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“Rock-Ad”, a new wild rocket (Diplotaxis tenuifolia) mutant with late flowering and delayed postharvest senescence
Scientia Horticulturae 174 (2014) 17–23
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“Rock-Ad”, a new wild rocket (Diplotaxis tenuifolia) mutant with late flowering and delayed postharvest senescence David Kenigsbuch ∗ , Alona Ovadia, Yelena Shahar-Ivanova, Daniel Chalupowicz, Dalia Maurer Department of Postharvest Science of Fresh Produce, Institute of Postharvest and Food Sciences, Agricultural Research Organization, The Volcani Center, PO Box 6, Bet Dagan 50250, Israel
a r t i c l e
i n f o
Article history: Received 4 February 2014 Received in revised form 28 April 2014 Accepted 30 April 2014 Keywords: Wild rocket Postharvest Senescence Flowering
a b s t r a c t Wild rocket (Diplotaxis tenuifolia) is a leafy herb in salads and cooked dishes originating in the Mediterranean basin. The leaves contain many nutrients, plus beneficial phytochemicals such as flavonoids and glucosinolates. Rocket suffers from two major limitations: early bolting during growth and leaf yellowing after harvest, both of which affect both yield and market quality. We have used ethyl methanesulfonate (EMS) to induce mutation in wild rocket, and selected a mutant line (registered as cv. “Rock-Ad”), which has a two week delay in flowering time compared to current commercial cultivars. “Rock-Ad” plants began to flower when more than 50% of commercial plants had flowered. Storage properties of the mutant were also better than the commercial cultivar. In storage, after simulation of shipment by sea, there was no yellowing in young “Rock-Ad” leaves, chlorophyll levels were higher, fewer flower buds developed, and hydrophilic antioxidant levels decreased less than the commercial cultivar. Holding leaves in 5 kPa CO2 in air decreased yellowing and decay significantly in both cultivars compared to air storage, while exposure to 2.23 mol L−1 ethylene increased yellowing and decay. When ethylene and CO2 were applied together (5 kPa and 2.23 mol L−1 , respectively), yellowing and decay decreased in both cultivars compared to air storage. 1-MCP treatment reduced yellowing and decay in leaves of the commercial variety, but had no effect on leaves of “Rock-Ad”. The respiration rate of the leaves increased after 7 days of storage in both cultivars, but was significantly higher in leaves of the commercial cultivar, and 1-MCP significantly moderated this increased respiration. Although treatment with 1-MCP led to increased ethylene levels on the first day after harvest, during storage, ethylene levels decreased in all treatments and cultivars. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Wild rocket (Diplotaxis tenuifolia) from the Brassicaceae family can be found growing freely in most Mediterranean countries. The plants have two rows of seeds in the seed pods, hence the name Diplotaxis, from Greek diploos (double) and taxis (row), and slender leaves, thus the species name tenuifolia from Latin tenuis (slender) and folium (leaf). It is considered a perennial plant since its roots can survive the winter and sprout again the next spring (Pignone, 1997). Wild rocket was thought to be an aphrodisiac and has many medical utilities (Bianco and Boari, 1997). The leaves contain vitamin C, fibers, flavonoids and glucosinolates (Crozier et al., 1997; Barillari et al., 2005; Bennett et al., 2006, 2007; Heimler et al., 2007; Martínez-Sánchez et al., 2007, 2008).
∗ Corresponding author. Tel.: +972 3 9683600; fax: +972 3 9683622. E-mail address: [email protected] (D. Kenigsbuch). http://dx.doi.org/10.1016/j.scienta.2014.04.038 0304-4238/© 2014 Elsevier B.V. All rights reserved.
There are two major limitations to commercial production of rocket: early bolting during spring and autumn, about 35 days after germination, and leaf yellowing after harvest. Both problems affect the yield and market quality. Another commercial type of rocket (Eruca sativa), a closely related species, flowers 55–68 days after sowing (Yaniv, 1995; Morales and Janick, 2002). A search for a latebolting rocket (E. sativa) germplasm resulted in a cultivar named “Adagio”, which has delayed flowering of up to four weeks, compared to the wild-type (Morales and Janick, 2002; Morales et al., 2006). Postharvest quality is the second problem of wild rocket. Leaves rapidly turn yellow and then are not suitable for marketing. Leaf yellowing occurs due to chlorophyll degradation. Ethylene is known to increase the rate of chlorophyll degradation, whereas controlled atmosphere (CA) in which CO2 level is elevated (5–8%), decreases the degradation rate (Watada et al., 1996; Kader, 1995; Kader, 1986; Yamauchi and Watada, 1993). Respiration rate is also affected by storage conditions, which in turn affects quality. CA
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conditions decrease respiration. In mint, for example, respiration was lower in conditions of 5 kPa CO2 than in air storage. Respiration was not affected by 1-MCP, an ethylene action inhibitor, but ethylene production was elevated, and was further increased under a combined treatment of 1-MCP together with 5 kPa CO2 . However, in the presence of exogenous ethylene, CO2 strongly suppressed the 1-MCP-induced ethylene production (Kenigsbuch et al., 2007a). In a preliminary report on the M2 generation of the wild rocket mutant we demonstrated a delay in flowering and a greater number of leaves at harvest time in the mutant compared to the commercial wild rocket cultivar (Kenigsbuch et al., 2009). In this work we present further analysis of flowering and post harvest leaf qualities of the “Rock-Ad” cultivar including chlorophyll and antioxidant levels, respiration rate and ethylene production, as well as the influence of exogenous ethylene, 1-MCP and controlled atmosphere.
USA) according to the supplier’s instructions, with concentrations determined by gas chromatograph to produce a stock of 1000 L L−1 concentration inside a sealed bottle. Treatment with 1MCP was achieved by injecting a single dose of 1-MCP into 2-L jars containing wild rocket leaves (about 15 cm long, 100 g) which were then maintained fully sealed for 4 h in the dark at 17 ◦ C to achieve a concentration of 0.5 mol L−1 . Control jars (CK) were sealed without 1-MCP. Following this initial period, air flow of 50 mL min−1 was commenced, with three replicates per treatment. The jars were held for 7 days at 17 ◦ C. Leaf yellowing and decay were evaluated according to the yellowing and decay indices (see Section 2.6), CO2 and ethylene levels were measured as described below (Section 2.5).
2. Materials and methods
CO2 was determined by gas chromatograph (GC) equipped with a thermal conductivity detector (GOW-MAC instruments Co. series 580, PA, USA) with a double-column CTR-1 (Altech, 6 ft × 1/4 in.), carrier gas helium, oven temperature 30 ◦ C. Ethylene levels were measured by a GC with flame ionization detector (FID) (Varian 3300; Walnut Creek, CA, USA). using packed alumina column (Altech 4 ft × 1/8 in.), carrier gas helium, oven temperature 100 ◦ C, injection temperature155 ◦ C, detector temperature 100 ◦ C.
