Plant tissue culture is a process where plant cells, tissues, or organs are cultured in an aseptic, nutrient-rich environment. This document discusses plant tissue culture, including its definition, history, requirements, processes, and applications. Specifically, it examines a study that analyzed phenolic content and antioxidant activity of extracts from rosemary (Rosmarinus officinalis) callus cultures, finding the highest yields using woody plant medium supplemented with specific hormones.
1. Definition
Refers to a process in which we culture a part of a
grown plant. That part could be a cell, protoplast,
tissue or an organ which are called the (explants).
This process is meant to be done in a microbe-
free plant material, supplied by all the nutrients
needed for survival of the living tissue, in an
aseptic environment.
Plant Tissue
Culture
Plant Tissue
Culture
Definition
Why?
What is Needed?
A Little From The Past
How it is all be done?
Applications
Plant Tissue
Culture
Plant Tissue
Culture
Definition
Why?
What is Needed?
A Little From The Past
How it is all be done?
Applications
Determination of phenolic content andDetermination of phenolic content and
antioxidant activity of extracts obtained fromantioxidant activity of extracts obtained from
RosmarinusRosmarinus officinalisofficinalis.’.’ callicalli
Ozlem Yesil-Celiktas, Department of Bioengineering, Faculty
of Engineering, EGE University, Bornova - Izmir, Turkey
5. A Little From The Past
Plant Tissue
Culture
Plant Tissue
Culture
Definition
Why?
A Little From The Past
This technology relies on
two main concepts:
DifferentiationTotipotency
It implies that every cell of the plant
contains all the information
necessary for growth and it is
capable of developing into an entire
plant if suitably stimulated. (the
meristemic cells are best able to
express this ability)
The ability of mature
cells to return to the
meristemic condition
and reorganize into
new organs.
What is Needed?
How it is all be done?
Applications
6. A Little From The Past
Plant Tissue
Culture
Plant Tissue
Culture
Definition
Why?
A Little From The Past
Haberlandt .. early 1900’s
proposed concept of Totipotency
(Cells cultured under right
conditions)
Gautheret, Nobecourt, Whire in the 1930s.
(Cells kept alive but did not develop)
The first commercial use of plant clonal propagation on
artificial media was in the germination and growth of orchid
plants, in the 1920’s
In the 1950’s and 60’s there was a
great deal of research, but it was only
after the development of a reliable
artificial medium (Murashige &
Skoog, 1962) that plant tissue culture
really ‘took off’ commercially
What is Needed?
How it is all be done?
Applications
8. What is Needed?Plant Tissue
Culture
Plant Tissue
Culture
Definition
Why?
A Little From The Past
What is Needed?
How it is all be done?
Applications
2) A Suitable Growth Medium
Culture Medium Constituents
Inorganic salt formulation
Organic supplement
Source of carbohydrates
Water
Plant Hormones
Solidifying/Gelling Agent
(This can be
either liquid
or ‘solid’
forms,
depending on
the type of
culture being
grown)
9. What is Needed?Plant Tissue
Culture
Plant Tissue
Culture
Definition
Why?
A Little From The Past
What is Needed?
How it is all be done?
Applications
2) A Suitable Growth Medium
Culture Medium Constituents
Inorganic salt formulation
Salt formulations are some of the
elements important for plant nutrition
and their physiological function. These
elements have to be supplied by the
culture medium in order to support the
growth of healthy cultures in vitro.
(This can be
either liquid
or ‘solid’
forms,
depending on
the type of
culture being
grown)
ElementElement FunctionFunction
Nitrogen proteins, nucleic acids
Calcium cell wall synthesis, membrane function
Magnesium component of chlorophyll
Phosphorus nucleic acids, energy transfer
Chlorine Required for photosynthesis
Iron Electron transfer as a component of
cytochromes
10. What is Needed?Plant Tissue
Culture
Plant Tissue
Culture
Definition
Why?
A Little From The Past
What is Needed?
How it is all be done?
Applications
2) A Suitable Growth Medium
(This can be
either liquid
or ‘solid’
forms,
depending on
the type of
culture being
grown)
Culture Medium Constituents
Organic supplement
supplying vitamins and/or amino acids
Only two vitamins,
thiamine (vitamin
B1) and myoinositol
are considered
essential for the
culture of plant cells
in vitro.
Amino acids provide a source of reduced
nitrogen and uptake causes acidification of
the medium. Casein hydrolysate can be
used as a relatively cheap source of a mix
of amino acids.
