somatic embryogenesis and selection regimes for particle
bombardment of friable embryogenic callus and somatic
cotyledons of cassava (Manihot esculenta Crantz)
Heredia F.F.2, Pinheiro C.B.2, Aragao
F.J.L.3 and Campos F.A.P.2
1Department of Biochemistry, Ahmadu Bello University,
2Department of Biochemistry and Molecular Biology, Federal
University of Ceará, P. O. Box 6039, Fortaleza, CE, Brazil.
Recursos Genéticos e Biotecnologia, PqEB W5 Norte,
70770-900, Brasilia, DF, Brazil.
*Corresponding author: E-mail:
Tel: +234 70 34589199.
Cassava somatic embryogenesis induction medium; CMM,
Cassava somatic embryogenesis maturation medium; FEC,
friable embryogenic callus;
GD medium + 2% sucrose and 12 mg/L picloram; MS2NAA5,
MS medium+ 2% sucrose and 1mg/L NAA;
secondary somatic embryogenesis.
Accepted 26 June, 2008
a number of transformation systems and selection regimes
have been developed for cassava (Manihot esculenta
Crantz), they have only been applied in a limited number of
genotypes. This limitation of the applicability of the
systems is due largely to variation in morphological
responses of different genotypes to the regeneration and
transformation procedures employed, which underscores the
need to study all possible parameters for any given
cultivar. Using two cultivars (“Rosinha” and “Bujá Preta”)
we made an attempt to improve the frequency of somatic
embryogenesis (SE), establish friable embryogenic callus (FEC)
lines from developing somatic embryos and evaluate the
effect of different concentrations of the antibiotics
kanamycin, on secondary somatic embryogenesis (SSE) and of
paromomycin, on FEC in proliferation and
histodifferentiation media. Further, we report on transient
expression of the visual marker gene uidA following
particle bombardment of FEC from ‘Bujá Preta’ and of cut
pieces of green somatic cotyledons from ‘Rosinha’, using the
plasmid pBI426. Higher number of embryos, which emerged as
early as 8 days after culture on MS medium (CIM), was
obtained when abaxial part of the explant was in direct
contact with the medium as against
the adaxial side, and this did not depend on the
explant size (0.5 cm² or
2.5 cm²). Highly friable
and embryogenic callus was obtained on GD medium
supplemented with 2% (w/v) sucrose and 12 mg/L picloram.
While paromomycin, at concentration of 60 mg/L arrested the
proliferation and histodifferentiation of FEC, kanamycin in
CIM containing explants undergoing SSE led to a decrease in
their embryogenic potential resulting in tissue death at 50
mg/L. The highest transient expression of uidA gene
in FEC was observed combining plasmolysis, M5 particle and a
helium pressure of 1,200 psi, while in somatic cotyledons,
the highest expression was observed when M5 particle was
used along with the helium gas pressure of 900 psi.
FEC, Manihot esculenta, somatic embryogenesis,
selective agent, transient expression.