Plant Breed. Biotech.

2018 (September) 6(3):233~244 Online ISSN: 2287-9366

https://doi.org/10.9787/PBB.2018.6.3.233 Print ISSN: 2287-9358
RESEARCH ARTICLE

Characterization of ‘GolSam’ Lines Developed from the Cross

between Samgwang and 5MT Resistant Lines in Rice

Franz Marielle Nogoy1, Yu Jin Jung2, Kwon Kyoo Kang2*, Yong-Gu Cho1*
1
Department of Crop Science, Chungbuk National University, Cheongju 28644, Korea
2
Department of Horticulture, Hankyong National University, Anseong 17579, Korea

ABSTRACT Rice grain quality is usually observed by its chalkiness and is affected by genetic effects of endosperm, cytoplasm and
maternal plant. Controlling the chalkiness in rice can be a very challenging task because it is affected by genotype and environmental
factors. The present study aimed to introduce 5-methyl tryptophan (5MT) resistance from the 5MT resistant mutants into Samgwang,
a high grain and eating quality Korean variety by introgression of, resulting to elevated tryptophan content in grains. The progenies
generated from single crosses of two different cross combinations were phenotyped based on agronomic traits and by 5MT growth
inhibition test. Through direct PCR sequencing, the inheritance of single base mutation (F124V) in OsASA was selected among the
progenies. The latter generations were used to analyze the grain and eating quality of the selected lines. Inbred lines (S4-10, S4-28, and
S5-11) carrying the point mutation in OsASA and with reduced chalkiness plus good eating qualities were successfully generated.
Tryptophan content in the milled grains of the selected lines showed 2 - 4 times higher (mg/100 mg) than the maternal parent. The three
selected lines, S4-10, S4-28, and S5-11, were later renamed as GolSam-1, GolSam-2, and GolSam-3 respectively.
Keywords Grain quality, Tryptophan, Nutritional rice, Palatability, Viscosity, Whiteness

INTRODUCTION tryptophan rice with good eating and grain quality.

The main objective of crop scientists is to help supply
enough food for our growing population. Today, a lot of the
food available in the market doesn’t supply enough
nutrients we need. It is important to provide highly
nutritious food with good grain and eating quality in the
market. Preservation of nutrients in rice grains adds to its
value. Bio fortification of rice is exemplified by famous
studies like golden rice (Ye et al. 2000) and iron-fortified
rice (Goto et al. 1999; Cho et al. 2009). However, even if
the rice grain has an added value and it has bad grain
appearance, it becomes undesirable for consumers. This is
the case in mutant rice lines with high-level tryptophan
content. This paper tackles on the development of high-level
Tryptophan (Trp) is one of the essential amino acids
needed by our body along with histidine, threonine, valine,
methionine, isoleucine, leucine, phenylalanine, and lysine
(Yang et al. 2016). As a review of concept, essential amino
acids are those that cannot be synthesized by living
organism but needed for normal functioning, so these
amino acids need to come from our diet. Dietary tryptophan
that goes in the liver proceeds to one of the two basic
metabolic pathways. First, it is used for protein synthesis
and other functions by cells throughout the body and
second, it can be degraded in the liver through a series of
metabolic steps known collectively as the kynurenine
pathway (Moffett and Namboodiri 2003). In the nervous
system and gut, tryptophan is needed as a substrate to

Received July 31, 2018; Revised August 9, 2018; Accepted August 9, 2018; Published September 1, 2018
*Corresponding author Kwon Kyoo Kang, kykang@hknu.ac.kr, Tel: +82-31-670-5104, Fax: +82-31-670-5109
*Corresponding author Yong-Gu Cho, ygcho@cbnu.ac.kr, Tel: +82-43-261-2514, Fax: +82-43-273-2242

