Food-grade sugar can promote differentiation in melon (Cucumis melo L.) tissue culture
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- Çürük, S., Çetiner, S., Yalçın-Mendi, Y. et al. In Vitro Cell.Dev.Biol.-Plant (2012) 48: 600. doi:10.1007/s11627-012-9453-0
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The objective of the present study was to investigate the origin of discrepancy between experimental results in in vitro culture of Turkish melon (Cucumis melo L.) cultivars, conducted by the same individual using the same protocol and same seed batches in two different laboratories. The difference in the sucrose source was found to be the major reason for the deviation in results between the two laboratories. The percentage of regenerating explants and the number of bud-like protuberances and/or shoots were significantly greater when a food-grade Turkish sucrose was used in the medium compared with analytical-grade sucrose. Media formulated with the food-grade sucrose regenerated 37 and 67 % more explants and bud-like protuberances and/or shoots per explant, respectively, than media containing analytical-grade sucrose. No meaningful differences were found in added elements or anions between the sucrose sources or by liquid chromatography/mass spectroscopy. The only significant chemical difference observed between the sucrose samples was the presence of melanoidins (Maillard reaction products) in the food-grade sucrose. The melanoidins were of high molecular weight (>3,000 Da determined by ultrafiltration), with characteristic ultraviolet–visible spectra and in vitro antioxidant activity. Melanoidin-containing sucrose can be differentiated by color and spectroscopy.
KeywordsSucrose qualityFood-grade sucroseBud regenerationMelonCucumis melo
Although regeneration of melon cultivars has been previously described (Moreno et al.1985; Niedz et al.1989; Fang and Grumet 1990; Vallés and Lasa 1994; Gaba et al.1999), none of these reports utilized Turkish melon cultivars. Since success of melon regeneration protocols is dependent on genotype, growth regulators, and explant source, size, and age (Moreno et al.1985; Niedz et al.1989; Fang and Grumet 1990; Vallés and Lasa 1994; Gaba et al.1999), it was not known whether these protocols would be effective for Turkish melon cultivars. In vitro regeneration of several Turkish melon cultivars has been successful using regeneration and transformation media developed for other melon cultivars (Moreno et al.1985; Niedz et al.1989; Fang and Grumet 1990; Vallés and Lasa 1994) in Adana, Turkey. However, great problems were encountered when attempting to repeat the work (Çürük et al.2002) in Bet Dagan, Israel, despite the same researcher employing a similar protocol with the same seed batches.
Difficulties associated with repeatability of published research of in vitro regeneration methods between different laboratories are well known but little discussed in the literature (Poonsapaya et al.1989; Meurer et al.2001; Dahleen and Bregitzer 2002; Amutha et al.2009). Although methodology is often transferable from one laboratory to another one, an exact transfer does not usually occur and a nearly exact transfer of methods may not result in repeatability. Therefore, each laboratory can have its own subtle differences in methodologies and techniques derived from local optimization of published protocols (Poonsapaya et al.1989).
A study of soybean regeneration through embryogenesis between laboratories in three different locations found that each genotype could be regenerated at each location, provided that care was taken to standardize the protocol (Meurer et al.2001). Threefold differences in regeneration of the same plant material with similar techniques were found by Dahleen and Bregitzer (2002). The type of culture vessels and the height above sea level were considered important variables in the discrepancy between laboratories studying rice culture and regeneration (Poonsapaya et al.1989). Regeneration of explants from seedlings of Cucurbita pepo was affected by seed storage period (Amutha et al.2009).
The objective of the current study was to investigate the origin of the discrepancy between laboratories in in vitro regeneration experiments of Turkish melon cultivars. Differences in response can be introduced by the researcher or may result from environmental variations (photon flux density, temperature, humidity, and condensation in culture vessels), water quality, season, location (height above sea level and atmospheric contamination), plant material (cultivar, seed source, and seed storage period), or media components such as sucrose and agar.
In the present study, a discrepancy between results obtained in two laboratories was found to result from use of different sucrose sources. There are reports describing the effects of different sugars or sugar concentrations on in vitro regeneration on a variety of species, including melon (Debeaujon and Branchard 1992), cucumber (Lou and Kako 1994), and tomato (El-Bakry 2002). However, to our knowledge, this is the first report to document the effects of different sources of sucrose on in vitro regeneration.
Materials and Methods
Preliminary experiments, conducted in Adana, Turkey, and Bet Dagan, Israel, were nearly at the same elevation, using the same plastic Petri dishes (9 cm diameter; Miniplast Ltd., Ein Shemer, Israel). The same researcher performed all the experiments with the same seed lots, separated only by a few weeks, with good-quality chemicals and the same agar (8 g L−1, cat. No. A1296, Sigma, St. Louis, MO). Therefore, only two major sources of variation remained to be examined: water and sucrose.
