Background/Introduction World Population during 2020 is 7.8 billion, is growing steadily, and it is predicted to reach 9.1 billion by the year 2050. (united nation population division). Per capita calorie consumption of Fruit & Vegetable is expected to more than triple by the year 2050 in south Asia as compared to 2010. To feed growing population FAO projects the need to increase agricultural output by at least 60 per cent in the next decade. Over the coming decades a changing climate, growing World Population, rising food prices, and environmental stresses will have significant yet impact on food security. To fed growing population, FAO projects the need to increase agricultural output by at least 60 per cent in the next decade. India is ranked 94th position out of 104 countries. And it is also leading producer of many vegetables, but it has estimated around 15 per cent population is under nourished and lack of inadequate food intake both in quality and quantity. So thus improvement of nutritive value of crop is the major gole of breeding programme. By use of conventional breeding method to get the product will take more number of years and labours, so if we start by modern breeding technologies like, Marker assisted selection, Molecular Breeding, Antisense RNA, and CRIPSR/cas-9 etc., along with conventional method of breeding we can save the lot of time to develop the product. Vegetable crops are rich in dietary micronutrients, antioxidants and functional phytochemicals. Among vegetables solanaceous vegetables are the good source of health- protective dietary elements such as vitamins, minerals and antioxidant in the human diet. These solanaceous family includes Tomato, Brinjal, Chilli and Potato. (Kumari et al., 2017) Classification of Quality traits Any degree of excellence is called quality. There are mainly 5 categories- 1. Morphological traits- Related to product appearance which includes fruit size, shape, color and thickness 2. Orgenoleptic traits- Concerned with palatability of the produce, examples are taste, aroma, flavor etc., 3. Nutritional traits (Neutraceuticals)- These are determine value of the product such as Beta carotene, ascorbic acid, TSS, Protein , vitamins, minerals 4. Undesirable traits- Includes Solanine content in brinjal and browning in potato 5. Other traits- Includes keeping quality, cooking quality Advantages of Quality Breeding  It enables grower to get the good product  Helps in consumer acceptability  Makes the product more suitable for processing  Provides Food and health security Quality traits of Solanaceous vegetables ADVANCED BREEDING METHODS E. GENETIC ENGINEERING F. RNA INTERFERENCE TECHNOLOGY (RNAi) G. CRISPR CAS9- A genome editor ACHIEVEMENTS Transgenic Approach for Bio fortification in vegetables crops Research Regulation  The square tomato is not only special in terms of shape, but it will also improve your quality of life. Because it contains more than 50% lycopene FUTURE THRUT

1. 1
College of Agriculture, UAS, Bangalore - 560065
DEPARTMENT OF HORTICULTURE
Seminar report on
Seminar-I: HRT- 681 (0+1) Year: 2022-23
Submitted to
Dr. Vidya, A.
Seminar Teacher
Associate Professor
Department of Horticulture
Submitted by
Pallavi Wani
PAMB 1076
II Ph.D
Department of Horticulture
Major Advisor
Dr. Srinivasappa, K. N.
Professor
Department of Horticulture,
CoA, UAS, GKVK, Bengaluru, 65
UNIVERSITY OF AGRICULTURAL
SCIENCES, GKVK, BANGALORE
Title: Advanced Breeding methods for
improvement of quality traits in Solanaceous crops

2. 2
Content
Sl. No. Flow of seminar PAGE
NO.
1. Introduction 1- 3
2. Quality traits classification 3
3. Gene action of important quality traits 3
4. Genetics of quality traits 4
5. Quality Traits of Solanaceous vegetables 5-6
6. Advanced Breeding methods and related
Case studies
6-25
7. Achievements 26-29
8. Future Prospects 30
9. Conclusion 30
10.
References
31-32

3. 3
Background/Introduction
World Population during 2020 is 7.8 billion, is growing steadily, and it is predicted to
reach 9.1 billion by the year 2050. (united nation population division). Per capita calorie
consumption of Fruit & Vegetable is expected to more than triple by the year 2050 in south
Asia as compared to 2010. To feed growing population FAO projects the need to increase
agricultural output by at least 60 per cent in the next decade. Over the coming decades a
changing climate, growing World Population, rising food prices, and environmental stresses
will have significant yet impact on food security. To fed growing population, FAO projects the
need to increase agricultural output by at least 60 per cent in the next decade. India is ranked
94th
position out of 104 countries. And it is also leading producer of many vegetables, but it
has estimated around 15 per cent population is under nourished and lack of inadequate food
intake both in quality and quantity. So thus improvement of nutritive value of crop is the major
gole of breeding programme.
By use of conventional breeding method to get the product will take more number of
years and labours, so if we start by modern breeding technologies like, Marker assisted
selection, Molecular Breeding, Antisense RNA, and CRIPSR/cas-9 etc., along with
conventional method of breeding we can save the lot of time to develop the product.
Vegetable crops are rich in dietary micronutrients, antioxidants and functional
phytochemicals. Among vegetables solanaceous vegetables are the good source of health-
protective dietary elements such as vitamins, minerals and antioxidant in the human diet.
These solanaceous family includes Tomato, Brinjal, Chilli
and Potato. (Kumari et al., 2017)