2.1. Plants Local commercial wild rocket (D. tenuifolia) and the “Rock-Ad” mutant (M4 generation and onward) (Kenigsbuch et al., 2009) were used for all experiments. Plants were grown commercially in net houses in the winter–spring seasons in the Negev in southern Israel. Seeds of the “Rock-Ad” mutant were given to commercial nursery to produce seedlings as the procedure for the commercial line, about twelve seeds per pot. Three weeks old Seedlings from both cultivars were planted in growers net houses. The seedlings density on beds was 60 seedlings per meter bed, 10 cm between the seedlings. Irrigation and fertilization were given according to the growers’ procedure. From each experiment 4–5 harvests were examined for quality parameters after shipment and shelf life simulation. For the flowering time experiments plants were grown in net house during the winter–spring seasons, one plant per 300 gr pot. The time of flowering and percentage of flowering plants was determined by examining the plants and counting every 3–4 days after the first flower appeared. 2.2. Packaging and temperature conditions Freshly cut leaves were placed in ventilated atmosphere (in polyethylene bags with multi microperforation (MMP)), with three replicate 1 kg bag for each sample. Bagged leaves were held for six days at 3 ◦ C followed by six days at 6 ◦ C and finally two days shelf life at 17 ◦ C as a simulation of conditions obtaining during shipment by sea. The leaves were either from young rosettes with maximum 12–15 cm long leaves or old rosettes with maximum 20–22 cm long leaves. 2.3. Ethylene and CO2 in storage Experiments were carried out with freshly harvested wild rocket leaves. All experiments were repeated three times with three replications per experiment. Each wild rocket bunch (about 15 cm long, 100 g) was enclosed in sealed 2-L jars fitted with inlet and outlet ports allowing the plant material to be maintained under controlled atmospheres (CA) comprising of either air; 5 kPa CO2 in air; 2.23 mol L−1 ethylene in air; or 5 kPa CO2 plus 2.23 mol L−1 ethylene in air, with a flow rate of 50 mL min−1 . Gases were bubbled through sterile water to maintain high relative humidity. 2.4. 1-MCP treatment 1-MCP was released from a commercial powder formulation (Ethylbloc, Biotechnologies for Horticulture, Inc., Burr Ridge, IL,
2.5. Gas measurements
2.6. Yellowing and decay indices Leaf yellowing and decay were evaluated by subjective indices on day 14 at the end of the storage and shelf life period. The yellowing scale allows quantitative assessment of yellowing in accordance with marketability: 0%—fresh green leaves with no color change; 25%—more substantial reduction in color quality—up to 10% of leaves showing some levels of light green appearance; 50%—produce with this level of color loss is no longer of marketable quality—20% of leaves with color loss; 75%—produce not of marketable quality—60% of leaves with color loss; 100%—produce not of marketable quality—100% of leaves turned yellow. The decay index during leaf senescence with respect to marketability was: 0%—fresh marketable product, all leaves symptom-free; 25%—up to 5% of leaves showing lesions 3—10 mm in diameter; 50%—produce with this level of damage were no longer of marketable quality, 20% of leaves showed physiological or pathological damage; 75%—produce not of marketable quality, 60% of leaves showing lesions representing physiological or pathological damage; 100%—produce not of marketable quality, all leaves showing lesions representing physiological or pathological damage.
2.7. Chlorophyll quantification Freshly cut leaves, about 40 g, were placed in plastic bags with one hole (0.7 mm), three repeats for each sample. Leaves were placed for six days at 3 ◦ C, six days at 6 ◦ C and two days in shelf life at 17 ◦ C. Leaf samples were taken on the first day after harvest (day 0), on day 12 of storage and after shelf life (day 14), lyophilized for three days and stored in the freezer (−20 ◦ C, dark). Ground freezedried leaves (10 mg) were placed in 1 mL 80% acetone in a 2 mL glass tube and placed in 4 ◦ C overnight. Samples were diluted 1:20 before reading absorbance of chlorophyll a and b by spectrophotometer at 663 nm and 645 nm, respectively, and calculating concentrations as described by Bruinsma (1963). There were three replicates per experiment.
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Fig. 2. Chlorophyll quantification after storage. Wild rocket leaves were stored in simulation of sea shipment. Leaf samples were taken on the first day of harvest (day 0), on day 12 of storage and after shelf life (day 14). Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test. Fig. 1. Flowering plant (%) after sowing. Plants were grown in a net house. The counting started when the first flower appeared and every 3-4 days thereafter.
3.2. Postharvest quality 2.8. Antioxidant capacity The free radical scavenging capacity was evaluated in leaf samples according to the method described by Vinokur and Rodov (2006). Leaves were sampled on the first day after harvest (day 0) and at the end of the experiment (day 14), ground and extracted into lipophilic and hydrophilic fractions. The free radical scavenging capacity was measured by the rate of decolorization of 2,2 -azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid), diammonium (ABTS) radical cation. 6-Hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid (Trolox, a water-soluble derivative of the vitamin E) was used as a standard, and the radicalscavenging activity of samples was expressed as Trolox equivalent antioxidant capacity (TEAC). 2.9. Statistical analysis Data are means of three experiments with three replicates per treatment. The results were submitted to analysis of variance and the mean values were compared by Tukey’s multiple range test.
In young rosette leaves (12–15 cm max, plant age 30 days) the “Rock-Ad” leaves showed no yellowing after 14 days of simulated shipment and marketing compared to the commercial leaves (Table 1). In old rosette leaves (20–22 cm max, plant age 36 days) the yellowing was increased in leaves of both cultivars, but was significantly higher in the commercial leaves than in “Rock-Ad”. The appearance of flower buds during storage is an undesirable phenomenon which can disqualify a shipment. The young “RockAd” rosette had no flowers after 14 days of storage compared to commercial plants, in which some flowers could be found (Table 1). In older rosette leaves, some flowers were found in the “Rock-Ad” but significantly fewer than the number of flowers found in the commercial variety. Freshly cut wild rocket leaves from the commercial and “RockAd” cultivars were held in simulated seas shipment conditions. Young rosette–the older leaves in the rosette were 12–15 cm. Old rosette—the older leaves were 20–22 cm. Mean values were compared by Tukey’s multiple range test. Capital letters indicate statistical difference in yellowing; Lower case letters indicate statistical difference in flowers at P = 0.05 standard deviation is given. 3.3. Chlorophyll content
3. Results 3.1. Flowering time The commercial cultivar plants began to flower on the 28th day after sowing and had 50% flowering by day 39 (Fig. 1). The “RockAd” plants began flowering two weeks after the commercial plants and reached 50% flowering on day 48 post-sowing. When the commercial cultivar was at 50% flowering, “Rock-Ad” had not flowered yet (Fig. 1). Plants were grown in a net house. The counting started when the first flower appeared and every 3–4 days thereafter.
Commercial and “Rock-Ad” leaves had the same level of chlorophyll on harvest day (Fig. 2). On day 12 there was a small decrease in chlorophyll level in the commercial leaves, while levels remained the same in “Rock-Ad”. At the end of the experiment (day 14), the chlorophyll content in the commercial leaves had decreased by 40% while in “Rock-Ad” the chlorophyll loss was only 20%. Wild rocket leaves were stored in simulation of sea shipment. Leaf samples were taken on the first day of harvest (day 0), on day 12 of storage and after shelf life (day 14). Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test.
