Bt Technology - Present Status And Future Prospectus

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2. Bt Technology - Present Status And Future
Prospectus
Dev Hingra
Ph D Scholar
RAJASTHAN COLLEGE OF AGRICULTURE
UDAIPUR (RAJASTHAN)
Department of Plant Breeding and Genetics
Mail Id- mail2devhingra@gmail.com

3. CONTENT
•Introduction
•Production Gm/Bt Crops
•Extracting The Desired DNA
•Cloning The Gene Of Interest
•Transformation Process
•Mode Of Action Bt-crop/Bt-gene
•Need Of Bt Transgenic
•Adverse Effect Of Pesticide Use
•Bt Formulated Product
•Crop –Bt Gene Used-against Pest
•Risk, Concern In Gm/ Bt Crop
•Future Aspect’s
•Conclusion

4. What is Bt ?
Bt (Bacillus thuringiensis)
- Living Organism Soil Bacterium
- Gram - Positive
- Family of bacteria
- Produces Spores
- Spores have Insecticidal crystal
Protein around it
- When insect eat this bacterium
get Killed

5. What are Bt-Crops ?
What are GM Crops?
How it (GM) is Produced? i.e. How Bt –crops are produced?
Bt –Crops –
GM Crops (Genetically Modified) Crops
GM Crop- in which Foreign genes of bacteria or
virus called gene of interest (GOI) is inserted
to host plant to alter their genetic make-up .
Bt –Crops Produced are the one of the Example
of it (GM)Crops .
In Bt –Crops gene responsible to produce
Insecticidal Proteins are Identified, Isolated,
Inserted into the host plant and GM / Bt –
crops produced

6. Example of GM Crops
Sr.
No.
GM Crop
Vegetables
Sr.
No.
GM Crop
Cereals , Oil Seed
Sr.
No.
GM Crop
Horticultural Crop
1. Black Pepper 14. Cotton 26. Ragi
2. Brinjal 15. Rice 27. Maize
3. Capsicum 16. Soyabean 28. Potato
4. Cardamom 17. Pearl Millet 29. Castor
5. Cauliflower 18. Finger millet 30. Bamboo
6. Cucumber 19. Groundnut 31. Apple
7. Chili 20. Sorghum 32. Pomegranate
8. Okra 21. Pigeon pea 33. Black Paper
9. Tomato 21. Sugarcane 34. Banana
10. Onion 22. Wheat 35. Coffee
11. Carrot 23. Sunflower 36. Papaya
12. Pea 24. Safflower 37. Watermelon
13. 25. Mustard 38. Tea
IGRIMOS DATA

7. Production GM/ Bt-Crops

8. EXTARCTING THE DESIRED DNA:
Before,
Extracting DNA From Donor organism i.e. Bacillus
thuringiensis
 Identify the particular gene responsible for
insecticidal toxin
 Unfortunately scientist today still know very
little about Identify the particular gene
 Most of the research in transgenic is focused on
identifying and sequencing these certain genes
Once, The Gene has Identified
The two main tools involved with isolating a gene
 restriction enzymes (as “scissors”)
 D.N.A ligase. (as “glue scissors”)
The restriction enzymes recognize and cut’s the
D.N.A at a specific region of the D.N.A, like
scissors
The ligase then attaches the two ends of D.N.A
fragments together, like glue

9. CLONING THE GENE OF INTEREST
Before, the specific D.N.A region can be
inserted into another organism,
we must obtain the D.N.A in a significant
amount.
That’s Why cloning the gene is necessary.
Here, A Plasmid is used - where the isolated
gene is to be put ,
Along with desired DNA (gene) an antibiotic-
resistance gene is also inserted into a
bacterial plasmid, which in turn is inserted
into a carrier cell. (usually a bacterial cell
such as E. coli)
Carrier cell (E. coli ) multiplies and along with
it also multiplies Desired DNA (Gene) in
Plasmid.

