Their Development, Uses and Implications

1
Stress Tolerant Plants

• Their Development, Uses and Implications

2
Introduction

• What Stresses Effect Plants?
• And
• Why there is a need for Stress Tolerant Plants
  (STPs)?

3
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
4
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
5
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.

6
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

7
The Development Of Stress Tolerant Plants

• Introduction To Previous Methods
• And
• Modern Techniques

8
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.

9
Modern Techniques for Developing STPs

• Transformation
• - Agrobacterium tumefaciens
• - Direct Gene Transfer Techniques (DGT)

10
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)

11
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.

12
Transformation DGT techniques

• Using physical, electrical or chemical means.
• -Direct Gene Uptake by Protoplasts
• -Microinjection
• -Electroporation
• -Liposome Mediated DNA Delivery
• -Microprojectile Gun method

13
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.

14
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.

15
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.

16
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.

17
Applications of the STPs

• Different approaches
• Examples of improved plants
• STPs for Phytoremediation

18
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.

19
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

20
Examples Of Foreign Genes Expressed In Transgenic
Plants
21
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.

22
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.

23
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).

24
Phytoremediation contd

• 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

25
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.

26
Example of Improved Plants For Phytoremediation
contd

• 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

27
Implications

• Present Outcomes
• Human tolerance
• Future Challenges

28
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.

29
Present Outcomes Contd

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

30
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.

31
Human Tolerance Contd

• 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.

32
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.

33
The Future contd

• 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.

34
The Future contd

• 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.

35
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