STRESS

 Stress can be defined as an influence that is outside the normal range

of homeostatic control. (Lerner, 1999)
 Any strain or interference that disturbs the functioning of an organism.
(Encyclopedia Britannica)
GENETIC ENGINEERING
 The artificial manupulation, modification and recombination of DNA or
other nucleic acid molecules in order to modify an organism or
population of organism.
(Encyclopaedia Britanica 15 edn.)
 The branch of biology dealing with the splicing and recombining of
specific genetic units from the DNA of living organisms as in order to
produce new species or biochemicals.
(Webster's New World College Dictionary )
1. Direct methods:
•protoplast microinjection
•particle bombardment
•polyethyleneglycol (PEG) method
•electroporation
•sonication
•macroinjection

2. Agrobacterium -mediated
transformation
•Agrobacterium tumefaciens
•Agrobacterium rhizogenes (Hairy Root)
A model for the Agrobacterium-mediated genetic transformation

Recognition and attachment

Vir genes expression by host signals
T-strand produce
T-complex export into host
Transport through cytoplasm and
nuclear
T-DNA uncoating and integration.
Plasmid pPM5 contained an EcoRI-HindIII fragment of 1.75 kb with the reinforced 35S
promoter, the HAL1 ORF, and the nos terminator

0
5 5 0Z1
IK2 tion
c
sele um
i
med

Carmina et al., 2000, plant physiology , 123: 393–402,

 Saccharomyces genes HAL1 and HAL3, which are
involved in theregulation of K1 and Na1 transport
respectively
 introduction of the yeast HAL1 gene (using a
modified plasmid with enhancer elements) in
tomato(Lycopersicon esculentum cv P-73)
 TG plant growth was evaluated by measuring
rooting capacity, shoot height, number of leaves,
and total fresh (FW) and dry (DW) weight of
plants after 28 d on culture media with or without
NaCl.

Carmina et al., 2000, plant physiology , 123: 393–402,

Table:
Effect of salt
stress on
shoot growth
after 28 d of
culture on B1
medium
supplemente
d with NaCl

Fig : The relative K1 to

Na1 ratio, which was
higher in the
transgenic families,
indicated higher K1
retention under saline
conditions
Carmina et al., 2000, plant physiology , 123: 393–402,
 Overexpression of HAL 1 gene in yeast
confers a high salt tolerance level by reducing
K loss and decreasing intracellular Na from
the cells upon salt stress
 HAL 1 ability to maintain K uptake in the
presence of external Na, as shown by the
transgenic families (especially TG3), is
noteworthy. This ability has been related to
salt tolerance in tomato
 The salt tolerance levels of transgenic tomato
plants assayed in this work were higher than
that previously observed in melon. This could
be due to genetically engineered plasmid
used in this work, which had a duplicated 35S
promoter.

Carmina et al., 2000, plant physiology , 123: 393–402,

Plasmid pBY520 with
bar gene

Xu et al., Plant Physiol. (1996) 11 O: 249-257

 HVA7, is a late embryogenesis abundant (LEA)
protein gene, from barley (Hordeum vulgare L.)

 This
gene was introduced into rice suspension cells
using the Biolistic-mediated transformation
method

 BarleyHVA 7 gene regulated by the rice actin 1 gene

promoter led to high-level, constitutive accumulation
of the HVAl protein in both leaves and roots of
transgenic rice plants

Xu et al., Plant Physiol. (1996) 11 O: 249-257

Structure of the plasmid pBY520 for
expression of HVA7 in transgenic rice.

Xu et al., Plant Physiol. (1996) 11 O: 249-257

Table II. estimated levels of HVA1 protein
accumulation in different transgenic lines
Using a partially purified HVAl protein
preparation as the standard

Xu et al., Plant Physiol. (1996) 11 O: 249-257

 Table III. Seed germination and growth of young
seedlings in medium under osmotic stress or salt
stress

• After 5 d in the stress medium, the germinated seeds

(with 0.2- to 0.5-cm-long shoot) were transferred onto MS
medium. Both transgenic and control seedlings recovered
and resumed normal growth, but transgenic seedlings
grew faster during this recovery period, their shoots were
significantly longer after 1 week (Table III),
Xu et al., Plant Physiol. (1996) 11 O: 249-257
 Comparison of transgenic plants and
NT plants grown
under drought and salinity stress
conditions. Two NT plants and two R,
transgenic plants from each of two lines
(numbers 36 and 41) are show here.

