The effect of developmental status and excision injury on the success of cryopreservation of germplasm from non-orthodox seeds.

Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2007.The zygotic germplasm of plant species producing desiccation-sensitive seeds can be conserved in the long-term only by cryopreservation. Usually the embryonic axis is excised from the cotyledons and is used as the explant for cryopreservation as it is small and provides a large surface area:volume ratio. However the shoot of the axis of most species studied does not develop after excision, with the result that survival after cryopreservation is often recorded as callus production or simply explant enlargement and/or greening. Thus, besides explant size, factors such as in vitro regeneration techniques, physical injury induced upon excision and developmental status of the seed could compromise the success of cryopreservation. This study investigated the effect of the factors mentioned above, with particular attention to the developmental status of the seeds on explant in vitro development (section 3.1), response to dehydration (section 3.2) and cryopreservation of the desiccation-sensitive embryonic axes (section 3.3) of two species: Trichilia dregeana, T. emetica and embryos of a third, Strychnos gerrardii. For all three species, investigations were conducted on the embryonic axes/embryos excised from mature seeds immediately after fruit harvesting and from mature seeds stored under hydrated conditions for different periods (in order to achieve different degrees of development). In addition, preliminary studies were carried out on axes of T. dregeana to assess whether generation of reactive oxygen species (ROS) occurs in response to wounding upon axis excision (section 3.4). Excised embryonic axes of T. dregeana and T. emetica did not develop shoots in vitro unless the explants included attached cotyledonary segments. Following the development associated with short-term storage, however, the excised axes could develop shoots after complete cotyledon excision. The embryos from the (endospermous) seeds of S. gerrardii which included the paper-thin cotyledons, developed normally in vitro, with percentage germination increasing with seed storage time. For all three species, in vitro axis germination was promoted when activated charcoal was included in the germination medium, regardless of the developmental stage of the seeds. When dehydrated to approximately 0.3 g H2O g-1 dry mass (g g-1), embryonic axes from all three species failed to develop shoots even though a minimum of 50% produced roots in all cases. Hence, shoot production was shown to be more sensitive to desiccation than was root production. Furthermore, the sensitivity of the shoot apical meristem to desiccation was not ameliorated with seed storage for T. dregeana and T. emetica, but did decrease for S. gerrardii when seeds were stored for 6 – 8 weeks. The application of certain cryoprotectants did facilitate production of shoots after dehydration by a few axes of both Trichilia spp. For T. dregeana explants, combination of glycerol and sucrose allowed for 10% of the axes to retain the ability for shoot production after dehydration while for T. emetica explants, the combination of DMSO and glycerol (10 - 20% shoot production after dehydration) was best. The efficacy of the cryoprotectants was not influenced by storage period. The provision of cryoprotectants still needs to be tested for S. gerrardii. Survival of subsequent cryopreservation of T. dregeana and S. gerrardii explants was best achieved with rapid cooling in nitrogen slush, with the cooling procedure for T. emetica explants still to be optimized. The highest post-cryopreservation survival of T. dregeana axes was achieved when seeds had been stored for three months, while the seed storage period did not affect post-thaw survival of the axes of T. emetica or S. gerrardii. A small proportion of S. gerrardii explants only, produced shoots after cryopreservation, whereas the surviving embryonic axes of T. dregeana and T. emetica regenerated only as non-embryogenic callus. Although callus production is less desirable than successful seedling establishment, it has the potential for micropropagation if embryogenicity can be induced. Ultrastructural examination of the shoot apical meristem of T. dregeana after a 3-d recovery period, following excision, revealed considerable cellular derangement, although damage of individual organelles could not be resolved microscopically. Preliminary studies on T. dregeana involving a colorimetric assay using epinephrine, confirmed the generation of ROS in response to wounding associated with axis excision. Reactive oxygen species generated appeared to persist over prolonged periods rather than occurring only as a single oxidative burst. Hence, ROS production at the wound site could be the primary factor contributing to lack of shoot development. Axes immersed in the anti-oxidant, ascorbic acid (AsA) immediately after excision, showed lower ROS production and 10% shoot development when cultured in vitro, indicating that the oxidative burst coincident with, and after excision might be counteracted if immediate ROS production can be adequately quenched. Future investigations should aim to identify the specific ROS associated with wounding and optimize an anti-oxidant treatment(s) that will facilitate shoot development. Thus, the successful cryopreservation of the germplasm of the species tested, and others producing recalcitrant seeds, depends on a spectrum of species-specific factors, some still to be elucidated