Genetic transformation of Brassica nigra by agrobacterium based vector and direct plasmid uptake

Summary Genetic transformation systems have been established for Brassica nigra (cv. IC 257) by using an Agrobacterium binary vector as well as by direct DNA uptake of a plasmid vector. Both the type of vectors carried nptII gene and gus gene. For Agrobacterium mediated transformation, hypocotyl tissue explants were used, and up to 33% of the explants produced calli on selection medium. All of these expressed B-glucuronidase gene on histochemical staining. Protoplasts isolated from hypocotyl tissues of seedlings could be transformed with a plasmid vector by FEG mediated uptake of vector DNA. A number of fertile kanamycin resistant plants were obtained using both the methods, and their transformed nature was confirmed by Southern blot analysis and histochemical staining for GUS. Backcrossed and selfed progenies of these transformed plants showed the presence of npt and gus genes.     

Effect of repeated DNA sequences on direct gene transfer in protoplasts of Nicotiana plumbaginifolia

Summary Highly repeated nuclear DNA sequences from leaves of Nicotiana plumbaginifolia were cloned in pBR322 and tested for their effect on direct gene transfer in protoplasts of the same organism. Protoplasts were prepared from suspension cultures and were incubated in the presence of the plasmid pHP23 carrying the kanamycin resistance gene APH(3)II and in the presence of the plasmids carrying the cloned sequence. DNA uptake was induced by a polyethyleneglycol (PEG) treatment. Out of the 22 tested clones, 3 significantly stimulated the frequency of appearance of transformed colonies. DNA was extracted from some of the kanamycin-resistant calli obtained by co-transformations. Dot-blots have shown that the stimulatory effect on transformation frequency is often accompanied by a consistent increase in integrated genes sequences.     

Efficient regeneration of Brassica oleracea hypocotyl protoplasts and high frequency genetic transformation by direct DNA uptake

Summary Efficient regeneration (80%) and high frequency genetic transformation (10–33%) were achieved by culturing protoplasts isolated from hypocotyl tissues of six day old Brassica oleracea seedlings and by subjecting these protoplasts to PEG mediated direct plasmid uptake. Three different plasmid vectors carrying marker genes for resistance to methotrexate (dhfr), hygromycin (hpt) and phosphinotricin (bar) were constructed and used for transformation. Large number of normal, fertile transformants were obtained with vectors carrying hpt and bar genes. No transformants could be regenerated for resistance to methotrexate as it severely suppressed shoot differentiation.Abbreviations bar/PAT bialaphos resistance gene/phosphinotricin acetyltransferase - 2,4-D 2,4-di-chlorophenoxyacetic acid - dhfr/DHPR dihydrofolate reductase gene/enzyme - gus/GUS -glucuronidase gene/enzyme - hpt/HPT hygromycin phosphotransferase gene/enzyme - Kn kinetin - PEG polyethylene glycol - RH relative humidity     

Efficient transformation of Arabidopsis thaliana using direct gene transfer to protoplasts

Summary Direct gene transfer has proved to be an efficient transformation method for arabidopsis thaliana, a member of the Brassicaceae. Transgenic Arabidopsis plants resistant to hygromycin B have been regenerated from mesophyll protoplasts treated with polyethylene glycol and plasmid DNA carrying the hygromycin phosphotransferase (HPT) gene under the control of the 35 S promoter of cauliflower mosaic virus. The transformation procedure reproducibly yields transformants at frequencies of approximately 1×10^-4 (based on the number of protoplasts treated) or 5% (based on the number of regenerating calli). DNA from plants regenerated from hygromycin resistant colonies was analysed by Southern blot hybridization demonstrating that the foreign gene is stably integrated into the plant chromosome. Genetic analysis of several hygromycin resistant plants showed that the HPT gene is transmitted to the progeny. Transformation experiments performed with a selectable and a non-selectable gene on separate plasmids resulted in a co-transformation rate of functionally active copies in about 25% of the transformants analysed. Hence this approach can be used to introduce non-selectable genes into the Arabidopsis genome.     

Direct gene transfer to protoplasts: Fate of the transferred genes

Direct gene transfer to protoplasts is one of several methods developed for the production of transgenic plants. This method utilizes the efficient uptake of DNA from the surrounding medium by protoplasts (cell wall-less plant cells). Where a suitable protoplast system exists large numbers of transformant clones can be efficiently produced and often regenerated to normal fertile plants. This review concentrates on the fate of the DNA which is taken up into the protoplasts. Particular emphasis is given to the factors which can influence the integration and form of the transferred DNA, the expression of transferred genes, and the inheritance in further generations of those genes. The information available suggests (1) that DNA is taken up by a large proportion of the cells in a transformation mixture, (2) that this DNA forms complexes sometimes involving carrier DNA, (3) that fewer cells actually take up DNA into the nucleus, and (4) that the complex may be rearranged and/or amplified and then integrated into the genome. If the DNA is arranged in such a way that a gene can be expressed it does so in a normal manner and is stably inherited both mitotically and meiotically.     

