by hairy root-cultures [bg-106] field application of ethephon...
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og ”Enhedens
Henrik Lütken, Assoc Prof.
Horticultural Science and Biotechnology
Department of Plant- and Environmental Sciences
Faculty of Science
University of Copenhagen
Denmark
Elicitation of anthocyanins in black carrots• by hairy root-cultures [BG-106]
• field application of ethephon [CP-202]
SYNTHETIC colors
Does it matter?
NATURAL colors
Source: http://www.foodmanufacture.co.uk/
Slide 2
Does it matter?
Source: http://sensientfoodcolors.com/market-trends/implications-of-the-nestle-decision/
Slide 3
Natural food colors
• Traditional natural colors: high price, instability, poor color range
• Southhampton study: certain synthetic colors can cause hyperactivity in children (McCann et al., 2007)
• 2015: natural food flavors and colors estimated market value:
~ 6 billion $ worldwide
• Raw materials from fruits and vegetables
• Main categories: Carotenoids (E160, E161, E164), Chlorophyllin (E140, E141) Anthocyanins (E163) Betanin (E162)
• To be competitive lower production cost are required
Market share, by region (2015)
North America
Europe
Asia
Rest
Source: MarketsandMarkets Analysis
Slide 4
• Daucus carota ssp. sativus var. atrorubens Alef.
• Origin: Middle Asia; Afghanistan
• Today: many new varieties and old landraces with high pigment contents
• Strong antioxidant activity
• High amounts of bioactive compounds
• Extracts of black carrots widely used in:
juices, confectionery, candies, ice cream and soft drinks
• How can we increase the profitability?
Black carrots as source of anthocyanin
Slide 5
6
To increase anthocyanin content in black carrot roots via:
• hairy root-cultures
• elicitation in the field
Objectives
punktopstilling, brug
”Sidehoved / Sidefod”.Indføj ”Sted og dato” i
Transformation using Agrobacterium rhizogenes
-Agropine strainsTL: rolA-rolD genesTR: aux1, aux2 genes
-Mannopine, Cucumopine, Mikimopine strains
One T-DNA with rolA-rolC genes
Hairy root-cultures
DEPARTMENT OF PLANT AND ENVIRONMENTAL SCIENCES
Plants regenerated from hairy roots often have altered phenotypes
(Ri phenotype), e.g.:
-stunted height
-changed morphology of leaves and flowers
-increased production of secondary metabolites
Slide 7
Application of various explants:
Co-cultivation
Hairy roots
Transformation
Taproots
Hypocotyls
Growth inliquid medium
A. rhizogenes mediated transformation
A. rhizogenes
Slide 8
punktopstilling, brug
”Sidehoved / Sidefod”.Indføj ”Sted og dato” i
Hairy root-cultures
DEPARTMENT OF PLANT AND ENVIRONMENTAL SCIENCES
Ono and Tian (2011), Plant Sci 180: 439–446Slide 9
Cultivar Pigment content
‘Deep Purple F1’
‘Purple Sun F1’
‘Night Bird F1’
‘Christian Hansen 43’
‘Christian Hansen 44’
‘Nairobi’ control
Kalanchoë control
Plant material for the transformation
Slide 10
Transformation efficiencies taproots
Slide 11
0%
2%
4%
6%
8%
10%
12%
14%
16%
‘Deep Purple
F1’
‘Night Bird F1’ ‘Christian
Hansen 43’
‘Christian
Hansen 44’
Orange
Contaminated Uncontaminated
___5 experiments___ ___2 experiments___ 1 experiment
%
0
10
20
30
40
50
60
70
80
90
100
‘Deep Purple F1’ ‘Christian Hansen
43’
‘Christian Hansen
44’
‘Purple Sun F1’
Chart Title
Contaminated Uncontaminated
Transformation efficiencies hypocotyls
Slide 12
_______________________2 experiments_______________________
%
0
2
4
6
8
10
5 15
Flask
Every 6 h
Every 1 h
0
2
4
6
0 5 10
Light, 20 ˚C
Darkness, 20 ˚C
Darkness, 28 ˚C
Gro
wth
rate
(rela
tive u
nits)
Flasks vs. TIS bioreactors
Effect of light and temperature (in flasks)
Bio
mass a
ccum
ula
tion
(gra
ms F
W)
Bioreactor flush frequency
Time (days)
Time (days)
Growth performance of a ‘Night Bird F1’ HR-line
Slide 13
• Preharvest management: Growth regulators, fertilization, salinity
• Postharvest environment: Temperature, hormone application, UV-light
Elicitation methods in the field
Slide 14
Ethephon
• Ethephon is the chemical name for 2-chloroethylphosphonic acid
(active ingredient)
• releases ethylene in the plant
• Used to accelerate ethylene responses in plants such as flower
induction, stimulation of latex flow, fruit ripening, fruit abscission
• Commercial names e.g. Cerone, Bromeflor, Arvest or Ethrel.
Slide 15
Field trial conditions
• Carrot: ‘Deep Purple’ F1
• Foliar application of Ethephon, sprayed at two concentrations:
360 and 720 g / ha, every third week
• Year 2014 and 2015
• Harvest over the whole growing period
Slide 16
Control + Ethephon
Longitudinal and transversal sections of ’Deep Purple’:
• darker color upon Ethephon treatment
• most evident in the cambium and xylem
Elicitation: darker carrot roots
Slide 17
Anthocyanin / FW
Anthocyanin / DW
Ethephon (g ha-1)
a a
b
AA
B
Experiment 1
0
1000
2000
3000
4000
mg g
-1D
W
µg g
-1FW
40
30
20
10
0
0 360 720
a a
b
AA
B
Experiment 2
0
1000
2000
3000
4000 40
30
20
10
0
mg g
-1D
Wµg g
-1FW
0 360 720
~ 30% increased anthocyanin content, week 21
FW: fresh weight
DW: dry weight
Different letters indicate significance according to Tukey’s test (p ≤ 0.05).
Slide 18
Conclusions and perspectives
• Black carrot are suitable source for the development of anthocyanin-based natural colorants
• Foliar applied Ethephon anthocyanin ~30%
• Anthocyanin level increases for several weeks after finished Ethephontreatment
• A. rhizogenes transformation is promising method to increase anthocyanin content
Ongoing experiments: elicitation (MeJA, Phenylealanine), regeneration
Slide 19
20
Gregorio Barba Espin
Renate Müller
Stephan Glied
Shih-Ti Chen
Mikkel KokJensen
Bjarne Jørnsgård
03/04/2017 21
Thank you for listening
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