Table 1 Yellowing and flowers after harvest. Freshly cut wild rocket leaves from the commercial and “Rock-Ad” cultivars were held in simulated seas shipment conditions. Young rosette – the older leaves in the rosette were 12-15 cm. Old rosette – the older leaves were 20-22 cm. Old rosette
Line
Commercial Rock-Ad
Young rosette
Flowers in 1 kg
Yellowing (%)
Flowers in 1 kg
Yellowing (%)
12.1 ± 0.4 a 0.7 ± 0.2 b
34.5 ± 7.1 A 9.4 ± 3.2 B
1.4 ± 0.6 b 0c
11.3 ± 1.6 B 0C
Mean values were compared by Tukey’s multiple range test. Capital letters indicate statistical difference in yellowing; Lower case letters indicate statistical difference in flowers at P = 0.05 Standard deviation is given.
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Fig. 3. Antioxidant quantification at harvest and after storage. Wild rocket leaves were stored in simulation of sea shipment. Leaf samples were taken on the first day of harvest (day 0) and after 14 days of storage (day 14). A- Antioxidant levels in the lipophilic fraction, B- Antioxidant levels in the hydrophilic fraction. Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test.
3.4. Antioxidants On the first day after harvest (day 0) commercial leaves and “Rock-Ad” had similar levels of antioxidants in both the lipophilic and hydrophilic fractions (Fig. 3). At the end of the experiment (day 14), the lipophilic fraction of the antioxidants decreased significantly in both commercial and “Rock-Ad”. In the hydrophilic fraction, there was a significant decrease in the commercial leaves, while in “Rock-Ad” there was no significant decrease. Wild rocket leaves were stored in simulation of sea shipment. Leaf samples were taken on the first day of harvest (day 0) and after 14 days of storage (day 14). (A) Antioxidant levels in the lipophilic fraction, (B) Antioxidant levels in the hydrophilic fraction. Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test. 3.5. The effects of ethylene and elevated CO2 A CO2 atmosphere of 5 kPa decreased yellowing significantly in both commercial and “Rock-Ad” leaves, compared to air or ethylene treatments (Fig. 4A). Ethylene increased yellowing compared to air storage in both cultivars. When ethylene was given in the presence of 5 kPa CO2 , the amount of yellowing was similar to CO2 without ethylene present. In both CO2 and ethylene plus CO2 treatments, the yellowing was much less pronounced in “Rock-Ad” leaves compared to the leaves of the commercial cultivar. CO2 atmosphere significantly decreased decay in both commercial and “Rock-Ad” plants, while having ethylene in the air slightly increased the level of decay of both varieties compared to air storage (Fig. 4B). Decay development on leaves in ethylene in the presence of CO2 was similar to decay in CO2 atmosphere alone.
Fig. 4. Ethylene and elevated CO2 effect on postharvest quality. Freshly cut leaves were stored in simulation of sea shipment. Leaf yellowing and decay were evaluated on day 14. A- Yellowing, B- Decay. Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test.
Freshly cut leaves were stored in simulation of sea shipment. Leaf yellowing and decay were evaluated on day 14. (A) yellowing, (B) decay. Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test. 3.6. The effects of 1-MCP After treatment with 1-MCP, the commercial wild rocket leaves were significantly less yellow and had less decay compared to the untreated leaves (Fig. 5). The 1-MCP treatment did not affect the “Rock-Ad” leaves. The “Rock-Ad” leaves, as noted before, were less yellow and had less decay than untreated commercial leaves. Freshly cut wild rocket leaves from the commercial and “RockAd” cultivars were placed in 2-L jars and 1-MCP was injected to a concentration 0.5 mol L−1 and sealed for 4 h. Control jars (CK) were sealed without 1-MCP. After treatment, air flow of 50 mL min−1 commenced for 7 days at 17 ◦ C. Leaf yellowing and decay were evaluated according to the yellowing and decay indices. (A) yellowing, (B) decay. Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test. 3.7. Respiration rate and ethylene production Respiration rate did not increase until seven days after harvest (Fig. 6A). The commercial leaves had a 230% increase in respiration rate compared to the first day, while 1-MCP significantly moderated this increase. “Rock-Ad” leaves had only 50% higher respiration than at the beginning of storage, with no moderating effect of 1-MCP. On the first day after harvest ethylene levels were high in leaves treated with 1-MCP (Fig. 6B). Ethylene levels were significantly lower in “Rock-Ad” leaves without 1-MCP and lowest in the commercial leaves. During storage, ethylene levels decreased in all leaves, regardless of source or treatment.
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Fig. 5. 1-MCP treatment. Freshly cut wild rocket leaves from the commercial and “Rock-Ad” cultivars were placed in 2-L jars and 1-MCP was injected to a concentration 0.5 mol L-1 and sealed for 4 h. Control jars (CK) were sealed without 1-MCP. After treatment, air flow of 50 ml/min commenced for 7 days at 17 ◦ C. Leaf yellowing and decay were evaluated according to the yellowing and decay indices. A- Yellowing, B- Decay. Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test.
Freshly cut wild rocket leaves from the commercial and “RockAd” cultivars were placed in 2-L jars and 1-MCP was injected to a concentration 0.5 mol L−1 and sealed for 4 h. Following this initial period, air flow of 50 mL min−1 was commenced. The jars were held for 7 days at 17 ◦ C and CO2 and ethylene levels were determined by gas chromatograph. A—respiration rate, B—ethylene level. Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test. 4. Discussion Wild rocket is an important crop which is increasingly considered a salad green rather than a culinary herb. The main factors limiting continued expansion of commercial production are early flowering and limited storability. A previous study with transgenic Arabidopsis plants found that they flowered later and had better leaf storability than wild type plants (Kenigsbuch et al., 2007b; Ovadia et al., 2010). Based on this study, we decided to mutagenize wild rocket and from the mutagenized lines selected the new cultivar “Rock-Ad”, to address the problems of flowering and storage (Kenigsbuch et al., 2009). We found, as previously reported with the M2 generation of the mutagenized line, that “Rock-Ad” flowering time was delayed by two weeks compared to the commercial cultivar, and had improved storage life (Kenigsbuch et al., 2009). These results are consistent with studies showing that flowering time and leaf senescence have common regulatory factors (Golldack et al., 2002), although the opposite, late flowering and early senescence, has also been reported (Diaz et al., 2005). It is important to note that the regulation system is very complex and it is possible that different signals
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Fig. 6. Respiration rate and ethylene levels in detached wild rocket leaves. Freshly cut wild rocket leaves from the commercial and “Rock-Ad” cultivars were placed in 2-L jars and 1-MCP was injected to a concentration 0.5 mol L-1 and sealed for 4 h. Following this initial period, air flow of 50 ml/min was commenced. The jars were held for 7 days at 17 ◦ C and CO2 and ethylene levels were determined by gas chromatograph. A- respiration rate, B- ethylene level. Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test.
