Biotechnological interventions for crop improvement in fruit crops

1. Topic:- Biotechnological interventions for crop
improvement in fruit crops
Submitted to
Dr. H S Dhaliwal
Dr. Monika Gupta
Dr. Karanbeer Singh
Submitted by
Anil Sharma
L-2019-H-89-D
CREDIT SEMINAR
Department of Fruit Science, PAU, Ludhiana

2. Fruits undoubtly , constitute the oldest food of mankind
 In India, fruits have remained in prominence from ancient times
[ banana, mango].
 Presently fruit production is becoming more important to
national as well as global economics.
(668.75 USD Millions)
 It helps us to earn the foreign exchange
Earliest known
cultivated fruits
INTRODUCTION
National Horticulture Database 2019-2020

3. • With time man cultivated majority of the fruit crops and transform
these fruit crops to such an extent that they are far removed from
their wild progenitors, as man have done intense selection of few
genotypes.
to fixation of unique
and desirable genotypes
• Narrowing germ plasm base for many fruit crops
• Depend upon narrow genetic base for production of fruits and
obstacles in system for improvement of fruit crops and imperial fruit
production
leads
leads
leads
Sugam Birthare, 2018

4.  After Mendels work on heredity in plants , breeding work was established on
genetic principles
When we go back to traditional methods for fruit improvement , it involves
Conventional methods take long time
Depends on naturally occurring variations
Induced mutations are often harmful, random and unstable
Detection of rare recombination is difficult during selection
Inability of sexual system to in corporate variations from unrelated
species
Lack of sufficient space to grow necessary population to recover superior
recombinants
• Identification of superior phenotypes
• Clonal propagation of best selected phenotypes
• Hybridization of best selections
• Standardization of best selections
(Xu et al 2019)
But has some limitations with special reference to

5. Biotechnology defined as 'any technological application that uses biological
systems, living organisms to make or modify products or processes for
specific use‘.
However plant biotechnology can provide many methods to
overcome some of the limitations encountered in fruit crop
improvement
Verma et al 2014

6. Advantages of biotechnological approaches in fruit
crop improvement
Biotechnology
Elimination of
unreliable phenotypes
Eliminate long term
field trials
Shorten breeding
cycle
No linkage drag
Overcome distant
hybridization
barriers
No species/genus
transfer barriers

7. Biotechnology for Fruit Improvement
Genetic
Engineering
Molecular
Markers
Other techniques
A. Transgenics
B. Cisgenics
C. Rna I
D. CRISPR cas
A. Marker
assisted
selection
B. Gene
mapping
A. Protoplast fusion
B. Embryo culture
C. Tissue Culture
D. Anther Culture
etc.

8. Molecular markers
A genetic marker can be defined as a chromosomal landmark or
allele that allows for the tracing of a specific region of DNA
Genetic markers that are located in close proximity to genes (i.e. tightly linked) may be
referred to as gene ‘tags’.
Dhillon W et al 2010

10. Morphological markers
These marker are related to the variation
in color, shape and size.
Advantages
• Readily available
• Sophisticated equipments not
required
• Direct measure of phenotype
Disadvantages
• Subject to environmental influences
• Limited in number
Biochemical markers
Depends on differences in enzymes,
secondary metabolites, proteins
Advantages
•Requires less sophisticated
equipments
•Robust as compared to
morphological markers
Disadvantages
•Influenced by stages of development
and tissues used
•Limited in number

11. Molecular markers
• Highly polymorphic
• Reproducible
• Dominant/Co-dominant
• Not subjected to environmental influences
• Direct genetic comparison
PCR based Non PCR based
PCR is used to amplify DNA fragments
AFLP [amplified fragment length polymorphism]
RAPD [ rapid amplified polymorphic DNA]
SCAR [ sequence characterized amplified region]
CAPS [ cleaved amplified polymorphic sequence]
ISSR [ inter simple sequence repeats]
SSR [ simple sequence repeats]
SNP [ single nucleotide polymorphism]
EST [ expressed sequence tags]
RFLP[restriction fragment
length polymorphism ]
Semagn K et al 2006

