Agriculture Reference
In-Depth Information
Wilkin, Woodworth, Williams, Wayne, Traverse, Swift, Steele, Sm-ie, Rampage, Provar,
Protana, Norman, Mokapu Summer, Magna, Mack, Kahala, Kailua, Hawkeye 63, Harosoy
63, Harosoy, Forrest, Evans, Essex, Disoy, Davis, Corsoy, Coles, Clay, Calland, Bragg,
Bonus, Beeson, Anoka, Amsoy 71, Ada, and Hodgson) and 19 were crossing varieties (Ap-
225 C, Clark Z, Clark E, Harosoy, Harosoy Dense, J-114, A 73-19084, Ix1-17, Clark Dt1e2,
Harosoy F, Harosoy Fe, Harosoy Lf1, Harosoy Dt1, Harosoy Dt2, Clark Dt1e1te2, Clark Fe,
Clark Lo, Clark Rj, Clark S, and Clark Dt1). Moreover, one commercial variety was from
Canada (Bellatti-4p), eleven were from different countries (France, Poland, Rumania,
Germany, Austria, Russia, and Old Yugoslavia) of Europe (10 commercial varieties (Ns-gr,
Fred, Srecka, Zolta Brzebedowska, Wilenska Brutnana, Mazowiecka II, Giesso, Flora,
Caloria, and A-100) and one crossing variety (Ns-mm)), 17 commercial varieties were from
Japan and Taiwan (Wasedaizu No. 1, Wase shiroge, Tokachi, Toyosuzu, Tokachi nagaha,
Tainung No. 4, Shiroge-9, Shinanomejiro, Shih-shih, Oshimashirome, Osage, Nagaba jiro,
Koganejiro, Kita musume, Kitamishiro, Kakushin 1, and Kaohsiung No. 3), and one
commercial variety was from Uganda (Merit). All these soybean cultivars presented an
almost identical aspect being not possible their differentiation based on seed color (yellow),
shape (round) or size.
Soybean solutions were prepared by pulverizing 50 mg of every variety with a domestic
miller, dissolving in 10 mL of extracting solution (80:20 (v/v), water:ACN), sonicating for 5
min in a bath sonicator (150W, 50Hz, Ultrasons-H, Selecta, Barcelona, Spain) and
centrifuging at 3662g (Heraeus Instruments, Megafuge 2.0R, Osterode, Germany). The
supernatant was removed and injected into the chromatographic system [69].
2. High-Performance Liquid Chromatography
A Hewlett-Packard 1100 series liquid chromatograph (Hewlett-Packard, Pittsburgh, PA,
USA) with a degassing system, a binary pump, a thermostated compartment for the column,
an injection system, and a diode-array detector was employed to carry out the separation. HP-
Chemstation software was used for data acquisition and processing. The chromatographic
separations were accomplished with the reversed-phase POROS R2/10 perfusion column (4.6
mm I.D. x 50 mm, 10 µm particle size) (Applied Biosystems, Foster City, CA, USA). The
separation conditions were: injection volume, 20µL; flow-rate, 3 mL/min; temperature, 60 ºC;
mobile phase A, 0.1% (v/v) TFA in Milli-Q water; mobile phase B, 0.1% (v/v) TFA in ACN;
binary gradient: 5-25% B in 1.7 min, 25-45% B in 0.8 min, and 45-5% B in 1 min; UV
detection, 280 nm [69].
3. Data Treatment
The area percentage for every peak was calculated as the average of two replicates (each
one injected in duplicate). The integration was performed by setting the baseline from valley
to valley. Multivariate classification techniques (cluster analysis and linear discriminant
analysis), Box-and-whisker diagram, and multiple linear regression (MLR) were performed
with the computer program Statgraphics Plus for Windows 4.0 (Statistical Corp., Rockville,
MD, USA). Principal components regressions (PCR) and partial least squares regressions
(PLS1) were performed with the computer program Unscrambler 9.7 (Camo Software, Oslo,
Norway).
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