MNL206.DOC
LLAVALLOL, ARGENTINA
INSTITUTO FITOTECNICO DE SANTA CATALINA, FACULTAD DE CIENCIAS AGRARIAS Y
FORESTALES, UNIVERSIDAD NACIONAL DE LA PLATA AND CIGEN (CONICET-CIC-UNLP)
MAIZE QUALITY BREEDING IN ARGENTINA. II. DETERMINATION OF LYSINE AND FATTY
ACIDS BY CHROMATOGRAPHY
Corcuera VR1, Giraudo M2, BernatenŽ EA3,
S‡nchez Tuero H2, Irene Malcowski2
1C.I.C.
2 Lab. F’sico-Qu’mica UNLa 3CONICET
Maize is an important source of
carbohydrates (74%) and also provides proteins (9%), oil (3.4%) and fiber (1%)
according to Paliwal (2001). Maize
endosperm proteins are normally deficient in lysine and triptophan. Protein quality does not only
constitute a determinative factor for man and swine«s nutrition, but even for
poultry when it is not possible or convenient to use balanced feed due to
costs. Protein quality is usually
controlled by simple mutant genes. Since
Mertz (1964) discovered the single recessive gene o2 (opaque-2) many efforts throughout the world
were done to obtain high lysine and high triptophan maize genotypes with at
least an acceptable field performance. Maize oil is exclusively obtained from
its germ through wet milling process. It is well known that the value and
primary utility of oils strongly depends on its fatty acids composition, so it
results necessary and convenient to determine it with commercial purposes. Within
the last decade at the Instituto Fitotecnico Santa Catalina and CIGEN located
in Llavallol, province of Buenos Aires, Argentina (22 m.a.s., 34ˇ 48«S; 58ˇ 31«W) a maize quality breeding program was
initiated. Normal genotypes previously developed in Argentina were reconverted
to quality protein maize through the incorporation of the o2, o5, o11 or o12
genes from Illinois and
Bergamo inbreds used as donors. After conveniently fitting the chromatography
conditions, lysine content in endosperm flour of three inbreds and a
single-cross via rp-HPLC was determined. Simultaneously, the germ fatty acids
composition of 4 inbreds and three single-crosses was analyzed through gas
chromatography. Actually, we are determining lysine and fatty acids composition
in many genotypes. Lysine content of endosperm flour was quantified by taking samples
of 50 mg each which are first hydrolized by adding 8 ml of HCl 6N and flowed back during 24 hs
at 100ˇC within a
nitrogen-rich atmosphere. Later, at room temperature, pH was fitted to 7 and
the samples kept in darkness at 4ˇC until chromatography analysis. An a-aminobutiric acid standard (AABA) and borate buffer were also added. An aliquote of this solution filtered through a
Millex LCR-13 filter (Millipore). The lysine standard and
the samples were derivatized by adding AccQ-Tag buffer and the AccQ-Fluor
derivatization reagent (Waters Corp). Chromatography analysis was performed
using HPLC grade water obtained from a Milli-Q system, HPLC grade acetonitrile
(J.T.Baker) and AccQ-Tag eluent A. The solvents were daily passed through a
Millipore GVWPO4700 filter. The chromatograph device (Shimadzu) was composed
by: quaternary bomb, on-line ungasifier, automatic injector, column furnace,
UV-VIS variable detector, and fluorescence detector. Hypersil C18 column (200
mm length x 2.1 mm inner diameter) filled with porous silica treated with
dimethyl-ODS was used. Particle diameter: 5 m and pore diameter: 12nm. Flow: 0.33 mL/min. Excitation wavelength: 250
nm. Emission wavelength: 395 nm. Temperature: 40ˇ C. Injection volume: 5
uL. On the other hand, to determine germ fatty
acids composition, 10 grams of each genotype were deffated by Soxhlet with hexane:ether
(2:1) during 24 hs and dried for 2 hs at 50ˇC. The fats obtained were analyzed using a Perkin Elmer Autosystem XL gas
chromatography device with an Altech capilar column EC-1000 (30 x 0.32 mm id x
0.25Mn. Temperature: 220ˇC; carrying gas: Nitrogen; Flow: 20 ml/min; FID detector; FID at 220ˇC; injector at 250ˇC.
Normally, maize has only a 0.3%
lysine in endosperm flour but the expression of o2 gene may double or treble it.
