Course Seminar Course Seminar onon

Bacterial gene for crop Bacterial gene for crop improvementimprovement

ChairmanChairman Dr.K.N. SinghProfessor & Head

Delivered byDelivered byAnurag MishraM.Sc.(Ag)IIIrd SemesterA-5016/09

DEPARTMENT OF P.M.B. & G.E.Narendra Deva University of Agriculture and Technology

Kumarganj Faizabad (U.P.)

HighlightsHighlights

IntroductionProperties of bacterial gene for

transformation Methods of gene transfer for crop

improvement Cry protein Bacterial gene for crop improvement Case study Achievements Conclusion References

The bacteria (singular: bacterium) are a large group of single-celled, prokaryote microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals.

Bacteria were first observed by Antonie van Leeuwenhoek in 1676, the name bacterium was introduced much later, by Christian Gottfried Ehrenberg in 1838.

Indica transgenic rice containing a synthetic gene from Bacillus thuringiensis (Bt) expressing Cry1Ac toxin had enhanced resistance to stem borer. One parental strain contained the xa21 gene conferring resistance to bacterial blight.

Xa21 Resistance to bacterial leaf blight IR72, IR64, IR68899B, MH63, BPT5204, Pusa Basmati-1, IR50, CO39, IR72 field-evaluated in China, India, and Phillipines

A stacked combination of Bt toxins Cry1Ab and Cry1Ac along with tolerance to the herbicide glufosinate.

Transgenic sugarcane plants with cry1Ab gene were produced through particle bombardment as well as by Agrobacterium-mediated transformation for shoot borrer.

A barley gene hva1 protects the cell membrane during drought, and when inserted in rice, could reduce drought damage.

Transgenic plants resistant to insects like those expressing Bacillus thuringiensis. Cry proteins (Bt plants), offer several advantages over their corresponding non transgenic cultivars like cotton, brinjal, maize etc.

Transgene expression can vary, depending on the transgene insertion site (Leeuwen et al., 2001), and the genes at or around the insertion site can be affected in expression rate.

Provitamin A carotenoids( β-carotene), are derived from plant foods and are a major source of vitamin A for the majority of the world’s population, production of ‘Golden Rice 2’ which contains high levels of provitamin A carotenoids to combat VAD.

Modified potatoes carrying gene Cry3A originating from bacteria Bacillus thuringiensis were produced to control this potato beetle (Leptinotarsa decemlineata).

Aluru M. et al., (2008). Reported the bacterial genes crtB (for phytoene synthase) and crtI (phytoene desaturase and carotene desaturase in plants), under the control of a ‘super gamma zein promoter’ for endosperm-specific expression, resulted in an increase of total carotenoids of up to 34-fold with a preferential accumulation of β-carotene in the maize endosperm.

B. Sharma (June 2010), presented the transformation and expression of cry2Aa gene in transgenic chickpeas which exhibited up to 98% protection against pod borer larvae.

Ideal properties of bacterial gene for transformation

It must be replicate in host cell. It must contain marker gene such as tetracycline, amplicine

and kanamycin etc. Unique cleavage site must be present in one of the marker

gene. It should contain specific control system, like promoters,

operators, ribosomal binding site etc. Easy transformation. Easy purification.

Methods of gene Methods of gene transfer for crop transfer for crop

improvementimprovement Chemical method

Electroporation

Particle gun method

Microinjection

Gene transfer through Agrobacterium Generally two method are used in crop

improvement Particle gun method and Gene transfer through Agrobacterium

PARTICLE GUN METHOD

This method was first used by Klein & co-worker for transient assay in onion epidermis.

In this method by using Helium pressure. Main component of particle gun method-Helium gasGas acceleration tubeRapture discStopping screen Coated DNATarget cell

Gene Gun

Gene transfer through Gene transfer through Agrobacterium tumefaciensAgrobacterium tumefaciens

Agrobacterium tumefaciens has Ti and Ri

plasmid responsible for crown gall

disease and hairy root disease.

The Ti plasmid of A. tumefaciens has

been developed as a vehicle for

introducing foreign genes into plants.

When infects plants, a region of the Ti

plasmid called the T-DNA is taken up by

the plant cell and incorporated into plant

genome.

T-DNA has both sides a 24 bp direct

repeat border sequence and contain the

gene for tumour inducing.

Onco region

Os region

Golden rice

Golden rice developed by Professor Ingo Potrykus & Dr. Peter Beyer

(August 1999), Swiss institute of Technology & University of Freiburg in

Germany.

