Tissue culture technique

This presentation is about to the Tissue Culture Technique like Culture room, Sterilization, Autoclaving, Nutrients media.

1. Tissue Culture Technique Presented By Vivek Kumar Singh M.Sc. (Botany) 4 th semester 2016-2017

2. Introduction The term tissue culture is commonly used in a very wide sense to include in vitro culture of plant cells, tissues as well as organs . Tissue Culture denotes the in vitro cultivation of plant cells in an unorganized mass, e.g. Callus Culture. Another term, cell culture is used for in vitro culture of single or relatively small group of plant cells, e.g. suspension culture. 18/04/2017 2

3. The culture of plant cells, tissue, and organs such as root, shoot tips and leaves in artificial nutrient media aseptically under defined physical and chemical condition is referred to as “ Plant Tissue Culture ”. 18/04/2017 3

4. “Tissue Culture” is commonly used as a broad term to describe all type of plant cultures, namely callus, cell, protoplast, anther, meristem, embryo and organ cultures . 18/04/2017 4

5. History of T issue Culture The in vitro technique were developed initially to demonstrate the totipotency of plant cells predicted by Gottlieb Haberlandt in 1902 . Totipotency- is the ability of a plant cell to perform all the function of development which are characteristic of zygote. 18/04/2017 5

6. The first embryo culture was carried out by Hanning in 1904; he cultured nearly mature embryo of certain crucifer and grew them to maturity. Haploid plants from pollen grain was first produced by Maheshwari and Guha in 1964 by culturing the anther of Datura. This marked the beginning of anther and pollen culture for production of haploid plant. 18/04/2017 6

7. Plant protoplast are naked cells from which cell wall has been removed. In 1960, Cocking produced large quantities of protoplast by using cell wall degradation enzyme. In 1972 Carlson and coworkers produced the first somatic hybrid plant by fusion the protoplast of Nicotiana glauca and Nicotiana longsdorfii . 18/04/2017 7

8. The first continuously growing callus culture were established from cambium tissue in 1939 independently by Gautheret , White and Nobecart . 18/04/2017 8

9. The General Technique The technique of in vitro cultivation of plant cells or organ is – Keep the plant cell free from microbes. Suitable nutrient media Environmental condition 18/04/2017 9

10. Laboratory Space- In general space for the following is needed. Washing, Drying and Storage of Vessels Preparation, Sterilization and Storage of Media Aseptic handling of explant and cultures Maintains of culture Observation of culture 18/04/2017 10

11. Culture Room The culture room should have the following facilities: Controlled temperature (usually 25°± 2°C ). Culture racks fitted with light. A shaker for agitation of liquid cultures. 18/04/2017 11

12. Culture Vessels and their Washing Generally, glass culture vessels are used as they are cheaper, reusable and autoclavable . It is desirable to use only borosilicate or pyrex glassware as ordinary soda glass may be toxic to some tissue. Tissue are generally cultured in culture tube, flasks and Petri plate but many specially designed dishes are also used. 18/04/2017 12

13. Culture vessels and other labware are generally soaked in a suitable nontoxic detergent solution overnight, washed with a suitable brush, thoroughly rinsed clean with tap water, followed by rinse with distilled water and dried in a hot air oven 70 - 80° C. Washed culture vessels should be stored in a dust proof cabinet. 18/04/2017 13

14. Sterilization All the material used in culture work must be freed from microbes. This is done by one of the following method. Dry heat Autoclaving Filter Sterilization Wiping with 70% Ethanol Surface Sterilization 18/04/2017 14

15. Dry Heat Glassware and Teflon plasticware , and instruments may be sterilized by dry heat in an oven at 160 - 180°C for 3 hours. 18/04/2017 15

16. Autoclaving Culture vessels, (both empty and containing media) are generally sterilized by heating in autoclave or a pressure cooker to 121° C at 15 pound per square inch for 15 to 40 minutes the time being longer for larger medium volume. Sterilization during autoclaving depends mainly on temperature. 18/04/2017 16

