#Biotechnology #bt cotton #plant technology

1. Plant Biotechnology
Plant Biotechnology
3.1 Genetic engineering of Plants
Plant transformation with Ti plasmids of A.tumefaciens,
Ti plasmid derived vector systems, physical methods of
transferring genes to plants.
3.2Uses of genetically engineered plants:
To overcome Biotic and abiotic stress: Insect resistance:
Increasing expression of the B.thuringiensis protoxin,
other strategies for protecting plants against insects,
preventing the development of Bacillus thuringeinsis
resistant insects, Herbicide resistant plants Oxidative
stress, Salt and drought stress, Modification of plant
nutritional content: Vitamin A

2. Reasons for developing transgenic
plants.
• First, the addition of a gene(s) often improves the
agricultural, horticultural, or ornamental value of
a crop plant.
• Second, transgenic plants can act as living
bioreactors for the inexpensive production of
economically important proteins or metabolites.
• Third, plant genetic transformation (transgenesis)
provides a powerful means for studying the
actions of genes during development and other
biological processes.

3. • Insecticidal activity, protection against viral
infection, resistance to herbicides, protection
• against pathogenic fungi and bacteria, delay of
senescence, tolerance of environmental
stresses, altered flower pigmentation,
improved nutritional quality of seed proteins,
increased postharvest shelf life, and self
incompatibility

4. • Transgenic plants can be made to produce a
variety of useful compounds, including
therapeutic agents, polymers, and diagnostic
tools, such as antibody fragments.
• Alternatively, they can be engineered to
synthesize viral antigenic determinants and,
after ingestion, can be used as edible vaccines.

5. • Plant biotechnology is having an enormous
• impact on plant-breeding programs because it
significantly decreases the 10 to 15 years that
it takes to develop a new variety using
traditional plant-breeding techniques.

7. Herbicide resistant
plants

8. Pest resistant plants

10. Plant transformation with Ti plasmids
of A.tumefaciens

11. Natural genetic engineer

18. Agropine

23. Ti Plasmid-Derived Vector Systems
• The simplest way to exploit the ability of the Ti
plasmid to genetically transform plants would
be to insert a desired DNA sequence into the
T-DNA region and then use the Ti plasmid and
A. tumefaciens to deliver and insert this
gene(s) into the genome of a susceptible plant
cell.

24. Although the Ti plasmids are effective as natural
vectors, they have several
serious limitations as routine cloning vectors.
• The production of phytohormones by transformed cells growing in culture
prevents them from being regenerated into mature plants.
• Therefore, the auxin and cytokinin genes must be removed from any Ti
plasmid-derived cloning vector.
• A gene encoding opine synthesis is not useful to a transgenic plant and
may lower the final plant yield by diverting plant resources into opine
production. Therefore, the opine synthesis gene should be removed.
• Ti plasmids are large (approximately 200 to 800 kb). For recombinant DNA
experiments, however, a much smaller version is preferred, so large
segments of DNA that are not essential for a cloning vector must be
removed

25. • Because the Ti plasmid does not replicate in
Escherichia coli, the convenience of perpetuating
and manipulating Ti plasmids carrying inserted
DNA sequences in that bacterium is not available.
• Transfer of the T-DNA, which begins from the
right border, does not always end at the left
border. Rather, vector DNA sequences past the
left border are often transferred, although the
transfer of these sequences is not often tested
for.

26. Vector design
• A selectable marker gene, such as neomycin phosphotransferase, that
confers kanamycin resistance on transformed plant cells.
• Because the neomycin phosphotransferase gene, as well as many of the
other marker genes used in plant transformation, is prokaryotic in origin,
it is necessary to put it under the control of plant (eukaryotic)
transcriptional regulation signals, including both a promoter and a
termination polyadenylation sequence, to ensure that it is efficiently
expressed in transformed plant cells.
• An origin of DNA replication that allows the plasmid to replicate in E. coli.
In some vectors, an origin of replication that functions in A. tumefaciens
has also been added.
• The right border sequence of the T-DNA region. This region is absolutely
required for T-DNA integration into plant cell DNA, although most cloning
vectors include both a right and a left border sequence.
• A polylinker (multiple cloning site) to facilitate insertion of the cloned
gene into the region between T-DNA border sequences.
• A “killer” gene encoding a toxin downstream from the left border to
prevent unwanted vector DNA past the left border from being
incorporated into transgenic plants. If this incorporation occurs, and the
killer gene is present, the transformed cells will not survive.

