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.

54. 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.

55. Biological nitrogen fixation in
sugarcane

56. 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

57. 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

58. 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

59. 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)

60. 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

61.  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;

62. 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.

63. 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

64. 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

65. 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

66. 3/13/201
Greenhouse culture of transgenic plants using
phosphomannose isomerase (PMI) as label for screening

67. Identification of transgenic plant resistance
to Chilo infuscatellus
Three larva of Chilo infuscatellus were inoculated into one
transgenic plants for experiment.

68. 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

69. Drought resistance related proteomics
greenhouse
Pot culture
Aquiculture

70. 2-DE profiles of PDQuest analyses
ROC22
ROC16

71. CK PEG PEG+Si
ROC22
CK PEG PEG+Si
ROC16
Profiles of parts of enlarged differential protein
spots

72. MALDI-TOF-TOF/MS identification
1st profile
2nd profile
MALDI-TOF-TOF/MS profile for spot 25

73. 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.

74. • 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.

75.  The program has completed small fragments
sequencing, and the data are under analyses.
 BAC library has been completed, and will be
used for sequencing.

76. 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).

77. 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
•

78. Guilin - Grand place with
wonderful landscape

80. Wonderful Seashores in Hainan province

81. 3/13/201