Application
1. The commercial production of plants which uses meristem & shoot culture to produce large numbers of identical individuals. |
2. To conserve rare or endangered plant species. |
3. A plant breeder may use tissue culture to screen cells rather than plants for advantageous characters. |
4. Large scale growth of plants in liquid culture in bioreactors for the production of valuable compounds. |
5. To cross distantly related species by protoplast fusion & regeneration of the novel hybrid. |
Types of In Vitro Culture (explant based)
1. Culture of intact plants (seed and seedling culture) |
2. Embryo culture (immature embryo culture) |
3. Organ culture (Hairy Root Culture, Shoot tip and meristem culture) |
4. Callus culture |
5. Cell suspension culture |
6. Protoplast culture |
Types of In Vitro Culture
1. Seed Culture
-growing seed aseptically in vitro on artificial media |
-Use: |
->Increasing efficiency of germination of seeds that are difficult to germinate in vivo |
->Precocious germination by application of plant growth regulators |
->Production of clean seedlings for explants or meristem culture |
->In vitro selection |
2. Embryo Culture
-growing embryo aseptically in vitro on artificial nutrient media |
-Use: |
->Rescue embryos (embryo rescue) from wide crosses where fertilization occurred, but embryo development did not occur |
->Production of plants from embryos developed by non-sexual methods (haploid production) |
->Overcoming embryo abortion due to incompatibility barriers |
->Overcoming seed dormancy and self-sterility of seeds |
->Shortening of breeding cycle |
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The advantages of growing an embryo isolated from the rest of the seed |
-To remove the immature plant from the endosperm and/or cotyledon(s) which may in particular cases prevent or modify the development of the plant. |
-As a means of propagating species which resist attempts to use standard methods of vegetative propagation. |
-Rescue of weak/ aborting embryos resulting from breeding/ crossing process |
3. Organ Culture
-Any plant organ can serve as an explant to initiate cultures |
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Organ |
Culture Types |
Shoot |
a) Shoot tip culture |
Root |
b) Root culture |
Leaf |
c) Leaf culture |
Flower |
d) Anther/ovary culture |
3a) Shoot Apical Meristem Culture
-Shoot tip can be cultured in vitro, producing clumps of shoots from either axillary or adventitious buds. |
-This method can be used for clonal propagation |
-Shoot meristem cultures are potential alternatives methods for cereal regeneration as they are less genotype-dependent and more efficient |
-Use: |
1. Production of virus free germplasm |
2. Mass production of desirable genotypes |
3. Facilitation of exchange between locations (production of clean material) |
4. Cryopreservation (cold storage) or in vitro conservation of germplasm |
3b) Root Organ Culture
-can be established in vitro from explants of the root tip of either primary or lateral roots and can be cultured on fairly simple media |
-growth of roots in vitro is potentially unlimited, as roots are indeterminate organs |
-Use: |
1. Production secondary metabolites |
2. Study the physiology and metabolism of roots, and primary root to determinate growth patterns |
3d) Anther/Ovary culture
-production of haploid plants |
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Ovary/Ovule Culture |
-Use: |
1. Production of haploid plants |
2. A common explant for the initiation of somatic embryogenic cultures |
3. Overcoming abortion of embryos of wide hybrids at very early stages of development due to incompatibility barriers |
4. In vitro fertilization for the production of distant hybrids avoiding style and stigmatic incompatibility that inhibits pollen germination and pollen tube growth |
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Anther and Microspore Culture |
1. Production of haploid plants |
2. Production of homozygous diploid lines through chromosome doubling, thus reducing the time required to produce inbred lines |
3. Uncovering mutations or recessive phenotypes |
4. Callus Culture
-un-organized mass of cells |
-tissue that develops in response to injury caused by physical or chemical means |
-Most cells of which are differentiated although may be and are often highly unorganized within the tissue |
Culturing Callus |
-Production of plantlets through somatic embryogenesis or organogenesis |
-Secondary metabolites production |
-Any plant tissue can be used as an explant |
-often performed in the dark as light can encourage differentiation of the callus |
-During long-term culture, the culture may lose the requirement for auxin and/or cytokinin - ‘habituation’ - common in callus cultures |
