HORTSCIENCE 53(10):1487–1493. 2018. https://doi.org/10.21273/HORTSCI13269-18

The Role of Sequestrene 138 inHighbush Blueberry (Vacciniumcorymbosum L.) MicropropagationDoina Clapa and Claudiu BuneaUniversity of Agricultural Sciences and Veterinary Medicine Cluj-Napoca/Horticulture Research Station, M�an�asxtur 3-5, 400372, Cluj-Napoca, RomaniaOrsolya Borsai1, Adela Pintea, Monica Hartxa, and R�azvan SxtefanUniversity of Agricultural Sciences and Veterinary Medicine Cluj-Napoca,M�an�asxtur 3-5, 400372, Cluj-Napoca, RomaniaAlexandru FiraIndustrial Plants Ltd., Kazanlak, Bulgaria

Additional index words. photosynthetic pigments, FeNaEDDHA, in vitro, woody plantmedium

Abstract. The current research was carried out to investigate the effects of iron source inthe culture media for Vaccinium corymbosum L. ʻBluerayʼ, ʻDukeʼ, and ʻPatriotʼ cultivarsgrown on five different types of medium (Woody Plant Medium supplemented with 1.0mg·LL1 zeatin and 0, 25, 50, 75, and 100 mg·LL1 Sequestrene 138). After 10 weeks ofculture, seven physiological parameters were measured, such as the number and lengthof 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 medialed to a slight decrease of the proliferation rate but increased the length of the shoots. Thechlorophyll and carotenoid content in all of the three cultivars was considerablyincreased as the iron concentration of the media increased. The shoots developed onthe Sequestrene 138–free medium were chlorotic and short, whereas at differentconcentrations 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 theEricaceae family, which includes �400 spe-cies. Of all the cultivated species and hybridsin this genus, highbush blueberry (V. corym-bosum L.) is the most commonly knownspecies that produces small, edible fruitscontaining high amounts of antioxidants, phe-nolic compounds, and vitamins with a remark-able ability to provide high yieldswithminimalinputs (L�opez et al., 2010; Prodorutti et al.,2007; Samad et al., 2014; Zheng and Wang,2003). Due to the valuable pharmaceuticalproperties of the fruits, blueberry productionhas been increased considerably in the recentyears. Therefore, to satisfy the high demand forblueberries, a new and commercially viable

way to produce homogeneous and disease-freeblueberry plants in a short time is required(Gao et al., 2018).

Research on highbush blueberry micro-propagation was started in the 1980s (Reedand Abdelnour-Esquivel, 1991) and thenbroadened and accelerated in recent years toimprove the existing micropropagation pro-tocols (Gao et al., 2018; Hung et al., 2016;Kudryashova et al., 2012; Ru�zi�c et al., 2012).In the beginning, research was focused onfinding the optimal culture media for micro-propagation and checking the influence ofdiverse growth regulators (Chandler andDraper, 1986; Da Silva et al., 2006; Eccherand Noe, 1989; Eccher et al., 1986;Gajdo�sov�a et al., 2006; Isutsa et al., 1994;Ostroluck�a et al., 2002, 2004; Reed andAbdelnour-Esquivel, 1991; Tetsumura et al.,2008; Zhang et al., 2006). The results showedthat the most suitable medium for this specieswas McCown’s Woody Plant Basal Salt Mix-ture, or Woody Plant Medium (WPM), asdescribed by Lloyd and McCown (1980). Inthe following years, the influence of differentplant hormones on the efficiency of in vitroregeneration of highbush blueberry was stud-ied (Kudryashova et al., 2012), and otherinvestigations also were made on the antiox-idant and anti-inflammatory properties ofV. corymbosum L. (Samad et al., 2014).

The latest scientific research was carriedout to investigate the hyperhydricity-induced

ultrastructural and physiological changes inblueberry (Gao et al., 2018), the effects ofLED lights used for in vitro culture (Hunget al., 2016), the effect of 24-epibrassinolideon in vitro micropropagation of highbushblueberry (Kudryashova et al., 2012), andthe use of a bioreactor in lowbush blueberrymicropropagation (Debnath, 2009).