4. 4
Classification of Quality traits
Any degree of excellence is called quality. There are mainly 5 categories-
1. Morphological traits- Related to product appearance which includes fruit size, shape,
color and thickness
2. Orgenoleptic traits- Concerned with palatability of the produce, examples are taste,
aroma, flavor etc.,
3. Nutritional traits (Neutraceuticals)- These are determine value of the product such as
Beta carotene, ascorbic acid, TSS, Protein , vitamins, minerals
4. Undesirable traits- Includes Solanine content in brinjal and browning in potato
5. Other traits- Includes keeping quality, cooking quality
Advantages of Quality Breeding
 It enables grower to get the good product
 Helps in consumer acceptability
 Makes the product more suitable for processing
 Provides Food and health security
Additive gene action refers to average effect of alleles. Non additive gene action
includes both additive and domiant epistasis Gene action of some quality traits are listed in
Table 1.
Table.1. Gene action of important quality traits
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, also Non-additive
Fruit size index Additive
Fruit length Additive
Fruit width Additive
Chilli Fruit Weight Additive, also Non-additive
Pungency Additive genes

5. 5
Table 2. Genetics of quality traits
Crops Traits Gene Features
Tomato Lycopene
Og
c
, 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
Quality traits of Solanaceous vegetables
1. Tomato
Tomato is the second highest produced and consumed vegetable in the world Today.
Tomato fruits are an important source of nourishment for the whole world’
s population. And it
is botanically called Solanum lycopersicum L. having its chromosome number is 24.
 Specific Breeding objectives for quality traits are-
 For fresh market-quality purpose- Breeding Objictives includes size, shape,
Appearance, color and smoothness
 If we want to breed for processing quality traits some of important breeding objictives
such as high Tss (5.5 Brix), Low pH (< 4.5) and low acidity.
 And also to develop variety rich in lycopene, vitamin C and Tomatine content.
2. Brinjal
 There is wide range of diversity in India based on fruit size, shape, color as well as
preference for consumption. Brinjal botanically called Solanum melongena L. It is an
important source of plant-derived nutrients like vitamins, minerals available throughout
the year and popular among the poor.
Specific Breeding objectives for quality traits are-
 Low glyco-alkaloid content. And to develop variety rich in vitamin A and anthocyanin
content.

6. 6
3. Chilli
Chilli is the most important spice in Indian dishes. It is botanically called Capsicum annum L.
chromosome number is 24. The breeding objectives like,
 We need to breed variety rich in high sugar to acid ratio, High vitamin C in bell
pepper, high capsaicin, capsanthin and oleoresin content in hot pepper fruits.
4. Potato
Potato is considered as the staple food crop, it has good source of CHO, vitamin C,
amino acids and minerals. It is botanically called Solanum tuberosum L. having its
chromosome number is 48. The breeding objectives for quality traits like include-
To breed variety which in Low glycoalkoloid content that is solanine, and if we want to
breed for processing purposes for example, French fries and chips the most important
breeding objectives includes tuber shape should be oval or round, dry matter content (21 %),
starch (21 %) and reducing sugars etc.,
ADVANCED BREEDING METHODS
Advance quality breeding needs a combination of genetic source, modern genomic tools and
biochemical diagnostic techniques. (Singh et al., 2021)
The Recent breeding methods includes,
 Mutation breeding
 Polyploidy breeding
 Molecular breeding
 SNP’s discovery
 Transgenic approach
 RNA interference
 Targeted genome editing
A. MUTATION BREEDING
 Mutation is sudden heritable change in DNA sequence which leads to change in
characteristics of an organism. The entire operation of mutation induction and isolation of
mutants for crop improvement is called mutation breeding. The term mutation was introduced
by Hugo de vries in 1900.

