Basics of PTC (1).pdf basics of tissue culture

BASICS OF PLANT TISSUE CULTURE
T.Y.B.Sc.
BO345 Plant Biotechnology
Sem. IV
BY
Dr. KIRTI M. NITNAWARE
ASSISTANT PROFESSOR
DEPARTMENT OF BOTANY
HUTATMA RAJGURU MAHAVIDYALAYA, RAJGURUNAGAR

Introduction
 What is plant tissue culture?
◦ The growth or regeneration of plant cells,
tissues, organs or whole plants in artificial
medium under aseptic conditions.
 In vitro culture
◦ From Latin- “within the glass” performing an
experiment in a test tube.
Dr. K. M. Nitnaware HRM

History of Plant Tissue Culture
First attempt of plant tissue culture
Callus culture
1902
1939
Haberdlandt
Maheshwari Guha
1964
First haploid culture

Murashige Skoog
1962
MS media
1966 Steward Totipotency carrot plant
1970 Power et al Protoplast fusion
1977 Chilton Integrated Ti plasmid
from Agrobacgerium
tumefaciens in plant
1984 Horshet et al Transgenic tobacco
1987 Kleinet Biolistic gene transfer

Terminology
Explant: A plant organ or piece of tissue used to initiate a culture.
Culture: Growing cells, tissue, plant, organs or whole plant in nutrient
medium under aseptic condition. Eg. cell culture, embryo culture, anther
culture
Aseptic culture : Raising cultures from a tissue or an organ after
freeing it of bacteria, fungi and other microorganism.
Callus: A tissue arsing from disorganized proliferation of cells either in
cultures or in nature. Plural calli
Cell culture: Culture of single cells or small groups of similar cells
Ref:PlantTissue CultureTheory and Practice,a Revised Edition S.S. Bhojwani and M.K Razdan

Contamination: Unwanted microorganism which may inhibit the
growth of the cells or tissues in culture.
 Subculture: Transfer of tissue or explant into fresh media
Regeneration-: In tissue cultures, a morphogenetic response that results
in the production of new organs, embryo or whole plants from cultured
explant or calli derived from them.
Dedifferentiation: Reversal of organized structures into an
undifferentiated state.
Differentiation : The development of different morphological
characteristics. In tissue culture the term is used to describe the formation
of different cell type, roots, shoots, embryos or any other orgsns in callus
or cell culture.
Ref:PlantTissue CultureTheory and Practice,a Revised Edition S.S. Bhojwani and M.K Razdan

Micropropagation: Asexual or vegetative propagation of plants in
vitro.
Organogenesis: Differentiation of organs from cultured cells or tissue.
Plantlet: A small rooted shoot or germinated embryo.
Somatic hybridization: The creation of hybrids by fusion of protoplast
derived from genetically different cells.
Totipotency: Potentiality or property of cell to produce a whole
organism.
Transgenic plant: Plants which have been genetically engineered to
modify certain characteristics by introducing one or a few selected genes.
Ref: PlantTissue CultureTheory and Practice,a Revised Edition S.S. Bhojwani and M.K Razdan

Requirement of PTC
 Appropriate tissue (Explant)
 Equipment and Glasswares
 A suitable growth medium
 Aseptic (sterile) conditions
 Culture room
 Incubation room

Explant
An explant is a piece of
tissue used to initiate a
tissue culture.
Explant should be disease
free.
Ref:https://www.slideshare.net/ojuederiebern/plant-tissue-culture-
methods-and-applications

Hot air Oven
Glasswares
kept in the
oven for drying
at 1600-2000C
 Equipment and Glasswares

Double Distillation
Unit
- FOR THE PURIFICATION OF TAP
WATER BY DISTILLATION
PROCESS.
- DISTILLED WATER HAS
NEUTRAL pH (pH = 7) IT IS SALT
FREE
AND PURE.

