Development of strategies towards the cryopreservation of germplasm of Ekebergia capensis Sparrm. : an indigenous species that produces recalcitrant seeds.
The conservation of germplasm of indigenous plant species is vital not only to preserve valuable genotypes, but also the diversity represented by the gene pool. A complicating factor, however, is that a considerable number of species of tropical and sub-tropical origin produce recalcitrant or otherwise non-orthodox seeds. Such seeds are hydrated and metabolically active when shed and cannot be stored under conventional conditions of low temperature and low relative humidity. This poses major problems for the longterm conservation of the genetic resources of such species. Presently, the only strategy available for the long-term conservation of species that produce recalcitrant seeds is cryopreservation. Ekebergia capensis is one such indigenous species that produces recalcitrant seeds. The aim of the present study was to develop methods for the cryopreservation of germplasm of this species. Different explant types were investigated for this purpose, viz. embryonic axes (with attached cotyledonary segments) excised from seeds, and two in vitro-derived explants, i.e. ‘broken’ buds excised from in vitro-germinated seedlings and adventitious shoots generated from intact in vitro-germinated roots. Suitable micropropagation protocols were developed for all explant types prior to any other experimentation. Before explants could be cryopreserved it was necessary to reduce their water content in order to limit damaging ice crystallisation upon cooling. All explants tolerated dehydration (by flash drying) to 0.46 – 0.39 g gˉ¹ water content (dry mass basis) with survival ranging from 100 – 80%, depending on the explant. In addition, penetrating and non-penetrating cryoprotectants were used to improve cryo-tolerance of explants. The cryoprotectants tested were sucrose, glycerol, DMSO and a combination of sucrose and glycerol. Explant survival following cryoprotection and dehydration ranged from 100 – 20%. Cryoprotected and dehydrated explants were exposed to cryogenic temperatures by cooling at different rates, since this factor is also known to affect the success of a cryopreservation protocol. The results showed that ‘broken’ buds could not tolerate cryogen exposure. This was likely to have been a consequence of the large size of explants and their originally highly hydrated condition. Adventitious shoots tolerated cryogenic exposure slightly better with 7 – 20% survival after cooling in sub-cooled nitrogen. Limited shoot production (up to 10%) was obtained when axes with attached cotyledonary segments were exposed to cryogenic temperatures. In contrast, root production from axes cooled in sub-cooled nitrogen remained high (67 – 87%). Adventitious shoots were subsequently induced on roots generated from cryopreserved axes by applying a protocol developed to generate adventitious shoots on in vitrogerminated roots. In this manner, the goal of seedling establishment from cryopreserved axes was attained. Each stage of a cryopreservation protocol imposes stresses that may limit success. To gain a better understanding of these processes the basis of damage was investigated by assessing the extracellular production of the reactive oxygen species (superoxide) at each stage of the protocol, as current thinking is that this is a primary stress or injury response. The results suggested that superoxide could not be identified as the ROS responsible for lack of onwards development during the cryopreparative stages or following cryogen exposure. The stresses imposed by the various stages of a cryopreservation protocol may affect the integrity of germplasm. Since the aim of a conservation programme is to maintain genetic (and epigenetic) integrity of stored germplasm, it is essential to ascertain whether this has been achieved. Thus, explants (axes with cotyledonary segments and adventitious shoots) were subjected to each stage of the cryopreservation protocol and the epigenetic integrity was assessed by coupled restriction enzyme digestion and random amplification of DNA. The results revealed little, if any, DNA methylation changes in response to the cryopreparative stages or following cryogen exposure. Overall, the results of this study provided a better understanding of the responses of germplasm of E. capensis to the stresses of a cryopreservation protocol and two explant types were successfully cryopreserved. Future work can be directed towards elucidating the basis of damage incurred so that more effective protocols can be developed. Assessment of the integrity of DNA will give an indication as to the suitability of developed protocols, or where changes should be made to preserve the genetic (and epigenetic) integrity of germplasm.