-Manipulation of the auxin to cytokinin ratio in the medium can lead to the development of shoots, roots or somatic embryos |
-Callus cultures can also be used to initiate cell suspensions |
Development of Callus |
-The proliferation can be maintained more or less indefinitely, if the callus is subcultured on to fresh medium periodically |
-Callus is usually composed of unspecialized parenchyma cells |
-During callus formation there is some degree of dedifferentiation |
-i.e. the changes that occur during development and specialization are, to some extent, reversed, both in morphology and metabolism |
-One major consequence of dedifferentiation: most plant cultures lose the ability to photosynthesize |
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Two categories of callus: |
a) Compact callus: the cells are densely aggregated |
b) Friable callus: the cells are only loosely associated with each other and the callus becomes soft and breaks apart easily |
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The friability of callus can be improved by: |
a) manipulating the medium components |
b) repeated subculturing |
c) culturing it on ‘semi-solid’ medium (medium with a low concentration of gelling agent) |
5. Cell Suspension Culture
-When callus pieces are agitated in a liquid medium, they tend to break up. |
-Suspensions are much easier to bulk up than callus since there is no manual transfer or solid support. |
-Friable callus provides the inoculum to form cell-suspension cultures |
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Growing of cell suspension from friable callus |
->liquid medium |
->agitated |
->single cells and/or small clumps of cells are released |
->continue to grow and divide |
->cell-suspension culture |
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Criteria in growing cell suspension from friable callus: |
-A relatively large inoculum should be used when initiating cell suspensions so that the released cell numbers build up quickly. |
-The inoculum should not be too large though, as toxic products released from damaged or stressed cells can build up to lethal levels. |
-Cell suspensions can be maintained relatively simply as batch cultures in conical flasks. |
-The degree of dilution during subculture should be determined empirically for each culture. |
-Too great a degree of dilution will result in a greatly extended lag period or, in extreme cases, death of the transferred cells. |
-After subculture, cells divide and culture biomass increases in a characteristic fashion, until nutrients in the medium are exhausted and/or toxic by-products build up to inhibitory levels ->‘stationary phase’ |
-If cells are left in the stationary phase for too long, they will die and the culture will be lost |
-cells should be transferred as they enter the stationary phase |
-important to determine batch growth-cycle parameters for each cell-suspension culture |
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Advantage |
1. it can ultimately provide a continuous, reliable source of natural products. |
2. synthesis of bioactive secondary metabolites |
3. running in controlled environment |
4. independently from climate and soil conditions |
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Importance and Application of cell suspension culture as an experimental technique |
-Contribute information about cell physiology, biochemistry, metabolic events at the level of individual cells and small cell aggregates |
-Develop understanding of an organ formation or embryoid formation starting from single cell or small cell aggregates |
-Suspension culture derived from medicinally important plants can be studied for the production of secondary metabolites |
-Mutagenesis studies may be facilitated by the use of cell suspension cultures to produce mutant cell clones from which mutant plants can be raised |
Introduction into Suspension
6. Protoplast Culture
-The living material of a plant or bacterial cell, including the protoplasm and plasma membrane after the cell wall has been removed. |
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Somatic Hybridization |
-development of hybrid plants through the fusion of somatic protoplasts of two different plant species/varieties |
Process: |
1. Isolation of protoplast |
2. Fusion of the protoplasts of desired species/varieties |
3. Identification and Selection of somatic hybrid cells |
4. Culture of the hybrid cells |
5. Regeneration of hybrid plants |
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How to make a protoplast? |
-Two general approaches to removing the cell wall (without damaging the protoplast): |
a) Mechanical isolation: although possible, often results in low yields, poor quality and poor performance in culture due to substances released from damaged cells |
