bt maize insect resistance to lepidopteran pest
TRANSCRIPT
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 Productivity
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)
Source: Science & technology, Deccan herald, Tuesday, August 5,2008.
Maize Area, Yield and Production for Top 10 Maize Countries 2006.
Sl.noCountry
Harvested Hectares(Millions)
YieldMT/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 8.510 Philippines 2.4 1.8 4.3
Distribution of World Maize Production
Source: CIMMYT, Mexico
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.
Sl.no Common Name Scientific Name Affected RegionsI Tropical stem borers 1 Spotted stem borer Chilo Partellus Asia, East Africa2 Oriental corn borer (Asian corn borer) Ostrinia furnacalis Asia3 Lesser cornstalk borer Elasmopalpus lignosellus Americas4 Pink stem borer Sesamia cretica Africa5 African pink stem borer Sesamia calamistis Africa6 African maize stalk borer Busseola fusca Africa7 African sugarcane borer Eldana saccharina Africa8 Asiatic rice borer Chilo suppresalis Asia9 Asiatic pink stem borer Sesamia inferens Asia
10 Sugarcane borer Diatraea saccharalis AmericasII Subtropical stem borers 1 European maize borer Ostrinia nubilalis North Africa, Mideast2 Lesser corn borer Elasmopalpus lignosellus Americas3 Oriental corn borer (Asian corn borer) Ostrinia furnacalis Asia4 Spotted stem borer Chilo partellus Africa5 African maize stalk borer Busseola fusca Africa6 Sugarcane borer Eldana saccharina Africa7 Sugarcane borer Diatraea saccharalis Americas8 Southwestern corn borer Diatraea grandiosella AmericasIII Temperate stem borers 1 Southwestern corn borer Diatraea grandiosella North America2 Sugarcane borer Diatraea saccharalis Southern Cone, S. America3 Lesser corn stalk borer Elasmopalpus lignosellus Southern Cone, South America4 Oriental corn borer Ostrinia furnacalis East Asia5 European corn borer Ostrinia nubilalis Europe, North America
Lepidopteran pest species of the world
Maize Borer Map of the World .
Source: CIMMYT, Mexico
Life cycle of stem borer
Sites of damage caused by pest in maize
Stem borers
Damage caused by borers
Symptoms of pest attack in maize
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
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.
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.
Bt Maize Events that have been Approved for Commercial Planting
Event Gene Year approved Country Product Name CompanyMON 810
cry1Ab
1996 USA Yield Gard®Corn borer
Monsanto
1997 Canada1997 South Africa1998 Argentina1998 EU2000 Bulgaria2002 Philippines2003 Uruguay
Bt 11
cry1Ab
1996 USA Yield Gard®
Syngenta
1996 Canada1996 Japan2001 Argentina
176
cry1Ab
1995 USA Knockout®
Syngenta
1996 Canada1997 EU1998 Argentina
MON 863
cry3Bb1
2003 USA Yieldgard®Rootworm
Monsanto 2003 Canada
TC 1507
cry1Fa2
2001 USA Herculex® 1
Pioneer Hi-BredMycogen Seeds -
Dow Agro Sci.2002 Canada 2002 Japan
History of Bt Corn
Outbreak in Minnesota in 1995 cost of 285 million $
Ist Hybrid is released in 1996 by Monsanto ,Yield Gard
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.
Genotype
pore-forming domain I
receptor binding domains II
crystals are made up of pore-forming toxins
Bacillus thuringiensis : produces proteins
that kill insectsThe proteins are called “Cry”, because they occur in crystals
sporulation induces synthesis of crystals
Domain III which protect the endotoxin
Physiological mechanism of Cry toxin action
Lepidoptera
Coleoptera
Diptera
Cry Toxin Specificity
Crickmore et al. 1998
Nematoda
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
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
undifferentiated callus
Transform cells
Select cells
Redifferentiate callus
Grow transgenic plant
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.
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 the main diet during the 1st and 2nd instars of the ECB.
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 methodsMaize 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)].
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)].
Backcross GM plant into high yield crops
GM plant = yyGG
High yield plant =
YYgg
YYgg x yyGG YyGg
YYgg x YyGg YYgGYygGYYggYygg
YYgG x YYgG YYgGYYggYYGgYYGG
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.
Bt crop Non Bt crop
Concerns Concerns Of Insect resistance
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.
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.
Pros and Cons of genetically engineering Pros and Cons of genetically engineering crops with Btcrops with Bt
Pros:1. Stable during several
years of storage2. 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 transgenics5. Development of
resistance because of persistent exposure