agricultural application o DNA technology.pdf

Agricultural DNA technology refers to the use of molecular biology tools to improve crop and livestock traits by manipulating their genetic material. It encompasses techniques such as DNA molecular markers, recombinant DNA technology (rDNA), and advanced gene editing tools like CRISPR. These technologies are employed to enhance agricultural productivity, resilience, and sustainability.

1. AGRICULTURAL
APPLICATIONS OF DNA
TECHNOLOGY
SAROJ KUMAR
School Teacher
B.Tech(FT), KUK

2. I. GENERATION OF
TRANSGENIC PLANTS

3. B. DNA INSERTED INTO PLANTS 
TRANSGENIC PLANT
•
Characteristics of transgenic plants
•
All cells in the plant are derived from one cell
•
All cells express the desired genetic information
•
Why make transgenic plants?
•
Genes from distantly related plant families can be
introduced without need for breeding (some families of
plants are incompatible)
•
To improve crop hardiness and characteristics of final plant
product
•
Protein content
•
Ripening rate
•
Drought resistance…..

4. •
Procedures for generating transgenic plants
•
Microinjection
•
DNA constructs injected using fine glass pipettes in combination
with phase contrast microscopy
•
Electroporation of protoplasts
•
Electric pulses of high field strength
•
Reversibly permeabilize cell membranes
•
Electric discharge gun –Gold beads
•
Firing DNA-coated pellets using a modified .22 caliber gun

5. •
“Whiskers” of silicon carbide –holes punched, DNA introduced
•
Agrobacterium tumefaciens
•
Viral vectors
•
Cauliflower mosaic virus vectors
•
Gemini virus vectors
•
Liposome-mediated transformation of protoplasts
•
Artificial lipid vesicles = Liposomes
•
Chemically-stimulated DNA uptake by protoplasts
•
Polyethylene glycol + CaCl
2

6. •
Many gene transfer techniques start with protoplasts
•
Cell wall is digested with cellulase and cells are separated using
pectinase
•
Plant cells are maintained in suspension
•
DNA is introduced, it integrates and expression of desired genes is
achieved
•
Electroporation
•
Microinjection

7. •
Protoplast fusion can also be used to fuse two different plant
types together New Plant Varieties (hybrid plantlet)•
Fused cell acquires some of the characteristic of both genetic
backgrounds and can be regenerated into a plant with some traits
from both parental plants
•
Fusigenic agents (polyethylene glycol) or electroporation used to fuse
membranes
•
Useful if species are sexually incompatible or cross with difficulty

8. •
Commercially important plants that can be grown from single somatic
(non-seed) cells•
Asparagus
•
Cabbage
•
Citrus fruits
•
Carrots
•
Alfalfa
•
Millet
•
Tomatoes
•
Potatoes
•
Tobacco
•
More than 30 different crop plants developed with rDNA techniques
are being tested in field studies

9. II. EXAMPLES OF CROP
IMPROVEMENT
MEASURES

10. A. NITROGEN FIXATION
•
To enable plants to fix atmospheric N
2
so that it can
be converted into NH
3
, NO
3
-
, and NO
2
-

providing a nitrogen source for nucleic acid and
amino acid synthesis
•
Thereby eliminating need to fertilize crops with nitrogen
•
Exploit N
2
fixation metabolic machinery of bacteria
and fungi•
Some live freely in soil and water
•
Some live in symbiosis
•
Rhizobium spp. live in symbiosis with leguminous species of
plants in root nodules (e.g. soy, peas, beans, alfalfa, clover)

11. B. FROST RESISTANCE
•
Ice-minus bacteria•
Ice nucleation on plant surfaces caused by bacteria that aid in
protein-water coalescence forming ice crystals @ 0
o
C (32
0
F)
•
Ice-minus Pseudomonas syringae
•
Modified by removing genes responsible for crystal formation
•
Sprayed onto plants
•
Displaces wild type strains
•
Protected to 23
o
F
•
Dew freezes beyond this point
•
Extends growth season
•
First deliberate release experiment –Steven Lindow –1987-sprayed
potatoes
•
Frost Ban
•
Different strain of bacteria –Julie Lindemann led different project –
1987
•
Strawberries in California

