Natural and Electroporation-Mediated Transformation of Methanococcus voltae Protoplasts

The lack of high-efficiency transformation systems has severely impeded genetic research on methanogenic members of the kingdom Archaeobacteria. By using protoplasts of Methanococcus voltae and an integration vector, Mip1, previously shown to impart puromycin resistance, we obtained natural transformation frequencies that were about 80-fold higher (705 transformants per μg of transforming DNA) than that reported with whole cells. Electroporation-mediated transformation of M. voltae protoplasts with covalently closed circular Mip1 DNA was possible, but at lower frequencies of ca. 177 transformants per μg of vector DNA. However, a 380-fold improvement (3,417 transformants per μg of DNA) over the frequency of natural transformation with whole cells was achieved by electroporation of protoplasts with linearized DNA. This general approach, of using protoplasts, should allow the transformation of other methanogens, especially those that may be gently converted to protoplasts as a result of their tendency to lyse in hypotonic solutions.     

Effect of Proteolytic Enzymes on Transfection and Transformation of Streptococcus lactis Protoplasts

With both chymotrypsin and mutanolysin used to form protoplasts, consistent transformation frequencies of 10⁴ to 10⁵ transformants and transfectants per μg of DNA were achieved. The procedure was used to transform protoplasts of Streptococcus cremoris CS224 at low frequency (5 transformants per μg of DNA).     

Transformation ofSchizophyllum commune: An analysis of specific properties

Several properties of transformation in the basidiomycete,Schizophyllum commune, were examined. The transformation efficiency of protoplasts made from germinating basidiospores is dependent upon the length of time that the spores are incubated under conditions that promote germination. Protoplasts prepared from ungerminated spores transform at least 10 times more efficiently than protoplasts prepared from germlings (25 μm in length) or from mycelium. Transformation frequencies of 1000 transformants/μg of control plasmid DNA and 10⁷ protoplasts are sufficient for obtaining transformants with 2 × 10⁷ protoplasts and 10 μg of bank DNA from a genomic plasmid library. The probability of cotransforming with two plasmids is dependent on the DNA concentrations of each; concentrations can be adjusted to yield nearly 100% cotrasformants. The presence of a nonselected plasmid in the reaction mix improves the transformation frequency of a selected marker carried on another plasmid; this is not true if linear fragments ofSchizophyllum genomic DNA are used as the nonselected DNA. Transformation of aSchizophyllum protoplast does not require its fusion to another protoplast.     

A new protoplast culture system in Daucus carota L. and its applications for mutant selection and transformation

Petiole protoplasts from in vitro-grown carrot plants are a very good alternative to traditionally obtained protoplasts from suspension cultures. High plating and regeneration efficiencies were obtained in most of the breeding lines that were tested. The embedding of the protoplasts in alginate was crucial for initiating cell division and further development. Several streptomycin resistant and chlorophyll-deficient plant lines were selected for using the petiole protoplast system. Maternally inherited streptomycin resistance was demonstrated by sexual crosses. Protoplast fusion of several chlorophyll-deficient lines did not result in complementation, indicating the cytoplasmic nature of the mutations. Petiole protoplasts were used for direct transformation with plasmid DNA pNUNV containing NPTII as a selectable marker. High transformation frequencies (up to 1%) were obtained after PEG treatment of the protoplasts. Kanamycin resistance was shown to be inherited as a single dominant nuclear trait.     

Isolation and Characterization of Cytotoxic Compounds from Corn

A host-vector system was constructed in Bacillus megaterium strain NK84–0128, an oxetanocin A producer. The replication origin of an endogeneous plasmid, P–4, was used to construct a potential plasmid vector, pSM5, which had a chloramphenicol resistance gene as a selective marker. Plasmid transformation by a protoplast method was used in B. megaterium strain NK84–0128. The maximum transformation frequency attained with the pSM5 plasmid was 2.0 x 10⁴cfu/µg DNA.     

Stability and modulated expression of a hygromycin resistance gene integrated in Botrytis cinerea transformants

When transformation of Botrytis cinerea occurred in mononucleated protoplasts the hygromycin resistance phenotype was stable and integrated plasmid DNA although rearranged was transmitted through meiosis. We observed that transformants were often heterokaryotic and using serial conidial transfer, we showed failure of expression of the entire copies of integrated plasmids in some conidial isolates. A non-Mendelian segregation of the hygromycin resistance phenotype was observed in most crosses between these transformants and sensitive strains. However, a 1:1 segregation ratio of plasmid DNA hybridisation was observed. Mechanisms of gene silencing in B. cinerea, in both the asexual and the sexual cycle, are discussed.     

