Studies on factors influencing viability after cryopreservation of excised zygotic embryos from recalcitrant seeds of two amaryllid species.
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Date
2010
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Abstract
Recalcitrant unlike orthodox seeds do not show a sharp border between maturation and germination
and remain highly hydrated and desiccation-sensitive at all developmental and post-harvest
stages. In contrast with recalcitrant seeds, orthodox types retain viability for predictably long
periods in the dry state and hence can be stored under low relative humidity and temperature
conditions. Storage of recalcitrant seeds under conditions allowing little to no water loss, at
moderate temperatures, allows for short- to medium-term storage but only facilitates viability
retention for a matter of a few weeks to months, at best, because the seeds are metabolically
active and initiate germination while stored. Cryopreservation, i.e. storage at ultra-low
temperatures (usually in liquid nitrogen [LN] at -196°C), is a promising option for the long-term
germplasm conservation of recalcitrant-seeded species but their seeds present some unavoidable
difficulties in terms of the amenability of their germplasm to cryopreservation. Pre-conditioning
treatments can reduce the amount of ‘free’ water available for freezing and may increase the
chances of cells or tissues surviving exposure to cryogenic temperatures. Such conditioning may
be imposed by physical dehydration or cryoprotection, i.e. exposure to compounds that depress
the kinetic freezing point of water and so reduce the likelihood of lethal ice-crystal formation
during cooling (i.e. exposure to LN at -196°C or sub-cooled LN at -210°C) and subsequent
thawing. Partial dehydration is presently a standard pre-treatment for the cryopreservation of
recalcitrant zygotic germplasm and explant cryoprotection has been shown to improve postthaw
survival in some recalcitrant-seeded species. However, there is a paucity of information on
the physiological and biochemical basis of post-thaw survival or death in recalcitrant seeds, and
this is the major focus of the current contribution. Additionally, in light of the lack of
understanding on how cryo-related stresses imposed at the embryonic stage are translated or
manifested during subsequent seedling growth, this study also investigated the effects of partial
dehydration and the combination of partial dehydration and cooling of recalcitrant zygotic
embryos on subsequent in and ex vitro seedling vigour. All studies were undertaken on the
zygotic embryos of two recalcitrant-seeded members of the Amaryllidaceae, viz. Amaryllis
belladonna (L.) and Haemanthus montanus (Baker); both of which are indigenous to South
Africa.
Studies described in Chapter 2 aimed to interpret the interactive effects of partial
dehydration (rapidly to water contents > and <0.4 g g-1), cryoprotection (with sucrose [Suc; nonpenetrative]
or glycerol [Gly; penetrative]) and cooling rate (rapid and slow) on subsequent
zygotic embryo vigour and viability, using three stress markers: electrolyte leakage (an indicator
of membrane integrity); spectrophotometric assessment of tetrazolium chloride-reduction (an
indicator of respiratory competence); and rate of protein synthesis (an indicator of biochemical
competence). These studies showed that in recalcitrant A. belladonna and H. montanus zygotic
embryos, stresses and lesions, metabolic and physical, induced at each stage of the
cryopreservation protocol appear to be compounded, thus pre-disposing the tissues to further
damage and/or viability loss with the progression of each step. Maximum post-thaw viability
retention in both species appeared to be based on the balance between desiccation damage and
freezing stress, and the mitigation of both of these via Gly cryoprotection. Post-thaw viabilities
in both species were best when Gly cryoprotected + partially dried zygotic embryos were
rapidly, as opposed to slowly, cooled. However, the rate at which water could be removed
during rapid drying was higher in A. belladonna and this may explain why the optimum water
content range for post-thaw survival was <0.40 g g-¹ for A. belladonna and >0.40 g g-¹ for H.
montanus. These results suggest that to optimise cryopreservation protocols for recalcitrant
zygotic germplasm, attention must be paid to pre-cooling dehydration stress, which appears to
be the product of both the ‘intensity’ and ‘duration’ of the stress.