Copyright ⓒ 2018 by the Korean Society of Breeding Science

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0)
which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
234 ∙ Plant Breed. Biotech. 2018 (September) 6(3):233~244
synthesize serotonin. In the pineal gland, it is required to
produce melatonin. Also, when niacin content in the diet is
insufficient for metabolic requirements, tryptophan is
necessary for the synthesis of the essential cellular cofactor,
nicotinamide adenine dinucleotide (NAD+) (Moffett and
Namboodiri 2003). Collectively, tryptophan is important
for the production of bioactive metabolites and regulation
of serotonin and melatonin. Lack of enough tryptophan can
increase the risk of pellagra disease, a type of nutrient
deficiency disease that may cause dermatitis, dementia, or
diarrhea (Hegyi et al. 2004). Synthetic tryptophan is
expensive due to the relatively low efficiency of industrial
production (Ishihara et al. 2007). Thus, Trp fortification in
rice for human food can contribute great health benefits.
Anthranilate synthase (AS) converts the substrate of
tryptophan synthesis, chorismate to anthranilate, the first
reaction in tryptophan biosynthesis (Ishikawa et al. 2003).
AS has alpha and beta subunits, the alpha subunit is
important for feedback inhibition of the enzyme by
tryptophan, thus, AS has a key role in regulating tryptophan
biosynthesis (Ishikawa et al. 2003). In previous studies,
ethylmethane sulfonate (EMS) treated rice generated
mutant rice with mutation in AS, resulting in resistance to
growth inhibition by tryptophan analog like 5-methyl
tryptophan (5MT). 5MT may fit into an allosteric site AS,
the key feedback player in the tryptophan biosynthetic
branch of the shikimate pathway, in the same way as
tryptophan (Kim et al. 2004). A sensitive enzyme fails to
recognize completely between tryptophan and 5MT. The
cell thus responds to 5MT by inhibiting further tryptophan
synthesis. In addition, the 5MT is unable to replace the
tryptophan in cellular protein synthesis, therefore arresting
cellular growth. However, tryptophan over-synthesizing
mutants are resistant to growth inhibition (Kim et al. 2007).
For the present study, we further characterized the
high-level tryptophan mutant rice by evaluating their
eating and grain qualities. The rice mutants were found to
have a low eating and grain quality. To develop rice line
with high tryptophan content and good grain and eating
quality, Samgwang, a good grain and eating quality Korean
cultivar was crossed with two high-level tryptophan rice
lines to introduce the point mutation in OsASA, resulting in
5MT resistance. Inbred lines (S4-10, S4-28, and S5-11)
carrying the point mutation in OsASA and with reduced
chalkiness plus good eating qualities were successfully
generated.
MATERIALS AND METHODS
Plant materials and growth conditions
Dehulled mature seeds were surfaced sterilized by
washing once with 70% ethanol for five minutes and
washed twice with 2.5% sodium hypochlorite for ten
minutes each. Seeds were then washed-rinse several times
using sterile distilled water. Seeds were then inoculated in
Murashige and Skoog (MS) medium containing 50 µM
5-Methyl-DL-tryptophan (M0534 Sigma) and grown for
10 days at 30°C under constant light.
Sequence analysis
PCR was performed using the primer set, Fw: GGGGA
AGCCAGAGGCAAG Rv: CCTGGGGATCTGCATAG
GAT, flanking the F124V point mutation. Direct sequencing
of PCR products was applied and aligned with Multiple
sequence alignment (Corpet 1988).
Anthranilate synthase assay
Leaves of 2-week-old seedlings grown in half strength
MS vitamins with 5MT were grounded using liquid
nitrogen with a mortar and pestle and suspended in one
volume of ice-cold anthranilate synthase (AS) extraction
buffer and 2 mg polyvinylpolypyrrolidone per 1 g weight
of fresh leaves. The solution was centrifuged for 15
minutes at 30,000 × g twice. One volume of the collected
supernatant was mixed with two volumes of saturated
ammonium sulphate, and centrifuged. The resulting pellet
was then re-dissolved in 500 µL AS extraction buffer for
later use in the AS assay. AS activity was measured by the
chorismate dependent production of anthranilate. The
re-suspended ammonium sulphate pellet was combined
with 1.5 mL of AS buffer 2 and 200 µL of a substrate
solution. Anthranilate levels were quantified fluorometrically
at 340 nm (excitation) and 400 nm (emission) using an
Aminco-Bowman spectrofluorimeter (SLM-Aminco, Urbana,
IL). Anthranilate formation was followed by continuous
 ‘GolSam’ Lines Developed between Samgwang and 5MT Mutants ∙ 235
fluorometric monitoring of the reaction mixture at 30°C,
with the stepwise addition of small volumes (1, 10, 100,
250, 500 and 1000 mM) of tryptophan stock solution (5
mM). The reaction was allowed to stabilize between each
addition.
mRNA expression
Quantification of relative mRNA expression of OsASA
in crossing parents and selected progenies was done. Leaf
samples were collected from two-week old seedlings and
crude extraction method of RNA was employed by using
RNAiso Plus (Total RNA extraction reagent). We used
®
ReverTra Ace qPCR RT master mix with gDNA remover
for cDNA synthesis. For qRT-PCR, the supermix we used
TM ®
was iQ SYBR Green in Bio-Rad real time PCR
instrument. ß-tubulin gene was used as internal control.
Physico-chemical analysis
After harvest, rice grains were oven dried at 55°C for
three days. After ensuring that the grains have around 14%
