PLANT TISSUE CULTURE

Plant tissue culture

1. PHARMACOGNOSY AND PHYTOCHEMISTRY-I Module-3 Prepared by GOWRI SANKAR CHINTAPALLI ASSISTANT PROFESSOR ARKA JAIN UNIVERSITY

2. PLANT TISSUE CULTURE Plant tissue culture is defined as culturing plant seeds, organs, explants, tissues, cells, or protoplasts on a chemically defined synthetic nutrient media under sterile and controlled conditions of light, temperature, and humidity. Types of Plant tissue culture Seed Culture. Embryo Culture. Callus Culture. Organ Culture. Protoplast Culture. Anther Culture.

3. PLANT TISSUE CULTURE Preparing Plant Tissue Culture Medium Media composition Plant tissue culture media should generally contain some or all of the following components: Macronutrients Micronutrients Vitamins Amino acids or nitrogen supplements Source(s) of carbon Undefined organic supplements Growth regulators Solidifying agents

4. PLANT TISSUE CULTURE Macronutrients: The essential elements in plant cell or tissue culture media include, besides C, H and O, macro elements:  nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulphur (S)  for satisfactory growth and morphogenesis. Micronutrients: The inorganic element required in small quantities but essential for proper growth of the plant cell or for tissue are , Boron (B), Copper (Cu), Iron(fe) , manganese (Mn), Zinc (Zn),  and molybdenum (Mo ). Vitamins: Plant synthesis required vitamin Essential for many biochemical reaction Cultured cell are capable to produce vitamins at some level They require an exogenous supply vitamin at some level They require an exogenous supply of different vitamin fir optimum growth Most Usable Vitamin are thiamine pyridoxine nicotinic acid vitamin B complex.

5. PLANT TISSUE CULTURE Amino acids or nitrogen supplements: Amino acid are added for substitution for augmentation of the nitrogen supply. If is noted that theonine, glycine and valine reduce ammonium utilisation by inactiving glumate synthase located in chloroplasts and cytoplasm. Source(s) of carbon: Carbohydrates are used as carbon sources. The standard carbon source is sucrose at a concentration of 2-5 per cent. Monosaccharides like glucose or fructose can also be used as carbon sources but are generally less suitable. Sucrose is the best source, since sucrose is dehydrolysed into usable sugars during autoclaving. Undefined organic supplements:  The requirements of tissue cultures for trace amounts of certain organic substances were satisfied by “undefined” supplements such as  fruit juices, coconut milk, yeast or malt extracts and hydrolysed casein . These supplements can contribute vitamins, amino acids and growth regulants to a culture medium .

6. PLANT TISSUE CULTURE Growth regulators: The growth and the development of higher plant tissue in Vitro is controlled by Endogenous plant growth regulators. There are 5 known groups of these growth regulators namely auxins, cytokinins, gibberellins, unsaturated hydrocarbon gases and growth inhibitor. The previous two group are of great importance for culture media preparation while the letter to group are represented by ethylene and abscisic acid.

7. PLANT TISSUE CULTURE Solidifying agents: Agar and phytagel  are the most commonly used solidifying agents in tissue culture. Agar is extracted from red algae, mainly from the species Gelidium amansii Lamouroux, and consists of a complex mixture of polysaccharides, especially agarose and agaropectins.

8. PLANT TISSUE CULTURE Steps in Plant tissue Culture: Initiation stage Multiplication stage Root formation stage Shoot formation stage Acclimatization stage

9. PLANT TISSUE CULTURE Initiation stage: The initiation phase is the first phase of tissue culture. Here,  the tissue of interest is obtained and introduced and sterilized in order to prevent any microorganism from negatively affecting the process . It is during this stage that the tissue is initiated in to culture. Multiplication stage: The multiplication phase is  the second step of tissue culture where the in vitro plant material is redivided and then introduced in to the medium . Here, the medium is composed of appropriate components for growth including regulators and nutrients. Root formation stage: Rooting or preplant stage.  Growing shoots can be induced to produce adventitious roots by including an auxin in the medium . Auxins are plant growth regulators that promote root formation. For easily rooted plants, an auxin is usually not necessary and many commercial labs will skip this step.

10. PLANT TISSUE CULTURE Shoot formation stage: A cytokinin is a plant growth regulator that promotes shoot formation from growing plant cells.  Rooting or preplant stage . Growing shoots can be induced to produce adventitious roots by including an auxin in the medium. Auxins are plant growth regulators that promote root formation. Shoot formation stage Acclimatization stage: Acclimatization here simply means  the adaptation of plantlets to a new environment . Plantlets or shoots in the culture vessels are accustomed to a different micro-environment. You customize this micro-environment in a way that the developing plants experience minimal stress and optimum conditions to grow and multiply. Acclimatization stage

11. PLANT TISSUE CULTURE

12. Procedure for plant tissue culture Sterilisation of glassware tools/vessels Preparation of explant Surface sterilisation of explant Production of callus from explant Proliferation of cultured callus Subculturing of callus Suspension culture

13. CARROT ROOT CULTURE

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