Hybrid genes in the analysis of transformation conditions

Direct gene transfer into plant protoplasts has been recently developed, and conditions for high frequency transformation of SR₁ tobacco protoplasts established. In this paper we analyse numerous transformation parameters in a comparative study on SR₁ Nicotiana tabacum and N. plumbaginifolia, and report on a simple chemical technique for very efficient protoplast transformation. It is based on the synergistic interaction of MgCl₂ and PEG. The technique yielded up to 1400 transformants per 3×10⁵ treated N. tabacum protoplasts (up to 4.8% of the survivors, late selected clones). Using N. plumbaginifolia, the frequencies were 10-fold lower, indicating that the competence for transformation has a species-specific component.Abbreviations PEG polyethyleneglycol - RTF relative transformation frequency - ATF absolute transformation frequency     

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     

Investigation of novel oligoelectrolyte polymer carriers for their capacity of DNA delivery into plant cells

Development of modern agriculture and biotechnology is closely connected with the use of novel and effective genetic engineering methods. Presently, non-viral nanoparticle-mediated plant transformation methods gain more attention because of their stability, safety, and convenience of performance. In this work, new polymeric dimethylaminoethyl metacrylate (DMAEM)-based polymers were synthesized and investigated for their properties in gene delivery. Formation of stable complexes between TN 83/6, TN 84/5, DLM-9-DM and LM-8-DM polymers and plasmid DNA, as well as the DNA protection by the PDMAEM polymers against nuclease degradation were confirmed by electrophoresis in agarose gel. In addition, model organisms Allium cepa and Nicotiana tabacum L. were studied to evaluate cytotoxic effect of the PDMAEM carriers. The created PDMAEM-based carriers were effective in delivery of plasmid DNA into moss and tabacco protoplasts (obtaining stable transformants of Ceratodon purpureus moss, as well as in transient expression of the reporter yfp gene product in N. tabacum protoplasts). Thus, novel PDMAEM-based polymers were shown to be promising carriers for delivery of DNA into plant cells, and carriers possess high potential for further applications in this field.     

Gene transfer by electroporation in betulaceae protoplasts: Alnus incana

The Escherichia coli -glucuronidase gene (GUS) was introduced into Alnus incana (L.) Moench protoplasts by electroporation. Level of GUS transient gene expression was increased by increasing DNA concentrations of pBI 221 plasmid and was affected by the amplitude and duration of the applied electric pulse as well as by the presence of polyethylene glycol (PEG) in the electroporation medium. An optimal level of GUS activity was obtained after electroporation with a capacitive discharge of 500 V/cm and 71 ms-duration. This transformation procedure is simple and efficient. These results motivated us to investigate this method as a possible way of achieving the stable transformation of actinorhizal alder.Abbreviations CaMV cauliflower mosaic virus - CPW salts, Cocking-Power-White salts - kb kilobase(s) - MU 4-methyl umbelliferyl - MUG 4-methyl umbelliferyl glucuronide - F microfarad(s) - NOS nopaline synthase     

Direct gene transfer to cells of a graminaceous monocot

Summary Definitive evidence is presented for the first time for stable gene transfer to cultured cells in a plant of the family Gramineae, Lolium multiflorum (Italian Ryegrass), using DNA transformation of protoplasts from a non-morphogenic cell culture. A construction consisting of expression signals from gene VI of Cauliflower Mosaic virus joined to the aminoglycoside (neomycin) phosphotransferase gene (APH(3)II) from transposon Tn5 conferred resistance to the antibiotic G-418 to cell colonies arising from transformed protoplasts. By demonstrating a tight correlation between the resistant phenotype, the physical presence of the foreign gene and the presence of the active gene product we have shown that these colonies are true transformants and that a gene which is expressed well in dicotyle-denous plants is also expressed in cells of graminaceous monocots.Dedicated to professor Georg Melchers to celebrate his 50-year association with the journal     

