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Breeding for colour development in vegetable crops : current approaches and achievements

DIVISION OF VEGETABLE SCIENCEINDIAN AGRICULTURAL RESEARCH INSTITUTE

NEW DELHI-110012

R AV I N D R A D A N G I

R O L L N O - 2 0 6 9 8

Content

Introduction

Pigments in Vegetables : Types & Distribution

Breeding Approaches for Colour Improvement

in Vegetables

Research Achievements

Edible colour rich varieties of vegetable crops

Case Studies

INTRODUCTION

Nutritionists of WHO-FAO suggested that vegetables

is essential for balance diet

Vitamin A deficiency (VAD) is recognized as a

serious public health problem in India

It is estimated that 25% of the 15 million blind

people globally are from India

Colourful fruits and vegetables have enormous

nutritional and medicinal value

PhotosynthesisEncourage pollination

Higher nutritional value Higher consumer preference

What are the significance of colours in vegetables?

Pigment class Main subgroups

Typical colors Examples

Carotenoids Carotenes, lycopene andxanthophylls

Orange, yellow,, red

Carrot, tomato, water melon,pepper, Leaf y Vegetables

Flavonoids Anthocyanins;flavonols

Purple, blue,red

Eggplant, redCabbage, onion

Betalains β-cyanins andβ-xanthins

Red,orange, yellow

Beet, SwissChard

Chlorophylls a and b Green Any green plants

Pigments in vegetable crops: Types & Distribution

(Chen,2015)

Carotenoids are lipophilic, tetraterpenoids organic pigments.

CAROTENOIDS

Carotenoids in Human NutritionSignificance of Provitamin A Carotenoids in Developing Countries

Fertility and Reproductive Success

Prevention of Oxidative Stress and Inflammation

Vision and Diseases of the Eye

Cognitive Decline and Alzheimer’s Disease

Cancer Prevention and Treatment

Metabolic Syndrome, Obesity, Cardiovascular Disease, and Diabetes

(Chen,2015)

(Cazzonelli et al., 2010)

CAROTENOIDS RICH VEGETABLES

β-carotene

α-carotene

Lycopene

Lutein + zeaxanthin

β-Cryptoxanthin

Antioxidant Properties

Cardiovascular Diseases Control

Role in Obesity

FLAVONOIDS

Water-soluble polyphenolic compounds.

Distribution of flavonoids in vegetables

Anthocyanins

Flavones

Flavonols

Isoflavones

Betalains

Betalains

β-cyanins

β-xanthin

Betalains are a class of water-soluble pigments that are found only in the order

Caryophyllales.

Betalains differ from anthocyanins in the chemical structures but share

similarities to anthocyanins in the color spectra, biological functions.

For example, betalains contain nitrogen but anthocyanins do not.

Similarly, betalains are also localized in vacuoles.

(Chen,2015)

Betalains

Swiss Chard

CactiAmaramths

Beet

Breeding for colour development in vegetables

Breeding Approaches For Colour Improvement In

Vegetable Crops

Selection

Mutagenesis

Hybridization

Interspecific hybridization

Somaclonal Variation

Genetic engineering (Singh, 2009)

Selection Most of the colour varieties are developed by this method

Selected on the basis of phenotype

Carrot cream colour variety Pusa Kulfi content high lutein and Pusa

Ashita have high anthocyanin are the two Classical example

Pusa Kulfi Pusa Ashita

MutagenesisCarotenoid pathway mutants in Pepper & Tomato

Species Mutant name Phenotype References

Tomato r (yellow flesh) Yellow fruit color Fray and Grierson,1993

delta Orange fruit color Ronen et al.,1999

tangerine Orange fruit color Isaacson et al.,2002

Beta Orange fruit color Ronen et al.,2000

Pepper y (yellow) Yellow fruit color Lefebvre et al., 1998

c2 Yellow fruit color Thorup et al., 2000

(Farré et al.,2010)

Hybridization

High-Carotene Cucumber Germplasm

Early Orange Mass 400, Early Orange Mass 402, Late Orange Mass 404

Developed by crosses between U.S. pickling cucumber lines (Cucumis

 sativus L. var. sativus) and the orange-fruited Xishuangbannan cucumber

(C. sativus L. var. xishuangbannanesis Qi et Yuan)

Fig. Cross sections of four mature fruits of LOM 404 high carotene cucumber (15-25 mg.kg-1carotenes).

Simon and Navazio, 1997

Interspecific Hybridization

M. L. Tomes(1958) , bred Caro-Red, a Provitamin -A rich tomato variety

Develeped by cross between common tomatoes, Lycopersicon esculentum

Mill., and the wild species, L. hirsutum Humb

Due to its orange colour it not got commercial acceptance and consumers

preference

Tissue culture derived plants show variation termed somaclonal variation

Chromosomal rearrangements are an important source of this variation

A cultivar of sweet potato ‘Scarlet’ having higher yield and disease

resistance characteristics similar to their parent but also have darker and

more stable skin colour

Somaclonal Variation

(Moyer and Collins, 1983)

Cauliflower Or gene

Semi-dominant, single-locus mutation

No alteration in carotenoid biosynthetic genes expression

Function- regulating the differentiation of some non-photosynthetic plastids

into chromoplasts, which provide the deposition “sink” for carotenoid

accumulation

The Or gene encodes a plastid membrane protein

Beta carotene range from 3 to 320 µg/ 100 gram fresh tissue

Achievements

(Li et al.,2012)

A. Normal cauliflower

B. Orange cauliflower (heterozygous) curd

C. Orange homozygous mutant

D. Apical shoot of three month old normal cauliflower

E. Apical shoot of three month old orange cauliflower

Continued….

