Enhancement of transformation rates in higher plants by low-dose irradiation: Are DNA repair systems involved in the incorporation of exogenous DNA into the plant genome?

Köhler, Friedrich; Cardon, Guillermo H.; Pöhlman, M.; Gill, R.; Schieder, O.

doi:10.1007/bf00020504

Cited by 86 publications

(46 citation statements)

References 38 publications

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“…Using total genomic DNA from B. nigra for PEG mediated transformation of B. napus, Golz et al (1990) transferred a marker gene for hygromycin resistance. Pretreating protoplasts with X-rays (lOGy) prior to PEG mediated DNA transformation increased the number of transformed calli five times, but no regeneration was achieved (Kohler et al 1989). …”

Section: Vectorless Transformationmentioning

confidence: 99%

Genetic transformation of Brassica

Poulsen

1996

Plant Breeding

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Tissue culture regeneration of plants from Brassica species can be achieved from various explant types. Their performance, however, varies with the genotype and age of the explant and is sensitive to medium supplements and Agrobacterium cocultivation. Most Brassica transformations were carried out using Agrobacterium and it was established that nopaline and agropine strains of .4. tumefaciens and A. rhizogenes, respectively, are the most efficient. Application of virulence gene induction is ambiguous. Binary vectors are mostly apphed in spite of indications that cointegrate vectors are more efficient. Cotransformation is successful with two different bacteria or with one bacterium carrying both plasmids, however, it has proved problematic to integrate the two transgenes into separate loci. Transformations with wild type rol genes develop hairy roots, from which transgenic plants can be regenerated. Vectorless transformation is feasible with microprojectile bombardment of microspore derived embryos being the most promising.Antibiotic markers providing resistance to kanamycin, hygromycin, and spectinomycin are applicable as selectable markers, while chloramphenicol is not suitable. Among herbicides, phosphinotricin gives good results while chlorsulfuron is unsuitable for in vitro selection. Major therapeutic antibiotics being successfully applied are carbenicillin, cefotaxime, amoxycillin, and ticarcillin. Standard methods for evaluating transgenic plants also have been applied to Brassica species. Some breeding objectives obtained through genetic transformation are presented.

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Section: Vectorless Transformationmentioning

confidence: 99%

Genetic transformation of Brassica

Poulsen

1996

Plant Breeding

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“…lonizing radiation has also been shown to increase the efficiency of foreign DNA integration into the plant genome (assumed to occur by illegitimate recombination); a threefold to sixfold increase in the recovery of transformants was observed when freshly isolated protoplasts were exposed to x-rays shortly after being transfected with a plasmid containing a chimeric selectable marker (Kohler et al, 1989). Because the stress of tissue culture appears not to induce recombination (Peterhans et al, 1990) and our irradiation experiments used in vitro cultured cells, it is clear that the increase in the number of Kmr calli recovered represents a genuine enhancement of homologous recombination frequency in these plant cells.…”

Section: Frequency Of Recombinants Recoveredmentioning

confidence: 99%

Somatic and Meiotic Chromosomal Recombination between Inverted Duplications in Transgenic Tobacco Plants.

Tovar

Lichtenstein

1992

Plant Cell

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Homologous recombination has been extensively studied in bacteria, yeast, and more recently in animal cells, but little is known about this process in plants. We present here an analysis of meiotic and somatic chromosomal recombination between closely linked inverted duplications located on a single chromosomal region in tobacco. Transgenic tobacco lines were constructed by Agrobacterium transformation with plasmid vectors containing a functional hygromycin phosphotransferase (hyg) selectable marker flanked by a pair of defective neomycin phosphotransferase (neo) genes positioned as inverted repeats. As each neo gene is mutated in a different site, recombination between the two defective genes can be detected following selection for kanamycin-resistant plant cells. The recombination substrates were designed to allow investigation into the nature of molecular events underlying homologous recombination by restriction endonuclease analysis. Chromosomal recombination was studied in mitotically dividing cells (cultured leaf mesophyll cells) and after meiosis (germinated seedlings). Spontaneous somatic recombinants were recovered at frequencies between ~3 x 10-5 to 10-6 events per cell. Low dose y irradiation of somatic cells resulted in a threefold maximum increase in the recovery of recombinants. Recombinants were also detected at low frequency when transgenic T3 seeds were germinated under kanamycin selection. DNA gel blot analyses demonstrated that homologous recombination occurred mainly as gene conversion unassociated with reciprocal exchange, although a variety of other events including gene coconversion were also observed.

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“…T-DNA integrates at random sites (Kim et al, 2007) and preferentially into double-strand breaks (DSBs) (Salomon and Puchta, 1998;Chilton and Que, 2003;Tzfira et al, 2003). T-DNA integration into DSBs was inferred from increased foreign gene insertions into plants irradiated by x-rays (Köhler et al, 1989) and from plants whose genomes were cut by rare-cutting endonucleases (Salomon and Puchta, 1998;Chilton and Que, 2003;Tzfira et al, 2003). The process of T-DNA integration is not well understood.…”

Section: Introductionmentioning

confidence: 99%

Agrobacterium May Delay Plant Nonhomologous End-Joining DNA Repair via XRCC4 to Favor T-DNA Integration

et al. 2012

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Agrobacterium tumefaciens is a soilborne pathogen that causes crown gall disease in many dicotyledonous plants by transfer of a portion of its tumor-inducing plasmid (T-DNA) into the plant genome. Several plant factors that play a role in Agrobacterium attachment to plant cells and transport of T-DNA to the nucleus have been identified, but the T-DNA integration step during transformation is poorly understood and has been proposed to occur via nonhomologous end-joining (NHEJ)-mediated doublestrand DNA break (DSB) repair. Here, we report a negative role of X-RAY CROSS COMPLEMENTATION GROUP4 (XRCC4), one of the key proteins required for NHEJ, in Agrobacterium T-DNA integration. Downregulation of XRCC4 in Arabidopsis and Nicotiana benthamiana increased stable transformation due to increased T-DNA integration. Overexpression of XRCC4 in Arabidopsis decreased stable transformation due to decreased T-DNA integration. Interestingly, XRCC4 directly interacted with Agrobacterium protein VirE2 in a yeast two-hybrid system and in planta. VirE2-expressing Arabidopsis plants were more susceptible to the DNA damaging chemical bleomycin and showed increased stable transformation. We hypothesize that VirE2 titrates or excludes active XRCC4 protein available for DSB repair, thus delaying the closure of DSBs in the chromosome, providing greater opportunity for T-DNA to integrate.

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Enhancement of transformation rates in higher plants by low-dose irradiation: Are DNA repair systems involved in the incorporation of exogenous DNA into the plant genome?

Cited by 86 publications

References 38 publications

Genetic transformation of Brassica

Genetic transformation of Brassica

Somatic and Meiotic Chromosomal Recombination between Inverted Duplications in Transgenic Tobacco Plants.

Agrobacterium May Delay Plant Nonhomologous End-Joining DNA Repair via XRCC4 to Favor T-DNA Integration

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