PLANT TISSUE CULTURE

Virginia Sarropoulou & Kortessa Dimassi-Theriou & Ioannis Therios

Received: 15 April 2013 /Accepted: 17 September 2013 /Published online: 8 October 2013 / Editor: J. Forster# The Society for In Vitro Biology 2013

Abstract Conventional multiplication of cherry (Prunuscerasus L.) rootstocks utilizes division, cuttings, and propa-gation through seed, which are relatively slow and laborintensive and result in genetic variability. Tissue culture, onthe other hand, ensures rapid, large-scale, and low-cost pro-duction of genetically identical, physiologically uniform, andpathogen-free plants. In the cherry rootstocks CAB-6P, Gisela6, and MxM 14, sodium nitroprusside (SNP) promoted callusinduction, in vitro shoot proliferation, and rooting from leafexplants in a medium containing 17.6 μM benzyladenine and2.68 μM α-naphthaleneacetic acid. CAB-6P explants treatedwith 10 μM SNP gave the maximum shoot number (5),whereas 30 μM SNP gave the longest shoots and the greatestshoot induction rate (26.67%). Best rooting was obtained with50 μM SNP. In Gisela 6 rootstock, the shoot number (10) andshoot length (20.5 mm) were maximal in the control groupwithout plant growth regulators. The shoot induction rate wasenhanced (40%) with 40 μM SNP. SNP at 40 μM resulted inroot formation, while 30 μM produced the largest callus size,and 10 μMSNP resulted in the maximum callus fresh weight.MxM 14 leaves treated with 30 μM SNP gave the maximumshoot number (3), root number (7.56), and shoot inductionrate (40%), whereas 40 μM SNP gave the longest shoots(12 mm) and roots (20 mm). Best results for callus size, callusfresh weight, and callus induction rate (100%) in the CAB-6Pand MxM 14 rootstocks were observed with 30 and 40 μMSNP, respectively. Rooted explants with shoots were graduallyacclimatized to the external environment with a high survival

percentage (85%). An efficient protocol of indirect organo-genesis was established for the three cherry rootstocks usingSNP.

Keywords Cherry rootstocks . Indirect organogenesis .

Rooting . Shoot proliferation . Sodium nitroprusside

Introduction

CAB-6P is a commercial cherry (Prunus cerasus L.) root-stock. All cherry varieties grafted on this rootstock have 30%less vigor, earlier cropping, better fruit quality and color, andhigher yield efficiency, in comparison to those grafted on wildcherry seedlings. Gisela 6 (P. cerasus × Prunus canescens )tolerates soils of poorer quality and reduced water supply, andits vigor is between that of Gisela 5 and of Prunus avium L.Whereas, MxM 14 (P. avium × Prunus mahaleb ) is a clonalselection from an open-pollinated population of P. mahaleb .Plants grafted on this rootstock are about 20% more vigorousthan those grafted on CAB-6P (P. cerasus L.) and about 30%less vigorous than those grafted on cherry seedlings.

Fruit rootstocks are traditionally propagated either by rela-tively slow and labor-intensive vegetative methods (tech-niques such as division and cuttings) or from seed, whichoften result in nonuniform material. Seedling propagation isthe most efficient and economical method as long as geneticvariability can be controlled within acceptable limits.Although seedlings are used extensively in nurseries, theplants produced from the tested rootstocks show genetic var-iability. Therefore, clonal methods of propagation, such ascuttings and tissue culture, are the most important means ofpropagation of the three studied cherry rootstocks.Propagation by cuttings is an inexpensive, rapid, and simplemethod. However, the rooting percentages of the cherry root-stocks in such studies have not yielded promising results.

