Abstract
The possibility of streamlining general cryoprotection procedures is investigated using aluminium cryoplates with wells, facilitating fluid handling and drying treatment. Precultured shoot tips of potato cultivar ‘Sayaka’ were embedded in calcium alginate gel in wells of the aluminium cryo-plates. In the V cryo-plate protocol, dehydration was performed for 30 min at 25 °C in plant vitrification solution after osmoprotection. In the D cryo-plate protocol, dehydration was performed by placing the cryo-plates for 2.0 h under an air current in a laminar flow cabinet after osmoprotection. In both protocols, cooling was performed by placing the cryo-plates in uncapped cryotubes, which were immersed in liquid nitrogen. For rewarming, the cryo-plates were immersed in liquid MS medium supplemented with 1.0 M sucrose and diluted for 15 min at room temperature. Under these conditions, regrowth rates of cryopreserved shoot tips in V cryo-plate and D cryo-plate were 96.7 and 93.3 %, respectively. Both protocols will facilitate efficient strategies for preservation, storage, and maintenance of genetic stability of important potato germplasm.
Resumen
Se investigá la posibilidad de realizer procedimientos generales de crioprotección mediante el uso de crioplacas de aluminio con cavidades, facilitando el manejo fluido y tratamiento de secado. Se colocaron ápices de tallo de papa pre-cultivados, de la variedad “Sayaca” en crio placas de aluminio y se embebieron en de alginato de calcio. En el protocolo de crio placa -V se hizo la deshidratación por 30 min a 25 °C en una solución de vitrificación de planta después de osmoprotección. En el protocolo de crio placa -D, la deshidratación se hizo colocándolas por 2 h bajo una corriente de aire en una cámara de flujo laminar después de la osmoprotección. En ambos protocolos, el enfriamiento se realizó mediante la colocación de las crio placas en crio tubos sin tapa, que fueron inmersos en nitrógeno líquido. Para la secuperacid se sumergieron las crio placas en un medio líquido MS suplementado con 1.0 M de sacarosa en dilución por 15 min a temperatura ambiente. Bajo estas condiciones, los niveles de secuperacion de los ápices de tallo criopreservados en crio placas -V y -D fueron de 96.7 y 93.3 %, respectivamente. Ambos protocolos facilitarán las estrategias eficientes para preservación, almacenamiento, mantenimiento y estabilidad genética de germoplasma de importacia de accesiones de papa.
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Acknowledgments
We are grateful to Dr. B. A. Antonio for critical reading the manuscript and to Ms. N. Nohara, Ms. A. Nishiuchi, Ms. N. Ishikura and Ms. Y. Sato for their excellent technical assistance. This work was mainly supported by a grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics-based Technology for Agricultural Improvement, CRS-1001, CRS-1002) and was partially supported by JST/JICA, SATREPS: Diversity Assessment and Development of Sustainable Use of Mexican Genetic Resources.
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Yamamoto, Si., Wunna, Rafique, T. et al. The Aluminum Cryo-plate Increases Efficiency of Cryopreservation Protocols for Potato Shoot Tips. Am. J. Potato Res. 92, 250–257 (2015). https://doi.org/10.1007/s12230-014-9425-5
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DOI: https://doi.org/10.1007/s12230-014-9425-5