weed biotech mesures

1. Biotechnological approaches – Development of
herbicide resistance in crops – genetic methods
and other methods
AGR 606 Advances in weed management (2+0)
Course teacher
Dr. C.R. Chinnamuthu
Professor and Head
Department of Agronomy
TNAU, Coimbatore
K. Vijay Aravinth
2019602021
I Ph.D Agronomy

2. Biotech crops
• Biotech crop area in 2018 attains new record- high adoption at 191.7 million hectares
worldwide.
• Biotech crop area increased 113 fold from 1996 - fastest adopted crop technology in the
world.
• In 2018, 70 countries adopted biotech crops- 26 countries planted and 44 additional
countries imported.
• Biotech soybeans reached 50% of the global biotech crop area.
• Herbicide tolerance remained dominant trait, occupied 45% of the global biotech crop
area.
• Stacked traits are favoured by farmers for all three major biotech crops: maize,
soybeans and cotton.

4. Herbicide resistance
“Herbicide resistance is the inherited ability of a plant to survive and reproduce
following exposure to a dose of herbicide normally lethal to the wild type. In a
plant, resistance may be naturally occurring or induced by such techniques as
genetic engineering or selection of variants produced by tissue culture or
mutagenesis.”

5. HR Crops
• There has been growing interest over the past three decades in the
development of crop varieties physiologically and biochemically resistant
to non-selective herbicides or selective to only specific crops effective
against a wide range of weed species.
• HR crops offer flexibility in management of weeds, reduce the number of
applications in a season in long duration crops, enable using low toxicity
compounds which do not remain active in the soil for long periods and
facilitate using no-tilll or conservation till systems.
• HR traits are used on >80% of the estimated 191 million hectares
transgenic crops grown annually in 25 countries.

7. Mechanism of herbicide resistance
• Exclusionary resistance mechanism -
Plant exclude the herbicide molecules
where they produce the toxic response
• Site of action response - specific site in
the plant become resistant against the
herbicide
• Site of action over production - plant
confer resistance by over production of
target site. It dilutes the herbicides that
reaches the target site and the over
produced enzyme thus remains active to
complete normal function and growth. Kandasamy et al., 2002

8. Source of Resistant genes and traits
• Microorganisms, higher plants and animals are potential sources of resistant
genes.
• The isolation of genes from microorganisms is often easier than from higher
plants, but the right organisms must be found first (Gressel, 1993).
• Crop plants are inherently resistant to many herbicides, and improvements can be
made with proper selection efforts in many instances.
• One of the richest sources of resistance against herbicides - resistant weeds
– Atrazine tolerance into foxtail millet was introduced from the weed green
bristle grass (Darmency, H., & Pernes, J. (1989))
– Sulfonylurea tolerance into domestic lettuce was introduced from prickly
lettuce (Mallory-Smith et al., 1993)

9. Techniques involved in developing herbicide resistance crop

10. Traditional plant breeding
• Inter and intra-specific variability in the herbicide tolerance of crop and
weeds are utilized.
• Source of the resistance genes are crop related weeds.
• Success rate is very limited in this method because of sexual
incompatibility, excessive time consumption, labour costs and slow
process.
• Triazine resistant gene of bird’s rape (Brassica campestris L.) was
transferred to canola (Brassica napus L.) through back crossing (Beversdorf
et al., 1980).

11. Germplasm screening for herbicide tolerance

12. Biotechnological techniques
In vitro mutant selection at the cell or tissue level:
• Somaclonal variation in cultured plant cells are exploited to select herbicide
resistance.
• Herbicide tolerant mutants can be selected by exposing them to a herbicide.
• Radin and Carlson (1978) sprayed immature leaves of mutagenized haploid
tobacco plants with the herbicides bentazon and phenmedipham.
• Following herbicide treatment and leaf expansion, the small green islands that
appeared on the otherwise yellow leaves were excised and placed on a culture
medium known to induce shoot regeneration.
• This method is also known as ‘Green island technique’.
• Bentazone and Phenmedipham tolerant tobacco, Glyphosate tolerant petunia
and L- phosphinothricin tolerant alfalfa was developed through this method.

