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PLANT TISSUE CULTURE :

MICROPROPAGATION
TECHNIQUES
Department of biotechnology
N.C.College of Engineering,Israna(Panipat)

Work carried out under


Dr. Subash Kajla
Senior Scientific Officer
Project Guide,Micropropagation
Project
At
Centre for Plant Biotech.,CCSHAU,Hisar
By
Ashish Katyal

Outline
Introduction
Need
Stages of Micropropagation
Experimental Work
Applications of Plant Tissue culture
Results & Discussions

Introduction
What is Plant Tissue
Culture
It is the technique of growing plant cells,
tissues, organs, seeds
or other plant parts in a sterile environment
on a nutrient medium

Plant Tissue Culture = Plant Cloning

Why We Need Tissue Culture?

Quick multiplication of plants that are rare, elite or


face extinction.
Progeny is disease free & true to type.
High multiplication rates
Export of high quality planting material

A Totipotent Cell
Morgan in 1901 gave the term Totipotent
Cell implying that a cell is capable of
developing into a whole organism.
Plant Cells are Totipotent
In 1902 Haberlandt :
Gave Practical shape to
Totipotency Concept
Attempted to culture
cells

Stages of Micropropagation
Stages 0 to 4
Stage 1: Explants in initiation medium
Stage 2: Newly formed bud, shootlet or callus
are cultured on multiplication media
Stage 3: Shootlets that are formed are
transferred to rooting media
Stage 4: Rooted plantlets are hardened
Stage 0: Grow the parent plants in sterile
conditions or glasshouse.

Tissue Culture Stages

GENERAL PROCEDURE
Explants Collected
Inoculated in Initial bud proliferation
medium
Transferred to Multiplication medium
Sub-cultured, rooted, hardened and
planted.

Micropropagation
Large scale multiplication of high value plant
material
Normally use meristem as explant
meristem is the growing point of a plant
consists of cells which are undifferentiated
All regenerated material are clones

Micropropagation
Allows for continuous production
Permits seasonal production
Allows for long term preservation of plants
Small space requirements
Because the meristem is starting material,
products are virus and disease free

Experimental Work
TISSUE CULTURE IN SUGARCANE
STEPS INVOLVED:
Washing
Explant Preparation
Media Preparation
Sterilization
Inoculation
Hardening

Washing

Explant Preparation

Media Preparation

Composition of MS media
(Murashige and Skoog, 1962)
For SUGER CANE cultures
Micronutrients
MnSO4.4H2O
ZnSO4.4H2O
H3BO3
Kl
Na2MoO4.2H2O
CuSO4.5H2O
CaCl2.6H2O

mg/l
22.3
8.6
6.2
0.83
0.25
0.025
0.025

Macronutrients
(NH4)NO3
KNO3
CaCl2.2H2O
MgSO4.6H2O
KH2PO4
Iron
Na2EDTA
FeSO4.7H2O

mg/1
1,650
1,900
440
370
170
mg/l
33.6
27.8

Vitamins
nicotinic acid
pyridoxine.HCl
thiamine.HCl
Cytokinin
Kinetin

mg/l
0.05
0.05
0.01
mg/l
0.1

Other components

Glycine
myo-inositol
IAA
Sucrose
Agar (0.8% w/v)
pH 5.7

mg/l
0.2
100
10
30,000

Sterilization

Sterilized Media Storage

Inoculation

Primary Storage

Secondary Storage

Hardening

Hardening Of Tissue Culture Plants

Applications of Plant Tissue culture


Has been referred to as biotechnology of developing
world
Low capital costs relative to other technologies
High labor input (labor is cheap in most developing
countries)
Micropropagation $$$$$$$$$
Artificial seeds and forestry
In vitro breeding
interspecific crosses
haploid breeding
Somaclonal variation and cell selection
Cell culture (similar to fermentation)

Results

Of Course
Challenges and
Opportunities
are always
two sides
of the same coin