The document discusses various techniques for genetic engineering and the transformation of plants, specifically focusing on the gene gun method, which uses biolistics to deliver DNA into plant cells. It outlines the advantages of this method, including applications in herbicide and insect resistance, altered oil content, and delayed fruit ripening. Additionally, the document mentions potential risks associated with genetically modified organisms (GMOs) and lists examples of GM crops.

1. Gene Gun
By
Allah Nawaz
Class: BS Botany
Presented to
Dr NOSHIA ARSHAD
Subject: Genetic Engineering

2. TRANSFORMATIONTECHNIQUE
AGROBACTERIUM
MEDIATEDGENE
TRANSFER(INDIRECT)
DIRECTGENETRANSFER
Used for engineering DICOTS For MONOCOT 2 methods are used
Chemical
Physical
Electroporation
Biolistics
Micro/Macro injection
6

3. Or the methods used for producing transgenic plants can be categorized as……………
I. INDIRECT
a) BIOLOGICAL
Agrobacterium mediated
Virus mediated
II. DIRECT
2. PHYSICAL
Gene gun/biolistics
Micro/Macro injection
Electrophoresis
Pressure
Laser mediated
Using pollen tubes
Silica/ carbon fibers (fiber mediated DNAdelivery)
3. CHEMICAL
Artificial lipids (lipofection)
PEG
Proteins
Dendrimers
Dextran
7

4. II. DIRECTGENETRANSFER
• Is a Vector less DNAtransfer systems
• Naked DNAis introduced into the
plant/animal cells
• DNAcan be introduced by the
following methods;
a. Chemical
b. Microinjection
c. Electroporation
d. Particle bombardment
(Biolistic)

5. d) Gene Gun Method
• Abiolistic particle delivery system, originally designed for plant transformation,
• Device for delivering exogenous DNA(transgenes) to cells
• It was invented and used by John C Sanford, Ed Wolf and Nelson Allen at Cornell university,
and Ted Klein of Dupont, between 1983 and 1986, to transform epidermal cells of Allium
cepa.
• This method is mainly used for cereal transformation
22

6. The basic steps to use the gene gun
are:
 Prepare the tungsten particles and coat them with
DNA.
 Load the particles onto macrocarriers.
 Prepare the target cells.
 Decide on your parameters and set the gene gun
accordingly for distance from target, pulse
duration, and CO2 pressure.

7. DNA is coated with gold or tungsten
How DNA is coated with gold or tungsten particles?
 This can be achieved with ballistic labeling, which refers to
the delivery of a cell label by means of carrier particles
(tungsten or gold) propelled from a pressurized gun. Ballistic
delivery can reach many dispersed cells in one shot and can
deploy a wide variety of cell markers to neurons in diverse
preparations.

8. Load the particles onto macrocarriers.
 Loading of coated gold particles onto macrocarriers for
bombardment in a PDS-10 0 0/He Biolistic Particle Delivery
System. A) Macrocarrier with coated gold microparticles in a
drying chamber. The yellow line shows the area where the coated
particles should be applied. B) Macrocarriers with dried DNA-
coated microparticles. C) Position of endosperm culture plate on
the target plate shelf of the biolistic bombardment system. D)
setup of the bombardment chamber.

9. Decide on your parameters and set the gene gun accordingly
for distance from target, pulse duration, and CO2 pressure.
 High-pressure solenoid ($75)
 Adjustable relay that provides pulses in the 10–500 millisecond range ($30–$60
on eBay)
 A bicycle CO2 inflator to attach the CO2 cartridge to the gene gun ($22)
 A valve to adjust the pressure ($21)
 A pressure gauge that reads from 0–1,000 psi ($15 on eBay)
 A fitting to suspend the tungsten particles above the target in front of the
target
 A compressed gas discharge (macrocarrier) ($5)
 Pipe fittings to hook up the Solenoid, CO2, and nozzle ($30)

10.  The four key parts that are
needed are the relay, the
solenoid, the valve, and a
CO2 tire inflator.
 Other than that, the rest of the
parts are 1/8″–1/2″ pipes and
fittings available from any
hardware store.
 See Figure 1-1.

