Enhancement of sugar production by modern biotechnological methods

Professor of genetics and molecular biology, Faculty of Agriculture, Ain shams University, Cairo, Egypt.

Fatthy M. Abdel-Tawab

Abdel-Wahab I. AllamProfessor of genetics, Director of Supreme

Council of Sugar Crops, Cairo, Egypt

List of contents1- Sugarcane1-1 Phylogenetic relationships in sugarcane1-2 Marker assisted selection in sugarcane

1-2-1 Molecular markers for smut resistance1-2-2 Molecular markers for sugar content (Brix)1-2-3 Functional genomic analysis for enhancement of sugar content by

RNAi approaches

2-Stevia2-1 MAS2-2 Genotoxity

3- Recommendations

Phylogenetic relationships in sugarcane

• The phylogenetic relationships between twelve sugarcane genotypes belonging to three different Saccharum spp. 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 .

• Microsatellites are valuable tools, 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.

• Fig. (1): Dendrogram showing genetic distances between 12 sugarcane genotypes based on 13 RAPD and 9 SSR markers combined.

Marker assisted selection in sugarcane

Traditional sugarcane breeding steps

1- Parental selection from a source population.2- Hybridization using bi-parental crosses and

polycrosses.3- Progeny selection at several stages.

Commercial cultivar 12-15 years

Sugarcane breeding difficulties

• Saccharum spp. are genetically large genome size ( 3.05-8.91 pg) .

• Complexity levels in commercial cultivars (2n=99-168 chromosomes) are aneuploid with various ploidy levels.

• Occurrence of somaclonal variation.• The challenge in plant breeding is identifying the superior progeny Molecular markers are valuable tool in indirect and early selection.

1- Molecular markers for smut resistance:

- Ten cultivars were used in this study including seven promising cultivars, i.e., G99/165, G95/19, G95/21, G98/28, G98/24, G84/47, G85/37, one susceptible cultivar NCo310 and the commercial cultivars; GT54/9 and Ph8013.

- The performance of the ten cultivars which were artificially infected with teliospores suspension was assessed under greenhouse conditions.

- The results revealed that nine cultivars were relatively resistant (R). Some molecular markers, using RAPD-PCR and ISSR-PCR techniques were positively associated with smut resistance.

- The molecular markers identified in this study could be used to accelerate selection programs for smut resistance in cost-effective way.

1- Evaluation of sugarcane progeny from different crosses for sugar content and some sugar-related traits.

2- Development of molecular markers associated with sugar content using RAPD, ISSR, R-ISSR and SSR-PCR techniques.

2- Molecular markers for sugar content(Brix):

1- Twenty two clones were chosen from 4000 clones resulted from crosses in green house of SCRI according to some vegetative traits.

2- Differences between means were compared using Duncan’s Multiple range test (Duncan 1955).

3-Brix values were used as an indicator of sugar content.

4- these clones were divided into two groups (according to Brix):

high sugar content; 13 (A) & low sugar content; 9 (B).

Formula to calculate percent pol (sucrose) in juice:% pol = {-6.517 + (25.3 X PR*) – 0.X (PR x PR) + 2.37 X brix) – 0.207 x (brix x brix) } / 100*PR = correction indices of brix from specific table

1- Stalk diameter had no significant differences while the other traits showed significant differences between individuals.

2-The two groups showed significant differences regarding Brix, sugar yield and number of stalks traits.

Table(1): Means of Brix & some sugar content-related traitsClone No. Brix*

No. of stalks / m2 Stalk height (cm)

Stalk diameter (cm2) Cane yield (ton/fed) Sugar yield (ton/fed)

Group (A)

190 22.5A 14.1A 298D 2.78A 52.500AB 5.7A

191 22.17AB 14.3A 290 F 2.86 A 53.000 A 5.68 A

189 21.75ABC 15.6 ABCD 291 F 3.00 A 48.889FGH 5.14 ABCD

209 21.67ABC 16.1 AB 299 CD 2.45 A 51.150BCD 5.38 AB

104 21.33ABC 18.3 ABC 290 F 2.56 A 50.600CDE 5.23 ABC

120 21.17ABC 17.8ABCDE 286 H 2.83 A 46.056J 4.72 ABCDEF

194 21.08ABC 16.0 AB 286 H 2.53 A 52.700AB 5.38 AB

121 21 ABC 13.06ABCD 280 I 3.06 A 50.000DEF 5.09 ABCD

87 20.85BC 8.55ABCDE 275 K 3.11 A 48.734FGH 4.93 ABCDE

198 20.83BC 9.15ABC 300 C 2.56 A 52.350ABC 5.29 ABC

122 20.83 BC 14.32ABC 299 CD 2.73 A 52.900 A 5.34 ABC

188 20.67 BC 10.32ABCDE 288 G 2.92 A 48.740FGH 4.89 ABCDE

193 20.33 C 8.00ABCD 308 A 2.40 A 51.750ABC 5.11 ABCD

Group (B)

