Parameters influencing transient and stable transformation of barley (Hordeum vulgare L.) protoplasts

Cat gene expression has been investigated following PEG-mediated plasmid uptake into barley protoplasts. The uptake conditions optimised for transient expression were employed for stable transformation. Transformed protoplast-derived calli of the cvs. Dissa and Igri, were selected on medium containing G418 at 40 g ml^–1 or kanamycin sulphate at 250 g ml^–1. Absolute transformation frequencies of 28.9×10^–5 and 21.3×10^–5 were recorded for Dissa with kanamycin sulphate and G418 selection, respectively. The frequency for Igri was 11.5×10^–5 with G418 selection. Antibiotic resistant protoplast-derived colonies expressed NPTII activity; Southern hybridisation confirmed integration of the nptII gene into barley genomic DNA.Abbreviations ABA abscisic acid - AC-CAP acetylated chloramphenicol - BAP 6-benzylaminopurine - cat chloramphenicol acetyltransferase gene - CAT chloramphenicol acetyltransferase activity - CaMV cauliflower mosaic virus - CAP chloramphenicol, 2,4-d-2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - G418 Geneticin - gus -glucuronidase gene - HEPES (N[2-hydroxyethyl] piperazine-N-[2-ethanesulphonic acid]) - IAA indole acetic acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid - npt II neomycin phosphotransferase gene - NPTH neomycin phosphotransferase activity - PEG polyethylene glycol - SCV settled cell volume     

Transformed calli obtained by direct gene transfer into sunflower protoplasts

Helianthus annuus protoplasts were transformed with the plasmid pCaMVNEO (Frommet al. 1986) conferring kanamycin resistance to plant. Transformed calli were selected with a frequency of 4 calli for 10⁶ treated protoplasts. DNA was extracted from kanamycin resistant calli. Analysis of this DNA shows the presence of the NPTII gene.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4 dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - NAA 1-naphtalenoacetic acid - NPT Neomycin phosphotransferase - PEG Polyethyleneglycol     

Expression of a chimaeric kanamycin resistance gene introduced into the wild soybeanGlycine canescens using a cointegrate Ri plasmid vector

Seedling hypocotyl explants ofGlycine canescens were inoculated withAgrobacterium rhizogenes carrying a chimaeric NPTII gene cointegrated into the TL-DNA of pRiA4. Transformed roots produced shoots on B5 based medium with 10.0 mgl^–1 BAP, 0.05 mgl^–1 IBA and 50 gml^–1 kanamycin. Cultured roots and regenerated plants expressed NPTII enzyme activity which was correlated with the presence of Ri TL-DNA and the structural sequence of the NPTII gene.Abbreviations BAP 6-benzylaminopurine - BSA bovine serum albumin - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - IBA indole-butyric acid - PAGE polyacrylamide gel electrophoresis - NPTII neomycin phosphotransferase II - PMSF phenylmethylsulphonyl fluoride - SDS sodium dodecylsulphate     

Regeneration of Transgenic Soybean (Glycine max) Plants from Electroporated Protoplasts

Transgenic soybean (Glycine max [L.] Merr.) plants were regenerated from calli derived from protoplasts electroporated with plasmid DNA-carrying genes for a selectable marker, neomycin phosphotransferase (NPTII), under the control of the cauliflower mosaic virus 35-Svedberg unit promoter, linked with a nonselectable mannityl opine synthesis marker. Following electroporation and culture, the protoplast-derived colonies were subjected to kanamycin selection (50 micrograms per milliliter) beginning on day 15 for 6 weeks. Approximately, 370 to 460 resistant colonies were recovered from 1 × 10⁶ electroporated protoplasts, giving an absolute transformation frequency of 3.7 to 4.6 × 10⁻⁴. More than 80% of the kanamycin-resistant colonies showed NPTII activity, and about 90% of these also synthesized opines. This indicates that the linked marker genes were co-introduced and co-expressed at a very high frequency. Plants were regenerated from the transformed cell lines. Southern blot analysis of the transformed callus and leaf DNA demonstrated the integration of both genes. Single-plant assays performed with different plant parts showed that both shoot and root tissues express NPTII activity and accumulate opines. Experiments with NPTII and mannityl opine synthesis marker genes on separate plasmids resulted in a co-expression rate of 66%. These results indicate that electroporation can be used to introduce both linked and unlinked genes into the soybean to produce transformed plants.     

