Micro-propagation | Online Biotech Notes

 

Micro-propagation

#Plant Biotech    #GATE     #MIcropropagation      # Micrografting

• In-vitro clonal propagation through tissue culture is mentioned as micro-propagation. The use of the tissue culture technique for micropropagation was first started by Morel (1960) for the propagation of orchids
• A tissue culture technique for plant propagation during which tissue is taken from a plant and grown during a laboratory to supply plantlets that are genetically just like the parent.
• The different techniques of a single cell and protoplast culture also enable a thousand plants to be derived within a short space and time. 

Methods of Micro-Propagation

• Shoot/Meristem-tip culture

• Single node culture

• Axillary bud culture

• Micro grafting

Shoot/Meristem-tip culture

Shoot tip culture could also be described because of the culture of terminal (0.1-1.0 mm) portion of a shoot comprising the meristem (0.05-0.1 mm) alongside primordial and developing leaves and adjacent stem tissue.

Shoot and Meristem Culture

Single node culture

• This is a natural method for the vegetative propagation of plants in both in vivo and in vitro conditions.
• The bud found within the axil of the leaf is like the stem tip, for its ability in micropropagation. A bud alongside a bit of stem is isolated and cultured to become a plantlet.
• Closed buds are wont to reduce the probabilities of infections.

Axillary Bud Culture

• In this method, a shoot tip along the side axillary bud is isolated. The cultures are administered with high cytokinin concentration. As a result of this, apical dominance stops,  and axillary buds develop.

• For an honest axillary bud culture, the cytokinin/ auxin ratio is around 10: 1. This is, however, variable and depends on the character of the plant species and therefore the developmental stage of the explant used. In general, juvenile explants require less cytokinin compared to adult explants. 

Axiallary bud Culture

Micro grafting

Micro-grafting is an in vitro grafting technique that involves the location of a meristem or shoots tip explant onto a decapitated rootstock that has been grown aseptically from seed or micropropagation cultures.

In vitro micrografting has been used for the improvement and multiplication of fruit trees because the technique has the potential to mix the benefits of rapid in vitro multiplication with the increased productivity that results from grafting superior rootstock and scion combinations.

Successful micrografting protocols are developed for various fruit crops including almond, apple, cherry, chestnut, Citrus, grapes, mulberry, olive, peach, pear, walnut, etc.

 It has been used on a commercial scale for the production of virus-free plants in fruit crops and viroid-free plants in Citrus.

Micrografting has also been utilized in the prediction of incompatibility between the grafting partners, histological studies, disease indexing, production of disease-free plants particularly immune to soil-borne pathogens, and multiplication of difficult to root plants.

Stages of Micro-Propagation

There are four stages to micro-propagation

1. Stage I Establishment

1. Stage II Multiplication

1. Stage III Rooting

1. Stage IV Acclimatization

Stage I - Establishment 

• During the establishment stage, the explant must be disinfested and stabilized.
• The explant is typically sterilized with a mixture of detergent and bleach. In difficult situations, alcohol or a fungicide may be used.
• The objective of this stage is to urge clean cultures which will begin the method of shoot multiplication.

Stage II Multiplication

• The objective of the shoot multiplication stage is to extend the number of shoots produced by the first explant.
• By subculturing these new shoots on to new medium, the amount of shoots produced in culture increases dramatically.
• A high cytokinin to auxin ratio is employed during the multiplication stage to induce axillary or adventitious shoot formation.
• Too high a degree of cytokinin will end in a high number of adventitious shoots that don't elongate.
• Common cytokinins utilized in culture are benzyl adenine and kinetin.

Stage III Rooting

• In the rooting stage, micro cuttings are induced to make roots - usually by application of auxin.
• In general, species root easier in tissue culture than they are doing from conventional cuttings.
• Micro cuttings can either be rooted in vitro or ex-vitro.
• In general, micro cuttings rooted ex vitro have a more normal rootage and acclimatize to a traditional growing environment better than cuttings rooted in vitro.

