Review

Received: 28 January 2016 Revised: 13 June 2016 Accepted article published: 4 July 2016 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.7875

Thirty-three years of 2-acetyl-1-pyrroline, a

principal basmati aroma compound in scented
rice (Oryza sativa L.): a status review
Kantilal Wakte,a Rahul Zanan,a Vidya Hinge,a Kiran Khandagale,a
Altafhusain Nadafa* and Robert Henryb

Abstract
Rice is the staple food of around 3 billion people, most of them in Asia which accounts for 90% of global rice consumption.
Aromatic rices have been preferred over non-aromatic rice for hundreds of years. They have a premium value in national as well
as international market owing to their unique aroma and quality. Many researchers were involved in identifying the compound
responsible for the pleasant aroma in aromatic rice in the 20th century. However, due to its unstable nature, 2-acetyl-1-pyrroline
(2AP) was discovered very late, in 1982. Buttery and co-workers found 2AP to be the principal compound imparting the pleasant
aroma to basmati and other scented rice varieties. Since then, 2AP has been identied in all fragrant rice (Oryza sativa L.) varieties
and a wide range of plants, animals, fungi, bacteria and various food products. The present article reviews in detail biochemical
and genetic aspects of 2AP in living systems. The site of synthesis, site of storage and stability in plant systems in vivo is of
interest. This compound requires more research on stability to facilitate use as a food additive.
2016 Society of Chemical Industry

Keywords: scented rice; 2-acetyl-1-pyrroline (2AP); biosynthesis; betaine aldehyde dehydrogenase 2 (BADH2)

INTRODUCTION cracked the fragrance and identied 2-acetyl-1-pyrroline (2AP) as

Rice is a staple food and major food grain crop for two-thirds
of the population in the world. China and India are major rice
producers. During 20142015, world rice production was 478.18
million tonnes, of which India contributed 100 million tonnes rice
grains [FAOSTAT (http://faostat.fao.org/site/339/default.aspx)].
Of the total rice production (20142015), India exported
3 702 260.12 Metric tonnes (MT) of basmati rice worth Rs.
275 978.7 Million and 8 274 046.06 MT of non-basmati rice worth
Rs. 204 285.1 Million to dierent countries of the world [APEDA
(http://agriexchange.apeda.gov.in/indexp/reportlist.aspx)].
Aroma is one of the most important characteristics of rice, espe-
cially when taking consumer acceptance as a criterion. Nowadays
consumers have become more conscious of the quality of the rice
that they consume. They often prefer fragrant rices due to their
characteristics and pleasant odour.1 Hence demand for aromatic
rice is increasing in both domestic and international markets.2
Thus, rice aroma has gained a leading importance as a quality char-
acter. Hence, basmati rice from India and Pakistan, and jasmine rice
from Thailand fetch a higher market price. Besides basmati rice, the
tropical countries have their own set of aromatic rice varieties that
are popularly consumed, e.g. KDML, Della etc.
During the past 33 years, various aspects of the compounds
responsible for the rice aroma have been studied. As early as
1965, an attempt to identify the aroma compounds in scented
rice found hydrogen sulde, ammonia and acetaldehyde in the
volatiles of cooked rice.3 Later, several compounds responsible
for the aroma were identied in cooked and un-cooked rice.4 6
The chemical nature of the aroma compound remained a mystery
until 1982, when Ron Buttery with his co-workers successfully
the principal aroma compound responsible for the pleasant aroma
in scented rice.7 The universality of this molecule was conrmed
by subsequent rice aroma analyses in several aromatic varieties
worldwide.7 10 The importance of 2AP was realised when 2AP was
detected beyond scented rice varieties from unicellular bacteria
to animals. The wide diversity of the molecule opened vistas for
the researchers to look into its biosynthetic, genetic, molecular
and synthetic aspects. Other studies reported more than 100
volatiles from aromatic rice grains and their contribution to the rice
aroma.11,12
2AP AS THE PRINCIPAL AROMA COMPOUND
2AP [IUPAC name: 5-acetyl-3,4-dihydro-2H-pyrrole and
1-(3,4-dihydro-2H-pyrrol-5-yl)ethanone, molecular weight
0.111145 kg mol1 , density 1090 kg m3 ] is 1-pyrroline in which
the hydrogen at position 2 is replaced by an acetyl group with a
methyl ketone group. The pyrroline ring makes the compound
highly unstable. It is a principal aroma and avour compound
present in scented rice and responsible for the popcorn aroma
in food products. It has a very low odour threshold (0.1 g kg1 )
Correspondence to: A Nadaf, Department of Botany, Savitribai Phule Pune
University, Pune 411007, India. E-mail: abnadaf@unipune.ac.in
a Department of Botany, Savitribai Phule Pune University, Pune 411007, India
b Queensland Alliance for Agriculture and Food Innovation (QAAFI), The Univer-
sity of Queensland, St Lucia, QLD 4072, Australia

J Sci Food Agric (2016) www.soci.org 2016 Society of Chemical Industry

 www.soci.org K Wakte et al.

Figure 1. Structure of 2AP.
in water and as a result it can be detected by the human nose
at very low concentrations. The pyrroline ring is non-reactive
and the methyl ketone group is reactive (Fig. 1) and reacts with
ketones detecting the reagent 2,4-dinitrophenyl hydrazine to
form a derivative, 2,4-dinitrophenylhydrazine. This property was
used to detect 2AP histochemically in scented rice.13
OCCURRENCE OF 2AP
2AP was identied for the rst time as the most important avour
compound of cooked rice.7 However, since its discovery, 2AP has
continued to reveal its presence in a large variety of living systems
and food products. Interestingly, besides living systems, in a large
number of heated and processed food products 2AP has been
found as an important Maillard avour compound.14,15 The diverse
occurrence of 2AP is depicted in Table 1. The detailed accounts of
2AP measured in living organisms following dierent methods of
extraction are listed in Table 2.
2AP DIVERSITY IN RICE
2AP content varies due to genotype, cultural practices,
post-harvest practices, environmental factors etc.17,96 98 The
range of variation in dierent rice types is depicted in Table 3.
BIOSYNTHESIS OF 2AP IN RICE
Genetic and molecular basis of 2AP biosynthesis
Before the onset of molecular techniques, the genetic basis of
aroma in general and 2AP in particular was attempted through
genetic mapping. Fragrance was found to be a highly heritable
trait. The genes controlling the aroma trait in rice have been
studied by genetic and molecular mapping. Siddiq and co-workers
studied an Indian scented rice variety and found that two recessive
genes for aroma on chromosome 5 and 9.103,104 Then the numbers
of genes controlling the inheritance of aromatic characters was
reported to vary from one to four.10,105 108 Repeated genetic
analysis with using dierent rice cultivars by several researchers
revealed that the primary fragrance trait is controlled by a recessive
monogenic inheritance, independent of cytoplasmic genes.107,108
Further studies were extended at the molecular level using
molecular markers that identied a single recessive gene located
on chromosome 8 by RFLP,109,110 translocation and trisomics
lines,111 molecular markers RG28 and RG1112 and by using SSR
markers RM210 and RM515.113 After rice genome sequencing,
a physical ne mapping study revealed that a candidate gene
(fgr/badh2/Os2AP) homologous to betaine aldehyde dehydro-
genase (BADH) on chromosome 8 is responsible for aroma
formation in aromatic rice.114 116 An eight base pair deletion in
exon 7 or a seven base pair deletion in exon 2 of badh2 gene
on chromosome 8 results in a loss of function of BADH2115 119
(Fig. 2) and, consequently, 2AP accumulates in fragrant rice. This
was conrmed by the absence of 2AP in an aromatic variety
after transformation with functional BADH120 and suppression
of the badh2 transcript in a non-aromatic rice increases 2AP
concentrations.121
Interestingly, in other plants where 2AP is synthesised, the
gene responsible for 2AP biosynthesis and the mechanism of
biosynthesis of 2AP was found to be similar having deletions in
badh2 at dierent positions in the gene. In the aromatic vegetable
soybean (Glycine max L.) genetic mapping conrmed the quan-
titative trait locus (QTL) position of fragrance and GmBADH2 for
2AP biosynthesis. Three single nucleotide polymorphism (SNP)
changes in exon 6 and 10 were found. This study also conrmed
that the nucleotide substitution of G to A in exon 10 resulted in
a change in amino acid from glycine to aspartic acid that made
Gmbadh2 non-functional.94 Yundaeng and co-workers identied
a continuous 1444 bp deletion encompassing exon 12 to 15 of
SbBADH2 in sorghum IS19912.24 Cucumber fragrance is controlled
by a single recessive gene.29 Sequencing of the BADH gene of