7. 7
In vegetable there are several quality mutant genes are reported for developing
neutrarich hybrids. For example in Tomato two mutant gene hp-1 and hp-2 have been
reported for carotenoid biosynthesis and used for developing carotene rich tomato hybrid.
The physical or chemical agents which enhance the mutation frequency are commonly
called as mutagens. Physical mutagens include ionizing radiations like α, β, γ, X-rays and
non-ionizing radiations like UV and infrared rays. The chemical mutagens consists of ethyl
methyl sulphonate, dimethyl sulphonate, bromouracil, etc., Singh et al. (2021).
CHLOROGENIC ACID
 Chlorogenic acid is the one of the most important phenolic compound found in brinjal.
And it ranges from 50- 70%.
 It has Health promoting activities like Anti-diabetic, Cardio protective, Anti-
carcinogenic, Anti-oxidant, Anti-inflammatory and analgesic, Neuroprotective, Anti-
obesity properties Chada et al., 1998.
Objective: The aim of 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.
Introduction- He-Ne laser (632.8 nm) is one of the significant lasers used extensively to
enhance the plant growth and development.
Till now influence of radation on growth parameters, biochemical characters are
reported on in-vitro condition but in this case study under taken determine the He-Ne laser on
Brinjal in field condition.
Materials and Methods includes the ripe fruits of brinjal (var. Mattu Gulla) were collected
from the fields of Mattu Village, Udupi, Karnataka. Prior to laser treatment uniform and
Healthy seeds were selected and soaked in running water for 2 h and anhydrated using tissue
Case study-1
Responses of He-Ne Laser Irradiation on Chlorogenic Acid
Content of Brinjal (Solanum melongena L.) var. Mattu Gulla
Swathy et al., 2021

8. 8
paper. Subsequently the seeds were exposed to He-Ne laser of assigned doses of 20, 25 and 30
J/cm2
. Later the sterilized seeds were aseptically transferred on the Murashige and Skoog (MS)
and solidifying agent agar.
RESULTS:
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
Chlorogenic acid content was found to be highest in laser dose of 20 J/cm2
as compared
to other treatments.
Inference:
This study showed that positive influence of the He-Ne laser on cholorogenic acid
content of Brinjal fruit tissue.
B. MOLECULAR BREEDING FOR QUALITY TRAITS
 Molecular breeding is refers to use of DNA markers that are tightly linked to phenotypic
traits to assist in selection scheme for particular breeding objective.
A marker is usually a DNA sequence located close to the gene of interest that can be used to
identify individuals or sps.
Molecular markers are widely used in breeding programme it includes RAPD, AFLP, SSR,
CAPS and SNP. Among these markers SNP are widely used due to their abundance,
stability and cost effectiveness.
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., 2021
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

9. 9
Features of DNA/Molecular Markers
Marker must be polymorphic in nature
It should be codominant in nature (so that it can differentiate between heterozygote’s allele
from homozygous allele.
It should be even distribution across the whole genome
 Easy, fast and cheap to detect and It should be reproducible
Table .4. Molecular breeding for quality traits
Reference Marker Trait Crop
Lixia et al. 2011 SCAR high-lycopene Tomato
Tanaka et al., 2016 CAPS High Pungency in C. Chinense Chilli
Fischer et al., 2013 SSR Cold induced sweetness Potato
Objective: To develop tomato fruit color is enriched for beta-carotene and chlorophyll
content.
Material and Method-
They have taken two inbred lines that is KNY2 (orange color) and KNB1 (brown color) were
crossed all F1 progeny produced orange color fruit, it means that orange color is dominant
over other fruit colors. And subsequently selfing F1 progeny they got F2 segregation in 9:3:3:1
that is orange, red, orange brown and brown respectively.
Case study-2
Molecular breeding of a novel orange- brown tomato fruit with
enhanced beta-carotene and chlorophyll accumulation
Manoharan et al., 2017

10. 10
Fig.1. a. Structure of the SGR gene showing the SNP location 371 bp from the start codon b.
Promoter region of CYC-B gene identifying the SNP position at −77
 Here SGR gene play important role in regulation of chlorophyll degradation in tomato
fruit and CYC-B gene responsible for orange color formation and involved in
improving carotenoid content in tomato fruit.
Fig.2. Expression of the CYC-B gene during fruit development in F2 generation plants
In these figure we can clearly see that level of CYC-B gene expression was significantly higher
at the turning stage in both orange and orange brown phenotypes as compared to other
phenotypes.

11. 11
Table.5. Mean value of Lycopene, β-carotene and chlorophyll content in F2 generation plants.
Result and Discussion-
High amount of Beta-carotene and low levels of lycopene was observed in orange
brown and orange phenotypes. And the chlorophyll content was found to be high in orange
brown and brown phenotypes. It mightily due to higher expression of CYC-B gene with
accumulation of Beta carotene and loss of SGR protein function which leads to inhibit
chlorophyll degradation.
Inference:
In this study they concluded that SGR and CYC-B genes play important role in the fruit
color of tomato and these study provides insight into development of genotypes with enhanced
beta-carotene accumulation and chlorophyll retention in tomato fruits.
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

12. 12
C. MARKER ASSISTED SELECTION
• MAS refers to the indirect selection for a desired plant phenotype based on the banding
pattern of linked molecular markers (Singh et al., 2021).
Steps in Marker Assisted Selection
1. Selection of Parents: The parents selected for the mapping population will differ for
one or more traits of interest. It means parents should be distantly related so that it
provides adequate polymorphism.
2. Development of mapping populations:
3. The mapping population should be larger in size. Several different populations may be
utilized for mapping: 1. F2 populations 2. Backcross (BC) populations, 3. Recombinant
inbred (RI) lines, 4. Doubled haploid (DH) populations.
4. Identification of Polymorphism
5. The construction of Linkage Map with Polymorphic Markers: Linkage map will be
constructed by using the computer software’s.
6. Identification of Molecular Markers linked to the Trait of interest: Based on the
Linkage maps constructed, the tightly linked markers to the gene of interest are
selected.
7. Marker Validation: Markers should also be validated by testing for the presence of
the marker on a range of cultivars and other important genotype.
D. MARKER-ASSISTED BACKCROSSING (MABC)
 Marker assisted selection refers to indirect selection for a phenotype based on banding
pattern of linked molecular markers. Molecular breeding is effective in introgression of
desirable genes from wild into cultivated genotypes (Singh et al., 2021).

