production of rosmarinic acid from in-vitro culture of ... · composition. plant tissue culture...
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8th Euro Biotechnoloy Congress, 18 – 20 August,
Frankfurt, Germany
Production of Rosmarinic Acid from in-vitro culture of
Basil (Ocimum bacilicum L.) and Lemon balm (Melissa officinalis L.)
Dr: Raoufa Ahmed Abdel Rahman
Associate professor of plant biotechnology
City of scientific Research & Technology Applications
New Borg ElArab, Alexandria, Egypt
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Intoduction
Rosmarinic acid & its medicinal uses.
Basil & Lemon balm.
Plant tissue culture.
Results.
Conclussions.
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Secondary Metabolites
Studies on plant secondary metabolites have been
increasing over the last 50 years.
Many higher plants are a major source of natural
products used as:
• Pharmaceuticals and Agrochemicals.
• Flavor and fragrance ingredients.
• Food additives and Pesticides.
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• First isolated from
Rosmarinus officinalis
by (Scarpati and
Oriente, 1958)
• it is an ester of caffeic
acid and (3, 4-
dihydroxyphenyl) lactic
acid.
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Antiviral
Antibacterial
Anti-inflammatory
Anti-allergic activities
Antioxidant activities
Prevents the proliferation
of human cancer cells
Rosmarinic
acid
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Basil
Ocimum bacilicum is a member of the Lamiaceae (mint)
family. The common name is sweet basil.
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Anti bacterial (gram positive &
gram negative bacteria).
Anti-inflammatory
Strong antioxidant activity
Anti proliferative effect
Anti cancer
Antiviral
Antifungal activity
medicinal properties of O.bacilicum
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Lemon balm
Melissa officinalis is a member of the Lamiaceae family. The
common name is lemon balm.
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medicinal properties of M.officinalis
Antioxidant
Antibacterial
Antifungal agent
Anti cancer
Antiviral properties (against HIV-1 and
the influenza virus).
useful in the treatment of Alzheimer’s
disease.
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Plant Tissue Culture
Plant tissue culture is a
collection of techniques used
to maintain or grow plant
cells, tissues or organs
(explants) under sterile
conditions on a nutrient
culture medium of known
composition.
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Plant tissue culture
Over the past three decades, plant cell cultures
techniques have proved to be a valuable tool for
study biosynthesis and production of secondary
natural products.
One of the plant tissue culture protocols that is
considered as an attractive alternative for the
production of many valuable natural products is hairy
root culture.
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Hairy root culture Hairy root is a transgenic
phenotype resulted from the
infection of suitable plant tissues
with the gram-negative soil
pathogen Agrobacterium
rhizogenes .
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Advantages of hairy root cultures
Hairy roots
high growth rate and genetic stability
produce high levels of secondary metabolites
produce stable levels of secondary
metabolites over long time
Enhance secondary metabolites production
scaling up in plant bioreactor
Elicitation
Metabolic Trapping
Metabolic
engineering
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A i m o f T h e w o r k
study the efficiency of using plant tissue culture
techniques for the production of rosmarinic acid
from basil and lemon balm.
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Materials and Methods
Seed germination
Lemon balm seedlings Basil seedlings
Callus &shoot &hairy root production
Rosmarinic acid extraction &determination
Data analysis
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Results
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Ms medium was used for germination of O.bacilicum
and M.officinalis seeds under aseptic condition
Seed germination:
Ocimum bacilicum Germination percentages
63%
Melissa officinalis Germination
percentages 84%
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Media type Leaf Stem Root
MS1(basal MS) ------ ------ ------
MS2(MS+1mg/l
BA+1mg/l NAA)
11.75±1.85 8.52±1.04 6.23±0.85
MS3(MS+3mg/l
BA+1mg/l NAA)
8.54±1.22 4.24±0.25 3.17±0.22
MS4(MS+5mg/l
BA+1mg/l NAA)
4.35±0.43 3.42±0.33 2.21±0.25
Callus production in lemon balm :
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Callus production in basil:
Media type Leaf Stem Root
MS1(basal MS) ------ ------ ------
MS2(MS+1mg/l
BA+1mg/l NAA)
6.55±0.64 4.90±0.54 5.74±0.67
MS3(MS+3mg/l
BA+1mg/l NAA)
7.33±0.82 2.28±0.74 4.82±0.42
MS4(MS+5mg/l
BA+1mg/l NAA)
10.45±1.01 5.88±0.83 4.21±0.32
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B A
A- Basil callus culture derived from leaf explants grow on
MS+1mg/l BA and 1mg/l NAA (MS2).