Table 1. Worldwide occurrence of 2AP

Group Organism/product

Plants Rice,7,12,16,17 Pandanus amaryllifolius,18 Bassia latifolia,19 Vallaris glabra,20 soybean,21 mung,22 sorghum,23,24 pearl millet,25
spinach,26 taro corms,27 French beans,28 cucumber,29 muskmelon,30 wood apple,31 cempedak and jackfruit.32
Animal Tiger pheromone.33
Fungi Spoilage yeasts Brettanomyces and Dekkera,34,35 Penicillium nalgiovense on Mediterranean dried sausages,36 bakers yeast,37 in
the culture of fungal strains like Aspergillus nigricans, Aspergillus awamori and Aspergillus oryzae and a yeast strain
Kluveromyces maxianus var. marianus (Hansen), JCM TISTR.38 Rice rhizosphere fungi like Aspergillus terreus, Aspergillus niger,
Aspergillus fumigatus, Rhizopus oryzae Aspergillus avus, Nigrospora sphaerica, Nigrospora oryzae, Trichoderma ssp., Penicillium
ssp., Fusarium oxyporum, Monilia sitophila and Thielavia terricola.39,40
Bacteria Bacillus cereus isolated from cocoa fermentation boxes41,42 and wine produced by heterofermentative wine lactic acid bacteria
Lactobacillus hilgardii, Lactobacillus brevis, Lactobacillus plantarum, Pediococcus sp.34,35
Food products Bread crust,43 popcorn,44 sweet corn based products,45 cooked blue crab,46 toast,47 moderately roasted sesame,48 cooked tail
meat of freshwater craysh (Procamarus clarkia),49 baguette crusts,50 maize our,51 taro volatiles [Colocasia esculanta (L.)
Schott],27 boiled crap llet (Cyprinus carpia L.),52 boiled potatoes,53 roasted seeds of wild mango (Irvingia gabonensis),54
oyster cooker euent,55 cooked tail meat of American lobster (Homarus americanus),56 heat-treated non-fat dry milk,57
cooked acha (Digitaria exilis Stapf ),58 Iberian dry-cured ham,59 cooked meat,60 thermally treated Theobroma grandiorum
pulp,61 pu pastries,62 mousy wines,63 roasted seasame,64 pan-red green teas,65 cured tobacco leaves,66 thermally treated
yeast extracts,67 pale lager beer,68 peanut and pumpkin seed oils,69 honey,70 soy-based products,71 Italian-type salami and
Parma ham,72 sweet whey powder,73 dry and fresh milk,74 Camembert and Swiss gruyere cheese,75 heated egg yolk,76
extruded potato snacks,77 rennet casein,78 liquid cheddar whey,79 boiled salmon,80 cod and trout,81 boiled prawns,82
cooked ripened anchovy,83 craysh-processing byproducts,84 tuna sauce85 and stored sardines.86

wileyonlinelibrary.com/jsfa 2016 Society of Chemical Industry J Sci Food Agric (2016)

Acetyl-1-pyrroline: a principal compound in scented rice www.soci.org

Table 2. 2AP content reported in living systems

Sr. no. Organism Source Method of extraction 2AP content (mg kg1 )

1 P. amaryllifolius Fresh leaves DSE 14.1087

2 P. amaryllifolius Fresh leaves DSE 10.2620
3 P. amaryllifolius Fresh leaves SDE 8.0087
4 P. amaryllifolius Fresh leaves SC-CO2 7.1688
5 P. amaryllifolius Fresh leaves SDE 3.1089
6 P. amaryllifolius Fresh leaves DSE 2.7790
7 P. amaryllifolius Freeze dried leaves SDE 1.0018
8 P. amaryllifolius Fresh leaves SC-CO2 0.7190
9 P. amaryllifolius Fresh leaves SDE 0.01590
10 P. amaryllifolius Fresh leaves SPME 6.8591
11 P. amaryllifolius Fresh leaves SPME 12.2592
12 Bassia latifolia Fresh owers SPME 3.3093
13 Vallaris glabra Fresh owers SDE 3.3620
14 Vallaris glabra Fresh leaves SDE 0.5320
15 Aspergillus nigricans Liquid culture LLE 2.0838
16 Aspergillus awamori Liquid culture LLE 1.1138
17 Lactobacillus hilgardi Liquid culture LLE 0.0250.32035
18 Bacillus cereus Liquid culture SDE 0.0300.08741
19 Bacillus cereus Liquid culture LN-SDE 0.00260.95742
20 Glycine max Grain SPME 0.66094
21 Sorghum bicolor Grain SPME 0.301924
22 Vigna radiata Grain SPME 0.1995
23 Vigna radiata Leaves SPME 0.11995

function of CsBADH was responsible for 2AP accumulation.128

Table 3. Variation in 2AP content in aromatic rice type
A non-functional PaBADH2 gene identied in P. amaryllifolius
Method of 2AP content conrmed similar molecular mechanism of 2AP to that found in
Rice type Variety detection (mg kg1 ) scented rice129 (Fig. 3).
Basmati Basmati 370 SPME 0.21499
Pusa basmati SDE 0.03015 Biochemical basis of 2AP biosynthesis
Basmati SPME 0.17899 Several attempts were made to understand the biochemical
Royal SPME 0.0003100 basis of 2AP biosynthesis. Buttery and co-workers suggested
Jasmine Golden Elephant SPME 0.0038100 that 2AP has a similar origin to the bread aroma compound,
(long-grained 2-acetyl-1,4,5,6-tetrahydropyridine.131 Thermal reactions between
Thai indica) proline, 1-pyrroline and several metabolites of the glycolytic
KDML 105 SDE 0.74517 pathway were shown to be responsible for 2AP formation in
Jasmine (ITC) DSE 0.810101
wheat bread crust.132 They also reported a signicant amount
Indica non-basmati Ambemohar 157 SPME 0.11599
scented (local)
of 2AP in a 1-pyrroline/methylglyoxal model system under boil-
Indrayani SPME 0.31499 ing condition.133 In B. cereus, an increase in 2AP content was
Basumati SPME 0.19099 reported when supplemented with proline, ornithine and glu-
Ghansal SPME 0.23799 tamate; further labelling experiments conrmed proline and
Raibhog SPME 0.25399 glutamate are the two nitrogen sources for 2AP formation.41 Later,
Kamod SPME 0.41899 proline was identied and conrmed as the precursor molecule
Japonica Hyangmibyeo 1 SPME 0.0031100 of 2AP.17,134 Tracer experiments with 15 N-proline, 15 N-glycine,
Hyangmibyeo 2 SPME 0.0002100 and proline-1-13 C indicated that the nitrogen source of 2AP
Goemjeongssal (black SPME 0.0049100 was proline, whereas the carbon source of the acetyl group
pigmented rice) was not its carboxyl group.17 Costello and Henschke suggested
Other A301 cultivar (America) DSE 2.053102 the role of ornithine as a nitrogen source for 2AP via -amino
B2-3 cultivar (Italy) DSE 2.746102 butyraldehyde (GABAld), a product of the putrescine degrada-
B5-3 cultivar (Italy) DSE 0.529102 tion pathway in Lactobacillus hilgardi. GABAld spontaneously
Kasmati (Rice Tec) DSE 0.496101 cyclises to 1 -pyrroline followed by acylation at C-2 position of
Texmati (Rice Tec) DSE 0.526101 acyl-CoA derivatives, leading to the synthesis of 2AP.35 Detailed
precursor studies by Adams and Kimpe revealed that the pro-
duction of 2AP by B. cereus ATCC 27522 proceeds via acylation
fragrant cucumber revealed 14 mutations, six single base substi- of 1-pyrroline, a metabolic degradation product of proline and
tutions and eight indels in dierent exons. However in exon 5 a ornithine.42 Cheetangdee and Chaiseri also showed glutamic
single base substitution caused a change in amino acid in a highly acid, proline, glucose and fructose are possible precursors of 2AP
conserved motif, Y163C. This also established that the defective in P. amaryllifolius,135 whereas L-proline was found to enhance

J Sci Food Agric (2016) 2016 Society of Chemical Industry wileyonlinelibrary.com/jsfa

 www.soci.org K Wakte et al.

Figure 2. Multiple alleles of BADH2 gene of aromatic rice. (a) 3-bp deletion in the 5 untranslated region and an 8-bp insertion in the promoter (1314
site upstream from the initiation codon).122 (b) A miniature interspersed transposable element (MITE) insertion in promoter region.123 (c) 7-bp deletion
in the 2nd exon.118 (d) 8-bp deletion in the 7th exon.116 (e) One FNP in the 7th exon.124 (f ) 7-bp insertion in the 8th exon.125 (g) One insertion/deletion,
1 SNP at the 10th exon.124 (h) 803-bp deletion between the exons 4 and 5.126 (i) Two SNPs in the 8th intron.127 (j) 3-bp insertion in the 13th exon.124 (k)
One SNP in the 14th exon.124

Figure 3. Mutations in BADH2 gene of other species, responsible for 2AP synthesis. (a) Various insertion, deletion and SNPs in BADH gene associated with
fragrance in cucumber.128 (b) 1444-bp deletion causes a premature stop codon in SbBADH2 of sorghum IS19912 is responsible for fragrance in sorghum.24
(c) Nucleotide substitution of G to A in the exon 10 results in an amino acid change of glycine to aspartic acid94 and a 2-bp (TT) deletion in exon 10 causes
a reading frame shift and introduces a premature stop codon leading to aroma in soybean.130 (d) Stop codon at exon 6 is the cause of basmati aroma in
Pandanus amaryllifolius.129

the in vitro synthesis of 2AP in semi-dierentiated callus of P.
amaryllifolius.87 GABald/1 -pyrroline serves as source for the
pyrroline ring in 2AP whereas 2-oxopropanal or methylgly-
oxal serves as a carbon source and precursor of acetyl group,
even in non-thermal conditions.21,136 2AP was synthesised
non-enzymatically at room temperature simply by stirring a
mixture of 1 -pyrroline-5-carboxylate (P5C) and methylglyoxal
for 30 min.136 Similarly, Wu and co-workers reported that, the
interaction of methylglyoxal (MG) and P5C are involved in the
formation of 2AP in aromatic vegetable soybean (Glycine max
L.).21 The examination of the expression level of P5CS and the
enhanced concentration of its product among fragrant varieties
revealed that 2AP is produced by the direct reaction of P5C
with methylglyoxal.2,136 Finally, proline and methylglyoxal were
identied as important precursors of 2AP biosynthesis.136,137
The accumulation of 2AP in rice is due to the absence of
BADH2 activity, leading to an increased level of its substrate,
GABald/1 -pyrroline, the immediate precursor of 2AP.93,120,138,139
GABald/1 -pyrroline has been observed to be the rate control-
ling factor in synthesis of 2AP, and is dependent on degradation
of polyamines and proline. Adams and Kimpe also reported
1 -pyrroline as an important precursor in the formation of
2AP in thermal as well as biological reactions.42 Consumption
of GABald/1 -pyrroline by converting it into GABA inhibits
2AP synthesis, whereas accumulation of GABald/1 -pyrroline
results in increased 2AP synthesis.120 Further, it was reported that
1 -pyrroline increased when inhibitors of aldehyde-metabolising
enzymes were added to the crude enzyme preparations and con-
cluded that when aldehyde-metabolising enzymes are inhibited,
GABald is entirely converted into 1 -pyrroline and vice versa.140
Inactivation of aminoaldehyde dehydrogenases (AADH) was
responsible for higher synthesis of 2AP in the detached eshy
corolla of Bassia latifolia owers compared with high AADH activ-
ity in the intact corolla.93 The eshy corollas of B. latifolia are
known to harbour very high sugar (glucose) contents, which
act as a precursor for methylglyoxal, the other immediate sub-
strate for synthesis of 2AP.141 The BADH2 enzyme usually oxi-
dises betaine aldehyde to the osmoprotectant glycine betaine,
but glycine betaine has not been detected in rice.142 However,
recent studies have shown that BADH2 is capable of metabolis-
ing a range of substrates including -aminoaldehydes, often more
eciently than betaine aldehyde.139,143,144 When expressed in E.
coli, BADH2 shows a greater anity for GABald than for betaine
aldehyde and it is capable of oxidising GABald to GABA.120,139
Thus, BADH2 participates in the pathway of GABA synthesis by
oxidising GABald/1-pyrroline. Some researchers reported that
AADH has high homology to BADH but no anity to betaine alde-
hyde, leading to the conclusion that BADH and AADH are the

wileyonlinelibrary.com/jsfa 2016 Society of Chemical Industry J Sci Food Agric (2016)

