1 research outputs found
Plant regeneration via indirect somatic embryogenesis and optimisation of genetic transformation in Coffea arabica L. cvs. Caturra and Catua\ued
A protocol for Coffea arabica L. cvs. Caturra and Catua\ued plant
regeneration via indirect somatic embryogenesis (ISE) was established.
Furthermore, a biolistic mediated genetic transformation protocol was
optimized for Catua\ued callus aggregates. Maximum callus induction
was obtained when Caturra (87%) and Catua\ued (67%) leaves were
cultured on Murashige and Skoog medium with 18.56 \u3bcM kinetin and
4.52 \u3bcM2,4-dichlorophenoxyacetic acid (2,4-D). Catua\ued
suspension cultures were established from embryogenic callus using
liquid proliferation CP and Sli media and diffused light and darkness.
The higher suspension cultures fresh weight was obtained using
Erlenmeyer (1425.4 \ub1 354.9 mg) than Recipient for Automated
Temporary Immersion System (RITA\uae) (518.6 \ub1 55.1 mg), whereas
the dry weight of suspension cultures was not significantly affected by
the culture system used. Higher number of embryos per vessel (307.6
\ub1 49.0) and their fresh weight (9.6 \ub1 1.5 mg) were obtained
with semisolid R medium than S3 medium. The highest somatic embryo
development (25.0 \ub1 2.7) and fresh weight (780.0 \ub1 85.4 mg)
were obtained with 1 min of immersion every 8 hrs. Higher fresh weight
of regenerated plantlets was obtained with liquid Yasuda medium in
RITA\uae (124.6 \ub1 16.3 mg) than semisolid media (36.3 \ub1
11.3 mg). For genetic transformation, the effect of helium pressure
(900 and 1550 psi), and target distance (9 and 12 cm) and plasmid
(pCAMBIA 1301, pCAMBIA 1305.2 and pCAMBIA 1301-BAR) on transient uidA
expression Catua\ued suspension cultures were evaluated. The highest
number of blue spots was obtained using 900 psi and 9 cm (125.8 \ub1
17.3). Stable uidA expression was observed on Catua\ued callus
aggregates transformed with pCAMBIA 2301 and cultured on 100 mg l-1 of
kanamycin