Plant Tissue Culture Conditions

The choice of medium is based on the types of plant species; explants are used for culture for optimal response. All the nutrients required for a plant’s proper growth and development should be present in the plant tissue culture media. Macronutrients, micronutrients, vitamins, other organic ingredients, plant growth regulators, a carbon source, and in the case of a solid medium, a few gelling agents make up the majority of its composition. Similarly, hormone levels and culture variables like temperature, pH, light intensity, and humidity also play an important role in the success of tissue culture.

• The minerals consist of macronutrients such as nitrogen, potassium, phosphorus, calcium, magnesium, and sulfur, and micronutrients such as iron, manganese, zinc, boron, copper, molybdenum, and cobalt. 
• Vitamins are necessary for the healthy growth of plant cultures. The vitamins like thiamine (vitamin B1), pyridoxine (B6), and nicotinic acid (niacin). Other vitamins such as biotin, folic acid, ascorbic acid (vitamin C), and vitamin E (tocopherol) are sometimes added to media formulations. 
• Plants also require an external carbon source; sugar. The most commonly used carbon source is sucrose. Other sources used are glucose, maltose, and sorbitol. 
• The pH of the culture medium remains vital as it influences the uptake of various components of the medium and regulates a wide range of biochemical reactions. Most media are adjusted to a pH of 5.2–5.8. A higher pH may be required for certain cultures.

Plant Tissue Culture Media

• The most popular medium for in vitro vegetative propagation of various plants is Murashige and Skoog medium (MS medium). For culturing, either a solid or liquid medium can be employed. 
• McCown’s woody plant medium (WPM) has been widely used for tree tissue culture.
• Knudson’s medium is used for orchid tissue culture and fern tissue culture.

Plant Tissue Culture Growth regulators

• Plant growth regulators (PGRs) are crucial for determining the development of plant cells and tissues in a culture medium. 
• The most commonly used plant growth regulators are auxins, cytokinins, and gibberellins.
• The high auxin concentration often favors the development of roots. The most commonly used auxins are IAA (indoleacetic acid), IBA (indolebutyric acid), NAA (naphthaleneacetic acid), and 2,4-D (2,4 dichlorophenoxyacetic acid). 
• Cytokinins promote cell division and shoot growth. The most commonly used cytokinins are BAP (benzylaminopurine), zeatin, Isopentenyl adenine (2-ip), and kinetin. Cytokinins are generally dissolved in dilute HCl or NaOH.
• A clump of undifferentiated cells called a callus develops when auxin and cytokinin levels are balanced.

Plant Tissue Culture Vessels 

• Another critical aspect in plant tissue cultures is the management of the gaseous hormone ethylene. In closed culture vessels used for in vitro plant growth, ethylene builds up and is often detrimental to the cultures. The addition of ethylene biosynthetic inhibitors such as silver nitrate, AVG (aminoethoxyvinylglycine), and silver thiosulphate have been shown to increase the formation of shoots.
• Cultures are grown in walk-in growth rooms or growth chambers. Humidity, light, and temperature must be controlled for the proper growth of cultures. 
• A 16-hour light photoperiod is optimal for tissue cultures, and a temperature of 22 – 25⁰C is used in most laboratories. 
• Cool white fluorescent lamps also supply a light intensity of 25–50 µmol m-2 s-1. 
• Relative humidity of 50–60% is maintained in the growth chambers. Some cultures are also incubated in the dark. 
• Cultures can be cultivated in various containers, including test tubes, flasks, Petri dishes, and bottles.

Sterility

• The preservation of a sterile environment is necessary for effective tissue culture. The laminar flow hood is used for all tissue culture work. A dust filter and a high-efficiency particulate air (HEPA) filter are used in the laminar flow hood to filter the air. The hood must be kept spotless, which can be accomplished by wiping it with alcohol that contains 70% of the alcohol.
• The surfaces of plant tissues naturally contain a variety of bacteria and fungi. Before tissue culture, it is crucial to thoroughly clean the explant since contaminants can proliferate in the culture media. They also compete with the plant tissue for nutrients, depriving it of those nutrients. Bacteria and fungi can rapidly surpass plant tissues and destroy them. 
• Explants are commonly surface-sterilized using sodium hypochlorite, ethanol, and fungicides when using field-grown tissues. 
• The type of tissue usually decides the time of sterilization. Leaf tissue requires a shorter sterilization time than seeds with a hard seed coat.