Cyropreservation of embryogenic calli of cassava using sucrose cryoprotection and air desiccation

A simplified technique which simultaneously induces and cryoprotects embryogenic calli using sucrose followed by dehydration was developed for the cryopreservation of cassava genetic resources. An initial experiment to optimise the sucrose concentration needed for both embryo production and cryoprotection showed that higher concentrations of sucrose-between 0.4 M and 0.5 M-significantly reduced the viability as well as the number of embryos produced by the embryogenic clumps in the absence of freezing. Post-thaw viability as well as embryogenic competence of clumps depended on the percentage moisture lost, duration of exposure to higher sucrose concentrations and the duration of induction of embryogenic clumps. Extending the period of cryoprotection to 21 days coupled with increased moisture loss (greater than 75%) significantly increased both post-thaw viability and the embryogenic competence of cryopreserved clumps to 95%, while reducing the duration decreased post-thaw viability. Cryopreserved callus clumps developed secondary and cyclic embryos similar to those of the non-cryopreserved controls. The optimised protocol was successfully applied to SM1-2075-1 Line 1 somatic embryos. The rate of plant recovery from cryopreserved embryos of both TME 9 and SM1-2075-1 Line 1 was comparable to that of the non-cryopreserved embryos. Successful cryopreservation of embryogenic clumps of cassava can be used to establish in vitro genebanks for long-term conservation of cassava genetic resources to complement field genebanks and other in vitro methods already being used.