Role of Biotechnological Approaches in Underutilized Tropical Fruit Improvements.pptx

Role of Biotechnological Approaches in Underutilized
Tropical Fruit Improvements
Name : Chavda Hardiksinh R. Course No. : MBB 591
Degree : M. Sc.(Agri.) PMBB Reg. No. : 04-AGRMA-02210-2020
Major Advisor : Dr. H. N. Zala Date : 01/10/2021
Minor Advisor : Dr. N. V. Soni Time : 3.00 to 4.00 PM
2

CONTENTS
Introduction
 Jack Fruit
 Guava
 Custard Apple
Biotechnological Approaches
 Tissue culture techniques and its studies
 Molecular markers and its studies
 Genetic engineering/ Genetic transformation
and its studies
Case Studies
3
Conclusion

INTRODUCTION :-
 Fruit trees are important as additional sources of revenue and are important diet
supplements.
 Fruits are the major source of several products of industrial importance and rich sources
of plant metabolites of medicinal value.
 Domestic demand for fresh fruits in tropical countries has been on the increase with
rising incomes, population growth and increased health consciousness among
consumers.
 Demand for fresh and processed fruits has been strong in recent years and this trend is
likely to continue in the coming years.
 Despite their significance these plants have not been researched thoroughly for their
propagation, cultivation and molecular characterization.
 Unlike the annual agricultural crops, fruit trees present certain unique problems in terms
of their improvement.
4
Bapat et al., 2020

INTRODUCTION :-
 To overcome problems like, long life cycle of fruit tree, many abiotic and biotic stress
conditions to reduce the productivity, quality of fruit yield is not assured every year.
 Biotechnological tools including tissue culture techniques, molecular markers associated
genetic diversity, genetic modification are used to enable plants tolerate the biotic and
abiotic stresses, and plant resistances to problematic disease, which may provide higher
nutritional contents, and extend the shelf-life of the produce.
 In India total production of fruits is estimated to be 102.76 million tones in 2020-21(NHB,
2021).
5
Crops
Year 2019-20 Year 2020-21
Area
(‘000 ha)
Production
(MT)
Area
(‘000 ha)
Production
(MT)
Jack fruit 187 1.739 189 1.931
Guava 292 4.361 304 4.433
Custard apple 50 0.395 45 0.390 (NHB, 2021)

Jack fruit :-
Botanical name: Artocarpus heterophyllus Lam.
Family : Moraceae
Chromosome Number: 2n =2x=56
Center of origin: Western ghats of southern India, Sri Lanka and Malaysia
Synonyms: Chakka, Kathhal, Pala, Halasina Hannu, Panasa Pandu Phanos etc.
6
 Jackfruit is the largest tree-borne fruit in the world, reaching up to 50 kg in weight and 60-90 cm in length.
 A mature tree produces up to 700 fruits per year, each weight 0.5-50 kg.
 Jackfruit pulp is rich in calories (84%), carbohydrates (18.9%), proteins (1.9%), vitamin A (540 IU) as well
as being the tree good source of iron and potassium.
 Ripe fruit flakes (bulb) contain total soluble solids (25-29º brix), carotene (500-580 IU), thiamin (30g),
pectin (1.5-6%) and minerals like phosphorus (30-40 mg) and calcium (20-30 mg)/100gm of edible portion
and ripe fruits are canned, used for preparation of jam, squash and candy.
 Lectine a natural protein from fruit is used in cancer treatment.
 The jackfruit are good source of “Jacalin” which is useful in preventing colon cancer, AIDS etc.
 Tree has got good timber value and is less attacked by white ants.

Guava :-
Botanical name: Pisidium guajava L.
Family : Myrtaceae
Chromosome Number: 2n=2x=22, 33(x=11)(Genome size: 443.76 Mb)
Center of origin: Northern south America
Synonyms: Jamphal, Amrut, etc.
7
 Guava generally known as ‘the apple of tropics’ consider one of the most important fruit of high
nutraceutical value in international trade and domestic economy of several tropical and subtropical
countries.
 Guava contains four times higher vitamin C than any other fruits and also good source of calcium and
phosphorus.
 Low in calories and fats but carry several antioxidant poly-phenolic and flavonoid compounds that play a
crucial role in prevention of many important diseases like cancer, aging, infections, etc.
 High concentration of several vitamins, dietary fiber, carotenoids, lectins, saponins, tannins, phenols,
triterpenes, and flavonoids altogether make guava therapeutically an important fruit.
 High pectin content make guava suitable for jelly making.
 The ripe fruit is used for making jams, guava syrup, guava juice and a number of recipies are made from
guava like cakes, sauce, ice- creams, butter and ketchups.

