Introduction

Potato (Solanum tuberosum L.) suffers from undesirable sprouting during storage for the fresh market, prior to industrial processing, and when tubers of early harvests are to be used as seed tubers. This serious problem occurs when dormancy is broken and sprouting is activated (Coleman 1987). Dormancy is a complex set of physiological states and conditions in which plants respond to a series of stresses, such as drought and overwintering, by entering into a state of growth suspension (Campbell et al. 2008). Growth suspension can result from different causes. The classification and degrees of dormancy found in plant meristems have been defined as endodormancy, paradormancy, and ecodormancy (Lang et al. 1987). Endodormancy is due to an endogenous signal that results in growth suppression. In some situations, such as in the case of stored potatoes, time is all that is required for endodormancy to terminate, although in most potato cvs endodormancy length is shortened by very low temperatures (Turnbull and Hanke 1985). Control of tuber dormancy is critical for both seed and ware potato storage (Sorce et al. 2005) since postharvest sprouting leads to alterations in weight, turgidity, and texture. Cold temperature storage (2–4°C) delays sprout development, but also results in unacceptable tissue sweetening (Coffin et al. 1987). Successful long-term storage of potatoes necessitates the use of a sprout inhibitor in combination with proper storage management. Chlorpropham (isopropyl N-(3-chlorophenyl) carbamate; CIPC) is the most effective post-harvest sprout inhibitor registered for use in potato storage. This product has been used successfully as a sprout inhibitor for more than 40 years. CIPC must be applied in the window between the postharvest wound-healing period and dormancy break or initiation of sprout growth. It inhibits sprout development by interfering with cell division through the specific inhibition of mitosis (Nurit et al. 1989) and is effective at maintaining long-term sprout control (Wiltshire and Cobb 1996). However, new limits on both total CIPC application and residue have now been put in place by the Advisory Committee on Pesticides in the UK. Random sampling has shown the extant potential to exceed the maximum residue limit of 10 mg kg−1, even when applications are performed according to best practices. Moreover, potato seed tubers cannot be treated or stored in CIPC storage rooms because of the chemical’s long-term negative effect on field germination (Conte et al. 1995). Due to increasing concern for consumer health and safety, there is considerable interest in finding effective potato-sprouting suppressants that have a negligible environmental impact. Previous research has concentrated on such compounds as ethylene (Prange et al. 1998), ozone (Daniels-Lake et al. 1996), H2O2 (Afek et al. 2000), volatile monoterpenes, aromatic aldehydes, and alcohols (Vaughn and Spencer 1993; Hartmans et al. 1995). To date, only one monoterpene (S)-(+)-carvone (S-5-isopropenyl-2-methyl-2-cyclohexenone), a chemical produced from caraway (Carum carvi) seeds that was described as a volatile sprout suppressant more than 30 years ago (Meigh 1969; Beveridge et al. 1981), has been developed commercially. However, higher production and application costs compared with traditional sprout suppressants such as CIPC have limited its use primarily to the Netherlands. S-carvone has been shown to prevent sprouting and reduce the development of microbial activity in potatoes (Oosterhaven et al. 1995). This plant-derived compound can be applied on certified organic crops and is expected to leave behind little or no residue, because of its high volatility (Hartmans et al. 1995). However, the mode of action of sprouting control by carvone remains unclear. Here, we examine the advantages of using mint essential oil (MEO; extracted from Mentha spicata L.) on sprout inhibition. The active component in this essential oil is R-(−)-carvone (R-carvone). Carvone has two mirror image forms or enantiomers: S-(+)-carvone smells like caraway; its mirror image, R-carvone, smells like spearmint (Leitereg et al. 1971). We found that MEO and synthetic R-carvone delay sprouting and maintain tuber firmness during storage (Eshel et al. 2008). In this study, we show specific tissue damage to the tuber apical buds caused by inhibitory doses of MEO and its active chemical, R-carvone, and induction of sprouting by low doses of the same.

