Rapid vegetable improvement can be achieved through biotechnological and molecular approaches.

1. ADVANCES IN VEGETABLE
BREEDING: BIOTECHNOLOGY AND
MOLECULAR TOOLS
NAOREM BIDYALEIMA CHANU
03H(P)-2018

2. Biotechnology:
“Any technological application that uses biological
systems, living organisms, or derivatives thereof, to
make or modify products or processes for specific use”
(UN Convention on Biological Diversity)
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Advances in vegetable breeding are
manifested in the form of transgenics,
cybrids, MAS, MABC etc.

3. Need for modern
breeding
Saves breeding cycles
Successful transfer of
only desirable genes
Overcomes the breeding
barriers in wide crosses
Rapid accomplishment of
objectives made
Feed the over growing
population
Nutritional security
Mitigate climate change

4. Outline...
BIOTECHNOLOGICAL AND MOLECULAR
MEANS OF VEGETABLE IMPROVEMENT
•Tissue culture: Meristem culture, anther culture, ovule
culture, etc.
•Embryo rescue
•Somatic hybrids: Protoplast fusion and cybridization
•Genetic engineering: Transgenics
•Molecular approaches: Diversity studies, genome
sequencing, genome mapping, MAS, gene tagging, MABC
etc.

5. TISSUE CULTURE:
Meristem culture, anther culture, pollen culture,
ovule culture and embryo rescue

6. Meristem culture
• In 1952 Morel and Martin were the first
scientists who developed the technique of
meristem culture for in-vivo virus eradication of
Dahlia.
• Orchid cymbidium was micropropagated using
this culture by Morel in 1965.

7. Meristem culture = VIRUS free plants???
Cells in this region are actively
dividing and lacks
plasmodesmatal connections
which limits the virus entry, thus
upon culture of such tissues
results in virus free plants.

8. Shoot tip or Apical meristem culture

9. Case study
Meristem culture and micropropagation of a variety of
ginger (Zingiber officinale Rosc.) with a high yield of
oleoresin
B. Bhagyalakshmi &Narendra S. Singh
 Liquid media (agitated or static) were less effective than a solid
(agar-gelled) medium for micropropagation. The in vitro plants
were successfully established in vivo and the rhizome yield
was comparable with that of plants grown by conventional
methods.
 Meristems of ginger with or without leaf primordia were induced to
form shoots on three-quarter strength Murashige-Skoog’s (MS)
medium containing sucrose 6%, coconut milk (CM) 20%, ascorbic acid
(AA) 100 mg l−1, glutamine (GL) 400 mg l−1, activated charcoal (AC)
250 mg l−1, 6-benzylaminopurine (BAP) 0.5 mg l−1, indolebutyric acid
(IBA) 0.4 mg l−1 and agar 0.8.

10. Virus Free Potato Tuber Seed
Production Through Meristem
Culture In Tropical Asia
Ahsan Nagib, S.A. Hossain , M.F. Alam , M.M.
Hossain , R. Islam and R.S. Sultana
In vitro culture was established using apical meristem of four widely
cultivated varieties of Bangladesh namely Diamant, Cardinal, Multa and
Lalpakri. Meristem of these varieties of potato were cultured for shoot
proliferation and root induction in media supplemented with different types
and concentration of phytohormones.
For high frequency of root formation, combination of GA3 (0.1 mg l-
1)+KIN (0.1 mg l-1) was most effective. Rooted plantlet were gradually
acclimatized and successfully established in the field. Visual evolution of
the morphological trials of the tissue culture derived plants showed
all plants were found normal and free from virus diseases.
Substantial yield increase was observed in meristem derived plants
over their source plants.
Case study

11. DOUBLE HAPLOIDS (DH)
• DH plants are produced by haploid cells
(pollen or egg cells) culture followed by undergoing
chromosome doubling.
•Artificial production of doubled haploids is important
in plant breeding to utilize as inbred lines in hybrid
breeding.
•Conventional inbreeding procedures take six generations
to achieve approximately complete homozygosity, whereas
doubled haploidy achieves it in one generation

15. Case study
Double Haploid Inbred lines USVL048 and
USVL131 of Heading Broccoli
 Two inbred lines of heading broccoli (Brassica
oleracea L. var. italica) designated USVL048 and
USVL131, were released by USDA (2012) which was
originally derived from anther cultures. These two
lines are uniform, homozygous in nature and can
be readily cross with other broccoli inbred lines to
derive a new hybrid.
Mark W Farnham

16. EMBRYO
RESCUE
Invitro techniques which promotes the
development of an immature or weak embryo
resulted from wide crosses into a viable plant

17. Case study
Production of embryo rescued hybrids between the landrace
“Friariello” ( Capsicum annuum var. annuum) and C.
baccatum var. pendulum
Gaetana Cremona et. al
 Seven interspecific hybrids between the
Italian landrace Friariello” ( Capsicum
annuum var. annuum) and C. baccatum var.
pendulum were obtained through embryo
rescue.