Botanical name: Annona spp.
Family : Annonaceae
Chromosome Number: 2n =2x=14 and 16
Center of origin: Tropical America
Synonyms: Sitaphal, Sugar apple, Sweetsop, Noina, Ata, etc.
Custard apple :-
8
 The white pulp of Annonas is rich in minerals and vitamins and also a potential source of dietary fibre (up
to 50 % w/w dry basis).
 High nutritive value of A. cherimola is due to fatty acids, edible fibres, carbohydrates, and minerals such as
calcium, phosphorous and potassium.
 Annona seeds, especially A. squamosa and A. muricata contain good amount of oil which can be exploited
for industrial purpose.
 Fatty acid composition in seeds, such as stearic acid (9.3%), oleic acid (37%), linoleic acid (10.9%,) and
isoricinoleic acid (9.8%).
 Extracts of the bark, leaves, and seeds possess anti-tumor activity.
 The fruit contains 1-b,17-dihyroxykauran-19-oic acid which has demonstrated anti-HIV activity.

Solution offered by fruit trees
Challenge Role of biotechnological interventions in fruit tree improvement
Limited
Productivity
 Identify and grow new species and
varieties that occupy new niches
 Able to be productive in more
marginal and/or degraded (e.g dry,
saline unfertile)
 Use molecular marker characterization of associated genetic diversity to
select the breed types that grow better in more marginal and/or degraded
areas
 Use genomics to understand the genetic pathways involved in abiotic stress
 Use GM to introduce new resistance to the pests and diseases
Propagation
 Genetic variability in the seeds
affects fruit plant characteristics.
 Use micropropagation and related multiplication methods to develop more
effective varieties
Post Harvest
Lost
 To develop new methodologies for
processing ripen fruits and develop
new applications to minimize post
harvest and production losses
 Use genomics to understand the genetic pathways involved in ripening
process and use GM to increase shelf life of fruits
 To develop new methodologies for processing ripen fruits
Plant
Derived
compounds
 It is possible that underutilized
species may provide valuable
sources of natural medicines
 Use micropropagation and plant cell, organ culture
 Resistant to abiotic and biotic stress
 Breeding for desirable traits
 Genetic transformation systems
Figure 1. Possible role of biotechnological strategies in solving current and emerging challenges of underutilized fruit
trees (Modified from Dawson et al. 2009)
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Figure 2. Biotechnological interventions in under-utilized tropical fruit tree improvement (Bapat et al., 2020)
10

Tissue culture techniques and its studies
11

1) Jack fruit (Artocarpus heterophyllus Lam.)
In vitro Rapid Propagation of Jackfruit (Artocarpus heterophyllus Lam.)
Harb et al., 2015
Case studies :-
12
 Explants (Shoot tips & Nodal segments) excised from healthy and juvenile shoots collected
during the four different seasons (Winter, Spring, Summer and Autumn) and cultured on MS
media.
For multiplication stage,
 Nodal segments cultured on MS medium supplemented with 6-benzlaminopurine (BA) at
different concentrations (0.0, 1.0, 2.0 and 5.0mg/l) in combination with kinetin at different
concentrations (0.0, 0.5, 1.0, 1.5mg/l).
For rooting,
 Elongated shoot excised and cultured on half strength MS medium supplemented with 0.5 and 1.0
mg/l IBA (Indole-3 butyric acid) and/or NAA (-Naphthalene acetic acid).
For shooting,
 Proliferated shoots excised and cultured on MS medium supplemented with concentrations of
gibberellic acid (1, 2 and 3 mg/l).
Egypt

Fig. 3. Vegetative propagation of
jackfruit by tissue culture technique
a. Jackfruit tree
b. Establishment of Jackfruit after 8 weeks
c. Multiple shoot proliferation after 6 weeks
d. Elongation of shoots after 8 weeks
e. In vitro rooted plantlets after 6 weeks
f. A plantlet acclimatized in sand and peat
Season Survival % Growth to survival %
Shoot tips Nodal
segments
Shoot tips Nodal
segments
Winter 100% 100% 90% 50%
Spring 70% 60% 60% 20%
Summer 20% 10% 40% 10%
Autumn 70% 50% 50% 20%
Table 1 : Effect of seasonal variations and explant type on survival and
growth to survival percentages of A. heterophyllus cultured on MS
medium.
13

