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