Criopreservação de embriões zigóticos de Coffea arabica por vitrificação

Abstract

Due to the economic and social importance of coffee to Brazil, it is necessary that measures must be taken for the conservation of their genetic resources. However, due the difficulty to coffee beans storage, biotechnological alternatives such as cryopreservation has been required. In this context, the objective was to develop a cryopreservation protocol by vitrification technique for zygotic embryos of Coffea arabica L. cv. Red Catuaí (IAC 144). First, we evaluated the seeds disinfection and zygotic embryos excision. In seeds disinfestation, seeds were immersed in different formaldehyde concentrations (0, 0.5, 1 and 1.5%) for different periods of time (5, 15 and 30 min). For embryos excision the seeds were disinfected and further immersed in a boric acid solution 0.5% for different periods of time (1, 2 and 3 days). For the study of cryopreservation were evaluated before freezing different immersion times in Plant vitrification solution 2 (PVS2) (0, 10, 25, 50, 100, and 250 min) at two temperatures (0 °C and 25 °C). Following was determined the best thawing time in water bath (1, 3, 5 or directly in Recovery solution (RS)). A anatomical study was also conducted in zygotic embryos not cryopreserved (control), and in zygotic embryos frozen with or without the use of PVS2. It was subsequently set up the acclimatization of plantlets from cryopreserved embryos and not cryopreserved. The complete seeds disinfection was achieved with 1.5 or 1% formaldehyde for 30 min. Soaking seeds for 2 or 3 days makes more flexible seeds for the subsequent embryo excision, providing 100% germination with normal seedlings. Cryopreservation of zygotic embryos was successfully obtained by vitrification. Immersion in PVS2 solution for 100 min at 0 °C temperature allows cryopreservation of embryos promoting 80% germination with 87% of normal seedlings. For thawing the frozen zygotic embryos can be thawed directly in RS solution, thus eliminating the need to use water bath. Anatomically, it was observed that PVS2 solution decreases the internal water content of the cells observed by plasmolysis, allowing there freezing and subsequent growth resumption after thawing. Regarding acclimatization further work is needed to improve the survival rate of the seedlings derived from embryos frozen, since only 13% of seedlings survived after acclimatization.