GA3
concentration
(mg/l)
Shoot length (cm)
Before treatment (a) After 8 weeks (b)
Increase in length
(cm) (b-a)
0.0 1.45 cm 3.56 cm 2.11 cm
1.0 1.40 cm 4.74 cm 3.34 cm
2.0 1.35 cm 6.40 cm 5.05 cm
3.0 1.40 cm 6.54 cm 5.14 cm
Auxins concentration (mg/l)
Rooting %
No. of
roots/shoots
Average root length
(cm)
IBA NAA
0.5 0.0 30% 1.60 2.20 cm
1.0 0.0 40% 1.75 2.80 cm
0.0 0.5 10% 1.00 2.50 cm
0.0 1.0 30% 1.00 2.20 cm
0.5 0.5 60% 3.30 3.60 cm
1.0 1.0 80% 4.30 4.00 cm
Table 2: Effect of GA3 in MS
medium on shoot
elongation of Artocarpus
heterophyllus.
Table 3 :Effect of IBA
and NAA in ½ MS
medium on rooting of in
vitro derived shoots of
Artocarpus heterophyllus.
14

2) Guava (Psidium guajava L.)
Recurrent Somatic Embryogenesis and Plantlet Regeneration in Psidium guajava L.
Bajpai et al., 2016 15
 Collected four commercial cultivars of guava (Allahabad safeda, Lalit, Sardar (L-49) and Sweta)
70 days post-anthesis immature fruits.
 Immature zygotic embryos isolated from fruits of guava and cultured on MS medium with 2, 4-D
(2 mg/l), L-glutamine(400 mg/l) and 6% sucrose and observation take at every 4 weeks up to 12
weeks in dark conditions.
For maturation,
 Somatic embryos placed in a MS basal
medium containing PEG (50 mg/l) and
3% sucrose.
For plantlet regeneration,
 Somatic embryos isolated from the clumps and
cultured on MS medium with BA (0.5 mg/l), NAA
(0.2mg/l), casein hydrolysate (100mg/l) and 3%
sucrose.
UP, India

Fig. 4. Different developmental stages of somatic embryo
induction in Psidium guajava L. a) 70 day zygotic embryo as
explant source b) Pre embryogenic mass of cells c)
protuberance of transparent PEMC in clump d) globular &
heart shaped embryo e) elongated embryo f) torpedo-shaped
embryos g) cordiform embryo h) embryo proliferation i)
recurrent embryogenesis.
Fig. 5. Different stages of plantlet regeneration in
guava through somatic embryogenesis pathway a)
Maturation of somatic embryo b) plantlet germination c)
rooting in plant d) elongation of plantlets e) shoot
proliferation f) plant maturation g) plant in coco-peat h)
hardening in poly-house i) Plants in soil-pots.
16

Fig. 7. - Effect of 2, 4-D and its interactive
effect on the four genotypes of guava.
Represents maximum embryogenesis
induction in terms of frequency and intensity
with reference to the four genotypes
Fig. 6. - Effect of sucrose and its interactive
effect on the four genotypes of guava.
Represents maximum embryogenesis
induction in terms of frequency and intensity
with reference to the four genotypes
17

In vitro culture of Annona emarginata: A rootstock for commercial annonaceae species
3) Custard apple (Annona spp.)
De freitas et al., 2018
 Nodal segments excised from three years old Annona emarginata plants.
For shoot induction,
 Nodal segments with single bud cultured on MS media with BA at different concentrations (0, 1, 3 or
5µM).
For shoot elongation,
 Axillary proliferation with four bud cultured on WPM supplement with 1µM BA in combination
with different concentrations (0, 1, 5 or 10µM) of gibberellic acid-3 (GA3).
For root induction,
 Shoots with four bud cultured on WPM media with 1µM BA for 60 days and transferred to a WPM
basal medium without Plant growth regulators or supplement with different auxins: IBA (Indole-3
butyric acid), IAA (Indole-3 acetic acid) and/or NAA (1-Naphthalene acetic acid) at different
concentrations (1, 10 and 100µM).
18
Belgium

Fig. 8. Number of buds (A) and leaves (B) after 45 days in MS and WPM
media supplemented with different concentrations of BA.
Fig. 10. Clump formation at the
base of a shoot (indicated by an
arrow) in the presence of 5 μM
BA after 30 days on WPM
medium.
Fig. 9. Shoot height in WPM
medium with 1 μM BA and
different concentration of
GA3 after 45 days of culture
19

