Tissue Culture Techiniques

2. Presented PresentedBy T,Ugandhar Asst.Prof of Botany GDC MHBD By Dr. Dr.T,Ugandhar

3. CONTENTS 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. APPLICATIONS 11. HAIRYROOTCULTURE 12. RECOGNITION OFTISSUE CULTUREFACILITIES INTRODUCTION TISSUE CULTURE? MURASHIGE & SKOOG MEDIUM MILE STONES IN PLANT TISSUE CULTURE ADVANTAGES& DISADVANTAGES TYPES OFTISSUE CULTURE CHOICE OFEXPLANT TECHNIQUES REGENERATION PATHWAYS Dept.ofDravyaguna 13. CONCLUSION 3

4. INTRODUCTION  Conservation of medicinal plants deals with the controlled utilization & official supervision in order to preserve or protect them.  Acc to WHO, as many as 80% of the world’s population depends on traditional herbal medicine for their primary health care needs.  Today many medicinal plants face extinction or severe genetic loss.  Tissue culture is one of the many techniques in biotechnology which can be used for the conservation of such medicinal plants. 4 Dept.ofDravyaguna

5. term tissue culture is commonly sens se e to to in inc cl lu ude • The The term v ve ery ry w wi id de e sen plant cells, tissues as well as organs. . • T Ti issue ssue C Cu ul lture ture denotes the in vit ro cul t ivation of plant cells in an unorganized mass, e.g. Callus Culture. • A An noth othe er r t te erm, rm, cel l cul ture is used culture of single or relatively small group of plant cells, e.g. suspension culture. is commonly used in de in vit ro cul ture of used in a a for in vitro is used for 18/04/2017 5

6. MILE STONES IN PLANT TISSUE CULTURE Haberlandt proposed concept of invitro cell culture 1902 Kolte & Robbins successfully cultured root & stem tips respectively 1922 Went discovered first plant growth hormone-Indole acetic acid 1926 Overbeek was first to add coconut milk for cell division in Datura 1941 Skoog & Miller discovered Kinetin as cell division hormone 1955 Skoog & Miller gave concept of hormonal control of organ formation 1957 Kanta & Maheswari developed test tube fertilization technique 1960 Murashige & Skoog developed MS medium with higher salt concentration 1962 Dept.ofDravyaguna 6

7. Reinhard introduced biotransformation in plant tissue cultures 1974 Chilton et al. successfully integrated Ti plasmid DNA from Agrobacterium tumefaciens in plants 1977 Melchers et al. carried out somatic hybridization of tomato & potato resulting in Pomato 1978 Larkin & Scowcroft introduced the term somaclonal variation. 1981 Rice genome sequenced under International Rice Genome Sequencing Project 2005 Dept.ofDravyaguna 7

10. WHAT WHAT DO DO WE MEAN WE MEAN BY BYTISSUE TISSUE CULTURE CULTURE??? ??? • Plant tissue culture is a collection of techniques maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture composition used to medium of known  మొక్క ల క్ణజాల సంసక ృతి అనేదీ తెలీసీన కూర్పు యోక్క పోషక్ సంసక ృతి పరిసీితులలో మొక్క క్ణాలు, , అవయవిలను నిర్వ హంచడానికీ ఉపయౌగంచే పద్ధతుల సమాహార్ం. . మాధ్య మంలో క్ణజాలిలు లేది శుభ్రమైన లేది పంచడానికీ

11.  Tissue Culture” is commonly used as a broad term to describe all type of plant cultures, namely callus,cell, Protoplast, Anther, Meristem, Embryo and cultures  క్ణజాల సంసక ృతి ” ”సిధార్ణంగా అనిి సంసక ృతులను వీవరించడానికీ ఉపయౌగసి త ర్ప, , అవీ కిలీస్, , సెల్, , భ్పోటోప్ల ా స్్, , యంథర్, , మెరిసె్మ్, , పండం మరియు అవయవ సంసక ృతులు organ ర్కిల మొక్క ల వీసతృత పద్ంగా

12. techni que to of plant w ere in vitro i ni ti al l y • The developed the predi cted by G ottl i eb H aberl andt in 1902. • Totipotency- is the plant cell to function of development characteristic of zygote. demonstrate totipotency cells ability of a perform the are all which 18/04/2017 12

13.  Gottlieb Haberlandt, Pioneerof Plant Tissue culture.  Murashige & Skoog medium, an important plant growth medium. 5 Dept.ofDravyaguna

15. Basic Methodology/technique of Plant Tissue Culture The general technique used in the isolation and growth of culture is described as follows: 1. Preparation of suitable nutrient medium: As per the selection of plant medium is autoclaved. 2. Selection of explant: Any excised part of health plant to be used e.g. Bud, leaf, root, seed etc. 3. Sterilisation of explants: by sodium hypochlorite, mercuric chloride etc. and washed asceptically for 6-10 times with sterilised water. 4. Inoculation (Transfer): The sterile explant is inoculated on solidified nutrient medium under asceptic condition. 5. Incubation: Cultures are incubated at of 25±2°C and at a relative humidity upto 50-70% fro 16 hrs of photo period. 6. Regeneration: Plantlets regenerated after transferring a portion of callus into another medium and induction of roots and shoots or directly from explants. 7. Hardening: Is the gradual exposure of plantlets for acclimatisation to environment condition. 8. Plantlet transfer: Plantlet are transferred to green house or field conditions. 15