18. Case study
Introgression of black rot resistance from Brassica
carinata to cauliflower (Brassica oleracea botrytis
group) through embryo rescue
Brij B Sharma, Pritam Kalia, Dinesh Singh, Tilak R sharma
 Interspecific hybrid and back cross progeny were generated
between cauliflower ‘Pusa sharad’ and Ethiopian mustard ‘NPC-9’
using in vitro embryo rescue technique.
 Direct ovule culture was found better. Hybrids were tested
using SSR markers. The interspecific hybrids and interspecific
BC1 population obtained through embryo culture were found to
be resistant to Xanthomonas campestris pv. Campestris race 1.

19. SOMATIC HYBRIDS:
PROTOPLAST FUSION& CYBRIDIZATION

20. • Protoplast fusion and cybrid production are
important somatic hybridization methods for
production of hybrids of distant origin
(interspecific/ intergeneric) without facing
crossing barriers.
PROTOPLAST FUSION CYBRIDIZATION

21. IMPROVEMENT OF IMPORTANT TRAITS IN
POTATO THROUGH SOMATIC HYBRIDIZATION

22. Case study
Improved resistance to bacterial soft rot by
protoplast fusion between Brassica rapa and B.
oleacea
J P Ren, M H Dickson, E D Earle
 Protoplast from B. rapa and B. oleracea genotypes are
selected for resistance to soft rot were fused in order to
combine different sources of resistance.
12 somatic hybrids were obtained and confirmed by
morphology and nuclear DNA through markers.
The offspring populations of the somatic hybrids showed
higher percent of resistance than parents

23. Case study
Cytoplasmic male sterility in rapeseed (Brassica
napus L.): female fertility of restored rapeseed with
“Ogura” and cybrids cytoplasms
R Pellan- Delourme, M Renard
 The study of Brassica napus L. plants carrying restorer
genes introgressed from radish (Raphanus sativus L.) showed
that these genes ensured restoration of male fertility in
rapeseed for all the male sterility-inducing cytoplasm studied,
i.e., "Ogura"-type cytoplasm and that of four cybrids obtained
by protoplast fusion.
 Plants with high levels of restored male fertility were
obtained.

24. GENETIC ENGINEERING:
GMOs

25. GENETIC ENGINEERING

26. TRANSGENIC DEVELOPMENT

27. EXPLOITATION OF GENETIC ENGINEERING

28. LIST OF IMPORTANT GM VEGETABLES
CROP TRAIT IMPROVED BY
TRANSGENE
SOURCE OF
TRANSGENE
TRANSGE
NIC
VARIETY
COMPANY
/INSTITU
TION
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Cry gene to control
colarado potato beetle
Bacillus
thuringiensis
New Leaf
(1995)
Monsanto
Cry gene to control
colarado potato beetle
and potato leafroll virus
Bacillus
thuringiensis
New Leaf
Y (1999)
Monsanto
Cry gene to control
colarado potato beetle
and potato leafroll virus
Bacillus
thuringiensis
New Leaf
Plus
(1998)
Monsanto
Tomato Altered ripening Bacteria FlavrSavr
(1994)
Calgene

29. CRO
P
TRAIT IMPROVED BY
TRANSGENE
SOURCE OF
TRANSGENE
TRANSGE
NIC
VARIETY
COMPANY/I
NSTITUTIO
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S
Q
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Resistance to watermelon
mosaic and zucchini
yellow mosaic virus
Bacteria
and virus
Freedo
m II
(1995)
Seminis
Resistance to watermelon
mosaic, zucchini yellow
mosaic virus, cucumber
mosaic virus
Bacteria and
virus
* (1997) Seminis
Resistance to glyphosate
herbicide
Bacteria and
virus
* (1999) Monsanto
Suga
rbeet
Altered ripening Bacteria and
virus
Endless
Summer
(1995)
DNA plant
technology
Brin
jal
Resistance to shoot and
fruit borer
Bacillus
thuringiensis
Bt Brinjal Monsanto

30. Case study
Functional expression of the taste modifying
protein, miraculin, in transgenic lettuce
Hyeon- Jin Sun, Min-long Cui, Biao Ma, Hiroshi Ezura
A synthetic gene encoding miraculin was placed under
the control of constitutive promoters and transferred to
lettuce. Expression of this gene in transgenic lettuce
resulted in the accumulation of significant amounts of
miraculin protein in the leaves.
The miraculin expressed in transgenic lettuce possessed
sweetness‐inducing activity. These results demonstrate that
the production of miraculin in edible plants can be a good
alternative strategy to enhance the availability of this
protein.

