Adavanced Breeding Methods for quailty improvement in solanaceous- Pallavi Wani

5/2/2023 2
Food & nutritional security

4
The improper diet and unbalanced nutrition are the major reasons of
malnutrition of about 3 billion people in our world (Kumari et al., 2021).
 In 2021 the GHI ranking India has been ranked 94th out of 107 countries.
(IFPRI 2021, Global Hunger Index: The challenge of hidden hunger)

Conventional Vs Non conventional

Advanced Breeding methods for improvement of
quality traits in Solanaceous crops
University of Agricultural Sciences,
GKVK, Bengaluru
PALLAVI WANI
PAMB1076
II ph.D
Department of Horticulture

Introduction
2
1
3
4
5
6
7
Gene action of important
quality traits
Quality traits classification
Genetics of quality traits
Solanaceous vegetables
Advanced breeding methods and
case study
Conclusion
Contents

Introduction
Review
Improving the quality of food crops- provide
nutrient security.
Solanaceous vegetables are cheaper sources of
nutrients and can easily meet the dietary
requirements.
Kumari et al., 2017
INTRODUCTION

• Shape, size, surface, color,
thickness
Morphological
traits
• Taste, aroma, flavour, sweetness
Orgenoleptic traits
• Beta carotene, ascorbic acid,
TSS
• Protein , vitamins, minerals
Nutritional traits
(Neutraceuticals)
• Solanine content, browning,
prickly ness
Undesirable traits
• keeping quality, cooking quality,
Other traits
10
Quality traits

More consumer
acceptability
Enables the grower to get
good return
Makes the produce more suitable
for processing
Food and health
security
Advantages
11

Crop Character Gene action
Tomato Fruit Weight Non-additive
TSS, Vit C, Carotenoids,
Lycopene
Non-additive
Pericarp thickness Additive
Locule number Additive
Brinjal Fruit Weight Additive
Fruit size index Additive
Fruit length Additive
Fruit width Additive
Chilli Fruit Weight Additive
Pungency Additive genes
Fruit length Additive
Table 1. Gene action of important quality traits
Bartlett et al., 1996

13
Crops Traits Gene Features
Tomato Lycopene Ogc, hp Red pigment
Vitamin C Vtc High Vit C
Shelf life Rin, nor, Nr Non-ripening
Chilli Capsaicin Pun, Cap High pungency
Capsanthin C High capsanthin
Brinjal Anthocyanin fap Anthocyanin
accumulation
Fruit stripe fst Striped fruits
Potato Zeaxanthin Chy Zeaxanthin accumulation
Anthocyanin Stan Anthocyanin
accumulation
Nicolia, 2001
Table 2. Genetics of quality traits

14
Quality traits of
Solanaceous vegetables

Breeding
objectives for
quality traits
Fresh Market-
Quality
Processing traits
(Tss,pH,Acidity)
Lycopene
(12-15%)
Vitamin-C
(120mg/100g)
Tomatine
(300mg/100g)
Tomato
Botanical name :Solanum lycopersicum L.
 Chromosome number : 2n=2x=24
Kumari et al., 2017

Botanical name : Solanum melongena L.
Brinjal
Breeding
objectives for
quality traits
Fruit size, shape,
colour, Soft flesh
Glycoalkaloid
content
(6mg /100g)
Anthocyanin
and Phenols
(0.44g/100g)
Vitamin- A
Ascorbic acid
(12mg/100g)
Kumari et al., 2017

Chilli
Botanical name : Capsicum annum L.
Breeding
objectives for
quality traits
Color
rention
sugar to acid
ratio
High Vit C
(111mg/100g)
High
capsaicin
(0.35mg/g)
High
oleoresin
Kumari et al., 2017

Potato
Botanical name :
Solanum tuberosum L.
Good source of
CHO, Vit C, amino
acids and minerals
Potato chips
and French
fries
Sensorial traits
(Flavor, Texture)
Industrial Traits
(Tuber size&
shape)
Low
glycoalkoloid
content
Breeding
objectives
for quality
traits
Kumari et al., 2017

Advanced breeding methods
19
Mutation Breeding
Molecular Breeding
Marker assisted selection
Marker assisted Back crossing
Genetic engineering
RNA interference
CRISPR cas9 mechanism
Singh et al., 2021

MUTATION BREEDING
20
Singh et al., 2021
Mutation is the sudden heritable change in the genetic
of an individual.
The term mutation was introduced by Hugo de Vries in 1900.
Tomato two mutant gene hp-1 and hp-2 have been reported for
carotenoid biosynthesis and used for developing carotene rich
tomato hybrid (Spair et al., 2008).

