Some investigations of the responses of Quercus robur and Ekebergia capensis embryonic axes to dehydration and cryopreservation.

Abstract

Recalcitrant seeds are those that are shed at high water contents, are actively

metabolic throughout development, when they are, and remain, desiccation-sensitive,

and may also be chilling sensitive. These properties preclude their

conventional storage. Because recalcitrant seeds lose viability rapidly (within a

few days to several months depending on the species) the long-term storage of

their germplasm is achievable only by cryopreservation [i.e. storage at very low

temperatures, generally in or over, liquid nitrogen at -196°C or -150°C,

respectively. Generally the seeds are far too large to be cryostored, thus explants

- most conveniently, excised zygotic embryonic axes - are used. As the axes of

recalcitrant seeds are highly hydrated, specific pre-treatments prior to freezing

have to be applied in order to avoid lethal ice crystal formation.

During the course of this study, cryopreservation protocols were developed for

excised zygotic embryonic axes of two different species (Quercus robur L. and

Ekebergia capensis Sparrm.). Surface-sterilisation regimes were tested for axes

of both species, with the use of a 1% sodium hypochlorite solution containing a

wetting agent, emerging as the best. For both species, the vigour and viability of

axes, assessed by in vitro germination performance, was tested after the

implementation of four different rates of desiccation (achieved by a laminar-airflow;

silica-gel-; flash- and fast flash-drying). The most rapid dehydration rate

(fast flash-drying) facilitated the best germination rates (vigour) for both Q. robur

and E. capensis axes after 240 and 20 min, when water contents were reduced

to 0.37 ±0.04 and 0.39 ±0.06 g g-1 (dmb), respectively.

Consequently, fast flash-drying was used in combination with three different

freezing rates (slow, intermediate and ultra-rapid cooling). While axis viability was

lost after slow or intermediate cooling, good survival was obtained for each

species after ultra-rapid cooling. In addition to the optimisation of culture

conditions, desiccation and freezing rates, the efficacy of different thawing media

(distilled water, mannitol, sucrose, full-strength MS medium supplemented with

sucrose and a 1 µM calcium/1 mM magnesium solution) was also assessed. The

only thawing medium that ensured normal seedling production was the Ca2+Mg2+

solution, in which electrolyte leakage was significantly curtailed.

In addition to vigour and viability assessment the responses of the embryos to

the various manipulations were monitored by light microscopy and/or

transmission electron microscopy. The results of the various manipulations are

discussed in terms of the stresses imposed on the excised axes, by each of the

procedures. For axes of Q. robu, the outcome of the presently developed

successful procedure and two unsuccessful protocols from the published

literature are compared and contrasted.

It is concluded that while in vitro germination media need to be assessed on a

species basis, use of the mildest effective surface-sterilant, in conjunction with

the most rapid means to achieve dehydration and cooling/freezing, are likely to

underlie generally successful cryopreservation. Additionally, thawing parameters

have emerged as being critically important.