Breeding for Quality in Onion

Saurabh SinghPh. D. Student 1st Year

L-2014-A-43-D

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.

• 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.

• 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.

• 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)

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.

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

• 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.

• 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.

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).

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)

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

CompoundsCSO

S-propyl-cysteine-sulphoxide

Flavonoids• Flavonol-glycosides - Quercetin-3gly• Anthocyanins - Cyanidine – 3-gly

• 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.

• 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.

• 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.

• 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)

• 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

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).

• 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.

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.

• 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.

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

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.

• 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).

• 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.

• 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)

• 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.

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).

• 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

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.

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.

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.

• 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.

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.

• 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.

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.

• 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.

• 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.

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.

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.

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.

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.

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.

• 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.