Biotchnological approaches in insect pest control viki

1. BIOTECHNOLOGICAL CONTROL
Submitted To :- Submitted By :-
Dr O. P. Chaudhary Vaibhav

2. BIOTECHNOLOGY
• The use of biological means to develop
processes and products by studying organisms
and their components.
• Biotechnology is any technique that uses living
organisms or substances from these organisms
to make or modify a product for a practical
purpose. Biotechnology can be applied to all
classes of organism - from viruses and
bacteria to plants and animals.

3. Agricultural biotechnology
• Agricultural biotechnology is a collection of
scientific techniques used to improve plants,
animals and microorganisms. Based on an
understanding of DNA, scientists have developed
solutions to increase agricultural productivity.

4. Biotechnological approaches in
Agriculture
 Tissue culture techniques
 rDNA technology
 Development of transgenic crops.

5. Tissue Culture
• Technique of growing plant tissues on
synthetic medium under controlled and
aseptic conditions.
• German plant physiologist G. Haberlandit, is
is known as the father of plant tissue culture.
• Plant tissue culture includes several
specialized areas, like micro propagation,
somaclonal variation, protoplast and anther
culture.

7. rDNA technology
• One of the most significant breakthroughs in
modern sciences is the development of
techniques to transfer genes from unrelated
sources into crop plants
• Recent advances in molecular biology have
made it possible to introduce genes from
diverse sources such as unrelated plants,
bacteria, fungi. Insects and even from
chemical synthesis.

8. • The whole process of introduction, integration
and expression of the foreign gene(s) in the
host is called gene transformation or
transgenesis.

9. • Unlike conventional plant breeding, in this
case only the cloned gene(s) of agronomic
importance are introduced into the plants
without the co-transfer of other undesirable
genes from the donor.
• The recipient genotype is less desturbed.

10. Foreign gene can be transferred by two
methods :
• Direct gene transfer
• Vector-mediated gene transfer

11. Direct gene transfer
• Since the host range of agro bacterium has been
largely limited to dicotyledonous plant species, some
other methods are used for monocotyledonous plants
such as cereals have been developed.
• Methods used in direct gene transfer tech.
are –
Direct uptake of DNA
Electroporation
Microinjection
Microprojectile Bombardment

12. Direct uptake of DNA
• It is based on the ability of protoplast to
uptake the foreign DNA from surrounding
solution.
• An isolated plasmid DNA (vector) is mixed
with protoplasts in the presence of
polyethylene glycol (PEG), which inhance the
uptake of DNA by protoplast.

13. • This method depends on the plant
regeneration ability of the protoplast and has
been successfully used to produce transgenic
plants in brassica, strawberry, lettuce, rice,
wheat and maize.

14. Electroporation
• Electroporation, or electro permeabilization, is
a molecular biology technique in which an
electrical field is applied to cells in order to
increase the permeability of the cell
membrane, allowing chemicals, drugs, or DNA
to be introduced into the cell.
• The cells recovered from the electric shock
can be regenerated into whole plants.

15. • Electroporation has been successfully
used for obtaining transgenes in
tobacco, maize, rice, wheat and
sugercane.

16. Microinjection
• During microinjection, DNA is injected directly into
the cell, or even into the cell nucleus via an inserted
cannula. The process is observed and controlled
under the microscope. The DNA is then integrated
into the plant genome – probably during the cell’s
own DNA repair processes.
Micro injection into the cell of DNA inserted into onion cells
a potato callus

17. Micro projectile Bombardment
• The Particle bombardment device, also known
as the gene gun, was developed to enable
penetration of the cell wall so that genetic
material containing a gene of interest can be
transferred into the cell.
• Today the gene gun is used for genetic
transformation of many organisms to
introduce a diverse range of desirable traits.

18. • The
transgenic
plants
have been
recovered
in banana,
barley,
bean,
cotton,
maize,
papaya
etc.

19. Vector-mediated gene transfer
• Among the various vectors used in plant transformation,
the Ti plasmid of Agrobacterium tumefaciens has been
widely used.
• This bacteria is known as “natural genetic engineer” of
plants because these bacteria have natural ability to
transfer T-DNA of their plasmids into plant genome
upon infection of cells at the wound site and cause an
unorganized growth of a cell mass known as crown gall.
• Ti plasmids are used as gene vectors for delivering
useful foreign genes into target plant cells and tissues.
• The foreign gene is cloned in the T-DNA region of Ti-
plasmid in place of unwanted sequences.