Leaf pigment content and composition alsoprovide important information about the phys-iological status of the in vitro plant. Severalstudies have been conducted on this topic toinvestigate the effects of iron chelates [FeEDTA(iron-ethylenediaminetetraacetate)andFeEDDHA(ferric sodium ethylenediamine di-2-hydroxy-phenylacetic acid)] on in vitro plants fordifferent plant species such as Rosa hybridaL. ʻMoneywayʼ (Van der Salm et al., 1994),Carica papaya L. (Castillo et al., 1997),Citrullus lanatus (Thomas et al., 2000), hybridhazelnut (Garrison et al., 2013; Nas and Read,2001), and in five different raspberry cultivars(Zawadzka and Orlikowska, 2006), analyzingthe chlorophyll content and chlorosis occur-rence in the in vitro plants. The results showthat iron chelates have beneficial effects on invitro plants, but to the best of our knowledge,no scientific reports have been published onthis topic in highbush blueberry in vitro plants.Therefore, the main aim of this paper was toinvestigate the influence of four differentconcentrations of Sequestrene 138 [ferric sodiumethylenediamine di-2-hydroxy-phenylacetic acid(FeNaEDDHA)] on the physiological status ofthe in vitro–propagated highbush blueberryplants represented by the number and length ofthe proliferated shoots, photosynthetic pigmentcontent 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 ScienceInstitute of University of Agricultural SciencesandVeterinaryMedicine Cluj-Napoca, Romania,for 5 months, from Jan. to May 2017.

The plant material used consisted of the invitro–propagated plants from three differenthighbush blueberry (Vaccinium corymbosumL.) cultivars as follows: Blueray, Duke, andPatriot. The mini-shoots with lengths of 1.5to 2 cm used in this experimentwere taken fromthe 10-week-old in vitro plantlets cultured ona medium prepared from stock solutions havingthe following composition: WPM (Lloyd andMcCown, 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, and3% (w/v) of sugar.

To check the influence of Sequestrene 138on the micropropagation success of highbushblueberry plants, WPM (Duchefa BiochemieBV, The Netherlands) was used supple-mented with 1 mg·L–1 zeatin, 3% (w/v) ofsugar, 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 componentswere purchased from Duchefa Biochemie

Received for publication 21 June 2018. Acceptedfor publication 7 Aug. 2018.This work was made possible with the financialsupport of the Romanian National Authority forScientific Research and Innovation, National Scien-tific Research Council/Advisory Board for Research,Development and Innovation-Executive Agency forHigher Education, Research, Development and In-novation Funding (CNCS/CCCDI-UEFISCDI), proj-ect number PN-III-P2-2.1.-BG-2016-0046, withinPNCDI III.1Corresponding author. E-mail: borsaiorsi@yahoo.com.This is an open access article distributed under theCC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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BV. The pH of the media was adjusted to 5.0before autoclaving. The autoclave programused for sterilizing the culture media was runat 0.11 MPa at 121 �C for 20 min.

The culture vessels used in this experimentwere 720 glass jars, with a diameter of 9 cm anda height of 13.5 cm with sponge-vented screwcaps. In each vessel, 100mL of culture mediumwas dispensed for culturing. The in vitrocultures were incubated in the growth roomfor a 16-h photoperiod with 32.4 mmol·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 2cmwere inoculated in suchway that two-thirds tothree-fourths of the basal part of the explants wasimmersed in the culture media. The length of theshoots and the multiplication rate were recordedand calculated after 10 weeks of culture. In total,50 randomly selected plantlets (five jars pertreatment per cultivar) were measured.