moisture content, grains were hulled through a rice huller to
make brown rice. Brown rice samples were then polished to
90% by a polishing machine (MC-90A, Toyoseiki). Fifteen
grams of polished rice samples were weighed and milled
through a 100-mesh screen by Cyclotec Sample Mill (Foss
North America [Tecator] No.1093-003). Ground samples
were used for viscosity test, amylose content and amino
acid content analyses (Lee et al. 2014).
Grain selector separated broken rice from the whole
grains. Grains selected were used for further analysis. The
grain inspector machine measures whole grains,
brokenness, chalky grains, and damaged grains. The
Infratec Grain Analyzer measures moisture, protein,
amylose, and whiteness of the grains.
Viscosity measurement
To measure the viscosity of rice samples in this study,
the Rapid Visco Analyzer (RVA) Japanese Rice method
was used. This method was developed by The Food
Agency in Japan, it provides a long profile to better
differentiate rice samples of similar quality. A canister was
filled with 25.0 mL of distilled water. Selected ground rice
samples were measured to three grams and transferred to
the canister. Into the canister, the paddle was placed
properly and the canister was inserted into the RVA
instrument. All viscosity parameters were measured in
Rapid Visco Units (RVU) with three replications for each
sample. For palatability, two sets of machines were used,
the first one was Satake Rice Taste Analyzer requiring
about 10 grams of milled rice and the other machine was the
TOYO taste meter system (MB-90A and MA-90B, Japan)
which uses 33 g of milled rice per sample (Lee et al. 2014).
Agronomic traits and statistical analysis
Agronomic traits of the three parents used and the
progenies were measured based on their field performance.
The data gathered were in three replications and analyzed
for significant difference, measurements followed the
Standard Evaluation System (SES) for rice. For this data,
significant tests were performed using Duncan Multiple
Range Test.
RESULTS
Development of high-level tryptophan rice
Two 5MT resistant lines generated by EMS mutation
were characterized and evaluated using molecular and
phenotype analysis. In this paper, the two high-level
tryptophan mutant lines were referred as 5MT-4 and
5MT-5. The wildtype (WT) or background of these lines is
Donganbyeo, a widely grown variety in Korea. The point
of interest in this study is the tryptophan content of the
generated inbred lines and the grain and eating qualities.
First, seeds were selected by growing in half strength MS
media with 50 µM of 5-methyl-tryptophan, an analog of
tryptophan that inhibits the synthesis of anthranilate
compounds that are the first steps in the biosynthesis of
tryptophan. Fig. 1A shows the well-developed shoots and
roots of 5MT-4 and 5MT-5 seedlings compared to its WT.
This insensitivity to tryptophan analog is caused by a single
base change resulting to amino acid change F124V in
anthranilate synthase (AS) protein (LOC_Os03g15780)
(Fig. 1B).
AS is the enzyme that converts the tryptophan precursor
chorismate to anthranilate, and it is susceptible to feedback
236 ∙ Plant Breed. Biotech. 2018 (September) 6(3):233~244
Fig. 1. Phenotype of rice grown in 5MT containing media
and partial amino acid sequence of anthranilate
synthase. (A) Various lines were screened by growing
seeds in half strength Murashige and Skoog (MS)
media with 50 µM 5-methyl-tryptophan. The wildtype
Donganbyeo have no well-developed roots while the
two high-level tryptophan mutant lines 5MT-4 and
5MT-5 showed well grown shoots and roots. (B)
Sequence alignment of anthranilate synthase (AS)
(LOC_Os03g15780.1) against the two mutant lines,
where mutation occurs in F124V (F [TTC] to V
[GTC]).
Fig. 2. Biosynthetic pathway of tryptophan in plants and AS activity. (A) The curved line from Trp to AS indicates
negative feedback regulation. (B) Feedback inhibition of anthranilate synthase activity by tryptophan. Anthranilate
synthase activity in the presence of tryptophan (0, 10, 100, 250, 500, and 1000 µM) with 10 mM glutamine and
100 µM chorismate as substrates.
inhibition (Fig. 2A). To determine if there was a change in
AS, its enzyme activity should have a difference between
the WT and the two mutant lines. AS activity was measured
in different concentrations of tryptophan (0, 10, 100, 250,
500, and 1000 µM) with 10 mM glutamine and 100 µM
chorismate as substrates. Fig. 2B shows the increasing
trend of AS from the extracts of two high tryptophan lines
as the tryptophan concentration increased, 5MT-5 showed
1.5 times higher activity compared to 5MT-4 at the end. In
contrast, their wildtype showed very low activity.
Grain and eating quality of high-level tryptophan rice
The amount of tryptophan in mature seeds of 5MT-4 and
5MT-5 lines was the highly desirable characteristic in the
said mutant lines. These lines showed stable reaction in
5MT containing medium in every generation. The trypto-
phan content reveals as much as 30-fold change in pmol
and 5-fold change in mg/100 mg. However, in this study,
high-level tryptophan mutant rice lines were found to have
a low grain and eating quality. Limited studies showed the
grain and eating quality of high-level tryptophan rice, most
studies have characterized the molecular aspect of AS
which causes the insensitivity of high-level tryptophan rice
in tryptophan analogs (Niyogi and Fink 1992; Tozawa et al.
 ‘GolSam’ Lines Developed between Samgwang and 5MT Mutants ∙ 237