A, Plants recovered from

drought stress. Photograph was taken
21 d after the beginning of initial water
stress (three cycles of 5 d of drought
stress followed by 2 d of recovery with
watering).
B, Plants recovered from
salt stress. Photograph was taken after
10 d of salt stress in 200 M NaCI and
10 d of recovery in tap water

Appearance and development of stress symptoms occurred much more slowly in

transgenic plants than in control plants.
Xu et al., Plant Physiol. (1996) 11 O: 249-257
Transgenic rice plants showed significantly increased tolerance
to water deficit and salinity.

Transformed Plants maintained higher growth rates than

nontransformed control plants under stress conditions.

The increased tolerance was also reflected by delayed

development of damage symptoms caused by stress and by
improved recovery upon the removal of stress conditions.

The extent of increased stress tolerance correlated with the

leves of the HVAl protein accumulated in the transgenic rice
plants
 Using a transgenic approach, this study provides
direct evidence supporting the hypothesis that LEA
proteins play an important role in the protection of
plants under water or salt-stress conditions

Xu et al., Plant Physiol. (1996) 11 O: 249-257

 Over expression of stress responsive gene SNAC1
(STRESS-RESPONSIVE NAC 1) significantly enhances
drought resistance in transgenic rice in the field under
severe drought stress conditions at the reproductive
stage
 The transgenic rice also shows significantly improved
drought resistance and salt tolerance at the
vegetative stage
 Compared with WT, the transgenic rice are more
sensitive to abscisic acid and lose water more slowly
by closing more stomatal pores, yet display no
significant difference in the rate of photosynthesis
 DNA chip analysis revealed that a large number of
stress-related genes were up-regulated in the SNAC1-
overexpressing rice plants

Hu et al., 2006, PNAS, 103(35):12987–12992

 STRESS-INDUCIBLE EXPRESSION OF
SNAC1.
(a) RNA gel blot analysis of expression of the
SNAC1 under drought (DT), salt (200mM),
cold (4°C), and ABA treatment (100 M)

Hu et al., 2006, PNAS, 103(35):12987–12992

 Thetemporal and spatial patterns of SNAC1
expression were investigated by
transforming a japonica cultivar Nipponbare
with a fusion gene of PSNAC1:GFP

Hu et al., 2006, PNAS, 103(35):12987–12992

A GFP signal was observed in callus, root,
ligule, stamen, and pistil from transgenic
plants under normal growth conditions

Hu et al., 2006, PNAS, 103(35):12987–12992

 When transgenic plants were drought-
stressed to the stage of leaf-rolling, strong
induction of GFP was detected in leaves and
minor induction was observed in roots and
nodes, whereas no obvious change of GFP
expression level was observed in callus,
ligule, stamen, and pistil after drought stress

Hu et al., 2006, PNAS, 103(35):12987–12992

 Further examination of the GFP signal in the
stressed leaves revealed that the signal is
localized predominantly in guard cells that
constitute the stomata

This finding suggests that SNAC1 gene expression was

specifically induced in guard cells
Hu et al., 2006, PNAS, 103(35):12987–12992
 The strongly localized induction of SNAC1 in
guard cells of WT plants suggests that
increased stomatal closure is a likely target
of regulation by SNAC1
 It is interesting to note that quite a few rice
homologs of genes related to stomatal
movement are up regulated in the SNAC1-
overexpressing plants

Hu et al., 2006, PNAS, 103(35):12987–12992

 Cosegregation of SNAC1-overexpressing
(RNA gel blot analysis) with the improved
drought tolerance in the T1 family of S19.
SS(%), seed-setting rate

Hu et al., 2006, PNAS, 103(35):12987–12992

 All of the SNAC1-overexpressing plants
produced significantly (t test, P 0.01) higher
spikelet fertility than the negative control
under all drought treatments
 no significant difference was detected between the transgenic
plants and the control as evaluated by a number of agronomic
traits such as plant height, number of panicles per plant,
number of spikelets per panicle, and grain yield per plant, as
well as root depth and root volume under unstressed
conditions
 This finding clearly indicates that
overexpression of SNAC1 does not affect
growth and productivity of the rice plant
 Besides the improved drought resistance, SNAC1-
overexpressing transgenic rice also showed enhanced salt
tolerance indicating that there was another potential
mechanism of salt tolerance regulated by SNAC1
Hu et al., 2006, PNAS, 103(35):12987–12992
 Introduced Arabidopsis thaliana hsp101
(Athsp101) cDNA into the Pusa basmati 1
cultivar of rice (Oryza sativa L.) by
Agrobacterium mediated transformation
 Diagrammatic representation of pUH-
Athsp101 construct employed for rice
transformation.

