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The role of reactive oxygen species and anti-oxidants during
pre-cooling stages of axis cryopreservation in recalcitrant Trichilia dregeana Sond.
Journal: Botany
Manuscript ID cjb-2015-0248.R1
Manuscript Type: Article
Date Submitted by the Author: 15-Feb-2016
Complete List of Authors: Naidoo, Cassandra; University of KwaZulu-Natal , School of Life Sciences
Berjak, Patricia; University of KwaZulu-Natal , School of Life Sciences Pammenter, Norman; University of KwaZulu-Natal, School of Life Sciences Varghese, Boby; University of Kwazulu-Natal, Westville campus, School of Life Sciences
Keyword: reactive oxygen species, cryopreservation, recalcitrant seeds, cathodic protection
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The role of reactive oxygen species and anti-oxidants during pre-
cooling stages of axis cryopreservation in recalcitrant Trichilia
dregeana Sond.
Cassandra Naidoo, Patricia Berjak, Norman W. Pammenter* and Boby
Varghese
School of Life Sciences, University of KwaZulu-Natal, Westville Campus, Durban,
4001 South Africa
*Corresponding author; tel.: +27 31 260 3097; fax: +27 31 260 1195
E-mail: pammente@ukzn.ac.za
Running title: Cryopreservation and oxidative stress
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Abstract
Cryopreservation is currently the only feasible method for long-term
conservation of non-orthodox germplasm. Previous attempts to cryopreserve
embryonic axes of Trichilia dregeana, indicated that an inability for shoot production,
suggestedly associated with a wound-induced burst of reactive oxygen species
(ROS) accompanied by declining anti-oxidant potential, consistently accompanied
procedures necessary for cryopreservation. The present study involved provision of
cathodic protection, both in the aqueous phase (electrolysis of a solution containing
dilute electrolytes) and dry state (rapid drying of axes on a grid on which a static field
is generated via the cathode of a power-pack) with the addition of other anti-oxidants
i.e. ascorbic acid and DMSO to counteract uncontrolled ROS activity. Superoxide
(.O2-), hydrogen peroxide (H2O2) and hydroxyl radical (.OH) production in conjunction
with the corresponding total aqueous anti-oxidant activity and viability was
quantitatively assessed after each stage of the cryo-procedure, including and
excluding cathodic protection and/or anti-oxidants. No shoot production occurred in
treatments without cathodic protection and/or anti-oxidants. In contrast, 80, 40 and
40% of axes produced seedlings after excision, cryoprotection and flash (rapid)
drying, respectively, when the treatments included a form of cathodic protection.
Keywords: anti-oxidants, cathodic protection, cryopreservation, reactive oxygen
species, recalcitrant seeds, Trichilia dregeana.
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Introduction
Cryopreservation is presently considered to be the only means of long-term
storage of the germplasm of desiccation-sensitive recalcitrant-seeded species, as
the seeds themselves cannot be stored under the low moisture and low temperature
conditions used for orthodox seeds (Engelmann, 2011; FAO, 2013). Successful
cryopreservation of the embryonic axes of some recalcitrant-seeded species, e.g.,
Cocos nucifera (Assy-Bah and Engelmann, 1992), Citrus sinensis (Santos and
Stushnoff, 2003), Poncirus trifoliata (Wesley-Smith et al., 2004), various amaryllids
(Sershen et al., 2007) and Ilex brasiliensis (Mroginski et al., 2008) have been
reported. However, more often than not, cryopreservation of embryonic axes of a
range of tropical/sub-tropical woody species has not been successful as shoot
production was either supressed (Poulsen, 1992; Beardmore and Whittle, 2005;
Ballesteros et al., 2014) or totally absent following cryogen exposure (Wesley-Smith
et al., 1992; Kioko et al., 1998; Perán et al., 2006; Goveia, 2007; Normah et al.,
2011). Critical parameters compromising seedling establishment after
cryopreservation include the stage of development of the axes: if under-developed,
germination competence could be diminished as indicated by Goveia et al. (2004), or
if already in the early stages of gemination, metabolic rate is enhanced with a
concomitant increase in desiccation sensitivity, as demonstrated for several
amaryllid species (Sershen et al., 2007). In the case of axes which dry slowly, failure
of seedling establishment may be because their survival is compromised by the
prolonged period of dehydration necessary to reduce their WC to a level appropriate
for cryogenic cooling (Sershen et al., 2012a; Ballesteros et al., 2014).
Successful cryopreservation of axes of Trichilia dregeana Sond., the subject of
the present study, has been elusive, with the lack of shoot production being the
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major problem. In an earlier work the suggestion was made that necrosis of the
shoot apical meristem could be associated with an excision-related burst of reactive
oxygen species (ROS) when the cotyledonary attachments, which are situated close
to the shoot apex, are severed upon axis excision (Goveia et al,, 2004). This has
since been established to be the case (Whitaker et al., 2010; Pammenter et al.,
2011). Additionally, the embryonic axes of these seeds have been reported to
respond badly to cryoprotection (Berjak et al., 1999), and many previous attempts to
cryopreserve them have been unsuccessful (Kioko et al., 1998; Goveia et al., 2004;
Whitaker et al., 2010).
Cryopreservation of embryonic axes from recalcitrant seeds requires that they
are rapidly dehydrated to relatively low WCs which should minimise or (ideally)
preclude intracellular ice crystallisation when they are cooled to cryogenic
temperatures (updated reviews by Berjak and Pammenter, 2014; Pammenter and
Berjak, 2014). However, after these procedures, establishment of seedlings naturally
producing both a root and a shoot by axes excised from recalcitrant seeds of
tropical/sub-tropical provenance has seldom been achieved (Engelmann, 2011;
Normah et al., 2011). In a break-through with T. dregeana axes, Naidoo et al. (2011)
reported that shoot production was inhibited by the very first stage of the cryo-
procedure, viz. excision. Those authors found that implementation of a pre-culture
step exposing axes to dimethyl sulphoxide (DMSO) prior to complete removal of the
cotyledons, and post-excision soaking in DMSO or 1% (w/v) ascorbic acid (AsA)
promoted the production of shoots by most of the excised axes. That study, together
with several others on T. dregeana axes (Goveia et al., 2004; Song et al., 2004;
Whitaker et al., 2010; Pammenter et al., 2011; Varghese et al., 2011) have led to the
suggestion that failure to produce shoots after excision (and hence cryopreservation)
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may be a consequence of physical injury to shoot meristematic cells situated in close
proximity to the point of excision (Ballesteros et al., 2014) combined with unregulated
activity of ROS (Goveia et al., 2004).
It is necessary , however, that embryonic axes have to be excised, rapidly
(flash) dried and rapidly cooled to cryogenic temperatures. All these steps, as well
as axis retrieval from, and thawing and rehydration after, cryogenic storage do - or
have the potential to – lead to the generation of ROS (Benson and Bremner, 2004;
Roach et al., 2008; Whitaker et al., 2010; Berjak et al., 2011). The ROS having
attracted the most attention in terms of oxidative damage are .O2– , .OH, and H2O2.
Although ROS are implicated in normal metabolism under conditions where they
must be strictly controlled by endogenous anti-oxidants (Mittler et al., 2002), any or
all of the cryo-procedures may be accompanied by the production of a ROS burst
with the potential to overwhelm the endogenous anti-oxidant capacity of small
explants such as embryonic axes (Berjak et al., 2011; Varghese et al., 2011). Under
such conditions, severe oxidative damage and death of all or part of the explant
tissues are the likely consequences conjectured to underlie poor cryo-survival
(Touchell and Walters, 2000; Goveia et al., 2004; Roach et al., 2008, 2010; Whitaker
et al., 2010; Pammenter et al., 2011).
