Plant regeneration via indirect somatic embryogenesis and optimisation of genetic transformation in Coffea arabica L. cvs. Caturra and CatuaíAndrés M. Gatica-Arias*#
Centro de Investigación en Biología Celular y Molecular
Universidad de Costa Rica
P.O. Box 2060, San Pedro, Costa Rica
Tel: 506 2075091
Fax: 506 2074216
Ana M. Espinoza Esquivel
Financial support: This research was supported by the Costa Rica - United States of America Foundation for Cooperation (CRUSA) and the University of Costa Rica.
Keywords: bioreactor, coffee, genetic transformation, somatic embryogenesis, suspension cultures.
Present address: #Escuela de Biología, Sede Universitaria Rodrigo Facio, Universidad de Costa Rica. P.O. Box 2060, San Pedro, Costa Rica. Tel: 506 207 50 91; Fax: 506 207 42 16.
A protocol for Coffea arabica L. cvs. Caturra and Catuaí plant regeneration via indirect somatic embryogenesis (ISE) was established. Furthermore, a biolistic mediated genetic transformation protocol was optimized for Catuaí callus aggregates. Maximum callus induction was obtained when Caturra (87%) and Catuaí (67%) leaves were cultured on Murashige and Skoog medium with 18.56 µM kinetin and 4.52 µM2,4-dichlorophenoxyacetic acid (2,4-D). Catuaí 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 ± 354.9 mg) than Recipient for Automated Temporary Immersion System (RITA®) (518.6 ± 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 ± 49.0) and their fresh weight (9.6 ± 1.5 mg) were obtained with semisolid R medium than S3 medium. The highest somatic embryo development (25.0 ± 2.7) and fresh weight (780.0 ± 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® (124.6 ± 16.3 mg) than semisolid media (36.3 ± 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 Catuaí suspension cultures were evaluated. The highest number of blue spots was obtained using 900 psi and 9 cm (125.8 ± 17.3). Stable uidA expression was observed on Catuaí callus aggregates transformed with pCAMBIA 2301 and cultured on 100 mg l-1 of kanamycin.