16. • General Techniques General Techniques The technique technique of of in plant plant cells cells or organ is or organ is – vitro cultivation cultivation of – • The of 1. 1. Keep Keep the plant the plant cell free 2. 2. Suitable nutrient Suitable nutrient media 3. 3. Environmental Environmental condition cell free from media condition from microbes. microbes. 18/04/2017 16

17. • Laboratory Space- • In In general space for the following general space for the following is 1. 1. Washing, Washing, Drying Drying and and Storage is needed. needed. Storage of of Vessels Vessels 2. 2. Preparation, Preparation, Sterilization Media Media Sterilization and Storage and Storage of of 3. 3. Aseptic handling Aseptic handling of of explant explant and and cultures cultures 4. 4. Maintains of Maintains of culture culture 5. 5. observation observation of of culture culture 18/04/2017 17

18. Infrastructure & Major Equipment's 5/14/202 S.R.R. GOVERNMENT ARTS & SCIENCE COLLEGE 18 0

19. The The following following facilities facilities culture culture room room sho shou uld ld h ha ave ve the the 1. 1. Contr Contro olled 2 2° °C) C). . 2 25 5° 2. 2. Culture Culture racks 3. 3. A A sha shak ker cultures cultures. . lled tempera temperat ture °± ± racks fitted fitted with er for for ure (usua (usual lly ly with light light. . a ag gitat itati ion on of of Liquid Liquid 18/04/2017 19

20. • Generally, used as they are cheaper, autoclavable. • It It is is desirable desirable to or or pyrex pyrex glassware glassware glass glass may may be be toxic • Tissue are generally cultured in culture tube, flasks and Petri plate but many specially designed dishes are also used. glass culture vessels reusable and are to use use only as as to some some tissue only borosilicate borosilicate ordinary ordinary tissue. . soda soda toxic to 18/04/2017 20

21. • Culture vessels and in a suitable nontoxic other labware are generally soaked detergent solution overnight, washed with a suitable brush, thoroughly rinsed clean with tap distilled water and dried oven 70 - 80° C. • Washed culture vessels should be stored in a dust proof cabinet. water, followed by rinse with in a hot air 18/04/2017 21

24. Vitamins- growth, required- Inositol pyridoxine (B-3) of which thiamine is essential and the rest are promotory. Pantothenic acid is also known to be promotory. For the optimum Vitamins callus are The following (B-8) , (B-6) and nicotinic thiamine (B-1), acid 18/04/2017 24

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

28. Complex Organic Additives like- 1. 1. Yeast Yeast extract, extract, 2. 2. Coconut Coconut milk, milk, 3. 3. Casein Casein hydrolysate, hydrolysate, 4. 4. Corn Corn milk, milk, 5. 5. Malt extract Malt extract and 6. 6. Tomato Tomato juice juice -were used growth. Such additive synthetic media fail. – Complex additive and to support plant tissue should be used only when 18/04/2017 28

29. Tissue Culture Media Preparation

30. • All All the be be freed freed from one one of of the 1. 1. Dry Dry heat heat 2. 2. Autoclaving Autoclaving 3. 3. Filter Filter Sterilization Sterilization 4. 4. Wiping Wiping with with 70 5. 5. Surface Surface Sterilization Sterilization the material material used from microbes the following following method used in microbes. . This method. . in culture culture work This is is done work must done by must by 70% % Ethanol Ethanol 18/04/2017 30

31. • Culture containing sterilized by heating in autoclave or a pressure cooker to 121° C at 15 pound per square inch for 15 to 40 minutes the longer time being volume. • Sterilization during autoclaving depends mainly on temperature. vessels, (both empty generally and media) are for larger medium 18/04/2017 31

33. TECHNIQUES PERFORMED UNDER ASEPTIC CONDITIONS UNDER HEPA FILTERED AIR PROVIDED BY ALAMINAR FLOW CABINET STERILIZATION OFEXPLANTS EXPLANTS ARE PLACED OVER SOLID/LIQUID MEDIUM PROFOUND EFFECT ON THE MORPHOLOGY OFTISSUES CULTURES GROW PIECES ARE TYPICALLY SLICED OFF &TRANSFERRED TO NEWMEDIA SHOOTS EMERGE FROM CULTURE 33

35. • All All plant are are sterilizing sterilizing agent present present on Surface Surface Sterilization Sterilization. . • It depends mainly of tissue determine tolerance of the sterilizing agent. plant materials materials to treated treated agent to on their their surface, to be with with to inactive inactive the surface, this be used used for an an for culture culture appropriate appropriate the microbes microbes this is is called called on the source and type of the the contamination explant which load will and 18/04/2017 35

36. • The sterilizing Agent The sterilizing Agent used disinfection disinfection are • Calcium Hypochlorite Calcium Hypochlorite (9 • Sodium Hypochlorite Sodium Hypochlorite (2%) • Mercuric Chloride Mercuric Chloride (0.1-1%) • Silver nitrate Silver nitrate (1%) • Bromine water Bromine water (1 • H2O2(10-12%) • Antibiotic (4-50 mg/l) used for surface for surface are- - (9- -10%) 10%) (2%) commonly used (1%) (1- -2%) 2%) 18/04/2017 36

38. Laminar air flow inoculation in Laminar Flow Tissue culture rack 38

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41. Preparation of an explant Inoculation Afterincubation Selection of plant Various stages of plant growth Plant ready to be transferredinto green house or hardeningstage Regeneration of a plant from an explant 41

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