Ex vitro rooting and acclimatization. Theshoots obtained in the multiplication stage wereacclimatized in a nonsterile mixture of peat[Klasmann-Deilmann, Lithuanian Peat MossSpecial Ericaceous plants, pH value (H2O):4.0–4.5, structure: medium, without fertiliza-tion] and perlite (‘BIOS’ Research and Devel-opment Center of Bio-stimulators, Cluj-Napoca,Romania) in 1:1 (v:v) ratio, inmini-greenhouses[Versay T1 (DEDEMAN SRL, Bac�au, Roma-nia), 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 acclimati-zation percentage of the in vitro plantlets, fiverepetitions with 32 shoots per treatment foreach cultivar and rooting substrate (160shoots per rooting substrate, 800 shoots percultivar, in total 2400 shoots) were used. Therooting and acclimatization data and percent-age were recorded and calculated after 30 d.

Chlorophyll content. To measure the freshchlorophyll content, leaves were harvestedfrom the in vitro plantlets before passing themto the acclimatization stage. Fresh samples (0.2g) were homogenized and extracted with 90%acetone in water using a magnetic stirrer untilthe residue was uncolored. The absorbance wasread at 645 and 663 nm using the V-530 UV/VIS Spectrophotometer (JASCO, Eason, MD).The following formulas were used to quantifychlorophyll a (Chl a) and chlorophyll b (Chl b)and to check the ratio between Chl a and b(Chl a/Chl b):

Chl a mg=gð Þ¼ 11:75 · A663� 2:35 · A645ð Þ · V=g

Chl b mg=gð Þ¼ 18:61 · A645� 3:96 · A663ð Þ · V=g

where A645 and A663 represent the opticaldensity at specific wavelength, V representsthe volume of the extract (mL), and grepresents sample s weight (mg).

Carotenoid (Caro) content. The extrac-tion of Caro was carried out with acetone

using fresh leaf samples and repeated untilthe residue became uncolored. The combinedextracts were partitioned in a separation funnelwith diethyl ether and saturated saline solu-tion. The upper organic phase was evaporatedto dryness under vacuum, using a rotary evap-orator, then the residue was dissolved in

a measured volume of diethyl ether andtreated with an equal volume of 30% potas-sium hydroxide in methanol for saponifica-tion. Furthermore, the samples were stirredfor 2 h, then transferred in a separationfunnel and washed with saline solution un-til neutral pH of the aqueous phase was

Fig. 1. The influence of Sequestrene 138 on the number of shoots per explant developed on the mediumsupplemented with the indicated Sequestrene 138 (mg·L–1) concentrations. The values shown aremeans ± SE (n = 5). Different lowercase letters above the bars indicate significant differences betweentreatments for each cultivar, and different capital letters indicate significant differences between thecultivars for each treatment, according to Tukey’s test (P # 0.05).

Fig. 2. The influence of Sequestrene 138 on the length of shoots grown on the medium supplemented withthe 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 eachcultivar, and different capital letters indicate significant differences between the cultivars for eachtreatment, according to Tukey’s test (P # 0.05).

Table 1. Acclimatization percentage of the in vitro–cultured plants in the three different cultivars.

Cultivar

WPM + Sequestrene 138

0 mg·L–1 25 mg·L–1 50 mg·L–1 75 mg·L–1 100 mg·L–1

Blueray 96.250 ± 0.583 98.125 ± 0.400 100 100 100Duke 95.000 ± 0.509 96.875 ± 0.547 100 100

98.125 ± 0.244Patriot 94.375 ± 0.583 99.375 ± 0.200 98.750 ± 0.244 100 100

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obtained. After the separation, the organicphase was dried over anhydrous sodiumsulfate until the solvent evaporated. Finally,the residue was dissolved in a known vol-ume of hexane and the measurments wererecorded at 450-nm absorbance level usingthe V-530 UV/VIS Spectrophotometer(JASCO) (Britton et al., 1995). The concen-tration of total Caro was calculated accord-ing to the formula to follow:

X mg carotenoidsð Þ¼ A · V · 1000ð Þ= A1%1 cm · 1 · 100ð Þ

where A represents absorbance at 450 nm, Vrepresents volume (mL), and A1% 1cm =2500 and expressed as mg Caro/g freshmaterial (Britton et al., 1995). All experi-ments were done under subdued light.

