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PubMed2026

Genetic transformation of forage crops: comparative barriers, evidence, and emerging strategies.

Forage crops include phylogenetically and biologically distinct legumes and grasses, and their genetic transformation is constrained by different combinations of host response, DNA-delivery efficiency, regeneration competence, genotype dependence, and genome stability. This review critically compares evidence from forage legumes and forage grasses rather than treating these groups as a single transformation category. We evaluate Agrobacterium-mediated transformation, protoplast-based delivery, particle bombardment, CRISPR/Cas-enabled applications, developmental regulators (DRs), viral vectors, and nanomaterial-mediated delivery according to four practical outcomes: reproducibility across genotypes, recovery of regenerated plants, heritable transmission, and genetic stability. Direct evidence in forage crops shows that protocol performance is strongly species-, genotype-, explant-, and endpoint-dependent; efficiencies based on transient reporters or resistant callus therefore cannot be directly equated with stable, fertile events. DR-assisted regeneration has direct proof of concept in recalcitrant forage grasses, whereas stable nanomaterial-mediated transformation and virus-induced heritable editing remain unvalidated in forage crops. We conclude that current progress is best interpreted as the engineering of interacting delivery and regeneration constraints, not as a universal transition to genotype-independent transformation. Priority should be given to standardized outcome reporting, multi-genotype and inter-laboratory validation, controlled DR expression, and rigorous molecular and phenotypic assessment of regenerated plants.

Crops, Agricultural↗

PubMed1981

Genetic transformation of Rhizobium meliloti by plasmid DNA.

A procedure for the genetic transformation of Rhizobium meliloti by plasmid DNA is described. It is an adaptation of a procedure used originally for Escherichia coli and later for Agrobacterium tumefaciens. Depending on the R. meliloti isolate used as the recipient, the efficiency of transformation was in the range of 7 x 10(1) to 3.7 x 10(3) per microgram of plasmid pRK248 DNA and 10(-6) to 10(-9) per viable cell. A number of different naturally occurring strains could be transformed. Plasmids related to pRK248 and varying in size from 9.6 to 56 kb could be used, suggesting that the procedure will be useful for molecular cloning of genes in R. meliloti.

Cloning, Molecular↗

PubMed1987

Combined genetic transformation and nutritional assay for identification of Moraxella nonliquefaciens.

A combined genetic transformation and nutritional assay is described that permits definitive identification of clinically isolated strains of Moraxella nonliquefaciens. Crude DNA preparations of strains of various Moraxella species were used to transform nutritional mutants of a stably competent strain of M. nonliquefaciens for ability to grow on a defined medium (Mn-B). DNA samples from 24 independently isolated strains of M. nonliquefaciens all resulted in massive (4+) transformation of each of two mutant assay strains. DNA samples from strains of M. bovis and M. lacunata frequently gave weak (1+) transformation of one of the mutant assay strains (Mn64) but almost always failed to transform another assay strain (Mn136). DNA samples from eight other Moraxella species failed completely to transform either of the mutant assay strains. When streaked on the defined medium used for the transformation assay (Mn-B), 23 of the 24 strains of M. nonliquefaciens grew well, but all strains of M. bovis and M. lacunata failed to grow on this medium.

Culture Media↗

PubMed1996

Genetic transformation of cassava (Manihot esculenta Crantz).

Genetic engineering can be used to complement traditional breeding methods in crop plant improvement. Transfer of genes from heterologous species provides the means of selectively introducing new traits into crop plants and expanding the gene pool beyond what has been available to traditional breeding systems. The prerequisites for genetic engineering are efficient transformation and tissue culture systems that allow selection and regeneration of transgenic plants. Cassava, an integral plant for food security in developing countries, has until now been recalcitrant to transformation approaches. We report here a method for regenerating stably transformed cassava plants after cocultivation with Agrobacterium tumefaciens, which opens cassava for future improvement via biotechnology.

Agrobacterium tumefaciens↗

PubMed2000

The potential for intraspecific horizontal gene exchange by natural genetic transformation: sexual isolation among genomovars of Pseudomonas stutzeri.

