealed two major mechanisms of uptake: (a) “endocytosis” by a single protoplast and (b) “cell fusion” between two or more protoplasts. Some bacterial cells disintegrated during the subsequent incubation period through a heterophagy-like process.     

Transformation of Bacillus Protoplasts by Plasmid pTP4 DNA

Polyethylene glycol (PEG)-induced protoplast transformation by plasmid pTP4 DNA encoded chloramphenicol resistance determinant was developed for Bacillus subtilis, B. amyloliquefaciens, B. licheniformis, B. megaterium and B. pumilus. Protoplasts were formed by treatment of cells with lysozyme and the transformation frequencies (transformants per regenerants) were in the range of 1.3 × 10^?2 to 7.1 × 10^?1. Reisolated plasmid DNA prepared from transformants exhibited covalently closed and open circular forms similar to those of the donor DNA. These results indicate that PEG-induced protoplast transformation is an adequate method for plasmid transformation and pTP4 is a useful plasmid as a cloning vector in a wide range of varieties of the genus Bacillus.     

Rapid Delivery of Foreign Genes into Plants by Direct Rub-Inoculation with Intact Plasmid DNA of a Tomato Bushy Stunt Virus Gene Vector

Tomato bushy stunt virus (TBSV) cDNA, positioned between a modified cauliflower mosaic virus 35S promoter and the hepatitis delta virus antigenomic ribozyme with a downstream nopaline synthase gene polyadenylation signal, established infections upon rub-inoculation of plants with intact plasmids. Application of this methodology produced a TBSV DNA-based gene vector which yielded readily detectable levels of localized foreign gene expression in inoculated leaves. This is the first demonstration of an infectious DNA from a member of the Tombusviridae which permits rapid TBSV-mediated foreign-gene expression upon direct rub-inoculation of miniprep DNA onto a variety of plant species.     

Yeast Spheroplasts Formed by Cell Wall-Degrading Enzymes from Oerskovia sp

Oerskovia sp. produces inducible extracellular enzymes which degrade the walls of various yeasts. Yeast spheroplasts are formed from both log- and stationary-phase cells.     

Binding of ColE1-kan Plasmid DNA by Tobacco Protoplasts: Nonexpression of Plasmid Gene

Protoplasts prepared from cultured tobacco cells were treated with ColE1-kan plasmid DNA, a hybrid of ColE1 and pSC105 plasmids bearing a gene for kanamycin resistance. The conditions employed permitted the uptake or irreversible binding of 2.9% of the added DNA in acid-insoluble form. Upon commencement of division, the treated cells were plated in agar medium containing kanamycin and differentiating hormones. Plantlets or shoots obtained as presumptive transformants were further tested on kanamycin medium by subculturing small leaf pieces. No evidence was obtained for expression of the kanamycin resistance gene of ColE1-kan in tobacco tissue.     

Transformation of Heat-Treated Clostridium acetobutylicum Protoplasts with pUB110 Plasmid DNA

Heat treatment of Clostridium acetobutylicum SA-1 protoplasts at 55°C for 15 min before transformation resulted in expression in this microorganism of the kanamycin resistance determinant associated with plasmid pUB110. No heat treatment, or heat treatment at 65 or 44°C for various time intervals, resulted in no kanamycin resistance transformants being recovered on selective kanamycin-containing regeneration medium. DNase plate assay indicated that treatment at 55°C for 15 min completely inactivated the DNase activity associated with SA-1 protoplasts. Treatment of protoplasts at 65 or 55°C for various periods under simulated transformation conditions had an inhibitory effect, although prolonged treatment at 55 or 44°C appeared to stimulate DNase activity. Inactivation of protoplast-associated DNase activity by heat treatment at 55°C for 15 min correlated with successful expression of kanamycin resistance and suggests that an extremely active, heatsensitive, protoplast-associated DNase may be a factor in the polyethylene glycol-induced transformation of C. acetobutylicum SA-1 protoplasts. Plasmid pUB110 DNA was isolated from C. acetobutylicum SA-1 kanamycin-resistant (Km^r) transformant cultures by a modification of the procedure used for C. perfringens plasmids. Detection of pUB110 DNA was possible only when diethyl pyrocarbonate was incorporated into isolation protocols to inactivate DNase activity. Restriction studies further verified the presence of pUB110 DNA in C. acetobutylicum SA-1 Km^r transformants.     