Statistical analysis. One-way analysis ofvariance (ANOVA) was performed for treat-ments within one cultivar and for all thecultivars within one treatment to investigatewhether the differences in physiological pa-rameters of the in vitro plants were affectedby the presence of different concentrations ofSequestrene 138 added to the culture media.Post hoc testing for the ANOVAs was per-formed using Tukey’s honestly significantdifference test (Tukey’s test) using a P #0.05 significance level to determine thestatistically significant differences betweenthe means. Values shown are means ± SE. Inaddition, all variables determined in plantssubmitted to iron treatments were correlatedby principal component analysis (PCA), in-dependently for each of the three studied V.corymbosum L. cultivars. Each principalcomponent was a linear combination of theoriginal variables with coefficients equal orgreater than eigenvectors of the correla-tion matrix. The results of these analyseswere represented graphically, for each spe-cies, as a biplot of the two main principalcomponents.

Results

Effect of Sequestrene 138 on in vitro shootmultiplication of Vaccinium corymbosum L.‘Blueray’, ‘Duke’, and ‘Patriot’. The greatestmultiplication rates were recorded on theWPM[containing 36.7 mg·L–1 ferric sodium ethyl-enediamine tetra acetic acid (FeNaEDTA)]as compared with the other culture mediasupplemented with different concentrationsof Sequestrene 138 used in this experiment.Among all the highbush blueberry cultivarstested, the greatest shoot proliferation rate(7.2 ± 0.9927) was observed in ʻBluerayʼ(Fig. 1) with the exception of the mediumsupplemented with 75 mg·L–1 Sequestrene138. ʻBluerayʼ had the greatest proliferationrates in all types of media tested, whereasthe lowest proliferation rates were recordedin ʻPatriot’, ranging from 4.08 ± 0.0902 to5.22 ± 0.1501.

Regarding shoot lengths, it was ob-served that in all cultivars the averagelength of the shoots obtained on the me-dium supplemented with Sequestrene was

significantly larger as compared with con-trol (Fig. 2).

On the WPM basal medium, whereas theshortest shoots were obtained in ʻPatriotʼ,with an average length of 2.84 ± 0.0655, thelongests were recorded in ʻDukeʼ, with anaverage length of 3.59 ± 0.0976 cm. Theconcentration given by 50 mg·L–1 Seques-trene 138 stimulated the growth of the shootsin ʻDukeʼ (4.28 ± 0.2278 cm).

The shortest shoots on the WPM basalmedium were recorded in ʻPatriotʼ, with anaverage length of 2.84 ± 0.0655 cm, but thelongests were measured in ʻDukeʼ, with anaverage length of 3.59 ± 0.0976 cm. InʻBluerayʼ, the average length of the shootsdeveloped was 3.07 ± 0.0897 cm. The 50mg·L–1 concentration of Sequestrene 138 hada beneficial effect on shoot growth also inʻDukeʼ (4.28 ± 0.2278 cm), but the longestshoots were recorded in ʻBluerayʼ (with anaverage length of 3.94 ± 0.1952 cm) on theWPM basal medium supplemented with 25mg·L–1 of Sequestrene 138. The longestshoots in ʻPatriotʼ were obtained on themedium supplemented with 75 mg·L–1 and100 mg·L–1 of Sequestrene (no statisticallysignificant differences were observed be-tween them).

Effects of Sequestrene 138 on ex vitrorooting and acclimatization of ‘Blueray’,‘Duke’, and ‘Patriot’ in vitro plantlets. Theex vitro rooting and acclimatization werecarried out simultaneously using the shootsresulting from the multiplication stage. Theshoots developed on the Sequestrene-freemedium had the lowest acclimatization per-centage, whereas those grown on the mediawith 75 and 100 mg·L–1 Sequestrene had thegreatest acclimatization percentage (Table1).ʻBluerayʼ had the most successful rooting andacclimatization percentages, ranging from96.25% to 100%; 96.25% was registered for

the shoots developed on the Sequestrene-freemedium; 98.12% for the shoots obtained onthe medium supplemented with 25 mg·L–1

Sequestrene; and 100% for the shoots grownon the medium supplemented with 50, 75,and 100 mg·L–1 Sequestrene, respectively.

