Cryopreservation of coffee zygotic embryos: dehydration and osmotic rehydration

Abstract

Conservation of plant genetic resources is important to prevent genetic erosion. Seed banks are the most common method of ex situ conservation; however, coffee seeds can not be stored by conventional methods. Cryopreservation is a viable alternative for long-term conservation of species that produce intermediate or recalcitrant seeds, as coffee. The aim of this work was to cryopreserve Coffea arabica L. cv Catuaí Vermelho IAC 144 zygotic embryos, and analyse the effects of dehydration prior cryopreservation and osmotic rehydration after thawing, in embryos germination and seedlings formation after cryopreservation. Prior to cryopreservation, different dehydration times (0, 15, 30, 60 and 120 min) were tested. Dehydrated embryos were cryopreserved in liquid nitrogen for 1 hour, and after thawing were rehydrated by osmotic solutions. Dehydrated and non-cryopreserved embryos were also analysed. The test with 2,3,5 triphenyl tetrazolium chloride (TTC) was used to evaluate the embryos viability. Non-dehydrated embryos did not survive after freezing. Embryos that were dehydrated until 20% of the moisture content did not germinate when osmotic rehydration was not performed. In contrast, cryopreserved embryos with the same moisture content presented 98% germination when they were rehydrated slowly in osmotic solution. According to tetrazolium tests, embryos presented maximum viability (75%) after dehydration for 60 minutes (23% moisture content). Therefore, coffee zygotic embryos (Coffea arabica L. cv. Catuaí Vermelho) can be successfully cryopreserved using physical dehydration in silica gel for 60 minutes (23% moisture content), followed by osmotic rehydration after thawing. This method allowed a germination of 98% of cryopreserved zygotic embryos.Index terms: Coffea arabica L.; ex situ conservation; embryos dehydration; long-term storage; gene bank. RESUMOA conservação dos recursos genéticos vegetais é uma medida importante para prevenção do processo de erosão genética. Bancos de sementes são as formas mais comuns de conservação ex situ, entretanto, sementes de cafeeiro não podem ser armazenadas por métodos convencionais. A criopreservação é uma alternativa viável para conservação em longo prazo de espécies, como o cafeeiro, que produzem sementes intermediárias ou recalcitrantes. O objetivo deste trabalho foi criopreservar embriões zigóticos de Coffea arabica L. cv Catuaí Vermelho avaliando o efeito da desidratação antes da criopreservação, e da reidratação osmótica após o descongelamento. Antes da criopreservação foram testados e determinados os teores de umidade em diferentes tempos de desidratação (0, 15, 30, 60 e 120 min). Após a desidratação os embriões foram criopreservados por uma hora em nitrogênio líquido, e após o descongelamento foram reidratados utilizando solução osmótica. Para avaliação da viabilidade de embriões desidratados e/ou criopreservados, utilizou-se o teste com o cloreto de 2,3,5-trifenil tetrazólio. Embriões não desidratados não sobreviveram ao congelamento. Embriões desidratados até...

Etapas del protocolo de crioconservación y su efecto sobre la viabilidad y regeneración de embriones cigóticos de vid (Vitis vinífera L.)

“…La regeneración de plántulas a partir de los embriones expuestos a las distintas etapas del protocolo (tratamientos) de crioconservación se puede observar en la Figura 4, dónde se muestra que el control mostró el crecimiento óptimo de plántulas (RP: 100 %), mientras que en el tratamiento con NL el crecimiento fue casi nulo (RP: 4 %), lo cual puede rela- prueba de TTC no son totalmente indicativos de la viabilidad final del tejido, ya que las enzimas encargadas de metabolizar el formazán pueden aún estar activas en células muertas (Nausch y Buyel, 2021), no obstante, este análisis puede ser de gran utilidad como primer acercamiento para discernir entre tejidos con potencial de germinación y regeneración. La prueba de viabilidad más confiable es sin duda la regeneración de los tejidos después del tratamiento (Pinto et al, 2016). Es importante destacar que la prueba de TTC se realizó inmediatamente después de cada uno de los tratamientos, en cambio, la regeneración de los embriones a plántulas se obtiene 15 d después de que el tejido es mantenido en cultivo de tejidos in vitro en medio de regeneración, por lo que en este periodo los embriones pudieron tener tiempo para poder recuperarse de alguna alteración debido a los tratamientos de crioconservación.…”

Advances in Plant Breeding Strategies: Nut and Beverage Crops

“…Recently, Coelho et al (2017) suggested that rapid drying to 20% moisture content (dry basis), followed by direct immersion in liquid nitrogen and later reheating in a water bath for 2 minutes allowed effective cryopreservation of coffee seeds. Pinto et al (2016) developed a protocol of cryopreserving coffee zygotic embryos using physical dehydration in silica gel, followed by osmotic rehydration after thawing. In another study, De Freitas et al (2016) demonstrated the successful cryopreservation and germination of zygotic embryos of Coffea arabica by immersing the zygotic embryos in plant vitrification solution.…”

Cryopreservation of Vasconcellea quercifolia St.-Hil (Caricaceae) Zygotic Embryos

“…So, for the successful of cryopreservation, it is essential that the plant material has been dehydration prior cryopreservation. A way to efficiently reduce the water content in the plant material is with the use of the silica gel dehydration that can be used for different material to physical desiccation (Pinto et al, 2016;Pérez-Rodríguez et al, 2017;Carmona-Martín et al, 2018). Since the silica gel technique is simple it becomes more reproducible.…”