A single seed batch of Cucumis melo L. cv. Kirkağaç 637 (Çağdaş Seeds Ltd., Adana, Turkey) was used.
Tissue culture conditions and media.
All tissue culture experiments reported here were performed in Adana, Turkey. Melon seed coats were removed and the seeds were washed in 70 % ethanol for 5 min, rinsed three times with sterile distilled water, and sterilized in a solution of 1 % sodium hypochlorite (20 % Domestos, Unilever, Port Sunlight, UK) for 45 min. The seeds were washed three times with sterile distilled water and soaked for 15 min in an aqueous solution containing 500 mg L−1 carbenicillin and 500 mg L−1 cefotaxime. The seeds were germinated on complete Murashige and Skoog (1962) (MS) basal medium with 8 g L−1 agar (Sigma A1296), and 3 % sucrose (Yonca Co., Adana, Turkey), in a growth room at 29 ± 3°C with a 16-h photoperiod of 85–120 μmol m−2 s−1 cool-white fluorescent light.
Cotyledons were excised from 4- or 5-d-old plants and cut crosswise to make three explants per cotyledon, trimming off the cotyledon tip but not the edges. Sucrose was supplied at 3 % in all subsequent media, from either Merck KGaA (Darmstadt, Germany; Sucrose for Microbiology cat. No. 1.07651) or Yonca Co. (a locally purchased Turkish food-grade beet sucrose). The adaxial sides of the explants were placed on the following two different regeneration media: (1) IK1560 medium (MS salts, 100 mg L−1 myo-inositol, 1 mg L−1 thiamine-HCL, 1.5 mg L−1 indole-3-acetic acid (IAA), 6 mg L−1 kinetin, 30 g L−1 sucrose (Merck or Yonca), and 8 g L−1 agar) (Moreno et al.1985), or (2) N medium (MS salts and vitamins, 30 g L−1 sucrose (Merck or Yonca), 8 g L−1 agar, 0.88 mg L−1 IAA, 1.13 mg L−1 benzyl adenine (BA), and 0.26 mg L−1 abscisic acid) (Niedz et al.1989). The explants were then incubated in the growth room for 28–36 d. The regeneration percentage (percent explants producing bud-like protuberances (protuberances typically observed to become buds on regeneration or elongation medium) and/or shoots) was determined as per Niedz et al. (1989). The culture vessels were 9 cm diameter plastic Petri dishes (Miniplast).
The regenerating parts of the explants (i.e., green callus, bud-like protuberances, and/or shoots) were excised and transferred onto elongation medium (NB00101 of Vallés and Lasa 1994) (MS salts, 100 mg L−1 myo-inositol, 1 mg L−1 thiamine-HCL, 0.1 mg L−1 BA, 0.01 mg L−1 naphthalene acetic acid (NAA), 30 g L−1 sucrose (Merck or Yonca), and 8 g L−1 agar). For each treatment, the sucrose in the elongation medium was from the same source as that used in the regeneration medium. The explants were cultured for 16 d on elongation medium and subcultured once for 28–32 d. The numbers of buds and/or shoots per explant were scored at the end of the subculture.
Effect of water source.
An experiment was conducted to investigate the effect of the water source. Melon cotyledon explants were excised from 5-d-old seedlings and cultured on two regeneration media (IK1560 and N) made with the two different water sources for 28 d. The experiment was performed in Adana, Turkey, with Yonca sucrose. The water in the Turkish laboratory was supplied by a GFL Double Distiller (2–3 MΩ cm−1, Gesellschaft Labortechnik, Burgwedel, Germany) and the water shipped from the Israeli laboratory was produced by Barnstead NANOpure (13–17 MΩ cm−1, Cole Palmer, Vernon Hills, IL).
Chemical analysis of sucrose samples.
Analysis of elements by inductively coupled plasma/atomic emission spectroscopy, anion analysis by ion chromatography, and analysis of semivolatile organic compounds by gas chromatography–mass spectrometry (GC-MS) were performed by the Interdepartmental Equipment Laboratory, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel. Analysis by liquid chromatography mass spectroscopy (LC-MS), ultraviolet (UV)–visible spectroscopy, and antioxidant potential were performed at the ARO Volcani Center, Bet Dagan, Israel.
Analysis of elements by inductively coupled plasma/atomic emission spectroscopy.
Batches of sucrose (500 mg) were digested in 5 mL 65 % HNO3 in a MLS 1200 Mega microwave digestion unit (Milestone S.r.l., Sorisole, Italy) at 500 W for 10 min and at 580 W for another 10 min. The volume was made up to 25 mL with deionized water. Elements were determined by inductively coupled plasma/atomic emission spectroscopy (Spectroflame Modula E, Spectro Analytical Instruments GmbH, Kleve, Germany) against Merck standards.