Materials and methods

Plant tissue

Eight potato cultivars that are commonly grown in Israel and differ in their length of dormancy, were used: ‘Bellini’, ‘Mondial’, ‘Désirée’, ‘Karlena’, ‘Eos’, ‘Nicola’, ‘Rodeo’, and ‘Winston’. Tubers were grown in two main areas in Israel, Sharon, and the northern Negev, under standard cultural conditions, from 2006 to 2009. Harvested tubers were cured for 2 weeks, during which time the temperature was gradually reduced from 25 to 8°C, and then stored at 8°C under 95% humidity generated by an ultrasonic humidifier (SMD Technology, Rehovot, Israel). To remove the effect of the MEO, tubers were washed in tap water for 2 min and kept in a 20°C room with circulating air.

MEO and R-carvone treatment

A commercially available product of raw material extracted from spearmint (Xeda International, Saint Andiol, France) was used for thermal fogging (Electro-fogger, Xeda International). Monthly thermal fogging with 30 ml MEO t−1 was initiated after 2 weeks of curing. During application, fresh-air inlets were closed and the humidifier shut off, and the storage air was recirculated for 24 h. In each treatment, an average 500 kg of tubers in net bags were stored in a pile to make MEO thermal fog penetration more difficult. Treatment was applied in 12-m3 storage rooms, at 8°C and 95% humidity generated by an ultrasonic humidifier. Experiments conducted in 2-l jars were performed by dripping MEO or R-carvone (Sigma-Aldrich, Rehovot, Israel) (0.5–9 μl l−1 air) on filter paper (Whatman #1; 2.3-cm diam.; Whatman, Maidstone, Kent, UK) which was placed inside the cover immediately before sealing the jar.

Tuber storage quality parameters

Sprouting was measured by weighing the sprouts in each tuber and dividing them by total tuber weight, thereby minimizing the effect of tuber size on sprout weight. We measured sprouting in 50 tubers (150 ± 25 g each) in four replicates monthly up to 6 months of storage. Weight loss was measured by weighing 50 tubers in the same box monthly in four replicates. Softening was characterized by manual pressure using a scale from 0, representing hard to 5, representing very soft tubers. Two people (the same throughout all experiments) performed the assessments on a whole-experiment basis and the scores were averaged between assessors. The accuracy of softening measurements was found to be higher than using Durometer (Shore Instrument and Manufacturing, Jamaica, NY, USA).

Histological analysis of MEO and R-carvone damage

Histological analyses were performed on 10-μm-thick bud meristem sections cut by microtome. Samples were handled according to the method described by Kamenetsky (1994) with minor modifications. Tissues were fixed overnight in FAA (10% formaldehyde, 5% acetic acid, 50% ethanol), then dehydrated in increasing concentrations of ethanol (25, 50, 75, 95, and 100%), cleared with histoclear, and embedded in paraffin. Prepared sections were spread on microscope slides, rehydrated, and stained with 1% (w/v) Safranin followed by 0.2% (w/v) Fast Green. Sections were dehydrated in the ethanol series and mounted with Permount (Fisher Scientific, Leicestershire, UK). Slides were examined at 16–40× magnification under bright-field using a Leica light microscope equipped with a camera.

To detect membrane damage, apical meristems of potato tubers from each treatment were sliced by hand and immediately stained with a final concentration of 25 μM FM4-64 (Molecular Probes Inc., Eugene, OR, USA) for 5–20 min (Bolte et al. 2004). An IX81/FV500 laser-scanning microscope (Olympus) was used to observe fluorescently labeled cells with the following filter sets: 488 or 515 nm excitation and BA660.

