Direct Gene Transfer method (gene gun method).

plant tissue culture topic- Transfer Of Gene by Direct Method.

1. Assignment topic- direct gene transfer by gene gun method . Submitted To- Submitted By- Dr. RAJESH K. Tiwari. Shaista Khan. Associate Professor. MSc.MB.3 rd Semester. AIB. A07199319007.

2. what is gene transfer? It is defined simply as a technique to efficiently and firmly introduce froeign gene into the genome of target cells. The insertion of unrelated, therapeutic genetic information in the form of DNA into target cells.

3. Types of gene transfer method-

4. Introduction- The word ‘direct gene transfer’ is applied to differentiate between processes of  plant transformation  which rely on the use of indirect methods ( Agrobacterium ) with those direct methods. Direct gene shift or transfer method rely on the delivery of huge quantitys of ‘naked’ DNA even as the plant cell is rapidly permeabilised. Biolistic transfection or gene gun is a mechanical method that is potentially relevant to a wide range of tissue and cell types. In the present day, the gene gun is used for hereditary transformation of several organisms to initiate a diverse collection of desirable attributes. This method contains following steps- 1) Isolation of the genes of concern from the source organism. 2) Development of a useful transgenic build including the gene of concern, promoters to impel expression, etc.

5. Contd.. 3) Incorporation of it into a functional plasmid. 4) Introduction of the transgene into plant cell. 5) Regeneration of the plants cells, and 6) Gene expression or test trait performance at lab, field and greenhouse level.  The gene gun device also known as the Particle bombardment, , was extended to facilitate dissemination of the cell wall so that hereditary material consisting of a gene of concern can be shifted into the cell.

6. What is gene gun method ? Biolistic or gene gun Particle bombardment is the most vital and valuable direct gene transfer process in habitual use. In this method, gold or tungsten particles are covered with the DNA which is to be used to alter the plant tissue. These particles are drived at high speed into the aimed plant material, where the DNA is delivered inside the cell and be able to add it into the genome. Two types of plant tissues are normally used- a proliferating embryogenic cultures which are bombarded and after that allowed to flourish further and consequently redevelop. explants which are bombarded and then persuaded to become embryogenic.

7. Mechanism- The recombinant plasmids are created in such a manner that it carries the gene of interest or selectable marker gene like hyg. and reporter gene e.g. gusA. The plasmid is covered on to gold particles for bombardment into the cell of plant. The microcarrier are after that mixed, washed with ethanol and at last resuspended in ethanol. The plant material which utilized for transformation is embryogenic callus obtained from full-grown seeds on MS medium. The embryogenic callus is assembled in a petridish including high concentration of maltose to produce a high osmotic pressure former to gene gun of the plant tissue.

8. Contd.. Once the vacuity in the inferior part of the device is established, the helium pressure over the shatter disc is boosted until at 1100 psi the shatter disc bursts. Pushes the pieces of the macrocarrier and the projectiles downward the chamber. The macrocarrier is ended at the ending plate and allowing just the microcarrier to overtake through and batter the plant material. After one day of bombardment, the embryogenic callus is shifted to MS media. After 1 week, the embryogenic callus is shifted to selection media (MS media+hygromycin) and then incubated in gloomy for 2 weeks. The occurrence and expression of the gene concern is confirmed by diverse means. Juvenile plantlets are adjusted in growth rooms and then can be shifted to soil and grown to development.

9. First genetically-engineered fruit crop by using gene gun method (papaya) - Papaya is one of the five leading crops in Hawaii, it has been overcome in latest years by PRSV, which decreases fruit quality and ultimately kills the trees. Investigators developed the genetically engineered papaya by using recombinant DNA methods to separate and clone a PRSV gene which encodes for making of the coat protein of viral. Investigators at Geneva chosen papaya as a "model system" to build up the skill for genetically engineered virus resistance in fruit crops. The transgenic papaya have no detrimental effects to humans as PRSV-infected fruits, which hold the gene, are normally eaten by buyers. The gene was "injected" into the cells of the plant papaya by using special gene gun developed at the trial station. Expression of the gene in the resultant papaya render it anti-virus. The first virus-resistant papaya began to come out in grocery stores in 1998.

10. Other importance of this method- DNA gun occupies the transformation of cell organelles. Every Cashmore’s experiments, which engage isolating the genes, injecting them into tobacco cells, and reproducing the cells to plants, gets from 4-6months. Delivery of DNA by using this skill has allowed short-lived gene expression to be broadly studied, but addition of the transgene happens very seldom.

11. Thank YOU