EVERYTHING; THERE IS A SEQUENCE
and connected to a metabolic pathway
Biofortification: Engineering the metabolic pathways
Swapan Datta, DDG (Criop Science), ICAR, New Delhi
Nutrition enriched food crop:
Engineering metabolic pathways
•
•
•
•
Importance of Nutrition Rice
Why genetic engineering to alter the pathways?
What and how do we understand the pathways
Can pathways relate to functional gene
expression?
• Plant breeding, Cross-talk and phenotyping
• Dream Nutrition-Rice
GLOBAL FOOD SECURITY AND
MALNUTRITION
•
•
•
•
•
•
1.1 billion are absolutely poor with incomes < 1U$ day
2.0 billion are marginally better off
840 million people are food insecure
200 million malnourished children
400 million have acute iron deficiency
125 million are affected by a lack of vitamin A
• Only 4% rice of the world supply is non-traded internationally
• Many of 8 billion people on the earth by 2020 will live outside
the market driven supply of food
1 Billion people of world is malnourished while
30% Indian population (mostly women and children) are malnourished : Food +
Nutrition Security come together & can easily be utilized with PDS
Improved protein-potato (Ama1)
Carotenoids enriched potato
Insulin promoting rice
Biofortified food crops for India?
Canola with b-carotene
Vitamin C food crop
High iron rice
b-carotene + Vit E rice
Vitamin E + b-carotene maize
am
bo
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M
ya a
nm
ar
L
B
an ao
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gl
ad
e
Vi sh
et
N
a
In
do m
ne
s
Th ia
ai
la
nd
Sr
iL
Ph ank
a
ili
pp
in
es
N
ep
al
K
In
or
ea dia
D
P
K
or Rp
ea
R
ep
M
al
ay
si
a
C
hi
na
Ja
A
p
fg
ha an
ni
st
Pa an
ki
st
an
C
% share
90
80
Nutrition from rice
Protein
Calories
70
60
50
40
30
20
10
0
IPP
Gibberellins
GGPP
Vitamin E
Chlorophyll
Common pathway in plants (rice)
PS (psy)
Phytoene (1)
PDS (crt1)
Lycopene (2)
LC (lyc)
-carotene
b-carotene (3)
Pathway in transgenic rice
Lutein
GGPP
Zeaxanthin
b-carotene biosynthesis
Fig. 1. Biosynthesis pathway of b-carotene
Sources of Vitamin E : Tocotrienols
 Primary sources of vitamin E are derived from
plants. Tocopherols and Tocotrienols are plastid
localised molecules.
 Oil seeds are richest source of vitamin E, having
total tocol levels ranging from 330 to 2,000 µg per
gram. Tocotrienols are the primary form of vitamin
E in seed endosperm of most monocots,
including cereals, such as wheat, rice, and barley.
 Tocotrienols are found in the seed endosperm of a
limited number of dicots, such as tobacco and
found rarely in vegetative tissues of plants
Strategies for increasing Vitamin E content in plant food
Recommended daily allowances of vitamin E is 40 I.U.
Much effort is currently aimed at identifying the genes
involved in tocol biosynthesis to improve vitamin E
levels in crop plants by metabolic engineering. Two
strategies can be taken in this regard.
1. Produce elevated levels of total tocols through
biosynthetic pathway.
2. Altering tocol composition in favor of α-tocopherol
The isolation of genes for nearly all the steps in tocopherols
and tocotrienols biosynthesis has fascilitated efforts to
alter metabolic flux in plant cells.
Biosynthetic pathway of Tocopherols & Tocotrienols
Vitamin E- Maize
 HGGT catalyzes an analogous reaction to
HPT, only it is highly specific for GGDP
whereas HPT uses PDP as its prenyl
substitute.
 Results from the expression of barley HGGT
in transgenic plants suggest that this enzyme
has strong substrate specificity for
geranylgeranyl diphosphate, rather than
phytyl diphosphate.
 Expression of HGGT enzyme in tobacco calli
and Arabidopsis leaves resulted in
accumulation of Vitamin E antioxidants in the
form of tocotrienols ,principally as γTocotrienols, and generated little or no
change in the content of Tocopherols (Cahoon
et al, 2003)
 Barley HGGT gene was over-expressed in
maize seeds, leading to a 20-fold increase in
tocotrienol level, which translated to an eightfold increase in total tocols (tocopherols and
tocotrienols) (Cahoon et al, 2003).