Analysis of volatiles in MEO and in the extracted potato sprouts

To prepare the MEO and R-carvone samples for GC–MS analysis, 30 μl of each sample was diluted in 1 ml petroleum ether (~1:30,000). Monoterpene derivatives from potato sprout samples were obtained by solvent extraction with HPLC-grade methyl-tert-butyl-ether (MTBE) containing 1 ppm isobutylbenzene as an internal standard. The solvent-to-plant material ratio was 10:1 (v/w). Samples were shaken for 24 h at 25°C in a closed scintillation vial and filtered through Pasteur pipettes containing silica gel (230–240 mesh, Merck, Darmstadt, Germany) and anhydrous sodium sulfate (Na2SO4). The analyses were performed on a gas chromatography–mass spectrometry (GC–MS) apparatus (Agilent Technologies, Palo Alto, CA, USA) with autosampler Combi PAL (CTC Analytic, Zwingen, Switzerland) using an Rtx-5SIL MS (“Restek”) 95% dimethyl-5% diphenyl polysiloxane column (30 m × 0.25 mm i.d. × 0.25 μm) (Larkov et al. 2007). Helium was used as the carrier gas at constant pressure with retention-time locking. The pressure range was 8–14 psi (linear velocity 32 cm s−1 at 8 psi) to achieve a constant retention time for internal standard of 7.5 min on a non-polar column. 1 μl MEO and R-carvone samples were injected in split mode at a ratio of 1:50; and 1 μl of the potato extract samples were injected in splitless mode. The injector was kept at 250°C and the transfer line at 280°C. The MS was operated in electron ionization (EI) mode at 70 eV in an m/z range of 42–350. Oven parameters were 50°C for 1 min, 50–200°C at 5°C min−1, 200–280°C at 20°C min−1, and maintaining at 280°C for 10 min. Identification was performed on the basis of two parameters: MS and linear retention index (LRI). For MS identification, the comparison was performed with MSDChem software (Agilent) with commercial libraries NIST 98, Wiley 7 and QuadLib 2005 (Adams 2001). The LRI determination was carried out by injecting a homologous series of n-alkanes under the same chromatographic conditions.

Results

MEO delays sprouting and maintains tuber quality

Over the 3 years of the study, we found that monthly thermal fogging with MEO inhibits sprouting for at least 6 months in all treated cultivars (Fig. 1a, b). To determine the breadth of MEO’s effect on sprout control, experiments were conducted in 4 replicas of 100 tubers each in 8 potato cultivars and experiment was repeated twice for each cultivar (Fig. 1a, b). Non-treated potato tubers stored at 8°C began to sprout after about 3 month of storage. The average number of sprouts per tuber was 2.2 ± 0.3 in all tested cultivars, and sprouts reached 1.5 ± 0.2 to 12.7 ± 0.9% of tuber weight after 6 months of storage in cvs. Nicola and Karlena, respectively (Fig. 1a). MEO fogging prevented sprouting completely while causing black necrotic symptoms in all tuber meristems with no visible damage to the tuber skin. Due to sprouting prevention, weight loss and softening were reduced (Fig. 1c, d). After 140 days of storage, non-treated ‘Désirée’ potato tubers had lost 7.2% of their weight as compared with their treated counterparts which lost only 2.68% (Fig. 1c). Similar results were obtained with other cultivars (data not shown). In addition to reducing tuber weight loss during storage, MEO maintained tuber firmness. On a scale of 0–5 (0 representing hard and 5, soft), we estimated that none of the treated cultivars ranked higher than level 2 (representing the limit of marketability) after 140 days of storage. Non-treated ‘Nicola’ and ‘Winston’ tubers tended to become softer than ‘Eos’ and ‘Rodeo’, but in all cases the cultivars were softer than their treated counterparts (Fig. 1d). We concluded that sprouting inhibition probably delays tuber softening and weight loss.

Fig. 1
figure 1

Effect of mint essential oil (MEO) thermal fogging on potato sprouting in storage. a Tubers from eight cultivars were stored for 6 months. b Typical cv. Nicola sprouting tubers after 6 months of storage (upper picture) compared to the MEO-treated tubers (lower picture). c Weight loss of cv. Désirée tubers during storage. d Softening of tubers during storage on a scale of 1 (hard) to 5 (soft); dashed line represents the level above which potatoes are no longer marketable. Tubers in c and d were stored for 140 days after curing. All tubers were stored at 8°C and 95% humidity and were thermally fogged monthly with MEO at 100 ml t−1 in the first application and 30 ml t−1 monthly in subsequent applications. Error bars represent SE, n = 4