Stable transformation of papaya via microprojectile bombardment

Summary Stable transformation of papaya (Carica papaya L.) has been achieved following DNA delivery via high velocity microprojectiles. Three types of embryogenic tissues, including immature zygotic embryos, freshly explanted hypocotyl sections, and somatic embryos derived from both, were bombarded with tungsten particles carrying chimeric NPTII and GUS genes. All tissue types were cultured prior to and following bombardment on half-strength MS medium supplemented with 10 mg 1^–1 2,4-D, 400 mg 1^–1 glutamine, and 6% sucrose. Upon transfer to 2,4-D-free medium containing 150 mg 1^–1 kanamycin sulfate, ten putative transgenic isolates produced somatic embryos and five regenerated leafy shoots. Leafy shoots were produced six to nine months following bombardment. Tissues from 13 of these isolates were assayed for NPTII activity, and 10 were positive. Six out of 15 isolates assayed for GUS expression were positive. Three isolates were positive for both NPTII and GUS,Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - X-gluc 5-Br-4-Cl-3-indolyl--D-glucuronic acid - CaMV cauliflower mosaic virus - NOS nopaline synthase - NPTII neomycin phosphotransferase II Journal Series no. 3448 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Fusion of Agrobacterium and E. coli spheroplasts with Nicotiana tabacum protoplasts — Direct gene transfer from microorganism to higher plant

Spheroplasts of Agrobacterium tumefaciens strains and E. coli were fused with protoplasts of Nicotiana tabacum. Fusion products were cultured in the presence of antibiotics to eliminate remaining bacterial spheroplasts. On hormone free medium, tobacco protoplasts treated with wild type Agrobacterium-strains formed colonies with an average frequency of 10^–4. Opine synthesis was detected in the tissues. Some calli derived from protoplasts treated with A. tumefaciens C58C1pRi15834 formed typical hairy roots. Kanamycin resistant calli were obtained after fusion with A. tumefaciens containing pLGVTi23 neo (frequency=10^–3). Fusion of E. coli spheroplasts containing a virulent pTiB6S3::RP4 co-integrate with tobacco protoplasts yielded two hormone independent growing calli producing octopine out of 10⁵ microcalli.Abbreviations PEG Polyethylene glycol - PVA Polyvinyl alcohol     

Physical,chemical and physiological parameters for electroporation-mediated gene delivery into rice protoplasts

Transformation of cereal protoplasts has been reported using several methods; however, the efficiencies of transformations are still very low. We have evaluated a number of parameters that influence electroporation-mediated DNA uptake and have also compared the efficiency of transient GUS activity and stable transformation obtained using an optimized electroporation method with that of the PEG method. The electroporation conditions tested were ionic composition of buffer, ionic strength, resistivity of buffer, type of anions, voltage, and capacitance.Protoplasts isolated from suspension cultures derived from immature embryos of rice (cvs Radon and IR-54) were used for this study. Stable transformation or transient GUS expression experiments were carried out using a plasmid construct containing the CaMV 35S promoter driving thebar gene and a rice actin promoter driving thegus A (uid A) gene (pAG35bar). Electroporation under optimized conditions resulted in about 13-fold higher GUS activities compared to the PEG method. Protoplast survival following optimized electroporation conditions was 55–60%, compared to 35–40% with the PEG treatment. Protoplasts isolated from a suspension culture at different ages gave substantially different levels of transient GUS expression following electroporation-mediated DNA uptake. In contrast, the age of the suspension culture did not influence PEG-mediated DNA uptake and transient GUS activities, which remained low throughout the culture period examined (21 months). Putatively transformed calluses were selected after three to four weeks on medium containing phosphinothricin as the selection agent. The transformation frequencies ranged from 6.2×10^–5 to 5.4×10^–4 with the electroporation method compared to 1.3×10^–5 to 5.3×10^–5 with the PEG method. Southern blot analysis of PPT-resistant calluses obtained by the electroporation-mediated transformation showed simple intergration patterns of integrated DNA in most of the transformants.     

