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BITTERNESS IN CITRUS
FRUIT-THE BIOCHEMISTRY,ANALYSIS AND
APPLICATIONS.
MANPREET KAUR
SAINI
21th march 2012
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CONTENTS OF PRESENTATION
BIOSYNTHESIS & BIOCHEMISTRY OF LIMONOIDS
EXTRACTION AND QUALITATIVE & QUANTITATIVE ANALYSIS OF CITRUS
BITTER PRINCIPLE.
GENE EXPRESSION AND TRANSCRIPTOME STUDIES
CLONING AND CREATION OF TRANSGENIC VARIETIES
APPLICATION OF THE BITTER PRINCIPLE
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PRIMARY AND SECONDARY METABOLITESMetabolites- compounds synthesized by organisms using enzyme-
mediated chemical reactions called metabolic pathways
PRIMARY METABOLITES
Essential to growth and development.
Present in all plants as are components or
products of fundamental metabolic
pathways or cycles.
SECONDARY METABOLITES
Not Essential to growth and development.
colored, fragrant, or flavorful compounds
typically mediate the interaction of plants
with other organisms.
EXAMPLES OF PRIMARY
METABOLITES
Energy rich fuel molecules, such as
sucrose and starch,Structural components such as cellulose,
informational molecules such a DNA
and RNA
Pigments such as chlorophyll.
EXAMPLES OF SECONDARY
METABOLITES
Alkaloids such as caffeine, nicotine, etc.
Terpenoids such as monoterpenes,diterpenes, triterpenes like Limonin and
tetraterpenes.
Phenolics such as flavonoids like naringin
and anthocyanins etc.
The main focus of this presentation will be triterpenoids mainly the Limonoids.
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INTRODUCTION TO BITTER PRINCIPLES
Kinnow mandarin, a hybrid of
Citrus nobilis and Citrus
deliciosa is considered one of
the major crops of Punjab.
But processing of Kinnow juice
faced formidable problems in
terms of bitterness and
delayed bitterness thusaffecting its consumer
acceptability.
Biochemical basis of bitterness
in kinnow:
bitterness due to flavonoids e.g.
naringin species related topumello. Threshold of bitterness
is 50 ppm.
delayed bitterness due to
limonoids e.g. limonin.
Threshold of bitterness is 6
ppm.
Critical reviews in
biotechnology(199
6)
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CITRUS BITTER PRINCIPLES IN DIFFERENT
PART OF THE FRUIT
Structure of citrus fruit showing concentration of limonoids and
flavonoids in different parts.
F- Flavedo, A- Albedo, SM-segment membrane, S- seeds, J- juiceKasetsart J. (Nat. Sci.) 43 : 28 - 36 (2009)
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BIOSYNTHESIS OF
LIMONOIDS
The Plant Cell, Vol. 7,
1015-1026, July (1995)
Acetyl Co A thiolase HMG Co A synthase
HMG Co A reductase
Mevalonate
kinase
PhosphoMevalonate
kinaseMVAP,Decarboxylase
GPP synthase
IPP Isomerase
FPP synthase
GGPP synthase
squalene
synthase
Terpene synthases
Prenyltransferase
s
MEVALONIC ACID PATHWAY AND TERPENOID SKELETON BIOSYNTHESIS( HMG Co A- 3 hydroxy 5 methyl glutaryl Co A, MVAP-Phosphomevalonate,MVAPP- 5-pyro Phosphomevalonate, IPP-Isopentyl Pyrophosphate, DMAPP-Dimethylallylpyrophosphate,
GPP-geranyl pyrophosphate, FPP- franesyl pyrophosphate, GGPP- geranyl geranyl pyrophosphate)
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BIOSYNTHESIS CONT.
SQUALENE
DEACETYLNOMILINIC ACIDNOMILIN
DEACETYLNOMILIN METHYL DEACETYLNOMILIC ACID
OBACUNONE
ICHANGENSIN(KETO) CALAMIN CYCLOCALAMIN
OBACUNOIC ACID
ICHANGIN ICHANGENSIN(KETAL)
LARL
LIMONIN DEOXYLIMONIN DEOXYLIMONIC ACID
LIMONOL
17 -D GLUCOPURANOSIDE
NOMILIN ACEYL
LYASE
HYDROLASE
DEHYDROGENASE
UDP-D-GLUCOSE:
LIMONOID GLUCOSYL
TRANSFERASE
Biol. Pharm. Bull. 29(2) 191201 (2006)
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BIOCHEMISTRY OF LIMONOIDS IN CITRUS
.