2001; Ishikawa et al. 2003).
To quantify the grain quality, moisture content, whole
grain, chalkiness, and broken grains were measured in
milled high-level tryptophan lines. The two high trypto-
phan rice lines have many damaged grains and were very
chalky (Fig. 3A) despite the standard moisture content. In
Fig. 3B the grain phenotype of WT Donganbyeo, 5MT-4
and 5MT-5 was shown. Based on the initial results on
average taste parameters (perception, hardness, stickiness,
balance and taste), the wildtype showed higher values than
the high tryptophan rice lines.
Development of good grain and eating quality rice
crossed with high-level tryptophan rice
To developed inbred lines having high tryptophan
content and good grain and eating quality, 5MT-4 and
5MT-5 as donors of 5MT resistant gene were crossed to
Samgwang variety, which is popular as high quality rice
variety in Korea. Two cross combinations were used to
increase the probability of generating rice lines with high
tryptophan and good eating and grain quality (Fig. 4). Each
selection focuses on screening the progenies in agar
medium containing 50 µM 5-methyl-tryptophan. Lines
showing well grown shoots and roots after 10-14 days (Fig.
5A) were forwarded in the next generation. Furthermore, the
introgression of mutation was confirmed by sequencing the
target sequence of ASA2 gene in the progenies. The single
point mutation (F124V) found in 5MT-4 and 5MT-5 was
successfully introgressed in the progenies generated from
the two cross combinations (Fig. 5B). Further, the relative

Fig. 3. Grain and eating quality of 5MT resistant lines. (A) Evaluation of grain quality traits in high-level tryptophan
mutant rice along with the wildtype. (B) Physical grain appearance, the first row is unpolished rice while the
second row is milled rice.
238 ∙ Plant Breed. Biotech. 2018 (September) 6(3):233~244

