International Conference on:
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Enhancement of sugar production by modern
biotechnological methods
Fatthy M. Abdel-Tawab(1), Abdel-Wahab I. Allam(2)
(1)
Faculty of Agriculture, Ain shams University, Egypt.
Chairman of Sugar Crops Council, Ministry of Agriculture, Egypt
(2)
Abstract:
Sugar in Egypt is processed from domestically grown sugar beets and
sugarcane. Sugarcane area for 2012/13 is projected at 112,000 hectares. There are
no expected increases to sugarcane area for the next 5 years due to national
efforts to conserve water resources. Sugar beet area for 2012/13 is projected at
148,000 hectares, up 2,000 hectares over 2011/12. Because sugar beets are less
intensive in their use of water, future Egyptian sugar production growth is
expected to come mostly from beet sugar and search for new sources of
sweeteners as stevia. Sugar consumption for 2012/13 is projected at 2.95 mmt, an
increase of 3.5 percent. With the Egyptian population growing at 1.5 million per
year, sugar demand should remain strong into the future. Therefore, it became
imperative to search for other sources of sweeteners.
The Stevia rebaudiana is commonly known as sugar leaf, sweet leaf, or
simply Stevia and is widely now grown for its sweet leaves. The sweet herb
Stevia is becoming a major source of natural sweetener as an alternative of table
sugar. It is rapidly replacing the artificial sweeteners like, Saccharine, Splenda
and Aspartame.
The aim of this presentation is the exploration and applications of some
advanced and unconventional approaches for the enhancement of sugar
production in Eygpt.
Unlike traditional breeding methods, marker-assisted selection has been
widely used in our laboratories as fast and reliable aid to select the promising
sugar cane genotypes with respect to sugar content and yield .
In addition, new natural sweeteners like Stevia has been under rigorous
testing for the enhancement of production and selection for high stevioside and
robidosides content using molecular markers to pinpoint the best lines in this
respect.
Tissue Culture techniques has been used as supplemental tool for the
improvement of sugar content and cane yield and as a prerequisite for
transformation of same specific genes as well as the utilization of somaclonal
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variation to obtain promising mutant types of sugar cane .
Most recently, the use of RNA in functional genomics analysis has been
successfully used for knowking down and/or up regulation of the expression of
some genes of interest.
Our dissection focus on how these different approaches can contribute to
the improvement of sugar crops in a fast and cost-effective ways.
Germplasm Introduction, Collection and Conservation:
Germplasm is a very important material for the improvement of crops.
Introduction of germplasm from one area to another continues to be an important
activity for breeding, particularly in developing countries. Adapting exotic
germplasm is, however, a long-term programme. Intermating should be carried
out for several generations and selection pressure applied gradually for desirable
gene combinations. Institutions around the world have undertaken research
and/or appraisal studies on sugarcane germplasm.
In our laboratories, the phylogenetic relationships between twelve
sugarcane genotypes belonging to three different Saccharum spp. (Table 1) were
elucidated based on RAPD and SSR molecular markers. The combined marker
analysis (RAPD and SSR) revealed some closely vs. distantly related taxa with
respect to phylogenetic relationships (Figure 1). This was in harmony with the
hypothesis of Roach and Daniels (1987) who suggested that S. officinarum
evolved from S. robustum, which evolved from S. spontaneum, and indicated that
S. spontaneum is the starting parent of Kassoer, the interspecific hybrid of S.
officinarum X S. officinarum is in independent cluster distant from S.
spontaneum and S. officinarum because it is F1 of another form from the two
species, i.e., S. officinarum and S. spontaneum (Glagah).
Microsatellites are valuable not only for their rapidity to generate markers
but also for their high polymorphism. This indicated that markers specific to a
genotypes could be easily identified with SSR markers. Therefore, such markers
seem to be an appropriate tool to follow the efficiency of introgression programs
in sugarcane. (Abdel-Tawab et al., 2004)
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Fig. (1): Dendrogram showing genetic distances between 12 sugarcane genotypes based
on 13 RAPD and 9 SSR markers combined.