Genotype screening for the carotenoids in brown and milled rice
Variety Name
Country of
Origin
HPLC
Chromatograph of
Unpolished Seed
HPLC
Chromatograph of
Polished Seed
I n d i v i d u a l S a m p le R e p o r t
R e p o rte d
b y
U s e r:
S y s t e m
P r o je c t
N a m e :
I n d i v i d u a l S a m p le R e p o r t
b e t a c a ro t e n e
R e p o rte d
b y
U s e r:
S y s t e m
P r o je c t
N a m e :
b e t a c a ro t e n e
0 .0 0 3 0
A m a r i ll o
C
u p ls
s e e d / 1
in
H e x a n e
A m a r i lo
0 .0 2 5
C
p ls d
s e e d / 1 g
+
M e O H
0 .0 0 2 5
0 .0 0 2 0
0 .0 2 0
0 .0 0 1 5
0 .0 1 5
A U
Cuba
A U
Amarillo C
0 .0 1 0
0 .0 0 1 0
0 .0 0 0 5
0 .0 0 0 0
0 .0 0 5
I n d i v i d u a l S a m p le R e p o r t
0 .0 0 0
R e p o rte d
b y
U s e r:
S y s t e m
P r o je c t
N a m e :
I n d i v i d u a l S a m p le R e p o r t
-0 . 0 0 0 5
b e t a c a ro t e n e
R e p o rte d
b y
U s e r:
S y s t e m
P r o je c t
N a m e :
b e t a c a ro t e n e
-0 . 0 0 1 0
2 .0 0
4 .0 0
6 .0 0
8 .0 0
1 0 .0 0
1 2 .0 0
M in u te s
1 4 .0 0
1 6 .0 0
1 8 .0 0
2 0 .0 0
2 2 .0 0
2 4 .0 0
2 .0 0
4 .0 0
6 .0 0
8 .0 0
1 0 .0 0
1 2 .0 0
M in u te s
1 4 .0 0
1 6 .0 0
1 8 .0 0
2 0 .0 0
2 2 .0 0
2 4 .0 0
0 .0 0 3 0
0 .0 0 3 0
B R 2 9 / 0 . 9 7 g , u n p ls h d
s e e d
;
B R 2 9 / 1 g , p ls h d
4 5 0 n m
0 .0 0 1 5
D e f a u lt
I n d iv id u a l R e p o r t
P r in t e d
9 :3 1 : 4 4
A M 7 /2 5 / 0 2
P a g e :
1
o f
1
R e p o rt
I n d i v i d u a l S a m p le R e p o r t
p o rte d
b y
U s e r:
S y s t e m
4 .0 0
6 .0 0
P r o je c t
8 .0 0
1 0 .0 0
N a m e :
1 2 .0 0
M i n u te s
1 6 .0 0
P r in t e d
9 :4 0 : 4 4
A M 7 /2 5 / 0 2
P a g e :
1
o f
1
2 0 .0 0
2 2 .0 0
2 4 .0 0
0 .0 0 1 8
0 .0 0 1 6
0 .0 0 1 4
0 .0 0 1 2
0 .0 0 1 2
0 .0 0 1 0
P r in t e d
9 :0 2 : 0 7
A M 7 /2 5 / 0 2
P a g e :
1
o f
1
R e p o rt
A U
A U
I n d iv id u a l R e p o r t
1 0 .0 0
N a m e :
1 2 .0 0
M in u te s
b e t a c a ro t e n e
1 4 .0 0
1 6 .0 0
1 8 .0 0
k r a n t i/ 0 . 9 5 g , p ls h d
M e t h o d :
0 .0 0 0 6
0 .0 0 0 4
0 .0 0 0 4
0 .0 0 0 2
0 .0 0 0 2
I n d i v i d u a l S a m p le R e p o r t
0 .0 0 0 0
D e f a u lt
I n d iv id u a l R e p o r t
P r in t e d
9 :0 4 : 1 0
2 0 .0 0
2 2 .0 0
2 4 .0 0
s e e d
;
P D A
4 5 0 n m
A M 7 /2 5 / 0 2
P a g e :
1
o f
1
I n d i v i d u a l S a m p le R e p o r t
0 .0 0 0 0
-0 . 0 0 0 2
-0 . 0 0 0 2
b y
U s e r:
S y s t e m
P r o je c t
N a m e :
b e t a c a ro t e n e
R e p o rte d
-0 . 0 0 0 4
b y
U s e r:
S y s t e m
P r o je c t
N a m e :
b e t a c a ro t e n e
-0 . 0 0 0 4
2 .0 0
4 .0 0
6 .0 0
8 .0 0
1 0 .0 0
1 2 .0 0
M in u te s
1 4 .0 0
1 6 .0 0
1 8 .0 0
2 0 .0 0
