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021614 yang-rui li--research and development priorities for sugar industry of china

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  • 1. Research and Development Priorities for Sugar Industry of China: Recent Research Highlights Dr. Yang-Rui Li, Professor and Director Sugarcane Research Center Chinese Academy of Agricultural Sciences Guangxi Academy of Agricultural Sciences International Conclave on Sugar Crops & SugarFest 2014 Sweeteners and Green Energy from Sugar Crops: Emerging Technologies February 15-17, 2014, Lucknow-India
  • 2. Distribution of Sugar Crops in China 1 Guangxi 2 Yunnan 3 Guangdong 4 Hainan 5 Hunan 6 Sichuan 7 Jiangxi 8 Fujian 9 Guizhou ① ③ ② ② ④ ⑤ ⑥ ⑦ ⑧⑨ Major Sugarcane Growing Area in Mainland China
  • 3. Sugar industry for 2012/2013 in China • Total sugar:13.07 million tons; • Cane sugar is 11.98 million tons; • Beet sugar 1.09 million tons. • Sugar mills: total 293 among them 248 for sugarcane and 45 mills for sugar beet.
  • 4. Sugarproduction(MillionTons) Milling year Sugar Production in China 0 2 4 6 8 10 12 14 16 98/99 99/00 00/01 01/02 02/03 03/04 04/05 05/06 06/07 07/08 08/09 09/10 10/11 11/12 12/13 Cane sugar Beet sugar Tatol sugar
  • 5. The cane sugar output (million ton) in the major provinces in China since 1998-99 Year Total Guangxi Yunnan Guang- Hainan Others dong 2002/03 9.40 5.61 1.89 1.17 0.42 0.31 2003/04 9.43 5.88 1.95 0.99 0.41 0.20 2004/05 8.57 5.32 1.59 0.90 0.39 0.37 2005/06 8.01 5.38 1.41 0.92 0.19 0.12 2006/07 10.75 7.09 1.83 1.28 0.38 0.17 2007/08 13.68 9.41 2.16 1.45 0.52 0.17 2008/09 11.53 7.64 2.23 1.06 0.46 0.14 2009/10 10.07 7.10 1.71 0.86 0.32 0.08 2010/11 9.66 6.73 1.76 0.87 0.23 0.07 2011/12 10.51 6.94 2.01 1.15 0.31 0.10 2012/13 11.98 7.93 2.24 1.21 0.50 0.10
  • 6. Challenge for Sugar Industry in China
  • 7. 1. More than 80% of sugarcane is grown in rain-fed upland fields, and easily affected by drought.
  • 8. 2. Cost increase for sugarcane production due to fast increase in labor, fertilizers, chemicals and other supplies. • Cost escalation Labor cost: 20-30% increase yearly. Cane harvest: 99% manual operation; 100-150 Yuan (USD$16.5-24.8) per ton. Fertilizer & chemical: 30% increase; especially 50-70% increase for potassium fertilizer.
  • 9. 3. Singleness of sugarcane variety It is suffered easily by biotic and abiotic stresses, such as low temperature, insect pest , diseases, etc. Serious damage by frost
  • 10. 4. Pests and diseases Ratoon stunting disease Smut Shoot rot
  • 11. Borer Thripid
  • 12. Aphis Scarab Longhorn beetle
  • 13. Sugarcane germplasm innovation Future research priorities of Chinese sugar industry
  • 14. Sugarcane germplasm innovation • Wild germplasm such as S. Spontaneum, Erianthus, Narenga distributes in various locations of China. • Incorporate wild germplasm such as Saccharum spontaneum L., Erianthus arundinacius (Retz.) Jesws. and Narenga porphyrocoma (Hance) Bor. into the commercial breeding parents for sugarcane improvement.
  • 15. SRC-CAAS/GXAAS has a sugarcane cross breeding base in Sanya city of Hainan province (18°35′ N, 109°68′E).
