Sweet Sorghum Improvement and Production in Brazil

1. Sweet Sorghum Improvement and Production in Brazil Consultation on Pro-poor Sweet Sorghum Development for Bio-ethanol - IFAD November 8-9, 2007 Robert E. Schaffert Embrapa Milho e Sorgo schaffer@cnpms.embrapa.br www.cnpms.embrapa.br

2. In the late 1970´s Brazil initiated a bio-energy program (Pro-Alcohol) anticipating an energy crises caused be a shortage of petroleum to meet Brazil's fuel needs. There was a strong incentive to develop technology for micro-distilleries (100L hr-1) and mini-distilleries (1000L hr-1) Embrapa´s sweet sorghum program was developed to provide raw material for these distilleries. Pilot Projects were successfully developed in the mid-1980’s at Sete Lagoas, Brasilia, and Pelotas to process sweet sorghum in micro-distilleries.

3. The potential uses of sweet sorghum for food, fiber, fertilizer, ethanol, and methane gas production (Embrapa Maize and Sorghum) Silage xxxx

4. Advantages of Using Sweet Sorghum vs. Sugarcane • Sweet sorghum may be harvested 3 – 4 months • after planting • Sweet sorghum production can be completely • mechanized • The sweet sorghum crop can be established • from seed • The grain from sweet sorghum can be used as • food, feed or fuel • The bagasse from sweet sorghum has a higher • biological value than the bagasse from • sugarcane when used as a forage for animals • Sweet sorghum is more water use efficient

5. Sweet Sorghum Sugarcane Micro- distillary Other uses Integrated rural energy system developed at Embrapa Maize and Sorghum 1980 Integrated Rural Energy System Developed at Embrapa Maize and Sorghum 1980 xxxxxxxx

6. Development of Sweet Sorghum Cultivars Breeding priorities depend upon how the product will be used • Panicles removed for food or feed • Small vs. large panicles • Juice is extracted from stalks and leaves • Not feasible to separate leaves and stalks • Juice fermented in micro- and mini-distilleries • Juice extraction less efficient in smaller • distilleries • Bagasse used for forage or fuel • Starch present in sorghum juice and stalk • Biological value of sweet sorghum bagasse is • greater for sweet sorghum than for sugarcane • Tillering vs. Non-tillering to control stalk diameter

7. A Simple Flow Diagram of a Roller Mill Micro-distillery; Used in Establishing Breeding Priorities at Embrapa Maize and Sorghum A - roller mill, B - boiler, C - yeast treatment and distribution tank, D - juice distribution tanks, E - fermentation tanks, F – beer holding tank, and G - distillation column (Embrapa Maize and Sorghum) Xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx X X x

8. Industrial Planning Sweet Sorghum Planting Harvesting Sugarcane Planting Harvesting Planting and harvesting periods for sweet sorghum and sugarcane in Brazil. (Embrapa Maize and Sorghum) J F M A M J J A S O N D Ratoon Harvest Irrigation Required

9. Yield and Quality Goals must be Established • Minimum Biomass Yield - 40 tha-1 • Minimum Total Sugar Extraction – 80 kg 1t-1 biomass • Considering 60-65% Extraction Efficiency • Minimum Total Sugar Content in Juice – 12.5% • Minimum Alcohol Yield – 40L t-1 biomass Considering: 60 -65% sugar extraction efficiency 90% Fermentation Efficiency 90% Distillation Efficiency or 81% Industrial Efficiency • Establish minimum parameters for determining Period of • Industrial Utilization (PIU - 80 kg 1t-1 biomass) • Minimum PIU – 30 days

10. Total Biomass Production of Two New Sweet Sorghum Varieties Developed at Embrapa for Micro-distilleries * * * * *

11. Sweet Sorghum Nomenclature Sweet sorghum variety names that begin with the letter or sound or R are high sucrose types and variety names that begin with other letters are low sucrose types. Redlan and Tx623 are non-sweet juicy stem female lines.

