A simple system for pea transformation

The lateral cotyledonary meristems of germinatingPisum sativum cv. Puget seeds were used to develop a reproducibleAgrobacterium tumefaciens-mediated transformation system. This procedure exhibits distinct advantages over those previously reported, in that it uses dry seed as starting material, and the highly regenerable cotyledonary meristems rapidly produce transgenic shoots without an intermediate callus phase. This transformation regime facilitates the rapid generation of phenotypically normal, self-fertile plants containing functional transgenes inherited in a Mendelian fashion.Abbreviations bar Bialaphos resistance gene - BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - IBA Indole-3-butyric acid - PAT Phosphinothricin acetyltransferase - PPT Phosphinothricin     

A protoplast transformation system for Listeria sp

We describe protoplast transformation of members of the genus Listeria with plasmid DNA. Shuttle vectors able to replicate in both Escherichia coli and Listeria have been constructed by fusing the E. coli plasmid pUC8 with the Bacillus subtilis plasmids pBD9 and pBD10.     

Mannose selection system used for cucumber transformation

The selectable marker system, which utilizes the pmi gene encoding for phosphomannose-isomerase that converts mannose-6-phosphate to fructose-6-phosphate, was adapted for Agrobacterium-mediated transformation of cucumber (Cucumis sativus L.). Only transformed cells were capable of utilizing mannose as a carbon source. The highest transformation frequency of 23% was obtained with 10 g/l mannose and 10 g/l sucrose in the medium. Molecular, genetic analysis, and PMI activity assay showed that the regenerated shoots contained the pmi gene and the gene was transmitted to the progeny in a Mendelian fashion. The results indicated that the mannose selection system, which is devoid of the disadvantages of antibiotic or herbicide selection, could be used for cucumber Agrobacterium-mediated transformation.     

Integrated bombardment andAgrobacterium transformation system: An alternative method for soybean transformation

Described here is a new method for soybean transformation, based on microwounding of embryonic clumps by particle bombardment prior to inoculation with anAgrobacterium suspension. The method combines the advantages of somatic embryogenesis and gene transfer through an integrated transformation system. This is the first report of application of this technique to transformation ofGlycine max.     

An efficient transformation system for Bifidobacterium spp.

This study describes a broad host transformation protocol that enables the uptake of plasmid DNA into 10 different species of Bifidobacterium , some of which have never been transformed before. The vector pNC7 (4·9 kb) was used to optimize the electroporation protocol. Transformation efficiencies ranged from 3·6×10⁻¹ to 1·2×10⁵ transformations per μg DNA. The impact of growth medium composition and electric field strength on transformation efficiency were independently optimized. Electrocompetent cells were grown in Iwata medium broth enriched with Actilight^RP 16%, harvested during the early exponential growth phase, and pulsed at 12·5 kV cm⁻¹, 100 Ω and 25 μF.     

A transformation system for the hypotrichous ciliate Stylonychia mytilus

We describe a transformation system for the ciliate Stylonychia mytilus. The neomycin resistance gene from Escherichia coli transposon Tn5, which codes for the enzyme phosphotransferase and confers resistance to the antibiotic G 418, was ligated into macronuclear `gene-size' DNA molecules. Using this recombinant DNA for transformation experiments we show that the gene is replicated and expressed in transformed cells.     

A highly efficient electroporation system for transformation of Yersinia

The various pathogenic Yersinia species are not readily and efficiently transformed by classical methods. For this reason, the electroporation technique was applied for genetic transformation of these species. Using optimal conditions, we were able to transform the six Yersinia strains studied with the two most widely used groups of plasmids: pSU2718 (a pACYC184 derivative) and pK19 (a pUC19 derivative). Only Yersinia enterocolitica (Y. e.) serotype 0:8 gave poor results (less than 5 x 10(2) transformants/microgram) DNA). Electrical transformation of the other species resulted in high efficiencies, up to 10(5) transformants/microgram DNA for Y. e. serotypes 0:3 and 0:9, 10(6) for Y. pseudotuberculosis and 10(7) for Y. pestis. The results varied for each strain with the type of plasmid used. Neither the introduced foreign plasmid nor the resident 72-kb virulence plasmid underwent detectable deletions. Transformation was most efficient with supercoiled DNA, decreasing by one and four orders of magnitude for relaxed circular and linearized plasmids, respectively. The ability to easily and efficiently transfer plasmid DNA via electroporation will greatly facilitate the application of recombinant DNA technology for direct cloning and analysis of significant genes into Yersinia.     

