Bt Maize

2.  Maize is called as “King of cereals” because of its high productivity
and adaptability.
 It is the 4th
most important crop next to rice, wheat and sorghum.
 Area, production and productivity of maize in 2007.
 This year, maize production has been severely affected due to
insufficient rainfall, which adversely influenced the epidemic of
stem borers. (source: Dr.C.P.Mallapur,entomologist)
Area Production Productivit
y
World 139 (mha) 590 (mt) 4229 (Kg/ha)
India 7.32 (mha) 14.93 (mt) 19.04(q/ha)
Karnataka 9.3 (lakh ha) 16.2 (lt) 29.5 (q/ha)

3. Source: Science & technology, Deccan herald, Tuesday, August 5,2008.

4. Maize Area, Yield and Production for Top 10 Maize Countries 2006.
Sl.no
Country
Harvested
Hectares
(Millions)
Yield
MT/Hectare
Production
(Millions MT)
1 USA 28.5 8 228.7
2 China 24.5 5.1 124.2
3 Brazil 11.8 3 35.5
4 Mexico 8 2.4 19
5 India 6.2 1.9 12
6 Nigeria 4.2 1.3 5.4
7 South Africa 3.3 2.7 9.1
8 Indonesia 3.3 2.8 9.3
9 Romania 2.9 2.9 8.5
10 Philippines 2.4 1.8 4.3

5. Distribution of World Maize Production
Source: CIMMYT, Mexico

6.  Stem borers causes 80% of losses in maize. It is a polyphagous
pest.
 Global level loss due to borers - $ 5.7 billion annually
Insecticides used - $550 million annually.
 In US 1$ billion per annum – control + losses.
 These borers belong to the order: Lepidoptera
family : Pyralidae
 There are different species of stem borers are their which
causes damage to the crop.

7. Sl.no Common Name Scientific Name Affected Regions
I Tropical stem borers
1 Spotted stem borer Chilo Partellus Asia, East Africa
2 Oriental corn borer (Asian corn borer) Ostrinia furnacalis Asia
3 Lesser cornstalk borer Elasmopalpus lignosellus Americas
4 Pink stem borer Sesamia cretica Africa
5 African pink stem borer Sesamia calamistis Africa
6 African maize stalk borer Busseola fusca Africa
7 African sugarcane borer Eldana saccharina Africa
8 Asiatic rice borer Chilo suppresalis Asia
9 Asiatic pink stem borer Sesamia inferens Asia
10 Sugarcane borer Diatraea saccharalis Americas
II Subtropical stem borers
1 European maize borer Ostrinia nubilalis North Africa, Mideast
2 Lesser corn borer Elasmopalpus lignosellus Americas
3 Oriental corn borer (Asian corn borer) Ostrinia furnacalis Asia
4 Spotted stem borer Chilo partellus Africa
5 African maize stalk borer Busseola fusca Africa
6 Sugarcane borer Eldana saccharina Africa
7 Sugarcane borer Diatraea saccharalis Americas
8 Southwestern corn borer Diatraea grandiosella Americas
III Temperate stem borers
1 Southwestern corn borer Diatraea grandiosella North America
2 Sugarcane borer Diatraea saccharalis Southern Cone, S. America
3 Lesser corn stalk borer Elasmopalpus lignosellus Southern Cone, South America
4 Oriental corn borer Ostrinia furnacalis East Asia
5 European corn borer Ostrinia nubilalis Europe, North America
Lepidopteran pest species of the world

8. Maize Borer Map of the World.
Source: CIMMYT, Mexico

9. Life cycle of stem borer

10. Sites of damage caused by pest in maize
Stem borers

11. Damage caused by borers

12. Symptoms of pest attack in maize

13. Control of stem borers
 Cultural methods
 Biological control.
 Application of Carbaryl 4% G or Endosulfan 8% G @ 7.5
– 15 Kg/ha into the leaf whorls.
 Spraying of endosulfan 35 EC @ 2ml/liter.
 Bt maize

14. History of Bt
 Bt was Ist
isolated by Japanese biologist, Ishiwatari when he was
investigating the cause of the Sotto disease (sudden collapse disease) in
silkworms.
 In 1915, Berliner reported the existance of a crystal within Bt, but
activity was not discovered at that time.
 1920, farmers started using Bt as a pesticides.
 But the Bt products such as spray are rapidly washed away by rain and
degrade under the sun’s UV rays.
 in 1956,Hannay, James and Angus found that the main insecticidal
activity was due to crystal. With this discovery came interest in the
crystal structure, biochemistry and general mode of action of Bt.

