Onion Breeding for Quality Traits

1. Breeding for Quality in Onion
Saurabh Singh
Ph. D. Student 1st Year
L-2014-A-43-D

2. Introduction
• Quality is perhaps one of the most elusive
breeding goals upon which so much depends
but about which limited information is
available.
• Traditionally, crop improvement programmes
have emphasized yield and disease resistance
with only a limited regard for quality.
However, the ultimate success of new cultivars
is measured by how well the consuming public
accepts them.

3. • The complexity of quality as a breeding target is
its likely cause for neglect.
• Sometimes, what may be good quality to some is
not adequate to others.
• The multiplicity of breeding objectives other than
quality is already a tremendous burden to the
plant breeder that very often consideration of
quality comes as an afterthought.

4. • Quality is define as ‘the sum total of all those
attributes which combine to make fruit and
vegetable acceptable, desirable and
nutritionally valuable as human foods’.
• Quality of fresh vegetable is combination of
characteristics, attributes and properties that
give the vegetable value to human for food
and enjoyment.

5. • Among the diverse attributes contributing to quality,
the most commonly used, though not necessarily the
most important, are yield, size, colour, texture,
flavour and absence of defects.
• Quality characteristics were grouped into three
classes:
• quantitative (which includes yield and net weight);
hidden (including nutritive value and toxic
substances) and sensory (subdivided into
appearance, e.g. colour, size, flavour including smell
and texture.
• Another important aspect determining the quality
parameters is the pre-harvest and post harvest
factors affecting the quality of the fruits.
(Knott , 2007)

6. Onion
Onion is widely grown in different parts of the
country. It is used as salad or cooked in various
ways in all curries, fried, boiled or baked.
Onion is also used in processed forms e.g. flakes,
powder paste, pickles etc.
It has very good medicinal value. Nutritive value
of onion varies from variety to variety. Its major
value is in its flavour.
The perpetual demand of onions within the
country and for the export has made it essential to
supply onions round the year either from fresh
harvest or from stocks.

7. Quality attributes of onion
• Appearance (size, shape and color)
• Shelf life
• Availability of biochemical compounds
(pungency and soluble solids) and nutritional
quality
• Processing quality (Dehydration): High TSS

8. • The consumer preference for shapes and colors varied all over the world.
• Bulbs may be white, red, brown or yellow; round, piriform or flat; and
soft or firm.
• Bulb quality mainly include: bulb shape, bulb size, scale color, scale
thickness, scale retention, number of scales, bulb firmness, number of
growing points, and neck thickness.
• The scale characteristics are important in the grading, packing, shipping
and appearance of the onion.
• Onion ring processors desire large, single-centered onion bulbs for
making onion rings. The neck thickness is important in the bulb curing
process and preventing disease entry into the bulb.

9. • Dormancy is important in onion for storage.
• Higher soluble solids are important for dehydrating industry to
produce onion chips or powder. The amounts of s-alkyl cysteine
sulfoxide precursors and the enzyme alliinase contribute to the
yield of volatile sulfur compounds that constitute the pungency of
the onion bulb.
• Consumers in the India often prefer strongly pungent onion,
whereas in USA and many European countries low pungency is
liked.

10. Qualitative genetics
• Inheritance of bulb colour in onion:
• Colour classes: White, Yellow, Red and Brown
• Dominant basic colour factor ‘C’ is necessary for either red or
yellow colour
(Clarke et al 1944).
• All the plants with ‘cc’ have white bulbs regardless of presence of
other colour factors.
• Dominant ‘R’ with ‘C’: red bulb colour
• ‘r’ with ‘C’: Yellow bulb
• ‘I’ colour inhibiting factor incompletely dominant over ‘i’.
• ‘II’ : White bulb colour regardlesss of presence or absence of C and
R factors.
• Five major genes ‘I’, C, G, L and R, interact and segregate
independently for 4 different colours. (Khar et al 2008).