It has also been postulated that axes separated from the rest of the tissues of
recalcitrant seeds by excision may not have fully functional endogenous anti-oxidant
systems (Varghese et al., 2011; Sershen et al., 2012b) and hence not be capable of
quenching stress-related ROS bursts induced by procedures involved in
cryopreservation. Such imbalance could lead to a state of oxidative stress which may
be an underlying cause of axis tissue necrosis – especially of shoot meristems –
during cryo-procedures.
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The present study examined the effectiveness of known chemical anti-oxidants
(Naidoo et al., 2011) and novel means of cathodic protection (Pammenter et al.,
1974; Berjak et al., 2011) to ameliorate the destructive consequences of unregulated
ROS generation accompanying the various steps involved in T. dregeana axis
cryopreservation. The premise of cathodic protection is that the free radicals/ROS
generated as a consequence of the various steps of cryopreservation will be
quenched, thereby obviating the cascade of events leading to oxidation of cellular
macromolecules, particularly lipids, proteins and nucleic acids (Hanaoka, 2001;
Hiraoka et al., 2004; Berjak et al., 2011).
Production of ROS and the corresponding anti-oxidant activity were chosen as
indicators of oxidative damage (Halliwell and Gutteridge, 2007). These parameters
were assessed in conjunction with in vitro germination of axes and particularly the
capacity for shoot development after each stage of cryopreservation to establish
whether poor seedling development could be attributed to disrupted cellular
homeostasis (Kranner et al., 2006; Varghese et al., 2011). The successful use of
exogenously applied anti-oxidants in facilitating shoot development by axes of T.
dregeana after excision (Naidoo et al., 2011) and recovery after cryopreservation of
shoot tips (Reed et al., 2012) prompted the further investigation of changes in
oxidative metabolism and the corresponding capacity for onwards development of
axes in the context of the provision of anti-oxidant treatments. These parameters
were evaluated in all cases on material that had been exposed to the conventional
treatments (no anti-oxidants) and on material that was exposed to the treatments
inclusive of an anti-oxidant or cathodic protection.Materials and Methods
The sequence of procedures and parameters investigated are summarised in
Figure 1.
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Seed collection, surface decontamination and hydrated seed storage: Seeds
were collected, decontaminated and stored as described by Naidoo et al. (2011).
Excision of primary and secondary explants: Primary explants consisted of the
embryonic axis with a portion (2 mm x 2 mm x 0.5 mm; 2 mm3) of the basal segment
of each cotyledon remaining attached to the axis (for the purpose of preventing injury
to the shoot meristem before priming with an anti-oxidant treatment). Primary
explants were excised using a sterile 11 pt blade and immediately subjected to anti-
oxidant pre-conditioning (pre-culture). Subsequently, the secondary explant was
isolated by removing the remaining attached cotyledonary segments using two
hypodermic needles (Naidoo et al., 2011).
Anti-oxidant pre-conditioning (pre-culture) of primary explants: All procedural
steps including pre-conditioning were conducted in a darkened laminar air-flow
cabinet. Primary explants were incubated on a pre-culture medium containing the
anti-oxidant, DMSO, for 6 h in the dark (n=20), the medium consisting of full strength
(4.4 g L-1) MS (Murashige and Skoog, 1962), 30 g L-1 sucrose and 10 g L-1 agar, pH
5.6 – 5.8. Filter-sterilised DMSO (1 ml L-1, Sigma, ≥ 99.5% [GC grade]) was added to
the medium after autoclaving.
Preparation of cathodic water: Cathodic water, which is a strong reductant
(Hanaoka, 2001) was prepared by the electrolysis of an autoclaved solution of 1 µM
CaCl2 and 1 mM MgCl2 (CaMg). The electrodes were platinum foil, the cathode and
anode were in separate chambers connected via a salt bridge (saturated KCl in 3%
agar) and electrolysis was conducted at 60 V for 1 h (Berjak et al., 2011). The non-
electrolysed solution of Ca+ and Mg+ (CaMg solution; Mycock, 1999) in distilled
water, served as the control.
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Post-excision soaking: Pre-cultured primary explants were immersed in a 1%
DMSO solution (v/v) in distilled water for cotyledon excision, leaving the axes only as
the secondary explants. Axes (n=20) were immersed in one of the following post-
excision soaking treatments for 30 min: i. distilled water; ii. cathodic water; iii. 1%
ascorbic acid in distilled water (w/v); iv. 1% ascorbic acid prepared in cathodic water
(w/v); or v. 1% DMSO in distilled water (v/v).
Cryoprotection: Subsequent to post-excision soaking in ascorbic acid prepared
in cathodic water (treatment showing the highest viability after post-excision
soaking), axes (n=20) were treated with the penetrating cryoprotectants, DMSO and
glycerol dissolved in either cathodic or distilled water. The cryoprotectants were
applied individually and in combination as 5 and 10% (v/v) solutions. Axes were
immersed in the 5% cryoprotectant solutions for 60 min, followed by exposure to the
10% solutions for a further 60 min.
Desiccation: Axes were rapidly desiccated in a flash dryer (Berjak et al., 1990;
Pammenter et al., 2002) with or without ‘dry’ cathodic protection, the former
connecting the cathode of the power source to the stainless steel wire grid on which
the axes were supported during flash drying (Pammenter et al., 1974). A potential
difference of 300 V was applied throughout the duration of drying. Three sets of
conditions were tested: i. excision of axes, pre-culture, post-excision soak and flash-
drying (90 min); ii. excision of axes, pre-culture, post-excision soak, cryoprotection
and flash-drying (120 min); iii. excision of axes and flash drying (75 min). In each
case, the duration of flash drying was the time necessary to reduce WC to a range of
0.35-0.4 g g-1 (i.e. the range avoiding desiccation damage sensu stricto, but which
should facilitate cryogenic cooling with no, or minimal, ice crystallisation).
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Rehydration after desiccation: To ascertain the impact of flash drying in each
case, axes were rehydrated (n=20) in solutions of CaMg (control) or cathodic water
for 30 min in the dark.
Cooling, thawing and rehydration: Naked axes (n=20) after respective
treatments were plunged directly into nitrogen slush (-210°C) in a polystyrene cup
(250 ml) where they were tumble-mixed with swirling for 5 min, during which the
slush reverted to the liquid form (-196°C). Axes were stored in LN overnight. Upon
retrieval they were transferred from cryovials directly into Petri dishes and thawed by
direct immersion in cathodic water or the CaMg solution at 40°C for 2 min and
thereafter transferred to the same solution at 25°C (room temperature) for 30 min
(Berjak et al., 1999) for rehydration.
Surface decontamination and germination of axes: Axes were decontaminated
by serial immersion in 2% (v/v) Hibitane® for 2 min, 70% ethanol for 2 min and 1%
(v/v) sodium hypochlorite (NaOCl) for 5 min. Axes were subsequently rinsed three
times with sterile distilled water before being immersed again for 5 min in 1% (w/v)
NaOCl, followed by three rinses with a sterile CaMg solution (Kioko, 2003). After
each stage of the protocol, axes were germinated on full strength (4.4 g L-1) MS
basal medium (Murashige and Skoog, 1962) containing sucrose (30 g L-1) and agar
(10 g L-1) and supplemented with the growth hormone, 6-benzylaminopurine (BAP;
0.1 mg L-1) at pH 5.6-5.8 (Perán et al., 2006).
