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PubMed2005

Agrobacterium tumefaciens-mediated sorghum transformation using a mannose selection system.

A dual-marker plasmid containing the selectable marker gene, manA, and the reporter gene, sgfp, was used to transform immature sorghum embryos by employing an Agrobacterium-mediated system. Both genes were under the control of the ubi1 promoter in a binary vector pPZP201. The Escherichia coli phosphomannose isomerase (PMI) gene, pmi, was used as the selectable marker gene and mannose was used as the selective agent. The sgfp gene encoding green fluorescence protein (GFP) was the reporter gene and served as a visual screening marker. A total of 167 transgenic plants were obtained from nine different embryogenic callus lines grown on a selection medium containing 1%-2% mannose. Embryoids and shoots regenerated via embryogenesis, that showed strong GFP fluorescence, were selected from two sorghum genotypes: C401, an inbred line, and Pioneer 8505, a commercial hybrid. The GFP accumulation in transgenic plants was observed with a dissecting stereomicroscope. The integration and expression of the manA gene was confirmed by Southern blot and Western blot analyses, and the feasibility of manA selection was demonstrated by the chlorophenol red (CPR) assay. Our results indicated that transgenes segregated in the Mendelian fashion in the T1 generation. The conversion of mannose to a metabolizable fructose carbon source is beneficial to plants. In addition, except in soybean and a few legumes, no endogenous PMI activity has been detected in plant species, indicating that PMI is useful in the transformation of sorghum. In addition, PMI has no sequence homology to known allergens. Optimization of this selection system for sorghum transformation provides an efficient way to produce transgenic plants without using antibiotic or herbicidal agents as selectable markers, and our results showed that the transformation efficiency reached 2.88% for Pioneer 8505 and 3.30% for C401, both values higher than in previously published reports.

Journal Article↗

PubMed2002

Molecular breeding of transgenic rice plants expressing a bacterial chlorocatechol dioxygenase gene.

The cbnA gene encoding the chlorocatechol dioxygenase gene from Ralstonia eutropha NH9 was introduced into rice plants. The cbnA gene was expressed in transgenic rice plants under the control of a modified cauliflower mosaic virus 35S promoter. Western blot analysis using anti-CbnA protein indicated that the cbnA gene was expressed in leaf tissue, roots, culms, and seeds. Transgenic rice calluses expressing the cbnA gene converted 3-chlorocatechol to 2-chloromucote efficiently. Growth and morphology of the transgenic rice plants expressing the cbnA gene were not distinguished from those of control rice plants harboring only a Ti binary vector. It is thus possible to breed transgenic plants that degrade chloroaromatic compounds in soil and surface water.

Biodegradation, Environmental↗

PubMed2003

Plant transformation by coinoculation with a disarmed Agrobacterium tumefaciens strain and an Escherichia coli strain carrying mobilizable transgenes.

Transformation of Nicotiana tabacum leaf explants was attempted with Escherichia coli as a DNA donor either alone or in combination with Agrobacterium tumefaciens. We constructed E. coli donor strains harboring either the promiscuous IncP-type or IncN-type conjugal transfer system and second plasmids containing the respective origins of transfer and plant-selectable markers. Neither of these conjugation systems was able to stably transform plant cells at detectable levels, even when VirE2 was expressed in the donor cells. However, when an E. coli strain expressing the IncN-type conjugation system was coinoculated with a disarmed A. tumefaciens strain, plant tumors arose at high frequencies. This was caused by a two-step process in which the IncN transfer system mobilized the entire shuttle plasmid from E. coli to the disarmed A. tumefaciens strain, which in turn processed the T-DNA and transferred it to recipient plant cells. The mobilizable plasmid does not require a broad-host-range replication origin for this process to occur, thus reducing its size and genetic complexity. Tumorigenesis efficiency was further enhanced by incubation of the bacterial strains on medium optimized for bacterial conjugation prior to inoculation of leaf explants. These techniques circumvent the need to construct A. tumefaciens strains containing binary vectors and could simplify the creation of transgenic plants.

Agrobacterium tumefaciens↗

PubMed2006

Transformation of rhizobia with broad-host-range plasmids by using a freeze-thaw method.

