Insect resistance Transgenic in Vegetable Crops

2. Insect resistance Transgenic in Vegetable Crops
Presented by:
Magar Sayali G.
CAU/CPGS/MBB/M17/01
School of Crop Improvement
College of Post-Graduate Studies, CAU (Imphal)
Umiam, Meghalaya
seminar

3. • Transgenics or GMOs are defined as those organisms with a gene or genetic construct of
interest that has been introduced through genetic engineering
• Technology - ‘gene technology’ or ‘recombinant DNA technology’
• Organism - ‘genetically engineered’ or ‘transgenic’
• genes transfer - algae, bacteria, viruses or animals to plants or between sexually
incompatible species
• Ability to move genes - organism to crop plants to impart novel characteristics
transgenics
Source: Kuruganti and Ramanjaneyulu, 2007

4. • Exchange of genes
within a species
• Many genes are
transferred
• Simple Technology
• Time consuming
method
• No barrier
• Only Gene of Interest
is transferred
• Intensive Technology
• Quick, specific &
efficient method
Transgenics over Conventional Breeding

5. • Hold 2nd rank in world
• Contributes 14% of the total world production of vegetables
• Area under production: 10.175 million hectares
• Production: 169.1 million metric tonnes
• Productivity: 17.7 million tonnes / ha
• loss due to insect: 15.7%
Vegetable status In india 2017
Source: Horticultural Statistics at A Glance 2017

6. Global Area of GM Crops In 2017
Source: ISAAA, 2017
TOP GM CROPS GROWING COUNTRIES : 2017 (million hectares)
US 75.0
BRAZIL 50.2
ARGENTINA 23.6
CANADA 13.1
INDIA 11.4
PARAGUAY 3.0
PAKISTAN 3.0
CHINA 2.8
SOUTH AFRICA 2.7
BOLIVIA 1.3
URUGUAY 1.1
TOTAL* 189.8
*Includes other countries

7. Transgenic STATUS in india
Source: ISAAA, 2017

8. Howto prepare transgenic
Transgenic
preparation
Gene Manipulation
Identification
Isolation
Insertion into vector
Gene Introduction
Direct Gene Transfer
Electroporation of protoplast
Biolistic or Particle Bombardment
(Gene Gun)
• Using different techniques
Indirect Gene Transfer
Agrobacterium tumefaciens
Agrobacterium rhizogenes
Microinjection
• Using different techniques

9. Agrobacterium Method

10. A. Microinjection

11. B. Electroporation of protoplast
Gene integrate into
genomic DNA
Gene inside cell

12. C. Biolistic or Particle Bombardment
(Gene Gun)

13. Applications of transgenics plants
Resistance to biotic
stress
Insect resistance
Virus
resistance
Fungal and
bacterial
resistance
Resistance to
abiotic stress
Drought
tolerance
Salinity
tolerance
Cold tolerance
For quality
improvement
Herbicide
Resistance

14. Insect resistance
Resistance gene from
microorganism
• Bt gene
Resistance gene from
higher plants
Protease inhibitor
• Eg. Cp TI gene
α- amylase inhibitor
• Eg. Adzuki beans
Lectins
• Eg. GNA from
Snowdrop
Resistance gene from
animals
Serine proteinase
inhibitors
• Eg. BPTI, SI

15. • Bacillus thuringiensis, gram positive soil bacteria
• Discovered by Ishiwata in 1901
• Produces ICP, Class: δ endotoxin
Resistance gene from microorganism

17. Cry protein

19. • Protease inhibitors are enzymes that inhibit the function of proteases present in insect midgut
Protease inhibitors
Malgorzata Rzychon, 2004

20. • Class:
• Serine PI
• Thiol PI
• Metallo PI
• Aspartyl-protease inhibitors
• Accumulated in storage tissue
• Effect on humans
• Eg. Cowpea trypsin inhibitor gene derived from cowpea and cloned into tobacco against
Helicoverpa armigera

21. • α- amylase inhibitor :
• Affects digestion of carbohydrate in insect
• Eg. Transgenic tomato expressing α- amylase inhibitor give resistance
against lepidopteron pest
• Lectin :
• Lectin are protein bind to carbohydrate and thus interfere with uptake
of nutrient in insect midgut
• Eg. gene encoding the pea lectin has been introduce into tobacco for
resistance against Helicoverpa armigera

22. CASE STUDY I
Objective :
• Standardization of genetic transformation protocol
• Diamondback moth resistance in broccoli using cry1Aa gene

23. • Material:
• Certified seeds of broccoli cv. Solan Green Head
• Explants: cotyledon and hypocotyl grown in half-strength MS
• Agrobacterium tumefaciens strain : LBA 4404, containing binary vector pBinAR-
1Aa
Materials and methods

24. • Method :
• Agrobacterium mediated gene transfer
• Transgene integration was confirmed by :
• Polymerase chain reaction
• Southern blot analysis
• Transgenic events had been confirmed:
• Reverse transcriptase-PCR
• Quantitative real time-PCR
• Insect bioassay

25. Results

30. • A pre-culturing period of 72 h followed by 48 h co-cultivation
period was found the best to obtain an efficient transformation
frequency
• This study yielded 4 transgenic line carrying cry1Aa for resistance
against diamondback moth
Conclusion