10. Desired DNA (Gene) is in Carrier cell OR Not?
For that, Two Mediums are used
1) One Medium With Specific Antibiotic
2) Second Medium Do not have Antibiotic
 Carrier Cell, then Placed onto both the Mediums
 Carrier Cells grow in the medium that did not have the antibiotic
would grow substantially
 While the medium with the antibiotic would only grow slightly
This is because, carrier cells that did not have the desired D.N.A
and antibiotic- resistance gene will not grow on the medium
which has the antibiotic on it.
 This ensures, that the carrier cells are all going to have the
desired D.N.A in it

11. Desired DNA (Gene) Will Express in Plant Genome?
Desired DNA is present in Plant cell?
For that,
Designing a gene has to be Done, so it can be easily inserted into a
crop
For the Successfully Insertion of Gene
It must be slightly modified As Follows:
 Promoter sequence –
must be added to the gene so that it can be correctly expressed (is
considered an on/off switch ,which controls, when and where the
specific gene will be expressed)
For Example: A common promoter is CaMV35S
 Promoter generally results in a high degree of expression in
plants

12. Conti...
Another, Must be added with Terminator Sequence
- Which sends a signal to the cellular machinery that the end of
a gene has been reached
Last thing that must be added a selectable marker gene
- in order to identify plant cells or tissues that have successfully
been inserted with the desired D.N.A gene or Not?

13. Transformation
Transfer of Desired DNA (Gene) into Plant cell
There are two Methods : 1) Gene gun method
2) Agro bacterium method
Most Common, Agrobacterium method:
 Includes Use of soil-dwelling bacteria known as
Agrobacterium tumefaciens
 Because, it has the ability to infect plant cells with a piece of
its D.N.A( Tumor Inducing Plasmid)
 And that DNA get Integrated into Plants Chromosomes
 Scientist inserted Desired DNA(Gene) at the Ti Plasmid of
Agrobacterium tumefaciens
 By using Above Bacterium’s ability Desired DNA is
Transformed into Host Plant

14. Transformation Process

15. Mode of action Bt –Crop/Bt-Gene

16. Mode of action Bt –Crop/Bt-Gene
Gene responsible produces toxin protein
Remains Inactive Until it get Solubllised by Proteases (alkaline Condition)
When Insect /pest –feed on such Plants
These toxin proteins enter in there Body
Toxin Get Solublised in Insects Midgut
Upon Proteolytic activation become Highly Specific in Insecticidal Activity
Activated Toxin Binds to only Specific Receptors on the apical brush Border
Of Susceptible Insect
Upon Binding to Specific receptors proteins Alpha-H elicaces penetrate the
Membrane
Forms pores in the membrane
these Disruption of gut lining leads to Death of Insect due to Starvation Or
Septicemia

18. Why to go towards Bt TRANSGENIC
Basic Objectives :
-To Increase Yield
- Provide Food to Growing Population
-Providing Ample Food Grain at Affordable
Price
- Not to Destroy Natural Resources
Above objectives can be achieve through following methods:
-Use of Conventional Breeding Methods
-Use of Pesticides to Control pest thereby
increasing Yield
-Use of Fungicides to control Diseases
thereby increasing Yield

19. Cont…
By Conventional Breeding Methods:
Mutation Breeding
Hybrid Production
Synthetic
Composite Variety
Increases Yield But has Following Demerits:
1) Time Consuming
2) Trial And Error Process
3) Result Frequency is very low For Example: In
Mutation Breeding Frequency of Desirable
mutation is Very Low i.e. 0.1% of total mutation
Again, Desirable Mutation is associated with
Undesirable Mutation

20. About Pesticide Use
Pesticides are use to control Pest
As Pest Causes tremendous loss in yield in Crop:
Sr. No. Crop Name % Loss Due to Pest
1. Wheat 52%
2. Soybean 58%
3. Maize 59%
4. Potato 74%
5. Rice 83%
6. Cotton 84%
Oerkret et al.(1994)

21. Productivity loss Caused by some Important Insect–Pest
Common Name Scientific Name Productivity Loss (%)
Chilli midge Aspordylia capsici 60-80
Cotton white fly Bemisia tabaci 53-80
Pulse stem fly Ophiomyia sp 50-72
Cotton boll worm Spodoptera sp 50-60
Brinjal fruit and stem borer Lucinodes orbonalis 48
Caster semilooper Achea janata 40
Mustard aphid Lipaphis erysimi 30-70
Sorghum shoot fly Atherigona soccata 30-60
Sorghum midge Contarinia sorghicola 30-50
Linseed budfly Dasineura lini 30
Maize pink borer Sesamia inferens 25-75
borerRice stem Scirpophaga incertulas 25-30
Pandey, et al.(1995)

22. Adverse Effect’s of Pesticide Use
 Adverse Effect on Beneficial Organisms
 Environmental Pollution
 Pesticide Residue in Food, Fodder and water
 Causing Health Problem

