Basic requirement for tissue culture

Basic Requirement For Tissue Culture

1. Basic Requirement For Tissue Culture

2. TISSUE CULTURE Def; Tissue culture is a technique that uses an artificial medium to grow and maintain cell and tissue or an organ under controlled environmental conditions.

3. Explant Explant is living tissue removed from an organism and placed in a medium for tissue culture. Totipotency Plant cell have a unique property call totipotency . That is , most of the plant cell have the capacity to regenerate into the entire plant when placed in proper environment with nutrient and hormonal support.

4. Basic Requirement For Tissue Culture Media Constituents The basic requirements of mineral elements such as potassium, calcium, magnesium, nitrogen, phosphorous and sulphur , which are required in large quantities (macronutrients/ macroelements / major elements) and others, like iron, manganese, copper, zinc, boron and molybdenum, are required in small quantities (micronutrients/ microelements/minor elements) for the growth of plant tissues are fulfilled by providing their common salts in the medium.

5. One type of ion may be contributed by more than one salt in the medium. For example, in MS medium, NO3– ions are contributed by NH4NO3, as well as KNO3, and K+ ions by KNO3 and KH2PO4.

6. 2. Inorganic Nutrients ( i ) Macronutrients . The macronutrients are required in millimolar ( mM ) quantities. Calcium (Ca2+), potassium (K+), magnesium (Mg2+), nitrogen (NO3-), sulphur (SO42-) and phosphorous (PO43-) are the macroelements and the essential ingredients of plant tissue culture media.

7. These are added to the medium as calcium nitrate, potassium dihydrogen phosphate and magnesium sulphate . Alternately, potassium and calcium may be provided as KCl or KNO3 and CaCl2.2H2O, respectively. Nitrogen is one of the vital elements required for the growth of plants in cultures as also in nature.

8. Inorganic nitrogen is usually supplied in the form of ammonium (NH4+) and/or nitrate (NO3-) ions.

9. (ii) Micronutrients Some micronutrients, although required in small quantities, are essential for tissue growth in cultures. They act as cofactors of enzymes. Micronutrients typically include boron (BO33-; from H3BO3), manganese (Mn2+; from MnSO4.

10. Organic Nutrients In order to achieve the best growth of plant tissues in cultures, the medium needs to be supplemented with organic compouApparently , the cultured plant cells synthesize them in sub-optimal quantities. Sucrose, a source of carbon or energy, is a very important constituent of plant tissue culture media. nds , such as vitamins and amino acids.

11. ( i ) Vitamins and amino acids. Vitamins that act as coenzymes are required to be added to the medium for healthy growth of tissue cultures. The most widely usedvitamins are those of B group, viz., thiamine (vitamin B1), nicotinic acid (also known as niacin or vitamin B3), pyridoxine (vitamin B6) and myo-inositol (sometimes referred to as meso-inositol ). Glycine , the simplest amino acid, is a common constituent of plant tissue culture media.

12. (ii) Carbon source Most plant tissue cultures are unable to photosynthesize because of the absence of chlorophyll or poorly developed chloroplasts, limited CO2 in the culture vessel due to poor gaseous exchange and absence of optimum light intensity. It is, therefore, obligatory to add to the culture medium an utilizable source of carbon necessary for various metabolic activities.

13. The most commonly used carbon source is sucrose at a concentration of 2–5 % (w/v). Generally, sucrose autoclaved along with the medium supports better growth of tissues than filter -sterilized sucrose. Autoclaving causes hydrolysis of sucrose into more efficiently utilizable sugars, such as glucose and fructose.

14. Plant Growth Regulators Addition of growth regulators to an otherwise complete medium, containing inorganic and organic nutrients and sucrose called Basal medium (BM), is invariably necessary to trigger various types of growth and differentiation. Although the explants may have certain levels of endogenous growth hormones (naturally synthesized growth regulators), it is often necessary to supplement them exogenously to evoke certain responses.

15. The growth regulators are generally required in very minute quantities (0.001–10 lM ). The nature and quantity of growth regulators in the medium is varied according to the variety of plant, nature of the tissue and also the stage of culture (initiation of callus, induction of somatic embryogenesis, shoot differentiation or multiplication, rooting of shoots, etc.).

16. In order to develop a tissue culture protocol for a new plant species, various types and concentrations of growth regulators in several permutation and combination need to be tested. The concentrations of growth regulators used are very often reported in mg L-1.

