Abstract
The current research was carried out to investigate the effects of iron source in the culture media for Vaccinium corymbosum L. ʻBluerayʼ, ʻDukeʼ, and ʻPatriotʼ cultivars grown on five different types of medium (Woody Plant Medium supplemented with 1.0 mg·L−1 zeatin and 0, 25, 50, 75, and 100 mg·L−1 Sequestrene 138). After 10 weeks of culture, seven physiological parameters were measured, such as the number and length of axillary shoots, rooting and acclimatization percentage, as well as chlorophyll (a, b, a/b) and carotenoid content of the leaves. Adding Sequestrene 138 to the culture media led to a slight decrease of the proliferation rate but increased the length of the shoots. The chlorophyll and carotenoid content in all of the three cultivars was considerably increased as the iron concentration of the media increased. The shoots developed on the Sequestrene 138–free medium were chlorotic and short, whereas at different concentrations of iron in the culture medium the shoots were dark green and vigorous, providing a greater acclimatization success than those grown in iron-free medium.
The genus Vaccinium is a member of the Ericaceae family, which includes ≈400 species. Of all the cultivated species and hybrids in this genus, highbush blueberry (V. corymbosum L.) is the most commonly known species that produces small, edible fruits containing high amounts of antioxidants, phenolic compounds, and vitamins with a remarkable ability to provide high yields with minimal inputs (López et al., 2010; Prodorutti et al., 2007; Samad et al., 2014; Zheng and Wang, 2003). Due to the valuable pharmaceutical properties of the fruits, blueberry production has been increased considerably in the recent years. Therefore, to satisfy the high demand for blueberries, a new and commercially viable way to produce homogeneous and disease-free blueberry plants in a short time is required (Gao et al., 2018).
Research on highbush blueberry micropropagation was started in the 1980s (Reed and Abdelnour-Esquivel, 1991) and then broadened and accelerated in recent years to improve the existing micropropagation protocols (Gao et al., 2018; Hung et al., 2016; Kudryashova et al., 2012; Ružić et al., 2012). In the beginning, research was focused on finding the optimal culture media for micropropagation and checking the influence of diverse growth regulators (Chandler and Draper, 1986; Da Silva et al., 2006; Eccher and Noe, 1989; Eccher et al., 1986; Gajdošová et al., 2006; Isutsa et al., 1994; Ostrolucká et al., 2002, 2004; Reed and Abdelnour-Esquivel, 1991; Tetsumura et al., 2008; Zhang et al., 2006). The results showed that the most suitable medium for this species was McCown’s Woody Plant Basal Salt Mixture, or Woody Plant Medium (WPM), as described by Lloyd and McCown (1980). In the following years, the influence of different plant hormones on the efficiency of in vitro regeneration of highbush blueberry was studied (Kudryashova et al., 2012), and other investigations also were made on the antioxidant and anti-inflammatory properties of V. corymbosum L. (Samad et al., 2014).
The latest scientific research was carried out to investigate the hyperhydricity-induced ultrastructural and physiological changes in blueberry (Gao et al., 2018), the effects of LED lights used for in vitro culture (Hung et al., 2016), the effect of 24-epibrassinolide on in vitro micropropagation of highbush blueberry (Kudryashova et al., 2012), and the use of a bioreactor in lowbush blueberry micropropagation (Debnath, 2009).
Leaf pigment content and composition also provide important information about the physiological status of the in vitro plant. Several studies have been conducted on this topic to investigate the effects of iron chelates [FeEDTA (iron-ethylenediaminetetraacetate) and FeEDDHA (ferric sodium ethylenediamine di-2-hydroxy-phenylacetic acid)] on in vitro plants for different plant species such as Rosa hybrida L. ʻMoneywayʼ (Van der Salm et al., 1994), Carica papaya L. (Castillo et al., 1997), Citrullus lanatus (Thomas et al., 2000), hybrid hazelnut (Garrison et al., 2013; Nas and Read, 2001), and in five different raspberry cultivars (Zawadzka and Orlikowska, 2006), analyzing the chlorophyll content and chlorosis occurrence in the in vitro plants. The results show that iron chelates have beneficial effects on in vitro plants, but to the best of our knowledge, no scientific reports have been published on this topic in highbush blueberry in vitro plants. Therefore, the main aim of this paper was to investigate the influence of four different concentrations of Sequestrene 138 [ferric sodium ethylenediamine di-2-hydroxy-phenylacetic acid (FeNaEDDHA)] on the physiological status of the in vitro–propagated highbush blueberry plants represented by the number and length of the proliferated shoots, photosynthetic pigment content of the leaves, and, last but not least, rooting and acclimatization rates.
