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seminar on Agrobacterium mediated gene transfer

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Agrobacterium mediated gene transfer in Plants:

Agrobacterium mediated gene transfer in Plants 1 Submitted by- Yashlok singh M.Sc.,Plant breeding and genetics NMCA,NAU,NAVSARI Submitted to - Dr. Ajay Narwade Deptt. Of Plant Physiology NMCA,NAU,NAVSARI

Gene Transfer:

2 Gene Transfer Recombinant DNA technology is based on the insertion of a DNA fragment (Gene) of interest into a suitable cloning vector and then its introduction into a suitable host to propagate the recombinant DNA.

Slide 3:

3 Gene transfer vehicle

Gene Carrier Vehicle:

4 Gene Carrier Vehicle Gene carrier vehicle: If a gene is to be introduced into a host cell, a carrier molecule that can transport the gene into the host cell is required. Such a molecule is called a cloning vehicle , carrier molecule or a vector.

Gene Carrier Vehicles:

5 Gene Carrier Vehicles Following are a few gene carrier vehicles Plasmids Bacteriophages Cosmids Viruses Virus vector for animal cells Simian Virus

Agrobacterium tumefaciens :

6 Agrobacterium tumefaciens Agrobacterium tumefaciens is a soil borne gram negative bacterium. It invades many dicot plants when they are injured at the soil level and causes grown gall disease .

Agrobacterium tumefaciens :

7 Agrobacterium tumefaciens The bacterium enters the plant through a fresh wound and attaches itself to the wall of the intact cell. This cell is genetically transformed by the bacterium. This transformation results in a tumour which synthesizes certain special metabolites required by the bacteria

Opines::

8 Opines: The tumour develops only at the site of the wound. Such tumours can be removed from the plant and cultured in vitro where they continue to grow indefinitely Continued presence of Agrobacterium is not required for tumour proliferation. Agraobacterium induced tumours synthesize a variety of unusual compounds called opines

Opines::

9 Opines: Octapine Nopaline Agropine These opines are catabolised by Agrobacterium to obtain energy . Thus AT genetically engineers the plant cell for its won purpose.

Tumour Inducing Principle:

10 Tumour Inducing Principle The tumour inducing principle of AT is a plasmid called tumour inducing plasmid or Ti Plasmid 200 kb long Has two essential regions: T-DNA & vir region. These two regions are essential for the transformation process.

Transfer of Tumour Inducing Principle:

11 Transfer of Tumour Inducing Principle T-DNA (Transferred DNA) is excised from the Ti-Plasmid and transferred to the nucleus of the plant cell. Here the T-DNA gets integrated into the DNA which is stable. The T-DNA can be passed on to daughter cells as an integral part of the plant chromosome.

Slide 12:

12 Induction of crown gall on a dicot plant by Agraobacterioum tumefaciens

Diagram of Agrobacterium tumifaciens:

13 Diagram of Agrobacterium tumifaciens

The Ti-Plasmids:

14 The Ti-Plasmids An axtra chromosomal double stranded circular DNA molecule. Tumour inducing. 200 kb in size and conjugative type. Encodes enzymes responsible for the synthesis and catabolism of certain opines. One of the opines is Nopaline . pTiC58 is present in Agrobacterium strain C58 . It is 192 kb long Only a small segment of the Ti Plasmid is transferred to the host plant cell and gets integrated with the genome. This is the T-DNA.

The T-DNA:

15 The T-DNA Only a small segment of the Ti Plasmid is transferred to the host plant cell and gets integrated with the genome. This is the T-DNA. It contains genes for tumour formation ( Tum ) and nopaline biosynthesis ( Nos ). Tum genes encode enzymes that catalyse the synthesis of phytohormones like the IAA and the cytokinin, isopentenyl adenosine that cause tumerous growth of cells in crown galls. The T-DNA is bordered by 25 bp repeats , required for the excision and transfer of T-DNA.

Nopaline Ti plasmid pTiC58:

16 Nopaline Ti plasmid pTiC58 The vir (virulence) region of the Ti- plasmid contains the genes required for the T-DNA transfer process. The genes in this region encode the DNA processing enzymes required for excision, transfer and integration of the T-DNA segment.

