8. Thailand

Rice Mutation Breeding for Various Grain Qualities in Thailand
S. Taprab, W. Sukviwat, D. Chettanachit, S. Wongpiyachon and W. Rattanakarn
Bureau of Rice Research and Development, Rice Department,
Ministry of Agriculture and Cooperatives, Thailand

8.1 Introduction
Rice breeding through mutagenesis in Thailand had been continuously done for long period of time.
Recommended varieties of mutated glutinous rice (RD6) and RD 15 an earlier matured mutant ofKDML 105 were released. They are photoperiod sensitive varieties that can be grown once a year inThailand. RD10 was later a glutinous mutated variety released. It was a photoperiod insensitive
variety that can be grown more than one crop per year. Those released mutants were purposed forhigher yield per unit area. Rice breeders are consequently carrying on mutation breeding for better
grain quality. We reviewed previous mutation breeding activities in Thailand prior to FNCA Mutation
breeding project. The results showed some success in improving grain qualities of various purposes.
Seeds of recommended varieties were widely used for mutation induction through gamma ray andfast neutron irradiations. We have joined FNCA Sub-project on improvement of composition orquality in rice since 2007. Using nuclear technique to induce mutation in rice for various grain
qualities was our breeding program proposal. We have just started by mutation induction by gamma
ray irradiation in 2008. We have not joined the Ion-beam mutation breeding project.
Collaborated with FNCA Mutation Breeding Project, five years plan of rice mutation breeding forimprovement of grain quality in Thailand started in 2007. Main objective is to develop mutants withvarious grain qualities such as amylose content, protein content and low phytic acid. Two groups ofvarieties used for mutation induction are low amylose content varieties (KDML105 and RD15) andhigh amylose content varieties (SPR1 and CNT1) including one international check variety, IR64.
Gamma ray with 200 and 300Gy, and fast neutron with 20 and 30Gy are used for irradiation.
KDML105 and RD15 are supreme grain quality aromatic rice in Thailand contain about 15%
amylose. They can be grown only one crop a year due to their photoperiod sensitivity. Mutants withvarious level of amylose content retained excellent physical grain quality with good cooking quality(aroma) and photoperiod insensitivity are intended to be obtained. KTH17, SPR1 and CNT1 are highyielding varieties and high amylose content of about 27%. They have very good physical grain
quality. Irradiated grains of those varieties were screened for low amylose content and low phyticacid. IR64 was used for international check variety of the FNCA mutation breeding project.

8.2 Materials and Methods
Rice seeds of varieties: KDML105, RD15, KTH17, SPR1, CNT1 and IR64
Mutation induction: -200 and 300Gy of Gamma ray irradiation,-20 and 30Gy of fast neutron irradiation
Grain analyses: -IRRI’s standard protocol for amylose content analyses,-SDS-PAGE technique (Laemmli, 1970) for protein and its component analyses,-Screening technique developed by Victor Raboy (2005) for low phytate analyses- 72 -

Mutant selection: -Pedigree selection method
Seeds had been irradiated and M 1 obtained in Wet season 2007. M2 plants of KDML105 and RD15
had been grown in Dry season 2008 where their wild types could not flower. During this period, M 2plants could be selected for photoperiod-insensitive mutants. Then, M3 seed of 200 mutant lines had
been separately harvested as single hill per line. Photoperiod sensitivity is an obvious trait and easy
to be selected in dry season whereas long day period presented. Those 200 photoperiod-insensitive
mutants were continuously screened for important agronomic traits and to be analysed for amylose
content. They were again tested for photoperiodism, tillering ability and maturity in Dry season 2009.
A half of single hill harvested M4 seeds had been grown in Wet season 2009 and another half of seed
had been analyzed for amylose content. In wet season, flowering time of the mutants were earlier
than their wild types but not much differ in plant height. Expected higher amylose content witharoma would be identified from those mutants. While, lower amylose mutants would be used forbreeding germplasm and for special purposes. Selected M5 mutants had been grown in Dry season 2010
and M6 and M7 seeds had been grown in Wet season 2010 and 2011. In 2008, we developed simple
technique for amylose content determination on breeding lines and distributed to FNCA members.
While amylose content was being analyzed, we developed a technique for protein analysis. Protein
extraction technique reported by Iida et al. (1993) and Tanaka et al. (2004) had been modified and tested
for indica rice protein analysis. Extracted proteins such as globulin, albumin, prolamine and glutelinfrom KDML105 and Koshihikari had been identified using SDS-PAGE technique (Laemmli, 1970).
In case of low phytic acid mutants, 2 mutant lines are going to be tested for bioavailability inartificial intestine digestion. New irradiated populations were screened for low phytic acid mutants.