Marker -Assisted Selection (MAS):
The development of molecular marker technology and consequent
identification of marker loci linked to important agronomic traits have created
exciting new opportunities for plant breeders. Marker-assisted selection provides
the potential for improving selection efficiency by allowing for earlier selection
and reduced plant population size. In the past decade the creation of genetic maps
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has been the foundation of this new plant breeding tool. One of the priorities of
plant genome mapping is the identification of genes associated with economically
important traits and the use of this information for further improvement of crops.
The value of molecular markers for the development of linkage maps and their
use in the analysis of economically important traits has been amply demonstrated
in both, field crops. Molecular linkage maps have been constructed for most
major crop plants (Abdel-Tawab et al., 2008 and Khaled et al., 2011). These
maps provide a more direct method for the selection of desirable qualitative and
quantitative traits through their linkage to easily detectable genetic markers
(Abdel-Tawab et al., 2004).
Smut resistance:
Smut is a serious disease that inflects heavy losses in sugarcane
field. Our studies were aimed to development of molecular markers (RAPD and
ISSR) associated with smut resistance in ten varieties of sugarcane. (Table 2)
(Abdel-Tawab et al., 2008).
Table (2): Code, name and pedigree of 10 cultivars of sugarcane
Some RAPD and ISSR markers were obtained which discriminated
between relatively smut resistant and susceptible genotypes are summarized in
Table (3 and 4).
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Table (3): RAPD-PCR profiles of the 10 sugarcane with 20 RAPD primers
Table (4): ISSR-PCR profiles of the 10 sugarcanes with 9 ISSR primers
Several scientists used RAPD markers for the identification of molecular
markers linked to head smut resistance gene (Shs) in Sorghum (Oh et al., 1994),
three hundred and twenty six RAPD markers were used for linkage analysis with
Shs. RAPD analysis revealed that one RAPD locus from primer OPG5 were
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linked to Shs. Procunier et al., (1997) identified molecular markers linked to a
race T10 loose smut (Ustilago tritici, U. segetum var. tritici]) resistance gene. A
RAPD marker and a RFLP marker were located on opposite flanks of the
resistance gene and were shown to be loosely linked. These markers can be used
for a faster and more reliable selection of T10 resistant plants than previous
conventional loose smut ratings. Multiple regression analysis was used to identify
putative markers associated with resistance to PCR technique to several viruses
and fungi such as smut. For the identification of molecular markers linked to
sugarcane disease, two RAPD fragments were consistently present in sugarcane
mosaic virus (SCMV) resistant and absent in susceptible cultivars, while 8
showed the reverse trends (Huckett and Botha, 1995; Barnes et al., 1997;
Zambrano et al., 2003). RAPD technique has also proved useful for diagnosing
new diseases such as yellow leaf disease (Dookun et al., 1996; Autrey et al.,
1998; Aljanabi et al., 2007). Moreover, RAPD has been developed for ratoon
stunting disease [caused by Clavibacter xyli subsp. xyli], Pachymetra
[Pachymetra chaunorhiza] root rot and white leaf. RAPD markers were used for
identification of molecular markers linked to hybrids or cultivars (Yang et al.,
2001; Ranade et al., 2002; Abde-Tawab et al., 2003; Zhang et al., 2008).
Sugar content:
The values of Brix (a relative indictor of sugar content) in sugarcane
represent the major component seeked by both farmers and sugar companies. In a
recent study, Khaled et al., (2011) reported that twenty two clones selected for
high and low Brix values (Table 5) were subjected to molecular analysis (RAPD,
ISSR, R-ISSR and SSR). They obtained marker associated with Brix values as
shown in Table (6). Molecular markers based on RAPD and ISSR combination
(R-ISSR) were suggested by Ye et al., (2005) indicated that such combination
would give a better resolution than one alone.
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International Conference on:
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Cane
yield
(ton/fed
)
Stalk
height
(cm)
Stalk
diamete
r (cm)
No. of
stalks /
m2
Brix
Clone
No.