Superoxide assessment: Extracellular production of .O2- was assessed by the
oxidation of epinephrine to adrenochrome, measured spectrophotometrically at 490
nm (Misra and Fridovich, 1972), using a UV-Vis spectrophotometer (Cary 50 Conc
UV Vis spectrophotometer, Varian, Palo Alto, CA). A 1 mM epinephrine solution (pH
7.0) in 1 M HCl was prepared, from which 0.5 ml was dispensed into 1.5 ml distilled
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water to make up the incubation medium. Immediately after treatments, axes were
divided into five replicate batches of four axes per treatment and placed in incubation
medium on an orbital shaker (Labcon, Instrulab CC, Maraisburg, South Africa) at 70
rpm, at room temperature, for 15 min in dark (after Roach et al., 2008). Absorbance
of each replicate was subsequently read. The extinction co-efficient of adrenochrome
at 490 nm, 4.47 mM-1 cm-1, was used to calculate .O2- production which was
expressed in µmol min-1 g-1 on a dry mass basis. The specificity of this assay was
confirmed via the 50% inhibition of O2- by 250 units ml-1 superoxide dismutase (SOD;
data not shown).
Hydroxyl radical assessment: After each treatment, five replicates of four axes
each were incubated in 1.5 ml of potassium phosphate buffer (prepared using 20
mM K2HPO4 and 20 mM KH2PO4, pH 6.0), containing 20 mM 2-deoxy-d-ribose, on
the orbital shaker at 70 rpm, at room temperature, for 45 min (as described by
Schopfer et al., 2001). Debris originating from the axes was cleared by centrifugation
at 9 000 rpm for 2 min. After centrifugation, a mixture of 0.5 ml of incubation medium
(from each replicate), 0.5 ml of 2-thiobarbituric acid (TBA; 10 g L-1 in 50 mM NaOH)
and 0.5 ml tricholoroacetic acid [TCA; 28 g L-1 in distilled water]) was used to
estimate the formation of the breakdown product malondialdehyde. The mixture was
heated in a water bath at 95°C for 10 min, and then cooled on ice for 5 min. Dèbris
was once again cleared by gentle spinning as described above. The reaction product
was measured by dispensing 300 µl of solution into each well of black Elisa® plates,
and subsequently reading the fluorescence using a fluorescence spectrophotometer
(FLx 800, Bio-Tek Instruments Inc.; excitation: 530 nm and emission: 590 nm).
Hydrogen peroxide assessment: Hydrogen peroxide levels were measured
using the xylenol orange assay (Gay and Gebicki, 2000; Bailly & Kranner, 2011),
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with a few minor modifications. Five replicates of four axes per treatment were
incubated in 1.0 ml distilled water on the orbital shaker and rotated at 70 rpm, at
room temperature, for 30 min. Thereafter, 300 µl incubation medium was added to
1.5 ml working reagent, prepared by mixing 0.1 ml of reagent A (containing 25 mM
FeSO4, 25 mM (NH4)2SO4, 2.5 M H2SO4) and 10 ml of reagent B (125 mM xylenol
orange (Sigma-Aldrich) and 100 mM sorbitol). The reaction mixture was incubated
for 20 min afterwhich the absorbance of samples were read at 560 nm (Cary 50
Conc UV Vis spectrophotometer, Varian, Palo Alto, CA). The extinction coefficient of
Fe3+ xylenol orange complex at 560 nm is 267 mM−1 cm−1, which was used in
calculations to estimate H2O2 content in axes on a dry mass basis. The specificity of
this assay was confirmed by the inhibition of > 80% H2O2 by 500 units ml-1 catalase
(CAT; data not shown).
All procedures subsequent to primary excision of the explant were conducted
under dark conditions to limit ROS generation initiated and perpetuated by photo-
oxidation (Touchell and Walters, 2000).
Total aqueous anti-oxidant activity (TAA): Total aqueous anti-oxidants
(enzymatic and non-enzymatic) was measured in treated and control axes (n=5)
using the 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid; ABTS, Sigma-
Aldrich) decolorisation assay, which is applicable to lipophilic and hydrophilic anti-
oxidants, including flavonoids, hydroxycinnamates and carotenoids (Re et al., 1999).
Four replicated batches of five axes were separated for each treatment and
extraction was effected immediately in 2 ml chilled extraction buffer (50 mM KH2PO4
buffer; pH 7.0) containing 1mM CaCl2, 1mM KCl and 1mM EDTA. Samples were
centrifuged for 15 min at 14 000 rpm, 9 m s-2 at 4° C. Supernatant was extracted and
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further centrifuged for 5 min after which the final supernatant was used for the total
antioxidant assay.
The assay was performed twice on 100 µl of extract from each of the four
replicates per treatment, in the dark (after Johnston et al., 2006). A standard curve
was generated using a 5 mM Trolox® (6-hydroxy-2.5.7.8-tetramethylchromeane-2-
carboxylic acid, 97%) solution prepared in the anti-oxidant extraction buffer.
Changes in absorbance were estimated spectrophotometrically and expressed on a
fresh mass basis as Trolox equivalents using the standard curve.
Oxidative parameters and viability assessments were conducted after each of
the treatments (with and without anti-oxidants) subsequent to secondary excision as
outlined in Figure 1.
Statistical analysis: Production of ROS (.O2-, .OH and H2O2) and TAA were
tested for significant inter-treatment differences between cathodic and non-cathodic
treatments using an independent samples t-test for all stages of cryopreservation,
except for post-excision soaking treatments where differences were tested by
analysis of variance (ANOVA). For the soaking treatments, the Tukey HSD post-hoc
test was analyzed to determine where differences were significant between
treatments. A Pearson Chi-Square test was done to assess significant inter-
treatment differences in viability between cathodic and non-cathodic treatments. A
Mann-Whitney-U test was performed in instances where the assumptions of
normality or equal variance were not met. All statistical analyses were performed at
the 0.05 level of significance using SPSS statistical package (Version 19; SPSS Inc.
Chicago, Illinois, USA).
Results
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Results presented here show selected treatments from each stage of
cryopreservation, the non-cathodic treatment in each case represents the control.
Post-excision soaking: Figures 2 and 3 show viability and biochemical data
respectively for freshly excised (non-treated) axes. Post-excision soaking in a 1%
solution of ascorbic acid prepared in cathodic water promoted shoot development by
the greatest number of excised axes of T. dregeana (p < 0.05; Fig. 2). Significant
differences occurred in .O2- and H2O2 production between the anti-oxidant soak and
distilled water treatments, and each of these treatments compared with freshly
excised axes (p < 0.05; Fig. 3). Each of these ROS was lower in axes treated with
ascorbic acid used in combination with cathodic water.
Cryoprotection: Results pertaining to cryoprotectant treatment showed
significant root and shoot production (p < 0.05) by 50 and 40% of axes, respectively,
compared with neither root nor shoot development when the solvent was distilled
water (Fig. 4). Viability results were observed to correspond with a significant
difference in H2O2 production such that levels were lower in axes treated with
cryoprotectants prepared in cathodic water (Fig. 5).
