1. 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 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 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 (Nigel 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 (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 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 Kpn1 and Sal1 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 ATZIP1NOS 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 A14ATZIP1NOSPATNOSAtMTP1 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 PALAtHM4NOSPATNOSAtMTP1 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 C1C2C9C3C5C4 C7C8 H₂0H₂0 C10C11C2 WT T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T12 T13 A14:ZIP H₂0H₂0 WT