A comparative study on the transformation of Aspergillus nidulans by microprojectile bombardment of conidia and a more conventional procedure using protoplasts treated with polyethyleneglycol

 An Aspergillus nidulans strain, auxotrophic for pyrimidine, was transformed to prototrophy by means of microprojectile bombardment. The transformation frequency was somewhat lower than conventional polyethyleneglycol-mediated transformation of protoplasts. However, the percentage of stable transformants was considerably higher with the biolistic approach. Typically, integrations of several copies of the plasmid introduced into chromosomal DNA were observed. The effect of several parameters, like the concentration of conidia, chamber pressure during bombardment and size of microprojectiles, on transformation frequencies were investigated and compared to previously published data on microprojectile bombardment of fungal conidia. Optimum results (6 transformants/μg plasmid DNA) were obtained when 10⁸ conidia were bombarded with a helium pressure of 5.5–8.3 MPa (800–1200 lb/in²). M5, M10 and M17 tungsten particles were equally efficient. Received: 9 August 1995/Received revision: 27 September 1995/Accepted: 4 October 1995     

Evaluation of selectable markers for obtaining stable transformants in the gramineae

Cell suspension cultures of Triticum monococcum, Panicum maximum, Saccharum officinarum, Pennisetum americanum, and a double cross trispecific hybrid between Pennisetum americanum, P. purpureum, and P. squamulatum were tested for resistance to kanamycin, hygromycin, and methotrexate for use in transformation studies. All cultures showed high natural levels of resistance to kanamycin, in excess of 800 milligrams per liter, and variable levels of resistance to hygromycin. Methotrexate was a potent growth inhibitor at low concentrations with all species. Kanamycin and hygromycin were growth inhibitory only if added early (within 5 days after protoplast isolation and culture). Protoplasts of T. monococcum, P. maximum, S. officinarum, and the tri-specific hybrid were electroporated with plasmid DNA containing hygromycin (pMON410), kanamycin (pMON273), or methotrexate (pMON806) resistance genes. Resistant colonies were obtained at low frequencies (1 × 10⁻⁵ to 2 × 10⁻⁶) when selected under conditions which were growth inhibitory to protoplasts electroporated without DNA. Southern blot hybridization confirmed stable integration of plasmid DNA into T. monococcum using hygromycin vectors and P. maximum using the methotrexate vector with 1 to 10 copies integrated per haploid genome.     

Efficient protoplast isolation and transient gene expression system for <Emphasis Type="Italic">Phalaenopsis</Emphasis> hybrid cultivar ‘Ruili Beauty’

With the release of the Phalaenopsis equestris (Schauer) Rchb.f. genome database, more in-depth studies of Phalaenopsis spp. will be carried out in the future. Transient gene expression in protoplasts is a useful system for gene function analysis, which is especially true for Phalaenopsis, whose stable genetic transformation is difficult and extremely time-consuming. In this study, juvenile leaves from aseptic Phalaenopsis seedlings were used as the starting material for protoplast isolation. After protocol refinement, the highest yield of viable protoplasts [5.94 × 10⁶ protoplasts g^?1 fresh weight (FW)] was achieved with 1.0% (w/v) Cellulase Onozuka R-10, 0.7% (w/v) Macerozyme R-10, and 0.4 M D-mannitol, with an enzymolysis duration of 6 h. As indicated by transient expression of green fluorescent protein (GFP), a transformation efficiency of 41.7% was achieved with 20% (w/v) polyethylene glycol (PEG-4000), 20 μg plasmid DNA, 2 × 10⁵ mL^?1 protoplasts, and a transfection duration of 30 min. The protocol established here will be valuable for functional studies of Phalaenopsis genes.     

Evidence for functional hemizygosity at the Emtr locus in CHO cells through segregation analysis

The hypothesis of functional hemizygosity at the emetine-resistant (Emt^r, a non-X-linked recessive marker) locus in Chinese hamster ovary (CHO) cells has been examined by segregation analysis. The frequencies and the rates of segregation of the Emt^r and Thg^r (thioguanine-resistant, an X-linked recessive mutation) markers were determined from hybrids constructed between an Emt^r-Thg^r CHO cell line and various other Chinese hamster lines (V79, M3-1, CHO, GM7S, CHW and CHL). Thg^r segregants were obtained at similar frequencies (10^?2–10^?3) from all the hybrids. The frequency of segregation of the Emt^r marker, however, was similar to that of Thg^r only in the CHO × CHO hybrids and was much lower (10^?4–10^?6) than the CHO × other Chinese hamster hybrids. Similar results were obtained when the segregation rates for the two markers from various hybrids were determined. These results are consistent with the hypothesis that in CHO cells, the gene responsible for Emt^r is present in a single (functional) copy, whereas two copies of this gene are present in other Chinese hamster lines examined.     