Types of
mutation
Induced
Physical
mutagens
Chemical
mutagens
spontaneous
Types of mutation
21
Singh et al., 2018

CHLOROGENIC ACID
 Brinjal is a good reservoir of phenolic compounds such as
chlorogenic acid.
 The chlorogenic acid was found to be the major phenolic with a
range of 50-70% of total phenolics in brinjal.
 Health promoting activities are Anti-diabetic effect, Cardio
protective, Anti-carcinogenic, Anti-oxidant and Anti-
inflammatory.
Rao et al., 2016

Responses of He-Ne Laser Irradiation on Chlorogenic
Acid Content of Brinjal (Solanum melongena L.) var.
Mattu Gulla
Objective- The study was to understand the influence of Helium-
Neon (He-Ne) laser irradiation on Chlorogenic acid of
Solanum melongena L. var. Mattu Gulla in the field
conditions.
J. Phytochem. Photobiogy
Swathy et al., 2021
Case study-1

Material and Methods
Seed material : Brinjal Var. Mattu Gulla
Healthy seeds were soaked in water for 2 hours
The seeds were irradiated with single exposure of He-Ne laser of
assigned doses of 20, 25 and 30 J/cm2.
The irradiated seeds were aseptically transferred on the Murashige
and Skoog (MS).
Assessment of chlorogenic acid content
Swathy et al., 2021

Fig.1. Mutation breeding in brinjal
Swathy et al., 2021

Table .3. Chlorogenic acid of brinjal (Solanum melongena L.) var. Mattu Gulla
irradiated with three doses (20, 25 and 30 J/cm2) of He-Ne laser
Sl.
No.
Laser dose (J/cm²) Chlorogenic acid (µg/g FW)
1 0 (Control) 1.09
2 20 1.23
3 25 0.66
4 30 0.44
26
Swathy et al., 2021

Positive influence of the He- Ne laser on cholorogenic acid
content of Brinjal.
5/2/2023 27

Marker
MOLECULAR BREEDING
DNA markers that are tightly linked to phenotypic traits to assist
in selection scheme for particular breeding objective.
Marker: Easily identifiable trait
Singh et al., 2021

DNA sequence dependent molecular markers (SNP )

SNPs :Single Nucleotide Polymorphisms (Snips)
A SNP is defined as a single base change in a DNA sequence at a
given locus that occurs in a significant proportion (>1 %) of a large
population
Single nucleotide polymorphisms (SNPs) are single nucleotide base variations,
caused by transitions (C/T or G/A) or transversions (C/G, C/A, or T/A, T/G)
Singh et al., 2018

Marker Trait Crop Reference
SCAR High-lycopene Tomato Lixia et al., 2011
CAPS High Pungency in C. Chinense Chilli Tanaka et al., 2016
SSR Cold induced sweetness Potato Fischer et al., 2013
SNP High Beta carotene and
Chlorophyll
Tomato Manoharan et al., 2017
Table 4. Molecular breeding for quality traits

5/2/2023
Molecular breeding of a novel orange- brown
tomato fruit with enhanced beta-carotene and
chlorophyll accumulation
Objective: To enhance beta carotene and chlorophyll
accumulation in tomato
Department of Horticulture, Sunchon National
University, Korea Manoharan et al., 2017
Case study-2

9(Red):3(orange):3 (brown):1(Orange brown)
KNB 1
Brown
KNY 2
Orange
F1
Orange
F2
34
Analysis of Carotenoids pigments through HPLC