17. ( i ) Auxins Auxins are involved with many developmental processes in plants, including elongation of stem and internodes, tropisms, apical dominance, abscission and rooting. In tissue cultures, auxins have been used to induce cell division, cytodifferentiation and organogenic and embryogenic differentiation. Generally, auxin at low concentration favours root initiation, whereas at higher concentration induces callus formation.

18. (ii) Cytokinins This is another major group of plant hormones. Natural cytokinins are N6substituted adenine derivatives and occur in plants as nucleosides and nucleotides. Roots are the possible sites of their synthesis.

19. In nature, cytokinins are concerned with cell division, modification of apical dominance and shoot differentiation. Incorporation of these compounds in culture media is mainly to trigger cell division, and to induce differentiation of adventitious shoots from callus and organs, and shoot proliferation by the release of axillary buds from apical dominance

20. (iii) Gibberellins These are less commonly used in plant tissue culture. There are over 20 known gibberellins, of which GA3 is used most often.

21. (iv) Ethylene and Abscisic acid Ethylene (ethane, C2H4) is an unusual, gaseous plant hormone. It is produced by ageing and stressed tissues.

22. Other Supplements ( i ) Polyamines , derived through decarboxylation of amino acids, have been used to promote organogenesis/somatic embryogenesis ( Mengoli and Bagni 1992; Rajam et al. 1998). Of the three polyamines ( putrescine , spermidine and spermine ), putrescine has proved most effective (Altman 1990; Litz 1993).

23. (ii) TIBA (2,3,5-triiodobenzoic acid) is an inhibitor of auxin polar transport. In carrot cultures, it arrests development of somatic embryos at the globular stage. (iii) Phloroglucinol has been shown to promote rooting in rosaceous fruit trees such as apple. (iv) Activated charcoal is usually added at 0.5 % to culture media to promote rooting and/or to adsorb toxic exudates from cultured tissues.

24. Undefined Supplements In order to promote growth of certain calli and evoke a desired morphogenic response in cultures, several complex, undefined organic supplements such as banana homogenate, CH, CM, corn milk, ME, TJ and YE have been used in the medium. These crude natural plant products are generally used when no other combination of defined components yield the desired results.

25. Gelling Agents In static liquid cultures, the tissue would get submerged and die of anaerobic conditions. To circumvent this problem, the medium is solidified with a suitable gelling agent. The most desirable properties of a gelling agent are that it should: ( i ) be inert (ii) withstand sterilization by autoclaving and (iii) be liquid when hot so that the medium could be dispensed in culture vessels in desired quantities.

26. ( i ) Agar. Agar, obtained from red algae, especially Gelidium amansii , is the most commonly used gelling agent. (ii) Agarose . Agarose , comprising b-D(1-3) and 3,6-anhydro-a-L(1-4) galactopyranose molecules, is obtained by further purifying agar to remove agaropectins with its sulphate groups.

27. (iii) Gellan gum. A linear polysaccharide obtained from the bacterium Pseudomonas elodea and marketed under different trade names, such as gelrite (C.P. Kelco & Co.) and phytagel (Sigma-Aldrich Chemical Co.), has become a popular substitute of agar. (iv) Isubgol . Indian scientists have shown that the husk of Plantago ovata seeds ( Isubgol ), used at 3 %, could be a cheaper alternative to gel plant tissue culture media (Jain and Babbar 1998).

28. pH of the Medium The pH of medium greatly influences the uptake of ingredients by the tissue in culture, solubility of salts and gelling efficiency of agar. The pH of medium is usually set at 5.8 as at this pH all the salts are maintained in a near buffered form.

29. In general, a pH higher than 6.0 gives a fairly hard medium and a pH below 5.0 does not allow satisfactory gelling of the agar due to its hydrolysis during autoclaving. The pH of culture medium generally drops by 0.3–0.5 units after it is autoclaved and then changesthroughout the period of culture both due to oxidation and differential uptake of ions such as NH4+ and NO3- and secretion of substances by the growing tissue.

30. Media Preparation Preparation of culture media is a critical step in tissue culture work wherein great precision is required on the part of the investigator. There are two possible ways to prepare medium. One method is to weigh and dissolve the required quantities of the ingredients separately and mix them every time a medium is to be prepared. But this is a cumbersome method and chances of committing error are more.

31. A more convenient and widely practiced method is to prepare, in advance, a series of concentrated stock solutions and store them in a refrigerator. Wheneveramediumistobeprepared , the required amounts are drawn from the stock solutions and mixed.For example,to prepare MS, four stock solutions are prepared .

32. Reference Auther ; Sant Saran Bhojwani Prem Kumar Dantu Book; Plant Tissue Culture: An Introductory Text