Cryoprotection and dehydration increased the chances of post-thaw survival in A.
belladonna and H. montanus zygotic embryos. However, transmission electron microscopy
studies on the root meristematic cells from the radicals of these embryos (described in Chapter
3) suggest that their practical benefits appear to have been realised only when damage to the
sub-cellular matrix was minimised: when (a) pre-conditioning involved the combination of
cryoprotection and partial dehydration; (b) the cryoprotectant was penetrating (Gly) as opposed
to non-penetrating (Suc); and (c) embryos were rapidly cooled at water contents that minimised
both dehydration and freezing damage.
The ability of A. belladonna and H. montanus embryos to tolerate the various
components of cryopreservation in relation to changes in extracellular superoxide (.O2
-)
production and lipid peroxidation (a popular ‘marker’ for oxidative stress) was investigated in
studies featured in Chapter 4. Pre-conditioning and freeze-thawing led to an increase in
oxidative stress and the accompanying decline in viability suggests that oxidative stress was a
major component of cryoinjury in the embryos presently investigated. Post-thaw viability
retention in Gly cryoprotected + partially dried embryos was significantly higher than noncryoprotected
+ partially dried embryos, possibly due to the relatively lower post-drying lipid
peroxidation levels and relatively higher post-drying and post-thawing enzymic antioxidant
activities in the former.
Exposure of certain plant tissues to low levels of oxidative or osmotic stress can improve
their tolerance to a wide range of stresses. In contrast, exposure of H. montanus zygotic
embryos to low levels of oxidative stress provoked by exogenously applied hydrogen peroxide
(H2O2) or exposure of A. belladonna embryos to low levels of osmotic stress provoked by low
water potential mannitol and polyethylene glycol solutions (in studies featured in Chapter 5)
increased their sensitivity to subsequent dehydration and freeze-thaw stresses. Exposure of Gly
cryoprotected and non-cryoprotected amaryllid embryos to such stress acclimation treatments
may pre-dispose A. belladonna and H. montanus embryos to greater post-drying and post-thaw
total antioxidant and viability loss than untreated embryos.
To assess the vigour of seedlings recovered from partially dried H. montanus embryos,
seedlings recovered from fresh (F) and partially dried (D) embryos in vitro were hardened-off ex
vitro, and subsequently subjected to either 42 days of watering or 42 days of water deficit (in
studies described in Chapter 6). In a subsequent study (described in Chapter 7), seedlings
recovered from fresh (F), partially dried (D) and cryopreserved (C) A. belladonna embryos were
regenerated in vitro, hardened-off ex vitro and then exposed to 12 days of watering (W) or 8
days of water stress (S) followed by 3 days of re-watering. Results of these studies suggest that
the metabolic and ultrastructural lesions inflicted on A. belladonna and H. montanus zygotic
embryos during cryopreservation may compromise the vigour (e.g. development of persistent
low leaf water and pressure potentials and reduced photosynthetic rates) and drought tolerance
of recovered seedlings, compared with seedlings recovered from fresh embryos. While the
adverse effects of freeze-thawing were carried through to the early ex vitro stage, certain
adverse effects of partial drying were reversed during ex vitro growth (e.g. the increased relative
growth rate of seedlings from partially dried embryos). The reduced vigour and drought
tolerance of seedlings recovered from partially dried and cryopreserved embryos in the present
work may therefore disappear with an extension in the period afforded to them for hardening-off
under green-house conditions, and in the field.
The results presented in this thesis reinforce the notion that each successive manipulation
involved in the cryopreservation of recalcitrant zygotic germplasm has the potential to inflict
damage on tissues and post-thaw survival in such germplasm relies on the minimisation of
structural and metabolic damage at each of the procedural steps involved in their
cryopreservation. The results also highlight the need to design research programmes aimed not
only at developing protocols for cryopreservation of plant genetic resources, but also at
elucidating and understanding the fundamental basis of both successes and failures.
Description
Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2010.
Keywords
Plant cells and tissues--Cryopreservation., Amaryllidaceae., Amaryllis (Genus), Theses--Botany.