Fig 2. a. Structure of the SGR gene “(Regulation of chlorophyll degradation)” showing the SNP
location 371 bp
b. Promoter region of CYC-B gene“(Responsible for orange color formation)”
identifying the SNP position at −77 bp
5/2/2023 35
Manoharan et al., 2017

Fig 3. Expression of the CYC-B gene during fruit development in F2 generation plants
5/2/2023 36

Selected plant in F2
generation
Stage Lycopene β-carotene
Chlorophyll
(a+b)
Red M 0.04 0.91 2.84
T 14.03 2.97 0.14
R 32.12 4.03 0.08
Orange M 0.02 1.16 2.54
T 0.16 7.94 0.22
R 0.58 11.07 0.07
Brown M 0.01 1.12 5.84
T 8.27 1.84 2.97
R 27.64 2.62 2.05
Orange-brown M 0.13 1.03 3.27
T 0.34 10.97 2.68
R 0.46 11.74* 2.03
Table .5. Lycopene, β-carotene, and chlorophyll content in F2 generation plants.
5/2/2023 37
High expression level of CYC-B gene coincide with
accumulation of beta-carotene and loss of SGR protein function
leads to inhibit chlorophyll degradation.

SGR and CYC-B genes play important role in the orange-brown
phenotype has high β-carotene content and retains chlorophyll
through ripening.
5/2/2023 38
Inference

MAS refers to the indirect selection for a desired plant phenotype based on the
banding pattern of linked molecular markers .
Marker Assisted Selection
Singh, 2018

Selection of parents
Development of mapping
population
Identification of polymorphism
Construct linkage maps with polymorphic markers
Identification of molecular markers linked to the trait of interest
Marker Validation
Steps in Marker Assisted Selection
Singh, 2018

Effective selection
of target loci
Minimize linkage
drag
Accelerated
recovery of
recurrent parent
FOREGROUND
SELECTION
BACKGROUND
SELECTION
Marker Assisted-Backcrossing (MAB)
MABC is a precise and an effective method to introgress a single locus controlling a trait of
interest while retaining the essential characteristics of the RP.
Debnath et al., 2017

Case study-3
Marker assisted backcrossing of alcobaca
gene into two elite tomato breeding lines
Objective- The aim of the study was to using
marker-assisted backcrossing (MABC) to
transfer shelf-life gene (alc) into the genetic
background of two elite tomato breeding lines.
Maxwell et al., 2022
Frontiers in Horticulture

Recurrent parents used : CSIR/CRI-P002 and
CSIR/CRI-ATS06
Donor parent used : Long shelf-life gene
derived from tomato mutant alcobaca
(Alc-LA3134)
Local check Varity
Marker assays- SNP Marker

Fig:4. Scheme of F1 development between two elite CSIR/CRI tomato breeding lines and
alc (Donor) line (3134).
Maxwell et al., 2022

P1 P2
F1
Foreground selection (Test for hybridity)
F1 P1
50 % recovery of genome of recurrent parent
Bc1F1
Bc1F1 P1
Foreground + background selection with SNP
79 % recovery of genome of recurrent parent
Bc2F1
92.2 % recovery of genome of recurrent parent
Bc2F1
Selfing
Bc2F2 Foreground + background selection with SNP
Inbred line
Fig. 5. Scheme of backcross population development between two elite Ghanaian breeding lines and
donor line with alc gene.
Foreground + background selection with SNP