2 2 .0 0
2 4 .0 0
2 .0 0
0 .0 0 7
4 .0 0
6 .0 0
8 .0 0
1 0 .0 0
1 2 .0 0
M in u te s
1 4 .0 0
1 6 .0 0
1 8 .0 0
2 0 .0 0
2 2 .0 0
2 4 .0 0
0 .0 0 7
L e u a n g
B a n g
B a i u p ls
L e u a n g
s e e d /1 g
0 .0 0 6
0 .0 0 6
0 .0 0 5
0 .0 0 5
0 .0 0 4
B a n g
B a i p ls
s e e d / 1 g
0 .0 0 4
D e f a u lt
I n d iv id u a l R e p o r t
P r in t e d
6 :1 1 : 4 8
P M 9 /1 3 / 0 2
P a g e :
1
o f
1
0 .0 0 3
R e p o rt
A U
A U
P r o je c t
8 .0 0
0 .0 0 0 8
0 .0 0 0 6
M e t h o d :
6 .0 0
0 .0 0 1 0
D e f a u lt
0 .0 0 0 8
R e p o rt
S y s t e m
4 .0 0
0 .0 0 2 0
0 .0 0 1 4
R e p o rte d
U s e r:
s e e d
0 .0 0 1 6
M e t h o d :
b y
2 .0 0
0 .0 0 1 8
R e p o rt
I n d i v i d u a l S a m p le R e p o r t
R e p o rte d
1 8 .0 0
K r a n t i/ 1 g , u n p l s h d
IRGC# 47858
I n d iv id u a l R e p o r t
0 .0 0 0 0
b e t a c a ro t e n e
1 4 .0 0
0 .0 0 2 0
Thailand
D e f a u lt
0 .0 0 0 5
2 .0 0
Leuang Bang Bai
M e t h o d :
0 .0 0 1 0
0 .0 0 0 5
0 .0 0 0 0
India
4 5 0 n m
0 .0 0 1 0
-0 . 0 0 R
0 5e
Kranti
P D A
0 .0 0 1 5
M e t h o d :
A U
R e p o rt
A U
Bangladesh
;
0 .0 0 2 0
0 .0 0 2 0
BR 29
s e e d
0 .0 0 2 5
0 .0 0 2 5
0 .0 0 2
M e t h o d :
D e f a u lt
I n d iv id u a l R e p o r t
P r in t e d
2 :3 4 : 0 0
P M 7 /2 4 / 0 2
P a g e :
1
o f
1
0 .0 0 3
0 .0 0 2
0 .0 0 1
0 .0 0 1
I n d i v i d u a l S a m p le R e p o r t
0 .0 0 0
I n d i v i d u a l S a m p le R e p o r t
0 .0 0 0
R e p o rte d
b y
U s e r:
2 .0 0
S y s t e m
4 .0 0
6 .0 0
P r o je c t
8 .0 0
1 0 .0 0
N a m e :
1 2 .0 0
M in u te s
b e t a c a ro t e n e
1 4 .0 0
1 6 .0 0
L e u a n g
0 .0 1 4
2 0 .0 0
Y a i u n p ls
2 2 .0 0
s e e d /1 g
;
2 4 .0 0
b y
U s e r:
2 .0 0
4 5 0 n m
S y s t e m
4 .0 0
6 .0 0
P r o je c t
8 .0 0
1 0 .0 0
N a m e :
1 2 .0 0
M in u te s
b e t a c a ro t e n e
1 4 .0 0
1 6 .0 0
1 8 .0 0
L e u a n g
0 .0 1 4
0 .0 1 2
Y a i p ls
2 0 .0 0
s e e d / 1 g
2 2 .0 0
;
2 4 .0 0
4 5 0 n m
0 .0 1 2
0 .0 1 0
0 .0 1 0
0 .0 0 8
0 .0 0 8
R e p o rt
M e t h o d :
D e f a u lt
I n d iv id u a l R e p o r t
P r in t e d
2 :4 9 : 1 6
P M 7 /2 5 / 0 2
P a g e :
1
o f
1
0 .0 0 6
R e p o rt
A U
Thailand
A U
Leuang Yai
74-4-20
R e p o rte d
1 8 .0 0
M e t h o d :
D e f a u lt
I n d iv id u a l R e p o r t
P r in t e d
2 :5 0 : 4 4
P M 7 /2 5 / 0 2
P a g e :
1
o f
1
0 .0 0 6
0 .0 0 4
0 .0 0 4
0 .0 0 2
IRGC# 44159
I n d i v i d u a l S a m p le R e p o r t
0 .0 0 0
R e p o rte d
b y
U s e r:
S y s t e m
P r o je c t
N a m e :
I n d i v i d u a l S a m p le R e p o r t
0 .0 0 2
0 .0 0 0
b e t a c a ro t e n e
R e p o rte d
b y
U s e r:
S y s t e m
P r o je c t
N a m e :
b e t a c a ro t e n e
