021614 yang-rui li--research and development priorities for sugar industry of china
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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
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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
⑧ ⑨
Major Sugarcane
Growing Area in
Mainland China
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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.
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Su
gar
pro
du
cti
on
(M
illi
on
To
ns)
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
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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
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Challenge for Sugar
Industry in China
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1. More than 80% of sugarcane is grown in
rain-fed upland fields, and easily affected by
drought.
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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.
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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
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4. Pests and diseases
Ratoon stunting disease
Smut
Shoot rot
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Borer Thripid
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Aphis
Scarab
Longhorn beetle
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Sugarcane germplasm innovation
Future research priorities of
Chinese sugar industry
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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.
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SRC-CAAS/GXAAS has a sugarcane cross breeding base in Sanya city of Hainan province (18°35′ N, 109°68′E).
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E. arundinaceus - S. spontaneum complex (AS complex) creating
E. arundinaceus × S. spontaneum
15 crosses, 8000 seedlings
Progeny detection : Phenotype + molecular (SSR-PCR\SRAP-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
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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
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Germplasm innovation with S. spontaneum and E. arundinaceus
AS complex:
GXAS07-6-1
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×
GT02-761
Sugarcane germplasm innovation
AS complex: GXAS07-6-1 Progeny
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×
GT02-761
Sugarcane germplasm innovation
AS complex: GXAS07-6-1 Progeny
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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
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AS complex genomic in situ hybridization (GISH)
50×Blocking 80×Blocking
Green: S. spontaneum
Red: E. arundinaceus
Sugarcane germplasm innovation
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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
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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
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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
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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
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Major traits investigation of GXASBC1
GXASBC1 11-1-11 GXASBC1 11-3-9
Stalk number: 14
Stalk diameter: 2.2
Brix: 19
Stalk number: 6
Stalk diameter: 2.5
Brix: 23
Sugarcane germplasm innovation
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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
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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.
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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.
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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
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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
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Released in 2011
Cross: ZZ92-126×CP72-2086
Mid maturity, high yield, good
ratoon ability, good drought
tolerance
GT37 (GT03-2357)
New sugarcane varieties
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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
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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
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Development of low cost
drought resistant cultivation
technologies
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Model for applying
vinasse in sugarcane:
Changli Farm, Shangsi
County
② ①
③ ④
Use vanasse as liquid fertilizer
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Vinasse application Conventional management
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• 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.
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(3)Water-saving
irrigation
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(3)Water-saving irrigation
Spray irrigation
Drip irrigation
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Trash mulching
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PC-Based fertilization
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(6)Comprehensive pests control
Sexual attractant application
Cuban fly application
•Borer control
Trichogramma application
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Longhorn beetle control
▲Beauveria
bassiana control
▲Manual control ▲Good food
•Larva
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Field trapping
•Adult:In May to early June
•Field trapping
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• Good for many pests.
Light trapping
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• Only good for young larva
Chemical control
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Healthy seedcane production
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Sterilized stem tip is used for tissue culture
Healthy seedcane production
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1st year healthy seedcane Good performance of healthy seedcane
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(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.
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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.
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Biological nitrogen fixation in
sugarcane
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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
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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
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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
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Proteomic analysis of interaction between N-fixing bacterium
and sugarcane
GT21, Control
pH4-7,loaded 300μg/gel,silver stained
1 1
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)
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AB1881
22.1)Azo
hydrom
onas la
taEU0
48175.1
)Uncult
ured bac
terium
EU5425
78.1)Ide
onella d
echlora
tans
AB1881
21.1)Azo
hydrom
onas au
stralica
SRSnifH
120609
CT51
SRSnifH
120609
CT33
EU048169
.1)Uncu
ltured ba
cterium
AJ50531
5.1)Sinor
hizobium
sp.
JX081993.1)Ensifer adhaerens
EU586055.1)Uncultured bacterium
AB188120.1)Pelomonas saccharophila
EU544203.1)Uncultured bacterium
FJ008185.1)Uncultured soil bacterium
AP012304.1)Azoarcus sp._seq2
AP012304.1)Azoarcus sp._seq1
FR850745.1)Rhizobium leguminosarum
381376528)Rubrivivax 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 propionicusSRSnifH120609CT57
HQ190142.1)Uncultured bacterium
CP001998.1)Coraliomargarita akajimensis
EU978427.1)Uncultured microorganism
JX545230.1)Stenotrophomonas maltophilia
CP002298.1)Desulfovibrio vulgaris
AY787578.1)Uncultured bacterium
JX268325.1)Uncultured bacterium
AY196392.1)Uncultured nitrogen-fixing bacterium
SRSnifH120
609CT17
JX2682
43.1)Un
cultured
bacteriu
m
JX2682
54.1)Un
cultured
bacteriu
m
GQ289
580.1)B
radyrhiz
obium ja
ponicum
DQ5203
42.1)Un
cultured
bacteriu
m
DQ7763
42.1)Un
cultured
soil ba
cterium
AY6010
51.1)Un
cultured
bacteriu
m
CP0011
24.1)Ge
obacter
bemidjie
nsis
CP00166
1.1)Geob
acter sp
.
CP002479
.1)Geob
acter sp
.
EU912951.1)Uncultured bacterium
CP000769.1)Anaeromyxobacter sp
.
CP001390.1)Geobacter daltonii
AE017180.2)Geobacter sulfurreducens
SRSnifH120609CT34
SRSnifH120609CT38
GQ289581.1)Bradyrhizobium japonicum
JX268268.1)Uncultured bacterium
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
SRSnifH120609CT6
SRSnifH120609CT19
SRSnifH120609CT42
SRSnifH120609CT47
SRSnifH120609CT53
SRSnifH120609CT22
SRSnifH120609CT40
SRSnifH120609CT31
CP000482.1)Pelobacter propionicus
GU121502.1)Uncultured bacterium
HQ586727.1)Uncultured alpha proteobacterium
0.1
Sequence alignment and
phylogenetic tree construction
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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;
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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.
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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
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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
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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
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Greenhouse culture of transgenic plants using
phosphomannose isomerase (PMI) as label for screening
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Identification of transgenic plant resistance
to Chilo infuscatellus
Three larva of Chilo infuscatellus were inoculated into one
transgenic plants for experiment.
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68
Proteomics related to sugarcane resistance
to adverse stresses
Materials Protein extraction
2-D eletrophoresis Scanning
Protocol for proteomic analyses
Software analysis Differential proteins
MS identification
Bioinformatics
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Drought resistance related proteomics
greenhouse
Pot culture
Aquiculture
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2-DE profiles of PDQuest analyses
ROC22
ROC16
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CK PEG PEG+Si
ROC22
CK PEG PEG+Si
ROC16
Profiles of parts of enlarged differential protein
spots
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MALDI-TOF-TOF/MS identification
1st profile
2nd profile
MALDI-TOF-TOF/MS profile for spot 25
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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.
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• 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.
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The program has completed small fragments
sequencing, and the data are under analyses.
BAC library has been completed, and will be
used for sequencing.
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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).
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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
•
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Guilin - Grand place with
wonderful landscape
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Wonderful Seashores in Hainan province
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