Ex Situ Conservation of Australian Citrus Species: Investigations on Seed Biology, Cryopreservation and in Vitro Culture

Abstract

Many potentially economically important taxa of Australia are threatened in situ and are vulnerable to erosion of genetic diversity and extinction. In this study, over one hundred rare and threatened Queensland edible plants and/or crop wild relatives were identified. Many of these species have subtropical to tropical distribution and may have non-orthodox seed storage behaviour, thus excluding standard seed banking approaches for long-term ex situ conservation. There is an urgent need to develop alternative ex situ conservation strategies to conserve this diversity. Establishment of ex-situ collections of this valuable germplasm in field collections would be prohibitive in cost and would be susceptible to environmental damage, including disease and pest attack. In vitro and cryopreservation techniques offer alternative strategies for medium and long-term storage of germplasm. However, there have been very few attempts to apply in vitro storage and cryopreservation techniques to any wild Australian tropical or subtropical species. Moreover, limitations exist for the development of alternative ex situ storage techniques due to a lack of basic research on plant ecology or biology, including seed physiology and morphology. Further restrictions to the development of ex situ conservation of these species occurs because of a lack of supporting techniques needed for cryostorage, such as in vitro culture, germination protocols, propagation and acclimation. The Australian wild species of the Citrus genus are a priority for investigation of ex situ conservation strategies because of their conservation priority, potential socioeconomic importance (e.g. novel genes and fruits), probable non-orthodox seed storage behaviour and lack of corresponding techniques for their long term ex situ conservation. This study reports on seed biology, cryopreservation and in vitro culture of three Australian wild Citrus species, C. australasica (finger lime), C. inodora (Russell River lime) and C. garrawayi (Mount White lime), to facilitate germplasm storage and as a regeneration system. Descriptors of mature seed morphology and anatomy are reported in C. australasica, C. inodora and C. garrawayi - vital to the development and application of effective seed storage protocols (i.e. seed lot quality). C. garrawayi seed shape and seed coat morphology was found to be different to C. australasica and C. inodora. In addition, seed topography, as viewed by scanning electron microscopy, was found to be a useful tool for taxa identification. In terms of both germination and seedling growth, temperatures of _20ºC were found to be sub-optimal for germination. Germination from seeds of all the three species was optimal at 30ºC and above 80%. Both C. australasica and C. inodora displayed ‘essentially’ orthodox seed storage behaviour, i.e. desiccation and cryopreservation tolerance, whilst C. garrawayi displayed more complex seed storage behaviour. Overall, it appears that seed banking of germplasm of these species could be by standard orthodox protocols (i.e. 5%MC at -20ºC). However, because of variation in seed responses and other storage constraints in these species, cryopreservation is recommended as the safest storage option to prevent seed deterioration (loss of viability). Cryobiology studies have allowed the determination of the unfrozen water content (WCu) of C .australasica (11%), C. inodora (est. 8%MC) and C. garrawayi (14%). Desiccation of seeds to well below the WCu resulted in high levels of germination (radicle emergence <1mm) after liquid nitrogen exposure, but negligible levels of germination were observed from seeds above the WCu. This data both supports other studies undertaken on cultivated citrus and provides evidence that seed of these species will tolerate cryostorage when below the WCu. In addition, this study demonstrated in vitro culture systems for the micropropagation and medium term storage of C. australasica, C. inodora and C. garrawayi, as well as shoot-tip cryopreservation in C. australasica using a vitrification- based method. In vitro embryogenic potential, using a range of culture media, was low to moderate in C inodora and low in C. australasica, whilst C. garrawayi was recalcitrant to in vitro embryogenesis. The addition of citric acid to the embryo induction medium resulted in the best quality and highest number of somatic embryos from callus proliferated through liquid culture in C. inodora. This is the first report of the promotive affect of citric acid on embryo formation in the Citrus genus. Cryopreservation of encapsulated C. inodora embryogenic callus gave high levels of recovery (69%). However, further optimisation of embryo formation and plantlet recovery is needed to improve efficiency to be suitable conservation purposes. Micropropagation provides a useful tool, for medium-term storage of rare and threatened germplasm and offers a valuable step in the implementation of horticultural and restoration programs. Establishment of an in vitro culture system for shoot-tips also provides a technique for producing virus free material for germplasm exchange or maintenance. The findings of this study facilitate the development of ex situ conservation of Australian wild Citrus, which is of significant interest to complement in situ conservation and secure sustainable access to this rich biodiversity.