32. Insect Resistance
• Biotic stress
• The genetic engineering of crop plants to
produce functional insecticides
• makes it possible to develop crops that are
intrinsically resistant to insect
• predators and do not need to be sprayed
(often six to eight times during a
• growing season) with costly and potentially
hazardous chemical pesticides.

33. Insect Resistance
• One approach involves a gene for an
insecticidal protoxin produced by one of
several subspecies of the bacterium Bacillus
thuringiensis.
• Other common strategies use genes for plant
proteins,
such as α-amylase inhibitors, protease
inhibitors, and lectins, that have been shown
to be effective against a wide variety of
insects

34. Plant made self sufficient
• Both simpler and less costly to express the genes
for B. thuringiensis toxins in plants than to spray
B. thuringiensis preparations onto the surface of
the plant. Target speciffic
This mode of insecticidal-toxin delivery limits the
environmental distribution of the toxin and avoids
problems associated with spraying B.
• thuringiensis preparations, such as limited
environmental stability and the timing of the toxin
application.

35. Insect Resistance
• Some of these parasporal crystals known as
δ-endotoxins (Cry and Cyt) confer the
pathogenic capacity against larvae of different
orders of insects, mostly Lepidoptera, Diptera,
Coleoptera and in some cases against species
of other phyla

36. Mode of action of toxin
• the toxic properties come from crystalline inclusions produced
during the sporulation of Bt.
• The crystals and their subunits are inert protoxins and are not
biologically active, and their mode of action can be plotted as
follows: the δ-endotoxins are ingested, the crystals are solubilized
by the alkaline pH of the intestine, the inactive protoxins are
digested by proteases of the midgut which produces an active
toxin of about 60–70 kDa resistant to proteases, and then the Cry
toxins come into contact with the N-aminopeptidase receptors and
cadherin on the surface of the membrane.
• The affinity between toxins and certain types of receptors results
in proteolysis of the Cry protein that causes structural changes in
the chains and forms oligomers that function as “pre-pores.”
• The N-aminopeptidase receptor anchors the pre-pore in the lipid
bilayer, pore formation affects integrity of the membrane, and
electrophysiological evidence and biochemistry suggest that the
pores cause an osmotic imbalance that causes cell death and lysis;
the intestine is paralyzed, the insect stops feeding, and there is
diarrhea, total paralysis, and finally death

38. Insect Resistance
• minimum sequence that encoded toxin activity
had to be determined.
• analysis showed that the N-terminal portion of
the protoxin molecule is highly conserved (~98%)
and the C-terminal region is more variable (~45%
conserved).
• Further work showed that all of the
insecticidal-toxin activity resides within the first
646 amino acids from the N terminus of the
1,156-amino-acid protoxin

39. • scientists truncated the gene so that only the
N-terminal portion of the insecticidal
protoxin—the part of the protoxin that
contains the toxin —was produced and
inserted a strong plant promoter to direct
gene expression

41. Insect Resistance
• One group of researchers expressed the fully
modified protoxin gene under the control of
the promoter for the gene that codes for the
small subunit of the plant enzyme ribulose
bisphosphate carboxylase and downstream
from the chloroplast transit peptide sequence
of this enzyme, so that the overproduced
• protoxin became localized within the
chloroplast.

42. Insect Resistance
• have introduced an insecticidal- protoxin gene directly
into the chloroplast DNA of the host plant.
• The B. thuringiensis protoxin gene was integrated into
a specific site on the chloroplast DNA by constructing a
vector that contained the protoxin gene flanked by
two single-copy chloroplast genes . Integration of the
• introduced genes occurs by homologous
recombination. Once integrated into the chloroplast
DNA, a protoxin gene under the transcriptional control
of a strong chloroplast promoter may be expressed at
high levels.