12. RFLP[restriction fragment length polymorphism ]
 Restriction enzymes (endonucleases) are bacterial enzymes (e.g., MseI, EcoRI,
PstI, etc.) that recognize specific four, six or eight base pair (bp) sequences in
DNA, and cleave double-stranded DNA whenever these sequences are
encountered.
 Co-dominant inheritance
SOUTHERN

13. RAPD[ random amplified polymorphic DNA]
1. Random sequence primers
2. Dominant
3. The band may be present or absent and the
brightness intensity of the band may be
different. Band intensity differences may
result from copy number or relative sequence
abundance

15. • Di-nucleotide repeat: CACACACA
Tri-nucleotide repeat: ATGATGATGATG
• Pure : CACACACACACACACACA
• Compound : CACACACAGAGAGA
• Interrupted : CACATTCACACATTCA
SSR locus are flanked by unique
sequence, primers are designed
to these unique flanking
sequence to amplify SSR locus
SSR [ simple sequence repeats ] / microsatellites
ISSR SSR ISSR SSR ISSR
SSR
PRIMER
Flanking regions
Flanking regions

16.  Co-dominant
 Highly variable
 Widely used
 Used in marker assisted selection, fingerprinting
 Reproducible (within species)
Charactertics of SSR Marker

17. SNP [single nucleotide polymorphism]
• SNPs are insertions and deletions (InDels) and are highly distributed
throughout the genome.
• SNP marker are widely used for mapping, marker-assisted breeding and
map-based cloning.
Single nucleotide
difference

18. Uses of Molecular Markers in Fruit Crop
Improvement
Genetic Mapping
Marker Assisted Selection

19. Importance of gene Mapping
 In genetics, the genome represents such an area, the
chromosomes being the key landmarks, while individual
genes represent the places or elements of interest.
 Many of plant breeders, researchers benefit from knowing
the genetic landscape of the genome as it pertains to the
relative position of genes along a chromosome.
 The ultimate goal of gene mapping is to clone the
mapped genes

20. Determination
of map order
of the genes.
Key steps in Genetic linkage map construction
Creation of a mapping population
Identification of polymorphism.
Calculation of pair-wise recombination
frequencies from the mapping
population.
Establishment of linkage groups and
estimation of map distances.
M1
M2
Collard et al 2005

21. Genotyping of population with polymorphic markers

22. R gene
M1
M2
0cM
1.64cM
2.10cM
Feed the data to software's such as JoinMap, Mapmaker, QTL crtogrpher etc

23. In this study,
 198-bp allele at the VMC7F2 microsatellite locus as a potential marker
for selection of seedless genotypes due to its close linkage to the major
effect seedless SDI gene. Cultivar Sultanina main source of
stenospermocarpic seedlessness in table grape breeding.
 Presence of the 198-bp allele at VMC7F2 allows the reduction of the
progeny size to 54%, selecting most of the seedless individuals.

24. The resistance donor was the
selection 1980-015-025, which
contains the Vf, V25, and Vd3 genes

25. For further mapping of Vd3, SSRs were tested. Seven out of nine SSRs screened were
polymorphic in the mapping population, and five of them were incorporated to the
1980-015-025 map
Map position of Vd3 gene on
chromosome with respective
markers
Near to Vd3 gene

26. Goal of a mapping Is to clone our target gene
in a desired plant
Ellis et al 1988

27. • Carotenoid pigments in fruits are indicated ripening process and potential
nutritional value.
• Papaya fruit flesh color is caused by the accumulation of lycopene or β-
carotene in chromoplasts.
• To uncover the molecular basis of papaya flesh color.
• Candidate gene (CpCYC-b) sequences were obtained from bacterial line
(BACs) containing the two alleles.

28.  CpCYC-b, is the gene controlling fruit flesh color. Sequence
analysis of wild and cultivated papaya accessions showed the
presence of this frame-shift mutation in all red flesh accessions
examined due to 2 bp insertion in the coding region.