b) Enzymatic isolation: usually carried out in a simple salt solution (with a high osmoticum) + cell wall degrading enzymes. |
-It is usual to use both cellulase and pectinase enzymes (must be of high quality and purity) |
-Protoplasts are fragile and easily damaged -> must be cultured carefully. |
-Liquid medium is not agitated and a high osmotic potential is maintained, at least in the initial stages. |
-The liquid medium must be shallow enough to allow aeration in the absence of agitation |
-Protoplasts can be plated out on to solid medium and callus produced |
-Whole plants can be regenerated by organogenesis or somatic embryogenesis from this callus |
-Protoplasts are ideal targets for transformation by a variety of means |
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Uses for Protoplast Fusion: |
1. Combine two complete genomes |
->another way to create allopolyploids |
2. In vitro fertilization |
3. Partial genome transfer |
->Exchange single or few traits between species |
->May or may not require ionizing radiation |
4. Genetic engineering |
->Micro-injection, electroporation, Agrobacterium |
5. Transfer of organelles |
->Unique to protoplast fusion |
->The transfer of mitochondria and/or chloroplasts between species |
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Plant Genetic Engineering
Genetic Transformation |
-Introduction of foreign DNA to generate novel genetic combinations. |
-Transfer of desirable genes for disease and pest resistance from related or unrelated plant species into high yielding susceptible cultivars. |
-Study of structure and function of genes. |
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Purposes of Introducing Novel Traits |
1. Biodiversity screening: To look for new traits in other or wild population |
2. Management of germplasm: Propagation of elite germplasm through micropropagation |
3. Interspecific or intergeneric crosses: Introduction of traits from other genus or species |
4. Overcome crossing barrier: Use protoplast fusion or somatic hybridization to produce hybrids |
5. Genetic engineering: Introduce genes from the same species, distantly related species or unrelated species (e.g. bacteria into plants) |
6. Incorporation into plant breeding: Use selected inbreeds for gene transformation |
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Creation of Novel Traits |
Somaclonal variation |
-Variation arise in culture (especially those undergone long period of culturing) due to genetic or epigenetic mutation |
Mutagenesis |
-Treatment of cultures with mutagens, such as ethyl methane sulfonate (EMS), UV and radioactive radiations |
-Dosage of mutagens can be vital, sub-vital or sub-lethal |
-Mild mutation - opt for vital |
-Heavy mutation – opt for subvital to sublethal |
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Purposes of Creating Novel Traits |
Propagate high yielders |
-Micropropagation of high yielders could increase yield |
-This could be a strategy until full inbreeds are produced |
Disease susceptibility |
-Reliance on one clone is dangerous to food security, since disease susceptibility could decimate the entire crop |
-At least a few clones of the high yielders must be grown in a field and best if they are separated by rows |
Plant Genetic Engineering Process
Germplasm Conservation
-Germplasm is living tissue from which new plants can be grown |
(google)-Germplasm is the term used to describe the seeds, plants, or plant parts useful in crop breeding, research, and conservation efforts. |
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An extension of micropropagation techniques through two methods: |
-Slow growth techniques e.g.: ↓ Temp., ↓ Light, media supplements ( growth retardants). |
-Medium-term storage (1 to 4 years) |
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Cryopreservation |
-Ultra low temperatures in liquid nitrogen at -196°C. |
-Stops cell division & metabolic processes |
-Very long-term (indefinite) |
Pros and Cons of Plant Tissue Culture
Advantages |
Disadvantages |
1. In plants prone to virus diseases, virus free explants (new meristem tissue is usually virus free) can be cultivated to provide virus free plants |
1. It is a labor intensive & expensive process. |
2. Plant “tissue banks” can be frozen, the regenerated through tissue culture |
2. Not all plants can be successfully tissue cultured - it is usually because the medium of growth is not known. |
3. 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 |
4. Tissue culture allows fast selections for crop improvement – explants are chosen from superior plants then cloned |
5. High degree of uniformity (true type plants) when compared to conventionally produced plants |
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