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1

Regeneration of winter oilseed rape and trials of transformation mediated by Agrobacterium tumefaciens

100%
Starzycki M., Starzycka E., Matuszczak M.
Biotechnologia
|
1999
|
issue 1
164-171
EN
The effects of one strain Agrobacterium tumefaciens LBA 4404/pBI 121, conditions of co-cultivation and growth regulators on regeneration were studied and trials of transformation were performed on winter oilseed rape Brassica napus L. cv. Lirajet, Valesca and Bor. Strong shoots regeneration was observed after co-cultivation without acetosyringone. Among antibiotics that were used in the experiments, kanamycin A and geneticin hampered whereas carbenicillin and rifampicin stimulated shoots regeneration. The presence of nptII gene was confirmed by PCR and K1, K2 primers which generate a fragment of 732 bp in length.
2

Efficiency of transformation of Polish cultivars of pea (Pisum sativum L.) with various regeneration capacity by using hypervirulent Agrobacterium tumefaciens strains

100%
Pniewski T., Kapusta J.
Journal of Applied Genetics
|
2005
|
vol. 46
|
issue 2
139-147
EN
An Agrobacterium-mediated transformation method of pea has been developed for several edible and fodder cultivars of pea (Pisum sativum L.), characterized previously in their potential for regeneration via organogenesis. The most appropriate explant, which was susceptible to Agrobacterium infection and capable of regenerating transgenic plants, turned out to be a slice of an immature embryo, including the embryo axis and the basal part of a cotyledon. Three hypervirulent strains of A. tumefaciens were tested: AgL0, AgL1 and EHA105. Each carried the binary vector pP35SGIB containing the uid gene, with an intron under control of the 35S promoter, and the bar gene conferring resistance to phosphinotricin. Strain AgL0 was found to be efficient for the majority of cultivars, followed by AgL1 and EHA105. Transformation efficiency varied from 0.7 to 4.1%, depending on cultivar and Agrobacterium strain. The transformation efficiency of particular pea cultivars did not clearly correspond to their regeneration capacity, which ? although indispensable ? was not a critical parameter of successful transformation. The presence of integrated genes in pea genomic DNA was detected by the PCR. T-DNA was stably transmitted to the progeny, as it was confirmed by Southern hybridization. The activity of introduced genes was analysed by the histochemical GUS assay and by painting leaves or by spraying transgenic plants with the herbicide Basta.
3

Genetic transformation of Rhododendron sp.

100%
Madej M., Czernicki W., Klein M.
Biotechnologia
|
2006
|
issue 3
36-43
EN
The aim of our study is to review the results of genetic transformation of rhododendrons which has been published in scientific literature or presented during scientific conferences so far. Despite complicated and work-consuming protocol, genetic transformation has great potential to improve future ornamental plants. Rhododendrons of tomorrow could have desired morphological architecture and flower pigmentation, resistance to diseases, pests and harmful environmental conditions. Gene transfer experiments that were carried out so far, proved successful. More and more significant factors are discovered during each investigation. However, that study has to be worked out in order to optimize the efficiency of genetic transformation. Then, we can speculate that traditional rhododendron breeding will be hastened. Breeders will have exclusive plants whose genomes will be transformed in terms of desirable features.
4

Agrobacterium T-DNA in the eukaryotic cell: nuclear import and integration into the plant genome

100%
Ziemienowicz A.
Biotechnologia
|
2000
|
issue 4
24-31
EN
Agrobacterium tumefaciens, a gram-negative soil bacterium, is able to transfer DNA to most plant species causing crown gall disease in dicotyledonous plants. Due to this activity Agrobacterium is widely used for plant transformation. The transferred DNA (T-DNA) that resides on a large Ti plasmid is processed within the bacterium and is exported to the plant where it is integrated into the chromosome. DNA transfer requires plasmid encoded virulence (vir) genes as well as several chromosomal genes. In vivo studies suggested that Agrobacterium proteins are involved in T-DNA transfer and integration. We study the function of virulence proteins VirD2 and VirE2 in T-DNA nuclear import and integration using in vitro systems. We found that the T-DNA is imported into the plant cell nucleus as a complex with VirD2 and VirE2 proteins. The C-terminal NLS of VirD2 has a piloting function in this process. Import of the T-DNA follows the classical NLS- and importin-dependent nuclear import pathway for proteins. For studies of integration of T-DNA into the plant DNA an in vitro integration/ligation assay has been designed. We have found out that VirD2 is not able to ligate the T-DNA to the plant DNA in vitro. Consequently, plant enzymes must be involved in this process. Indeed, we found an activity responsible for the ligation of T-DNA in extracts from tobacco BY2 suspension cultured cells and from pea axes. This activity is likely to originate from plant DNA ligase, since the T-DNA ligation shows the same requirements for hydrolysis of ATP to AMP as ligation mediated by any ATP-dependent DNA ligase. This does not, however, exclude the involvement of other plant enzymes in T-DNA integration.
5

Effect of initial explant on effectivity of Agrobacterium tumefaciens-mediated transformation of Gerbera hybrida