29. MAS [ marker assisted selection]
• Marker-assisted selection (MAS) is a method whereby a
phenotype is selected on the genotype of a marker
• There are following steps: high resolution mapping,
validation of markers and marker conversion
High-resolution mapping
• For marker assisted selection a marker must be at least <5
cM but ideally <1 cM away from the gene
Melese Lema et al 2018

30. Validation of markers
• Markers should be validated by testing their effectiveness in determining the
target phenotype in independent populations and different genetic backgrounds,
which is referred to as ‘marker validation’
Marker conversion
RAPD/AFLP/
RFLP
SCA
R
Or
STS

31. In this study
SSR markers were developed and validated.
A total of 116,453 (72.6%) of all identified SSR markers
were successfully mapped.
Primer pairs were designed for markers and assembled a list
of 300 genes potentially involved in fruit ripening in
papaya.

32. Distribution of SSR type according Genomic location
• This study showed primer pairs (257 SSR) for 113 cell wall-related and 187
ethylene-related genes (528 SSRs). These two groups comprise very good
candidate pulp softening and pigmentation control genes.
• This study resulted these SSR markers can be used in marker assisted
selection and cloning for fruit ripening in papaya

33. • A total of 12 strawberry cultivars were used in this study, Florida
Elyana, Sweet Sensation, Florida 127, Florida Radiance, Sweet
Charlie, Winterstar, FL10-121, 11.28-34, 11.31- 54, Strawberry
Festival, Florida Ninety, Winter Dawn and FL10-46
• For FaFAD1 gene for aroma, SSR marker were used determine
the presence or absence of this gene.

34. Three markers, UFGDHRM2, UFGDHRM4 and UFGDHRM5 were developed to
detect FaFAD1 in strawberry.
Primer
positions
Blue line indicate
presence of
FaFAD1 gene for
aroma, all the
three primer for
shows positive
resulted
Red line indicate
absent of FaFAD1
gene

35. Which does not
have FaFAD1
gene for aroma
 The development of three markers to detect FaFAD1 locus in octoploid
strawberry can be effectively used in fruit crop breeding programs

36. TRANSGENICS
Transgenic plants are plants that have their genomes modified through genetic
engineering techniques by the addition of a foreign gene from different species or
even kingdom.
Application of transgenic plants
• Resistance to biotic or abiotic stresses
Bacillus thuringiensis (Bt) crops are an example of transgenic plant
produced through this method.
• Improving crop yield and nutritional value
(Biofortification) a well-known example would be the golden rice, a
variety of Oryza sativa, produced to biosynthesize beta-carotene through genetic
modification (Narcissus pseudonarcissus).
• Transgenic plants as bioreactors for recombinant proteins
The recombinant proteins produced in transgenic plants include antibodies,
metabolites or catabolites, proteins, and vaccines
Sathish et al 2019

37. Process
Gene constructs
Vectors for the production of transgenic plants
Transformation techniques
Integration and inheritance of the transgenes
Analysis and confirmation of transgene integration
Manoj et al 2013

38. Gene constructs
Reporter genes/ Marker
It allow for detection of the transgene expression. The common
reporter genes include green fluorescent protein (GFP),
chloramphenicol acetyltransferase (CAT), beta-galactosidase
(LacZ), luciferase (Luc), and beta-glucuronidase (GUS).
Promotor
Gene of
interest
Terminator
Marker Gene casset
Promoters/enhancers Promoters
have function like on/off the gene
Terminator sequence ,
after transcription stops
the gene.
Lee-Yoon et al 2018

39. Vectors for the production of transgenic plants
A vector acts as a vehicle that transports the gene of interest into a
target cell for replication and expression.
Common vector consists of three components:
1. An origin of replication (initiates the replication of the vector )
2. Multicloning site (allows the insertion of the gene of interest)
3. Selectable marker (allow differentiation between transformed
and non-transformed cells Ti plasmid vector)
Anuradha Upadhyay, 2018

40. Transformation Techniques
Method
Vector mediated Direct gene transfer
Agrobacterium-
Mediated gene
transfer
Physical Methods
i. Electroporation
/Microinjection
ii.Particle
bombardment/micr
oprojectile
Chemical methods
Polyethylene glycol (PEG)-
mediated used for destabilizing the
cell membrane in the presence of a
divalent cation, thus increasing the
permeability of the cell
membrane, allowing for the
uptake of foreign DNA.