Fig. 12. A) Callus formation at the base of a shoot (indicated by
an arrow) in the presence of 100 μM NAA after 45 days of
culture. B) Leaf abscission and bud necrosis (indicated by an
arrow) in the presence of 10 μM NAA after 30 days of culture.
Fig. 11. Annona emarginata control supplemented with
only 1μM BA, compared to plants supplemented with 1μM
BA plus 5 μM GA3 under dark conditions with irregular
shapes and color of leaves after 45 days of culture.
20

Crop Research title Result References
Jack
fruit
The growth of jackfruit (Artocarpus
heterophyllus l.) Shoots on various
concentration benzyl amino purine
(BAP) in vitro
The result of tukey’s HSD test showed that concentration 2, 5 ppm gave the
fastest shoot emergence with an average of 2,875 days after planting, in
concentration 2,0 ppm gave the highest number of shoots with an average of
2.125 shoots per explant.
Bulo et al.,
2018
Protocol optimization for in vitro shoot
multiplication of Jackfruit (Artocarpus
heterophyllus L.)
The results show the combination of BAP and NAA resulted in significant
(P<0.01) differences for shoot number, Shoot length and leaf number,
whereby 2 mg/L BAP alone was found to be the best with a mean shoot
Number of 5.12 and length of 0.89 cm.
Ali et al.,
2016
Guava Evaluation of root-to-shoot de novo
organogenesis in wild guava Species,
Psidium schenckianum and P.
Guineense (myrtaceae)
The highest percentage of responsive explants (61.66%) and number of
shoots per Explant (9.25) was observed culture medium supplemented With
2.22 μm BA.
Dos santos
et al., 2021
Custard
apple
In vitro clonal propagation from adult
material of a savannah species of
socio-economic Importance: Annona
senegalensis pers.
The best average numbers of shoots (2.3) and Nodes (5.3) are obtained in
MS + BAP 2 mg·/l, with a reactivity of 91.66%, The addition of 0.1 mg·/l
NAA gave the best average length (8.25 cm) of in vitro plants.
Oumar et
al., 2021
Micropropagation of juvenile and
mature Annona Muricata L.
Increasing concentrations of BAP added to the basic woody plant medium
stimulated up to four or five shoots per explant in nodal cuttings or
hypocotyls, respectively. The addition of NAA with BAP was essential for
outgrowth of buds.
Lemos et
al., 2015
Other Tissue culture studies
21

22
Molecular markers and its studies
Markers
Biochemical
Isozyme
Protein banding
pattern
Morphological Molecular
Non PCR
based
RFLP
PCR
based
SSR
RAPD
AFLP
ISSR

Development of novel g-SSR markers in guava (Psidium guajava L.) cv. Allahabad Safeda
and their application in genetic diversity, population structure and cross species
transferability studies
Kumar et al., 2020 23
 Selected 40 guava genotypes: five wild species, nine hybrid and 26 commercial cultivar.
For molecular genetic diversity,
 Microsatellite-enriched libraries constructed which used for the design 38 novel g-SSRs
markers.
For phylogenetic and cross-transferability analysis,
 Neighbor-joining (N-J) tree was constructed of Psidium genotypes including the wild
species by the used of novel g-SSR markers.
For population structure analysis,
 The grouping of fourty guava genotypes including wild species by using bar plot diagram.
New Delhi, India
Case studies :-

24
SI
No
Marker ID Annealing
temp.
Allele size PIC
1. GUV-3 45.9 140-160 0.36
2. GUV 13-2 62.6 180-200 0.35
3. GUV-19 63.3 320-420 0.70
4. GUV 38-2 60 150-170 0.21
5. GUV 45-2 63.9 210-280 0.37
6. GUV 26-1 57.1 280-360 0.37
7. GUV 31 60 230-280 0.74
8. GUV 36-2 60 220-300 0.57
9. GUV 32 43 200-280 0.75
10. GUV 32-2 43 170-185 0.35
11. GUV 1-37 40.9 200-420 0.75
12. GUV 1-38-1 40.9 150-175 0.30
13. GUV4-10 46.9 380-520 0.54
SI
No
Marker ID Annealing
temp.
Allele size PIC
14 GUV 4-33 46.9 160-200 0.30
15. GUV2-19 50.9 190-210 0.37
16. GUV2-21 56.9 230-260 0.34
17. GUV2-36-2 56.9 400-550 0.14
18. GUV2-37 56.9 240-270 0.32
19. GUV2-43 56.9 170-390 0.87
20. GUV4-33 57.6 220-230 0.26
21. GUV4-42 60 420-470 0.37
22. GUV4-42-2 50.9 200-260 0.62
23. GUV4-45 50.9 280-380 0.55
24. GUV4-50 56.9 190-320 0.10
25. GUV4-50-2 56.9 250-400 0.68
26. GUV4-53 56.9 200-260 0.62
Table 4. Genetic variability indices of the g-SSRs among the set of guava genotypes.

