research article factors affecting isoflavone content in...

Hindawi Publishing CorporationInternational Journal of AgronomyVolume 2013 Article ID 163573 11 pageshttpdxdoiorg1011552013163573

Research ArticleFactors Affecting Isoflavone Content in Soybean SeedsGrown in Thailand

Supanimit Teekachunhatean12 Nutthiya Hanprasertpong1

and Thawatchai Teekachunhatean3

1 Department of Pharmacology Faculty of Medicine Chiang Mai University Chiang Mai 50200 Thailand2 Center of Thai Traditional and Complementary Medicine Faculty of Medicine Chiang Mai University Chiang Mai 50200 Thailand3 School of Crop Production Technology Institute of Agricultural Technology Suranaree University of TechnologyNakhon Ratchasima 30000 Thailand

Correspondence should be addressed to Supanimit Teekachunhatean steekachmedcmuacth

Received 11 July 2013 Accepted 5 September 2013

Academic Editor Patrick J Tranel

Copyright copy 2013 Supanimit Teekachunhatean et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Soybeans are the most common source of isoflavones in human foods The objectives of this study were to determine the effects ofThai soybean variety planting date physical seed quality storage condition planting location and crop year on isoflavone contentas well as to analyze the relationship between seed viability and isoflavone content in soybean seeds grown inThailand Isoflavonecontent in Thai soybeans varied considerably depending on such factors as variety physical seed quality crop year planting date(even in the same crop year) and planting location Most varieties (except for Nakhon Sawan 1 and Sukhothai 1) had significantlyhigher isoflavone content when planted in early rather than in late dry season Additionally seed viability as well as long-termstorage at 10∘C or at ambient condition seemed unlikely to affect isoflavone content in Thai soybean varieties Isoflavone contentin soybean seeds grown inThailand depends on multiple genetic and environmental factors Some varieties (Nakhon Sawan 1 andSukhothai 1) exhibited moderately high isoflavone content regardless of sowing date Soybeans with decreased seed viability stillretained their isoflavone content

1 IntroductionSoybeans (Glycine max (L) Merrill) are the most commonsource of isoflavones in human foods especially in manyAsian countries Isoflavones represent the most commongroup of phytoestrogens which are structurally similar toestradiol-17120573 the most potent mammalian estrogen [1]Isoflavones exhibit three aglycone structures (daidzein genis-tein and glycitein) which enter into three 120573-glycoside con-jugates (daidzin genistin and glycitin) each with a corre-sponding acetyl and malonyl glycoside conjugate (Figure 1)Since glycitein and its glycoside conjugates account for lessthan 5ndash10 of the total isoflavones in soy-based productsmost studies have focused on daidzein and genistein and theirrespective glycoside conjugates [2 3] This study adopted asimilar approach

Epidemiological and clinical studies of postmenopausalwomen have suggested that isoflavones decrease their risk

of osteoporosis and cardiovascular diseases while alleviatingvasomotor symptoms [4ndash8] However the beneficial effectsof isoflavones remain controversial Discrepancies in overallstudy design differences in isoflavone type and dosagevariations in participant demographics and differing out-come parameters might all have contributed to contradictorystudy results [9] However a consistent finding is that theselection of isoflavone-rich soybeans as raw materials in themanufacturing of soy or soy-based products is a crucial factorin their clinical effectiveness

Isoflavone content in soybeans depends on both geneticand environmental factors including climate planting loca-tion crop year planting dates within a given crop yearand storage conditions [10ndash13] Although findings exist for avariety of genotypes in a variety of environments there areno firm data regarding the isoflavone content of Thai-certi-fied soybeans grown in Thailand This study remedied that

2 International Journal of Agronomy

Aglycones

Daidzein H HGenistein OH H

Glycitein H

R1

R1

R2

R2

OCH3

OHO

OH

O

(a)

Glycosides

Daidzin (120573-glycoside) H H HGenistin (120573-glycoside) OH H H

Glycitin (120573-glycoside) H H

H H

OH H

H

H H

OH H

H

6998400998400-O-Malonlydaidzin

6998400998400-O-Malonlygenistin

6998400998400-O-Malonlyglycitin

6998400998400-O-Acetyldaidzin

6998400998400-O-Acetylgenistin

6998400998400-O-Acetylglycitin

R3

R3

R4

R4

R5

OCH3

OCH3

OCH3

COCH2COOH

COCH2COOH

COCH2COOH

COCH3

COCH3

COCH3

O

O

OOOH

OHOHHO

CH2OR5

(b)

Figure 1 Structure of isoflavones in the forms of aglycones (a) 120573-gly-cosides 610158401015840-O-acetyl-120573-glycosides and 610158401015840-O-malonyl-120573-glyc-osides (b)

deficiency by investigating 14 Thai soybean varieties includ-ing Chiangmai 60 one of the most popular varieties

The study consisted of two basic parts The first partsought to determine the isoflavone content of the 14 varietiesgrown at the same location but with variations in plantingdate physical seed quality and storage conditionThe secondpart targeted sound seeds of Chiangmai 60 Specificallyit examined the effects of planting location and crop yearon total isoflavone content and analyzed the relationshipbetween isoflavone content and seed viability

2 Materials and Methods

21 Description and Preparation of 14 SoybeanVarieties Grownat the Same Location Covering a broad range of pedigreesand physical types the 14 soybean varieties represented the

vast majority of soybeans grown inThailand In alphanumer-ical order they were Chiangmai 2 Chiangmai 5 Chiangmai60 Chakkrabhanhu 1 KKU 35 Nakhon Sawan 1 SJ 1 SJ 2SJ 4 and SJ 5 Srisamrong 1 Sukhothai 1 Sukhothai 2 andSukhothai 3 Significant characteristics are summarized inTable 1 [14 15]

The 14 soybean varieties were collected from the Surana-ree University of Technology (SUT) Farm located in NakhonRatchasima Province in northeasternThailand about 250 kmeast of Bangkok (14∘ 531015840N 102∘ 011015840E) All seeds were grown atthe farm itself using Lob Buri Series soil black in color withclay texture and high holding capacity Measuring 72 pH thesoil contained 335 organic matter 365mgkg of P

2O5 and

197mgkg of K2O All planting took place in dry season on

three dates 19 December 2009 26 December 2009 and 15January 2010

Cultural practicewas the same for all varieties on all datesEach variety occupied a two-row plot 10m long and 05mapart The seeds were planted 3 per hill without rhizobialinoculant Fourteen days after planting the seedlings werethinned to 2 plants per hill The crop received fertilizer at15 and 35 days after planting at the rates of 1875 5000 and1875 kgha of N P

2O5 and K

2O respectively Drip irriga-

tion was applied over the planting season Hand weedingand application of appropriate chemicals controlled weedsdiseases and pests The plants were hand-harvested at R8maturity (brown pod) and sun dried Depending on plantingdate and varietal type harvesting occurred between 8 Marchand 28 April 2010Threshing was done manually followed bywinnowing for seed precleaning

The dried seeds of each soybean variety and plantingdate were divided according to physical quality into threecategories undersized unsound and sound Undersizedseeds were separated by oblong screens withmeshmeasuring38mm times 191mm After the removal of these substandard-sized seeds the remainder was visually inspected and hand-sorted for damage (mechanical insect and fungal) andfor immaturity (green seeds) After the removal of theseldquounsoundrdquo seeds the remaining portion was characterized asldquosoundrdquo seeds suitable for planting or human consumptionAll dried seeds of all categories were then refrigerated at 10∘Cinheat-sealed plastic bags until analysis for isoflavone contentin mid-August 2010

211 Sample for Studying Effect of Soybean Genotype on Iso-flavone Content This sample comprised sound seeds of all 14varieties planted on 19 December 2009

212 Sample for Studying Effect of Planting Date on IsoflavoneContent This sample was drawn from the sound seeds of all14 varieties planted on all three dates

213 Sample for Studying Effect of Physical Seed Qualityon Isoflavone Content The planting date of 19 December2009 contributed the soybeans for determining the effectof physical seed quality on isoflavone content The samplecomprised all 14 soybean varieties and all three categories ofphysical seed quality (undersized unsound and sound)

International Journal of Agronomy 3

Table1Va

rietalcharacteristicso

f14certified

varie

tieslowast

ofsoybeanused

inthee

xperim

ents[14

15]

(a)

Varie

tyCh

iang

mai2

Chiang

mai5

Chiang

mai60

Chakkrabhand

hu1

KKU35

Nakho

nSawan

1SJ

1Certifi

edyear

1998

2006

1987

1998

1994

1986

1965

Daysto50flo

wering

31ndash35d

33ndash35d

32ndash34d

43d

37d

26ndash32d

35-36d

Daystoharvest

77d

85ndash100

d89ndash92d

111d

104d

75ndash80d

85ndash9

5dMaturity

grou

pEa

rlyMod

erate

Mod

erate

Late

Late

Early

Mod

erate

100seedsw

eight

25g

13ndash15g

16ndash20g

11g

16g

16ndash20g

11ndash15g

Proteinoilcon

tent

398202

334201

391

229

413222

466201

36791

40216

6

Dise

aser

esistance

Dow

nymild

ewRu

stRu

stbacterial

pustu

leanddo

wny

mild

ew

Dow

nymild

ewandantracno

seBa

cterialpustule

Bacterialpustuleand

soybeanmosaicv

irus

Non

e

Dise

ases

usceptibility

Rust

Non

eNon

eRu

stDow

nymild

ewand

rust

Dow

nymild

ewrust

andantracno

seRu

st

Seed

yield

1463

kgha

2419k

gha

1750ndash218

8kgha

1800

kgha

1913kgha

1538

kgha

15625ndash1875

kgha

Recommended

plantin

glocatio

nAllregion

sAllregion

sAllregion

sAllregion

sLo

wer

northern

centraland

northeastern

region

s

Lower

northern

and

upperc

entralregion

s

Northern

central

andno

rtheastern

region

sRe

commended

plantin

gseason

Allseason

sAllseason

sAllseason

sEa

rlyrainyanddry

season

sAllseason

sRa

inyseason

Rainyseason

lowast

Certified

byDepartm

ento

fAgricultureM

inistry

ofAgricultureTh

ailand

(b)

