It was evident that developed biotechnological approaches have the potential to enhance the yield, quality, and shelf-life of fruits and vegetables to meet the demands of the 21st century. However, the developed biotech approaches for fruits and vegetables were more of academic jargon than a commercial reality

1. Master’s Seminar- FSC-599
on
Role of biotechnology in enhancing crop production
and quality
Ankit Gavri
2017A57M
Dept. of Horticulture
CCS Haryana Agricultural University, Hisar-125004

2. Biotechnology may be defined as “generations of useful
products or services from plant cells, tissue and often,
organs. Such cell, tissue and organ either continuously
maintained in vitro or they pass through a variable in vitro
phase enable regeneration from them of complete plantlets
which are ultimately transferred to the field.
BIOLOGY TECHNOLOGY BIOTECHNOLOGY

3. Biotechnology
No Species/
genus gene
transfer barrior
Eliminates
long term field
trials
100 %
Achievement
of gene
transfer
Short breeding
cycles
Elimination of
unreliable
phenotypic
evaluation
No Linkage
drag
Production of
true to types
Overcome
Distant
hybidization
barriors

4. Tools and technologies of biotechnology for
enhancing fruit production and quality
Tissue
culture
Genetic
Engineering
Molecular
markers

5. Abano and Buahb, 2015

6. Plant tissue culture is a collection of techniques used to
maintain or grow plant cells, tissues or organs under sterile
conditions on a nutrient culture medium of known
composition.

7. TOTIPOTENCY
The capacity of a plant cell to regenerate into a
whole plant. Vochting, 1878

8. PLANT TISSUE
CULTURE
MICRO
PROPAGATION
MERISTEM
CULTURE
ANTHER
CULTURE
PROTOPLAST
CULTURE
EMBRYO
RESCUE
SOMATIC
HYBRIDIZATION
SOMACLONAL
VARIATION

10. It may be described as the culture of terminal (0.1-
1.0mm) portion of a shoot comprising the meristem
(0.05 -0.1mm) together with primordial and
developing leaves and adjacent stem tissue.

11. To get genetically true to type plants
To propagate male sterile, self incompatible, Inbred
lines and haploids that are not easily propagated by
seed.
Clonal propagation of F1 hybrids in vegetatively
propagated plants.
To propagate superior manually produce hybrids for
several generations without any change in the
genetic make up (without loss in heterosis).
Propagation of virus free plants.

12. Manganaris et al.,2003
Regeneration and virus elimination in nectarine meristem-tip explants

13. Maliogka et al., 2009
Survival rates and virus elimination post-thermotherapy.

14. A. Surviving shoot tips
B. Elongated shoots
C. In vitro plantlets
D. Plants established under
greenhouse conditions
from shoot tips
V.I. Maliogka et al., 2009

15. It is the technique in which in vitro culturing of anthers
containing microspores or immature pollen grains
cultured on a nutrient medium for the purpose of
generating haploid plantlets.

16. • Development of homozygous lines.
• Fixation of heterosis.
• For production of biotic and abiotic stress resistant
plants.
• For cytogenetical research.
• For induction of genetic variability at haploid level.
• For evolutionary studies.
• For genome mapping as genetic maps

17. Variation in leaf morphology and fruit shape among haploid (a and d), octoploid (b and e), and
doubled-octoploid (c and f) plants.
Naguyen et al., 2012

18. It is the genetic variability present among cultured cells and plants
derived from such cells or progeny of such plant is called somaclonal
variants.
• Somaclonal variation is one of the useful source of introducing genetic
variations that could be of value to plant breeders.
• Single gene mutation in nuclear or organelle genome may give the best
available variety in vitro that has a specific character.
• Various cell lines selected in vitro may prove potentially applicable to
agriculture and industry like resistance to herbicide, pathotoxin, salt or
aluminium.
• Variability in cell cultures has played a useful role in synthesis of secondary
metabolites on a commercial scale.