Stable transformation of maize: the impact of feeder cells on protoplast growth and transformation efficiency

Summary The importance of cell culture conditions, including the use of feeder cells, on protoplast growth and transformation in maize (Zea mays L.) was investigated. Total GUS activity, measured two days after transformation, was five-fold higher in protoplasts cultured on feeder cells compared to those grown in the absence of feeder cells. Since the specific activity of GUS was only slightly higher in the transformed protoplasts plated over feeder cells, the stimulation in transient gene expression resulted mainly from the improved environment provided by the feeder system. For stable transformation, either PEG treatment or electroporation of protoplasts was used to introduce the neo gene. When PEG was used, over 85% of the putative transformants (resistant to kanamycin) contained the neo gene. The combination of PEG transformation and the optimized cell culture protocol using feeder cells enabled the selection of about 100 stably transformed lines per gFW of cells. Electroporation was less efficient.     

Potato granule-bound starch synthase promoter-controlled GUS expression: regulation of expression after transient and stable transformation

Chimaeric genes of promoter sequences from the potato gene encoding granule-bound starch synthase (GBSS) and the -glucuronidase (GUS) reporter gene were used to study GBSS expression and regulation. Analysis of stable transformants revealed that a GBSS promoter sequence of 0.4 kb was sufficient to result in tissue-dependent GUS expression: levels in stably transformed microtubers exceeded levels in corresponding leaves by orders of magnitude. GBSS-GUS constructs could be transiently expressed in leaf protoplasts from wild-type and amylose-free potato lines, etuberosumSolanum brevidens, Nicotiana tabacum andArabidopsis thaliana. Transient expression levels in potato leaf protoplasts were clearly lower than in corresponding suspension cell protoplasts. This lower expression in leaf protoplasts could not be elevated by increasing DNA concentrations during transfection. Light incubation of electroporated suspension cell protoplasts reduced transient GBSS-GUS expression, whereas incubation of transfected protoplasts in media with different sucrose concentrations did not affect transient expression levels. However, electroporated protoplasts, isolated from suspensions, which had been grown on media with increasing amounts of sucrose showed a sucrose concentration-dependent transient expression profile. This indicates that studying GBSS regulation by transient expression experiments needs pre-treatment of the protoplast source. Sequence data of the GBSS promoter were compared to those of two other potato alleles.     

Targeted integrative transformation of Candida tropicalis by electroporation

Summary A method for integrative transformation of the diploid yeast Candida tropicalis by electroporation has been developed. By linearizing the transforming plasmid DNA containing the URA3 gene prior to electroporation of recipient cells, its integration was targeted to a specific locus in the genome, resulting in single or multiple tandem integrations. The optimal electroporation conditions for this yeast were established and include an electric pulse of 2.25 kV/cm for a duration of 50 ms. Using these conditions, Ura⁺ transformants were readily obtained at a high frequency (45 transformants/g DNA) as the result of targeted integration of the URA3 gene containing plasmid DNA at the chromosomal ura3 locus. The number of transformants resulting from this procedure is comparable to that achieved with a recently reported spheroplast transformation procedure for C. tropicalis; in addition, it offers the advantages of being simple, rapid and reproducible.     

Transformation of wheat (Triticum aestivum L.) through electroporation of protoplasts

Protoplasts isolated from embryogenic suspension cultures of wheat (Triticum aestivum cv. Hartog) were electroporated in the presence of plasmid pEmuGN and/or pEmuPAT, which contained the reporter gene gus and selectable marker gene bar, respectively. Under optimised electroporation conditions, up to 0.9% of viable protoplasts displayed gus activity two days after electroporation. To select for phosphinothricin (PPT) resistant colonies, electroporated protoplasts were incubated for six weeks in a medium containing 10 g/ml PPT. The cells surviving the selection were maintained as individual colonies on solid medium or as suspension cultures. More than 60% of these colonies exhibited tolerance to 40 g/ml PPT when tested 10 months after initial selection. To date, 57 green plants have been regenerated from these colonies and 24 have been transferred to soil. Southern blot analyses of colonies and plants, using the bar gene sequence as the probe, confirmed transformation of the cells. Positive PAT assays of both regenerated colonies and plants indicated the presence of the bar gene product. These results provide a basis for the establishment of routine procedures for transformation of wheat by direct gene transfer into protoplasts.Abbreviations gus -glucuronidase - PAT phosphinothricin N-acetyltransferase - PPT phosphinothricin - MS Murashige and Skoog medium     