25
Fig. 16. Gel image of SSR locus GUV31 showing allelic
variation among the Psidium genotypes
Fig. 17. N-J tree of guava genotypes including wild
Psidium species using scored data of the 26 developed
g-SSRs

26
Fig. 18. Bar plot of population structure
(K = 3) of 40 Psidium genotypes based
on 26 g-SSRs.
Fig. 19. Analysis of molecular
variance (AMOVA) of 40 Psidium
genotypes based on 26 g-SSRs.
Percentages of Molecular Variance

5) Custard Apple (Annona spp. )
Molecular diversity of Annona species and proximate fruit composition of selected
genotypes
Anuragi et al., 2016 27
For molecular genetic diversity analysis,
 Total 20 Annona genotypes belong to
different five species are used.
 The RAPD amplification and SSR
amplification was carried out using a 11
and 12 primers respectively.
For biochemical analysis,
 Out of 20 Annona genotypes, only nine
matured enough to produce fruit and
proximate fruit composition analysis in
this nine Annona genotypes.
Gujarat, India

Fig. 20. DNA amplification profile of RAPD (OPB-7
and OPB-16) and SSR (LMCH-43 and LMCH-128)
markers
The PIC for RAPD ranged from 0.86 to 0.92 and
PIC for SSRs ranged from 0.169 to 0.694
28
Fig. 21. Dendrogram of 20 Annona genotypes based on
combined RAPD and SSR data. (1. A. cherimola, 2. A.
atemoya, 3. A. reticulata, 4. A. muricata, 5. Red Sitaphal, 6.
Anand Selection, 7. Sindhan, 8. Balanagar, 9. GJCA-1, 10.
Vidyanagar Local, 11. ACC-1, 12. ACC-2, 13. ACC-3, 14.
ACC-4, 15. ACC-5, 16. ACC-6, 17. Sindhan 9 Anand
Selection, 18. Sindhan 9 Balanagar, 19. Anand Selection 9
Balanagar 20. Balanagar 9 Red Sitaphal)

29
Genotypes
MC
(%)
AC
(%)
TC
(%)
TSS
(%)
RS
(%)
FC
(%)
PC
(%)
PhC
(%)
TA
(%)
Asc(mg/
100g)
OC
(%)
A. cherimola 79.26 1.56 17.54 14.41 4.33 2.22 1.69 0.35 0.36 19.60 29.33
A. reticulata 73.00 1.48 22.71 14.89 4.80 2.18 1.85 0.41 0.38 23.36 29.05
A. muricata 81.23 1.69 16.48 8.92 3.27 2.34 1.15 0.50 0.65 39.24 32.50
A. atemoya 76.63 1.62 20.71 14.62 6.67 3.82 1.84 0.40 0.37 32.74 28.41
A. squamosa 73.99 1.36 23.24 16.62 7.81 3.28 1.97 0.3 0.22 31.53 25.39
Red sitaphal 74.48 1.41 22.51 16.08 7.49 3.28 1.78 0.34 0.29 28.41 23.13
Anand
selection
74.42 1.32 22.66 16.29 7.61 3.18 1.81 0.31 0.24 32.21 24.41
Sindhan 73.65 1.43 23.04 16.69 7.81 3.38 2.13 0.26 0.23 32.06 26.56
Balanagar 74.85 1.32 23.95 17.44 8.39 3.24 2.14 0.29 0.17 33.39 26.52
GJCA-1 72.55 1.32 24.05 16.63 7.75 3.32 1.98 0.30 0.19 31.57 26.35
Mean 75.56 1.46 21.52 15.11 6.46 3.00 1.82 0.35 0.32 30.29 27.36
CD @ 5% 5.37 0.14 2.16 1.31 0.48 0.38 0.34 0.05 0.05 3.50 3.10
SD 2.94 0.14 2.74 2.54 1.84 0.59 0.29 0.07 0.15 5.81 2.81
S.Em 1.79 0.05 0.72 0.44 0.16 0.13 0.11 0.02 0.02 1.17 1.03
CV (%) 4.11 5.67 5.80 5.02 4.30 7.35 10.72 8.86 9.59 6.67 6.55
Table 5. :- Statistics
of fruit (pulp)
quality parameters
(fresh weight basis)
and seed oil content
(OC) of nine
Annona genotypes
MC moisture content,
AC ash content,
TC total carbohydrates,
TSS total soluble sugars,
RS reducing sugars,
FC fibre content,
PC protein content,
PhC phenol content,
TA titratable acidity
ASC ascorbic acid
content,
OC seed oil content

