DEVELOPMENT OF BIOTECHNOLOGY FOR SUGARCANE

The importance of biotechnology to deal with climate change:
Case studies on biotechnology for droughttolerant and high-sugar yield of sugarcane
Bambang Sugiharto
Center for Development of Advanced Science and Technology (CDAST)
University of Jember
Jl. Kalimantan No 37 Kampus Tegalboto, Jember 68121
INDONESIA
Tel/Fax : +62-331-321825, Email: [email protected]
Outline Presentation :
Introduction of sugarcane and climate change
Development of Agrobacterium-mediated
transformation in sugarcane
Drought-tolerant sugarcane with a gene
encoding for choline dehydrogenes (CDH)
Creation of high-sugar production for
sugarcane using genes encoding proteins for
sucrose synthesis and translocation
INTRODUCTION
Back ground
1. Sugarcane is main plant for sugar production and
its sugar production was decreased
2. Sugarcane is C4 plant that well adapted in
tropical climate
3. Its has high efficiencies of photosynthetic rate,
water up take and nitrogen assimilation
4. Its growth and biomass production very high
Climate Changes – Potential Loss in Sugarcane Productivity
Flooding
Temperature
increased
Drought Stress
Formation of
adventives root
(Begum et al, 2013)
Climate changes
FOOD
SECURITY
Increased
population
THE BENEFITS OF SUGARCANE
Processing of 100 ton
Sugar
sugarcane produce :
1. 10 ton sucrose
2. 4 ton molasses
3. 3 ton filter mud
Paper-plates
4. 30 ton bagasse
5. 1500 kWh electricity
Bagasse
Bio-ethanol
Sugarc
ane
Sugar wax
Molasses
Limitation of Sugarcane Breeding :
 Poor flowering in recent sugarcane cultivars
 Difficulties in cross pollination among sugarcane
cultivars
LIght
The pollen is quickly dried and can not be use for
hybridization
Sugarcane seed is very small and difficult to
germinate
Dark
Seed
Seedling
DEVELOPMENT OF Agrobacterium-MEDIATED
TRANSFORMATION METHOD FOR SUGARCANE
Particle bombardment
Agrobacterium-mediated
Outline of Agrobacetrium-mediated transformation in sugarcane
Agrobacterium
infection
Cocultivation
for 3 days +
Acetosyringone
Explants :
28oC for
30 min
EC and SC
shoot tips
in vitro plants
multiple shoots
GUS assay
Selection
medium
LBA4404
GUS assay and
PCR analysis
Micropopagation
putative
transformant
Regeneration
(selection
medium)
Transient gus expression using
various sugarcane explants
Explants
Transient
gus expr.
callus
spindle
leaf
in vitro
plants
Explant callus : Selection and regeneration of the infected
callus
• Infected calli were inoculated on
selection medium containing
appropriate antibiotics.
Untransformed calli were brown
and died.
• Resistant calli regenerated shoots
into plantlet on the selection
medium.
• After 5 successive cycles on the
selective and rooting medium
putative transformants were
obtained.
• Problems : somaclonal variation
Explant : sugarcane meristematic leaf tissues
(spindle leaf) isolated from field grown sugarcane
Co-cultivation
Callus induction
Regeneration
Isolated meristematic leaf tissues (spindle leaf) were cocultivated with Agrobacterium, then incubated in callus
induction and regeneration media containing selection
antibiotic. Problem: Agrobacterium overgrowth
Explant : Base segments of in vitro
sugarcane for transformation
Axillary buds were isolated
from apical portion of
sugarcane stalk and
inoculated on appropriate
medium to develop in vitro
plants.
Shoots
propagation
Shoots rooting
Base segments
Detection of gus gene in transgenic sugarcane
PCR analysis
Southern Blot analysis
All sugarcane explants can produced transgenic sugarcane.
PCR and Southern Blot analysis showed the presence of inserted gus
gene in genome DNA isolated from leaf of the transgenic sugarcane.
