INTRODUCTION
Sorghum [Sorghum bicolor (L.) Moench] is the fifth most important cereal
crop grown on over 44 million hectares in both temperate and tropical regions.
Sorghum is mainly grown as rainfed crop by subsistence farmers in the semi-arid
tropical regions of Africa and Asia as well as by other farmers in the USA,
China and Latin America. About 88% of sorghum production is used as human food
and animal feed. Nearly 150 insect species have been reported as pests on sorghum.
Sorghum is a host for more than 100 plant pathogens, including fungi, bacteria,
virus and nematodes. Some of these pathogens cause more serious damage and are
more widespread than others (ICRISAT, 2007).
Phenols (mainly condensed tannins) in sorghum kernel are considered a desirable
agronomic trait since it can protect sorghum from being damaged by birds, insect
pests and diseases (Waniska et al., 2001). But
in the view of nutritive value, tannins are considered undesirable due to its
capacity of binding proteins and make them less digestive as well as producing
astringent taste (Ambula et al., 2003). Therefore,
debates on the necessity and amount of tannins in sorghum kernels exist between
nutritionists and agronomists and consumers and growers. Sorghum breeders have
been wandering between the two extremes. How to take the advantages of tannins
in fields and to minimize their disadvantages of reducing nutritive value of
sorghum grain is a knotty task for sorghum breeders.
However, in ruminants, tannins can induce beneficial effects. For example,
higher retention of nitrogen has been observed in sheep and cattle with low
to moderate levels of tannins in forages. In these cases, the lower digestibility
of nitrogen was compensated for by reduced urinary loss of hydrogen. Moderate
levels of tannins (The preferred tannin content is between 0.1-0.6%) in forage
legumes can have beneficial responses in ruminants, resulting in higher growth
rates and milk yield (Hagerman and Klucher, 1986).
Less than 1% tannin in sorghum grain is generally considered moderate amount
(Hancock, 2000). Mild amount of tannins is also necessary
for high quality liquors in many regions in China (Lu and
Sun, 2005). Therefore, mild amount of tannins in sorghum grain is needed
in many cases even in view of feed nutrition and grain quality.
Sorghum containing condensed tannins has dominant B1B2 genes that produce a
thick, pigmented testa layer in the kernel upon maturation (Earp
and Rooney, 1986). This layer varies in thickness, intensity and color.
Peircarp color is determined by R-Y-genes whether the pericarp is red (R-Y-),
colorless or white (R-yy, rryy), or lemon yellow (rrY-).
The most widely accepted method of determining condensed tannins in sorghum
is the vanillin-HCl method (Price et al., 1978)
when the blanks are subtracted to eliminate background of non-tannin materials.
However, it requires significant time and is not readily applied in routine
grading of sorghum. The kernels can also be dissected and visually evaluated
for the presence of a testa layer which can be difficult without sufficient
experience.
In the present study, we investigated the relationships between sorghum
kernel pericarp and testa colors with tannin contents and their relevance
in breeding programs. The study was initiated at Carlsberg Research Center,
Demark and completed at The Agri-Biotechnology Research Center of Shanxi
Province, China in 2006.
MATERIALS AND METHODS
Twenty-four sorghum entries, varying in pericarp color, absence or presence
of testa and color of testa, were from China (Shanxi Academy of Agricultural
Science), Africa (Ford Research Center at Katum, Sudan and a few local varieties
from Tanzania and Rwanda) and USA (Purdue University and Texas A and M University).
Tannin content analysis was conducted with modified Vanilin-HCl method (Hahn
and Rooney, 1986). The method is based on the ability of condensed tannins
to react with vanilin in the presence of mineral acids to produce a red color.
Grain samples were ground at same day with tannin content analysis. Ground samples
(1 g was extracted with 20 mL of 1% HCl in methanol) for 20 min at 30°C
in a water bath. The samples were centrifuged at 2000 rpm for 4 min. The supernatant
(1.0 mL) was reacted with 5 mL vanillin solution (0.5% vanillin + 2% HCl in
methanol) for 20 min at 30°C. Blanks were run with 4% HCl in methanol in
place of vanillin reagent and subtracted. Each sample was assayed for 3 replications
and the mean was calculated to represent its tannin content. The entries were
grouped according to the official United States Standards for Grain (UMSDA,
1987). Genotypes of entries were visually determined by the description
of Rooney and Miller (1981).
Absorbance was read at 500 nm and determined by subtracting blank readings
from sample readings. The reference was developed by using commercial
catechin to prepare a standard curve.
Photos of kernels were taken to represent the various types of pericarp
and testa.
RESULTS AND DISCUSSION
The tannin contents of group I entries varied between 0-0.2% with
the average of 0.08% CE; those of group II varied between 0.1-5.8% CE
with the average of 1.8% CE and those of group III varied between 3.6-12.8
CE with the average of 5.9% CE. The differences among the three groups
were obvious but without a clear-cut (Table 1).
Some entries in group I did not have detectable tannin by the present
test and the others in the same group did contain trace amount of tannins.
The two entries in the group I (without testa) with brown or red pericarps
all produced faint positive reaction, an indication of the existence of
minor amount of tannins. The results implied that the dark pericarp per
se was also responsible for producing a little amount of tannins.