12. C. RESISTANCE TO BIOLOGICAL
AGENTS
•
Anti-Insect Strategy -Insecticides
•
From Bacillus thuringensis•
Toxic crystals found during sporulation
•
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

13. •
Monsanto Chemical Company –1991Trials•
BT into cotton plants using A. tumefaciens vector
•
Cottton bollworms protection in 6 loctions, 5 different states,
consistent results
•
First crops –1996
•
Corn
•
Cotton
•
Seed potatoes
•
Soybean
•
Others

14. •
Cloned BT toxin gene into a different bacterium that lives harmlessly
in corn plants
•
Pressure applied to introduce modified bacterium into seeds
•
Corn stalks protected from corn borers
•
BT in poplar and white spruce catepillar resistance
•
BT-resistant strains are beginning to emerge in some catepillars

15. •
Anti-Viral Strategy•
TMV-coat protein inserted into tobacco and tomato plant cells using
Ti plasmid
•
Viral capsids inhibit viral replication of TMV when infected
•
Grape fan-leaf virus (GFLV)
•
Causes yellowing and deformation of grape leaves
•
Transmitted in soil by nematodes
•
Viral capsid genes introduced into champagne grape vines using T
plasmid
•
Resistance to virus acquired
•
Other trials using capsid proteins: potato leaf-roll virus, cantaloupe
mosaic virus, rice strip virus
•
Concerns that recombination events may lead to new plant virus
strains

16. •
Anti-Bacterial Strategies
•
Resistance to Xanthomonas oryzae(rice wilting)•
Conferred by cloning resistance genes from wild rice strains
•
Anti-Worm Strategies (Animal pest)
•
Nematode resistance gene from wild beet plants
•
To protect sugar beet

17. RESISTANCE TO HERBICIDES
•
Glyphosate resistance
•
Glyphosate = “Roundup”, “Tumbleweed” = Systemic herbicide
•
Glyphosate inhibits EPSP synthase (S-enolpyruvlshikimate-3
phosphate –involved in chloroplast amino acid synthesis)
•
Escherichia coliEPSP synthase = mutant form less sensitive to
glyphosate
•
Cloned via Ti plasmid into soybeans, tobacco, petunias
•
Increased crop yields of crops treated with herbicides

18. •
Bromoxynil
•
= bromine-based herbicide
•
Bromoxynil resistant cotton
•
Concern over movement of resistance genes into weeds
making compounds useless

19. BIOENGINEERED FOODS

20. FLAVR-SAVR TOMATO
•
“Rot-Resistant Tomato”
•
Calgen, Inc.
•
Anti-sense gene complementary to polygalacturonase
(PG)
•
PG = pectinase accelerates plant decay/rotting

21. LAURATE CANOLA OIL
•
Canola plant modified with thioesterase gene obtained
from California bay laurel tree
•
Enzyme produces lauric acid (up to 40% in oil from genetically
modified (GM) canola seeds)
•
Low saturated fat content
•
Heat tolerant
•
Does not break down
•
Excellent for high temperature cooking processes

22. BIOPHARMING

23. WHAT IS BIOPHARMING?
•
Drug production in
genetically modified plants•
Tobacco
•
Alfalfa
•
Potatoes
•
Corn
•
Soybeans
•
Wheat
•
Rice
•
Oilseed rape
•
Ethiopian mustard

24. •
Drugs = Biopharmaceuticals
•
Drugs synthesized organically•
Many drugs are made naturally in plants•
Aspirin (originally isolated from willow bark)
•
Vincristine and vinblastine (periwinkle)
•
Taxol (Pacific yew)
•
Digitalis (foxglove)
•
Recombinant DNA techniques enable many more drugs to be made
artificially in plants
•
Human proteins in plants = xenogenic proteins

