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Enhancement of sugar production by modern biotechnological methods

Enhancement of sugar production by modern biotechnological methods. Fatthy M. Abdel-Tawab. Professor of genetics and molecular biology, Faculty of Agriculture, Ain shams University, Cairo, Egypt. Abdel- Wahab I. Allam.

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Enhancement of sugar production by modern biotechnological methods

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  1. Enhancement of sugar production by modern biotechnological methods Fatthy M. Abdel-Tawab Professor of genetics and molecular biology, Faculty of Agriculture, Ain shams University, Cairo, Egypt. Abdel-Wahab I. Allam Professor of genetics, Director of Supreme Council of Sugar Crops, Cairo, Egypt

  2. List of contents 1- Sugarcane 1-1 Phylogenetic relationships in sugarcane 1-2 Marker assisted selection in sugarcane 1-2-1 Molecular markers for smut resistance 1-2-2 Molecular markers for sugar content (Brix) 1-2-3 Functional genomic analysis for enhancement of sugar content by RNAi approaches 2-Stevia 2-1 MAS 2-2 Genotoxity 3- Recommendations

  3. Phylogenetic relationships in sugarcane

  4. 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.

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

  6. Marker assisted selection in sugarcane

  7. 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

  8. 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.

  9. 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.

  10. 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):

  11. 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

  12. 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.

  13. Table(1): Means of Brix & some sugar content-related traits

  14. Table: Summary of molecular markers associated with sugar content (BRIX) (+ve) = Positive marker for high sugar content (-ve )= Positive marker for low sugar content

  15. Functional genomic analysis for enhancement of sugar content by RNAi approaches

  16. siRNA 3’ 5’ 3’ 5’

  17. RISC RNA-Induced Silencing Complex Translation Initiation Factor RNA/DNA Helicase (is required to unwind the dsRNA) RNA-Dependent RNAPolymerase (RdRP) Transmembrane Protein

  18. Effector Step RISC (RNA-Induced Silencing Complex( • siRNA binding • siRNA unwinding • RISC activation

  19. RNA interference is a powerful reverse genetic tool to study gene function by the interference with gene activity.

  20. 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.

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

  22. Steps of study • 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) )

  23. 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

  24. Candidate genes location and size from NCBI site

  25. Sucrose phosphate synthase 5) LOCUS: EU278618 Size :7382 bp DNA linear PLN 11-DEC-2007 6) LOCUS: EU269038 Size: 3186 bp mRNA linear PLN 03-DEC-2007 7) LOCUS :AB001337 Size : 3322 bp mRNA linear PLN 13-FEB-1999 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

  26. Sucrose synthase II 5) LOCUS: AF263384 Size: 2717 bp mRNA linear PLN 03-SEP-2003 6) LOCUS : AY118266 Size: 7771 bp DNA linear PLN 15-MAR-2005 7) LOCUS: AY670698 Size: 3634 bp DNA linear PLN 15-MAR-2005 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

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

  28. What is Stevia? • 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’ahe’ê’’ • Source of a high-potency natural sweetener • It is safe for diabetics, as it does not affect blood sugar levels. • Used in Paraguay for centuries, Japan for decades • Discovered centuries ago, FDA approval in 2008 • There 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 • 1-

  29. What Makes Stevia Sweet? • 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).

  30. What is Stevioside?

  31. 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) • Stevioside • 100% natural

  32. 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

  33. Food sweetened with Stevioside

  34. Yield – related traits and stevioside content: In a study on fifteen stevia accessions, Allamet 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 , esteraseisoenzymes and randomly amplified polymorphic DNA ( RAPD). These markers could be efficiently used to assist selection for accessions with high stevioside content .

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

  36. Molecular marker associated with some stevia traits. (+) = Positive marker, (-) = Negative marker

  37. Assessment of genotoxicity: Stevia rebaudianaBertoni, 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.

  38. Assessment of genotoxicity: 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 nomutagenic effect on bone marrow cells or lower weight. In vivo study on Drosophiliamelanogaster 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).

  39. Means and standard errors for males and females organs weights as percentage from body weight for treated and control parental groups.

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