Mastering the techniques and procedures of plant tissue culture is crucial for achieving successful in vitro cultivation. This chapter delves into the essential methodologies ​used in plant tissue culture, from sterilization and media preparation to explant selection and culture maintenance. By understanding and applying these techniques, ​researchers can ensure the optimal growth and development of plant tissues under controlled conditions. This chapter provides detailed, step-by-step instructions, practical ​tips, and troubleshooting advice to guide you through each procedure. Whether you're a novice or an experienced practitioner, these techniques are fundamental to advancing ​your skills in plant tissue culture.

Surface Sterilization Methods

Surface sterilization is crucial to prevent contamination in in vitro ​cultures. Common methods include:

• Chemical Sterilants: Ethanol (70%), sodium hypochlorite (bleach), ​and mercuric chloride are frequently used.

• Procedure: Typically, explants are first rinsed in running water, ​followed by immersion in a detergent solution. They are then ​treated with a chemical sterilant for a specific duration, followed by ​several rinses in sterile distilled water to remove any residual ​sterilant.

• Optimization: Sterilization protocols may vary depending on the ​type of explant and plant species to avoid tissue damage while ​ensuring effective sterilization. For new species, the concentration ​of bleach and the duration of exposure to the bleach must be ​determined empirically to develop an effective decontamination ​regime. This involves conducting trials to identify the optimal ​conditions that achieve thorough sterilization without harming the ​plant tissues or compromising their regeneration potential.

Explants Selection and Preparation

Types of Explants

Explants are small pieces of plant tissue used to initiate in vitro ​cultures. Common types of explants include:

• Leaves: Leaf discs or segments, particularly from young, healthy ​leaves, are often used due to their high regenerative capacity.

• Stems: Internodal segments, nodal segments, and shoot tips are ​commonly used stem explants.
  

• Roots: Root tips or sections are used less frequently but can be ​important for certain species or specific culture requirements.

• Flowers, Seeds, and Buds: These are occasionally used, ​especially for reproductive tissue cultures or when somatic ​embryogenesis is desired.

The choice of explant depends on the species, the objective of the ​culture (e.g., micropropagation, genetic modification), and the ​availability of plant material.

Citrus stem cuttings undergoing ​surface sterilization in a solution ​of detergent and bleach within a ​laminar air flow (LAF) hood: ​Ensuring a sterile environment to ​prevent contamination in vitro ​cultures.

In Vitro Culture Techniques

1. Callus Culture

Callus culture involves the proliferation of undifferentiated plant cells from ​explants. Key steps include:

Induction: Explants are placed on a nutrient medium containing ​appropriate concentrations of plant growth regulators (auxins and ​cytokinins) to induce callus formation.

Subculturing: The callus is periodically transferred to fresh media to ​maintain growth and prevent necrosis.

Applications: Callus culture is used for genetic studies, plant breeding, ​and production of secondary metabolites.

3. Organ Culture

Organ culture refers to the in vitro culture of plant organs to study ​growth, development, and differentiation:

Root Culture: Isolated roots are cultured to study root development, ​nodulation, and secondary metabolite production.

Shoot Culture: Shoot tips or nodal segments are cultured to produce ​shoots, often used in micropropagation.

Leaf Culture: Leaves or leaf segments are cultured to study leaf ​development or for regeneration studies.

Nodal segments cultured for ​inducing axillary buds to form ​shoots in citrus.

Callus forming on in vitro ​sugarcane leaves: Demonstrating ​the initial stages of plant tissue ​culture and regeneration.

2. Suspension Culture

Suspension cultures involve growing plant cells in a liquid medium with continuous agitation.

Establishment: Callus tissue is broken down into smaller cell aggregates and transferred to a liquid medium with ​continuous agitation, typically using a rotary shaker at a speed of 80-100 rpm.

Applications: Suspension cultures are utilized in various advanced applications within plant tissue culture, including:

• Large-Scale Production of Secondary Metabolites: Suspension cultures are ideal for producing valuable secondary ​metabolites on a large scale, which can be used in pharmaceuticals, agriculture, and other industries.
  

• Genetic Transformation Studies: These cultures provide a suitable environment for studying genetic transformation ​processes, enabling researchers to introduce and express new genes within plant cells.
  

• Cell Biology Research: Suspension cultures are extensively used in cell biology research to study cell growth, ​differentiation, and physiological responses under controlled conditions.
  

• Clonal Micropropagation via Somatic Embryogenesis: They are highly effective for clonal micropropagation through ​somatic embryogenesis, allowing for the mass production of genetically identical plants from somatic cells.
  

Embryogenic cell suspension ​culture of taro.

4. Micropropagation

Micropropagation is the practice of rapidly multiplying plant material to produce a large number of progeny plants using modern plant tissue ​culture methods.

Stages of Micropropagation

Initiation: Selection and sterilization of explants, which are then placed on an initiation medium to induce growth.

Multiplication: Proliferation of shoots from the initial explant by subculturing on a multiplication medium.

Rooting: Induction of roots on the microshoots using a rooting medium.

Acclimatization: Gradual adaptation of in vitro grown plants to external conditions by transferring them to soil or other substrates in a controlled ​environment.

Multiple shoot formation on ​internode explants in potato: ​Demonstrating the multiplication ​stage in micropropagation.

Banana plants being acclimatised ​in shade.

Advantages and Applications

Micropropagation offers numerous advantages, including:

Rapid Multiplication: Production of a large number of plants in a relatively short time.

Disease-Free Plants: Produces plants that are free from pathogens.

Conservation: Preservation of rare and endangered plant species.

Genetic Uniformity: Clonal propagation ensures uniformity in the progeny.

Establishing in vitro culture, from the choice of explants, through surface sterilization or ​decontamination of explants, to callus formation and beyond leading to plant formation ​requires an empirical approach; once established, diligent maintenance to prevent ​contamination and a regular subculture regime pave the way towards successfully ​cultivating your desired plants.

Rooting in tobacco ​micropropagated shoots.

Banana plants regenerated from ​embryogenic cell suspension ​culture through somatic ​embryogenesis.