Plant Tissue Culture

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Transcript Plant Tissue Culture

Biotechnology Application
in Agriculture
By Parawita Dewanti
‘Green’ Biotechnology
• Green biotechnology is defined as the application of biological
techniques to plants with the aim of improving the nutritional
quality, quantity and production economics.
• The most recent application of biotechnology in respect to this
area is genetic modification (GM), also known as genetic
engineering, genetic manipulation, gene technology and/or
recombinant DNA technology.
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DNA, Chromosomes, Genoms
Plant Transformation
Modern Plant Breeding
Plant Tissue Culture
Molecular Marker
What is Plant Tissue Culture?
‘……the aseptic culture of plant protoplasts, cells,
tissues or organs under conditions which lead to
cell multiplication or regenation of organs or whole
plants
In Vitro Developmental Pathways
Explant:
• Piece of tissue put into culture
• Tissue selection depends on purpose,
species, many factors
• Pieces of organs: Leaves, Stems, Roots,
Cotyledons, Embryos, Other
• Specific cell types: Leaf tissue, Embryo,
Pollen, Endosperm, Nucellus
What for?
• Tissue culture produces clones, in which all
product cells have the same genotype
(unless affected by mutation during culture)
• A more recent advance is the use of [lant
and animal tissue culture along with
genetic modification using viral and
bacterial vectors and gene guns to create
genetically engineered organisms
First commercial use
• The first commercial use of plant clonal
propagation on artificial media was in the
germination and growth of orchid plants, in the
1920’s
• In the 1950’s and 60’s there was a great deal of
research, but it was only after the development of
a reliable artificialmedium (Murashige & Skoog,
1962) that plant tissue culture really ‘took off’
commercially
Young cymbidium orchid
What is needed?
Tissue culture has several critical requirements:
• Appropriate tissue (some tissues culture better
than others)
• A suitable growth medium containing energy
sources and inorganic salts to supply cell growth
needs.This can be liquid or semisolid
• Aseptic (steril) conditions, as microorganisms
grow much more quickly than plant and animal
tissue and can over run a culture
• Growth regulators-in plants, both auxins &
cytokinins
• Frequent subculturing to ensure adequate
nutrition and to avoid the build up of waste
metabolites
Culturing Plant Tissue-the steps
• Selection of the plant tissue (explant) from a
healthy vigorous ‘mother plant’- thisis often the
apical bud, but can be other tissue
• This tissue must be sterilized to remove
microbial contaminants
A
B
Elimination of microbial contaminants
Surface contaminants, principally microbial
saprophytesthat are eliminated by surface
disinfestation
Internal contaminants, principally pathogens that
are eliminated by thermotherapy (35-40oC) and
culture of explants free of organisms or by
antibiotics
Maintenance of asepsis (free from
microorganisms), during excision and
culture,procedures are carried out in sterilelaminar
flow positive pressure hoods (0.3 µm filters)
Culture Medium constituents
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Inorganic salt formulation
Source of carbohydrate
Vitamins
Water
Plant hormones-auxin, cytokinin,
GA’s
• Solidifying agents
Composition of tissue
culturemedium is complex
Two Hormones affect Plant Differentiation:
• Auxin: Stimulates Root Development
• Cytokinin: Stimulates Shoot
Development
Generally, the ratio of these two hormones
can determine plant development:
Effect of different auxine and cytokinine
concentration on tissue development
Success Story of Transgenic crops
Important Traits for Crop Improvement
• High crop yield
• High nutritional quality
• Abiotic stress tolerance
• Pest resistance
• Adaptation to inter-cropping
• Nitrogen Fixation
• Insensitivity to photoperiod
• Elimination of toxic compounds
Requirements for plant transformation
DNA delivery systems must be
- simple,
- efficient and
- preferably inexpensive.
The method must be available for use either because it is in
the public domain or because it can be licensed.
One’s system of choice depends on
- the target plant,
- its regeneration system,
- the position or affiliation of the researcher.
Elements of transformation
A. Uptake of DNA into competent host cells
 Integration
 Transient selection
Requirements for plant transformation
A. Cell culture and plant regeneration system
B. Cloned DNA to be introduced
1. selectable marker gene
2. promoter (constituitive or inducible),
coding region, polyadenylation site
C. Method of delivery of DNA into the cell
D. Proof of transformation of plant
Method of Delivery of DNA Into the Cell
1. Agrobacterium
-mediated transformation
Agrobacterium tumefaciens
Agrobacterium rhizogenes (Hairy Root)
- cultured cells, wounded explants, vacuum infiltration
2. Direct
methods
-protoplast polyethyleneglycol (PEG) method
-protoplast electroporation
-silicon carbide fibers
-protoplast microinjection
-particle bombardment
Overview of the Infection Process
The big five successful
traits
 Insect Resistance
 Delayed Fruit Ripening
 Nutritional Enhancing
 Herbicide Resistance
 Virus Resistance
Roundup Ready™ Soybeans
A problem in agriculture is the reduced growth of crops
imposed by the presence of unwanted weeds. Herbicides such
as RoundupTM and Liberty LinkTM are able to kill a wide range
of weeds and have the advantage of breaking down easily.