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MEO inhibitory action may be nullified

Histological observation of the tuber apical meristem revealed that MEO treatment causes damage to the vascular tissue in the first stage, probably leading to total bud necrosis (Fig. 2). During storage, the bud meristem generally remained viable with only minor tip necrosis as storage progressed, probably as a result of oxidation (Fig. 2a). Two days after thermal fogging with MEO, apical meristems stained with Safranin/Fast Green showed damage in the meristem tips and vascular tissue (Fig. 2b) leading to necrosis of the entire emerging bud meristem and some of its lower cortex in the following 5–7 days (Fig. 2c). Four weeks after treatment MEO inhibitory effect has been decreased and axillary bud (AX) was observed next to necrotic apical meristem (Fig. 2d). Washing potato tubers with water a few days to after treatment almost completely eliminated MEO’s effect in all eight cultivars, and axillary meristem sprouting occurred with only a few days’ delay. In cv. Nicola, which is characterized by strong apical dominance during sprouting (Fig. 2e), washing the tubers with tap water led to sprouting of most of the lateral eyes of the tuber simultaneously (Fig. 2f).

Fig. 2
figure 2

Effect of mint essential oil (MEO) on potato apical meristem and sprouting. a Untreated meristem. b Limited damage to bud meristem and vascular tissue (red arrows) caused by MEO 2 days after treatment. c Bud meristem treated with MEO 7 days after treatment. d Axillary bud (AX) growth associated with apical meristem necrosis 4 weeks after treatment with MEO. Samples were stained with Safranin/Fast Green and observed under a light microscope. Scale bar is 200 μm (a, b), 500 μm (c, d). e, f Potato tubers after washing with water to remove the effect of MEO, and incubation at 20°C for 21 days: e non-treated, f after treatment with MEO. Note that removal of MEO caused loss of apical dominance. Inset in f is an enlargement of the apical meristem

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Active component of MEO

We analyzed MEO components by GC–MS and found 73% R-carvone. We compared sprout-control effects of MEO and R-carvone: 6–9 μl l−1 of synthetic R-carvone (98%) or the equivalent amount of MEO for 24–96 h in 2-l jars caused the same sprout-inhibition effect (data not shown). Surprisingly, we found that applying very low doses of MEO or R-carvone (0.5–1 μl l−1 air for 96 h) induces earlier AX sprouting in the treated tubers (Fig. 3). Based on previous studies by us and others (Hartmans et al. 1995; de Carvalho and da Fonseca 2006; Elmastas et al. 2006), we assume that R-carvone is the active material in MEO. A sprout-inducing dose (0.5 μl l−1 air) caused necrosis of only the apical meristem tip and induced AX induction and usually, growth of three to four meristems in the same sprout eye (Fig. 3e). The sprout-inhibiting dose (4.5–9 μl l−1 air) caused necrosis of the entire apical meristem and some of its underlying cortex (Fig. 3f).

Fig. 3
figure 3

Differential dose effect of R-carvone on potato sprouting. Tubers from cv. Nicola were exposed to four doses of R-carvone (a, b, c, d represent 0, 0.5, 1.5 and 4.5 μl l−1 of air, respectively) for 5 days at 24°C. R-carvone was added every 24 h after ventilation of 2-l jars with fresh air. e Axillary meristem growth induction by low dose of R-carvone (0.5 μl l−1). f Total meristem necrosis caused by inhibitory dose of R-carvone (9 μl l−1). Scale bar is 100 μm for e and f

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R-carvone mode of action

Since R-carvone is lipophilic and therefore cannot be dissolved in water, we hypothesized that it affects the cell membrane. Bud meristems were stained with membrane-selective dye (FM4-64) (Bolte et al. 2004) and scanned by confocal laser scanning microscopy after exposing the tuber to R-carvone every 24 h for 96 h: cell membranes were found to be damaged mainly in the meristem tip (Fig. 4). In non-treated tubers and at low doses of R-carvone, minor damage to the membranes was found (Fig. 4). Equivalent dose of MEO had the same effect on membrane integrity (data not shown).

