Cryopreservation in Coffea canephora Pierre seeds: Slow and fast cooling

ABSTRACT Coffee is one of the main agricultural commodities in the country, and it is important to conservation of plant material for breeding programs. Cryopreservation is a promising alternative for preserving in the long-term the germplasm of species considered recalcitrant. However, studies should be performed to achieve maximum survival of seedlings after immersion in liquid nitrogen. The objective of this work was to find a cryopreservation protocol for storing seeds of Coffea canephora, studying two methods of cryopreservation, slow and fast cooling. Seeds were subjected to drying in silica gel up to the water content of 0.25 g g-1. In the first experiment, dried seeds were subjected to treatments of slow cooling at speeds of -1 ºC min-1,-3 ºC min-1 and -5 ºC min-1 until the end temperatures of -40 ºC, -50 ºC and -60 ºC, by means of a bio freezer and subsequently immersed in liquid nitrogen. In the second experiment, the best result was selected of the first experiment and compared with the rapid cooling, in which dried seeds, with 0.25 g g-1 of water content, were immersed directly into liquid nitrogen. Physiological and biochemical alterations occurring in the seeds after cryopreservation were evaluated. Coffea canephora seeds respond better to cryopreservation by rapid cooling, when compared to slow cooling. Drying, one of the cryopreservation steps does not affect the viability of Coffea canephora Pierre seeds, when these seeds are dried to 0.25 g g-1 of water content. Catalase and esterase enzymes are good biochemical markers for cryopreserved coffee seeds and their activity is greater in larger seed physiological quality.摘要 咖啡是该国主要农业大宗商品之一，对于育种项目的植物材料保存而言至关重要。超低温保存（cryopreservation）是实现顽拗型物种种质资源长期保存的极具潜力的方案，但仍需开展相关研究以实现液氮浸没后幼苗存活率的最大化。本研究旨在开发一套适用于刚果咖啡（Coffea canephora）种子的超低温保存规程，对比评估慢速降温与快速降温两种超低温保存方法。实验将种子置于硅胶干燥剂中干燥至含水量为0.25 g·g⁻¹。第一组实验中，经干燥处理的种子通过生物冷冻仪以-1 ℃·min⁻¹、-3 ℃·min⁻¹和-5 ℃·min⁻¹的降温速率分别降温至-40 ℃、-50 ℃和-60 ℃，随后浸没于液氮中。第二组实验选取第一组实验的最优结果，与快速降温法进行对照：将含水量为0.25 g·g⁻¹的干燥种子直接浸没于液氮中。本研究同时评估了超低温保存后种子发生的生理与生化变化。结果显示，相较于慢速降温法，刚果咖啡种子通过快速降温法进行超低温保存的效果更佳。当将种子干燥至含水量0.25 g·g⁻¹时，作为超低温保存步骤之一的干燥处理并不会影响刚果咖啡（Coffea canephora Pierre）种子的生活力。过氧化氢酶与酯酶可作为咖啡超低温保存种子的优良生化标记物，且种子生理品质越高，其酶活性越强。