Download Intro. Plant Tissue Culture

ERT 103
PLANT TISSUE CULTURE AND
MEDIA FORMULATION
BY
DR. ZARINA BT ZAKARIA
WEEK 12-13
PLANT TISSUE AND CELL CULTURE
1. Introduction
- The purpose of practicing plant and cell culture
includes for regeneration/propagation, embryogenesis
and secondary metabolites production.
- In this technique, culture medium components, explant
source and plant growth regulator is the important
factors.
- Procedures should be follows includes laboratory set
up, medium preparation, sterilization, initiation and
maintenance of cultures.
- Refers to technique of growing plant cells, tissues,
organs, seeds or other plant parts in a sterile
environment on a nutrient medium
2. Terminology
- Explants – segments of plant organs used to initiate
culture
- Callus cultures – tissues arising by proliferation from
explants on agar medium/Mass of undifferentiated cells
produced in tissue culture is called callus. The callus is
highly vacuolated and unorganised cells.
- Suspension cultures – cells or small cells aggregates
growing dispersed in liquid medium
- Aseptic transfer – to move the tissue/cell while
maintain the sterile condition
- Basal medium - Most of the media contain inorganic
salts of major and minor elements, vitamins and sucrose.
A medium with these ingredients is called basal medium.
- Inoculation - Transfer of explant to culture medium is
called inoculation.
- In vitro - Cells/tissues removed from the intact
organism and grown in controlled condition in laboratory.
- Embryogenesis – the origin of plantlets closely
resembling the normal embryology from fertilized ovum
- Organogenesis – the origin of shoot buds or roots
from tissue cultures or suspension cultures/Process of
differentiation of callus initially into embryo like
structure (embryoids) and then showing organ like
roots/shoots
- Somatic embryogenesis - Process of production of
embryo from somatic cells is called somatic
embryogenesis and such embryo is called embryoid.
- Sub-culture - Callus produced and cultured again for
production of big mass of callus is called sub-culture.
3. Laboratory organization
- Tissue and cell cultures work needs clean
environmental conditions.
- Basics set up include an autoclave, laminar flow
chamber, incubator, wash-up area, media preparation
area, dissecting set and laboratory facilities (glasswares,
chemicals, refrigerator etc.).
- The culture maintenance and tissue transfer rooms
may be fumigated with formalin regularly against fungal
and bacterial contaminants prior to use.
- To prevent the entry of microbes at the time of
inoculation, laminar air flow cabinet (chamber used for
culture) is sterilized with ultra violet light or filter
sterilized air.
- The principal advantage of working in these cabinets is
that the flow of air does not hamper the use of spirit
lamp.
- Under tropical and subtropical areas, where dust
particles are very high, it must be kept in double door
fitted culture room in order to prolong the effective life
of the filter.
4. Preparation of culture media
- All plant culture media components can be divided into
six groups. These are
- major inorganic nutrients
- trace elements
- iron source
- vitamins
- carbon source
- plant growth regulators
- Culture media can be solidified (semi-solid) as agar is
added, or liquid media without agar.
- Solidified media usually used for callus cultures and
liquid media is used for suspension cultures.
- Media can be prepared or obtained from ready made in
packaging such as Murashige and Skoog, Gamborg, B5,
Nitsch etc.
- All components are mixed at required concentration
prior autoclaving to get rid of contaminants.
- Sterile media later on are poured into containers (vials,
Erlenmeyer flasks, petri dish, test tube etc.) according
to size of explants.
- Culture media usually prepared a few days before use
to ensure sterility and dry. For longer keeping, they can
be stored in refrigerator.
5. Sterilization
- For all types of materials (media, glasswares, utensils),
sterilization by autoclaving at 15 psi, 121oC for 15-20
minutes are commonly used.
- Thermo-labile substances such as certain plant growth
regulators and vitamins, are sterilized by membrane
filtration (0.22 to 0.45 m pore size).
- Dry-heat sterilization in an oven is used for glasswares
and instruments wrapped in aluminium foil and treated
for 1-2 hr at 160oC.
- Routinely used utensils such as scalpels and forceps
kept sterile by immersing in 80% ethanol and flamed
prior using.
- Source of explants brought from the field is invariably
heavily contaminated with dust and microorganisms.
- They are surface-sterilization using disinfectant
solution such as calcium hypochlorite or sodium
hypochlorite.
- The concentration used must be both harmless to the
tissue and effective to combat bacteria.
All American Electric Sterilizer
6. Initiation of callus
- Explant is extracted with a sharp scalpels - tissues
remain undamaged from suitable parts of a plant.