  • 16. E. arundinaceus - S. spontaneum complex (AS complex) creating E. arundinaceus × S. spontaneum 15 crosses, 8000 seedlings Progeny detection : Phenotype + molecular (SSR-PCRSRAP-PCR)+cytology AS complex (GXAS) AS complex F1 (GXASF1) AS complex BC1 (GXASBC1) Backcross: S. hybrid Backcross: S. hybrid Germplasm innovation with S. spontaneum and E. arundinaceus
  • 17. AS complex: GXAS07-6-1 (GXA87-36 × GXS79-9) 1:Female:GXA87-36 2:Male: GXS79-9 3: GXAS07-6-1 400 300 200 100 1 2 3 1 2 3 1 2 3 M SSR identification mSSCIR36 mSSCIR67 mSSCIR19 Germplasm innovation with S. spontaneum and E. arundinaceus
  • 18. Germplasm innovation with S. spontaneum and E. arundinaceus AS complex: GXAS07-6-1
  • 19. × GT02-761 Sugarcane germplasm innovation AS complex: GXAS07-6-1 Progeny
  • 20. × GT02-761 Sugarcane germplasm innovation AS complex: GXAS07-6-1 Progeny
  • 21. chromosome observation GXS79-9 (2n=64) GXA87-36(2n=60) GXAS07-6-1 (2n=62) GXASF1 08-2-33 (2n=80) GXASF1 08-1-10 (2n=76) GXASF1 08-3-1 (2n=76) Deduced transmission pattern: n + n Chromosome transmission of AS complex
  • 22. AS complex genomic in situ hybridization (GISH) 50×Blocking 80×Blocking Green: S. spontaneum Red: E. arundinaceus Sugarcane germplasm innovation
  • 23. Similarity coefficient between GXASF1 and male parent ♀YT93-159 ♂and F1 ♀GT01-53 ♂and F1 ♀GT02-761 ♂and F1 GXAS07-6-1 0.488 GXAS07-6-1 0.535 GXAS07-6-1 0.531 08-1-1 0.753 08-2-1 0.713 08-3-1 0.723 08-1-2 0.736 08-2-2 0.748 08-3-2 0.746 08-1-3 0.719 08-2-5 0.742 08-3-3 0.750 08-1-4 0.701 08-2-6 0.717 08-3-5 0.753 08-1-5 0.737 08-2-10 0.754 08-3-6 0.730 08-1-6 0.738 08-2-22 0.734 08-3-7 0.736 08-1-7 0.725 08-2-24 0.725 08-3-8 0.717 08-1-8 0.718 08-2-33 0.703 08-3-10 0.733 08-1-9 0.726 08-2-37 0.742 08-3-11 0.712 08-1-10 0.720 08-2-52 0.707 08-3-12 0.729 08-1-11 0.712 08-2-54 0.731 08-3-13 0.736 08-3-15 0.713 Genetic background of AS complex F1 progeny: Molecular Marker Assisted Selection Germplasm innovation with S. spontaneum and E. arundinaceus
  • 24. M 1 2 3 4 5 6 M AFLP genetic background of GXASBC1 1:GXASF1 08-1-11 2, 5:Co649 3: GXASBC1: GXASF1 08- 1-11 × Co649 4:GXASF1 08-1-1 6: GXASBC1: GXASF1 08- 1-1 × Co649 500 400 300 200 Sugarcane germplasm innovation
  • 25. DNA inheritance of E. arundinaceus and S. spontaneum in their progenies of different generation 4.1 genetic relationship of GXAS07-6-1 and its parents by SRAP 600 500 400 300 200 100 M 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 M 1:Male: GXA87-36 2:Female: GXS79-9 3: GXAS07-6-1 Sugarcane germplasm innovation
  • 26.  24 particular loci of female parent GXA87-36 (E. arundinaceus) all pass to both F1 and BC1.  16 particular loci of male parent GXS79-9 (S. spontaneum) pass 16 to F1, 15 to BC1. DNA inheritance of E. arundinaceus and S. spontaneum in their progenies of different generations Sugarcane germplasm innovation
  • 27. Major traits investigation of GXASBC1 GXASBC1 11-1-11GXASBC1 11-3-9 Stalk number: 14 Stalk diameter: 2.2 Brix: 19 Stalk number: 6 Stalk diameter: 2.5 Brix: 23 Sugarcane germplasm innovation
  • 28.  Design more BC2, BC3 crosses by good BC1 or BC2 clones;  Keep tracking molecular marker in breeding process;  Keep tracking chromosome transmission in breeding process; Work on going: Sugarcane germplasm innovation
  • 29. Germplasm exchanges  Germplasm exchanges with different countries are also our priority.  We hope to improve the genetic variation of sugarcane crossing parents through utilizing the germplasm from different countries.  The accumulation of multiple germplasm would increase heterogeneous in the hybrids.
  • 30. Sugarcane breeding and variety propagation  Producing high productivity, high sugar, strongly resistant and nitrogen efficient sugarcane cultivars is one of our major priority in our sugarcane breeding program.  Ratoon ability is also one of the important selections for sugarcane improvement in China.
