Biotechnological interventions for improvement of fruit

1. Topic:- Biotechnological interventions for improvement
of fruit crops
Tajamul Farooq Wani
PhD Fruit Science
SKUAST-K
CREDIT SEMINAR
1

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)
2

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
3

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
But has some limitations with special reference to
4

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
5

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
6

7. Biotechnology for crop Improvement
Genetic
Engineering
Molecular
Markers
Tissue Culture
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. Anther Culture
etc.
7

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 et al. (2010) 8

9. 9

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
10

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 et al. (2006)
11

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 and small quantity of DNA is required
SOUTHERN
12

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
13

14. 14

15. SSR [ simple sequence repeats ] / microsatellites
15

16. co
16

17. 17

18.  Co-dominant
 Highly variable
 Widely used
 Used in marker assisted selection, fingerprinting
 Reproducible (within species)
Charactertics of SSR Marker
18

19. 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
19

20. Uses of Molecular Markers in Fruit Crop
Improvement
Genetic Mapping
Marker Assisted Selection
20

21. Marker assisted selection for seedlessness in table grape
breeding
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.
21
Karaagae et al. (2012)
Haplotype
name
S1 S2 S1r S2r MA1 MA2 MA1r MA2r
Allele size
(bp)
157 153 157 153 149 151 149 149
Allele size
(bp)
198 200 198 200 198 205 198 198
Allele size
(bp)
212 200 200 212 242 236 236 236
Phenotype seedless seedless seedless seeded seeded seeded seeded seeded
Number of
accessions
17 1 2 31 14 35 9 3
% seedless 100 100 100 0 14 3 11 0

22. Strain Without V25, Vd3, Vf Vf V25+Vd3+
Vf
V25+Vd3 Vd3
Elsatr Gala GD 041 Priscilla 025 D3 2000-012
EU-B05 (1) S S S S R R R S
EU-
NL19(1)
S S S S R R R S
1639 (2) S S S S R R R S
US2 (3) S S S S R R R S
1638 (4) S S S S R R R S
EU-D42 (6) S S S S S R R S
EU-NL05
(7)
S S R S S R R S
1066 S S R S S R R S
22
Identification and mapping of the novel apple scab resistance
gene Vd3
Soriano et al. (2009)
Table: 1 Strains of V. inaequalis used in the disease tests, and their sporulation on
cultivars and selections containing Vf, V25 or Vd3

23. mapping of Vd3, SSRs were tested.
Map position of Vd3 gene
on chromosome with
respective markers 23
SSR marker SSR marker 1980-015-025
Map (cM)
χ2 SSR origin
CH03g12 4.2 0 0.19 Liebhard et al.
(2002)
Hi21g05 7.6 - 1.35 Silfverberg-
Dilworth et al.
(2006)
Hi02c07 23.1 - - Silfverberg-
Dilworth et al.
(2006)
Hi12c02 42.0 31.7 1.90 Silfverberg-
Dilworth et al.
(2006)
NZ03c1 53.5 - - Guilford et al.
(1997)
KA4 42.6 34.4 0.55 Silfverberg-
Dilworth et al.
(2006)
CH-Vf1 55.9 37.4 0.04 Vinatzer et al.
(2004)
Hi07d08 67.8 - 2.84 Silfverberg-
Dilworth et al.
(2006)
CH05g08 77.4 58.9 4.54 Liebhard et al
(2002)
Table 2: SSRs tested in 1980-015-025 parent, their position in genetic maps of Discovery
(Silfverberg-Dilworth et al. 2006) and 1980-015-025, and the χ2 statistical analysis of the segregation
for monogenic inheritance

24. 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 stresses : Нerefore, growing insect-pest and disease
resistance GM fruits such as papaya, grapes, and apple etc. not only reduces the
economic losses but also ensure to provide chemical free fruits.
• Cold tolerance : Unexpected frost can destroy sensitive seedlings in many fruit
crops. Genetic transformation of guava with cold hardiness genes (CBF1, CBF2
and CBF3) make these plants able to tolerate cold temperatures.
• Rapid method of crop improvement: Stable transgenic plant can be developed in
3- 4 years, whereas it takes 12-15 years to develop a new variety through
conventional methods of breeding.
24

25. Process
Gene constructs
Vectors for the production of transgenic plants
Transformation techniques
Integration and inheritance of the transgenes
Analysis and confirmation of transgene integration

26. 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)
26

27. 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.
27

28. 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
28

29. Overall Process in Transgenics
GMO [genetically
modified organism]
Tissue culture
29

30. Particle bombardment
Electroporation
High Voltage
30

31. Table.3 Permits and notifications of transgenic fruits approved
CROP TRAIT GENES DEVELOPER
Apple Reduced polyphenol
oxidase
PPO suppression
transgene, nptII
Gebber farms
Non browning
reduced polyphenol
oxidase
Polyphenol oxidase
antisense, PGAS1,
PGAS2
Cornell university
Banana Bunchy top
resistance
Replicase associated
protein, replicase
inverted repeat,
nptII
University of hawaii
Grape root stock Grapevine
fanleafneo virus
resistance
Coat protein
gene,heat shock 90
homologous gene,
nptII
Cornell university
Grape fruit Aphid resistance Agglutin coat
protein, GUS.
Texas agrilife
research
31

