This document discusses plant tissue culture, including its history, essential facilities and equipment needed, composition of culture media, preparation of stock solutions and media, sterilization methods, measurement of growth, and types of culture. It notes that tissue culture developed from embryology techniques and involves growing small tissue explants aseptically on defined media. Essential facilities include an incubator, autoclave, microscope, and culture room, while media contains major and trace nutrients, vitamins, carbon source, and plant growth regulators.

1. G.R.Sitaphale -Assistant Professor
Anuradha college of Pharmacy.Chikhli

2. Development of the Biotech 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 of biotechnology 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 towards the 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 requirements for
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-air circulation 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 can make 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-70 0
 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 culture media
 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
 Required in 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 plant thrives 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
 Most common 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 stock solution
 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
 Many companies produce ready to use salts
and vitamin mixture which can be easily
handled by adding proper amount of powder
to water.

18. Vitamin PGRS and others
 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 in media 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 growth parameters
 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 Plant tissue culture
 Production of phytopharmaceuticals
 Biochemical conversion
 Colonal propagation
 Production of immobilized plant cells.