A binary-BAC system for plant transformation with high-molecular-weight DNA

A binary-BAC (BIBAC) vector suitable for Agrobacterium-mediated plant transformation with high-molecular-weight DNA was constructed. A BIBAC vector is based on the bacterial artificial chromosome (BAC) library vector and is also a binary vector for Agrobacterium-mediated plant transformation. The BIBAC vector has the minimal origin region of the Escherichia coli F plasmid and the minimal origin of replication of the Agrobacterium rhizogenes Ri plasmid, and thus replicates as a single-copy plasmid in both E. coli and in A. tumefaciens. The T-DNA of the BIBAC vector can be transferred into the plant nuclear genome. As examples, a 30-kb yeast genomic DNA fragment and a 150-kb human genomic DNA fragment were inserted into the BIBAC vector; these constructs were maintained in both E. coli and A. tumefaciens. In order to increase the efficiency of transfer of unusually large BIBAC T-DNAs, helper plasmids that carry additional copies of A. tumefaciens virulence genes virG and virE were constructed. These helper plasmids are compatible with, and can be present in addition to, the BIBAC vector in the A. tumefaciens host. This report details the components of the BIBAC system, providing information essential to the general understanding and the application of this new technology.     

Genetic transformation system for the aflatoxin-producing fungus Aspergillus flavus

A heterologous transformation system was developed for Aspergillus flavus with efficiencies greater than 20 stable transformants per micrograms of DNA. Protoplasts of uracil-requiring strains of the fungus were transformed with plasmid and cosmid vectors containing the pyr-4 gene of Neurospora crassa. Transformants were selected for their ability to grow and sporulate on medium lacking uracil. Vector DNA appeared to integrate randomly into the genome of A. flavus with a tendency for multiple, tandem insertion. Transformants with single or multiple insertions were stable after five consecutive transfers on medium containing uracil. Uracil-requiring recipient strains were obtained either by UV-irradiating conidia and selecting colonies resistant to 5-fluoroorotic acid or by transferring the mutated pyr locus to strains by parasexual recombination. This is the first report of a transformation system for an aflatoxin-producing fungus. The transformation system and the availability of aflatoxin-negative mutants provide a new approach to studying the biosynthesis and regulation of aflatoxin.     

农杆菌转化系统研究进展

农杆菌转化系统是一种优良的转化系统。作者综述了农杆菌转化系统的原理、菌株及载体系统的发展,并概括了植物农杆菌转化的发展历程及与转化效率有关的因素,最后指出了农杆菌转化系统的优点。     

Development of an efficient Agrobacterium-mediated transformation system for Brassica carinata

Shoot organogenesis and plant regeneration were readily achieved from cotyledonary petioles and hypocotyls of Brassica carinata. These explants were used for Agrobacterium-mediated transformation. A construct containing the selectable marker genes, neomycin phosphotransferase II, phosphinothricin acetyl transferase and the reporter gene β-glucuronidase, under the control of a tandem 35S promoter, was used for transformation. Although transformation was achieved with both cotyledonary petioles and hypocotyls, cotyledonary petioles responded best, with 30–50% of the explants producing GUS-positive shoots after selection on 25 mg/l kanamycin. Direct selection on L-phosphinothricin also produced resistant shoots but at a lower frequency (1–2%). Received: 9 April 1997 / Revision received: 3 July 1997 / Accepted: 30 July 1997     

Shuttle vector system for Methanococcus maripaludis with improved transformation efficiency

We have identified an open reading frame and DNA element that are sufficient to maintain shuttle vectors in Methanococcus maripaludis. Strain S0001, containing ORF1 from pURB500 integrated into the M. maripaludis genome, supports a significantly smaller shuttle vector, pAW42, and a 7,000-fold increase in transformation efficiency for pURB500-based vectors.     

A highly efficient electroporation system for transformation of Bacillus licheniformis

Summary Transformation of intact cells of Bacillus licheniformis 5A24 with plasmids pLM6 (2.8 kb), pC194 (2.9 kb) and pCP49 (7.1 kb) by electroporation resulted in 1.5 × 10⁶, 1.2 × 10⁶ , and 5.2 × 10⁴ transformants per μg DNA, respectively. Transformation was possible with plasmid DNA, which was religated after restriction endonuclease digestions. In addition, evidence is presented that indicates that B. licheniformis possesses DNA restriction and modification systems.     

Establishment of a micro-particle bombardment transformation system for Dunaliella salina

In this study, we chronicle the establishment of a novel transformation system for the unicellular marine green alga, Dunaliella salina. We introduced the CaMV35S promoter-GUS construct into D. salina with a PDS1000/He micro-particle bombardment system. Forty eight h after transformation, via histochemical staining, we observed the transient expression of GUS in D. salina cells which had been bombarded under rupture-disc pressures of 450 psi and 900 psi. We observed no GUS activity in either the negative or the blank controls. Our findings indicated that the micro-particle bombardment method constituted a feasible approach to the genetic transformation of D. salina. We also conducted tests of the cells' sensitivity to seven antibiotics and one herbicide, and our results suggested that 20 microg/ml of Basta could inhibit cell growth completely. The bar gene, which encodes for phosphinothricin acetyltransferase and confers herbicide tolerance, was introduced into the cells via the above established method. The results of PCR and PCR-Southern blot analyses indicated that the gene was successfully integrated into the genome of the transformants.     

