MNL106.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. I. CHEMICAL
ANALYSIS
OF WAXY MAIZE STARCH
Corcuera VR1,
Caro Sol’s C2, Garcia-Rivas G2, Tortoriello C2,
Salmoral EM2
1 C.I.C. 2GIB-F.I.-UBA
Traditionally, plant breeding has contributed to increase
the obtention of nutritional elements as well as to improve the efficiency of
their production, but the carefull improvement of nutritional topics
constitutes a more recient goal in plant breeding. In 1990, with the purpose of
obtaining new starch and protein quality genotypes, a breeding program was
initiated at the Instituto FitotŽcnico Santa Catalina and CIGEN located in
Llavallol, province of Buenos Aires, Argentina (22 m.a.s., 34¡ 48«S; 58¡ 31«W). During the first stage of the program several maize
inbreds were developed, tested and selected following the classic methodology.
These inbreds carry single recessive genes (wx, o2, o5, 09. 011, sh4) or may be double recessive (wxsh4;
wxo2). Using some
of the inbreds developed, single-crosses were obtained and tested in several
complete randomized block design field trials with three replicates conducted
in Llavallol. During the last year, the starch composition of twenty-six
inbreds and four single-crosses was analyzed through molecular fractionation
and spectrophotometry. Starch content, amylose% and amylopectin% were
determined in all the genotypes. Starch molecular fractionation was performed
according to Cur‡ & Krisman (1990) method, based on the differential
solubility of starch components in a water:butanol mixture. So, 5g of milled and dried seeds were defatted by the
Soxhlet method and suspended in four volumes of 3 % Hg Cl2 pH 7 at 28 ¡C and stirred for an hour. The
suspension was centrifuged at 5,000 x g for 15 min and the sediment was
resuspended in 1-butanol: water (1: 7), autoclaved for 3 hours at 1 atmosphere,
110¡C. After centrifugation at 3,000 x g for 20 min. two fractions were
obtained: a) the supernatant named butanol soluble fraction (BS) containing the
amylopectin and b) the sediment termed butanol insoluble fraction (BI)
including the amylose. Polysaccharides from both fractions were recovered after
adding three volumes of 96% ethanol, then repeated washes with acetone and
ether, dried and weighed for later characterization. All the fractions were
additionally purified by gel filtration chromatography on Biogel P6 Amersham
Biotech (100- 200 mesh) in a
0.8 x 10 cm diameter column in 0.1M buffer pyridine-acetate pH 5. For
these studies, 10 mg of each sample were solubilized with 0.1 N NaOH and
neutralized to pH 5 with 0.1N ClH, then passed through the column. Fractions of
1 ml were recovered, the amylose and amylopectin content were controlled in the
presence of yodine reagent following Krisman«s methodology (1962) and the lmax of the colored
complex was determined using a Shimatzu UV spectrophotometer in
seven inbreds and four single-crosses out of the whole. The results of the starch
molecular fractionation performed in inbreds and single-crosses are shown in
Table 1 and 2. As known, normal maize starch has a 25 to 30% amylose and 70 to
75% amylopectin. This ratio of proportionality between both a-D-glucanes is clearly expressed in the starch
molecular constitution of the flint inbred 3043b used as tester. Likely, an
analogous ratio was found in the normal inbreds 3132a and 3115 which do not
carry the wx
gene in their genetic background. Nevertheless, high amylopectin content (³75%) was found in the rest of the
genotypes analyzed. The highest amylopectin content was found in the inbreds 3074a,
3078a and 3022a/1. Though the wx gene is included within the background of the inbred 3016b
and also in the single-cross CIG 66, xenia prevented the expression of a higher amylopectin
content as expected because of the contamination with foreign pollen containing
the Wx allele during fertilization. It must be remarked that in the inbreds
3002a, 3022b, 3096b and also in the single-cross CIG47 we found resistent
starch. If really, this resistent starch were only amylopectin the value found
for this a-D glucane in these genotypes could therefore be higher and
then it would be convenient to confirm the total glucose content of each
polysaccharide through the phenol sulphuric acid method according to Dubois or
Somagi Nelson. It is also interesting to consider the ratio (R) between
amylopectin and amylose content and our results point out that when R ³3,2 we
are in presence of a waxy genotype. According to the peculiar nature of the R
ratio, we verified a highly significant correlation (r: 0.93) between R and amylopectin % and
whether also highly significant, negative between R and amylose % (r: -0.93). The results of the spectrophotometric analysis are shown in
Table 3. The methodology used is based on the fact that in presence of I2:KI
in CaCl2 saturated solution reagent (Krisman,1962) the a1,4 a1,6 glucopolysaccharides show
absorbance values (lmax) around 380 to 700 nm. A lmax higher than 560 nm
indicates the presence of the linear glucopolysaccharide (amylose) giving a
blue-colored complex. Instead, a lmax value below 560 nm
points out the existence of amylopectin that stains purple-brown. Both
starch components have a linear response within the concentration range of 100-
400 ug. mL−¹. By computing the ratio (ÒAÓ) between the maximun absorbance value found
for the polysaccharide and the maximun absorbance value corresponding to its
shoulder as shown in the spectrophotometer profile, the length of the external
chains of each a-D glucanes may be estimated. Long external chains of glucopolysaccharides
(amylose) usually yield a high ÒAÓ value (around 2-4). Smaller values of ÒAÓ
(around 1-1.8) are typical of the
short and branched
chains of amylopectin. Our results pondered through the ÒAÓ ratio, lower
than usually expected, show that the amylose contained in these genotypes has
some degree of ramification and therefore is not completely linear. On the
other hand, the data presented in Table 3 also evidence that the amylopectin of
the different genotypes has a similar degree of branching and that may also be
considered moderately branched. Undoubtedly, the single-crosses CIG10, CIG25
and CIG45 have the higher amylopectin content (81 to 88%). As CIG10 was
obtained by crossing a ÒnormalÓ inbred
(female) x waxy (male), its endosperm genotype is WxWxwx. Considering that the amylopectin
content in normal maize (WxWxWx) is about 70 to 75%, the expression of only one recessive
allele (wx)
seems to arise the content of this a-D Glucane in ca. 8 to 10 %.