Varie

tySJ

2SJ

4SJ

5Srisa

mrong

1Sukh

othai1

Sukh

othai2

Sukh

othai3

Certifi

edyear

1965

1976

1980

2007

1986

1995

1999

Daysto50flo

wering

36d

34-35d

33ndash35d

26ndash28d

30ndash35d

29d

39-40d

Daystoharvest

92ndash9

7d89ndash9

8d88ndash9

6d72ndash77d

83ndash88d

82ndash88d

90ndash93d

Maturity

grou

pMod

erate

Mod

erate

Mod

erate

Early

Mod

erate

Mod

erate

Mod

erate

100seedsw

eight

11ndash15g

15ndash18g

13ndash16g

13ndash15g

9ndash12g

13ndash15g

11ndash13g

Proteinoilcon

tent

388213

390205

41919

3

35914

5

364232

37490

39512

2

Dise

aser

esistance

Bacterialpustule

Rustand

antracno

se

Rust

soybean

mosaicv

irus

antracno

seand

purpleseed

stain

Dow

nymild

ewBa

cterialpustuleand

soybeanmosaicv

irus

Bacterialpustule

downy

mild

ewand

soybeanmosaicv

irus

Antracnosea

nddo

wny

mild

ew(m

oderate)


(b)Con

tinued

Varie

tySJ

2SJ

4SJ

5Srisa

mrong

1Sukh

othai1

Sukh

othai2

Sukh

othai3

Dise

ases

usceptibility

Rustanddo

wny

mild

ewBa

cterialpustule

Bacterialpustule

Non

ePu

rpleseed

stain

and

rust

Rust

Bacterialpustule

Seed

yield

1250ndash

1875

kgha

1875

kgha

200

0kgha

1819kgha

1538

kgha

1813ndash1938

kgha

1863

kgha

Recommended

plantin

glocatio

nAllregion

sNorthernand

centralregions

Allregion

sLo

wer

northern

region

Upp

ercentralregion

Lower

northern

and

centralregions

Allregion

s

Recommended

plantin

gseason

Laterainyand

dryseason

sAllseason

sWidely

adapted

varie

tyNA

05568

1384

Allseason

sbut

not

suitablefor

middlea

ndlater

ainy

season

sin

uppern

orthernregion

Allseason

sbut

not

suitablefor

middleand

later

ainy

season

sin

uppern

orthernregion

Allregion

s

lowast

Certified

byDepartm

ento

fAgricultureM

inistry

ofAgricultureTh

ailand


214 Sample for Studying Effect of Storage Condition onIsoflavone Content Sound seeds of the 14 soybean varietiesgrown on 19 December 2009 were randomized into 2 por-tions One portion was packed in heat-sealed plastic bags andrefrigerated at 10∘C The other portion was packed in paperbags and stored under ambient condition After 18 monthsof storage on 7 February 2012 both portions were analyzedfor isoflavone content The results were then compared withbaseline values obtained in mid-August 2010 (see above)

22 Description of Chiangmai 60 from4 Agricultural Research Centers

221 Sample for Studying Effect of Planting Location on Iso-flavone Content and Its Correlation to Seed Viability Thesample consisted of 38 lots of sound seeds of Chiangmai60 grown in dry season of crop year 2010 at 4 agriculturalresearch centers located in 4 different regions of ThailandThe name location and contribution of the centers wereas follows Chiang Mai Field Crops Research Center uppernorthern region (18∘141015840N 92∘31015840E) 17 lots Phitsanulok Agri-cultural Research and Development Center lower northernregion (16∘151015840N 100∘231015840E) 15 lots Loei Agricultural Researchand Development Center north-eastern region (17∘241015840N101∘441015840E) 4 lots and Lop Buri Agricultural Research andDevelopment Center central region (14∘171015840N 100∘501015840E) 2lots All seeds were refrigerated in heat-sealed plastic bagsuntil the determination of isoflavone content and seedviability in mid-August 2010 (see below) The relationshipbetween seed viability (determined by percentage of seedfield emergence) and isoflavone contentwas then analyzed forcorrelation

222 Sample for Studying Effect of Crop Year on IsoflavoneContent Chiang Mai Field Crops Research Center provided30 lots of sound seeds of Chiangmai 60 all grown in dryseason as follows crop year 2010 (17 lots) crop year 2009 (5lots) and crop year 2008 (8 lots) All seeds were refrigeratedin heat-sealed plastic bags until analysis in mid-August 2010

223 Determination of Seed Field Emergence Seed viabilitywas expressed as percentage of seed field emergence All test-ing took place in concrete plots 190m times 080m in a plastic-roofed house at SUTFarmThe various soybean samples werehand-planted in three rows of 50 seeds each The soil wassandy clay loam The plots were watered immediately afterplanting and every day thereafter Healthy seedlings werecounted at 15 days after sowing

23 Extraction and Quantification of Isoflavones in All Soy-bean Samples Preparation of the soybean samples for isofla-vone quantification followed amodification ofmethods takenfrom Barnes et al [16] and from the AOAC Method [17]Briefly 200mg of raw soybeans with seed coat was groundmixed with 10mL of 80 methanol in water sonicated for30 minutes and stirred for 24 h at room temperature Next350 120583L of 2M of sodium hydroxide (NaOH) was added andmixed followed by 250120583L of 100 acetic acid One mL ofthis solutionwas centrifuged Ten 120583L of clear supernatant was

then mixed with 30 120583L of mobile phase B (see below) andspiked with 10 120583L of internal standard (IS 50000 ngmL offluorescine) Five 120583L of this mixture was injected into a highperformance liquid chromatography (HPLC) system Eachsoybean sample was extracted and assayed in triplicate

The samples were analyzed on a C18 column (Inertsil150mm times 46 ID 5 120583m GL Science Tokyo Japan) with aC18 guard column (Inertsil ODS-3 10mm times 40 ID 5 120583mGL Science Tokyo Japan) The chromatography condit-ion consisted of two mobile phases Mobile phase A was55mM ammonium acetate in distilled wateracetonitrilemethanol (250 45 45 vvv) Mobile phase B was 55mMammonium acetate in distilled wateracetonitrilemethanol(250 255 220 vvv) Both mobile phases contained 29120583Lof perchloric acid and 250 120583L of 144mM sodium dodecylsulfate Separationwas performed at 25∘CTheflow rate of themobile phase was maintained at 1mLmin and analyses weredetected by UV absorption at 259 nm Standard isoflavones(daidzin genistin daidzein and genistein) were spiked inserial dilution to obtain a standard calibration curve of 93751875 375 750 1500 3000 and 6000 ngmL The regressionequations for testing the linearity of standard calibrationcurves were as follows

119910 = 02373119909 + 00003 (1199032

= 1) for daidzin119910 = 05115119909 + 25752 (119903

2

= 1) for genistin119910 = 02994119909 + 04664 (119903

2

= 09998) for daidzein119910 = 0396119909 + 04456 (119903

2

= 09999) for genistein

The chromatogram of isoflavones and their retentiontimes is shown in Figure 2(a) The isoflavone concentrationsof unknown samples were determined by using a calibrationcurve of the peak height ratios of isoflavones and IS versusrespective isoflavone concentrations with use of linear regres-sion

24 Assay Validation Five quality control (QC) samples spi-ked at 3 different concentrations of daidzin genistin daid-zein and genistein (28125 2750 and 5500 ngmL) were ana-lyzed for intraday and interday assay validation The averagecoefficient of intraday assay validation for daidzin genistindaidzein and genistein was 241 plusmn 164 163 plusmn 100 301 plusmn202 and 203 plusmn 047 respectively The average coefficientof interday assay validation for daidzin genistin daidzeinand genistein was 143 plusmn 058 190 plusmn 068 199 plusmn 135and 170 plusmn 073 respectively The Lower limit of quantifi-cation (LLQ) was defined as the lowest concentration on thecalibration curve (9375 ngmL) that could be measured withacceptable precision (coefficient of variation (CV) of less thanplusmn 20)

25 Statistical Analysis The isoflavone content in the soy-bean samples was presented as mean plusmn standard deviationDifferences among the sample means were analyzed by usingone-way analysis of variance (ANOVA) with the post hocleast significant difference (LSD) test 119875 lt 005 being consi-dered significant The coefficient of determination value (1199032)calculated by linear regression analysis was used to evaluate


(min)0 2 4 6 8 10 12 14 16

(mAU

)

(mAU

)