11. Bulb colour:
1. Homozygous red i/i C/C R/R
2. Heterozygous red i/i C/c R/R
3. Heterozygous red i/i C/C R/r
4. Heterozygous red i/i C/c R/r
5. Homozygous yellow i/i C/C r/r
6. Heterozygous yellow i/i C/c r/r
7. Homozygous recessive white i/i c/c R/R
8. Heterozygous recessive white i/i c/c R/r
9. Homozygous recessive white i/i c/c r/r
10. Homozygous dominant white I/I C/C/R/R
11. Heterozygous dominant white I/I C/c R/r
(Swarup V 2006)

12. Onion and onion by-products bioactive phytochemicals content
Vitamins
• Vit. C 65 mg/kg
• Vit. B6 1.18 µg/kg
• Folic 190 µg/kg
Minerals
• Ca 230 mg/kg
• Mn 1.4mg/kg
• K 1.4 g/kg
• Selenium 6 µg/kg
• Chromium 150 µg/kg
Fibres
• 18 g/kg
• Inulin 6-10%
• Neokestose
Onions &
onions
by-poducts
Sulphur
Compounds
CSO
S-propyl-cysteine-
sulphoxide
Flavonoids
• Flavonol-glycosides
- Quercetin-3gly
• Anthocyanins
- Cyanidine – 3-gly

13. • Dehydrated Onions: Important characteristics of onions to
be dehydrated are:
• The onions should be high in solid contents which range
from 5 to 20 per cent in common varieties and up to 25 per
cent in highly pungent ones,
• Large bulbs are desired for economy in field harvesting .
• The onions with small neck and root zones and of full globe
to tall globe shape are preferred to the flat types, to permit
greater efficiency in rooting and topping, The onions should
be high in pungency because dehydrated product is
primarily used as flavouring agent.
• The bulbs must remain good in common storage for at least
2 to 3 months with a minimum of rot, shrinkage or
sprouting.
• The bulbs should have white flesh and preferably a white
skin. Yellow or red varieties have been used but
commercially they are less desirable.

14. • Pickled Onions:
• A pickle is defined as an edible product that has been preserved and
flavoured in a solution of brine and edible acid such as vinegar.
• The ideal pickling onion is white, globe-shaped, has small neck and root
zone and uniform small size with a diameter of about 2 to 3 cm.
• French Fried Onions:
• French fries, viz. onion rings fried in deep fat, sometimes after being
dipped in butter, are packed by the frozen food industry. Medium-sized
onion with fleshy rings and single centers are required and mild types
are preferred.
• Freeze Preservation of Onion:
• Both whole and chopped onions have become important frozen items.
Small whole onions processed by the frozen food industry are a delicacy
in hotels and restaurants for making creamed onions.
• Onion Juice:
• Onion juice is extracted and preserved in high vinegar and salt
concentrations, with a pH of 4.2. Succulent bulbs with high pungency are
more desirable.

16. • Bulb colour :
• White colour is preferable for dehydration because
pigmented dehydrated products are not preferred.
• Bulb shape :
• Globe shape is preferred as it leads to reduced waste
during tailing and toppling.
• Bulb size :
• 5.0 to 7.5 cm bulb diameter is preferable
• Pungency:
• High pungency is preferred
• Density :
• Firm bulbs with higher TSS, dry matter are preferred
• Bolting and twins :
• Low bolting and twins is preferred as it leads to firm
bulbs.

17. • The acceptable Indian onion varieties for dehydration
among white flesh onions are
• Bombay White
• No-36-1-3-4 ‘
• Udaipur-102’,
• ‘S-74’,
• ‘Pb-48’,
• ‘L-131’,
• ‘L-124’,
• ‘L-106’,
• ‘Pusa White Round’,
• ‘Pusa White Flat’, ‘
• N-257-9-1’ etc.
• (Mitra et al 2012)

18. • Generally, Indian white onion varieties have low TSS (10-14%),
which is not suitable for dehydration.
• After assessing Indian varieties and land races which do not
have high TSS, Jain Food Park Industries, Jalgaon, introduced
White Creole, which was further subjected to selection
pressure for high TSS and they developed V-12 variety with
TSS range of 15-18%.
• TSS in any variety is a function of genotype, environment and
cultural practices. Long day onion grown under mild climate is
high in TSS, whereas, short-day onion maturing under short
winters does not develop high TSS. Internationally, long-day
and intermediate short-day varieties have been developed
mostly from USA, Spain, Israel, Mexico, etc.
Lawande et al 2009

19. Molecular markers for colour improvement in onion
• Bulb colour is one of the important traits in onion (A. cepa L).
Three major colours of white, yellow, red and a variety of other
bulb colours such as chartreuse and gold exist in onion
germplasm.
• The bulb colour is due to flavonoid compounds and 54 kinds of
flavonoids have been reported in onion (Slimestad et al. 2007).
• Kim et al. (2004) identified critical mutations in the chalcone
isomerase (CHI) gene causing gold onions.
• The inactivation of dihydroflavonol 4-reductase (DFR) in the
anthocyanin synthesis pathway was responsible for colour
differences between yellow and red onions, and two recessive
alleles of the anthocyanidin synthase (ANS) gene were responsible
for a pink bulb colour (Kim et al. 2005b).