Molecular
marker
Results References
RAPD Six RAPD primers used to distinguish 9 breadfruit cultivars (A. altilis, P.) from
Indonesia.
Ifah et al., 2018
Hard-fleshed and soft-fleshed fruit types of A. heterophyllus distinguished using OPB-
01–1.0 RAPD primer
Pushpakumara et al.,
2007
RFLP Eleven Artocarpus spp. were characterized using 20 restriction enzymes Kanazaki et al., 1997
AFLP Fifty jackfruit accessions from three provinces in China were analyzed using AFLP
markers. A total of 320 unambiguous bands were produced by eight primer pair
combinations, and 65 (20.3%) of them were found to be polymorphic.
Li et al., 2010
SSR Using 22 SSR primers for the 20 jackfruit genotypes out of which six primers (SSR 9,
SSR 10, SSR 30, SSR 34, SSR 45 and SSR 48) showed polymorphism among twenty
genotypes.
Kavya et al., 2019
Thirty-four primer pairs successfully amplified and 10 of them displayed clear
polymorphisms across the 67 individuals from four populations of A. hypargyreus.
Liu et al., 2016
ISSR Seventy-six accessions of jackfruit (A. heterophyllus Lam.) were analyzed for ISSR
markers among which 477 bands were produced by 24 ISSR primers, 427 were
polymorphic (89.52%).
Ye et al., 2009
Other studies on Molecular markers in Jack Fruit
30

Molecular
marker
Results References
RAPD Four primers, A02, A03, S07, and S08, were able to amplify and yield a total of 252 band
patterns from 33 P. guajava germplasm from three selected south-western location of
Bangladesh of which 33.19% were polymorphic.
Ahmed et al.,
2011
Six polymorphic RAPD fragments were identified in P. guajava among which three could be
correlated with quercetin content.
Feria-Romero
et al., 2009
AFLP Forty-eight guava accessions cultivated in Mexico were genetically characterize using four
AFLP primers.
Hernández-
Delgado et al.,
2007
SSR The genetic relationships of 45 individuals of Psidium guajava from six cultivars were
analyzed by screening SSR primers with high polymorphism and good repeatability.
Zehua et al.,
2019
Eleven SSR markers used to identify Psidium guajava in the Galapagos islands. Urquía et al.,
2019
24 SSR markers were utilized to evaluate the genetic variation across thirty-six guava
varieties including wild species and revealed a polymorphism of 95.7%.
Kherwar et al.,
2018
ISSR Used to confirm the genetic fidelity of somatic embryogenesis regenerated guava (Psidium
guajava L.)
Kamle et al.,
2014
Other studies on Molecular markers in Guava
31

Molecular
marker
Results References
RAPD Eight primers used to find out the genetic diversity amongst the A. muricata accessions. Brisibe et al., 2017
Genetic diversity determined in a total of 70 individuals collected from 7 A. muricata
populations in Java (Indonesia) using 6 RAPD primers.
Suratman et al.,
2015
Sixty-four sugar apple accessions collected from different municipalities in northern Minas
Gerais was assessed using 20 RAPD primers that produced 167 fragments of which 48 were
polymorphic.
Guimarães et al.,
2013
AFLP Three primer combinations that produced a total of 276 fragments were used to assess the
genetic diversity 106 Annona crassiflora individuals.
Egydio-Brandão et
al., 2016
SSR The genetic diversity of the Annona muricata L. species using eleven SSR markers. Thirty-
four accessions of A. muricata leave samples.
Thanachseyan et
al., 2017
Nine SSRs were used to analyze a total of 1504 cherimoya accessions 395 from Bolivia, 351
from Ecuador and 758 from Peru.
Van Zonneveld et
al., 2012
Fifty-two single locus polymorphic microsatellites were developed in cherimoya cv. Fino de
Jete.
Escribano et al.,
2008
ISSR Genetic differentiation among populations of A. squamosa collected from Rajasthan (India)
analyzed using 15 ISSR primers.
Nagori et al., 2018
Other studies on Molecular markers in Custard apple
32

