zinc biofortification of cassava tubers

twoeggfinnishBiotechnology

Dec 14, 2012 (4 years and 6 months ago)

279 views

ZINC BIOFORTIFICATION OF CASSAVA TUBERS

Shuaibu Kahya
,Narayanan N. Narayanan

1

,

Eliana Gaitan
-

solis , Martin Fregene¹ and Richard
T. Sayre
1

1
Donald Danforth Plant Science Center, St. Louis, MO 63132, USA


ABSTRACT

Agrobacterium Mediated Transformation

(Cassava FEC
)

A14
-
AtZIP1
-
tNOS in Cassava


Path

of Transition Metals and


Genetic Engineering Target

Introduction

1


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.

.

A14ZIP

A14ZIP+PATZAT

p2300

A14ZIP
-
P2301

PATZAT
-
P2301

Water control

A14

ZIP

PAT

ZAT

Use

Use

Uptake

Phloem transport

Storage and detoxification


(d)

Storage and

detoxification



(a)

Mobilization


(b)

Uptake


(c)


Xylem


loading

Symplastic


passage

Symplastic


passage




Xylem

transport

Apoplastic


passage

unloading

PCR AMPLIFICATION

CLONING

DIGESTION

SEQUENCING

AGRO
-
TRANSFORMATION

CASSAVA FEC SYSTEM

MS
-
BAP

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.




Objectives

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

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


Cloning and Construct Vectors

(Stephan et al; 2002)

840
-
PAL is found to express rapidly in vascular tissues especially into xylem parenchyma, tyloses both in leaves and roots (Nige
l Taylor) .AtHMA4 enhance the zinc loading into xylem tissue
and increase root


shoot translocation.

Screening of clones by PCR with different
primers

A14
-
AtZIP1
-
tNOS construct in p2301 was given as a
gift from Eliana Gaitan
-
solis, DDPSC. Primers with
restriction enzymes (
EcoR
I and
Kpn
I) 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

Patatin (1kb) is a root specific promoter from
Solanum tuberosum,
while
AtMTP1
(1kb ) was
amplified from
Arabidopsis thaliana
leaves. PAT
-
AtMTP1
-
tNOS in p2301 was digested with
Kpn
1 and

Sal
1 and cloned into plasmid of pCambia
-
A14
-
AtZIP
-
tNOS. This double construct was also introduced into
cassava (FEC) via
Agrobacterium

-

mediated
transformation


PCR of 9 Tobacco Transgenic lines

FEC

Germination
media

Acknowledgement

RESTING

SPREAD PLATE

STAGE 1

STAGE 2

C0
-
CULTURE

MS2

MS
-
BAP

GFP

Shoot

Root

Constructs Used

A14


ATZIP1

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)

GFP Expression

A14
-
AtZIP1
-
tNOS in Tobacco seedlings

Conclusions

Acknowledgements

Rooting media

Selection

Green house

Soil

Germination
media

PCR results of nine Tobacco Transgenic lines

Dot blot Analysis

A14


ATZIP1

NOS

PAT

NOS

AtMTP1

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

PAL


AtHM4

NOS

PAT

NOS

AtMTP1

840
-
PAL is found to express rapidly in vascular tissues especially into xylem parenchyma,
tyloses both in leaves and roots. AtHMA4 enhance the zinc loading into xylem tissue and
increase root


shoot translocation
(
Verret et al; 2004).

WT


A

Cassava
invitro

seedlings transformed with p2300
-
GFP showing GFP fluorescence in

root and shoot tissues. Pictures were taken 8 weeks after transformation.



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)

as shown in the figure above. Nine lines show the presence of the gene.



Leaves, roots and seeds from T
0

generation will be collected and

mineral analysis will be performed.



Homozygous lines will be obtained and be used to study zinc

homeostasis.

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.



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.


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 .


B


C


D


E


F


G


H



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.



C6


A14ZIP

C1

C2

C9

C3

C5

C4


C7

C8

H

0

C10

C11

C2

WT

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T12 T13


A14:ZIP

H

0

WT