19. Sahijram et al., 2003

20. Induction of somaclone using different tissue culture methods.
A–C: micropropagation
D–F: meristem culture Biswas et al., 2009

21. Induction of somaclone using different tissue culture methods
G–I: plant regeneration via callus culture;
J–L: directplant regeneration from leaf
M–O: plant regeneration via somatic embryogenesis. Biswas et al., 2009

22. Biswas et al., 2009

23. Field performance of tissue culture derived clones.
Biswas et al., 2009

24. Production of hybrid plants through the fusion of protoplsts of two
different plant species is called somatic hybridization, and such
hybrids are known as somatic hybrids.
• Production of novel interspecific and intergenic hybrid.
• Production of fertile diploids and polypoids from sexually sterile
haploids, triploids and aneuploids
• Transfer gene for disease resistance, abiotic stress resistance,
herbicide resistance and many other quality characters
• Production of heterozygous lines in the single species which cannot be
propagated by vegetative means

25. Examples of seedless fruits from triploid hybrids produced by interploid crosses using
somatic hybrid pollen parents.
Grosser et al., 2011
‘Sugar
Belle X Nova + Succari
‘Todo del Ano
Lemon X Mexican lime +
Valencia
‘Todo
del Ano lemon X Hamlin +
Femminello

26. Rootstock Seeds/ fruit Yield (B/T)
Hamlin + Flying Dragon 10 2.33/1.50
Cleopatra + Flying Dragon 1 1.39/1.96
Flying Dragon (diploid) 20+ 1.83/1.33
Sour orange + Rangpur 14 2.48/2.2
Cleopatra + Arg. Tri. Orange 1 2.18/2.10
Somatic hybrid Seeds/fruit Yield (B/T)
White Grapefruit + 50–7 20 2.03/1.50
Changsha + 50-7 17 2.25/1.96
Sour orange + Carrizo 13 2.10/1.33
Changsha + Benton 19 2.42/2.2
Tree size controlling somatic hybrid rootstocks and standard Flying Dragon in an initial trial of
somatic hybrid rootstocks with ‘Roble’ sweet orange (C. sinensis)
Tree size controlling somatic hybrid rootstocks producing nucellar seedlings; tree size and
‘Valencia’ sweet orange yield
Grosser et al., 2011

27. Dambier et al., 2011

28. Rootstock Flhorag1 Carrizo citrange Volkamer lemon
Yield
2007-2008 (kg/tree) 40 37 49.7
2008-2009 (kg/tree) 40.1 33.5 51
2 years cumul 80.1 70.5 100.7
Avg yield
efficiency/year(kg/m3)
14.13 8.45 5.65
Fruit quality 2007-2008
Fruit weight (g) 266 224 240
% juice 43.4 40.27 35.4
% citrate 1.14 1.1 1.2
TSS 10.8 11 1O.8
Dambier et al., 2011
Behaviour of Flhorag1 allotetraploid somatic hybrid rootstock grafted with sweet orange cv ‘Valencia’
compared with Volkamer lemon and Carrizo citrange

29. Fruiting sweet orange tree grafted on the allotetraploid somatic hybrid ‘Flhorag1’ in Morocco

30. Genetic engineering:- Refers to application of technique
available in molecular genetic and molecular biology for the
genetic manipulation, modification and analysis of genetic
material for various purpose including genetic
transformation of organism.

31. • Improved Nutritional Quality
• Insect resistance
• Disease resistance
• Herbicide resistance
• Salt tolerance
• Delayed Fruit Ripening
• Biopharmaceuticals and Vaccines

32.  Identification of gene of interest
 Isolation of the gene of interest
 Insertion of the gene to the transfer vector
 Transfer of the vector to the organism to be modified
 Transformation of the cells of the organism
 Selection of the genetically modified organism
 Regeneration of cells

33. Vector mediated or
indirect gene method
• Agrobacterium
mediated gene
transfer
Direct gene methods
• Chemical methods
• Electroporation
• Particle gun delivery
• Lipofection
• Microinjection
• Laser induced
• Fiber mediated

34. Agrobacterium tumefaciens
• Gram-negative soil bacterium that cause crown gall (cancer)
tumors
• Tumor formation is the result of the transfer, integration and
expression of genes on a specific segment of A. tumefaciens
plasmid DNA called the T-DNA (transferred DNA)
• The T-DNA resides on a large
plasmid called the Ti (tumor inducing)
plasmid found in A. tumefaciens

36. Shekhawat et al, 2014
A summary of the generation of ihpRNA-Rep- and ihpRNA-ProRep-transformed banana
plants and their bioassay for BBTV resistance following in vitro and ex vivo viruliferous
aphid inoculation

37. A. BBTV infected banana plant.
B. Multiplication of banana aphid on in
vitro banana plants.
C. Untransformed control banana plants
showing characteristic BBTD symptoms
2 months after inoculation with
viruliferous banana aphid.
D. Transgenic banana plants expressing the
two ihpRNAs (targeted against
replication initiation protein and its
upstream regulatory region) show
efficient resistance towards BBTV after a
2 months long bioassay with banana
aphid.
Shekhawat et al, 2014

