Plant Tissue Culture

Tissue culture could be defined as the method of ‘in Vitro’ culture of plant or animal cells, tissue, or organ on nutrient medium under aseptic conditions usually in a glass container. It is also sometimes referred to as ‘sterile culture’ or ‘in vitro culture’. The culture media is provided with water, minerals, vitamins, hormones, carbon sources, and certain antibiotics depending upon the plant being cultured. It should be noted that most plant cells are totipotent and scientists use this characteristic to manipulate plant cells by genetic engineering to regenerate an entire plant. Tissue culture techniques are used to generate large numbers of genetically identical plants for agricultural applications and also grow rare plants.

Nutritional Requirements of Plant Tissue Culture

The culture media for plant tissue culture consists of various nutritional components to sustain the plant’s growth. Different plants do need different media, however, specific media have been devised for specific tissue and organs. Some of the important media are:

• White’s Medium

• MS (Murashige and Skoog) Medium

• B5 (Gamborg’s) Medium

• LS (Linsmaier and Skoog) Medium

Some of the Organic Nutritional Components are:

• Vitamins like thiamine (B1), Pyridoxin (B6), Nicotinic Acid (B3), etc.

• Antibiotics like Streptomycin, Kanamycin

• Amino Acids like Arginine, Asparagine

Inorganic Nutrients that are added are:

Some of the micronutrients are Manganese (Mn), Iron (Fe), Molybdenum (Mo), Zinc (Zn), Copper (Cu), Boron (B).

Six major macronutrients that are included are Nitrogen (N), Sulphur (S), Phosphorus (P), Potassium (K), Magnesium (Mg), Calcium (Ca).

The Components that are used as Carbon and Energy Sources are:

• Lactose

• Maltose

• Galactose

• Raffinose

• Cellobiose

Growth Hormones:

• Auxins- Generally used to induce cell division

• Cytokinins- Used for modifying apical dominance and shoot differentiation

• Abscisic Acid (ABA)- Used occasionally

• Gibberellins- Used occasionally

Plant Tissue Culture Steps / Plant Tissue Culture Procedure

The following is the general process of plant tissue culture. There are specific steps for the regeneration of a complete plant from an explant cultured on the nutrient medium. These steps are: 

1. Selection and Sterilisation of Explant: A suitable explant is chosen and excised from the donor plant and the explant is sterilized using disinfectants.

2. Preparation and Sterilisation of the Culture Media: A suitable culture media is prepared with specific components for the growth of the explant, the culture is then sterilized.

3. Inoculation: The sterilized plant is inoculated on the culture medium under aseptic conditions.

4. Incubation: The cultures are then incubated in the culture room with appropriate conditions of light, temperature, and humidity for successful culturing.

5. Sub-Culturing: Cultured cells are transferred to a fresh nutrient medium to obtain the plantlets.

6. Transfer of Plantlets: After the hardening process (i.e., acclimatization of plantlets to the environment), the plantlets are transferred to the greenhouse or in pots.

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What are the Types of Plant Tissue Culture?

There are 6 types of plant tissue culture techniques. These are:

• Seed Culture: Seeds may be cultured in-vitro to generate fully developed plants. It is one of the best methods of tissue culture for raising sterile seedlings. The seed culture is done to get the different types of explants from aseptically grown plants which help in better maintenance of aseptic tissue.

• Embryo Culture: Embryo culture is the sterile isolation and growth of an immature or mature embryo in-vitro to grow a viable plant. In some plants, seed dormancy can also be due to mechanical resistance, chemical inhibitors or structures covering the embryo. Excision of embryos and culturing them in nutrient media help in developing seedlings.

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• Meristem Culture: The apical meristem of shoots of gymnosperms and angiosperms can be cultured to get disease-free plants. Meristem tips, which are between 0.2-0.5 mm, frequently produce virus-free plants and this method is referred to as meristem-tip culture.

This method is more successful in the case of herbaceous plants than woody plants. In the case of woody plants, success is obtained when the explant is taken after the dormancy period is over. After the shoot tip proliferation, the rooting is done and then the rooted plantlet is potted.

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• Bud Culture: Buds contain active meristems in the leaf axils which are capable of growing into a shoot. Single node culture is where each node of the stem is cut and allowed to grow on a nutrient medium to be developed into a shoot tip from the axil which ultimately develops into a new plantlet. In the axillary bud method, the axillary buds are isolated from the leaf axils and developed into shoot tips under little high cytokinin concentration.

