Describe the protocol involved in rooting and acclimatization of plants produced using in vitro culture. Elaborate on the various problems and solutions associated with this technique.

Rooting Protocol

Rooting is typically induced by altering the hormone balance in the culture medium. Shoots are transferred to a rooting medium, generally containing a lower concentration of auxins compared to the shoot multiplication medium. Common auxins used include Indole-3-butyric acid (IBA) and α-Naphthaleneacetic acid (NAA). The concentration varies depending on the plant species, with IBA generally preferred for its ease of metabolism and reduced phytotoxicity.

• Media Composition: MS (Murashige and Skoog) medium is commonly used, supplemented with 0.5-2 mg/L IBA or NAA. Activated charcoal (0.2-0.5%) is often added to adsorb inhibitory substances and promote root formation.
• Environmental Control: Rooting is usually carried out under controlled conditions: 25 ± 2°C, 16/8 h photoperiod (light/dark), and humidity maintained at 60-70%.
• Root Development: Roots typically emerge within 2-4 weeks. Plants with well-developed root systems are selected for acclimatization.

Acclimatization Protocol

Acclimatization is the gradual transition of in vitro-grown plants to ex vitro conditions. This is a crucial step as plants lack a functional cuticle, underdeveloped stomata, and a poorly developed vascular system, making them highly susceptible to desiccation and pathogen attack.

• Stage 1: Humidity Control: Plants are initially transferred to a high-humidity environment (80-90%) in a controlled growth chamber or greenhouse. This can be achieved using misting systems or covering plants with transparent plastic bags.
• Stage 2: Gradual Humidity Reduction: Over several weeks, humidity is gradually reduced to 60-70%. This allows plants to develop a functional cuticle and stomata.
• Stage 3: Substrate and Light: Plants are transplanted into a well-draining substrate (e.g., peat moss, perlite, vermiculite) and exposed to gradually increasing light intensity.
• Stage 4: Hardening: Plants are further hardened by reducing humidity and increasing light intensity, preparing them for field conditions.

Problems and Solutions

Problem	Solution
Hyperhydricity (Vitrification) – Plants appear glassy and swollen due to excessive water uptake.	Reduce humidity, lower auxin concentration, add activated charcoal to the medium, pulse treatment with abscisic acid (ABA).
Ex Vitro Shock – Plants fail to establish after transfer to the ex vitro environment.	Gradual acclimatization, use of anti-transpirants (e.g., kaolin), maintaining high humidity initially, optimizing substrate composition.
Rooting Inhibition – Failure to induce root formation.	Optimize auxin type and concentration, add activated charcoal, ensure proper media sterilization, use of plant growth regulators like cytokinins in combination with auxins.
Contamination – Bacterial or fungal growth in the culture.	Strict aseptic techniques, use of antibiotics or antifungal agents (as a last resort), regular subculturing.

Furthermore, genetic variability can sometimes arise during long-term in vitro culture (Somaclonal variation). Careful monitoring and selection of plants are necessary to maintain the desired genotype.