V. Sarropoulou (*) :K. Dimassi-Theriou : I. TheriosLaboratory of Pomology, Department of Horticulture,School of Agriculture, Aristotle University of Thessaloniki,54124 Thessaloniki, Greecee-mail: vsarrop@gmail.com

V. Sarropouloue-mail: vsarropo@agro.auth.gr

In Vitro Cell.Dev.Biol.—Plant (2014) 50:226–234DOI 10.1007/s11627-013-9565-1

Ιn vitro plant regeneration from leaf explants of the cherryrootstocks CAB-6P, Gisela 6, and MxM 14 usingsodium nitroprusside

Therefore, the application of tissue culture techniques hasbecome an important alternative to more conventional propa-gation procedures. Using shoot tip culture resulted in virus-free plants and impressive rates of mass propagation in arelatively short time. The basis for this in vitro advantageappears to be the progressive increase in propagation potentialwith repeated subculturing. However, particular skills arerequired to carry out the in vitro procedures. The applicationof tissue culture methods for vegetative propagation of tem-perate fruit tree rootstocks started in the mid-70s, and a con-siderable number of improved protocols have since beendeveloped. Generally, the goal of micropropagation isobtaining rapid, large-scale, and low-cost production of ge-netically identical, physiologically uniform, and pathogen-free plants (Rathore et al. 2004). Successful in vitro clonalpropagation methods are reported in many rootstocks, includ-ing cherry (Sedlák et al. 2008).

In this research, sodium nitroprusside (SNP), which pro-duces nitrous oxide (NO), was used. NO is a highly reactivegas which is a ubiquitous bioactive molecule that plays acentral role as a signal in plant response to biotic and abioticstresses (Crawford and Guo 2005; Arasimowicz andWieczorek 2007) and was recently recognized as a new typeof phytohormone (Leterrier et al. 2012). The accumulation ofNO in roots mediates auxin induction of lateral root formation(Correa-Aragunde et al. 2006), adventitious root growth(Tewari et al. 2008), and root hair development (Lombardoet al. 2006). SNP is the most common NO donor (Floryszak-Wieczorek et al. 2006; Ederli et al. 2009) because of itsrelative low cost, its well-documented application(Zandonadi et al. 2010), and the long-lasting NO production(Floryszak-Wieczorek et al. 2006).

Pagnussat et al. (2002) and Huang and She (2003) foundthat exogenous NO and/or SNP could induce adventitiousroot formation in cucumber and mung bean hypocotyl cut-tings, respectively. Furthermore, Correa-Aragunde et al.(2004) demonstrated that NO and/or SNP played a key rolein determining lateral root development in tomato. Tunet al. (2001) reported that cytokinin could induce the releaseof NO in plant cell cultures, such as Arabidopsis , tobacco,and parsley. In addition, NO and/or SNP could also promotethe growth of pea leaves (Leshem 1996) and induce elon-gation of root tips in maize (Gouvêa et al. 1997). All thesemight indicate that NO and/or SNP plays a role in celldivision, as well as in adventitious shoot regeneration andmultiplication. However, the way SNP interacts with auxinsto induce adventitious rooting is not known; thus, this studywas undertaken to determine whether a synergistic actionbetween SNP and IBA exists and whether the combinationof the two yields better rooting results than an individualapplication.

In view of the particularity of the cherry rootstocks CAB-6P, Gisela 6, andMxM 14 and the significance of SNP in plant

physiology and horticulture, this study was conducted toelucidate SNP effects on plant regeneration from leaf explants.Another aim was to achieve concurrent shoot proliferation androoting, by shortening the in vitro propagation cycle of thethree cherry rootstocks in order to reduce the cost of theirproduction.