Rapid and high frequency shoot regeneration from hypocotyl protoplasts of Brassica nigra

Protoplasts, isolated from etiolated hypocotyls of seven day old seedlings of Brassica nigra, were cultured in Kao's liquid medium containing 7.2% glucose, 2,4-d (1 mg 1^-1), NAA (0.1 mg 1^-1) and zeatin riboside (0.5 mg 1^-1). After initial incubation for 3 days in dark at 25±1°C, cultures were transferred to a photoperiod cycle of 16/8 h and diluted on seventh and tenth day with MS medium containing 3.4% sucrose, 2,4-d (0.1 mg 1^-1) and BAP (1 mg 1^-1). About 62% of the cells divided at least once and 46% of them reached 8–16 cell stage in one week. The dividing cell clusters could be plated on agarose medium on the fifteenth day to obtain proliferating minicalli with a plating efficiency of 1.8%. 56.8% of minicalli, regenerated shoots on a regeneration medium containing 2 IP and IAA at 1 and 0.2 mg 1^-1 respectively. The in vitro produced shoots were rooted in MS medium containing 1 mg 1^-1 IBA and established in soil without difficulty. The time taken for protoplasts to develop into plants varied from 9 to 10 weeks.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - 2 IP 2-isopentenyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - Kn kinetin     

Direct gene transfer in potato: A comparison of particle bombardment of leaf explants and PEG-mediated transformation of protoplasts

Direct gene transfer methods in potato would facilitate the transfer of multiple genes and the manipulation of metabolic pathways in this species. In this study, up to 1.8 transformation events per shot (=0.5 per bombarded leaf) and 67.2 events per million protoplasts treated were obtained with particle bombardment and PEG-mediated direct DNA uptake, respectively. Limited disassociation of both HPT and GUS genes appeared to occur during the process of integration in only 19% of transformants. A large number of transformed potato plants with transgene expression at levels comparable to Agrobacterium-mediated transformation was obtained. High levels of GUS expression were only obtained in lines derived from PEG treatment. No correlation between the number of gene insertions and gene expression levels was found, suggesting that multiple insertions may have little or no effect on transgene expression. W. Craig and D. Gargano contributed equally to this work     

Cultivar dependence of transformation rates in moth bean after co-cultivation of protoplasts with Agrobacterium tumefaciens

Summary A simplified protoplast regeneration system for Vigna aconitifolia was developed. A plating efficiency of 60% was obtained using mesophyll protoplasts from 10-day-old seedlings. By co-cultivation of protoplasts with Agrobacterium tumefaciens containing the Ti plasmid derivative pGV 38501103 neo kanamycin-resistant colonies were obtained; 23% of the transformed lines showed expression of the nonselected co-transferred nopaline synthase gene. Transformation was confirmed by Southern blot analysis using a nonradioactive detection system. The plant cultivar used was an important factor in determining transformation frequencies since one of the cultivars had an 85 fold higher transformation rate than the other.On deputation from: Bhabha Atomic Research Centre, Bombay, India, under the Indo-FRG Bilateral Programme     

Introduction of plasmid pC194 into Bacillus thuringiensis by protoplast transformation and plasmid transfer

The Staphylococcus aureus plasmid pC194 which codes for resistance to chloramphenicol was introduced into six Bacillus thuringiensis strains representing five varieties by protoplast transformation. Six other varieties could not be transformed. pC194 could be identified in transformed strains as autonomous plasmid. The transformed clones contained in addition a new extrachromosomal element of somewhat lower electrophoretic mobility hybridizing with pC194, and pC194 in multimeric forms. pC194 was also transferred from one B. thuringiensis variety to another and from Bacillus thuringiensis to Bacillus subtilis and vice versa by a conjugation-like process, requiring close cell-to-cell contact.Non-standard abbreviations BSA bovine serum albumin - CAT chloramphenicol acetyltransferase - Cm^R chloramphenicol resistant - PAB Penassay broth - SDS sodiumdodecylsulfate - Tc^R tetracycline resistant     