Materials and Methods
Plant material and culture conditions.
This research was carried out in the Life Science Institute of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Romania, for 5 months, from Jan. to May 2017.
The plant material used consisted of the in vitro–propagated plants from three different highbush blueberry (Vaccinium corymbosum L.) cultivars as follows: Blueray, Duke, and Patriot. The mini-shoots with lengths of 1.5 to 2 cm used in this experiment were taken from the 10-week-old in vitro plantlets cultured on a medium prepared from stock solutions having the following composition: WPM (Lloyd and McCown, 1980), 100 mg·L−1 Sequestrene 138 (FeNaEDDHA 6%), 100 mg·L−1 myo-inositol, 2 mg·L−1 vitamin B1, 1 mg·L−1 vitamin B6, 1 mg·L−1 nicotinic acid, 1 mg·L−1 zeatin, and 3% (w/v) of sugar.
To check the influence of Sequestrene 138 on the micropropagation success of highbush blueberry plants, WPM (Duchefa Biochemie BV, The Netherlands) was used supplemented with 1 mg·L−1 zeatin, 3% (w/v) of sugar, and 0, 25, 50, 75, and 100 mg·L−1 Sequestrene 138, respectively.
The culture medium was solidified with 0.35% (w/v) plant agar. All the components were purchased from Duchefa Biochemie BV. The pH of the media was adjusted to 5.0 before autoclaving. The autoclave program used for sterilizing the culture media was run at 0.11 MPa at 121 °C for 20 min.
The culture vessels used in this experiment were 720 glass jars, with a diameter of 9 cm and a height of 13.5 cm with sponge-vented screw caps. In each vessel, 100 mL of culture medium was dispensed for culturing. The in vitro cultures were incubated in the growth room for a 16-h photoperiod with 32.4 μmol·m−2·s−1 light intensity (Cool white fluorescent lamps, 36 W; Philips, Amsterdam, The Netherlands) at 23 ± 3 °C and 50% ± 2% humidity.
Ten explants per jar with a length of 1.5 to 2 cm were inoculated in such way that two-thirds to three-fourths of the basal part of the explants was immersed in the culture media. The length of the shoots and the multiplication rate were recorded and calculated after 10 weeks of culture. In total, 50 randomly selected plantlets (five jars per treatment per cultivar) were measured.
Ex vitro rooting and acclimatization.
The shoots obtained in the multiplication stage were acclimatized in a nonsterile mixture of peat [Klasmann-Deilmann, Lithuanian Peat Moss Special Ericaceous plants, pH value (H2O): 4.0–4.5, structure: medium, without fertilization] and perlite (‘BIOS’ Research and Development Center of Bio-stimulators, Cluj-Napoca, Romania) in 1:1 (v:v) ratio, in mini-greenhouses [Versay T1 (DEDEMAN SRL, Bacău, Romania), sizes 39 × 25 × 7.5 cm, polyvinyl chloride], in laboratory conditions. For acclimatization, only shoots longer than 2.5 cm were used.
To determine the rooting and acclimatization percentage of the in vitro plantlets, five repetitions with 32 shoots per treatment for each cultivar and rooting substrate (160 shoots per rooting substrate, 800 shoots per cultivar, in total 2400 shoots) were used. The rooting and acclimatization data and percentage were recorded and calculated after 30 d.
Chlorophyll content.
Carotenoid (Caro) content.
Statistical analysis.
One-way analysis of variance (ANOVA) was performed for treatments within one cultivar and for all the cultivars within one treatment to investigate whether the differences in physiological parameters of the in vitro plants were affected by the presence of different concentrations of Sequestrene 138 added to the culture media. Post hoc testing for the ANOVAs was performed using Tukey’s honestly significant difference test (Tukey’s test) using a P ≤ 0.05 significance level to determine the statistically significant differences between the means. Values shown are means ± se. In addition, all variables determined in plants submitted to iron treatments were correlated by principal component analysis (PCA), independently for each of the three studied V. corymbosum L. cultivars. Each principal component was a linear combination of the original variables with coefficients equal or greater than eigenvectors of the correlation matrix. The results of these analyses were represented graphically, for each species, as a biplot of the two main principal components.
