Cryopreservation of Clonal Crops: a Review of Key Parameters

For a cryopreservation protocol to be successful or to provide optimal results, experimental conditions for each of its successive steps must be optimized. This paper presents some of the parameters, which have to be considered during the establishment of a cryopreservation protocol for clonal crop species. A prerequisite to the development of a cryopreservation protocol for any plant species is the availability of efficient in vitro culture protocols. Relevant in vitro protocols including micropropagation, shoot tip and in some cases meristem culture have been developed for numerous clonal crops. The physiological state of in vitro mother-plants needs to be optimized. In case of temperate species, a cold acclimation period, which triggers cold adaptation mechanisms, is often beneficial. For tropical, cold-sensitive species, this cold acclimation period can be efficiently replaced by a culture period on medium with high sucrose concentration. In order to produce large numbers of homogeneous and actively growing apices, in vitro plantlets can be cut into single node microcuttings, from which shoot tips are excised after a determined period. The composition of the recovery medium for excised shoot tips has to be optimized. Of particular importance is the hormonal composition of the recovery medium. Accessions differing in their in vitro reactivity should be included in all experiments to observe the effect of this parameter on their response to cryopreservation. Among the various techniques available, encapsulation-dehydration and droplet-vitrification are particularly well adapted for cryopreserving shoot tips. The recently developed D-cryoplate and V-cryoplate techniques should also be considered due to their high efficiency and operational simplicity. Various parameters should be taken into consideration for the selection of the optimal technique(s), including notably the type of explants, their sensitivity to cryoprotectants and the environmental conditions. A series of alternative loading and vitrification solutions have been developed recently, which can be efficiently used when the classical solutions prove to be toxic or do not afford the dehydration and desired cryopreservation tolerance. In conclusion, the development of cryopreservation protocols for clonal crops and their large scale application can be expected in a foreseeable future, thanks to the availability of relevant in vitro culture and highly efficient cryopreservation techniques. INTRODUCTION The number of publications on plant cryopreservation has increased steadily since the beginning of the 90s from around 10 in 1994 to around 70 in 2012. This is due to the development of two vitrification-based techniques, the so-called vitrification technique established by the late Professor Sakai (Sakai et al., 1990) and the encapsulationdehydration developed by the late Professor Dereuddre (Dereuddre et al., 1990). A number of vitrification-based techniques have been developed since that time, including vitrification, encapsulation-dehydration, pregrowth-desiccation, encapsulationvitrification, droplet-vitrification and use of cryoplate. Logging on the Web of knowledge and typing the name of the technique and plant as topics provides a more precise idea of Proc. II IS on Plant Cryopreservation