REFRIGERATOR
- IT IS USED TO STORE THE STOCK
SOLUTIONS OF MACRONUTRIENTS,
MICRONUTRIENTS,
VITAMINS AND GROWTH HORMONES
AT 40C

CAPACITY : 0.001 TO
200 gm IS SUITABLE
FOR TISSUE
CULTURE
TECHNIQUES.
- USED TO MEASURE
MINUTE QUANTITY
OF CHEMICALS
USED IN NUTRIENT
MEDIA
DIGITAL BALANCE

pH Meter
- used to check or adjust
the pH of nutrient
medium
- 5.8 pH is prefered for
tissue culture media .
- If pH is alkaline, it is
adjusted by addition of
0.1 N HCl.
- if pHis acidic, it is
adjusted by addition of
1N NaOH.

CONDITIONS :
- 1210C TEMPERATURE
- 15 lb PRESSURE
- 20 MINUTES
For glassware and
media sterilization
AUTOCLAVE

For cell culture
Orbital Shaker

LAMINAR AIR FLOW :
 Used for inoculation purpose.
 It works on the principle of
application of high efficiency
particulate air (HEPA) filters
It creates aseptic condition and positive pressure by blowing
sterile air towards the operator.
Hepa filter is of 0.3 micrometer
size, hence it prevents microorganisms and
dust particles from passing
through, thus creates sterile air and
aspetic conditions.
VERTICAL LAMINAR AIR FLOW
HORIZONTAL LAMIANR AIR FLOW

Culture media composed of inorganic, organic
nutrient, vitamins and plant hormones.
Major – C, H, O, N, P, K, Ca, Mg, S
Minor- Fe, Cu, Mn, Bo, I, Zn, Co
Major types of Media
Whites medium – White (1963)
MS medium – Murashige and Skoog (1962)
B5 medium – Gamborg et al (1968)
Nitsch’s medium – Nitsch (1969)
Nutrient medium

Plant growth regulators
Auxins – Stimulate root formation.
Cytokinins –stimulate shoot formation
 Auxin ↓Cytokinin = Root Development
 Cytokinin ↓Auxin = Shoot Development
Class Name Abbreviation
Auxins Indo3 lacetic acid * IAA
1 Napthaleneacetic acid NAA
2,4 dichlorophenoxyacetic acid 2,4-D
Indol 3 butyric acid IBA
Cytokinin 6 Benzylaminopurine BAP
Kinetin Kin
* Indicates natural auxin Dr. K. M. Nitnaware HRM

Culture rack
Inoculation

Steps of Plant Tissue Culture
Preparation of Nutrient media
Selection of suitable explant
Surface sterilization of explant
Inoculation (Transfer of explant on nutrient media under aseptic condition)
Incubation
Regeneration
Hardening/Acclimatization
Sodium hypochlorite (1%)
Mercuric chloride (0.1%)
Temp. 25±20C
Relative humidity -50-70%
Light =16 hr light/8 hr dark
Light Intensity =45 μmol m−2 s−1
Germinating seeds, root, shoot, leaf, node, ovary,
embryo, anther etc

Embryo culture
Plant tissue culture
Seed culture
Protoplast culture
Pollen culture
Meristem culture
Callus culture
Cell culture
Anther culture
Types of PlantTissue culture

Organogenesis
 The production of roots,
shoots or leaves.
 These organs may arise out
of pre-existing meristems or
out of differentiated cells.
 This, like embryogenesis,
may involve a callus
intermediate but often
occurs without callus.

Somatic Embryogenesis
 The production of
embryos from
somatic or “non-
germ” cells.
 Usually involves a
callus intermediate
stage which can
result in variation
among seedlings

Globular stage of Somatic Embryos

Cotyledonary Stage of S.E.

CALLUS CULTURE

Regeneration - The process whereby a part
of a plant can be turned into a whole new plant
sprouting
new shoots

Hardened plantlets

Importance of plant tissue culture
 Production of improved crop varieties
Disease free and desired propagules
Production of secondary metabolites
Production of salt tolerant, drought tolerant and heat
stress tolerant variety
Biotransformation
Biosynthetic pathway
Haploid production

Basics of PTC (1).pdf basics of tissue culture

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