Heterologous transformation of Agrocybe aegerita with a bacterial neomycin-resistance gene fused to a fungal promoter-like DNA sequence

DNA sequences of the basidiomycete Agrocybe aegerita were cloned in E. coli based on their ability to drive the expression of the bacterial promoterless tetracycline (Tc)-resistance gene. A 0.48% frequency of the cloned sequences promoted antibiotic-resistance. The sequence conferring the highest Tc resistance (40 μg/ml) was selected to drive the expression in E. coli of two other promoterless genes encoding chloramphenicol and neomycin resistance. One of the derivative vectors, pN13-A2, carrying a chimeric neomycin-resistance gene, was used to transform an A. aegerita neomycin-sensitive strain by protoplast electroporation. Transformation frequencies ranged from 1 to 2.8 transformants per μg of DNA per 10³ viable cells, in a relatively high background of spontaneous-resistant colonies (2% of the surviving protoplasts). Molecular analyses showed that transformation had occurred by the integration of pN13-A2 sequences, either ectopically or at the resident locus carrying the A. aegerita promoter-like sequence, with probable molecular rearrangements. The nucleotide sequence of the promoter-like fragment revealed the presence of a CT motif that is known to be involved in a promoter function in some highly expressed genes of filamentous fungi.     

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation¹. Due to its capacity to undergo efficient mitotic homologous recombination, Physcomitrella patens has emerged as an important model system in recent years². This capacity allows high frequencies of gene targeting^3-9, which is not seen in other model plants such as Arabidopsis. To take full advantage of this system, we need an effective and easy method to deliver DNA into moss cells. The most common ways to transform this moss are particle bombardment¹⁰ and PEG-mediated DNA uptake¹¹. Although particle bombardment can produce a high transformation efficiency¹², gene guns are not readily available to many laboratories and the protocol is difficult to standardize. On the other hand, PEG mediated transformation does not require specialized equipments, and can be performed in any laboratory with a sterile hood. Here, we show a simple and highly efficient method for transformation of moss protoplasts. This method can generate more than 120 transient transformants per microgram of DNA, which is an improvement from the most efficient protocol previously reported¹³. Because of its simplicity, efficiency, and reproducibility, this method can be applied to projects requiring large number of transformants as well as for routine transformation.     

Single-stranded DNA used as an efficient new vehicle for transformation of plant protoplasts

In relation to the question which DNA form (single- or double-stranded) is transferred by Agrobacterium tumefaciens to plant cells, we studied the behaviour of single-stranded DNA, as compared to double-stranded DNA, when it is introduced into plant protoplasts by electroporation. To this end, we cloned a construct with a plant NPTII gene as well as a CAT gene in the M13 vectors tg130 and tg131. We found that both complementary single-stranded molecules gave rise to substantial CAT activity in plant protoplasts, suggesting that single-stranded DNA is converted into double-stranded DNA by the plant cell replication machinery. Unexpectedly, we found that single-stranded DNA leads to a 3–10 fold higher frequency of stable transformation (selection for kanamycin resistance) than double-stranded DNA. These results indicate that the use of single-stranded DNA might be considered in experiments in which optimal transformation frequencies are needed, e.g. with protoplasts form recalcitrant plant species.Abbreviations ss single-stranded - ds double-stranded - CAT chloramphenicol acetyl transferase - NPTII neomycin phosphotransferase II - RT room temperature     

A polyethylene glycol-mediated protoplast transformation system for production of fertile transgenic rice plants

We have established an efficient procedure for protoplast transformation and regeneration of fertile transgenic plants of rice (Oryza sativa L.) cultivars Nipponbare and Taipei 309. Protoplasts were mixed with a plant-expressible hygromycin resistance gene and treated with 25% (w/v) polyethylene glycol. Stringent selection of transformed colonies was applied to 14-day-old regenerated protoplasts in the presence of 95 micromolar of hygromycin B for 12 days. After selection, 450 and 200 resistant colonies were recovered per million treated Taipei 309 and Nipponbare protoplasts, respectively. Southern hybridization analysis of hygromycin-resistant cell lines and regenerated plants indicated that 1 to 10 copies of transferred DNA were integrated at 1 to 4 loci of the rice genome. Southern DNA analysis suggests that the introduced plasmid DNA may form concatemers by intermolecular recombination prior to integration. Four Taipei 309 and 39 Nipponbare transgenic rice plants were regenerated and grown to maturity in the greenhouse. Two Taipei 309 and 35 Nipponbare plants set viable seeds. Agronomic traits of Taipei 309 transgenic plants and inheritance of the hygromycin resistance trait by progeny of the selfed transgenic plants were analyzed.     