Acetyl-1-pyrroline: a principal compound in scented rice
Figure 4. Schematic representation of the 2AP biosynthetic pathway.
same enzymes.139,143 145 Hence, it is more appropriate to refer to
the enzyme as -aminobutyraldehyde dehydrogenase (-AMADH)
than BADH/AADH. Arikit and co-workers studied the association
between amino aldehyde dehydrogenase (AMADH/AADH) de-
ciency and 2AP biosynthesis in soybean and suggested that de-
ciency in the GmAMADH2 product, AMADH, plays a similar role in
soybean as in rice, which is to promote 2AP biosynthesis. They also
proposed that this phenomenon might be a conserved mecha-
nism among plant species.146 The schematic representation of the
possible biosynthetic pathway of 2AP is given in Fig. 4.
CHEMICAL SYNTHESIS OF 2AP
Chemically, 2AP was synthesised with a yield less than
10% by using rhodium on an alumina catalyst, reducing
2AP for 15 h followed by oxidation of the intermediate
2-(1-hydroxyethyl)1-pyrrolidine with silver carbonate.131 A
straightforward synthesis of 2AP was reported by De Kimpe
and co-workers.147 They also reported synthesis of 2AP by depro-
tonation of a vicinal diamine and subsequent alkylation with
an N,N-diprotected -bromoalkylamine, followed by deprotec-
tion and intermolecular transamination of the functionalised
intermediate.148 In addition they also synthesised diethylacetal
of 2AP which was more stable. Duby and Huynh-Ba synthesised
2AP by hydrolysing 2-(1-alkoxyethenyl)-1-pyrroline with an acid
and then added an equimolar amount of a base to neutralise
the reaction medium containing 2AP.149 2AP was prepared by
penicillin acylase mediated hydrolysis of 1-[(N-phenylacetyl)
amino] 4,5-dioxohexane to 1-amino 4,5-diketone followed by
spontaneous ring closure of the latter.150 A novel straightfor-
ward synthesis involving a four-step reaction starting from the
N-shielded cyclic -amino acids.151 Srinivas and Gurudatta syn-
thesised a 2AP derivative that is more stable than 2AP using
L-pyrroline passing through a Grignard reaction.152 A synthetic
procedure for preparation of 2AP from 2-pyrrolidinone with an
overall yield of 22% was developed.153 Fuganti and co-workers154
and Srinivas and co-workers155 synthesised 2AP by catalytic SeO2
oxidation of the 2-alkyl pyrroline using TBHP as co-oxidant.
EXTRACTION AND QUANTIFICATION OF 2AP
Methods for the determination of aroma compounds and their
quantication are based on the common principles of extraction,
concentration, separation, and quantication. Perhaps the lim-
itations of the method of isolation and quantication might be
J Sci Food Agric (2016) 2016 Society of Chemical Industry
www.soci.org
responsible for the considerable delay in detection of the 2AP
molecule by previous researchers. The methods of extraction
can be grouped into traditional and modern methods. Tradi-
tional methods include direct distillation, purge and trap, steam
distillationsolvent extraction and direct solvent extraction.156
The modern methods include headspacesolid phase micro
extraction (HS-SPME), static headspace (SHS) followed by sep-
aration of aroma compounds using gas chromatography (GC)
with ame ionisation detector (FID) or mass spectroscopy (MS).
The conrmation of 2AP was on the basis of the mass spectrum
showing molecular mass at 111 (5) and other mass ions at 43 (100),
41 (50), 42 (24), 69 (11), 55 (2), 52 (0.9), 54 (0.5), 67 (0.5) and 67 (0.5)
(Fig. 5). 2AP was conrmed as the principal aroma compound in
volatile oil of freeze-dried P. amaryllifolius leaves having 10 times
more than rices.18 Later, several rice varieties were explored for
2AP contents (Table 3).
Widjaja and co-workers compared the volatiles of non-fragrant
and fragrant rice and identied 70 compounds that are involved in
the fragrance of scented rice.12 Interestingly, 2AP was also found to
be present in non-scented rice at very low levels, 10 times less than
scented rice.18 Later, dierent extraction methods were attempted
for analysis and quantication of 2AP (Table 3). In P. amaryllifolius
leaves when comparing extraction using three dierent methods,
viz. supercritical carbon dioxide extraction, simultaneous steam
distillation and ethanol extraction, 2AP recovery was 0.72 mg kg1 ,
0.002 mg kg1 and 2.771 mg kg1 , respectively.90 Bhattacharjee
and co-workers found that in traditional methods such as solvent
extraction (SE) and LikensNickerson extraction, 2AP yield are
low (1 mg kg1 ) compared to SC-CO2 extraction (7.16 mg kg1 ).88
Thus, these studies indicate that the extraction method has a prin-
cipal role in the reliable quantication of 2AP. This is because
2AP is highly unstable and thermo-labile and is degraded, result-
ing in lower yields of 2AP in distillation methods. For the same
reason direct solvent extraction yields are higher. In the last
decade HS-SPME has become a popular, eective and reliable
method and has been used to assess 2AP and other aroma
compounds,20,91,157 159 while, in rice, headspace sorptive extrac-
tion (HSSE)160 was eective. SPME-GC/MS was further modied
to distinguish aromatic from non-aromatic rice using single ker-
nels of brown or milled rice.161 Further, a modied method for
the synthesis of 2AP was studied and the presence of a tau-
tomer of 2AP was conrmed. A stable isotope dilution assay
(SIDA) was developed, involving HS-SPME combined with gas
chromatographypositive chemical ionisationion traptandem
mass spectrometry (GC-PCI-IT-MS-MS) for 2AP quantication.162
HS-SPME enables faster sampling, extraction, concentration, sam-
ple introduction and analysis within an hour with high sensitivity
and accuracy.163 Therefore, HS-SPME can be taken as a method of
choice for quantication of 2AP and other aroma compounds.
BIOTECHNOLOGICAL STRATEGIES FOR
IDENTIFICATION OF THE FRAGRANT TRAIT
AND ENHANCEMENT IN RICE AROMA
Molecular markers and biotechnological tools have been devel-
oped for detection and enhancement of 2AP content in aro-
matic rice.
Genetic mutations and molecular markers linked with 2AP
biosynthesis
The high economic value of fragrant rice has encouraged rice
breeders to discover fragrance genes and to develop fragrant
wileyonlinelibrary.com/jsfa
 www.soci.org
Figure 5. GC graph depicting the peak of 2AP and mass spectrum showing various mass ions of 2AP in leaves of P. amaryllifolius.
varieties. Based on this recessive gene, PCR-based co-dominant
and functional markers have been developed and exploited for
selection of genotypes in rice breeding programmes.164 However,
the deletion in the BADH2 gene is not universal to all the aroma
rice varieties; aromatic rice of dierent origins may have various
regulation165 or other mutations that drive the accumulation of
2AP.119
An RFLP marker (RG28) linked to fragrance of rice locus on chro-
mosome 8 was the rst reported.109 RG28 was converted into a
PCR-based STS (sequence tagged site) marker166 but these mark-
ers are very far from the gene so they were not eective in MAS. To
overcome this, Bradbury and co-workers developed the rst func-
tional marker which dierentiated scented and non-scented rice
based upon the 8 bp deletion in the 7th exon of the BADH2 gene.116
Further, several functional markers were developed for various
scented rice varieties which exploited dierent mutations in the
BADH2 gene.118,122,125,167 These BADH2 alleles and functional mark-
ers provided an important genetic resources for development and
identication of new fragrant rice varieties using marker assisted
selection and bioinformatics analyses.120
Over-expression of the P5CS gene
Many studies proved that proline synthesis is mediated through
the key enzyme P5CS and further used as a precursor amino
acid for 2AP biosynthesis.17,87,168 The P5CS gene expression was
positively correlated with 2AP content in aromatic rice cultivars
and soybean.21,136 For the rst time, Keyghobad and co-workers
reported a more than two-fold increase in 2AP content after
over-expressing the P5CS gene in aromatic rice cultivars.169
Methylglyoxal augmentation
Methylglyoxal, a precursor of 2AP in aromatic rice and other
organisms, is produced through the glycolysis pathway.170
Down-regulation of glyceraldehyde-3-phosphate dehydrogenase
C (GapC) and up-regulation of triose (TPI) in aromatic vegetable
soybean results in the accumulation of triose phosphate and thus
leads to increased MG levels and thereby results in increased
wileyonlinelibrary.com/jsfa 2016 Society of Chemical Industry
K Wakte et al.
2AP.21 Thus, similar strategies could be applied to increase 2AP
content in aromatic rice.
Proline supplementation
Proline was identied as the preferred precursor for 2AP biosyn-
thesis in aromatic rice.17,134 The L-proline degradation rate is higher
in rice callus leading to higher synthesis of GABald/1 -pyrroline,
which results in higher synthesis of 2AP.171 In B. cereus cultures 2AP
was increased when supplemented with proline, ornithine and
glutamate.41
Down-regulation of the functional BADH2 gene
A hairpin RNAi was constructed from the BADH2 exons 6 to 9
and joined in the opposite direction to its cDNA under the 35S
promoter. The levels of 2AP increased up to 20-fold in transgenic
Nipponbare rice.117 Similarly the OsBADH2 disrupted by hpRNA
signicantly increased 2AP production showing altered expression
levels of the OsBADH2 gene inuence aroma accumulation.121
Further fragrance was induced in a non-aromatic rice variety
by suppressing the OsBADH2 using an articial microRNA under
a maize ubiquitin promoter; the 2AP content in grain was sig-
nicantly elevated. It was found that proline content is higher in
transgenic BADH2 down-regulated lines than in wild type.172 Dis-
ruption of BADH2 with TALEN increased 2AP in grain from 0 to
0.350.75 mg kg1 , which was similar to the positive control aro-
matic variety.173
AGRONOMIC AND POST-HARVEST PRACTICES
AFFECTING AROMA
Soil conditions, nutrients and cultivation practices
Nitrogen is an important factor which increased 2AP formation in
aromatic rice during the development.17,96 Application of nitroge-