Fig. 4. Diagram of breeding history. A single cross descent was used to introduce the single point mutation from mutant
rice to Korean variety with good eating and grain quality. Method of selection in each generation is shown. A
total of two cross combinations were used, namely Samgwang X 5MT-4 and Samgwang X 5MT-5. Breeding
history of Samgwang is also shown as derivatives from other rice cultivars.

mRNA expression of OsASA encoding anthranilate synthase
was determined. Since anthranilate synthase is the key
regulator of tryptophan biosynthesis, it will be helpful to
know the relative expression of OsASA gene between
parents and selected high-level tryptophan lines. Samgwang,
having no point mutation showed relatively higher
expression compared to the high-level tryptophan lines
carrying the point mutation (Fig. 5C).
After screening progenies via selection in 5MT
containing media and good phenotype selection in the field,
the seeds in F6 generation were harvested in bulk for each
line and used for grain and eating quality experiments. The
most notable characteristic of a bad grain quality were the
brokenness and chalkiness, which was observed in 5MT-4
and 5MT-5 grains. As expected, reduced chalkiness percentage
were observed from the lines selected in F6 generation (Fig.
6A). Grain phenotypes of the selected breeding lines were
exemplary in terms of no chalkiness in grains (Fig. 6B).
The progenies showed low level of whiteness showing
clearness compared to the high-level tryptophan parents
thereby creating a good grain appearance.
This phenotype was supported for good grain and eating
quality by the quantitative data on protein content,
viscosity, amylose content (AC), and palatability. To
measure the general nutritional content of the generated
lines, protein content was determined and found out that the
most progenies are significantly lower than their parents,
5MT4 and 5MT5, which was expected for improving the
eating and grain quality of new rice lines (Fig. 7A). For the
final viscosity, S5-3, showed the highest significant difference
among other progenies (Fig. 7B). In terms of amylose
content, all breeding lines showed lower than 20% AC but
5MT4 and 5MT5 showed higher amylose contents than F6
progenies except for S5-3 line (Fig. 7C). For palatability,
lines S4-9, S4-10, S4-28, S5-7, and S5-11 showed higher
values compared to their maternal parent Samgwang (Fig.
7D).
Rice powder samples were used to measure the amino
acid contents of the selected F6 lines along with their
parents. All twenty amino acid contents are presented
individually in Table 1. For our interest, not all F6 lines
inherited the high-level tryptophan, however, three
 ‘GolSam’ Lines Developed between Samgwang and 5MT Mutants ∙ 239

Fig. 5. Phenotypic response to 5MT containing media, ASA2 gene sequence alignment and relative mRNA expression. (A)
Responses of crossing parents and F5 progenies in 5MT inhibition media. (B) Sequence alignment of ASA2 gene
in rice, crossing parents and progenies (S5-11 and S4-28) DNA sequence giving the mutation F124V (F [TTC]
to V [GTC]). (C) Relative mRNA expression of OsASA between Samgwang, 5MT line and two selected progenies.
As shown, those lines carrying the point mutation showed relatively lower expression of OsASA compared to
Samgwang with no mutation.

promising lines, namely, S4-10, S4-28 and S5-11, all from
Samgwang background have significantly elevated tryptophan
content in their mature grains. These three selected lines
were renamed as GolSam-1, GolSam-2 and GolSam-3 for
possible new candidate varieties of rice. Remarkably, other
amino acids in other F6 lines showed significant higher
content compare to maternal parent Samgwang. Finally,
we considered the agronomic traits and yield characteristics
of the selected lines by pairwise comparison and found out
that no significant changes were found (Table 2). The
selected lines can be used further as materials for studying
transcriptome expression of agronomically important
genes and/or for varietal candidate evaluation. The
development of good grain quality lines with 5MT
resistance represented by the GolSam series led to a new
source of high tryptophan rice. The potential benefits of
GolSam series to farmers are the improvement in nutrition
and higher quality of rice.
DISCUSSION
In this study we showed the development of high-level
tryptophan rice in terms of grain and eating quality by
making crosses between the high-level tryptophan rice
with popular Korean variety having good grain and high
eating quality. Previous studies have characterized the
insensitivity of different crops in 5-methyltryptophan (Lee
and Kameya 1991; Kang and Kameya 1993; Tam et al.
1995; Matsuda et al. 2005) and described the genes regulating
anthranilate synthase (Niyogi and Fink 1992; Tozawa et al.
2001; Hanafy et al. 2006; Chun et al. 2012). Part of our
240 ∙ Plant Breed. Biotech. 2018 (September) 6(3):233~244