11. What is Needed?Plant Tissue
Culture
Plant Tissue
Culture
Definition
Why?
A Little From The Past
What is Needed?
How it is all be done?
Applications
3) Aseptic (Sterile) Conditions
(As
microorganisms
grow much
more quickly
than plant and
animal tissue
and can over
run the culture(
SurfaceSurface
contaminantscontaminants
eliminated by surface
disinfectants (e.g., Na
hypochlorite, Ca
hypochlorite, ethanol).
InternalInternal
contaminantscontaminants
(Pathogens) eliminated
by thermotherapy,
culture of explants free
of organisms or by
antibiotics.
MaintenanceMaintenance of asepsisof asepsis
(During excision and culture) procedures are
carried out in sterile laminar air flow
12. In Plant Tissue Culture:
Auxins: Stimulate Root Development
Cytokinins: Stimulate Shoot Development
The General Role Of:
Auxins ------------- Cell Enlargement
Cytokinins ------------- Cell Division
Gibberellins ------------- Cell Elongation
Abscisic acid ------------- Inhibits Cell Division
What is Needed?Plant Tissue
Culture
Plant Tissue
Culture
Definition
Why?
A Little From The Past
What is Needed?
How it is all be done?
Applications
4) Growth Regulators
Naturally
occurring or
synthetic
compounds.
Compounds that have main roles in the
organizing and controlling all the
physiological and metabolic processes
in the plant tissues (Also known as the
Plant Hormones)
Four Main Classes:
Auxins, Cytokinins, Gibberellins, Abscisic acid
The ratio of Auxins to Cytokinins, also the
interaction between both with each other and
between them and other hormones and/or other
media components, determine the plant
development and the final result or product
acquired in the culture process.
13. What is Needed?Plant Tissue
Culture
Plant Tissue
Culture
Definition
Why?
A Little From The Past
What is Needed?
How it is all be done?
Applications
5) Frequent Subculturing
(To ensure
adequate
nutrition and to
avoid the build
up of waste
metabolites)
14. What is Needed?Plant Tissue
Culture
Plant Tissue
Culture
Definition
Why?
A Little From The Past
What is Needed?
How it is all be done?
Applications
5) Frequent Subculturing
4) Growth Regulators
3) Aseptic (Sterile) Conditions
2) A Suitable Growth Medium
1) Appropriate Tissue
15. How it is all be done?How it is all be done?
Plant Tissue
Culture
Plant Tissue
Culture
Definition
Why?
A Little From The Past
What is Needed?
Applications
The Desired Pathway
Callus Induction
Organogenesis
(Morphogenesis)
Embryogenesis
16. Plant Tissue
Culture
Plant Tissue
Culture
Definition
Why?
Callus Induction
How it is all be done?
A Little From The Past
What is Needed?
Applications
How it is all be done?
The Desired Pathway
Callus: Refers to the
undifferentiated, unorganized mass
of cells produced from the explants
cultured due to rapid proliferation
of the explants’ cells.
19. Plant Tissue
Culture
Plant Tissue
Culture
Definition
Why?
How it is all be done?
How it is all be done?
A Little From The Past
What is Needed?
Applications
Selection of The
Plant Tissue
Sterilizing
Selecting &
Preparing The
Proper Medium
Establishment
20. Plant Tissue
Culture
Plant Tissue
Culture
Definition
Why?
Important Factors InvolvedImportant Factors Involved
During ProcessingDuring Processing
1.1. Explant SourceExplant Source
Usually, the younger, less differentiated the
explant, the better for tissue culture
3.3. Environmental FactorsEnvironmental Factors
Light, Temperature, Photoperiod, Sterility, Media
2.2. Growth MediaGrowth Media
Minerals, Growth factors, Carbon source,
Hormones
4.4. GeneticsGenetics
Different species show differences in
amenability to tissue culture
In many cases, different genotypes within
a species will have variable responses to
tissue culture
How it is all be done?
A Little From The Past
What is Needed?
Applications
How it is all be done?
22. Determination of phenolic content andDetermination of phenolic content and
antioxidant activity of extracts obtainedantioxidant activity of extracts obtained
fromfrom RosmarinusRosmarinus officinalisofficinalis.’.’ callicalli
Ozlem Yesil-Celiktas, Department of Bioengineering, Faculty
of Engineering, EGE University, Bornova - Izmir, Turkey
Journal of Plant Physiology
Received 22 March 2007; Revised 24 April 2007;
accepted 14 May 2007
24. Rosmarinus officinalisRosmarinus officinalis..
UsageUsage:
flavor foods, treat gout, improving memory.