Desiccation: The influence of dry cathodic protection during flash drying was
investigated at three stages of the protocol and the most effective cathodic and non-
cathodic regime was selected for comparative purposes (Figs. 6 and 7). Dry cathodic
protection was observed consistently and significantly to reduce .O2- and H2O2 levels
across the regimens (p < 0.05; Fig. 7). However, no root or shoot production
occurred unless the full spectrum of pre-culture, post-excision soaking,
cryoprotection and dry cathodic protection during dehydration was applied, under
which circumstances only 10% of the axes each produced a root (Fig. 6).
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Rehydration: Further positive results were attained after rehydration of axes.
Following the drying regime that resulted in 10% root production and subsequently
rehydrating axes in cathodic water for 30 min facilitated significantly enhanced
onwards development of the excised axes (p < 0.05; Fig. 8), exemplified by root and
shoot production by 50 and 40% of the axes, respectively. There was significant
reduction (p < 0.05) of both .O2- and H2O2 in axes rehydrated in cathodic water (Fig.
9).
Cooling: Processing of axes with cathodic water after rapid cooling viz.
rewarming (thawing) and rehydration, resulted in significantly lowered levels (p <
0.05) of the three ROS measured compared with the situation when CaMg was used
(Fig. 10). Nevertheless, despite the seemingly efficient reduction of oxidative
indicators, neither roots nor shoots were produced by axes after exposure to
cryogenic temperatures (-210 followed by -196°C; data not shown). In this regard,
.O2- and .OH production was considerably higher after cooling (Fig. 10) compared
with levels in axes that had been only rapidly dried and rehydrated (Fig. 7).
No significant differences occurred in anti-oxidant activity at any stage of
cryopreservation or in .OH production except after retrieval from cooling.
Discussion
An oxidative burst has been defined as the rapid production of ROS as a
defence mechanism in response to an external stimulus (Ross et al., 2006). It has
been well documented that excision of the cotyledons from the embryonic axis of
many recalcitrant seeds (Roach et al., 2008; Berjak et al., 2011; Pammenter et al.,
2011) including T. dregeana seeds (Goveia et al., 2004; Whitaker et al., 2010;
Varghese et al., 2011) elicits an oxidative burst in response to wounding thus
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precluding shoot development (Goveia et al., 2004) by possibly causing injury to the
shoot mersitatic cells (Ballesteros et al., 2014). The high levels of ROS observed in
freshly excised axes were significantly reduced by post-excision soaking in the
ascorbic acid and cathodic water solution (Fig. 3) and the amelioration of detrimental
ROS production seems to have facilitated the highest percentage of shoot
development by excised axes (Fig. 2). Levels of ROS were reduced even by the
distilled water post-excision soak (Fig. 3) but this result was obtained in conjunction
with the use of a pre-culture treatment containing DMSO prior to excision of
cotyledons (see Fig. 1). Dimethyl sulphoxide has been documented to have powerful
radical scavenging properties and is particularly potent as a .OH scavenger
(Rosenblum and El-Sabban, 1982; Yu and Quinn, 1994), and the use of DMSO in
the concentrations applied in this study is unlikely toxic, compared to the
concentrations used in Plant Vitrification Solutions (PVS) where toxicity to plant cells
is possible. The observed results for germination (Fig. 2) and oxidative indicators
(Fig. 3) after treatment of axes using the anti-oxidant treatment supports the action of
cathodic water as an enhancer of proton donors, suggesting that .O2- and H2O2 are
efficiently scavenged by this treatment as a result of the increased dissociation
activity of AsA when dissolved in cathodic water, in accordance with Hanaoka
(2001).
Responses of cells to cope with oxidative stress may not always involve
increasing levels of endogenous anti-oxidants (Halliwell, 2006), external provision of
protection can act in inhibiting ROS-producing systems and enhancing other
mechanisms such as chaperones for transport of anti-oxidants (Halliwell, 2006). The
details of the mechanism by which AsA (in cathodic water) best promoted shoot
development (Fig. 2) and reduced .O2- and H2O2 levels after excision (Fig. 3), remain
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to be resolved. Nevertheless, this combination for a post-excision soaking solution
was selected as the treatment of choice in view of the results obtained in this study
and in consideration of the synergistic function of AsA with an array of other anti-
oxidants in ROS-scavenging, its role in maintaining membrane and lipoprotein
integrity and in promoting cell growth and development (Halliwell, 1994; Potters et
al., 2002; Shao et al., 2008).
While the application of anti-oxidants pre- and post-excision was adequate to
reduce ROS and facilitate significant establishment of seedlings (i.e. showing both
root and shoot development) by excised axes (Fig. 2), successive steps of
cryopreservation viz., cryoprotection, partial dehydration, cooling and rewarming are
likely to induce stresses far more difficult to counteract. Excised axes of T. dregeana
had not previously been reported to produce shoots or to show substantial root
development after cryoprotection (Kioko, 2003; Goveia, 2007) where those authors
described callus production or swelling to be the most common indications of
survival. In the present study it was believed that preparation of the cryoprotectants,
DMSO and glycerol, as solutions in cathodic water may have had a positive effect on
survival with 55% root and 40% axes exhibiting shoot production (Fig. 4).
Considering both cryoprotective treatments exposed axes to the same
concentration of the cryoprotectants for the same duration, the inability of axes to
survive conventional cryoprotection (when the solvent was distilled water) was not a
consequence of cryoprotectant toxicity. Dimethyl sulphoxide and glycerol are
penetrating cryoprotectants that act by increasing membrane permeability to aid in
water removal from cells and facilitate protective dehydration during freezing (Fuller,
2004). Increased membrane permeability could facilitate an influx of ROS,
specifically H2O2 that travels freely across membranes (Halliwell, 2006), making the
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uncontrolled generation of this species all the more harmful during cryoprotection.
The unregulated generation of H2O2 during conventional cryoprotection, as shown in
this study (Fig. 5), possibly had markedly cytotoxic effects, and the scavenging of
this ROS alone by cathodic water (Hanaoka, 2001) is suggested to have facilitated
survival during cryoprotection (Fig. 4).
In terms of cryopreservation, these results are significant as it is beneficial to
have T. dregeana axes survive the step of cryoprotection where both DMSO and
cathodic water are present: i.e. both may provide the means to regulate oxidative
metabolism. Cryoprotection is a stress inducing stage during cryopreservation of a
variety of organs (Best, 2015) and the preparation of cryoprotectants in a highly
reducing solution can be applied to mediate ROS production and improve viability.
During desiccation, water from the cytoplasm is removed and WC is lowered.
These circumstances in recalcitrant axes induces ROS generation and hinders anti-
oxidant defences (Varghese et al., 2011), where even the slightest removal of water
causes a large decrease in the ascorbate pool (Smirnoff, 1993). This initiates
metabolic derangement (Pukacka and Ratajczak, 2006) and the situation is
exacerbated in the shoot pole by the extended duration needed to dehydrate the
whole axis to an appropriate, minimally-injurious WC range (Ballesteros et al., 2014).
Additionally, effects of possible mechanical damage accompanying drying could
induce secondary oxidative events such as lipid peroxidation (not investigated in this
study), while concomitantly reducing anti-oxidant capacity (Bailly, 2004; Varghese et
al., 2011), thus severely affecting first the shoot meristematic region and
subsequently, the root pole.
The results observed after desiccation of T. dregeana (Fig. 6) suggest that the
long periods of drying axes to reduce WC to that amenable for cooling imposes so
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severe a stress to the shoot meristem that none of the potentially ameliorative
measures involving cathodic protection was adequate to counteract oxidative events.