Fig. 6. Comparison of grain quality appearance of parents (Samgwang, 5MT-4, and 5MT-5) and three selected lines in F6
generation from each cross combination. (A) Whiteness level of milled grains of parents and the breeding lines. (B)
Grain phenotype of polished grains of parents and selected breeding lines.

Fig. 7. Eating quality measurements of three parents used in crossing (Samgwang, 5MT-4, and 5MT-5) and their selected
progenies in F6 generation. Each histogram shows the comparison of parents among their progenies based on some of
the important eating quality measures (A) protein – for nutritional value present in grains (B) viscosity – as a means
of measuring how viscous the rice starch (C) amylose content – tells about the firmness and stickiness of rice and (D)
palatability – quantitative measurement of cooked rice taste.
 ‘GolSam’ Lines Developed between Samgwang and 5MT Mutants ∙ 241

Table 1. Amino acid content of Samgwang, 5MT resistant lines, and selected breeding lines (mg/100 mg).
Amino Sam-
5MT-4 5MT-5 S4-1 S4-9 S4-10 S4-28 S5-1 S5-3 S5-7 S5-11
acid gwang
Asp 1.18 2.12 2.14 1.43 1.11 1.27 1.68 1.10 1.38 0.92 1.38
Glu 2.20 2.08 1.82 1.19 1.22 1.32 1.90 1.15 1.26 1.38 1.75
Asn 1.17 1.95 1.52 0.69 0.61 1.01 1.24 1.15 1.23 0.65 1.36
Ser 0.24 0.24 0.20 0.10 0.17 0.18 0.25 0.15 0.09 0.16 0.19
Gln 0.28 0.57 0.50 0.44 0.32 0.36 0.29 0.40 0.33 0.43 0.39
Gly 0.04 0.05 0.05 0.04 0.07 0.06 0.09 0.08 0.03 0.07 0.11
His 0.16 0.17 0.14 0.15 0.14 0.16 0.15 0.13 0.12 0.14 0.11
Arg 0.25 0.48 0.43 0.21 0.14 0.16 0.48 0.24 0.23 0.21 0.49
Thr 0.09 0.09 0.10 0.04 0.05 0.05 0.09 0.05 0.03 0.05 0.06
Ala 0.55 0.46 0.46 0.34 0.35 0.43 0.42 0.31 0.35 0.44 0.48
Pro 0.22 0.19 0.18 0.18 0.13 0.19 0.20 0.25 0.18 0.31 0.21
Tyr 0.18 0.18 0.15 0.12 0.10 0.17 0.17 0.11 0.10 0.13 0.17
Val 0.08 0.07 0.06 0.08 0.05 0.05 0.11 0.08 0.04 0.11 0.07
Met 0.03 0.03 0.04 0.04 0.03 0.04 0.03 0.03 0.04 0.04 0.03
Cys2 0.04 0.03 0.02 0.03 0.02 0.02 0.04 0.03 0.03 0.03 0.04
Ile 0.05 0.06 0.05 0.06 0.04 0.04 0.08 0.07 0.04 0.07 0.05
Leu 0.04 0.05 0.04 0.04 0.03 0.03 0.06 0.07 0.03 0.07 0.03
Phe 0.02 0.05 0.04 0.03 0.02 0.02 0.04 0.04 0.02 0.04 0.04
Trp 0.14 5.28 4.91 0.07 0.08 1.45 4.17 0.05 0.04 0.07 2.95
Lys 0.14 0.15 0.14 0.19 0.11 0.12 0.16 0.16 0.12 0.22 0.13
TOTAL 7.09 14.29 12.98 5.48 4.77 7.12 11.63 5.65 5.67 5.53 10.03

Table 2. Agronomic traits of parents and F5 progenies.