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TABLE
1: Inbreds
starch content and composition |
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Genotype |
Starch % |
Amylose % |
Amylopectin % |
R |
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3002
a |
68.8 |
24.2 |
75.5 |
3.2 |
|
3008 |
67.8 |
26.4 |
73.6 |
2.8 |
|
3014a |
69.3 |
22.0 |
78.0 |
3.5 |
|
3016a |
70.1 |
20.1 |
80.0 |
4.0 |
|
3016
b |
69.2 |
29.3 |
69.9 |
2.4 |
|
3020/1 |
69.5 |
21.0 |
79.0 |
3.8 |
|
3020a |
70.7 |
20.3 |
79.7 |
3.9 |
|
3022a |
73.1 |
18.0 |
82.0 |
4.6 |
|
3022a/1 |
70.0 |
11.1 |
89.0 |
8.0 |
|
3022a/2 |
70.7 |
16.0 |
84.0 |
5.3 |
|
3022
b |
69.7 |
15.8 |
84.2 |
5.3 |
|
3022c |
69.5 |
18.0 |
82.0 |
4.6 |
|
3043
b |
70.9 |
29.9 |
70.1 |
2.3 |
|
3074a |
69.9 |
9.0 |
91.0 |
10.1 |
|
3074b |
71.3 |
17.1 |
82.8 |
4.8 |
|
3074c |
71.9 |
20.0 |
80.0 |
4.0 |
|
3078a |
71.6 |
9.0 |
91.0 |
10.1 |
|
3088 |
70.1 |
25.0 |
75.0 |
3.0 |
|
3092 |
69.0 |
20.0 |
80.0 |
3.0 |
|
3096
b |
70.2 |
17.0 |
82.0 |
4.8 |
|
3098 |
69.1 |
24.0 |
76.0 |
3.2 |
|
3109 |
69.4 |
22.0 |
78.0 |
3.6 |
|
3115 |
70.4 |
29.0 |
71.0 |
2.3 |
|
3119 |
69.0 |
21.6 |
78.5 |
3.6 |
|
3132
a |
70.7 |
27.1 |
72.8 |
2.7 |
|
3139a |
70.2 |
24.0 |
76.0 |
3.2 |
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TABLE
2: Single-crosses
starch content and composition |
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Genotype |
Starch % |
Amylose % |
Amylopectin % |
R |
|
CIG10 |
69.6 |
18.8 |
80.7 |
4.3 |
|
CIG24 |
73.4 |
20.0 |
79.9 |
4.0 |
|
CIG25 |
71.1 |
12.2 |
87.8 |
7.2 |
|
CIG45 |
71.0 |
11.1 |
88.9 |
8.0 |
|
CIG47 |
70.0 |
22.0 |
78.0 |
3.5 |
|
CIG50 |
72.4 |
20.3 |
78.7 |
3.9 |
|
CIG66 |
71.4 |
29.0 |
71.0 |
2.4 |
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TABLE 3: Spectrophotometric analysis of
starch molecular
components. |
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AMYLOSE |
AMYLOPECTIN |
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lmax |
lmax shoulder |
A |
lmax |
lmax shoulder |
A |
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Inbred |
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3002a |
590.0 |
415.0 |
1.43 |
497.5 |
408.0 |
1.22 |
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3016b |
614.0 |
415.0 |
1.48 |
521.0 |
411.0 |
1.27 |
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3022b |
560.0 |
415.0 |
1.35 |
494.5 |
410.0 |
1.21 |
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3043b |
596.0 |
415.0 |
1.44 |
506.0 |
410.0 |
1.23 |
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3096b |
617.0 |
414.0 |
1.50 |
490.0 |
408.0 |
1.20 |
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3115 |
589.0 |
414.0 |
1.42 |
525.5 |
411.0 |
1.28 |
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3132a |
620.0 |
415.0 |
1.49 |
500.5 |
411.0 |
1.22 |
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Single-cross |
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