Development of herbicide resistant crops allows the
elimination of surrounding weeds without harm to the crops.
Insect Resistance
Various insect resistant crops have been produced. Most of
these make use of the Cry gene in the bacteria Bacillus
thuringiensis (Bt); this gene directs the production of a protein
that causes paralysis and death to many insects.
Corn hybrid with a Bt gene
Corn hybrid susceptible to European
corn borer
Virus resistance
a) Plants may be engineered with genes for resistance to viruses,
bacteria, and fungi.
b) Virus-resistant plants have a viral protein coat gene that is
overproduced, preventing the virus from reproducing in the host
cell, because the plant shuts off the virus’ protein coat gene in
response to the overproduction.
c) Coat protein genes are involved in resistance to diseases such as
cucumber mosaic virus, tobacco rattle virus, and potato virus X.
Delayed Fruit Ripening
a) Allow for crops, such as tomatoes, to have a higher shelf
life.
b) Tomatoes generally ripen and become soft during
shipment to a store.
c) Tomatoes are usually picked and sprayed with the plant
hormone ethylene to induce ripening, although this does
not improve taste
d) Tomatoes have been engineered to produce less ethylene
so they can develop more taste before ripening, and
shipment to markets
First biotech plant product – Flav’r Sav’r
tomato
“Rot-Resistant Tomato”
Anti-sense gene  complementary to polygalacturonase (PG)
PG = pectinase  accelerates plant decay/rotting
Nutritionally Enhanced
Plants
1.More than one third of the world’s population relies on
rice as a food staple, so rice is an attractive target for
enhancement.
2.Golden Rice was genetically engineered to produce high
levels of beta-carotene, which is a precursor to vitamin
A. Vitamin A is needed for proper eyesight.
3.Other enhanced crops include iron-enriched rice and
tomatoes with three times the normal amount of betacarotene
Golden Rice
Normal rice
Transgenic technology produced a type of rice that
accumulates beta-carotene in rice grains. Once inside the body,
beta-carotene is converted to vitamin A.
“Normal” rice
“Golden” rice
Molecular Farming
1.A new field where plants and animals are genetically
engineered to produce important pharmaceuticals, vaccines,
and other valuable compounds.
2.Plants may possibly be used as bioreactors to mass-produce
chemicals that can accumulate within the cells until they are
harvested.
3.Soybeans have been used to produce monoclonal antibodies
with therapeutic value for the treatment of colon cancer.
Drugs can also be produced in rice, corn, and tobacco
plants
4.Plants have been engineered to produce human antibodies
against HIV. Pharmaceuticals has begun clinical trials with
herpes antibodies produced in plants.
Why Plants?
Plants offer unique benefits for the
production of pharmaceutical proteins:
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Ease of Scale-up
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Low Cost
Reduced Capital Expenditures
No Animal Contaminants (virus)
Molecular Farming
(Vaccines)
a) Making plants that produce vaccines
b) Potatoes have been studied using a portion of the E.
coli enterotoxin in mice and humans.
c) Other candidates for edible vaccines include banana
and tomato, and alfalfa, corn, and wheat are possible
candidates for use in livestock.
d) Edible vaccines may lead to the eradication of
diseases such as hepatitis B and polio.
Pharmaceutical Production in Plants
Genetically modified plants have been used as “bioreactors” to produce
therapeutic proteins for more than a decade. A recent contribution by
transgenic plants is the generation of edible vaccines.
Edible vaccines are vaccines produced in plants that can be administered
directly through the ingestion of plant materials containing the vaccine. Eating
the plant would then confer immunity against diseases.
Edible vaccines produced by transgenic plants are
attractive for many reasons. The cost associated
with the production of the vaccine is low, especially
since the vaccine can be ingested directly, and
vaccine production can be rapidly up scaled should
the need arises. Edible vaccine is likely to reach
more individuals in developing countries.
The first human clinical trial took place in 1997.
Vaccine against the toxin from the bacteria E.coli
was produced in potato. Ingestion of this transgenic
potato resulted in satisfactory vaccinations and no
adverse effects.
Edible Vaccines
One focus of current vaccine effort is on hepatitis B, a virus responsible
for causing chromic liver disease. Transgenic tobacco and potatoes were
engineered to express hepatitis B virus vaccine. During the past two years,
vaccines against a E.coli toxin, the respiratory syncytial virus, measles virus,
and the Norwalk virus have been successfully expressed in plants and
delivered orally. These studies have supported the potential of edible
vaccines as preventive agents of many diseases.
There is hope to produce edible vaccines in bananas, which are grown
extensively throughout the developing world.
Molecular Farming
(Biopolymers and Plants)
a) Plant seeds may be a potential source for plastics
that could be produced and easily extracted.
b) A type of PHA (polyhydroxylalkanoate) polymer
called “poly-beta-hydroxybutyrate”, or PHB, is
produced in Arabidopsis, or mustard plant.
c) PHB can be made in canola seeds by the transfer
of three genes from the bacterium Alicaligenes
eutrophus, which codes for enzymes in the PHB
synthesis pathway.
d) A polymer called PHBV produced through
Alicaligenes fermentation, which is sold under the
name Biopol.
