1. Laboratory Arrangements, Aseptic Technique, Media, Plant Growth Hormones Jebunnesa Chowdhury

2. Components of Plant Tissue Culture Lab. 1.Cleaning and storage of supplies 2.Media preparation, sterilization and storage of sterilized material 3.Aseptic manipulation of plant material 4.Growth of the culture under controlled environmental condition 5.Observation or data collection area In addition to this 6. Adequate place for hardening of plant tissue culture derived material

3. Cleaning and Storage Supplies 1.Water connection for washing 2.Automatic dish washer or brushes 3.Detergent bath 4.Tap water and distilled water 5.Oven to dry the glassware 6.Autoclave Q. What is the need of distilled water? Q. What is the need of autoclaving the discards?

4. Media preparation, sterilization and storage of sterilized material 1.All the media components 2.A fine balance 3.pH meter 4.Microwave or hot water bath 5.Refrigerator 6.Distilled water 7.A combination of hot plate and magnetic stirrer 8. Sterilization equipments: autoclave or pressure cooker, filters Q. What needs to be filter sterilized?

5. Area for Aseptic Manipulation 1.Transfer room: very clean and dry atmosphere (why?) 2.Laminar flow: vertical and horizontal 3.Spirit lamp or bunsen burner with gas connection 4.Ethyl alcohol (96%) 5.Tiles or glass plates for using during sterile cutting 6.Forceps and scalpels with sterile blades 7.Hypochlorite solution (why?)

6. Growth Room: Incubation of the Cultures 1.Temperature controller 2.Controlled light: duration or photoperiod, light quality, intensity 3.Humidifier 4.Plant culture shelves 5.Shakers for liquid cultures

7. Observation examination and evaluation of plant growth 1.Hand lens 2.Dissecting microscopes 3.Compound microscope with photomicrograph accessories

8. Growth of Tissue Culture Derived Plant Materials

9. 1.Glassware (hot air oven/ autoclave) 2.Instruments (autoclave & ethanol) 3.Culture room and transfer area (surfactants: detol, savlon, UV light) 4.Nutritional media (autoclave, filter) 5.Plant materials (disinfectants: ethanol, hypochlorite sol., mercuric chloride) Aseptic Techniques Sterilization of Q. What is the difference of detergent and disinfectants?

10. Aseptic Techniques Autoclave (15 lbs/in 2 at 121º C for 15 min) Filter sterilization Laminar Air Flow Cabinet (working bench) Chemical sterilization Ethanol Na/ Ca-Hypochlorite Clorox/ bleach Mercuric chloride (HgCl 2 ) Tobacco seeds: 5 min ethanol Peanut seeds: 5 min NaOCl Lentil seeds: detergent wash then 30 sec ethanol wash then 25 min HgCl 2 Personal hygiene Q. What is the condition for autoclaving? Q. Why to use autoclaved distilled water after clorox usage?

11. Media and Plant Growth Hormones Media provides optimum nutritional support for growth and morphogenesis of in vitro grown plant tissue. Principal components 1.Inorganic nutrients 2.Carbon source 3.Organic supplements 4.Growth hormones 5.A gelling agent (in case of solid medium) medium containing only ‘chemically defined compounds’ Examples: Murashige and Skoog medium or MS medium White’s medium Synthetic medium:

12. Media: Inorganic Nutrients Two types: 1. Macronutrients, and 2. Micronutrients Apart from C, H and O there are 12 inorganic elements are known to be essential for plants growth Macronutrients: N, P, S, Ca, K, Mg required by the plants more than 0.5 mmol/l MS medium: MgSO 4.7H 2 O, KH 2 PO 4, KNO 3, NH 4 NO 3, CaCl 2.2H 2 O Micronutrients: Fe, Mn, Cu, Zn,, B, Mo required by the plants less than 0.5 mmol/l MS medium: H 3 BO 3, MnSO 4.4H 2 O, ZnSO 4.7H 2 O, Na 2 MoO 4.2H 2 O, CuSO 4.5H 2 O, CoCl 2.6H 2 O, KI, FeSO 4.7H 2 O, Na-EDTA.2H 2 0 Q. Why there are two sources of Nitrogen?

