Low temperature storage of plant tissue cultures

Abstract

Plant tissue culture is a technique with massive potential for exploitation in the fields of plant physiology, horticulture and agriculture, somatic cell genetics, secondary metabolite production and genetic conservation. Important stock cultures for all of these applications can be maintained by serial subculture. However, to ensure the cessation of growth and therefore prevention of genetic change, it is necessary to store the stocks at ultra-low temperatures. Conveying plant material to and from the storage temperature (normally at or near — 196 °C) requires specific precautions to prevent damage. Cryoprotective compounds must normally be applied before freezing at a chosen slow or rapid rate. Thawing is usually carried out rapidly. Cryoinjury is thought to be due to a number of factors including intracellular ice formation and growth, ‘solution effects’ resulting from increased intra-cellular solute levels, and deplasmolysis stress after thawing. The condition of a frozen and thawed specimen can be ascertained by viability tests, ultrastructural examination and a capacity for regrowth. Modification of the culture conditions in the pregrowth period before preservation and in the post-thaw phase can increase survival potential greatly. In this article, the current stage of expertise in the cryopreservation of cultured plant tissues is assessed and important areas for emphasis in future work are indicated. Protocols are offered for the preservation of specimens including suspension cultures, callus, somatic embryos and meristems. Limitations to the use of freeze-preservation for genome storage are discussed, with an evaluation of alternative storage methods.