33
Genetic engineering or Genetic transformation and its studies
Transgenic Plants
Biotic Stress
Tolerance
Nutritional
Quality
Abiotic Stress
Tolerance
Industrial
Products
Enhanced
Shelf Life
Hybrid Development
For Higher Yield
Pharmaceuticals &
Edible Vaccine

An Agrobacterium mediated transformation system of guava (Psidium guajava L.) with
endochitinase gene
Case studies :-
Mishra et al.,2014 34
 In vitro grown shoot tip explant co-cultivated with
Agrobacterium tumefaciens strain LBA4404 harbouring
binary vector pIIHR-JBMch with endochitinase and nptII
genes.
For infection,
 Prepared Agrobacterium tumefaciens fresh culture in YEB
medium containing 50 mg/L kanamycin and 25 mg/L
rifampicin and overnight growth at 28 °C.
For co-cultivation,
 Shoot tips infected with Agrobacterium
suspension with 100 μM
acetosyringone for 30, 45 and 60
minutes.
 After blotting, the explants placed again
on co-cultivation medium for 24, 48 and
72 hours under dark conditions.
After co-cultivation,
 Agrobacterium infected shoots apics selected and culture on
MS medium with 2 mg/l BAP, 0.1 mg/l IAA and different
concentrations of kanamycin (50, 100, 200 and 300 mg/l).
 For PCR confirmation analysis the
gene specific primers of nptII and
endochitinase use it.
New Delhi, India

Infection
time
(min)
Co-
cultivation
time
(Hrs)
No.
of explant
targeted
Kanamycin
resistant
shoots (%)
Transformation efficiency
(%)
30 24 150 0.00±0.00 0.00±0.00
48 150 0.33±0.33 0.66±0.66
72 150 0.66±0.33 1.33±0.66
45 24 150 1.00±0.00 2.00±0.00
48 150 1.33±0.33 2.66±0.66
72 150 2.66±0.33 5.33±0.66
60 24 150 0.66±0.33 1.33±0.66
48 150 0.33±0.33 0.66±0.66
72 150 0.00±0.00 0.00±0.00
Table 6. Effect of infection and co-cultivation duration on transformation efficiency of guava
35

Table 7. Effect of wounding methods on transformation
efficiency
Wounding methods No. of
explant
targeted
Kanamycin
resistant shoots
(%)
Transformati
on efficiency
(%)
No wounding 150 0.00±0.00 0.00±0.00
Silicon carbide
mediated wounding
150 3.00±0.57 2.00±1.15
Tungsten mediated
microprojectile
wounding
150 6.33±0.88 4.66±0.6
Notching of the shoot
meristem
150 2.00±0.57 1.33±0.6
36
Fig. 22. In vitro selection of putative transformants
of guava: a) In vitro bud induction in guava b) pre-
culture of explant c) co-cultivation of explant with
Agrobacterium d) selection of explant in
kanamycin 200 mg/l in 8 weeks e) selection of
explant in kanamycin 200 mg/l in 12 weeks f)
acclimatized guava plant.

Fig. 23. PCR confirmation of transformed plants: a) Lines 1-4
showing transformed plant analyzed for presence of
endochitinase gene (1200 bp) (b) Lines 1-3 showing
transformed plant analyzed for nptII gene +ve: positive
control, M: 100bp DNA ladder (Marker).
37
Table 8. Effect of different doses of kanamycin on
selection of putative transformants
Kana
mycin
(mg/l)
No. of
expla
nt
target
ed
No. of
explant
survived
after 4
weeks
Survival of
explant
after 8
weeks
Survival of
explant
after 12
50 50 48.00±0.57 46.33±0.88 42.66±0.6
6
100 50 43.66±1.20 39.33±1.33 35.00±1.5
2
200 50 28.00±2.30 13.33±2.40 5.33±0.66
300 50 35.33±1.76 7.00±0.57 0.33±0.33