Effect of Sequestrene 138 on chlorophylland 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) todetermine the relationship between the pig-ment content in the leaves of the in vitroplants and the concentration of Sequestrene138 (Figs. 3–5). In all three V. corymbosumL. cultivars, the photosynthetic pigment con-tent of the leaves was increased by increasingthe concentration of Sequestrene 138 in theculture media. In all the three cultivars,the greatest Chl a content was registered inthe leaves developed on the medium supple-mented with 100 mg·L–1 Sequestrene 138,but the lowest statistically significant valuesof the Chl a content in all the cultivars wererecorded in the leaves of the plantlets grownon the Sequestrene 138-free medium. Both,the highest and the lowest Chl a contentamong the cultivars studied were recordedin ʻDukeʼ, with a mean value of 2.729 ±0.0083 mg/g and 0.260 ± 0.0070 mg/g, re-spectively (Fig. 3).

Except for ʻBluerayʼ, the Chl b content inthe leaves analyzed showed a similar in-creasing tendency in all the three investigatedcultivars on the medium supplemented with100 mg·L–1 Sequestrene 138. In ʻBluerayʼ, themaximum Chl b content was recorded in theleaves developed on the WPM basal mediumsupplemented with 50 mg·L–1 Sequestrene138. Similar to the Chl a content, in ʻDukeʼboth were recorded, the highest and thelowest Chl b content, in the leaves developedon the WPM basal medium supplemented

Fig. 3. The influence of Sequestrene 138 on chlorophyll a content of the leaves of the in vitro plantlets grown onthe medium supplemented with the indicated Sequestrene 138 (mg·L–1) concentrations. The values shownare means ± SE (n = 5). Different lowercase letters above the bars indicate significant differences betweentreatments for each cultivar, and different capital letters indicate significant differences between thecultivars for each treatment, according to Tukey’s test (P # 0.05).

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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, thegreatest values were recorded in the 0 and 25mg·L–1 Sequestrene 138 treatments, which in-dicate iron deficiency in plants and also a lowerchlorophyll content as previously confirmed byother researches in other species such as Betavulgaris (Spiller and Terry, 1980) and Corylusavellana L. (Garrison et al., 2013).

It also was observed that the increasingconcentration of Sequestrene 138 in theculture media led to a greater accumulation

of Caro content in the leaves. The greatestCaro content for the in vitro plantlets wasrecorded in ʻDukeʼ, 0.443 ± 0.0004 mg/g onthe WPM basal medium supplemented with100 mg·L–1, whereas the lowest Caro contentwas recorded in the leaves of the ʻBluerayʼ invitro plantlets grown on the iron chelate-freeWPM basal medium (0.059 ± 0.0009 mg/g).

Principal component analyses. The ana-lyses of principal components were performedseparately for all the three cultivars, includingall the measured parameters to emphasize thevariations and obtain strong patterns in our

dataset. In the three PCAs shown in Fig. 6, twocomponents with an eigenvalue equal to orgreater than 1 expressed a cumulative percent-age of variance of about 92%. The firstcomponent (X axis) was determined by theSequestrene 138 concentrations of the me-dium (Table 1) and alone explains 63% to 71%of the variance. The iron chelate concentrationof the medium was weakly correlated, nega-tively, with the number of shoots developedand positively with the other parameters ana-lyzed. The small angles of the loading vectorsof ACL and Caro with the X-axis indicate thestrongest, positive correlations. Positive cor-relations, albeit weaker, also were registeredbetween Chl a, Chl b, and LS and the X-axis.