Daffodil plant (Norcissus pseudonorcissus)

psy - phytoene synthase

lcy - lycopene beta-cyclase

Erwinia uredovora

crt I - Phytoene desaturase

Vector

Agrobacterium tumefaciens

Cry proteinCry protein B. thuringiensis was first discovered in 1901 by Japanese

biologist Shigetane Ishiwatari. In 1911, B. thuringiensis was rediscovered in Germany by

Ernst Berliner, who isolated it as the cause of a disease called Schlaffsucht in flour moth caterpillars.

Cry toxins have specific activities against insect species of the orders Lepidoptera (moths and butterflies), Diptera (flies and mosquitoes), Coleoptera (beetles), hymenoptera (wasps, bees, ants and sawflies) and nematodes.

B. thuringiensis an important reservoir of Cry toxins for production of biological insecticides and insect-resistant genetically modified crops.

Insects ingest toxin crystals, the alkaline pH of their digestive tract activates the toxin. Cry inserts into the insect gut cell membrane, forming a pore. The pore results cell lysis and eventual death of the insect.

Introduction of Bt cotton in Introduction of Bt cotton in India: 1994-2002India: 1994-2002

Cry-proteins. Any of several crystalline proteins found in Bt spores that are activated by enzymes in the insect’s midgut. These proteins attack the cells lining the gut, cause gut paralysis, and subsequently kill the insect.

Bt toxins were classified into 14 distinct groups and 4 classes (Hofte and Whiteley, 1989)

CryI (active against Lepidoptera) CryII (Lepidoptera and Diptera), CryIII (Coleoptera), and CryIV (Diptera)

2002-2008 – 135 hybrids cry 1Ac (MON 531 event) cry 1 Ac and cry 2 Ab (MON 15985 Event) cry 1 Ac (Event 1), cry 1 Ab + cry Ac –GFM and cry 1Ac (CICR event), cry 1 Ac (Event 9124),

Mode of action for Bt toxin after eaten by a Mode of action for Bt toxin after eaten by a tobacco budworm larva, (Ostlie et al. 1997).tobacco budworm larva, (Ostlie et al. 1997).

Mode of action of Bt

Cotton boll damage from Cotton boll damage from budworm/bollworm larvaebudworm/bollworm larvae

Heliothis virescens (larva)

Trail

Bacterial gene for crop Bacterial gene for crop improvementimprovement

The bacteria (singular: bacterium) are a large group of single-celled, prokaryote microorganisms. Many gene are used for crop improvement

S.No. Bacterial gene Crop Project institute

1 Bean alpha AI Chick pea To generate plants resistant to bruchids

AAU, Jorhat , Assam

3 Bt, cry gene(s) Cotton To generate plants resistant to lepidoteran pests

CICR, Nagpur

4 Bt, cry I A (b) Potatoe To generate plants resistant to lepidoteran pests

CPRI, Shimla

5 Bt, cry I A (b) and cry 1 c Tobacco To generate plants resistant to Helicoverpa armigera and Spodotera litura

CTRI , Rajahmundry

6 Bt, cry I A (b), Xa 21 Rice To generate plants resistant to lepidoteran pests , bacterial blight/ desease

CRRI, Cuttak

S.No. Bacterial gene Crop Project institute

7 bar, HVA1, PIN2 Wheat Resistant against biotic and abiotic stresses

Delhi University, South Campus, New

Delhi8 Xa 21, cry I A (b) Rice To generate plants

resistant to lepidoteran pestsand bacterial and fungal deseases

Directorate of Rice Research,

Hyderabad

9 Bt, cry I A (b) Brinjal To generate plants resistant to lepidoteran pests

IARI, New delhi

10 Bt, cry I A (b) Tomato To generate plants resistant to lepidoteran pests

IARI, New delhi

11 Bt, cry I A (b) Cauliflower plants resistant to Plutella scylostella

IARI, New delhi

12 Bt, cry I A (b) Cabbage plants resistant to Plutella scylostella

IARI, New delhi

13 Bt, cry I A (b) Rice To generate plants resistant to lepidopteran pests

IARI, New delhi

14 Bt, cry I A (b) Rice Yellow stem borer IARI, substation Shillong

15 Cry 1A(b) gene Rice Lepidopteran pest, bacterial and fungal disease

NDUA & T, kumarganj,

Faizabad

BtBt Brinjal Brinjal

Development of Development of BtBt brinjal brinjal

Cotton, brinjal and chickpea are the three crops highly infested with insect pests.

Damage by fruit and shoot borer (FSB) a major problem in brinjal production.

Yield losses estimated to be 60 to 70% even after repeated insecticide sprays.

Intensive use of pesticides not very effective due to mode of action of FSB.

Increased dependence on pesticides leading to adverse effects of higher cost of production, environmental pollution, destruction of natural enemies and health problems due to pesticide residues.