29 11.5H 11.30G 295 E 2.80 A 51.100BCD 3.04G

131 12.83 GH 9.61FG 288 G 2.75 A 47.553HIJ 3.12FG

36 13.33 FG 7.13FG 285 H 2.86 A 52.250ABC 3.55DEFG

97 13.67EFG 13.61FG 280 I 3.02 A 50.400DEF 3.51DEFG

133 14 EFG 10.16FGH 270 M 3.01 A 47.000IJ 3.34EFG

94 14.67 EF 9.71EFG 303 B 2.85 A 51.900ABC 3.85BCDEFG

160 15 DE 15.16FGH 290 F 2.98 A 51.500ABCD 3.9BCDEFG

139 15 DE 12.30FGHI 270 M 2.99 A 49.231EFG 3.73CDEFG

4 16.5 D 10.86ABCDEFG 300 C 2.36 A 49.900DEF 4.13ABCDEFG

Marker type No. of primers or combinations +ve markers -ve Markers

RAPD-PCR 9 Primers 22 7

ISSR-PCR 5Primers 8 5

R-ISSR-PCR 20 Combinations 28 16

SSR-PCR 6 Primers 6 6

Table: Summary of molecular markers associated with sugar content (BRIX)

(+ve) = Positive marker for high sugar content (-ve )= Positive marker for low sugar content

Functional genomic analysis for enhancement of sugar content by RNAi approaches

siRNA5’

3’ 5’

3’

RISC RNA-Induced Silencing Complex

RNA/DNA Helicase (is required to unwind the dsRNA)

Translation Initiation Factor

RNA-Dependent RNA Polymerase (RdRP)

Transmembrane Protein

Effector Step

• siRNA binding• siRNA unwinding• RISC activation

RISC (RNA-Induced Silencing Complex(

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 synthase(SUC SYN) and sucrose phosphate synthase ﴾SPS ﴿ are involved in regulation of accumulation and / or breakdown of sucrose.

• Both SAI and SUC SYN 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.

• Sucrose -6-phosphate synthetase(EC2.4.1.12)• Sucrose synthase(EC2.4.1.13)• Soluble acid invertase

Sucrose synthesis

• Isolation of some genes responsible for sucrose content in sugarcane.

• Down regulation of genes responsible of sucrose breakdown in sugarcane. (invertases)

• Up regulation of genes which increase sucrose percentage in sugarcane. ( sucrose phosphate synthase) )

Steps of study

• Using databases to detect the sequence of genes affecting sucrose content.

• Isolation, cloning and sequencing of the candidate genes

• Comparing the obtained sequences with the related genes using bioinformatics approaches.

• Designing SiRNA sequence for targeted genes and insert it in suitable expression vector

• Transform it in sugarcane plant callus• Evaluating the transformed plants for the sucrose

content trait in GM and control plants

Candidate genes location and size from NCBI site

1) LOCUS: HQ117935 SIZE: 3252 bp mRNA linear PLN 02-SEP-2011 2) LOCUS: JN584485 Size: 3481 bp mRNA linear PLN 19-SEP-2011 3) LOCUS: AB001338 Size: 3287 bp RNA linear PLN 17-OCT-2008 4) LOCUS: EU278617 Size: 6493 bp DNA linear PLN 11-DEC-2007

5) LOCUS: EU278618 Size :7382 bp DNA linear PLN 11-DEC-20076) LOCUS: EU269038 Size: 3186 bp mRNA linear PLN 03-DEC-2007 7) LOCUS :AB001337 Size : 3322 bp mRNA linear

PLN 13-FEB-1999

Sucrose phosphate synthase

1) LOCUS: AY670701 Size: 3632 bp DNA linear PLN 15-MAR-2005 2) LOCUS: AY670699 Size: 3857 bp DNA linear PLN 15-MAR-2005 3) LOCUS: AY670702 Size: 3857 bp DNA linear PLN 15-MAR-2005 4) LOCUS: AY670700 Size:3867 bp DNA linear PLN 15-MAR-2005

5) LOCUS: AF263384 Size: 2717 bp mRNA linear PLN 03-SEP-2003

6) LOCUS : AY118266 Size: 7771 bp DNA linear PLN 15-MAR-20057) LOCUS: AY670698 Size: 3634 bp DNA linear PLN 15-MAR-2005

Sucrose synthase II

1) LOCUS :AF083855 Size: 494 bp mRNA linear PLN 17-SEP-1998 2) LOCUS: AF062734 Size :1808 bp mRNA linear PLN 18-MAY-1998 3) LOCUS: AF062735 Size: 1808 bp mRNA linear PLN 18-MAY-1998

4) LOCUS : AF083856 Size: 1402 bp mRNA linear PLN 17-SEP-1998 5) LOCUS : AY302083 Size: 2274 bp mRNA linear PLN 12-JAN-2010

Soluble acid invertase

What is Stevia?