The outline of the experiment Agrobacterium-mediated transformation.
Harbour construc SoSPS1 gene
Explants :
Agrobacterium
Infection
28oC
for 30
min
cokultivasi
pSMAB-SoSPS1
Selection media
-
Analysis PCR
Analysis SPS activity
Analysis sucrosa
Analysis Western Blot
Regeneration
media
GERMINATION
The surface-sterilized tomato seeds are placed in a petri
dish with nutrient medium for germination
CUT EXPLANTS
After 7 days, the tomato seedings are at the stage where
the cotyledons (explants) can be cut for use in
transformation.
CUT EXPLANTS
The cotyledon explants are cut from the seedlings in liquid MS.
The wounded cells made by cutting will be the site of DNA transfer
from the Agrobacterium.
EXPOSE TO AGROBACTERIUM
After overnight preconditioning on the feeder plates, the explants
are competent for transformation and are exposed to the
agrobacterium.
BLOT EXCESS AGROBACTERIUM
Excess agrobacterium is blotted from the explants on filter paper to
avoid excess growth of the bacteria during co-cultivation.
DNA TRANSFER
The explants are co-cultivated with the agrobacterium for 48 hours
to allow time for the agrobacterium to transfer the engineered DNA
to the wounded plant cells.
TOMATO PLANT REGENERATION
The explants are placed on a medium containing antibiotics for
selection and control of the Agrobacterium. The medium also
contains the growth regulator, zeatin riboside.
Here the formation of an initial callus can be seen on the
explant. The callus occurs at the site of wounding and is a result
of stimulated plant cell growth caused by the growth regulator,
zeatin riboside.
Time point: 3 weeks
SHOOTS BEGIN
After 6 weeks the cotyledon explants are cut from the calli and
discarded.
Shoots are beginning to form and are transferred to fresh MSZ media.
ROOTING MEDIA
The tomato shoots have regenerated from the calli and are ready to be
transferred to rooting media. This media lacks zeatin, but contains
kanamycin for selection of transformants.
Time point: 9 weeks
TRANSGENIC PLANTS
These are fully differentiated transgenic tomato plants rooting
in MS media with kanamycin.
Time point: 11 weeks
TRANSFER TO SOIL
Rooted plantlets are then transferred to boxes containing soil.
The plants must be acclimated to the air, or "hardened off".
The process takes 4-5 days.
Time point: 13 weeks
TO THE GREENHOUSE
These transgenic plants are ready for transfer to the greenhouse.
Shown are two cultivars of tomato; Moneymaker (left), and Motelle
(right). Both contain engineered genes.
Time point: 15 weeks
A GREENHOUSE OF TRANSGENIC PLANTS.
Plants and growth conditions are carefully monitored and controlled.
The plants are used for analysis, seed production, or are
transplanted to the field.
Transgenic tomato with wheat germin
two days after inoculation
with the Sclerotonia
Transgenic
Wild-type
Plant enhanced with vitamins
Gene engineering is a possibility
to add to a plant a totally new traits
that are not characteristic for plant at all
Vaccines are being produced in bananas
BetaSweet® carrot
Contains approximately 50% more
Beta Carotene than normal carrot
Beta carotene
is a potent cancer-fighting antioxidant.
dark maroon-purple color
(as also anthocyanine (another antioxidant) is added)
taste similar to regular carrots,
but have a very crispy texture,
which is easier to chew
Produced by Texas A&M University.
“Golden rice”
Rice normally do not produces vitamine A.
On the other hand,
rice endosperm anyway contains
geranylgeranyl diphosphate (GGDP)
(progenitor of vitamine A)
GGDP  vitamin-A
Unfortunately,
production is too low.
2 genes from daffodils
1 gene from bacterium Erwinia uredovora
All 3 genes are expressed in endosperm
(major part of the rice grain)
Normal serving of rice (300 g)
provides just a few percent
of daily diet
Anyway, Syngenta supports the humanitarian use of golden rice,
for individuals receiving $10,000 or less income from developing country
Geniered Eternal Flowers
Made by antisense disruption of ACC synthase
Antisense gene is expressed in the plant
8 days after ethylene treatement.
9 days after pollination
transgenic
Non-transgenic
non-transgenic
transgenic
Transgenic long life carnation
Produced by Florigene Pty Ltd
Melbourn –based company
Long lived flower by itself
No need in expensive refrigeration;
No need in silver thiosulfate (STS) or
EthylBlock compaund
The growth condition of explant at 5 cycle in selection medium
containing 50 ppm kanamycin or 5 ppm phosphinotricyn and 500
ppm cefotaxime
A
B
C
D
E
A
A
B
C
D
E
B
Representing the growth of explants on 5 selection cycle MS medium
containing 50 mgl-1 kanamycin (A) and 5mgl-1 phosphinotricyn (B).
Each cycle for 3 weeks incubation
Tearless Onion
Colorful Cauliflowers
Purple tomatoes
Blue Roses