-0 . 0 0 2
2 .0 0
4 .0 0
6 .0 0
8 .0 0
1 0 .0 0
1 2 .0 0
M in u te s
1 4 .0 0
1 6 .0 0
1 8 .0 0
2 0 .0 0
2 2 .0 0
2 4 .0 0
2 .0 0
0 .0 0 3 5
4 .0 0
6 .0 0
8 .0 0
1 0 .0 0
1 2 .0 0
M in u te s
1 4 .0 0
1 6 .0 0
1 8 .0 0
2 0 .0 0
2 2 .0 0
2 4 .0 0
0 .0 0 3 5
S e re n d a h
K u n in g
u n p ls
s e e d / 1 g
S e re n d a h
K u n in g
p ls d
s e e d /1 g
0 .0 0 3 0
0 .0 0 3 0
0 .0 0 2 5
0 .0 0 2 5
0 .0 0 2 0
IRGC# 47740
R e p o0 r. 0t 0M1 0e t h o d :
D e f a u lt
I n d iv id u a l R e p o r t
P r in t e d
1 2 : 1 1 : 0 4
P M7 / 3 1 / 0 2
P a g e :
1
o f
1
R e p o0 .r0t 0 M
1 5e t h o d :
A U
Indonesia
A U
Serendah Kuning
0 .0 0 2 0
0 .0 0 1 5
D e f a u lt
I n d iv id u a l R e p o r t
P r in t e d
1 2 : 1 3 : 1 0
P M
7 /3 1 / 0 2
P a g e :
1
o f
1
0 .0 0 0 5
0 .0 0 1 0
0 .0 0 0 0
0 .0 0 0 5
-0 . 0 0 0 5
-0 . 0 0 1 0
0 .0 0 0 0
-0 . 0 0 1 5
-0 . 0 0 0 5
2 .0 0
4 .0 0
6 .0 0
8 .0 0
1 0 .0 0
1 2 .0 0
M in u te s
1 4 .0 0
1 6 .0 0
1 8 .0 0
2 0 .0 0
2 2 .0 0
2 4 .0 0
2 .0 0
4 .0 0
6 .0 0
8 .0 0
1 0 .0 0
1 2 .0 0
M in u te s
1 4 .0 0
1 6 .0 0
1 8 .0 0
2 0 .0 0
2 2 .0 0
2 4 .0 0
0.012
0.012
0.010
0.010
0.008
0.008
0.006
0.006
AU
AU
Gradual Decrease of Carotenoids with
the Increasing of Polishing Time (SECONDS)
0.004
0.004
0.002
0.002
0.000
0.000
-0.002
5.00
10.00
15.00
20.00
Minutes
25.00
30.00
5.00
35.00
0.012
0.012
0.010
0.010
10.00
15.00
20.00
Minutes
25.00
30.00
35.00
0.008
0.008
AU
AU
0.006
0.006
0.004
0.004
0.002
0.002
0.000
0.000
-0.002
5.00
10.00
15.00
20.00
Minutes
25.00
30.00
35.00
5.00
10.00
15.00
20.00
Minutes
25.00
30.00
35.00
Selected historical developments in carotenoid metabolism
in relation to plant metabolic engineering
Genes
involved in
carotenoid
biosynthesis
Cloned/
transferred
Crop
species
Y1
cloned
crtI (Phytene
desaturase)
cloned/
transformed
crtE
cloned
A gene cluster
cloned
Erwinia
herbicola
psy
transformed
Tomato
lcy
cloned
Daffodil
psy
cloned/
transformed
Daffodil/
Rice
crtB
(phytoene
synthase)
transformed
Brassica
Maize
Erwinia
uredovora/
Tobacco/
Erwinia
herbicola
Remarks
Importance of
such regulatory
gene in rice is
conceptualized
Herbicide
resistance;
Increased
coding for GGPP
synthase
For complete
carotenoid
pathway
Resulted in
dwarfism
redirecting the
metabolites from
gibberellin
pathway
Lycopene to
beta-carotene
Accumulation of
phytoene in rice
endosperm
Overexpression
led to increase
in carotenoids
and other
metabolites
Reference
Buckner et al. 1990
Misawa et al 1990,
1993
Math et al 1992
To et al 1994
Fray et al 1995
Al-Babili et al 1996
Scheldz et al 1996;
Burkardt et al 1997
Shewmaker et al
1999