38. Shekhawat et al, 2014

39. Chemical method: using certain chemicals like PEG, PVA &
Calcium phosphate enhance the uptake of DNA by plant
protoplasts.
Electroporation: This method was introduced by From and his
co-workers in 1986. In this technique, short pulses of high
voltage are applied to protoplasts which make temporary pores
in the plasma membrane to increase their permeability and
facilitate the uptake of foreign gene

40. Genegun/biolistic/microprojectile: The process of partical
acceleration (or) biolistics acceleration of DNA into cells with
sufficient force such that a part of it gets integrated into DNA of target
cells.
Microinjection: In this method DNA can be introduced into cells or
protoplast with the help of very fine needles or glass micropipettes
having the diameter of 0.5 to 10 µm.

41. Lipofection: These are artificial vesicles that can act as
delivery agents for exogenous materials including trans-
genes. These liposomes are able to interact with the
negatively charged cell membrane more readily than
uncharged lipo-somes.
Srinivas et al., 2009

42. Method and crop References
Electroporation
Citrus
Mango
Avocado
Niedz et al., 2003
Litz et al., 2008
Tucker et al., 1987
Gene Gun
Banana and plantain
Pineapple
Papaya
Sagi et al., 1995; Becker et al., 2000
Sripaoraya et al., 2001& 2006
Sanford et al., 1992
Polyethylene glycol mediated transfection
Citrus
Avocado
Guo et al., 2005
Huber et al.,2001

43. Agrobacterium mediated
Transformation
References
Mango
Citrus
Banana and plantain
Pineapple
Papaya
Avocado
Kiwifruit
Passionfruit
Persimmon
Mathews et al., 1992 & 1993
Bellaster et al., 2007
Ganpathi et al., 2001; Khana et al.,2004
Smith et al.,2002, 2005& 2008
Gonsalves et al., 1998
Fitch et al., 1992
Raharjo et al., 2008
Rugini et al., 1989
Hood et al., 1986

44. Fruit Crop Trait Research work
Apple
M. domestica
• Reduced polyphenol oxidase
• Ethylene suppression
• Altered sorbitol levels
• Juvenile stage reduced
• Resistance to fire blight
• Scab resistance
• PPO suppression transgene,
• ACC oxidase, ACC synthase S6PDH sorbitol
• phosphate dehydrogenase, GUS, nptII
• BpMADS4, NPTII attE, nptII, gusA
• ech42, nag70,
• npt II
Plum • Non-browning; resistance to
Plum pox virus (PPV)
• PPV coat protein -
Papaya • Female to male or
hermaphrodite
• PRSV resistant
• EST116, EST5, FSH11, FSH19, Gene11Y,
Gene5, GM183, nptII
• Coat Protein gene
Sharma et al., 2016

45. Grape
Rootstock
• Grapevine fanleaf nepovirus
resistance
• Grapevine leafroll-associated
ampelovirus resistance
• Grapevine leafroll-associated
closterovirus resistance
• Coat protein gene, heat shock 90
homologous
• nptII gene
Banana
Musa spp.
• Bunchy top resistance
• Resistant to Xanthomonas wilt
• Tolerance to Sigatoka
• leaf spot
• Resistance to virus
• Resistance to Fusarium wilt
• Replicase associated protein, replicase
inverted repeat, nptII
• Hrap and Pflp
• pYC39
• pAB6, pAHC17, pH1
• pflp, nptII
Grapefruit
Citrus
paradisi
• Aphid resistance
• Citrus tristeza virus resistance
• agglutinin, coat protein, GUS, nptII
Sharma et al., 2016

46. Grapevine
V. vinifera
• Xylella fastidiosa resistance
• Powdery mildew resistance
• Increased anthocyanin
• Increased seedlessness
• Resistance to viruses,
• crown gall, fungal pathogen
• Endogenous grapevine antifungal
gene, Albgene, defensin gene,
EGFP/NPTII, Lima-A, Lima-B, PR1
gene, Snakin gene, SuSy
antisense,VvMybA1, VVTL-1, rice
chitinase gene, hgt
• Mutant virE2,nptII
• GLRaV-3cp; chitinase, rip, nptII
Guava • Endochitinase gene against
guava wilt
• nptII and GUS
• genetic transfor-mation of guava
with cold hardiness genes (CBF1,
CBF2 and CBF3)
• Genetic transformation of guava
(Psidium guajava L.) was developed
for the first time using in vitro grown
shoot tip explant cocultivated with
A. tumefaciens strain LBA4404
harbouring binary vector pIIHR-
JBMch with endochitinase and nptII
genes
Sharma et al., 2016

