PTC media widely used for the propagation of plants

preparation of media-Plant tissue culture (PTC) is a method of growing and maintaining plant cells, tissues, or organs under sterile and controlled conditions, often in a nutrient medium. This technique is based on the principle that plant cells can regenerate into a whole plant under the right conditions. It is widely used for the propagation of plants, conservation of endangered species, and genetic modification.

1. DISCOVER . LEARN . EMPOWER UNIT-1 UNIVERSITY INSTITUTE OF AGRICULTURAL SCIENCES DEPARTMENT OF AGRICULTURE Bachelor of Science (Agriculture) AGB-314 (MICROPROPAGATION TECHNOLOGIES) Dr Shabnam Katoch (E8861)

2. Culture media for tissue culture 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 and solidifying agents. 2

3. 3

4. MACRONUTRIENTS   Need of macro nutrients is higher . It is present in milli molar ( mM ) quantities (more than 30 ppm /1 or mg/1 ) Macro nutrients provide both anions and cations for the plant cells . nitrogen (as NO3 and NH4 ), phosphorus(PO4), potassium(K), sulphur (a s SO4 ), magnesium(Mg), and calcium(Ca ) DEFINE - Macronutrients are those required in relatively high concentrations for the purposes of plant growth. The essential elements in plant cell or tissue culture media include, besides C, H and O, macroelements : nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulphur (S). Macro nutrients have structural and functional role in protein synthesis, cell wall synthesis enzyme Co-factors and membrane integrity. 4

5. NITROGEN In organic form used as amino acids, different organic acids and casein hydrolysate . In inorganic form used as Nitrate or ammonia . Nitrogen is major component of all plant tissue culture media . Nitrogen helps to synthesis complex organic molecule . Potassium K ion is present in high concentration in the cytoplasm (100-200 mM ) and in chloroplast(20-200 mM ). K+ is essential for mentaining the ion balancing, activation of many enzymes. Maintaining osmotic pressure and osmotic regulation of cells. 5

6. CALCIUM Calcium functions with different enzymes as Co- factor and bound to the cell wall and cell membrane . It gives strength to cell wall . It helps in the regulation of the cell membrane structure . Deficiency causes disintegration of the membrane and shoot tip necrosis . Important in cell and root multiplication . Supplied as calcium chloride and calcium nitrate. 6

7. PHOSPHORUS Very important for energy metabolism . Essential element for DNA & RNA Deficiency may cause delayed growth and dark green colour of leaves . Supplied as sodium hydrogen phosphate or potassium hydrogen phosphate . MAGNESIUM Essential for enzymatic reactions, energy metabolism(ATP synthesis ). Supplied as magnesium sulphate . Sulphur Deficiency of Sulphur inhibits protein synthesis and decreases Chlorophyll in leaves. Supplied as magnesium Sulphate and Potassium Sulphate 7

8. Nitrogen- essential element of all the amino acids in the plant structure. Potassium is required for cell growth of most plant species. Phosphorus plays key roles in many plant processes such as energy metabolism, the synthesis of nucleic acids and membranes, photosynthesis, respiration, nitrogen fixation and enzyme regulation.   Calcium - Promotes proper plant cell elongation and strengthen cell wall structure. Magnesium -  magnesium's well-known roles is in the photosynthesis process, as it is a building block of the Chlorophyll, which makes leaves appear green. Sulphur -  Formation of chlorophyll, the green substance in leaves that permits photosynthesis 8

9. Micronutrients- are the ones required in very small amounts by the plants. These include iron, zinc, boron etc. Iron : It is required in larger amounts as compared to other micronutrients. It is an important constituent of protein and helps with the activation of enzymes. Manganese : Enzymes involved in photosynthesis, respiration etc are activated by this. It helps in the splitting of water during photosynthesis. Zinc and copper  are also involved in the activation of certain enzymes. interstitial fluid helps in the uptake and utilization of calcium, functioning of the membrane, pollen germination, cell elongation, germination etc. 9