Results
Effect of Sequestrene 138 on in vitro shoot multiplication of Vaccinium corymbosum L. ‘Blueray’, ‘Duke’, and ‘Patriot’.
The greatest multiplication rates were recorded on the WPM [containing 36.7 mg·L−1 ferric sodium ethylenediamine tetra acetic acid (FeNaEDTA)] as compared with the other culture media supplemented with different concentrations of Sequestrene 138 used in this experiment. Among all the highbush blueberry cultivars tested, the greatest shoot proliferation rate (7.2 ± 0.9927) was observed in ʻBluerayʼ (Fig. 1) with the exception of the medium supplemented with 75 mg·L−1 Sequestrene 138. ʻBluerayʼ had the greatest proliferation rates in all types of media tested, whereas the lowest proliferation rates were recorded in ʻPatriot’, ranging from 4.08 ± 0.0902 to 5.22 ± 0.1501.
The influence of Sequestrene 138 on the number of shoots per explant developed on the medium supplemented with the indicated Sequestrene 138 (mg·L−1) concentrations. The values shown are means ± se (n = 5). Different lowercase letters above the bars indicate significant differences between treatments for each cultivar, and different capital letters indicate significant differences between the cultivars for each treatment, according to Tukey’s test (P ≤ 0.05).
Citation: HortScience horts 53, 10; 10.21273/HORTSCI13269-18
Regarding shoot lengths, it was observed that in all cultivars the average length of the shoots obtained on the medium supplemented with Sequestrene was significantly larger as compared with control (Fig. 2).
The influence of Sequestrene 138 on the length of shoots grown on the medium supplemented with the indicated Sequestrene 138 (mg·L−1) concentrations. The values shown are means ± se (n = 5). Different lowercase letters above the bars indicate significant differences between treatments for each cultivar, and different capital letters indicate significant differences between the cultivars for each treatment, according to Tukey’s test (P ≤ 0.05).
Citation: HortScience horts 53, 10; 10.21273/HORTSCI13269-18
On the WPM basal medium, whereas the shortest shoots were obtained in ʻPatriotʼ, with an average length of 2.84 ± 0.0655, the longests were recorded in ʻDukeʼ, with an average length of 3.59 ± 0.0976 cm. The concentration given by 50 mg·L−1 Sequestrene 138 stimulated the growth of the shoots in ʻDukeʼ (4.28 ± 0.2278 cm).
The shortest shoots on the WPM basal medium were recorded in ʻPatriotʼ, with an average length of 2.84 ± 0.0655 cm, but the longests were measured in ʻDukeʼ, with an average length of 3.59 ± 0.0976 cm. In ʻBluerayʼ, the average length of the shoots developed was 3.07 ± 0.0897 cm. The 50 mg·L−1 concentration of Sequestrene 138 had a beneficial effect on shoot growth also in ʻDukeʼ (4.28 ± 0.2278 cm), but the longest shoots were recorded in ʻBluerayʼ (with an average length of 3.94 ± 0.1952 cm) on the WPM basal medium supplemented with 25 mg·L−1 of Sequestrene 138. The longest shoots in ʻPatriotʼ were obtained on the medium supplemented with 75 mg·L−1 and 100 mg·L−1 of Sequestrene (no statistically significant differences were observed between them).
Effects of Sequestrene 138 on ex vitro rooting and acclimatization of ‘Blueray’, ‘Duke’, and ‘Patriot’ in vitro plantlets.
The ex vitro rooting and acclimatization were carried out simultaneously using the shoots resulting from the multiplication stage. The shoots developed on the Sequestrene-free medium had the lowest acclimatization percentage, whereas those grown on the media with 75 and 100 mg·L−1 Sequestrene had the greatest acclimatization percentage (Table1). ʻBluerayʼ had the most successful rooting and acclimatization percentages, ranging from 96.25% to 100%; 96.25% was registered for the shoots developed on the Sequestrene-free medium; 98.12% for the shoots obtained on the medium supplemented with 25 mg·L−1 Sequestrene; and 100% for the shoots grown on the medium supplemented with 50, 75, and 100 mg·L−1 Sequestrene, respectively.
Acclimatization percentage of the in vitro–cultured plants in the three different cultivars.