13. 13
FOREGROUND SELECTION- Is used to identify our target locus with help of linked
marker
BACKGROUND SELECTION- Is used to test recovery of recurrent parent
Advantages of MABC over conventional method of breeding-are
• Effective selection of target loci
• Minimize linkage drag
• Accelerated recovery of recurrent parent
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.
Materials and Methods- In these study they used total three parents, two are recurrent parent
such as CSIR/CRI-P002 and CSIR/CRI-ATS06. Long shelf-life gene derived from tomato
mutant alcobaca (Alc-LA3134) as a donor parent. Local check Varity is used for comparison
with MABC derived lines.
Fig.3. Scheme of F1 development between two elite CSIR/CRI tomato breeding lines and alc
(Donor) line (LA3134).
Case study-3
Marker assisted backcrossing alcobaca gene into two elite tomato
breeding line
Maxwell et al., 2022

14. 14
.Fig. 4. Scheme of backcross population development between two elite Ghanaian breeding
lines and donor line with alc gene
Population development between recurrent and Donor parent- Here recurrent parent will
be used as female and donor parent as male. Each recurrent parents are crossed with donor
parent which is carrying gene of interest that is alc to generate F1 seeds. (Fig. 4). Fig number 5
shows the general population development between RP and DP. Here they crossed RP with DP
to obtain F1 seeds, next as we can see F1 plants will be subjected to true hybridity gene test by
using foreground selection. By this will get to know about F1 plants which are in heterozygous
nature. After the conformation of target gene present in F1 plants they will used these F1 for
backcross with RP to generate BC1F1 seeds. In generation they will go for foreground and
background selection. In BC1F1 generation they got 79 per cent of recovery of RP. The
selected BC1F1 plants again backcross with RP to generate BC2F1 seeds. Here they got 92.2
per cent recovery of genome of RP. And after these BC2F1 plants carrying gene of interest so
they will go for continue selfing for 2 generation to produce BC2F2 and BC2F3 population.
The final BC2F3 family called inbred line or near isogenic lines these will be evaluated for
agronomic performance.

15. 15
Table. 6. Mean performance of parents and MABC derived lines of BC2F3.
Result shows that there were significant differences observed between MABC derived lines
and recurrent parents along with local check with respect to fruit firmness, number of locules,
pericarp thickness and shelf life which confers by genetic regulation of alc gene.
Inference: Use of marker-assisted backcross selection was possible to introgress the ripening
mutant donor parent Alc-LA3134 into the two elite breeding lines. Use of markers allows the
rapid recovery of genome of RP and also we can reduce the number of generation to 3 to 4.

16. 16
The spicy taste of the pepper is mainly due to the compound called
capsaicin, This capsaicin and its anologs are called as capsaicinoids.There
is another compound called capsinoid which are smiliar to capsacin but
mild pungent which are present mostly in sweet peppers.
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
Fig.5. Biosynthesis pathway of capsaicin
This capsacinoids and casinoids are mainly biosynthesised through the phenyl propinoid
pathway, the major enzyme called pAMT which biosynthesis capsacinoids from precursor
called vanilylamine, if mutation in pAMT enzyme it will leads to biosynthesize vanilyl alcohol
instead of vanilylamine which is the capsinoid precursor.
Case study- 4
Breeding Capsicum chinense Lines with High Levels of
Capsaicinoids and Capsinoids in the Fruit
Lee et al., 2021

17. 17
Materials and Methods- In these study they used three parental lines of Capsicum spp. that
is “SNU11–001, Habanero and Bhut Jolokia” these parents were used for HJ and SJ
population development. The F2 population are taken for QTL mapping through genotyping
by sequencing, for QTL they used QTL cartographer programme Then the quantification of
capsicinoids and capinoids on the selected line were done by HPLC method.
Fig A showes ‘HJ’ population it was developed by crossing ‘Habanero’ and ‘Bhut jolokia’. In
this HB parent which has normal pAMT enzyme this popultion is majorly used to increase
capascionoids content. In fig B the SJ population was obtained by crossing ‘Bhut jolokia’ with
‘SNU11001’ line, in this SNU11-001 parent has mutant pMAT enzyme. This population is
majorly used to increase the capsinoid contents. Both the populations are advanced up to F6
generation and from F6 three lines are selected from HJ population and 2 superior lines were
selected from SJ population which having high capasicinoid and capsinoid content
respectively.