Materials and Methods

Plant material and culture conditions. The P. cerasus L.explant material was excised leaves from shoot tip explantsof previous in vitro cultures. The leaves were injured with ascalpel by making two to five parallel incisions to the midribin the abaxial (lower) leaf surface. The initial material wascertified as virus free. The explants were grown in glass flatbottom test tubes, 25×100 mm, containing 10 mL ofMurashige and Skoog (MS) medium (Murashige and Skoog1962) supplemented with 30 g/L sucrose, and 6 g/L agar(Bacto agar). The pH of the culture medium was adjusted to5.8 before adding agar and was sterilized by autoclaving at121°C for 20 min. One explant was aseptically transferred toeach test tube, which was capped with aluminum foil. All thecultures were incubated in a growth room under controlledenvironmental conditions with a light intensity of 150 μmol/m2/s provided by cool white fluorescent lamps (36W, Philips,Amsterdam, Netherlands), a photoperiod of 16 h, and a tem-perature of 22±1°C.

Plant regeneration medium. The basal regeneration mediumwasMS supplemented with 17.6μMbenzyladenine (BA) and2.68 μM α-naphthaleneacetic acid (NAA). Leaves were re-moved from shoot tips and were transferred after midribwounding, as described above, to the regeneration mediumcontaining 0, 10, 20, 30, 40, or 50 μM SNP. Τhe growthregulators (BA and NAA) excluding SNP were added beforeautoclaving (1.4 kg/cm, 121°C, 18 min). Filter-sterilized SNPwas added to the sterilized medium after the temperaturereached 65°C. The medium containing SNP was used imme-diately. Mean number of shoots per explant, mean shootlength, shoot induction frequency (percent), root number perrooted explant, root length, root fresh weight (FW), rootingpercentage (percent), callus size, callus FW, and callus induc-tion frequency (percent) were recorded after 24 wk. Every4 wk the culture medium was replaced with fresh mediumthroughout the 24-wk experimental period.

Rooting and acclimatization ex vitro . Plantlets with well-developed shoots and roots were removed from the glass testtubes, washed thoroughly with tap water, and transferred to apeat and perlite (1:1) substrate. Potted plantlets were coveredwith transparent polyethylene bags to ensure high humidityand were maintained for 2 wk in a growth room at 22±1°C

ORGANOGENESIS OF CHERRY ROOTSTOCKS WITH SNP 227

and light intensity of 150 μmol/m2/s, and watered every 3 dwith quarter-strength MS nutrient solution lacking organicconstituents. For plant acclimatization, the polyethylene bagswere removed after 2 wk to lower the relative humidity (70%),while the plants developed an efficient root system and be-came photosynthetically active. The conditions in the green-house during acclimatization were temperature 25°C, lightintensity 250 μmol/m2/s to provide enough energy for plantletgrowth and establishment, and a 12-h photoperiod. Light wasmodulated by appropriate whitewash of the greenhouse. After5 wk from planting and hardening in the greenhouse, theacclimatized plants were transplanted to pots filled with or-chard soil and transferred to the field for evaluation of theirmorphologic stability and survival.

Statistical analysis. The experimental layout was completelyrandomized, and the data were analyzed with analysis ofvariance (ANOVA) using the statistical package SPSS 17.0(SPSS Inc, Chicago, IL). The experiment was repeated twice,and the reported data are the means of the two experiments,which included six treatments, with 15 replications (test tubes)per experiment giving a total of 30 replications. For thestatistical analysis of the rooting percentage and shoot andcallus induction frequency, three replications (five tubes perreplication) were used giving a total of six replications for thetwo times that the experiment was conducted, which was

sufficient to conduct the statistical analysis. Both experimentsentailed six treatments each, and each of those treatmentsconsisted of ten replicates. To establish significant differencesamong the treatments, the Duncan multiple range test wasused at P ≤0.05 for mean comparison.

