Abstract
As a consequence of the difficulty in conventional coffee seed storage, biotechnological alternatives such as cryopreservation have been investigated. The objective of this study was to develop a protocol for the cryopreservation of Coffea arabica L. (cv. ‘Catuaà Vermelho’ - IAC 144) zygotic embryos by vitrification. For the cryopreservation study, the embryos were immersed in Plant Vitrification Solution 2 at different times (0, 10, 25, 50, 100, and 250 min) and two temperatures (0 and 25 °C). Subsequently, the best thawing time was determined in a water bath (1, 3, 5 minutes or directly in Recovery Solution). An anatomical study was conducted on non-stored and stored embryos, with or without the use of Plant Vitrification Solution 2. The immersion in cryoprotectant solution for 100 min at 0 °C allows embryo cryopreservation. Embryos can be directly thawed in Recovery Solution after storage in liquid nitrogen. It was observed that Plant Vitrification Solution 2 reduced internal water content in the cells, allowing subsequent embryo growth resumption.
Highlights
Brazil is the largest producer and exporter of coffee, which is the fifth most exported agricultural product (Conab, 2016)
The low viability in tetrazolium salt and the nongermination of embryos treated with Plant Vitrification Solution 2 (PVS2) at immersion times higher than 25 minutes at 25 °C show that, under these conditions, the cryoprotectant was toxic to embryos
Similar results were obtained by (Lambardi et al, 2000) in the vitrification of Populus alba L. apical buds treated with PVS2 solution for a time period of over 20 minutes at 25 °C or above 60 minutes at 0 °C, which showed clear damage symptoms caused by toxicity or osmotic stress, such as bleaching or lack of growth recovery
Summary
Brazil is the largest producer and exporter of coffee, which is the fifth most exported agricultural product (Conab, 2016). PVS2 showed 100% germination and 100% formation of normal seedlings, regardless of their immersion time in the cryoprotectant solution In treatments in which the embryos remained in PVS2 for longer than 25 minutes, germination or normal seedlings did not occur, and the embryos showed reduced viability in tetrazolium salt (Fig. 1A).
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