0

20

40

0

20

40

631

6 da

idzi

n 11

592

872

0 ge

nisti

n 24

485

988

6 IS

786

4

114

21 d

aidz

ein

1454

4

131

83 g

enist

ein

1930

5Retention timeNameHeight

(a)

0 2 4 6 8 10 12 14 16

0

20

40

0

20

40

631

4 da

idzi

n 63

726

741

1562

872

7 ge

nist

in 1

6794

939

0 59

99

923

IS 8

070

(Dai

dzei

n)

(Gen

istei

n)

(min)

(mAU

)

(mAU

)

Retention timeNameHeight

(b)

Figure 2 (a) Chromatogram of isoflavones in a standard mixture of daidzin genistin daidzein and genistein (6000 ngmL each) andinternal standard (IS fluorescein 10000 ngmL) (b) Chromatogram of soybean seed sample (SJ 2 grown at SUT Farm) extracted with 80methanol plus 2M NaOH

the correlation between the percentages of field emergenceand isoflavone content

3 Results and Discussion

31 Isoflavone Profiles The 120573-glycosides daidzin and genistinwere the predominant forms of isoflavones found in all soy-bean samplesThe corresponding aglycone forms of daidzeinand genistein were undetectable or detectable in negligibleamounts in some soybean samples Glycitin was not deter-mined in this study The chromatogram for soybean varietySJ 2 is representative of the studyrsquos findings (Figure 2(b))

Previous studies have demonstrated that twomalonyl gly-cosides (610158401015840-O-malonlydaidzin and 610158401015840-O-malonlygenistin)are the major isoflavone derivatives in soybean seeds [18ndash20] whereas aglycone forms which derive from enzymatichydrolysis during fermentation are the predominant formsin fermented soy foods such as miso and tempeh [16 21] Forthis reason none or only negligible amounts of the aglyconeforms were detected in the unfermented soybean samplesreported here and elsewhere [3]

However the predominant forms of isoflavones extractedin the present study were the 120573-glycosides daidzin andgenistin instead of the malonyl glycosides as previouslyreported elsewhere This discrepancy resulted from the con-version of mostly malonyl glycosides to 120573-glycosides underthe increased pH condition elicited by the application ofNaOH during sample extraction [22] Glycitein and its glyco-side derivatives were not determined in this study due to theirnaturally smaller occurrence in soybeans [2 3] Given thestudyrsquos assay methodology and overall goals the summationof daidzin and genistin contents would seem to provide asufficiently accurate measure of the total isoflavone contentof the soybean samples under analysis The assay method issimple and easy to use Its inability to differentiate malonyland acetyl glycoside conjugates from 120573-glycoside conjugatesseems a minor limitation considering that such conjugatecompounds are finally hydrolyzed to aglycone forms by 120573-glucosidase enzyme prior to human gastrointestinal absorp-tion

32 Effect of Seed Variety on Isoflavone Content Amongsound seeds of 14 soybean varieties grown under the samecultural practice on 19 December 2009 mean isoflavone con-tent varied widely (Figure 3) Aglycone forms were undete-ctable except for varieties SJ 2 Sukhothai 3 and KKU 35which showed trace amounts (approximately 0017ndash0039mgg)

The lowest isoflavone yields were 034mgg of daidzinand 031mgg of genistin in the Sukhothai 1 variety whereasthe highest were 091mgg of daidzin and 108mgg of geni-stin in the SJ 2 variety The mean isoflavone content in 11 outof 14 soybean varieties was significantly higher than that ofthe Sukhothai 1 variety

Although environmental factors cannot be ruled out thedata derived from this study continue to emphasize the sig-nificant influence that soybean genotypes exert on isoflavonecontent Previous work with 60 Korean varieties grown inKorea reported mean isoflavone values ranging from 162to 252mgg [23] while a study of 210 soybean varietiesgrown in South Dakota United States found variationsfrom 116 to 274 [24] and still another study of 7 Ameri-can soybean varieties reported concentrations ranging from205 to 422mgg [18] In keeping with these previousresults the mean isoflavone content of Thai soybean vari-eties grown from sound seeds in Thailand was found torange widely from 065mgg (Sukhothai 1 grown at SUTFarm) to 275mgg (Chiangmai 60 obtained from Phitsan-ulok Agricultural Research and Development Center) (seebelow)

Morever the daidzin genistin ratio in 14 Thai soybeanvarieties investigated in this study varied from 1 09 to 1 21which was comparable to the ratio of 1 15 to 1 2 in varioussoybean products as reported in previous research [25]Indeed the proportion of genistin and its glycoside conju-gates in soybeans might be of significant pharmacologicalimportance since genistein is a relatively potent agonist forthe 120573-isoform of the estrogen receptor (ER120573) which mightexplain its clinical efficacy especially in postmenopausalhealth [26]


00

05

10

15

20

25Daidzin genistin ratio

Isofl

avon

e con

tent

s1(m

gg)

1 09lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowastlowastlowast

Sukh

otha

i 1

Chia

ngm

ai 6

0

Chak

krab

hand

hu 1

Chin

gmai

5

SJ 4

Chin

gmai

2

Nak

hon

Swan

1

Srisa

mro

ng 1

SJ 5

SJ 1

KKU

35

Sukh

otha

i 2

Sukh

otha

i 3 SJ 2

GenistinDaidzin

1 15 1 11 1 1 1 12 1 121 12 1 211 13 1 09 1 09 1 1 1 1 1 17

Figure 3 Isoflavone content in sound seeds of 14 soybean varieties planted at SUT Farm on 19 December 2009 Sound seeds of each soybeanvariety were extracted and assayed in triplicate (119899 = 3) Bars and error bars represent mean plusmn standard deviation lowast119875 lt 005 compared toSukhothai 1 1Isoflavone content was the summation of daidzin and genistin contents

Table 2 Isoflavone content1 (mgg) in sound seeds of 14 soybean varieties planted on different dates in the same crop year at SUT Farm

Variety Planting date19 December 2009 26 December 2009 15 January 2010

Chiangmai 2 108 plusmn 004 068 plusmn 002lowast

038 plusmn 001lowastdagger





Chakkrabhandhu 1 076 plusmn 010 064 plusmn 004 NDKKU 35 165 plusmn 009 122 plusmn 002

lowast NDNakhon Sawan 1 113 plusmn 004 152 plusmn 004

lowast

150 plusmn 005lowast

SJ 1 149 plusmn 004 136 plusmn 001lowast


SJ 2 199 plusmn 003 202 plusmn 004 070 plusmn 005lowastdagger




Srisamrong 1 115 plusmn 001 119 plusmn 003lowast


Sukhothai 1 065 plusmn 003 168 plusmn 006lowast






Mean plusmn SD 127 plusmn 044 113 plusmn 042 072 plusmn 034lowastdagger

Each soybean sample was extracted and assayed in triplicate (119899 = 3) Data represent mean plusmn standard deviation lowast119875 lt 005 compared to 19 December 2009dagger

119875 lt 005 compared to 26 December 2009 ND not determined due to lack of soybean seed samples 1Isoflavone content was the summation of daidzin andgenistin contents

33 Effect of Planting Date in the Same Crop Year on IsoflavoneContent in 14 Soybean Varieties Isoflavone content in soundseeds of 14 soybean varieties planted under the same cul-tural practice on 19 December 2009 26 December 2009and 15 January 2010 is shown in Table 2 Mean isoflavone

content ranged from 072 to 127mgg with the cohort of15 January 2010 generally expressing a significantly lowervalue than those of 19 December 2009 and 26 December2009 However 2 soybean varieties (Nakhon Sawan 1 andSukhothai 1) planted on 15 January 2010 had significantly


00

04

08

12

16

20

Undersized seeds Unsound seeds Sound seeds

GenistinDaidzin

Isofl

avon

e con

tent

s1(m

gg)

lowast

lowastdagger

Figure 4 Isoflavone content in 14 soybean varieties planted at SUTFarm on 19 December 2009 categorized according to physical seedquality Each soybean sample was extracted and assayed in triplicate(119899 = 3) Bars and error bars represent mean plusmn standard deviationlowast

119875 lt 005 compared to undersized seeds dagger119875 lt 005 compared tounsound seeds 1Isoflavone content was the summation of daidzinand genistin contents

higher mean values than those planted on 19 December2009

The Thai Department of Agriculture has generally enco-uraged farmers to plant soybeans early in dry season [15] aslate sowing increases the risk of exposing the crop to rain andhigher temperatures thereby leading to seed deteriorationand smaller harvests Early sowing in dry season also usuallymeans that the soybean crop will be exposed to lowertemperatures during the seed-ripening phase As Tsukamotoet al [27] have found that such climatic conditions cansignificantly increase total isoflavone yields The presentstudy appeared to support these findings by demonstratinghow earlier planting dates of only a few weeks during theDecember-January dry season of 2009-2010 significantly affe-cted mean isoflavone content To achieve maximum isofla-vone yields it therefore seems imperative to match soybeanvariety with appropriate planting date In addition NakhonSawan 1 appears to be of special interest because in thisstudy it persistently exhibited moderately high isoflavonecontent regardless of sowing date or environment Seguinet al [13] also identified soybean varieties with consistentlyhigh and low isoflavone content when grown across a rangeof environments Further studies are warranted to confirmthe advantages of Nakhon Sawan 1 and to identify the mostappropriate planting dates for Thai soybean varieties in eachseason