Several species of rhizobia were successfully transformed with broad-host-range plasmids of different replicons by using a modified freeze-thaw method. A generic binary vector (pPZP211) was maintained in Mesorhizobium loti without selection and stably inherited during nodulation. The method could extend the potential of rhizobia as a vehicle for plant transformation.

Alphaproteobacteria↗

PubMed1986

Activity of T-DNA borders in plant cell transformation by mini-T plasmids.

By using a binary vector system, we examined the requirements for border sequences in T-DNA transformation of plant genomes. Mini-T plasmids consisting of small replicons with different extents of pTiT37 T-DNA were tested for plant tumor-inducing ability in Agrobacterium tumefaciens strain LBA4404 containing helper plasmid pAL4404 (which encodes virulence genes needed for T-DNA transfer). Assays of these bacteria on carrot disks, Kalanchoë leaves, and SR1 Nicotiana tabacum plantlets showed that mini-T plasmid containing full length T-DNA including left and right borders was highly virulent, as were mini-T plasmids containing all onc (oncogenicity) genes and only the right border. In contrast, mini-T plasmids containing all onc genes and only the left border induced tumors only rarely, and a mini-T plasmid containing all onc genes but no T-DNA borders was completely avirulent. Southern hybridization analyses of tumor DNA showed that T-DNA border sequences delimited the extent of the two-border mini-T plasmid transferred and integrated into the plant genome. When only one T-DNA border was present, it formed one end of the transferred DNA, and the other end mapped in the vector sequences. The implications of these results for the mechanism of T-DNA transfer and integration are discussed.

Cell Transformation, Neoplastic↗

PubMed1986

The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA.

We used a binary-vector strategy to study the hypervirulence of Agrobacterium tumefaciens A281, an L,L-succinamopine strain. Strain A281 is hypervirulent on several solanaceous plants. We constructed plasmids (pCS65 and pCS277) carrying either the transferred DNA (T-DNA) or the remainder of the tumor-inducing (Ti) plasmid (pEHA101) from this strain and tested each of these constructs in trans with complementary regions from heterologous Ti plasmids. Hypervirulence on tobacco could be reconstructed in a bipartite strain with the L,L-succinamopine T-DNA and the vir region on separate plasmids. pEHA101 was able to complement octopine T-DNA to hypervirulence on tobacco and tomato plants. Nopaline T-DNA was complemented better on tomato plants by pEHA101 than it was by its own nopaline vir region, but not to hypervirulence. L,L-Succinamopine T-DNA could not be complemented to hypervirulence on tobacco and tomato plants with either heterologous vir region. From these results we suggest that the hypervirulence of strain A281 is due to non-T-DNA sequences on the Ti plasmid.

Amino Acids↗

PubMed1987

Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281.

Agrobacterium tumefaciens A281 induces large, rapidly appearing tumors on a variety of plants and has a wider host range than other strains of A. tumefaciens. By using Tn3HoHo1 transposon mutagenesis and complementation analysis, a 2.5-kilobase DNA fragment which is responsible for the supervirulence phenotype was identified in the virulence (vir) region of the Ti plasmid. This fragment contains the virG locus, as well as the 3' end of the virB operon. A clone of this fragment conferred the supervirulence phenotype on A348, a nonsupervirulent strain. The increased virulence was correlated with an increased expression of vir genes, which could be achieved by introducing an extra copy of the transcriptional activator virG or the supervirulence region for maximum virulence. The virulence of the supervirulent strain A281 could be increased even further if the entire virB operon was added in addition to the virG operon. A plasmid, pToK47, containing virB and virG increased the virulence of all A. tumefaciens strains into which the plasmid was introduced. These data suggest that a highly virulent binary vector system can be constructed which might prove especially useful in the transformation of certain higher plants.

Cloning, Molecular↗

PubMed1995

Isolation and characterization of Bacillus subtilis groE regulatory mutants: evidence for orf39 in the dnaK operon as a repressor gene in regulating the expression of both groE and dnaK.