31. FLAVR SAVR TOMATO

32. Bt crops

35. • The use of Bt technology is not the only way to eliminate the usage of pesticides having
several drawbacks such as risk and hazards to human and animals (impact on lactation
periodofcows, delayedfood consumption inrabbits and healthimpacts on rat).
• There is no any data available on its impact on human health. But we would not turn our
people into"labRats"
• Brinjal belongs to Solanacae family and are often cross pollinated crop, so the gene can
easily transferredto othervarietiesleadinggreatthreattobio diversity.
• The issue of seed being controlled by international agencies such as Monsanto, whose
public perception in India has influenced much of the debate. Although the company has
made investments in India and many scientists work here, we must be able to derive
maximum benefit from the findings. However, lack of competition in terms of publically
funded agricultureagencies has ledtomonopolistic situations.

36. Can you identify a transgenic crop???
GMCROP

37. MOLECULAR
APPROACHES

39. Molecular Markers
• A molecular marker is a fragment of DNA that
is associated with a particular sequence of
DNA within a genome.
• Major applications: Assessment of Genetic
Diversity, Gene Tagging, DNA Fingerprinting
for Varietal Identification, genome mapping,
genome sequencing, marker based hybrid seed
production, MABC, MAS etc.

40. Applications of markers in potato breeding
Breeding
Strategy
Type of
breeding
objective
Breeding objective Type of
marker
References
Introgression;
MABC
Disease/
pest
Resistance
Broad-spectrum potato
late blight resistance
derived from Solanum
bulbocastanum
PCR
marker
Colton et al.
2006
Pyramiding Disease/
pest
resistance
Combination of resistances
to potato virus Y, root cyst
nematode , Potato virus X ,
and potato wart
CAPS,
SCAR
PCR-
based
markers
Gebhardt et
al. 2006
MAS Pest
resistance
Potato Virus Y resistance PCR
marker
Fulladolsa
et al. 2015

41. Applications of markers in Tomato breeding
Breeding
Strategy
Type of breeding
objective
Breeding objective Type of
marker
References
Introgression;
MABC
Disease/ pest
resistance
Acylsugar-mediated
pest resistance from
Lycopersicon pennellii
RFLP, RAPD Lawson et al.
1997
Introgression;
MABC
Quality traits Introgression of 5
QTLs controlling fruit
quality traits '
RFLP, RAPD
Lecomte et al.
2004
Introgression
Disease/ pest
resistance
Black mold resistance
QTLs from
Lycopersicon
cheesmanii
RFLP, CAPS Robert et
al.2001
MABC;
Introgression
Disease/ pest
resistance
Identification of QTLs
for early blight
resistance from
Lycopersicon
hirsutum
RFLP Fooled et al.
2002

42. Breeding
Strategy
Type of
breeding
objective
Breeding objective Type of
marker
References
MABC
Disease/ pest
resistance
Resistance to Tomato Spotted
Wilt Virus (TSWV) CAPS
Langella et al.
2004
Pyramiding
Disease/ pest
resistance
Resistances to Tomato Yellow
Leaf Curl Virus (TYLCV),
Fusarium oxysporum f sp.
lycopersici, Verticillium wilt
DNA
markers
Atamain et al.
2009)
Pyramiding
Disease/ pest
resistance
Resistance to Tomato Mosaic
Virus, Fusarium oxysporum f.s.
lycopersici, Fusarium
oxysporum f.s. radicis-
lycopersici: and the nematode
Meloidogyne incognita
CAPS,
RFLP
Barone et al.
2005
MABC
Disease/ pest
resistance
Resistance to Tomato Spotted
Wilt Virus (TSWV) CAPS
Langella et al.
2004
Contd…

43. Genome sequencing
Genome sequencing is figuring out the order of DNA
nucleotides, or bases, in a genome—the order of As, Cs,
Gs, and Ts that make up an organism's DNA.
Role in crop improvement:
• The sequenced data obtained will help identify genes
determining different economic traits. Thus, traits of
interest can be transferred by transferring gene of
interest to the recipient plant

44. Genome sequencing
Sequencing machine

45. LIST OF GENOME SEQUENCED VEGETABLES

46. GENE TAGGING
• Gene tagging refers to mapping of genes of
economic importance (example biotic and abiotic
resistance) with known markers.
• The most recent application of molecular
markers is to facilitate transfer of gene of
interest.

