T-DNA transfer, integration, expression and inheritance in rice: effects of plant genotype and Agrobacterium super-virulence (original) (raw)
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Agrobacterium-mediated transformation of indica rice cultivars using binary and superbinary vectors
Functional Plant Biology, 1999
The Agrobacterium-mediated transformation system was extended to two indica cultivars: a widely cultivated breeding line IR-64 and an elite basmati cultivar Karnal Local. Root tips and shoot tips of seedlings, and scutellar-calli derived from mature seeds showed high-efficiency Agrobacterium tumefaciens infection and stable transformation. In addition to the superbinary vector pTOK233 in Agrobacterium strain LBA4404, almost equally high levels of transformation were achieved with a relatively much smaller (13.1 kb) binary vector (pCAMBIA1301) in a supervirulent host strain AGL1. In both cases, as well as in both cultivars, while 60-90% of the infected explants produced calli resistant to the selectable agent hygromycin, 59-75% of such calli tested positive for GUS. A high level (400 µM) of acetosyringone in the preinduction medium for Agrobacterium and a higher level (500 µM) in the cocultivation medium was necessary for an enhancement in transformation frequency of the binary vector to levels comparable to a superbinary. Hygromycin-resistant calli could be produced from all the explants used. Transformants could be regenerated for both cultivars using the superbinary and binary vector, but only for calli of scutellar origin. In addition to the molecular confirmation of hpt and gus gene transfer and transcription, absence of gene sequences outside the transferred DNA (T-DNA) region confirmed absence of any long T-DNA transfer.
Plant Cell Reports, 2011
Rice chitinase (chi11) and tobacco osmotin (ap24) genes, which cause disruption of fungal cell wall and cell membrane, respectively, were stacked in transgenic rice to develop resistance against the sheath blight disease. The homozygous marker-free transgenic rice line CoT23 which harboured the rice chi11 transgene was sequentially re-transformed with a second transgene ap24 by co-transformation using an Agrobacterium tumefaciens strain harbouring a single-copy cointegrate vector pGV2260::pSSJ1 and a multi-copy binary vector pBin19DnptII-ap24 in the same cell. pGV2260::pSSJ1 T-DNA carried the hygromycin phosphotransferase (hph) and b-glucuronidase (gus) genes. pBin19DnptII-ap24 T-DNA harboured the tobacco osmotin (ap24) gene. Co-transformation of the gene of interest (ap24) with the selectable marker gene (SMG, hph) occurred in 12 out of 18 T 0 plants (67%). Segregation of hph from ap24 was accomplished in the T 1 generation in one (line 11) of the four analysed co-transformed plants. The presence of ap24 and chi11 transgenes and the absence of the hph gene in the SMG-eliminated T 1 plants of the line 11 were confirmed by DNA blot analyses. The SMG-free transgenic plants of the line 11 harboured a single copy of the ap24 gene. Homozygous, SMG-free T 2 plants of the transgenic line 11 harboured stacked transgenes, chi11 and ap24. Northern blot analysis of the SMG-free plants revealed constitutive expression of chi11 and ap24. The transgenic plants with stacked transgenes displayed high levels of resistance against Rhizoctonia solani. Thus, we demonstrate the development of transgene-stacked and marker-free transgenic rice by sequential Agrobacteriummediated co-transformation with the same SMG. Keywords Agrobacterium Á Homozygous transgenic plants Á Marker-free transgenic plants Á Oryza sativa Á Sequential co-transformation Á Transgene stacking Communicated by H. Ebinuma.
Expression and Inheritance of GUS Gene in Transgenic Tobacco Plants
journals.tubitak.gov.tr
Agrobacterium tumefaciens-mediated transformation of tobacco leaf discs was performed with plant transformation vectors of pBI 121 and pGus-Int. Both carry the neomycin phosphotransferase II (npt II) gene and, unlike the pBI 121, pGus-Int carries a portable plant intron inside the beta-glucuronidase (GUS) gene. In addition to the integration of the marker genes into the genomes of primary transformants, R1 and R2 progenies were confirmed by polymerase chain reaction (PCR) and Southern blot analyses. Histochemical and fluorometric analyses were also performed to determine the activity of the GUS gene. Segregation of progenies on kanamycin-resistance trait was also shown as evidence for the transformation. The analyses showed that the transgenes were transmitted to subsequent generations in a Mendelian manner and intron sequences enhanced the expression of the GUS gene by 25-30% in some transformants.
Molecular and General Genetics MGG, 1999
We have identi®ed T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium tumefaciens (rat mutants). These mutants are highly recalcitrant to the induction of both crown gall tumors and phosphinothricin-resistant calli. The results of transient GUS (b-glucuronidase) assays suggest that some of these mutants are blocked at an early step in the Agrobacterium-mediated transformation process, whereas others are blocked at a step subsequent to translocation of T-DNA into the nucleus. Attachment of Agrobacterium to roots of the mutants rat1 and rat3 was decreased under various incubation conditions. In most mutants, the transformation-de®cient phenotype co-segregated with the kanamycin resistance encoded by the mutagenizing T-DNA. In crosses with susceptible wild-type plants, the resistance phenotype of many of these mutants segregated either as a semi-dominant or dominant trait.