are affecting the phenomena. A number of different Arabidopsis mutants have been shown to have increased leaf longevity and late flowering compared with the wild type (Ellis et al., 2005; Wu et al., 2008). However, one mutant in a metalloproteinase gene had late flowering, but early senescence and degradation of chlorophyll (Golldack et al., 2002). In studies not involving mutants, different ecotypes of Arabidopsis that bolted late also senesced late (Levey and Wingler, 2005). Our results with the “Rock-Ad” mutation in rocket demonstrate a link between late flowering and delayed leaf senescence postharvest. To our knowledge, this is the first time a connection between late flowering and postharvest traits have been shown. “Rock-Ad” had significantly less yellowing of leaves in all the storage conditions, air, 5% CO2 , ethylene and a combination of CO2 and ethylene, than the commercial cultivar. The increased yellowing of both in the presence of ethylene is in agreement with changes during storage of other leafy products including parsley (Lers et al., 1998; Yamauchi and Watada, 1993), broccoli (Aharoni et al., 1985; Watada et al., 1996), and mint (Kenigsbuch et al., 2007a). However, since 1-MCP did not prevent the yellowing, but only reduced it in control leaves, ethylene is not the only regulatory process which leads to chlorophyll loss. Other processes may be involved in promoting chlorophyll loss, similar to what occurs in dark induced senescence in Arabadopsis leaves (Rossenwasser et al., 2011) In a study with salad rocket (E. sativa Mill.) stored at 10 ◦ C, it was found that 1-MCP in the absence of ethylene had only a small effect
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in prolonging shelf life. However, if 1-MCP was given before ethylene exposure, yellowing and chlorophyll loss were prevented (Koukounaras et al., 2006). It is possible that chlorophyll loss in wild rocket is under somewhat different regulatory control than wild rocket. Leaf yellowing and decay incidence in both plants were decreased in a 5 kPa CO2 atmosphere, nonetheless they were significantly lower in “Rock-Ad” plants than the commercial cultivar. This beneficial effect of high CO2 is consistent with the role of CO2 in maintaining visual quality (Martínez-Sánchez et al., 2006), delaying decay and acting as an ethylene antagonist (Aharoni et al., 2010). In parsley, CO2 treatment retarded chlorophyll and protein degradation, amino acid accumulation and respiration (Lers et al., 1998; Yamauchi and Watada, 1993). In mint, senescence phenomena include yellowing, browning, decay and leaf abscission, and increased levels of CO2 in a CA system inhibited these phenomena (Kenigsbuch et al., 2007a). In both salad rocket (Koukounaras et al., 2007) and wild rocket (Martínez-Sánchez et al., 2006) storage in low O2 and high CO2 partial pressures helped preserve sensory and microbiological quality of the leaves. The lipophilic antioxidant levels in wild rocket leaves decreased about 30% during simulated shipment and shelf life, while the hydrophilic antioxidant levels decreased 25% in the commercial cultivar and only 5% in “Rock-Ad”. Rocket leaves contain a wide range of compounds that contribute to antioxidant activity, including vitamin C, glucosinolates, flavonoids and phenolics (Jin et al., 2009). Martínez-Sánchez et al. (2006) found a decrease in the antioxidant capacity of wild rocket during 14 d at 4 ◦ C storage to a similar extent as in our study, and this was positively correlated with vitamin C content, and poorly correlated with total phenolics or with flavonoids. Jin et al. (2009) reported that in both salad and wild rocket gluocosinolates were reduced during 14 d at 4 ◦ C, while flavonoids did not decrease, and Kim and Ishii, 2007 also found decreases in glucosinolates of salad rocket during storage at either 4 ◦ C or 15 ◦ C. The higher levels of hydrophilic antioxidants in “Rock-Ad” leaves may indicate greater retention of vitamin C and glucosinolates during simulated shipment and shelf life. Respiration of the leaves was low during the first days of 17 ◦ C storage, and only increased on day 7, while ethylene was initially high and decreased steadily over time. Koukounaras et al. (2007) found the same kinetics of ethylene for salad rocket leaves held at 10 ◦ C, while the respiration had an initially high level which declined 50% after 3 days and remained steady. In contrast, Martínez-Sánchez et al. (2006) found a steady increase in respiration of wild rocket leaves held at 4 ◦ C and four fold higher CO2 evolution after 14 d than after 1 d. The differences in these reports may be partially due to the age of the leaves, as Koukounaras et al. (2007) found somewhat different kinetics of respiration and ethylene if the leaves were young, fully expanded, or old. Exposure to 1-MCP ameliorated the respiration increase on d 7 in the commercial leaves but had little effect on “Rock-Ad”. It is unclear yet whether the insensitivity of leaves of “Rock-Ad” to 1-MCP is caused by changes in the ethylene biosynthesis pathway or in the sensitivity to ethylene. On the other hand, 1-MCP increased ethylene production in both leaves in the first days of storage to a similar extent. This stimulating effect on ethylene production has been found in other leafy commodities (Kenigsbuch et al., 2007a; Jiang et al., 2002). It has been suggested that the feedback control of ethylene synthesis in leaves is induced by 1-MCP, even though the inhibitor blocks ethylene action (Jiang et al., 2002). In conclusion, the “Rock-Ad” wild rocket mutant is a promising new cultivar. It has later flowering than the commercial cultivar, and slower senescence. It retains chlorophyll and antioxidant contents longer in storage than the commercial cultivar. Altogether, it has better storage potential, making it suitable for sea shipment.
Acknowledgments We are very grateful to Dr. Susan Lurie and Dr. Joshua Klein for their critical review and editing. This manuscript is Contribution no. 674/3 from the Agricultural Research Organization, The Volcani Center, PO Box 6, Bet Dagan 50250, Israel.
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The effect of CO2 and 1-methylcyclopropene on the regulation of postharvest senescence of mint Mentha longifolia L. Postharvest Biol. Technol. 43, 165–173. Kenigsbuch, D., Ovadia, A., Tabibian-Keissar, H., Chalupowicz, D., 2007b. Circadian rhythm of ethylene related genes under postharvest conditions in transgenic Arabidopsis plants. In: Ramina, A., Chang, C., Giovannoni, J., Klee, H., Perata, P., Woltering, E. (Eds.), Advances in Plant Ethylene Research. Proceedings of the Seventh International Symposium on the Plant Hormone Ethylene. Springer, Netherlands, pp. 285–291. Kenigsbuch, D., Ovadia, A., Ivanova, Y., Chalupowicz, D., Maurer, D., Aharon, Z., Vinokur, Y., Aharoni, N., 2009. Wild Rocket (Diplotaxis tenuifolia) mutant with late flowering and delay in postharvest senescence. Acta Hortic. 830, 91–96. Kim, S.J., Ishii, G., 2007. Effect of storage temperature and duration on glucosinolate, total vitamin C and nitrate contents in rocket salad (Eruca sativa Mill.). J. Sci. 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D. Kenigsbuch et al. / Scientia Horticulturae 174 (2014) 17–23 Koukounaras, A., Siomos, A.S., Sfakiotakis, E., 2007. Postharvest CO2 and ethylene production and quality of rocket (Eruca sativa Mill.) leaves as affected by leaf age and storage temperature. Postharvest Biol. Technol. 46, 167–173. Lers, A., Jiang, W., Lomaneic, E., Aharoni, N., 1998. Gibberellic acid and CO2 additive effect in retarding postharvest senescence of parsley. J. Food Sci. 63, 66–68. Levey, S., Wingler, A., 2005. Natural variation in the regulation of leaf senescence and relation to other traits in Arabidopsis. Plant Cell Environ. 28, 223–231. Martínez-Sánchez, A., Marin, A., Llorach, R., Ferreres, F., Gil, M.I., 2006. Controlled atmosphere preserves quality and phytonutrients in wild rocket (Diplotaxis tenuifolia). Postharvest Biol. Technol. 40, 26–33. Martínez-Sánchez, A., Llorach, R., Gil, M.I., Ferreres, F., 2007. Identification of new flavonoid glycosides and flavonoid profiles to characterize rocket leafy salads (Eruca vesicaria and Diplotaxis tenuifolia). J. Agric. Food Chem. 55, 1356–1363. Martínez-Sánchez, A., Gil-Izquierdo, A., Gil, M.I., Ferreres, F., 2008. Comparative study of flavonoid compounds, vitamin C, and antioxidant properties of baby leaf brassicaceae species. J. Agric. Food Chem. 56, 2330–2340. Morales, M., Janick, J., 2002. Arugula: a promising specialty leaf vegetable. In: Janick, J., Whipkey, A. (Eds.), Trends in New Crops and New Uses. ASHS Press, Alexandria, VA, pp. 418–423. Morales, M.R., Maynard, E., Janick, J., 2006. ‘Adagio’: a slow-bolting arugula. Hortscience 41, 1506–1507.