Transfer of Isolated Nuclei into Protoplasts of Trichoderma harzianum

Protoplasts released from young hyphae of Trichoderma harzianum contained 0 to 10 nuclei per protoplast, and most (about 80%) contained from 4 to 6 nuclei. Most protoplasts were larger than 3 μm in diameter. Nuclei were isolated from protoplasts of an auxotrophic mutant of T. harzianum and transferred into protoplasts obtained from another auxotroph of the same strain. This intrastrain nuclear transfer gave rise to numerous progeny which were stable, prototrophic, and heterokaryotic. Interstrain transfers in which nuclei from a wild-type prototroph of one strain were transferred into protoplasts from a lysine-deficient auxotroph of a second strain were also done. Heterokaryotic progeny were recovered from these interstrain transfers when the regenerating protoplasts were provided with a low concentration of lysine 48 h after the initial plating. Heterokaryotic progeny contained 11 to 17% of donor-type nuclei. Progeny homokaryotic for donor-type nuclei were obtained as single-spore isolates. These homokaryotic isolates expressed the isozyme pattern and colony morphology phenotype of the nuclear donor. When regenerating protoplasts were provided with lysine 10 days after the initial plating, only a single progeny was obtained. However, single-spore subprogeny of this nuclear transfer were prototrophic and exhibited a wide range of unstable morphological phenotypes.     

Surface Structure of Yeast Protoplasts

The fine structure of the yeast cell wall during protoplast formation was studied by means of phase-contrast microscopy and the freeze-etching technique. The freeze-etching results indicated that at least in some cases the entire wall substance was not removed from the surface of the protoplasts. After a treatment of 30 min to 3 hr with 2% snail enzymes, an innermost thin wall layer as well as remnants of the fibrillar middle layer sometimes could be demonstrated.     

用酚-氯仿直接从克隆中提取质粒DNA

目的:对用酚-氯仿直接从克隆中提取质粒DNA的方法进行了研究。方法:采用不同次数的酚-氯仿抽提,在不同RNaseA作用温度及作用时间下提取了三种质粒。结果:用酚-氯仿抽提2次,RNase A45℃作用8min,质粒纯度(OD260/OD280)为1.85左右、产量大于0.8μg/克隆。结论:本法不但快速,且所提质粒均能满足后续实验如酶切鉴定、测序等方面的需要。对于大批量重组克隆的筛选尤为适合。     

Exploitation of Plasmid pMRC01 To Direct Transfer of Mobilizable Plasmids into Commercial Lactococcal Starter Strains

Genetic analysis of the 60.2-kb lactococcal plasmid pMRC01 revealed a 19.6-kb region which includes putative genes for conjugal transfer of the plasmid and a sequence resembling an origin of transfer (oriT). This oriT-like sequence was amplified and cloned on a 312-bp segment into pCI372, allowing the resultant plasmid, pRH001, to be mobilized at a frequency of 3.4 × 10⁻⁴ transconjugants/donor cell from an MG1363 (recA mutant) host containing pMRC01. All of the resultant chloramphenicol-resistant transconjugants contained both pRH001 and genetic determinants responsible for bacteriocin production and immunity of pMRC01. This result is expected, given that transconjugants lacking the lacticin 3147 immunity determinants (on pMRC01) would be killed by bacteriocin produced by the donor cells. Indeed, incorporation of proteinase K in the mating mixture resulted in the isolation of transformants, of which 47% were bacteriocin deficient. Using such an approach, the oriT-containing fragment was exploited to mobilize pRH001 alone to a number of lactococcal hosts. These results demonstrate that oriT of pMRC01 has the potential to be used in the development of mobilizable food-grade vectors for the genetic enhancement of lactococcal starter strains, some of which may be difficult to transform.     

Introduction of Functional RNA into Plant Protoplasts by Electroporation

A simple apparatus was constructed for producing electric dischargeof varying intensities between two electrodes in a spectrophotometercuvette. This apparatus was used to study the conditions forefficient introduction of functional RNA into plant protoplastsusing RNAs of tobacco mosaic virus (TMV) and cucumber mosaicvirus (CMV). Electroporation under optimal conditions resultedin the production of TMV and CMV in 80% of the protoplasts fromtobacco cell line BY2. Infection by TMVRNA occurred in 60% ofVinca rosea suspension culture protoplasts and in 40% of tobaccomesophyll protoplasts. Electroporation in the presence of TMVand CMV particles also resulted in infection, suggesting thatpores larger than 30 nm are formed. The advantages and usesof the electrical method for introduction of functional RNAare discussed. (Received December 21, 1985; Accepted March 1, 1986)     

