indirect mode of gene tranfer
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Gene transfer methods in plants Transformation :- Transfer of desirable gene from one plant species to another Transgene : Transferred gene Transgenic plant : Transformed plantReasons for developing transgenic plants :-To improve the agriculture , horticulture or
ornamental value of plantsTo study the action of gene in plants during
development & various biological process
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To develop plant bioreactors for inexpensive manufacture of commercially important products
e.g:- proteins , medicine , pharmaceutical compoundGenetic traits introduced into plants :-• Resistance to herbicides• Protection against viral infections• Insecticidal activity• Improved nutritional quality • Altered flower pigmentation • Tolerance to environmental stresses• Self incompatibility
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Gene transfer techniques:-The gene transfer technique in plant genetic transformation is broadly divided in two categories :- 1.Vector mediated / indirect gene
transfer2.Vector less mediated / direct
gene transfer
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Vector mediated :-Carried by agrobacterium or by use of plant virus as vectors
Most vectors carried marker genes which allow recognition of transformed cells – selectable markers
E.g:- npt is a common selectable marker providing kanamycin resistance Common features of vectors :-1. Multiple unique restriction sites2. Ori
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Agrobacterium ti plasmid is preferred all over other vectors because of wide range & capacity to transfer genes due to presence of T DNA border sequence
Agrobacterium mediated gene transfer :-soil born gram negative bacterium Rod shaped Family – rhizobiaceae A natural genetic eng capable of integrating its DNA into plant genome
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Species of agrobacterium :- A. tumefaciens - crown gall disease A. rhizogenes - hairy root disease Characteristics of ti plasmid :-200 kb plasmid Varying length of 12 – 24 kb 4 major regions • T DNA region • An ori• Virulence region • Opine catabolism region
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T DNA region is a part of Ti plasmid it can be integrated in to the plant genome . T DNA has right & left borders
Virulence region genes helpful for T DNA transfer in to plant genome . Mutations in this region abolishes virulence
The opine catabolism region synthesis opine
Ori is responsible for replication
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General structure of T DNA :- T DNA consists of 1.Onc (oncogenisity region) :-Onc region consists of tms 1 & tms 2 , tmr These are responsible for biosynthesis of 2 phytohormones namely IAA , isopentyl adenosine 5ˡ monophosphate ( cytokine )
These genes causes crown gall disease2.Nos region :-Responsible for the synthesis of aa or sugar derivatives called opines
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Depending upon opine, the bacteria is octopine type or nopaline type
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Virulence region :-Organized in to 6 operons ( a,b,c,d,e,g )A,B,D,G – required for virulence C,E – tumor formationA - located on the inner membrane of bacteria . Chemoreceptor for plant hormones
B - form a pore between bacteria & plant cell
D - cutting of T DNA E - preventing degradation of T DNA , covers T DNA
G - induces all virulence gene
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Mechanism of T DNA transfer
production of acetosyrengone binding of bacteria to plant cell vir genes activated by phenolic compounds vir A & vir G & all vir genes are activated vir D cleaves T DNA tube formed between bacteria & plant cell DNA is transferred in to plant cell Recombination occurs
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Production of dis armed Ti plasmid:- In T DNA the genes b/w the L&R border are removed
Any fragment can be transformed in to plant genome by replacing T DNA with our gene of interest
Prerequisites• L&R borders • Vir genes • Chromosomal genes
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Hairy root disease A.rhizogenes infect plants results in hairy
root . These plasmids are referred as Ri plasmids
Some of these Ri plasmids posses genes that are homologous to Ti plasmid
e.g:- auxin biosynthetic genesInstead of virulence genes Ri plasmid
contains a series of open reading frames on TDNA
Hairy root system are used in the production of secondary metabolites particularly pharmaceutical products
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Advantages :-Natural method of gene transferAgrobacterium can conveniently infect any
explantLarge fragment of DNA can be effectively
transferredTransformed plant can be effectively
regenerated
Limitations:-Extensively utilized for dicotyledonous species monocotyledons could not be successfully utilized for agrobacterium gene transfer(failure of gene response in monocotyledons)
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Cells that regenerate more efficiently are often difficult to transformed
Deep layers are not easy target
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