Effect of Sequestrene 138 on chlorophyll and Caro content of the leaves in ‘Blueray’, ‘Duke’, and ‘Patriot’ in vitro plantlets.
The photosynthetic pigments were quantified (Chl a, Chl b, Chl a/Chl b, and Caro) to determine the relationship between the pigment content in the leaves of the in vitro plants and the concentration of Sequestrene 138 (Figs. 3–5). In all three V. corymbosum L. cultivars, the photosynthetic pigment content of the leaves was increased by increasing the concentration of Sequestrene 138 in the culture media. In all the three cultivars, the greatest Chl a content was registered in the leaves developed on the medium supplemented with 100 mg·L−1 Sequestrene 138, but the lowest statistically significant values of the Chl a content in all the cultivars were recorded in the leaves of the plantlets grown on the Sequestrene 138-free medium. Both, the highest and the lowest Chl a content among the cultivars studied were recorded in ʻDukeʼ, with a mean value of 2.729 ± 0.0083 mg/g and 0.260 ± 0.0070 mg/g, respectively (Fig. 3).
The influence of Sequestrene 138 on chlorophyll a content of the leaves of the in vitro plantlets grown on the medium supplemented with the indicated Sequestrene 138 (mg·L−1) concentrations. The values shown are means ± se (n = 5). Different lowercase letters above the bars indicate significant differences between treatments for each cultivar, and different capital letters indicate significant differences between the cultivars for each treatment, according to Tukey’s test (P ≤ 0.05).
Citation: HortScience horts 53, 10; 10.21273/HORTSCI13269-18
The influence of Sequestrene 138 on chlorophyll b content of the leaves of the in vitro plantlets grown on the medium supplemented with the indicated Sequestrene 138 (mg·L−1) concentrations. The values shown are means ± se (n = 5). Different lowercase letters above the bars indicate significant differences between treatments for each cultivar, and different capital letters indicate significant differences between the cultivars for each treatment, according to Tukey’s test (P ≤ 0.05).
Citation: HortScience horts 53, 10; 10.21273/HORTSCI13269-18
The influence of Sequestrene 138 on the carotenoid content of the leaves of the in vitro plantlets grown on the medium supplemented with the indicated Sequestrene 138 (mg·L−1) concentrations. The values shown are means ± se (n = 5). Different lowercase letters above the bars indicate significant differences between treatments for each cultivar, and different capital letters indicate significant differences between the cultivars for each treatment, according to Tukey’s test (P ≤ 0.05).
Citation: HortScience horts 53, 10; 10.21273/HORTSCI13269-18
Except for ʻBluerayʼ, the Chl b content in the leaves analyzed showed a similar increasing tendency in all the three investigated cultivars on the medium supplemented with 100 mg·L−1 Sequestrene 138. In ʻBluerayʼ, the maximum Chl b content was recorded in the leaves developed on the WPM basal medium supplemented with 50 mg·L−1 Sequestrene 138. Similar to the Chl a content, in ʻDukeʼ both were recorded, the highest and the lowest Chl b content, in the leaves developed on the WPM basal medium supplemented with 100 mg·L−1 Sequestrene 138 as follows: 0.790 ± 0.0105 mg/g and 0.057 ± 0.0000 mg/g, respectively. Regarding Chl a/Chl b ratios, the greatest values were recorded in the 0 and 25 mg·L−1 Sequestrene 138 treatments, which indicate iron deficiency in plants and also a lower chlorophyll content as previously confirmed by other researches in other species such as Beta vulgaris (Spiller and Terry, 1980) and Corylus avellana L. (Garrison et al., 2013).
It also was observed that the increasing concentration of Sequestrene 138 in the culture media led to a greater accumulation of Caro content in the leaves. The greatest Caro content for the in vitro plantlets was recorded in ʻDukeʼ, 0.443 ± 0.0004 mg/g on the WPM basal medium supplemented with 100 mg·L−1, whereas the lowest Caro content was recorded in the leaves of the ʻBluerayʼ in vitro plantlets grown on the iron chelate-free WPM basal medium (0.059 ± 0.0009 mg/g).
Principal component analyses.