Tumour induction by Agrobacterium:

17 Tumour induction by Agrobacterium Recognition of susceptible wounded plant cell: Plant exudates: act as signals by inducing genes in the Vir Genes of the Ti Plasmid. Acetosyringone (AS), - hydroxy acetosyringone (OH-AS). Binding to wounded cells: Controlled by two chromosomal genes of Agrobacterium : chv -A and chv -B.

Tumour induction by Agrobacterium:

18 Tumour induction by Agrobacterium Excision, transfer and integration : The border repeats of the T-DNA play an important role. Any DNA sequence located between the border repeats is transferred to the host plant The T-DNA region is excised from the plasmid by the enzymes encoded by the Vir -region. These enzymes specifically recognize the T-DNA borders. The T-DNA enters the plant cell and integrates into the host genome, mediated by host enzymes.

Ti-Plasmid as a Vector:

19 Ti-Plasmid as a Vector The Ti-Plasmid has an innate ability to transmit bacterial DNA into plant cells. This potential is exploited by the genetic engineers to use this as a vector. The gene of a donor organism can be introduced into the Ti plasmid at the T-DNA region This plasmid now becomes a recombinant plasmid. By Agrobacterium infection, the donor genes can be transferred from the recombinant Ti- Plasmid and integrated into the genotype of the host plant. This results in the production of a transgenic plant

Slide 20:

20 Ti-Plasmid mediated transfer of gene into a plant

Disarmed Ti Plasmid:

21 Disarmed Ti Plasmid Disarmed Ti-plasmids Deletion of T-DNA region PGV3850 is constructed from pTiC58 It has pBR 322 with AmpR Has Border repeats and NOS genes

Disarmed Ti Plasmid:

22 Disarmed Ti Plasmid Agrobacterium having this PGV 3850 can transfer the modified T-DNA into plant cells. But the recipient cells will not produce tumour, but could produce nopaline. This can be used as an efficient vector for introducing foreign genes into plants

Construction of a Cointegrate:

23 A foreign gene cloned into an appropriate plasmid ( pBR322 ) can be integrated with the disarmed Ti-Plasmid by a homologous recombination. A compound plasmid called a cointegrate is formed Construction of a Cointegrate

Transformation of Tissue Explants by Co-cultivation with Agrobacterium:

24 Transformation of Tissue Explants by Co-cultivation with Agrobacterium A co-integrate plasmid derived by the recombination of pGV3850 and pBR322 loaded with the foreign gene is now used to transfer the foreign gene into many crop plants The methodology is:…….

Transformation of Tissue Explants by Co-cultivation with Agrobacterium:

25 Transformation of Tissue Explants by Co-cultivation with Agrobacterium Small discs ( a few mm diameter) are punched from leaves of Petunia, tobacco, tomato or other dicot plants. These discs are inoculated in a medium containing Agrobacterium carrying the recombinant disarmed T-DNA as a co-integrate. The cointegrate plasmid has the foreign gene and also the gene for resistance to kanamycin ( KmR )

Transformation of Tissue Explants by Co-cultivation with Agrobacterium:

26 Transformation of Tissue Explants by Co-cultivation with Agrobacterium The discs are cultured for two days. The Agrobacterium infects the cut edges of the disc. The discs are then transferred to a shoot inducing solid medium (high cytokinin) contining kanamycin to select the transferred kanamycin gene. Corbenicillin in the medium kills Agrobacterium . After 2-4 weeks the shoot develops.

Transformation of Tissue Explants by Co-cultivation with Agrobacterium:

27 Transformation of Tissue Explants by Co-cultivation with Agrobacterium The callus having the shoot is transferred to root inducing solid medium (high in auxin content). After 4-7 weeks roots appear. The rooted plantlets are transferred to soil.

Slide 28:

28 Transformation of leaf disc explants by co-cultivation with Agrobacterium having the Cointegrate Ti plasmid

Uses::

29 Uses: Employing this method, genes from entirely unrelated plants can be transferred to other dicots. Transgenic plants with the following attributes were developed: Resistance- to antibiotics. pesticides and insect pests drought and herbicides.

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