Analysis of anions by ion chromatography.
Samples were dissolved at 10 mg mL−1 in water, and the solutions were filtered through a reverse-phase filter and a 0.2-μm filter. Anions were determined with a DX-300 Ion Chromatography system (Dionex Corp., Sunnyvale, CA), equipped with an AS4A analytical column and guard column and an Anion Micromembrane Suppressor. A carbonate/bicarbonate buffer was used as the eluent. Measurements were calibrated with standards from Dionex and Alltech (Grace Corp., Deerfield, FL).
Semivolatile organic compounds detected by GC-MS.
Batches of sucrose (2 g) were mixed with 2 g sodium sulfate and 10 mL methylene chloride, and the mixture was sonicated at 750 W for 3 min with 1-s pulses and a 50 % duty cycle. The extract was filtered through glass wool and a 5-ml volume was concentrated to 1 mL in a heating block. Analysis was carried out with a Varian (Palo Alto, CA) Saturn-2000 ion-trap GC-MS and 1 μL of the sample was injected into the Varian Star 3800 gas chromatograph. An HT-8 capillary column was used (25 m long, 0.25 mm (i.d.), 0.25 μm (d.f.)) with a flow rate of 1.5 mL min−1. The mass spectrometer was operated in the electron impact mode, and electron energy set to 70 eV.
Ultrafiltration and UV–visible spectroscopy.
Aqueous sucrose solutions (20 mg mL−1 with Barnstead C water) were ultrafiltered using Amicon Ultra 3K centrifuge filter units (Millipore, Billerica, MA). UV–visible spectra (190 – 900 nm) of the original solutions, filtrates, and retentates were recorded on a GENESYS 10S spectrophotometer (Thermo Fisher Scientific, Waltham, MA), compared with a water blank.
Sucrose antioxidant activity.
Antioxidant activity of aqueous sucrose solutions (55 mg mL−1), filtrates, and retentates was quantified against gallic acid standards using the Folin–Ciocalteu method for total phenols. This method measures total reducing capacity by electron transfer (Huang et al.2005).
Sucrose analysis by LC-MS.
Sucrose samples (∼0.1 mg mL−1 water, ULC-MS-Grade, Bio-Lab Ltd., Jerusalem, Israel) were filtered through a Millex-HV Durapore PVDF (Millipore) membrane (0.22 μm) before injection into the LC-MS instrument. Mass spectral analyses were carried out by the ultraperformance LC-quadrupole time-of-flight (UPLC-QTOF) instrument (Waters Premier QTOF, Waters Inc., Milford, MA), with the UPLC column connected online to a photo diode array detector (Waters Acquity), and then to the mass spectroscopy detector equipped with an electrospray ion (ESI) source (performed in ESI-positive and ESI-negative modes). Separation was performed on a 2.1 × 50 mm (i.d.), 1.7 μm UPLC BEH C18 column (Waters Acquity).
The chromatographic and mass spectroscopy parameters were as follows: the mobile phase consisted of 0.1 % formic acid in water (phase A) and 0.1 % formic acid in acetonitrile (phase B). The linear gradient program was as follows: 100 to 95 % A over 0.1 min, 95 to 5 % A over 9.7 min, held at 5 % A over 3.2 min, then returned to the initial conditions (95 % A) over 4.2 min. The flow rate was 0.3 mL min−1, and the column temperature was kept at 35°C.
The following settings were applied during UPLC-MS runs: capillary voltage at 3.2 kV(ESI+) and 2.6 kV(ESI−), cone voltage at 30 eV(ESI+) and 30 eV(ESI−), and collision energy at 5 eV. Argon was used as the collision gas. The m/z range was 100 to 1,000 Da. The mass spectroscopy equipment was calibrated using sodium formate, and Leu-enkephalin was used as the lock mass. A mixture of standard compounds was used for instrument quality control. The MassLynx software version 4.1 (Waters) was used to control the instrument and calculate accurate masses. LC-MS analyses were repeated four times.
The tissue culture experiments were designed and analyzed according to a completely randomized or split-plot factorial design (Compton 1994) (three to four replicates and six explants per replicate). All experiments were performed twice. Datasets containing a large number of zeros or values less than 15 were transformed ((Y + 0.5)0.5) (Bartlett 1936), and all values expressed as percentages were transformed (arcsin (P = original percentage value)0.5) (Bartlett 1947), and analyzed by analysis of variance. Tukey’s honestly significant difference (HSD value) test was used to compare the treatments (Compton 1994) at the 0.05 level. Untransformed data were used to present the mean value.