A group of highly oxygenated tetracyclic
triterpenoids in Rutaceae and Meliaceae
plant families causes delayed bitterness in
citrus and is a secondary metabolite.
Two forms in citrus:
1. Limonoid aglycones (LA) -- >50 isolated
from the Rutaceae (36 from Citrus & related
genera)
2. Limonoid glucosides (LG) -- 17 isolated
3. LARL and NARL- the precursors for
limonoid synthesis can also be considered
Relation between LA and LG1. LA: bitter, insoluble in water
2. LG: non-bitter, water-soluble
3. LA glucosidated to LG-during fruit maturation
and this is known as natural debittering.
Food review. Int, 12(4),(1996).
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LIMONOID AGLCONES J. Agric. Food Chem., Vol.55, No. 21, 2007 Review s
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LIMONOID GLUCOSIDES AND SYNTHETIC LIMONIN CARBOXYMETHOXIME
J. Agric. Food Chem., Vol.
55, No. 21, 2007 Review s
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SITES OF BIOSYNTHESIS OF LIMONOIDS
3 forms of limonoids
monolactone dilactone glucosides
Mature peel and fleshtissues In leavesand seeds
Limonin glucoside Nomilin glucoside
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ACCUMULATION OF LIMONOIDS
acetate, mevalonate, or farnesylpyrophosphate
Nomilin in stem phloem region
translocated to fruit
tissues, peels, seeds
and leaves
other limonoids
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MAJOR BIOSYNTHETIC GROUPS
Four groups of Limonoid Aglycones
1. Limonin group
2. Calamin group
3. Ichangensin group
4. 7-acetate limonoid group
Biosynthetic pathways of each group ofthese limonoids have been elucidated:
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THE LIMONIN BIOSYNTHETIC
GROUP in all citrus species, citrus hybrids and many non citrus members of family
rutaceae.
Limonin, nomilin, deacetylnomilin, Ichangin and obacunone are the major
limonoids
Food review. Int, 12(4),(1996).
BIOSYNTHETIC PATHWAYS OF LIMONOIDS: THE LIMONIN GROUP
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CALAMIN BIOSYNTHETIC GROUP Found only in tissues of Fortunella and its hybrids like calamondin. And major limonoids
include calamin, retrocalamin, methyl iso-obacunoate diosphenol, 6-keto-7b-
deacetylnomilol and 6-keto-7b-nomilon.
6-keto-7b-nomilon contains structural features of both calamin and limonin groups-
represent a biosynthetic link between them.
Food review. Int, 12(4),(1996).
BIOSYNTHETIC PATHWAYS OF LIMONOIDS: THE CALAMIN GROUP
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GROUP Found in tissues of Citrus ichangenesis and its hybrids Yuzu, Sudachi, Kabosu,Hanayu
and Ichang lemon, ichangenesin, deacetylnomilin, and deacetylnomilinic acid being the
major compounds.
present as ketal and keto group in chloroform solution but as ketal only in citrus.
Nomilin converted to deacetylnomilin by nomilin deacetylase, enzyme not found in any
other citrus species.
Food review. Int, 12(4),(1996).
Nomilin
deacetlylase
BIOSYNTHETIC PATHWAYS OF LIMONOIDS: THE ICHANGENSIN GROUP
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THE 7-ACETATE LIMONOID BIOSYNTHETIC
GROUP Found only in tissues of Poncirus and its hybrids
This limonoid group includes 7a-obacunol, limonyl acetate [20] and 7a-obacunyl
acetate..
Food review. Int, 12(4),(1996).BIOSYNTHETIC PATHWAYS OF LIMONOIDS: THE 7-ACETATE lIMONOID GROUP
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DELAYED BITTERNESS
Most citrus fruits do not taste bitter if eaten fresh or if freshly squeezed juiceis consumed but within a few hours after juice extraction, the juice becomes
bitter. This phenomenon is generally referred to as delayed bitterness.
The two classes of chemical compound namely flavonoids and limonoids
were found responsible for bitterness in citrus juices. But there is a difference
between flavonoid and limonoid bitterness.
The fruits containing high flavonoids are bitter even when consumed as
fresh. The peel (rind) of the citrus fruit contain very high amount of flavonoids
like naringin, neohesperidine etc. making it highly bitter.
The limonoids are present in the form of non-bitter compound (limnoate - A-
ring lactone), which is converted to bitter limonin and other bitter limonoids in
the presence of enzyme limonoate-D-ring lactone hydrolase on storage.