The analyses of principal components were performed separately for all the three cultivars, including all the measured parameters to emphasize the variations and obtain strong patterns in our dataset. In the three PCAs shown in Fig. 6, two components with an eigenvalue equal to or greater than 1 expressed a cumulative percentage of variance of about 92%. The first component (X axis) was determined by the Sequestrene 138 concentrations of the medium (Table 1) and alone explains 63% to 71% of the variance. The iron chelate concentration of the medium was weakly correlated, negatively, with the number of shoots developed and positively with the other parameters analyzed. The small angles of the loading vectors of ACL and Caro with the X-axis indicate the strongest, positive correlations. Positive correlations, albeit weaker, also were registered between Chl a, Chl b, and LS and the X-axis.
Principal component analysis. Changes in all the measured parameters with respect to the control, iron-free medium in Vaccinium corymbosum L. Blueray in correlation to the iron concentration of the culture medium. The parameters examined were length of the shoots (LS), number of shoots (NS), Chlorophyll a (Chl a), Chlorophyll b (Chl b), Carotenoids (Caro), and acclimatization percentage (ACL): ‘Blueray’ (A), ‘Duke’ (B), and ‘Patriot’ (C).
Citation: HortScience horts 53, 10; 10.21273/HORTSCI13269-18
Discussion
In all the three Vaccinium corymbosum L. cultivars cultured on the standard WPM basal medium supplemented with 1 mg·L−1 zeatin, 3% (w/v) sugar, gelled with 0.35% (w/v) plant agar with a pH adjusted to 5, it was observed that iron chlorosis was predominant (Fig. 7). This phenomenon could be explained by the iron deficiency of the culture medium as observed in other in vitro cultures as well. Thus, it was found by Zawadzka and Orlikowska (2006) that supplementing the multiplication media with FeNaEDDHA eliminated leaf chlorosis in raspbery in 4 weeks. The leaves harvested from these shoots maintenaned their green color on the regeneration medium longer than those from FeEDDHA-free medium (Zawadzka and Orlikowska, 2006). The results obtained by Nas and Read (2001) indicate that Sequestrene 138 is a suitable chelating agent for hazelnut tissue culture. The enhanced leaf color and morphology on medium containing Sequestrene 138 compared with ferrous sulfate and Sequestrene 330 are likely a consequence of improved Fe availability for the explants. A previous study has shown that Sequestrene 138 yielded better results than FeNaEDTA in blueberry in vitro culture, conferring dark green color to the plants and increasing the multiplication rate and plant vigor in ʻBlue Cropʼ (Clapa et al., 2007). Similar studies made on blueberry indicate that supplementing the basal WPM medium with 100 mg·L−1 Sequestrene 138 (WPMm) in combination with growth regulators led to a high proliferation rate in Elliot bluberry cultivar and the greatest number of shoots developed per explant (14.85) on the WPMm + 2 mg·L−1 zeatin (Vescan et al., 2012).
In vitro cultured V. corymbosum L. ʻPatriotʼ plants on the five different types of medium supplemented with Sequestrene 138 (from the left to the right: 0, 25, 50, 77, and 100 mg·L−1 Sequestrene 138) (A); shoots of ‘Patriot’ grown on the iron-free WPM medium (B), shoots grown on basal WPM + 25 mg·L−1 Sequestrene 138 (C), shoots grown on basal WPM + 50 mg·L−1 Sequestrene 138 (D), shoots grown on basal WPM + 75 mg·L−1 Sequestrene 138 (E), and shoots grown on basal WPM + 100 mg·L−1 Sequestrene 138 (F).