Conventional plant breeding not successful in controlling FSB; need for alternate strategies

BtBt Brinjal – fact Brinjal – fact Developed by M/s Mahyco; also public private

partnership with TNAU, Coimbatore and UAS, Dharwad. Contains the cry1Ac gene derived from Bacillus

thuringiensis to produce an insecticidal protein. Has an in-built mechanism of protection against target

pest viz. fruit and shoot borer. Transformation and greenhouse evaluation initiated in

2000. Extensive biosafety studies and field trials undertaken

over a period of six years as per the protocols prescribed by RCGM.

Based on the biosafety data and results of multilocational trials, RCGM had recommended LST to GEAC in 2006.

Expressed Bt protein is highly specific to lepidopteran pests.

Expression of cry1Ac gene is consistent during the entire life of the crop and the levels of Cry1Ac protein are sufficient for effective control of FSB in various agro-climatic conditions.

The Cry1Ac protein expressed in Bt brinjal is 100% identical to one expressed in approved Bt cotton event MON 531.

Introgression of cry1Ac gene has in no way affected outcrossing potential and weediness characteristics.

RECOMMENDATIONS Bt brinjal event EE-1 is safe for environmental

release in India. Bt brinjal event EE-1 has been extensively tested

for its biosafety and no additional studies/review are necessary.

Status of ApprovalGEAC approved Bt brinjal for environmental

release on 14.10.2009.Minister has invited comments upto 31.12.2009.Final decision of Govt after national consultations

during Jan –Feb 2010.

Expression of bacterial Expression of bacterial genes in transgenic tobaccogenes in transgenic tobacco

Bacterium Gene Expressed protein

Function References

Pseudomonas syringae

argK ROCT ornithineCarbamoyl transferase

Resistance to Pseudomonas.syringae pv. phaseolicola

Hatziloukas and Panopoulos.(1992)

Halobacterium halobium

bO Bacterio-opsin (BO)

Resistance to Pseudomonas.syringae pv. tabaci

Rizhsky and Mittler (2001)

Bacillus thuringiensis

cry2Aa Crystal protein (Cry2Aa2)

Insect resistance Kota et al. (1999)

Actinomyces A19249

choM choM Resistance to boll weevil.larvae

Corbin et al. (2001)

Agrobacterium.tumefaciens

ipt Cytokinin isopentenyl transferase

Resistance to tobacco.hornworm

Smigocki et al. (1993)

Escherichia coli

betA CDH Enhance salt tolerance

Holmström et al. (2000)

Escherichia coli

betB BADH Enhance salt tolerance

Holmström et al. (2000)

Synechococcus vulcanus

desC Acyl-lipid desaturase

Enhance cold tolerance

Orlova et al. (2003)

Erwinia uredovora

crtZ β-carotene hydroxylase

Enhance UV tolerance

Götz et al. (2002)

Bacillus licheniformis

amyl α-amylase Alpha-amylase production

Pen et al. (1992)

Acidothermus.cellulolyticus

e1 Cellulase endo-1,4-β-D-glucanase (E1)

Cellulase production

Jin et al. (2003)

Streptomyceshygroscopicus

bar PPT acetyltransferase

Bialaphos tolerance

Lutz et al. (2001)

Resistance of Resistance of Helicoverpa armigeraHelicoverpa armigera to to Cry1Ac Cry1Ac toxin toxin from from Bacillus thuringiensisBacillus thuringiensis is due to improper is due to improper

processing of the protoxin,processing of the protoxin, Rajagopal R. Rajagopal R. et al.et al.,(2009).,(2009).

The bacterium Bacillus thuringiensis produces ICPs (insecticidal crystal proteins) that are deposited in their spore mother cells.

ICPs get solubilized in the alkaline gut environment, insecticidal protein Cry1Ac has been applied extensively as the main ingredient of spray formulation.

The 135 kDa Cry1Ac protein, upon ingestion by the insect, is processed successively at the N- and C-terminus by the insect midgut proteases to generate a 65 kDa bioactive core protein.

The 135 kDa protoxin-susceptible insect larval population processed the protein to the biologically active 65 kDa core protein, while the resistant insect larval population yielded a mixture of 95 kDa and 68 kDa Cry1Ac polypeptides.

N-terminal sequencing of these 95 and 68 kDa polypeptides produced by gut juices of resistant insects revealed an intact N-terminus.

Protease gene transcription profiling by semi-quantitative RT (reverse transcription)–PCR led to the identification of a down-regulated HaSP2 (H. armigera serine protease 2) in the Cry1Ac-resistant population.

The larval population resistance to Cry1Ac, do not show an altered sensitivity against another insecticidal protein, Cry2Ab.