In vitro sugarcane can grow and develop faster than other explants,
but meristematic leaf tissue produced more transgenic sugarcane
Study on drought stress in sugarcane
Protein profile separated by 2D electrophoresis that extracted from sugarcane leaf grown under normal condition (A)
and without watering for 8 days (B). Inset is the protein SoDip22 that increased during water stress
Regulatory manner of SoDip22 gene expression in sugarcane leaf
Accumulation pattern of the transcript for SoDip22 gene (A) and the
polypeptide (B) during drought stress (0 – 8 days). The effect of PEG6000 (0.9 MPA) (C) and administration of various hormone (D) on the transcript
for SoDip22.
DEVELOPMENT OF DROUGHT-TOLERANT SUGARCANE USING
GENE ENCODING FOR CHOLINE DEHYDROGENASE (betA)
Synthesis of glicine betaine
(GB) from choline in bacteria
Synthesis of choline and GB in plants.
Not all plants synthesize GB.
Bacteria
Plants
Choline dehydrogenase (CDH)
Choline monooxygenase (CMO)
Betaine aldehyde
dehydrogenase (BADH)
Compatible solute (osmoprotectant)
Gene encoding for choline dehydrogenase (CDH) (betA) was
cloned from Rhizobium meliloti by Ajinomoto Company Japan
Agrobacterium-mediated transformation was performed by PT.
Perkebunan Nusantara XI (Persero) in collaboration with
Jember University
SacI
XbaI
LB
PNOS
NPT II
TNOS
E1E2
P35S
betA
TNOS
P35S
HPT
T35S
RB
pMLH2113-betA
Explant
callus
Screening
Regeneration
Aclimatization
GREEN HOUSE TRIALS :
Growth respond of drought-tolerant sugarcane during water stress
Sugarcane
Cultivars
Lenght of water stress (days)
Wilting
Permanent
Wilting
Dried
NXI-3T
12
> 30
> 30
NXI-4T
19
> 36
> 36
NXI-5T
14
> 30
> 30
BL579-NT
(Control)
6
19
19
PSJT 941
(non GM drought
tolerant cane)
19
> 36
> 36
Drought responds for 8 days after stop
watering, from the left GM cane 3T,
Drought stress was treated to sugarcane by stop watering 4T, 5T, control cane (BL)
Root profile of wild
type (left) and GM
cane (right)
Root profile of cane
of GM cane 4T (left),
drought tolerant
cane PSJT (midle),
and control cane
(right)
Source : PTPN XI
Detection and expression of betA gene in GM sugarcane
planted in green house trials
1T 4T 6T NT
betA
Detection of inserted
gene by Southern Blot
Analysis showed single
inserted betA gene in
GM sugarcane event 6T,
1T, and 4T, but no in
control (K) sugarcane
CaMV
PCR analysis to detect inserted gene in
the GM sugarcane 1T, 4T, 6T and control
(NT) with a pair of primer for betA gene
(upper) and CaMV (lower)
Data:03061017.D01 Method:03061017.M01
Chrom:03061017.C01 Atten:6
mAbs
Ch=1
Betain in
sugarcane juice
60
Betain
Standard
40
5.322
5.324
40
20
5
0
min
2
4
9.529
0
10
4.598
2.289
10.825
9.662
10.408
7.877
0
7.042
6.068
5.726
20
0
Source: PTPN XI
Data:07061011.D01 Method:07061011.M01
Chrom:07061011.C01 Atten:6
mAbs
60
2.375
The presence of
glycine betain in
GM sugarcane juice
detected by HPLC
with Inertsil ODS3
4.6Ø x 250 mm
column
Ch=1
6
8
10
min
Growth and sugar yield of GM sugarcane in
confined field trial
Variety
Germin Number
ation
of tillers
(%)
57
68
GMsugarcane
Non GM- 60
sugarcane
Main field road
65
Number Biomass Sugar
of stalk (ton/Ha) content
(%)
62
54.0
8.47
Sugar
yield
(ton/Ha)
4.59
Betain
content
(ppm)
457,37
64
4.06
Not
detected
51.7
7.83
Adjacent field
with different
planting period
Source: PTPN XI
Risk assessment of drought-tolerant GM sugarcane
PT. Perkebunan Nusantara XI, Surabaya (PP 21 / 2005)
Environmental safety assessment :
 There is no evidence for gene flow
to other organism. No possible cross
breeding among Saccharum
species.