African Journal of Biotechnology, 2011
Preliminary steps in the genetic transformation of indica rice MR219 was investigated in the plant-Agrobacterium tumefaciens interaction. Agrobacterium tumefaciens strain LBA 4404 carrying a binary vector pCAMBIA 1305.2 harboring the modified GUS gene driven by the CaMV 35S promoter was used. Various transformation parameters influences were optimized using embryogenic calli via βglucuronidase (GUS) as a reporter marker. Various transformation parameters were optimized including bacterial concentration, age of embryogenic callus, pre-culture period, wounding technique, cocultivation period, immersion time and dry time before co-cultivation, acetosyringone (AS) concentration, pH of co-cultivation media and temperature of the co-cultivation period. The expression of the transient gusA gene in the plant genome was preliminary confirmed by histochemical GUS assay activity (as blue spots). The results from transient gusA gene expression of calli suggested that the Agrobacterium-mediated transfer system of T-DNA in indica rice MR219 was highly efficient. Therefore, the investigation of factors that influence T-DNA delivery is an important first step in the utilization of Agrobacterium in the transformation of indica rice MR219 calli.
Horizontal gene transfer from Agrobacterium to plants
Frontiers in Plant Science, 2014
Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana and Linaria. In these natural examples of horizontal gene transfer from Agrobacterium to plants, the T-DNA donor is assumed to be a mikimopine strain of A. rhizogenes. A sequence homologous to the T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30 years ago, and was named "cellular T-DNA" (cT-DNA). It represents an imperfect inverted repeat and contains homologs of several T-DNA oncogenes (NgrolB, NgrolC, NgORF13, NgORF14) and an opine synthesis gene (Ngmis). A similar cT-DNA has also been found in other species of the genus Nicotiana. These presumably ancient homologs of T-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. Recently T-DNA has been detected and characterized in Linaria vulgaris and L. dalmatica. In Linaria vulgaris the cT-DNA is present in two copies and organized as a tandem imperfect direct repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. All L. vulgaris and L. dalmatica plants screened contained the same T-DNA oncogenes and the mis gene. Evidence suggests that there were several independent T-DNA integration events into the genomes of these plant genera. We speculate that ancient plants transformed by A. rhizogenes might have acquired a selective advantage in competition with the parental species. Thus, the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this review we focus on the structure and functions of cT-DNA in Linaria and Nicotiana and discuss their possible evolutionary role.
Plant Biotechnology, 2006
Southern hybridization-based zygosity analysis was done in a transgenic rice plant (Oryza sativa L. cv Pusa Basmati 1) generated by Agrobacterium-mediated transformation with a rice chitinase (chi11) gene. A T 0 plant with two unlinked T-DNA insertions (A and AЈ), was chosen for the application of Southern hybridization analysis to study genetic separation of the two loci by segregation and to identify the homozygous and hemizygous plants in T 1-generation. The T 1 plants showed differences in band intensities that reflected the homozygous and hemizygous status of each of the two integration events. The predictions of zygosity of T 1 plants were confirmed by analyzing segregation in T 2 plants. Southern hybridization analysis is demonstrated as a simple and effective method to distinguish hemizygous and homozygous plants in the T 1 generation itself.
The EMBO Journal
Communicated by C.J.Leaver We determined whether T-DNA molecules introduced into plant cells using Agrobacterium are suitable substrates for homologous recombination. For the detection of such recombination events different mutant versions of a NPII construct were used. In a first set of experiments protoplasts of Nicotiana tabacum SR1 were cocultivated with two Agrobacterium tumefaciens strains. Each strain contained a different T-DNA, one carrying a 5' deleted NPTII gene and the other a NPMII gene with a 3' deletion. A restored NPMII gene was found in 1-4% of the protoplasts that had been cotransformed with both T-DNAs. Restoration of the NPMI gene could only be the consequence of homologous recombination between the two different T-DNAs in the plant cell, since the possibility of recombination in Agrobacterium was excluded in control experiments. In subsequent experiments we investigated the potential use of Agrobacterium for gene targeting in plants. A transgenic tobacco line with a T-DNA insertion carrying a defective NPTII gene with a 3' deletion was transformed via Agrobacterium with a T-DNA containing a defective NP1'l repair gene. Several kanamycin resistant plant lines were obtained with an intact NPTIM gene integrated in their genome. In one of these lines the defective NPTII gene at the target locus had been properly restored. Our results show that in plants recombination can occur between a chromosomal locus and a homologous T-DNA introduced via A.tumefaciens. This opens the possibility of using the Agrobacterium transformation system for site directed mutagenesis of the plant genome.