It has been reported that survival after desiccation and subsequent cooling, is
significantly higher in species where axes rapidly lose water (Ballesteros et al., 2014)
and are therefore exposed to desiccation for a much shorter period, as exemplified
by Strychnos gerarrdii (Berjak et al., 2011) and certain monocot Amaryllid species
(Ballesteros et al., 2014). Extensive periods of drying hold axes at intermediate WCs
for a longer duration, during which time ROS production is exacerbated (Walters et
al., 2001; Varghese et al., 2011). This potentially inhibited root production (10% only)
by the majority of axes and precluded shoot development by all (Fig. 6). In species
such as T. dregeana, where axes are inherently resistant to water loss, long periods
of desiccation cannot be avoided. A secondary complication is the differential drying
rates of the root and shoot meristems (Ballesteros et al., 2014). Kioko (2003) also
showed that the axis shoot tip dries considerably more rapidly than does the root
pole: hence the shoot apical meristem may be far more adversely affected as a
result of the duration of the applied stress (shoot meristem may be dried to a WC far
lower than that which would permit survival). These results highlight that cathodic
protection, while successful in regulating oxidative stress during stages prior to
desiccation of axes of T. dregeana, was not adequate to mitigate ROS-induced (or
other) damage, presumably particularly of the shoot meristematic region.
Superoxide is known to inactivate a spectrum of enzymes that are fundamental
in energy and amino acid metabolism (Halliwell, 2006), hence elevated levels of this
ROS could result in inhibition of metabolic pathways crucial to growth and repair, and
ultimately could result in death. It is possible that the levels of the major scavengers
of.O2- and H2O2 i.e. superoxide dismutase (SOD) and catalase (CAT), respectively
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(Halliwell, 2006), might decline or become dysfunctional under water stressed
conditions in recalcitrant tissues (Chaitanya and Naithani, 1994; Varghese et al.,
2011). The results of cryo procedures thus far, indicate that external provision of
anti-oxidants, even when dry cathodic protection is provided, is inadequate to
overcome the oxidatively-stressed state as shown by the levels of .O2- and H2O2
immediately after desiccation (Fig. 7), which bring about localised necrosis in, or
even death of, axes. Regardless of the minimal capacity for ongoing development
(10% of axes producing roots; Fig. 6) obtained using the selected drying treatment
which incorporated wet (post-excision soaking and cryoprotection) and dry cathodic
protection, this was the procedure selected for assessment of rehydration and
cooling on each parameter.
Rehydration of axes in cathodic water facilitated 50 and 40% root and shoot
production respectively (Fig. 8): such success after desiccation and rehydration has
never before been achieved with T. dregeana (Kioko, 2003; Goveia, 2007). The
rehydration step could impose a stress disrupting the oxidative balance (Smirnoff,
1993) in desiccated recalcitrant axes such that survival is uncertain. Nevertheless,
the use of cathodic water at this stage seemed to curb excessive production of ROS
adequately, and is suggested to alleviate oxidative stress (Fig. 9) to facilitate survival
(Fig. 8). However, it should be noted that it may not be the use of cathodic water
alone at the rehydration stage that acts as a key promoter of survival. The axes had,
up to this point in the procedure, been exposed to protection in the form of AsA,
DMSO and dry cathodic protection. It is the cumulative effect of protective
mechanisms at each procedural stage that fine-tuned oxidative metabolism such that
survival of axes after rapid dehydration was attained.
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Inspite of 50% and 40% of axes producing roots and shoots respectively after
the cryo-procedure preceeding cryogen immersion (Fig. 8), none of the axes
exhibited root or shoot formation after retrieval from LN and thawing with CaMg or
cathodic water. Two possible causes could underlie the lack of capacity for onward
development by axes after cooling . Firstly, oxidative stress here caused axes to
respond in a “plastic” manner, where irreversible damage as a result of failed repair
mechanisms resulted in cell death (Kranner et al., 2010). This response was initiated
by the level of ROS production (Fig. 10) completely overwhelming the anti-oxidant
capacity within the cells (Benson and Bremner, 2004) even when reducing power
was provided by the cathodic water. This is in contrast to pre-cooling stages where
axes displayed an “elastic response” (Kranner et al., 2010) i.e. changes were
reversible and could be repaired such that function and viability were somewhat
maintained. Secondly, physical damage could have been incurred as a result of
intracellular ice crystallisation (Benson, 1990; Pegg, 2001), particularly in terms of
lethal numbers, dimensions and locations of the ice crystals (Wesley-Smith et al.,
2013). It is probable that cell/tissue death resulted from a combination of both, but
detailed quantitation of individual ROS and anti-oxidants will be necessary, as will be
electron microscopical examination of freeze-substituted and freeze-fractured
specimens. The latter investigation will determine the size range, frequency and
distribution of intracellular ice crystals commensurate with survival or death, as
demonstrated by Wesley-Smith et al. (2013).
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Concluding comments
It is acknowledged that the diminished capacity for embryonic axes to survive
various stages of the cryo-procedure could be attributed to causes other than the
oxidative state; however, results presented here show significant differences in ROS
production and shoot development between treatments including exogenously-
provided anti-oxidants or not. Furthermore, there were significant differences in
oxidative parameters and survival of embryos between stages of cryopreservation.
The higher production of ROS post-cooling (Sershen et al., 2012b) compared
with any other procedural stage is suggested to be a function of cumulative stress.
While provision of anti-oxidant protection was adequate to mitigate oxidative damage
after each individual stage prior to cryogen exposure, the accumulated stress after
cooling was too great to overcome.
The freezing process itself is highly injurious to recalcitrant germplasm and, in
the WC range, 0.40 – 0.35 g g-1, it is probable that residual intracellular solution
(freezable) water was present. This possibility is to be resolved using sub-ambient
differential scanning calorimetry.
The present study has advanced progress in understanding oxidative stress
associated with the entire cryo-procedure, and the positive influence of cathodic
water on viability has significantly moved forward the goal of cryopreservation of
axes of tropical/sub-tropical recalcitrant-seeded species as exemplified here by T.
dregeana.
Acknowledgements: The authors are pleased to acknowledge ongoing financial
support from the National Research Foundation (South Africa).
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References
Assy-Bah, B., and Engelmann, F. 1992. Cryopreservation of mature embryos of
coconut (Cocos nucifera L.) and subsequent regeneration of plantlets. CryoLetters
13: 117-126.
Bailly, C. 2004. Active oxygen species and anti-oxidants in seed biology. Seed Sci.
Res. 14: 93-107.
Ballesteros, D., Sershen., Varghese, B., Berjak, P., and Pammenter, N.W. 2014.
Uneven drying of zygotic embryos and embryonic axes of recalcitrant seeds:
challenges and considerations for cryopreservation. Cryobiology 69: 100-109.
Beardmore, T., and Whittle, C-A. 2005. Induction of tolerance to desiccation and
cryopreservation in silver maple (Acer saccharinum) embryonic axes. Tree Physiol.
25: 965-972.
Benson, E.E. 1990. Free radical damage in stored plant germplasm. International
Board for Plant Genetic Resources, Rome.
Benson, E.E., and Bremner, D. 2004. Oxidative stress in the frozen plant: a free
radical point of view. In Life in the Frozen State. Edited by B. Fuller., N. Lane., and
E.E. Benson. CRC Press, Florida. pp. 205-242.
Berjak, P., and Pammenter, N.W. 2014. Cryostorage of germplasm of tropical
recalcitrant-seeded species: approaches and problems. Int. J. Plant Sci. 175: 29-39.