17. Filter Sterilization Some growth regulators, e.g. GA3 , urea, certain vitamins, and enzyme are heat labile. Such compound are filter sterilized by passing their solution through a membrane filter of 0.45 micron or lower pore size. Laminar air flow cabinets are used to create an aseptic working space by blowing filter – sterilized air through an enclosed space. 18/04/2017 17

18. Wiping with 70% Ethanol The surface that can not be sterilized by other techniques are sterilized by wiping them thoroughly with 70% ethyl alcohol and the alcohol is allowed to dry. 18/04/2017 18

19. Surface Sterilization All plant materials to be used for culture are treated with an appropriate sterilizing agent to inactive the microbes present on their surface, this is called Surface Sterilization. It depends mainly on the source and type of tissue of the explant which will determine the contamination load and tolerance of the sterilizing agent. 18/04/2017 19

20. The sterilizing Agent used for surface disinfection are- Calcium Hypochlorite (9-10%) Sodium Hypochlorite (2%) Mercuric Chloride (0.1-1%) Silver nitrate (1%) Bromine water (1-2%) H 2 O 2 (10-12%) Antibiotic (4-50 mg/l) commonly used 18/04/2017 20

21. Nutrients Media A synthetic medium consist of only chemically defined compounds. Inorganic Nutrients- In addition to C, H, and O, all nutrient media provide the 12 element essential for plant growth, viz., N, P, K, Ca, S, Mg ( These are macronutrients). Fe, Zn, Mn , Cu, B, Mo (these are micronutrients). The different nutrients media provide different concentration of the inorganic nutrients. 18/04/2017 21

22. Vitamins - For the optimum callus growth, The following Vitamins are required- Inositol (B-8), thiamine (B-1), pyridoxine (B-6) and nicotinic acid (B-3) of which thiamine is essential and the rest are promotory . Pantothenic acid is also known to be promotory . 18/04/2017 22

23. Carbon Source- Sucrose (20-25 g/l) is the most commonly used carbon source for all culture material, including even green shoots. Autoclaving hydrolyses sucrose, which enhances its availability to plants. In some System such as in monocots Glucose may be superior to sucrose. 18/04/2017 23

24. Growth Regulators- The following growth regulators are used in plant tissue culture. Auxin e.g. IAA, (indole-3-acetic acid) IBA , (indole-3-butyric acid) NAA , (naphthalene acetic acid) NOA , ( naphthoxy acetic acid), 2,4-D, (2,4- dichlorophenoxy acetic acid) are commonly used to support cell division and callus growth (especially 2,4-D) ,somatic embryo induction, rooting etc. 18/04/2017 24

25. Cytokinins like kinetin ( furfurylamino purine), BAP ( benzylamino purine), Zeatin , 2- ip (isopentenyl adenine), TZD ( Thidiazuron , a compound having cytokinin activity) are employed to promote cell division, regeneration of shoots, often somatic embryo induction and to enhance proliferation and growth of auxillary buds. 18/04/2017 25

26. Abscisic acid promotes somatic embryo and shoot bud regeneration in many species and markedly improves somatic embryo maturation. Gibberellins (over 20 types known), GA 3 is almost exclusively used; it promote shoot elongation and somatic embryo germination. Concentration are as follows- auxin, 0.1-3 mg/l; cytokinins , 0.1- 3mg /l; ABA, 0.2mg /l; and GA 3 , 0.1- 1mg /l. 18/04/2017 26

27. Complex Organic Additives – Complex additive like- Yeast extract, Coconut milk, Casein hydrolysate, Corn milk, Malt extract and Tomato juice -were used to support plant tissue growth. Such additive should be used only when synthetic media fail. 18/04/2017 27

28. How to select a suitable medium? A simple process is to first test several concentration of auxin and cytokinin to identify a suitable combination of the two. Now check the ½, full, even higher salt conc. of the selected medium as well as different sucrose conc. Alternatively worker find the various conc. Of low, medium and high conc. of four solution viz ; minerals, organics, auxin & cytokinin . 18/04/2017 28

29. Reference BIOTECHNOLOGY- B. D. SINGH Plant Tissue Culture- M. K. Razdan Plant Tissue Culture –P. K.Gupta 18/04/2017 29

30. Thank You ! 18/04/2017 30