8.3 Results
Wet Season 2010 and Dry Season 2011
8.3.1 Protein component analyses
Using SDS-PAGE technique adapted from Laemmli (1970) for protein and its component analyses todetermine total protein, albumin, globulin, glutelin and prolamine of KDML105 and Koshihikari.
The result showed clear bans of albumin, globulin and glutelin but not of prolamine (Figure 1). We
could distinguish KDML105 from Koshihikari by their pattern of protein components on SDS-PAGE
algarose gel (Figure 2).

Figure 1. SDS-PAGE analyses on total protein, globulin, albumin, prolamine and glutelin of rice variety, KDML105 .

- 73 -Figure 2. Agarose gel electrophoresis of Koshihikari and KDML 105, lane M, DNA marker (GeneRuler™
1 kb DNA Ladder, Fermentas), lane 1, the PCR product amplified from genomic DNA with the primer set
F2 and F8, lane 2, the nested RT-PCR product amplified with the primer set F2 and F8, lane 3, the PCR
product amplified from genomic DNA with the primer set FC and F5, lane 4, the nested RT-PCR productamplified with the primer set FC and F5.

Molecular cloning of Glu-B from KDML105 was done by Nested reverse transcriptase-mediated
PCR. The cDNA was made from total RNA of 20 days seedling using RetroTools ® Two Step Kit(BIOTOOLS B&M LABS., S.A.) and random hexamers (pd(N)6 )as primers for 1st – Step Reverse
Transcription

(RT).

The

secondroundF2(5’-GTTTTGGAACGTTAATGCCCATAG-3’)-F8(5’-ATGAGCACCAAAAGATCCAC-3’) primers were used to amplified the read through LCC1
and the PCR product amplified from LCC1 genomic DNA with the primer set FC (5’CTCCTAGATATCAACAACAGAC -3’) and F5(5’- AGTTGTTGCTCTATATGTCTTCGACT-3’).
Then the PCR product was process to insert in pGEM®-T Easy(Promega, USA). Nucleotides
sequences analysis of Glu-B product were done by ABI 3130 automatic sequencer (AppliedBiosystems) using BigDye ® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Results
show the nucleotide sequences with 600 bps are homologous to Oryza sativa glutelin precursor(GluB4) gene exon 1 through 4 and complete cds.
8.3.2 Mutant lines selection
Since 2008, mutant lines with photoperiod insensitiveness had been achieved. In the year 2010, twohundreds M 5 mutant lines of both KDML105 and RD15 derived from 2008 had been analyzed for
amylose content and protein components. Low amylose mutant lines had been achieved from KTH17
which is originally high amylose content local variety. Various amylose content mutant lines fromoriginal low amylose content varieties KDML105 and RD15 had been obtained. Some of thosemutants contained lower and higher amylose content than did of their wild types. 293 mutant lines ofRD15 showed wide variation of amylose contents from 10.58 – 28.08 % (Figure 3). 241 mutant lines
showed less variation of amylose contents than did of RD15, from 11.96-16.02 % (Figure 4). 191
mutant lines of KTH17 showed wide variation of amylose contents from 13.30 – 29.98 % (Figure 5).
Protein contents of those mutant lines are being analyzed.
- 74 -

Figure 3. Number of mutant lines of RD15 with various amylose contents.

Figure 4. Numbers of mutant lines of KDML105 with various amylose contents.

Numbers of mutant lines

Numbers of mutant lines of KTH17 with various amylosecontents

Amylose contents (%)
Figure 5. Numbers of mutant lines of KTH17 with various amylose contents.

- 75 -

When photoperiodism and dosages of gamma ray were considered, it was found that 20Kr (200Gy)
and 30Kr (300Gy) induced different frequency of mutation on amylose content. Photoperiod
sensitive mutants of RD15, KDML105 and KTH17 mostly contained lower amylose than wild types
did. From RD15, photoperiod sensitive mutants derived from 20Kr irradiation contained lower
amylose while 30Kr mutants contained slightly higher amylose (Figure 6). Highest frequency of20Kr mutants of RD15 was 45 lines with 12% amylose content while 30Kr mutants was 43 lines with
13% amylose content. From KDML105, photoperiod sensitive mutants derived from 20Kr and 30Kr
were similar distribution to RD15 mutants. But 20Kr created wider range of amylose with 1 line
contained 18% amylose while 30 Kr had highest amylose of 15%. Both 20Kr and 30Kr had highest
frequency at 13% amylose content with 45 and 61 lines, respectively (Figure 7). Similarly, highamylose variety KTH17 had been also induced lower amylose mutants. KTH17 wild type contained
27-28% amylose while its mutants derived from both 20 Kr and 30 Kr contained lower amylose.
Their highest frequencies are 24 and 16 lines at 23% amylose content (Figure 8). In contrast,
photoperiod insensitive mutants showed different distribution from photoperiod sensitive mutants.
From RD15, lower dose of 20Kr induced higher amylose content than higher dose of 30Kr. It
produced very higher amylose content up to 28% mutants compared to its 15% amylose wild type. It
had highest frequency at 25% amylose with 32 mutant lines. The dose of 30Kr induced lower
amylose to the lowest of 10% amylose content. Its highest frequency was 40 mutant lines at 12%
amylose content (Figure 9).