Katiyar-Agarwal et al., (2003) Pt .Mol. Biol. 51: 677–686

 Stable
integration and expression of the
transgene into the rice genome was
demonstrated by Southern, northern and
western blot analyses

 Northernanalysis of Athsp101 expression

untransformed (C2) and transgenic rice (15, 19
and 43).

Katiyar-Agarwal et al., (2003) Pt .Mol. Biol. 51: 677–686

 Comparison of survival of transgenic lines after
exposure to different levels of high-temperature stress
with the untransformed control plants
 (a)45 ◦C for 3 h and then were placed at 28 ◦C

 The optimum temperature for rice growth throughout its life cycle is 25–31 ◦C

untransformed (C2) and transgenic lines (15and 43)

Katiyar-Agarwal et al., (2003) Pt .Mol. Biol. 51: 677–686

 (B) 47 ◦C for 2 h
and, after the
stress, leaves
were excised
and the cut
plants were
placed at 28 ◦C

(C) 50 ◦C for 40
min and, after
the stress,
leaves were
excised and
the plants were
then placed at
28 ◦C for re-
growth

untransformed (C2) and transgenic lines (15and 43) Katiyar-Agarwal et al., (2003) Pt .Mol. Biol. 51: 677–686
The transgenic rice lines showed significantly
better growth performance in the recovery
phase following the stress
The results showed that all transgenic rice
plants survived in the high-temperature range
of 45–50◦C exhibiting vigorous growth during
the subsequent recovery at 28◦C, whereas
most of the untransformed plants succumbed.
These tests revealed that AtHsp101 imparts
basal high temperature tolerance possibly by
acting in the post-stress recovery period

Katiyar-Agarwal et al., (2003) Pt .Mol. Biol. 51: 677–686

 Glycine betaine is an osmoprotectant that plays an important role and accumulates
rapidly in many plants during salinity or drought stress

 Choline monooxygenase (CMO) is a major catalyst in the synthesis of glycine betaine.

Zhang et al., 2009, Mol Breeding, 23:289–298

 CMO gene (AhCMO) cloned from Atriplex hortensis
was introduced into cotton (Gossypium hirsutum L.)
via Agrobacterium mediation

AhCMO
AhCMO
Glycine betain

GMO with
high glycine
AhCMO
AhCMO betain
 Two transgenic AhCMO cotton lines used to study
their salinity tolerance in both greenhouse and field
under salinity stress

Zhang et al., 2009, Mol Breeding, 23:289–298

 Greenhouse study showed that on average,
seedlings of the transgenic lines accumulated 26
and 131% more glycine betaine than those of
non-transgenic plants under normal and salt-
stress (150 mmol l-1 NaCl) conditions
respectively

Zhang et al., 2009, Mol Breeding, 23:289–298

 Theosmotic potential, electrolyte leakage
and Malondialdehyde (MDA) accumulation
were significantly lower in leaves of the
transgenic lines after salt stress

•SM3, non-transgenic cotton,

•L2 and L4; AhCMO transgenic lines 2 and 4.
•Bars carrying the same letter are not
significantly different (P = 0.05)

Zhang et al., 2009, Mol Breeding, 23:289–298

 Thenet photosynthesis rate in transgenic
cotton leaves were less affected by salinity
than in nontransgenic cotton leaves

Zhang et al., 2009, Mol Breeding, 23:289–298

 Therefore, transgenic cotton over-expressing AhCMO was
more tolerant to salt stress due to elevated accumulation of
glycine betaine, which provided greater protection of the
cell membrane and photosynthetic capacity than in non-
transgenic cotton.

Zhang et al., 2009, Mol Breeding, 23:289–298

 Abiotic stress’ are major cause of concern for the
global food security
 Conventional knowledge has almost saturated in
finding the solutions for the sprawling abiotic
stress’ resulting due to climatic change and other
causes
 GE has proved its worth in tweaking the plants’
ability to cope with the various abiotic stresses
 The main advantage of GE is that it can transcend
across the species barrier
 Although much progress has been made through
GE in taming stress’ much is need to be done to
realize the full potentiality of this technology
THANKS