nous fertiliser strengthens the aroma, by increasing free proline
content in rice whereas low fertiliser tends to result in lower 2AP
content.174 Supplementation of N, P, Ca, Mg, Mn, and Zn increased
J Sci Food Agric (2016)
Acetyl-1-pyrroline: a principal compound in scented rice
2AP content. The highest content of 2AP was recorded on addition
of P (two-fold) as compared to other nutrients.175
Irrigation, draining and harvesting time
The eect of dierent irrigation modes on aroma content was
studied and showed that soil water potential maintained at 0 to
25 (5) kPa could signicantly increase free proline content and,
ultimately, aroma in Guixiangzhan and Meixiangzhan cultivars.176
The eects of varying draining and harvesting time on rice sensory
properties was studied and stable avour was observed with
timing of eld draining (14-day span) and harvesting (3248 days
after owering).177
Planting density
Association of the highest 2AP concentrations with lower planting
densities was reported.98 This study recommended that planting
density could be reduced from 28 hills m2 to 1916 hills m2 while ROLE OF 2AP AS A BIOSYNTHETIC MOLECULE
maintaining optimum yield and quality.
Eect of temperature
The temperature during developmental stages, harvesting and
drying of paddy greatly inuences the grain aroma. Aroma forma-
tion and retention in grain is enhanced at low temperature during
the grain lling stage. Basmati requires relatively cooler temper-
ature (25 C/21 C day/night during crop maturity) for best reten-
tion of aroma.178 Further, Singh also reported that a Basmati variety
will be more aromatic if cultivated in an area having relatively cool
temperatures in the afternoon (2532 C) and night (2025 C) MG non-enzymatically reacts with another unstable accumulated
with humidity of about 7080% during primordial and grain lling
stages.179 Drying at higher temperature (100150 C) enhanced
three-fold 2AP content in the grain along with other volatile com-
pounds compared to the normal sun drying in Thai jasmine rice.97
A higher rate of reduction in 2AP content at the beginning of stor-
age was reported and this emphasised the need to focus on stor-
age conditions of aromatic rice.180 Higher 2AP was reported during
storage of 3 months at 4 C.98
Enrichment and stabilisation of aroma
Coating of a modied spouted bed with 30% sorbitol-plasticised
rice-starch lm containing 25% pandan (Pandanus amaryllifolius)
leaf extract to rice grain eectively stabilises 2AP and reduces
n-hexanol in stored grains over without coating and also add 2AP
content in non-scented rice cultivars (PTT1).181
Phytohormone
Kong and Zhao treated 15-day-old seedlings of Kam sweet and
Kam rice cultivars with 120 mg L1 jasmonic acid and found that
2AP and other volatiles were elevated at the time of grain lling.182
STABILISATION OF 2AP
The pyrroline ring in 2AP makes the molecule highly unstable.7
For long-term storage and commercial usage, there is a need to
preserve synthetic 2AP in an acidic form.7 Eorts by Brahmachary
showed that 2AP could be preserved for more than 6 months as
2AP citrate salt. They further claimed that 2AP can remain stable
for up to 2 years as a picrate.183 De Kimpe and Keppens synthesised
the diethylacetal of 2AP which was claimed to be more stable.148
Srinivas and co-workers successfully stabilised this compound for
more than 1 year by making a derivative of 2AP using acids.155
J Sci Food Agric (2016) 2016 Society of Chemical Industry
www.soci.org
In the avour industry encapsulation is a popular modern tech-
nique for converting a volatile aroma concentrate into a sta-
ble powder form.184 They found that micro-encapsulation can
reduce loss of 2AP after 72 days of storage. The stability of
2AP was enhanced by formation of an inclusion complex with
-cyclodextrin.150 The vacuum-dried powder of starch and 2AP
in ethanol was useful for the avouring of rice and other food
products.185 The unstable nature of the compound remained as
a main constraint for its commercial synthesis and its use in the
avour industry. Therefore, in countries like Thailand and Malaysia
crude extracts of P. amaryllifolius are used to impart 2AP avour
to the food items. Among all the natural sources, Kawista fruit
(wood apple) and fresh bread owers (Vallaris glabra Ktze) are
the strongest candidates as a raw material for synthesis of 2AP in
industry, especially in Indonesia.186
The natural synthesis of this compound in a variety of organisms
was correlated with defence. Aroma plays an important role in
insect perception of a food.7 Knowledge of the identity of an
important aroma component of a food is useful in preventing
insect infestation. Interestingly, tigers use 2AP as a component of
their pheromonal, communicatory signal.33
2AP synthesis may be a result of methylglyoxal detoxication in
plants. Environmental stresses such as salinity induce the produc-
tion of methylglyoxal (MG) in plant cells, hence considered to be a
cytotoxic compound.187 To avoid cytotoxicity in plant cell, reactive
compound 1 -pyrroline to form 2AP, a non-cytotoxic compound
that can be easily vaporised into the atmosphere thereby prevent-
ing cytotoxicity. However, this hypothesis demands more experi-
mentation for its conrmation.
UTILITY OF 2AP AS AN AROMATIC
COMPOUND
The aromatic nature due to the presence of 2AP makes aromatic
rices highly preferable amongst consumers. GABA, the oxidation
product of GABald, has been shown to have natural pesticide
properties in plants.188 This may explain why some fragrant rice
cultivars are particularly susceptible to insect pests.112,189 The
non-functional BADH2 enzyme in fragrant plants would be unable
to convert GABald into GABA so the plants may accumulate less of
this natural pesticide than non-fragrant plants.
The leaves of P. amaryllifolius have a strong aroma and are
widely used for avouring various food products.89 Traditionally, in
India and other parts of Asia, P. amaryllifolius leaves are added to
ordinary rice to impart aroma resembling costly scented rices. 2AP
has now been included in GRAS 22 (the 22nd publication by the
Expert Panel of FEMA) describing the recent avouring ingredients
recognised as safe under the Food Additives Amendment, and
average usual and maximum use levels in dierent food categories
are dened.190
CONCLUSIONS
Food aroma additives are often sought. Among them, 2AP has a
wide range of occurrence and is an excellent molecule for this
purpose. It adds to the quality of rice and thus scented rice
is preferred by consumers and fetches a higher price. Aroma
wileyonlinelibrary.com/jsfa
 www.soci.org
characters due to 2AP can be taken as a unique example of human
selection and domestication through which the BADH2 recessive
gene has been carried forward over several generations even
though the plants face several metabolic challenges to survive
in the absence of functional BADH2. As far as commercial use is
concerned, the unstable nature makes it unsuitable for commercial
synthesis. It is a matter of interest in living systems to work out the
site of synthesis and the site of storage for this molecule and its
stability in plant systems in vivo. Hence this compound demands
more research on stability to facilitate use as a food additive in a
variety of foods.
ACKNOWLEDGEMENTS
K.W. and A.N. acknowledge the nancial assistance from the
Department of Atomic Energy, Board of Research in Nuclear
Sciences, Mumbai, India (Sanction No. 2006/37/45/BRNS). R.Z.
and V.H. acknowledge the Department of Science and Tech-
nology, India [Sanction No. SR/FT/LS-350/2012 and Sanction
No. SR/WOS-A/LS433/2011(G)]. K.K. acknowledges the Coun-
cil of Scientic and Industrial Research, India (Sanction No.
09/137/(0541)/2012-EMR-1) for the award of SRF.
REFERENCE
1 Del Mundo AM and Juliano BO, Consumer preference and properties
of raw and cooked milled rice. J Texture Stud 12:107120 (1981).
2 Sakthivel K, Sundaram RM, Shobha Rani N, Balachandran SM and
Neeraja CN, Genetic and molecular basis of fragrance in rice.
Biotechnol Adv 27:468473 (2009).
3 Obata Y and Tanaka H, Studies on the photolysis of L-cysteine. Agric
Biol Chem 19:191 (1965).
4 Ayano Y and Furuhashi T, Volatiles from cooked kaorimai rice. Chiaba
Diagaku Engeigakubu Gakujutsu Hokoku 18:53 (1970).
5 Bullard RW and Holguin G, Volatile components of unprocessed rice
(Oryza sativa). J Agric Food Chem 31:99 (1977).
6 Yajima I, Yanai T, Nakamura M, Sakakibara H and Habu T, Volatile
avor components of cooked rice. Agric Biol Chem 42:12291233
(1978).
7 Buttery RG, Ling LC and Juliano BO, 2-acetyl-1-pyrroline: An impor-
tant aroma component of cooked rice. Chem Ind (London UK)
12:958959 (1982).
8 Buttery RG, Ling LC and Mon TR, Quantitative analysis of
2-acetyl-1-pyrroline in rice. J Agric Food Chem 34:112114 (1986).
9 Buttery R, Turnbaugh J and Ling L, Contributions of volatiles to rice
aroma. J Agric Food Chem 36:10061009 (1988).
10 Lin CF, Hsieh RCY and Ho BJ, Identication and quantication of the
popcorn-like aroma in Louisiana Aromatic della rice (Oryza sativa).
J Food Sci 35:14661467 (1990).
11 Paule CM and Powers J, Sensory and chemical examination of aro-
matic and nonaromatic rices. J Food Sci 54:343347 (1989).
12 Widjaja R, Craske J and Wooton M, Comparative studies on volatile
components of non-fragrant and fragrant rices. J Sci Food Agric
70:151161 (1996).
13 Nadaf AB, Krishnan S and Wakte KV, Histochemical and biochemical
analysis of major aroma compound (2-acetyl-1-pyrroline) in Bas-
mati and other scented rice (Oryza sativa L). Curr Sci 91:15331536
(2006).
14 Mahatheeranont S, Keawsaard S and Dumri K, Quantication of the
rice aroma compound, 2-acetyl-1-pyrroline, in uncooked Khao
Dawk Mali 105 brown rice. J Agric Food Chem 49:773779 (2001).
15 Adams A and Kimpe ND, Chemistry of 2-acetyl-1-pyrroline,
6-acetyl-1,2,3,4-tetrahydropyridine, 2-acetyl-2-thiazoline and
5-acetyl-2,3-dihydro-4 h-thiazine: extraordinary Maillard avor
compounds. Chem Rev 106:22992319 (2006).
16 Demyttenaere J, Tehrani KA and De Kimpe N, The chemistry of the
