BITTERNESS IN CITRUS FRUIT-THE BIOCHEMISTRY, ANALYSIS AND APPLICATIONS.

MANPREET KAUR SAINI 21th march 2012

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

PRIMARY AND SECONDARY METABOLITES

Metabolites- compounds synthesized by organisms using enzymemediated 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.

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 thus affecting its consumer acceptability. Biochemical basis of bitterness in kinnow:
bitterness due to flavonoids e.g. naringin species related to pumello. 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)

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- juice
Kasetsart J. (Nat. Sci.) 43 : 28 - 36 (2009)

BIOSYNTHESIS OF LIMONOIDS
Acetyl Co A thiolase HMG Co A synthase HMG Co A reductase PhosphoMevalonate kinase MVAP,Decarboxylase Mevalonate kinase

IPP Isomerase GPP synthase Prenyltransferase s

FPP synthase
squalene synthase GGPP synthase

Terpene synthases

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)
The Plant Cell, Vol. 7, 1015-1026, July (1995)

BIOSYNTHESIS CONT.
SQUALENE
NOMILIN ACEYL LYASE

DEACETYLNOMILINIC ACID
DEACETYLNOMILIN ICHANGENSIN(KETO) METHYL DEACETYLNOMILIC ACID CALAMIN CYCLOCALAMIN

NOMILIN
HYDROLASE

OBACUNONE OBACUNOIC ACID ICHANGIN ICHANGENSIN(KETAL)

LARL
DEHYDROGENASE

LIMONIN LIMONOL

DEOXYLIMONIN
UDP-D-GLUCOSE: LIMONOID GLUCOSYL TRANSFERASE

DEOXYLIMONIC ACID

17 -D GLUCOPURANOSIDE
Biol. Pharm. Bull. 29(2) 191201 (2006)

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 LG

1. 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).

LIMONOID AGLCONES

J. Agric. Food Chem., Vol. 55, No. 21, 2007 Reviews

LIMONOID GLUCOSIDES AND SYNTHETIC LIMONIN CARBOXYMETHOXIME

J. Agric. Food Chem., Vol. 55, No. 21, 2007 Reviews

SITES OF BIOSYNTHESIS OF LIMONOIDS

3 forms of limonoids monolactone dilactone glucosides

Mature peel and flesh tissues

In leaves and seeds

Limonin glucoside

Nomilin glucoside

ACCUMULATION OF LIMONOIDS
acetate, mevalonate, or farnesyl pyrophosphate

Nomilin

in stem phloem region

other limonoids

translocated to fruit tissues, peels, seeds and leaves

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 of these limonoids have been elucidated:

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

BIOSYNTHETIC PATHWAYS OF LIMONOIDS: THE LIMONIN GROUP

Food review. Int, 12(4),(1996).

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-7bdeacetylnomilol and 6-keto-7b-nomilon. 6-keto-7b-nomilon contains structural features of both calamin and limonin groupsrepresent a biosynthetic link between them.

BIOSYNTHETIC PATHWAYS OF LIMONOIDS: THE CALAMIN GROUP

Food review. Int, 12(4),(1996).

THE ICHANGENSIN BIOSYNTHETIC 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.

Nomilin deacetlylase

BIOSYNTHETIC PATHWAYS OF LIMONOIDS: THE ICHANGENSIN GROUP

Food review. Int, 12(4),(1996).

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..

BIOSYNTHETIC PATHWAYS OF LIMONOIDS: THE 7-ACETATE lIMONOID GROUP

Food review. Int, 12(4),(1996).

DELAYED BITTERNESS

Most citrus fruits do not taste bitter if eaten fresh or if freshly squeezed juice is 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 - Aring 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.

FACTORS RESPONSIBLE FOR DELAYED BITTERNESS

Cultivar Location Maturation time Storage temperature Acidic medium Field Freeze injure Mechanical damage

Different varieties has different level of bitterness like in valencia orange and navel orange. Different temperature conditions affect the bitternesss content of same fruit.

With more time bitterness decreases.

Less the storage temperature, less the bitterness as enzyme activity reduced
Promotes enzme activity

Initiates conversion of LARL to bitter limonoid aglycone

MECHANISM OF DELAYED BITTERNESS

Food review. Int, 12(4),(1996).

