Obtaining embryos in the cotyledon phase of Robusta Coffee (Coffea canephora) using a temporary immersion system, using the somatic embryogenesis technique from leaf segments

Abstract

Standard methods were developed to obtain cotyledon-stage embryos of Coffea canephora in the laboratory. The stablishment phase was standardized using a chlorine concentration of 1.5 % and 20 minutes immersion time. This method was effective because it reduced contamination and produced low percentages of oxidation. In the callus induction phase, the medium used was M&S (Murashige & Skoog) with saccharose (3 %) and agar (7 %). We tested different concentrations of 2,4-D (2,4- dichlorophenilacetic acid), BAP (benzylaminopurine) and KIN (kinetin). The best medium was: M&S with 0.5 mg/L of 2,4-D and 2 mg/L of KIN. The embryos were grown in M&S medium at half strength without growth regulators. This medium was also tested as a liquid in large tubs in two systems, the home immersion system and Automated Temporary Immersion System (TIS). There was embryo growth after 20 days in the home inmmersion system. We produced globular stage embryos in the system and after 12 weeks we had 611 embryos, 15 of these embryos were in the cotyledon stage. The automated temporary immersion system did not produced embryos.