Results

Effect of SNP on in vitro shoot proliferation from leafcallus. In the CAB-6P rootstock, no shoot induction wasobserved in the control or in the 20-μM SNP treatments(Table 1; Fig. 1a). The remaining treatments (10, 30, 40, and50 μM SNP) were found to enhance shoot induction (Fig. 1b,g–i). More specifically, 10 μMSNP gave the maximum shootnumber (5), while 30 μM SNP produced the longest shoots(18.33 mm). The greatest shoot induction rate was 26.67%with 30 μM SNP. In the Gisela 6 rootstock, the maximumshoot number/explant (10) and shoot length (20.5 mm) wererecorded in the control treatment (Table 1; Fig. 2e, g–i).However, the shoot induction rate was greatest (40%) when40 μM SNP was added to the medium (Fig. 2c, d, f). In theMxM 14 rootstock, concentrations higher than 10 μM SNPresulted in multiple shoot production (Table 1; Fig. 3c, d, f–h).Best results regarding the shoot number/explant (3) and shoot

Table 1. Effect of SNP on shootand root growth through callusfrom leaves of cherry rootstocksCAB-6P, Gisela 6, and MxM 14

The medium contained 17.6 μMΒΑ and 2.68 μM NAA plus SNP(0–50 μM). Treatments denotedby the same letter in each columnand in each rootstock, separately,are not significantly differentaccording to Duncan’s multiplerange test at P ≤0.05

SNP (μΜ) Shootnumber/explant

Shootlength(mm)

Shootinductionrate (%)

Rootnumber/explant

Rootlength(mm)

Root freshweight (g)

Rootingpercentage(%)

CAB-6P

0 0.00 a 0.00 a 0 a 2.00 b 10.00 b 0.011 b 10 b

10 5.00 e 17.00 c 20 b 5.00 c 20.50 c 0.030 c 10 b

20 0.00 a 0.00 a 0 a 0.00 a 0.00 a 0.000 a 0 a

30 2.60 c 18.33 e 26.67 c 0.00 a 0.00 a 0.000 a 0 a

40 3.50 d 17.50 d 20 b 0.00 a 0.00 a 0.000 a 0 a

50 1.00 b 15.00 b 20 b 9.00 d 20.56 c 0.065 d 30 c

Gisela 6

0 10.00 f 20.50 f 10 b 0.00 a 0.00 a 0.000 a 0 a

10 3.50 c 20.00 e 20 c 0.00 a 0.00 a 0.000 a 0 a

20 0.00 a 0.00 a 0 a 0.00 a 0.00 a 0.000 a 0 a

30 2.00 b 15.00 c 10 b 0.00 a 0.00 a 0.000 a 0 a

40 3.75 d 14.00 b 40 e 3.00 b 25.00 b 0.105 b 10 b

50 5.00 e 18.00 d 30 d 0.00 a 0.00 a 0.000 a 0 a

MxM 14

0 0.00 a 0.00 a 0 a 4.88 e 13.21 a 0.101 f 88.89 b

10 0.00 a 0.00 a 0 a 3.00 a 13.33 a 0.061 b 50.00 a

20 2.50 c 7.50 b 40 c 4.44 d 18.00 b 0.074 c 80.00 b

30 3.00 d 10.33 c 40 c 7.56 f 17.47 b 0.097 e 80.00 b

40 2.00 b 12.00 d 20 b 3.80 c 20.00 c 0.092 d 50.00 a

50 2.00 b 10.00 c 14.29 b 3.40 b 13.42 a 0.055 a 57.14 a

228 SARROPOULOU ETAL

induction rate (40%) were exhibited in explants treated with30 μMSNP, whereas those treated with 40 μMSNP producedthe longest shoots (12 mm).

Effect of SNP on in vitro rooting from leaf callus. In the CAB-6P, root formation was observed with 0, 10, and 50 μM SNPconcentrations (Table 1; Fig. 1c, f). Best rooting in terms ofroot number/rooted explant (9), root length (20.56 mm), rootfresh weight (0.065 g), and rooting percentage (30%) wereobtained with 50 μM SNP (Fig. 1d, e). In the Gisela 6 root-stock, only the 40-μMSNP treatment produced roots from leafcallus with three roots per rooted explant which were 25 mmlong, and rhizogenetic capacity was rather low (10%). In the

MxM 14 rootstock, explants treated with 30 μMSNP gave themaximum root number/rooted explant (7.56), whereas 40 μMSNP gave the longest roots (20 mm). The maximum root freshweight and rooting percentage (88.89%) were in the controltreatment. The minimum values in all rooting parameters wereobserved at the lowest and the highest SNP concentrations of10 and 50 μM, respectively (Fig. 3b).

