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By I.Mary Thanuja

Under the guidance ofMr.R.Karthikeyan M.Pharm.

Asst. professor,Dept. of Pharmacognosy,

VIGNAN PHARMACY COLLEGE(Approved by AICTE & PCI Affiliated to JNTU KAKINADA)

VADLAMUDI, GUNTUR DIST, ANDHRA PRADESH, INDIA, PIN: 522 213

PLANT TISSUE CULTURE

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CONTENTS:

Definition History Nutrient’s requirement Preparation of medium Sterilization of medium Basic requirement of tissue culture laboratory Establishment of plant tissue culture Growth profile Growth determination Types of culture Advantages Applications

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PLANT TISSUE CULTURE

Definition

Tissue culture is in vitro cultivation of plant cell or tissue under aseptic and controlled environmental conditions, in liquid or on

semisolid well defined nutrient medium for the production of primary and secondary metabolites or to regenerate plant.

Tissue culture relies on three fundamental abilities of plant there are:

Totipotency

Dedifferentiation

competency

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Haberlandt early 1900’S• proposed concept of totipotency• cells cultured under right conditions• Callus cultured from tree cambium

Gautheret, Nobecourt, Whire

In the 1930s.• cells kept alive but did not develop

HISTORY

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NUTRIENT’S REQUIREMENT

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COUMPOUNDS Mg/MlNH4NO3 1,650.00

KNO3 1,900.00

CaCl2 (anhyd) 332.20

MgSO4 (anhyd) 180.70

KH2PO4 170.00

Na2EDTA 37.25

FeSO4.7H2O 27.80

H3BO3 6.20MnSO4.H2O 16.90

ZnSO4.H2O 5.37

KI 0.83

Na2Mo4.2H2O 0.25

Sucrose 30,000.00

i-Inositol 100.00

Thiamine.HCl 0.40

INORGANIC & ORGANIC SUPLLEMENTS

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Antibiotics : Stertomycin,kanamycinActivated charcoal

Other organic supplements : Protein, coconut milk,yest,malt extract, orange juice, and tomato juiceGrowth regulators : Auxins,cytokininsWater : Demineralized or distilled waterSolidifying agents : Agar, gelatin.pH adjusters : 5 - 6 it is considered to be optimum.

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PREPARATION OF CULTURE MEDIA:

Stock solution 1 : MgSo4,KH2Po4,KNO3,NH4No3,CaCl2

Stock solution 2 : H3Bo3,MnSo4,ZnSo4,CuSo4,Cocl2

Stock solution 3 : FeSo4,sodium EDTA

Stock solution 4 : ionositol,thiamine,pyridamine,nicotinic acid,glycine

To prepare 1 liter of medium:

Take 50 ml of stock solution 1 + 5ml of stock solution 2 & 4 in a beaker. The stock solution 3 prepared separately in a other 450ml flask by adding double distilled water and heat with constant stirring.Mix two solutions and adjust PH to 5.5.

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STERILISATION OF MEDIA

•The prepared media should be sterilized by ISI mark Autoclave( for large amounts) at 121º Domestic pressure cookers( for small amounts)

•For the sterilization of glassware and metallic equipments Hot air oven with adjustable tray is required.

Incubator Hot air oven

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BASIC REQUIREMENT FOR A TISSUE CULTURE LABORATORY

For the successful achievement, the following general basic facilities are required:

Equipment & apparatus Washing and storage facilities Media preparation room Sterilization room Aseptic chamber for culture Culture rooms or incubators fully equipped with temperature, light and humidity control devices Observation or recording area well equipped with computer for data processing

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TISSUE CULTURE LABORATORY

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EQUIPMENT & APPARATUS

VESSELS & GLASS WARE :

• All the glassware should be of Pyrex.• Large test tubes,flasks,graduated pipettes etc.. are used.

EQUIPMENT :

• Scissors,scapels,foreceps are used for explants preparation.• A spirit burner for flame sterilization.• Hot air oven.• A PH meter.• A BOD incubator.• Laminar air flow chamber.• A balance to weigh nutrients.• Data collection and recording room.

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Laminar air flow chamber

Knife

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DATA COLLECTION & RECORDING ROOM

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ESTABLISHMENT OF PLANT TISSUE CULTUREIn vitro culturing of plant tissue culture involves the following steps. Collecting & sterilization of glassware tools/vessels. Preparation of explant. Surface sterilization of Explant. Production of callus from explant. Proliferation of culture. Sub culturing of callus. Suspension culture

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EXPLANT PREPARATION

EXPLANT : It is defined as a portion of plant body, which has been taken from the plant to establish a culture• Explant may be taken from any part of the plant like root,stem,leaf,or meristematic tissue like cambium, floral parts like anthers, stamens etc..•Age of the explant.• Homozygous plants are preferred.