Cultivation of Rice Protoplasts and Their Transformation Mediated by Agrobacterium Spheroplasts

Improvement of the cultivation of rice (Oryza saliva L.) protoplastsisolated from suspension cultures led to their division at afrequency of 5 to 10%. Rapidly growing colonies were obtainedon a hormone-free medium when Agrobacterium tumefaciens spheroplastswere introduced into the protoplasts by polyethylene glycoltreatment. Opines corresponding to the strains of A. tumefaciensused for the spheroplast treatments were detected in some ofthese colonies at a frequency of about 10^–4. Using radioactiveprecursors, [¹⁴C]--ketoglutaric acid and [³H]-arginine, activitiesof nopaline synthase, a marker enzyme of nopaline-type crowngall, were also detected in some of these clones. These resultsshow that the rice cells were transformed by Ti plasmid introducedby the spheroplast method. (Received September 6, 1985; Accepted January 24, 1986)     

DNA Binding and Uptake by Nuclei Isolated from Plant Protoplasts: Fate of Single-stranded Bacteriophage fd DNA

Binding and uptake of exogenous DNA by nuclei isolated from Glycine max L. Merr were studied using ³H-labeled single-stranded DNA of bacteriophage fd. A comparison of single-stranded with double-stranded DNA for binding and uptake by nuclei was also made.     

Evolutionary Capture of Viral and Plasmid DNA by Yeast Nuclear Chromosomes

A 10-kb region of the nuclear genome of the yeast Vanderwaltozyma polyspora contains an unusual cluster of five pseudogenes homologous to five different genes from yeast killer viruses, killer plasmids, the 2μm plasmid, and a Penicillium virus. By further database searches, we show that this phenomenon is not unique to V. polyspora but that about 40% of the sequenced genomes of Saccharomycotina species contain integrated copies of genes from DNA plasmids or RNA viruses. We propose the name NUPAVs (nuclear sequences of plasmid and viral origin) for these objects, by analogy to NUMTs (nuclear copies of mitochondrial DNA) and NUPTs (nuclear copies of plastid DNA, in plants) of organellar origin. Although most of the NUPAVs are pseudogenes, one intact and active gene that was formed in this way is the KHS1 chromosomal killer locus of Saccharomyces cerevisiae. We show that KHS1 is a NUPAV related to M2 killer virus double-stranded RNA. Many NUPAVs are located beside tRNA genes, and some contain sequences from a mixture of different extrachromosomal sources. We propose that NUPAVs are sequences that were captured by the nuclear genome during the repair of double-strand breaks that occurred during evolution and that some of their properties may be explained by repeated breakage at fragile chromosomal sites.It is well known that the nuclear genomes of most eukaryotes contain integrated fragments of organellar DNA called NUMTs (nuclear copies of mitochondrial DNA) and NUPTs (nuclear copies of plastid DNA, in plants) (26, 29, 44, 45, 57). These fragments are usually pseudogenes, although some NUMTs and NUPTs have become incorporated into functional nuclear genes (38). The NUMTs present in the nuclear genomes of Saccharomycotina yeast species were recently analyzed by Sacerdot et al. (48).In addition to their mitochondrial genomes, yeast species contain a variety of other extranuclear DNA and RNA elements, including viruses and plasmids. These extrachromosomal elements are usually considered to be autonomous entities that do not interact with nuclear DNA. When our laboratory sequenced the genome of the yeast Vanderwaltozyma polyspora (synonym: Kluyveromyces polysporus) (49), we were therefore surprised to find the genomic region we describe here, which contains integrated fragments of several plasmid- and virus-like sequences. We propose that this region was formed by the capture of plasmid and viral sequences by the same mechanism that captures mitochondrial DNA to form NUMTs (43, 65). In a literature search, we could find only one previous report of a similar finding: Utatsu et al. (59) reported the sequences of two regions of nuclear DNA from Zygosaccharomyces rouxii that were highly similar to parts of the 2μm-like plasmid pSR1 from that species, but rearranged.Before describing the V. polyspora region, and similar regions found in other species, we will first briefly introduce the extrachromosomal RNA and DNA entities that are known to exist in yeasts. Extrachromosomal nucleic acids are relatively uncommon in yeasts: a broad survey of 1,800 strains from 600 species by Fukuhara (14) found that 196 strains (11%) contained some sort of extrachromosomal entity. Among these, 105 strains had a double-stranded RNA (dsRNA), 28 had a linear dsDNA plasmid, and 53 had a circular DNA plasmid of the 2μm family. These elements typically also have a patchy distribution within a species, being found in some individuals or strains but not in others. For instance, Nakayashiki et al. (37) surveyed 70 “wild” strains of Saccharomyces (mostly S. cerevisiae) for the presence of five extrachromosomal elements (2μm DNA plasmid, L-A and L-BC helper RNA viruses, and W and T RNA entities) and found each element to be present in between 1 and 38 of the strains, with 1 strain even containing all five elements simultaneously.     