20. • Based on mutations in recessive alleles of these two genes
Kim et al. (2007) developed PCR based markers for
identification of polymorphisms between pink and red
alleles of the ANS gene. Most pink onions were
homozygous recessive for the ANS gene indicating the
homozygous recessive. The two pink onions, heterozygous
for the ANS gene, were also heterozygous for the
dihydroflavonol 4-reductase (DFR) gene indicating that the
pink colour was produced by incomplete dominance of a
red colour gene over that of yellow colour.
• Park et al. (2013b) developed functional CAPS markers for
two inactive DFR-A alleles, DFR-APS and DFR-ADEL, for
detection of inactive DFR-A alleles responsible for a failure
of anthocyanin production in onions. Of these two alleles,
DFR-APS predominantly occurs in yellow onion cultivars.

21. Breeding for yellow onion
• Indians do not prefer yellow onion but these find
international market in European. Minimum
requirements for export are: bigger sized (>60 mm
diameter), less pungent and single-centered types.
(Lawande et al 2009).
• “NuMex Starlite”, a new yellow-onion variety
developed by Corgan and Holland (1993), was
resistant to bolting and the short-day type was
obtained by 5 recurrent selections from Texas Grano
502 PRR.

22. • Texas ‘Grano 1015 Y’, a mildly pungent, sweet, short-day yellow onion variety, was
developed by Pike et al (1988) through original, single-bulb selection from Texas Early
Grano 951 through 5 generations of selections.
• Similarly, “Texas Grano 1030 Y” was developed from F2 selections of Texas Early Grano
502 x Ben Shemen by Pike et al (1988), which is a late maturing mildly pungent short-
day onion variety.
• Only two varieties were developed, viz., Phule Swarna from MPKV, Rahuri and Arka
Pitambar from IIHR, Bangalore and were released at the state / institute level. Yield of
these varieties was comparatively less than in commercial red onion varieties.
Mohanty et al (2000) assessed 12 varieties of onion during kharif season and found
lowest bulb diameter of 4.2 cm in Arka Pitambar, along with low yields.

23. Plant Physiology, August 2008, Vol. 147, pp. 2096–2106,

24. Introduction
• Allium species synthesize a unique set of secondary sulfur
metabolites derived from Cys. Most notable are the S-
alk(en)yl-L-Cys sulfoxides, including S-2-propenyl- L-cysteine
sulfoxide (alliin; 2-PRENCSO) and trans-S-1- propenyl-L-
cysteine sulfoxide (isoalliin; 1-PRENCSO; Rose et al., 2005).
• When the tissues of any Allium species are disrupted, these
amino acid derivatives are cleaved by the enzyme alliinase
into their corresponding sulfenic acids, and volatile sulfur
compounds are produced that give the characteristic flavor
and bioactivity of the species.

25. • In onion (Allium cepa), 1-PRENCSO is the major sulfoxide
(Fritsch and Keusgen, 2006). This would be predicted to
produce di-1-propenyl thiosulfinate and di-1-propenyl
disulfide.
• Instead, propanthial S-oxide (lachrymatory factor [LF]), 1-
propenyl methane thiosulfinate, and di-propyl disulfide are
dominant (Block et al., 1992a, 1992b; Rose et al., 2005).
• It is the chemical responsible for inducing tearing in onion, an
undesirable irritant, and it is hypothesized that LF production
causes the absence of otherwise predicted sulfur volatiles
(Randle and Lancaster, 2002), analogues of which in garlic are
known for their health attributes (Griffiths et al., 2002).