High level of GUS gene expression driven by pollen-specific promoters in electroporated lily pollen protoplasts

Gene constructs that contained the -glucuronidase (GUS) gene under the control of a pollen-specific Zm13 promoter from maize and a LAT52 promoter from tomato were introduced by electroporation into pollen protoplasts isolated from bicellular pollen grains of Lilium longiflorum. After 20 h in culture, the pollen protoplasts exhibited the apparent expression of GUS in a fluorometric assay. The GUS activity induced under the control of the Zm13 promoter was over 10 000 times higher than activity in the control (with no DNA or without electroporation). By contrast, the GUS gene was nearly silent in the lily microspore protoplasts and generative cell protoplasts. The GUS activity driven by the Zm13 and LAT52 promoters was also detected by a cytochemical assay. The frequency of blue-staining pollen protoplasts was about 70% in the case of the Zm13 promoter. The efficiency of gene transfer by electroporation was much higher than by particle bombardment. This protoplast-specific electroporation system is suitable for rapid and reliable examination of pollen-specific promoters, being as good as the particle bombardment system.     

PEG- and electroporation-induced transformation in Nicotiana tabacum: influence of genotype on transformation frequencies

Summary Experimental parameters for direct gene transfer with recombinant DNA encoding neomycin phosphotransferase II (NPTII) under control of eukaryotic expression signals were established. The introduced gene was shown by the growth of transformants on media containing kanamycin, by genomic blotting and by assaying NPTII activity. Leaf protoplasts from three green genotypes of varieties xanthii and petit havanna, and from four plastome-encoded albino genotypes of Nicotiana tabacum were analyzed with respect to cell division kinetics and yield of kanamycin-tolerant colonies after direct gene transfer. No clear correlation was found between the time of onset of cell division and transformation frequency.     

Electroporation of rapeseed protoplasts – transient and stable transformation

Protoplasts of Brassica napus hypocotyls were transfected using electroporation. Parameters such as discharge potential, protoplast density and buffer constituents were tested to determine the most suitable conditions for gene transfer. To monitor the introduction of DNA into protoplasts a plasmid containing the β-glucuronidase (EC 3.2.1.31), and the neomycin phospotransferase (EC 2.7.1.95) genes was used. By using this construct, expression of a screenable marker gene for transient expression analysis as well as an antibiotic resistance marker gene for selection of stable transformants were obtained. Refined electroporation conditions resulted in a frequency of 0.1% transiently transformed protoplasts. Microcalluses were cultured under selective conditions in a bead-type culture system. Resistant callus, with an absolute transformation frequency of 4.9 × 10⁻⁵ and a relative transformation frequency of 0.3% could be achieved. X-ray irradiation of newly electroporated protoplasts did not enhance absolute transformation frequencies. From some of the resistant calluses, transgenic plants could be regenerated which were characterized by molecular analysis.     