Pollen irradiation and gene transfer in Capsicum

Summary The aim of the experiment was to study the possibility of facilitating the gene transfer and reducing the number of required backcrosses through pollen irradiation and subsequent selection of F₁M₁ plants containing a very high proportion of sterile pollen as male parent for backcrossing. Anthers of a donor line, C-3-1, were irradiated with 1,500 rad -rays and the pollen used for pollination of a recipient genotype W-8 which posseses a number of recessive marker genes. Five F₁M₂ plants containing more than 80% sterile pollen grains and one semi-sterile plant were selected and used for backcross to W-8. The segregation pattern of four characters expressed in the first backcross generation [W–8×(W–8×C–3–1)] was assessed and compared with the non-irradiated control. A changed segregation pattern was observed (in some cases even non-transfer of a paternal allele) as well as a shift towards more plants possessing the investigated maternal alleles. A scheme for backcross procedure in combination with pollen irradiation is discussed.     

Transformation efficiencies and progeny analysis after varying different parameters of direct gene transfer of Nicotiana tabacum protoplasts

Nicotiana tabacum protoplasts were transformed by polyethylene glycol (PEG)-mediated uptake and electroporation, with circular and linear DNA, and with or without X-ray irradiation. We investigated the influence on the transient expression by these parameters as well as on the frequencies for stable transformation. Plants were regenerated and selfed, and the progenies of the transformed plants were analysed and used to compare the pattern of gene integration by these different variations in transformation methods. The results from the transient expression as judged by glucuronidase (GUS) activity, showed electroporation to give higher and more reproducible results than PEG-mediated uptake. Using linear instead of circular DNA increased the rate of stable transformation about 3 times. Including a mild X-ray treatment gave an increase in the same range. When the inheritance of the transferred trait was investigated, it was found that protoplasts transformed with linear DNA resulted in the highest number of plants with single-copy insertions. Protoplasts transformed with circular DNA showed the highest incidence of losing the trait, while plants in which the transformation included an X-ray treatment, had the highest frequency of multicopy insertion events.     

Transformation efficiencies and progeny analysis after varying different parameters of direct gene transfer of Nicotiana tabacum protoplasts

Nicotiana tabacum protoplasts were transformed by polyethylene glycol (PEG)-mediated uptake and electroporation, with circular and linear DNA, and with or without X-ray irradiation. We investigated the influence on the transient expression by these parameters as well as on the frequencies for stable transformation. Plants were regenerated and selfed, and the progenies of the transformed plants were analysed and used to compare the pattern of gene integration by these different variations in transformation methods. The results from the transient expression as judged by glucuronidase (GUS) activity, showed electroporation to give higher and more reproducible results than PEG-mediated uptake. Using linear instead of circular DNA increased the rate of stable transformation about 3 times. Including a mild X-ray treatment gave an increase in the same range. When the inheritance of the transferred trait was investigated, it was found that protoplasts transformed with linear DNA resulted in the highest number of plants with single-copy insertions. Protoplasts transformed with circular DNA showed the highest incidence of losing the trait, while plants in which the transformation included an X-ray treatment, had the highest frequency of multicopy insertion events.     

Plant regeneration from callus and protoplasts of Brassica nigra (IC 257) through somatic embryogenesis

A method to obtain plants from embryogenic callus of Brassica nigra and protoplasts of hypocotyl expiants is described. Callus was initiated on Murashige and Skoog medium containing kinetin (kn) and 2,4-dichlorophenoxy acetic acid (2,4-D). Lowering of auxin induced embryo formation. Supplementation with gibberellic acid (GA₃) enhanced embryogenic response tenfold. Passage through liquid medium devoid of growth regulators was essential for the growth of embryos. Secondary embryos were produced on transfer to solid basal medium. Embryogenic callus retained its morphogenic ability even after 12 subcultures. Both primary and secondary embryos produced fertile plants. Hypocotyl-derived protoplasts were also regenerated to plants following the same protocol. The survival of plants on transfer to soil was about 80%. The seeds from plants derived from callus and protoplasts were viable.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA naphthalene acetic acid - IAA indole acetic acid - kn kinetin - GA₃ gibberellic acid     

Characterization of chromosome and plasmid transformation in Bacillus subtilis using gently lysed protoplasts

Competent cells of Bacillus subtilis were transformed with DNA from gently lysed protoplasts. Significant linkages among markers separated by distances of approximately 2.3% of the total chromosome were found, which have not been detected for conventional transformation. In comparison to previous reports, enhanced plasmid transformation was observed [4.0×10⁷ transformants per g DNA (one transformant per 5×10⁴ molecules added)], when competent cells were transformed with DNA from lysed protoplasts harboring pUB110.     