Citation: HortScience horts 53, 10; 10.21273/HORTSCI13269-18
In a previous investigation, differences were observed regarding the color of the shoots in the initiation stage of in vitro cultures in blueberry cultivars such as Berkeley, Bluecrop, and Earliblue on three different culture media, as follows: MS (Murashige and Skoog, 1962), WPM, and MW (a mixture of equal parts of MS and WPM). The color of the shoots varied from one cultivar to another; 80% of shoots from ‘Bluecrop’ and ‘Earliblue’ explants were yellowish green, and 30% of ‘Berkeley’ shoots were red regardless of the medium (Tetsumura et al., 2008). Our results show that the basal WPM medium supplemented with the four different Sequestrene 138 (FeNaEDDHA) concentrations had a significant impact on the shoot length reached in the multiplication stage in all the cultivars studied (Fig. 2). Studies also suggest that there is a slight difference between the responses of the blueberry cultivars to the iron chelate concentrations applied to the culture medium (Reed and Abdelnour-Esquivel, 1991; Ružić et al., 2012; Tetsumura et al., 2008). In this research, it was observed that the length of the shoots varied among the cultivars on all types of media; the longest shoots developed being recorded in ʻDukeʼ, followed by ʻBluerayʼ, whereas the shortest shoots were measured in ʻPatriotʼ. A slight decrease also was observed in the number of shoots per explant on the media supplemented with iron chelate (Fig. 1). Although the proliferation rate was greater on the Sequestrene 138-free medium, the shoots developed were shorter and yellowish. For this reason, some of the shoots (<2 cm) were eliminated from the ex vitro rooting stage, and others didn’t regenerate roots due to chlorosis. In this context, in ʻBluerayʼ, the average number of shoots per explant that occurred was greater (7.2 ± 0.1621) on the basal WPM medium in comparison with the number of shoots per explant obtained on the medium supplemented with 100 mg·L−1 Sequestrene 138, reaching an average number of 6.12 ± 0.2191/explant. The rooting and ex vitro acclimatization percentage recorded in the shoots obtained from the iron-free medium was 96.250 ± 0.583%, whereas the acclimatization percentage of the shoots obtained from the medium supplemented with 100 mg·L−1 Sequestrene 138 was 100 (Table 1).
Blueberry plants have a remarkable ex vitro rooting capacity, which permits one to eliminate the in vitro rooting stage from the multiplication process, lowering the production costs in micropropagation (Isutsa et al., 1994). The ex vitro rooting percentages recorded in all the cultivars on all types of media supplemented with iron chelate are similar to the acclimatization percenteges of blueberry plants reported by Isutsa et al. (1994) in ʻBerkeleyʼ (>99%); 100% rooting was reported by Hung et al. (2016) in the ex vitro rooting stage of ‘Huron’ shoots in a nonsterile mixture of perlite and peatmoss (Top Blueberry Co., Cheonan, South Korea) (v/v = 1:6). These results are in accordance with our results obtained in the shoots grown on the culture medium supplemented with 75 mg·L−1 and 100 mg·L−1 Sequestrene 138.
The results reveal that chlorophyll a, b, Chl a/Chl b, and the Caro content of the leaves of the in vitro plants were significantly influenced by the increasing concentration of iron chelate added to the culture medium. The chlorophyll content of the leaves is one of the most important and reliable indicators of physiological status of the plants. This aspect is very important for the evolution of the in vitro cultures; namely, the low chlorophyll content can affect photosynthesis, leading to a low acclimatization rate and unsatisfactory plant growth and development (Rafi et al., 2018). Similar results also were reported by Van der Salm et al. (1994), who investigated the effects of iron chelates in Rosa hybrida L. ‘Moneyway’ in vitro cultures. The authors claim that the use of FeEDDHA is more effective. We can hypothesize that the replacement of FeNaEDTA by FeNaEDDHA in media also will improve the in vitro culture of other plant species that are susceptible to iron deficiency. Also, in vitro–grown Vaccinium pahalae and V. myrtillus plants as reported by Shibli et al. (1997) had a considerably better performance. Chlorosis and hyperhydration were eliminated when the iron chelate (FeNaEDTA) was replaced with or supplemented by FeNaEDDHA (Sequestrene 138–50, 100, and 150 µM). Castillo et al. (1997), in one of their studies carried out on Carica papaya L. in vitro culture, suggest that supplementing the culture media with 100 µM concentration of iron provided by FeNaEDDHA may be sufficient to markedly improve in vitro culture performance.
The length of the shoots obtained in the in vitro multiplication stage, the color, and the morphology of the in vitro plants were considerably improved on the culture media supplemented with Sequestrene 138, as compared with those obtained on the basal WPM medium, as a probable consequence of the fact that the iron in this compound is more readily available to the plants. The use of Sequestrene 138 in the culture media of all cultivars resulted in a greater chlorophyll content in the leaves, a more equilibrated Chl a/Chl b ratio, and a greater micropropagation rate as compared with control.