43. • First, the protoxin gene does not have to be modified, because the
• chloroplast transcriptional and translational apparatuses are typically
• prokaryotic.
• Second, because there are many chloroplasts per cell and many copies
of chloroplast DNA per chloroplast, the protoxin gene is present in
multiple copies and therefore is more likely to be expressed at a high
level.
• Third, in most plants, chloroplasts are transmitted only through the egg
and not through pollen, which means that plants receive all of their
chloroplast DNA from their female parent. Consequently, there is no
risk of unwanted transfer of the protoxin gene to other plants in the
environment by pollen.
• The disadvantage of expressing the B. thuringiensis protoxin in
chloroplasts
• is that insects that attack stems or fruit will not encounter the protoxin,
since
• these tissues do not have any chloroplasts.
Chloroplasts as site of insertion
and production

44. Other Strategies for Protecting Plants
against Insects
• Protease inhibitors--cowpea trypsin inhibitor
• some plants produce protease inhibitors that,
when ingested, prevent the feeding insect from
hydrolyzing plant proteins, thereby effectively
starving the predator insect.
• transgenic plants that expressed more than 2 mg
of cowpea trypsin inhibitor

45. Other Strategies for Protecting Plants
against Insects
• potato proteinase inhibitor II gene provides
rice plants with protection against the pink
stem borer

46. Other Strategies for Protecting Plants
against Insects
• α-Amylase inhibitor in the seed proteins of the common bean.
• The cowpea weevil and the azuki bean weevil are
seed-feeding beetles that cause considerable economic loss of
these legume crops.(pea)
• strong seed-specific promoter for the bean
phytohemagglutinin gene used to transform pea plants

47. Other Strategies for Protecting Plants
against Insects
• Cholesterol oxidase.
• Cholesterol oxidase, which is present in a range of different
bacterial genera, catalyzes the oxidation of 3-hydroxysteroids
to ketosteroids and hydrogen peroxide.
• Cholesterol oxidase probably acts by disrupting the insect’s
midgut epithelial membrane, thus killing the insect.

48. Other Strategies for Protecting Plants
against Insects
• Preventing the Development of B. thuringiensis-Resistant
Insects --- assumes that resistance to two control methods is
much less likely to develop simultaneously
• Using two or more different B. thuringiensis insecticidal toxins ( gene
stacking, gene pyramiding) or fusing portions of the active regions of two
different toxin genes to generate novel hybrid protein
Transforming plants with both a B. thuringiensis insecticidal-toxin
gene and another form of biological insecticide (e.g., an α-amylase
inhibitor gene.

49. Other Strategies for Protecting Plants
against Insects
• Preventing the Development of B. thuringiensis-Resistant
Insects
• the expression of the insecticidal toxin in transgenic plants
was limited to a short period
❖ the promoter of a gene from tobacco called the
pathogenesis-related protein 1a (PR-1a) gene.
• induced by any one of a variety of pathogenic organisms or by
chemicals, such as salicylic acid and polyacrylic acid.
❖ the ricin B-chain binds with very high affinity
toN-acetylgalactosamine residues--fusion protein

50. • Gossypol is a yellow polyphenolic aldehyde
• that permeates cells and acts as an inhibitor of several of the
insect’s dehydrogenase enzymes.
• The cotton bollworm protects itself from the toxic effects of
gossypol by inactivating the gossypol with the enzyme
cytochrome P450 monooxygenase.
• transgenic plants were constructed to
synthesize an RNAi molecule that would
silence the insect’s gene for the cytochrome
P450 monooxygenase.

51. Virus Resistance
• Viral Coat Protein-Mediated Protection
• an RNA molecule that is complementary
• to a normal gene transcript (mRNA) is called
antisense RNA.
• Most plant viruses have double stranded RNA
as their genetic material.--a strain of wheat
was engineered to express the E. coli gene
(rnc) for ribonuclease (RNase) III, an enzyme
that cleaves only double-stranded RNA;

52. Antiviral plant proteins.
• Pokeweed (Phytolacca americana) has three antiviral proteins in its cell
wall:
• pokeweed antiviral protein (PAP), which is found in spring leaves; PAPII,
which is found in summer leaves; and PAP-S, which appears in seeds.
Although they are only 40% identical at the protein level, and antibodies
directed against PAP do not react with PAPII, they employ similar modes
of action.
• Both PAP and PAPII are ribosome-inactivating proteins that remove a
specific adenine residue from the large ribosomal RNA of the 60S subunit
of eukaryotic ribosomes.
• When pokeweed plants are infected with viruses, either PAP or PAPII is
synthesized, depending on the season, and the ribosomes in the infected
cells are inactivated.
• Based on their mode of action, PAP and PAPII are good candidates for
developing transgenic plants that are resistant to a broad spectrum of
plant viruses.

