Some invetsigations on the responses to desiccation and exposure to cryogenic temperatures of embryonic axes of Landolphia kirkii.
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Date
2011
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Abstract
Landolphia kirkii is scrambling shrub forming an integral part of the flora along the
coastal areas of north-eastern South Africa. The non-sustainable harvesting of fruit as
food source, by monkeys and rural communities and the highly recalcitrant nature of their
seeds threatens the continuation of the species. In addition, the ability of the plants to
produce high quality rubber makes its long-term conservation highly desirable.
Previously, attempts have been made to cryopreserve germplasm of L. kirkii, but no
survival had been recorded at cryogenic temperatures of below -140ºC.
The present study reports on the effects of rapid dehydration, chemical cryoprotectants
and various cooling rates, thawing and imbibition treatments on survival of embryonic
axes excised with cotyledons completely removed, as well as with 3 mm portion of each
cotyledon attached, from fresh, mature, recalcitrant seeds of L. kirkii. Survival was
assessed by the ability for both root and shoot development in in vitro culture, the
tetrazolium test and electrolyte leakage readings.
At seed shedding, embryonic axes were at the high mean water content of 2.24 g gˉ¹ (dry
mass basis). All axes (with and without attached cotyledonary segments) withstood rapid
(flash) drying to a water content of c. 0.28 g gˉ¹; however, the use of chemical
cryoprotectants, singly or in combination, before flash-drying was lethal. Rapid cooling
rates were detrimental to axes flash-dried to 0.28 g gˉ¹, with no explants showing shoot
production after exposure to -196ºC and -210ºC. Ultrastructural examination revealed
that decompartmentation and loss of cellular integrity were associated with viability loss
after rapid cooling to cryogenic temperatures, although lipid bodies retained their
morphology regardless of the thawing temperature employed. Furthermore, analysis of
the lipid composition within embryos of L. kirkii revealed negligible amounts of capric
and lauric acids, suggested to be the medium-chained saturated fatty acids responsible for
triacylglycerol crystallisation when lipid-rich seeds are subjected to cryogenic
temperatures. Hence, lipid crystallisation was not implicated in cell death following
dehydration, exposure to cryogenic temperatures and subsequent thawing and
rehydration. Rapid rehydration of embryonic axes of L. kirkii by direct immersion in a
calcium-magnesium solution at 25ºC for 30 min (as apposed to slow rehydration on
moistened filter paper or with rehydration in water) was associated with highest survival
post-dehydration. Cooling at 1ºC minˉ¹ and 2ºC minˉ¹ facilitated survival of 70 and 75%
respectively of axes with attached cotyledonary segments at 0.28 g gˉ¹ after exposure to -
70ºC. Viability retention of 40 and 45% were recorded when embryonic axes with
attached cotyledonary segments were cooled at 14 and 15ºC minˉ¹ to temperatures below
-180ºC. However, no axes excised without attached cotyledonary segments produced
shoots after cryogenic exposure. The use of slow cooling rates is promising for
cryopreservation of mature axes of L. kirkii, but only when excised with a portion of each
cotyledon left attached.
Description
Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.
Keywords
Apocynaceae., Plant cells and tissues--Cryopreservation., Germplasm resources, Plant--Cryopreservation., Seeds--Preservation., Theses--Botany.