100%
Nowak E., Rojek Z., Kucharska D., Orlikowska T.
Biotechnologia
|
1997
|
issue 4
27-37
EN
An efficient method for genetic Agrobacterium tumefaciens - mediated transformation of five cultivars Gerbera hybrida was established. The youngest leaves and shoot tips from proliferating in vitro cultures were co-cultivated with disarmed strain LBA 4404/pBI121 carrying the chimaeric genes nptII and gus. The inoculated explants were repeatedly cultured on regeneration medium with kanamycin and Biotaxym. After 9-10 months from 0.04 to 0.25 independently transformed shoots from one inoculated leaf and from 0.06 to 0.65 from one inoculated shoot were obtained, depending on cultivar and additional treatment. Transformed shoots accounted for 5.0 to 50.0% of all regenerated shoots. Additional wounding` of leaves before inoculation by squeezing and scratching off increased transformation efficiency. Better results for recalcitrant to regeneration cultivars were obtained if squeezed shoot tips were inoculated. Regenerating shoots were selected on kanamycin, screened for gus expression with X-gluc and tested by PCR for nptII gene. All together 162 transgenic shoots were obtained in cvs Amber, Boy, Ferrari, Mariola and Tamara, but only 24 relatively stable lines were established.
6

Mechanism of Agrobacterium-mediated genetic transformation of plant cell

100%
Nadolska-Orczyk A.
|
EN
Agrobacterium-mediated genetic transformation is the only known example of horizontal gene transfer from bacteria to eucaryota including plants, fungi and animal cells. The knowledge of the basic mechanism of this process is the key to understand problems concerning methods of plant transformation and transgene expression. The main element of the system is Ti plasmid (tumor-inducing) containing T-DNA (transferred DNA) delimited by 25. nucleotide sequences (left and right borders). Any DNA located on Ti plasmid and flanked by these two borders might be recognised by Agrobacterium as a T-DNA and integrated into plant chromosome. The process is controlled by ten vir operons located on Ti plasmid. The most important products of the vir genes are VirA and VirG controlling the expression of all the other vir genes. VirD1 and VirD2 recognise 25 bp border sequences and take part in endonucleolitic cleavage. Additionally, VirD2 covalently attached to the 5?-end of single stranded (ss) T-DNA targets it into the nucleus of a plant cell. T-strand is coated by VirE2 molecules, each containing two sites of nuclear localisation signals (NLS). Eleven of VirB and VirD4 proteins are required to form a transmembrane channel and transfer T-strand to the plant cell. Some genes of the bacterial chromosome are responsible for bacterial attachment and colonisation of the plant cell.
7

Agrobacterium-mediated genetic transformation of cereal crops

88%
Nadolska-Orczyk A., Przetakiewicz A., Orczyk W.
Biotechnologia
|
2000
|
issue 4
93-98
EN
The results published in recent years proved that Agrobacterium based system for genetic transformation was also suitable for cereal crops. Several groups were able to obtain transgenic rice, corn, wheat and barley using hipervirulent strains Agl1 and EHA101 (or EHA105) or 'regular' LBA4404 strain with superbinary vector pTOK233. The first phase of our research was designed to establish transformation susceptibility of two wheat, two barley and one triticale cultivars using three different bacterial systems. Two of those systems were based on hipervirulent strains: Agl1 (pDM805) and EHA101 (pGAH). The third one combined strong virulence of pTOK233 vector and commonly-used LBA 4404 strain. Putative transgenic plants (regenerated and rooted under selective pressure of appropriate factor and further confirmed with GUS or PCR) were obtained for barley (cultivar Scarlett), wheat (Torka and Kontesa) and triticale (Wanad) with Agrobacterium strain Agl1. Kontesa's putative transgenics were also obtained with the strain EHA101. The highest rate of selection of putative transgenics was for Agl1 / phosphinothricine and ranged from 9 to15% for wheat cultivars. The lowest rate for the same strain and selection was 0,5% for barley cv. Scarlett.Inoculation of 700 immature embryos of barley cv. Lot with three bacterial systems (strains, vectors and selection factors) failed to produce putative transformants. Also no putative transgenics of barley Scralett, wheat Torka and triticale Wanad were obtained after transformation with EHA101 and selection on higromycine. Selection with kanamycin and hygromycin + kanamycin after transformation with EHA101 and LBA4404 respectively also failed to give positive results.
8

Transformation of plants using Agrobacterium tumefaciens

87%
Orlikowska T.
Biotechnologia
|
1994
|
issue 1
32-43
9

Agrobacterium-mediated transformation of yellow lupin to generate callus tissue producing HBV surface antigen in a long-term culture

75%
Pniewski T., Kapusta J., Plucienniczak A.
Journal of Applied Genetics
|
2006
|
vol. 47
|
issue 4
309-318
EN
The idea of an oral vaccine administered as a portion of plant tissue requires a high level of antigen production. An improved protocol for the induction of transgenic yellow lupin calli or tumours, reaching 44% of transformation rate, is presented here. It has been developed by using the nptII marker gene and the uidA reporter gene as well as various Agrobacterium strains and plant explants. This method of seedling and hypocotyl transformation was applied to raise calli or tumours producing a small surface antigen of Hepatitis B Virus (S-HBsAg). Lupin tissue lines were long-term cultured on selection media maintaining the growth rate and high expression level of the native form of S-HBs, up to 6 mug per g of fresh tissue.
10

Alfalfa (Medicago sativa) as an object for transformation by Agrobacterium tumefaciens and in vitro regeneration

75%
Lisova O., Legocki A. B., Legocki A. B.
Biotechnologia
|
2002
|
issue 3
74-90
EN
From the economical point of view alfalfa is a very valuable plant. It has very high yield potential, it is rich in protein, vitamins and minerals, so it is prized as very important feed for horses, beef cattle, sheep, goats and other domestic animals. That is why the improving of alfalfa quality by the methods of molecular biotechnology is a very interesting issue. In this review information about the crucial elements of alfalfa transformation and regeneration are gathered together. Among other following elements, genetic background of plant, A. tumefaciens strains, explant types, in vitro culture medias, plant development stages are discussed. It seems that optimalisation of the transformation and regeneration procedure are individual for every plant genotype and A. tumefaciens strain.
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