26. • Imai et al. (2002) discovered that the conversion of 1-propenyl
sulfenic acid to LF is mediated by an enzyme they named
lachrymatory factor synthase.
• The production of LF could be reduced by genetic
manipulation of the LFS transcript using RNA interference
(RNAi) silencing.
• Eady, et al. silenced the gene for the lachrymatory factor
enzyme by using RNA inter-ference, to produce tearless
onions.
• This feat of genetic engineering reduces levels of
lachrymatory factor up to 30-fold but does not diminish the
overall levels of organosulphur compounds in the bulb.

27. • These “tearless onions” have potential health
benefits for consumers as they do not produce
tears, but retain their health-promoting
properties.
• Vidalia: Tearless onion (Randle and Lancster
2002)

28. • By reducing LFS and stopping the conversion of 1-
propenyl sulfenic acid to the undesirable LF, we
tested the hypothesis that this would allow 1-
propenyl sulfenic acid to be available for
spontaneous conversion into thiosulfinate and
thiosulfinate-derived sulfur compounds,
analogues of which are renowned for their
desirable sensory and health-promoting
attributes.

29. MATERIALS
• A mild hybrid mid-daylength fresh onion
(Allium cepa ‘Enterprise’), a pungent open-
pollinated fresh onion (‘Pukekohe
LongKeeper’), and a pungent dehydration
mid-daylength onion (Sensient Dehydrated
Flavors) were transformed, regenerated, and
ex-flasked according to the method of Eady et
al. (2000).

30. • The T-DNA cassette designed for silencing in
onion was contained within a pArt binary
vector (Gleave, 1992) with a m-gfpER reporter
gene under the control of a CaMV 35S
promoter (Haseloff et al., 1997) and a nptII
gene under the control of a nos promoter for
ease of detection and selection. It contained
the pHANNIBAL-based RNAi cassette (Wesley
et al., 2001) containing a 512-bp hairpin of the
lfs gene sequence under CaMV 35S
promotional control

32. Results
• Three onion cultivars were studied: a mild hybrid (H)
mid-daylength fresh onion (‘Enterprise’), a pungent
open-pollinated (O) fresh onion (‘Pukekohe
LongKeeper’), and a pungent dehydration (D)
middaylength onion (Sensient Dehydrated Flavors).
• Eleven plants were evaluated, three nontransgenic
plants (HN, ON, and DN) and eight transgenic plants
(H1, H2, H3, O1, O2, O3, D1, and D2), from the
hybrid, open-pollinated, and dehydration cultivars as
indicated.

33. All transgenic plants grew and formed morphologically similar plants and bulbs to
their nontransgenic counterparts. Seed set and F1 progeny had been obtained from
two lines by selfing or crossing onto nontransgenic counterparts.

34. T-DNA Integration and Integrity
• Southern-blot analysis of
onion plants, using a gfp gene
probe (Eady et al., 2000),
revealed that plants H1 and D1
contained two copies of the T-
DNA construct at different loci
and that plant O1 contained a
multiple insert at a single
locus.
• The remaining five plants, H2,
H3, O2, O3, and D2, contained
single-copy inserts integrated
at different locations from
each other, confirming the
nonclonal nature of the
transgenic events.

35. • PCR data (Table I) indicated that the T-DNA cassette
was not complete in all plants evaluated.
• In plant O3, the nptII gene sequence could not be
detected. Initial identification of this transgenic event
by GFP expression and rescue to nonselective
medium resulted in the maintenance of this plant.
• In plant H2, the 5’ region of the lfsRNAi cauliflower
mosaic virus (CaMV) 35S promoter sequence was
truncated.
• However, this did not compromise transcription or
small interfering RNA (siRNA) production.

37. siRNA Production
• Detection of lfsRNAi transcript by reverse transcription
(RT)-PCR was used to indicate functionality of the
transgene. All transgenic plants except O1 produced
lfsRNAi transcript (Table I).
• Such observation of transgene inactivation due to
multiple-copy inserts at a single locus is common
(Muskens et al., 2000; Tang et al., 2007).
• Detection of lfs siRNA using a lfs probe showed that six
plants (H1, H2, H3, O2, O3, and D2) produced siRNA
fragments corresponding to the LFS gene sequence.

38. • Interestingly, plant D1, which produced
lfsRNAi transcript, failed to produce lfs siRNA
at detectable levels.
• In this case, we can assert that the cause was
not transcriptional inactivation.