Hence the fresh citrus juice does not taste bitter but turns highly bitter on
storage.
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FACTORS RESPONSIBLE FOR
DELAYED BITTERNESS
Cultivar
Location
Maturation time
Storage temperature
Acidic medium
Field Freeze injure
Mechanical damage
Initiates conversion of LARL to bitter
limonoid aglycone
Promotes enzme activity
With more time bitterness decreases.
Less the storage temperature, less the
bitterness as enzyme activity reduced
Different varieties has different level of
bitterness like in valencia orange and navel
orange.
Different temperature conditions affect the
bitternesss content of same fruit.
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MECHANISM OF DELAYED
BITTERNESS
Food review. Int, 12(4),(1996).
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NATURAL LIMONOID DEBITTERING PROCESS
Limonin bitterness occurs in early to mid season harvested fruits only
Natural debittering process occurs when in late stages of fruit growth and
maturation limonoid aglycones were converted to their respectiveglucosides .
Limonoid glucosides contain one D- glucose molecule attached to the C-17
position of each corresponding aglycone via a beta- glucosidic linkage such
as limonin 17- beta D- glucopyranoside.
Enzyme involved is UDP-D_glucose transferase, isolated from citrus albedo
tissues, found only in mature fruit tissues and seeds .
Food review. Int, 12(4),(1996).
ENZYMES INVOLVED IN BIOSYNTHESIS AND BIODEGRADATION
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ENZYMES INVOLVED IN BIOSYNTHESIS AND BIODEGRADATION
OF LIMONOIDS IN CITRUS
ENZYME GROUP NAME OF ENZYME SITE OF ACTION FUNCTIONS
E-1 Various enzymes ofterpenoid pathway likethiolase, synthase,
reductase, kinase,
isomerase, etc.
phloem region ofcitrus stem tissues biosynthesis of nomilinfrom acetate and
mevalonate
E-2 Enzymes like lyase,hydrolase,
dehydrogenase,
esterase, etc.
all citrus tissues
including leaves,
stems, fruit tissues,
fruit peels and seeds
biosynthesis of other
limonoids from Nomilin.
E-3 limonoid UDP- D-glucopyranoside
tranferase
seeds convert monolactones
to glucosides during
late stages of fruit
growth and maturation
E-4 limonoid D- ring lactone
hydrolase
seeds lactonization of the D-
ringconverts monolactones
to dilactones
E-5 limonoid 17- beta-D-glucopyranoside beta-
glucosidase
dormant seeds and
germinating seeds
limonoid biodegradation
catalyzes the hydrolysis
of limonoid glucosides
and liberates limonoids
and glucoseFood review. Int, 12(4),(1996)
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DEBITTRING METHODSDEBITTERING METHODS
column and batch
methods using
adsorbent and
ion exchange
resins
blend bitter
juice with non
bitter juice,
diluting out the
bitter taste
Creating new
citrus varieties
through
Genetic
engineering
Transgenic citrus varieties free from limoninbitterness
Three specific target enzymes
Insertion of one of the three gene
codings
Yields nonbitter fruits in
transgenic plants
Limonoid UDP-D-glucose
transferase
Limonoate Dehydrogenase
Nomilin deacetylase
Food review. Int, 12(4),(1996)
C t
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Cont
Nomilin deacetylaseLimonoid UDP-D-glucose
transferaseLimonoate Dehydrogenase
Converts limonoid aglycones
to glucosides
Convert limonin to nonbitter
limonoids
Convert nomilin to
deacetylnomilin
Enhancement of enzyme
activity through genetic
Engineering
reduces aglycone
concentration that reduces
delayed bitterness
In low level in citrus fruits
so isolated from bacteria
N terminal sequence
determined
Gene being from cDNA
library prepared from
bacterium
diverts biosynthetic
pathway of limonin
Nonbitter deacetylnomilin
accumlated instead of
limonin
Enzyme not isolated yet
Isolated from albedo tissues by a
combination of ammonium slfate
fractionation, affinity
chromatography,
and ion-exchange high-
performance liquid
chromatography (HPLC)
Isolated by ammonium
slfate fractionation, Blue
dye-ligand affinity
chromatography,
and ion-exchange HPLC
Food review. Int 12 4 1996
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EXTRACTION & QUALITATIVE &QUANTITATIVE ANALYSIS OF
CITRUS BITTER PRINCIPLE.