Fig. 4
figure 4

Effect of R-carvone treatment on cell membranes of apical meristem of potato tuber. a, b, c Untreated meristem. d, e, f Meristem treated with 0.5 μl R-carvone oil l l−1 air. g, h, i Meristem treated with 9 μl R-carvone oil l−1 air every 24 h for 96 h. Samples were stained with FM4-64 (Bolte et al. 2004) and observed with a confocal microscope. Scale bar is 200 μm (a, b, d, e, g, h) and 20 μm (c, f, i). a, d and g Merged light and confocal images. b, c, e, f, h and i Confocal images. Arrows in b, e and h indicate membrane damage caused by R-carvone in the apical tip. Organelles (arrow) are observed in i as a result of membrane damage caused by R-carvone

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Extraction and analysis of potato sprouting eyes exposed to R-carvone showed bioconversion of the monoterpene to its reduced compounds (Fig. 5). Potato tubers exposed to 0, 0.5, 9 and 100 μl R-carvone l−1 air for 72 h were washed and their sprouting eyes were extracted. R-carvone was detected in all treated sprouting eyes in amounts that were correlated to the applied dose (Table 1). The analysis also showed the bioconversion of R-carvone [(4R)-carvone] to its reduced compounds (4R,6S)-transcarveol and (1R,2S,4R)-neo-dihydrocarveol (Fig. 5). Although conversion products were detected, R-carvone still constituted 100, 75, and 73.6% of the total carvone products when 0.5, 9, and 100 μl R-carvone l−1 air was applied, respectively (Table 1). (4R,6S)-transcarveol constituted 7.9 and 12.5% and (1R,2S,4R)-neo-dihydrocarveol was found at slightly higher levels, 17.3 and 13.9% of the total R-carvone products when 9 and 100 μl R-carvone l−1 air was applied, respectively (Table 1). Reduced R-carvone compounds were not detected when tubers were exposed to the sprout-inducing dose of 0.5 μl R-carvone l−1 air for 72 h.

Fig. 5
figure 5

Structure of R-carvone [(4R)-carvone] and its reduced compounds upon bioconversion

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Table 1 Concentration of R-carvone and its derivatives in potato sprouting eyes after treatment with different amounts of R-carvone in the gassed phase for up to 72 h
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Discussion

Potato dormancy is defined as the physiological state of the tuber in which autonomous sprout growth will not occur, even under conditions that are ideal for sprouting. Dormancy release and sprouting during storage constitute a major problem in tubers supplied to the fresh, industrial, and seed markets. MEO was able to inhibit sprouting in all eight cultivars tested, indicating a general effect. Inhibition of potato sprouting during storage by thermal fogging with MEO probably delays metabolic changes in potato tubers, leading to most of the treated tubers being marketable after more than 6 months of storage. Sprout inhibition was strongly correlated to minimization of softening, probably because most of the tuber water loss is due to evaporation via the sprouts. During sprouting, tubers undergo a functional transition from active sink for assimilates to source of nutrients for the developing sprouts (Prat et al. 1990; Visser et al. 1994). The mobilization and transport of carbohydrates and other nutrients from the storage parenchyma into the growing buds is believed to cause weight loss and softening as well (Fernie and Willmitzer 2001).

MEO and its active chemical, the monoterpene R-carvone, inhibited sprout growth, probably via physical damage to the meristem tip; after the inhibitor’s removal, the tuber sprouts through AX growth. Inhibition of seed germination by several alicyclic and heterocyclic compounds, including some monoterpenes, has been documented (Asplund 1968; Fischer 1986; Reynolds 1987). The biological activity of alicyclic compounds is related to their lipophilicity (Reynolds 1987). Indeed, 3-day exposure of Allium cepa roots to the monoterpene cineole resulted in disturbances in membrane integrity (Lorber and Muller 1976). The functional α,β-unsaturated keto group of carvone, including its spatial orientation, has also been shown to be important for germination inhibition (Asplund 1968; Reynolds 1989). Initial exposure of tubers to R-carvone probably results in significant membrane damage and apical meristem stress, and the tuber recovers from this stress by inducing AXs. We can assume that repetition R-carvone treatment is necessary to physically damage the newly induced AX leading to inhibition of sprouting for as long as needed to preserve tuber quality.

