AbstractsBiology & Animal Science

Submitted in fulfilment of the requirements for the degree of Master of Applied Sciences in Biotechnology, Durban University of Technology, Durban, South Africa, 2015. In contrast to orthodox seeds, recalcitrant seeds are short-lived, shed at relatively high water contents (WCs), and are desiccation sensitive. Presently, the only option for long-term conservation of genetic resources of such plant species is by cryostorage in liquid nitrogen (LN; -196°C) or in the vapour phase over LN (at -150⁰C to -160⁰C). A number of cryopreservation protocols applied for recalcitrant zygotic embryos or embryonic axes of tropical/sub-tropical species have reported survival as either root or shoot development or callus formation, with no shoot or root production after cryopreservation. This is a consequence of the challenges encountered when optimising the WC for successful cryopreservation across species. Other shortcomings may also be the formation of ice or the sensitivity to desiccation resulting in lethal damage or poor re-growth. However, for successful cryopreservation, a normal plantlet with a shoot and a root needs to be obtained post-cryo. Specimens required for successful cryopreservation must be small; therefore embryonic axes excised from seeds have been often used as the explants of choice. However, in some cases, excised embryonic axes of mature recalcitrant seeds are too large to be cryopreserved, or, even if small, may be adversely affected by excision, dehydration and/or immersion in LN, thus failing to produce plantlets after cryopreservation. As a result, in such cases, there is a need to develop explants alternative to zygotic axes such as buds derived from in vitro shoots, shoot meristems, or shoot apices and somatic embryos. These alternative explants must have a high capacity for plantlet formation before and after cryopreservation. The present study aimed to successfully cryopreserve shoot apices of Trichilia emetica and T. dregeana, tropical recalcitrant-seeded tree species, and monitor the responses or effects of some of the procedural steps involved in cryopreservation on the survival and shoot production from these shoot apices. The main foci of the investigation were to produce vigorous plantlets after cryopreservation and ultimately develop a protocol for the successful cryopreservation of germplasm of these species. Furthermore, this study reports on a number of factors that may affect survival after cryopreservation, viz. WC of the explants, PVS2 treatment, production of reactive oxygen species (ROS) and levels of endogenous total aqueous antioxidants (TAA) during the various steps of cryopreservation. The effects of the various steps of cryopreservation on the ultra-structure of the shoot apices were also observed. Cathodic protection (by using highly reducing cathodic water; CW) of the explants was attempted to improve vigour and shoot production from the surviving shoot apices after cryopreservation as cathodic water has been reported to ameliorate the excessive burst…