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 respective glucosides . 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 OF LIMONOIDS IN CITRUS

ENZYME GROUP NAME OF ENZYME SITE OF ACTION FUNCTIONS

E-1

Various enzymes of terpenoid pathway like thiolase, synthase, reductase, kinase, isomerase, etc. Enzymes like lyase, hydrolase, dehydrogenase, esterase, etc. limonoid UDPglucopyranoside tranferase D-

phloem region of citrus stem tissues

biosynthesis of nomilin from acetate and mevalonate

E-2

all citrus tissues including leaves, stems, fruit tissues, fruit peels and seeds seeds

biosynthesis of other limonoids from Nomilin.

E-3

convert monolactones to glucosides during late stages of fruit growth and maturation lactonization of the Dring converts monolactones to dilactones limonoid biodegradation catalyzes the hydrolysis of limonoid glucosides and liberates limonoids and glucose
Food review. Int, 12(4),(1996)

E-4

limonoid D- ring lactone hydrolase

seeds

E-5

limonoid 17- beta-Dglucopyranoside betaglucosidase

dormant seeds and germinating seeds

DEBITTRING METHODS
DEBITTERING 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 limonin bitterness Limonoid UDP-D-glucose transferase Three specific target enzymes Limonoate Dehydrogenase Nomilin deacetylase Insertion of one of the three gene codings

Yields nonbitter fruits in transgenic plants

Food review. Int, 12(4),(1996)

Cont
Limonoid UDP-D-glucose transferase Limonoate Dehydrogenase Nomilin deacetylase

Converts limonoid aglycones to glucosides

Convert limonin to nonbitter limonoids

Convert nomilin deacetylnomilin

to

Isolated from albedo tissues by a combination of ammonium slfate fractionation, affinity chromatography, and ion-exchange highperformance liquid chromatography (HPLC)

Isolated by ammonium slfate fractionation, Blue dye-ligand affinity chromatography, and ion-exchange HPLC

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
Food review. Int, 12(4),(1996)

EXTRACTION & QUALITATIVE & QUANTITATIVE ANALYSIS OF CITRUS BITTER PRINCIPLE.

Analytical Methods of Citrus Limonoids Thin-layer chromatography (TLC) -- for limonoid detection Nuclear Magnetic Resonance (NMR) -- determination of limonoid structure HPLC -- detection & quantification Radioimmunoassay detection & quantification HPLC-MS detection & quantification

EXTRACTION OF LARL & NARL FROM

CITRUS JUICE
Juice 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)

Juice (150 l)+internal calibrator solution(15 l)

Juice (150 l)+ water (1.26 ml)

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).

JOURNAL OF CHROMATOGRAPHY A, 1064(2005) 187-191

EXTRACTION OF BITTER LIMONOIDS/ LIMONOID AGLYCONES FROM CITRUS JUICE

Juice obtained by hand squeezing

2 ml juice transferred to test tube & 40 l internal standard added (167 ng/l)

Juice gently liquid/liquid extracted with CHCl3 (2*2.5 ml) for 30 s

CHCL3 extracts combined & 100 l of it removed.

It was evaporated to dryness and redissolved in MeOH or THF(300 l)

3 l diluted extract was injected into LC-MS

J. AGRIC. FOOD CHEMISTRY 2005, 51, 3709-3714

EXTRACTION OF LIMONOID GLUCOSIDES

Solid samples (peel, seeds, etc) oven-dried but pulp samples dried overnight at 60 C and ground to pass 2mm mesh screen. Wet samples (juice, molasses, wash water) centrifuged at 16000 * g for 5 min at 10 C.

100 mg weighed into 10 * 50 mm cellulose extraction thimbles.

Supernatant collected and filtered through a 0.45 um filter.

Sample extracted overnight in soxhlet extracter with 25 ml MeOH.