32. Table :4 Approved transgenic in fruit crops
Crop Variety Trait Developer Approval
Papaya (Carica
papaya)
SunUp PRSV
resistant
Cornell
University
USA (1996)
Canada
Papaya (Carica
papaya)
Rainbow PRSV
resistant
University of
Florida
USA (2008)
Plum (Prunus
domestica)
Honey
Sweet
PPV resistant USDAARS USA (2009)
Apple (Malus x
domestica)
Arctic apple Non-
browning
Okanagan USDA (2015)
32

33. Development of Transgenic Papaya through Agrobacterium-
Mediated Transformation
Azad 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.
33
Treatment Survivability (%)
(Mean ± SE)
Callus formation
(%) (Mean ± SE)
No. of GUS
positive response
(%) (Mean ± SE)
No. of somatic
embryos
produced/explants
(Mean ± SE)
No. of regenerated
plantlets/explants
(Mean ± SE)
C. papaya cv. Shahi
Hypocotyls 6.83 ± 0.26 42.41 ± 2.72 27.48 ± 1.40 8.41 ± 0.65 5.36 ± 0.47
Zygotic embryos 5.50 ± 0.21 34.46 ± 1.12 20.06 ± 0.93 6.53 ± 0.60 3.38 ± 0.60
C. papaya cv. Ranchi
Hypocotyls 4.73 ± 0.32 38.80 ± 0.79 22.80 ± 1.50 7.60 ± 0.49 4.06 ± 0.35
Zygotic embryos 4.50 ± 0.15 28.90 ± 1.64 28.90 ± 1.64 28.90 ± 1.64 2.11 ± 0.17
Table : 5 Transformation efficiency and GUS activities of hypocotyls and immature zygotic
embryos of Carica papaya cv. Shahi and Ranchi infected by Agrobacterium tumefaciens
strains LBA-4404.

34. (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
34

35. Over - expression of the apple spermidine synthase gene in
pear confers multiple abiotic stress tolerance by altering
polyamine titers
• 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.
35
Wen et al. (2006)

36.  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
36

37. 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 201337

38. • 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.
38
Krens et al. (2015)
Cisgenic apple trees; development, characterization, and
performance

39. 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.
39

40. 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 40

41. 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 41

42. 42

43. 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 (2020)
43

44. • 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.
44
RNAi- mediated silencing and overexpression of the FaMYB1
gene and its effect on anthocyanin accumulation in strawberry
fruit
Ishikawa et al. ()

45. 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.
45

46. PaCYP78A9, a Cytochrome P450, Regulates Fruit
Size in Sweet Cherry (Prunus avium L.)
Sweet cherry is an important fruit crop in which fruit
size is strongly associated with commercial value
They characterized genePaCYP78A9that is involved
in the regulation of fruit size
Overexpression and silencing of this gene
(PaCYP78A9) was done.
RNAisilencingof PaCYP78A9 producedsmall
cherryfruits,Overexpressionof PaCYP78A9 resulted
inincreased seed size
Qi et al. (2015) 46

47. RNAi silencing of PaCYP78A9 gen
RNAi
overexpression
of
PaCYP78A9
gene
In this study showed that PaCYP78A9 gene is responsible for fruit size in cherry
47

48. CRISPR Cas9
(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
Hille F & Charpentier E 2016
48

49. 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
49

50. 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
50

51. 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
51
Funtional analysis of TM6 MADS - box gene in the octoploid
strawberry by CRISPR/Cas 9 directed mutagenesis

52. 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
52

53. 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
53

54.  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
54
Jia et al. (2017)
Genome editing of the disease susceptibility gene CsLOB1 in citrus
confers resistant to citrus canker

55. 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
55

56. 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
56

57. • 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.
Table: 6 Number of microcallus, tetraploid and somatic hybrid PEMs line and somatic hybrid plants
obtained following the protoplast fusion of mango at three parental combinations at cultivar level: ‘Tommy
Atkins’ + ‘Kensington Pride’, ‘Keitt’ + ‘Kensington Pride’ and ‘Haden’+ ‘Kensington Pride’
57
Intraspecific somatic hybridization of mango (Mangifera
indica L.) through protoplast fusion
Rezazadeh et al. (2011)
Parental
combination
(‘cultivar’)
Fusion drop Micro callus obtained Tetraploid
PEMs line
Somatic hybrid
Subcultu
re 1
Subcultu
re 2
PEMs
line
Plant
‘Kensington
Pride’ + ‘Haden’
10 1500 138 27 4 3
Kensington
Pride’ + ‘Tommy
Atkins
10 1200 50 6 0 0
Kensington
Pride’ +’ Keitt
10 1320 54 8 0 0
Total 30 4020 242 41 4 3

58. (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
58

59. 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
59

60. • 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)
60

61. 61
Culture
components
Media
M1 M2 M3 M4 M5 M6 M7 M8
Macroelements MSa MSa MSa MSa MSa MSa 1/2MSa MSa
Microelements MSa MSa MSa MSa MSa MSa 1/2MSa MSa
Sucrose (g/ L) 30 15 30 30 30 30 15 30
Vitamins Morelb Morelb Morelb Morelb Morelb Morelb Morelb MA1
6-BA (mg/ L) - - 0.1 0.5 - - - -
Kinetin (mg/ L) - - - - 0.1 0.5 - -
IAA (mg/ L) - - - - - - - 1.0
2,4-D (mg/ L) - - - - - - - 4.0
NAA (mg/ L) - - - - - - - 1.0
Table :7 Composition of media used in study

62. 62
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 .

63. 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.
63

64. Thank you
64