34 Effect of Physical Seed Quality on Isoflavone Content in14 Soybean Varieties Mean isoflavone content in undersizedseeds unsound seeds and sound seeds of 14 soybean varietiesplanted on 19 December 2009 under the same culturalpractice is presented in Figure 4 The mean value in soundseeds was significantly greater than in unsound seeds and

00

04

08

12

16

20

Baseline After storage in

for 18 months

After storage at ambientcondition in paperbags for 18 months

Aglycones120573-glycosides

Isofl

avon

e con

tent

s1 (mg

g)

refrigerator (10∘C)

Figure 5 Isoflavone content in sound seeds of 14 soybean varietiesplanted at SUT Farm on 19 December 2009 at baseline and afterstorage under different conditions Sound seeds of each soybeanvariety were extracted and assayed in triplicate Bars and error barsrepresent mean plusmn standard deviation 1Isoflavone content was thesummation of daidzin and genistin contents

undersized seeds Similarly the mean value in unsound seedswas significantly greater than in undersized seeds Thesefindings suggest that selection of sound seeds as rawmaterialsin the manufacturing of soy and soy-based products is essen-tial for isoflavone-rich food products Generally undersizedseeds are young seeds at R5-R6 (beginning seed to green full-size seed) stages These stages coincide with the beginning ofisoflavone production [28] Total isoflavone concentrationsare lowest at R5 but increase steadily reaching their peakat R8 (harvest maturity) stage Therefore lower isoflavoneyields should be expected from soybean varieties producinghigh percentages of undersized seeds whether as a responseto environmental conditions inhibiting the development ofseed maturity or as a result of indeterminate growth habitDeterminate soybean varieties start flowering at all nodealmost at the same time when the plants have reachedmaximum height and the addition of node number on themain stem ceases abruptly at R1 In contrast indeterminatevarieties start flowering when the plants have reached 30ndash50 of their maximum height and still continue to increaseboth vegetative growth and height while the node numberon the main stem stops at R5 This results in a longerduration of flowering and prolonged ripening period in theindeterminate varieties [29 30] Accordingly variation inseed size within a seed lot is greater in indeterminate varietiesthan that of determinate varieties [31]

35 Effect of Storage Condition on Isoflavone Content in 14Soybean Varieties Mean isoflavone contents in sound seedsof 14 soybean varieties stored under different conditionsfor 18 months were compared to mean values at base-line (Figure 5) The various samples showed no significant


0

1

2

3

4

Isofl

avon

e con

tent

s1(m

gg)

Phitsanulok Loei Lob Buri

dagger

Dagger lowastdaggerDagger

Chiang Mai Loei

Phitsanulok

Lob Buri

Chiang Mai(n = 17 lots) (n = 15 lots) (n = 4 lots) (n = 2 lots)

Figure 6 Isoflavone content in sound seeds of Chiangmai 60soybean variety grown in dry season crop year 2010 and obtainedfrom 4 agricultural research centers in Thailand The soybeansamples from each site were from different lots Each lot wasextracted and assayed in triplicate lowast119875 lt 005 compared to ChiangMai province dagger119875 lt 005 compared to Loei province Dagger119875 lt 005compared to Phitsanulok province Bars and error bars representmean plusmn standard deviation 1Isoflavone content was the summationof daidzin and genistin contents

differences whether they were refrigerated at 10∘C for 18months or stored at ambient condition for the same periodThis observation is in agreement with previous researchwhich investigated the effects of soybean storage at 5∘C for18 months [32]

However long-term exposure to ambient condition cau-sed a slight increase in the aglycone forms of isoflavones (dai-dzein and genistein) in comparison to the negligible amountsdetected at baseline or after equivalent long-term refrigera-tion The increased proportion of aglycones after long-termstorage at ambient condition in this study suggests that thisphenomenon possibly results from a spontaneous conversionof some glycoside conjugates to aglycones This conversionseems unlikely to affect the benefits of soybeans consumptionbecause the oral bioavailability of aglycones and glycosideconjugates is equivalent [33] In contrast a Korean studyof soybean varieties has shown that a longer storage periodof 3 years does significantly decrease isoflavone content forsamples stored at both a lower temperature of minus30∘C and atambient condition [20] This research should be replicatedusingThai varieties

36 Effect of Planting Location on Isoflavone Content in Chi-angmai 60 Mean isoflavone content in sound seeds of Chia-ngmai 60 grown in the crop year 2010 at 4 different researchcenters is presented in Figure 6 Values varied significantly

0

1

2

3

4

GenistinDaidzin

Isofl

avon

e con

tent

s1(m

gg)

The year 2008(n = 8 lots)


The year 2010

Crop year(n = 17 lots)

lowast lowast

Figure 7 Isoflavone content in sound seeds of Chiangmai 60soybean variety grown in different crop years and obtained fromChiang Mai Field Crops Research Center The soybean samplesfrom each crop year were obtained from different lots Each lot wasextracted and assayed in triplicate lowast119875 lt 005 compared to theyear 2008 Bars and error bars represent mean plusmn standard deviation1Isoflavone content was the summation of daidzin and genistincontents

from 122 plusmn 124 to 275 plusmn 033mgg Such values were com-parable to previously reported variations of 118 to 175mggfor a single variety (Vinton 81) from different locations inIowa USA [18] The highest isoflavone concentrations werefound in the samples from Phitsanulok Province followedby Loei Province Chiang Mai Province and Lop BuriProvince respectively Soybean seeds obtained fromLob BuriAgricultural Research and Development Center in 2010 gavethe lowest isoflavone content perhaps reflecting the highertemperatures customary at Lob Buri during soybean matu-ration as compared to temperatures at the other locationssituated at lower latitudes

37 Effect of Crop Year on Isoflavone Content in Chiangmai60 Mean isoflavone content in sound seeds of Chiangmai 60grown in different crop years at the same location is shownin Figure 7 Values ranged from 105 to 121mgg in 2008227 to 295mgg in 2009 and 179 to 317mgg in 2010Plantings from crop year 2008 were significantly lower inmean isoflavone content than those from crop years 2009 and2010These data suggest the remarkable influence of differingcrop years on isoflavone content [11] Again such values werein accordance with previously reported variations of 118 to330mgg for soybeans grown in Iowa in different years [18]

38 Correlation between Percentage of Seed Field Emergenceand Isoflavone Content in Chiangmai 60 Figure 8 depicts thecorrelation between percentage of seed field emergence andisoflavone content in 38 different lots of Chiangmai 60 grownin the crop year 2010 The percentage of field emergencein these soybean samples ranged from 17 to 79 (average5688 plusmn 1601) There was no correlation between theseparameters (1199032 = 0186) Furthermore Thai soybeans inthis study remained stable in isoflavone content regardless of


0

20

40

60

80

100

0 05 1 15 2 25 3 35

Fiel

d em

erge

nce (

)

Chiang MaiPhitsanulok

LoeiLob Buri

Isoflavone contents1 (mgg)

y = 1339x + 2285 r2 = 0186

Figure 8 The coefficient of determination value (1199032) calculated bylinear regression analysis between percentages of seed field emer-gence and total isoflavone content in sound seeds of Chiangmai 60soybean variety grown in dry season crop year 2010 (119899 = 38 differentlots) and obtained from 4 agricultural research centers in ThailandEach lot was extracted and assayed in triplicate 1Isoflavone contentwas the summation of daidzin and genistin contents

storage conditions within 18 months (see above) It thereforeappears that leftover soybean seeds from seed shops orwarehouses that are no longer suitable for planting can stillbe used as rawmaterials in themanufacturing of soy and soy-based products without sacrificing isoflavone content

39 Study Limitations Although isoflavone values reportedhere seemed sufficiently accurate for the studyrsquos comparativepurposes it must be noted that the lack of measurement ofglycitin and its derivatives underestimated total isoflavonecontent by 5ndash10 Similarly the use of NaOH in the quan-tification assay probably depressed isoflavone values in somedegree because of possible incomplete conversion of malonyland acetyl glycoside conjugates to their respective daidzinor genistin forms Finally the investigation of differentialplanting dates on isoflavone content was conducted withina short period of time represented by dry season December2009 to January 2010 Other planting dates in other seasonsshould be further investigated

4 Conclusions

Isoflavone content in Thai soybeans varied considerablydepending on multiple genetic and environmental factorssuch as variety physical seed quality crop year planting date(even in the same crop year) and planting location Nonethe-less some striking varieties (Nakhon Sawan 1 and Sukhothai1) exhibited moderately high isoflavone content regardlessof sowing date Furthermore soybeans with decreased seedviability were shown to retain their isoflavone content

Acknowledgments

This work was supported by the Faculty of Medicine ChiangMai University Thailand The authors are grateful for theeditorial assistance of Jeffrey Hess an English language con-sultant under contract with the Faculty of Medicine ChiangMai University The authors would also like to acknowledgeMs Sujitra Techatoei for her assistance in extraction andquantification of isoflavones in all soybean samples

References

[1] KD R Setchell ldquoPhytoestrogens the biochemistry physiologyand implications for human health of soy isoflavonesrdquo TheAmerican Journal of Clinical Nutrition vol 68 no 6 pp 1333Sndash1346S 1998