13. Somatic hybridization:
• The lack of sexual compatibility between many crop plants and weeds was
an important factor limiting the application of classic breeding techniques
in genetic manipulations for the development of crops tolerant to selected
herbicides.
• This problem can be bypassed by the use of somatic cell genetic
procedures such as protoplast fusion.
• Atrazine resistant potato was developed from black nightshade (Solanum
nigram L.) (Binding et al., 1982).

14. Microspore and seed mutagenesis:
• This technique require several backcrosses of the isolated, resistant
mutant lines to eliminate undesirable mutations resulting from random
mutagenesis.
• Many mutagens including physical (gamma irradiation) and chemical
(ethyl methanesulfonate, N-nitroso-N-methylurea, ethylnitrosourea and
sodium azide)
Type of mutagenesis Resistant mutants Crop
EMS Imidazolinones, Sulfonylureas Soybean, tobacco
Sodium azide Trans-4-hydroxy-L-proline Barley
Gamma irradiation Chlorsulfuron Lentil

15. d) Plant transformation:
• The direct transfer of cloned genes into cells of sensitive plants. There are
different of this technique:
 Engineered vector technique:
 Gene gun technique
 Microinjection
 Direct gene transfer or electroporation
 Pollen vector system

16. Engineered vector technique
• Various vectors can be used to transfer DNA from one plant to another.
• The most promising and widely used is the Ti plasmid of Agrobacterium
tumefaciens.
• This bacterium has the unique ability to enter a plant cell and insert a small
piece of its plasmid DNA into the chromosomes of the host plant causing a
plant disease known as crown gall (Caplan et al., 1983).
• By means of recombinant DNA technology, the gene to be transferred is
spliced into the Ti plasmid of Agrobacterium tumefaciens.
• The engineered bacterium, carrying the recombinant DNA in its plasmid, is
then introduced into excised leaf disks or plant cells growing in culture.
• The bacterium enters the cells and the plasmid inserts a part of its DNA into
the chromosomes of the plant cells, introducing thus the cloned gene into the
plant genome.

17. Transfer of cloned genes into susceptible plants

18. Gene gun technique:
• Another tool which facilitates gene transfer is the “gene gun” developed
1986 at the Cornell University, USA.
• The gene gun shots DNA segments into cell at high speed and some of the
DNA segments are incorporated into plant genome.

19. Microinjection
• In microinjection, DNA is injected directly into the nucleus of a specific
plant cell, using a fine glass needle which is less than a micron wide at its
tip.
• It is a delicate process and sophisticated instrumentation is needed for the
application of this technique.

20. Direct gene transfer or electroporation
• Direct gene transfer (electroporation) describes the phenomenon of uptake
of genes into plant cells (protoplasts) through the naked plasma membrane
and the functional integration of these genes into the host genome.
• The method is independent of biological vectors and it makes use of the
fact that DNA can pass through plasma membrane and that protoplasts can
be totipotent.
• The method requires protoplasts - totipotent protoplasts are not readily
available for every desired plant species, especially the cereal crops.
• An electroporator is used to apply short high-voltage pulses to facilitate the
entrance of the foreign DNA into the shocked protoplasts.

21. Multiple HRC
• HR crops need to be resistant to more than just glyphosate.
• Currently, Monsanto, Dow, Bayer, Syngenta and BASF are developing new
crop herbicide resistance traits in combination with glyphosate resistance

22. Herbicide tolerant GM Crop Events approved in India
Crop Gene introduced Gene source GM trait Method of
transformation
Trade name
Soybean pat Streptomyces
viridochromogenes
Glufosinate
herbicide
tolerance
Microparticle
bombardment
of plant cells or
tissue
Liberty Link®
csr1-2 Arabidopsis thaliana Sulfonylurea
herbicide
tolerance
Microparticle
bombardment
of plant cells or
tissue
Cultivance
cry1Ac+cp4
epsps
Bacillus thuringiensis
+Agrobacterium
tumefaciens
Lepidopteran
insect+Glyphosa
te herbicide
tolerance
Conventional
breeding - cross
hybridization
and selection
Intacta™
Roundup
Ready™ 2 Pro
cp4 epsps Agrobacterium
tumefaciens
Glyphosate
herbicide
tolerance
plant
transformation
Genuity®
Roundup Ready
2 Yield™

