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International Conference on: “New Role for the World Sugar Economy in a Changed Political and

Economic Environment ”

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.(2)Chairman of Sugar Crops Council, Ministry of Agriculture, Egypt

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 variation to obtain promising mutant types of sugar cane .

10-13 November 2012, Aswan, Egypt Fatthy M. Abdel-Tawab et al 1

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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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Table (5): Means of some cane and sugar traits of the twenty two clones

Clo

ne

No.

Bri

x

No.

of

stal

ks /

m 2

Stal

k he

ight

(c

m)

Stal

k di

amet

er

(cm

)C

ane

yiel

d (t

on/fe

d)

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

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.

RNA interference:10-13 November 2012, Aswan, Egypt Fatthy M. Abdel-Tawab et al

8

Primer No. Primer name TAF PF MF UF P % SM

+ve -veRAPD-PCR

1 OP-A01 19 18 1 2 94.74 4 32 OP-A04 23 22 1 3 91.30 2 13 OP-A18 17 16 1 3 94.11 2 24 OP-A19 13 13 0 2 100 1 ---5 OP-B08 15 14 1 2 93.33 2 16 OP-B10 17 16 1 2 94.11 4 17 OP-B18 18 18 0 4 100 3 ---8 OP-O10 21 21 0 1 100 1 19 OP-O14 20 19 1 2 95 3 1

ISSR-PCR10 17898 A 7 4 3 --- --- 4 ---11 17899 B 11 3 8 --- --- 1 212 HB 12 7 1 6 --- --- 1 ---13 HB 10 6 3 3 --- --- 1 214 HB 13 5 2 3 --- --- 1 1

SSR-PCR15 Xtxp04 19 19 0 12 100 1 ---16 Xtxp08 5 4 1 --- 80 --- 217 Xtxp10 9 7 2 --- 77.78 1 118 Xtxp 12 13 11 2 1 84.62 2 118 Xtxp19 17 16 1 5 94.12 2 ---19 Xtxp61 19 19 0 2 100 --- 2

R-ISSR PCR20 A01 + 844A 6 3 3 --- --- 2 121 A01+17898A 6 2 4 --- --- 1 122 A01+17899B 11 4 7 --- --- 3 123 A01 + HB 12 10 3 7 --- --- 3 ---24 A01 +844 B 4 2 2 --- --- 2 ---25 A01 +HB 10 9 3 6 --- --- 3 ---26 A04 + 844A 5 3 2 --- --- --- 327 A04+17898A 5 3 2 --- --- --- 328 A04 + 844B 6 3 3 --- --- --- 329 A04 + HB10 6 3 3 --- --- 3 ---30 B18 + 844 A 9 4 5 --- --- 4 ---31 B18+17898A 9 5 4 --- --- 4 132 B18+17899B 9 2 7 --- --- 2 ---33 B18 + 844 B 6 2 4 --- --- --- 234 B18 + HB 10 6 3 3 --- --- 1 2

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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-19-oic acid. However, Vis and Fletcher (1956) proposed that, stevioside is the main sweet glycoside (6-8%, in dry stevia leavesYield – 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 Figure 2) which allowed selection to make substantial improvements in this natural sweetner. The stevioside content were highly associated with leaves dry


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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.

(+) = 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 non-caloric 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).


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(9)

(10)

(11)

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(12)

(3)

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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


(4a)

(4b)

(4c)

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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.

Liver% Kidneys% Heart% Spleen% Lung% Testes%

Male

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

Female

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.

GroupsMean of litter size

1st mating 2nd mating

NM X NF

TM X TF

NM X TF

TM X NF

8.9 ± 0.85

7.1 ± 1.03

8.0 ± 0.68

8.8 ± 0.70

5.1 ± 0.44

5.5 ± 0.68

5.0 ± 0.38

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.,

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. Abdel-Tawab 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

Stevioside feedingTreatments

Aspartame feedingAspartame immersionTreatments

Total % mutantsTotal % mutantsBrood2Brood1Brood2Brood1Brood2Brood1

0.1960.178Control0.1960.1780.1960.178Control1.03**0.89**Colchicine1.03**0.89**1.03**0.89**Colchicine0.0520.275mg/ml0.230.180.1890.1604mg/ml0.1240.1520m/ml0.0750.1690.1540.17112m/ml0.0540.05250mg/ml0.1900.2420.1540.10720mg/ml

* 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.

Treatments

Overall mutants rate %

Treatments

Overall mutants rate %

Aspartame immersion

Aspartame feeding

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 cost- effective 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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