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Ovadia, A., Tabibian-Keissar, H., Cohen, Y., Kenigsbuch, D., 2010. The 5 UTR of CCA1 includes an autoregulatory cis element that segregates between light and circadian regulation of CCA1 and LHY. Plant Mol. Biol. 72, 659–671. Pignone, D., 1997. Present status of rocket genetic resources and conservation activities. In: Padulosi, S., Pignone, D. (Eds.), Rocket: A Mediterranean Crop for the World. Report of a workshop, 13–14 December 1996. Legnano, Padova, Italy. International Plant Genetic Resources Institute, Rome, Italy, pp. 2–12. Rossenwasser, S., Rot, I., Sollner, E., Meyer, A.J., Smith, Y., Leviatan, N., Fluhr, R., Friedman, H., 2011. Organelles contribute differentially to reactive oxygen species-related events during extended darkness. Plant Physiol. 156, 185–201. Vinokur, Y., Rodov, V., 2006. Method for determining total (hydrophilic and lipophilic) radical-scavenging activity in the same sample of fresh produce. Acta Hortic. 709, 53–60. Watada, A.E., Ko, N.P., Minott, D.A., 1996. Factors affecting quality of fresh-cut horticultural products. Postharvest Biol. Technol. 9, 115–125. Wu, K., Zhang, L., Zhou, C., Yu, C.-W., Chaikam, V., 2008. HDA6 is required for jasmonate response, senescence and flowering in Arabidopsis. J. Exp. Bot. 59, 225–234. Yamauchi, N., Watada, A.E., 1993. Pigment changes in parsley leaves during storage in controlled or ethylene containing atmosphere. J. Food Sci. 58, 616–618. Yaniv, Z., 1995. Preliminary report on major activities initiated within the framework of Network activities. In: Padulosi, S. (Ed.), Rocket: Genetic Resources Network. Report of the first meeting, 13–15 November 1994, Lisbon, Portugal. International Plant Genetic Resources Institute, Rome, Italy, pp. 2–6.
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“Rock-Ad”, a new wild rocket (Diplotaxis tenuifolia) mutant with late flowering and delayed postharvest senescence David Kenigsbuch ∗ , Alona Ovadia, Yelena Shahar-Ivanova, Daniel Chalupowicz, Dalia Maurer Department of Postharvest Science of Fresh Produce, Institute of Postharvest and Food Sciences, Agricultural Research Organization, The Volcani Center, PO Box 6, Bet Dagan 50250, Israel
a r t i c l e
i n f o
Article history: Received 4 February 2014 Received in revised form 28 April 2014 Accepted 30 April 2014 Keywords: Wild rocket Postharvest Senescence Flowering
a b s t r a c t Wild rocket (Diplotaxis tenuifolia) is a leafy herb in salads and cooked dishes originating in the Mediterranean basin. The leaves contain many nutrients, plus beneficial phytochemicals such as flavonoids and glucosinolates. Rocket suffers from two major limitations: early bolting during growth and leaf yellowing after harvest, both of which affect both yield and market quality. We have used ethyl methanesulfonate (EMS) to induce mutation in wild rocket, and selected a mutant line (registered as cv. “Rock-Ad”), which has a two week delay in flowering time compared to current commercial cultivars. “Rock-Ad” plants began to flower when more than 50% of commercial plants had flowered. Storage properties of the mutant were also better than the commercial cultivar. In storage, after simulation of shipment by sea, there was no yellowing in young “Rock-Ad” leaves, chlorophyll levels were higher, fewer flower buds developed, and hydrophilic antioxidant levels decreased less than the commercial cultivar. Holding leaves in 5 kPa CO2 in air decreased yellowing and decay significantly in both cultivars compared to air storage, while exposure to 2.23 mol L−1 ethylene increased yellowing and decay. When ethylene and CO2 were applied together (5 kPa and 2.23 mol L−1 , respectively), yellowing and decay decreased in both cultivars compared to air storage. 1-MCP treatment reduced yellowing and decay in leaves of the commercial variety, but had no effect on leaves of “Rock-Ad”. The respiration rate of the leaves increased after 7 days of storage in both cultivars, but was significantly higher in leaves of the commercial cultivar, and 1-MCP significantly moderated this increased respiration. Although treatment with 1-MCP led to increased ethylene levels on the first day after harvest, during storage, ethylene levels decreased in all treatments and cultivars. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Wild rocket (Diplotaxis tenuifolia) from the Brassicaceae family can be found growing freely in most Mediterranean countries. The plants have two rows of seeds in the seed pods, hence the name Diplotaxis, from Greek diploos (double) and taxis (row), and slender leaves, thus the species name tenuifolia from Latin tenuis (slender) and folium (leaf). It is considered a perennial plant since its roots can survive the winter and sprout again the next spring (Pignone, 1997). Wild rocket was thought to be an aphrodisiac and has many medical utilities (Bianco and Boari, 1997). The leaves contain vitamin C, fibers, flavonoids and glucosinolates (Crozier et al., 1997; Barillari et al., 2005; Bennett et al., 2006, 2007; Heimler et al., 2007; Martínez-Sánchez et al., 2007, 2008).
∗ Corresponding author. Tel.: +972 3 9683600; fax: +972 3 9683622. E-mail address: [email protected] (D. Kenigsbuch). http://dx.doi.org/10.1016/j.scienta.2014.04.038 0304-4238/© 2014 Elsevier B.V. All rights reserved.
There are two major limitations to commercial production of rocket: early bolting during spring and autumn, about 35 days after germination, and leaf yellowing after harvest. Both problems affect the yield and market quality. Another commercial type of rocket (Eruca sativa), a closely related species, flowers 55–68 days after sowing (Yaniv, 1995; Morales and Janick, 2002). A search for a latebolting rocket (E. sativa) germplasm resulted in a cultivar named “Adagio”, which has delayed flowering of up to four weeks, compared to the wild-type (Morales and Janick, 2002; Morales et al., 2006). Postharvest quality is the second problem of wild rocket. Leaves rapidly turn yellow and then are not suitable for marketing. Leaf yellowing occurs due to chlorophyll degradation. Ethylene is known to increase the rate of chlorophyll degradation, whereas controlled atmosphere (CA) in which CO2 level is elevated (5–8%), decreases the degradation rate (Watada et al., 1996; Kader, 1995; Kader, 1986; Yamauchi and Watada, 1993). Respiration rate is also affected by storage conditions, which in turn affects quality. CA
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conditions decrease respiration. In mint, for example, respiration was lower in conditions of 5 kPa CO2 than in air storage. Respiration was not affected by 1-MCP, an ethylene action inhibitor, but ethylene production was elevated, and was further increased under a combined treatment of 1-MCP together with 5 kPa CO2 . However, in the presence of exogenous ethylene, CO2 strongly suppressed the 1-MCP-induced ethylene production (Kenigsbuch et al., 2007a). In a preliminary report on the M2 generation of the wild rocket mutant we demonstrated a delay in flowering and a greater number of leaves at harvest time in the mutant compared to the commercial wild rocket cultivar (Kenigsbuch et al., 2009). In this work we present further analysis of flowering and post harvest leaf qualities of the “Rock-Ad” cultivar including chlorophyll and antioxidant levels, respiration rate and ethylene production, as well as the influence of exogenous ethylene, 1-MCP and controlled atmosphere.
