Increasing Phytoene and ß-carotene content in cassava roots by expression of an exogenous
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Poster67: Increasing Phytoene and B-carotene content in cassava roots by expression of an exognous phytoene synthase (CrtB) gene


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Poster for CIAT 2009 Knowledge Sharing Week

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Poster67: Increasing Phytoene and B-carotene content in cassava roots by expression of an exognous phytoene synthase (CrtB) gene

  1. 1. Increasing Phytoene and ß-carotene content in cassava roots by expression of an exogenous phytoene synthase (CrtB) gene Beltrán J1, Ladino J1, Vacca O1, López D1, Arango J2, Chávez AL1, Al-Babili S2, Beyer P2, Chavarriaga P1 and Tohme J1 1. Conservation and Use of Tropical Genetic Resources, CIAT, AA 6713 , Cali, Colombia 2. Albert-Ludwigs-Universitat Freiburg, Center for Applied Biosciences, Schanzlestr 1, D-79104, Freiburg, Germany INTRODUCTION • Carotenoid Analysis A current strategy to improve the nutritional quality of cassava Phytoene: ranged from 3.49 to 15.55 ng/mg of DW (control = 1.17) storage roots is the overexpression of carotenogenic genes in a tissue- trans β-carotene: ranged from 0.72 to 9.58 ng/mg of DW specific manner. In transgenic plants, the bacterial phytoene synthase (control = 0.98). CrtB gene catalyzes the conversion of Geranyl-Geranyl Diphosphate (GGPP) to phytoene in the first step of carotenoid biosynthesis 487- Transgenic cassava roots ( from soil) High Performance Liquid Chromatography (HPLC) to We developed cassava transgenic plants using the CrtB gene fused to detect trans β-carotene 2- Transgenic cassava roots ( in vitro) the CP1+ promoter. The promoter was isolated from a Glutamic Acid-Rich Protein (GARP) gene from cassava roots. Although fibrous and not storage roots were analyzed, most transgenic lines had increased the content of Phytoene and trans β-carotene respect to the The lines Cl and Cl-509 were the most promising with the highest levels non-transgenic control. of trans β-carotene (9,589 and 3,490 ng / mg DW, respectively) 18 METHODS 16 14 12 Phytoene and trans β- Carotene contents in nine ng/mg DW 10 Trans-ß-carotene 8 Total Phytoene transgenic lines (WtS = 6 storage root from wild type; 4 WtF = fibrous root from wild 2 • pCasPhyt plasmid: CP1+ promoter from cassava Glutamic Acid-rich root type) 0 protein; CrtB gene; 35S-hpt II. Wt S Wt F Cl Cl 509 Cl 88 Cl 143 Cl 146 Cl 60 122 487 220 Cassava Transgenic Lines Transformation: In a group of four different transgenic events it was clearly seen that an increase in phytoene (early intermediary in the route) was directly Cassava: Friable Embryogenic proportional to the increase in β -carotene (late intermediary) Callus , genotype 60444, Agrobacterium Strain: Agl1 Phytoene Vs β-Carotene content CRTB 3.000 Cassava transgenic plants were obtained by standard protocols at CIAT 2.500 β-Carotene (ng/mg DW) 2.000 RESULTS AND DISCUSSION 1.500 * transgenic lines * Wild type 1.000 • Real-time PCR transgene detection 0.500 0.000 0.00 5.00 10.00 15.00 20.00 15 transgenic events were confirmed by real-time PCR amplification Phytoene (ng/mg DW) of the crtB and hptII genes Our results suggest that the endogenous enzymes that follow CRTB are 1kb 12 17 60 84 139 146 220 509 NT H2O P active to convert part of the new phytoene into β-carotene. CrtB The question that remains is if accumulation of β-carotene will also hpt II increase during the formation of the storage roots in plants. • Quantifying CrtB transcripts in fibrous roots • Field testing • Fifteen transgenic lines were transferred Roots registered a varied pattern of expression of the CrtB transgene, to the field (May 2008) to analyze carotene with differences up to 13.16 times. While CrtB expression was accumulation in mature, field-grown detected in roots, it was also observed in leaves, suggesting that the plants. CP1+ promoter is not driving the expression in roots only. CrtB expression in roots CONCLUSIONS AND PERSPECTIVES 14 13.16 12 CrtB expression in roots and • The expression of CrtB in cassava roots evidently generates an leaves of different transgenic lines increment in trans β -carotene content in fibrous roots, that ranged from Relative mRNA level 10 8 5.49 CrtB 1.4 to 10-fold. 6 4 2 1 1.09 • We are currently focused in the production of transgenic cassava 0 509 487 122 60 plants that carry the entire mini-pathway, which contains the genes CrtB, CrtI and CrtY to augment the conversion of GGPP into β – Cassava transgenic lines carotene. Date prepared July 2008