15.  In US, Bt was commercially started in 1958.
 In 1961, Bt was registered as a pesticide in EPA (Environmental Protection
Agency) .
 Until 1977, only 13 Bt strains had been described, which are toxic to
lepidopteron larvae. By the end of 1983, new strain was discovered which
was toxic to species of coleopteran (beetles).
 Today, there are thousands of strains of Bt. Many of them have genes
that encode unique toxic crystals n their DNA.
 The Ist Genetically engineered plant, Corn was registered with the EPA in
1995.
 Presently available GM Crops like Soybean, Cotton, Papaya, Tomato, Potato,
Okra, Brinjal.

16. Bt Maize Events that have been Approved for Commercial Planting
Event Gene Year approved Country Product Name Company
MON 810 cry1Ab 1996 USA Yield Gard®
Corn borer
Monsanto
1997 Canada
1997 South Africa
1998 Argentina
1998 EU
2000 Bulgaria
2002 Philippines
2003 Uruguay
Bt 11 cry1Ab 1996 USA Yield Gard® Syngenta
1996 Canada
1996 Japan
2001 Argentina
176 cry1Ab 1995 USA Knockout® Syngenta
1996 Canada
1997 EU
1998 Argentina
MON 863 cry3Bb1 2003 USA Yieldgard®
Rootworm
Monsanto
2003 Canada
TC 1507 cry1Fa2 2001 USA Herculex® 1 Pioneer Hi-Bred
Mycogen Seeds -
Dow Agro Sci.
2002 Canada
2002 Japan

17. History of Bt Corn
Outbreak in
Minnesota in 1995
cost of 285 million $
Ist
Hybrid is released in 1996
by Monsanto ,Yield Gard

19. Bt (Bacillus thuringiensis)
 Gram +ve bacteria
 cry gene
 produces protein called delta-endotoxin
 which is active against a specific species
of target pest.
 Harmless to human, birds, animals and
other non target organisms.

20. Genotype

21. pore-forming
domain I
receptor binding
domains II
crystals are made up
of pore-forming
toxins
Bacillus thuringiensis : produces proteins
that kill insects
The proteins are called “Cry”,
because they occur in crystals
sporulation
induces
synthesis of
crystals
Domain III which
protect the endotoxin

22. Physiological mechanism of Cry toxin action

23. Lepidoptera
Coleoptera
Diptera
Cry Toxin Specificity
Crickmore et al. 1998
Nematoda

24. The Expression of a Synthetic cry1a(b) Gene in
Transgenic Maize Confers Resistance to European Corn
Borer.
Insect resistance maize:Recent advance and utilization (1994),CIMMYT.
Estruch.J.J, Carozzi.N.B, Desai.N, Warren.G.W, Duck.N.B and Koziel.M.G ,
CIBA Agricultural Biotechnology, USA.
Material and method:
 Vector used are derivatives of pUC 18 or pUC 19.
 Contain truncated- synthetic version of the cry1A(b) genes from Bt
var.kurstaki.
 Placed under the control of either the CaMV 35S promoter or
tissue-specific prompters.
CaMV 35S Synthetic cry1A (b) [648aa]
Maize PEPC Synthetic cry1A (b) [648aa]
Maize pollen Synthetic cry1A (b) [648aa]
Maize pith Synthetic cry1A (b) [648aa]
Event 171
Event 176

26.  Immature embryos were excised 2 weeks after
pollination & planted in 2DG4 + 5 mg/l chloramben.
 Plasmid DNA was deposited on microprojectiles at
rate of 6mg of DNA /50 ml of microcarrier.
 Delivery of the microprojectiles is performed
using PDS-1000He Biolistic gun with rupture disks
of 1550 psi.
 After bombardment, embryos are kept for 1 day in
the dark at 25 °c.
 Transformed to callus induction medium, 3mg/l of
phosphinothricin (PPT)
 Embryogenic callus was transferred to 2DG4 with
0.5mg/l of 2,4-D.
 12 weeks later, tissue transferred to MS medium
containing 3% sucrose & harmones.
Isolate plant cells
Grow
undifferentiat
ed callus
Transform cells
Select cells
Redifferentiate
callus
Grow transgenic plant

27. Cont……..
 Transformed plants were identified by
PCR for sequences in the promoters and
synthetic cry1A(b) gene.
 Positive plants were moved to
greenhouse for additional tests and
crosses with various inbreeds.
 Quantitative determination of the levels
of cry1A (b) protein were performed by
ELISA.