 No potential to be an invasive weed
or weedness
Certificate No :
B-7945/MENLH/08/2011
Food safety assessment :
 Sugar is refined products
resulted from purification
process
 There is no toxicity and no
allergen, easily digested in
gastric and intestinal fluid.
Certificate No:
HK.04.1.52.10.12.6489 / 2012.
Field visits of Deputy Ministry
of Agriculture
CARBON ASSIMILATION AND SUCROSE SYNTHESIS IN PLANTS
Exported
Study on activity of carbon assimilating and sucrose
metabolizing enzymes in leafs of Saccharum species.
Lines
Ni9
NiF8
NCo310
PEPC NADP
-ME
1.68
1.08
1.47
1.11
1.14
0.98
SPS
Sucrose
37.74
38.70
25.77
77.50
84.44
55.90
Molokai
Babakan
Bois R
1.67
1.75
1.29
0.8
0.72
1.16
31.53
28.45
23.50
59.20
52.70
51.10
28NG
IN84
1.45
1.99
0.92
0.90
24.51
21.94
52.70
55.82
SES186
JW94
2.10
1.71
0.90
1.24
20.51
15.69
46.00
39.10
Ni9 NiF8
Bois
NCo
Mol
Bbkn
28NG IN84 SES JW94
Levels of LSU-Rubisco
protein in leafs of
Saccharum species
PEPC : phosphoenolpyruvate
carboxylase
NADP-ME: NADP malic enzyme
SPS : sucrose-phosphate synthase
Rubisco : ribulose-1,5-biphosphate
carboxylase/oxygenase
The enzyme activities are expressed as unit/mg protein
Sucrose contents
(μg Suc/g FW)
Induction of SPS activity during drought stress in sugarcane
Dry season is the best
condition for harvesting
sugarcane
SPS activity
(μg Suc/min/prot)
ABA induced SPS levels
1 Day
SPS levels
Water stress (days)
2 Days
Cloning gene for sucrose-phosphate synthase
Cloning and expression analysis of genes for sucrosephosphate synthase from sugarcane
Relative levels of mRNAs
SoSPS1
10
8
6
4
2
0
0
12
SoSPS2
24
48
Time after illumination (h)
Comparison of the amino acid sequences deduced
from cDNAs for SPS from sugarcane. Box sequences
I, II, III are functionally important regions. Dotted
Ser residues are the regulatory phosphorylation sites.
Detection of
transcripts of
SoSPS1 and
SoSPS2 in
different organ
of sugarcane
Sugiharto et al (1997) [Plant Cell Physiology 38(8):961-965]
Overexpression of SoSPS1 gene :
Increasing of sweetness (Brix) and SPS
protein contents in trangenic tomato
Clones
Plant
Flowering
Fruit
Height (cm) time (week) number
Fruit
Sweetness
weight (g) (Brix)
Clone -2
122
8.33
6.33
26.38
7.33
Clone-3
119
7.33
7
20.85
8
Clone-4
118
7
5.33
17.12
8
Clone-5
120.67
9
15.33
21.04
8
Wild type
115.67
11.33
5.33
16.33
6.33
WT 2.1
Transgenic tomato
2.2
3.1
3.3
4.1
5.4
5.5
Western Blot Analysis with antibody against SPS
Overexpression of SoSPS1 gene increased SPS activity and
sucrose content in leaves of transgenic sugarcane
SPS activities in leafs
(µg suc/min/mg prot)
Western blot analysis of
the protein leafs
WT
3
4
5
6
20
7
16.7
15
11.7
13.1
11.4
8.7
10
5
0
WT
G4
G5
G6
G7
Galur tebu
Sucrose content in leafs
(µg suc / g FW)
6
4.9
5
4.3
4
3
3.8
4.2
2.4
2
1
0
Kontrol
Galur 4
Galur 5
Galur 6
Galur 7
Increases of sucrose accumulation in stem of
transgenic sugarcane (mg sucrose/g FW)
Galur 4
Galur 5
Galur 6
Galur 7
kontrol
13
12
11
10
9
8
7
6
5
4
3
2
1
0
2
3
4
5
6
7
8
9
10
Internodes number (from young to mature)
The increases of sucrose contents in stem were not as high as the increase
in sugarcane leaves.