Tomato genotypes
Fruit firmness
(N/cm2)
Pericarp
Thickness
(mm)
NO.Locules
Shelf Life
(days)
BC2 F2.3 -A-13-39-30 54.83 5.15 4.33 39.66
BC2 F2.3 -A-13-39-33 54.83 5.15 4.33 41.00
BC2 F2.3 -A-13-39-65 54.83 5.13 4.00 40.00
BC2 F2.3 -A-80-70-23 54.8 5.17 4.33 45.00
BC2 F2.3 -A-80-70-4 54.8 5.16 4.33 45.33
BC2 F2.3 -A-80-70-8 54.8 5.17 4.33 45.00
BC2 F2.3 -E-22-27-1 54.67 5.25 4.33 47.66
BC2 F2.3 -E-22-27-23 54.67 5.24 4.66 46.66
BC2 F2.3 -E-22-27-8 54.67 5.26 4.33 45.00
BC2 F2.3 -E-80-19-26 54.7 5.19 4.33 50.33
BC2 F2.3 -E-80-19-30 54.7 5.2 4.66 42.33
BC2 F2.3 -E-80-19-4 54.47 5.22 4.33 41.33
CSIR/CRI-ATS06(check) 54.31 4.74 5.00 28.66
CSIR/CRI-P002(check) 54.53 4.9 5.00 23.66
ALC-LA3134(check) 54.82 5.54 4 50.33
Local(check) 51.94 4.37 6.66 16.33
Mean 54.52 5.11 4.56 40.52
CV 0.25 2.97 9.49 3.83
LSD 0.23 0.25 0.72 2.59
MSE 0.01 0.02 0.18 2.41
TABLE .6. Mean performance of parents and MABC derived lines of BC2F3.
Maxwell et. al., 2022

Inference
Use of marker-assisted backcross selection was possible to
introgress the ripening mutant donor parent Alc-LA3134
into the two elite breeding lines

Case study-4
Breeding Capsicum chinense Lines with High Levels
of Capsaicinoids and Capsinoids in the Fruit
Objective: To Develop lines with high capsaicinoid and capsinoid
contents in the whole fruit by introducing the genetic
factors that regulate capsaicin biosynthesis in the pericarp
Lee et al., 2021

Background
Mutant
pAMT
pAMT
Fig.6. Biosynthesis pathway of
capsaicin
Capsaicinoid Capsinoid
Lee et al., 2021

Materials and Methods
C. chinense accessions- “SNU11–001, Habanero and Bhut Jolokia”
HJ Population-Habanero × Bhut Jolokia
SJ Population- SNU11–001 ×Bhut Jolokia
QTL Analysis- For capsinoid contents of the ‘SJ F2’ plants and for pungency
content of the ‘HJ’ population
QTL cartographer programme – QTL mapping
Analysis of Capsaicinoid and Capsinoid Contents on the selected lines -HPLC method.
Lee et al., 2021

Habanero
×
Bhut jolokia
F1
F2
F3
F4
F5
F6
HJ -3-4-1-3-1
HJ-3-1-3-1-2
HJ-3-1-3-1-1
(A) Selection of highly pungent lines from the ‘HB’
‘BJ’ population
F1
F2
F3
F4
F5
F6
×
SJ- 103-4334
SJ-103-4335
(B) Selection of lines containing high levels of
capsinoids from the ‘SNU11–001’ ‘BJ’
population
Bhut jolokia SNU11-001
Continuous selfing
and selection of
plants
Continuous selfing
and selection of
plants

Fig.7. Capsaicinoid contents of the placenta (A), pericarp (B), and fruit (C) in the ‘HJ’ F7 lines

Fig.8. Capsinoid contents of the placenta (A), pericarp (B), and fruit (C) in the ‘SJ’ F7 lines

Inference
Lee et al., 2021
Gene regulating
capsaicinoid biosynthesis in
extremely pungent pepper
pericarps can be a useful
tool in the breeding of
varieties with increased
capsaicinoid and capsinoid
contents

Genetic Engineering
1
Paul berg is considered as “ Father of
genetic engineering”
2
Paul Berg
56
Genetic engineering is the manipulation of
plant genome through recombinant DNA
technology to alter plant characteristics.
Fig.9. Steps followed in Genetic engineering Singh, 2018

Genetically modified plants – Reduced mal-nutrition problem

Genetic engineering to improve β-carotene
content in pepper
Objective :To introduce the Arabidopsis lycopene beta-cyclase
(β-Lcy) gene via Agrobacterium mediated vector gene
transformation
Nagar and Mekawi, 2018
Plant biotechnology
Case study-5

