ZINC BIOFORTIFICATION OF CASSAVA TUBERS

1. 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 (d) Storage and detoxification PCR AMPLIFICATION A14 A14 PAL ATZIP1 AtHM4 ATZIP1 NOS NOS NOS PAT PAT AtMTP1 AtMTP1 NOS NOS A14ZIP+PATZAT Water control Use PATZAT-P2301 A14ZIP-P2301 SPREAD PLATE A14ZIP p2300 C0-CULTURE RESTING Apoplastic passage Symplastic passage CLONING A14 Germination media Use Introduction A14-AtZIP1-tNOS in Tobacco seedlings Uptake unloading Phloem transport Cloning and Construct Vectors Xylem transport DIGESTION ZIP (c) Xylem loading STAGE 1 PAT • 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. SEQUENCING 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 Symplastic passage ZAT STAGE 2 MS2 AGRO-TRANSFORMATION (a) Mobilization (b) Uptake Storage and detoxification ABSTRACT CASSAVA FEC SYSTEM Objectives 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 Germination media 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 Rooting media Path of Transition Metals and Genetic Engineering Target Selection A14:ZIP H₂0 WT T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T12 T13 1 PCR results of nine Tobacco Transgenic lines Soil Green house • 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 T0 generation will be collected and • mineral analysis will be performed. • Homozygous lines will be obtained and be used to study zinc • homeostasis. PCR of 9 Tobacco Transgenic lines Screening of clones by PCR with different primers A14-AtZIP1-tNOS in Cassava Agrobacterium Mediated Transformation (Cassava FEC) A Dot blot Analysis B 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 . C D E FEC F G (Stephan et al; 2002) 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. WT A14ZIP H₂0 WT C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C2 Constructs Used MS-BAP GFP • 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. GFP Expression MS-BAP 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) Shoot 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. 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 Acknowledgements Acknowledgement 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. 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). 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. Cassava invitro seedlings transformed with p2300-GFP showing GFP fluorescence in root and shoot tissues. Pictures were taken 8 weeks after transformation. .