41. Particle bombardment
Electroporation
High Voltage

42. Overall Process in Transgenics
GMO [genetically
modified organism]
Tissue culture

43.  In apple it is difficult to obtain transgenic plants by Agrobacterium-
mediated transformation using in vitro leaves as explants.
 In this study, we developed an efficient regeneration and Agrobacterium-
mediated transformation system for crab apple (Malus micromalus) using
cotyledons as explants.
 A. tumefaciens contains vector pCAMBIA2301 was used for
transformation.

44. Therefore, proximal cotyledons
were selected as the explants
for the following
transformation experiments.
a) Seedling of Apple
b) Four types of explants
c) Adventitious buds
d) Adventitious buds regenerated

45. Development of Transgenic Papaya through Agrobacterium-
Mediated Transformation
Dayarani et al 2013
Transgenic papaya plants were regenerated from hypocotyls and immature
zygotic embryo after co-cultivation with Agrobacterium tumefaciens carrying a
plasmid vector containing 𝛽-glucuronidase (GUS) as the reporter gene.
The result of this study showed that the hypocotyls of Papaya cv. Shahi and cv.
Ranchi are better explants for genetic transformation compared to immature
embryos.

46. (a) Callus formation (b) Embryogenic callus (c) Transgenic expression
indicating blue colour on callus. (d) Blue colour showing the transgenic
expression in cross section of transformed callus. (e) Transgenic plant in culture
tube. (f) Ex-vitro condition of transgenic plant.
Genetic transformation of hypocotyls of Papaya cv. Shahi

47. • In apple MdSPDS1 gene is involved in abiotic stress.
• To obtain transgenic fruit trees tolerant to abiotic stresses in European pear,
transgenic pear plant were created by Agrobacterium-mediated transformation
by over-expressing this gene (MdSPDS1) in pear.
• The selected lines were exposed to salt (150 mM NaCl), osmosis (300 mM
mannitol), and heavy metal (500 mM CuSO4) for evaluating their stress
tolerances.

48.  Transgenic line no. 32, showed highest expression
level of MdSPDS1 and showed the strongest
tolerance to these stresses.
This study showed, MdSPDS1 gene over-
expressing transgenic pear plants could be used to
improve desert land and/or to repair polluted
environments .
limited or no sign of
wilting or
necrosis was
observed in line no.
32

49. Cisgenics
Cisgenesis is a term in which genes are artificially
transferred between closely related organisms.
• It uses the same technology that are used to
produce transgenic organisms, making
cisgenesis similar in nature to transgenesis.
Ratan et al 2013

50. • For the generation of cisgenic apples.
• The MdMYB10 gene for red fleshed apple (anthocyanin
biosynthesis) red plantlets were obtained and were grafted
and grown in a greenhouse. After 3years, the first flowers
appeared, showing red petals and red flesh colour .
• Also the introduction of the scab resistance gene Rvi6,
derived from resistant apple. Transformed plant were
grafted onto rootstocks. Young trees from four cisgenic lines
containing Rvi6 gene, were planted in an orchard. Fruits
from cisgenic lines were free of scab.

51. Different steps in the process of gene rating cisgenic MdMYB10 apple plants based
on purely visual selection
(A) Callus showing red coloration by anthocyanin production (B) The onset of regenerating red shoots (C) Red-
colored shoots ready for propagation (D) Micro grafted, in vitro propagated red shoot (E) Plant showing scion
(top, red) and rootstock (bottom, green) (F) Developing red-colored flower buds on a cisgenic line (G) Wild-
type flowering (H) wild-type and cisgenic red-fleshed apple.

