Plant Tissue Culture – Requirements and Callus culture

Content

• General Introduction

• Types of Tissue Culture

• Plant Tissue Culture

• Callus Culture

Objective

At the end of the lecture the student will able to:

• Discuss the role of tissue culture in production of sec
metabolites

• Discuss the technique of callus culture

• Explain the role of nutrients of tissue culture media

Explant
Preparation

• Explant: Initial piece of the plant introduced in vitro or
detached portion of plant body used in tissue culture

• Age of explant                              

• Optimum success – explants – healthy vigorous plants                               

• Meristems, shoot tip, auxillary bud and immature, rapidly
growing tissues – must be selected as explants

• Surface
sterilization Disinfection – explant – surface microorganisms – presence of
any contaminant – interfere with the growth of explant

Explant

• Spore dormant microorganisms – resistant – disinfectants –
antibiotics incorporated – medium or explant is dipped

• Explants –priory washed with soap solution – dipped in
ethanol – disinfectants solution for 10-20 min – rinsed – sterile water –
remove residual disinfectants

Eg: Mercuric chloride (0.1- 1 %) – 2-10 min

Silver nitrate (1%) – 5-30 min

Bromine solution (1-2%) – 2-10 min

Hydrogen peroxide (10-12%) – 5-15 min

Sodium hypochlorite (1-2%) – 5-30 min

Sterilization of
seeds

Seeds washed with sterile distilled water à 70 % alcohol, 1 min
Sterilizing agent for specific period à
Wash with sterile distilled water à
moist cotton/filter paper à
germinated part transferred

Sterilization of
other vegetative part

Explants washed with sterile distilled water à 70 % alcohol, 1 min à Sterilizing agent for
specific period à Wash
with sterile distilled water à
transferred to the media

Types –
Plant Tissue Culture Techniques

1. Organ culture or
differentiated tissue culture

Excised root tip culture

Excised meristem culture

Embryo culture

Leaf primordia culture

2. Undifferentiated
tissue culture in solid media or callus culture

3. Undifferentiated
tissue culture in liquid media

Suspension culture

Protoplast culture

Pollen culture

Callus
culture

• Callus – mass of undifferentiated unorganised
parenchymatous cells proliferate on explant – grown in solid medium

• Each callus cell – has all the genetic information –
similar to parent cells – under suitable condition – give rise to new plant

• Addition of suitable growth hormones à Differentiate into
root and shoot system à
Entire seedlings

• Possible to produce secondary metabolites by adding
precursors

Technique of Callus culture:

• Selection, cleaning and sterilization of apparatus

• Selection and preparation of media

• Selection and sterilization of explant

• Establishment of culture / Inoculation of media

• Growth measurement

1. Selection,
cleaning and sterilization of apparatus

• Callus is three dimensional / irregular growth

• Petri plates are not suitable, conical flasks or culture
tubes made of glass or plastic are suitable

• Glass apparatus – Borosilicate glass, plastic apparatus –
High quality plastic

• To close the containers nonabsorbent cotton/ silicone
rubber/ aluminium foil should be used

• Apparatus must be cleaned using detergents (soap water
teapol 1 hr), conc. HCl- 2 hrs – remove traces – washed with sterile distilled
water

• Cleaned glass apparatus sterilized by dry heat sterilization
/ hot air oven – 140-160˚C – 2 hrs – Plastic apparatus do not require
sterilization

2. Selection and
preparation of media

• Media should be selected in such a way that it should
provide maximum growth with less nutrients

• Media – auxins – high level – induce callus formation +
cytokinins

3. Selection and
sterilization of explant

• Explant – buds, leaves, stem, roots, seeds, embryos,
cotyledons, anthers, pollens and hypocotyls

4. Inoculation of explant/Establishment
of culture

• Sterilized explant is transferred to medium under aseptic
conditions

• Care must be taken i.e, it must not be submerged inside
the media

• Incubate at suitable temperature (25-35 0C)

• Light – Aerial parts – Fluorescent light, subterranean
parts – Dark condition

• Acidity – medium – PH – 5.5 – 6.0 – depends upon the
strain of tissues employed

• Callus initiation occurs within 2-3 weeks, reaches maximum
growth after 5-6 weeks

• Growth curve of callus involves Lag phase, Steady phase,
Exponential phase and Decline/death phase

Callus culture Growth Curve:

• Lag phase:
Inoculation of explant – Lag time before the cells undergo cell division – Slow
growth

• Exponential phase:
Vigorous growth and rapid cell division – tissues consume nutrients – media –
deplete

• Decline phase: Depletion
of elements – media – starvation of cells – decline in the growth of cells

Subculture: Decline
phase of cells can be prevented by sub culturing

• Process in which a small piece of callus is transferred to
a fresh medium for maintaining steady growth

5. Growth measurement

a.   Determination of fresh wt/dry wt of callus

• Callus cells are collected on pre weighed nylon membrane
and weighed, gives fresh wt

• Collected cells dried in oven at 80 0C and weighed to a
constant weight, gives dry wt

b. Cell viability

• Capacity of a cell or an organism to live and grow

• Rather than growth measurement – metabolic activity is
measured cells exhibit green fluorescence under UV light

c. Determination of
cell count/cell number

• Gives accurate information – cell growth

• Disruption of clumps – single cell – 1% pectinase or
macerating fluid, consists equal volume of 10% chromic acid and 10% nitric acid
before counting

• Separated cells are stained with suitable staining reagent
and counted on cell counting chamber – Determined using haemocytometre

• Expressed as number of cells per original volume of
culture

Callus Culture from tap root of Carrot

Summary

• Tissue culture is growing of tissues or cells on a
suitable nutrient medium under aseptic conditions in vitro, may be plant or
animal tissue culture

• Types include callus, suspension, hairy root, single cell,
haploid, organogenesis, embryo culture etc

• Callus is also called as biomass, grown on semi solid
media, mass of undifferentiated parenchymatous cells

• Technique include selection and sterilization of
apparatus, media, and explant, establishment of culture and growth measurement