  • 31.  Released in 2010  Cross: YC94-46×ROC22  Early maturity, high yield, high sucrose content, good smut resistance, strong ratoon ability, strong tillering, good cold tolerance GT29( GT02-761) New sugarcane varieties
  • 32.  Released in 2011  Cross: YT91-976×ROC1  Early maturity, high yield, high sucrose content, good cold and drought tolerance, good ratoon ability GT32 (GT02-208) New sugarcane varieties
  • 33.  Released in 2011  Cross: ZZ92-126×CP72-2086  Mid maturity, high yield, good ratoon ability, good drought tolerance GT37 (GT03-2357) New sugarcane varieties
  • 34.  Released in 2013  Cross:YT85-177 × CP84- 1198  Early maturity, high sucrose content, high yield, good smut resistance, good cold and drought tolerance, good ratoon ability GT40 (GT02-1156) New sugarcane varieties
  • 35.  Released in 2013  Cross: ROC22 × GT92-66  High yield , high sucrose content, good ratoon ability, good disease resistance, good drought tolerance, broad adaptability GT42 (GT04-1001) New sugarcane varieties
  • 36. Development of low cost drought resistant cultivation technologies
  • 37. Model for applying vinasse in sugarcane: Changli Farm, Shangsi County ②① ③ ④ Use vanasse as liquid fertilizer
  • 38. Vinasse application Conventional management
  • 39. • Recently, we have successfully used vinasse for making granule fertilizer, which is good for storage, transportation and application. • Combining popularization of the two technologies will completely avoid vanasse from pollution, and recycle the nutrient into agricultural fields.
  • 40. (3)Water-saving irrigation
  • 41. (3)Water-saving irrigation Spray irrigation Drip irrigation
  • 42. Trash mulching
  • 43. PC-Based fertilization
  • 44. (6)Comprehensive pests control Sexual attractant application Cuban fly application •Borer control Trichogramma application
  • 45. Longhorn beetle control ▲Beauveria bassiana control ▲Manual control ▲Good food •Larva
  • 46. Field trapping •Adult:In May to early June •Field trapping
  • 47. • Good for many pests. Light trapping
  • 48. • Only good for young larva Chemical control
  • 49. Healthy seedcane production
  • 50.  Sterilized stem tip is used for tissue culture Healthy seedcane production
  • 51. 1st year healthy seedcane Good performance of healthy seedcane
  • 52. (10)Machine operation  It is urgent to develop mechanization for sugarcane production;  Large scale of farm will be necessary for mechanization;  Machine operation must match with farming practice.  New standard for millable cane should be made and used to promote the mechanization development.
  • 53. Biological nitrogen fixation in sugarcane • We are trying to develop methods for detecting the bacterial nifH gene expression in sugarcane stems based on the established associative sugarcane- diazotroph systems, use qRT-PCR to measure the nifH expression activity in sugarcane stems from 25 cultivars grown under conditions suitable for associative N2 fixation, and use RT-PCR to amplify the nifH transcriptomes from cultivars showed high nifH expression activity.