An inverted repeat sequence known as CIRCE (controlling inverted repeat of chaperone expression) in the Bacillus subtilis groE operon has been suggested to function as an operator. To identify the regulatory gene directly or indirectly involved in CIRCE-mediated heat-inducible groE expression, B. subtilis WBG2, carrying an integrated groE-bgaB transcription fusion in the amyE locus, was mutagenized. Dark blue colonies formed at 37 degrees C represent mutants which constitutively produce BgaB (a thermostable beta-galactosidase) at high levels. Seven mutants (WBG101 to WBG107) were selected for further characterization. They all overproduced BgaB, GroEL, and DnaK simultaneously at 37 degrees C. These mutants could be restored to normal by introducing a plasmid carrying a functional copy of orf39, the first gene in the B. subtilis dnaK operon. Genomic sequencing of these mutants demonstrated that they all carried a single mutation in orf39. These mutations can be divided into three groups: (i) Gly-307 to Asp, (ii) Ser-122 to Phe, and (iii) Gly-63 to Glu. By using a binary vector system in E. coli, production of ORF39 was found to negatively regulate the expression of groE-bgaB in a CIRCE-specific manner. Under the heat shock condition, the negative regulation mediated by ORF39 was abolished. Mobility shift of the CIRCE-containing probe was also observed with the crude extract prepared from the E. coli strain that overproduced ORF39. Therefore, ORF39 is the negative regulatory factor which regulates both groE and dnaK expression in B. subtilis. It is likely to function as a CIRCE-specific repressor.

Amino Acid Sequence↗

PubMed2005

Efficient genetic transformation of Sorghum using a visual screening marker.

To transform grain sorghum (Sorghum bicolor (L.) Moench) with a visual reporter gene (gfp) and a target gene (tlp), three genotypes (two inbreds, Tx 430 and C401, and a commercial hybrid, Pioneer 8505) were used. We obtained a total of 1011 fertile transgenic plants from 61 independent callus lines, which were produced from 2463 zygotic immature embryos via Agrobacterium-mediated transformation. The reporter gene, gfp, encoding green fluorescent protein (GFP), was used as a visual screening marker, and the target gene, tlp, encoding thaumatin-like protein (TLP), was chosen for enhancing resistance to fungal diseases and drought. Both genes were under the control of the maize ubi 1 promoter in the binary vector pPZP201. A total of 320 plants showing GFP expression, derived from 45 calli, were selected and analyzed by Southern blot analysis. There was a 100% correlation between the GFP expression and the presence of the target gene, tlp, in these plants. Transgenic plants showing strong TLP expression were confirmed by Western blotting with antiserum specific for TLP. The transgene segregated in various ratios among progeny, which was confirmed by examining seedlings showing GFP fluorescence. The progeny also showed different copy numbers of transgenics. This report describes the successful use of GFP screening for efficient production of stably transformed sorghum plants without using antibiotics or herbicides as selection agents.

Agrobacterium tumefaciens↗

PubMed2005

In vivo performance of a dual genetic marker, manA-gfp, in transgenic bentgrass.

A dual-marker combination, manA-gfp, comprising 2 independent expression cassettes of genes encoding an Escherichia coli phosphomannose isomerase (PMI) and a synthetic green fluorescent protein (GFP), was incorporated into the binary vector pPZP201. Agrobacterium tumefaciens-mediated transfer was used to introduce the manA-gfp into the mature-seed derived calli of Agrostis stoloifera L. 'Crenshaw'. The putative transgenic bentgrass calli were screened in Murashige and Skoog medium containing 15 g mannose/L, in conjunction with a visual examination of the GFP expression with a fluorescence stereomicroscope. Calli with GFP fluorescence grew well on the mannose selection media. A total of 24 transgenic plants derived from a single piece of callus lobe were studied for the genomic integration, expression, and function of the transgene. Genomic integration of the dual markers manA and gfp was confirmed by Southern blotting analysis, and the expression of manA also was validated by using PMI-specific antiserum. The inheritance and expression of the dual marker, manA-gfp, was demonstrated in the T1 generation. This study on the environmentally friendly markers further documented the feasibility of using alternative selection methods without using herbicide- or antibiotic-resistance markers.

Agrostis↗

PubMed1994

[Molecular genetics and biotechnology in medicinal plants: studies by transgenic plants].

