Introduction
plant cells re-differentiate |
plant tissues can be regenerated from explants |
Application
increase crop yield (developing countries) |
produce consistent yield quality (private at-home grower) |
produce exact replicas of species for profit (businesses) |
Plasticity
-plant's ability to adapt and cope with changes in the environment |
-plant's ability to different developmental pathways (alter their phenotype) in response to a particular stimuli/changes in the environment |
-alter its metabolism, growth, and development which suit the current environment the best |
Plant cells and tissues with high plasticity is needed for plant tissue culture. |
EXAMPLE: Fanwort (aquatic weed) |
1. feathery underwater leaves |
2. floating surface leaves |
-both leaf types are genetically identical cells, but the dissimilar environments cause certain genes involved in leaf formation to be expressed or unexpressed in different environments |
Plant Adaptation/Response
plastic structural responses to specific environment |
etc. growth of plant towards sunlight source and growth of root towards source of water |
morphological adaptations in specific environment |
etc. cactus's leaves are reduced to spines and a stem to reduce water lost in desert |
Fundamental Abilities of Plants
1. Totipotency |
potential of a cell/group of cells to develop into an entire organism if suitably stimulated |
2. Dedifferentiation |
development of differentiated explant into a undifferentiated callus (mature cells return to meristematic condition) |
3. Competency?? |
endogenous potential of a given cells or tissue to develop in a particular way |
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*Differentiation |
physiological and morphological changes that occur in a cell, tissue or organ during development |
*Meristematic Condition |
unmatured plant which does not have specific differentiated meristematic tissues (etc. apical meristem, lateral meristem) and all meristem tissues function for the 'simple growth' of the plants |
*Redifferentiation |
development of undifferentiated callus into planta differentiated |
Plant Regeneration Pathway Image
Plant Regeneration Pathway
1. Organogenesis |
-initiation and development of tissues and organ from cells which is not meristems |
a) Dedifferentiation |
-starts shortly after the isolation of explant |
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-rapid cell division and formation of undifferentiated cells (callus) |
b) Redifferentiation (budding) |
-starts after the first callus cell forms |
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-tissue named organ primordia is differentiated from callus cells |
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-organ primordia will give rise to small meristems (cells densely filled with protoplasm and strikingly large nuclei) |
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-different types of specialized cells will further differentiate |
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-vascular system formed will connect new organs with the parent explant/callus mass |
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2. Somatic Embryogenesis |
-dedifferentiation of plant somatic cell into totipotent embryonic stem cell then to differentiated embryos |
-embryonic stem cell need to have the ability to give rise to an embryo which can further develop into a whole new plant without sexual fertilization of zygotic embryos |
a) initiated directly |
from explants |
b) initiated indirectly |
from callus |
Process |
1) induction of embryogenic cultures from zygotic seed, leaf or stem segment |
2) further multiplication of embryos |
3) mature embryos are then cultured for germination and plantlet development |
4) transferred to soil |
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3. Histogenesis |
-differentiation of undifferentiated cells and their component cell types into specific tissues and organ |
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*Somatic embryos - embryos form from ordinary plant cells(2n) which normally are not involved in embryo development |
Plant Tissue Culture
collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition |
1. Appropriate tissue |
2. Sterile conditions with aseptic techniques |
3. Suitable growth medium |
Factors Affecting Plant Tissue Culture
1. Growth Media |
-minerals, growth factors, carbon source, hormones |
2. Environmental Factors |
-light, temperature, photoperiod, sterility, media |
3. Explant Source |
-usually, younger, less differentiated explant is a better explant source |
4. Genetics |
-different species show difference in amenability to tissue culture |
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-different genotype within a species will have variable response to tissue culture |
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Explant
-small pieces of plant parts or tissues that are aseptically cut from a matured plant and used to initiate a culture in a nutrient medium |
-almost all parts of plant are amenable to in vitro plant regeneration provided that they are able to dedifferentiate into totipotent cells |
-to grow, it require a nutrient medium consisting of mineral salts mixture, a carbon source, (usually sucrose) and vitamins |
-to initiate and maintain cell division, it need phytohormones (auxins and cytokines) in the nutrient medium |
-occasionally, to ensure the prolonged growth of the excised tissue to give an established callus, other organic supplements (amino acids or hexitols) is also needed |
correct choice of explant material can have an important effect on the success of a tissue culture experiment |
Explants used in Micropropagation
shoot tip |
leaf tip |
axillary bud |
shoot tip |
inflorescence segment |
nodal segment |
lateral bud |
flower stalk segment |
leaf base |
root tips |
Plant Explant Selection
-correct choice of explant material can have an important effect on the success of a tissue culture experiment |
1. Season in which the explant is obtained |
season of the year can affect on the contamination and response in culture |
2. Position/part of plant |
explants of various organs of a same parent plant vary in their rate of growth & regeneration |
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in certain plants some organs may be more regenerative than the others |
3. Quality of the source plant |
best to obtain explants from healthy plants compared to plants under nutritional or water stress or plants which are exhibiting disease symptoms |
4. Size of explant (commonly: 1-1.5 x 104 cells/ml) |
minimum inoculation size of explant varies according to the genotype of the plant being cultured and the cultural conditions |
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-large explants generally survive more frequently and grow more rapidly at the outset than very small ones |
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-large explants probably contain more nutrient reserves and plant growth regulators to sustain the culture |
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-smaller explant harder to culture where the medium of culture has to have additional components |
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-smaller explant increase the chance of virus elimination from subsequent cultures |
5. The purpose/ goal of the proposed culture |
choice of explant tissue will vary depending on what type of a response is desired from the cell culture |
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a) clonal propagation |
lateral or terminal shoot or bud |
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b) callus induction |
cotyledon, hypocotyl, stem, leaf, or embryo |
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c) protoplast isolation |
leaf tissue from aseptically germinated seed |
6. The kind of culture to be initiated |
choice of explant material also determines if the plantlets developed via tissue culture are |
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a) haploid/diploid |
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b) cell/organ |
7. Physiological condition/age of the explant source |
younger tissue is more responsive in vitro, usually the newest formed and is easier to surface disinfect and establish clean cultures |
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older tissue will not form callus that is capable of regeneration |
Micropropagation
practice of rapidly multiplying stock plant material to produce a large number of progeny plants, using modern plant tissue culture methods |
Advantage of Plant Tissue Culture
In plants prone to virus diseases, virus free explants (new meristem tissue is usually virus free) can be cultivated to provide virus free plants |
Plant “tissue banks” can be frozen, the regenerated through tissue culture |
Plant culture in approved media are easier to export than soil-grown plants, as they are pathogen free and take up little space (most current plant export is now done in this manner |
Tissue culture allows fast selections for crop improvement – explants are chosen from superior plants then cloned |
High degree of uniformity (true type plants) when compared to conventionally produced plants |
Disadvantage of Plant Tissue Culture
It is a labor intensive & expensive process. |
There is a chance that the propagated plants will be less resilient to diseases due to the type of environment they are grown in. |
It is imperative that, before being cultured, the material is screened; failure to pick up any abnormalities could lead to the new plants being infected. |
While the success rate is high if the correct procedures are followed, success with the tissue culture is not a guarantee. There is still a chance that the process triggers a secondary metabolic chemical reaction, and the new explants or cells' growth gets stunted, or even die off |
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