Cauliflower Pr gene

(Chiu et al.,2010)

Anthocyanins are responsible for purple color.

Purple (Pr) gene mutation is a spontaneous mutation.

The cauliflower purple mutation controlled by a single, semidominant

gene

Phenotype - Intense purple color in curds and a few other tissues

A commercial purple cauliflower cultivar Graffiti (Harris Seeds)

cultivated in USA.

The curds accumulated approximately 3.75 mg cyanidin diglucoside

equivalent per g fresh weight

Figure. Phenotypic comparison between wild type and the cauliflower Pr mutant. A, Young seedlings of 5-d-old plants. B, Young plants of 3-week-old. C, Curds of cauliflower plants grown in field. D, Young flower buds.E, Flowers. F, Young (inner) and old siliques of G, Seed endosperms wild type (left)and mutant (right).

Continued….

Edible colour rich varieties of vegetable crops

Carrot

Pusa Rudhira(High lycopene)

Pusa Meghali(High β-carotene)

Pusa Ashita (High anthocyanin)

Radish

Pusa Mridula (Lycopene)

Pusa Jamuni (Anthocyanin )

Pusa Gulabi(Anthocyanin

)Tomato

Pusa Rohini (Lycopene) Pusa Uphar (Lycopene)

Amaranths Pusa Lal Chaulai

(Anthocyanin) Onion Pusa Ridhi

Rich in antioxidant (quercetin 107.42 mg/100g)

Pumpkin Pusa Vikas (β-carotene) Continued….

Case Study -1

To enrich the anthocyanin content of the fruits of a cultivated tomato cultivar

To enhance the antioxidant value of tomato fruits

objectives

Materials and Methods

Two gene - delila and rosea1 from antirrhinum majus

Transformation of tomato with pGAntho construct was carried out by Agrobacterium-mediated gene transformation

Figure1 . Schematic diagram of the pGAntho gene construct in pGreen II.

Gene cloning and vector construction

Results and Discussion Transgenic tomato plants accumulating high amounts (70–100 fold) of anthocyanin in the fruit were developed

Figure1. Biochemical analysis of tomato fruit. a, Anthocyanin content; b, Antioxidant capacity.

WT, Wild type; C, Commercial; V, vector and pGAntho, Transgenic tomato.

a b

Figure 2. Biochemical analysis of tomato fruit. c, Total carotenoid content; d, Lycopene content.

WT, Wild type; C, Commercial; V, vector and pGAntho, Transgenic tomato.

c d

Continued….

Gene Expression Analysis

Figure 3. Expression analysis of Del and Ros1 and their targets:

a, Relative mRNA levels of Del and Ros1 at three developmental stages of fruit.

b, Relative mRNA levels of F3H and CHI induced by Del and Ros1 at three developmental stages of fruit.

a b

Figure . Tomato fruit colour in pGAntho (E8:Ros/Del) transgenic, WT-control and vector control .a Comparison of ripened fruits of pGAntho, WT-control and Vector control. b Transverse section of the ripened fruits of pGAntho, WT-control and Vector control.

Fig. Fruit colour during developmental stages of the fruit, from left green, breaker and red-ripe of WT-control (top) and pGAntho (bottom).

b

a

In conclusion, they have developed transgenic tomato

producing fruits rich in anthocyanins

This approach has achieved high levels of anthocyanin

content through-out the fruit

The fruit is of value for its health benefits as it is rich in

anthocyanins along with naturally occurring carotenoids.

In addition to the direct consumption, these fruits can also

be utilized to extract biological food colourants and

anthocyanin compounds used as a nutraceuticals.

INFERENCE

Case Study -2

To develop carotenoids and anthocyanin rich transgenic sweet potato with

high antioxidant capacity.

To characterize carotenoid contents and antioxidant activity in transgenic

sweet potato

Objective

Materials and MethodsPlant materials and expression vectors

Take anthocyanin rich sweet potato cultivar Sinzami (SZM) as wild type (WT). Under the control of the cauliflower mosaic virus (CaMV) 35S promoter was constructed. Agrobacterium -mediated transformation

Fig.Schematic diagram of the T-DNA region of the IbOr constructs used for plant transformation. LB and RB, left and right T-DNA borders, respectively; 35S Pro, CaMV 35S promoter; gray boxes, cloning site of gateway vector systems; FLAG, FLAG octapeptide.

Fig. Phenotypes of storage roots and leaves of three IbOr transgenic and wild-type (WT) lines.

Fig. Expression of IbOr in leaves and storage roots

Results and Discussion

Carotenoids contents in the storage roots of transgenic and wild-type sweet potato plants (μg /g DW).

Fig. Analysis of total anthocyanin contents.

Continued….

Photograph of aerial plant parts and storage roots

Fig. Average yields of storage roots

INFERENCE

In conclusion, anthocyanin-rich transgenic sweet potato plants with high

carotenoids were successfully generated

Carotenoids contents, and antioxidant activity in storage roots were

characterized

Transgenic plants is expected to contribute to human health by increasing

the contents of carotenoids

Transgenic sweet potato show tolerance to environmental stresses .This

would be beneficial for sustainable agriculture on marginal lands

worldwide.