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.     

Requirement for Cell Dispersion Prior to Selection of Induced Azaguanine-Resistant Colonies of Chinese Hamster Cells

With V79 Chinese hamster cell cultures treated with a mutagen, the maximum frequency of colonies resistant to 8-azaguanine (AZG) was attained when the cells were dispersed after a suitable expression time before adding the selection medium. V79–4 cells were exposed to 500 µM MMS, 7 µM AFAA, or 10 µM MNNG and allowed to multiply before being reseeded at 4 x 10⁴ cells/60 mm dish and selected with 10 µg/ml AZG. Maximum frequencies of 4 x 10^-5, 4 x 10^-4, and 2.4 x 10^-3 were obtained about 100, 130, and 200 hrs after exposure to MMS, AFAA, and MNNG, respectively. The maximum frequencies following MMS or MNNG treatments were about 10-fold greater than those obtained when induction and selection of AZG-resistant colonies were performed in the same culture dish. The reseeding of treated cells eliminated the possibility of metabolic cooperation within mosaic colonies of wild-type and mutant cells and achieved expression of the induced changes before intercolony crossfeeding reduced the frequency of resistant colonies.—AZG-resistant colonies were selected in medium containing dialyzed fetal bovine serum, and the selection medium was replaced at least twice. Both serum dialysis and selection medium replacement were necessary for consistent achievement of background frequencies of resistant colonies near 10^-6. Reconstruction experiments with AZG-resistant V79 lines showed that the efficiency of recovery of resistant cells in the selection medium was constant over a range of 0–20 colonies observed/dish. A mixed population of V79 and AZG-resistant cells was also correctly analyzed by the procedure used in mutagenesis studies.     

Isolation of L-ethionine resistant cell lines in Vigna sinensis L. after mutagen treatment

Chemical mutagenesis was employed for the isolation of variant cell lines resistant to L-Ethionine (L-Eth) in Vigna sinensis L. We have measured cell survival after treatment of Vigna sinensis cell suspensions with different mutagens: Ethyl methanesulfonate (EMS), N-methyl-N-nitrosoguanidine (NTG) and Acridine Orange (AO). NTG was more toxic than EMS and AO.L-Eth resistant colonies were isolated by plating on selective medium after NTG treatment. The frequencies of appearance of resistant cells of MS-3 media supplemented with 20 g/ml L-Eth were 1.3 × 10^-6 to 1.8 × 10^-5. The highest number of L-Eth resistant calli were recorded in cells treated with 10 g/ml NTG. Few resistant colonies also appeared spontaneously from non-mutagenized cultures with a frequency of 2.9 × 10^-7 to 5.7 × 10^-7. However, a number of isolated colonies were discarded after successive retesting. The number of resistant calli dropped from 204 to 22 during successive retests. The significance of these observations has also been discussed.     

Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO₃ and 0.5–2 mg/L BA in combination with 0.01–0.05 mg/L 2,4-D or 0.1–1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All R_o transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R₁ seed progeny and showed co-segregation.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - NPTII neomycin phosphotransferase type II - GUS -glucuronidase - CaMV cauliflower mosaic virus - MS Murashige and Skoog - X-Gluc 5-bromo-4-chloro-3-indolyl-D--glucuronic acid - IBA indolebutyric acid - SDS sodium dodecyl sulfate     

Root hair protoplasts ofLotus corniculatus L. (birdsfoot trefoil) express their totipotency

Treatment of the roots of 24–48 h old seedlings of the forage legumeLotus corniculatus with 1.0% Cellulase YC, and 0.1% Pectolyase Y-23 in 4.2% mannitol solution released protoplasts from the tips of root hairs within 30–40 sec of enzyme incubation. Roots from approximately 1000 seedlings yielded 1.7×10⁵ protoplasts. Ten percent of protoplasts divided to form cell colonies when cultured at 1.0×10⁵ ml^–1 in droplets of KM8P medium with 0.6% Sea Plaque agarose. Colonies formed callus on UM agar medium; protoplast-derived tissues produced shoots on B5 medium containing 0.05 mg 1^–1 of BAP. Regenerated plants were phenotypically and cytologically normal (2n=2x=24±2), and produced nitrogen fixing root nodules following inoculation withRhizobium. These results confirm the totipotency of protoplasts isolated from specialised epidermal cells of seedling roots ofLotus corniculatus.     

Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts

Transgenic rice plants have been regenerated by somatic embryogenesis from cell suspension derived protoplasts electroporated with plasmid carrying the NPTII gene under the control of the 35S promoter from cauliflower mosaic virus. Heat shock of protoplasts prior to electroporation maximised the throughput of kanamycin resistant colonies. Omission of kanamycin from the medium for plant regeneration was essential for the recovery of transgenic rice plants carrying the NPTII gene. This report of the production of kanamycin resistant transgenic rice plants establishes the use of protoplasts for rice genetic engineering.Abbreviations NPTII neomycin phosphotransferase - SDS sodium dodecyl sulphate     

Isolation and characterization of streptomycin-resistant mutants in Nicotiana plumbaginifolia

Summary Streptomycin-resistant colonies were isolated from protoplast cultures of haploid Nicotiana plumbaginifolia based on their ability to green in medium containing 1 mg/ml streptomycin sulfate. The frequency of resistant colonies was 0.9×10^–5 in nonmutagenized culture, and increased ten-fold following treatment of culture with 10 g/ml N-methyl-N-nitro-N-nitrosoguanidine. Of a total of 52 resistant clones isolated, 2 gave rise to haploid, 15 to diploid, and 3 to tetraploid plants upon transfer of calli to differentiation medium. Leaf-segment and protoplast assays showed that all diploid regenerates were resistant to streptomycin but sensitive to chloramphenicol, kanamycin, lincomycin, neomycin, and spectinomycin. Plants in most diploid clones were fertile and able to set seeds when self-fertilized and crossed reciprocally to wild-type plants. Inheritance of streptomycin resistance was studied in the diploid clones and, without exception, the resistance was transmitted maternally. Comparative studies of the ultrastructure of organelles and protein synthesis in isolated chloroplasts between wild-type and resistant clones in the presence of streptomycin suggest that streptomycin resistance is controlled by chloroplasts.     

Plant regeneration from callus-derived protoplasts of Pelargonium x domesticum

A protocol for regenerating plants from callus-derived protoplasts of Pelargonium x domesticum (rega l geranium cv. Melissa) has been developed. Protoplasts were isolated from leaf-derived callus tissue on MS medium supplemented with 3.0 mg/l naphthalene acetic acid, 2.0 mg/l 6- benzylaminopurine, and 3.0% sucrose. This callus yielded 2.7×10⁵ protoplasts/gram of tissue after a 6 hr incubation in an enzyme solution consisting of 2.0% cellulysin, 0.5% macerase, and 0.5 M sucrose. Protoplasts were plated at 1×10⁵ protoplasts/ml in a mixture (11 v/v) of KMP8/KP liquid medium layered on the same medium solidified with 0.6% agarose. Protoplast division was initiated within 2 days, and colonies of 15 to 50 cells developed 8 wk after plating. P-calli 1–2 mm³ developed 15 wk after plating, and plants regenerated from the p-calli have been transferred to the greenhouse.Abbreviations NAA naphthaleneacetic acid - 6-BAP 6-benzylaminopurine - CW Calcofluor White - FDA fluorescein diacetate     

Toxicity of antibiotics on zygotic embryos of white spruce (Picea glauca) cultured in vitro

The toxicity of kanamycin, hygromycin B, geneticin, methotrexate and cefotaxime on zygotic embryos ofPicea glauca was studied. Embryos placed on bud induction medium produced approximately 20 adventitious buds per embryo under control conditions. Addition of antibiotics reduced the number of bud-forming embryos. Using the percentage of embryos with buds as an indication of antibiotic toxicity, two-day-old explants were found to be more sensitive than nine-day-old. Kanamycin toxicity was enhanced by cefotaxime and this effect increased with increases in concentration of either antibiotic. Although no morphological difference was observed after 21 days, embryos growing on medium containing 20 g ml^–1 kanamycin showed a decrease of 73% in dry weight and 23% in protein content per embryo when compared to control embryos. Similarly, a decrease of 38% in dry weight and 40% in protein content per embryo was found in embryos on medium containing 300 g ml^–1 cefotaxime.Abbreviations BA 6 benzyl-aminopurine - EDTA ethylenediamine-tetraacetic acid - PVP 10 polyvinylpyrrolidone (MW 10,000) - Tris-HCl Tris [hydroxymethyl] amino hydrochloride NRCC No. 30262     