B- Lemon balm callus cultures derived from leaf explants grow on
MS+ 5mg/l BA + 1mg/l NAA (MS4).
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Shoot production in lemon balm :
Media type No. of shoots/explant
Leaf Stem Root
M1 (basal MS) 0 0 0
M2 (MS+1mg/l KIN+1mg/l 2,4-D)
0 12±1.5 0
M3(MS+ 3mg/l KIN+1mg/l 2,4-D) 0 7±0.8 0
M4(MS+5mg/l KIN+ 1mg/l 2,4-D) 0 3±0.4 0
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A B
Shoot culture culture of basil and lemon balm.
A-Basil shoot culture derived from shoot tip explants grow
on MS+ 0.4mg/l thiamine HCl + 1mg/l BA.
B-Lemon balm shoot culture derived from stem explants
grow on MS+1mg/l of both Kin and 2,4-D (M2).
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Leaf discs, stem discs,
or seedling segments A. rhizogenes
Inoculation 1
2
Co-cultivation (1-5 days)
3
Selection on antibiotic
containing medium
4
Hairy root formation
5
Cultivation in liquid medium
Method of transformation. Procedure 1
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A B C
D E F
G H
Agrobacterium mediated transformation of basil and hairy root culture
production using procedure (1).
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Effect of explant type on transformation frequency and
hairy root production in basil infected with R 1000 strain.
Explant
type
Incubation
period
Total
number of
infected
explants
Number of
explants
Producing
roots
Number of
hairy roots
per explant
Root
length
(cm)
Transformation
frequency (%)
Leaf 1hr 143 110 branched 1.0 ±
0.2
77
Stem 1hr 47 0 0 0 0
Data collected after 15 days on selection plates
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Effect of explant type on transformation frequency and
hairy root production in basil infected with A4 strain.
Explant
type
Incubation
period
Total
number of
infected
explants
Number of
explants
Producing
roots
Number
of hairy
roots per
explant
Root
length
(cm)
Transformation
frequency (%)
Leaf 1hr 154 134 8.0 ±
0.2
1.5 ±
0.5
87
Stem 1hr 52 0 0 0 0
Data collected after 15 days on selection plates
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In the method a suspension of Agrobacterium is
injected into a plant tissues, where it transfers the
desired gene to plant cells.
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Agrobacterium mediated transformation of lemon balm and hairy root
culture production using procedure (2). Arrows showed hairy roots
produced after stem injection with Agrobacterium R1000 strain.
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Effect of explant type on transformation frequency
and hairy root production in lemon balm infected
with R 1000 strain.
Explant
type
Incubation
period
Total
number
of
infected
explants
Number of
explants
Producing
roots
Number
of hairy
roots per
explant
Root
length
(cm)
Transformation
frequency (%)
Leaf injection 56 0 0 0 0
Stem injection 40 15 7.0 ±
0.2
2.0 ±
0.5
37.5
Data collected after 30 days after infection
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Effect of explant type on transformation frequency and
hairy root production in lemon balm infected with A4
strain.
Explant
type
Incubation
period
Total
number of
infected
explants
Number of
explants
Producing
roots
Number
of hairy
roots per
explant
Root
length
(cm)
Transformation
frequency (%)
Leaf injection 48 0 0 0 0
Stem injection 36 12 2.0 ±
0.1
1.5 ±
0.5
33.3
Data collected after 30 days after infection
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M +Con 1 2 3 - Con
308bp
PCR amplification bands of WT and transformed hairy roots.
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Rosmarinic acid levels (ug/mg DW) in control, callus, shoot and hairy root cultures of basil and lemon balm.
Basil Lemon balm
Control 9.42±1.27 12.82±0.83
Callus 13.20±1.21 28.12±0.39
Shoot 25.30±0.24 38±25±0.73
Hairy Root 68.52±1.83 42.77±0.98
Each treatment is average of three replicates ± SE
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Conclusions
Plant tissue culture techniques specially Agrobacterium
Mediated transformation can be used successfully to
produce natural products such as rosmarinic acid.
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City of Scientific Research & Technology Applications
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Thank You for Attention