18. 18
Fig.6. Capsaicinoid contents of the placenta (A), pericarp (B), and fruit (C) in the ‘HJ’ F7 lines
In this fig we can see the three lines were selected from the HJ populations such as HJ- 3-1-3-
1-1, 3-1-3-1-2 and 3-4-1-3-1 contained significantly more capsaicinoids in the placenta,
pericarp, and fruit than parental lines
.
Fig.7. Capsinoid contents of the placenta (A), pericarp (B), and fruit (C) in the ‘SJ’ F7 lines
The two lines selected from the ‘SJ’ population that is SJ 103-4-3-3-4 and SJ 103-4-3-3-5,
contained significantly more capsinoids in the placenta, pericarp and fruit than parental line.
Inference- 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

19. 19
E. GENETIC ENGINEERING
It refers to direct or artificial manipulation of one or many genes, most often foreign gene is
inserted into an organism to get a desired phenotype.
Ex: Golden rice, Flavr savr tomato etc.,
Steps involved in genetic engineering are
1. Extraction of plasmid from bacterial cell which is followed by isolation our gene of interest
2. After that cutting DNA at specific location with help of certain enzymes called restriction
enzymes or molecular scissors
3. After the second step that is cutting DNA segment followed by the ligases process it is
nothing but joining of DNA fragments of desired sequences along with plasmid molecule to
form a specific type of hybrid DNA is called recombinant DNA technology
4. In next step the rDNA inserted into the host cell to form a transformed bacterial cell these
transformed bacterial cell allowed them to grow inside a media
5. Last step is selection and screening of transformed cells with desired character.
Fig.8. Steps followed in Genetic engineering

20. 20
Pepper fruits an often be a major dietary source of cartenoids especially
beta-carotene there is a considerable variation in Beta-carotene content in
pepper fruits
Objective: To introduce the Arabidopsis lycopene beta-cyclase (β-Lcy) gene via
Agrobacterium mediated vector transmission method
Materials and Methods- In these study they used two different pepper genotype Balady, and
Topepo rosso, were analyzed with regard to their efficiency for regeneration and genetic
transformation in vitro. Cotyledons were used in vitro from young seedlings as target tissue for
regeneration and transformation of pepper. Over expression of β-Lcy gene via Agrobacterium-
mediated transformation was used. The presence of the transgene was assayed on leaf DNA
via PCR and biochemical analysis using HPLC.
Fig.9. Beta-carotene and Total carotenoids content in wild and transgenic pepper fruits during
different maturity stages
Case study-5
Genetic engineering to improve β-carotene content in pepper
Nagar and Mekawi, 2018

21. 21
Results showed that total amount of beta carotene and total carotenoids was found highest in
transgenic plants as compared to wild plants in both genotypes during all stages from green to
ripe stage.
Inference-
The transgenic pepper plants showed a significant increase in β-carotene content reaching 7 to
10 folds than the control
F. RNA INTERFERENCE TECHNOLOGY (RNAi)
RNA interference technology (RNAi): sequence specific gene silencing phenomenon caused
by the presence of double stranded RNA. These RNAi allows down regulation in gene in a
more precise manner without effecting expression of other genes. It was first observed by
Richard in 1990.
Fig.10. Mechanism of the RNAi pathway.
Due to action of dicer enzyme on dsRNA is chopped and converted into fragments called
small interfering RNAs (siRNAs). With the help of argonout enzyme siRNA forms the RISC
(RNA induced silencing complex). The complex of SiRNAs and RISC binds to the target
mRNA. Later this will inhibit the mRNA synthesis and silence the gene (Fig. 12). So with
these same process utilized in the ethylene biosynthesis pathway. So that RNAi block ACC
oxydise which is involved in the ethylene biosynthesis since ACC was blocked ethylene

22. 22
synthesis will be downregulated, thus ethylene production in tomato will be delayed that will
lead to the delay of ripening of fruit (Fig. 13)
Fig. 11. Ethylene biosynthesis pathway in tomato
Table.7. Status of RNAi for quality improvement in vegetable crops

23. 23
G. CRISPR CAS9- A genome editor
It has been successfully applied to various plants, including Rice, Wheat, Maize and Tomato
(clustered regularly interspaced short palindromic repeats) is a types of DNA sequences
found in the genomes of prokaryotic organisms such as bacteria. It is nothing but acquired
immune system present in prokaryotes, which is identified in early 1987 in E. coli.Later,
scientists identified and modified as a powerful gene editing tool and got a broad application in
genome research field due to its ease of use and cost-effectiveness. Emmanuelle Charpentier
and Jennifer Doudna got nobel prize for genome editing discovery.
Fig.12: Schematic view of gene silencing by RNAi technology in plants
The synthetic guide RNA (sgRNA) contains a region (usually 20 bp in length)
complementary to the target site on the genomic loci and stem loops that mediate the binding
of the Cas9 protein. The protospacer adjacent motif (PAM, NGG) required for cleavage.
Induced DSBs of the target DNA are repaired either by NHEJ (non-homologous end joining)
or HDR (homology-directed repair) resulting in gene mutations that include nucleotide
insertion, deletion, or substitution around the cleavage site.