[2] T Song K Barua G Buseman and P A Murphy ldquoSoy iso-flavone analysis quality control and a new internal standardrdquoThe American Journal of Clinical Nutrition vol 68 no 6 pp1474Sndash1479S 1998

[3] S Jung P A Murphy and I Sala ldquoIsoflavone profiles of soy-milk as affected by high-pressure treatments of soymilk andsoybeansrdquo Food Chemistry vol 111 no 3 pp 592ndash598 2008

[4] A Atmaca M Kleerekoper M Bayraktar and O Kucuk ldquoSoyisoflavones in the management of postmenopausal osteoporo-sisrdquoMenopause vol 15 no 4 pp 748ndash757 2008

[5] D-F Ma L-Q Qin P-Y Wang and R Katoh ldquoSoy isoflavoneintake increases bone mineral density in the spine of meno-pausal women meta-analysis of randomized controlled trialsrdquoClinical Nutrition vol 27 no 1 pp 57ndash64 2008

[6] S-H Li X-X Liu Y-Y Bai et al ldquoEffect of oral isoflavonesupplementation on vascular endothelial function in post-menopausal women a meta-analysis of randomized placebo-controlled trialsrdquo The American Journal of Clinical Nutritionvol 91 no 2 pp 480ndash486 2010

[7] C R Cederroth and S Nef ldquoSoy phytoestrogens and metabo-lism a reviewrdquo Molecular and Cellular Endocrinology vol 304no 1-2 pp 30ndash42 2009

[8] R D rsquoAnna M L Cannata H Marini et al ldquoEffects of the phy-toestrogen genistein on hot flushes endometrium and vaginalepithelium in postmenopausal women a 2-year randomizeddouble-blind placebo-controlled studyrdquoMenopause vol 16 no2 pp 301ndash306 2009

[9] V S Lagari and S Levis ldquoPhytoestrogens and bone healthrdquoCurrent Opinion in Endocrinology Diabetes and Obesity vol 17no 6 pp 546ndash553 2010

[10] D Zhu N S Hettiarachchy R Horax and P Chen ldquoIsoflavonecontents in germinated soybean seedsrdquo Plant Foods for HumanNutrition vol 60 no 3 pp 147ndash151 2005

[11] J A Hoeck W R Fehr P A Murphy and G A WelkeldquoInfluence of genotype and environment on isoflavone contentsof soybeanrdquo Crop Science vol 40 no 1 pp 48ndash51 2000

[12] S J LeeW Yan J K Ahn and IM Chung ldquoEffects of year sitegenotype and their interactions on various soybean isoflavonesrdquoField Crops Research vol 81 no 2-3 pp 181ndash192 2003

[13] P Seguin W Zheng D L Smith and W Deng ldquoIsoflavonecontent of soybean cultivars grown in eastern Canadardquo Journalof the Science of Food and Agriculture vol 84 no 11 pp 1327ndash1332 2004

[14] B Jumroonpong W Somprasong and P Pankhaew Plant Ger-mplasm Database Department of Agriculture Ministry of


Agriculture and Cooperatives The Agricultural CooperativesFederation of Thailand 2004

[15] S Inthipong and R Sopa Soybean Cultivation Departmentof Agriculture Ministry of Agriculture and Cooperatives IdeaSquare Thailand 2004

[16] S Barnes M Kirk and L Coward ldquoIsoflavones and their conj-ugates in soy foods extraction conditions and analysis byHPLC-mass spectrometryrdquo Journal of Agricultural and FoodChemistry vol 42 no 11 pp 2466ndash2474 1994

[17] AOAC official method 200110 Determination of Isoflavones inSoy and Selected Foods Containing Soy Extraction Saponifica-tion and Liquid Chromatography Official Methods of Analysis17th edition 2000

[18] H-J Wang and P A Murphy ldquoIsoflavone composition of Ame-rican and Japanese soybeans in Iowa effects of variety crop yearand locationrdquo Journal of Agricultural and Food Chemistry vol42 no 8 pp 1674ndash1677 1994

[19] S Kudou Y Fleury D Welti et al ldquoMalonyl isoflavone glyco-sides in soybean seeds (Glycine max Merrill)rdquo Agricultural andBiological Chemistry vol 55 pp 2227ndash2233 1991

[20] J J Kim S H Kim S J Hahn and I M Chung ldquoChangingsoybean isoflavone composition and concentrations under twodifferent storage conditions over three yearsrdquo Food ResearchInternational vol 38 no 4 pp 435ndash444 2005

[21] H-J Wang and P A Murphy ldquoIsoflavone content in commer-cial soybean foodsrdquo Journal of Agricultural and Food Chemistryvol 42 no 8 pp 1666ndash1673 1994

[22] V Yerramsetty K Mathias M Bunzel and B Ismail ldquoDetec-tion and structural characterization of thermally generatedisoflavonemalonylglucoside derivativesrdquo Journal of Agriculturaland Food Chemistry vol 59 no 1 pp 174ndash183 2011

[23] K J Yang and I M Chung ldquoYearly and genotypic variationsin seed isoflavone content of local soybean cultivarsrdquo KoreanJournal of Crop Science vol 46 pp 139ndash144 2001

[24] C Wang M Sherrard S Pagadala R Wixon and R A ScottldquoIsoflavone content among maturity group 0 to II soybeansrdquoJournal of the American Oil Chemistsrsquo Society vol 77 no 5 pp483ndash487 2000

[25] J J B Anderson M Anthony M Messina and S C GarnerldquoEffects of phyto-oestrogens on tissuesrdquo Nutrition ResearchReviews vol 12 no 1 pp 75ndash116 1999

[26] M F McCarty ldquoIsoflavones made simplemdashgenisteinrsquos agonistactivity for the beta-type estrogen receptormediates their healthbenefitsrdquoMedical Hypotheses vol 66 no 6 pp 1093ndash1114 2006

[27] C Tsukamoto S Shimada K Igita et al ldquoFactors affecting iso-flavone content in soybean seeds changes in isoflavones sapo-nins and composition of fatty acids at different temperaturesduring seed developmentrdquo Journal of Agricultural and FoodChemistry vol 43 no 5 pp 1184ndash1192 1995

[28] J-A Kim and I-M Chung ldquoChange in isoflavone concen-tration of soybean (Glycine max L) seeds at different growthstagesrdquo Journal of the Science of Food and Agriculture vol 87no 3 pp 496ndash503 2007

[29] S Belfield C Brown and M Martin Soybean Guide to UplandCropping in Cambodia ACIAR Monograph no 146 AustralianCentre for International Agricultural Research Canberra Aus-tralia 2011

[30] P Pedersen ldquoSoybean physiology yield maturity groups andgrowth stagesrdquo 2013 httpwwwplantmanagementnetworkorg

[31] D B Egli R AWiralaga and E L Ramseur ldquoVariation in seedsize in soybeanrdquo Agronomy Journal vol 79 no 3 pp 463ndash4671987

[32] H J Hou and K C Chang ldquoInterconversions of isoflavones insoybeans as affected by storagerdquo Journal of Food Science vol 67no 6 pp 2083ndash2089 2002

[33] X Xu H-J Wang P A Murphy and S Hendrich ldquoNeitherbackground diet nor type of soy food affects short-term iso-flavone bioavailability in womenrdquo Journal of Nutrition vol 130no 4 pp 798ndash801 2000

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Food ScienceInternational Journal of

Agronomy


International Journal of



Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in


PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of


ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of


Plant GenomicsInternational Journal of


Biotechnology Research International


Forestry ResearchInternational Journal of


Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of


Veterinary Medicine International


Cell BiologyInternational Journal of


Evolutionary BiologyInternational Journal of



Aglycones

Daidzein H HGenistein OH H

Glycitein H

R1

R1

R2

R2

OCH3

OHO

OH

O

(a)

Glycosides

Daidzin (120573-glycoside) H H HGenistin (120573-glycoside) OH H H

Glycitin (120573-glycoside) H H

H H

OH H

H

H H

OH H

H

6998400998400-O-Malonlydaidzin

6998400998400-O-Malonlygenistin

6998400998400-O-Malonlyglycitin

6998400998400-O-Acetyldaidzin

6998400998400-O-Acetylgenistin

6998400998400-O-Acetylglycitin

R3

R3

R4

R4

R5

OCH3

OCH3

OCH3

COCH2COOH

COCH2COOH

COCH2COOH

COCH3

COCH3

COCH3

O

O

OOOH

OHOHHO

CH2OR5

(b)

Figure 1 Structure of isoflavones in the forms of aglycones (a) 120573-gly-cosides 610158401015840-O-acetyl-120573-glycosides and 610158401015840-O-malonyl-120573-glyc-osides (b)

deficiency by investigating 14 Thai soybean varieties includ-ing Chiangmai 60 one of the most popular varieties

The study consisted of two basic parts The first partsought to determine the isoflavone content of the 14 varietiesgrown at the same location but with variations in plantingdate physical seed quality and storage conditionThe secondpart targeted sound seeds of Chiangmai 60 Specificallyit examined the effects of planting location and crop yearon total isoflavone content and analyzed the relationshipbetween isoflavone content and seed viability