52. An impression of phenotypes that were observed in the field trial
(B) Diseased leaf of wild-type (C) Flowering of a cisgenic tree (D) Scab symptoms
on an apple fruit (E) Unaffected apples on the cisgenic line

53. RNA I technology/post transcriptional gene silencing
• Technology in which RNA molecule inhibits gene expression or
translation.
• DNA RNA PROTEIN
– It selects target mRNA and chop it off
transcription translation
Saurabh S et al 2019
Which resulted protein
synthesis & gene
expression will STOP

54. RNA is normally single strand , if somehow in a cell it produces double
strand RNA , it could be dangerous. WHY??
It produces Si rna, sh rna, mi rna
Silencing of mRNAs
Using this technology we can silence our target gene.
pathogen
Diseased gene
If we identify that mRNA of
pathogen and specifically design si
RNA or ds mRNA and inject it into
the plant , it will cleave that mRNA
and silence the gene
Protiva and Sherif M 2020

55. Dicer enzyme
Slicer enzyme

56. • Strawberry contains anthocyanins, which are important antioxidant and
contribute nutritional value of the fruit.
• Down-regulation of FaMYB1 gene in plant a using Agrobacterium-
mediated RNA interference. As a result, FaMYB1-RNAi fruits increase
in anthocyanin content.
• Conversely, overexpression of FaMYB1 resulted in a decrease in
anthocyanin content.

57. RNA-i FaMYB1 gene
down regulation
RNA-i FaMYB1
gene overexpression
Anthocyanin FaMYB1
gene when overexpressed
Anthocyanin when
FaMYB1 gene down
regulated
FaMYB1 gene
down regulation
FaMYB1 gene
overexpression
Control
These data suggest that this gene (FaMYB1) negatively control anthocyanin
biosynthesis in the strawberry fruit.

58. Sweet cherry is an important fruit crop in which fruit
size is strongly associated with commercial value
They characterized gene PaCYP78A9 that is
involved in the regulation of fruit size
Overexpression and silencing of this gene
(PaCYP78A9) was done.
RNAi silencing of PaCYP78A9 produced small cherry
fruits, Overexpression of PaCYP78A9 resulted in
increased seed size

59. RNAi silencing of PaCYP78A9 gen
RNAi
overexpression
of
PaCYP78A9
gene
In this study showed that PaCYP78A9 gene is responsible for fruit size in cherry

60. CRISPR Cas
(clustered regularly interspaced short palindromic repeats)
This system composed of
Cas protein is a DNA cutting protein and locate a sequence in the genome called PAM
[protospacer adjacent motiff usually NGG seq]. Then
They form complex, identify and
cut specific sections of a DNA
Cas 9 protein
Single guide RNA (sg RNA)
Guide RNA unwinds double helix of DNA,
RNA is designed to match the sequence of DNA
and binds to DNA and cut the DNA
Cell tries to repair this break but
process is error prone and leads to
mutation and disable the gene
1) So, a good tool to knock out the specific gene
2) Some researchers deactivate cas 9 cutting ability & other enzymes and
transport to a specific DNA sequence & replaces one base with other . This is
called genome editing.
Hille F & Charpentier E 2016

61. PAM Sequence
Cas 9 protein cut in the DNA
sg RNA unwind
the genomic DNA
Genomic DNA
This represent the
cut in the DNA &
insert with new DNA
Gnome
Editing

62. Lulu and Nana, twin sisters
• CRISPR cas has been recently used in humans.
• He Jiankui who made the first genome-edited human babies in 2018.
• He is working at the Southern University of Science and Technology
(SUSTech) in China, started a project to help people with fertility problems,
involving HIV-positive fathers and HIV-negative mothers. He uses CRISPR
gene editing in vitro fertilization and embryos were edited of
their CCR5 gene to give genetic resistance to HIV.
WORLD’S FIRST GENOME EDITED BABIES

63. In this study, they use CRISPR/Cas9 to functionally characterize
the role of gene, FaTM6, in strawberry for anther
development.
Martın-Pizarro et al 2018

64. The mutant lines failed to develop any fruit due to a lack of fertile anthers
Phenotype of CRISPR knockout lines of FaTM6 in garden strawberry
A) Control : no mutagenesis has been done B) tm6-1, tm6-7 and tm6-9 are mutant line
shows no anthers and fruit development

