Plant Tissue Culture

Plant tissue culture (PTC) means growing plant cells, tissues or organs outside the plant body on an artificial nutrient medium under sterile and controlled conditions. It uses the property of totipotency, by which a plant cell can divide, differentiate, and regenerate into a whole plant.

The culture media used for plant tissue culture contains the following components:

1. Inorganic components
- Macronutrients: needed in large amounts, e.g. carbon, calcium, hydrogen, potassium, magnesium, nitrogen, oxygen, phosphorus and sulphur.
- Micronutrients: needed in small amounts, e.g. boron, cobalt, copper, iron, manganese, molybdenum and zinc.

2. Organic supplements
- Mainly amino acids and vitamins.
- Amino acids such as arginine, asparagine, glycine and proline act as nitrogen sources.
- Vitamins commonly used are thiamine (B1), nicotinic acid (B3) and pyridoxine.

3. Carbon source
- Usually sucrose at about 2–5%.
- Other sources include glucose, fructose and mannose.

4. Plant growth hormones
- Five main categories are auxin, cytokinin, gibberellin, abscisic acid and ethylene.
- In tissue culture, auxins and cytokinins are most important because their ratio controls root or shoot formation.

5. Gelling agent
- Used for solid media.
- Agar is the most commonly used gelling agent.

6. Antibiotics
- Used to suppress bacterial contamination and antifungal agents for mould infections.

The media pH is usually adjusted to about 5.8–6.0.

The general steps of plant tissue culture are:

1. Selection and sterilisation of nutrient medium
- The medium is selected and sterilised by autoclaving or by passing through micropore filters.

2. Selection of explant
- A suitable starting material such as leaf, apical meristem, cotyledon, hypocotyl or immature embryo is chosen.

3. Surface sterilisation of explant
- The explant is disinfected, commonly with sodium hypochlorite, and washed with sterile distilled water.

4. Inoculation
- The sterilised explant is transferred onto the nutrient medium.

5. Growth of cultures
- Cultures are kept in a tissue culture room under suitable light, temperature and humidity.

6. Transfer for shoot regeneration and elongation
- Growing cultures are moved to suitable media to form and elongate shoots.

7. Rooting
- Regenerated shoots are excised and transferred to rooting medium.

8. Hardening and transfer to soil
- Plantlets are transferred to sterilised soil in pots, hardened in a greenhouse or growth room, and then moved to field conditions.

Plant tissue culture has many applications:

- Micropropagation: rapid multiplication of genetically identical plants.
- Artificial seed production: somatic embryos are encapsulated to form synthetic seeds.
- Haploid and double haploid production: useful in breeding and producing homozygous plants.
- Somatic hybridisation: fusion of protoplasts from different species to produce hybrids.
- Production of virus-free plants: using apical or axillary meristems.
- Somaclonal variation: useful for crop improvement and selecting novel traits.
- Production of secondary metabolites: such as taxol, shikonin, azadirachtin, quinine, etc.

These applications are important in agriculture, horticulture, forestry and industry.

Somatic hybrids are developed by somatic hybridisation.

- The cell wall of plant cells is digested using enzymes such as cellulases and pectinases.
- This releases protoplasts.
- Protoplasts from two different plants are brought into close contact.
- They are induced to fuse using polyethylene glycol (PEG).
- The fused protoplasts are then cultured and regenerated into hybrid plants.

When the fusion combines the nucleus of one parent with the cytoplasmic organelles of the other, the product is called a cybrid. Somatic hybridisation helps produce hybrids even from distantly related species where sexual crossing is not possible.

The correct answer is somaclonal variations. These are the genetic variations seen in plants regenerated from cultured somatic tissues or long-term in vitro cultures.

An explant is any plant tissue, organ or part used as the starting material in plant tissue culture to regenerate a mass of dedifferentiated cells, tissues, an organ or a whole plant.

Five commonly used explants are:

1. Root apical meristem
2. Shoot apical meristem
3. Leaves
4. Cotyledons
5. Hypocotyls

Other commonly used explants include immature embryos.

Somatic embryogenesis is the process in which an embryo develops from somatic cells instead of from gametes. The embryos formed are called somatic embryos.

These somatic embryos follow the same basic embryogenic pathway as zygotic embryos, but they differ in origin and are usually larger.

### Application in synthetic seed production
- Somatic embryos are produced on callus tissue using suitable nutrient media and hormones.
- They are then encapsulated with a protective coating such as calcium alginate.
- Nutrients and growth regulators may also be added to the coating.
- This mimics a normal seed and forms an artificial seed or synthetic seed.

Synthetic seeds can be stored for a long time and used for rapid mass propagation of elite or hybrid plants.

The pH of nutrient media is usually adjusted to about 5.8 to 6.0.

Its role is important because:
- If the pH increases, the medium becomes harder.
- If the pH decreases, the medium may show poor solidification.
- pH also affects the uptake of nutrients by plant cells.
- It influences the solubility of media salts.

So, proper pH is necessary for correct medium consistency and nutrient availability.

Somatic hybridisation is a method in which protoplasts from two different plants are fused to produce a hybrid.

### Method
1. Cell wall digestion
- Plant cell walls are removed using enzymes like cellulases and pectinases.

2. Isolation of protoplasts
- Naked protoplasts are obtained from the different parent plants.

3. Fusion of protoplasts
- Protoplasts are brought together and fused using PEG.

4. Culture and regeneration
- The fused protoplasts are cultured.
- They regenerate into somatic hybrid plants.

### Advantages
- Allows hybrid formation between distantly related species.
- Helps overcome species barriers that prevent sexual crossing.
- Useful for combining desirable traits from different plants.
- Can also combine nuclear genome of one parent with the cytoplasmic genome of another, producing cybrids.