Diluted to 30 ml with MeOH

Sample for injection prepared by combining 75 l sample, 925 l water and 300 l MeOH and 200 l internal standard solution

300 l of above extract + 700 l water + 200 l carminic acid solution(30 mg/l) added to autosampler vial

Samples analyzed by ESI-LC-MS

Sample analyzed by ESI-LC-MS

J. AGRIC. FOOD CHEMISTRY 2005, VOL.49, NO 3, 2001

GENE EXPRESSION AND TRANSCRIPTOME STUDIES

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

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

RNA ISOLATION AND cDNA LIBRARY PREPARATION

BIOINFORMATICS USAGE OF SOFTWARES FOR ILLUMINA GENOME ANALYSER 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 BETWEEN SAMPLES 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

CITRUS FRUIT TRANSCRIPTOME SEQUENCING USING MICROARRAY ANALYSIS

RNA ISOLATION AND cDNA LIBRARY PREPARATION EST PROCESSING AND FUNCTIONAL ANNOTATION MICROARRAY 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

CLONING AND CREATION OF TRANSGENIC VARIETIES

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 FREE FROM LIMONIN BITTERNESS

GENETIC 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 EFFICIENT TRANSFORMATION IN CITRUS

GENE STACKING. 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..

GENE REMOVAL REMOVING GENES THAT ARE NO LONGER USEFUL USEFUL 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.

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 (KNOCKOUTS). RESTORATION OF FUNCTION OF DEFECTIVE GENES (GENE THERAPY) REPLACEMENT OF A GENE WITH A NEW OR ALTERED VERSION.

TECHNOLOGIES CONT....
TECHNOLOGIES FOR GENETIC TRANSFORMATION OF CITRUS

PROMOTER LIBRARY. REPERTOIRE OF PROMOTER & CIS-ACTING ELEMENTS 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 LONG JUVENILITY PERIOD. DIRECT TESTING OF PUTATIVE GENE CANDIDATES.

VIRAL-MEDIATED TRANSFORMATION. REQUIRE A NONPATHOGENIC 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.

APPLICATIONS OF THE BITTER PRINCIPLES

BIOLOGICAL ACTIVITY OF CITRUS LIMONOIDS

Anticarcinogenesis Activity (Inducers for Glutathione STransferase Activity) Antifeedant Activity Taxonomic Markers

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.

Major limonoids with GST activity includes nomilin,Obacuone, Iso-obacunoic acid and ichangin

Limonoids acts as insecticides against Colorado beetle, corn earworm, fall armyworm, spruce budworm and tobacco bud worm

Can be used as taxonomic markers as certain biosynthetic pathways are unique to species and genus. Information as such can be used to evaluate existing classification schemes or to modify those schemes

J. Agric. Food chem 2007, 55, 8285-8294

Cont
HYPOCHOLESTEROLEMIC ACTIVITY ANTIVIRAL ACTIVITY

The results of the rabbit study, expanded the anecdotal evidence that secondary metabolites, including flavonoids and limonoids, could function endogenously to lower LDL cholesterol.

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 of in Vitro HIV-1 protease activity.

J. Agric. Food chem 2007, 55, 8285-8294

REFERENCES
1. 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. 6. Thomas K. Schoch, Garry D. Manners, Shin HasegawaAnalysis of Limonoid Glucosides from Citrus by ESI LC MS, J.agric. Food Chem, 49 (3), 1102-1108, 2001. 7. Gary D. Manners, Andrew P. Breska , Thomas K. Schoch, Marlene B. Hidalgo, Analysis Of bitter Limonoids in Citrus Juices by APCI and EI -LC-MS, J.agric. Food Chem, 51,3709-3714, 2003. 8. J. Forment etal, Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies, Plant Molecular Biology (2005) 57:375391,2005. 9. Manuel Talon and Fred G. gmitter jr.,Citrus Genomics, International Journal of Plant Genomics, volume 2008. 10. Keqin Yu, Qiang Xu, Xinlei Da, Fei Guo, Yudon Ding, Xiuxin Deng, Transcriptome Changes during fruit development and ripening of sweet orange(Citrus sinesis) BMC Genomics, 2012 11. M. Omura, M. Kita, . Endo-Inagaki, T. Moriguchi, R. Matsumoto, C. Suhayda, and Shin Hasegawa, Genetic Evaluation and Modification of the Accumulation of Limonoids in Citrus, ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

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