The results of a study suggest that carnosic acid, found in
rosemary, may shield the brain from free radicals, lowering
the risk of strokes and neurodegenerative diseases like
Alzheimer.
The medical uses of rosemary mainly due to its high content of
antioxidants such as carnosic acid and rosmarinic acid.
The antioxidant activity of plant extracts is due primarily to phenolic
compounds. In rosemary extracts, these are categorized into three
groups: phenolic diterpenes, flavonoids, and phenolic acids.
25. The aim of the studyThe aim of the study
The aim of this study was to report the best medium and
conditions to obtain high phenolic yield and antioxidant
activity of the methanolic extracts of rosemary callus cultures.
26. Materials & MethodsMaterials & Methods
Explant Source: Three well-grown R. officinalis plants located
at Bornova, Izmir, Turkey under Aegean climatic conditions.
Explants: sections of young shoots were collected from June
2004 to September 2004.
Explants were cultured on the day in which they were
collected.
Media Used: woody plant medium (WPM) and Murashige and
Skoog (MS) media supplemented with 7 g/L agar, 30 g/L
sucrose, and 1 and 3 mg/L naphthaleneacetic acid (NAA) for
callus initiation. (pH: 5.8(
Treatments
Names
MS WPM
1mg/L
naphthaleneacetic
acid (NAA(
MS 1 WPM 1
3mg/L
naphthaleneacetic
acid (NAA(
MS 3 WPM 3
27. Establishment of in vitro callus culturesEstablishment of in vitro callus cultures
Young shoots
Tap water
Hold in distilled
water for an hour
Remove leaves
Take stem sections
5-7 cm length
Stir in 70% ethyl
alcohol (5 min(
Stir in 0.5% sodium
hypochlorite (20 min(
Rinse with
autoclaved distilled
water 3 times
Cut stems into
1.0-1.5 cm
Put in vessels
(150 ml) + 23-
25 ml medium
Calli induction
was observed
within 10–15
days on the
surface at the cut
edges of the
sections
Incubation in
the dark
28. Establishment of in vitro callus culturesEstablishment of in vitro callus cultures
After calli became 1.0–1.5 cm in diameter,
they were subcultured on the same medium
4 times with intervals of 7–10 days
The calli collected from each vessel were gently
pressed on filter paper to remove excess water
and their fresh weights (FWs) were recorded
After holding at -18o
C for 1 night, they were
freeze-dried (lyophilized) and their dry weights
(DWs) were recorded
29. Preparation of methanolic extractsPreparation of methanolic extracts
1g of each lyophilized callus was transferred to
a vial and 20.0 ml of methanol was added
After that all the samples were sonicated at 35
kHz (Ultrasonic LC 30) for 45 min
Half of the samples sonicated with 50o
C and the
other half without heat treatment
HPLC analysis for the extracts & Antioxidant
assays
30. Results and discussionResults and discussion
Calli culturesCalli cultures
Media
Percentages of
explants
producing
callus)%(
Biomass (fresh
weights) (g(
MS 1 65.0 18.6
MS 3 40.0 15.4
WPM 1 55.2 16.7
WPM 3 50.2 16.0
On the basis of these findings, MS medium
supplemented with 1 mg/L NAA would prove to
be the most effective medium for biomass
production among the samples tested.
31. Results and discussionResults and discussion
Phenolic contentPhenolic content
Callus extracts
Content of rosmarinic acid
mg/g dry callus
MS 1 0.5
MS 1T 2.9
MS 3 1.8
MS 3T 1.1
WPM 1 3.1
WPM 1T 5.9
WPM 3 0.2
WPM 3T 0.8
32. ConclusionConclusion
The extracts from the WPM1 sample treated with
temperature provided particularly high amounts of RA,
a higher total phenol value, and a higher percentage of
radical scavenging activity, although MS1 proved to be
the ideal medium for callus induction
Therefore, woody plant medium (WPM) supplemented with
7 g/L agar, 30 g/L sucrose, and 1 mg/L naphthaleneacetic
acid (NAA) proved to provide ideal conditions for RA
accumulation and higher antioxidant activity.
33. Great Wishes & Thank You For Your Attention.Great Wishes & Thank You For Your Attention.
Thank YouThank You
Prepared by: Elmontasser Bellah AhmedPrepared by: Elmontasser Bellah Ahmed
Under Supervision of: Dr. Hoda ElmokademUnder Supervision of: Dr. Hoda Elmokadem
