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Plant tissue culture for b.pharm
- 1. G.R.Sitaphale -Assistant Professor Anuradhacollege of Pharmacy.Chikhli
- 2. Development of theBiotech is undoubtedly one of the most significant contribution of modern biologist.Tissue culture had developed quite naturally from some of techniques of embroyology. It is a proess whereby small piece of living tissues called as explants are isolated from an organism and grown aseptically for indefinite period of time on a defined nutrient medium.
- 3. The definition ofbiotechnology includes the development of methods by which biological process may be controlled such that their rate of production enables economic industrial production. Totipotency-Ability of plant material to grow and divide invitro is called totipotency. Cell totipotency is the potentiality of cell to regenerate the whole organism from a single cell.It was first discovered by Hyberlant in 1902.
- 4. First step towardsthe technique for the cultivation of tissues outside the body of an organism was a realization of internal environment. History 1860-Sach and Knop 1878-Vochtung Moral 1960 WIMBER 1963
- 5. Facitities and requirementsfor PTC Essential Facilities Beneficial facilitied Useful facilities ESSENTIAL FACILITIES are the bare minimum requirement without which the job cannot be performed.
- 6. ESSENTIAL FACILITIES Incubator-aircirculation temp AUTOCLAVE Refrigrater Microscope Washing up equipments Oven for dry sterilization Water purification Centrifuge Shelves Culture room and cabinate Laminar air flow cabinate
- 7. Beneficial equipments-equipments which canmake laboratory easier to use and more efficient Laminar flow hood Cell counter Vaccum pump CO2 incubator Osmometer Densitometer Microscope roller raks Magnetic stirrer Phase contrast fluroscent microsc. Separate sterilization oven
- 8. Useful facilities Freezer-700 Colony counter Glassware washing machine
- 9. Plant Tissue Culture Historical view of the advances in plant tissue culture revels a closer relationship with the progress in research and new information on the nutritional requirement of cultural cells. Although the general nutriyional requirement of PTC have been well established even today there are still a Considreable number of problems to be evacuated
- 10. Composition of culture A perfect balance of nutritional and hormonal requirement. Cell culture,cell suspension culture,somatic embryo or protoplast culture. A large number of different culture media are described. But of these only few like MS,SH B5 have found wide spread usage for a range of plant species.
- 11. Composition of culturemedia The components of nearly all plant culture media can be divided in to six groups Major inorganic nutrients(macronutrients,Macroelements) Trace elements(microelements) Iron source Vitamins Carbon source Plant growth Regulators
- 12. Major inorganic elements Nitrogen-N-enhance somatic embryogenesis Phosphrous-P-essential element of nucilc acid DNA RNA Potassium-K-catalytic process of cell Calcium-Ca-organisation of cell wall Magnesium-Mg-constituent of chlorophyll Sulphur-S-catalytic process of cell
- 13. Trace elements Requiredin small quantities. BO-boron Mn-Magnese Zn-Zinc Cu-Copper Cl-Chlorine Mo-Molybdenum Ni-Nickel Al-Aluminium Co-cobalt I-Iodine Fe-Ferrous Na-Sodium
- 14. In general plantthrives on 17 elements ,4 (C,H,O,N) from atmosph. And rest 13 from soil.(K,Ca,Mg,P,S,Fe,Cu,Mn,Mo,Zn,B,Co,Ni) Iron source Vitamins Carbon source Sucrose PGRS
- 15. Media Formulation Mostcommon media formulation is MS medium formulated for culture of Tobacco explants. Gamberg,s B5 medium made for soyabean callus culture high nitrates. Hiberlants 1972 SH medium for monocotyledons and Dicotyledons culture.
- 16. Preparation of stocksolution Most efficient way of preparing PTC media is first to prepare stock solution of mineral salt and vitamins which are ranging from 10-1000 times the final conc. Stock solution can be prepared as two solutions contain all micronutrients. Salts stock solutions keep dilutions 100x.
- 17. Prepared Mixtures Manycompanies produce ready to use salts and vitamin mixture which can be easily handled by adding proper amount of powder to water.
- 18. Vitamin PGRS andothers Most organic addenda added to medium in relatively low conc.As it is very difficult to weigh out less than 10mg accurately it is adventagenous to prepare stock solution.
- 19. Water Water inmedia is type-Iiwater as it contains very little solids and is produced through distillation ion exchange reverse osmosis.Tap water should not be used. AGAR For semisolid agar medis Agar conc 6-10g/lit prior to autoclaving. PH of most nutrient media should be 5.5-6.0.
- 20. Sterilization of media. Autoclave sterlization 121 degree celcius for 15 min 1.05 Kg/cm2 Filter sterlization 0.22 mm membrane made of nitrocellulose. PREPRATION OF EXPLANTS- For initation of cultures.
- 21. Measurement of growthparameters Growth of any tissue callus or cell suspension culture can be monitered by increase in fresh or dry weight or increase in cell number. 1)Fresh or dry weight measurement.(preweighed nylon fabric,preweighed centrifuge tube) 2)Increase in cell number or cell count.(Haemocytometer) 3)Packed cell volume% of volume of pallet to entire cult volume 4)Cell viability-Microscopic exam.cytoplasmic streaming. 5)Protein & DNA content. 6)Mitotic index. 7)Plating of cell suspension.
- 22. Types of culture Root tip culture Leaves or leaf primordia culture Shoot tip culture Complete flower culture Anther and pollen culture Ovule and embryo culture Protoplast culture Hairy root culture Callus culture Suspension culture
- 23. Applications of Planttissue culture Production of phytopharmaceuticals Biochemical conversion Colonal propagation Production of immobilized plant cells.