Table (5): Means of some cane and sugar traits of the twenty two clones
Group (A)
190
22.5A
14.1A
298D
2.78A
52.500AB
191
22.17AB
14.3A
290 F
2.86 A
53.000 A
189
21.75ABC
15.6 ABCD
291 F
3.00 A
48.889FGH
209
21.67ABC
16.1 AB
299 CD
2.45 A
51.150BCD
104
21.33ABC
18.3 ABC
290 F
2.56 A
50.600CDE
120
21.17ABC
17.8ABCDE
286 H
2.83 A
46.056J
194
21.08ABC
16.0 AB
286 H
2.53 A
52.700AB
121
21 ABC
13.06ABCD
280 I
3.06 A
50.000DEF
87
20.85BC
8.55ABCDE
275 K
3.11 A
48.734FGH
198
20.83BC
9.15ABC
300 C
2.56 A
52.350ABC
122
20.83 BC
14.32ABC
299 CD
2.73 A
52.900 A
188
20.67 BC
10.32ABCDE
288 G
2.92 A
48.740FGH
193
20.33 C
8.00ABCD
308 A
2.40 A
51.750ABC
Group (B)
29
11.5H
11.30G
295 E
2.80 A
51.100BCD
131
12.83 GH
9.61FG
288 G
2.75 A
47.553HIJ
36
13.33 FG
7.13FG
285 H
2.86 A
52.250ABC
97
13.67EFG
13.61FG
280 I
3.02 A
50.400DEF
133
14 EFG
10.16FGH
270 M
3.01 A
47.000IJ
94
14.67 EF
9.71EFG
303 B
2.85 A
51.900ABC
160
15 DE
15.16FGH
290 F
2.98 A
51.500ABCD
139
15 DE
12.30FGHI
270 M
2.99 A
49.231EFG
4
16.5 D
10.86ABCDEFG
300 C
2.36 A
49.900DEF
Means within columns followed by the same letter(s) are not significantly different
(P≤0.05) by Duncan's new multiple range test.
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Table (6):The total number of amplified and polymorphic fragments,
polymorphism % and the specific markers for sugar content in the
clones using RAPD, ISSRs, R-ISSRs and SSRs analysis
Prime
r No.
Primer name
TAF
1
2
3
4
5
6
7
8
9
OP-A01
OP-A04
OP-A18
OP-A19
OP-B08
OP-B10
OP-B18
OP-O10
OP-O14
19
23
17
13
15
17
18
21
20
10
11
12
13
14
17898 A
17899 B
HB 12
HB 10
HB 13
7
11
7
6
5
15
16
17
18
18
19
Xtxp04
Xtxp08
Xtxp10
Xtxp 12
Xtxp19
Xtxp61
19
5
9
13
17
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
A01 + 844A
A01+17898A
A01+17899B
A01 + HB 12
A01 +844 B
A01 +HB 10
A04 + 844A
A04+17898A
A04 + 844B
A04 + HB10
B18 + 844 A
B18+17898A
B18+17899B
B18 + 844 B
B18 + HB 10
6
6
11
10
4
9
5
5
6
6
9
9
9
6
6
PF
MF
RAPD-PCR
18
22
16
13
14
16
18
21
19
ISSR-PCR
4
3
1
3
2
SSR-PCR
19
4
7
11
16
19
R-ISSR PCR
3
2
4
3
2
3
3
3
3
3
4
5
2
2
3
SM
+ve -ve
UF
P%
1
1
1
0
1
1
0
0
1
2
3
3
2
2
2
4
1
2
94.74
91.30
94.11
100
93.33
94.11
100
100
95
4
2
2
1
2
4
3
1
3
3
1
2
--1
1
--1
1
3
8
6
3
3
-----------
-----------
4
1
1
1
1
--2
--2
1
0
1
2
2
1
0
12
----1
5
2
100
80
77.78
84.62
94.12
100
1
--1
2
2
---
--2
1
1
--2
3
4
7
7
2
6
2
2
3
3
5
4
7
4
3
-------------------------------
-------------------------------
2
1
3
3
2
3
------3
4
4
2
--1
1
1
1
------3
3
3
----1
--2
2
TAF=total amplified fragments, PF= polymorphic fragments, MF=monomorphic
fragments, UF=Unique fragments for specific clone, P%=Polymorphism
percentage, SM=Specific marker, (+ve)=marker for high sugar content, (-ve)
=marker for low sugar content.