USA) according to the supplier’s instructions, with concentrations determined by gas chromatograph to produce a stock of 1000 L L−1 concentration inside a sealed bottle. Treatment with 1MCP was achieved by injecting a single dose of 1-MCP into 2-L jars containing wild rocket leaves (about 15 cm long, 100 g) which were then maintained fully sealed for 4 h in the dark at 17 ◦ C to achieve a concentration of 0.5 mol L−1 . Control jars (CK) were sealed without 1-MCP. Following this initial period, air flow of 50 mL min−1 was commenced, with three replicates per treatment. The jars were held for 7 days at 17 ◦ C. Leaf yellowing and decay were evaluated according to the yellowing and decay indices (see Section 2.6), CO2 and ethylene levels were measured as described below (Section 2.5).
2. Materials and methods
CO2 was determined by gas chromatograph (GC) equipped with a thermal conductivity detector (GOW-MAC instruments Co. series 580, PA, USA) with a double-column CTR-1 (Altech, 6 ft × 1/4 in.), carrier gas helium, oven temperature 30 ◦ C. Ethylene levels were measured by a GC with flame ionization detector (FID) (Varian 3300; Walnut Creek, CA, USA). using packed alumina column (Altech 4 ft × 1/8 in.), carrier gas helium, oven temperature 100 ◦ C, injection temperature155 ◦ C, detector temperature 100 ◦ C.
2.1. Plants Local commercial wild rocket (D. tenuifolia) and the “Rock-Ad” mutant (M4 generation and onward) (Kenigsbuch et al., 2009) were used for all experiments. Plants were grown commercially in net houses in the winter–spring seasons in the Negev in southern Israel. Seeds of the “Rock-Ad” mutant were given to commercial nursery to produce seedlings as the procedure for the commercial line, about twelve seeds per pot. Three weeks old Seedlings from both cultivars were planted in growers net houses. The seedlings density on beds was 60 seedlings per meter bed, 10 cm between the seedlings. Irrigation and fertilization were given according to the growers’ procedure. From each experiment 4–5 harvests were examined for quality parameters after shipment and shelf life simulation. For the flowering time experiments plants were grown in net house during the winter–spring seasons, one plant per 300 gr pot. The time of flowering and percentage of flowering plants was determined by examining the plants and counting every 3–4 days after the first flower appeared. 2.2. Packaging and temperature conditions Freshly cut leaves were placed in ventilated atmosphere (in polyethylene bags with multi microperforation (MMP)), with three replicate 1 kg bag for each sample. Bagged leaves were held for six days at 3 ◦ C followed by six days at 6 ◦ C and finally two days shelf life at 17 ◦ C as a simulation of conditions obtaining during shipment by sea. The leaves were either from young rosettes with maximum 12–15 cm long leaves or old rosettes with maximum 20–22 cm long leaves. 2.3. Ethylene and CO2 in storage Experiments were carried out with freshly harvested wild rocket leaves. All experiments were repeated three times with three replications per experiment. Each wild rocket bunch (about 15 cm long, 100 g) was enclosed in sealed 2-L jars fitted with inlet and outlet ports allowing the plant material to be maintained under controlled atmospheres (CA) comprising of either air; 5 kPa CO2 in air; 2.23 mol L−1 ethylene in air; or 5 kPa CO2 plus 2.23 mol L−1 ethylene in air, with a flow rate of 50 mL min−1 . Gases were bubbled through sterile water to maintain high relative humidity. 2.4. 1-MCP treatment 1-MCP was released from a commercial powder formulation (Ethylbloc, Biotechnologies for Horticulture, Inc., Burr Ridge, IL,
2.5. Gas measurements
2.6. Yellowing and decay indices Leaf yellowing and decay were evaluated by subjective indices on day 14 at the end of the storage and shelf life period. The yellowing scale allows quantitative assessment of yellowing in accordance with marketability: 0%—fresh green leaves with no color change; 25%—more substantial reduction in color quality—up to 10% of leaves showing some levels of light green appearance; 50%—produce with this level of color loss is no longer of marketable quality—20% of leaves with color loss; 75%—produce not of marketable quality—60% of leaves with color loss; 100%—produce not of marketable quality—100% of leaves turned yellow. The decay index during leaf senescence with respect to marketability was: 0%—fresh marketable product, all leaves symptom-free; 25%—up to 5% of leaves showing lesions 3—10 mm in diameter; 50%—produce with this level of damage were no longer of marketable quality, 20% of leaves showed physiological or pathological damage; 75%—produce not of marketable quality, 60% of leaves showing lesions representing physiological or pathological damage; 100%—produce not of marketable quality, all leaves showing lesions representing physiological or pathological damage.
2.7. Chlorophyll quantification Freshly cut leaves, about 40 g, were placed in plastic bags with one hole (0.7 mm), three repeats for each sample. Leaves were placed for six days at 3 ◦ C, six days at 6 ◦ C and two days in shelf life at 17 ◦ C. Leaf samples were taken on the first day after harvest (day 0), on day 12 of storage and after shelf life (day 14), lyophilized for three days and stored in the freezer (−20 ◦ C, dark). Ground freezedried leaves (10 mg) were placed in 1 mL 80% acetone in a 2 mL glass tube and placed in 4 ◦ C overnight. Samples were diluted 1:20 before reading absorbance of chlorophyll a and b by spectrophotometer at 663 nm and 645 nm, respectively, and calculating concentrations as described by Bruinsma (1963). There were three replicates per experiment.
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Fig. 2. Chlorophyll quantification after storage. Wild rocket leaves were stored in simulation of sea shipment. Leaf samples were taken on the first day of harvest (day 0), on day 12 of storage and after shelf life (day 14). Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test. Fig. 1. Flowering plant (%) after sowing. Plants were grown in a net house. The counting started when the first flower appeared and every 3-4 days thereafter.
3.2. Postharvest quality 2.8. Antioxidant capacity The free radical scavenging capacity was evaluated in leaf samples according to the method described by Vinokur and Rodov (2006). Leaves were sampled on the first day after harvest (day 0) and at the end of the experiment (day 14), ground and extracted into lipophilic and hydrophilic fractions. The free radical scavenging capacity was measured by the rate of decolorization of 2,2 -azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid), diammonium (ABTS) radical cation. 6-Hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid (Trolox, a water-soluble derivative of the vitamin E) was used as a standard, and the radicalscavenging activity of samples was expressed as Trolox equivalent antioxidant capacity (TEAC). 2.9. Statistical analysis Data are means of three experiments with three replicates per treatment. The results were submitted to analysis of variance and the mean values were compared by Tukey’s multiple range test.