47. Disease resistance gene tagging in
vegetables
crop pathogen Resistant
gene
Marker
used
Reference
Cucumber F. Oxysporum f. Sp.
melonis
Fom2 SSP Wechter et al
(1998)
tomato Tomato spotted wilt
virus
Tsw RAPD Jahn et al
(2000)
Bean Common bean mosaic
virus
I RAPD Melotto et al
(1996)
Pea mosaic virus Mo RFLP Dirlewanger et
al (1994)
Erysiphe polygone Er RAPD Dirlewanger et
al (1994)

48. Assessment of Genetic Diversity
• Molecular markers have provided very useful information
about the overall genetic range of crop germplasm. For
breeders this information is important to take decisions
regarding the utility of germplasm.
• Vegetable crops in which DNA markers have been
developed for the assessment of genetic diversity and
construction of genetic maps are Allium sp. (Berg et al.,
1996), Amaranthus sp. (Lanoue et al., 1996), common bean
(Nodari et al., 1993), Crucifers (James et al., 1993), Sweet
potato (Paul et al. 1997), Tomato (Smolders et al., 1997),
watermelon (Lee et al., 1996) and pea (Timmerman et al.,
1993)

49. Genome mapping
Genome mapping is the creation of a genetic map assigning DNA
fragments to chromosomes.
• Navot et al., 1990 found to determine genetic linkage map between
genes affecting bitterness and flesh colour in watermelon by isozymes.
• Garcia et al., 1998 determine genetic relationship among melon
breeding lines revealed by RAPD markers and agronomic traits.
• Li et al., 2001 developed sequence related amplified polymorphism
(SRAP) and its application to mapping and gene tagging in Brassica.
• Farinho et al., 2004 constructed a genetic map of broccoli including
downey mildew resistance locus using AFLP, RAPD and SSR
• Ohara et al., 2005 constructed genetic linkage map of Japanese
bunching onion based primarily on AFLP markers using reciprocally
backcrossed progenies.
Case study

50. G
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P
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51. Use of molecular marker in hybrid
seed production
Molecular marker technology can be utilized for identification of a
distinct ‘new’ variety from the segregating sources.
• Cabbage: Use of marker gene for purple stem pigmentation for
identification of F1 Hybrids.
• Watermelon: Use of non- lobbing leaf character as a single recessive
marker gene for production of hybrid seed is commercially exploited.
• Pumpkin: Inbred lines with yellow spots (recessive ) and white spot
(dominant) of pumpkin are allowed to grow and seedlings with white
spots on their leaves are marked as hybrids
Case study

53. MAS
• Marker Assisted Selection, refers to
the technique of using DNA markers
that are tightly linked to phenotypic
traits to assist in a selection scheme
for a particular breeding objective.

55. • Osborn et al. , 1987 first reported on the development
of the markers for QTL in vegetable crops has been on
development of RFLP markers for soluble solids content
in tomato.
• Walter et al., 1994, identified RAPD markers associated
with canning quality in beans
• Jiang and Sink, 1997 developed SCAR markers in
Asparagus which is linked to the sex locus at distance
of 1.6cM.
Case study

56. Major Pros and Cons
PROS CONS
Resolves hungerandmalnutrition
Saves breedingcycles
Successful transfer of only desirable
genes
Overcomes the breeding barriers in
widecrosses
Rapid accomplishment of breeding
objectives
Nutritional security
Mitigateclimate change
Luxurybreeding: highcapital
requirement
Development of super weeds
Deterioration of Horizontal
resistance
Question onbiosafety
Fear on possible health hazards
Effects on environment
Ethicalarguments

57. CONCLUSION

58. A MULTIDISCIPLINARY
approach of traditional plant breeding,
plant biotechnology, and molecular
biology would be strategically ideal for
developing new improved crop
varieties worldwide to feed the
world.

59. References
 Bhagyalakshmi, B., & Singh, N. S. (1988). Meristem culture and
micropropagation of a variety of ginger (Zingiber officinale Rosc.) with a high yield of
oleoresin. Journal ofHorticultural Science,63(2), 321-327.
 Cremona, G., Iovene, M., Festa, G., Conicella, C., & Parisi, M. (2018).
Production of embryo rescued hybrids between the landrace “Friariello”(Capsicum annuum
var. annuum) and C. baccatum var. pendulum: phenotypic and cytological
characterization. Euphytica, 214(8), 129.
 Nagib, A., Hossain, S. A., Alam, M. F., Hossain, M. M., Islam, R., & Sultana, R.
S. (2003). Virus free potato tuber seed production through meristem culture in tropical
Asia. Asian J.Plant Sci,2(8), 616-622.
 Farnham, M. W. (2013). Doubled Haploid Inbred Lines USVL048 and
USVL131 of Heading Broccoli. HortScience, 48(7), 907-908.
 Sun, H. J., Cui, M. L., Ma, B., & Ezura, H. (2006). Functional expression of the
taste‐modifying protein, miraculin, in transgenic lettuce. FEBSletters, 580(2), 620-626.

60. Thank you
Grow smart
reap better