12. RIO PIU – 32 days XXX Xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx x The interaction of refractometry Brix and percent total reduced sugars in the juice and percent fiber, percent juice extraction, and percent sugar extraction of sorghum stalks during the maturity phase for the varieties Rio and Wray grown in Brazil, (Embrapa Maize and Sorghum) xxxxxxxxxxxxxxxxxxx WRAY PIU – 58days Xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx Zzzz

13. -1 Brix, Total Sugars, Fiber and Percent Water of four sweet sorghum cultivars at Embrapa Maize and Sorghum, Sete Lagoas, Brazil, 1986/87. BR 501 = Brandes, BR 505 = Wray, BR506 and BR507 are new derived varieties Sugar extracted ( Kg 100 Kg biomass) Brix (% juice)) xxx Percent fiber of fresh biomass xxx BR 505 Total sugars (% juice) Total Sugars (%) Percent water in the biomass Xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx

14. Period for Industrial Utilization Wray 9% Total sugars Wray 8% Total sugars xxxx Wray PUI 32 days Wray PUE 51 days xxxxxxxxxx

15. PUI 28 days The interaction of refractometry Brix and percent total invert sugars in the juice and percent fiber, percent juice extraction, and percent sugar extraction of sorghum stalks during the maturity phase for the variety Rio grown in Brazil (Embrapa Maize and sorghum) .

16. Brix, total sugars (%), fiber (%), and juice extraction (%) Sugar extraction (%) PUI – 35 days The interaction of refractometry Brix and percent total invert sugars in the juice and percent fiber, percent juice extraction, and percent sugar extraction of stalks during the maturity phase for the variety Brandes grown in Brazil (Embrapa Maize and Sorghum).

17. CMSXS 623 (Brandes Derivative) Brix, total sugars (%), fiber (%), and juice extraction (%) Sugar extraction (%) PUI 37 days The interaction of refractometry Brix and percent total invert sugars in the juice and percent fiber, percent juice extraction, and percent sugar extraction of stalks during the maturity phase for the variety Brandes grown in Brazil (Embrapa Maize and Sorghum). Days after planting The interaction of refractometry Brix and percent total invert sugars in the juice and percent fiber, percent juice extraction, and percent sugar extraction of sorghum stalks during the maturity phase for the variety CMSXS 623 grown in Brazil (Embrapa Maize and sorghum).

18. PUI 54 days The interaction of refractometry Brix and percent total invert sugars in the juice and percent fiber, percent juice extraction, and percent sugar extraction of stalks during the maturity phase for the variety Brandes grown in Brazil (Embrapa Maize and Sorghum). The interaction of refractometry Brix and percent total invert sugars in the juice and percent fiber, percent juice extraction, and percent sugar extraction of sorghum stalks during the maturity phase for the variety Wray grown in Brazil (Embrapa Maize and sorghum) .

19. Biomass and Alcohol Production of two new Cultivars, BR506 and BR507, Compared to Brandes and Wray Embrapa Maize and Sorghum 1986/87

20. Current Plant Breeding Strategies and Priorities • Develop sweet sorghum 3 dwarf A and B lines (female) to be used • in hybrid development and production • - Identify molecular markers for juicy stem and sweet juice • - Incorporate genes for multiple stress (biotic and abiotic) • resistance • Evaluate existing new sweet sorghum cultivars (approx. 50) and • develop new sweet sorghum R lines (male) to be used in • hybrid development and production • - Incorporate genes for multiple stress (biotic and • abiotic) resistance • Identify molecular markers for tillering - Non-tillering desired to be • able to control stalk diameter with plant population (better • extraction with large stem diameter) • Develop transgenic sweet sorghum with sucrose isomerase gene • (SI), a gene that regulates the transformation of sucrose to • isomaltulose and thus can increase the sink capacity of sugar • storage (Wu and Birch, Plant Biotechnology Journal (2007) 5 • (pp109-117)