24. 24
Objective: To develop Reduced PPO activity lines in potato using StPPO2 gene
● The polyphenol Oxidase (PPO) is the ezymes which catalyze the oxidation of Phenol
compound into quinones. These quinones reacts with amino acids leads to brown
pecipitates. Thus causes browning which leads to reduction in quality of certain vegetables.
● In potato enzymatic browning is a severe problem, because the tubers can be affected
during harvest and post-harvest procedures.
● Therefore, the development of modern technologies to control PPOs activity in plants is the
most promising and safest approach to avoid undesirable browning.
Material and Method-
1. sgRNA design on StPPO2 gene: coding sequence of stPP02 was amplified from S.
tuberosum cv. Desiree and sequenced. later the Two sgRNAs were selected on the resulting
sequence and named sgRNA157 and sgRNA564.
2. Protoplast Transfection With RNPs and Mutation Screening of Regenerated Lines
CRISPR/Cas9 was delivered in the form of Ribonucleoprotein complexes into protoplasts by
transfections. Then HRFA analysis was done in the regenerated lines. This revealed totally 9
edited lines displayed mutations in the target alleles.
3. Sequence Analysis of StPPO2 in Selected Lines
Sequence analysis was performed on selected lines to confirm HRFA results.
4. Analysis of Off Target Mutations in StPPO Genes
HRFA was performed on StPPO1 and StPPO4 genes in selected lines carrying mutations in
alleles of StPPO2 gene.
5. Enzymatic Browning and PPO Activity Analysis in Tubers
Case study-6
Reduced Enzymatic Browning in Potato Tubers by Specific
Editing of a Polyphenol Oxidase Gene via Ribonucleoprotein
Complexes Delivery of the CRISPR/Cas9 System
Andersson et al. (2020)

25. 25
.
Fig.13. (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: 14. Discoloration development of selected edited lines at times 0, 24, and 48 h after
cutting

26. 26
Results-
The tubers were cut, exposed to air and discoloration development was registered at times 0,
24, and 48 h after cutting. After 24 h of air exposure, the typical brown discoloration related to
oxidation was visible in lines Desiree RC and M08003, but not in the rest of the analyzed lines
fig. 15. The relative enzymatic browning in lines M08001 and M08002 ranged between 0.26
and 0.27 demonstrating a reduction of around 73% related to the control line. Relative PPO
activity was significantly lower in lines M08001 and M08002 in comparison to the control line
Desiree RC.
Inference
 This system proved to be specific for the target gene, without affecting the coding
sequence of other StPPO family members.
ACHIEVEMENTS
Table.8 .Wild Sources for quality traits

27. 27
Table.9. Varieties released with improved quality traits.
(Kallo, 1986)
Transgenic Approach for Bio fortification in vegetables crops
Biofortification refers to process of improving nutritional quality of food crops, this can be
achieved through agronomic practices, conventional breeding or biotechnological approaches
like genetic engineering and genome editing by Kumar et al., 2020
Table.10. Biotechnological approaches for quality traits in vegetable crops

28. 28
 Some of examples of transgenic plants are in tomato Flavr savr, was th first
commercially grown genetically engineered food for human consumation. Followed by
Introgression of AmA1 gene from Amaranthus hypochondriacus tend to increase 60 %
protein content in potato. And The scientists identified two genes (SlFdAT1 and Solyc12)
from snapdragon that induce the production of anthocyanins concentration in purple
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
Organizations involved in biofortification
 Consortium of international Agricultural Research (CGIAR) International Food Policy
Research Institute (IFPRI)
 International Crops Research Institute for semi-Arid Tropics (ICRISAT)
 International Potato Centre (CIP)
Anoop pur : India’s First Biofortified Village
 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.
Research Regulation
 The departments of biotechnology and the ICAR have reconginsed the potential use of
gene-edting technology. The DBT’s national Agri food biotechnology institute Punjab
was one among the first in India to use CRIPSR/cas 9 to carry out a change in the
phytoene desaturase (fruit ripening) gene of banana cv Rasthalli .
 ICAR institute are involved in the application of CRIPSR technology for enhancing
stress tolerance and nutritional quality in a number of crops.

