Download Plant tissue culture

GENE 251/351
Plant tissue culture
Definitions
The sterile culture of plant cells, tissues or organs under conditions which lead to cell
multiplication or regeneration of organs or whole plants.
Tissue culture relies on three fundamental abilities of pla nts:
1- Totipotency is the potential or inherent capacity of a plant cell or tissue to develop into an
entire plant if suitably stimulated. Totipotency implies that all the information necessary for
growth and reproduction of the organism is contained in the cell. Although theoretically all plant
cells are totipotent the meristematic cells are best able to express it.
2- Dedifferentiation is the capacity of mature cells to return to meristematic condition and
development of a new growing point
3- Competency describes the endogenous potential of a given cell or tissue to develop in a
particular way. For example, as embryogenically competent cells are capable of developing into
fully functional embryos. The opposite is non-competent or morphogenetically incapable.
Types of in vitro culture:
1.
culture of intact plants
e.g. seed culture in orchids
2.
embryo culture
e.g. immature embryo culture
3.
organ culture
e.g. meristem culture,
shoot tip culture
root culture
anther culture
4.
callus culture
5.
single cell culture
6.
Protoplast culture.
Historical perspectives:
from theory of cell totipotency to molecular biotechnology
Advantages of tissue culture:
• genetically identical
• virus - indexed
• germplasm maintenance
• hybrid production for incompatible species
• haploid plants
• year round production
• difficult to propagate species
• New variants
• research tool
Disadvantages:
• requires expensive facilities
• particular skills are required
• error in maintenance of identity
• introduction of unknown pathogens or appearance of an unobserved mutant
Acram Taji - 2003
GENE 251/351
Aspects of pollination biology and in vitro seed set
1.
IN VITRO POLLINATION AND FERTILISATION
Success of in vitro pollination depends upon two basic considerations:
(i)
(ii)
correct stage of development of pollen grain and ovule
nutrient medium
Success of in vitro fertilisation (measured by production of viable seeds) is dependent upon
the following factors:
(i)
(ii)
(iii)
(iv)
(v)
age of the explants - particularly that of the ovule
adequate pollen germination
successful microsporogenesis
success in pollen tube entry into ovules
others
Application of in vitro pollination and fertilisation:
(i)
(ii)
(iii)
(iv)
2.
production of hybrids
overcoming sexual self-incompatibility (homomorphic incompatibility)
induction of haploid plants
research tool to study pollen physiology and fertilisation
EMBRYO CULTURE
The underlying principle of embryo rescue is the aseptic excision of the embryo and its transfer
to a suitable medium for development under optimum conditions.
Applications of embryo culture:
(i)
(ii)
(iii)
(iv)
(v)
(vi)
(vii)
overcoming embryo inviability
germination of seeds of obligatory parasite without the presence of host
overcoming seed dormancy
monoploid production in barley
prevention of embryo abortion with early ripening stone fruits
vegetative propagation
tool for research in experimental embryogenesis
Problem of embryo culture.
precocious germination
Acram Taji, 2003
GENE 251/351
Aspects of androgenesis
1.
METHODS OF HAPLOID PLANT PRODUCTION IN VIVO
(i)
Gynogenesis
Development from eggs penetrated by sperm but not fertilised.
(ii) Androgenesis
Development of a haploid individual from a pollen grain.
(iii) Genome elimination
Fertilisation occurs but soon afterwards one genome is eliminated.
(iv) Semigamy
In this case the nucleus of the egg-cell and the generative nucleus of the
germinated pollen grain divide independently,resulting in a haploid chimera.
(v) Chemical treatment
Chromosome elimination can be induced by addition of certain chemicals.
(vi) Shock treatment
With high or low temperatures.
(vii) Irradiation
With X-rays or UV light.
2.
METHOD OF HAPLOID PLANT PRODUCTION IN VITRO
Androgenesis is the production of haploid plants originating from very young pollen cell. In
this process, which virtually takes place in vitro, the vegetative or generative nucleus of a
pollen grain is stimulated to develop into a haploid individual without undergoing fertilisation.
3.
SUCCESS OF ANDROGENESIS INVOLVES TWO IMPORTANT
CONSIDERATIONS
(i)
(ii)
physiological state of the donor plant
the aptitude and the conditions necessary to modify the normal development
of the pollen and to force it towards embryogenesis. This embryogenesis
may be:
(a) direct
(b) indirect
4.
CONDITION OF DONOR PLANT
(i)
nutrition
(ii) light regime
(iii) watering
(iv) fungicide and pesticide application
(v) plant variety
5.
CONDITIONS AFFECTING ANDROGENESIS
(i)
(ii)
low temperature treatment
nutrition of medium
(iii)
(iv)
(v)
6.
REGENERATION OF DIPLOID PLANTS
(i)
(ii)
7.
major elements
minor elements
sugar
plant growth regulators
environmental conditions
liquid vs solid medium
light quality
activated charcoal
spontaneous doubling of chromosomes by endomitosis
treatment with colchicine
(a) at the time of the first pollen mitosis, just before the in vitro culture
(b) after the production of haloid plants in vitro
IMPORTANCE OF ANDROGENESIS
The time necessary to have an isogenic line is reduced from 5 or 6 generations to 1 or 2 using
the technique of androgenesis.
Acram Taji, 2003
______________________________________________________________________
Anther Squash Te chnique
To determine the stage of microspore (pollen) development within the anther it is necessary to
examine the microspores.
1.
Collect anthers from flower buds at various stages of development.
2.
Place anther on a clean slide, add a drop of 0.8% aceto-orcein stain in 40% acetic acid.
(Use1% orcein in 50% acetic acid or 1 g orcein in 50 mL glacial acetic acid. Boil in a
flask with a funnel on it, and immediately remove from heat. Cool; add 50 mL distilled
water. Stock: to 100 mL of preceding solution add 25 mL water).
3.
Gently macerate tissue with scalpel.
4.
Push large pieces of anther wall to the side using a scalpel.
5.
Gently heat slide, being careful not to scorch it. (Touch the slide to the back of your
hand to test how hot it is. If it is too hot for your hand, it is too hot!) As stain
evaporates, add more; continue for 5-10 minutes. This allows nuclear material to take
up stain.
6.
Let slide sit for 5-10 minutes.
7.
Place cover slip over material.
8.
Place slide on paper towel, and fold towel over slide; gently and evenly press cover
slip. Do not break cover slip.
9.
Observe under microscope.