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

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

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

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

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

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

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

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

Depending upon opine, the bacteria is octopine type or nopaline type

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

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

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

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

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)

Cells that regenerate more efficiently are often difficult to transformed

Deep layers are not easy target