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.