23. Publicly announced transgenic multiple-herbicide-resistant crops
Herbicide types Crops
Glyphosate and glufosinate Soybeans, corn and cotton
Glyphosate and ALS inhibitors Soybeans, corn and canola
Glyphosate, glufosinate and 2,4-D Soybeans and cotton
Glyphosate, glufosinate and dicamba Soybeans, corn and cotton
Glyphosate, glufosinate and HPPD
inhibitors
Soybeans and cotton
Glyphosate, glufosinate, 2,4-D and
ACCase inhibitors
Corn
Glufosinate and dicamba Wheat
Green JM 2011

24. Transgenic varieties/hybrids approved for commercial
cultivation in USA
ISAAA Website

25. Non-transgenic varieties tolerant to herbicide released for
cultivation

26. Advantages of transgenic herbicide tolerant crops
• Facilitate low or no tillage.
• Broader spectrum of weeds controlled.
• Reduced crop injury.
• Reduced herbicide carry-over.
• Use of herbicides that are more environmentally friendly.
• New mode of action for resistance management.
• Crop management flexibility and simplicity.
• Superweeds - A wild plant that has been accidentally pollinated by a
genetically modified plant.
• Examples of weeds resistant to Glyphosate are Common Ragweed, Italian
Ryegrass etc.

27. Disadvantages of herbicide tolerant crops
• Mammalian toxicity due to increased usage of herbicide.
• Ecotoxicity (side effects on soil microorganisms and
• agricultural flora or fauna).
• Raising herbicide resistant weeds and volunteers’ crop.
• Yield performance is affected.
• Single selection pressure and weed resistance.
• Shifts in weed species (minor weeds may become major).
• Gene escape (transfer of transgenic trait into related wild
• weedy species by pollination).
• Gene flow and contamination to organic crops.
• Drift and non-target movement of resistance

28. Induction of Herbicide resistance via seed-derived rice (Oryza sativa) Calli
Regenerated plantlets with glyphosate resistance at different stages
Ekanyaka et al., 2016

29. The development of herbicide-resistant maize:
stable Agrobacterium-mediated transformation of maize using explants of
type II embryogenic calli
Rizwan et al. 2017

30. Glufosinate leaf-painting assay, using Basta
with concentration 200 mg ml-1 glufosinate.
The production of transgenic plants using
Agrobacterium mediated transformation

31. References
• Green, J. M. (2014). Current state of herbicides in herbicide‐resistant crops. Pest Management
Science, 70(9), 1351-1357.
• Green, J. M. (2011). Outlook on weed management in herbicide-resistant crops: need for
diversification. Outlooks on Pest Management, 22(3), 100-104.
• Green, J. M. (2012). The benefits of herbicide‐resistant crops. Pest Management Science, 68(10),
1323-1331.
• Kandasamy, O. S., Chinnusamy, C., & Chitdeshwari, T. (2002). Herbicide resistance in crop plants–A
review. Agricultural Reviews, 23(3), 208-213.
• Darmency, H., & Pernes, J. (1989). Agronomic performance of a triazine resistant foxtail millet
(Setaria italica (L.) Beauv.). Weed Research, 29(2), 147-150.
• Mallory-Smith, C., Thill, D. C., & Dial, M. J. (1993). ID-BR1: sulfonylurea herbicide-resistant lettuce
germplasm. HortScience, 28(1), 63-64.
• Ekanyaka, E. M. S., Weerakoon, S. R., Silva, T. D., & Somaratne, S. (2016). Induction of herbicide
resistance viaseed-derived rice (Oryza sativa) calli. IRA-International Journal of Applied
Sciences, 3(3), 2455-4499.
• Saini, Gurpreet & Rana, Surinder. (2018). Transgenic herbicide resistance crops.
10.13140/RG.2.2.14194.61127.

32. Thank you