28. Results
 Increasing the GC content of Bt insecticidal protein genes leads to
better expression in plants.
 The insect control group at CIBA decided to make a synthetic
version of the cry1A(b) gene by increasing GC content from 38% in
native gene to 65%.
 Transgenic maize for the cry1A (b) gene under
PEPC specific promoter – 1000 ng /mg of crude protein.
Pollen specific promoter – 400 ng / mg of crude protein
Pith specific promoter- 35 ng/mg of crude protein.
 Not detected in the kernels.
 The presence of cry1A(b) protein in pollen is important because it is

29. Screening Cry Proteins By Bt Maize Leaves For Activity
Against Kenyan Maize Stem Borers.
S.Mugo, D.Bergvinson, D.Hoisington, S.McLean, C.Taracha, B.Odhiambo, J.songa, I.Ngatia and M.Gethi.
Seventh Eastern And Southern African Regional Maize Conference, 11th
-15th
feb,2001
 Materials and methods
Maize leaves: The Bt maize tissues that were introduced from the following 6
transgenic lines:
1. leaves from plants containing Event 176 [cry1Ab driven by the maize pollen
specific promoter and maize PEPC promoter (plasmid pCIB4431) co-tansformed
with the bar gene driven by the CaMV 35s promoter (plasmid pCIB3064)].
2. Leaves from 5th
generation plants containing Event 5207 [cry1Ac driven by maize
ubiqitin promoter (plasmid pU02) co-transformation with the bar gene driven by
the enhanced CaMV 35s promoter (plasmid pHO620)].

30. Cont….
3. Leaves from 5th
generation plants containing Event 5601 [cry1B driven by the
rice actin promoter and bar driven by the CaMV 35S promoter (plasmid
pCIRAD3)].
4. Leaves from 2nd
generation plants containing Event 1835 [cry1B driven by
the maize ubiquitin promoter and bar driven by the CaMV 35S promoter
(plasmid pCIRAD4) co-transformed with the bar/gus genes driven by the
maize ubiquitin promoter (plasmid pACH25)].
5. Leaves from 2nd
generation plants containing Event 7 [cry1B-1Ab driven by
the rice actin promoter and bar driven by the CaMV 35S promoter (plasmid
pCIRAD 7)].
6. Leaves from 2nd
generation plants containing Event 602 [cry1E driven by the
rice actin promoter and bar driven by the CaMV 35S promoter (plasmid
pCIRAD 58)].

33. Backcross GM plant into high yield crops
GM plant = yyGG
High yield plant =
YYgg
YYgg x yyGG YyGg
YYgg x YyGg YYgG
YygG
YYgg
Yygg
YYgG x YYgG
YYgG
YYgg
YYGg
YYGG

34. Two farmers stand among rows of
non-Bt corn, which has suffered
insect damage and on either side are
rows of Bt corn.
“Management” means that a
certain acreage must be set
aside for the non-GM crop so
that the insects will thrive
there.
This will reduce the selection
pressure and the occasional
mutant that evolves will find a
non-mutant mate.
This greatly delays the
emergence of resistance.

35. Bt crop Non Bt crop

36. ConcernsConcerns Of Insect resistance

37.  Infection of corn by Fusarium (ear rot) and Aspergillus (kernal rot)
is more common when corn is damaged by insects. Fusarium produces
fumonisin and Aspergillus produce Aflatoxins,a potent mycotoxin.
 FDA “Guidance for Industry” for fumonisin levels of 2 to 4 µg/g in
human food and animal feeds.
 Fatal to animals & carcinogens in humans.

38. Insecticides
 Difficult to control.
 Residues for only short period.
 Useful when larvae have just
hatched or migrate to
neighboring plants.
 Proper timing of spray is crucial
for success.
 Harmful to other non target
organism.
Bt maize
 Easy to control.
 Produces crystal like protein that
selectively kill specific group of
insects.
 Effective to all stages of pest.
 Depending upon the gene inserted
the protein cry1Ab, cry1Ac, cry1B,
cry9c are produced.
 Harmless to other non target
organism.

40. Pros and Cons of genetically engineeringPros and Cons of genetically engineering
crops with Btcrops with Bt
 Pros:
1. Stable during several
years of storage
2. Resistance of the
crystals to inactivation
by UV light
3. Reduction in insecticide
sprays (labor and
chemical costs).
4. Increased activity of
natural enemies.
5. Biological control can be
used on secondary pests
 Cons:
1. Specificity of strains;
activity against some
pests, but poor activity
against others
2. Takes longer to kill
pests, perceived as less
effective
3. You still need to control
the “secondary” pests
4. Cost of transgenics
5. Development of
resistance because of
persistent exposure