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RNA interference:
RNA interference is a powerful reverse genetic tool to study gene function
by the interference with gene activity.Three major enzymes, soluble acid
invertase ﴾SAI﴿, sucrose phosphate phosphatase ﴾SPP﴿ and sucrose phosphate
synthase ﴾SPS﴿ are involved in negative regulation of accumulation and / or
breakdown of sucrose. Both SAI and SPP are implicated in the degradation of
sucrose while SPS is involved in sucrose biosynthesis and accumulation
(Chandra et al., 2012 ﴿. Down - regulation of SAI gene expression can be
effectively achieved by RNAi approach to minimize its role of inversion of
sucrose into glucose and fructose which represents a major problem due to
significant loss of sucrose content. On the other hand Up - regulation of SPS gene
expression by introducing one copy of that gene by the appropriate
transformation procedure with efficient promoter may lead to significant
accumulation of sucrose in the plant. Our on – going research has been exploring
this approach and some promising progress is anticipated.
Stevia as a natural sweetener:
Stevia rebaudiana Bertoni (Compositae) is a small native herb to South
America and has been used for sweetening beverages and foods since 1600
(Glinsukon et al., 1988). Stevia has become popular and commercialized by
Japanese. The plant has been distributed to several Southeast Asia countries
including Thailand [as "Ya wan"] (Sararom et al., 1982). More than 750 tons of
stevia leaves per year are used as crude extract for consumption. The sweetening
compound was isolated from stevia leaves by Rebaudi and Resenac (Crammer
and Ikan, 1986), and was named as "stevioside" (Bell, 1954; Bridel and
Lavielle, 1931). Stevioside has very high sweetening potency, 250-300 times that
of sucrose, with little caloric value (Kinghorn and Sojarto, 1985). Its sweetness
is stable to heat and yeast fermentation. Stevia and stevioside have been applied
as a sugar substitute and used by those with obesity, diabetes mellitus, heart
disease, and dental caries (Fujita and Edahiro, 1979). Stevioside can also inhibit
the growth of certain bacteria as reported by Tama Biochemical Co. Ltd.
(1981). Eight different sweetening ent-kaurene glycosides (from about 88
compounds in stevia leaves) were isolated (Mosetting et al., 1963). The common
alycone of those glycosides is steviol, chemically ent-13-hydroxy kaur-16-en-19oic acid. However, Vis and Fletcher (1956) proposed that, stevioside is the main
sweet glycoside (6-8%, in dry stevia leaves
Yield – related traits and stevioside content:
In a study on fifteen stevia accessions, Allam et al., (2000) reported
marked variations in yield components and stevioside content (Table 7 and
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Figure 2) which allowed selection to make substantial improvements in this
natural sweetner. The stevioside content were highly associated with leaves dry
weight, leaves / stem ratio and plant vigor (visual ranking). Molecular markers
for some stevia yield components were detected using acid phosphase ,
peroxidase , esterase isoenzymes and randomly amplified polymorphic DNA (
RAPD). These markers could be efficiently used to assist selection for accessions
with high stevioside content (Table 8).
Table (7): Means of some yield-related traits and stevioside content for the 15
stevia accessions
Table (8): Molecular marker associated with some stevia traits.
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(+) = Positive marker, (-) = Negative marker
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Assessment of genotoxicity:
Stevia rebaudiana Bertoni, a plant originated from Paraguay, contains the
natural sweeteners, stevioside and rebaudioside A. Stevioside is 300 times
sweeter than sugar. Therefore, stevioside is considering a good resource as a noncaloric sweetener in human foods for different proposes. However, the
genotoxicity and safety of stevioside has been subjected to critical debates.