Wild typeFigure 6. Amylose content (%) of RD15 photoperiod sensitive mutants irradiated with Gamma Ray 20Krand 30Kr- 76 -

Figure 7. Amylose content (%) of KDML105 photoperiod sensitive mutants irradiated with Gamma Ray 20Kr and 30Kr

Figure 8. Amylose content (%) of KTH17 photoperiod sensitive mutants irradiated with Gamma Ray 20Kr and 30Kr

Figure 9. Amylose content (%) of RD15 photoperiod insensitive mutants irradiated with Gamma Ray 20Kr and 30Kr- 77 -8.3.3 Grain analyses of mutants
Twenty five M7 -mutant lines of KDML105 and RD15 had been randomly selected to be analyzed ongrain physical properties and chemical quality. Grain size had been measured on brown rice in length,width and thickness. Grain shape was determined by Length/Width ratio (L/W). The ratio of L/W thatwas greater than 3.0 indicated slender grain shape. Visually observation on chalkiness, pericarp colorand hull color had been also practiced. Means and standard deviations of mutants and wild types
were shown in Table 1.
Table1. Grain physical properties of 25 M 7 mutants of KDML105 and 25 M7 mutants of RD15 compare toits wild type.
Designation

Size of brown rice (mm)LengthWidthThick

L/W
ratio

Chalkiness Pericarp
color

Hullcolor

M7-KDML105 mutants

7.16 ± 0.08 2.05 ± 0.02

1.68 ± 0.03

3.34 ± 0.05

dull

White

Straw

M7-RD15 mutants

7.46 ± 0.09 2.14 ± 0.02

1.75 ± 0.03

3.48 ± 0.04

dull

White

Straw

KDML105 (wild type)

7.40

2.12

1.76

3.49

dull

White

Straw

RD15 (wild type)

7.49

2.11

1.74

3.55

dull

White

Straw

It had been found that all mutants shown dull endosperm as well as their wild type. Chalkiness could
not be identified in these samples. We have an experience that aromatic rice especially KDML105
and RD15 shown dull endosperm when grown in acid soil. Since our experimental field was acidsulfate soil, dull endosperm occurred. All mutants had white color pericarp and straw color hull aswell as their wild types. Mutants of KDML105 had shorter grain length (7.16mm) than their wild
type (7.40mm) and shown slightly bold shape (3.34 L/W) compared to their wild type (3.49 L/W).
Mutants of RD15 shown almost same grain length but they were wider and thicker that gave smaller
L/W ratio. This indicated that mutants of RD15 had bigger grain size than their wild type (Table 1).
Grains from 25 mutants of KDML105 and 25 mutants of RD15 that had been divided to be analyzed
on physical properties were synchronously analyzed on chemical quality. Amylose content, Gelconsistency, Alkali spreading value, Elongation ratio were important chemical quality that had beenanalyzed. Total protein (%) was analyzed by Kjaldal Method. Aroma or odor of milled rice had beentested by human sensory test. Aroma expressed in 4 levels of sensory test are 0 = odorless (notaroma), 1 = mild aroma, 2 = aroma and 3 = strong aroma. 2-Acetyl-1-pyrroline (2 AP) (Fig 10) was
also analyzed through gas chromatography. 2-Acetyl-1 pyrroline is a pyrroline that is 1-pyrroline inwhich the hydrogen at position 2 is replaced by an acetyl group. It is an aroma and flavor compound
present in jasmine rice and basmati rice. It is responsible for the 'popcorn' aroma in a large variety ofcereal and food products. It is one of the key odourants of the crust of bread and considered to be
responsible for the cracker-like odour properties. In bread, it is primarily generated during baking but
amounts are influenced by ingredient composition and fermentation conditions.