2 Materials and Methods

21 Description and Preparation of 14 SoybeanVarieties Grownat the Same Location Covering a broad range of pedigreesand physical types the 14 soybean varieties represented the

vast majority of soybeans grown inThailand In alphanumer-ical order they were Chiangmai 2 Chiangmai 5 Chiangmai60 Chakkrabhanhu 1 KKU 35 Nakhon Sawan 1 SJ 1 SJ 2SJ 4 and SJ 5 Srisamrong 1 Sukhothai 1 Sukhothai 2 andSukhothai 3 Significant characteristics are summarized inTable 1 [14 15]

The 14 soybean varieties were collected from the Surana-ree University of Technology (SUT) Farm located in NakhonRatchasima Province in northeasternThailand about 250 kmeast of Bangkok (14∘ 531015840N 102∘ 011015840E) All seeds were grown atthe farm itself using Lob Buri Series soil black in color withclay texture and high holding capacity Measuring 72 pH thesoil contained 335 organic matter 365mgkg of P

2O5 and

197mgkg of K2O All planting took place in dry season on

three dates 19 December 2009 26 December 2009 and 15January 2010

Cultural practicewas the same for all varieties on all datesEach variety occupied a two-row plot 10m long and 05mapart The seeds were planted 3 per hill without rhizobialinoculant Fourteen days after planting the seedlings werethinned to 2 plants per hill The crop received fertilizer at15 and 35 days after planting at the rates of 1875 5000 and1875 kgha of N P

2O5 and K

2O respectively Drip irriga-

tion was applied over the planting season Hand weedingand application of appropriate chemicals controlled weedsdiseases and pests The plants were hand-harvested at R8maturity (brown pod) and sun dried Depending on plantingdate and varietal type harvesting occurred between 8 Marchand 28 April 2010Threshing was done manually followed bywinnowing for seed precleaning

The dried seeds of each soybean variety and plantingdate were divided according to physical quality into threecategories undersized unsound and sound Undersizedseeds were separated by oblong screens withmeshmeasuring38mm times 191mm After the removal of these substandard-sized seeds the remainder was visually inspected and hand-sorted for damage (mechanical insect and fungal) andfor immaturity (green seeds) After the removal of theseldquounsoundrdquo seeds the remaining portion was characterized asldquosoundrdquo seeds suitable for planting or human consumptionAll dried seeds of all categories were then refrigerated at 10∘Cinheat-sealed plastic bags until analysis for isoflavone contentin mid-August 2010

211 Sample for Studying Effect of Soybean Genotype on Iso-flavone Content This sample comprised sound seeds of all 14varieties planted on 19 December 2009

212 Sample for Studying Effect of Planting Date on IsoflavoneContent This sample was drawn from the sound seeds of all14 varieties planted on all three dates

213 Sample for Studying Effect of Physical Seed Qualityon Isoflavone Content The planting date of 19 December2009 contributed the soybeans for determining the effectof physical seed quality on isoflavone content The samplecomprised all 14 soybean varieties and all three categories ofphysical seed quality (undersized unsound and sound)


Table1Va


f14certified

varie

tieslowast

ofsoybeanused

inthee

xperim

ents[14

15]

(a)

Varie

tyCh

iang

mai2

Chiang

mai5

Chiang

mai60

Chakkrabhand

hu1

KKU35

Nakho

nSawan

1SJ

1Certifi

edyear

1998

2006

1987

1998

1994

1986

1965

Daysto50flo

wering

31ndash35d

33ndash35d

32ndash34d

43d

37d

26ndash32d

35-36d

Daystoharvest

77d

85ndash100

d89ndash92d

111d

104d

75ndash80d

85ndash9

5dMaturity

grou

pEa

rlyMod

erate

Mod

erate

Late

Late

Early

Mod

erate

100seedsw

eight

25g

13ndash15g

16ndash20g

11g

16g

16ndash20g

11ndash15g

Proteinoilcon

tent

398202

334201

391

229

413222

466201

36791

40216

6

Dise

aser

esistance

Dow

nymild

ewRu

stRu

stbacterial

pustu

leanddo

wny

mild

ew

Dow

nymild

ewandantracno

seBa

cterialpustule

Bacterialpustuleand

soybeanmosaicv

irus

Non

e

Dise

ases

usceptibility

Rust

Non

eNon

eRu

stDow

nymild

ewand

rust

Dow

nymild

ewrust

andantracno

seRu

st

Seed

yield

1463

kgha

2419k

gha

1750ndash218

8kgha

1800

kgha

1913kgha

1538

kgha

15625ndash1875

kgha

Recommended

plantin

glocatio

nAllregion

sAllregion

sAllregion

sAllregion

sLo

wer

northern

centraland

northeastern

region

s

Lower

northern

and

upperc

entralregion

s

Northern

central

andno

rtheastern

region

sRe

commended

plantin

gseason

Allseason

sAllseason

sAllseason

sEa

rlyrainyanddry

season

sAllseason

sRa

inyseason

Rainyseason

lowast

Certified

byDepartm

ento

fAgricultureM

inistry

ofAgricultureTh

ailand

(b)

Varie

tySJ

2SJ

4SJ

5Srisa

mrong

1Sukh

othai1

Sukh

othai2

Sukh

othai3

Certifi

edyear

1965

1976

1980

2007

1986

1995

1999

Daysto50flo

wering

36d

34-35d

33ndash35d

26ndash28d

30ndash35d

29d

39-40d

Daystoharvest

92ndash9

7d89ndash9

8d88ndash9

6d72ndash77d

83ndash88d

82ndash88d

90ndash93d

Maturity

grou

pMod

erate

Mod

erate

Mod

erate

Early

Mod

erate

Mod

erate

Mod

erate

100seedsw

eight

11ndash15g

15ndash18g

13ndash16g

13ndash15g

9ndash12g

13ndash15g

11ndash13g

Proteinoilcon

tent

388213

390205

41919

3

35914

5

364232

37490

39512

2

Dise

aser

esistance

Bacterialpustule

Rustand

antracno

se

Rust

soybean

mosaicv

irus

antracno

seand

purpleseed

stain

Dow

nymild

ewBa

cterialpustuleand

soybeanmosaicv

irus

Bacterialpustule

downy

mild

ewand

soybeanmosaicv

irus

Antracnosea

nddo

wny

mild

ew(m

oderate)


(b)Con

tinued

Varie

tySJ

2SJ

4SJ

5Srisa

mrong

1Sukh

othai1

Sukh

othai2

Sukh

othai3

Dise

ases

usceptibility

Rustanddo

wny

mild

ewBa

cterialpustule

Bacterialpustule

Non

ePu

rpleseed

stain

and

rust

Rust

Bacterialpustule

Seed

yield

1250ndash

1875

kgha

1875

kgha

200

0kgha

1819kgha

1538

kgha

1813ndash1938

kgha

1863

kgha

Recommended

plantin

glocatio

nAllregion

sNorthernand

centralregions

Allregion

sLo

wer

northern

region

Upp

ercentralregion

Lower

northern

and

centralregions

Allregion

s

Recommended

plantin

gseason

Laterainyand

dryseason

sAllseason

sWidely

adapted

varie

tyNA

05568

1384

Allseason

sbut

not

suitablefor

middlea

ndlater

ainy

season

sin

uppern

orthernregion

Allseason

sbut

not

suitablefor

middleand

later

ainy

season

sin

uppern

orthernregion

Allregion

s

lowast

Certified

byDepartm

ento

fAgricultureM

inistry

ofAgricultureTh

ailand










119910 = 02373119909 + 00003 (1199032

= 1) for daidzin119910 = 05115119909 + 25752 (119903

2

= 1) for genistin119910 = 02994119909 + 04664 (119903

2

= 09998) for daidzein119910 = 0396119909 + 04456 (119903

2






(min)0 2 4 6 8 10 12 14 16

(mAU

)

(mAU

)

0

20

40

0

20

40

631

6 da

idzi

n 11

592

872

0 ge

nisti

n 24

485

988

6 IS

786

4

114

21 d

aidz

ein

1454

4

131

83 g

enist

ein

1930


(a)

0 2 4 6 8 10 12 14 16

0

20

40

0

20

40

631

4 da

idzi

n 63

726

741

1562

872

7 ge

nist

in 1

6794

939

0 59

99

923

IS 8

070

(Dai

dzei

n)

(Gen

istei

n)

(min)

(mAU

)

(mAU

)


(b)












00

05

10

15

20


Isofl

avon

e con

tent

s1(m

gg)

1 09lowast

lowastlowast

lowast

lowastlowast


Sukh

otha

i 1

Chia

ngm

ai 6

0

Chak

krab

hand

hu 1

Chin

gmai

5

SJ 4

Chin

gmai

2

Nak

hon

Swan

1

Srisa

mro

ng 1

SJ 5

SJ 1

KKU

35

Sukh

otha

i 2

Sukh

otha

i 3 SJ 2

GenistinDaidzin

1 15 1 11 1 1 1 12 1 121 12 1 211 13 1 09 1 09 1 1 1 1 1 17












lowast






















00

04

08

12

16

20


GenistinDaidzin

Isofl

avon

e con

tent

s1(m

gg)

lowast

lowastdagger






00

04

08

12

16

20


for 18 months



Isofl

avon

e con

tent

s1 (mg

g)






0

1

2

3

4

Isofl

avon

e con

tent

s1(m

gg)


dagger


Chiang Mai Loei

Phitsanulok

Lob Buri






0

1

2

3

4

GenistinDaidzin

Isofl

avon

e con

tent

s1(m

gg)