25. WHY FARM FOR PHARMACEUTICALS
IN PLANTS?
•
Cheaper than producing pharmaceutical proteins in
cell culture
•
Could reduce the cost of medicine
•
Example:
•
Newest factories producing GM proteins in mammalian cell
culture costs ~$100 million/300 kg, costing ~$1000/g
•
Biopharming producing GM proteins in plants costs ~$10
million capital investment/300 kg, costing ~$200/g
(according to Monsanto’s Integrated Protein Technologies)

26. •
However, costs of extracting and purifying
biopharmaceuticals can be high and processing
strategies need to be improved

27. •
Fewer complications than producing proteins in animals
(e.g. cell culture or milk from “pharm” animals)
•
Possible transmission of animal viruses –zoonoses
•
Plant viruses cannot infect animals
•
Plants do not serve as hosts for infectious agents
such as HIV, HepB, prions
•
Ethical considerations (animal welfare concerns)

28. •
Plants effectively transcribe, translate and assemble
proteins derived from eukaryotic sources
•
Improved quality of life

29. •
Produce beneficial pharmaceuticals in tobacco rather
than cigarettes
•
“If we can actually find a medical use for tobacco that saves
lives, what a turnaround for the much-maligned tobacco plant.”
•
Christopher Cook, CEO of ToBio

30. •
Tobacco is favored for many reasons
•
Easy to genetically engineer (Agrobacterium-mediated
transformation)
•
Excellent biomass producer
•
~1 million seeds can be isolated from a single plant (scale-up
benefits)
•
Number one cash crop in Virginia

31. EXAMPLES OF BIOPHARMACEUTICALS
•
Hepatitis B and other subunit vaccines
•
Urokinase (clot dissolving drug)
•
Human serum albumin (liver cirrhosis treatment)
•
Hemoglobin
•
Human erythropoietin
•
Glucocerebrosidase (Gaucher’s disease)

32. •
Blood coagulants
•
Proteases (e.g. trypsin)
•
Protease inhibitors (e.g. aprotinin -used by surgeons)
•
Growth promoters
•
HIV viral coat protein (HIV therapy)
•
Nutraceuticals (Vitamin A and E, amino acids)

33. •
Neurologically active agents (human enkephalins)
•
Protein based sweetener (Brazzein)
•
Avidin
•
Beta-glucoronidase
•
Indirect thrombin inhibitor (Hirudin –anticoagulant originally
isolated from the leech Hirudo medicinalis)
•
Human epidermal growth factor
•
Human interferon-alpha (Hepatitis B and C treatment)

34. •
Bacterial enterotoxins
•
Human insulin
•
Norwalk virus capsid protein
•
“Natural” plastic (plastic-like polymers) (Biopol)
•
Human GM-CSF
•
Human alpha-1 antitrypsin (cystic fibrosis/liver treatment)
•
Angiotensin-1-converting enzyme (hypertension)

35. •
Edible Vaccines –Ongoing Research Areas
•
Hepatitis B
•
Dental caries -Anti-tooth decay Ab (CaroRxTM) (anti-
Streptococcus mutans)
•
Autoimmune diabetes
•
Cholera
•
Rabies
•
HIV
•
Rhinovirus
•
Foot and Mouth
•
Enteritis virus
•
Malaria
•
Influenza
•
Cancer

36. ENVIRONMENTAL RISKS
•
Pharmaceutical products may inadvertently be introduced
into the general food supply
•
Cross-pollination
•
Pollen from a drug-containing crop fertilizes a neighboring related
crop (or wild relatives) used for animal consumption
•
Wind
•
Insects

37. •
Consumption of GM plant by insects Food chain•
Accumulation in birds –extinction? (e.g. DDT and bald eagle)
•
Deleterious effects on non-target organisms (NTO’s)
•
NTO’s = organisms in the environment that are affected by the
product unintentionally
•
Insects, arthropods
•
Risk to NTO’s
•
Depends on recombinant protein involved
•
Risk assessment carried out case-by-case