Berjak, P., Farrant, J.M., Mycock, D.J., and Pammenter, N.W. 1990. Recalcitrant
(homoiohydrous) seeds: the enigma of their desiccation-sensitivity. Seed Sci.
Technol. 18: 297-310.
Page 22 of 43
https://mc06.manuscriptcentral.com/botany-pubs
Botany
Draft
23
Berjak, P., Sershen, Varghese, B., and Pammenter, N.W. 2011. Cathodic
amelioration of the adverse effects of oxidative stress accompanying procedures
necessary for cryopreservation of embryonic axes of recalcitrant-seeded species.
Seed Sci. Res. 21: 187-203.
Berjak, P., Kioko, J.I., Walker, M., Mycock, D.J., Wesley-Smith, J., Watt, P., and
Pammenter, N.W. 1999. Cryopreservation – an elusive goal? In Recalcitrant seeds.
Proceedings of the IUFRO Seed Symposium, Kuala Lumpur, Malaysia. 12 October –
15 October 1998. Edited by M. Marzalina., K.C, Khoo., N. Jayanthi., F.Y. Tsan., and
B. Krishnapillay. Forest Research Institute. pp. 96-105.
Best, B.P. 2015. Cryoprotectant toxicity: facts, issues, questions. Rejuvenation Res.
18: 422-436.
Chaitanya, K.S.K., and Naithani, S.C. 1994. Role of superoxide, lipid peroxidation
and superoxide dismutase in membrane perturbation during loss of viability in seeds
of Shorea robusta Gaertn. F. New Phytol. 126: 623-627.
Einset, J., Winge, P., and Bones, A. 2007. ROS signalling pathways in chilling
stress. Plant Signaling Behav. 5: 365-367.
Engelmann, F. 2011. Cryopreservation of embryos: an overview. In Plant embryo
culture: methods and protocols. Edited by T.A. Thorpe and E.C. Yeung. Humana
Press, Totowa, NJ. pp. 155-184.
Food and Agriculture Organisation of the United Nations. 2013. Genebank
Standards for Plant Genetic Resources for Food and Agriculture.Rome.
Page 23 of 43
https://mc06.manuscriptcentral.com/botany-pubs
Botany
Draft
24
Fuller, B.J. 2004. Cryoprotectants: the essential antifreezes to protect life in the
frozen state. CryoLetters 25: 375-388.
Gay, C., and Gebicki, J.M. 2000. A critical evaluation of the effect of sorbitol on the
ferric-xylenol orange hydroperoxide assay. Anal. Biochem. 284: 217-220.
Goveia, M. 2007. The effect of developmental status and excision injury on the
success of cryopreservation of germplasm from non-orthodox seeds. M.Sc. thesis,
University of KwaZulu-Natal,Westville, South Africa. http://hdl.handle.net/10413/1052
Goveia, M., Kioko, J.I., and Berjak, P. 2004. Developmental status is a critical factor
in the selection of excised recalcitrant axes as explants for cryopreservation: a study
on Trichilia dregeana Sond. Seed Sci. Res. 14: 241-248.
Halliwell, B. 1994. Free radicals and anti-oxidants: a personal view. Nutr. Rev. 52:
253-265.
Halliwell, B. 2006. Reactive species and anti-oxidant. Redox biology is a
fundamental theme of aerobic life. Plant Physiol. 141: 312-322.
Halliwell, B., and Gutteridge, J.M.C. 2007. Free radicals in biology and medicine.
Oxford University Press, New York.
Hanaoka, A. 2001. Anti-oxidant effects of reduced water produced by electrolysis of
sodium chloride solutions. J. Appl. Electrochem. 31: 1307-1313.
Hiraoka, A., Takemoto, M., Suzuki, T., Shinohara, A., Chiba, M., Shirao, M., and
Yoshimura, Y. 2004. Studies on the properties and real existence of aqueous
solution systems that are assumed to have anti-oxidant activities by the action of
‘active hydrogen’. J. Health Sci. 50: 456-465.
Page 24 of 43
https://mc06.manuscriptcentral.com/botany-pubs
Botany
Draft
25
Imlay, J.A. 2003. Pathways of oxidative damage. Annu. Rev. Microbio. 57: 395-418.
Johnston, J.W., Dussert, S., Gale, S., Nadarajan, J., Harding, K., and Benson, E.E.
2006. Optimisation of the azinobis-3-ethyl-benzothiazoline-6-sulphonic acid radical
scavenging assay for physiological studies of total anti-oxidant activity in woody plant
germplasm. Plant Physiol. Biochem. 44: 193–201.
Karuppanapandian, T., Moon, J-C., Kim, C., Manoharan, K., and Kim, W. 2011
Reactive oxygen species in plants: their generation, signal transduction, and
scavenging mechanisms. Aust. J. Crop Sci. 9: 709-725.
Kioko, J.I. 2003. Aspects of post harvest seed physiology and cryopreservation of
the germplasm of three medicinal plants indigenous to Kenya and South Africa.
Ph.D. thesis, University of Natal, South Africa. http://hdl.handle.net/10413/4661
Kioko, J.I., Berjak, P., Pammenter, N.W., Watt, M.P., and Wesley-Smith, J. 1998.
Desiccation and cryopreservation of embryonic axes of Trichilia dregeana Sond.
CryoLetters 19: 15-26.
Kranner, I., Birtić, S., Anderson, K.M., and Pritchard, H.W. 2006. Glutathione half-cell
reduction potential: a universal stress marker and modulator of programmed cell
death? Free Radical Biol. Med. 40: 2155-2165.
Kranner, I., Minibayeva, F.V., Beckett, R.P., and Seal, C.E. 2010. What is stress?
Concepts, definitions and applications in seed science. New Phytol. 188: 655-673.
Misra, H.R., and Fridovich, I. 1972. The role of superoxide anion in the autoxidation
of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247:
3170-3175.
Page 25 of 43
https://mc06.manuscriptcentral.com/botany-pubs
Botany
Draft
26
Mittler, R. 2002. Oxidative stress, anti-oxidants and stress tolerance. Trends Plant
Sci. 7: 405-410.
Mroginski, L.A., Sansberro, P.A., Scocchi, A.M., Luna, C., and Rey, H.Y. 2008. A
cryopreservation protocol for immature zygotic embryos of Ilex (Aquifoliaceae).
Biocell 32: 33-39.
Murashige, T., and Skoog, F. 1962. A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497.
Mycock, D.J. 1999. Addition of calcium and magnesium to a glycerol and sucrose
cryoprotectant solution improves the quality of plant embryo recovery from
cryostorage. CryoLetters 20: 77-82.
Naidoo, C., Benson, E., Berjak, P., Goveia, M., and Pammenter, N.W. 2011.
Overcoming a critical limiting step in developing cryopreservation protocols for
excised embryonic axes of recalcitrant seeds: exploring the use of DMSO and
ascorbic acid to promote shoot development. CryoLetters 32: 166-174.
Neill, S., Desikan, R., and Hancock, J. 2002. Hydrogen peroxide signalling. Curr.
Opin. Plant Biol. 5: 388-395.
Normah, M.N., Choo, W.K., Vun, Y.L., and Mohamed-Hussein, Z.A. 2011. In vitro
conservation of Malaysian biodiversity – achievements, challenges and future
directions. In vitro Cell. Dev. Biol. Plant 47: 26-36.
Pammenter, N.W., and Berjak, P. 2014. The physiology of desiccation-sensitive
seeds and the implications for cryopreservation. Int. J. Plant Sci. 75: 21-28.