The potential for natural genetic transformation among the seven genomovars (gvs) of Pseudomonas stutzeri was investigated. Of the 12 strains originating from a variety of environments, six strains (50%) from five gvs were competent for DNA uptake (Rif(R) marker). The transformation frequencies varied over more than three orders of magnitude. With three highly transformable strains (ATCC 17587, ATCC 17641, JM300) from two gvs and all other strains as DNA donors, sexual isolation from other pseudomonad species (Pseudomonas alcaligenes, Pseudomonas mendocina) and also from other P. stutzeri gvs was observed (i.e. heterogamic transformation was reduced). For ATCC 17587 (gv 2) and ATCC 17641 (gv 8), heterogamic transformation was up to two and three orders of magnitude lower with other P. stutzeri gv and the other species employed, respectively, than in homogamic transformations. Interestingly, whereas with ATCC 17587 and ATCC 17641 heterogamic transformation with donors of the same gv was as high as homogamic transformation, JM300 (gv 8) was sexually isolated from its nearest relative (ATCC 17641). Also, sexual isolation of JM300 from other P. stutzeri gvs was most pronounced among the recipients tested, in some cases reaching the highest levels found with the other species as DNA donors (reduction of heterogamic transformation by 4000-fold). Results obtained here from nucleotide sequence analysis of part (422 nt) of the gene for the RNA polymerase ss subunit (rpoB) from various strains indicated that sexual isolation of ATCC 17641 increased with nucleotide sequence divergence. Implications of the observed great heterogeneity in transformability, competence levels and sexual isolation among strains are discussed with regard to the evolution of P. stutzeri.

Conjugation, Genetic↗

PubMed2005

Genetic transformation of conifers utilizing somatic embryogenesis.

Over the last 5 yr, the production of transgenic conifers has been greatly facilitated by the ability to transform somatic embryonal tissues (somatic embryos) via cocultivation with Agrobacterium tumefaciens. This has allowed us to develop protocols for the genetic transformation of several spruce species. Furthermore, these procedures can produce an average of 20 independent transgenic lines (translines) per gram fresh mass of embryonal tissue, providing for the first time the magnitude-of-scale required for implementing large-scale functional genomics studies in conifers. Combined with efficient regeneration of transgenic trees via somatic embryos, the potential for genetic engineering of conifers has been demonstrated by stable reporter gene expression (GUS or GFP) resulting from single insert T-DNA integration events.

Agrobacterium tumefaciens↗

PubMed2002

Stable genetic transformation of garlic plants using particle bombardment.

The improvement of garlic plants (Allium sativum L.) via biotechnological approaches is currently limited by the lack of an applicable direct gene transfer system. In this paper, we present the development of a genetic transformation system using particle bombardment for gene delivery and immature clove-derived callus as the gene target. Plasmid DNA (pBI221.23), containing the selectable "hpt" gene for hygromycin resistance and the reporter "gus" gene, was delivered into callus tissue that had been previously treated with aurintricarboxylic acid as an endogenous nuclease inhibitor. The transformed calli were selected using hygromycin B, regenerated, and analysed at the molecular level using DNA hybridization, transgenome rescue and histochemical beta-glucuronidase assay. The results indicated that biolistic transformation can lead to the transfer, expression and stable integration of a DNA fragment into garlic chromosomal DNA. The relative simplicity of this system is a good recommendation for its future use in the production of genetically modified garlic plants.

Biolistics↗

PubMed2001

Recent advances in the genetic transformation of trees.

As the commercial production of transgenic annual crops becomes a reality in many parts of the world, many people wonder if the genetic engineering of perennial trees will allow their eventual commercialization. Not long ago, trees were considered to be recalcitrant material for most molecular biology techniques, including genetic transformation. However, transgenes for shortening the juvenile phase or for phytoremediation purposes have now been incorporated, and the alteration of lignin biosynthesis and increased cellulose accumulation in forest trees have also been accomplished. For long-lived tree species, new questions arise regarding the stability of integration and expression of foreign genes. Biosafety considerations, including transgene dispersion through the pollen and advances in strategies to avoid this, are also important.