The path of sucrose translocation by sucrose transporter proteins
(SUT) from source to sink tissue in apoplastic loader (Sauer, 2007)
Sucrose synthesis in
leaves by SPS
Sucrose exported to
sink tissue by SUT
Sucrose accumulation
in stem
Cloning of the cDNA encoding sucrose transporter (SoSUT1)
Phylogenetic tree of the SUC/SUT family
Over-expression of SoSUT1 gene in sugarcane and the effect
on translocation and accumulation of sucrose
Comparison the transcripts for
SoSUT1 (upper) and protein contents
(lower) in leaf of transgenic sugarcane
Pattern of sucrose contents in the
leaf (upper) and stalk of 7 months
transgenic sugarcane (lower)
DOUBLE OVEREXPRESSION OF THE GENES FOR SoSPS1
AND SoSUT1 IN TOMATO AND SUGARCANE
RB
LB
nptII
T
SoSPS1
T
SpR
Rep
pNos
pRiceUbiq
pUbi-SoSPS1
ColE1 ori
St
LB
RB
hptII
T
SoSUT1
KanR
pACT-SoSUT1
ColE1 ori
T
Rep
PNos
pRiceActin
Sta
Construction of SoSPS1 and
SoSUT1 genes into different
vector and antibiotic resistant
Double-overexpression of the genes encoding for
sugarcane SPS1 and SUT1 protein in transgenic tomato
135
Plant Height
135
Tinggi Tanaman (cm)
130
130
128 128
130
125
125
127
Number of fruit
125 125
124
122
120
120
115
110
Klon Tomat
Kontrol
SPS
Weight per fruit
SUT
SPS-SUT
Total production of fruits
Double overexpression of the genes encoding for SPS1
and SUT1 proteins in sugarcane
A
B
Positive PCR analysis for insertion of
SoSUT1 gene (upper) and SoSPS1 gene
(lower) transgenic sugarcane
C
D
E
In vitro selection of putative transgenic
sugarcane in the media containing double
antibiotic hygromicine and kanamicine
Characterization of the
transgenic sugarcane with
double overexpression of
SoSUT1 and SoSPS1 genes
Increases of SPS1 activity and SUT1 contents in the transgenic sugarcane
Activities of SPS in leaf of wild type (WT) and transformant sugarcane. Total soluble
protein was extracted from the sugarcane leaf and used for the enzyme activity. The SPS
activity was measured by the amount of suc per prot per min with spectrophotometer
wt
32a
31c
26ba
26bb
33aa
212bd
26bc
31b
212bc
32b
33ab 212bd 212ba
32c
wt
Level of SUT1 protein in leaf of wild type and transformant sugarcane. Total insoluble
protein was extracted from the sugarcane leaf with a extraction buffer contining 2% SDS
and used for Western Blot analysis using specific antibody against sugarcane SUT1 protein
Sucrose Content in Sugarcane Leaf
(% of Fresh Weight)
1,40
1,20
1,00
0,80
0,60
0,40
0,20
0,00
26ba 26bb 26bc 212ba212bc212bd 31a
31b
31c
32a
32b
32c 33aa 33ab
wt
Sucrose Content in Sugarcane Stalk
(% of Fresh Weight)
Sugarcane Lines
9,0
8,0
7,0
6,0
5,0
ruas 3
4,0
ruas 8
3,0
2,0
1,0
0,0
Sugarcane Lines
SUCROSE IS AN
OSMOPROTECTANT
HIGHER SUCROSE LEAD
TO ENHANCE
DROUGHT TOLERANCE
CONCLUSION :
1. Development of sugarcane biotechnology is
needed to improve sugarcane cultivars
2. Drought-tolerant sugarcane cultivars were
developed to increase sugarcane productivity
in water limited environments
3. Genetic manipulation of sucrose metabolism
resulted in a higher sucrose productivity of
sugarcane.
THANKS YOU VERY MUCH
These works supported by Ministry of Education and Culture;
Ministry of Research and Technology, PT. Perkebunan Nusantara XI

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