most important avour compounds of bread and cooked rice. ACS
Symposium Series 826:150165 (2002).
17 Yoshihashi T, Huong NTT and Inatomi H, Precursors of
2-acetyl-1-pyrroline, a potent avor compound of an aromatic rice
variety. J Agric Food Chem 50:20012004 (2002).
wileyonlinelibrary.com/jsfa 2016 Society of Chemical Industry
K Wakte et al.
18 Buttery RG, Juliano BO and Ling LC, Identication of rice aroma com-
pound 2-acetyl-1-pyrroline in Pandan leaves. Chem Ind (London UK)
23:478 (1983).
19 Midya S and Brahmachary R, The aroma of Bassia ower. Curr Sci
71:430 (1996).
20 Wongpornchai S, Sriseadka T and Choonvisase S, Identication and
quantitation of the rice aroma compound, 2-acetyl-1-pyrroline, in
bread owers (Vallaris glabra Ktze). J Agric Food Chem 51:457462
(2003).
21 Wu ML, Chou KL, Wu CR, Chen JK and Huang TC, Characterization and
the possible formation mechanism of 2-acetyl-1-pyrroline in aro-
matic vegetable soybean (Glycine max L.). J Food Sci 74:192197
(2009).
22 Brahmachary RL and Ghosh M, Vaginal pheromone and other
unusual compounds in Mung bean aroma. Curr Sci 78:1410 (2000).
23 Ayyanger GRR, Studies in sorghum. J Madras Univ 11:131143 (1938).
24 Yundaeng C, Somta P, Tangphatsornruang S, Wongpornchai S and
Srinives P, Gene discovery and functional marker development for
fragrance in sorghum (Sorghum bicolor (L.) Moench). Theor Appl
Genet 126:2897906 (2013).
25 Seitz LM, Wright RL, Waniska RD and Rooney LW, Contribution of
2-acetyl-1-pyrroline to odors from wetted ground pearl millet. J
Agric Food Chem 41:955958 (1993).
26 Masanetz C, Guth H and Grosch W, Fishy and hay-like o-avours of
dry spinach. Z Lebensm Unters Forsch 206:108113 (1998).
27 Wong KC, Chong FN and Chee SG, Volatile constituents of Taro
(Colocasia esculenta (L.) Schott). J Essent Oil Res 10:9395 (1998).
28 Hinterholzer A, Lemos T and Schieberle P, Identication of the key
odorants in raw French beans and changes during cooking. Z
Lebensm Unters Forsch 207:219222 (1998).
29 Pramnoi P, Somta P, Chankaew S, Juwattanasomran R and Srinives P,
A single recessive gene controls fragrance in cucumber (Cucumis
sativus L.). J Genet 92:147149 (2013).
30 Hayata T, Sakamoto T, Maneerai C, Li X, Kozuka H and Sakamoto
K, Evaluation of aroma compounds contributing to muskmelon
avor in Porapak Q extracts by aroma extract dilution analysis. J
Agric Food Chem 5:34153418 (2003).
31 Apriyantono A and Kumara B, Identication of character impact
odorants of wood apple fruit (Feronia limonia). J Teknol dan Indust
Pangan 15:3546 (2004).
32 Wong KC, Lim CL and Wong LL, Volatile avour constituents of
Chempedak (Artocarpus polyphema Pers.) fruit and jackfruit
(Artocarpus heterophyllus Lam.) from Malaysia. Flavour Fragr J
9:319324 (1992).
33 Brahmachary RL, Sarkar MP and Dutta J, The aroma of rice and
tiger. Nature 344: 26 (1990).
34 Grbin PR and Henschke PA, Mousy o-avour production in grape
juice and wine by Dekkera and Brettanomyces yeasts. Aust J Grape
Wine Res 6:255 (2000).
35 Costello PJ and Henschke PA, Mousy o-avor of wine: Pre-
cursors and biosynthesis of the causative N-heterocycles
2-ethyltetrahydropyridine, 2-acetyltetrahydropyridine, and
2-acetyl-1-pyrroline by Lactobacillus hilgardii DSM 20176. J
Agric Food Chem 50:70797087 (2002).
36 Stahnke LH, 2-Acetyl-1-pyrroline key aroma compound in Mediter-
ranean dried sausages, in Frontiers of Flavor Science, ed by
Schieberle P and Engel KH. Deutsche Forschungsanstalt fur
Lebensmittelchemie, Garching, pp. 361365 (2000).
37 Snowdon EM, Bowyer MC, Grbin PR and Bowyer PK, Mousy o-avor:
A review. J Agric Food Chem 54:64656474 (2006).
38 Rungsardthong V and Noomhoom A, Production of
2-acetyl-1-pyrroline by microbial cultures. Flavour Fragr J
20:710714 (2005).
39 Shaikh MN and Nadaf AB, Production of 2-acetyl-1-pyrroline by rhizo-
sphere fungi of aromatic rice varieties. Int J Adv Life Sci 6:282286
(2013).
40 Shaikh MN and Nadaf AB, Qualitative analysis of 2-acetyl-1-pyrroline
from the rhizosphere fungal species of basmati rice varieties by
GC-FID. Int J Curr Res 5:16631665 (2013).
41 Romanczyk Jr LJ, McClelland CA, Post LS and Aitken WM, Formation of
2-acetyl-1-pyrroline by several Bacillus cereus strains isolated from
cocoa fermentation boxes. J Agric Food Chem 43:469475 (1995).
42 Adams A and Kimpe ND, Formation of pyrazines and
2-acetyl-1-pyrroline by Bacillus cereus. Food Chem 101:12301238
(2007).
J Sci Food Agric (2016)
Acetyl-1-pyrroline: a principal compound in scented rice
43 Schieberle P and Grosch W, Identication of volatile avour compo-
nents of wheat bread crust: Comparison with rye bread crust. Z
Lebensm Unters Forsch 180:474478 (1985).
44 Schieberle P, Primary odorants in popcorn. J Agric Food Chem
39:11411144 (1991a).
45 Buttery RG, Stern DJ and Ling LC, Studies on avour volatiles of some
sweet corn products. J Agric Food Chem 42:791795 (1994).
46 Chung HY and Cadwallader KR, Aroma extract dilution analysis of
blue crab claw meat volatiles. J Agric Food Chem 42:28672870
(1994).
47 Rychlik M and Grosch W, Identication and quantication of potent
odorants formed by toasting of wheat bread. LWT Food Sci Tech-
nol 29:515525 (1996).
48 Schieberle P, Odour-active compounds in moderately roasted
sesame. Food Chem 55:145152 (1996).
49 Cadwallader KR and Baek HH, Aroma-impact compounds in cooked
tail meat of freshwater craysh (Procambarus clarkii). Develop Food
Sci 40:271278 (1998).
50 Zehentbauer G and Grosch W, Crust aroma of baguettes I. Key
odorants of baguettes prepared in two dierent ways. J Cereal Sci
28:8192 (1998).
51 Bredie WLP, Mottram DS and Guy RCE, Aroma volatiles gener-
ated during extrusion cooking of maize our. J Agric Food Chem
46:14791487 (1998).
52 Schluter S, Steinhart H, Schwarz FJ and Kirchgessner M, Changes in
the odorants of boiled carp llet (Cyprinus carpio L.) as aected
by increasing methionine levels in feed. J Agric Food Chem
47:51465150 (1999).
53 Mutti B and Grosch W, Potent odorants of boiled potatoes.
Nahrung/Food 43:302306 (1999).
54 Tairu AO, Hofmann T and Schieberle P, Studies on the key odorants
formed by roasting of wild mango seeds (Irvingia gabonensis). J
Agric Food Chem 48:23912394 (2000).
55 Kim DS, Baek HH, Ahn CB, Byun DS, Jung KJ, Lee HG, et al., Develop-
ment and characterization of a avoring agent from oyster cooker
euent. J Agric Food Chem 48:48394843 (2000).
56 Lee GH, Suriyaphan O and Cadwallader KR, Aroma components of
cooked tail meat of American lobster (Homarus americanus). J Agric
Food Chem 49:43244332 (2001).
57 Karagul-Yuceer Y, Drake M and Cadwallader KR, Aroma-active com-
ponents of non-fat dry milk. J Agric Food Chem 49:29482953
(2001).
58 Laseken OO, Feijao Teixeira JP and Salva TJG, Volatile avour
compounds of cooked acha (Digitaria exilis Stapf ). Food Chem
7:333337 (2001).
59 Carrapiso AI, Ventanas J and Garcia C, Characterization of the most
odor-active compounds of Iberian ham headspace. J Agric Food
Chem 50:19962000 (2002).
60 Gasser U and Grosch W, Identication of volatile avor compounds
with high aroma values from cooked beef. Z Lebensm Unters Forsch
186:489494 (1988).
61 Fischer N, Hammerschmidt FJ and Brunke EJ, Analytical Investigation
of the Flavor of Cupuau (Theobroma gradiorum Spreng.), in Fruit
Flavors, ACS Symposium Series, 596, pp. 820 (1995).
62 Gassenmeier K and Schieberle P, Comparison of important odorants
in pu-pastries prepared with butter or margarine. Lebensm Wiss
Technol 27:282288 (1994).
63 Herderich M, Costello PJ, Grbin PR and Henschke PA, Occurrence
of 2-acetyl-1-pyrroline in mousy wines. Nat Prod Lett 7:129132
(1995).
64 Hofmann T, Schieberle P and Grosch W, Model studies on the oxida-
tive stability of odor-active thiols occurring in food avors. J Agric
Food Chem 44:2 (1996).
65 Kumazawa K and Masuda H, Identication of potent odorants in
dierent green tea varieties using avor dilution technique. J Agric
Food Chem 50:56605663 (2002).
66 Peng F, Sheng L, Liu B, Tong H and Liu S, Comparison of dierent
extraction methods: steam distillation, simultaneous distillation
and extraction and headspace co-distillation, used for the analysis
of the volatile components in aged ue-cured tobacco leaves. J
Chromatogr A 104:117 (2004).
67 Munch P, Hofmann T and Schieberle P, Comparison of key odorants
generated by thermal treatment of commercial and self-prepared
yeast extracts: Inuence of the amino acid composition on odorant
formation. J Agric Food Chem 45:13381344 (1997).
J Sci Food Agric (2016) 2016 Society of Chemical Industry
www.soci.org
68 Schieberle P, Primary odorants of pale lager beer dierences to
other beers and changes during storage. Z Lebensm Unters Forsch
193:558565 (1991).
69 Matsui T, Guth H and Grosch W, A comparative study of potent
odorants in peanut, hazelnut, and pumpkin seed oils on
the basis of aroma extract dilution analysis (AEDA) and gas
chromatographyolfactometry of headspace samples (GCOH).
Fett/Lipid 100:5156 (1998).
70 Blank I and Fischer KH, Intensive neutral odorants of linden honey.
Dierences from honeys of other botanical origin. Z Lebensm
Unters Forsch 189:426433 (1989).
71 Wu YFG and Cadwallader KR, Characterization of the aroma
of a meat-like process avoring from soybean-based
enzyme-hydrolyzed vegetable protein. J Agric Food Chem
50:29002907 (2002).
72 Blank I, Devaud S, Fay LB, Cerny C, Steiner M and Zurbriggen B,
Odor-active compounds of dry-cured meat: Italian-type salami
and parma ham. ACS Symp Ser 794:920 (2001).
73 Mahajan SS, Goddik L and Qian MC, Aroma compounds in sweet
whey powder. J Dairy Sci 87:40574063 (2004).
74 Bendall JG, Aroma compounds of fresh milk from New Zealand cows
fed dierent diets. J Agric Food Chem 49:48254832 (2001).
75 Kubckova J and Grosch W, Evaluation of potent odorants of camem-
bert cheese by dilution and concentration techniques. Int Dairy J
7:6570 (1997).
76 Cerny C and Guntz R, Evaluation of potent odorants in heated
egg yolk by aroma extract dilution analysis. Eur Food Res Technol
219:452454 (2004).
77 Majcher MA and Jelen HH, Identication of potent odorants formed