39. LFS Levels
• lfs transcript levels were
compared in cDNA samples
from transgenic and
nontransgenic plants by
quantitative RT-PCR. Low
levels of transcript
corresponded well with the
presence of lfs siRNA
fragments.
• No LFS protein could be
detected in plants that
produced lfs siRNA. Plants O1
and D1, with no observable lfs
siRNA, had LFS protein levels
that fell well within the range
of their respective control
nontransgenic plants.

40. • Assays of LFS activity in both leaf and bulb measured by in vitro
generation of LF demonstrated that plants with no detectable
LFS, as measured by western blot analysis, also had significantly
reduced LFS activity.
• This activity in plants H1, H2, and H3 was reduced by between
21- and 103-fold in leaf tissue and by between 18- and 1,168-
fold in bulb tissue.
• Activity in plants O2 and O3 was reduced by between 38- and
70-fold in leaf tissue and by between 1,515- and 1,544-fold in
bulb tissue.

41. • Activity in plant D2 was
reduced by 396-fold in leaf
and by 501-fold in bulb
tissue. The more
pronounced reduction
observed in bulb tissue
over leaf tissue suggests
that LFS is probably a
relatively major protein
within aestivating storage
bulb tissue compared with
leaf material.
• Plants D1 and O1 failed to
produce the LFS silencing
signal or to reduce LFS
activity.

42. Precursor 1-PRENCSO Levels and Alliinase Activity
• Biochemical analysis showed that the 1-PRENCSO
levels in the transgenic and control plants were
between 4 and 13 mg/g dry weight.
• Alliinase activity was between 15.8 and 42.4 nkat
/mg protein.
• These substrate and enzyme levels are within the
normal physiological range reported for onion
(Kitamura et al., 1997; Kopsel and Randle, 1999).
• This suggests that in transgenic onions, silencing lfs
transcripts did not affect alliinase activity or 1-
PRENCSO levels.

43. Phenotype Analysis of Secondary Sulfur Chemistry
• In order to identify all of the possible changes to
onion secondary sulfur metabolism, three
established techniques were used:
• gas chromatography (GC) with flame photometric
detection,
• solid-phase microextraction (SPME) GC-mass
spectrometry (MS), and
• solvent extraction GC-MS.
• In addition, to detect the previously undetected in
onion di-1-propenyl thiosulfinate, a novel
colorimetric (‘‘pinking’’) assay was developed and
used.

44. Volatile Sulfur Compounds
• GC-flame photometric detection
analysis of LF levels from freshly
crushed leaf material
demonstrated that H1, H2, and
H3 were reduced by 13.5-, 35.5-,
and 30-fold, respectively,
compared with HN, that O2 and
O3 were reduced by 30- and 67-
fold compared with ON, and that
D2 was reduced by 36-fold
compared with DN.
• In bulb material, LF was reduced
by 10.2- and 28.2-fold for H1 and
H3 compared with HN (H2 was
not measured, as the bulb was
infected), by 6.4- and 28-fold for
O2 and O3 compared with ON,
and by 12.8-fold for D2 compared
with DN.

45. GC analysis of solid-phase microextraction sulfur components from the head space of vials
containing cut onion leaf material. Peak 1, Dipropyl disulfide; peak 2, 1-propenyl propyl disulfide;
peak 3, di-1-propenyl disulfide isomer 1; peak 4, di-1-propenyl disulfide isomer 2; peak 5, di-1-
propenyl disulfide isomer 3; peak 6, syn-2-mercapto-3,4-dimethyl-2,3-dihydrothiophene; peak 7,
anti-2-mercapto-3,4-dimethyl-2,3- dihydrothiophene.

46. Conclusion
• Allium sulfur compounds are renowned for their human
health-giving attributes. We predict that the altered profiles
present in the reduced-LF plants are likely to have significant
consequences for these attributes; as such, they are being
further investigated.
• For example, thiosulfinates have antiasthmatic activity
(Griffiths et al., 2002).
• The unsaturated 1-propenyl-containing thiosulfinate in the
reduced-LF onions may confer health properties to onion that
have previously been associated with the unsaturated allicin
thiosulfinate in garlic.

47. • In summary, these reduced-LF onions are a unique
resource for understanding the role of specific sulfur
secondary metabolites in plant biology, in human
health, and in terms of their potential value to the
agrifood industry.