The results of the PCAs clearly and briefly summarize the very similar pattern in the three studied cultivars, which responded very similarly to the increasing iron chelate concentration in the media. Among the three cultivars under investigation, ‘Patriot’ had a positive response to iron added to the culture medium, showing a strong positive correlation between iron concentration and growth parameters, whereas ‘Blueray’ and ‘Duke’ demonstrated a negative effect of the iron chelate on the number and the length of the shoots developed as the iron concentration of the medium increased. The photosythetic pigment concentrations in all three cultivars were considerably increased as the iron concentration of the medium increased. The acclimatization success of the in vitro plants grown on the medium supplemented with iron chelates was considerably greater than in those grown on iron-free medium. Therefore, based on the results of our study, we highly recommend the use of Sequestrene 138 as the main iron source in the culture medium for a succsessful micropropagation of blueberry plants.
Literature Cited
Britton, G., Schiedt, K. & Liaaen-Jensen, S. 1995 Isolation and analysis, p. 104–107. In: Carotenoids, Vol. 1A. Birkhauser Verlag, Basel
Castillo, B., Smith, M.A.L., Madhavi, D.L. & Yadava, U.L. 1997 Interactions of irradiance level and iron chelate source during shoot tip culture of Carica papaya L HortScience 32 1120 1123
Chandler, C.K. & Draper, A.D. 1986 Effect of zeatin and 2ip on shoot proliferation of three blueberry clones in vitro HortScience 21 1065 1066
Clapa, D., Fira, A. & Rusu, T. 2007 The use of Isubgol and Sequestrene 138 for the in vitro propagation of the highbush blueberry (Vaccinium corymbosum L.) J. Food Agr. Environ. 6 1 145 147
Da Silva, L.C., Schuch, M.W., De Souza, J.A., Erig, A.C. & Antunes, L.E.C. 2006 Nutritive medium, growth regulators and cold in the in vitro establishment of blueberry (Vaccinium ashei Reade) cv. ‘Delite’ R. Bras. Agrociência, Pelotas 12 4 405 408
Debnath, S.C. 2009 A scale-up system for lowbush blueberry micropropagation using a bioreactor HortScience 44 1962 1966
Eccher, T. & Noe, N. 1989 Comparison between 2iP and zeatin in the micropropagation of highbush blueberry (Vaccinium corymbosum) Acta Hort. 241 185 190
Eccher, T., Noe, N., Piagnani, C. & Castelli, S. 1986 Effects of Increasing Concentration of BAP and 2iP on in vitro culture of Vaccinium corymbosum Acta Hort. 179 II 879 881
Gajdošová, A., Ostrolucká, M.G., Libiaková, G., Ondrušková, E. & Šimala, G. 2006 Microclonal propagation of Vaccinium sp. and Rubus sp. and detection of genetic variability in culture in vitro J. Fruit Ornam. Plant Res. 14 1 103 119
Gao, H., Li, J., Ji, H., An, L. & Xia, X. 2018 Hyperhydricity-induced ultrastructural and physiological changes in blueberry (Vaccinium spp.) Plant Cell Tissue Organ Cult. 133 65 76
Garrison, W., Dale, A. & Saxena, P.K. 2013 Improved shoot multiplication and development in hybrid hazelnut nodal cultures by ethylenediamine di-2-hydroxy-phenylacetic acid (Fe-EDDHA) Can. J. Plant Sci. 93 3 511 521
Hung, C.D., Hong, C.H., Kim, S.K., Lee, K.H., Park, J.Y., Nam, M.W., Choi, D.H. & Le, H.I. 2016 LED light for in vitro and ex vitro efficient growth of economically important highbush blueberry (Vaccinium corymbosum L.) Acta Physiol. Plant. 38 6 152
Isutsa, D.K., Pritts, M.P. & Mudge, K.W. 1994 Rapid propagation of blueberry plants using ex vitro rooting and controlled acclimatization of micropropagules HortScience 29 1124 1126
Kudryashova, O.A., Volotovich, A.A., Vasilevskaya, T.I., Varavina, N.P., Rupasova, Z.A. & Khripach, V.A. 2012 Effects of 24-epibrassinolide on in vitro micropropagation of highbush blueberry Russ. J. Plant Physiol. 59 4 586 593