Discussion

In all the three Vaccinium corymbosum L.cultivars cultured on the standardWPM basalmedium 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 wasobserved that iron chlorosis was predomi-nant (Fig. 7). This phenomenon could beexplained by the iron deficiency of the culturemedium as observed in other in vitro culturesas well. Thus, it was found by Zawadzka andOrlikowska (2006) that supplementing themultiplication media with FeNaEDDHAeliminated leaf chlorosis in raspbery in 4weeks. The leaves harvested from theseshoots maintenaned their green color on theregeneration medium longer than those fromFeEDDHA-free medium (Zawadzka andOrlikowska, 2006). The results obtained byNas and Read (2001) indicate that Seques-trene 138 is a suitable chelating agent forhazelnut tissue culture. The enhanced leafcolor and morphology on medium containingSequestrene 138 compared with ferrous sul-fate and Sequestrene 330 are likely a conse-quence of improved Fe availability for theexplants. A previous study has shown thatSequestrene 138 yielded better results thanFeNaEDTA in blueberry in vitro culture,conferring dark green color to the plantsand increasing the multiplication rate andplant vigor in ʻBlue Cropʼ (Clapa et al., 2007).Similar studies made on blueberry indicatethat supplementing the basal WPM mediumwith 100 mg·L–1 Sequestrene 138 (WPMm)in combination with growth regulators led toa high proliferation rate in Elliot bluberrycultivar and the greatest number of shootsdeveloped per explant (14.85) on theWPMm+2 mg·L–1 zeatin (Vescan et al., 2012).

In a previous investigation, differenceswere observed regarding the color of theshoots in the initiation stage of in vitrocultures in blueberry cultivars such as Berke-ley, Bluecrop, and Earliblue on three differ-ent culture media, as follows:MS (Murashigeand Skoog, 1962), WPM, andMW (amixtureof equal parts of MS andWPM). The color ofthe shoots varied from one cultivar to an-other; 80% of shoots from ‘Bluecrop’ and‘Earliblue’ explants were yellowish green,and 30% of ‘Berkeley’ shoots were redregardless of the medium (Tetsumura et al.,

Fig. 5. The influence of Sequestrene 138 on the carotenoid content of the leaves of the in vitro plantletsgrown on the medium supplemented with the indicated Sequestrene 138 (mg·L–1) concentrations. Thevalues shown are means ± SE (n = 5). Different lowercase letters above the bars indicate significantdifferences between treatments for each cultivar, and different capital letters indicate significantdifferences between the cultivars for each treatment, according to Tukey’s test (P # 0.05).

Fig. 4. The influence of Sequestrene 138 on chlorophyll b content of the leaves of the in vitro plantlets grown onthemedium supplementedwith the indicated Sequestrene 138 (mg·L–1) concentrations. The values shown aremeans ± SE (n = 5). Different lowercase letters above the bars indicate significant differences betweentreatments for each cultivar, and different capital letters indicate significant differences between the cultivarsfor each treatment, according to Tukey’s test (P # 0.05).

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2008). Our results show that the basal WPMmedium supplemented with the four differentSequestrene 138 (FeNaEDDHA) concentrationshad a significant impact on the shoot lengthreached in the multiplication stage in all the

cultivars studied (Fig. 2). Studies also suggestthat there is a slight difference between the re-sponses of the blueberry cultivars to the ironchelate concentrations applied to the culturemedium (Reed and Abdelnour-Esquivel, 1991;

Ru�zi�c et al., 2012; Tetsumura et al., 2008). Inthis research, it was observed that the length ofthe shoots varied among the cultivars on alltypes of media; the longest shoots developedbeing recorded in ʻDukeʼ, followed by ʻBlue-rayʼ, whereas the shortest shootsweremeasuredin ʻPatriotʼ. A slight decrease also was observedin the number of shoots per explant on themedia supplemented with iron chelate (Fig. 1).Although the proliferation rate was greater onthe Sequestrene 138-free medium, the shootsdeveloped were shorter and yellowish. For thisreason, some of the shoots (<2 cm) wereeliminated from the ex vitro rooting stage,and others didn’t regenerate roots due tochlorosis. In this context, in ʻBluerayʼ, theaverage number of shoots per explant thatoccurred was greater (7.2 ± 0.1621) on thebasal WPM medium in comparison with thenumber of shoots per explant obtained onthe medium supplemented with 100 mg·L–1