Stable transformation of barley via PEG-induced direct DNA uptake into protoplasts

Summary Protoplasts isolated from a barley cell suspension (cv Dissa) were transformed with plasmid DNA containing the neomycinphosphotransferase II (NPT) and -glucuronidase (GUS) genes, using polyethyleneglycol (PEG) to induce DNA uptake. Transformed microcalli were selected in media containing G418 sulphate. NPT activity was detected in all antibiotic-resistant cell lines, but not all NPT-positive cell lines had GUS activity. Southern analysis confirmed the presence of sequences homologous to the APT and GUS genes in DNA of G418-resistant callus.     

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.     

Transplantation of isolated nuclei into plant protoplasts

The uptake of isolated nuclei from Vicia hajastana Grossh. cells into protoplasts of an auxotrophic cell line of Datura innoxia P. Mill. was induced under the influence of polyethylene glycol and Ca²⁺ at pH 6.8. The frequency of nuclear uptake varied from 0.8 to 2.3% and that of the recovery of prototrophic clones from 10^-5 to 6·10^-4. The prototrophic nuclear fusion products following nuclear uptake could be rescued by initial culture of the protoplasts in non-selective conditions and by the subsequent use of feeder cell layers to support the growth of surviving colonies on a selective medium. The presence of Vicia genomic DNA in some prototrophic clones was confirmed by dot-blot hybridization using Datura and Vicia DNA probes. In certain transformed clones, the recovery of prototrophy was accompanied by the restoration of morphogenetic potential. Welldeveloped shoots typical of wild-type Datura could be regenerated employing an appropriate regeneration medium.Abbreviations MS Murashige and Skoog (1962) - PEG polyethylene glycol     

Agrobacterium-mediated transformation of white clover using direct shoot organogenesis

Summary White clover (Trifolium repens L.) plants from the cultivars Grasslands Huia and Grasslands Tahora have been transformed using Agrobacterium-mediated T-DNA transfer. Transgenic plants regenerated directly from cells of the cotyledonary axil. To transform white clover, shoot tips from 3 day old seedlings were co-cultivated with A. tumefaciens strain LBA4404 carrying the plasmid vector pPE64. This vector contains the neomycin phosphotransferase II gene (nptII) and -glucuronidase reporter gene (gus) both under the control of the CaMV 35S promoter. Kanamycin-resistant plants regenerated within 42 days after transfer onto selective media. Integration of the nptII and gus genes into the white clover genome was confirmed using Southern blotting, and histochemical analysis indicated that the gus gene was expressed in a variety of tissues. In reciprocal crosses between a primary transformant and a non-transformed plant the introduced gus gene segregated as a single dominant Mendelian trait.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - MS Murashige and Skoog - GUS -glucuronidase - X-GLUc 5-bromo-4-chloro-3-indolyl--D-glucuronide - MUG methylumbelliferyl--D-glucuronide - CaMV Cauliflower Mosaic Virus - NPTII neomycin phosphotransferase II - OCS octopine synthase - 4-MU 4-methyl umbelliferone     

Studies on plant regeneration from cotyledonary protoplasts in Brassica campestris

Protoplasts isolated from both 7-day-old light-grown and 4-day-old dark/dim light-grown cotyledons of four Brassica campestris varieties (Arlo, Sonja, Bunyip and Wonk Bok) were cultured in three liquid media: modified K8P, modified MS and modified Pelletier's B to compare the capacities for cell division and plant regeneration. Following cell wall regeneration the cultured protoplasts from dark/dim light-grown cotyledons of four varieties showed rapid division and high frequency of cell division compared with those isolated from light-grown cotyledons. The frequencies of cell division were significantly influenced by varieties and culture media but only in cultured protoplasts isolated from dark/dim light-grown cotyledons. The interaction between varieties and media was also significant. Cell colonies formed within 7–14 days in protoplast cultures from dark/dim light-grown cotyledons, and calli subsequently grown on a solid medium developed shoots when transferred onto a regeneration medium. Three of four tested varieties (Arlo, Sonja and Bunyip) showed shoot regeneration within 2–3 months after protoplast isolation, with a high degree of reproducibility in Arlo and Bunyip. Regenerated shoots, which were induced to root on half-strength MS medium with 0.1 mg.l^–1 IBA, survived in soil and grew to produce siliques and set viable seeds in the greenhouse. The present report is the first to document the production of regenerated plants that set seeds in Brassica campestris from cotyledonary protoplasts.Abbreviations BAP benzylaminopurine - CPW Composition of Protoplast Washing-solution - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediamine-tetraacetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - KT kinetin - FDA fluorescein diacetate - SDS sodium dodecyl sulfate