29. 29
 Scientist He jianukui created shock to the world in the year 2018 with the stunning
claim that he had altered the genetic makeup of IVF embryos and implanted them into
a women’s uterus, leading to the birth of twin girls name called 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.
 The square tomato is not only special in terms of shape, but it will also improve your
quality of life. Because it contains more than 50% lycopene, it will protect you from
many diseases. Ready to release for commercial purpose
 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

30. 30
FUTURE THRUST
 In future more systematic work should be needed in Vegetable crops with respect to
Nutraceuticals.
 There is need to make the molecular marker technology more price, productive and
cost effective in order to study various gene of interest
 And need to be create awareness on Nutraceuticals and Bio fortified varieties.
CONCLUSION
 Nutritional qualities and associated health benefits of vegetable crops is becoming
important criteria for their increase in consumer preference. Molecular tools and
technologies showed effectiveness to speed up the classical breeding methods for
quality trait. Utilization of advanced breeding methods such as molecular markers,
Marker assisted selection, genetic engineering and CRISPR-cas-9 along with
conventional breeding method have great promise in reducing time and cost involved
in breeding for improvement of quality traits in solanceous vegetables.

31. 31
References:
ANDERSON, M., GONZALEZ, M. N., MASSA, G. A., TURESSON, H., OLSSON, N.,
FALT, A. S. AND FEINGOLD, S. E., 2020, Reduced enzymatic browning in potato
tubers by specific editing of a polyphenol oxidase gene via ribonucleoprotein
complexes delivery of the CRISPR Cas-9 system. Front. Plant Sci., 10(1): 1-12.
BEHERA, T. K. AND SINGH, S., 2019, Advances in vegetable breeding for nutraceuticals
and quality traits. Indian J. Genet., 79(1): 216-226.
CHADHA, M. L., HEDGE, R. K. AND BAJAJ, K. L., 1998, Heterosis and combining ability
studies of pigmentation in brinjal (Solanum melongeona L.). Vegetable Sci., 15: 64-71.
KALLOO, G., 1986, Tomato (Lycopersicon esculentum Miller). Allied Publishers Private Ltd.
New Delhi-110002.
KUMAR, A., KARDILE, H. B., RAIGOND, P., CHANGAN, S. S., THAKUR, N., LAL, M.
K. AND SINGH, B., 2020, Biofortification of vegetables. Advances in agri-food
biotech., 105-129.
KUMARI, S., ANOKHE, A. AND KUMAR, R. 2017, Nutritional quality and health benefits
of solanaceous vegetables. Indian J. Genet., 29(1): 200-218.
LEE, D. G., JANG, S., PARK, M., LIM, J. H., JUNG, J. W. AND KANG, B. C., 2021,
Breeding Capsicum chinense lines with high levels of capsaicinoids and capsinoids in
the fruit. Agric., 11(9): 1-12.
MANOHARAN, R. K., JUNG, H. J., HWANG, I., JEONG, N., KHO, K. H., CHUNG, M. Y.
AND NOU, I. S., 2017, Molecular breeding of a novel orange-brown tomato fruit with
enhanced beta-carotene and chlorophyll accumulation. Hereditas, 154(1): 1-8.
MAXWELL, D. P., KWABENA OSEI, M., DANQUAH, A., DANQUAH, E. Y., BLAY, E.
AND MASSOUDI, M., 2022, Marker Assisted Backcrossing of alcobaca gene into
two elite tomato breeding lines. Front. Hortic., 6(1): 1-10.
NAGAR, M. M. AND MEKAWI, E. M., 2018, Genetic engineering to improve β-carotene
content in pepper. Plant Biotechonol., 56(1): 119-126.

32. 32
SINGH, S., YADAV, S. AND SINGH, A. 2021, Recent approaches for breeding vegetable
crops for quality, rich in nutrients and nutraceuticals. J. Pharmacom.
Phytochem., 10(1): 2061-2071.
SWATHY, S. P., KIRAN, K. R., RAO, M. S., MAHATO, K. K. AND RADHAKRISHNA,
M., 2021, Responses of He-Ne laser irradiation on chlorogenic acid content of brinjal
(Solanum melongena L.) var. Mattu Gulla. J. Phytochem. Photobio., 164(57): 182-190.

