Stress Tolerant Plants

Their Development, Uses and
Implications

Introduction

•
What Stresses Effect Plants?


And


•
Why there is a need for Stress Tolerant
Plants (STPs)?

What Stresses Effect Plants?

•
Most plants complete their life
cycle in a single location and are
therefore plagued by challenges
such as nutrient acquisition,
pathogen attack and
environmental stresses.

•
Environmental stresses include
Light, Oxidative Stress, Cold,
Heat, Nutrition, Water, Salinity,
Toxic concentrations of Metals
and Pathogens.

Flooding can cause stress
through waterlogging

Why Is There A Need For Stress
Tolerant Plants?

•
“Drought stress accounts
for more production losses
than all other factors
combined”

John Cushman,
Biochemistry Professor at the
University of Nevada, Reno.

•
Agricultural plant science has
had two main goals for
decades: to increase yield and
quality of agricultural products
and to improve the protection
of crops to stresses

Maize is a typical crop
which scientists are
trying to improve

Why Is There A Need For Stress
Tolerant Plants?

-
Pressure From The Environment

•
As the Earths
population
increases, new
means of improving
crop productivity
must be found to
increase the
resources available.


Pressure From The Environment

•
Intensive irrigation and
agriculture has led to severe
problems such as increased
salinity in the soil.

•
Global Climate change is
altering environmental
conditions


The Development Of Stress
Tolerant Plants

•
Introduction To Previous Methods


And


•
Modern Techniques

Developing STPs
–
the classical
way

•
Classical breeding programs develop new
traits by combining different germ plasms
in order to exploit natural or artificially
induced diversity and, subsequently, to
select for desired properties.

•
The problem with traditional plant breeding
is that it is time consuming and laborious;
it is difficult to modify single traits; and it
relies on existing genetic variability.

Modern Techniques for
Developing STPs


•
Transformation



-

Agrobacterium tumefaciens


-

Direct Gene Transfer Techniques (DGT)

Transformation

•
Steps using Genetic
Engineering

•
Using
Agrobacterium

as a
biological vector

OR

•
Using physical, electrical or
Chemical means of transfer
-
Direct Gene Transfer




Methods (DGT)

Transformation

•
Using
Agrobacterium





tumefaciens


A. tumefaciens

has been used
extensively for genetic
engineering of plants. This is
achieved by engineering selected
genes into the T
-
DNA of the
bacterial plasmid in laboratory
conditions so that they become
integrated into the plant
chromosomes when the T
-
DNA
is transferred.

Transformation
–

DGT techniques

•
Using physical, electrical or chemical
means.


-
Direct Gene Uptake by Protoplasts


-
Microinjection


-
Electroporation


-
Liposome Mediated DNA Delivery


-
Microprojectile Gun method

DGT Techniques

•
Direct Gene Uptake by Protoplasts


Protoplasts are cells without rigid cellulose walls.
It has been shown that plant protoplasts treated
with polyethylene glycol, commonly used to
induce protoplast fusion, will take up DNA from
their surrounding medium. More importantly, this
can then be stably integrated into the plant
chromosomal DNA.

DGT Techniques

•
Microinjection


A delivery system that involves the direct
injection of foreign DNA into plant cells using
minute needles. Microinjection of DNA into the
nuclei of isolate protoplasts could be an efficient
means of gene transfer.


DGT Techniques

•
Electroporation


This is a technique using electrical fields to make
protoplasts temporarily permeable to DNA, and offers an
effective alternative to vectors.


•
Liposome Mediated DNA Delivery


Liposomes are small artificial lipid vesicles prepared
from phosphatidyl choline and stearylamine by a process
known as reverse
-
phase evaporation. Nucleic acid
entrapped in such liposomes renders them highly
tolerant to attack by nucleases. Techniques for fusing
these liposomes to plant cell protoplast have been
evolved.

DGT Techniques

•
Microprojectile Gun method


To overcome the limitations of protoplast
regeneration, high velocity microprojectiles are
being used to deliver nucleic acids directly into
intact plant cells or tissues. In this method DNA
is coated on the surface of tungsten particles
which are projected by means of a particle gun
into intact cells or tissues. The particles can
penetrate through several layers of cells and can
transform cells within tissue/explants. Soybean,
tobacco, and maize have been transformed by
this method.

Applications of the STPs

•
Different approaches


•
Examples of improved plants


•
STPs for Phytoremediation

Different Approaches To
Improving Stress Tolerance

•
Several different approaches to improve
the stress tolerance of plants by foreign
gene transfer have been attempted. The
most consistently successful approach is
the introduction of genes encoding
enzymes that catalyse the conversion of a
naturally occurring substrate into a product
with osmoprotective properties.

