Plant Tissue Culture

Bookmark added to your notes.
View Notes

What is 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 on 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 specifics tissue and organs. Some of the important media are:

  • White’s Medium, etc.

  • 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 sterilised 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 sterilised.

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

  4. Incubation: The cultures are then incubated in the culture room where 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., acclimatisation of plantlets to the environment), the plantlets are transferred to the greenhouse or in pots.

(image will be uploaded soon)

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 seedling. 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 with the objective of growing 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.

(image will be uploaded soon)

  • Meristem Culture: The apical meristem of shoots of gymnosperms and angiosperms can be cultured to get the 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, the 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.

(image will be uploaded soon)

  • 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 media to be developed into a shoot tip from the axil which ultimately develops into a new plantlet. In the axillary bud method, where the axillary buds are isolated from the leaf axils and develop into shoot tip under little high cytokinin concentration.

(image will be uploaded soon)

  • Callus Culture: Callus is basically more or less unorganised de-differentiated mass of cells arising from any kind of explant under in vitro cultural conditions. The cells in callus are parenchymatous in nature, 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 habit. 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.

(image will be uploaded soon)

  • Cell Suspension Culture: The growth of individual cells that have been obtained from any kind of explant tis­sue or callus referred to as cell suspension culture. These are initiated by transferring pieces of tissue explant/callus into a liquid medium (without agar) and then placed 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 for biomass production.

(image will be uploaded soon)

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 utilised 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 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 years 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 developing countries that are improved and propagated by tissue culture include:

  • Cassava, sweet potato, and yam.

  • Commercial Plantation Crops: coffee, cocoa, suage 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 programmes are Gabon, Kenya, Nigeria and Uganda.

Some of the greatest successes with tissue culture has 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 to3 fold increase in seed health whilst generating more than $4 million in revenues.

Farmers in Vietnam have participated 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.

FAQ (Frequently Asked Questions)

Q 1: Give an Example of Plant Tissue Culture.

A: One example of plant tissue culture is the protoplast culture. It is the culture of plant protoplasts that’s is, the culture of cells devoid of the cell wall. Protoplasts are isolated from soft parenchymatous tissue by the enzymatic method and then viable proto­plasts are purified and cultured.

The protoplast culture is aimed mainly to develop a genetically transformed plant where the transgenic is put successfully within the plant proto­plast and the transgenic plant is regenerated from that transformed protoplast. Another aspect of protoplast culture is the somatic hybridization of two plant species through proto­plast fusion.

Q 2: What are the Different Plant Tissue Culture Techniques?

A: Some of the different tissue culture techniques are:

  • Seed culture

  • Embryo culture

  • Meristem culture

  • Bud culture

  • Callus culture

  • Cell suspension culture

  • Anther culture

  • Protoplast culture

Q 3: Explain the Plant Tissue Culture Procedure.

A: Please have a look at the plant tissue culture procedure section.