Plant height Culm length Panicle length 1000 grain
Entries No. of tillers Yield (t/ha)z)
(cm) (cm) (cm) weight (g)
Samgwang 103.6 ± 2.00ay) 77.5 ± 8.1a 20.3 ± 3.4a 18 ± 2a 20.07 ± 0.15g 5.30a
5MT-4 106.1 ± 4.10a 68.5 ± 7.7a 19.2 ± 2.1a 13 ± 1ab 24.60 ± 0.26a 5.96a
5MT-5 96.9 ± 23.52a 64.3 ± 6.0abc 18.7 ± 7.5a 11 ± 1bcd 25.10 ± 0.10a 5.43a
S4-1 105.3 ± 6.00ab 72.6 ± 12.1a 20.7 ± 2.4a 13 ± 1ab 23.87 ± 0.18ef 5.31a
S4-9 108.5 ± 7.84a 74.9 ± 4.2a 20.7 ± 4.4a 12 ± 1bcd 25.10 ± 0.00a 5.54a
a a a ab def a
S4-10 107.2 ± 10.58 74.0 ± 10.8 18.3 ± 1.3 14 ± 1 21.00 ± 0.40 4.92
a a
S4-28 105.3 ± 8.74 76.6 ± 4.3 18.4 ± 1.0a 11 ± 1bcd 21.73 ± 0.20 cd
5.30a
S5-1 104.1 ± 8.29a 80.2 ± 10.3a 17.4 ± 2.6a 12 ± 1bcd 22.60 ± 0.25b 5.52a
S5-3 107.4 ± 10.50a 80.0 ± 8.7a 18.9 ± 0.3a 13 ± 1abc 21.20 ± 0.06def 5.77a
a a a bcd fg a
S5-7 104.7 ± 3.18 70.2 ± 6.3 19.6 ± 7.6 12 ± 1 20.60 ± 0.25 5.56
S5-11 105.0 ± 4.24a 84.8 ± 2.3a 19.7 ± 2.9a 12 ± 1bcd 25.60 ± 0.00a 5.30a
z)
Yield was calculated based on 200,000 plants per hectare.
Different letters indicate significant difference at P＜0.05 by least significant difference.
y)