• Balady and topepo rosso
Plant material
• Cotyledons were used as explants and
cultured in MS media.
In vitro regeneration
• The cultured explants transformed with
Agrobacterium containing constructs
Transformation
• The regenerated transgenic plants are
analysed through PCR by specific Primer
Molecular Analysis
• β-carotene and total carotenoids content
are analysed during different maturity
stages
Biochemical Analysis
59
29/01/20
MATERIAL AND METHOD

Fig.10. Total carotenoids content in wild and transgenic pepper fruits during
different maturity stages
5/2/2023 60

Fig.11. Beta carotene content in wild and transgenic pepper fruits during different
maturity stages

Inference
5/2/2023 62
The transgenic pepper plants showed a significant
increase in β-carotene content reaching 7 to 10 folds than
the control

Fig. 12. Mechanism of the RNAi pathway.

CROP Targeted genes silencing Targeted traits
Tomato 1-aminocyclopropane-1carboxylate (ACC)
oxidase
Suppressed Ethylene
production and Improve
shelf life
α-Man and βHex 9 (Ethylene ripening
inducer)
MiR156 Enhanced shelf life
Colorless never ripe (CNR) gene
Enhanced shelf life
Potato β-carotene hydroxylase gene
Enhanced β-carotene
content
Table.7. Status of RNAi for quality improvement in
vegetable crops
Enhanced shelf life.
Singh et al., 2021

CRISPR-Cas9
 Clustered regularly interspaced short palindromic
repeats.
 A new class of genome-editing technology.
 The principle of the CRISPR-Cas9 system was derived from
bacterial adaptive immune system.
70

 CRISPRs were firstly identified in the Escherichia coli
genome.
71
It consisting of a series of 29-nucleotide “repeats”
separated by unique 32-nucleotide ‘‘spacer’’ sequences.

Fig.3. Various Types of Targeted Mutations by Different Mechanisms.

Fig:4. General application of CRISPR in genome editing
73

Fig.3: Microbial adaptive immune system through
CRISPR-Cas9 74

Fig:5. Applications of CRISPR in crop improvement

 2012- Idea of using CRISPR- Cas9
as a genome engineering tool was
published. by Jennifer Doudna
and Emmanuelle Charpentier
Awarded the 2020 Nobel prize in
chemistry for their contribution in
“harnessing an ancient mechanism of
bacterial immunity into a powerful
technology for editing genomes.”
Jennifer Doudna
UC, Berkeley
Emmanuelle Charpentier
Umea University, Sweden
76

Fig.13. Schematic illustration of the CRISPR-Cas9 system structure in plants.
77
Singh et al., 2018

Objective: To develop Reduced PPO activity lines in
potato using StPPO2 gene
Reduced Enzymatic Browning in Potato Tubers by Specific
Editing of a Polyphenol Oxidase Gene via Ribonucleoprotein
Complexes Delivery of the CRISPR/Cas9 System
Oxidation
PPO Enzyme
Andersson et al. (2020)
Case study-6

• Variety used : S. tuberosum cv. Desiree (tetraploid)
1.
• 2 sgRNA were designed sgRNA157 & sgRNA564
2.
• CRISPR/Cas9 was delivered in the form of Ribonucleoprotein
complexes into protoplasts by transfections
3.
• Identification of Edited Lines and Sequencing Analysis: Genomic
DNA of regenerated plants was extracted from leaves.
4.
• Off Target Analysis The presence of putative off target mutations in
StPPO1and StPPO4 genes was determined by HRFA
5.
• Enzymatic Browning and PPO Activity Determinations:
6.
80

Fig:14. (a) sgRNA design on the StPPO2 gene and
b)off target prediction sgRNA564 on StPPO1 and StPPO4 genes,
(C) Alignment of sgRNA157 with StPPO1 and StPPO4 genes

Fig:15.Discoloration development of selected edited lines at times 0, 24, and 48 h after cutting

This system proved to be specific for the target gene, without
affecting the coding sequence of other StPPO family members.
Inference