53. Single-chain antibodies.
• Plant viruses are RNA viruses, and many of them
contain positive-stranded RNA as the genetic material.
• These viruses all encode RNA-dependent RNA
polymerases that are essential for their replication.
• Thus, a single-chain Fv antibody that recognizes
epitopes that are common to the RNA-dependent RNA
polymerases from several different viruses should be
an effective means of inhibiting the replication of all of
these viruses, thereby making transgenic plants that
express these single-chain Fv antibodies resistant to
these viruses .

54. Fungus and Bacterium Resistance
• Plants often respond to fungal or bacterial pathogen invasion or other
• environmental stresses

55. PR proteins
• The PR proteins include β-1,3-glucanases,
chitinases, thaumatin-like proteins (thaumatin
is a small, very sweet protein), and protease
• inhibitors that protect the plant-invading
pathogens

56. Strategies
• 1. Overexpression of the NPR1 gene can lead to
the generation of broad-spectrum disease
resistance against both fungal and bacterial
pathogens.
• 2. overproducing salicylic acid
• Salicylate is synthesized from chorismate,
• which is produced in large amounts in the
chloroplast
• .

57. • 2. overproducing salicylic acid
• Salicylate is synthesized from chorismate,
• which is produced in large amounts in the
chloroplast
isochorismate synthase and isochorismate
pyruvate lyase, which catalyze
• salicylate synthesis

58. • to constitutively express chitinase under the
control of the cauliflower mosaic virus 35S
promoter include rice, tobacco, and canola.
• a cDNA encoding chitinase from the
biocontrol fungus Trichoderma harzianum
• beneficial root fungus Glomus mosseae to the
plant roots was not affected.

59. Pathogenic fungi belonging to the
genus Fusarium
different antimicrobial peptides (one from the
radish Raphanus sativus and
• one from the mold Aspergillus giganteus) and
a chitinase (from wheat) to a
• single-chain Fv antibody that binds to a
Fusarium cell wall protein

60. pathogenic soil bacterium Erwinia
carotovora
• Cauliflower mosaic virus 35S promoter
• potato plants that actively express
bacteriophage T4 lysozyme

61. Herbicide Resistance
• A number of different biological manipulations that would
cause a crop plant to be herbicide resistant can be envisioned.
• 1. Inhibit uptake of the herbicide.
• 2. Overproduce the herbicide-sensitive target protein so that
enough of it remains available for cellular functions despite
the presence of the herbicide.
• 3. Introduce a bacterial or fungal gene that produces a
protein that is not sensitive to the herbicide but performs the
same function as the plant (herbicide-sensitive) protein.
• 4. Reduce the ability of a herbicide-sensitive target protein to
bind to a herbicide.
• 5. Endow plants with the capability to metabolically inactivate
the herbicide.

62. Crops that have been engineered to be resistant to
glyphosate by this approach are said to be “Roundup
ready.”
• Glyphosate, trademarked as Roundup by the
Monsanto Corporation, inhibits a key enzyme in the
shikimate pathway,
• 5-enolpyruvylshikimate-3-phosphate synthase
(EPSPS), that plays an important role in the synthesis
of aromatic amino acids in both bacteria and plants.
• Plants resistant to this herbicide have been developed
by putting an EPSPS-encoding gene from a
glyphosate-resistant strain of E. coli under the control
of plant promoter and transcription
termination–polyadenylation sequences and cloning
the construct into plant cells.

65. Oxidative Stress
• Unlike many animals, plants cannot physically
avoid adverse environmental conditions, such as
high levels of light, ultraviolet (UV) irradiation,
• heat, high salt concentrations, or drought, so
physiological strategies have evolved to cope with
these stresses.
• At the molecular level, one of the undesirable
• consequences of physiological stress is the
production of oxygen radicals.

66. Oxidative Stress
• Factors like salt, freezing, and drought, as well as
exposure to pollutants, stimulate the formation of
reactive oxygen
• species in plant cells.
• These toxic molecules damage membranes,
membrane- bound structures, and macromolecules,
including proteins and nucleic acids, especially in the
mitochondria and chloroplast, resulting in
• oxidative stress.
• A common type of potentially damaging oxygen
radical is the superoxide anion.

67. Oxidative Stress
• Within a cell under oxidative stress, the
enzyme superoxide dismutase detoxifies
superoxide anion by converting it to hydrogen
peroxide, which in turn is broken down to
water by various cellular peroxidases or
catalases

68. Oxidative Stress
• tobacco plants that were transformed with a
superoxide dismutase gene that was under
the control of the 35S promoter from
cauliflower mosaic virus had reduced oxygen
radical damage under stress conditions
compared with control plants.