7) Custard Apple (Annona spp.)
Transcriptome Analysis and Identification of Genes Associated with Floral Transition and
Flower Development in Sugar Apple (Annona squamosa L.)
Liu et al., 2016 38
A 10-12 years old A.
squamosa “Bendi” plant
selected.
The inflorescent meristem (IM),
Flower buds (FB) & two stages of
flowers (FL1 & FL2) were collected
from A. squamosa
RNA isolation and
Library Preparation
The amplified fragments
were sequenced using
Illumina HiSeq™ 2500
The sequence direction of
the unigenes was alignment
from NR, Swiss-port
databases
Screening of
Differentially Expressed
Genes (DEGs)
Real-Time PCR
Validation
Analysis and Mapping of
Digital Gene Expression
(DGE) Tags
China

39
Fig. 24. Analysis of the differentially expressed unigenes (DEGs) during the floral transition and flower development
process in sugar apple. (A) The expression profiling of stage-preferential unigenes in sugar apple. (B) Numbers of DEGs
in different comparisons, including FBvs.IM, FL1vs.FB, FL2vs.IM, FL1vs.FB, FL2vs.FB, and FL2vs.FL1. The red
indicated up-regulated unigenes and green indicated down-regulated unigenes. (C) Venn diagram showed the number of
DEGs in different stages of flower development.
Fig. 25. Heat map for cluster analysis of the
differentially expressed unigenes. Red indicates
up-regulated genes and green indicates down-
regulated genes.

40
Fig. 26. Identification and
analysis of floral
transition and flower
development-associated
transcription factor genes.
A heat map depicting the
overall trend of the
differential expression
profiles of the transcription
factor genes during flower
development was
constructed using MeV.
The number of each
transcription factor was
showed in parentheses.

41
Fig. 27. Validation of the expression of flowering-related Genes in sugar apple. Expression level of 20 flower
development related genes in different stages of flowering process was validated by qRT-PCR.

Other Genetic engineering/ Genetic transformation studies :-
Crop Research Title Result References
Jack
fruit
Third-generation sequencing and
metabolome analysis reveal candidate
genes and metabolites with altered levels
in albino jackfruit seedlings.
Identified 8,202 differentially expressed genes (DEGs), including 225
genes encoding transcription factors (TFs), from 82,572 full-length
transcripts and also identified 298 significantly changed metabolites
(SCMs) in albino A. heterophyllus seedlings from a set of 692 metabolites
in A. heterophyllus seedlings.
Meng et al.,
2021
Transcriptome and selected metabolite
analyses reveal points of sugar metabolism
in jackfruit (Artocarpus heterophyllus
Lam.)
The comprehensive transcriptome analysis were uses for the sucrose
accumulated during ripening, when the expression levels of genes for
sucrose synthesis were elevated and high enzyme activity was observed.
HU et al.,
2016
Guava RNA-sequencing based gene expression
landscape of guava cv. Allahabad Safeda
and comparative analysis to colored
Cultivars
Transcriptome for guava consisting of 84,206 genes comprising 279,792
total transcripts with a N50 of 3603 bp. Blast2GO assigned annotation to
116,629 transcripts and Pfam based HMM profile annotated 140,061
transcripts with protein domains. Differential expression with EdgeR
identified 3033 genes in Allahabad Safeda tissues and other 68 genes are
developing pulp colour in guava.
Mittal et al.,
2020
Custar
d apple
Transcriptome analysis of atemoya
pericarp elucidates the role of
polysaccharide metabolism in fruit
ripening and cracking after harvest
The results revealed that alpha-amylase 1(AMY1), alpha-amylase 3
(AMY3), beta-amylase 1 (BAM1), beta-amylase 3 (BAM3), beta-amylase
9 (BAM9), pullulanase (PUL), and glycogen debranching enzyme (glgX),
were the major genes involved in starch degradation.
Jingjing et
al., 2019
42

Conclusion
 Fruit tree species represent an incredible source of biological active molecules such as,
carbohydrates, protein, vitamins, minerals, fiber, fatty acids and antioxidant compounds.
 Micropropagation in jackfruit, somatic embryogenesis in guava and other tissue culture
application are used for the development of more effective cultivar.
 Molecular marker characterization of associated genetic diversity in guava and custard apple
are used for the genetic improvement.
 Genetic transformation in guava plants expressing endochitinase gene which displayed
resistance to fusarium wilt disease.
 Transcriptome studies in custard apple are used to understand the genetic pathways
involved in flower development and ripening process.
43

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