47. • The strawberry (Fragaria x ananassa) fruit undergoes an
extensive and fast softening that limit its shelf life and
postharvest and it has also been considered that PG plays a
minor role on this process, due to the low PG activity found
in ripened strawberry fruits.
• Transgenic strawberry plants expressing an antisense
sequence of the ripening-specific PG gene FaPG1 have been
generated to get an insight into the role of this gene in
softening.
• In these firmer lines, FaPG1 was silenced to 95%, but total
PG activity was only minor reduced.
Quesada et al., 2009

48. Quesada et al., 2009
Control and transgenic strawberry fruit
expressing the antisense FaPG1 gene
(AntiPG) harvested at the full ripened stage
Firmness of control and transgenic AntiPG fruits
at the full ripened stage

49. Fruit crop Gene Fruit tissue References
Grape VvMYB5a Berry skin Deluc et al., (2006)
Apple MdMYB1 Red Peel colour Bogs et al., (2007)
Citrus MAC12.2 Significantly less seeds Tan et al., (2009)
Strawberry FvMYB10 Flesh colour Wang et al., (2010)
Strawberry ANS Anthocyanin biosynthesis Fischer et al (2014)
Strawberry FaEG3 Increased fruit firmness Mercado et al., (2010)
Sweet
Orange
CsLCYb1 Increase in flavedo Zhang et al., (2013)
Papaya ACC oxidase Delay ripening Gomez et al.,(2009)
Genetic transformation with improved fruit quality traits:

50. Gonsalves, 1998
Resistant to
papaya ring spot
virus
Commercial
cultivation in hawaii
1992: Start of the Transgenic Field Trial;
PRSV invades Puna

51. Cornell University used the parasite derived resistance (PDR) to
manage PRSV.
Genes from coat protein of mild strain of PRSV HA5-1 was
transferred to transgenic cv. To make them resistant to PRSV
under field conditions with no effect on pomological features.
The target cv. Sunset and Kapoho embryogenic tissues were
bombared with tungsten particles with gene gun, resulted in
number of transgenic plants micro propagated plants named as
55-1 with superior resistance
55-1 × non-transgenic Sunset
50% of progeny was transgenic
Homozygous line 55-1 named as Sunup is red fleshed
Sunup ×Kopoho (non transgenic)
Yellow fleshed Rainbow
GONSLAVES, 20O4

52. GONSLAVES, 20O4
Transgenic papaya test field

53. 53
Seeds of the transgenic cultivars “SunUp”
and “Rainbow” became available to
Hawaii’s farmers in 1998, and growing
cultivar “Rainbow” led to a significant
increase in papaya production.
SUNUP RAINBOW
GONSLAVES, 20O4

54. Scorza et
al.,2013

55. HS1 = HoneySweet inoculated with PPV-Rec + ACLSV + PDV
HS2 = HoneySweet inoculated with PPV-Rec + PDV
HS3 = HoneySweet inoculated with PPV-Rec + ACLSV
HS4 = HoneySweet inoculated with PPV-Rec
HS5 = HoneySweet non-inoculated control trees Scorza et al.,2013

56. Ethylene production in papaya transgenic fruitsCO2 production in transgenic papaya fruits
Gomez et al., 2009

57. Changes in flesh firmness of transgenic fruitChanges in peel color of transgenic fruit
Gomez et al., 2009

59. Molecular marker: A molecular selection technique of
DNA signposts which allows the identification of
differences in the nucleotide sequences of the DNA in
different individuals. Or any genetic element (locus,
allele, DNA sequence or chromosome feature) which
can be readily detected by phenotype, cytological or
molecular techniques, and used to follow a chromosome
or chromosomal segment during genetic analysis.

60. • Genetic Linkage Maps
• Assessment of Genetic Diversity
• Gene Tagging
• DNA Fingerprinting for Varietals Identification
• Detection of QTLs

61. Hybridization
based
markers
• Restriction Fragment
Length Polymorphisms
(RFLPs)
Polymerase
Chain
Reaction
(PCR) based
• Randomly Amplified
Polymorphic DNAs
(RAPDs)
• Simple Sequence Repeats
(SSRs)
• Amplified Fragment Length
Polymorphic DNA (AFLPs)
Sequence
based
markers
• Expressed Sequence Tags
(ESTs),
• Sequence Tagged Sites
(STSs)
• Single Nucleotide
Polymorphism (SNPs)
MOLECULAR
MARKERS