Electric field mediated stable transformation of carrot protoplasts with naked DNA

We have developed an electroporation procedure for the transformation of carrot protoplasts with Ti-plasmid DNA from Agrobacterium tumefaciens. The uptake of pTiC58 into carrot protoplasts was mediated by high voltage electrical pulses at field strengths from 0.5 to 3.8 kV/cm. Protoplast regeneration, somatic embryogenesis and plantlet regeneration were unaffected by the electroporation conditions selected for DNA uptake. Uptake of plasmid pTiC58 resulted in hormone independent regeneration of carrot protoplasts. Transformed somatic embryos were detected in carrot cultures 45 days after electroporation. The transformed somatic embryos developed into teratomas which synthesized nopaline. Hybridization was obtained between a labeled T-DNA fragment from pTiC58 and DNA fragments from 4 month old teratomas regenerated from electro-transformed protoplasts. Based on the number of somatic embryos regenerated after electro-transformation, a frequency of 1.6×10² transformants/10⁴ somatic embryos/g pTiC58 DNA was obtained.Abbreviations PEG polyethylene glycol - 2,4-D 2,4-dichlorophenoxyacetic acid - MES morpholinoethane sulfonic acid - DMSO dimethyl sulfoxide - HSV Herpes Simplex virus - TK thymidine kinase     

pEmu: an improved promoter for gene expression in cereal cells

Summary A recombinant promoter, pEmu, has been constructed to give a high level of gene expression in monocots. It is based on a truncated maize Adh1 promoter, with multiple copies of the Anaerobic Responsive Element from the maize Adh1 gene and ocs-elements from the octopine synthase gene of Agrobacterium tumefaciens. The pEmu promoter was one of 12 different promoter constructs that were linked to the -glucuronidase (GUS) marker gene. Promoter activity was measured 48 h after introduction of the constructs into protoplasts of five different monocot species [wheat, maize, rice, einkorn (Triticum monococcum), and Lolium multiflorum] and one dicot (Nicotiana plumbaginifolia). In suspension cell protoplasts, the most highly expressing construct (pEmuGN) gave 10- to 50-fold higher expression than the CaMV 35S promoter in all the monocot species. The pEmu promoter should be valuable where a high level of gene expression is required in monocots. The pEmu promoter showed instability in several widely used Escherichia coli strains but was stable in a recA, recD strain AC001, which is described. Another construct, p4OCS35SIGN, gave a tenfold increase in expression over the CaMV 35S promoter in dicot (Nicotiana plumbaginifolia) protoplasts.     

Expression of a foreign gene in electroporated pollen grains of tobacco

Summary The incorporation of genetically engineered DNA into pollen and subsequent fertilization of eggs by the transformed pollen would be a convenient method for producing genetically engineered seed. This method of pollen transformation would circumvent the need for other types of gene transfer methods such as the use of Agrobacterium tumefaciens, which has a limited host range and thus a limited capability for genetically engineering plants. It would also avoid the problems associated with the regeneration of some plants from tissue, cell, or protoplast culture after receiving foreign DNA. To this end, the genetically engineered plasmid DNA vector pBI221 containing the gene encoding -glucuronidase (GUS) was introduced by electroporation into germinating pollen grains of tobacco (Nicotiana gossei L.). Transient expression of the GUS gene was demonstrated by the presence of GUS activity in fluorometric assays of pollen extracts 24 h after the introduction of pBI221 via electroporation. Intact pBI221 was detected by Southern blotting procedures as a distinct DNA band in pollen extracts 1 h after electroporation. In addition, pBI221 was detected as a diffuse band of higher molecular weight DNA 24 h after electroporation, suggesting that some of the pBI221 was incorporated into the genome of the pollen.     

Tissue-specific expression of the rolC promoter of the Ri plasmid in transgenic rice plants

Summary Transgenic rice plants were obtained from protoplasts treated with two plasmids by electroporation. Primary transformants were selected on the basis of resistance to hygromycin, conferred by one of the co-transferred plasmids. Out of 26 hygromycin-resistant plants 2 showed the reporter gene activity due to another plasmid possessing a chimeric gene consisting of the promoter (about 900 by upstream non-coding region) of the ORF12 gene (roIC of the Ri plasmid and the coding region for -glucuronidase (GUS). Using a colorimetric reaction, the GUS enzyme was found to be localized in vascular tissues, demonstrating the similar expression of the roIC gene promoter in monocots and dicots (Sugaya et al. 1989; Schmülling et al. 1989).     