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Analytical Methods of Citrus
Limonoids
Thin-layer chromatography (TLC)
-- for limonoid detection
Nuclear Magnetic Resonance (NMR)
-- determination of limonoid structure
HPLC -- detection & quantification
Radioimmunoassaydetection &
quantification
HPLC-MS detection & quantification
EXTRACTION OF LARL & NARL FROM
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EXTRACTION OF LARL & NARL FROM
CITRUS JUICEJuice sample centrifuged at16000*g for 5min at 10 C.
Supernatant collected and filtered through 0.45 um filter.
Filtered liquid used to prepare three samples.
Juice(150 l)+CA internal standard
solution(75 l, 100 ppm)
Samples mixed and loaded(1 ml) onto strata X solid phase extraction columns
that was washed with MeOH(1 ml) and preconditioned with water(1 ml).
Thereafter column washed first with water(0.5 ml) and then CHCL3 (0.5ml) .
LARL finally eluted with solution B (CH3CN-MeOH-Water) (1 ml).
Juice (150 l)+internal
calibrator solution(15 l)
Juice (150 l)+
water (1.26 ml)
JOURNAL OF CHROMATOGRAPHY A, 1064(2005) 187-191
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EXTRACTION OF BITTER LIMONOIDS/ LIMONOID
AGLYCONES FROM CITRUS JUICE
Juice obtained by hand squeezing
Juice gently liquid/liquid extracted with CHCl3 (2*2.5 ml) for 30 s
3 l diluted extract was injected into LC-MS
2 ml juice transferred to test tube & 40 l internal standard added (167 ng/l)
CHCL3 extractscombined & 100 l of it removed.
It was evaporated to dryness and redissolved in MeOH or THF(300 l)
J. AGRIC. FOOD CHEMISTRY 2005, 51, 3709-3714
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EXTRACTION OF LIMONOID GLUCOSIDES
Wet samples (juice, molasses, wash
water) centrifuged at 16000 * g for 5 min
at 10 C.
Supernatant collected and filtered through
a 0.45 um filter.
Sample for injection prepared bycombining 75 l sample, 925 l water and
300 l MeOH and 200 l internal standard
solution
Samples analyzed by ESI-LC-MS
Solid samples (peel, seeds, etc) oven-dried
but pulp samples dried overnight at 60 C
and ground to pass 2mm mesh screen.
100 mg weighed into 10 * 50 mm cellulose
extraction thimbles.
Sample extracted overnight in soxhlet
extracter with 25 ml MeOH.
Diluted to 30 ml with MeOH
300 l of above extract + 700 l water + 200
l carminic acid solution(30 mg/l) added to
autosampler vial
Sample analyzed by ESI-LC-MS
J. AGRIC. FOOD CHEMISTRY 2005, VOL.49, NO 3, 2001
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GENE EXPRESSION AND
TRANSCRIPTOME STUDIES
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STUDY OF TRANSCRIPTOME CHANGES DURING
FRUIT DEVELOPMENT AND RIPENING FROM
DIFFERENT PARTS OF THE FRUIT.
STRUCTURE OF CITRUS
FRUIT
Braz. J. Plant Physiol., 19(4):333-362, 2007
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CITRUS FRUIT STRUCTURES TARGETED FOR ENZYMES
INDUCING NATURAL DEBITTERING IN CITRUS
SEED FLESH ALBEDO FLAVEDO
RNA ISOLATION AND CHARACTERIZATION AT DIFFERENT
STAGES OF FRUIT DEVELOPMENT
ANALYZING THE MECHANISM OF LIMONOID GLUCOSIDE
ACCUMULATION IN CITRUS
DEVELOPMENT OF TRANSGENIC PLANT WITH LOW LEVEL OF
BITTER LIMONOIDS TROUGH ENHANCEMENT OF NATURAL
DEBITTERING PROCESS
CITRUS FRUIT TRANSCRIPTOME SEQUENCING- HTS
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CITRUS FRUIT TRANSCRIPTOME SEQUENCING HTS
RNA ISOLATION AND cDNA LIBRARY PREPARATION
BIOINFORMATICS USAGE OF SOFTWARES FOR ILLUMINA GENOMEANALYSER DATA ASSEMBLY(Velvet, Oases, CLC GENOMICS)
TRANSCRIPTOME COMPARISON OF CITRUS FRUIT UNIGENE WITH
OTHER PLANT SPECIES
SEQUENCE ANALYSIS AND FUNCTIONAL ANNOTATION THROUGH GENE
ONTOLOGY(GO) CLASSIFICATION
TRANSCRIPT PER MILLION(TPM)-STATISTICAL CALCULATION BETWEENSAMPLES FOR DATA NORMALIZATION AND COMPARISON PURPOSE AND
ITS EXPRESSION PROFILING
DIFFERENTIAL EXPRESSION OF SELECTION OF GENES VALIDIATED BY
APPLYING REAL TIME QUANTITATIVE RT-PCR(q RT-PCR)Yu et al. BMC Genomics 2012, 13:10
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CITRUS FRUIT TRANSCRIPTOME SEQUENCING
USING MICROARRAY ANALYSIS
RNA ISOLATION AND cDNA LIBRARY PREPARATION
EST PROCESSING AND FUNCTIONAL ANNOTATIONMICROARRAY HYBRIDIZATION,SCANNING AND DATA ANALYSES
TRANSCRIPT PROFILING- first step in correlating gene expression with specific
biological processes, microarray being the high throughput method for it.