High frequency callus formation from maize protoplasts

Summary A solid feeder layer technique was developed to improve callus formation of Black Mexican Sweet maize (Zea mays L.) suspension culture protoplasts. Protoplasts were plated in 0.2 ml liquid media onto a cellulose nitrate filter on top of agarose-solidified media in which Black Mexican Sweet suspension feeder cells were embedded. Callus colony formation frequencies exceeding 10% of the plated protoplasts were obtained for densities of 10³–10⁵ protoplasts/ 0.2 ml, which was 100- to 1,000-fold higher than colony formation frequencies obtained for conventional protoplast plating methods such as liquid culture or embedding in agarose media. Compared with conventional methods, the feeder layer method gave higher colony formation frequencies for three independently maintained Black Mexican Sweet suspension lines. Differences among the three lines indicated that colony formation frequencies might also be influenced by the suspension culture maintenance regime and length of time on different 2,4-dichlorophenoxyacetic acid concentrations. The callus colony formation frequency reported is an essential prerequesite for recovering rare mutants or genetically transformed maize protoplasts.     

Protoplast isolation and genetically true-to-type plant regeneration from leaf- and callus-derived protoplasts of Albizia julibrissin

Protoplast isolation and subsequent plant regeneration of Albizia julibrissin was achieved from leaf and callus explants. Leaf tissue from 4 to 5-week-old in vitro seedlings was the best source for high-yield protoplast isolation. This approach produced 7.77?×?10⁵ protoplasts (Pp) per gram fresh weight with 94?% viability; after 60 min pre-plasmolysis with 0.7 M sorbitol followed by digestion in a solution of cell and protoplast wash plus 0.7 M mannitol, 1.5?% cellulase Onozuka R10, and 1?% pectolyase Y-23 for 6 h. Liquid Kao and Michayluk medium containing 2.7 μM α-naphthaleneacetic acid (NAA) and 2.2 μM 6-benzylaminopurine (BA) was best for sustained cell division and microcolony formation from both leaf- and callus-derived protoplasts at a density of 3–5?×?10⁵ Pp ml^?1. Protoplast-derived microcalli became visible after 3–4 weeks on semi-solid medium of the same composition. Microcalli were then cultured on Murashige and Skoog (MS) medium containing Gamborg B5 vitamins or woody plant medium supplemented with different concentrations of NAA plus 4.4 μM BA for further growth. Proliferated leaf- and callus-protoplast-derived calli differentiated into microshoots on MS medium containing 13.2 μM BA plus 4.6 μM zeatin after 2–3 weeks, with an overall shoot organogenesis efficiency of 78–93?%. Rooting of microshoots on half-strength MS medium containing 4.9 µM indole-3-butyric acid was successful, and plantlets were acclimatized to the greenhouse with a survival rate of >62?%. Using ten start codon targeted and ten inter-simple sequence repeat primers, the genetic integrity of nine leaf- and six callus-protoplast-based plants was validated along with the mother seedlings.     

Beauveria bassiana protoplast regeneration and transformation using electroporation

Summary Regeneration of Beauveria bassiana GK2016 protoplasts demonstrated three phases: (1) enlargement, (2) formation of a chain of budding cells, and (3) development of a true germ tube from the original protoplast. Regeneration frequencies of up to 80% were obtained when using the stabilizer ammonium sulphate. Using electroporation, protoplasts were transformed to methyl 1,2-benzimidazole carbamate (MBC) resistance with the Neurospora crassa MBC-resistant -tubulin gene. A transformation efficiency of one of three transformants per 5 g vector DNA was obtained. The MBC phenotype was stable and transformants grew in the presence of 5 g MBC/ml. Southern DNA-DNA hybridization analysis demonstrated that integration of the vector into the chromosomal DNA had occurred. Correspondence to: G. G. Khachatourians     

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.     

DNA-mediated transformation system in a bipolar basidiomycete, <Emphasis Type="Italic">Pholiota microspora</Emphasis> (<Emphasis Type="Italic">P. nameko</Emphasis>)

We cloned a gene encoding the succinate dehydrogenase iron-sulfur protein subunit (sip) from a bipolar mushroom, Pholiota microspora, and introduced a point mutation that confers carboxin resistance into this gene. Using this homologous selective marker and also a heterologous drug selective marker, the hygromycin B phosphotransferase gene (hph), we successfully constructed a DNA-mediated transformation system in P. microspora. Both these selection markers have high transformation efficiency: the efficiency of carboxin resistance transformation was about 88.8 transformants/μg pMBsip2 DNA using 5 × 10⁶ protoplasts in regeneration plates containing 1.0 μg/ml carboxin, and the efficiency of hygromycin B resistance transformation was about 122.4 transformants/μg pMBhph1 DNA using 5 × 10⁶ protoplasts in regeneration plates containing 150 μg/ml hygromycin B. Southern hybridization analysis showed that the introduced sequence (mutant sip or hph) was integrated into the chromosomal DNA in these transformants with a copy number of one or more.