20. Transgenic crops
• Transgenic crops or Genetically modified
crops (GMCs, GM crops, or biotech crops)
are plants used in agriculture, the DNA of
which has been modified using genetic
engineering techniques. In most cases the aim
is to introduce a new trait to the plant which
does not occur naturally in the species.

21. Development of insect-resistant transgenic
crop cultivars has focused on two distinct
approaches :-
Integration of bacterial genes from Bacillus
thuringienesis (Bt)
Integration of plant genes for the production
of enzyme inhibitor and suger binding lectins.

22. Bt Endotoxins
• The use of genes encoding endotoxins
from Bacillus thuringiensis is now a well-
established technology for producing transgenic
plants with enhanced resistance to the larvae of
lepidopteran insect pests.
• Bt cotton was first released for commercial
production in the USA in 1996 and subsequently
grown in several countries including Argentina,
Australia, China, Colombia, Indonesia, Mexico,
South Africa, and India.

23. • Since then other transgenic crop species
producing Bt toxins have been commercialized
including maize, tomato and potato.
• The adoption of Bt crop varieties by farmers
has been rapid reflecting the benefits of these
crops such as reduced insecticide use, lower
production costs and higher yields.

24. • B. thuringiensis, a Gram-positive soil bacterium, produces
a proteinaceous parasporal crystalline inclusion during
sporulation.
• There are two main categories of Bt toxins: Cry and Cyt.
• These two groups are classified further by a detailed
nomenclature system that describes groups Cry1 to Cry55 and
Cyt1 to Cyt2.
• The larvae of insect orders primarily affected by Bt toxins are
Lepidoptera (butterflies and moths), Diptera (mosquitoes) and
Coleoptera (larval and adult beetles).

25. • However, Bt toxins are not toxic to people,
wildlife, or most beneficial insects and
therefore the opportunities for biological
control are great.
• The effect of Bt toxin on a range of
lepidopteran insects has been studied
including:
Bombyx mori,
Helicoverpa armigera,
Heliothis virescens ,
Manduca sexta,
Ostrinia nubilalis,
Plutella xylostella,
Sesamia nonagrioides,
Spodoptera exigua,
Spodoptera frugiperda and
Spodoptera littoralis .

31. MECHANISM OF ACTION
• The Bt toxin mechanism of action is described
by two models:
1. The pore formation model and
2. the signal transduction model.

32.  The initial steps of both models are the same.
 Upon ingestion by insects the crystalline inclusion is
solubilised in the midgut .
 Most target insects have a high gut pH that is crucial for the
efficacy of Bt toxins since
 Most Bt-protoxins are only soluble above pH 9.5. The 130
kDa protoxins are activated by insect gut proteases.
 In the pore formation model the activated toxins bind to the
 primary receptors in the brush border membrane of the
midgut epithelium columnar cells .

33.  The major receptors for Cry toxins in lepidopterans are
cadherin-like proteins.
 The toxins then interact with secondary receptors in the
midgut larval membrane.
 Following secondary receptor binding, the toxin inserts into
the membrane creates pores.
 These pores lead to the disruption of membrane integrity
and cause an electrolyte
 imbalance that ultimately leads to death by starvation or
septicaemia.

34. • An alternative model for the Bt toxin mechanism of action
proposes that Cry toxins trigger a signalling cascade pathway.
This model differs from the pore formation model in that it
does not involve secondary receptors or the formation of
pores in the membrane. Instead, in this model, binding to the
cadherin receptor initiates a Mg2+ dependent signal cascade
pathway that includes a guanine nucleotide-binding protein,
adenylyl cyclase, and protein kinase A which ultimately results
in cell death.

36. Protease inhibitors
• Protease inhibitors are one component of a plant’s natural
defence mechanism against herbivores and pathogens.
• Plants protect themselves directly by constitutively
expressing protease inhibitors and by inducing protease
inhibitors in response to mechanical wounding or insect
attack.
• They may also release volatile compounds after insect
damage that function as potent attractants for predators of
insect herbivores.
• The release of volatile compounds after wounding, such as
methyl jasmonate also triggers the production of
proteinase inhibitors in neighbouring unwounded plants
essentially prearming the local population against insect
attack.

