This document discusses breeding for quality in onions. It begins by explaining that quality is an elusive but important breeding goal that influences consumer acceptance. The complexity of quality makes it a neglected target. Various attributes contribute to onion quality, including yield, size, color, texture, flavor, and lack of defects. The document then discusses specific quality attributes and breeding objectives for different uses and varieties of onions, such as for dehydration, pickling, and freezing. It also covers the genetics and inheritance of onion bulb color. The conclusion discusses developing tearless onions through genetic engineering to reduce lachrymatory factor while maintaining health-promoting compounds.
New England Veg & Fruit Conference, 2017- Breeding for Flavor and Late Blight...Emily Haga
Presentation given at New England Veg & Fruit Conference, 2017- Tomato Session. Also available at:
https://newenglandvfc.org/past-conference-proceedings-presentations/2017-proceedings-and-presentations
Classification of Vegetable Crops
There are many numbers of Vegetables. Different parts are used for consumption. They are botanically different and their climatic and cultural requirements are different.
There are four Main Methods of Classification Based on:
1. Education Botanical relation
2. Based on Hardiness (Tolerance to low temp)
3. Parts Used for consumption
4. Methods of Culture
1. Botanical Classification:
This method of classification is based on botanical relationships of crops in responding cytology, morphology, taxonomy and cross ability. This is useful to breaf for crop improvement and seed producer for deciding isolation distance. The cuntroal requirements may vary ex. Solanaceous family includes potato, chili and brampal, There are same cultural requirements. At the same time cucurbitaceous have similar cultural requirements and common pests and diseases. Botanical name avoids infusion in name as common names are different but scientific names are common all over would. It gives information on class, family, genus, species, variety etc Ex. Solanaceous family, Potato, brinjal, chili, cucurbitaceous family: Melons, gourds. Not useful to grower. Thus botanical classification is useful to breeder, seed producer and to avoid confusion in common name.
2. Classification Based on Hardiness:
Vegetables are grouped as hardy or tender on the basis of tolerance to frost, tolerance to lower temperature. This gives information of season of growing i.e. summer or winter. Not useful to grower, since soil and climatic requirements are
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Hardy
Semi Hardy
Tender
Asparagus
Beet Root
Okra
Cabbage
Carrot
Brinjal
Garlic
Cauliflower
Tomato / Chili
Onion
Palak
Beans
Peas
Potato
Cucurbit
Radish
Sweet Potato
Spinach
Sweet Potato
Spinach
Amaranthus
Turmp
3) Based on Parts Used for Consumption:
From roots to fruits different parts of vegetables are consumed. On that basis vegetables are classified.
Leafy Vegetable
Palak, Amaranthus Methi (leaves)
Root
Carrot, Radish, Turnip’, beet root
Fruit
Tomato, Biinjal; Cucurbit
Bulb
Onion, Garlic
Tuber
Potato, Sweet Potato
4) Based on Method of Culture:
In this method all those crops having similar cultural requirements are grouped together. They" are botanically different. System has practical utility for vegetable grower. In this method one can generalise cultivation practices for one group and thus avoid repetition individually for all crops. There are 11 classes. In some cases they are botanically also similar Ex. cucurbits. On excises for convenience they are grouped as under.
I.
Perennial Vegetable
Asparagus, cocinia (Tondali), Parwal, Drumstick
2
Greens
Spinach
3
Salad Crops
Lettuce, celery
4
Cole Crops
Cabbage, Cauliflower
5
Bulb Crops
Onion, Garlic
6
Root Crops
Carrot / radish, turnip
7
Tuber
a) Potato
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
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Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
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A brief information about the SCOP protein database used in bioinformatics.
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Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
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Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
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)
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.
15.
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.
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
31.
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.
36.
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.