Multiple strategies to enhance the accumulation of pro-Vit A in cassava roots
Chavarriaga P1, Beltrán J1, Ladino J1, Vacca O1, López D1, Arango J2, Al-Babili S2, Beyer P2, and Tohme J1
Beltrá Ló Al-
1. Conservation and Use of Tropical Genetic Resources, CIAT, AA 6713 , Cali, Colombia 2. Albert-Ludwigs-Universitat Freiburg, Center for Applied
6713 Albert- Ludwigs-
Biosciences, Schanzlestr 1, D-79104, Freiburg, Germany
D-
INTRODUCTION • CrtB, Y and I transcripts from in vitro roots
We are introducing three genes (crtB, crtY and crtI) of the carotene
The expression of the three genes was detected in in-vitro roots and
synthesis pathway from E. urodevora into cassava cultivars to increase β-
leaves, indicating that the Patatin and Patatin I promoters may be active
carotene in roots. These genes are under the control of root specific
on both organs in young plants.
promoters isolated from cassava, sugar beet and potato. Based on
evidence from other crops like potato, it is expected that the expression 1kb 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
of this pathway, which converts GGPP into β-carotene, may turn out to CrtB
be a more efficient way of increasing the pro-VitA content in cassava Expression in
than introducing just one crtB gene. Plants have been regenerated and CrtY in vitro fibrous
confirmed transgenic via real time RT-PCR. Some lines are planted in roots
CrtI
the field.
18S
METHODS
Gene constructs provided by The Albert-Ludwigs-Universitat
Freiburg. • Orange Embryos
35S npt II Pat CrtY Pat I CrtB Pat CrtI
1)
Some transgenic lines developed
orange embryos that seemed to
LB RB accumulate high levels of carotenes
in cotyledons, an starch-
35S hpt II Ext CrtY Ext CrtB Ext CrtI
2) accumulating organ. Evidence of
the activity of the promoters
LB RB
Patatin and Patatin I in this organ
was therefore observed.
35S hpt II Mll CrtY Mll CrtB Mll CrtI
3)
LB RB • T0-pPatErwII plants in vitro, greenhouse and field
T0 plants were regenerated mostly from green embryos and are being
1) pPatErwII Patatin from Potatoes planted in the field.
2) pCAExtThree Extensin from Cassava Roots
3) pCASBGolden Major Latex Like Protein from Sugar Beet
Transformation of Friable Embryogenic Callus
Model cultivar 60444 was
transformed using
Agrobacterium tumefaciens
(Agl1). Over 150 cell lines
were established from orange,
yellow and creamish-colored
calli
CONCLUSIONS AND PERSPECTIVES
• Besides introducing a single crtB gene, we are complementing the
strategy to increase β-carotene content in cassava roots by introducing
three (crtY, crtB, crtI) genes of the pathway.
RESULTS AND DISCUSSION
•The Patatin and Patatin I promoter seem to be active in leaves, roots
• Real-time PCR transgene detection in eleven T0-pPatErwII and cotyledons of cassava.
plants
Transgenic
pPatErwII
• It has been possible to regenerate transgenic plants from green
H2O
embryos carrying the three genes. They are being planted in the field for
NT
1kb --------------------------TRANSGENICS----------------------------
further analysis.
crtB
•Our strategy is being complemented by transforming yellow-rooted
cultivars from Brazil and Colombia, and one ACMD resistant cultivar
crtY from Nigeria.
crtI
Date prepared: July 2008