during the preparation of extruded potato snacks. J Agric Food
Chem 53:64326437 (2005).
78 Karagul-Yuceer Y, Vlahovich KA, Drake MA and Cadwallader KR,
Characteristic aroma components of rennet casein. J Agric Food
Chem 51:67976801 (2003).
79 Karagul-Yuceer Y, Drake MA and Cadwallader KR, Aroma-active com-
ponents of liquid cheddar whey. J Food Sci 68:12151219 (2003).
80 Milo C and Grosch W, Changes in the odorants of boiled salmon and
cod as aected by the storage of the raw material. J Agric Food
Chem 44:23662371 (1996).
81 Milo C and Grosch W, Changes in the odorants of boiled trout (Salmo
fario) as aected by the storage of the raw material. J Agric Food
Chem 41:20762081 (1993).
82 Morita K, Kubota K and Aishima T, Sensory characteristics and volatile
components in aromas of boiled prawns prepared according to
experimental designs. Food Res Int 34:473481 (2001).
83 Triqui R and Reineccius GA, Flavor development in the ripening of
anchovy (Engraulis encrasicholus L.). J Agric Food Chem 43:453458
(1995).
84 Baek HH and Cadwallader KR, Volatile compounds in avor concen-
trates produced from craysh-processing by products with and
without protease treatment. J Agric Food Chem 44:32623267
(1996).
85 Cha YJ and Cadwallader KR, Aroma-active compounds in skipjack
tuna sauce. J Agric Food Chem 46:11231128 (1998).
86 Triqui R and Bouchriti N, Freshness assessments of Moroccan
sardine (Sardina pilchardus): Comparison of overall sensory
changes to instrumentally determined volatiles. J Agric Food Chem
51:75407546 (2003).
87 Thimmaraju R, Bhagyalakshmi N, Narayan MS, Venkatachalam L and
Ravishankar GA, In vitro culture of Pandanus amaryllifolius and
enhancement of 2-acetyl-1-pyrroline, the major avouring com-
pound of aromatic rice, by precursor feeding of L-proline. J Sci Food
Agric 85:25272534 (2005).
88 Bhattacharjee P, Kshirsagar A and Singhal RS, Supercritical carbon
dioxide extraction of 2-acetyl-1-pyrroline from Pandanus amaryl-
lifolius Roxb. Food Chem 91:255259 (2005).
89 Wakte KV, Nadaf AB, Krishnan S and Thengane RJ, Studies on lower
epidermal papillae, a site of storage for potent basmati rice aroma
in Pandanus amaryllifolius Roxb. Curr Sci 93:238242 (2007).
90 Laohakunjit N and Noomhorm A, Supercritical carbon dioxide extrac-
tion of 2-acetyl-1-pyrroline and volatile components from pandan
leaves. Flavour Fragr J 19:251259 (2004).
91 Wakte KV, Thengane RJ, Jawali N and Nadaf AB, Optimization of
HS-SPME conditions for quantication of 2-acetyl-1-pyrroline and
study of other volatiles in Pandanus amaryllifolius Roxb. Food Chem
121:595600 (2010).
wileyonlinelibrary.com/jsfa
 www.soci.org
92 Wakte KV, Biochemical characterization of aroma volatiles and molec-
ular genotyping in Indian Pandanus amaryllifolius Roxb. clones. PhD
thesis, University of Pune, Pune, Maharashtra (2010).
93 Wakte KV, Kad TD, Zanan RL and Nadaf AB, Mechanism of
2-acetyl-1-pyrroline biosynthesis in Bassia latifolia Roxb. owers.
Physiol Mol Biol Plants 17:231237 (2011).
94 Juwattanasomran R, Somta P, Chankaew S, Shimizu T, Wongpornchai
S, Kaga A, et al., A SNP in GmBADH2 gene associates with fragrance
in vegetable soybean variety Kaori and SNAP marker develop-
ment for the fragrance. Theor Appl Genet 122:533541 (2011).
95 Attar UA, Biochemical characterization of aroma volatiles in scented
Sona mung bean (Vigna radiata (L.) Wilczek). MPhil thesis, University
of Pune, Pune, Maharashtra (2014).
96 Itani T, Tamaki M, Hayata Y, Fushimi T and Hashizume K, Variation
of 2-acetyl-1-pyrroline concentration in aromatic rice grains col-
lected in the same region in Japan and factors aecting its con-
centration. Plant Prod Sci 7:178183 (2004).
97 Sunthonvit N, Srzednicki GS and Craske J, Eects of high temperature
drying on the avor components in Thai fragrant rice. Drying
Technol 23:14071418 (2005).
98 Goufo P, Duan M, Wongpornchai S and Tang X, Some factors aecting
the concentration of the aroma compound 2-acetyl-1-pyrroline in
two fragrant rice cultivars grown in South China. Front Agric China
4:19 (2010).
99 Mathure SV, Wakte KV, Jawali N and Nadaf AB, Quantication of
2-acetyl-1-pyrroline and other rice aroma volatiles among Indian
scented rice cultivars by HS-SPME/GC-FID. Food Anal Method
4:326333 (2011).
100 Yang DS, Shewfelt RL, Lee KS and Keys SJ, Comparison of odor-active
compounds from six distinctly dierent rice avor types. J Agric
Food Chem 56:27802787 (2008).
101 Bergman CJ, Delgado JT, Bryant R, Grimm C, Cadwallader KR and
Webb BD, Rapid gas chromatographic technique for quantifying
2-acetyl-l-pyrroline and hexanal in rice (Oryza sativa L.) Cereal
Chem 77:454458 (2000).
102 Tava A and Bocchi S, Aroma of cooked rice (Orvza sativa): Comparison
between commercial Basmati and Italian line 1353. Cereal Chem
76:526529 (1999).
103 Siddiq EA, Sadananda AR and Zaman FU, Use of primary trisomic
of rice in genetic analysis, in Rice Genetics, Proceedings of the
International Rice Genetics Symposium. International Rice Research
Institute, Manila, pp. 185197 (1986).
104 Tsuzuki E and Shimokawa E, Inheritance of aroma in rice. Euphytica
46:157159 (1990).
105 Pinson SRM, Inheritance of aroma in six rice cultivars. Crop Sci
34:11511157 (1994).
106 Brijal JS and Gupta BB, Inheritance of aroma in Saanwal Basmati. Ind
J Genet 58:117119 (1998).
107 Berner DK and Ho BJ, Inheritance of scent in American long grain
rice. Crop Sci 26:876878 (1986).
108 Yano M, Shimosaka E, Sato A and Nakagahra M, Linkage analysis of
a gene for scent in indica rice variety, surjamkhi, using restriction
fragment length polymorphism makers (in Japanese). Jpn J Breed
41:338339 (1991).
109 Ahn SN, Bollich CN and Tanksley SD, RFLP tagging of a gene for aroma
in rice. Theor Appl Genet 84:825828 (1992).
110 Garland S, Lewin L, Blakeney A, Reinke R and Henry R, PCR-based
molecular markers for the fragrance gene in rice (Oryza sativa L.).
Theor Appl Genet 101:364371 (2000).
111 Li J, Ku DF and Li LF, Analysis of fragrance inheritance in scented
rice variety Shenxiangjing-4. Shanghai Nong Ye Xue Bao 12:7881
(1996).
112 Lorieux M, Petrov M, Huang N, Guiderdoni E and Ghesquiere A,
Aroma in rice: Genetic analysis of a quantitative trait. Theor Appl
Genet 93:11451151 (1996).
113 Ren JS, Xiao PC, Chen Y, Huang X, Wu XJ and Wang XD, Study on
heredity of aroma genes in several maintainer lines of aromatic
rice. Seed 23:2428 (2004).
114 Cordeiro GM, Christorper MJ, Henry RJ and Reinke RF, Identication
of micro-satellite markers for fragrance in rice by analysis of the rice
genome sequence. Mol Breed 9:245250 (2002).
115 Wanchana S, Kamolsukyunyong W, Ruengphayak S, Toojinda T, Tra-
goonrung S and Vanavichit A, A rapid construction of a physical
contig across a 4.5 cm region for rice grain aroma facilitates marker
enrichment for positional cloning. Sci Asia 31:299306 (2005).
wileyonlinelibrary.com/jsfa 2016 Society of Chemical Industry
K Wakte et al.
116 Bradbury LMT, Fitzgerald TL, Henry RJ, Jin Q and Waters DLE, The
gene for fragrance in rice. Plant Biotechnol J 3:363370 (2005).
117 Vanavichit A, Yoshihashi T, Wanchana S, Areekit S, Saengsraku
D, Kamolsukyunyong W, et al., Positional cloning of os2ap,
the aromatic gene controlling the biosynthetic switch of
2-acetyl-1-pyrroline and gamma aminobutyric acid (GABA) in
Rice, 5th International Rice Genetics Conference. International Rice
Research Institute, Manila, p. 44 (2005).
118 Shi W, Yang Y, Chen S and Xu M, Discovery of a new fragrance allele
and the development of functional markers for the breeding of
fragrant rice varieties. Mol Breed 22:185192 (2008).
119 Fitzgerald MA, Hamilton NRS, Calingacion MN, Verhoeven HA and
Butardo VM, Is there a second gene for fragrance in rice? Plant
Biotechnol J 6:416423 (2008).
120 Chen S, Yang Y, Shi W, Ji Q, He F, Zhang Z, et al., Badh2, Encoding
betaine aldehyde dehydrogenase, inhibits the biosynthesis of
2-acetyl-1-pyrroline, a major component in rice fragrance. Plant
Cell 20:18501861 (2008).
121 Niu X, Tang W, Huang W, Ren G, Wang Q, Luo D, et al.,
RNAi-directed downregulation of OsBADH2 results in aroma
(2-acetyl-1-pyrroline) production in rice (Oryza sativa L.). BMC
Plant Biol 8:100 (2008).
122 Shi Y, Zhao G, Xu X and Li J, Discovery of a new fragrance allele and
development of functional markers for identifying diverse fragrant
genotypes in rice. Mol Breed 33:701708 (2013).
123 Bourgis F, Guyot R, Gherbi H, Tailliez E, Amabile I, Salse J, et al.,
Characterization of the major fragance gene from an aromatic
japonica rice and analysis of its diversity in Asian cultivated rice.
Theor Appl Genet 117:353368 (2008).
124 Kovach MJ, Calingacion MN, Fitzgerald MA and McCouch SR, The
origin and evolution of fragrance in rice (Oryza sativa L.). Proc Natl
Acad Sci U S A 106:1444414449 (2009).
125 Amarawathi Y, Singh R, Singh AK, Singh VP, Mohapatra T, Sharma TR,
et al., Mapping of quantitative trait loci for basmati quality traits in
rice (Oryza sativa L.). Mol Breed 21:4965 (2008).
126 Shao GN, Tang A, Tang SQ, Luo J, Jiao GA, Wu JL, et al., A new
deletion mutation of fragrant gene and the development of three
molecular markers for fragrance in rice. Plant Breed 130:172176
(2011).
127 Sun SX, Gao FY, Lu XJ, Wu XJ, Wang XD, Ren GJ, et al., Genetic analysis
and gene ne mapping of aroma in rice (Oryza sativa L. Cyperales,
Poaceae). Genet Mol Biol 31:532538 (2008).
128 Yundaeng C, Somta P, Tangphatsornruang S, Chankaew S and
Srinives P, A single base substitution in BADH/AMADH is
responsible for fragrance in cucumber (Cucumis sativus L.),
and development of SNAP markers for the fragrance. Theor Appl
Genet 128:18811892 (2015).
129 Kad TD, Gene expression analysis of 2 acetyl-1-pyrroline biosynthesis
gene(s) in Pandanus amaryllifolius Roxb. PhD thesis, University of
Pune, Pune, Maharashtra (2013).
130 Juwattanasomran R, Somta P, Kaga A, Chankaew S, Shimizu T, Soraj-
japinun W, et al., Identication of a new fragrance allele in soybean
and development of its functional marker. Mol Breed 29:1321
(2012).
131 Buttery RG, Ling LC, Juliano BO and Turnbaugh JG, Cooked rice aroma