Sequestrene 138, reaching an average numberof 6.12 ± 0.2191/explant. The rooting and exvitro acclimatization percentage recorded in theshoots obtained from the iron-freemediumwas96.250 ± 0.583%, whereas the acclimatizationpercentage of the shoots obtained from themedium supplemented with 100 mg·L–1

Sequestrene 138 was 100 (Table 1).Blueberry plants have a remarkable ex

vitro rooting capacity, which permits one toeliminate the in vitro rooting stage from themultiplication process, lowering the produc-tion costs in micropropagation (Isutsa et al.,1994). The ex vitro rooting percentagesrecorded in all the cultivars on all types ofmedia supplemented with iron chelate aresimilar to the acclimatization percenteges ofblueberry plants reported by Isutsa et al.(1994) in ʻBerkeleyʼ (>99%); 100% rootingwas reported by Hung et al. (2016) in the exvitro rooting stage of ‘Huron’ shoots ina nonsterile mixture of perlite and peatmoss(Top Blueberry Co., Cheonan, South Korea)(v/v = 1:6). These results are in accordancewith our results obtained in the shoots grownon the culture medium supplemented with 75mg·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 theleaves of the in vitro plants were significantlyinfluenced by the increasing concentration ofiron chelate added to the culture medium.The chlorophyll content of the leaves is oneof the most important and reliable indicatorsof physiological status of the plants. Thisaspect is very important for the evolutionof the in vitro cultures; namely, the lowchlorophyll content can affect photosynthe-sis, leading to a low acclimatization rate andunsatisfactory plant growth and development(Rafi et al., 2018). Similar results also werereported by Van der Salm et al. (1994), whoinvestigated the effects of iron chelates inRosa hybrida L. ‘Moneyway’ in vitro cul-tures. The authors claim that the use ofFeEDDHA is more effective. We can hypoth-esize that the replacement of FeNaEDTA byFeNaEDDHA in media also will improve thein vitro culture of other plant species thatare susceptible to iron deficiency. Also, in

Fig. 6. 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 theculture 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).

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vitro–grown Vaccinium pahalae and V. myr-tillus plants as reported by Shibli et al. (1997)had a considerably better performance. Chlo-rosis and hyperhydration were eliminatedwhen the iron chelate (FeNaEDTA) wasreplaced with or supplemented by FeNaED-DHA (Sequestrene 138–50, 100, and 150 mM).Castillo et al. (1997), in one of their studiescarried out onCarica papayaL. in vitro culture,suggest that supplementing the culture mediawith 100 mM concentration of iron provided byFeNaEDDHA may be sufficient to markedlyimprove in vitro culture performance.

The length of the shoots obtained in the invitro multiplication stage, the color, and themorphology of the in vitro plants wereconsiderably improved on the culture mediasupplemented with Sequestrene 138, as com-pared with those obtained on the basal WPMmedium, as a probable consequence of thefact that the iron in this compound is morereadily available to the plants. The use ofSequestrene 138 in the culture media of allcultivars resulted in a greater chlorophyllcontent in the leaves, a more equilibratedChl a/Chl b ratio, and a greater micropropa-gation rate as compared with control.

The results of the PCAs clearly and brieflysummarize the very similar pattern in thethree studied cultivars, which responded verysimilarly to the increasing iron chelate con-centration in the media. Among the threecultivars under investigation, ‘Patriot’ hada positive response to iron added to theculture medium, showing a strong positivecorrelation between iron concentration andgrowth parameters, whereas ‘Blueray’ and‘Duke’ demonstrated a negative effect of theiron chelate on the number and the length ofthe shoots developed as the iron concentra-tion of the medium increased. The photo-sythetic pigment concentrations in all threecultivars were considerably increased as the

iron concentration of the medium increased.The acclimatization success of the in vitroplants grown on the medium supplementedwith iron chelates was considerably greaterthan in those grown on iron-free medium.Therefore, based on the results of our study,we highly recommend the use of Sequestrene138 as the main iron source in the culturemedium for a succsessful micropropagationof blueberry plants.

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