THE CONSTRUCTION OF A GENE- specific oligonucleotide chip based on
EST/genomic sequence data will expand microarray applications
METABOLOMICS AND PROTEOMICS DONE TO
1. correlate between compounds and/or proteins and a given trait or process.
2. to compare between cultivars displaying different traits, and to identify the compound(s)
and/or protein(s) associated with them.
3. to identify compounds of pharmaceutical, industrial and commercial values, such as
antioxidants, and unique aroma and flavor molecules
Plant Molecular Biology (2005) 57:375391
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CLONING AND CREATION OFTRANSGENIC VARIETIES
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DEVELOPMENT OF CITRUS VIRUS
VECTOR
sequencing plant genomes resulted in identification of large numbers of novel open reading
frames (ORFs) using large-scale functional genomic.
virus vectors for expression or silencing of plant genes.
Inoculation of plants with virus vectors a direct way to assay the function of specific genes
without the time consuming process of plant transformation and regeneration.
useful in woody plants like citrus, with long juvenile periods (up to 6-8 years).
complete sequence of a new virus, the citrus leaf blotch virus (CLBV) obtained.
used as a vector for expression or silencing genes in citrus plants because:
1.viruses like CTV being phloem limited, CLBV replicates in all citrus tissues.
2.accumulates mainly in meristematic tissues,offering an interesting model system to study
genes involved in growth and development of leaves and fruits;
3.monopartite genome of 8747nt containing three ORFs & easy to manipulate
4.mechanically transmissible to citrus & facilitate inoculation of engineered versions carrying the
foreign genes
CREATION OF TRANSGENIC CITRUS
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CREATION OF TRANSGENIC CITRUS
FREE FROM LIMONIN BITTERNESSGENETIC TRANSFORMATION OF CITRUS
EFFICIENT TRANSFORMATION IN CITRUS REQUIRED FOR TWO
MAIN REASONS:
TO IDENTIFY GENE FUNCTION
DEVELOPMENT OF NEW AND IMPROVED CITRUS VARIETIES
DEVELOPMENT OF TECHNOLOGIES FOR EFFICIENTTRANSFORMATION IN CITRUS
GENE REMOVAL
REMOVING GENES THAT ARE NO
LONGER USEFULUSEFUL WHEN THE PLANT IS
SMALL AND JUVENILE, NOT
REQUIRED IN THE MATURE TREE;
THIS WOULD PROVIDE THE
BENEFITS OF TRANSGENIC TRAITS
BUT RESULT IN NONTRANSGENIC
FRUIT.
GENE STACK ING.
MODIFYING MULTIPLE OR COMPLEX
CHARACTERISTICS OF A TREE
:INSERTION OF MULTIPLE GENES.
AND WOULD TAKE A LONG TIME.
GENE STACKING REQUIRES
TRANSFORMATION WITH MULTIPLE
GENES OR THE SEQUENTIAL
TRANSFORMATION..
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TECHNOLOGIES CONT
TECHNOLOGIES FOR GENETIC TRANSFORMATION OF CITRUS
NON-ANTIBIOTIC
SELECTION SYSTEMS
SELECT
TRANSFORMED
CELLS & TISSUES
WITHOUT USING
ANTIBIOTICS.
NEGATIVE (E.G.,
PHOSPHINOTHRICIN)AND POSITIVE (E.G.,
MANNOSE)
SELECTION
COMPOUNDS BE
DEVELOPED
LINKAGE GROUP
TRANSFORMATION.