Page 26 of 43
https://mc06.manuscriptcentral.com/botany-pubs
Botany
Draft
27
Pammenter, N.W., Adamson, J.H., and Berjak, P. 1974. Viability of stored seeds:
extension by cathodic protection. Science 186: 1123-1124.
Pammenter, N.W., Berjak, P., Wesley-Smith, J., and Vander Willigen, C. 2002.
Experimental aspects of drying and recovery. In Desiccation and survival in plants:
drying without dying. Edited by M. Black., and Pritchard, H.W. Wallingford,United
Kingdom, CABI Publishing. pp. 93-110.
Pammenter, N.W., Berjak, P., Goveia, M., Sershen., Kioko, J.I., Whitaker, C., and
Beckett, R.P. 2011. Topography determines the impact of reactive oxygen species
on shoot apical meristems of recalcitrant embryos of tropical species during
processing for cryopreservation. Acta Hortic. 908: 83-92.
Perán, R., Berjak, P., Pammenter, N.W., and Kioko, J.I. 2006. Cryopreservation,
encapsulation and promotion of shoot production in embryonic axes of a recalcitrant
species Ekebergia capensis Sparrm. CryoLetters 27: 5-16.
Pegg, D.E. 2001. The current status of tissue cryopreservation. CryoLetters 22: 105-
114.
Potters, G., Gara, L.D., Asard, H., and Horemans, N. 2002. Ascorbate and
glutathione: guardians of the cell cycle, partners in crime? Plant Physiol. Biochem.
40: 537-548.
Poulsen, K.M. 1992. Sensitivity to desiccation and low temperatures (-196°C) of
embryo axes from acorns of the pedunculate oak (Quercus robur L.). CryoLetters 13:
75-82.
Page 27 of 43
https://mc06.manuscriptcentral.com/botany-pubs
Botany
Draft
28
Pukacka, S., and Ratajczak, E. 2006. Antioxidative response of ascorbate-
glutathione pathway enzymes and metabolites to desiccation of recalcitrant Acer
saccharinum seeds. J. Plant Physiol. 163: 1259-1266.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., and Rice-Evans, C.
1999. Anti-oxidant activity applying an improved ABTS radical cation decolorization
assay. Free Radical Biol. Med. 26: 1231-1237.
Roach, T., Ivanova, M., Beckett, R.P., Minibayeva, F.V., Green, I., Pritchard, H.W.,
and Kranner, I. 2008. An oxidative burst in embryonic axes of recalcitrant sweet
chestnut seeds as induced by excision and desiccation. Physiol. Plant. 133: 131-
139.
Roach, T., Beckett, R.P., Minibayeva, F.V, Colville, L., Whitaker, C., Chen, H., Bailly,
C., and Kranner, I. 2010. Extracellular superoxide production, viability and redox
poise in response to desiccation in recalcitrant Castanea sativa seeds. Plant Cell
Environ. 33: 59-75.
Rosenblum, W.I., and El-Sabban, F. 1982. Dimethyl sulfoxide (DMSO) and glycerol,
hydroxyl radical scavengers, impair platelet aggregation within and eliminate the
accompanying vasodilation of, injured mouse pial arterioles. Stroke 13: 35-39.
Ross, C., Küpper, F.C., and Jacobs, R.S. 2006. Involvement of reactive oxygen
species and reactive nitrogen species in the wound response of Dasycladus
vermicularis. Chem. Biol. 13: 353-364.
Reed, B.B., Uchendu, E., and Normah, M.N. 2012. Are anti-oxidants effective for
reducing oxidative stress during cryopreservation. Proceedings of the first
Page 28 of 43
https://mc06.manuscriptcentral.com/botany-pubs
Botany
Draft
29
International Symposium for In Vitro Conservation and Cryopreservation. Tepatitlan
de Morolos, Jailisco, Mexico.
Santos, I.R., and Stushnoff, C. 2003. Desiccation and freezing tolerance of
embryonic axes from Citrus sinensis [L.] osb. pretreated with sucrose. CryoLetters
24: 281-92.
Schopfer, P., Plachy, C., and Frahry, G. 2001. Release of reactive oxygen
intermediates (superoxide radicals, hydrogen peroxide, and hydroxyl radicals) and
peroxidase in germinating radish seeds controlled by light, gibberellin and abscisic
acid. Plant Physiol. 125: 1591-1602.
Sershen, Pammenter, N.W., Berjak, P., and Wesley-Smith, J. 2007.
Cryopreservation of embryonic axes of selected amaryllid species. CryoLetters 28:
387-399.
Sershen, Berjak, P., Pammenter, N.W., and Wesley-Smith, J. 2012a. Rate of
dehydration, state of subcellular organisation and nature of cryoprotection are critical
factors contributing to the variable success of cryopreservation: studies on
recalcitrant zygotic embryos of Haemanthus montanus. Protoplasma 249: 171-186.
Sershen, Varghese, B., Pammenter, N.W., and Berjak, P. 2012b. Cryo-tolerance of
zygotic embryos from recalcitrant seeds in relation to oxidative stress – a case study
on two amaryllid species. J. Plant Physiol. 169: 999-1011.
Shao, H-B., Chu, L-Y., Lu, Z-H., and Kang, C-M. 2008. Primary anti-oxidant free
radical scavenging and redox signaling pathways in higher plant cells. Int. J. Biol.
Sci. 4: 8-14.
Page 29 of 43
https://mc06.manuscriptcentral.com/botany-pubs
Botany
Draft
30
Smirnoff, N. 1993. Tansley review no. 52: the role of active oxygen in the response
of plants to water deficit and desiccation. New Phytol. 125: 27-58.
Song, S-Q., Berjak, P., and Pammenter, N.W. 2004. Desiccation sensitivity of
Trichilia dregeana axes and anti-oxidant role of ascorbic acid. Acta Bot. Sin 46: 803-
810.
Touchell, D., and Walters, C. 2000. Recovery of embryos of Zizania palustris
following exposure to liquid nitrogen. CryoLetters 21: 261-270.
Varghese, B., Sershen, Berjak, P., Varghese, D., and Pammenter, N.W. 2011.
Differential drying rates of recalcitrant Trichilia dregeana embryonic axes: a study of
survival and oxidative stress metabolism. Physiol. Plant. 142: 326-338.
Walters, C., Pammenter, N.W., Berjak, P., and Crane, J. 2001. Desiccation damage,
accelerated ageing and respiration in desiccation tolerant and sensitive seeds. Seed
Sci. Res. 11: 135-148.
Wesley-Smith, J., Walters, C., Berjak, P., and Pammenter, N.W. 2004. The influence
of WC, cooling and warming rates upon survival of embryonic axes of Poncirus
trifoliata. CryoLetters 25: 129-138.
Wesley-Smith, J., Berjak, P., Pammenter, N.W., and Walters, C. 2013. Intracellular
ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer
saccharinum L.: an ultrastructural study of factors affecting cell and ice structures.
Ann. Bot. 1-15. doi:10.1093/aob/mct284.
Wesley-Smith, J., Vertucci, C.W., Berjak, P., Pammenter, N.W., and Crane, J. 1992.
Cryopreservation of desiccation-sensitive axes of Camellia sinensis in relation to
Page 30 of 43
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dehydration, freezing rate and the thermal properties of tissue water. J. Plant
Physiol. 140: 596-604.