Stage IV Acclimatization

• Finally, after roots became well established on the microcutting, plantlets must be acclimatized to a traditional growing environment in Stage IV.
• This involves gradually moving to open-air conditions where the humidity is reduced and therefore the light levels increased.
• This is a vulnerable stage for plantlet survival where the propagator can see large losses without proper acclimatization.

Factors Affecting Micro-propagation 

1. Genotype of the plant:

Selection of the right genotype of the plant species (by screening) is necessary for improved micro propagation. In general, plants with vigorous germination and branching capacity are more suitable for micro- propagation.

2. Physiological status of the explants:

Explants from more recently produced parts of plants are more effective than those from older regions. Good knowledge of donor plants’ natural propagation process with special reference to growth stage and seasonal influence will be useful in selecting explants.

3. Culture media:

The standard plant tissue culture media are suitable for micro propagation during stage I and stage II. However, for stage III, certain modifications are required. Addition of growth regulators (auxins and cytokinins) and alterations in mineral composition are required. This is largely dependent on the type of culture (meristem, bud etc.).

4. Culture environment:

• Light:

The photosynthetic pigment in cultured tissues does absorb light and thus influence micropropagation. The quality of sunshine is additionally known to influence in vitro growth of shoots, e.g blue light-induced bud formation in tobacco shoots. Variations in diurnal illumination also influence micropropagation. In general, an illumination of 16 hours a day and eight hours night is satisfactory for shoot proliferation.

• Temperature:

The Majority of the culture for micropropagation requires an optimal temperature of around 25°C. There are some exceptions e.g. Begonia X Cheimantha hybrid tissue grows at a coffee temperature (around 18°C).

• Composition of gas phase:

The constitution of the gas phase in the culture vessels also influences micropropagation. Unorganized growth of cells is generally promoted by ethylene, O2, CO2 ethanol and acetaldehyde.

Advantages of Micro-Propagation

1. The technique of micropropagation is an alternative approach to conventional methods of vegetative propagation, which has an enhanced rate of multiplication.
2. A million of shoot tips can be obtained from a small, microscopic piece of plant tissue within a short period of time and space.
3. The advantage in this type of propagation is that shoot multiplication usually has a short cycle (2-6 weeks) and each cycle results in a logarithmic increase in the number of shoots.
4. This method of propagation is more advantageous in the case of bulb or corm pro­ducing plants.
5. The smaller size of propagules is advantageous for storing and transporting as it takes lesser space.
6. The propagules can be maintained in the soil-free environment which facilitates their storage on a large scale.
7. Stocks of germplasm can be maintained for many years using this method of propagation.
8. This method is more applicable where disease-free propagules are wanted. This in vitro technique helps to raise pathogen-free plants and to maintain them.
9. Micro-propagation is very useful in the case of dioecious plants because there the seed progeny yield is 50% male and 50% female, but this technique helps to get the progeny according to the desired sex.
10. A major advantage of micro-propagation happens to be the minimum growing space required in commercial nurseries. Thousands to millions of plantlets can be maintained within the culture vials. This is especially useful for maintaining the horticultural species.
11. This method is more helpful in the case of slow-growing plants where the seeds are produced after a long term and the seeds are the only propagule. This method can overcome the difficulty to obtain the propagule
12. Through seed production, the genetically uniform progeny is not possible always. But the micropropagation method will help to maintain the genetic uniformity in the propagules.

Disadvantages of Micro-Propagation

1. It is very expensive and can have a labor cost of more than 70%.
2. A monoculture is produced after micropropagation, leading to a lack of overall disease resilience, as all progeny plants may be vulnerable to the same infections.
3. An infected plant sample can produce infected progeny. This is uncommon as the stock plants are carefully screened and vetted to prevent culturing plants infected with virus or fungus.
4. Not all plants can be successfully tissue-cultured, often because the proper medium for growth is not known or the plants produce secondary metabolic chemicals that stunt or kill the explant.
5. Sometimes plants or cultivars do not come true to type after being tissue cultured. This is often dependent on the type of explant material utilized during the initiation phase or the result of the age of the cell or propagule line.
6. Some plants are very difficult to disinfect of the fungal organism

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