The pCLEAN dual binary vector system for Agrobacterium-mediated plant transformation

The development of novel transformation vectors is essential to the improvement of plant transformation technologies. Here, we report the construction and testing of a new multifunctional dual binary vector system, pCLEAN, for Agrobacterium-mediated plant transformation. The pCLEAN vectors are based on the widely used pGreen/pSoup system and the pCLEAN-G/pCLEAN-S plasmids are fully compatible with the existing pGreen/pSoup vectors. A single Agrobacterium can harbor (1) pCLEAN-G and pSoup, (2) pGreen and pCLEAN-S, or (3) pCLEAN-G and pCLEAN-S vector combination. pCLEAN vectors have been designed to enable the delivery of multiple transgenes from distinct T-DNAs and/or vector backbone sequences while minimizing the insertion of superfluous DNA sequences into the plant nuclear genome as well as facilitating the production of marker-free plants. pCLEAN vectors contain a minimal T-DNA (102 nucleotides) consisting of direct border repeats surrounding a 52-nucleotide-long multiple cloning site, an optimized left-border sequence, a double left-border sequence, restriction sites outside the borders, and two independent T-DNAs. In addition, selectable and/or reporter genes have been inserted into the vector backbone sequence to allow either the counter-screening of backbone transfer or its exploitation for the production of marker-free plants. The efficiency of the different pCLEAN vectors has been assessed using transient and stable transformation assays in Nicotiana benthamiana and/or Oryza sativa.     

Genetic transformation system for the aflatoxin-producing fungus Aspergillus flavus.

A heterologous transformation system was developed for Aspergillus flavus with efficiencies greater than 20 stable transformants per micrograms of DNA. Protoplasts of uracil-requiring strains of the fungus were transformed with plasmid and cosmid vectors containing the pyr-4 gene of Neurospora crassa. Transformants were selected for their ability to grow and sporulate on medium lacking uracil. Vector DNA appeared to integrate randomly into the genome of A. flavus with a tendency for multiple, tandem insertion. Transformants with single or multiple insertions were stable after five consecutive transfers on medium containing uracil. Uracil-requiring recipient strains were obtained either by UV-irradiating conidia and selecting colonies resistant to 5-fluoroorotic acid or by transferring the mutated pyr locus to strains by parasexual recombination. This is the first report of a transformation system for an aflatoxin-producing fungus. The transformation system and the availability of aflatoxin-negative mutants provide a new approach to studying the biosynthesis and regulation of aflatoxin.     

A transformation system for an ectomycorrhizal basidiomycete, Lyophyllum shimeji

A transformation vector, pLS-hph, was constructed with the promoter and terminator of the glyceraidehyde-3-phosphate dehydrogenase (GPD) gene derived from an ectomycorrhizal basidiomycete, Lyophyllum shimeji, and with the hygromycin B (HmB) phosphotransferase (hph) gene from Escherichia coli. This vector was introduced into protoplasts of L. shimeji and 3.4 transformants per microg plasmid DNA were obtained. In most of the transformants, multiple copies of the vector were integrated into the genomic DNA. The results indicate that pLS-hph is a useful vector for L. shimeji.     

Development of reproducible regeneration and transformation system for Sesamum indicum

Thalictrum foliolosum is an endemic herb known for its medicinal properties and used for various clinical applications including ophthalmic, skin disease and dyspepsia. Due to its medicinal properties, the plants are uprooted hence can be prone to extinction. In the present study, a reproducible in vitro propagation protocol has been developed using axillary shoot buds and nodal segments. Seedling derived axillary shoot buds were cultured in Murashige and Skoog’s (MS) medium supplemented with 2.24 µmol of 6-benzylaminopurine (BAP) and readily produced maximum shoot (7.2?±?0.40) with the highest percentage of response (91.42%). Also, nodal explants (field-grown plant) developed maximum shoots (3.2?±?0.48) on MS medium containing 4.49 µmol BAP with a combination of 0.54 µmol α-naphthaleneacetic acid (NAA). Best growth and foliage development was achieved at 2.24 µmol BAP with 0.54 µmol NAA in presence of 0.3% activated charcoal and 113.4 µmol ascorbic acid. Micropropagated shoots showed maximum percentage (63.30%) of rooting in half-strength MS medium containing 1.23 µmol indole-3-butyric acid (IBA) and acclimatized in soilrite and leaf manure (2:1) during 4 weeks. Monomorphic bands developed by random amplification of polymorphic DNA (RAPD) and simple sequence repeats (SSR) markers confirmed the genetic stability of in vitro established plants. Additionally, HPLC analysis showed higher benzylisoquinoline (BIQ) alkaloids content in in vitro established plant root extracts. The micropropagation protocol developed in this study provides an alternative strategy for germplasm conservation and protection which at the same time can also be exploits for the production of pharmacologically active compounds.