transgenic techniques

TISSUE CULTURE & APPLICATIONS

PRESENT BY

NASEEM BHURGRI

PH.D. SCHOLAR (BIOTECHNOLGY DEP UNIVERSITY OF SINDH)

WHAT IS IT?

• THE TERM TISSUE CULTURE IS

COMMONLY USED IN A VERY WIDE

SENSE TO INCLUDE IN VITRO CULTURE

OF PLANT CELL, TISSUE AS WELL AS

ORGAN

• THE TISSUE CULTURE INVOLVES BOTH

ANIMAL AND PLANT CELL.

HISTORY OF TISSUE CULTURE

Tissue culture had itsorigins at the beginning ofthe 20th century with theworkof Gottleib Haberlandt(plants) and Alexis Carrel(animals

THE BACKGROUND,I

• the first commercial use of plantclonal propagation on artificialmedia was in the germinationand growth of orchid plants, inthe 1920’s

• in the 1950’s and 60’s there wasa great deal of research, but itwas only after the developmentof a reliable artificial medium(murashige & skoog, 1962) thatplant tissue culture really ‘tookoff’ commercially

THE BACKGROUND, II

•a more recent advance is the use of

plant and animal tissue culture

along with genetic modification

using viral and bacterial vectors and

gene guns to create genetically

engineered organisms

“

”

DESIGN AND LAB SETUP OF TISSUE CULTURE LABORATORY

SETTING OF OF A TISSUE CULTURE LAB REQUIRES PROPER PLANNING

IT IS DIVIDES INTO 5 AREAS

Acclimatization

roomAnalytical

room

Aseptic

transfer

area

Culture

room

Media preparation room

MEDIA PREPARATION ROOM

• REFRIGERATOR AND FREEZER

• WATER PURIFICATION AND STORAGE SYSTEM

• GLASS WARE WASHING FACILITY

• CONTINUOUS SUPPLY OF SINGLE AND DOUBLE DISTILLED

WATER

• CULTURE MEDIA, WASHING POWDER DISINFECTANTS

• CABINETS OR SHELVES

ASEPTIC TRANSFER AREA

Laminar air flow

Dissecting microscope

Dissection instruments

Gas outlets vacuum facility

Sterilization

CULTURE ROOM

•Environmentally

controlled

• Incubators with

controlled

temperature

•Rotary shakers

•Space for culturing

requiring complete

darkness

Culture

Room

ANALYTICAL ROOM

COLORIMETER

LOW SPEED

CENTRIFUGED

INVERTED

CENTRIFUGED

CHEMICAL REAGENT

RACKS

VISCOSITY METER

GAS OUTLETS

ACCLIMATIZATION ROOM

•HIGH ILLUMINATION

(4,000-10,000 LUX)

•HIGH HUMIDITY

OTHER ITEMS

• AIR CONDITIONS

• UNINTERRUPTED POWER SUPPLY

• BUNSEN BURNER

• ALUMINUM FOILS

• FLUORESCENTS LAMPS

• FIRE FIGHTING EQUIPMENT'S

MICRO ELEMENTS

ORGANIC COMPOUNDS

GROWTH REGULATORS

CONTI:

ROLE OF PLANT HORMONE IN GROTH OF PLANTS

A PLANT HORMONE CAN BE DEFINED AS A

SMALL ORGANIC MOLECULES THAT ELICITS

A PHYSIOLOGICAL RESPONSE AT VERY LOW

CONC.

PLANT GROWTH HORMONE PLAY AN

IMPORTANT ROLE IN THE PHENOTYPES

ACT AS MESSENGER BETWEEN

ENVIRONMENT AND THE GENOME

AUXIN

• essential for cell division, cell

elongation, cell differentiation,

organogenesis and embryo genesis,

callus formation.

• natural form auxin are iaa, iba,.

• synthetic form of auxin are naa,

2,4d.

CYTOKINIS

• cytokine promoting cell division, shoot

proliferation and influence the cell cycle

• embryogenesis and inhibit roof formation

• synthetic form is 2-ip which is most

active form of cytokine

• natural forms are bap and kinetin

GIBBERELLINS

• it promotes stem elongation, bulb corn formation,

embryo maturation, but can inhibit callus and root

induction

• ga3 is most common gibberellins.

ABSCISIC ACID

it inhibit root growth and

germination of embryo

it is thermostable but light

sensitive.

TYPES OF PLANT TISSUE CULTURE

Plant

tissue

culture

Embryo

culture

Meriste

ms

culture

Protopl

ast

cultureOrgan

culture

Callus

culture

Cell

culture

CALLUS CULTURE

callus culture means anorganized proliferative massof cells produced romisolated plant cells, tissue oforgan when grown asceticallyon artificial nutrient media.

it produced when explant areculture on the appropriatesolid media, with both ofauxin and cytokine in correctcondition. 2.4 d arecommonly used.

EMBRYO CULTURE

• DEFINATION :

• embryo culture can be defined as the in vitro culture

either of the polarized egg, zygote, proembryo or mature

embryo.

CELL CULTURE

• cell culture refers to the

removal of cells from an

animal or plant and their

subsequent growth in a

favorable artificial

environment.