Biodegradation, Environmental↗

PubMed1993

Aflagellated mutants of Helicobacter pylori generated by genetic transformation of naturally competent strains using transposon shuttle mutagenesis.

Three out of 10 Helicobacter pylori clinical isolates were found to be naturally competent for genetic transformation to streptomycin resistance by chromosomal DNA extracted from a spontaneous streptomycin-resistant H. pylori mutant. The frequency of transformation varied between 5 x 10(-4) and 4 x 10(-6), depending on the H. pylori isolate used. Transposon shuttle mutagenesis based on this natural competence was established using the flagellin gene flaA as the target. The cloned flaA gene was interrupted by insertion of TnMax1, a mini-Tn1721 transposon carrying a modified chloramphenicol-acetyltransferase gene, the catGC cassette. Natural transformation of competent H. pylori strains with plasmid constructs harbouring a catGC-inactivated flaA gene resulted in chloramphenicol-resistant transformants at an average frequency of 4 x 10(-5). Southern hybridization experiments confirmed the replacement of the chromosomal H. pylori flaA gene by the cat-inactivated cloned gene copy via homologous recombination resulting in allelic exchange. Phenotypic characterization of the mutants demonstrated the absence of flagella under the electron microscope and the loss of bacterial motility. Immunoblots of cell lysates of the H. pylori mutants with an antiserum raised against the C-terminal portion of recombinant H. pylori major flagellin (FlaA) confirmed the absence of the 54 kDa FlaA protein. This efficient transposon shuttle mutagenesis procedure for H. pylori based on natural competence opens up new possibilities for the genetic assessment of putative H. pylori virulence determinants.

Bacterial Proteins↗

PubMed2003

[Establisment of a genetic transformation method of coffee (Coffea arabica cv. Catimor) and incorporation of bar gene for ammonium glufosinate resistance].

In order to establish a successful method of genetic transformation in Coffea arabica cv. Catimor, different conditions of generation and electroporation were evaluated on different plant tissues. Cell suspension system was improved using one hormone only (BA), obtaining high yields of primary and secondary somatic embryo production. For selection of viable and potentially transformed cells, MTT (1%) method and ammonium glufosinate concentration (1 mg/L in leaf, callus and embryos; and 5 mg/L in cells) were established. Different conditions were evaluated to electroporate different explants (embryogenic callus, vitroplants leaves, globular and torpedo embryos). The highest gus gene expression percentage by explant were found on enzymatic treated tissues at 375 V/cm in callus, and at 625 V/cm in leaves and embryos. Torpedo embryos cultured on liquid medium were the only type of tissue that could regenerate into plants, where secondary somatic embryos were obtained. Those embryos were positive to the gus gene histochemical test and to the gus and bar genes amplification on a PCR reaction.

Aminobutyrates↗

PubMed2007

Genetic transformation of chickpea (Cicer arietinum L.) with insecticidal crystal protein gene using particle gun bombardment.

Here, we report the establishment of an efficient particle gun bombardment mediated genetic transformation in chickpea (Cicer arietinum L.) using cryIAc gene of Bacillus thuringiensis. Explants were bombarded with recombinant plasmids engineered for the expression of cryIAc transgene in plants and stable transformants regenerated in presence of benzyladenine, kinetin and kanamycin. Transformation frequency showed dependence on explant type, cultivars, plasmids, helium pressure and microcarrier type used. Integration of transgenes was demonstrated using polymerase chain reaction and Southern blot hybridization approaches in T (0) plants. The expression of CryIA(c) delta-endotoxin and GUS enzyme was ascertained by enzyme linked immunosorbent assay and histochemical assays, respectively. These transgenic plants (T (0)) showed more protection and high mortality for Heliothis armigera and Spodoptera litura larvae as compared to control plants. The results of the present study indicate that highest transformation frequency (18%) could be achieved by use of gold as a microcarrier in combination with helium pressure of 900 psi. Among the other factors tested, plasmid pHS 102 was the most efficient plasmid, while epicotyl explant was the best explant source for particle gun bombardment. Among the different cultivars of chickpea tested, cultivar ICCC37 and PG-12 produced higher frequency of transformation frequency compared to others.

