A simple and rapid method for determining cell survival in the cryopreserved shoot apex using luciferin–luciferase ATP assay

• Published in: Plant science (Limerick) Vol. 130; no. 1; pp. 107 - 112
• Main Authors: Sugawara, Yasutake, Takeuchi, Masayuki
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier Ireland Ltd 05-12-1997; Elsevier Science
• Subjects: Agronomy. Soil science and plant productions; ATP assay; Biological and medical sciences; Cryopreservation; Fundamental and applied biological sciences. Psychology; Generalities. Genetics. Plant material; Genetic resources, diversity; Genetics and breeding of economic plants; Luciferin-luciferase; Pisum sativum; Plant material; Shoot apex; Survival determination; DMSO, dimethylsulfoxide; Luciferin-luciferase; Cryopreservation; Pisum sativum; Shoot apex; NAA, α-naphthaleneacetic acid; Survival determination; ATP assay; ATP, adenosine 5′-triphosphate; BAP, 6-benzylaminopurine; Enzyme; Luciferase; Survival; Grain legume; Leguminosae; Luciferin; Investigation method; Dicotyledones; Angiospermae; Spermatophyta; Oxidoreductases; ATP; Apex
• ISSN: 0168-9452; 1873-2259
• DOI: 10.1016/S0168-9452(97)00226-4

A new method was developed to estimate the survival rate of cells in the shoot apex using an ATP assay with luciferin–luciferase. Shoot apices isolated as uniformly in size as possible from germinated pea seedlings were frozen after treatment with cryoprotectant to temperatures as low as −50°C and then transferred to liquid nitrogen temperature (−196°C) by a two-step freezing method. The frozen-thawed or unfrozen control shoot apices were washed with fresh medium and an individual shoot apex was suspended in a small volume of distilled water and then heated for 1.5 min on a boiling water bath. To release ATP more completely, the heated shoot apex was rapidly frozen in liquid nitrogen and thawed at room temperature. The content of released ATP was determined with the luciferin-luciferase assay. The percentage of cells in the pea shoot apex which survived freezing and thawing under present conditions was found to be 10% as estimated by this method.