High lysine contents were found in the first inbreds studied (3088:
1.3%; 3098: 0.9% and 3139 II:
0.6%). These inbreds have a high oil
content as previously detected by NIR using a Isotec 1227 device (3088:
5.8%; 3098: 6.0% and 3139 II:
7.3%). Undoubtedly the reconvertion of
normal inbreds through the incorporation of opaque-mutant genes was a complete success as may be
seen in the lysine values observed. Also, the single-cross 3152 obtained by
crossing one of these inbreds as female x a wxo2 double
recessive male was a complete success in relation to its high lysine content (0.7%).
This hybrid also has a good agronomic performance as demonstrated through its
average yield during three years running in multilocation trials (9,100 kg/ha).
The fatty acids composition of the different genotypes studied may be seen in
Tables 1 to 3. Generally, normal
endosperm maize (without expression of single-mutant genes) has a 60% of linoleic acid and 20 to 27% oleic acid
(3:1 ratio). In the case of the high lysine and double recessive wxo2 genotypes analyzed, we found a 1.3:1 to 2.2:1 ratio between
linoleic and oleic acid. The
narrower ratio (1.3:1) was expressed by the o11 mutant inbred 3115. Poly-insaturated fatty acids (linoleic and linolenic) reduce
cholesterol levels in blood and this is a very important reason by which maize
oil is so required. Instead, oleic acid (monoinsaturated) has a neutral action
on cholesterol levels. All the inbreds and single-cross hybrids studied showed
very low levels of araquidonic and linolenic acid. The levels of miristic,
palmitic, heptadecanoic and araquiric acids found in the single-cross 3257 (see
Table 3) obtained after crossing the inbreds 3096b (♀) x 3135a (♂)
let us deduce that their inheritance is completely dominated by the female
parent. Opposite, the contents of oleic, linoleic and linolenic acids seem to
be clearly dominated by the male parent. On the other hand, as the estearic
acid content of the hybrid does not differ significantly from the mid-parent
value, we suppose and additive way
of inheritance for it.
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TABLE
1: Fatty
acids composition in single-mutant gene inbreds. |
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% content |
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Fatty acid |
3115* |
3016b** |
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16:0 |
Palmitic |
10,04 |
7,47 |
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16:1 |
Palmitoleic |
1,25 |
0,21 |
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18:0 |
Estearic |
2,44 |
1,05 |
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18:1 |
Oleic |
32,16 |
35,25 |
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18:2 |
Linoleic |
42,85 |
50,18 |
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18:3 |
Linolenic |
1,04 |
0,8 |
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20:0 |
Araquiric |
0,56 |
0,53 |
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20:1 |
Araquidonic |
0,32 |
0,4 |
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22:0 |
Behenic |
0,39 |
0,41 |
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22:1 |
Erucic |
0,32 |
0,21 |
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24:1 |
Lignoceric |
1,37 |
0,95 |
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*: o11 inbred |
**: waxy inbred |
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TABLE
2: Fatty
acids composition in single-cross hybrids. |
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% content |
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Fatty acid |
3165* |
3166** |
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16:0 |
Palmitic |
9,36 |
12,83 |
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16:1 |
Palmitoleic |
1,05 |
no data |
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18:0 |
Estearic |
2,68 |
3,77 |
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18:1 |
Oleic |
33,2 |
27,49 |
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18:2 |
Linoleic |
52,2 |
38,36 |
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18:3 |
Linolenic |
0,33 |
no data |
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20:0 |
Araquiric |
0,68 |
no data |
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20:1 |
Araquidonic |
no data |
no data |
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22:0 |
Behenic |
1,06 |
no data |
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22:1 |
Erucic |
0,64 |
no data |
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24:1 |
Lignoceric |
4,54 |
no data |
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*: wxo2 |
**: waxy |
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Table
3: Fatty
acids composition of a single-cross and its parents |
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% content |
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Fatty acid |
3096b* |
3135** |
3257 |
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14:0 |
Miristic |
3,00% |
0,04 |
0,03 |
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16:0 |
Palmitic |
8,66 |
12,25 |
9,46 |
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16:1 |
Palmitoleic |
0,09 |
0,09 |
0,08 |
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17:0 |
Heptadecanoic |
0,06 |
0,1 |
0,05 |
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18:0 |
Estearic |
2,03 |
1,8 |
1,56 |
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18:1 |
Oleic |
35,84 |
25,12 |
28,53 |
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18:2 |
Linoleic |
45,13 |
55,45 |
55,87 |
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18:3 |
Linolenic |
0,94 |
0,82 |
0,8 |
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20:0 |
Araquiric |
0,54 |
0,35 |
0,58 |
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*: wxo2 female parent |
**: wxo2 male parent |
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