  • 54. Biological nitrogen fixation in sugarcane
  • 55. Morphological identification (22000×) L03: Klebsiella plantica rod shaped bacterium with multiple flagella, size: (0.3~0.5)μm×(0.9~1.2)μm A01:Pantoea agglomonarens Rod, single and pairwise aligment, multiple flagella; size:(0.6~0.8)μm×(1.8~2.5)μm L09: Klebsiella axytoca rod shaped bacterium with multiple flagella, size: (0.3~0.5)μm×(0.9~1.2)μm
  • 56. Endophytic N-fixing bacteria invasion and colonization E. coli S17-1pir strain with pFAJ1819 vector (GFP gene) pFAJ1819 vector was transferred into nitrogen-fixing bacterium, Klebsiella plantica (L03) Strain with GFP
  • 57. Day 1 root hair zone Day 2 root cells Day 4 root column Day 7 stalk parenchyma Day 8 leaf Kranz Root cap Root hair zone Lateral root formation Root cortex Leaf sheath cells Detection of N-fixing bacterium, Microbacterium sp. (16SH) contained GFP gene colonization in sugarcane under a laser scanning confocal microscopy Tissue cultured seedling of ROC22 inoculated with N-fixing bacterium, Klebsiella plantica (L03) under a fluorescence microscopy
  • 58. Proteomic analysis of interaction between N-fixing bacterium and sugarcane GT21, Control pH4-7,loaded 300μg/gel,silver stained 11 4 3 17 4 3 10 6 5 2 2 5 6 9 8 7 7 8 9 10 13 15 14 12 11 11 12 13 14 15 17 16 16 GT21 inoculated with Klebsiella sp. for 20 days up-regulated:1 (spot #10); down-regulated: 10 (#1,2,3,4,6,7,8,11,13,15; novel: 4 (#5,9,14,17)
  • 59. AB188122.1)Azohydromonaslata EU048175.1)Unculturedbacterium EU542578.1)Ideonelladechloratans AB188121.1)Azohydromonasaustralica SRSnifH120609CT51SRSnifH120609CT33 EU048169.1)Unculturedbacterium AJ505315.1)Sinorhizobiumsp. JX081993.1)Ensiferadhaerens EU586055.1)Unculturedbacterium AB188120.1)Pelomonassaccharophila EU544203.1)Unculturedbacterium FJ008185.1)Uncultured soilbacterium AP012304.1)Azoarcus sp._seq2 A P 012304.1)A zoarcus sp._seq1 F R 850745.1)R hizobium legum inosarum 381376528)R ubrivivax gelatinosus AY196462.1)Uncultured nitrogen-fixing bacterium SRSnifH120609CT15 FJ593866.1)Klebsiella sp. EU048149.1)Uncultured bacterium EU048059.1)Uncultured bacterium AY544164.1)Delftia tsuruhatensis HQ404304.1)Klebsiella pneumoniae AJ563957.1)Methylobacter luteus DQ481036.1)Uncultured bacterium FJ822995.1)Agrobacterium tumefaciens JN648883.1)Uncultured bacterium CP001157.1)Azotobacter vinelandii AM406670.1)Azoarcus sp.JX154793.1)Uncultured nitrogen-fixing bacterium JX154830.1)Uncultured nitrogen-fixing bacterium JX154844.1)Uncultured Dechloromonas sp. AJ563286.1)Dechloromonas sp. GU121498.1)Uncultured bacterium SRSnifH120609CT14 AY231551.1)Uncultured nitrogen-fixing bacterium AY768421.1)Tolypothrix sp. JN162465.1)Uncultured bacterium CP003548.1)Nostoc sp._seq1 CP003548.1)Nostoc sp._seq2 GU111829.1)Uncultured soil bacterium DQ142699.1)Uncultured bacterium EF408200.1)Uncultured cyanobacterium HQ836199.1)Anabaena sp. SRSnifH120609CT29 CP002364.1)Desulfobulbus propionicus S R S nifH 120609C T 57 H Q 190142.1)U ncultured bacterium CP001998.1)Coraliom argarita akajim ensis EU978427.1)Unculturedmicroorganism JX545230.1)Stenotrophomonasmaltophilia CP002298.1)Desulfovibriovulgaris AY787578.1)Unculturedbacterium JX268325.1)Unculturedbacterium AY196392.1)Unculturednitrogen-fi SRSnifH120609CT17 JX268243.1)Unculturedbacterium JX268254.1)Unculturedbacterium GQ289580.1)Bradyrhizobiumjaponicum DQ520342.1)Unculturedbacterium DQ776342.1)Unculturedsoilbacterium AY601051.1)Unculturedbacterium CP001124.1)Geobacterbemidjiensis CP001661.1)Geobactersp. CP002479.1)Geobactersp. EU912951.1)Unculturedbacterium CP000769.1)Anaeromyxobactersp. CP001390.1)Geobacterdaltonii AE017180.2)Geobactersulfurreducens SRSnifH120609CT34 S R S nifH 120609C T38 G Q 289581.1)B radyrhizobium japonicum JX 2 6 8 2 6 8 .1 )U n cu ltu re d b a cte riu m JX268291.1)Uncultured bacterium GQ289574.1)Bradyrhizobium japonicum FJ381622.1)Uncultured bacterium FJ381624.1)Uncultured bacterium JX268505.1)Uncultured bacterium SRSnifH120609CT20 SRSnifH120609CT27 SRSnifH120609CT24 GU117592.1)Uncultured bacterium SRSnifH120609CT62 SRSnifH120609CT45 SRSnifH120609CT23SRSnifH120609CT30 SRSnifH120609CT18 SRSnifH120609CT28 SRSnifH120609CT50 SRSnifH120609CT43 SRSnifH120609CT49 SRSnifH120609CT32 SRSnifH120609CT37 SRSnifH120609CT59 S R S nifH 120609C T 6 S R S nifH 120609C T19 SRSnifH120609CT42 SRSnifH120609CT47 SRSnifH120609CT53 SRSnifH120609CT22 SRSnifH120609CT40 SRSnifH120609CT31 0.1 Sequence alignment and phylogenetic tree construction
  • 60.  Use high-throughput sequencing and bioinformatics to analyze the diversity of the functional diazotrophs and to determine the major functional diazotrophs in sugarcane;  Isolate the major functional diazotrophs from sugarcane plants or select ones from the available culture collection for diazotrophs in China;  Use the major functional diazotrophs to inoculate the sugarcane cultivars shown high nifH expression activity;
  • 61. Use qRT-PCR and 15N isotope dilution methods to determine the efficient sugarcane N2-fixing systems; Use in situ hybridization assay to find the N2- fixing location in sugarcane plants. Detect the effects of soil pH, nitrogen and phosphorous levels, and microbes on nitrogen fixation efficiency in sugarcane.
