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