23.  Need of replacing Agrochemicals harmful to Human
Health and Environment
 IPM practices to provide adequate Crop Protection for
Sustainable Agriculture (Hazra et al., 1998)
 Same is the Case in Fungicide use.
 Bt/Transgenic Crop is the Solution To Above Problem
(Krattiger., 1997)
Bt/Transgenic Crop:
 Not Require More Time
 No Problem of Residue like Pesticide
 Cost of Production is Save
 GM/Bt Increase the Nutritional value of the food
For Example: Bt Corn Shows Increase amount of Vitamin
A

24. COST OF PRODUCTION OF Bt /GM CROP IS LESS
 Cost on Pesticide use is decreases
 Number of Application of Insecticidal Spray also
decreases
 The research published in Science in 2003 has shown that
the use of genetically modified Bt cotton in India
increased yields by 60% over the period 1998-2001.
 While the number of applications of insecticides against
bollworm were three times less on average. (Qaim,
Matim; Zilberman, 2003).
 Insecticide application was reduced by 67% in China
(Huang et al., 2002) and 66% in south Africa (Ismael et
al., 2002) by the use of Bt transgenic cotton
 Insecticide applications on Bt crops were reduced by 14-
76% and 72% of farmers worldwide Carpenter, J. E.
(2010).and Carpenter, Janet (2010).

25. Instead of Bt Transgenic, Why not to Use Bt Formulation
Products
Bt-Formulation ?
History of Bt–bacteria Formulated Products:
In 1901, Japanese Biologist – Shigetane Ishiwatari
-Investing The Cause of Sotto Disease (Sudden-Collapse
Disease)
- Killing Large Population Of Silkworm
Cause of above Disease was the Bacterium
In 1911, Ernst Berliner – Isolated same Bacterium which had
killed Mediterranean Flour Moth

26. How Bacterium named as Bacillus thuringensis ?
Ishiwatari – Named Bacterium – Bacillus Sotto-1901
Ernst Berliner-Name Bacterium Bacillus thuringensis-1911
(Germen Town Thuringia where Mediterranean Moth was
found )
Name Bacillus thuringensis Ruled Permanently to Bacterium
Berliner – Reported The existence of Crystal Protein within that
Bacterium
After Knowing, Spores of Bt Bacterium Contains Crystal Protein
- Which has Insecticidal Property
- Bt- Formulated Products started to prepare and
Farmers also started to use it.

27. Bt- Formulated Products
Bt-Active Ingredient Products Maufacturer
Bacillus thuringensis var.
galleriae
WAXMOTH
CERATAN
Sandoz
B.t.var isralensis Mosquitoes and Black flies
Bactimos Philips Dupher
Batics CRC
Skeetal Novo Biocontrol
Tknar Sandoz
Catterpiller, Loopers
(Bio-Control Symposium ,2003)

28. Farmers using Bt- Forsmulated Products
But,
These Bt- Biopestcides has following Disadvantages :
1) Bt –Products such as sprays are rapidly washed by rain
2) Become ineffective within 24 hours
3) Require to spray continuously,
(Because bt products (crystal protein) degraded under the sun’s UV ray’s)
4) Failure to penetrate tissues and therefore to reach insects in all parts of the
plant.
(Bt sprays are non-systemic insecticides and are therefore ineffective against
insects that do not come into direct contact with the crystals, such as sap
sucking and piercing insects, against root dwelling pests, or larvae that after
hatching rapidly burrow or bore into plant tissues). (McGaughey and Whalon,
1992)
That’s why Bt transgenic are only way to solve above problem

29. As Bt are Specific in action’s
i.e. For Specific insect -Specific Bt -Bacterium
(Gene of Insecticidal Property of Specific Bacterium to Specific Pest)
Bt has different strains – Against Different Pest
Cry gene designation Toxic to these insect orders
CryIA(a), CryIA(b), CryIA(c) Lepidoptera
Cry1B, Cry1C, Cry1D Lepidoptera
CryII Lepidoptera, Diptera
CryIII Coleoptera
CryIV Diptera
CryV Lepidoptera, Coleoptera
Bio-Control Symposium ,2003

30. Bt- Crops With Gene used
Sr.
No.
Crop Name
Vegetable &
Year
Company
Name
Trait Gene/Event
1. Tomato -2010 Indian Institute
of Vegetable
Research
Insect
resistance
cry1Ac
2. Cauliflower and
Cabbage-2010
Nunhems India
Pvt Ltd
Insect
Resistance
cry1Ba, cry1Ca
3. Brinjal-2010 Indian Institute
of Vegetable
Research
Insect
resistance
cry1Ac
4. Brinjal -2009
Bejo Sheetal
Seeds Pvt. Ltd.
Insect
Resistance
cry1Fa1
University of of
Agricultural
Sciences
cry1Ac
( IGRIMOS DATA ,2010)