21. Plant Breeding Strategies and Priorities (cont.) • Develop high yielding biomass cultivars for cellulose conversion • to bio-energy • - Identify molecular markers for maturity genes Ma5 and Ma6 • to be able to develop photosensitive (PS) sorghum biomass • hybrids Ma5Ma5ma6ma6 is photo-insensitive (PIS) and flowers in approximately 60 days regardless of day length. ma5ma5Ma6Ma6 is photo-insensitive and flowers in approximately 60 days regardless of day length. The hybrid between these genotypes, Ma5ma5Ma6ma6 is photo-sensitive (PS) and floral initiation is only induced with day lengths less than 12h and 20min. This can be useful in both sweet sorghum and biomass sorghum in locations farther from the equator where there is more variation in day lengths (McCollum et al. reported average yield increases of 25% of PS hybrids over PIS hybrids at Amarillo, Texas).

22. Plant Breeding Strategies and Priorities (cont.) • Develop high yielding biomass cultivars for cellulose conversion • to bio-energy • - Identify molecular markers for brown midrib low lignin • genes genes brm-6 and brm-12 Homozygous brm-6 hybrids and brm-12 hybrids have been reported to reduce lignin content in sorghum biomass by 50%

23. The price of ethanol today (November 5, 2007) is about US$ 0.35 L-1 at the distillery gate and US$ 0.75 to 0.90 at the pump, depending on the distance from distilleries

24. Incorporação de Tolerância ao Al em Cultivares de Sorgo Elite e Desenvolvimento de Linhagens Isogênicos Seleção p/ Tolerância ao Al em Solução Nutritiva BR 007B SC 283 Tol x Suc F3 Progenies Retrocruzamento Assistido para Desenvolver Linhagens de Sorgo com Tolerância ao Al Tóxico. Estamos incorporando o gene para tolerância ao Al em nossas linhagens elites em seis gerações usando a casa de vegetação em dois anos. O parâmetro usado é crescimento de raiz em solução nutritiva por sete dias. Usando este tecnologia estamos empilhando genes úteis em genótipos com tolerância ao estresse múltiplo. Estamos com ensaios no campo com três níveis de Al para quantificar o efeito dos genes para tolerância ao Al tóxico na produtividade e estabilidade de produção. Linhagem Recorrente Fonte Susceptível x Tolerante Cross tt x TT F1 Tt BC1 tt x Tt BC1F1 ½ Tt (½ tt eliminado em solução nutritiva) BC2 tt x Tt BC2F1 ½ Tt (½ tt eliminado em solução nutritiva) . . . BC4F1 ½ Tt (½ tt eliminado em solução nutritiva) BC4F2 ¼ TT ½ Tt (½ tt eliminado em solução nutritiva) BC4F3 100% TT and 97%Igual ao pai recorrente

25. COELHO & SCHAFFERT, 2005

26. Intra-Specific Diversity for Al Tolerance in Sorghum • SC283 and SC566 rely on the same locus AltSB for Al tolerance • Genetic basis apparently narrow • 8 different Al tolerant sources x BR012 • Inheritance study and linkage analysis to markers in the AltSB region • SSR-based phylogenetic analysis • Al tolerance gene diversity for pyramidation via breeding • Stronger alleles of AltSB • Different haplotypes at AltSB for candidate gene validation • Foundation for future association tests

27. Desenvolvimento de Sorgo Mais Eficiente na Aquisição de P Xxxx BR007B (IR) BR 005R (EN) SC 283 (EN) + P - P + P - P + P - P SC283 BR007 Pelos de raízes dos genótipos SC283 (eficiente) e BR007 (responsivo) em estresse de P • A identificação e conhecimento das mudanças no rhizosfero, mecanismos de aquisição e utilização de P facilitarão a manipulação destes parâmetros. • Não existe conhecimento de parâmetros nos cereais correlacionados a eficiência de aquisição/utilização de P. xx Plantas de Sorgo com 21 Dias em Rhizobox xxx • Tem a necessidade de identificar ou desenvolver genótipos com diferenças na habilidade de adquirir e utilizar P eficientemente • Utilizando condições de campo, solução nutritiva, e casa de vegetação, identificamos genótipos de sorgo contrastantes para aquisição e resposta ao P BR007B SC 283 +P -P