37. examples :-
Protease
inhibitor
Protease
family
Proteases
inhibited
Transforme
d plant
Insect species
used
bioassayEffe
ct of PI on
larval
growth
Barley
trypsin
inhibitor
[BTI]
Cereal
trypsin
inhibitor
Trypsin
Tobacco
Spodoptera
exigua
29%
reduction
in survival
Wheat
Sitotroga
cerealella
No effect
on growth
or
mortality
Soybean
Bowman-
Birk
trypsin
inhibitor
Bowman
-Birk
Trypsin,
chymotrypsin
Sugarcane
Diatraea
saccharalis
Growth
severely
retarded

38. PIS IN TRANSGENIC PLANTS : SUCCESS AND FAILURE

39. • The main problem of inadequate levels of PI expression is best
exemplified by studies with P. xylostella, the diamondback moth.
When larvae of the diamondback moth consumed transgenic
plants expressing the chymotrypsin and trypsin specific potato
type II proteinase inhibitor, Pot II, they suffered lower growth
rates. However, this did not confer an advantage to the plants
because the larvae consumed more tissue to compensate for
their decrease in metabolism. As a result, the insects maintained
population growth rates similar to those of larvae on non-
transgenic plants.

40. Lectins
• These are the plant derived proteins that binds to
oligo and polysaccharides, cause agglutination
and cell aggregation.
• Lectins have been isolated and characterized
from a wide variety of plants such as pea, rice,
wheat, caster, soybean, garlic, tobacco and
chickpea.
• A no. of plant lectins exhibits insecticidal
property.
• The precise mode of action of of insecticidal
lectins is unknown.

42. Transgenic crop transgene origin Target insect pest
Rice
GNA Snow drop Nilaparvata lugens,
Nephotettics
virescens
SFII and GNA Snow drop and
spider (Segestria
florentina)
N. legens
Wheat GNA Snow drop Sitobion avenae
Potato GNA Snow drop Lecanobia oleracea,
Myzus persicae
Mustard WGA Wheat Lipaphis erysimi
Insect resistant transgenic crops expressing plant lectins :-

43. Alarm Pheromone
• Alarm pheromones are defined as chemical
substances, produced and released by an organism,
that warn or alert another of the same species of
impending danger.
• This is common in the social Hymenoptera; for
example, the honeybee, Apis mellifera (Hymenoptera:
Apidae), and many ant species respond aggressively to
their alarm pheromones.
• Many aphid species produce the sesqiuterpene , (E)-β-
fernesens (EFB) as a component of alarm pheromone.

44. • Scientists at Rothemsted Research (UK) have
developed GM wheat, by transferring EFB synthase
gene from peppermint, to the genome of a spring
wheat strain.
• Lab trials have shown that EBF emmiting wheat not
only repels aphids, but also attracts their natural
enemies.
• This is the world first crop which repels insects
instead of killing them.

45. RESISTANCE OF LEPIDOPTERAN
INSECTS TO BT TOXINS
• More recently there have been reports of field
resistance to Bt crops in pink bollworm
(Pectinophore gosspiella, cotton bollworm
(Helicoverpa spp, armyworm (Spodoptera
frugiperda and western corn rootworm
Diabrotica virgifera virgifera.
• A decrease in field performance of Bt corn
against S. frugiperda was observed in Puerto Rico
and against Busseola fusca in South Africa. In
southeastern US problems with control of H.
zea on Bt cotton have also been reported.

46. MANAGEMENT OF RESISTANCE TO BT
CROPS
• There are two main strategies for management of insect
resistance to Bt crops:
Refuge and
pyramiding.
• The main approach for delaying evolution of resistance to
Bt crops is the refuge strategy.
• Farmers are mandated to maintain an abundance of host
non-Bt crops as a refuge surrounding their Bt crops.
• The theory behind this strategy is that any Bt resistant
larvae that arise on the Bt crops will mate with
susceptible individuals from neighbouring non-Bt crops.
• As long as inheritance of resistance remains recessive the
offspring will be susceptible to Bt crops.

47. • The other major strategy to combat the evolution of Bt
resistance is gene pyramiding.
• For example, the development of second generation Bt
cotton that has at least two Bt toxins such as the Monsanto
Bollgard II cotton variety, but up to six Bt toxins.
• Another resistance management strategy which is still in the
research phase of development is the use of insecticidal
genes with completely different modes of action such as
proteinase inhibitors. The success of combining multiple Bt
genes for resistance management is contingent on the
individual toxins having different targets to prevent cross
resistance developing.
• This information can be used to design combinations of Cry
toxins that complement each other to delay the
development of resistance to Bt crops.