31. Sr.No. Crop Name
Vegetable &
Year
Company Name Trait Gene/Event
5. Brinjal-2007 MAHYCO Insect Resistance cry 1 Ac
University of Agricultural
Sciences
Insect Resistance cry 1 Ac
Sungro Seeds Research Ltd. Insect Resistance cry 1 Ac
Tamil Nadu Agricultural
University
Insect Resistance cry 1 Ac
6. Okra -2007 MAHYCO Insect Resistance cry1Ac
7. Brinjal-2006 IARI, New Delhi
Insect Resistance
cry1Aa and cry1Aabc
Sungro Seeds Ltd, New Delhi
Insect Resistance
cry1Ac
Mahyco, Mumbai cry1Ac
8. Okra-2006 Mahyco, Mumbai
Insect Resistance
cry1Ac, cry2Ab
9. Cauliflower-2006 Sungro Seeds Ltd, New Delhi,
Insect Resistance
M/s. Nunhems India Pvt. Ltd.,
Gurgaon
cry1Ac, cry1Ba and
cry1Ca
Cont… Bt- Crops With Gene used
IGRIMOS DATA-2010

32. www.globalresearch.ca/index.php

33. (ISAAA DATA ,2007)

34. Adoption of Bt – Cotton in Major Growing Countries:
James, C. (2003)
Country 1996 1997 1998 1999 2000 2001 2002 2003
USA Yes Yes Yes Yes Yes Yes Yes Yes
Australia Yes Yes Yes Yes Yes Yes Yes Yes
China Yes Yes Yes Yes Yes Yes Yes
India Yes Yes
Indonesia Yes Yes
Mexico Yes Yes Yes Yes Yes Yes Yes Yes
Argentina Yes Yes Yes Yes Yes Yes

35. Crop –Bt-Gene used- Against Pest:
Source: Kumar(2007)

36. Crop –Bt-Gene used- Against Pest:
Source: Kumar(2007)

37. 2002 -2003 2004-2005 2006-2007
Bt Conventional Bt Conventional Bt Convention
al
Yield
(Kg/ha)
1627.94 1212.92 1835.80 1360.33 2079.72 1457.71
Insectcide
Use Kg/ha
5.11 10.30 5.06 10.35 3.01 3.83
Net Revenue
(/ha) in Rs.
13082.02 7741.62 12161.84 5317.79 17595.55 10331.89
Yield, insecticide use and Net Revenue from Bt and Conventional
Cotton plots:
(Subramanian and Qaim,2009)

38. Sr.n
o.
Year Name of Hybrid Name of
Compony
Gene event Zone
1. 2002 MECH12 Bt M/s Mahyco Cry 1ac Central
2. 2002 MECH 162 Bt M/s Mahyco Cry 1ac Central and
South
3. 2004 RCH 2 Bt M/s Rasi Seed
Ltd.
Cry 1ac Central and
South
4. 2005 NCs-207 M/s
Nausiveedu
Seed Ltd.
Cry 1ac Central and
South
Commercially Realeased Bt –Cotton Varieties in India :
Zone : Central – Gujrat , Madhya Pradesh , Maharashtra
South- Andhra Pradesh ,Karnatak, Tamil Nadu

39. Risk, Concern in GM/Bt Crops
The introduction of transgenic crops and foods into the existing food
production system has generated a number of Risk and Concern Can
be Categorized in Major Two Categories:
1. Damage to human health
Allergen city
Horizontal transfer and antibiotic resistance
Cauliflower mosaic virus promoter
Changed nutrient levels
2. Damage to the natural environment
Monarch butterfly
Crop-to-weed gene flow
Antibiotic resistance
Leakage of GM proteins into soil

40. Allergencity
However, there is no evidence so far that genetically
engineered foods are more likely to cause allergic
reactions than are conventional foods But,
Has Found Allergenic Reaction in GM-Soyabean and Star
Link corn
GM Soyabean is produced by the introduction of a gene
from the Brazil nut - to improve Nutritional Quality
Brazil Nut Shows Allergies, so GM Soyabean also Tested
For Allergencity : Shows Allergy same as Brazil Nut
Government Banned to Produce, Store GM Soyabean
In the Star Link Corn – Has the Allergic Strar Link-
Protein
Risk : Like the other Crops May Show Gene Linked
Character in Doner Plant Which May be Allergenic to
Human Being