11. 1

12. APPLICATION
 Herbicide resistance
 Insect resistance
 Virus resistance
 Altered oil content
 Delayed fruit ripening
 Drought, cold, salinity resistance
 Pollen control
 Enhanced shelf life
 Pharmaceutical &edible vaccines
 Biotic &Abiotic stress tolerance
 Nutritional quality
23

13. 1. HERBICIDERESIST
ANCE
a. BromoxynilResistance
i. A gene encoding the enzyme Bromoxynil nitrilase (BXN) is transferred from Klebsiella
pneumoniae bacteria to plants.
ii. Nitrilase inactivates the bromoxynil before it kills the plant.
a. Sulfonylurea
i. Kills plants by blocking an enzyme needed for synthesis of the amino acids valine, leucine,
and isoleucine.
ii. Resistance generated by mutating a gene in tobacco plants, and transferring the mutated
gene into crop plants.
2. INSECTRESIST
ANCE
• The Bt toxin isolated from Bacillus thuringiensis has been used in plants. The gene has been
placed in corn, cotton, and potato, and has beenmarketed.
• Alkaline protein degrades gut wall of lepidopteran larvae
Corn borer catepillars
Cotton bollworm catepillars
Tobacco hornworm catepillars
Gypsy moth larvae
• Sprayed onto plants – but will wash off
24

14. 3. VIRUSRESIST
ANCE
• Chemicals are used to control the insect vectors of viruses, but controlling the disease
itself is difficult because the disease spreads quickly.
• Plants may be engineered with genes for resistance to viruses, bacteria, and fungi.
• Virus-resistant plants have a viral protein coat gene that is overproduced, preventing
the virus from reproducing in the host cell, because the plant shuts off the virus’
protein coat gene in response to the overproduction.
• Coat protein genes are involved in resistance to diseases such as cucumber mosaic
virus, tobacco rattle virus, and potato virus x.
4. AL
TEREDOILCONTENT
• Oil content in plants are altered by modifying an enzyme in the fatty acid synthesis
pathway (oils are lipids, which fatty acids are a partof).
• Varieties of canola and soybean plants have been genetically engineered toproduce
oils with better cooking and nutritional properties.
• Genetically engineered plants may also be able to produce oils that are used in
25
detergents, soaps, cosmetics, lubricants, and paints.

15. 5. DELA
YEDFRUITRIPENING
• Allow for crops, such as tomatoes, to have a higher shelflife.
• Tomatoes generally ripen and become soft during shipment to a store.
• Tomatoes are usually picked and sprayed with the plant hormone ethylene to induce ripening,
although this does not improve taste
• Tomatoes have been engineered to produce less ethylene so they can develop more taste before
ripening, and shipment to markets.
6. POLLENCONTROL
• Hybrid crops are created by crossing two distantly related varieties of the same crop plant.
• The method may generate plants with favorable traits, such as tall soybean plants that make
more seeds and are resistant to environmental pressures.
• For success, plant pollination must be controlled. This is usually done by removing the male
flower parts by hand before pollen is released. Also, sterilized plants have been genetically
engineered with a gene from the bacteria Bacillus amyloliqueifaciens (barnase gene). This gene is
dominant gene for male sterility 26

16. RISKSOFGMO/GMCs……..
• Can be dangerous &cause allergies
E.g..Soybean containing gene of brazil nut
• Indirectly promote Antibiotic resistance (Resistance of microbes)
• Weed shows herbicide resistance &resistance to viral disease
• Change in chemistry of soil
• Genetically engineered plant cross pollinate non- engineered plants
27

17. EXAMPLESOFGM CROPS……….
2. Corn
3. Canola.
4. Cotton.
5. Papaya, rice,
6. Tomato,
7. Sugar beet, and
8. Red heart chicory.
9. Golden rice
Transgenic technology produced a type
of rice that accumulates beta-carotene in rice
grains. Once inside the body, beta-carotene is
converted to vitamin A.
Normal Rice
Golden Rice
28

18. 29