In young rosette leaves (12–15 cm max, plant age 30 days) the “Rock-Ad” leaves showed no yellowing after 14 days of simulated shipment and marketing compared to the commercial leaves (Table 1). In old rosette leaves (20–22 cm max, plant age 36 days) the yellowing was increased in leaves of both cultivars, but was significantly higher in the commercial leaves than in “Rock-Ad”. The appearance of flower buds during storage is an undesirable phenomenon which can disqualify a shipment. The young “RockAd” rosette had no flowers after 14 days of storage compared to commercial plants, in which some flowers could be found (Table 1). In older rosette leaves, some flowers were found in the “Rock-Ad” but significantly fewer than the number of flowers found in the commercial variety. Freshly cut wild rocket leaves from the commercial and “RockAd” cultivars were held in simulated seas shipment conditions. Young rosette–the older leaves in the rosette were 12–15 cm. Old rosette—the older leaves were 20–22 cm. Mean values were compared by Tukey’s multiple range test. Capital letters indicate statistical difference in yellowing; Lower case letters indicate statistical difference in flowers at P = 0.05 standard deviation is given. 3.3. Chlorophyll content
3. Results 3.1. Flowering time The commercial cultivar plants began to flower on the 28th day after sowing and had 50% flowering by day 39 (Fig. 1). The “RockAd” plants began flowering two weeks after the commercial plants and reached 50% flowering on day 48 post-sowing. When the commercial cultivar was at 50% flowering, “Rock-Ad” had not flowered yet (Fig. 1). Plants were grown in a net house. The counting started when the first flower appeared and every 3–4 days thereafter.
Commercial and “Rock-Ad” leaves had the same level of chlorophyll on harvest day (Fig. 2). On day 12 there was a small decrease in chlorophyll level in the commercial leaves, while levels remained the same in “Rock-Ad”. At the end of the experiment (day 14), the chlorophyll content in the commercial leaves had decreased by 40% while in “Rock-Ad” the chlorophyll loss was only 20%. Wild rocket leaves were stored in simulation of sea shipment. Leaf samples were taken on the first day of harvest (day 0), on day 12 of storage and after shelf life (day 14). Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test.
Table 1 Yellowing and flowers after harvest. Freshly cut wild rocket leaves from the commercial and “Rock-Ad” cultivars were held in simulated seas shipment conditions. Young rosette – the older leaves in the rosette were 12-15 cm. Old rosette – the older leaves were 20-22 cm. Old rosette
Line
Commercial Rock-Ad
Young rosette
Flowers in 1 kg
Yellowing (%)
Flowers in 1 kg
Yellowing (%)
12.1 ± 0.4 a 0.7 ± 0.2 b
34.5 ± 7.1 A 9.4 ± 3.2 B
1.4 ± 0.6 b 0c
11.3 ± 1.6 B 0C
Mean values were compared by Tukey’s multiple range test. Capital letters indicate statistical difference in yellowing; Lower case letters indicate statistical difference in flowers at P = 0.05 Standard deviation is given.
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Fig. 3. Antioxidant quantification at harvest and after storage. Wild rocket leaves were stored in simulation of sea shipment. Leaf samples were taken on the first day of harvest (day 0) and after 14 days of storage (day 14). A- Antioxidant levels in the lipophilic fraction, B- Antioxidant levels in the hydrophilic fraction. Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test.
3.4. Antioxidants On the first day after harvest (day 0) commercial leaves and “Rock-Ad” had similar levels of antioxidants in both the lipophilic and hydrophilic fractions (Fig. 3). At the end of the experiment (day 14), the lipophilic fraction of the antioxidants decreased significantly in both commercial and “Rock-Ad”. In the hydrophilic fraction, there was a significant decrease in the commercial leaves, while in “Rock-Ad” there was no significant decrease. Wild rocket leaves were stored in simulation of sea shipment. Leaf samples were taken on the first day of harvest (day 0) and after 14 days of storage (day 14). (A) Antioxidant levels in the lipophilic fraction, (B) Antioxidant levels in the hydrophilic fraction. Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test. 3.5. The effects of ethylene and elevated CO2 A CO2 atmosphere of 5 kPa decreased yellowing significantly in both commercial and “Rock-Ad” leaves, compared to air or ethylene treatments (Fig. 4A). Ethylene increased yellowing compared to air storage in both cultivars. When ethylene was given in the presence of 5 kPa CO2 , the amount of yellowing was similar to CO2 without ethylene present. In both CO2 and ethylene plus CO2 treatments, the yellowing was much less pronounced in “Rock-Ad” leaves compared to the leaves of the commercial cultivar. CO2 atmosphere significantly decreased decay in both commercial and “Rock-Ad” plants, while having ethylene in the air slightly increased the level of decay of both varieties compared to air storage (Fig. 4B). Decay development on leaves in ethylene in the presence of CO2 was similar to decay in CO2 atmosphere alone.
Fig. 4. Ethylene and elevated CO2 effect on postharvest quality. Freshly cut leaves were stored in simulation of sea shipment. Leaf yellowing and decay were evaluated on day 14. A- Yellowing, B- Decay. Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test.
Freshly cut leaves were stored in simulation of sea shipment. Leaf yellowing and decay were evaluated on day 14. (A) yellowing, (B) decay. Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test. 3.6. The effects of 1-MCP After treatment with 1-MCP, the commercial wild rocket leaves were significantly less yellow and had less decay compared to the untreated leaves (Fig. 5). The 1-MCP treatment did not affect the “Rock-Ad” leaves. The “Rock-Ad” leaves, as noted before, were less yellow and had less decay than untreated commercial leaves. Freshly cut wild rocket leaves from the commercial and “RockAd” cultivars were placed in 2-L jars and 1-MCP was injected to a concentration 0.5 mol L−1 and sealed for 4 h. Control jars (CK) were sealed without 1-MCP. After treatment, air flow of 50 mL min−1 commenced for 7 days at 17 ◦ C. Leaf yellowing and decay were evaluated according to the yellowing and decay indices. (A) yellowing, (B) decay. Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test. 3.7. Respiration rate and ethylene production Respiration rate did not increase until seven days after harvest (Fig. 6A). The commercial leaves had a 230% increase in respiration rate compared to the first day, while 1-MCP significantly moderated this increase. “Rock-Ad” leaves had only 50% higher respiration than at the beginning of storage, with no moderating effect of 1-MCP. On the first day after harvest ethylene levels were high in leaves treated with 1-MCP (Fig. 6B). Ethylene levels were significantly lower in “Rock-Ad” leaves without 1-MCP and lowest in the commercial leaves. During storage, ethylene levels decreased in all leaves, regardless of source or treatment.
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Fig. 5. 1-MCP treatment. Freshly cut wild rocket leaves from the commercial and “Rock-Ad” cultivars were placed in 2-L jars and 1-MCP was injected to a concentration 0.5 mol L-1 and sealed for 4 h. Control jars (CK) were sealed without 1-MCP. After treatment, air flow of 50 ml/min commenced for 7 days at 17 ◦ C. Leaf yellowing and decay were evaluated according to the yellowing and decay indices. A- Yellowing, B- Decay. Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test.