The year 2010


lowast lowast






0

20

40

60

80

100

0 05 1 15 2 25 3 35

Fiel

d em

erge

nce (

)


LoeiLob Buri


y = 1339x + 2285 r2 = 0186




4 Conclusions


Acknowledgments


References






































Journal of




Agronomy





Microbiology




Volume 2014

























Table1Va


f14certified

varie

tieslowast

ofsoybeanused

inthee

xperim

ents[14

15]

(a)

Varie

tyCh

iang

mai2

Chiang

mai5

Chiang

mai60

Chakkrabhand

hu1

KKU35

Nakho

nSawan

1SJ

1Certifi

edyear

1998

2006

1987

1998

1994

1986

1965

Daysto50flo

wering

31ndash35d

33ndash35d

32ndash34d

43d

37d

26ndash32d

35-36d

Daystoharvest

77d

85ndash100

d89ndash92d

111d

104d

75ndash80d

85ndash9

5dMaturity

grou

pEa

rlyMod

erate

Mod

erate

Late

Late

Early

Mod

erate

100seedsw

eight

25g

13ndash15g

16ndash20g

11g

16g

16ndash20g

11ndash15g

Proteinoilcon

tent

398202

334201

391

229

413222

466201

36791

40216

6

Dise

aser

esistance

Dow

nymild

ewRu

stRu

stbacterial

pustu

leanddo

wny

mild

ew

Dow

nymild

ewandantracno

seBa

cterialpustule

Bacterialpustuleand

soybeanmosaicv

irus

Non

e

Dise

ases

usceptibility

Rust

Non

eNon

eRu

stDow

nymild

ewand

rust

Dow

nymild

ewrust

andantracno

seRu

st

Seed

yield

1463

kgha

2419k

gha

1750ndash218

8kgha

1800

kgha

1913kgha

1538

kgha

15625ndash1875

kgha

Recommended

plantin

glocatio

nAllregion

sAllregion

sAllregion

sAllregion

sLo

wer

northern

centraland

northeastern

region

s

Lower

northern

and

upperc

entralregion

s

Northern

central

andno

rtheastern

region

sRe

commended

plantin

gseason

Allseason

sAllseason

sAllseason

sEa

rlyrainyanddry

season

sAllseason

sRa

inyseason

Rainyseason

lowast

Certified

byDepartm

ento

fAgricultureM

inistry

ofAgricultureTh

ailand

(b)

Varie

tySJ

2SJ

4SJ

5Srisa

mrong

1Sukh

othai1

Sukh

othai2

Sukh

othai3

Certifi

edyear

1965

1976

1980

2007

1986

1995

1999

Daysto50flo

wering

36d

34-35d

33ndash35d

26ndash28d

30ndash35d

29d

39-40d

Daystoharvest

92ndash9

7d89ndash9

8d88ndash9

6d72ndash77d

83ndash88d

82ndash88d

90ndash93d

Maturity

grou

pMod

erate

Mod

erate

Mod

erate

Early

Mod

erate

Mod

erate

Mod

erate

100seedsw

eight

11ndash15g

15ndash18g

13ndash16g

13ndash15g

9ndash12g

13ndash15g

11ndash13g

Proteinoilcon

tent

388213

390205

41919

3

35914

5

364232

37490

39512

2

Dise

aser

esistance

Bacterialpustule

Rustand

antracno

se

Rust

soybean

mosaicv

irus

antracno

seand

purpleseed

stain

Dow

nymild

ewBa

cterialpustuleand

soybeanmosaicv

irus

Bacterialpustule

downy

mild

ewand

soybeanmosaicv

irus

Antracnosea

nddo

wny

mild

ew(m

oderate)


(b)Con

tinued

Varie

tySJ

2SJ

4SJ

5Srisa

mrong

1Sukh

othai1

Sukh

othai2

Sukh

othai3

Dise

ases

usceptibility

Rustanddo

wny

mild

ewBa

cterialpustule

Bacterialpustule

Non

ePu

rpleseed

stain

and

rust

Rust

Bacterialpustule

Seed

yield

1250ndash

1875

kgha

1875

kgha

200

0kgha

1819kgha

1538

kgha

1813ndash1938

kgha

1863

kgha

Recommended

plantin

glocatio

nAllregion

sNorthernand

centralregions

Allregion

sLo

wer

northern

region

Upp

ercentralregion

Lower

northern

and

centralregions

Allregion

s

Recommended

plantin

gseason

Laterainyand

dryseason

sAllseason

sWidely

adapted

varie

tyNA

05568

1384

Allseason

sbut

not

suitablefor

middlea

ndlater

ainy

season

sin

uppern

orthernregion

Allseason

sbut

not

suitablefor

middleand

later

ainy

season

sin

uppern

orthernregion

Allregion

s

lowast

Certified

byDepartm

ento

fAgricultureM

inistry

ofAgricultureTh

ailand










119910 = 02373119909 + 00003 (1199032

= 1) for daidzin119910 = 05115119909 + 25752 (119903

2

= 1) for genistin119910 = 02994119909 + 04664 (119903

2

= 09998) for daidzein119910 = 0396119909 + 04456 (119903

2






(min)0 2 4 6 8 10 12 14 16

(mAU

)

(mAU

)

0

20

40

0

20

40

631

6 da

idzi

n 11

592

872

0 ge

nisti

n 24

485

988

6 IS

786

4

114

21 d

aidz

ein

1454

4

131

83 g

enist

ein

1930


(a)

0 2 4 6 8 10 12 14 16

0

20

40

0

20

40

631

4 da

idzi

n 63

726

741

1562

872

7 ge

nist

in 1

6794

939

0 59

99

923

IS 8

070

(Dai

dzei

n)

(Gen

istei

n)

(min)

(mAU

)

(mAU

)


(b)












00

05

10

15

20


Isofl

avon

e con

tent

s1(m

gg)

1 09lowast

lowastlowast

lowast

lowastlowast


Sukh

otha

i 1

Chia

ngm

ai 6

0

Chak

krab

hand

hu 1

Chin

gmai

5

SJ 4

Chin

gmai

2

Nak

hon

Swan

1

Srisa

mro

ng 1

SJ 5

SJ 1

KKU

35

Sukh

otha

i 2

Sukh

otha

i 3 SJ 2

GenistinDaidzin

1 15 1 11 1 1 1 12 1 121 12 1 211 13 1 09 1 09 1 1 1 1 1 17












lowast






















00

04

08

12

16

20


GenistinDaidzin

Isofl

avon

e con

tent

s1(m

gg)

lowast

lowastdagger






00

04

08

12

16

20


for 18 months



Isofl

avon

e con

tent

s1 (mg

g)






0

1

2

3

4

Isofl

avon

e con

tent

s1(m

gg)


dagger


Chiang Mai Loei

Phitsanulok

Lob Buri






0

1

2

3

4

GenistinDaidzin

Isofl

avon

e con

tent

s1(m

gg)



The year 2010


lowast lowast






0

20

40

60

80

100

0 05 1 15 2 25 3 35

Fiel

d em

erge

nce (

)


LoeiLob Buri


y = 1339x + 2285 r2 = 0186




4 Conclusions


Acknowledgments


References






































Journal of




Agronomy





Microbiology




Volume 2014

























(b)Con

tinued

Varie

tySJ

2SJ

4SJ

5Srisa

mrong

1Sukh

othai1

Sukh

othai2

Sukh

othai3

Dise

ases

usceptibility

Rustanddo

wny

mild

ewBa

cterialpustule

Bacterialpustule

Non

ePu

rpleseed

stain

and

rust

Rust

Bacterialpustule

Seed

yield

1250ndash

1875

kgha

1875

kgha

200

0kgha

1819kgha

1538

kgha

1813ndash1938

kgha

1863

kgha

Recommended

plantin

glocatio

nAllregion

sNorthernand

centralregions

Allregion

sLo

wer

northern

region

Upp

ercentralregion

Lower

northern

and

centralregions

Allregion

s

Recommended

plantin

gseason

Laterainyand

dryseason

sAllseason

sWidely

adapted

varie

tyNA

05568

1384

Allseason

sbut

not

suitablefor

middlea

ndlater

ainy

season

sin

uppern

orthernregion

Allseason

sbut

not

suitablefor

middleand

later

ainy

season

sin

uppern

orthernregion

Allregion

s

lowast

Certified

byDepartm

ento

fAgricultureM

inistry

ofAgricultureTh

ailand










119910 = 02373119909 + 00003 (1199032

= 1) for daidzin119910 = 05115119909 + 25752 (119903

2

= 1) for genistin119910 = 02994119909 + 04664 (119903

2

= 09998) for daidzein119910 = 0396119909 + 04456 (119903

2






(min)0 2 4 6 8 10 12 14 16

(mAU

)

(mAU

)

0

20

40

0

20

40

631

6 da

idzi

n 11

592

872

0 ge

nisti

n 24

485

988

6 IS

786

4

114

21 d

aidz

ein

1454

4

131

83 g

enist

ein

1930


(a)

0 2 4 6 8 10 12 14 16

0

20

40

0

20

40

631

4 da

idzi

n 63

726

741

1562

872

7 ge

nist

in 1

6794

939

0 59

99

923

IS 8

070

(Dai

dzei

n)

(Gen

istei

n)

