We have been working with recalcitrant seeds since the early 1980s, and have made a significant contribution to answering fundamental questions about the nature of seed recalcitrance, for example; why these seeds will die even if kept hydrated; the responses to sub-lethal desiccation; the characteristics of the lethal lesions; biophysical characteristics of the tissue water at various hydration levels; effects of drying rate on the proportion of water that can be 'safely' lost; and the nature, effects and minimisation or elimination of the ubiquitous seed-associated fungi. In regard to drying rate, we ascertained that if water is removed slowly (over many hours to days), seeds will die at a very high water concentration (~0.8 g/g), whereas the more rapidly water can be removed, the greater is the proportion of water loss tolerated (in the short term). This finding has been of the greatest significance, as it facilitated our developing a process we call flash-drying (dehydration in 15 - 120 minutes, depending on the species), which is intrinsic to the success of embryonic axis cryopreservation (whole seeds generally being far too large).

The understanding emanating from our basic research has led us, firstly, to being able to extend hydrated storage life, and secondly and more significantly, to develop methods that have the potential to store the genetic resources of species producing recalcitrant seeds. This is through cryopreservation, the process by which part (rarely all) of the seed is stored cryogenically - usually in liquid nitrogen (LN) at -196ºC - theoretically indefinitely. Generally, it is the embryonic axis (shoot-root continuum of the embryo) which is excised and cryostored, as it effectively has the potential to form a new plant. In-depth studies in our laboratory have assessed the effects of various cooling (freezing) rates of flash-dried axes, and have developed the means of achieving cooling at rates ≥500°C per second.

Our recent research has revealed that the stage of axis development prior to excision is pivotal to successful cryostorage. Equally important is the manner of rehydrating axes: we have found that rehydration in water (as conventionally practiced) may underlie the failure of an axis to develop normally into a seedling, because starch synthesis is extensively compromised. In our experiments we first rationalised the use of a calcium/magnesium solution for rehydration, which, for some species, has resulted in normal seedling establishment. Most recently, we have employed the cathodic fraction of electrolysed Ca/Mg solution, with far greater success. This is attributed to amelioration of the consequence of ROS-mediated damage by the cathodic water.

In the studies outlined above (as well as all others), success with a particular manipulation has emerged because of our highly analytical approach, examining microscopically, biochemically, biophysically and physiologically as well as by germination and seedling establishment, effects of approaches that succeed and of those that fail.

As seeds of unknown characteristics are acquired, they are subjected to a methodical screening protocol, enabling us to categorise them as orthodox or not. Those that emerge as non-orthodox are more finely characterised and intensive research is undertaken to develop a cryostorage protocol for the embryonic axes. Currently, development of such protocols proceeds on an empirical basis; however, our analyses at every stage of the procedure will ultimately result in the understanding necessary to facilitate generalised guidelines for the various axis types.

For any species:

· Initially, in vitro procedures for producing plants from excised axes must be developed which must be preceded by ascertaining the parameters for surface-sterilisation and systemic fungicide application (to remove external and internal seed-associated fungi, which thrive and over-run axes on culture medium);

· the drying characteristics (how rapidly water can be removed), and the water concentration at which vigour and viability are adversely affected must be determined. Axis responses to different water concentrations are also assessed physiologically, microscopically and biochemically, as appropriate;

· prior to cryopreservation flash drying procedures have to be optimised; the least amount of drying that permits survival of rapid cooling needs to be established;

· for cryopreservation, the effects of cooling rates need to be ascertained, as there are some species for which slower, or more rapid, cooling than at 500°C are optimal. Again, analyses are conducted as outlined for drying.

· finally, thawing and rehydration responses need to be analysed and optimised;

· only after all the procedures have been optimised, can one be relatively sure of success;

· additionally, the vigour of young plants produced from axes subjected to cryopreservation has to be assessed to determine any special requirements that may be necessary for re-introduction programmes.

Another necessity is to ensure that the genetic integrity of the axes has not been compromised by the various manipulations: this is ascertained not only by seemingly phenotypically-normal plant production, but also by DNA analyses, particularly for any epigenetic changes.

There are cases where the embryonic axis is too large to be cryopreserved, as this procedure requires very small specimens to ensure rapid dehydration and cooling (freezing). For such species, alternative explants need to be used: these could be buds derived from shoots developed from axes germinated in vitro (as sterility is of fundamental importance), shoot apical meristems or somatic embryos. For this type of explant, often an additional procedural step needs to be assessed and introduced, viz. application of a cryoprotectant solution(s).

We are also currently exploring the production of synthetic seeds (synseeds) where we alginate-encapsulate axes (or other explants) recovered after cryostorage. The object of synseed production is to facilitate dissemination of material for planting in such a way that a stasis is imposed on ongoing axis germination, so allowing transport to distant sites. Our aim is that synseeds should be easy to handle and be able to be planted directly into soil, without an intervening germination stage in vitro.