indirect mode of gene tranfer
- 1. Transformation :- Transfer of desirable gene from one plant species to another Transgene : Transferred gene Transgenic plant : Transformed plant Reasons for developing transgenic plants :- To improve the agriculture , horticulture or ornamental value of plants To study the action of gene in plants during development & various biological process
- 2. To develop plant bioreactors for inexpensive manufacture of commercially important products e.g:- proteins , medicine , pharmaceutical compound Genetic traits introduced into plants :- • Resistance to herbicides • Protection against viral infections • Insecticidal activity • Improved nutritional quality • Altered flower pigmentation • Tolerance to environmental stresses
- 3. The gene transfer technique in plant genetic transformation is broadly divided in two categories :- 1. Vector mediated / indirect gene transfer 2. Vector less mediated / direct gene transfer
- 4. 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 sites 2. Ori
- 5. 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
- 6. 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
- 7. 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
- 8. 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 disease 2.Nos region :- Responsible for the synthesis of aa or sugar derivatives called opines
- 9. Depending upon opine, the bacteria is octopine type or nopaline type
- 10. Virulence region :- Organized in to 6 operons ( a,b,c,d,e,g ) A,B,D,G – required for virulence C,E – tumor formation A - 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
- 11. 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
- 13. 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
- 17. 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 genes Instead 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
- 18. Advantages :- Natural method of gene transfer Agrobacterium can conveniently infect any explant Large fragment of DNA can be effectively transferred Transformed 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
- 19. Cells that regenerate more efficiently are often difficult to transformed Deep layers are not easy target