Results and Discussion
Influence of sucrose source on melon cotyledon explant regeneration: regeneration frequency on regeneration media
Regenerating explants on regeneration medium (%)
HSD (5 %)
Influence of sucrose source on melon cotyledon explant regeneration: number of buds and shoots per explant on elongation medium
Medium of origin
Number of bud-like protuberances and/or shoots per explant on elongation medium
HSD (5 %)
Concentrations of elements and anions added to the media (in milligrams per liter) by each of the sucrose sources
Element or anion
Antioxidant activity of sucrose (Yonca and Merck) solutions and their ultrafiltration filtrates and retentates
1.1 ± 0.1
1.2 ± 0.1
2.2 ± 0.2
Despite similar methodologies being used in separate laboratories, significant differences in results were obtained between locations with respect to melon regeneration, which were found to be due to differences in the source of the sucrose used in the culture medium. The only significant difference between the sucrose samples was the presence in the Yonca sucrose of a small amount of high molecular weight-colored substance(s) with antioxidant activity. These colored polymeric substances are thought to be melanoidins formed during the sugar production process.
Sugar liquors from cane or beet contain colorants formed during early refining stages; these colorants include melanoidins, caramels, and polyphenols combined with iron (Grandison and Lewis 1996). Melanoidins are products formed during the final stage of the Maillard reaction, which occurs upon heating amino acids and reducing sugars together. Such colored products are removed during the production of white sugar.
In vitro functions reported for melanoidins include antioxidant activity (Ruiz-Roca et al.2008; Brudzynski and Miotto 2011; Vignoli et al.2011), antimicrobial activity (Rufian-Henares and Morales 2007), metal ion chelating (Rufian-Henares and de la Cueva 2009), and free radical scavenging (Morales and Jimenez-Perez 2004). Closely related humic substances (Maillard 1912, 1917; Ikan et al.1986, 1994; Ishiwatari et al.1986) have been long reported to have positive effects on plant nutrition, seed germination, root initiation, and total plant biomass, as well as to possess auxin-like activity (Pinton et al.1992; Nardi et al.1994; Facanha et al.2002; Muscolo et al.2002; Canellas et al.2009; Elena et al.2009; Trevisan et al.2011).
Melanoidins are a complex mixture of brown, nitrogenous, polymeric, polydisperse and polyanionic macromolecules (Morales 2002) that cannot be individually speciated. The structures of melanoidins are unknown and the presence of true polymeric repeating units is uncertain. Spectroscopic techniques such as Fourier transform infrared spectroscopy and solid-state 13C-NMR give wide absorption bands that provide information about functional groups and the chemical environments of the carbon atoms but do not enable identification of specific melanoidin compounds (Ikan et al.1986; Bosetto et al.2006). The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry methods (similar to those used here) employed to obtain molecular masses frequently do not give spectra for melanoidins, or when they do, present multiple peaks at high molecular masses (>6,000 Da, above the resolution limit of the equipment used in this study) (Borrelli et al.2002). As a result, melanoidins are conventionally detected by measuring absorbance at 420 and 360 nm (Brands et al.2002).
The Yonca sucrose solution had three times the absorbance at 360 nm and 1.7 times the absorbance at 420 nm of the Merck sucrose solution (Fig. 5), indicating the presence of colored substances absorbing at the wavelengths used for quantifying melanoidins. For the high molecular weight retentates, the Yonca sucrose had 6.5 times the absorbance at 360 nm and 5.8 times the absorbance at 420 nm of the Merck sucrose (Fig. 5), demonstrating that the Yonca sucrose had a larger concentration of absorbing high molecular weight compounds than the Merck sucrose. The Yonca retentate also had twice the antioxidant activity of the Merck retentate (Table 4), supporting the identification of the UV–visible absorbing high-molecular-weight fraction as melanoidins.
The work reported here confirms anecdotes from commercial plant tissue culture researchers that food-grade sucrose (sugar) may promote in vitro growth and development, presumably due to the presence of melanoidins. This is commercially important, as sucrose is expensive for industrial producers, who purchase large quantities of food-grade sugar rather than expensive reagent-grade sucrose (e.g., Tyagi et al.2007). Melanoidin-containing sucrose can be identified by color and spectroscopy.
The role of melanoidins in stimulation of in vitro plant development should be confirmed using chemically synthesized melanoidins. Such efforts are underway in our laboratories.
Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 510/11. This work was supported by a fellowship and grant to S. Çürük from the Scientific and Technical Research Council of Turkey (TUBITAK/BAYG) and to VG by the Chief Scientist of the Ministry of Agriculture, Israel. We are grateful to Dr. B. Steinitz for review of an early version of the manuscript, and to Dr. A.A. Schaffer for helpful comments and assistance.