62. Bhat et al., 2015

63. Eibach et al., 2007

64. RAPD markers closest to the disease related locii in kinchaku based on 2 pt. analysis
Terakami et al., 2001

65. • Plant biotechnology has opened new avenues and opportunities,
especially tissue culture and genetic engineering in horticulture to
combat all kind of challenges.
• Transfer of desirable genes irrespective of cultivars, species, genera,
and even taxa is now possible through the wonderful techniques of
genetic engineering.
• Tissue culture techniques have a variety of application in fruit crops.
Among the tissue culture techniques, micropropagation allows rapid
multiplication of new or elite genotypes.
• In vitro screening could help in isolating new and improved cell
lines, from which plants with improved traits can be regenerated.
• Recent advances in molecular biology are providing unique methods
for gene mapping, and hold the potential to develop new transgenic
genotypes modified by the addition of one or few useful genes.

66. • Abano, E. E., & Buah, J. N. (2014). Biotechnological approaches to improve
nutritional quality and shelf life of fruits and vegetables. Int. J. Eng. Technol, 4, 11.
• Bhat, Z. A., DHILLON, W. S., Rashid, R., Bhat, J. A., Dar, W. A., & Ganaie, M. Y. (2010).
The role of molecular markers in improvement of fruit crops. Notulae Scientia
Biologicae, 2(2), 22-30.
• Biswas, M. K., Dutt, M., Roy, U. K., Islam, R., & Hossain, M. (2009). Development
and evaluation of in vitro somaclonal variation in strawberry for improved
horticultural traits. Scientia horticulturae, 122(3), 409-416.
• Dambier, D., Benyahia, H., Pensabene-Bellavia, G., Kaçar, Y. A., Froelicher, Y.,
Belfalah, Z., & Yesiloglu, T. (2011). Somatic hybridization for citrus rootstock
breeding: an effective tool to solve some important issues of the Mediterranean
citrus industry. Plant cell reports, 30(5), 883-900.
• Eibach, R., Zyprian, E., Welter, L., & Topfer, R. (2007). The use of molecular markers
for pyramiding resistance genes in grapevine breeding. VITIS-GEILWEILERHOF-
, 46(3), 120.

67. • Gonsalves, D. (2004). Transgenic papaya in Hawaii and beyond. AgBioForum,
7(1&2). 36-40
• Grosser, J. W. & Gmitter, F. G. (2011). Protoplast fusion for production of tetraploids
and triploids: applications for scion and rootstock breeding in citrus. Plant Cell,
Tissue and Organ Culture (PCTOC), 104(3), 343-357.
• López-Gómez, R., Cabrera-Ponce, J. L., Saucedo-Arias, L. J., Carreto-Montoya, L.,
Villanueva-Arce, R., Díaz-Perez, J. C., ... & Herrera-Estrella, L. (2009). Ripening in
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• Maliogka, V. I., Skiada, F. G., Eleftheriou, E. P., & Katis, N. I. (2009). Elimination of a
new ampelovirus (GLRaV-Pr) and Grapevine rupestris stem pitting associated virus
(GRSPaV) from two Vitis vinifera cultivars combining in vitro thermotherapy with
shoot tip culture. Scientia Horticulturae, 123(2), 280-282.
• Manganaris, G. A., Economou, A. S., Boubourakas, I. N., & Katis, N. I. (2003).
Elimination of PPV and PNRSV through thermotherapy and meristem-tip culture in
nectarine. Plant cell reports, 22(3), 195-200.

68. • Nguyen, T. X., Song, Y. S. & Park, S. M. (2012). Haploid plant production through
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• Quesada, M. A., Blanco-Portales, R., Posé, S., García-Gago, J. A., Jiménez-Bermúdez,
S., Muñoz-Serrano, A., & Muñoz-Blanco, J. (2009). Antisense down-regulation of
the FaPG1 gene reveals an unexpected central role for polygalacturonase in
strawberry fruit softening. Plant Physiology, 150(2), 1022-1032.
• Sahijram, L., Soneji, J. R., & Bollamma, K. T. (2003). Analyzing somaclonal variation
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Biology-Plant, 39(6), 551-556.
• Scorza, R., Callahan, A., Dardick, C., Ravelonandro, M., Polak, J., Malinowski, T. &
Kamenova, I. (2013). Genetic engineering of Plum pox virus resistance:
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69. • Shekhawat, U. K., Ghag, S. B. & Ganapathi, T. R. (2014).
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70. SYMBOL OF TRUST