Whitaker, C., Beckett, R.P., Minibayeva, F.V., and Kranner, I. 2010. Production of
reactive oxygen species in excised, desiccated and cryopreserved explants of
Trichilia dregeana Sond. S. Afr. J. Bot. 76: 112-118.
Yu, Z.W., and Quinn, P.J. 1994. Dimethyl sulfoxide: a review of its applications in cell
biology. Biosci. Rep. 14: 259-281.
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Captions to Figures
Fig. 1: Flow diagram illustrating steps of the cryopreservation protocol after each of
which selected redox parameters were assessed. CaMg, solution containing 0.5mM
CaCl2.2H2O and 0.5µM MgCl2.6H2O; cathodic water, cathodic fraction of electrolysed
solution of CaMg; DMSO, dimethyl sulphoxide; nitrogen slush (-210°C), produced by
applying a vacuum to liquid nitrogen. See Materials and Methods for detailed
description of cryoprotection.
Fig. 2: Axes of T. dregeana producing roots and shoots or roots only after exposure
to pre-culture and AsA + cathodic water post-excision soaking (n=20). AsA, ascorbic
acid.
Fig. 3: Reactive oxygen species production and total aqueous anti-oxidant (TAA)
levels in excised axes of T. dregeana after pre-culture and post-excision soaking
treatments (n=4 for ROS estimations and n=10 for TAA activity). AsA, ascorbic acid.
Error bars represent mean ± standard deviation. Letters above bars represent
significant differences between treatments.
Fig. 4: Axes of T. dregeana producing roots and shoots after exposure to pre-culture,
post-excision soaking (AsA + cathodic water) and cryoprotection (n=20). AsA,
ascorbic acid; PC, pre-culture.
Fig. 5: Reactive oxygen species production and TAA levels by excised axes of T.
dregeana after exposure to pre-culture, AsA + cathodic water post-excision soaking
and cryoprotection (n=4 for ROS estimations and n=10 for TAA activity). Error bars
represent mean ± standard deviation. Letters above bars symbolise significant
differences. AsA, ascorbic acid; PC, pre-culture.
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Fig. 6: Axes of T. dregeana producing roots after exposure to dry cathodic and non-
cathodic desiccation treatments (n=20). Axes in both treatement regimes rehydrated
in CaMg. AsA, ascorbic acid; PC, pre-culture; FD, flash dried.
Fig. 7: Reactive oxygen species production and TAA levels by excised axes of T.
dregeana after exposure to dry cathodic and non-cathodic desiccation treatments
(n=4 for ROS estimations and n=10 for TAA activity). AsA, ascorbic acid; FD, flash
dried; PC, pre-culture. Error bars represent mean ± standard deviation. Letters
above bars symbolise significant differences.
Fig. 8: Axes of T. dregeana producing roots only or shoots and roots after exposure
to a selected drying treatment and wet cathodic or non-cathodic rehydration (n=20).
AsA, ascorbic acid; PC, pre-culture; FD, flash dried.
Fig. 9: Reactive oxygen species production and TAA levels by excised axes of T.
dregeana after exposure to a selected drying treatment and wet cathodic or non-
cathodic rehydration (n=4 for ROS estimations and n=10 for TAA activity). PC, pre-
culture; FD, flash dried. Error bars represent mean ± standard deviation. Letters
above bars symbolise significant differences.
Fig. 10: Reactive oxygen species production and TAA levels by excised axes of T.
dregeana after exposure to cathodic water or CaMg thawing and rehydration after
the selected drying and cooling treatment (n=4 for ROS estimations and n=10 for
TAA activity). PC, pre-culture; FD, flash dried; LN, liquid nitrogen. Error bars
represent mean ± standard deviation. Letters above bars symbolise significant
differences.
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Primary excision of explant from seed (axis with a cotyledonary block)
DMSO pre-culture for 6 h
Final excision of explant
(Removal of axis from cotyledonary block under a 1% [v/v] solution of DMSO)
Post-excision soak for 30 min in selected anti-oxidant solutions
(One of DMSO, AsA, cathodic water or distilled water {control})
Cryoprotection of axes: 10% DMSO + 10% glycerol* (made up in cathodic water or cryoprotectant
prepared in distilled water {control})
Flash drying of axes to appropriate WCs (cathodic flash drying; conventional flash drying {control})
Cooling (nitrogen slush)
Thawing & Rehydration (cathodic water; CaMg {control})
Decontamination
Regeneration on appropriate medium
Rehydration (cathodic water;
CaMg {control})
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Fig. 2: Axes of T. dregeana producing roots and shoots or roots only after exposure to pre-culture and AsA + cathodic water post-excision soaking (n=20). AsA, ascorbic acid.
254x190mm (300 x 300 DPI)
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Fig. 3: Reactive oxygen species production and total aqueous anti-oxidant (TAA) levels in excised axes of T. dregeana after pre-culture and post-excision soaking treatments (n=4 for ROS estimations and n=10 for TAA activity). AsA, ascorbic acid. Error bars represent mean ± standard deviation. Letters above bars
represent significant differences between treatments. 254x190mm (300 x 300 DPI)
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Fig. 4: Axes of T. dregeana producing roots and shoots after exposure to pre-culture, post-excision soaking (AsA + cathodic water) and cryoprotection (n=20). AsA, ascorbic acid; PC, pre-culture.
254x190mm (300 x 300 DPI)
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Fig. 5: Reactive oxygen species production and TAA levels by excised axes of T. dregeana after exposure to pre-culture, AsA + cathodic water post-excision soaking and cryoprotection (n=4 for ROS estimations and n=10 for TAA activity). Error bars represent mean ± standard deviation. Letters above bars symbolise
significant differences. AsA, ascorbic acid; PC, pre-culture. 254x190mm (300 x 300 DPI)
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Fig. 6: Axes of T. dregeana producing roots after exposure to dry cathodic and non-cathodic desiccation treatments (n=20). Axes in both treatement regimes rehydrated in CaMg. AsA, ascorbic acid; PC, pre-
culture; FD, flash dried.
254x190mm (300 x 300 DPI)
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Fig. 7: Reactive oxygen species production and TAA levels by excised axes of T. dregeana after exposure to dry cathodic and non-cathodic desiccation treatments (n=4 for ROS estimations and n=10 for TAA activity). AsA, ascorbic acid; FD, flash dried; PC, pre-culture. Error bars represent mean ± standard deviation. Letters
above bars symbolise significant differences. 254x190mm (300 x 300 DPI)
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Fig. 8: Axes of T. dregeana producing roots only or shoots and roots after exposure to a selected drying treatment and wet cathodic or non-cathodic rehydration (n=20). AsA, ascorbic acid; PC, pre-culture; FD,
flash dried.
254x190mm (300 x 300 DPI)
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Fig. 9: Reactive oxygen species production and TAA levels by excised axes of T. dregeana after exposure to a selected drying treatment and wet cathodic or non-cathodic rehydration (n=4 for ROS estimations and n=10 for TAA activity). PC, pre-culture; FD, flash dried. Error bars represent mean ± standard deviation.
Letters above bars symbolise significant differences. 254x190mm (300 x 300 DPI)
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Fig. 10: Reactive oxygen species production and TAA levels by excised axes of T. dregeana after exposure to cathodic water or CaMg thawing and rehydration after the selected drying and cooling treatment (n=4 for ROS estimations and n=10 for TAA activity). PC, pre-culture; FD, flash dried; LN, liquid nitrogen. Error bars
represent mean ± standard deviation. Letters above bars symbolise significant differences. 254x190mm (300 x 300 DPI)
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