zinc biofortification of cassava tubers

Report
To increase by six-fold the content and bioavailability of zinc in cassava tubers and to demonstrate its viability in the field and efficacy in humans
ZINC BIOFORTIFICATION OF CASSAVA TUBERS
Shuaibu Kahya,Narayanan N. Narayanan 1 , Eliana Gaitan- solis , Martin Fregene¹ and Richard T. Sayre1
1Donald Danforth Plant Science Center, St. Louis, MO 63132, USA
Germination
media
Introduction
A14-AtZIP1-tNOS in Tobacco seedlings
Cloning and Construct Vectors
Cassava (Manihot esculenta), being the major staple food crop for more than 300 million people
in Africa lacks important micronutrients such as Vitamin A, Iron and Zinc. However, zinc
deficiency is a widespread nutrition and health problem in the world especially in the developing
countries.
Zn deficiency in humans is widespread and is estimated to affect more than 25% of the world’s
population and rank the 5th among the most important health risk factors in developing Countries
and 11th worldwide, and it is equally as important as iron (Fe) and vitamin A deficiency.
 Genetic engineering approach for biofortification of staple crops are currently used to
combat Zinc deficiency.
A14-AtZIP1-tNOS construct in p2301 was given as a
gift from Eliana Gaitan-solis, DDPSC. Primers with
restriction enzymes (EcoRI and KpnI) were designed to
pull out the construct and cloned it in pCAMBIA2300.
AtZIP1 driving by A14- root epidermal promoter was
introduced into cassava (FEC) via Agrobacterium mediated transformation
SEQUENCING
PAT
Apoplastic
passage
AGRO-TRANSFORMATION
ZAT
Use
Uptake
unloading
Phloem transport
(c)
Xylem
loading
Xylem
transport
Screening of clones by PCR with different
primers
CASSAVA FEC SYSTEM
Agrobacterium Mediated Transformation
(Cassava FEC)
1
Symplastic
passage
(a)
Mobilization
(b)
Uptake
FEC
C0-CULTURE
RESTING
Constructs Used
STAGE 1
ATZIP1
MS2
MS-BAP
GFP
STAGE 2
MS-BAP
NOS
Preliminary analysis shows that A14 is expressed in root epidermis and leaves (Elisa
LeyvaGuerrero, unpublished data). This should increase the transport of zinc into the root
epidermis and not concentrate in the cortex there by preventing the accumulation of zinc into the
root alone.AtZIP1 is a Zinc transporter expressed in the root(Natasha et al; 1998)
A14
ATZIP1
NOS
PAT
AtMTP1
PAL
AtHM4
NOS
PAT
AtMTP1
Shoot
NOS
AtMTP1 is already known to mediates Zn detoxification and storage by vacuolar sequestration
of Zn in plant cell (Anne- Garlonn et ;al 2005) . Using this gene with the patatin promoter will
balance zinc homeostasis in the plant and maintain high zinc concentration in the target root
tissue
Root
H₂0
T10 T12 T13
WT
T9
A14:ZIP
T8
Dot blot Analysis
A
100ng of genomic DNA (both WT
and transgenic) were loaded.
Hybridized with 2X35S probe,
Samples loaded in triplicates .
Out of 24 lines screened,
preliminary analysis indicate six
transgenic lines show 2 or 3 copy
numbers .
WT
 A14-AtZIP1-tNOS binary plasmid was introduced into cassava using
Agrobacterium transformation. Seventy independent transgenic
lines were obtained . Twelve lines were screened for PCR as shown in the
figure above. Four lines show the presence of the gene.
 Leaves and roots will be collected and mineral analysis will be
performed.
Conclusions
 Tobacco transgenic lines carrying A14-AtZIP1-tNOS shows a promising
phenotype in shoots indicating a balanced Zn homeostasis. ICP mineral
analysis are in progress.
 Constructs with different promoters are made and transformed into
cassava. Molecular analysis and mineral analysis will be performed.
Acknowledgements
NOS
840-PAL
found
express
rapidly
in parenchyma,
vascular
tissues
especially
into.AtHMA4
xylem
parenchyma,
840-PAL is found tois
express
rapidly to
in vascular
tissues especially
into xylem
tyloses
both in leaves
and roots (Nigel Taylor)
enhance
the zinc loading into xylem tissue
and increase root – shoot translocation.
tyloses both in leaves and roots. AtHMA4 enhance the zinc loading into xylem tissue and
.
increase root – shoot translocation (Verret et al; 2004).
T7
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C2
GFP Expression
A14
T6
A14-AtZIP1-tNOS in Cassava
F
G
H
Transition metal from the soil to the sites of use and storage in the leaf. (a) to enhance
mobilization by secretion of organic acids, (b) to increase uptake by over expression or
deregulation of transporters, (c) to stimulate uptake into the root and translocation via the xylem
by overproduction of intracellular chelators, (d) to increase the strength of metal sinks in the
leaves by over expression of storage and detoxification mechanisms.
T5
 A14-AtZIP1-tNOS binary plasmid was introduced into tobacco using
leaf-disc Agrobacterium transformation. Twelve independent transgenic
lines were obtained and screened for the presence of transgene (AtZIP1)
PCR of 9 Tobacco Transgenic lines
as shown in the figure above. Nine lines show the presence of the gene.
 Leaves, roots and seeds from T0 generation will be collected and
mineral analysis will be performed.
 Homozygous lines will be obtained and be used to study zinc
homeostasis.
SPREAD PLATE
(Stephan et al; 2002)
T4
PCR results of nine Tobacco Transgenic lines
B
C
D
E
Storage and detoxification
T3
H₂0
ZIP
T2
WT
DIGESTION
Symplastic
passage
T1
Water control
PATZAT-P2301
A14
Soil
Green house
A14ZIP
CLONING
A14ZIP-P2301
AMPLIFICATION
p2300
PCR
A14ZIP+PATZAT
Path of Transition Metals and
Genetic Engineering Target
A14ZIP
Patatin (1kb) is a root specific promoter from
Solanum tuberosum, while AtMTP1 (1kb ) was
amplified from Arabidopsis thaliana leaves. PATAtMTP1-tNOS in p2301 was digested with Kpn1 and
Sal1 and cloned into plasmid of pCambia-A14-AtZIPtNOS. This double construct was also introduced into
cassava (FEC) via Agrobacterium - mediated
transformation
To increase by six-fold the content and bioavailability of zinc in cassava tubers and to demonstrate
its viability in the field and efficacy in humans
Use
Rooting media
Germination
media
ABSTRACT
Objectives
(d)
Storage and
detoxification
Selection
Acknowledgement
Cassava invitro seedlings transformed with p2300-GFP showing GFP fluorescence in
root and shoot tissues. Pictures were taken 8 weeks after transformation.
We would like to thank Dr. Eliana Gaitan solis for giving the constructs and
support and Kevin Lutke, Tissue Culture Facility, DDPSC for transforming
into Tobacco. Funding from Gates Foundation and support from
Biocassava plus and NRCRI Umudike is greatly appreciated.

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