The advances in molecular genetics and biotechnology in the field of medicinal plant research are discussed with focusing on the works using transgenic plants. Differentiated organ cultures and transgenic teratomas, incited by the infection with mutants of Agrobacterium Ti and Ri plasmids, were established in quinolizidine-alkaloid producing plants and Solanaceae plants. These cultured cells were used for the production and bioconversion of specific alkaloids produced in these plants. The methods of integration of foreign genes into medicinal plants were developed using an Ri binary vector. The mode of gene expression driven by TR1'-2' promoters was elucidated in transgenic medicinal plants, e.g., Nicotiana tabacum, Glycyrrhiza uralensis, Digitalis purpurea and Atropa belladonna. The genes for herbicide resistance, mammalian cytochrome P450 and bacterial beta-hydroxydecanoylthioester dehydrase were transferred and expressed in plants either to confer herbicide-resistant trait or to change the pattern of metabolites. The cDNA clones encoding cysteine synthase responsible for sulfur assimilation and biosynthesis of non-protein amino acids were isolated and characterized from Spinacea oleracea and Citrullus vulgaris. The functional lysine residue was identified by site-directed mutagenesis experiments. An over-expression system in Escherichia coli was constructed for the bacterial production of the plant specific non-protein amino acids. We made transgenic N. tabacum integrated with sense- and antisense-constructs of cysteine synthase cDNA driven by cauliflower mosaic virus 35S promoter for the purpose of genetic manipulation of biosynthetic flow of cysteine in plants. The future prospects of medicinal plant research are also discussed in the context of modern plant molecular biology.

Alkaloids↗

PubMed2005

Development of simple and efficient in planta transformation method for rice (Oryza sativa L.) using Agrobacterium tumefaciens.

Seeds of rice (Oryza sativa L. var. Koshihikari) were soaked in water for 2 d. Thereafter, the embryo containing an apical meristem was inoculated with Agrobacterium tumefaciens by piercing a site of the husk overlying the embryonic apical meristem with a needle that had been dipped in an A. tumefaciens inoculum. The inoculated seeds were then grown to maturation (T0 plants) and allowed to pollinate naturally to set seeds (T1 plants) in pots under nonsterile conditions. To examine the transformation by various means, three different strains of A. tumefaciens were used for transformation: an M-21 mutant, which is an avirulent mutant with a Tn5 insertion in the iaaM gene, and two LBA4404 strains each with a different binary vector. Five different lines of evidence were demonstrated the transformation: the altered phenotype and its inheritance by the next generation, histochemical detection of beta-glucuronidase, resistance to hygromycin B, detection of the transgene by PCR and rescue of a plasmid consisting of the integrated T-DNA and the flanking rice genome DNA. Transformation efficiency of T1 plants was estimated to be 40% and 43% by PCR and a histochemical assay of beta-glucuronidase, respectively.

Agrobacterium tumefaciens↗

PubMed2006

[BcMF4 gene, encoding a leucine-rich repeat protein, plays a role in male fertility in Chinese cabbage-pak-choi].

The BcMF4 (Brassica campestris Male Fertility 4) gene was previously isolated from the fertile B line of Chinese cabbage-pak-choi (Brassica campestris ssp. chinensis var. communis, syn. B. rapa ssp. chinensis var. communis). In the present paper, based on the cDNA sequence of BcMF4, primers were designed and used to amplify two fragments from the cDNA of flower buds of Chinese cabbage-pak-choi. Two produced fragments were introduced separately into binary vector pBI121 in antisense and sense orientations. The generated RNA interference (RNAi) vector was then mobilized into Agrobacterium tumefaciens strain LBA4404. The A. tumefaciens harboring the BcMF4 fragments was transformed to flowering Chinese cabbage (B. campestris ssp. chinensis var. parachinensis) via tissue culture. Approximately 45.8% of the pollen grains from 72.2% of RNAi plants exhibited abnormal in their shapes, and only 23.7% of the pollen grains from these plants germinated normally. Northern blotting demonstrated that the phenotypic change of pollen grains resulted from the inhibition of expression of the BcMF4 due to the insertion of the transgene. This indicates that functional interrupting of BcMF4 by RNAi resulted in partial pollen abortion in flowering Chinese cabbage, suggesting that the product of BcMF4 gene plays an important role during pollen development of Chinese cabbage such as Chinese cabbage-pak-choi and flowering Chinese cabbage.