Biosafety of stevioside was studied in different biological systems e.g., mice,
drosophila, and human lymphocytes. In vivo study on mice (both sexes) revealed
that it had no mutagenic effect on bone marrow cells (Table 9 and Figure 3) or
lower weight (Table 10). In vivo study on Drosophilia melanogaster showed no
mutagenic effect since there were no significant differences in mutation
frequencies between the treated and the control insects (Table 11). In vitro study
on human lymphocytes revealed no significant differences between the treated
cells and controls ones (Table 12 and Figure 4). In general, all the tested systems
revealed no probable mutagenic effect of stevioside, which makes it safe for
human consumption. (Abdel – Tawab et al., 2000).
(9)
(10)
(11)
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(12)
(3)
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(4a)
(4b)
(4c)
Khalil (2004 ﴿ studied both acute and chronic potential genotoxic effect of
feeding mice on some doses of stevioside with respect to some body organs and
growth rate and reported that no significant effects were induced due to these
doses ( Table 13& 14 and Fig 5﴿ . Furthermore, no chromosomal structural
observations were observed (Fig 6﴿. Electrophoresis banding patterns for seven
isoenzymes,ie GOT , ACP , ALP , α,β-EST . LDH MDH and ADH did not reveal
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any changes in fingerprints of treated mice compared with their respective
control. DNA profile of the tumor suppressor gene (P53﴿ indicated that no
carcinogenic effect was observed after feeding mice on stevia (fig 7)
Table (13): Means and standard errors for males and females organs weights as
percentage from body weight for treated and control parental groups.
Female
Male
Liver %
Kidneys % Heart %
Spleen %
Lung %
Testes %
Control 4.99 ± 0.26
1.62 ± 0.06
0.54 ± 0.02
0.68 ± 0.43
1.22 ± 0.07 0.49 ± 0.07
Treated 5.10 ± 0.30
1.81 ± 0.07
0.62 ± 0.04
0.26 ± 0.04
1.27 ± 0.05 0.41 ± 0.05
Control 6.18 ± 0.30
1.37 ± 0.05
0.60 ± 0.04
0.36 ± 0.06
1.23 ± 0.07
-
Treated 5.55 ± 0.14
1.47 ± 0.05
0.59 ± 0.02
0.35 ± 0.05
1.18 ± 0.04
-
Table (14): Average litter size and standard errors for treated and control groups
in two matings.
Mean of litter size
Groups
1st mating
2nd mating
NM X NF
8.9 ± 0.85
5.1 ± 0.44
TM X TF
7.1 ± 1.03
5.5 ± 0.68
NM X TF
8.0 ± 0.68
5.0 ± 0.38
TM X NF
8.8 ± 0.70
4.3 ± 0.53
(5)
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The possible mutagenic hazardous of stevioside has been investigated by
two efficient mutagenicity systems (Saccharomyces cereviciae and Drosophila
melanogaster) as an in vivo biological systems for testing different genetic end
points. The yeast S. cereviciae D7 strain was treated with three different
concentrations of stevioside (5, 10 and 15 mg/ml) to evaluate its genotoxic effect.
The survival rate was increased with the increasing of stevioside concentration
than the concurrent negative control. The mutagenicity assay using S. cereviciae
D7 strain revealed that stevioside has no mutagenic activities including the
induction of mitotic gene conversion, mitotic crossing-over and reversion.
Instead, the frequencies of the three end points were lower than the spontaneous
levels. In addition the obtained results revealed that stevioside has non mutagenic
effects in all tested genetic end points on Drosophila; moreover, it decreased the
spontaneous mutation rate than the concurrent negative control (Table 25 and
Figure 8). Therefore, the possible antimutagenic effect of stevioside has been
tested against the mutagenic activities of colchicines and mitomycin C (MMC)
using the well defined antimutagenicity assays on Drosophila. The reduction of
mutagenic activity of MMC indicated that, stevioside has a strong antimutagenic
activity on Drosophila. (Abdel – Tawab et al., 2009).