33. 33
UNIVERSITY OF AGRICULTURAL SCIENCES, BENGALURU
DEPARTMENT OF HORTICULTURE
HRT- 681 (1+0)
SEMINAR-I
Name: Pallavi Wani Date: 08-04-2022
ID No.: PAMB1076 Time: 09:15 AM
Class: 2nd
Ph.D. Horticulture Venue: Seminar Hall
Synopsis
Advanced Breeding methods for improvement of quality traits in solanaceous crops
Vegetable crops are rich in dietary micronutrients, antioxidants and functional
phytochemicals. Among these, solanaceous vegetables are key sources of health protective
dietary constituents such as minerals, vitamins and antioxidants in human diet. Some of the
elements are of public health concern such as β-carotene, lycopene, anthocyanin, calcium and
folic acid which attracts breeders interest to improve their content in vegetable crops so that
their dietary intake could be increased. The attempts through conventional breeding methods
have been slow and inadequate to fulfill the above mentioned traits. Hence newly development
in molecular and biochemical diagnostic tools and techniques have been used for improving
the other quality traits (Behera and Singh, 2019).
Manoharan et al. (2017) studied on developing a new tomato fruit color that is enriched
with β-carotene and chlorophyll content. The expression of CYC-B mRNA coincided with the
accumulation of β-carotene and loss of SGR protein function leads to inhibit chlorophyll
degradation. They revealed that highest amount of β-carotene and chlorophyll content were
found in orange brown and brown phenotypes respectively.
Nagar and Mekawi (2018) conducted an experiment on in vitro regeneration and
transformation methods for increasing β-carotene content in pepper fruits. β- Lcy gene was
inserted via Agrobacterium mediated transformation. The transgenic pepper plants showed a
significant increase in fruit β-carotene reaching seven to ten folds than the control.
Andersson et al. (2020) studied the application of the CRISPR Cas-9 system to induce
mutations in the StPPO2 gene in the tetraploid potato cultivar Desiree. They revealed that
relative enzymatic browning was significantly lower in all the edited lines in comparison to the
control line. The mutant edited lines showed reduction of up to 69 and 73 per cent in tuber
PPO activity and enzymatic browning respectively as compared to that of control.
Lee et al. (2021) developed Capsicum chinense lines with high capsaicinoid and
capsinoid contents in the whole fruit by introducing the genetic factors that regulate capsaicin
biosynthesis in the pericarp. Result revealed that ‘SJ’ and ‘BJ’ lines found with high capsinoid
and capsaicinoid contents, respectively, in both the placenta and pericarp.
To study the response of Helium-Neon (He-Ne) laser irradiation on chlorogenic acid
content of Solanum melongena L. var. Mattu Gulla, seeds were irradiated with different doses
(20, 25 and 30 J/cm2
) of He-Ne laser showed significant enhancement on the chlorogenic acid.

34. 34
Seeds irradiated with dosage of 20 J/cm2
resulted with maximum chlorogenic acid content as
compared to other treatments (Swathy et al., 2021).
Maxwell et al. (2022) performed marker assisted backcross breeding to transfer shelf-
life gene (alc) into the two elite tomato breeding lines. All the MABC-derived lines showed
significant extended shelf life compared to the checks. The introgression of the alc gene into
cultivated tomatoes for extended shelf-life could reduce post-harvest losses of tomato and
considerable increased the shelf-life of MABC-derived lines.
Conclusion:
Molecular tools and genome sequencing techniques showed effectiveness to speed up
the classical breeding methods for quality traits of complex pathways. Utilization of
conventional breeding techniques along with powerful tools of biotechnology to pace the
development program for redesigning of crops. Molecular markers, RNA interference (RNAi),
genetic engineering and CRISPR cas-9 have great promise in reducing time and cost involved
in breeding for improvement of quality traits in solanaceous vegetables.
References:
ANDERSON, M., GONZALEZ, M. N., MASSA, G. A., TURESSON, H., OLSSON, N.,
FALT, A. S. AND FEINGOLD, S. E., 2020, Reduced enzymatic browning in potato
tubers by specific editing of a polyphenol oxidase gene via ribonucleoprotein
complexes delivery of the CRISPR Cas-9 system. Front. Plant Sci., 10(1): 1-12.
BEHERA, T. K. AND SINGH, S., 2019, Advances in vegetable breeding for nutraceuticals
and quality traits. Indian J. Genet., 79(1): 216-226.
LEE, D. G., JANG, S., PARK, M., LIM, J. H., JUNG, J. W. AND KANG, B. C., 2021,
Breeding Capsicum chinense lines with high levels of capsaicinoids and capsinoids in
the fruit. Agric., 11(9): 1-12.
MANOHARAN, R. K., JUNG, H. J., HWANG, I., JEONG, N., KHO, K. H., CHUNG, M. Y.
AND NOU, I. S., 2017, Molecular breeding of a novel orange-brown tomato fruit with
enhanced beta-carotene and chlorophyll accumulation. Hereditas, 154(1): 1-8.
MAXWELL, D. P., KWABENA OSEI, M., DANQUAH, A., DANQUAH, E. Y., BLAY, E.
AND MASSOUDI, M., 2022, Marker Assisted Backcrossing of alcobaca gene into
two elite tomato breeding lines. Front. Hortic., 6(1): 1-10.
NAGAR, M. M. AND MEKAWI, E. M., 2018, Genetic engineering to improve β-carotene
content in pepper. Plant Biotechonol., 56(1): 119-126.
SWATHY, S. P., KIRAN, K. R., RAO, M. S., MAHATO, K. K. AND RADHAKRISHNA,
M., 2021, Responses of He-Ne laser irradiation on chlorogenic acid content of brinjal
(Solanum melongena L.) var. Mattu Gulla. J. Phytochem. Photobio., 164(57): 182-190.