65. Hence this FaTM6 gene is important for fruit formation in Strawberry and
also for formation of pollen, anther development.
A) Control : Petals , anthers and pollen grains developed B) tm6-1, tm6-7 and tm6-
9 are mutant line shows no development of petals, anthers and pollens development

66.  Citrus is a highly valued tree crop worldwide, while, citrus
production faces many biotic challenges, including bacterial canker.
 Here, they used CRISPR/Cas9/sgRNA technology to modify the
canker susceptibility gene CsLOB1 in grapefruit

67. This study indicates that genome editing using CRISPR technology will provide a
promising pathway to generate disease resistant citrus cultivar.
DLOB2, DLOB3 shows low
mutation rate (31 % and 23
% and showed canker
symptoms on leaves )
Whereas, DLOB9, 10, 11, 12
shows high mutation rate (89 %,
88 %, 46 % 51 %) and showed low
canker symptoms after inoculation
with Xanthomonas citri

68. Protoplast fusion/ Somatic fusion
Protoplast fusion is a type of genetic modification in which two
distinct species of plants are fused together to form a
new hybrid plant.
Somatic fusion involves following steps
1. The removal of the cell wall by
cellulase enzyme to form protoplast.
2. The cells are fused using electric
shock (electrofusion) or chemical
treatment. The resulting
fused nucleus is called heterokaryon.
3. Then nuclii are fused and cell wall is
induced using hormones.
4. The cells are grown
into calluses then, to plantlets and
finally to a full plant. This plant
known as a somatic hybrid
Nitin Verma et al 2008

69. • Only Haden + Kensington Pride (3 plants) where found to formed somatic hybrid.
• Hence, Somatic hybridization could be used to introduced of the desirable
traits like tolerance to biotic and abiotic stresses from cultivars and wild
species of mango into cultivars of mango rootstocks.
Somatic hybridization of mango via protoplast fusion at cultivars (Tommy
Atkins, Keitt and Haden) and from pro embryonic masses (PEMs) of cv.
Kensington Pride were used.

70. (a) Young leaves of cv. Haden (b) PEM induction (c) PEM suspension culture (d)
Isolated protoplasts (e, f) PEG-induced binary protoplast fusion (g) Early cell division
(h) PEM formation (i-j) Heart and torpedo-shape embryo production (k,l,m)
Germination of embryos (n) Regenerated somatic hybrid plantlets (n,o,p) Regeration -
acclimatization stage.
Somatic hybrid regeneration of cvs. Kensington Pride + Haden

71. Embryo culture is the technique in which we culture isolate immature or
mature embryos
Embryo culture
o Overcoming seed dormancy
o Shortening of breeding cycle
o Overcoming seed sterility
Application of embryo culture
Embryos develops from
zygote, the single cell
resulting from fertilization of
the female gamete and male
gamete

72. • Seed set in Musa spp.
germinate at low rate in soil
thus making breeding bananas
difficult.
• In this study, Seeds were
harvested at 60, 80 and 100%
maturity after that embryos
were removed under aseptical
conditions and cultured in test
tubes .
Dayarani et al 2014
Embryo culture and embryo rescue studies in wild Musa spp.
(Musa ornata)

73. Conclusion:- Good embryo recovery was found in seeds from 80 and 100%
mature fruits and M8 media rich in auxins led to callus formation at all
maturity levels .
 This table shows various medias used with different cultural components

74. Conclusion
• Biotechnology techniques contributed major role in fruit
crops improvement to overcoming barriers in conventional
improvement practices.
• The widespread use of molecular marker and their
application in plant breeding , genetic selection and genome
editing bring a novel strategy to boost crop improvement.
• Transgenic technology will be a valuable alternating in
solving food security problem that happens in as world of
growing human population.
• Use of RNAi technology could be the gate for the regulation
of genes related to diseases management , plant
development and crop improvement.
• Technology like CRISPR Cas for genome editing led to
advance for fruit crop improvement and result in making a
way to breed for any kind of genomic trait.

75. Thank you