Animals↗

PubMedDate not supplied

[Regeneration of cybrid Lycopersicon peruvianum x (Solanum rickii) plants with genetically transformed chloroplasts].

The hybrid plants with transformed plastids were regenerated after PEG fusion of chlorophyll-deficient Lycopersicon peruvianum leaf mesophyll protoplasts and leaf mesophyll protoplasts of Solanum rickii, which were previously genetically transformed and as the result were resistant to streptomycine and spectinomycine. The hybrid callus selection was based on the inability of the Lycopersicon peruvianum minicalli to have the green coloration and on the inability of gamma-preirradiated Solanum rickii protoplasts to divide. The hybrids were identified on the base of PCR analyses of nuclear and plastid DNA.

Cell Fusion↗

PubMed1979

Competence for genetic transformation in pneumococcus depends on synthesis of a small set of proteins.

In bacterial genetic transformation the uptake of DNA and its integration into the resident chromosome is dependent on a special cellular state, termed competence. In those species where appearance of competence has been studied, specific (but often poorly defined) growth conditions lead to a simultaneous development of competence in a substantial fraction of the cells in a culture. In Bacillus subtilis, and in Haemophilus species, competence appears in the stationary phase of growth or in certain other growth-limiting conditions. Streptococcus pneumoniae (pneumococcus) is perhaps unusual in that virtually all cells of a culture become competent, for a short period at a specific cell density during logarithmic growth, without perturbing the growth rate. The synchronous appearance of competence in pneumococcal cultures results from an autocatalytic effect of a small protein released by the cells that induces competence. The response to competence factor has been shown to require protein synthesis. We report here additional information on the nature of competence in pneumococcus: pulse-labelling studies show that for the brief period of competence protein synthesis is restricted to a few specific polypeptides.

Bacterial Proteins↗

PubMed1977

Genetic transformation assays for identification of strains of Moraxella urethralis.

Studies of 31 strains of Moraxella urethralis have shown that 20 of them are competent for genetic transformation. This finding has led to the development of transformation assays for identification of newly isolated strains of this organism. Crude deoxyribonucleic acid (DNA) samples from all strains of M. urethralis readily transform auxotrophic mutants of competent strains to prototrophy, whereas DNA samples from unrelated bacteria such as Acinetobacter, Moraxella, and Neisseria species uniformly fail to elicit positive transformation of mutant tester strains. One of the competent strains of M. urethralis investigated is a naturally occurring mutant defective in its ability to utilize citrate as a carbon and energy source. DNA samples from 29 of the 30 remaining strains of utilization; the one nonreacting strain is citrate negative and probably possesses the same genetic lesion as the citrate-negative mutant. Three organisms originally identified as strains of M. urethralis, because of their phenotypic properties, are probably incorrectly designated, since DNA samples from these strains failed to transform any of the tester mutant strains used in the present study. The transformation assay for M. urethralis is very simple and can be performed readily in a clinical laboratory. The entire procedure can be carried out in less than 24 h.

Bacterial Infections↗

PubMed1988

Characterization of genetic transformation in Streptococcus oralis NCTC 11427: expression of the pneumococcal amidase in S. oralis using a new shuttle vector.

We have worked out conditions for the study of competence development and genetic transformation in Streptococcus oralis NCTC 11427 (type strain), a species that contains choline in the cell wall. The peak of competence was found at the early exponential phase of growth and the optimal conditions for transformation were achieved with shuttle plasmids prepared from S. pneumoniae or from Escherichia coli serving as donor DNA. Transformation with dye-buoyant density gradient purified plasmid preparations followed first-order kinetics. The pneumococcal amidase can be expressed in S. oralis harbouring a plasmid carrying the lytA gene. This enzyme lysed the cell wall of the transformed cell in the presence of detergents.