13. Media: Inorganic Nutrients Carbon and Energy Source Most preferred ‘C’ source is Sucrose. Glucose supports equally good while fructose is less efficient. Sucrose Glucose + Fructose Utilization: first glucose, then fructose Autoclave Hydrolysis Plant cell and tissue in culture condition can not trap carbon, i.e. lack autotrophic ability. Therefore, need external carbon for energy.

14. Media: Organic Nutrients 1. Vitamins 2. Amino acids 3. Complex organic supplements

15. Media: Organic Nutrients Vitamins Plant synthesis vitamins endogenously at sub- optimal quantities. For this, reason to achieve best growth of tissue it is needed to add vitamins as supplements in the media. Vitamins used: 1.Thiamine (vitamin B1) 2.Nicotinic acid (vitamin B3) 3.Pyridoxine (vitamin B6) 4.Calcium pantothenate (vitamin B5) 5.Myo-inositol Vitamins have catalytic function in metabolic processes.

16. Media: Organic Nutrients Amino acids: Only Glycine is added. Complex organic supplements: these are organic extracts which have constituents of an undefined nature. Examples: Casein hydrolysate Fruit juices: tomato juice Fruit extracts: banana fruit extract Coconut milk Yeast extract Factors: Age of the tissue and the variety of the donor organism Q. Why use of complex organic mixture should be avoided though it is nutritive for the plant?

17. Media: Plant Growth Hormone Mainly 3 plant hormones are used: 1. Auxins 2, Cytokinins 3. Gibberellins

18. Plant Growth Hormone: Auxins Functions of Auxins: 1.Elongation of stem and internodes 2.Apical dominance 3.Tropism 4.Rooting In nature In tissue culture

19. Auxins used in Tissue culture: 1.IBA (Indole-3-butyric acid) 2.NAA (naphthalenacetic acid) 3.IAA ((Indole-3-acetic acid) (naturally occurring) 1.2,4-D (dichloro-phenoxyacetic acid) Rooting Callus induction and growth MS + 0.2 mg/l NAA Plant Growth Hormone: Auxins Peanut

20. Plant Growth Hormone: Cytokinins Functions of Cytokinins : 1.Cell division 2.Modification of apical dominance 3.Shoot differentiation 4.Cell division and differentiation of adventitious shoots from callus and organ 5.Shoot proliferation In nature In tissue culture

21. Cytokinins used in Tissue culture: 1.BAP (benzylamino purine) 2.Kinetin (furfurylamino purine) 3.Zeatin (naturally occurring) Plant Growth Hormone: Cytokinins MS + 5 mg/l BAP Peanut MS + 5 mg/l BAP +0.5 mg/l Kn

22. Plant Growth Hormone: Gibberellins Function: 1.Very rarely used 2.Elongation of shoot under in vitro condition Example: GA 3

23. Hormone Habituation Habituation Biosynthesis of auxins or cytokinins by the culture tissue which lead to autonomous state is called habituation. These cultures do not need external supplies for that specific hormone.

24. Medium matrix Commonly used gelling agent: AGAR Advantage of Agar: 1.agar gel do not react with media constituent 2.They are not digested by plant enzymes and 3.remain stable in all culture incubation temperature Gelling or solidifying agents are commonly used for preparation of solid tissue culture medium. Q. What will happen if the correct concentration of agar is not used? If medium consistency is hard then it does not allow the diffusion of nutrients into tissue. Agar is not an essential component of the nutrient medium.

25. Medium pH Plant cells and tissues require optimum pH for growth and development in culture. The pH affect uptake of ions. Optimum pH is 5.8 set before autoclaving. So pH is adjusted between 5.0 and 6.0 before autoclaving. In general, a pH higher than 6.0 gives hard medium and a pH below 5.0 makes the medium soft. Q. What are the two reasons that resulted into hard medium?