Carotenoids biosynthesis in plants
Datta K et al (2003) Plant Biotech J
(Transgenic IR64, several other cultivars using Mannose selection
system)
Hoa et al (2003) Plant Physiol (Transgenic indica rice )
Parkhi et al (2005) Mol Genet Genomics
(Marker free BR29 GR by Agrobacterium)
Paine et al (2006) Nature Biotech (High carotenoids in US cultivar)
Datta K et al. (2006) Current Sci (High carotenoids indica rice)
Parkhi et al (2006) Plant Sci (Protection against draught)
Krishnan et al (2009) Plant Science
VPBR29-9
56
59
61 64
65
66
69
VPBR29-32
70
71
72
74
1
2
3
19
27 47 51 57 NT P
3.2 kb
(crtI)
1.5 kb
(hph)
Fig 3
P NT
VPBR29-9
VPBR29-31
3.2 kb
(crtI)
1.5 kb
(psy)
Fig 4
Golden BR29 rice without a marker gene (Mol Gen Genomics 2005)
Datta K et al PBJ, 2003/2005,2006
Parkhi et al MGG, 2005,2006
Rai et al 2003,2006
Ye et al Science, 2000
Painie et al Nature Biotech, 2005
Commercial right of GR remains with Syngenta
3.0-9.1 mg/g, DH homozygous lines developed
Golden Rice (BR29) developed at IRRI is now in Bangladesh soil
Syngenta-Golden Rice (GR2) is now in field at Louisiana, USA
BR29
β-crt
0.12
BR29
0.10
Lui
AU
0.08
0.06
α-crt
0.04
β-cry
0.02
0.00
5.00
10.00
15.00
Minutes
20.00
25.00
30.00
Fig. HPLC chromatograms showing beta carotene peaks in
the carotenoid extract from polished seeds of one progeny
of BR29 in T1 generation
LBA4404/pZPsC
+
LBA4404/pZLcyH
Co-transformation
Hemizygous T309 GoldenRice
(Ye et al. 2000)
Molecular analysis
Phenotyping
Dihaploid homozygous T309 GoldenRice
(Baisakh et al. 2001b)
x
IR64
Anther culture
PCR analysis
Flow chart for the
IR64
Development of
x
F1
1st Backcrossing
Marker-free
Near-isogenic golden
BC1F1
PCR screening
and Southern confirmation
BC1F1 progenies
Marker-free
x
IR64
Selection of hph negative
transgenic progenies
Rice lines of IR64
2nd Backcrossing
BC2F2
Marker-free
Molecular
analysis
Selfing
Phenotyping
HPLC
IR64 NILs
Marker-free
BC2F1
Marker-free
Agronomic performance of transgenic Golden rice
(cv. IR64) vis-à-vis the IR64 control
Plant
height
(cm)
No. of
panicles
per plant
No. of
grains
per
panicle
No. of
unfilled
spikelets
per panicle
Spikelet
fertility
(%)
1,000grain
weight (g)
Biological
yield per
plant (g)
Grain
yield per
plant (g)
Harvest
index (%)
Mean
107.13
9.13
88.81
34.16
71.46
25.86
109.25
13.49
13.66
SEm
0.745
0.358
2.460
1.364
1.078
0.168
5.953
0.661
0.610
Mean
108.80
8.65
86.05
28.75
74.67
25.77
98.98
13.74
14.86
SEm
1.733
0.539
5.558
3.312
2.635
0.223
9.309
1.350
1.290
0.950ns
0.391ns
0.242ns
2.881ns
1.627 ns
0.060ns
0.702 ns
0.030ns
0.770 ns
Characters
Treatments
TRANSGENIC
CONTROL
F-value
(transgenic vs.
