The document summarizes a seminar presentation on using bacterial genes for crop improvement. It introduces some key bacterial genes used in transgenic crops, such as Bt cry genes which provide insect resistance. Methods of gene transfer discussed include particle gun and Agrobacterium-mediated transformation. Examples are given of crops improved through bacterial genes, including Bt brinjal, Bt cotton, and 'Golden Rice' containing genes for vitamin A production. The document also discusses properties needed for effective bacterial transformation genes and the mode of action of Bt toxins in insects.

2. Course SeminarCourse Seminar
onon
Bacterial gene for crop improvementBacterial gene for crop improvement
ChairmanChairman
Dr.K.N. Singh
Professor & Head
Delivered byDelivered by
Anurag Mishra
M.Sc.(Ag)IIIrd
Semester
A-5016/09
DEPARTMENT OF P.M.B. & G.E.
Narendra Deva University of Agriculture and Technology
Kumarganj Faizabad (U.P.)

3. HighlightsHighlights
Introduction
Properties of bacterial gene for
transformation
 Methods of gene transfer for crop
improvement
 Cry protein
 Bacterial gene for crop improvement
 Case study
 Achievements
 Conclusion
 References

4.  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.

5.  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( -carotene), are derived from plant foods and are aβ
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.

6. 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.

7. Methods of gene transferMethods of gene transfer
for crop improvementfor crop improvement
 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

8. 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 gas
♦Gas acceleration tube
♦Rapture disc
♦Stopping screen
♦ Coated DNA
♦Target cell

9. Gene Gun

10. Gene transfer throughGene transfer through AgrobacteriumAgrobacterium
tumefacienstumefaciens
 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

11. 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

12. 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.

13. Introduction of Bt cotton in India:Introduction of Bt cotton in India:
1994-20021994-2002
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),

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

15. Mode of action of Bt

16. Cotton boll damage from budworm/bollwormCotton boll damage from budworm/bollworm
larvaelarvae
Heliothis virescens (larva)

17. Trail

18. Bacterial gene for cropBacterial 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

19. S.No. Bacterial gene Crop Project institute
7 bar, HVA1, PIN2 Wheat Resistant against
biotic and abiotic
stresses
Delhi University,
South Campus, New
Delhi
8 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

20. BtBt BrinjalBrinjal

21. Development ofDevelopment of BtBt brinjalbrinjal
 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

22. BtBt Brinjal – factBrinjal – 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.

23. 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.

24. 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 Approval
GEAC 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.

25. Expression of bacterial genes inExpression of bacterial genes in
transgenic tobaccotransgenic tobacco
Bacterium Gene Expressed
protein
Function References
Pseudomonas
syringae
argK ROCT ornithine
Carbamoyl
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)

26. 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 -caroteneβ
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)
Streptomyces
hygroscopicus
bar PPT acetyltransferase Bialaphos
tolerance
Lutz et al.
(2001)

27. Resistance ofResistance of Helicoverpa armigeraHelicoverpa armigera toto Cry1AcCry1Ac toxin fromtoxin from BacillusBacillus
thuringiensisthuringiensis is due to improper processing of the protoxin,is due to improper 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.

28. Pyramiding additional bacterial blight resistance genes inPyramiding additional bacterial blight resistance genes in
basmati rice backgroundbasmati 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

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

30. Potato carrying a gene Cry3A fromPotato carrying a gene Cry3A from BacillusBacillus
thuringiensisthuringiensis (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.

31. AchievementsAchievements
Improved nutritional quality
Insect resistance
Disease resistance
Herbicide resistance
Drought resistance
Salinity resistance

32. 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( -carotene), are derived from plant foods and are aβ
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

33. 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.

34. ReferencesReferences
Keshamma Entoori, Rohini Sreevathsa, Manoj Kumar Arthikala, Polumetla Ananda
Kumar, Amrita Raja Vinoda Kumar, Basavaraj Madhusudhan, Udayakumar Makarla (2008),
EurAsia J BioSci 2, 53-65
Madigan M, Martinko J (editors) (2005). Brock Biology of Microorganisms (11th ed.).
Prentice Hall. ISBN 0-1
Maneesha Aluru, Yang Xu, Rong Guo, Zhenguo Wang, Shanshan Li, Wendy White, Kan
Wang and Steve Rodermel (2008), Journal of Experimental Botany, Vol. 59, No. 13, pp.
3551–3562.
Manju Sharma, K.S.Charak and T.V.Ramanaiah (2003). Agricultural biotechnology
research in India:Status and policies. Current Science, Vol. 84:1-6.
Purohit,S.S (2001) Agrobacterium mediated gene transfer,Biotecnology Fundamental and
Application. 204-205.
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