10. 10

11. 11

12. Hormones - are the organic compounds, other than nutrients, which influence growth, differentiation and multiplication. They required in very minute quantity in the media. The different groups of PGRs commonly used in the media are: Auxins - for cell division and root differentiation. Cytokinins - for cell division, differentiation of adventitious shoots from callus and organ & shoot proliferation. Gibberellins - Naturally occurring plant hormones involved in internode elongation, enhancement of flower, fruit and leaf size, germination and vernalization in plants. Ethylene - A gaseous plant hormone involved in fruit maturation, abscission, and senescence. * Additional substances gaining recognition as hormones are polyamines, jasmonates , salicylic acid etc.   12 What are plant growth regulators/hormones?

13.  1. Auxin : In nature, the hormones of this group are involved with elongation of stem, internodes, tropism, apical dominance, abscission, rooting etc. In tissue culture, auxins have been used for cell division and root differentiation. The commonly used auxins in tissue culture are 1. Indole-3-acetic acid (IAA) 2. Indole-3-butyric acid (IBA) 3. Naphthalene acetic acid (NAA) 4. Dichlorophenoxyacetic acid (2, 4-D) Auxins are usually dissolved in either ethanol or dilute NaOH . 13

14.  2. Cytokinins : These hormones are essential for cell division, modification of apical dominance, shoot differentiation etc. In tissue culture media, cytokinins are incorporated mainly for cell division, differentiation of adventitious shoots from callus and organ & shoot proliferation. Commonly used cytokinins are: 1. Benzylamino purine (BAP) 2. Isopentenyl adenine (2-ip) 3. Furfurylamino purine (kinetin) 4. Zeatin Cytokinins are generally dissolved in dilute HCl or NaOH . 14

15.  3. Gibberellins:  Naturally occurring plant hormones involved in internode elongation, enhancement of flower, fruit and leaf size, germination and vernalization in plants. Among the 20 known gibberellins, GA3 is used widely. Compared to auxins and cytokinins , gibberellins are used very rarely. They stimulate normal development of plantlets from in vitro formed adventitious embryos. They are soluble in cold water.  4. Ethylene: A gaseous plant hormone involved in fruit maturation, abscission, and senescence. All kinds of plant tissue cultures produce ethylene and the rate of production increases under stress conditions. Use of ethylene precursor (2-chloroethyl phosphonic acid) in tissue culture may be inhibitory for the same process in different species. 15

16. 5. Abscisic acid:  A plant hormone involved in abscission, enforcing dormancy and regulating early stages of embryo development. It is required for normal growth and development of somatic embryos and promotes morphogenesis.  6. Brassinosteroids : It promotes shoot elongation at low concentrations and strongly inhibits root growth and development. It also promotes ethylene biosynthesis and epinasty . 7. Jasmonates :   Jasmonates are represented by jasmonic acid and it is a methyl ester. Jasmonic acid is considered to be a new class of plant growth substance. It is involved in differentiation, adventitious root formation, breaking of seed dormancy and pollen germination. 16

17.  8. Polyamines: There is some controversy as to whether these compounds should be classified with hormones. They appear to be essential in growth and cell division.  9. Salicylic acid: It is thought to be a new class of plant growth substances . It promotes flowering, inhibits ethylene biosynthesis and reverses the effects of ABA. 17

18. References Bhojwani , S.S. & Razdan , M.K., 1996, Plant Tissue Culture: Theory and Practice, a Revised Edition, Elsvier , Amsterdam. Gupta PK. 2004. Biotechnology and Genomics. Rastogi Publ. Singh BD. 2012. Biotechnology: Expanding Horiozon . Kalyani . Chawla HS. 2004. Introduction to Plant Biotechnology. Science Publishers, Inc., UK. Notes at - https://ecourses.icar.gov.in/ https://www.youtube.com/watch?v=xuwV3ywCxW8&t=102s 18

19. THANK YOU