1-

Stevia is a branched bushy shrub of the Asteraceae (Compositae) family, native to the Amambay region in the north east of Paraguay. Known as a ‘‘ sweet herb ’’ or called ‘‘ka’a he’ê ’’ Source of a high-potency natural sweetenerIt is safe for diabetics, as it does not affect blood sugar levels.Used in Paraguay for centuries, Japan for decadesDiscovered centuries ago, FDA approval in 2008There are many advantages of using Stevia : - Stevia leaves are 20-30 times sweeter than sugar. - Stevia leaves can be dried and stored. - Stevia can be used in raw form. - Stevia is short duration crop. - It is harvested 3/4 times a year. - The yearly yields can be in the range of 3-4 ton

• Steviol Glycosides• Variety of sweet-flavored molecules within the leaf

• 9 Steviol glycosides recognized by Joint FAO/WHO Expert Committee on Food Additives (JECFA).Structure of the major glycosides of Stevia rebaudiana leaves. Glc, Xyl, and Rha represent, respectively, glucose, xylose, and rhamnose sugar moieties (Geuns, 2003).

What Makes Stevia Sweet?

What is Stevioside ?

heat-stable

300 times sweeter

non-caloric

Stevioside, the major sweet substance of stevia plant (5-10% of dry weight), is 300 times as sweet as sucrose, having steviol as its aglycone and attached to three glucose molecules .Stevioside has the chemical formula of a diterpene glycoside (C38H60O18)

Stevioside100% natural

Potential Uses for Stevia– Soft drinks, teas, fruit juices– Table top sweeteners– Hot and cold cereals– Granola and snack bars– Yogurt– Flavored milk– Ice cream– Salad dressing– Baked goods– Chewing gum– Canned fruit and jams– Desserts– Alcoholic beverages

Food sweetened with Stevioside

In a study on fifteen stevia accessions, Allam et al., (2000) reported marked variations in yield components and stevioside content 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 .

Yield – related traits and stevioside content:

Means of some yield-related traits and stevioside content for the 15 stevia accessions

Molecular marker associated with some stevia traits.

(+) = Positive marker, (-) = Negative marker

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 or lower weight. 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 . In vitro study on human lymphocytes revealed no significant differences between the treated cells and controls ones.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).

Assessment of genotoxicity:

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%

MaleControl 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 -

(7)

DNA profile of the tumor suppressor gene (P53 ﴿ indicated that no carcinogenic effect was observed after feeding mice on stevia.

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.

Genetic activities of three different concentrations of stevioside in Saccharomyces cerevisiae strain D7.

C= Control T= Treatment -=<2 control level.

No mutagenic activities including the induction of mitotic gene conversion, mitotic crossing-over and reversion were obtained. Instead, the frequencies of the three end points were lower than the spontaneous levels.

In conclusion, the biological effectiveness of the three different concentrations of stevioside proved to have no effect on the survival percentages and no mutagenic activity on the D7 strain

In addition the obtained results revealed that stevioside has no mutagenic effects in all tested genetic end points on Drosophila; moreover, it decreased the spontaneous mutation rate than the concurrent negative control . Therefore, the possible antimutagenic effect of stevioside has been tested against the mutagenic activities of 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).

Negative control Stevioside (St) 5 mg/ml

MMC 20μg/ml Pre-treatment Post-treatment0

0.5

1

1.5

2

2.5

Frequency (No. of Tumor/ fly)

NO

. of T

umor

/ fly

Diagram represents the frequencies of spontaneous and induced warts epithelial tumors in wts/+ flies after treatments with Mitomycin C (MMC), stevioside (St) and combinations of MMC and stevioside (St) in post and pre treatments

- The frequency of induced tumor after MMC treatment of pretreated larvae with stevioside (5 mg/ml) was highly significant reduced (0.81 per fly), which showed 64% reduction of induced tumors.in post-treatment experim-

ent, larvae exposed to stevioside (5 mg/ml) after MMC treatment showed highly significant reduction of induced tumors (66 %) with a tumor average of 0.77 tumor/fly

Fig. (7): Diagram represents the frequencies of spontaneous and induced warts epithelial tumors in wts/+ flies after treatments with Mitomycin C (MMC), stevioside (St) and combinations of MMC and stevioside (St) in post and pre treatments in addition to the reduction rate of induced tumor frequencies due to antimutagenic activity of stevioside

Warts (Wts) phenotype

Negative Control

Stevioside (St) 5 mg/ml

MMC 20μg/ml

Warts (Wts) phenotype

MMC 20μg/ml

Pre-treatments (Stevioside - MMC)

post-treatments (MMC -Stevioside)

Abdel-Tawab et al., (2008) reported that Stevioside has no mutagenic effect in all tested genetic end points in Drosophila. PCR-based RAPD analysis was used to assess 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 this world wide uses of sweetener indicated no hazards to the health and welfare of the consumers.

antineoplasticeffect

improves cell regeneration

Antioxidant activity

Antimicrobial activity Stevia

antidiabetic

anti-obesity

Antihyper-glycemic

anti human rota-virusactivities

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 were encountered.

Recommendations :