Regeneration of transformed shoots from electroporated soybean (Glycine max (L.) Merr.) protoplasts

Stable transformation of soybean (Glycine max (L.) Merr.) protoplasts isolated from immature cotyledons was achieved following electroporation with plasmid DNA carrying chimeric genes encoding ß-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transformed colonies were stringently selected by growing 15-day-old protoplast-derived cells in the presence of 40 g/ml of hygromycin-B for 6 weeks. Over 93% of the resistant cells and colonies exhibited GUS activity, indicating that the two marker genes borne on a single plasmid were co-introduced and co-expressed at a very high freguency. This transformation procedure reproducibly yields transformants at frequencies of 2.9–6.8 × 10^–4 (based on the number of protoplasts electroporated) or 23.0% (based on the number of control microcalli formed) counted after 6 weeks of selection. After repeated subculturing on regeneration medium, shoots were induced from 8.0% of the transformed calli. Southern hybridization confirmed the presence of both the GUS and hygromycin genes in the transformed calli and shoots.     

Transient gene expression in electroporated Solanum protoplasts

Electroporation was used to evaluate parameters important in transient gene expression in potato protoplasts. The protoplasts were from leaves of wild potato Solanum brevidens, and from leaves, tubers and suspension cells of cultivated Solanum tuberosum cv. Désirée. Reporter enzyme activity, chloramphenicol acetyl transferase (CAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, depended on the field strength and the pulse duration used for electroporation. Using field pulses of 85 ms duration, the optimum field strengths for maximum CAT activity were: S. brevidens mesophyll protoplasts –250 V/cm; Désirée mesophyll protoplasts –225 V/cm; Désirée suspension culture protoplasts –225 V/cm; and Désirée tuber protoplasts –150 V/cm. The optimum field strengths correlated inversely with the size of the protoplasts electroporated; this is consistent with biophysical theory. In time courses, maximum CAT activity (in Désirée mesophyll protoplasts) occurred 36–48 h after electroporation. Examination at optimised conditions of a chimaeric gene consisting of a class II patatin promoter linked to the -glucuronidase (gus) gene, showed expression (at DNA concentrations between 0–10 pmol/ml) comparable to the CaMV 35S promoter in both tuber and mesophyll protoplasts. At higher DNA concentrations (20–30 pmol/ml) the patatin promoter directed 4–5 times higher levels of gus expression. Implications and potential contributions towards studying gene expression, in particular of homologous genes in potato, are discussed.     

Factors affecting transient gene expression in electroporated Glycine max protoplasts

Electroporation was used to evaluate parameters affecting transient gene expression in Glycine max protoplasts. Protoplast viability and reporter enzyme activity for chloramphenicol acetyl transferase (CAT) and ß-glucuronidase (GUS) depended on the field strength employed. Maximum CAT and GUS activity was obtained when a field strength of 500 V/cm at 1000 F and a protoplast concentration of 1–3 × 10⁶/ml was used. Transformation efficiencies up to approximately 1.6% GUS positive protoplasts were obtained. Transient gene expression increased with increasing plasmid DNA concentration and with the time after electroporation, reaching a maximum after 48 hr. Addition of polyethylene glycol at 5.6% and heat shock (5 rain at 45 °C) given to the protoplasts before adding DNA further enhanced the transformation efficiency. Under the optimized experimental conditions, CAT and GUS activity increased simultaneously, thereby indicating that the increased expression is caused by DNA uptake by more cells rather than greater DNA uptake by the same cells. Our results demonstrate that both GUS and CAT can be used as efficient screenable markers for transformation studies in soybean.Abbreviations CAT chloramphenicol acetyl transferase - GUS ß-glucuronidase - PEG polyethylene glycol