Traditional plant breeding has provided many improvements in crops (such as
increase in the yield, herbicide resistance and disease resistance etc.) but
it is limited by the traits available in the sexually compatible species.
The development of methods for transferring genes into plants has made genes
derived from recombinant DNA technologies accessible for practical uses. The
application of these techniques has produced transgenic plants that are,
resistant to various bacterial, viral or fungal infections, insect pests and
also tolerant to the application of various herbicides, have higher
nutritional content in the seeds, delayed fruit ripening or a greater degree
of stress protection, due to the incorporation of new genes.
Agrobacterium tumefaciens,
the highly effective vector for dicots either does not infect monocots or
does so very inefficiently, as described for asparagus and rice.
Introduction of DNA into protoplasts mediated by polyethylene glycol or
electroporation as a means for obtaining transgenic plants has been
examined extensively for many cereals. However, the protoplast-to-plant
method
of
obtaining transgenic plants is laborious, requires very high regeneration
capacity, is strongly dependent on the genotype, and seems applicable
routinely only for rice. In view of the difficulties involved in the
transformation and regeneration of monocots using protoplasts, other
approaches have been tried for introducing DNA into plant cells. These
include macroinjection, embryo imbibition, silicon-carbide fiber, and
particle bombardment etc,.Of all these, particle bombardment has proved to
be the most successful and reproducible methods, leading to the production
of transgenic plants of a wide range of species, including most of the
cereals, legumes, cotton, papaya, cranberry and populus. One advantage of
microprojectile bombardment is that it can be used to target a wide range of
tissues or organs, such as embryogenic cell suspensions or calli, immature
embryos, meristems, pollen, or leaf tissue. Particle bombardment allows the
introduction of DNA into live cells or tissues without previous preparation,
making it possible to evaluate transient expression of different gene
constructs in intact organs as well as the recovery of stably transformed
tissue and plants.
Microprojectile bombardment of DNA delivery involves the acceleration of
high-density metal particles, coated with DNA, to a speed such that they are
able to penetrate the cell wall. These small particles (0.4 to 3.0μm in
diameter), usually made of gold or tungsten, are coated with DNA. The
adsorption of DNA directly to the surface of the microprojectile particle is
essential for efficient DNA delivery. Both calcium chloride and spermidine
are necessary for good DNA precipitation onto tungsten particles. The
concentrations of these are critical and it has been found that the optimum
ranges are 0.24 to 2.5M for calcium chloride and 100mM for spermidine. For
highest transient expression 1.2μm tungsten particles were used. The
velocity with which the microprojectiles are propelled towards the target
cells and the distance that they travel before striking the target, both
affect the extent of cell injury. Velocity can be controlled by altering the
accelerating force (such as gas pulse pressure), vacuum in the target
chamber or distance traveled by the microprojectile particles. Cells
bombarded will transiently express the genes that are introduced and some of
these will incorporate the genes into the plant genome and will become
stably transformed. Transient expression of the transgene is detected within
two days after introduction of foreign DNA into cells, with the help of
gus A
reporter gene.
Tissue preparation:
Target tissue has to be arranged on petriplates containing osmotic medium
for 4 hours prior to the bombardment. After successfully bombarding, the
target
tissues are transferred onto same medium and incubated overnight in dark.
Bombarded calli selected randomly are transferred into Gus buffer solution
and incubated overnight at 37˚C. Next day the bombarded calli are observed
under microscope for transient GUS expression.
Working of particle in-flow gun:
Switch on the particle-inflow-gun. Open both the helium cylinder valve
completely and regulator valve to the required pressure and then open the
inlet valve. Aliquot 4μl of tungsten/DNA preparation into the filter holder
and place the remaining mixture back onto the ice. Screw the filter holder.
Take out the petriplate containing pre-treated callus material, and then
place the baffle over the callusing material. Place this on the eleventh
shelf directly underneath the filter holder (7 cms). Immediately open the
vacuum valve and allow the vacuum to reach 600 Hg. Now release the timer
relay switch. The tungsten will be propelled into the tissue by the force.
Immediately open the vacuum valve to release the vacuum, and then open the
door. Remove the bombarded material from the chamber.
After bombarding PIG must be closed down in the following way: Ensure that
the filter holder is being removed, then shut both the valves that are
linked to the vacuum pump. Close the helium cylinder valve completely.
Partially close the regulator valve and then release the timer rely switch
until all the pressure is released. This clears the piping. Now close the
regulator valve completely and put off all the switches and shut the door.
Preparation of tungsten stock:
Weigh about 50mg. of tungsten in a microcentrifuge tube and add 500μl of
100% ethanol to it. Vortex every 5 minutes and repeat this step thrice. Spin
at 3000 rpm for 5 min and remove the supernatant carefully with a pipette.
Then, resuspend tungsten in 500μl of sterile distilled water. Vortex every 5
minutes and repeat this step thrice. Spin at 3000 rpm for 5 min and remove
the supernatant carefully with a pipette. Resuspend the tunsgten in 500μl of
sterile distilled and store at 4˚C. For each bombardment 50μl of tungsten
stock is used.
Adsorption of plasmid DNA onto tungsten particles:
To
50μl tungsten, add 10μl of plasmid DNA and vortex it. Then, vortex it
immediately after adding 50μl of 2.5M calcium chloride and 20μl. of 100mM
spermidine. Place this sample in ice for about 5 minutes. Carefully remove
100μl of the supernatant from the prepared sample and discard it. Then place
the tungsten/DNA preparation back on to ice.
Four μl of tungsten/DNA preparation is used for each bombardment.
Preparation of Gus solution:
Buffer
NaPO4
(50mM,pH7.0) 10ml
Triton X-100 (0.3% v/v) 30μl
To
make a 0.1M sodium phosphate buffer (pH 7.0), mix 57.7ml 1M Na2HPO4
and 42.3ml NaH2PO4,
and make upto one litre with water. Dissolve 5mg X-Gluc in 50 μl DMF (dimethylformamide).
Add to 10ml buffer. Filter sterilize the solution. Incubate tissue at 37
0C
overnight. Cells and tissue expressing β-glucuronidase will turn blue.
Preparation of 2.5M calcium chloride:
Weigh 3.675 g of calcium chloride and dissolve in 10ml of autoclaved
distilled water. Filter sterilize the solution.
Preparation of 100mM spermidine:
Weigh 69 mg of spermidine and dissolve in 1 ml of autoclaved distilled
water. Filter sterilize the solution.
Preparation of osmotic medium:
To
LS medium, add 36.44g of sorbitol and 36.44 g of mannitol. Then add 30 g of
sucrose, adjust pH to 5.8. Make up the volume to 1 litre with distilled
water and add 9 g of agar powder, then autoclave the medium.
Materials:
Baffles (autoclaved)
Target tissue
Ethanol (100%)
Whatman Filter papers (sterile 5.5 or 7-cm diameter)
High
purity Helium tank
Pipettes
13mm
filter holder (autoclaved)
Osmoticum medium (LS+0.2M Sorbitol and 0.2M Mannitol)
Particle-inflow-gun
Vacuum pump
Vacuum grease
Vortex
Laminar flow
Calcium chloride
Spermidine
Tungsten particles/Gold particles
Foreign DNA Constructs (plasmid)
LS
macro and micro nutrients
LS
vitamins
Sorbitol
Mannitol
Sucrose
Agar
powder
Distilled water