(min)

(mAU

)

(mAU

)


(b)












00

05

10

15

20


Isofl

avon

e con

tent

s1(m

gg)

1 09lowast

lowastlowast

lowast

lowastlowast


Sukh

otha

i 1

Chia

ngm

ai 6

0

Chak

krab

hand

hu 1

Chin

gmai

5

SJ 4

Chin

gmai

2

Nak

hon

Swan

1

Srisa

mro

ng 1

SJ 5

SJ 1

KKU

35

Sukh

otha

i 2

Sukh

otha

i 3 SJ 2

GenistinDaidzin

1 15 1 11 1 1 1 12 1 121 12 1 211 13 1 09 1 09 1 1 1 1 1 17












lowast






















00

04

08

12

16

20


GenistinDaidzin

Isofl

avon

e con

tent

s1(m

gg)

lowast

lowastdagger






00

04

08

12

16

20


for 18 months



Isofl

avon

e con

tent

s1 (mg

g)






0

1

2

3

4

Isofl

avon

e con

tent

s1(m

gg)


dagger


Chiang Mai Loei

Phitsanulok

Lob Buri






0

1

2

3

4

GenistinDaidzin

Isofl

avon

e con

tent

s1(m

gg)



The year 2010


lowast lowast






0

20

40

60

80

100

0 05 1 15 2 25 3 35

Fiel

d em

erge

nce (

)


LoeiLob Buri


y = 1339x + 2285 r2 = 0186




4 Conclusions


Acknowledgments


References






































Journal of




Agronomy





Microbiology




Volume 2014

































119910 = 02373119909 + 00003 (1199032

= 1) for daidzin119910 = 05115119909 + 25752 (119903

2

= 1) for genistin119910 = 02994119909 + 04664 (119903

2

= 09998) for daidzein119910 = 0396119909 + 04456 (119903

2






(min)0 2 4 6 8 10 12 14 16

(mAU

)

(mAU

)

0

20

40

0

20

40

631

6 da

idzi

n 11

592

872

0 ge

nisti

n 24

485

988

6 IS

786

4

114

21 d

aidz

ein

1454

4

131

83 g

enist

ein

1930


(a)

0 2 4 6 8 10 12 14 16

0

20

40

0

20

40

631

4 da

idzi

n 63

726

741

1562

872

7 ge

nist

in 1

6794

939

0 59

99

923

IS 8

070

(Dai

dzei

n)

(Gen

istei

n)

(min)

(mAU

)

(mAU

)


(b)












00

05

10

15

20


Isofl

avon

e con

tent

s1(m

gg)

1 09lowast

lowastlowast

lowast

lowastlowast


Sukh

otha

i 1

Chia

ngm

ai 6

0

Chak

krab

hand

hu 1

Chin

gmai

5

SJ 4

Chin

gmai

2

Nak

hon

Swan

1

Srisa

mro

ng 1

SJ 5

SJ 1

KKU

35

Sukh

otha

i 2

Sukh

otha

i 3 SJ 2

GenistinDaidzin

1 15 1 11 1 1 1 12 1 121 12 1 211 13 1 09 1 09 1 1 1 1 1 17












lowast






















00

04

08

12

16

20


GenistinDaidzin

Isofl

avon

e con

tent

s1(m

gg)

lowast

lowastdagger






00

04

08

12

16

20


for 18 months



Isofl

avon

e con

tent

s1 (mg

g)






0

1

2

3

4

Isofl

avon

e con

tent

s1(m

gg)


dagger


Chiang Mai Loei

Phitsanulok

Lob Buri






0

1

2

3

4

GenistinDaidzin

Isofl

avon

e con

tent

s1(m

gg)



The year 2010


lowast lowast






0

20

40

60

80

100

0 05 1 15 2 25 3 35

Fiel

d em

erge

nce (

)


LoeiLob Buri


y = 1339x + 2285 r2 = 0186




4 Conclusions


Acknowledgments


References






































Journal of




Agronomy





Microbiology




Volume 2014

























(min)0 2 4 6 8 10 12 14 16

(mAU

)

(mAU

)

0

20

40

0

20

40

631

6 da

idzi

n 11

592

872

0 ge

nisti

n 24

485

988

6 IS

786

4

114

21 d

aidz

ein

1454

4

131

83 g

enist

ein

1930


(a)

0 2 4 6 8 10 12 14 16

0

20

40

0

20

40

631

4 da

idzi

n 63

726

741

1562

872

7 ge

nist

in 1

6794

939

0 59

99

923

IS 8

070

(Dai

dzei

n)

(Gen

istei

n)

(min)

(mAU

)

(mAU

)


(b)












00

05

10

15

20


Isofl

avon

e con

tent

s1(m

gg)

1 09lowast

lowastlowast

lowast

lowastlowast


Sukh

otha

i 1

Chia

ngm

ai 6

0

Chak

krab

hand

hu 1

Chin

gmai

5

SJ 4

Chin

gmai

2

Nak

hon

Swan

1

Srisa

mro

ng 1

SJ 5

SJ 1

KKU

35

Sukh

otha

i 2

Sukh

otha

i 3 SJ 2

GenistinDaidzin

1 15 1 11 1 1 1 12 1 121 12 1 211 13 1 09 1 09 1 1 1 1 1 17












lowast






















00

04

08

12

16

20


GenistinDaidzin

Isofl

avon

e con

tent

s1(m

gg)

lowast

lowastdagger






00

04

08

12

16

20


for 18 months



Isofl

avon

e con

tent

s1 (mg

g)






0

1

2

3

4

Isofl

avon

e con

tent

s1(m

gg)


dagger


Chiang Mai Loei

Phitsanulok

Lob Buri






0

1

2

3

4

GenistinDaidzin

Isofl

avon

e con

tent

s1(m

gg)



The year 2010


lowast lowast






0

20

40

60

80

100

0 05 1 15 2 25 3 35

Fiel

d em

erge

nce (

)


LoeiLob Buri


y = 1339x + 2285 r2 = 0186




4 Conclusions


Acknowledgments


References






































Journal of




Agronomy





Microbiology




Volume 2014

























00

05

10

15

20


Isofl

avon

e con

tent

s1(m

gg)

1 09lowast

lowastlowast

lowast

lowastlowast


Sukh

otha

i 1

Chia

ngm

ai 6

0

Chak

krab

hand

hu 1

Chin

gmai

5

SJ 4

Chin

gmai

2

Nak

hon

Swan

1

Srisa

mro

ng 1

SJ 5

SJ 1

KKU

35

Sukh

otha

i 2

Sukh

otha

i 3 SJ 2

GenistinDaidzin

1 15 1 11 1 1 1 12 1 121 12 1 211 13 1 09 1 09 1 1 1 1 1 17












lowast






















00

04

08

12

16

20


GenistinDaidzin

Isofl

avon

e con

tent

s1(m

gg)

lowast

lowastdagger






00

04

08

12

16

20


for 18 months



Isofl

avon

e con

tent

s1 (mg

g)






0

1

2

3

4

Isofl

avon

e con

tent

s1(m

gg)


dagger


Chiang Mai Loei

Phitsanulok

Lob Buri






0

1

2

3

4

GenistinDaidzin

Isofl

avon

e con

tent

s1(m

gg)



The year 2010


lowast lowast






0

20

40

60

80

100

0 05 1 15 2 25 3 35

Fiel

d em

erge

nce (

)


LoeiLob Buri


y = 1339x + 2285 r2 = 0186




4 Conclusions


Acknowledgments


References






































Journal of




Agronomy





Microbiology




Volume 2014

























00

04

08

12

16

20


GenistinDaidzin

Isofl

avon

e con

tent

s1(m

gg)

lowast

lowastdagger






00

04

08

12

16

20


for 18 months



Isofl

avon

e con

tent

s1 (mg

g)






0

1

2

3

4

Isofl

avon

e con

tent

s1(m

gg)


dagger


Chiang Mai Loei

Phitsanulok

Lob Buri






0

1

2

3

4

GenistinDaidzin

Isofl

avon

e con

tent

s1(m

gg)



The year 2010


lowast lowast






0

20

40

60

80

100

0 05 1 15 2 25 3 35

Fiel

d em

erge

nce (

)


LoeiLob Buri


y = 1339x + 2285 r2 = 0186




4 Conclusions


Acknowledgments


References






































Journal of




Agronomy





Microbiology




Volume 2014

























0

1

2

3

4

Isofl

avon

e con

tent

s1(m

gg)


dagger


Chiang Mai Loei

Phitsanulok

Lob Buri






0

1

2

3

4

GenistinDaidzin

Isofl

avon

e con

tent

s1(m

gg)



The year 2010


lowast lowast






0

20

40

60

80

100

0 05 1 15 2 25 3 35

Fiel

d em

erge

nce (

)


LoeiLob Buri


y = 1339x + 2285 r2 = 0186




4 Conclusions


Acknowledgments


References






































Journal of




Agronomy





Microbiology




Volume 2014

























0

20

40

60

80

100

0 05 1 15 2 25 3 35

Fiel

d em

erge

nce (

)


LoeiLob Buri


y = 1339x + 2285 r2 = 0186




4 Conclusions


Acknowledgments


References






































Journal of




Agronomy





Microbiology




Volume 2014















































Journal of




Agronomy





Microbiology




Volume 2014
























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