The role of reactive oxygen species and anti-oxidants during role of reactive oxygen species and...

Draft

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

https://mc06.manuscriptcentral.com/botany-pubs

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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: [email protected]

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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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.


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.


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.


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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Draft

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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Botany

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