Brassica↗

PubMed2006

Barley (Hordeum vulgare L.).

Crop improvement is limited by the availability of valuable traits in sexually compatible species. Access to new characters using genetic engineering would be of great value. Barley has been transformed using microprojectile bombardment and by direct gene transfer to protoplasts, but neither method has been able to produce fertile transformants in large numbers with simple transgene integration characteristics. Agrobacterium-mediated transformation was first achieved in 1997, and it has become the method of choice. Using immature embryos of the barley variety Golden Promise as the target organ, the binary vector pWBVec8 containing the intron-interrupted hygromycin resistance gene hph as the selectable marker, and selection of transformed cells on hygromycin, the Agrobacterium method is efficient, and the transgene insertion characteristics are superior to other methods. However, the procedure is strongly genotype dependent. In this report, we describe a transformation protocol giving details of plant culture, embryo isolation and preparation, vector details, Agrobacterium culture, infection methods, subsequent procedures for callus generation and plantlet production, and analysis of transgenic plants.

Drug Resistance↗

PubMed2006

Maize (Zea mays L.).

Agrobacterium tumefaciens-mediated transformation is an effective method for introducing genes into maize. In this chapter, we describe a detailed protocol for genetic transformation of the maize genotype Hi II. Our starting plant material is immature embryos cocultivated with an Agrobacterium strain carrying a standard binary vector. In addition to step-by-step laboratory transformation procedures, we include extensive details in growing donor plants and caring for transgenic plants in the greenhouse.

Agrobacterium tumefaciens↗

PubMed2006

Sorghum (Sorghum bicolor L.).

This chapter describes a stepwise protocol for Agrobacterium-mediated sorghum genetic transformation. Immature embryos from sorghum plants were used as the target explants. The Agrobacterium strain LBA4404, carrying a "super-binary" vector, was used in this protocol. Agrobacterium co-transformation vectors, one T-DNA containing the selectable marker gene and another T-DNA containing the trait gene(s), were also introduced in sorghum transformation for eliminating the selectable marker gene in the resulting transgenic plants. This chapter provides recommendations for analysis of the transgenic plants to confirm T-DNA integration into the sorghum genome and segregation of the selectable marker gene from the trait gene(s).

Agrobacterium tumefaciens↗

PubMed2006

Wheat (Triticum aestivum L.).

This chapter describes a procedure for Agrobacterium-mediated wheat transformation. Freshly isolated immature embryos, precultured immature embryos, or embryogenic calli are inoculated with a disarmed A. tumefaciens strain C58 (ABI) harboring the binary vector pMON18365 containing the beta-glucuronidase (GUS) gene with an intron, and a selectable marker, neomycinphosphotranferase (NPT) II gene. The inoculated explants are selected on callus induction medium with the selective agent G418 for approximately 2 wk. The resistant callus pieces that develop are then transferred onto medium with the selective agent and reduced plant growth regulators for plant regeneration and further selection. It takes approximately 2.5 to 3 mo from inoculation to the establishment of R0 plants in soil. All the transformants should be morphologically normal and set seeds. Approximately 35% of the transgenic plants have a single copy of the transgene based on data obtained from previous experiments.

Agrobacterium tumefaciens↗

PubMed2006

Three methods for the introduction of foreign DNA into Agrobacterium.

The genetic manipulation of Agrobacterium tumefaciens is used to facilitate studies of bacterial gene functions or as a first step in introducing genetic material into transformable plant cells through the use of T-DNA binary vectors. Three methods are commonly used. Transformation with purified plasmid can be done with either electroporation or a simple freeze/thaw transformation method. Alternatively, a mobilizable plasmid can be placed into Agrobacterium using the triparental mating method. Here we present three detailed protocols for Agrobacterium strain construction using electroporation, the freeze/thaw method of transformation, and triparental mating.

Agrobacterium tumefaciens↗