28. Produção diferencial de Sorgoleone em Sorgo BR 007 B – SEM FÓSFORO SC 283 – SEM FÓSFORO Sorgoleone Production by Various Sorghum Genotypesa a- three replicates of 25 seedlings each Fonte: Chandrashekhar I, et al.; J. Agric. Food Chem. 1996, 44, 1343−1347 Photomicrograph of Sorghum bicolor roots showing a sorgoleone-rich oily exudate secreted from the root hairs (Bar=80 μm) and b closer view of a root hair with sorgoleone exuding at the tip (Bar=15 μm). Sorgoleone-rich oil from tip of root hairs also exudes from secondary roots originating either from c roots (Bar=125 μm) or d stem (adventitious roots) (Bar=350 μm) Fonte: F.E. Dayan, USDA-ARS Natural Products Utilization Research Unit, P.O. Box 8048, University, MS 38677, USA

29. Desenvolvimento de Cultivares de Sorgo Forrageiro de Alta Qualidade (bmr6 bmr6) Hibrido Sorgo Forrageiro de Corte Tx635bmr6A x Tx2785bmr6R Hibrido Sorgo Forrageiro de Corte Tx635bmr6A x Tx2784bmr6R Linhagens R bmr6 Linhagens A e B bmr6 Desenvolvimento de novas linhagens bmr6 A/B e R com tolerância ao múltiplo estresse A/BTx 635 A/B BR007 A/B CMSXS205 A/B CMSXS206 A/B BR008 A/B CMSXS156 A/B CMSXS 157 RTx2784 RTx2785 CMSXS912 CMSXS225

30. Final Remarks • Embrapa has a relative large number (50 -60) of sweet sorghum varieties • available for characterization for quality and utilization in developing • experimental hybrids. • The variety BR501 (Brandes) is tolerant to Al toxicity and is the restorer • parent in two commercial forage sorghum hybrids. Brandes probably • was selected 25 years ago because it was tolerant to Al toxicity. • Brandes was successfully used in two pilot sweet sorghum distilleries in • Jundiai, SP and Pelotus, RS. • The proof of concept of sweet sorghum as a biofuel source has been • determined. The next step is an economic evaluation for • competitiveness with other available raw materials. • Embrapa is currently evaluating the return to an active research and • development program with sweet sorghum as a source for bio-fuels.

31. Thank You Robert Schaffert Embrapa Maize and Sorghum reschaffert@hotmail.com schaffer@cnpms.embrapa.br Tel: +55-31-3779-1076

34. Hibrido Sorgo Forrageiro de Corte Tx635bmr6A x Tx2785bmr6R

35. Percent fiber and juive extraction of cultivar Brandes at Araras, CNPMS/EMBRAPA, 1981 and 1982 (Schaffert et al. 1986). Percent fiber and juive extraction of cultivar Wray at Araras, CNPMS/EMBRAPA, 1981 and 1982 (Schaffert et al. 1986). Fiber Increases and Juice Extraction Decreases with Time Brandes Wray

36. Juice Extraction Varies Between Cultivars Percent juice extraction of four sweet sorghum cultivars at CNPMS/EMBRAPA, Sete Lagoas, Brazil, 1986/87, BR 501 = Brandes; BR 505 = Wray.

40. Mean yield of stalk, fermentable sugar, alcohol, fresh biomass and seed of sweet sorghum in experiments at the Beijing Botanical Garden

47. Sucrose vs. Total sugars

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50. Wray Percent total sugar (Juice) Rio Brandes CMSXS623 Days after planting The differences between four cultivars grown in Brazil for total invert sugars of the juice during the maturity phase of production (Embrapa Maize and sorghum).