These result of the possibility of development of resistance and its management in H. armigera to Cry1Ac through transgenic crop cultivation.

Pyramiding additional bacterial blight Pyramiding additional bacterial blight resistance genes in basmati rice backgroundresistance genes in basmati rice background

Background analysis revealed that Improved Pusa Basmati inherited most of the regions from Pusa Basmati 1, which are linked to Basmati quality traits.

Possibility of linkage drag was also minimum in respect of chromosomes 8 and 11, carrying genes Xa 13 and Xa 21 for BB resistance respectively.

Marker-based analysis suggested that this variety can be used as a combiner in Basmati hybrid-breeding programme. With the objective of adding more BB resistance genes in the Basmati background, a large segregating population was generated using Basmati 370 and IRBB 60, a non-Basmati rice line, carrying four genes Xa4, Xa5, Xa13 and Xa21.

This population will now be screened for identification of suitable recombinants possessing all the 4 BB resistance genes and Basmati traits.

Source: DARE/ICAR Annual Report 2007–2008

Engineering resistant corn. The insertion of a gene from the bacteria Engineering resistant corn. The insertion of a gene from the bacteria Bacillus thuringiensis, corn becomes resistant to corn borer Bacillus thuringiensis, corn becomes resistant to corn borer infection. infection.

Potato carrying a gene Cry3A from Potato carrying a gene Cry3A from Bacillus thuringiensisBacillus thuringiensis (Perlak (Perlak et al., et al., 1993)1993)

The most consequential potato plant pests is the potato beetle (Leptinotarsa decemlineata), which often becomes resistant to chemical insecticides.

Modified potatoes carrying gene Cry3A originating from bacteria Bacillus thuringiensis were produced to control this beetle.

This gene product is a toxic protein formed in leaves of these plants; after ingestion by a potato beetle, it passes on to its intestines and thus causes the death of the pest.

It is a great advantage that the protein affects all developmental stages of potato beetles in the same way; however it does not affect their natural enemies.

AchievementAchievementss

Improved nutritional quality

Insect resistance

Disease resistance

Herbicide resistance

Drought resistance

Salinity resistance

Xa21 Resistance to bacterial leaf blight IR72, IR64, IR68899B, MH63, BPT5204, Pusa Basmati-1, IR50, CO39, IR72 field-evaluated in China, India, and Phillipines.

Provitamin A carotenoids( β-carotene), are derived from plant foods and are a major source of vitamin A for the majority of the world’s population, production of ‘Golden Rice 2’ which contains high levels of provitamin A carotenoids to combat VAD.

Modified potatoes carrying gene Cry3A originating from bacteria Bacillus thuringiensis were produced to control this potato beetle (Leptinotarsa decemlineata).

With the help of PEG transformation, in Petunia 40% transformation calli (mesophyll protoplast). Transformation efficiency have been observed, 0.0004% in embryonic protoplast of rice, 0.7-1.0% was soybean and tobacco.

With the help op electroporation, Tobacco, with 0.2% of electroporated mesophyll protoplast . Low transformation efficiency recorded in rice 0.002%.

Rajagopal R. et al.,(2009). development of resistance and its management in H. armigera to Cry1Ac through transgenic crop cultivation.

CONCLUSION

Transgenic sugarcane plants with Cry1Ab gene were produced through particle bombardment as well as by Agrobacterium-mediated transformation and Cry1Ab gene was integrated. Cry1Aa, Cry1Ab and Cry1Ac resistance to sugarcane shoot borer.

In Potatoes carrying gene Cry3A originating from bacteria Bacillus thuringiensis were produced to control this beetle (Leptinotarsa decemlineata).

Bt brinjal contains the cry1Ac gene derived from Bacillus thuringiensis to produce an insecticidal protein to control lepidopteron pests.

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Manju Sharma, K.S.Charak and T.V.Ramanaiah (2003). Agricultural biotechnology research in India:Status and policies. Current Science, Vol. 84:1-6.

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R.ai Z. (1979). "Plasmid DNA from Bacillus thuringiensis". Microbiologiya 48 (2): 226–229.

Rajagopal, R., Arora, N. Sivakumar, S. Nagarjun G. V. RAO, Sharad A. and Raj K. Bhatnagar (2009). Resistance of Helicoverpa armigera to Cry1Ac toxin from Bacillus thuringiensis is due to improper processing of the protoxin. Biochem. J. 419, 309–316.

Sumerford, D.V., D.D. Hardee, L.C. Adams, and W.L. Solomon (2001). Tolerance to CryIAc in populations of Helicoverpa zea and Heliothis virescens (Lepidoptera: Noctuidae): Three-year summary. Journal of Economic Entomology. In press