Vectors Based Ti Plasmids, Promoters, Polyadenylation Signals, Disarming, Antibiotics, Kanamycin, Tumour Induction Property, Cointegrative Vectors

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Gene Transfer Methods in Plants Gene Transfer Methods in Plants Introduction Target Cells for Transformation Theoretical Routes for Production of Transgenic Plants Vectors of Gene Transfer Vectors based on Ti and Ri Plasmids of Agrobacterium Structure and Function of Ti and Ri Plasmids Selectable Marker Genes in Vectors Used for Gene Transfer T-DNA Transfer Process Vectors based on Ti and Ri Plasmids Virus Vectors Transformation Techniques Using Agrobacterium Prerequisites for Production of Transgenic Plants Explants Used for Transformation Marker Genes for Selection and Scoring of Tranformed Cells/Calli or Shoots Reporter Genes Used as Scoreable Markers and their Enzyme Assays Neomycin Phosphotransferase Gene (nptII) Chloramphenicol Acetyl Transferase Gene (cat)

Home >> Plant Biotechnology and Genomics >> Gene Transfer Methods in Plants >>Vectors Based On Ti and Ri Plasmids

Vectors based 00 Ti and Ri plasmids.
The following properties of Ti plasmids did not allow their direct use as vectors: (i) large size, (ii) absence of unique restriction enzyme sites and (iii) tumour induction property. Therefore, only the useful attributes of Ti plasmid have been used in designing several plant transformation vectors. Since tumour cells can not develop into normal shoots, disarming of Ti plasmids was an essential step in designing useful vectors. This was achieved by replacement of tumour inducting genes in T-DNA (T-DNA is found in both Ti and Ri plasmids) by selectable markers (like nptIl),providing resistance against antibiotics like kanamycin. Promoters and polyadenylation signals isolated from octopine or nopaline synthase genes were used for expression of selectable markers. Other powerful promoters included CaMV3SS and CaMV19S isolated from cauliflower mosaic virus.

As is obvious from the discussion in the previous section, T-DNA (disarmed) and virgenes are two essential elements for designingtransformation vectors. While T-DNA with border sequences allows manipulation of DNA sequences intended to be transferred, virgenes allow infection of host plant. Since vir genes can function even in trans configuration, it is not essential that virgenes be present on the same plasmid, which functions as a vector and carries DNA segment to be transferred. In view of this, basically two types of Agrobacteriumvectors are currently in use: (i) Cointegrative vectors recombine, via DNA homology, with an intermediate cloning vector, which is used for manipulation and cloning of the gene in E. coli.

(a) Use of the cointegrate vector pGV3850 with the intermediate pGV1103.

(b)Preparation of a cointegrative Vector for the transfer of a desired DNA segment.

Preparation of a Cointegrative Vector for the transfer of a desired DNA segment

Agrobacteriumcontaining cointegrative vector and E. coli containing intermediate cloning vector are allowed to undergo conjugation, since the intermediate vector can not replicate in Agrobacterium.The intermediate vector has to transfer the marker genes as well as the DNA segment to the resident Ti plasmid (cointegrative vector) through recombination in the region of DNA homology. One of the first cointegrative vectors (pGV3850) was developed from a nopaline-type Ti plasmid (C58), where almost all T-DNA has been deleted and replaced by pBR322, a common small E. colicloning vector. The intermediate vector (e.g. pGV1103) based on pBR322.undergoes recombination with pGV3850 in the region of pBR322 homology. After cointegration, pGV3850 : 1103 T-DNA contains the whole of intermediate vector including the unwanted DNA, which is also transferred to host plant along with the gene intended to be transferred. To overcome this difficulty the right border repeat or both borders are introduced to flank a cloning site in the intermediate vector, so that only a part of intermediate vector cointegrates. The utility of this vector system is however limited. (ii) Binary vectors are based on the principle that vir genes may be located on a 'helper'Ti plasmid having the whole of T-DNA deleted (e.g. pAL4404), because virgenes can function even in transconfiguration.

In this case, T-DNA is found on a separate vector (binary vector) designed to replicate in both E. coli and Agrobacterium and capable of conjugal transfer between these two bacterial species. Many binary vectors have been developed, which differ in size and the source of 25bp repeat sequences, plant selection marker, bacterial selection marker and cloning sites. pBin19, which was designed in 1984, is still popular and is based on wide host-range replicon pRK252.

Typical Binary Vector Strategy

Typical Binary Vector Strategy

1.Hind Sal Xba Bam Sma Kpn Eco

2.Multicloning site

3. Nos-npt-II

4.Bacterial selectable marker gene (APH-1)

5. (a) Origin of transfer and bom site

(b) wide host range

6.binary vector pBin19

7.Binary Vector System

8.Provided by RK2 sequences

9. Virulence-helper-Ti-plasmid-pAL 4404

This vector contains the following elements: (i) kanamycin resistance gene (APH-l)for selection .in bacteria, (ii) T-DNA borders derived from pTiT37, (iii) a plant-selectable transformation marker (nptIl) isolated from transposon Tn5 (this marker is associated with promoter and polyadenylation signal derived from nopaline synthesis gene or nos),(iv) a multiple cloning site derived from pUC19 and housed within lacZ(b -galactosidase gene) region. Bacterial colonies containing pBin19 are recognized by loss of blue colour on IPTG/X-GAL plates. Unlike the cointegrative vectors, binary vectors need not have any homology with resident Ti plasmid and are capable of autonomous replication. Binary vectors based on Ri plasmid of A. rhizogeneshave also been prepared. One such binary vector (pARS8) in a virulent strain (A4) of A. rhizogeneswas used for the production of transgenic plants in tomato. Other examples of vectors based on Ri plasmids are also available in literature.

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β-Glucuronidase (GUS)Gene (uidA) Luciferase Gene (lux) Green Fluorescent Protein Gene Phosphomannose Isomerase Gene (pmi) Agrobacterium-Mediated Transformation in Monocots Agroinfection and Gene Transfer Physical Delivery or DNA Mediated Gene Transfer (DMGT) Chemically Stimulated DNA Uptake by Protoplasts Microinjection and Macroinjection Microprojectiles for Gene Transfer Electroporation Method for Gene Transfer Liposome Mediated Gene Transfer Calcium Phosphate Precipitation Method Transformation Using Pollen or Pollen Tube Incubation of Dry Seeds,Embryos,Tissues or Cell in DNA Transformation by Ultrasonication Removal of Selectable Marker Gene from Transgenic Plants Need for Removal of Selectable Marker Gene Genetic Systems Used for Removal of Marker Gene Examples of North American Plant Transformation Facilities Plant Transformation Facilities (PTFs)