and 2-acetyl-1-pyrroline. J Agric Food Chem 31:823826 (1983).
132 Schieberle P, Formation of 2-acetyl-1-pyrroline and other impor-
tant avor compounds in wheat bread crust. ACS Symp Ser
409:268275 (1989).
133 Schieberle P, The role of free amino acids present in yeast
as precursors of the odorants 2-acetyl-1-pyrroline and
2-acetyltetrahydropyridine in wheat bread crust. Z Lebensm
Unters Forsch 191:206209 (1990).
134 Suprasanna P, Ganapathi TR, Ramaswamy NK, Surendranathan KK
and Rao PS, Aroma synthesis in cell and callus cultures of rice,
in Rice Genetics, Proceedings of the International Rice Genetics Sym-
posium, vol. 15. International Rice Research Institute, Manila, pp.
123125 (1998).
135 Cheetangdee V and Chaiseri S, Free amino acid and reducing sugar
composition of pandan (Pandanus amaryllifolius) leaves. Kasetsart
J Nat Sci 40:6774 (2006).
136 Huang TC, Teng CS, Chang JL, Chuang HS, Ho CT and Wu ML, Biosyn-
thetic mechanism of 2-acetyl-1-pyrroline and its relationship with
1-pyrroline-5-carboxylic acid and methylglyoxal in aromatic rice
(Oryza sativa L.) callus. J Agric Food Chem 56:73997404 (2008).
J Sci Food Agric (2016)
Acetyl-1-pyrroline: a principal compound in scented rice
137 Vanavichit A and Yoshihashi T, Molecular aspects of fragrance and
aroma in rice. Adv Bot Res 56:4973 (2010).
138 Nadaf AB, Wakte KV and Zanan RL, 2-Acetyl-1-pyrroline biosynthesis:
from fragrance to a rare metabolic disease. J Plant Sci Res 1:102
(2014).
139 Bradbury LM, Gillies SA, Brushett DJ, Waters DLE and Henry RJ,
Inactivation of an aminoaldehyde dehydrogenase is responsible
for fragrance in rice. Plant Mol Biol 68:439449 (2008).
140 Fogel WA, Bieganski T and Maslinski C, Eects of inhibitors of alde-
hyde metabolizing enzymes on putrescine metabolism in guinea
pig liver homogenates. Agents Actions 9:4244 (1979).
141 Swain MR, Kar S, Sahoo AK and Ray RC, Ethanol fermentation of
mahula (Madhuca Latifolia L.) owers using free and immobilized
yeast Saccharomyces cerevisiae. Microbiol Res 162:9398 (2007).
142 Ishitani M, Arakawa K, Mizuno K, Kishitani S and Takabe T, Betaine
aldehyde dehydrogenase in the gramineae: levels in leaves both
betaine-accumulating and non-accumulating cereal plants. Plant
Cell Physiol 34:493495 (1993).
143 Livingstone JR, Maruo T, Yoshida I, Tarui Y, Hirooka K and Yamamoto
Y, Purication and properties of betaine aldehyde dehydrogenase
from Avena sativa. J Plant Res 116:133140 (2003).
144 Trossat C, Rathinasabapathi B and Hanson AD, Transgenically
expressed betaine aldehyde dehydrogenase eciently cat-
alyzes oxidation of dimethylsulfoniopropionaldehyde and
omega-aminoaldehydes. Plant Physiol 113:14571461 (1997).
145 Sebela M, Brauner F, Radova A, Jacobsen S, Havlis J, Galuszka P,
et al., Characterization of a plant aminoaldehyde dehydrogenase.
Biochim Biophys Acta 1480:329341 (2000).
146 Arikit S, Yoshihashi T, Wanchana S, Uyen TT, Huong NT, Wong-
pornchai S, et al., Deciency in the amino aldehyde dehydroge-
nase encoded by GmAMADH2, the homologue of rice Os2AP,
enhances 2-acetyl-1-pyrroline biosynthesis in soybeans (Glycine
max L.). Plant Biotechnol J 9:7587 (2011).
147 De Kimpe NG, Stevens CV and Keppens MA, Synthesis of
2-acetyl-1-pyrroline, the principle rice avour component. J
Agric Food Chem 41:14581461 (1993).
148 De Kimpe N and Keppens M, Novel syntheses of the major avour
components of bread and cooked rice. J Agric Food Chem
44:15151519 (1996).
149 Duby P and Huynh-Ba T, Preparation of 2-acetyl-1-pyrroline. US
patent 5446171 A (1995).
150 Favino TF, Fronza G, Fuganti C, Fuganti D, Grasselli P and Mele
A, Penicillin acylase-mediated synthesis of 2-acetyl-1-pyrroline
and 2-propionyl-1-pyrroline, key roast-smelling odorants in food.
Inclusion complexes with -cyclodextrin and their NMR and MS
characterization. J Org Chem 61:89758979 (1996).
151 Hofmann T and Schieberle P, ZS2-oxopropanal,
hydroxy-2-propanone, and 1-pyrroline, important intermediates
in the generation of the roast-smelling food avor compounds
2-acetyl-1-pyrroline and 2-acetyltetrahydropyridine. J Agric Food
Chem 46:22702277 (1998).
152 Srinivas P and Gurudatta KN, Process for the preparation of
2-acetyl-1-pyrroline, the basmati rice avorant. US patent
6,723,856 B1 (2004).
153 Harrison TJ and Dake GR, An expeditious, high-yielding construction
of the food aroma compounds 6-acetyl-1,2,3,4-tetrahydropyridine
and 2-acetyl-1-pyrroline. J Org Chem 70:1087210874 (2005).
154 Fuganti C, Gatti FG and Serra S, A general method for the synthesis of
the most powerful naturally occurring maillard avors. Tetrahedron
63:47624767 (2007).
155 Srinivas P, Sulochanamma G, Raghavan B and Gurudutt K, Process for
the stabilization of 2-acetyl-1pyrroline, the basmati rice avourant.
US patent 20060147596 A1 (2006).
156 Reineccius G, Flavor Chemistry and Technology. Taylor & Francis
Group, Boca Raton, FL, pp. 391418 (2006).
157 Grimm CC, Bergman C, Delgado JT and Bryant R, Screening for
2-acetyl-1-pyrroline in the headspace of rice using SPME/GC-MS.
J Agric Food Chem 49:245249 (2001).
158 Laguerre M, Mestres C, Davrieux F, Ringuet J and Boulanger R, Rapid
discrimination of scented rice by solid phase microextraction,
mass spectrometry and multivariate analysis used as a mass sen-
sor. J Agric Food Chem 55:10771083 (2007).
159 Zeng Z, Zhang H, Chen JY, Zhang T and Matsunaga R, Direct extrac-
tion of volatiles of rice during cooking using SPME. Cereal Chem
84:423427 (2007).
J Sci Food Agric (2016) 2016 Society of Chemical Industry
www.soci.org
160 Grimm CC, Champagne ET, Lloyd SW, Easson W, Condon B and
McClung A, Analysis of 2-acetyl-1-pyrroline in rice by HSSE/GC/MS.
Cereal Chem 88:271277 (2011).
161 Bryant RJ, Mcclung AM and Grimm CC, Development of a single
kernel analysis method for detection of 2-acetyl-1-pyrroline in
aromatic rice germplasm. Cereal Chem 5:147154 (2011).
162 Maraval I, Sen K, Agrebi A, Menut C, Morere A, Boulanger R, et al.,
Quantication of 2-acetyl-1-pyrroline in rice by stable isotope dilu-
tion assay through headspace solid-phase microextraction cou-
pled to gas chromatographytandem mass spectrometry. Anal
Chim Acta 675:148155 (2010).
163 Eisert R and Pawliszyn J, New trends in solid-phase microextraction.
Crit Rev Anal Chem 27:103135 (1997).
164 Sakthivel K, Shobha Rani N, Pandey MK, Sivaranjani AKP, Neeraja
CN, Balachandran SM, et al., Development of a simple functional
marker for fragrance in rice and its validation in Indian Basmati and
non-Basmati fragrant rice varieties. Mol Breed 2:185190 (2009).
165 Kuo SM, Chou SY, Wang AZ, Tseng TH, Chueh FS, Yen HE, et al., The
betaine aldehyde dehydrogenase (BAD2) gene is not responsible
for the aroma trait of SA0420 rice mutant derived by sodium azide
mutagenesis. in 5th International Rice Genetics Symposium and 3rd
International Rice Functional Genomics Symposium. International
Rice Research Institute Press, Manila, pp. 166167 (2005).
166 Lang NT and Buu BC, Initial marker-assisted selection in rice breeding
at Cuu Long Delta Rice Research Institute. Omonrice 17:821
(2010).
167 Xu XL, Zhao GC and Li JY, Development of molecular markers used
to identify two types of fragrant rice and analysis of mutation sites
of Badh2 gene in 24 varieties of fragrant rice. Plant Divers Resour
33:667673 (2011).
168 Suprasanna P, Bharati G Ganapathi TR and Bapat VA, Aroma in rice:
Eect of proline supplementation and immobilization of callus
cultures. Rice Genet Newslett 19:911 (2002).
169 Keyghobad K, Kad TD, Zanan RL and Nadaf AB, 2-Acetyl-1-pyrroline
augmentation in scented indica rice (Oryza sativa L.) varieties
through 1-pyrroline-5-carboxylate synthetase (P5CS) gene trans-
formation. App Biochem Biotechnol 177:14661479 (2015).
170 Philips SA and Thornalley PJ, The formation of methylglyoxal from
triose phosphates. Eur J Biochem 212:101105 (1993).
171 Naik SD, In vitro expression analysis of 2 acetyl-1-pyrroline in scented
rice (Oryza sativa L.). M. Phil. thesis, University of Pune, Pune,
Maharashtra (2011).
172 Chen M, Wei X, Shao G, Tang S, Luo J and Hu P, Fragrance of the rice
grain achieved via articialmicroRNA-induced down-regulation of
OsBADH2. Plant Breed 131:584590 (2012).
173 Shan Q, Zhang Y, Chen K, Zhang K and Gao C, Creation of fragrant
rice by targeted knockout of the OsBADH2 gene using TALEN
technology. Plant Biotechnol J 13:791800 (2015).
174 Yang DS, Lee KS and Kays SJ, Characterization and discrimination
of premium-quality, waxy, and black-pigmented rice based on
odor-active compounds. J Sci Food Agric 90:25952601 (2010).
175 Monggoot S, Sookwong P, Mahatheeranont S and Meechoui S, Inu-
ence of single nutrient element on 2-acetyl-1-pyrroline contents
in Thai fragrant rice (Oryza sativa L.) cv. Khao DawkMali 105 grown
under soilless conditions, in Proceedings of the 26th Annual Meeting
of the Thai Society for Biotechnology and International Conference.
Mae Fah Luang University, Chiang Rai, pp. 642647 (2014).
176 Wang P, Tang X, Tian H, Sheng-gang P,Mei-yang D, Nie J, et al., Eects
of dierent irrigation modes on aroma content of aromatic rice at
booting stage. Guangdong Agric Sci, 08 (2013).
177 Champagne ET, Bett-Garber KL, Thompson J, Mutters R, Grimm CC
and McClung AM, Eects of drain and harvest dates on rice sensory
and physicochemical properties. Cereal Chem 82:369374 (2005).
178 Mann RF, Basmati rice: Wonder of Pakistans agriculture. IRRC Newslett
XXXVI:2328. (1987).
179 Singh VP, The Basmati rice of India, in Aromatic Rices, ed.by Singh RK,
Singh US and Khush GS. Oxford & IBH Publ. Co. Pvt. Ltd, New Delhi,
pp. 135153 (2000).
180 Wongpornchai S, Dumri K, Jongkaewwattana K and Siri B, Eects
of drying methods and storage time on the aroma and milling
quality of rice (Oryza sativa L.) cv. Khao Dawk Mali 105. Food Chem
87:407414 (2004).
181 Laohakunjit N and Kerdchoechuen O, Aroma enrichment and the
change during storage of non-aromatic milled rice coated with
extracted natural avor. Food Chem 101:339344 (2007).
wileyonlinelibrary.com/jsfa
 www.soci.org K Wakte et al.