Amidohydrolases↗

PubMed1986

Genetic transformation of the extreme thermophile Thermus thermophilus and of other Thermus spp.

Genetic transformation of auxotrophs of the extreme thermophile Thermus thermophilus HB27 to prototrophy was obtained at high frequencies of 10(-2) to 10(-1) when proliferating cell populations were exposed to chromosomal DNA from a nutritionally independent wild-type strain. The transformation frequency was proportional to the DNA concentration from 10 pg/ml to 100 ng/ml. T. thermophilus HB27 cells did not require chemical treatment to induce competence, although optimal transformation was obtained by the addition of a divalent cation (Ca2+ or Mg2+). Competence was maintained throughout the growth phase, with the highest transformation frequencies at pH 6 to 9 and at 70 degrees C. T. thermophilus HB27 and four other typical Thermus strains, T. thermophilus HB8, T. flavus AT62, T. caldophilus GK24, and T. aquaticus YT1, were also transformed to streptomycin resistance by DNA from their own spontaneous streptomycin-resistant mutants. A cryptic plasmid, pTT8, from T. thermophilus HB8 was introduced into T. thermophilus HB27 Pro- at a frequency of 10(-2).

Culture Media↗

PubMed1963

ELECTROKINETICS AND CELL PHYSIOLOGY. II. RELATIONSHIP OF SURFACE CHARGE TO ONSET OF BACTERIAL COMPETENCE FOR GENETIC TRANSFORMATION.

Jensen, Roy A. (The University of Texas M. D. Anderson Hospital and Tumor Institute, Houston) and Felix L. Haas. Electrokinetics and cell physiology. II. Relationship of surface charge to onset of bacterial competence for genetic transformation. J. Bacteriol. 86:79-86. 1963.-A reliable cell fractionation scheme, which is sensitive to the electrokinetic properties of Bacillus subtilis cells, has been described in detail. Recipient cell populations, characterized by a wide range of competency for transformation to independence of nutritional markers, were subjected to electrokinetic fractionation. Results indicated that (i) physiological competency is directly related to the electrical charge on the cell surface, (ii) newly competent cells carry a maximal negative charge, (iii) the newly competent cell appears with spontaneous abruptness, (iv) a kinetic flow of competent cells from the highly charged fractions to the lower charged fractions indicates the progressive loss of the surface charge maintained by a competent cell, and (v), by token of the latter statement, cells competent to undergo transformation do so within a range of surface-charge values.

Bacillus subtilis↗

PubMed2002

Engineering a genetic transformation system for Colletotrichum acutatum, the causal fungus of lime anthracnose and postbloom fruit drop of citrus.

Postbloom fruit drop (PFD) of citrus is caused by Colletotrichum acutatum. PFD isolates infect flower petals, induce abscission of small fruit and can cause severe yield loss on most citrus cultivars. Isolates from Key lime anthracnose (KLA) cause that disease on the Mexican lime, but also cause PFD on sweet orange. Both PFD and KLA isolates exhibited resistance to the common selection agents including hygromycin, bialaphos, benomyl and geneticin/G418. A genetic transformation system was developed for C. acutatum to confer resistance to sulfonylurea (chlorimuron ethyl) by expressing an acetolactate synthase gene (sur) cassette from Magnaporthe grisea. The protocol was tested on 11 different KLA and PFD isolates. The transformation frequencies were highly variable among isolates and among experiments (0-17.9 per microg circular DNA using 10(7) protoplasts). Southern blot analysis of transformants indicated that the plasmid vector was randomly integrated in multiple copies into the genome of C. acutatum. Addition of restriction enzymes or use of a vector with homologous sequences did not change the transformation frequencies, but tended to reduce the number integrated. Over 97% of the transformants retained the sulfonylurea resistance phenotype under non-selective conditions. Of 300 transformants tested, three were unable to cause necrotic lesions on detached Key lime leaves. The transformation method opens up opportunities for the genetic manipulation of C. acutatum.

Blotting, Southern↗