control)
ns= nonsignificant at p  0.05 (Rai et al. RGN 2004)
T3 progenies of transgenic golden IR64
NT PC
3.8-kb
Fig. 1. Southern blot showing homozygous progenies of Golden indica rice (cv. IR64) with integration of a 3.8-kb fragment
T
C
C
T
Fig. 2. Transgenic Golden indica rice (T) and control rice (cv. IR64; C) showing uniformity in overall phenotype (left panel)
and grain filling (right panel) grown under screenhouse conditions at IRRI, Philippines.
URP1
URP2
URP3
URP4
12 3 4 5 6 7 8 M 12 3 4 5 6 7 8 M 12 3 4 5 6 7 8 M 12 3 4 5 6 7 8 M
URP5
URP6
URP7
URP8
URP10
URP11
12 3 4 5 6 7 8 M 12 3 4 5 6 7 8 M 12 3 4 5 6 7 8 M 12 3 4 5 6 7 8 M M1 2 3 4 5 6 7 8 M 1 2 3 4 5 6 7 8 M
Fig. 3. Transgenic Golden indica rice of NHCD (lanes 1 and 2 in each panel) and IR64 (lanes 4, 5, 6, and 7 in each panel)
showing no polymorphism with Universal rice primers (URP) vis-à-vis their respective controls (lanes 3 and 8 in each panel). M
= 1 kb-plus molecular weight marker.
Essential Minerals: Iron
Iron deficiency is the most widespread micronutrient
deficiency worldwide.
Approx. 30% of world population suffers from
serious nutritional problems caused by insufficient
intake of iron (WHO 1992).
It is the important constituent of hemoglobin, the
oxygen carrying component of blood, and also a part
of myoglobin that helps muscle cells to store oxygen.
It is present in food in both inorganic (ferric and
ferrous) and organic (heme and nonheme) forms.
Highly bioavailable heme iron is derived primarily from
animal source.
Biofortified iron rice
Screening for iron- Mutational
rich rice varieties
breeding
Transgenic plant
strategy
1. High iron and enhanced carotenoids/beta-carotene rice
2. Reduced content of phytate in rice grains
Increased bioavailabillity of Fe and Zn
Sst I
nos
Bam HI
ferritin
GluB-1
Sst I
nos
35S
bar
Bam HI Kpn I
ferritin
Sst I
nos
Hind III
Glo-P
Bam HI Kpn I
ferritin
Pro-P
Vasconcelos et al Plant Sci 2003
Tan et al Int J Food Sci Tech 2004
Khalekuzzaman et al In J Biotech 2006
g7
The Aspartate-Family Biosynthetic Pathway
Aspartate
AK
b-aspartyl phosphate
aspartic b-semialdehyde
Threonine
Methionine
DHDPS
2-3 dihydropicicolinate
5 steps
Lysine
Technologies Ready for transfer
1. 30 Normal and 8 QPM SCH
Normal maize
Q PM
2. Baby corn, Sweet corn,
popcorn single cross hybrids
available
3. Technology for Single Cross
Hybrid Seed Production and
commercial cultivation for
normal QPM and specialty
corn
Sweet corn hybrid
HSC-1
Pop corn hybrid
Hyd 14-3 X
HKIPC5
SCH Seed production
Value added Dream-RICE
• High
iron rice (after polishing)
Provitamin A rice
Other micronutrient-rich rice
Development of Value added rice for both
favorable and unfavorable ecosystems.
combination of high yield with value-added rice
Green revolution
saved famine in Asia
Molecular breeding for Nutrition food
may help in reducing malnutrition
provided FTO (Govt supp.) is in place