Stabilisation of the Biosensor Properties of Protoplasts Used as the Biological Units of the Protoplast Biosensor

Abstract

The aim of this study was to develop a method to advance the applicability of the biosensor under field conditions. The sunflower (Helianthus annuus) was used, as it reached higher yields of vital mesophyll protoplasts than the broad bean (Vicia faba). A freezing method was developed, which prolonged the time over which the protoplasts could be used as biological units of the biosensor. Acclimating sunflower plants to cold condi-tions led to an increased preservation of the biosensor properties of the biological units after freezing. Another important step in optimising the freezing process was the develop-ment of the freezing medium. The developed protocols for H. annuus plant growth and protoplast freezing led to a clearly measurable respiration after freezing them in 10% (v/v) glycerol solution over a period of three months. In addition to giving evidence of respiration inhibiting substances (KCN and SHAM), the protoplast biosensor can also indicate the site- and mode-of-action of substances, as demonstrated by using Fluazinam, a substance utilised in agriculture. It is expected that the freezing protocol can be optimised further by using evacuolated protoplasts (MPP), which have a higher mechanical stability than normal protoplasts. First results are presented in this study. The protocols developed in this study contribute substantially to stabilising the biosensor properties of the protoplasts as the biological units of the biosensor over longer time periods. The biological units are, consequently, made available for use at any time, presenting a clear progress towards employing the protoplast biosensor routinely in the field.