Meiotic stability of transgene expression is unaffected by flanking matrix-associated regions

DNA repeats are associated with gene instability and silencing phenomena in plants. Therefore, the presence of a direct repeat of matrix-associated region (MAR) DNA, that considerably reduced position effects between independent transformants, may increase (epi)genetic instability. To investigate the influence of such a repeat on the stability of the expression of embedded transgenes, the meiotic stability of transgene expression was assessed in eighteen homozygous 1-locus transgenic tobacco lines carrying the kanamycin resistance (NPTII) and the -glucuronidase (GUS) gene. Half of the lines carry a 3 kb direct repeat of MAR DNA flanking the transgenes. Large progeny populations, totalling over a million seedlings, were screened for kanamycin resistance with the help of a newly developed high-density seedling screen. Kanamycin-sensitive seedlings were detected in selfed progeny at a frequency of 0.5-5.9×10^-4. The frequency became as high as 2×10^-2 when embryo development occurred under heat and/or drought stress. In backcrossed progeny only, a joint loss of NPTII and GUS gene expression was observed at an average frequency of 2.9×10^-5. In selfed and backcrossed progeny we observed similar frequencies of reversion to kanamycin sensitivity, indicating that epigenetic silencing mechanisms rather than MAR repeat-related homologous recombination underlie the reversal to kanamycin sensitivity. Different lines, hence different areas of the tobacco genome, differed in their genetic stability. No significant differences in reversal frequencies were apparent between lines with or without the MAR elements. The use of the MAR repeat is, therefore, not compromised by any increased (epi)genetic instability.     

Plant regeneration from stem cortex protoplasts of Brassica napus

Cell layer strips composed of the epidermis and 7–9 layers of subepidermal cells were isolated from the 3–4 terminal internodes of Brassica napus cv Westar plants at the early flowering stage. The strips were precultured for one day in modified liquid MS [11] medium and subsequently incubated for 17–18 h in a 0.4 M mannitol solution containing 1% Macerozyme and 1% Cellulase Onozuka R-10. Protoplast yield was 2–2.8×10⁶ per 1.0g of tissue. Protoplasts were cultured at 1×10⁵/ml in three different media: S₁ [13], B [12] and L[8]. The first cell divisions occurred after 2–8 days of culture at frequencies of 20–54%. The highest growth rate of colonies was obtained in L medium containing 0.4 M sucrose and 2% Ficoll. After 4 weeks, green calli, 1–2 mm in diameter were transferred onto B₅ [2] medium with 3 mgl^-1 zeatin, 1% sucrose, 0.1 M mannitol and 0.5% agarose for shoot regeneration. Up to 20% of the calli regenerated shoots which subsequently were rooted and established in soil in the greenhouse.     

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     

Wheat protoplast culture: embryogenic colony formation from protoplasts

Wheat (Triticum aestivum L. cv Chinese Spring) protoplasts were isolated from immature embryos or embryogenic calli (3–4 weeks of culture on MS medium with 32 mg/1 dicamba) and cultured in R2 medium containing 2 mg/1 2,4-D by the nurse culture methods originally developed for rice protoplasts (Kyozuka et al. 1987). Protoplasts isolated from embryogenic calli started to divide within 3–5 days and formed colonies at frequencies up to 2% after 3–4 weeks of culture, while protoplasts isolated from immature embryos formed colonies at much lower frequency (less than 0.1%). Some of these colonies were embryogenic, and they appeared at a frequency of approximately 0.5% of colonies formed when callus-derived protoplasts were used. From two of those embryogenic colonies, calli were regenerated and albino shoots and roots were obtained.