The entries in group I were most variable in tannin contents. Some entries,
e.g., Feterita and Kafinam B, with obvious testa contained very little amount
of tannins, implying the presence of testa did not necessarily result in high
amount of tannins.
| Table 1: |
Kernel characteristics, their genotypes, grouping and tannin
contents |
 |
| (a) A: Absent; P: Present; (b) Testa color was determined
by visual observation of kernels with pericarp scratched off with a pocket
knife; (c) According to Rooney and Miller (1981)
and (d) CE |
|
| Fig. 1: |
Testa of sorghum kernels: Top: purple testa; bottom left:
brown testa; bottom right: no testa |
We noticed that the testa color of both varieties was purple instead of being
brown as in most other type 2 sorghum varieties (Fig. 1).
The purple testa is controlled by a pair of recessive gene, tptp and its dominant
counterpart, Tp, produces brown testa (Rooney and Miller,
1981). Thus, sorghum breeders intending to select for low tannin varieties
should not discard materials solely by the presence of testa, but also observe
the color of the testa. On the contrary, some cultivars without testa, e.g.,
P954063 and BTX 623, did contain detectable tannins, implying that tannins do
not only locate in testa, but also in pericarp to certain extent. Therefore,
breeders should not select tannin-free lines solely by the absence of testa.
The entries in group III contained much higher amount of tannins than the previous
two groups, implying the presence of dark pericarp [designed as spreader(SS)
by Rooney and Miller (1981), will increase tannin amount
drastically when testa is present.
Tannin content in sorghum kernel varies in a great range, which could
be related to its pericarp and testa characteristics. Sorghum varieties
with dark pericarp but without testa contain a little amount of tannins
and may have certain resistance to bird, insects and diseases in the regions
where bird damage and grain diseases are not severe, thus to maintain
the advantages of tannins in the fields and do not produce significant
adverse effects on nutritive value of sorghum grain.
Bird damages and grain molds are very severe in experimental plots in Liaoning
and Hebei provinces in northern China. But two group I hybrids with dark pericarp,
i.e., Jinza No. 1 (TX3197A X Jinfu No. 1) and Liaoza No. 1 (TX622A X Jinfu No.
1) have been successively grown in millions of ha sorghum production fields
for more than 20 years (Lu and Sun, 2005). These facts
indicated that dark pericarp varieties (without testa) do possess certain resistances
to birds and grain molds. Although there is only trace amount, tannins in pericarp
are more effective in protecting sorghum grains compared with those in testa
since it is in the out-most defense layer. Still, tannins in the pericarp are
also easier to be eliminated simply by removing the pericarp. The range of tannin
contents in sorghum pericarp was narrow (0-0.2%) in the present study. But we
may find some varieties with higher tannin content for higher protection effects
by screening through the germplasm and breeding efforts.
It is very interesting in our study to find that two group II varieties,
Feterita and Kafinam B, contained tiny amount of tannins. This result
was not consistent with the common view that the presence of testa is
related with high tannin content. We assayed samples of 6 more Feterita
type varieties with purple testa in the following test and got very similar
results. Their tannin contents varied between 0.2-0.5% CE, implying that
purple testa did relate with low tannin content. Purple testa mostly exists
in Feterita and Hegri type sorghum varieties. Selecting for purple testa
cultivars (hybrids) may be one of the solutions to cop with the contradiction
between the nutritive value and field resistance of grain sorghum.
The trace amount of phenols detected in the group I entries in present study
might not be tannins, but flavanols and dihydrochalcones (Price
et al., 1978). These phenolic compounds are also effective in protecting
sorghum grain form bird and disease damages (Lu and Sun, 2005).
Based on the above results, we propose to subdivide the present group
I into group I (L) and group I (D) and the group II into group II (P)
and group II (B). The group I (L) includes testa-free entries with colorless,
white and yellow pericarp (most probably with 0.1% CE tannins or below)
and group I (D) contains the testa-free entries with darker pericarps
(most likely with tannin content of around 0.2%). The group II (P) includes
the sorghum entries with purple testa (most probably with tannin contents
of 0.5% CE or below), while the group II (B) is consisted of sorghum entries
with brown testa with light pericarp (most likely with 1.0-6.0% CE of
tannin). These subgroups are easily to be distinguished from another one
in the same group. And the subdivision of the two groups makes the entries
within the subgroup more uniform not only in pericarp and testa color,
but in their tannin content as well. For instance, in the present study,
the subgroup I (D) contained 0.2% CE tannins in average, while those in
subgroup I (L) contained only 0.07% and subgroup II (P) contained averagely
0.15% CE tannins whereas the subgroup II (B) contained 2.8% tannins in
average. The subdivision of the two groups will facilitate sorghum breeders
in their efforts to manage their breeding stocks and to use germplasm.
Sorghum breeders will be able to confidently select their breeding stocks
for a narrow range of tannin content simply by observing the grain color
and scratching the kernels with a pocket knife in fields. All the subgroup
II (P) entries in the present study had light pericarp. We may also combine
the dark pericarp with purple testa traits to breed for group II (DP)
type cultivars (hybrids), which should possess acceptable resistances
to birds, diseases and insects in fields and have little negative effects
on sorghum grain nutritive value. It would be helpful to further characterize
phenols present in the purple testa and study their biological effects
to both human and animal nutrition and sorghum pathogens.
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