  • 62. Gene cloning and transformation 1. Cloning and function analyses of different families of SPS gene in sugarcane 2. Transformation of insect resistance gene (Bt) into sugarcane 3. Transformation of cold resistance gene into sugarcane
  • 63. Cloning of SPS genes Full length of cDNA sequences of 4 families of SPS genes in sugarcane have been cloned: SofSPSA (HM854011) SofSPSB (JN584485) SofSPSDⅢ (HQ117935) SofSPSDⅣ
  • 64. Infection, co-culture and screening Differentiation and propagation Transformation of insect resistance gene (Bt gene) through Bar screening Hardening and rooting Root growth of Bt transgenic plants
  • 65. 3/13/201 Greenhouse culture of transgenic plants using phosphomannose isomerase (PMI) as label for screening
  • 66. Identification of transgenic plant resistance to Chilo infuscatellus Three larva of Chilo infuscatellus were inoculated into one transgenic plants for experiment.
  • 67. 68 Proteomics related to sugarcane resistance to adverse stresses Materials Protein extraction 2-D eletrophoresis Scanning Protocol for proteomic analyses Software analysisDifferential proteins MS identification Bioinformatics
  • 68. Drought resistance related proteomics greenhouse Pot culture Aquiculture
  • 69. 2-DE profiles of PDQuest analyses ROC22 ROC16
  • 70. CK PEG PEG+Si ROC22 CK PEG PEG+Si ROC16 Profiles of parts of enlarged differential protein spots
  • 71. MALDI-TOF-TOF/MS identification 1st profile 2nd profile MALDI-TOF-TOF/MS profile for spot 25
  • 72. Genome research  Although sugarcane genomes are very complex and the genome research progresses very slowly, it is important to sequence the whole genome.  After years of preparation, Sugarcane Research Center, CAAS/ GXAAS decided to begin the whole sugarcane genome sequencing program in China: F13TSFSCKF1043.  This is being conducted with a local collection of Saccharum spontaneum, GXS87-16 (2n=64) with the cooperation of Beijing Genome Institute (BGI) –Shenzhen.
  • 73. • The first term is planning to be completed in 3 years since July 2013. In the first term of the project, 100 X (100 Gb) of WGS sequencing will be done, 10 X BAC clones will be constructed and 1 X (about 10,000 clones) will be selected for BAC library construction and sequencing. These data will be used to estimate complexity of sugarcane genome, and the primary sequence map will be obtained.
  • 74.  The program has completed small fragments sequencing, and the data are under analyses.  BAC library has been completed, and will be used for sequencing.
  • 75. Acknowledgement • China 863 Program (2013AA102604), China International Cooperation Program (2013DFA31600), National Natural Science Foundation of China (31171504, 31101122, 31240056); Natural Science Foundation of Guangxi Provence (2011GXNSFF018002, 2011GXNSFA018076, 2013NXNSFAA019073, 2013NXNSFAA019082), and Guangxi Key R & D Program (GKC1123008-1, GKG1222009-1B).
  • 76. 5th IAPSIT International Conference (IS-2014) Green Technologies for Sustainable Growth of Sugar & Integrated Industries in Developing Countries November 25-28, 2014 Nanning, P.R. China •
  • 77. Guilin - Grand place with wonderful landscape
  • 78. Wonderful Seashores in Hainan province
  • 79. 3/13/201