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  Section: General Biotechnology / Genes & Genetic Engineering
 
 
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Techniques of Genetic Engineering

 
     
 

Particle bombardment gun

It was developed by Prof. Stanford and coworkers of Cornell University (USA) in 1987. As the term denotes, it shoots foreign DNA into plant cells or tissue at a very high speed. This technique is also known as particle bombardment, particle gun method, biolistic process, microprojectile bombardment or particle acceleration.

  Working system of particle bombardment gun.  
 

Fig. 4.13. Working system of particle bombardment gun.

 

This technique is most suitable for those plants which hardly regenerate and do not show sufficient response to gene transfer through Agrobacterium for example, rice, wheat corn, sorghum, chickpea and pigeon-pea.

The apparatus consists of a chamber connected to an outlet to create vacuum (Fig. 4.13). At the top, a cylinder is temporarily sealed off from the rest of chamber with a plastic rupture disk. Helium gas flows into the cylinder. A plastic microcarrier is placed close to rupture disk. It contains DNA coated tungsten particle, the microscopic pellets (i.e. coated microprojectiles). When to work the apparatus is placed in Laminar flow just to maintain sterile conditions. The target cells/tissue are placed in the apparatus. A stopping screen is put between the target cells and microcarrier assembly. Helium gas is flown in the cylinder at high velocity. When pressure of cylinder exceeds the bursting point of plastic disk, it gets ruptured. Helium shock waves propel the plastic mircrocarrier containing DNA coated micropellets. The stopping screen allows the micropellets to pass through and deliver DNA into target cells. The transformed cells are regenerated onto nutrient medium. The regenerated plant tissues are selected over culture media containing cither antibiotics or herbicide. The selected plants are then analyzed for expression of foreign DNA.

Using this technique scientist have got success in delivering foreign DNA into epidermal tissues of Allium cepa, scutellar tissues of maize and leaf and cell culture of many crops (Peters, 1993). In addition to bacterial cells, algae, fungi plant organelles (e.g. chloroplast and mitochondria), an animal and human cells and fruitfly embryos have been successfully transformed.

 

Content

Gene cloning in prokaryotes

 

Isolation of DNA to be cloned

 

Insertion of DNA fragment into vector

 

 

Use of restriction Linkers

 

 

Use of homopolymer tails

 

Transfer of recombinant DNA into bacterial cells

 

Selection of clones

 

 

Colony hybridization techniques

 

 

In vitro translation technique

 

 

Immunological tests

 

 

Blotting Techniques

 

Recovery of cells

 

Expression of cloned DNA

 

 

Shine-Dalgano sequence

 

 

Expression vectors

Gene cloning in eukaryotes

 

Plant cells

 

 

Yeasts

 

 

Filamentous fungi

 

 

Agrobacterium plasmids

 

 

Plant cell transformation

 

 

Plant cell transformation by ultrasonication

 

 

Liposome mediated gene transfer

 

Animal cell  

 

 

Animal viruses

 

 

Electroporation

 

 

Particle bombardment

 

 

Microinjection

 

 

Direct transformation

Site directed mutagenesis

 

Methods of mutagenesis

Table 4.1. Bombardment conditions employed on Basmati Indica rice callus.

1.

Distance between rupture disc and microcarrier

0.5 inch + 15 mm

2.

Size of gold particles

1 mm

3.

Distance between microcarrier and target

6 and 9 cm

4.

Density of particles/shot

9 mg/shot

5.

Bombardment medium

MS medium supplemented with 2, 4-D at 2 mg/1 (rice callus medium)

6.

DNA concentration

1 mg/ml

7.

Physical parameters

 

(i) helium pressure

450-1300 pounds/inch2

(ii) vacuum

26 mg Hg

8.

Post bombardment culture

On callus medium till GUS assay (48h)

Source: Minhas et al. (1996).

(i) Conditions for bombardment. There are several conditions required for introduction of foreign gene into the desired cell/tissue/pollen, etc. The conditions required for introduction of b-glucuronidase (GUS) reporter gene (for detail see section, Transgenic plants) for transformation of Basmati Indica rice have been given in Table 4.1. Manihas et al. (1996) has reported for the first time the transient expression of GUS reporter gene through biolistic delivery in mature embryo-derived embryonic callus cultures of Oryza sativa cv Basmati 370. In 1998, scientists of Plant Transformation Group at ICGEB (New Delhi) have got success in transferring human interferon gamma gene into chloroplasts of tobacco, maize, etc. using a particle gum mediated gene transformation.


(ii) Pollen transformation through particle bombardment. Genetic improvement in alfalfa has been made by plant tissue culture methods. This method is time consuming, requires special techniques and efficiency of getting stable regenerants is low. Ramaiah and Skinner (1997) produced transgenic alfalfa through direct delivery of DNA into pollen grains by particle bombardment method. Plasmid pBI121 bearing GUS reporter gene was introduced into pollen grains. The microprojectile bombarded liquid pollen suspension was allowed to fertilize the tagged and tripped recipient female flower. Pollinated flowers set seeds in about a month. Thirty per cent of plants derived from fertile seeds showed integration of GUS plasmid. It was confirmed by Southern analysis. After 10 vegetative generations some transgenic plants lost the integrated GUS plasmid, whereas in few others number, copies of GUS gene decreased due to unknown reasons. An outline of production of transgenic alfalfa plant through pollen transformation is given in Fig. 4.14.


  Outline of pollen transformation to obtain transgenic alfalfa plants.
 

Fig. 4.14. Outline of pollen transformation to obtain transgenic alfalfa plants.

 
 
     
 
 
     



     
 
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