TRANSFORMATION BY
LARGE DNA INSERTS
CONTAINING MULTIPLE
GENES IN LINKAGE
GROUPS
FACILITATING
POSITIONAL CLONING &FUNCTIONAL ANALYSIS
INTRODUCING MULTIPLE
GENES FOR SECONDARY
PRODUCT PATHWAYS
INTO CITRUS
.
GENE TARGETING.
TARGETING GENE
INSERTION BY
HOMOLOGOUS
RECOMBINATION INTO
THE CITRUS GENOME.
ALLOW DISRUPTION OF
GENE FUNCTION (KNOCK-
OUTS).
RESTORATION OF
FUNCTION OF DEFECTIVE
GENES (GENE THERAPY)
REPLACEMENT OF A
GENE WITH A NEW OR
ALTERED VERSION.
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TECHNOLOGIES CONT....
VIRAL-MEDIATED
TRANSFORMATION.
REQUIRE ANONPATHOGENIC VIRULENT
VIRAL VECTOR & A METHOD
TO DISPERSE THE VIRUS,
PRESUMABLY AN INSECT
VECTOR.
PHENOTYPE OF THE TREE
MODIFIED BY SIMPLY
DEPLOYING A DIFFERENT
ENGINEERED FORM OF THE
VIRUS.
TECHNOLOGIES FOR GENETIC TRANSFORMATION OF CITRUS
PROMOTER LIBRARY.
REPERTOIRE OF
PROMOTER & CIS-ACTINGELEMENTS CONTROLING
GENE EXPRESSION.
COORDINATION OF
APPROPRIATE EXPRESSION
LEVELS IN SPECIFIC
TISSUES OR CELL TYPES,
AT SPECIFIC
DEVELOPMENTAL STAGES,
AND UNDER VARIOUS
ENVIRONMENTAL
INDUCTION CONDITIONS.
MATURE TISSUE
TRANSFORMATION.
BYPASS THE LONGJUVENILITY PERIOD.
DIRECT TESTING
OF PUTATIVE GENE
CANDIDATES.
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APPLICATIONS OF
THE BITTER PRINCIPLES
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BIOLOGICAL ACTIVITY OF CITRUS
LIMONOIDS
Anticarcinogenesis Activity
(Inducers forGlutathione S-Transferase Activity)
Antifeedant Activity TaxonomicMarkers
Citrus limonoids posses furan
moiety attached to the D-ring at
the C-17 position that induces
GST activity.
Larvicidal effects.
AdUlt repellent and
oviposition deterrent
effect of limonoids.
Can be used as
taxonomic markers
as certainbiosynthetic
pathways are
unique to species
and genus.
Information as
such can be usedto evaluate existing
classification
schemes or to
modify those
schemes
Limonoids acts as
insecticides against
Colorado beetle, corn
earworm, fallarmyworm, spruce
budworm and
tobacco bud worm
Major limonoids with GST activity
includes nomilin,Obacuone,
Iso-obacunoic acid and ichangin
J. Agric. Food chem 2007, 55, 8285-8294
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Cont
The results of the rabbit study,expanded the anecdotal
evidence that secondary
metabolites, including
flavonoids and limonoids,
could function endogenously
to lower LDL cholesterol.
HYPOCHOLESTEROLEMIC
ACTIVITYANTIVIRAL ACTIVITY
limonin and nomilin have
also been evaluated for their
capacity to act as antiretroviral
agents.
They inhibit viral replication,
themechanism of action being
the inhibition ofin Vitro HIV-1
protease activity.
J. Agric. Food chem 2007, 55, 8285-8294
REFERENCES
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REFERENCES1. Douglas J. McGarvey and Rodney Croteau, Terpenoid Metabolism , The Plant Cell, Vol. 7, 1015-1026,
1995
2. Shin Hasegawa & Masaki Miyake, Biochemistry and biological functions of citrus limonoids, Food Rev.
Int,12(4),413-435,1996.
3. Gary D. Manners, Citrus limonoids: analysis, bioactivity and biomedical prospects, J.agric. Food Chem,
55, 8285-8294, 2007.
4. Munish puri, S.S. marwaha, R. M. Kothari, J.F. Kennedy ,Biochemical basis of bitterness in citrus fruit
juices and biotech approaches for debittering, Critical Reviews in Biotechnology, 16(2):145-155, 1996.
5. Andrew P. Breska, Audrius A. Zukas, Gary D. Manners, Detemination of LARL and NARL in citrus juices
by liquid chromatography-ESI MS, Journal of chromatography A, 1064, 187-191,2005.
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