- 78 -Figure 10. Structural formula of 2-Acetyl-1-Pyrroline

It found that M7 mutants of KDML105 had low amylase content similar to their wild type. They
contained amylose within range of 15.03 to 16.06% of amylose or 15.50≠0.2% in average, while theirwild type contained 15.42% amylose. In contrast, M 7 mutants of RD15 contained higher amount ofamylase than did of their wild type. The mutants had wider range of amylose content. Their amylase
content varied from 15.40 to 27.79% while their wild type contained 15.55% of amylase. Out ofthese 25 mutants, there were three mutants that contained 15.40, 15.87 and 15.93% of amylose.
Mutants of RD15 had harder gel than mutants of KDML105 and their wild type. Mutants of
KDML105 had softer gel than their wild type. Alkli spreading value of all mutants was almost notdiffered from their wild type as well as elongation ratio. Total protein content of mutants averagely
higher than their wild type in both KDML105 and RD15. But it was still in normal level of thosevarieties. Unfortunately, we could not find low protein content mutants in these samples. Mutants of
KDML105 contained 9.1% while those of RD15 contained 9.8% protein. Aroma tested by smellingshown different levels of aroma scented between KDML105 and RD15 mutants. Mutants ofKDML105 had strong aroma as well as their wild type with score of 3. Mutants of RD15 showedaroma in score 2 of sensory test level as well as their wild type. When 2-acetyl-1-pyrroline had been
detected it was found that all mutants of KDML105 contained 2 AP in higher amount than their wild
type in both milled rice and brown rice. Brown rice and milled rice of KDML105 mutants contained
2.00 and 2.32ppm of 2 AP, respectively. Their wild type KDML105 contained 2.06 and 2.24ppm of 2AP in its brown rice and milled rice. We could detect 2 AP in only 3 mutants of RD15 that contained
low amylase content. The mutants of RD15 those contained high amylose were not aroma with 2 AP
detection, but sensory test detected aroma from those mutants. It would be an expression of othervolatile compound. One mutant with 15.87% amylase content showed 2.18 and 2.16ppm 2 AP in its
brown rice and milled rice which was almost similar to its wild type (2.99 and 2.06ppm 2 AP) (Table2). It was very rare opportunity to find aromatic rice with high amylose content in our breeding
material. This will be new chance for us to produce various products from our aromatic mutants forworld market.
Table 2. Chemical quality of M7 mutants of KDML105 and RD15 compare to their wild type
Designation

amyloseGel1(%)(mm)

Alkali

E.R.2

ProteinAroma3 2 AP4 (ppm) 2 AP4 (ppm)(%)

Milled Rice

Brown Rice

M7-KDML105 mutants

15.5±0.2 72.9±3.9 6.9±0.28 1.6±0.03 9.1±0.39 2.7±0.48

2.32±0.35

2.00±0.28

M7-RD15 mutants

25.6±3.8 54.0±9.9 7.0±0.0 1.7±0.03 9.8±0.62 2.0±.00

0.1±0.43

0.1±0.44

KDML105 (wild type)15.4269

7.0

1.66

8.77

32.24

2.06

RD15 (wild type)15.5571

7.0

1.66

9.24

2

2.06

2.99

1Gel

E.R.

= Gel consistency
2= Elongation Ratio- 79 -Aroma
42 AP

3= aroma by sensory test
= 2 Acetyl-1-pyrroline detectionIn other purpose, low phytate mutant lines achieved from SPR1 are being evaluated on their grain
yield and other agronomic characters. Forty eight mutant lines of low phytic SPR1 are going to be
examined their protein content.

8.4 Conclusion
Gamma ray irradiation can be applied for mutation induction in rice. Dosages of 20Kr (200Gy) and30Kr (300Gy) showed advantage on mutation induction for grain quality improvement. Thai rice
varieties namely, SPR1, KTH17, KDML105 and RD15 had been irradiated by 20Kr and 30Kr ofgamma ray to induce mutation for various grain qualities such as phytic acid, amylose content andprotein and its compositions. Amylose content had been analyzed using standard method for ricedeveloped by IRRI. Globulin, albumin, prolamine, and glutelin were detected using SDS-pageanalyses. Low phytate mutants had been screened using technique developed by Raboy (2004). Low
amylose mutant lines had been achieved from KTH17 which is originally high amylose content localvariety. Various amylose content mutant lines from original low amylose content varieties KDML105
and RD15 had been obtained. Some of those mutants contained lower and higher amylose content
than did of their wild types. Photoperiod sensitive mutants from RD15, KDML105 and KTH17 hadlower amylose content than did of their wild types. Both 20Kr and 30Kr induced similar distributionof mutants in case of photoperiod sensitive. Among photoperiod insensitive mutants derived fromRD15, 20Kr induced higher amylose while 30Kr induced lower amylose than its wild type. M7mutants shown bigger grain size but shorter length than did of their wild types. Aromatic rice withlow amylose content obtained from KDML105 mutants. Newly found aromatic rice with highamylose content derived from RD15 mutants could not be detected on its 2-acetyl-1 pyrroline
compound. Those mutants might contain other volatile aromatic compound.

- 80 -