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El-Attar et al., (2011) assessed efficiency of mutagenicity test systems
using Drosophila as a genetic model to detect different genetic endpoints such as
translocations, partial and/or complete chromosome loss or gain and aneuploidy
in germ line cells as well as in somatic cells seems to be effective. The
frequencies of mutations were not affected by treatment with aspartame and
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stevioside which revealed the absence of mutagenic effect of aspartame and
stevioside. Moreover, stevioside may even have antimutagenic effect in germ-line
aneuploidy and chromosomal aberrations tests (ATE) system (Table 16 and
Figure 9) but in somatic mutation and recombination test (wts), aspartame
showed some genotoxic effect especially in the feeding treatments (Table 17 and
Figure 10). Our results are in partial agreement with those reported by Rabbani
et al., (2005) who tested citral as an antimutagenic agent and reported that it
produced a mild anti-clastogenic effect evident from the decreased micronuclei
frequency observed in polychromatic and normochromatic erythrocytes. It
prevented the nuclear damage induced by cyclophosphamide, mitomycin-c and
nickel chloride in both bone and peripheral blood micronucleus tests. AbdelTawab et al., (2008) reported that Stevioside has no mutagenic effect in all tested
genetic end points in Drosophila. PCR-based RAPD analysis indicated the
possibility of detecting molecular markers associated with genotoxic effect. In
summation, it is evident from the aforementioned discussion that from the stand
points of both the cytogenetic analysis (chromosomal aberrations) and molecular
analysis (RAPD) that the biomarkers obtained in this study indicated that we can
get reliable
Evidences regarding the biosafety of these two world wide uses of sweeteners
without any serious hazards to the health and welfare of their consumers.
Table (16): Frequencies of spontaneous and induced of aneuploidy and structural
chromosomal aberrations in Drosophila male germ line cells of ATE
strain after larval immersion or feeding treatments with colchicines and
three concentrations of aspartame and stevioside
Treatments
Control
Colchicine
4mg/ml
12m/ml
20mg/ml
Aspartame
Aspartame feeding
immersion
Total % mutants
Brood1 Brood2 Brood1 Brood2
0.178
0.89**
0.160
0.171
0.107
0.196
1.03**
0.189
0.154
0.154
0.178
0.89**
0.18
0.169
0.242
0.196
1.03**
0.23
0.075
0.190
Stevioside feeding
Treatments
Control
Colchicine
5mg/ml
20m/ml
50mg/ml
Total % mutants
Brood1 Brood2
0.178
0.89**
0.27
0.15
0.052
0.196
1.03**
0.052
0.124
0.054
* and ** significant and Highly significant difference from the negative control at P<0.05
and P <0.01 using χ2 test.
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Table (17): Frequencies of induced tumor in trans-heterozygous (wts/+) after
larvae immersion or feeding treatments with three concentrations of
aspartame and stevioside comparing with the (MMC) and control.
Overall mutants
rate %
Overall mutants rate %
Treatments
Aspartame
immersion
Aspartame
feeding
Treatments
Stevioside
feeding
Control
0.078
0.078
Control
0.078
MMc
0.97**
0.97**
MMc
0.97**
4mg/ml
0.09
0.04*
4mg/ml
0.03*
12mg/ml
0.05
0.11*
12mg/ml
0.05
20mg/ml
0.09
0.09
20mg/ml
0.11*
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Conclusion:
It is evident from the aforementioned discussion that there are good
opportunities for improvement of sugarcane biomass and sucrose content as well
as enhancement of smut tolerance by modern molecular breeding methods
(MAS). In addition RNA interference is a powerful reverse genetics tool to study
gene function by the interference with gene activity. Our on – going research has
been exploring this approach and some promising progress is anticipated which
enable the breeder to achieve substantial improvement in fast, reliable and costeffective way.
Furthermore, introducing new unconventional natural sweetners such as
stevia can contribute to filling the gap between supply and demand. As for the
debate about the safety of stevia for human consumption, it is evident from our
extensive tests on several biological systems that no risks on human health could
be encountered.
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