study described the growth and selection of high tryptophan
rice (5MT-4 and 5MT-5) in 5-methyltryptophan (5MT), a
tryptophan analog which would inhibit the production of
tryptophan because 5MT can fit to the allosteric site of AS.
AS is the first-step enzyme in the tryptophan pathway. But
5MT-4 and 5MT-5 contain F124V mutation in ASA2
(anthranilate synthase gene2) (Fig. 1B) that gives its
insensitivity to 5MT. Previous reports showed D323N in
ASA1 (Tozawa et al. 2001) and S126F/L530D, Y367A/
L530D, and A369L/L530D in ASA2 (Kanno et al. 2005) in
242 ∙ Plant Breed. Biotech. 2018 (September) 6(3):233~244
rice also allows insensitivity to tryptophan.
We showed here that 5MT-4 and 5MT-5 have high-level
of tryptophan content but the grain quality is low and in
effect, the eating quality turns out low as well. Some studies
that showed the importance of sensory evaluation in
fortified rice is on the development of a simple procedure to
fortify milled rice with calcium, the end product is
calcium-fortified cooked rice with harder texture suitable
for canned rice products (Lee et al. 1995) and that of
Moretti et al. (2005) where they developed iron-fortified
rice with comparable sensory characteristics to natural rice
by using iron compounds of high bioavailability. So far,
there are no studies that evaluated the grain and eating
quality of high-level tryptophan rice.
To generate lines with improved grain and eating quality
and high-level tryptophan, conventional breeding was
employed. The maternal parent for high grain quality and
high eating quality was Samgwang. Samgwang was rec-
ommended in Gangrung and Gangwon provinces based on
high yield, quality and suitability (Jeong et al. 2015). A
portion of ASA2 was sequenced using the DNAs of selected
lines and confirmed the introgression of a single point
mutation (F124V). The initial selection was based from the
growth of seeds in 5MT containing medium. Only those
seeds that grew well in the media were advanced in the
field. Plants were selected in the field based on their
agronomic and yield performance. To enable stable response
in 5MT medium and uniform phenotype, we advanced all
populations selected up to F6 generation (Fig. 4).
Using bulk harvested F6 seeds, we performed grain and
eating quality tests in order for us to finally select lines with
improved quality. Our analysis has shown reduced chalkiness
in the grains (Fig. 6) and improved in other parameters like
palatability, viscosity, amylose content and whiteness
became like Samgwang. We used Samgwang as high-quality
variety control (Fig. 7). For palatability, since we used
TOYO taste meter we can only described the quantitative
values of the changes in the glossiness of the cooked rice.
Among the F6 lines used for analysis, three lines were
significantly higher than those of their parents and the rest
are relatively the same values. Molecular structural
mechanism was suggested as a new tool to select cultivars
with desirable palatability. A cultivar which leaches more
amylopectin with shorter amylopectin chains and bigger
molecular size would be sticky after cooking. On the other
hand, a cultivar which leaches more amylose should be less
sticky but have a harder texture (Li et al. 2017). In terms of
viscosity, which also indicates rice quality, our data
showed low variability among the parents and progenies
except for three F6 lines that have the highest final
viscosity. No correlation is shown in our data between
viscosity and amylose content. In other studies, rapid visco
analysis parameters namely, pasting onset and peak
temperature correlated positively with amylose content in
native rice starches (Li et al. 2008) and is also similar for
maize starches (Lim et al. 2002). The ideal amylose content
in Korean rice is less than 20%, here, several high-level
tryptophan rice lines were not significantly different from
Samgwang variety. We also tested the protein content and
found out that the generated F6 lines have relatively lower
protein content than 5MT-4 and 5MT-5 parents. Interestingly,
we have obtained three F6 lines with high-level of trypto-
phan from Samgwang background. Since we focused on
increasing the tryptophan content in mature grains, we
looked if there is an effect in the regulation of AS in
tryptophan biosynthesis pathway. We measured the mRNA
expression of OsASA gene encoding AS and found out that
the trend of expression this gene was lower in high-level
tryptophan lines than Samgwang. This suggests that the
single point mutation that also causes alteration in amino
acid sequence results in low mRNA expression. Regarding
about the roles of the two subunits if OsASA, ASA2
expression is the same in many tissues and can be induced
by a chitin heptamer, suggesting a role in secondary
metabolism while ASA1 has higher expression in the
panicles and it is important in Trp regulation (Tozawa et al.
2001). Different roles are also suggested in Escherichia
coli, when TrpR and tnaA genes were knocked out, the
tryptophan production increased up to 20-folds (Yu et al.
2008).
Finally, this study has shown the development of high
tryptophan rice with improved grain and eating quality by
using conventional breeding. Through the two cross
combinations used, a large selection of population was
generated and compared the lines among the three parents.
Although this method was proven useful, our selection may
 ‘GolSam’ Lines Developed between Samgwang and 5MT Mutants ∙ 243
have been less laborious if there was a developed marker
for F124V point mutation. Another impediment of our
study is the selection from the early generation based on
grain and eating quality because of the need for large
amount of seeds for analysis. Nevertheless, after several
generations of advancing in field condition and analysis,
we were able to produce lines with high-level tryptophan
with good eating and grain qualities. Overall, the GolSam
series with high-level tryptophan and good grain and eating
quality can be candidates for new variety testing or can be
used for yield improvement or development with resistance
to various stresses. In this way, GolSam can contribute as
good source of high-level tryptophan which can enable
farmers to get higher income.
ACKNOWLEDGEMENTS
This research was supported by the Next-Generation
BioGreen 21 Program (Plant Molecular Breeding Center,
No. PJ013207), Rural Development Administration, Republic
of Korea.
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