- - Expants are cut into small sizes to increase the
surface area and to ensure the absorption of nutrients.
- Damaged tissues release various compounds which are
air oxidized.
- explant surface is sterilized with bleaching agents such
as chlorine water or sodium hypochlorite, HgCl2 (0.1 %),
then it is washed with sterilled distilled water to remove
adherent chemical particles.
- explant is transferred to sterilised agar-solidified
culture medium kept in test tubes or flasks in culture
chamber.
- This chamber is also pre-sterilized with ultra violet ray
emitting tubes.
- Callus produced in above stage is taken out, cut into
pieces and each piece is transferred to fresh culture
medium in separate tubes or flasks.
- After some time these pieces develop into big masses
of callus.
- This is called sub-culture or multiplication stage as
callus get multiplied here.
7. Initiation of Suspension Cultures
- Suspension cultures are form readily after transfer of
friable (soft) callus to shaken flasks of the liquid media.
- The preparation of friable callus is similar to callus
initiation technique with medium modification.
- A platform shaker is used to give a circular/orbital
motion in a variable speed control (a range form 30-150
rpm).
- For large scale suspension cultures, bioreactors system
is being used for the purpose of producing high value
plant products.
- For maintenance of cultures, callus or suspension
cultures have to transfer into a fresh medium
(subculture) at interval period (exp every 4 to 6 weeks).
- In order to growth, cultures need constant ambient
temperature of 25  2oC (for the tropics).
- A low level of lighting (100-1000 lux) would suffice for
maintenance of tissue culture of different plants. Some
cultures need to be incubated in the dark for other
purposes. Light is essential for plant regeneration only.
- For those purposes, incubators are used, and some
incubator equipped with shaker for suspension cultures.
- Callus formation is observed after two weeks of
inoculation, i.e. incubation period is of two weeks.
8. Acclimatization
- Cultured plants are taken out of test tubes/flasks and
thoroughly washed under running tap water to remove
adhered agar molecules.
- Plants are kept at low minimal salt medium for 24 - 48
hours and transferred to pots containing autoclaved
sterilized mixture of clay and core soil leaf moulds in
equal proportions.
- Plantlets of the pots are covered with transparent
polythene to maintain humidity for 15-30 days.
- Plantlets then are transferred to green house for a
week and then to field.
- In the field agricultural techniques are applied like
other crops/ plants.
9. Biotechnological application
a. To propagate new plantlets in mass and in a relatively
short time or micropropagation.
- Micropropagation occurs in five stages:
1. selection of explant source and prepagation
- the explant must be in good physiological condition and
disease free.
- Size of explant, its location on the plant, its age, or its
developmental phase are among factors that affect the
success of micropropagation.
2. Initiation and aseptic establishment
- The explant is surface sterilized before placing it on
the medium. Exogenous plant growth regulators may be
added to the medium
3. Proliferation of axillary shoota
- Cytokinin-enriched medium enhances axillary shoot
proliferation. The shoots may be subcultured at an
interval of about four weeks.
4. Rooting
- The purpose of this stage is to prepare shoots for
transfer into the soil. This requires application of auxin
in the medium. Some commercial producers bypass this
stage and plant directly into the soil.
5. Transfer to natural environment
- Plants are put through the process of hardening off to
ready them for natural environment. They are gradually
moved from ideal lab conditions to more natural
conditions by reducing the relative humidity and
increasing light intensity.
b. Until now cell culture technique enable us to evolve cell
lines with special attributes such as to obtain droughtresistance, disease resistance and salt-tolerance crops
and natural dye (shikonin).
c. The production of high value natural product such as
terpenoids, alkaloids and fenolic acid can be increased by
this technique. The use of suspension cultures of Morinda
citrifolia for example, for the production of
anthraquinones has give few advantages.
 Solve production problems obtaining these highvalue substances from natural plants such as the
decreased of plant resources and increases in labour
cost.
 Not affected by changes in environmental conditions
such as climate or natural depredation.
 Cell cultivation in culture systems are easy to
manipulate in order to improve the production. This
can be done in any place or season.
10.
It Is Worth to Adapt Tissue Culture
Technique?
a. High cost of tissue culture system
highly specialized equipments (laminar flow,
autoclave)
specific media formulation for specific plant, time
consuming to study.
High media cost
High skills for the operator (time, cost)
b. Factors have to consider
supply and demand
difficult to propagate
high value compounds
growth condition (bad/well) in certain climate
less tissue culture expertise