High Yield Isolation of Nuclei from Plant Protoplasts

Nuclei were isolated from protoplasts obtained from Parthenocissus tricuspidata Crown Gall callus tissues. The effect of various isolation procedures, detergent or ultrasonication, on yield and quality of nuclei was studied. A standard procedure, based on the use of 5 × 10^?3% Triton × 100 — 6% PVP — 20% glycerol, may be carried out in 30 min and gives 80 to 90% yield of nuclei in which RNA polymerase activity is retained.     

在烟草中酵母脯氨酸基因B的转化(英文)

重组质粒PYP22带有一从酵母基因文库分离到的4.6 kb DNA片段,此片段含有脯氨酸(Pro)合成途径必须的基因B(ProB)。用BamHⅠ酶解PYP22,回收ProB基因,重组入pGA471的Bg Ⅲ切口,形成含有ProB的双质粒载体PBYU4。pBYU4含有能在植物中表达的新霉素磷酸转移酶基因Ⅱ(NPT—Ⅱ),可做转基因植株的筛选标记。借助于辅助质粒pRK2013,pBYU4经过三亲结合转移到农杆菌LAB4404,在含有四环素12.5μg/ml、链霉素100μg/ml和利福平50μ/ml加的AB培养基上筛选出转接合子。提取筛选得到的农杆菌总DNA,用ECoR 1酶解,1％琼脂糖电泳,Southern转移DNA到硝酸纤维素膜上。用α—~(32)P-dCTP标记的ProB片段,与转移好的硝酸纤维素进行Southern杂交。Southern杂交证明含有ProB基因的农杆菌,在加有乙酸丁香酮(acetosyringone)125μg/ml和章鱼碱(octopine)125μg/ml的MS液体培养基中,诱导过夜。用叶圆片法转化革新1号烟草(Nicotana tabacum var.Gexin No.1),叶片与菌液共培养2～5d。从叶片分化再生出的芽转移到含有卡那霉素(K_m)80μg/ml、6—苄基腺嘌呤(6BA)1μg/ml和头孢氨噻腭钠(Cef)500μg/ml的MS培养基,筛选3周,将绿色的芽转移到含l％NaCl,6BA 1μg/ml和Cef 500μg/ml的MS培养基,进行复筛。测定经过这样筛选的再生芽的NPT—Ⅱ活性。     

Plant Regeneration from Mesophyll Protoplasts of Echinacea Purpurea

An efficient plant regeneration system was developed from isolated protoplasts of Echinacea purpurea L. using an alginate block/liquid culture system. Viable protoplasts could be routinely isolated from young leaves of Echinacea seedlings in an isolation mixture containing 1.0% cellulase Onozuka R-10, 0.5% pectinase and 0.3 mol l^–1 mannitol. Purified protoplasts were embedded in 0.6% Na-alginate block at a density of 1 × 10⁵/ml and cultured in a modified MS medium containing 0.3 mol l^–1 sucrose, 2.5 µmol l^–1 BA and 5.0 µmol l^–1 2,4-D. Cell colonies were observed after 4 weeks of culture, and the protoplast-derived colonies formed calluses when transferred onto 0.25% gellan gum-solidified MS medium supplemented with 1.0 µmol l^–1 BA and 2.0 µmol l^–1 IBA. Shoot organogenesis from protoplast-derived callus was induced on MS medium supplemented with 5.0 µmol l^–1 BA and 2.0 µmol l^–1 IBA. Complete plantlets were obtained from the regenerated shoots on MS basal medium. The protoplast to plant regeneration protocol developed in this study provides the prerequisite for creating novel genotypes of this valuable medicinal species through genetic manipulation.