In cv. Nicola, which is characterized by apical dominance during sprouting, washing the tubers probably leads to MEO removal from the tuber surface, which results in sprouting of most of the lateral eyes simultaneously. Growth of axillary meristems is usually inhibited by the shoot apical meristem, in a process termed apical dominance (Shimizu-Sato and Mori 2001). Although these interactions between the apical and axillary meristems are genetically determined, they are also mediated by internal and external cues such as hormone levels, light, or mechanical stimuli (Sussex and Kerk 2001). Auxin has been reported to restrict and cytokinin to promote, the growth of axillary meristems (Rosin et al. 2003). The origin of axillary meristems differs among plant species. In potato and tomato, axillary meristems are derived from the shoot apical meristem (Sussex 1955), whereas in Arabidopsis, they are initiated in leaf axils from cells on the adaxial surface of the substanding leaf (Schmitz and Theres 1999). There are two stages of axillary meristem development: initial formation and subsequent growth. After axillary meristem initiation, the apical meristem maintains its role as the primary site of growth by inhibiting growth of the axillary meristems. Working with a volatile essential oil such as MEO makes it difficult to treat only one eye of the tuber, and thus two explanations may be valid for the loss of apical dominance after treatment: one is alteration of the auxin-to-cytokinin ratio between the apical and lateral eyes in the same tuber. Since each meristem has the ability to induce AXs, the second possibility is that axillary growth from either the lateral or apical meristem is prevented by the physical damage imposed by MEO, which causes loss of hormone-producing ability.

Low doses of MEO or synthetic R-carvone induced earlier sprouting of treated tubers, as a result of induction of AX growth. To the best of our knowledge, this is the first report of an essential oil vapor inducing early sprouting in potato tubers. A similar differential dose effect has been observed with H2O2, used to control sprouting (Bajji et al. 2007). Applying H2O2 at a high dose four times during storage caused complete sprouting inhibition via physical damage to the bud tips (Afek et al. 2000). In contrast, tubers that were treated with the low dose, of 20 mM H2O2, showed a reduced dormancy period and rapid synchronized sprouting of the treated tuber (Bajji et al. 2007). Those authors concluded that manipulation of H2O2 metabolism controls tuber dormancy and sprouting in potato via inhibition of catalase activity (Bajji et al. 2007). Chemically induced dormancy cessation can also be achieved using other toxic compounds such as hydrogen cyanamid (Northcott and Nowak 1988) and bromoethane (Law and Suttle 2003; Alexopoulos et al. 2009). Bromoethane is believed to cause significant stress, from which tissue recovers in a non-dormant state (Campbell et al. 2008). The mechanism by which low doses of R-carvone induce sprouting still remains to be elucidated, but might be related to its sublethal toxicity to plant tissue.

Bioconversion of the active component R-carvone to its reduced compounds might explain its nullified and adverse effect on sprouting. When R-carvone was applied at a low, sprout-inducing dose, the reduced compounds were not detected in the sprouts, possibly because their amounts were below the limits of detection or because different pathways were triggered by the very low dose. Oosterhaven et al. (1995) reported that S-carvone extracted from caraway is converted by potato sprouts mainly into neo-isodihydrocarveol, but the other diastereomer isodihydrocarveol was also detected. The conversion occurred via dihydrocarvone and isodihydrocarvone, intermediates that were detected in significant amounts in the sprouts (Oosterhaven et al. 1995). However, our results showed a stereoselective reduction whereas only transcarveol and neo-dihydrocarveol were detected. The ability of potato tuber to convert R-carvone, probably via an enzymatic pathway, might be related to a reservoir of metabolic capabilities that normally remains hidden or unused (Dudai et al. 2000; Lewinsohn and Gijzen 2009).