Freshly cut wild rocket leaves from the commercial and “RockAd” cultivars were placed in 2-L jars and 1-MCP was injected to a concentration 0.5 mol L−1 and sealed for 4 h. Following this initial period, air flow of 50 mL min−1 was commenced. The jars were held for 7 days at 17 ◦ C and CO2 and ethylene levels were determined by gas chromatograph. A—respiration rate, B—ethylene level. Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test. 4. Discussion Wild rocket is an important crop which is increasingly considered a salad green rather than a culinary herb. The main factors limiting continued expansion of commercial production are early flowering and limited storability. A previous study with transgenic Arabidopsis plants found that they flowered later and had better leaf storability than wild type plants (Kenigsbuch et al., 2007b; Ovadia et al., 2010). Based on this study, we decided to mutagenize wild rocket and from the mutagenized lines selected the new cultivar “Rock-Ad”, to address the problems of flowering and storage (Kenigsbuch et al., 2009). We found, as previously reported with the M2 generation of the mutagenized line, that “Rock-Ad” flowering time was delayed by two weeks compared to the commercial cultivar, and had improved storage life (Kenigsbuch et al., 2009). These results are consistent with studies showing that flowering time and leaf senescence have common regulatory factors (Golldack et al., 2002), although the opposite, late flowering and early senescence, has also been reported (Diaz et al., 2005). It is important to note that the regulation system is very complex and it is possible that different signals
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Fig. 6. Respiration rate and ethylene levels in detached wild rocket leaves. Freshly cut wild rocket leaves from the commercial and “Rock-Ad” cultivars were placed in 2-L jars and 1-MCP was injected to a concentration 0.5 mol L-1 and sealed for 4 h. Following this initial period, air flow of 50 ml/min was commenced. The jars were held for 7 days at 17 ◦ C and CO2 and ethylene levels were determined by gas chromatograph. A- respiration rate, B- ethylene level. Standard deviation is shown, and the mean values were compared by Tukey’s multiple range test.
are affecting the phenomena. A number of different Arabidopsis mutants have been shown to have increased leaf longevity and late flowering compared with the wild type (Ellis et al., 2005; Wu et al., 2008). However, one mutant in a metalloproteinase gene had late flowering, but early senescence and degradation of chlorophyll (Golldack et al., 2002). In studies not involving mutants, different ecotypes of Arabidopsis that bolted late also senesced late (Levey and Wingler, 2005). Our results with the “Rock-Ad” mutation in rocket demonstrate a link between late flowering and delayed leaf senescence postharvest. To our knowledge, this is the first time a connection between late flowering and postharvest traits have been shown. “Rock-Ad” had significantly less yellowing of leaves in all the storage conditions, air, 5% CO2 , ethylene and a combination of CO2 and ethylene, than the commercial cultivar. The increased yellowing of both in the presence of ethylene is in agreement with changes during storage of other leafy products including parsley (Lers et al., 1998; Yamauchi and Watada, 1993), broccoli (Aharoni et al., 1985; Watada et al., 1996), and mint (Kenigsbuch et al., 2007a). However, since 1-MCP did not prevent the yellowing, but only reduced it in control leaves, ethylene is not the only regulatory process which leads to chlorophyll loss. Other processes may be involved in promoting chlorophyll loss, similar to what occurs in dark induced senescence in Arabadopsis leaves (Rossenwasser et al., 2011) In a study with salad rocket (E. sativa Mill.) stored at 10 ◦ C, it was found that 1-MCP in the absence of ethylene had only a small effect
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in prolonging shelf life. However, if 1-MCP was given before ethylene exposure, yellowing and chlorophyll loss were prevented (Koukounaras et al., 2006). It is possible that chlorophyll loss in wild rocket is under somewhat different regulatory control than wild rocket. Leaf yellowing and decay incidence in both plants were decreased in a 5 kPa CO2 atmosphere, nonetheless they were significantly lower in “Rock-Ad” plants than the commercial cultivar. This beneficial effect of high CO2 is consistent with the role of CO2 in maintaining visual quality (Martínez-Sánchez et al., 2006), delaying decay and acting as an ethylene antagonist (Aharoni et al., 2010). In parsley, CO2 treatment retarded chlorophyll and protein degradation, amino acid accumulation and respiration (Lers et al., 1998; Yamauchi and Watada, 1993). In mint, senescence phenomena include yellowing, browning, decay and leaf abscission, and increased levels of CO2 in a CA system inhibited these phenomena (Kenigsbuch et al., 2007a). In both salad rocket (Koukounaras et al., 2007) and wild rocket (Martínez-Sánchez et al., 2006) storage in low O2 and high CO2 partial pressures helped preserve sensory and microbiological quality of the leaves. The lipophilic antioxidant levels in wild rocket leaves decreased about 30% during simulated shipment and shelf life, while the hydrophilic antioxidant levels decreased 25% in the commercial cultivar and only 5% in “Rock-Ad”. Rocket leaves contain a wide range of compounds that contribute to antioxidant activity, including vitamin C, glucosinolates, flavonoids and phenolics (Jin et al., 2009). Martínez-Sánchez et al. (2006) found a decrease in the antioxidant capacity of wild rocket during 14 d at 4 ◦ C storage to a similar extent as in our study, and this was positively correlated with vitamin C content, and poorly correlated with total phenolics or with flavonoids. Jin et al. (2009) reported that in both salad and wild rocket gluocosinolates were reduced during 14 d at 4 ◦ C, while flavonoids did not decrease, and Kim and Ishii, 2007 also found decreases in glucosinolates of salad rocket during storage at either 4 ◦ C or 15 ◦ C. The higher levels of hydrophilic antioxidants in “Rock-Ad” leaves may indicate greater retention of vitamin C and glucosinolates during simulated shipment and shelf life. Respiration of the leaves was low during the first days of 17 ◦ C storage, and only increased on day 7, while ethylene was initially high and decreased steadily over time. Koukounaras et al. (2007) found the same kinetics of ethylene for salad rocket leaves held at 10 ◦ C, while the respiration had an initially high level which declined 50% after 3 days and remained steady. In contrast, Martínez-Sánchez et al. (2006) found a steady increase in respiration of wild rocket leaves held at 4 ◦ C and four fold higher CO2 evolution after 14 d than after 1 d. The differences in these reports may be partially due to the age of the leaves, as Koukounaras et al. (2007) found somewhat different kinetics of respiration and ethylene if the leaves were young, fully expanded, or old. Exposure to 1-MCP ameliorated the respiration increase on d 7 in the commercial leaves but had little effect on “Rock-Ad”. It is unclear yet whether the insensitivity of leaves of “Rock-Ad” to 1-MCP is caused by changes in the ethylene biosynthesis pathway or in the sensitivity to ethylene. On the other hand, 1-MCP increased ethylene production in both leaves in the first days of storage to a similar extent. This stimulating effect on ethylene production has been found in other leafy commodities (Kenigsbuch et al., 2007a; Jiang et al., 2002). It has been suggested that the feedback control of ethylene synthesis in leaves is induced by 1-MCP, even though the inhibitor blocks ethylene action (Jiang et al., 2002). In conclusion, the “Rock-Ad” wild rocket mutant is a promising new cultivar. It has later flowering than the commercial cultivar, and slower senescence. It retains chlorophyll and antioxidant contents longer in storage than the commercial cultivar. Altogether, it has better storage potential, making it suitable for sea shipment.
Acknowledgments We are very grateful to Dr. Susan Lurie and Dr. Joshua Klein for their critical review and editing. This manuscript is Contribution no. 674/3 from the Agricultural Research Organization, The Volcani Center, PO Box 6, Bet Dagan 50250, Israel.
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