gene trnasfer to plants

Views:
 
     
 

Presentation Description

gene transfer techniques

Comments

Presentation Transcript

DIRECT GENE TRANFER TO PROTOPLASTS : 

NARAYANAPPA. M HEAD, DEPT. OF BIOTECHNOLOGY GOVT. SCIENCE COLLEGE, HASSAN DIRECT GENE TRANFER TO PROTOPLASTS

Slide 2: 

2/25/2010 Agricultural Biotechnology 2 Gene introduction Selection and screening

PROTOPLAST : 

PROTOPLAST 2/25/2010 Agricultural Biotechnology 3 Naked plant cell with all components of plant cell excluding the cell wall Hanstein introduced the term protoplast in 1880 to designate the living matter enclosed by the plant cell membrane

DIRECT GENE TRANSFER : 

DIRECT GENE TRANSFER 2/25/2010 Agricultural Biotechnology 4 PHYSICAL GENE TRANSFR METHODS CHEMICAL GENE TRANSFER METHODS

PHYSICAL GENE TRANSFR METHODS : 

PHYSICAL GENE TRANSFR METHODS 2/25/2010 Agricultural Biotechnology 5 This is also referred to as DNA mediated gene transfer (DMGT) Electroporation Particle bombardment/ micro projectile/ biolistics Macro injection Microinjection Liposome mediated gene transfer Silicon carbide mediated gene transfer Ultra sound mediated gene transfer DNA transfer via pollen

2. CHEMICAL GENE TRANSFER METHODS : 

2. CHEMICAL GENE TRANSFER METHODS 2/25/2010 Agricultural Biotechnology 6 This involves plasma membrane destabilizing and /or precipitating agents PEG-mediated gene transfer Calcium phosphate co precipitation The polycation DMSO technique DEAE dextran procedure

ELECTROPORATION : 

ELECTROPORATION 2/25/2010 Agricultural Biotechnology 7 Electroporation is the process where electrical impulses of high field strength are used to reversibly permeabilize cell membranes to facilitate uptake of large DNA molecules Plant material is incubated is incubated in a buffer solution containing the desired foreign DNA and subjected to high voltage electricalimpulses

Slide 8: 

2/25/2010 Agricultural Biotechnology 8 This results in the formation of pores in the plasma membrane through which DNA enters and gets integrated into the host cell genome ADVANTAGES simple, convenient, rapid and cost effective The transformed cells are at the same physiological state after electroporation Efficiency can be improved by more strength and addition of spermidine

DISADVANTAGES : 

DISADVANTAGES 2/25/2010 Agricultural Biotechnology 9 Under normal conditions the amount of DNA delivered into plant cells is very low Regeneration of plants is not very easy particularly when protoplasts are used Efficiency is variable depending upon plant material

PARTICLE BOMBARDMENT/ MICRO PROJECTILE/ BIOLISTICS : 

PARTICLE BOMBARDMENT/ MICRO PROJECTILE/ BIOLISTICS 2/25/2010 Agricultural Biotechnology 10 The microprojectile bombardment method was initially known as biolistics by its inventor Sanford (1988) Biolistics is a combination of biological and ballistics. There are other names for this technique particle gun, gene gun, bioblaster

Slide 11: 

2/25/2010 Agricultural Biotechnology 11

Mechanism : 

Mechanism 2/25/2010 Agricultural Biotechnology 12 Microcarriers (microprojectiles), the tungsten or gold particles coated with DNA, are carried by macrocarriers These macrocarriers are inserted into apparatus and pushed downward by rupturing the disc The stopping plate does not permit movement of macrocarriers while the microcarriers (with DNA) propelled at a high speed into the plant material Here the DNA segments are released which enter the plant cell and integrate with the genome

Advantages : 

Advantages 2/25/2010 Agricultural Biotechnology 13 Gene transfer can be efficiently done in organized tissues Different species of plants can be used to develop transgenic plants

DISADVANTAGES : 

DISADVANTAGES 2/25/2010 Agricultural Biotechnology 14 The major complication is the production of high transgene copy number. This may result in instability of transgene expression due to gene silencing The target cell/tissue may get damged due lack of control of bombardment velocity Some times undesirable chimeric plants may be regenerated

MICROINJECTION : 

MICROINJECTION 2/25/2010 Agricultural Biotechnology 15 Microinjection is a direct physical method involving the mechanical insertion of the desirable DNA into a target cell This technique involves the gene transfer through a micropipette (0.5-10µm tip) into the cytoplasm or nuclei of plant cell or protoplast While gene transfer the cells held by suction holding pipette

Silicon carbide fibre mediated transformation : 

Silicon carbide fibre mediated transformation 2/25/2010 Agricultural Biotechnology 16 The silicon carbide fibres (SCF) are about 0.3-0.6µm in diameter and 10-100µm in length These fibres are capable of penetrating the cell wall and plasma membrane and thus can deliver DNA into cells Procedure is simple does not involve costly equipments Direct delivery of DNA into intact walled cells, this avoids the protoplast isolation Silicon carbide fibre mediated transformation

Slide 17: 

2/25/2010 Agricultural Biotechnology 17 Silicon carbide fibres are carcinogenic therefore have to be carefully handled Embryonic cells are hard and resistant for SCF penetration

Liposome mediated gene transfer : 

Liposome mediated gene transfer 2/25/2010 Agricultural Biotechnology 18 Liposomes are artificially created lipid vesicles containing phospholipid membrane Liposomes carrying genes can be employed to fuse with protoplast and transfer the genes Adhesion of liposomes to the protoplast surface, its fusion at the site of attachment and release of plasmids inside the cell

CHEMICAL GENE TRANSFER METHODS : 

CHEMICAL GENE TRANSFER METHODS Polyethylene glycol mediated transformation 2/25/2010 Agricultural Biotechnology 19 Polyethylene glycol (PEG), in the presence of divalent cations, destabilize the plasma membrane of protoplasts and renders it permeable to naked DNA

PROCEDURE : 

PROCEDURE 2/25/2010 Agricultural Biotechnology 20 Isolation protoplasts and their suspension Addition of plasmid DNA Slow addition of 40% PEG dissolved in mannitol and calcium nitrate solution As this mixture incubated protoplasts get transformed Transformed protoplasts are selected Plants are regenerated to produce transgenic plants

authorStream Live Help