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