MERISTEM CULTURE

• a cell culture

developed from a

small portion of the

meristem (growing

tip) tissue of a plant.

either a stem shoot or

root meristem can be

used

ORGAN CULTURE

• organ culture is a development

from tissue culture methods of

research, the organ culture is able

to accurately model functions of

an organ in various states and

conditions by the use of the

actual in vitro organ itself

Organ culture

Vegetative

organ

Root culture Leaf cultureShoot tip

culture

Reproductive

organ

Complete

flower culture

Isolated ovary

culture

Isolated ovule

and embryo

culture

SUSPENSION CULTURE

Can be initiated from any part of the plant.

Usually initiated from friable callus already growing in culture.

Transferred into liquid medium

PROTOPLAST CULTURE

• ISOLATED PROTOPLAST HAVE BEEN DESCRIBED AS A NAKED CELLS BECAUSE THE

CELL WALL HAS BEEN REMOVED BY EITHER A MECHANICAL AL AND ENZYMATIC

METHOD

CONTI:

Isolation of protoplast Separation of protoplast from

plant tissue

Enzymatic method

Mechanical

method

MECHANICAL METHOD

ENZYMATIC METHOD

ANTHER CULUTRE

APPLICATION OF TISSUE CULTURE

Medicine

and

research

There are various application of tissue

culture in a wide array of scientific

industries

Cancer cells,, are analyzed in vitro to

produced appropriate medicines that will

eliminate the abnormal cells

Skin grafting,, where severely burned

tissues are replaced with new ones using

culture cells

APPLICATION

Plant fiber culture

One of the most common types of tissue

culturing is conducted in botanical and

agricultural research.

Plant tissue is much easier to grow in a

nutrient rich suspension than human or

animal tissue, and with the right nutreints

different tissue samples can often be

encouraged to develop as they would on a

normal plant suspension

CONTI;

APPLICATION OF PLANT TISSUE CULTURE IN INDUSTIRES

PLANT CELL CULTURE IS USED FOR BIOTRANSFORMATION (MODIFICATION OF

FUNCTIONAL GROUPS OF ORGANIC COMPOUNDS BY LIVING CELLS.

FOOD AND AGRICULTURAL BIOTECHNOLOGISTS ARE INVOLVED IN USING TOOLS

OF MOLECULAR BIOLOGY TO ENHANCE THE QUALITY AND QUANTITY OF FOODS

AND ECONOMIC CROPS.

CONTI:

• FOR EXAMPLE, GOLDEN RICE WAS GENETICALLY ENHANCED WITH ADDED BETA

CAROTENE, WHICH IS A PRECURSOR TO VITAMIN A IN THE HUMAN BODY. PLANT

CELLS CAN BE CULTURED IN FERMENTERS FOR THE INDUSTRIAL PRODUCTION OF

SECONDARY METABOLITES USING CELL CULTURE.

BENEFITS OF TISSUE CULTURE

CONTI;

CONCLUSION

REFERENCES

REFERENCES D.DUMET,A.ADEYEMI,O.B.OJUEDERIE (2008). YAM INVITRO

GENEBANKING. GENEBANK MANUAL.

HTTP://WWW.IITA.ORG/GENEBANK/MANUAL

D.DUMET,A.ADEYEMI,O.B.OJUEDERIE (2008).

CASSAVA IN VITRO PROCESSING AND THE GENEBANKING.GENEBANK

MANUAL. HTTP://WWW.IITA.ORG/GENEBANK/MANUAL HORT689/AGRO689

BIOTECHNIQUES IN PLANT BREEDING H.S CHAWLA .2002 INTRODUCTION TO

PLANT BIOTECHNOLOGY 2ND EDITION. OXFORD & IBH PUBLISHING C./ PVT. LTD

NEW DELHI INDIA MURASHIGE T. AND SKOOG F. (1962). A

CONTI:

• A REVISED MEDIUM FOR RAPID GROWTH AND BIOASSAY WITHTOBACCO TISSUE CULTURE. PHYSIOLOGIAPLANTARUM 15: 473-497.

• HTTP://AGGIE-HORTICULTURE.TAMU.EDU /TISSCULT/PLTISSUE/PLTISSUE.HTML

• HTTP://WWW.LIV.AC.UK/~SD21/TISSCULT/WHAT.HTM

• WWW-PLB.UCDAVIS.EDU/COURSES/S99/PLB111I/TCMEDIUM.HTML

• HTTP://MEMBERS.AOL.COM/MRDJREED/PRIVATE/PTC.HTML

• HTTP://WWW.ACCESSEXCELLENCE.ORG/LC/ST/ST2BGPLANTPREP.HTML

• WWW.OSMS.OTAGO.AC.NZ/MAIN/BURSARY

• HTTP://WWW.KITCHENCULTUREKIT.COM/HISTORYTC.HTM

• HTTP://WWW.SIGMAALDRICH.COM/AREA_OF_INTEREST/LIFE_SCIENCE/CELL_CULTURE/HELPFUL_RESOURCES/CE

LL_CULTURE_HANDBOOK,HTM