Different Approaches To
Improving Stress Tolerance

Other important genes encode

:

•
Production of osmoprotective
compounds


•
Improved membrane flexibility


•
Stress
-
induced proteins

•
Scavenging reactive intermediates


•
Hypoxia
-

and anoxia
-
reducing proteins



Examples Of Foreign Genes
Expressed In Transgenic Plants

Improved Plants


•
Tobacco
-

Konstantinova et al




2002



Tobacco is a model culture for
biotechnology studies. It is a relatively
drought stress tolerant
plant.Konstantinova et al 2002 used
tobacco, which genes were already
proven to be involved in improving
abiotic stress tolerance, and developed
tolerance for low temperatures at early
growth stage.



Improved Plants

•
Arabidopsis

-

Yamaguchi
-
Shinozaki and Shinozaki 2001



Yamaguchi
-
Shinozaki and Shinozaki
showed that overexpression of the cDNA
encoding DREB1A in transgenic
Arabidopsis

plants activated the
expression of many of the stress
tolerance genes under normal growing
conditions and resulted in improved
tolerance to drought, salt loading and
freezing. As the DRE
-
related regulatory
element is not limited to
Arabidopsis

the
DREB1A cDNA and the rd29A promoter
may be useful for improving stress
tolerance of agriculturally important crops
by gene transfer.

Phytoremediation

•
Phytoremediation is a relatively
new approach to removing
contaminants from the
environment. It may be defined
as the use of plants to remove,
destroy or sequester hazardous
substances from the
environment. Unfortunately,
even plants that are relatively
tolerant of various environmental
contaminants often remain small
in the presence of the
contaminant (Glick, B.R. 2003).

Phytoremediation cont’d

•
Genetic modification of plants has been
useful in bio
-
remediation. Some plants
have been specially bio
-
engineered to
enable them remove toxic waste from the
environment. Several researchers have
reported encouraging results using plants
like mustard greens, alfalfa, river reeds,
poplar trees, and special weeds to clean
up the ravages of industries, agriculture,
and petroleum production

Example Of An Improved Plant
For Phytoremediation

•
Tomato plant genes used to increase metal
stress tolerance of Canola plants for
Phytoremediation (Nie, L. et al. 2002).




Transgenic tomato plants that express the Enterobacter
cloacae UW4 1
-
aminocyclopropane 1
-
carboxylate
(ACC) deaminase (EC 4.1.99.4) gene, and thereby
produce lower levels of thylene, were partially
protected from the deleterious effects of six different
metals.

Example of Improved Plants For
Phytoremediation cont’d


•
However, since tomato plants are unlikely to be utilized in the
phytoremediation of contaminated terrestrial sites, transgenic
canola (Brassica napus) plants that constitutively express the
same gene were generated and tested for their ability to
proliferate in the presence of high levels of arsenate in the soil
and to accumulate it in plant tissues.

•
In the presence of arsenate, in both the presence and absence of
the added plant growth
-
promoting bacterium, transgenic canola
plants grew to a significantly greater extent than non
-
transformed
canola plants

Implications

•
Present Outcomes


•
Human tolerance


•
Future Challenges

Present Outcomes

•
On the 12 March 2004 CIMMYT planted
for the first time transgenic drought
tolerant wheat and in field
-
like conditions
in Mexico

•
The wheat carries the DREB1A gene
from the plant Arabidopsis thaliana.

•
If the results are positive, there are major
implications for its use in other cereal
crops, such as rice, maize and barley.

Present Outcomes Cont’d

•
A comparison of DREB and
control wheat plants (DREB
plants on left, control on the
right), after 10 days without
water.

Human Tolerance Of Genetically
Modified Organisms

•
Although genetic modification of plants is
important and beneficial, it should be
adopted under conditions that avoid
potential risks.

•
Time and effort must be devoted to field
testing before the re
-
lease of any new
genetically engineered organism.


Human Tolerance Cont’d

•
The large agrobiotech companies should
establish measures to prevent movement
of transgenes from pollens to relatives of
GM crops or to weeds in nearby farms.

•
The public needs to be sufficiently
educated on genetic engineering of any
product to enhance acceptability.

The Future

•
Now it needs to be known how
plant roots sense environmental
stress and how stress signals
are transduced into altered gene
expression.

•
Plant hormones, such as ABA
and 1
-
aminocyclopropane
-
1
-
carboxylic acid (ACC), play
important roles, but their actions
are still not fully understood.

The Future cont’d

•
One approach for engineering extreme
stress tolerance may be to introduce
genes from different stress responses into
a single plant.

•
This could be achieved either by
transformation with multiple genes or by
crossbreeding plants containing different
stress
-
tolerance genes.

The Future cont’d

•
These are theoretically straightforward options,
but there may be severe perturbances to the
metabolic network of plants containing several
foreign enzymatic activities.

•
Thus, it is of paramount importance to target the
location, control the level and time of expression,
and ensure precursor availability for each
enzyme in order to avoid negative effects.

Summary

•
Stress Tolerant Plants are essential for
future food resources


•
New technology is making development of
Stress Tolerant Plants more possible


•
Public awareness of GM organisms needs
to be increased