Table.8 .Wild Sources for quality traits
CROP Wild species Trait
Solanum pimpinellifolium,
Solanum peruvianum
Ascorbic Acid
Solanum chmielewskii Total soluble solids
Solanum cheesmaniae β-Carotene
Solanum torvum
Solanum khasianum
Solanum aviculare
Phenols
S. vernei Starch
S. phureja spp. phureja Carotene
Capsicum frutescens Capsaicin
Kumar et al., 2015

Varieties Attributes Method
TOMATO :
Co-3 Vitamin C (25 mg/100g),
TSS (5.2ºbrix).
Mutation
PKM.1 (1978) TSS 5.6°brix and acidity is
0.99% and 23.7 mg/100g
vitamin C
Mutant of Annaji
BRINJAL:
PKM.1 0.29% acidity and 14mg of
ascorbic acid per 100g of
pulp
Mutantion
Table.9. Varieties released with improved quality traits.
86
Singh et al. (2021)

Agronomic approaches
Genetics approaches
Conventional breeding
Transgenic/Biotechnological
approaches
Transgenic Approach for Bio fortification in
vegetables crops
Singh et al. (2021)

Table.10. Biotechnological approaches for quality traits in
vegetable crops
Crops Genes Improved quality traits
Tomato Psy-1, Crt B, CYC-B, LCY-B, Carotenoid rich tomato
Ros1 and Del Anthocyanin-rich tomato
CHI, CHS Flavanols rich tomato
S-adenosylmethionine decarboxylase
gene
Folate-rich tomato
Potato AmA1 (Amaranthus
hypochondriacus) Protein rich potato
Tar1 (tarin) gene
ZEP gene Enhanced Zeaxanthin
xyIA (glucose isomerase) High tuber fructose
Or gene Enhance B-carotene and
phytoene
Singh et al. (2021)

Tomato
• Flavr Savr , a genetically modified tomato, was
the first commercially grown genetically
engineered food for human consumation.
Potato
• Introgression of AmA1 gene from Amaranthus
hypochondriacus tend to increase 60 % protein
content.
Purple
Tomato
• The scientists identified two genes (SlFdAT1 and
Solyc12) from snapdragon that induce the
production of anthocyanins concentration in
tomato
Singh et al. (2021)

Special initiatives in India
 Balwadi nutrition programme
 Pradhan Mantri Matru Vandana Yojana
 Integrated Child Development Services (ICDS)
 Mid-day meal
 Nutri-smart village
 Agri-Nutri smart village
 Weekly iron and folic acid supplementation programme for
adolescents
 POSHAN Abhiyan (National Nutrition Mission)
 Chiranjivi Yojana
 Akshaya Patra
 Annapurna Scheme
 Antyodaya Anna yojna
2-May-23
90
Kumar et al., 2019

Organizations involved in biofortification
2-May-23 91

 Recently, in Uttar Pradesh Chandra Shekhar Azad University of Agriculture and
Technology, Kanpur has developed a completely biofortified village named
Anoop pur in district Kanpur.
Anoop pur : India’s First Biofortified Village
https://agri.bot/first-biofortified-village-of-india/

• The DBT’s National Agri-Food Biotechnology Institute,
Mohali (Punjab): First in India to use CRISPR/Cas9 to
carry out a change in the phytoene desaturase (fruit ripening)
gene of banana cv Rasthali.
• ICAR institutes are involved in application of CRISPR
technology for enhancing stress tolerance and nutritional
quality in a number of crops.
Research and regulation in India

The most remarkable product
introductions of today!
Topember
The new way to have all your favourite salad
items in one bite.
Square tomato
https://www.hortidaily.com/
CRISPR

He Jiankui shocked the world when he
described the implantation of edited
embryos that led to the birth of twin girls,
Lulu and Nana
Second International Summit on Human
Genome Editing, which was to be
organized at the University of Hong
Kong during 27–29 November 2018

More systematic work should be needed in
Vegetable crops with respect to Nutraceuticals.
To make the molecular marker technology more
price, productive and cost effective
Need to be create awareness on Nutraceuticals
and Bio fortified varieties.
Future thrust

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