41. Risk of Horizontal transfer and Antibiotic Resistance
Use of Antibiotic Resistance Marker in the
development of Transgenic /Bt Crop, has Raised
Question .
Antibiotic Used against illness shows antibiotic
Resistance Because of Eating GM Food having
Antibiotic Resistance Marker used in it.
Horizontal transfer :
Transfer of DNA from one organism to another outside
of the parent-to-offspring channel i,e. By Eating
GM/Bt Food
Risk : Transfer of antibiotic Resistance marker into the
micro-organism present in stomach and Intestine
May cause serious attack to Human Kind
Study shows that Feeding of GM Food Shows
Decrease in Body Length

42. Risk of Cauliflower Mosaic Virus promoter
 Generally CaMV promoter or the 35S promoter mainly
use in the Production Process of GM /Bt-Crops
 These Promoter was obtained from the virus that causes
Cauliflower Mosaic disease in several vegetables, such as
cauliflower, broccoli, cabbage and canola
 It is Found that CaMV promoter might be harmful if it were to
invade our cells and turn on our genes.
 If such food is eaten there might be risk of invade this
Promoter Human cells and there have been no tests to
determine whether the CaMV promoter has invaded human
tissues, such Experiment has done on mice
Risk of changed nutrient levels :
By producing Roundup ready resistant (Herbicide Resistant)
soyabean shows deacrease in Nutrient Level than
Conventional Soyabean i.e. isoflavone level decreases,
Necessary for preventing heart disease, breast cancer and
osteoporosis

43. Damage to the natural environment
Monarch butterfly:
Losey et al. (1999) reported that larvae of
Monarch Butterfly (Harmless) were
poisoned by Bt Corn Pollens
Most colorful Monarch butterfly feed on the
leaves of the milkweed plants
Done the Experiment By Feeding Bt-Corn
Pollen to of Monarch Butterfly (Harmless)
–Dies (Kill)
Same Result was found on black swallowtail
Butterfly (Harmless)
Larva of a Monarch butterfly
feeding on a milkweed leaf
Black swallowtail butterfly
Monarch butterfly on alfalfa
flower Photo: Marlin E. Rice

44. Risk of crop-to-weed gene flow
There Might be risk of Gene flow from Crop to weed
It is Possible Because:
Gene flow from crops to weeds requires:
1. Presence of sexually compatible wild or weedy relatives close
to the crop
2. An overlap of flowering times between the crop and the wild
relatives
3. Presence of a pollinating agent such as a bird or an insect
unless pollination is accomplished by the wind
All the above condition are present in closely related weed
So, there is a chance to flow Herbicide Resistant Gene From GM
–Crop to Weed and weed become “SUPERWEED ”
- Not affected by Herbicide
- Difficult to Control

45. Leakage of GM proteins into soil
Many Plants Leak Chemical Compound into the soil
through their roots
Transgenic plants may leak different compounds into the
soil
Study has attempted in Canada to see whether
Transgenic Crops Leaks any Chemical into Soil:
Result’s are positive
Research was carried on Bt Corn – Shows that Bt Corn
root leaks Bt-toxin into Soil and remain potential
more than 200 days. (Saxena et. al.,1999)
One Report also shows that Bt –toxin move towards river
through soil (Professor Jean-Francois Narbonne of the
University of Bourdeaux) i.e. Micro organism
from soil and river may be affected severely.

46. Future Aspect’s
 Development of Tomato Plant
- With increased in content of Lycopene
- Salt Tolerant Tomato
- Late ripening Tomatoes
 Development of crop rich in Vitamin –A
Canola is a major oilseed crop
Nutritional quality of canola oil by enhancing the
Vitamin E content
 To Produce Crop with Edible Vaccines – Research
is in progress in Banana
 Development of Sclerotinia Resistant Sunflower

47. Conti…
 Development New Promoter Sequence other than CaMV
Promoter
 Development of GM /Bt –Crop With Male Sterile

48. Conclusion:
 As population of world is increasing tremendously,
our objectives are to:
Increase Yield , Provide Food to Growing Population, Providing
Ample Food Grain at Affordable Price
Not to Destroy Natural Resources .
 To achieve above Objectives we have:
Conventional Breeding Methods , New technology in breeding,
Use of Pesticides . Fungicides etc.,
- Has lot of disadvantages .
 This disadvantages can be minimized by the Bt/GM –Crops
better than above methods
- has the risk to health and Environment .

49. Cont…
 Finally there are :
 two ways to achieve our main objective
 either by Improving Efficiency of
breeding methods,
 minimizing hazards due to pesticides,
fungicides
or
 by doing more and research in Bt/GM-
crops i. e. using /developing techniques
not harming Health and Environment.