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• Callus Culture: Callus is a more or less unorganized de-differentiated mass of cells arising from any kind of explant under in vitro cultural conditions. The cells in the callus are parenchymatous but may or may not be a homogenous mass of cells.

The callus tissue from various plant species may be different in structure and growth habits. The callus growth is also depen­dent on factors like the type of explant and the growth conditions. After callus induction, it can be subcultured regularly with an appropriate new medium for growth and maintenance.

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• Cell Suspension Culture: The growth of individual cells that have been obtained from any kind of explant tis­sues or callus refers to as the cell suspension culture. These are initiated by transferring pieces of tissue explant/callus into a liquid medium (without agar) and then placing them on a gyratory shaker to provide both aeration and dispersion of cells. Like callus culture, the cells are also sub-cultured into the new medium.

Cell suspension cultures may be done in batch or continuous culture systems. In a continuous culture system, the culture is continuously sup­plied with nutrients by the inflow of fresh medium with subsequent draining out of used medium but the culture volume is constant. This culture method is mainly used for the synthesis of specific metabolites or biomass production.

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Application of Plant Tissue Culture

The uses of tissue culture are:

• In-plant biotechnology, the useful product is a plantlet and they are used for many purposes.

• All the cells in callus or suspension plant tissue culture are derived from a single explant by mitotic division.

• Hence, all plantlets regenerated from a callus or suspension culture have the same genotype and constitute a clone. These plantlets are utilized in rapid clonal propagation.

• A genetic variation that is observed amongst plant cells of culture is called somaclonal variation.

• A gene that is transferred into an organism by genetic engineering is known as a transgene and it can be introduced into individual plant cells.

• An organism that contains and expresses a transgene is called a transgenic organism.

• The plantlets can be generated from these cells and give rise to highly valuable transgenic plants.

• Mutagens are added to single-cell liquid cultures for the induction of mutations.

• Tolerance to stress like toxins, salts, drought, pollutants, flooding, etc. can also be obtained by providing them in a culture medium by increasing dosage. The surviving healthy cells are taken to a solid medium for raising resistant plants.

Application of Plant Tissue Culture in Agriculture

Tissue culture has been widely applied for more than half a century and is now used to improve many crops important to developing country food security including major staples such as rice, potato, and banana.

Crops that are important to develop countries that are improved and propagated by tissue culture include:

• Cassava, sweet potato, and yam

• Commercial Plantation Crops: coffee, cocoa, sugar cane, oil palm, and tea

• Horticultural Crops: cardamom, artichoke, garlic, ginger, and vanilla

• Fruit Trees: almond, citrus, coconut, date palm, grape, lemon, olive, pistachio, pineapple, etc.

Some of the countries with well-developed tissue culture programs are Gabon, Kenya, Nigeria, and Uganda.

Some of the greatest successes with tissue culture have been demonstrated with vegetatively propagated root crops. For example, disease-free sweet potatoes have been adopted on 500,000 hectares in Shandong Province in China, increasing yields between 30%–40% and incomes for 7 million sweet potato producers by 3.6%–1.6%.

In India, potato breeders have started using tissue culture to detect viruses at the initial stages of seed production. This has led to an estimated 2 to 3 fold increase in seed health whilst generating more than $4 million in revenues.

Farmers in Vietnam participate in the use of tissue culture for high-yielding, late-blight resistant potatoes. This has enabled them to double their yields from 10 to 20 tonnes per hectare.

Fun Facts about Plant Tissue Culture

• It was invented by a German Botanist named Gottlieb Haberlandt in 1898.

• It takes about 10-14 weeks to develop beginning from clipping to cell multiplication.

• Medicinal plants are cultured to obtain active compounds for the herbal and pharmaceutical industries.

• The media used for Plant tissue culture is agar. It is obtained from plant seaweeds. It is used universally as a gelling agent for various tissue culture formation. 

Conclusion

After reading this write-up you are in a position to answer any question asked in the exam related to Plant Tissue Culture - Nutritional Requirements, Culture Steps, and Application. Here we have discussed the definition of plant tissue culture, its requirements, steps of growing such a culture, and its applications.

After learning so much about plant tissue culture and its impact on various developing nations, one can easily say that this new development in the field of biotechnology is here to stay. It can help in increasing the productivity of various crops thereby meeting the demands of an ever-increasing population. 

Also, it is understood that there is a large scope of further research and development in this field and a lot remains to be discovered.