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flower

leaf

SURFACE STERILISATION OF EXPLANTFor surface sterilization chromic acid, Hgcl(0.11%),calcium hypochlorite, sodium hypochlorite(1-2%),alcohal(70%) are used.Process depends on the type of explant.SEED : absolute ethyl alcohol calcium hypochlorite bromine water sterile waterFRUIT : ethyl alcohol sodium hypochlorite sterile waterSTEM : running water sodium hypochlorite sterile waterLEAF : surface clean Hgcl2 sterile water dried

explant

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PRODUCTION OF CALLUS FROM EXPLANT

• Sterilized explant is transferred aseptically onto defined medium.• Transfer to BOD incubator.• Temperature (25 ± 2 G) and light is necessary for callus production.• Callus produced with in 3-8 days.

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PROLIFERATION OF CULTURE

• if callus is well developed, it should cut into small pieces & transferred to another fresh medium containing hormones, which supports growth.•The medium used for production of more amount of callus is called proliferation medium.

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CALLUS GROWTH15 DAYS

30 DAYS 50 DAYS 80 DAYS

SUBCULTURING OF CALLUS

•After sufficient growth of callus it should be periodically transferred to fresh medium to maintain viability of cells.

•This subculture will be done at the interval of 4-6 weeks.

•After a maximum growth transfer into a pottling soil under required condition.

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SUSPENSION CULTURE

•It contains a uniform suspension of separate cells in a liquid medium callus

liquid medium

agitated continuously

finally cells separated sub-culture the cells

•This can be achieved by rotary shaker attached within the incubator at a rate of 50-150 rpm.

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GROWTH PROFILE OF PLANT TISSUE CULTURE

They are classified as :

Single cell cultureCallus culture

SINGLE CELL CULTURE

The single cell culture exhibits various stages of growth.

a) Lag phase: Tissue starts to grow.

b) Exponential phase: This phase is characterized by rapid cell multiplication.

c) Linear phase: The growth follows a linear pattern with respect to time

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d) Progressive declaration phase: Aging of culture increase cell division decreasese) Stationary phase: No growth of cells occur Rate of production of cells = rate of their death f) Senescent phase: Cell death occurs to lack of nutrients

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IN CALLUS CULTURE

a) Lag phase: In this phase cell trying to adjust the new environment condition.b) Exponential phase : By utilizing nutrients rapid multiplication occurs.c) Decline phase : Due to starvation some cells leads to decline in the callus culture.d) Stationary phase : No growth is evident, requires sub culturing.

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GROWTH DETERMINATIONMethods used to determine are..

CELL NUMBER• By counting the cell number in haemocytometer under a microscope.•Suspension culture is preferable.

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PACKED CELL VOLUME

•Cell suspension is transfer to graduated centrifuge.•Centrifuged at 2000 rpm for 5mints.•Cell will form pellets called biomass volume, expressed by ml-1

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FRESH CELL WEIGHT

•When cells increase in number, the liquid will be turbid.•As a result optical density altered, detected by colorimeter.

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VIABLE CELL TEST

•The staining method such as fluorescein di-acetate is used for accessing the cell viability.•Dead cells appear as fluorescein red.

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TYPES OF CULTURECallus culture

Suspension culture

Root tip culture

Leaf or leaf primordial culture

Shoot tip culture

Complete flower culture

Anther & pollen culture

Ovule & embryo culture

Protoplast culture

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Callus culture Suspension culture

Pollen culture

Ovule culture Root tip culture Shoot tip culture

Protoplast culture Leaf primordial culture Flower culture

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ADVANTAGES :

Availability of raw materials.

Fluctuation in supplies & quantity.

Patent rights.

Political reasons.

Easy purification of the compound.

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Modifications of chemical structure.

Disease free & desired propagule.

Crop improvement.

Bio-synthetic pathway.

Immobilization of cell.

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APPLICATIONS alkaloids virus-free plants forest trees

saponins apical meristem culture fruit crops secondary metabolite PTC in industry micro propagation steroids anther or pollen culture vegetatively propagated plants antitumor haploid & homozygous lines plantation crop Bio pesticides food additives essential oil yielding plants chemicals

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REFERENCES :

• Trease and evans pharmacognosy.W.C evans-page num :72,73,74.

•A textbook of industrial pharmacognosy by A.N Kalia.Page num-105 to 114.

• A textbook of pharmacognosy by M.K Gupta & P.K Sharma. Page num-171 to 185.

• Pharmacognosy and photochemistry-part 2 by Vinod D Rangari. Page num-51 to 56.

• Internet source.

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THANK YOU..

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Who is the father of tissue culture ? Haberlandt

The production of secondary metabolites requires the use of ?Cell suspension

Synthetic seed is produced by encapsulating somatic embryo with Sodium alginate

Hormone pair required for a callus to differentiate are ?Auxin & cytokine

DMSO (dimethyl sulfoxide) is used as ?Cryoprotectant

The most widely used chemical for protoplast fusion, as fusogen isPoly ethylene glycol (PEG)

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Callus is ?Unorganized actively dividing mass of cell, maintained in culture

Which of the plant cell will show totipotency ?Meristem

To obtain haploid plant, we culture ?Entire anther

Growth hormone produce apical dominance ?Auxin

The vector mostly used in crop improvement ?Agro bacterium

A media which contains chemically defined compound is called ?Synthetic medium

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