31. Objective:
• To study the sporamin gene role in other variants of cabbage
• To study inheritance patterns and expression of the sporamin in transgenic progenies
CASE STUDY II

32. • Material:
• ‘Youdonger’ and ‘Shanghaiqing’, common variety of Chinese cabbages
• Agrobacterium tumefaciens strain GV3101 harbouring the vector pCAMBIA1300-
PMSNPMCN
• Seeds were grown on half MS media, leaf petiole used as explant
• Methods :
• Plants transformation
• Agrobacterium mediated gene transfer
• Transgene integration was confirmed by :
• Polymerase chain reaction
• Southern blot analysis
• Transgenic events had been confirmed:
• Reverse transcriptase-PCR
• Quantitative real time - PCR
Material and methods

33. Results
• PCR detection of sporamin and hygromycin in transgenic A) Youdonger and B) Shanghaiqing. lane 1 indicates the
molecular marker, lane 2 indicates the amplification band from the positive control pCAMBIA1300PMSNPMCN
plasmid, lane 3 indicates the amplification band from wild type plants and lane 4–23 in A) and 4- 13 in B) indicates
amplification band from 20 and 10 different transgenic lines, respectively
B)
A)

34. • Southern Blot hybridization of sporamin
• Lane 1 indicates the signal from the positive control pCAMBIA1300PMSN-PMCN
plasmid, lane 2–9 indicates the signal from eight different transgenic lines,
respectively. Lane 10 indicates the signal form wild type plants.
A) Youdonger B) Shanghaiqing

35. • Lane 1–17 indicates amplification band from
17 different transgenic lines, and lane 18
indicates the amplification band from wild
type plants. The UBC10 gene is selected as the
internal control
B) Shanghaiqing
A) Youdonger
• Lane 1–8 indicates amplification band from
eight different transgenic lines, and lane 9
indicates the amplification band from wild
type plants. The UBC10 gene is selected as the
internal control.
• Sporamin expression in transgenic was verified by semi-quantitative RT-PCR analysis.

36. • Quantitative RT-PCR analysis of sporamin expression in transgenic
A) B) Shanghaiqing
Youdonger

37. • In vitro insect bioassay in transgenic Chinese cabbage ‘Youdonger’ (Brassica campestris ssp.
chinensis var. communis cv. Youdonger).
a) Diamondback moth (Plutella
xylostella) larvae photographed
after being removed from wild type
and transgenic ‘Youdonger’ leaves
they had been feeding on for 4
days.
b) Damage caused by second-instar diamondback
moth to transgenic ‘Youdonger’ leaves over-
expressing sporamin. WT is the wild type plants,
and the names of T0 lines are indicated in the
figure

38. • In vitro insect bioassay in transgenic Chinese cabbage ‘Shanghaiqing’
(Brassica campestris ssp. chinensis var. communis cv. Shanghaiqing).
a) Diamondback moth (Plutella
xylostella) larvae photographed
after being removed from wild type
and transgenic ‘Shanghaiqing’
leaves they had been feeding on for
4 days.
b) Damage caused by second-instar diamondback
moth to transgenic ‘Shanghaiqing’ leaves over-
expressing sporamin. WT is the wild type plants,
and the names of T0 lines are indicated in the
figure

39. • All southern blot confirmed transformant self to produce seed
• Then PCR analysis was done in T1 progeny
• This confirms mendelian genetics there by it confirms stable
expression in transgenic progeny

40. Segregation analysis of T1 progeny population derived from transgenic Chinese cabbage
Youdonger’ (Brassica campestris ssp. chinensis var. communis cv. Youdonger) line T0-2
inserted by sporamin. M: molecular marker, P+: positive control pCAMBIA1300PMSN-PMCN
plasmid, WT: wild type plants, 1-30: T1 progeny plants of line T0-2.
Segregation analysis of T1 progeny population derived from transgenic Chinese cabbage ‘Shanghaiqing’
(Brassica campestris ssp. chinensis var. communis cv. Shanghaiqing) line T0-9 inserted by sporamin. M:
molecular marker, P+: positive control pCAMBIA1300PMSN-PMCN plasmid, WT: wild type plants, 1-30: T1 progeny
plants of line T0-9.
Result of study inheritance patterns and expression of the sporamin in transgenic progenies
A)
B)

41. Sporamin expression in T1 progeny population of transgenic Chinese cabbage (Brassica campestris
ssp. chinensis var. communis) was verified by semi-quantitative RT-PCR analysis. (A) Sporamin
expression in T1-2 population of transgenic ‘Youdonger’. (B) Sporamin expression in T1-9 population of
transgenic ‘Shanghaiqing’. WT: wild type plants, numbers indicate the different T1 progeny plants

42. • The analysis of inheritance pattern of exogenous sporamin in the
progenies demonstrated that sporamin could be inherited and
expressed stably in transgenic progenies.
• This study strongly suggests that sporamin is an efficient candidate
gene for insect-resistant genetic engineering in Chinese cabbage.
Conclusion

43. • Reduce pesticide usage
• Health benefits
• Lower production cost
• Increased yield
• Low pesticide residues on food
• Lowers environmental pollution
• Maintain ecology balance
Advantages of insect resistance

44. Thank you