182 Kong Z and Zhao D, The enhancing eect of jasmonic acid on
fragrance of Kam sweet rice. J Food Nutr Res 2:395400 (2014).
183 Brahmachary RL, The expanding world of 2-acetyl-1-pyrroline. Curr
Sci 71:257258 (1996).
184 Apintanapong M and Noomhorm A, The use of spray drying to
microencapsulate 2-acetyl-1-pyrroline, a major avour compo-
nent of aromatic rice. Int J Food Sci Technol 38:95102 (2003).
185 Srinivas P, Sulochanamma G, Raghavan B and Gurudutt K, An
improved process for the stabilization of 2-acetyl-1-pyrroline, the
basmati rice avourant. WIPO Patent, WO/2004/056202 (2004).
186 Dianningrum LW and Adhi TP, Raw Material for 2-acetyl-1-pyrroline
Synthesis As Flavor Constituent of Pandan Aromatic Rice in Industry.
Task 02, TK 4102 Perancangan Proses, Kimia. Available: http://
www.scribd.com/doc/36386747/Raw-Material-for-Synthesis-of-
2-Acetyl-1-Pyrroline-as-Flavor-Constituent-of-Pandan-Aromatic-
Rice-in-Industry. [12 March 2014].
187 Yadav SK, Singla-Pareek SL, Ray M, Reddy MK and Sopory
SK,Methylglyoxal levels in plants under salinity stress are depen-
dent on glyoxalase I and glutathione. Biochem Biophys Res Comm
337:6167 (2005).
188 Ramputh AL and Bown AW, Rapid g-aminobutyric acid synthesis and
the inhibition of the growth and development of oblique-banded
leaf-roller larvae. Plant Physiol 111:13491352 (1996).
189 Ghareyazie B, Alinia F,Menguito CA, Rubia LG, De Palma JM, Liwanag
EA, et al., Enhanced resistance to two stemborers in an aromatic
rice containing a synthetic Cry IA (b) gene. Mol Breed 3:401414
(1997).
190 Smith RL, Cohen SM, Doull J, Feron VJ, Goodman JI, Marnett LJ, et al.,
GRAS avoring substances, 22. Food Technol 59:2462 (2005).

wileyonlinelibrary.com/jsfa 2016 Society of Chemical Industry J Sci Food Agric (2016)