69. • Oxidative stress may also be reduced if the level of
oxidized glutathione within a plant is increased.
• Glutathione peroxidase catalyzes the
• conversion of glutathione to oxidized glutathione by
reacting with organic
• peroxides and reducing them to organic alcohols

70. Oxidative Stress
• Transgenic tobacco plants that expressed
glutathione peroxidase were created using
• the isolated cDNA under the control of the
35S promoter, and the construct was
introduced into plants with a binary Ti plasmid
system.

71. Salt and Drought Stress
• Many plants synthesize low-molecular-weight
nontoxic compounds collectively called
osmoprotectants.
• These compounds facilitate both water uptake
and retention and also protect and stabilize
cellular macromolecules from damage by high salt
levels.
• Some well-known osmoprotectants are sugars,
alcohols, the amino acid proline and quaternary
ammonium compounds.

72. Salt and Drought Stress
• To create more salt-tolerant
• plants, scientists have tried to engineer an
increase in the cellular accumulationof the
following osmoprotectants: trehalose, proline,
d-ononitol, mannitol, sorbitol, glycine betaine,
and 3-dimethylsulfoniopropionate.

73. Water stress or
high salinity.
• Betaine is a highly effective osmolyte that
accumulates in some plants during periods of
water stress or high salinity.
• To create a more salt-tolerant tobacco, plant
cells were transformed with a Ti plasmid
vector carrying the E. coli betA gene, which
encodes choline dehydrogenase, under the
control of the cauliflower mosaic virus 35S
promoter.

74. high levels of salt in the soil
• Increase the trehalose
• In E. coli, trehalose-6-phosphate is first formed
from uridine diphosphate (UDP)-glucose and
glucose-6-phosphate, and then the
trehalose-6-phosphate is converted to
trehalose

75. Salt tolerant
• Researchers have engineered the plant A.
thaliana to be salt tolerant by sequestering
sodium ions in the large intracellular vacuole.
• By concentrating the salt in the plant’s large
vacuole, water that is free of salt should
• be driven into the plant cells, resulting in
plants that use water more efficiently.

76. Drought-tolerant transgenic plants
• These approaches have included introducing
genes encoding overproduction of various
osmolytes (e.g., trehalose, proline, glycine
betaine, and polyamines), plant stress
proteins (e.g., chaperones and heat shock
proteins), reactive-oxygen-scavenging
proteins

77. Golden Rice
• Rice (Oryza sativa) is the staple food of
approximately half of the world’s population,
it is a poor source of several nutrients and
vitamins, including vitamin A.
• About 124 million children worldwide are
• deficient in vitamin A; this deficiency leads to
1 million to 2 million deaths per year and is a
leading cause of vision impairment, including
night blindness and total blindness.

78. Golden Rice
• Mammals synthesize vitamin A from
β-carotene, which is a common carotenoid
pigment normally found in plant
photosynthetic membranes.

79. Golden Rice
• Group of scientists reported using Agrobacterium-mediated
transformation to introduce the entire β-carotene
biosynthetic pathway
• into rice.
• The phytoene synthase and phytoene desaturase genes
• were introduced on a construct that did not contain any
selectable marker.
• The lycopene β-cyclase gene was part of a separate
construct that contained a selectable marker. The
frequency of insertion of all three genes into the rice
genome and their subsequent expression were quite high.
• Thus, the engineered rice produces β-carotene, which, after
ingestion, is converted to vitamin A.

80. The transgenic
rice that produces
β-carotene has a
yellow or golden color
and has been
called “golden rice”
Biosynthesis of β-carotene in rice and vitamin A in humans. The daffodil phytoene synthase
gene (psy) was controlled by a promoter from the rice seed storage protein glutelin. The
phytoene desaturase (crt) gene was from the bacterium Erwinia uredovora and was
controlled by the 35S promoter. The lycopene β-cyclase (lcy) gene originated from daffodil
and was controlled by the rice glutelin promoter. All three genes were fused to transit
peptides so that the proteins that they encoded would be transported into the plastid. GGPP,
geranylgeranyl pyrophosphate
Golden Rice

81. Page No to study from books
• Glick ----page no and chapters
• Chapter 19 page no 759-795
• Chapter 18 page no 725-741
• Golden rice Page no 811
• Also B.D Singh chapter pages 446-451,453-472
This chapter on the class room