Lloyd, G. & McCown, B. 1980 Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture Comb. Proc. Intl. Plant Prop. Soc. 30 421 427
López, J., Uribe, E., Vega-Gálvez, A., Miranda, M., Vergara, J., Gonzalez, E. & Di Scala, K. 2010 Effect of air temperature on drying kinetics, vitamin C, antioxidant activity, total phenolic content, non-enzymatic browning and firmness of blueberries variety O Neil Food Bioprocess Technol. 3 5 772 777
Murashige, T. & Skoog, F. 1962 A revised medium for rapid growth and bioassay with tobacco tissue cultures Physiol. Plant. 15 473 497
Nas, M.N. & Read, P.E. 2001 Micropropagation of hybrid hazelnut: Medium composition, physical state and iron source affect shoot morphogenesis, multiplication and explant vitality Acta Hort. 556 251 258
Ostrolucká, M.G., Gajdošová, A. & Libiaková, G. 2002 Influence of zeatin on microclonal propagation of Vaccinium corymbosum L Propag. Ornam. Plants 2 14 18
Ostrolucká, M.G., Libiaková, G., Ondrušková, E. & Gajdosová, A. 2004 In vitro propagation of Vaccinium species. Acta Universitatis Latviensis Biology (Basel) 676 207 212
Prodorutti, D., Pertot, I., Giongo, L. & Gessler, C. 2007 Highbush blueberry: Cultivation, protection, breeding and biotechnology Eur. J. Plant Sci. Biotechnol. 1 1 44 56
Rafi, S., Kamili, A.N., Ganai, B.A., Mir, M.Y. & Parray, J.A. 2018 In vitro Culture and Biochemical Attributes of Bergenia ciliata (Haw.) Sternb Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 88 2 609 619
Reed, B.M. & Abdelnour-Esquivel, A. 1991 The use of zeatin to initiate in vitro cultures of Vaccinium species and cultivars HortScience 26 1320 1322
Ružić, D., Vujović, T., Libiakova, G., Cerović, R. & Gajdošova, A. 2012 Micropropagation in vitro of highbush blueberry (Vaccinium corymbosum L.) J. Berry Res. 2 2 97 103
Samad, N.B., Debnath, T., Ye, M., Hasnat, M.A. & Lim, B.O. 2014 In vitro antioxidant and anti–inflammatory activities of Korean blueberry (Vaccinium corymbosum L.) extracts Asian Pac. J. Trop. Biomed. 4 10 807 815
Shibli, R.A., Smith, M.A.L. & Nasr, R. 1997 Iron source and cytokinin mitigate the incidence of chlorosis and hyperhydration in vitro J. Plant Nutr. 20 773 781
Spiller, S. & Terry, N. 1980 Limiting factors in photosynthesis: II. Iron stress diminishes photochemical capacity by reducing the number of photosynthetic units Plant Physiol. 65 1 121 125
Tetsumura, T., Matsumoto, Y., Sato, M., Honsho, C., Yamashita, K., Komatsu, H., Sugimoto, Y. & Kunitake, H. 2008 Evaluation of basal media for micropropagation of four highbush blueberry cultivars Scientia Hort. 119 1 72 74
Thomas, P., Mythili, J.B. & Shivashankara, K.S. 2000 Effects of photo-oxidative loss of FeNa2EDTA and of higher iron supply on chlorophyll content, growth and propagation rate in triploid watermelon cultures In Vitro Cell. Dev. Biol. Plant 36 6 537 542
Van der Salm, T.P., Van der Toorn, C.J., ten Cate, C.H.H., Dubois, L.A., de Vries, D.P. & Dons, H.J. 1994 Importance of the iron chelate formula for micropropagation of Rosa hybrida L.‘Moneyway’ Plant Cell Tissue Organ Cult. 37 1 73 77
Vescan, L.A., Pamfil, D., Clapa, D., Fira, A., Sisea, C.R., Pop, I.F., Petricele, I.V., Ciuzan, O. & Pop, R. 2012 Efficient micropropagation protocol for highbush blueberry (Vaccinium corymbosum L.) cv. ‘Elliot’ Rom. Biotechnol. Lett. 17 1 6893 6902
Zawadzka, M. & Orlikowska, T. 2006 Increae in the quality of raspberry cultures under the influence of FeEDDHA Acta Hort. 725 161 164
Zhang, Z., Liu, H., Wu, L. & Li, Y. 2006 Technical system of blueberry micropropagation in China Acta Hort. 715 421 426
Zheng, W. & Wang, S.Y. 2003 Oxygen radical absorbing capacity of phenolics in blueberries, cranberries, chokeberries, and lingonberries J. Agr. Food Chem. 51 2 502 509