Effect of SNP on callus induction from leaf explants. In theCAB-6P and MxM 14 rootstocks, best results in terms ofcallus size, callus fresh weight, and callus induction frequencywere obtained with 30 and 40 μM SNP, respectively (Table 2;Fig. 3i). In the Gisela 6 rootstock, callus size was maximum

Figure 1. In vitro callus induction, shoot differentiation, and rooting ofCAB-6P explants in a medium containing 17.6 μM ΒΑ and 2.68 μMNAA. (a) Leaf callus formation in the control treatment (0 μMSNP), (b)shoot differentiation from leaf callus with 30 μMSNP, (c) root formation

through callus from the leaf petiole with 50μMSNP, (d , e) shoot and rootinduction at the same time with 50 μM SNP, (f) root formation from leafcallus with 10 μM SNP, (g , h) production of multiple shoots with 10 μMSNP, and (i) shoot differentiation from leaf callus with 40 μM SNP.

ORGANOGENESIS OF CHERRY ROOTSTOCKS WITH SNP 229

(31.5 mm) with 30 μM SNP. SNP substantially enhancedcallus fresh weight with its maximum value observed at40 μM. The callus induction frequency was 100% in alltreatments for all rootstocks (Fig. 2a).

In the three cherry rootstocks, callus formation was ob-served after 3 wk of culture in all the treatments, both at theleaf petiole and on the adaxial and abaxial surface of the leafblade. In the CAB-6P rootstock, the callus was large, initiallya pale yellow that gradually turned a dark brown color by theend of the experiment. With time, callus swelling became

apparent. Shoot proliferation from callus was observed after5 wk of culture mainly from the leaf petiole. In the Gisela 6rootstock, rooting of shoots produced from leaf callus wasinitiated 6 wk after transferring the explants to the regenera-tion medium. The callus was a yellowish brown color andsmaller than the CAB-6P rootstock; it was produced primarilyfrom leaf petiole and to a lesser extent from the intersectionpoints of the upper and lower leaf blade surface with themidrib. In theMxM 14 rootstock, indirect organogenesis fromleaves was obvious after 6 wk of culture in regeneration

Figure 2. In vitro callus induction, shoot differentiation, and rooting ofGisela 6 explants in a medium containing 17.6 μM ΒΑ and 2.68 μMNAA. (a) Leaf callus formation in the control treatment (0 μMSNP), (b)shoot differentiation through callus from leaf petiole with 10μMSNP, (c ,

d) shoot induction with 40 μM SNP, (e) shoot production through callusfrom the leaf petiole with 50 μM SNP, (f) and with 40 μM SNP, (g , h)production of multiple shoots in the control treatment (0 μM SNP), and(i) shoot induction with 30 μM SNP.

230 SARROPOULOU ETAL

medium. The callus was of moderate size, brown, and formedat the leaf petiole, more precisely at the juncture of the petiolewith the leaf blade. Although the roots from the upper leafblade surface were white, those from the lower leaf surfacepresented a yellowish–white color. Furthermore, the deploy-ment of roots was observed from the upper and lower surfacesof the leaf, while their formation from the leaf petiole wasalmost negligible.

Over 100 plantlets with fully developed roots were suc-cessfully acclimatized, initially in a peat and perlite (1:1)

substrate in the greenhouse and afterwards transplanted inorchard soil, following the procedure described in the“Materials and Methods.” All the regenerated plantletsappearedmorphologically normal and grewwell. The survivalpercentage was high (85%), and the acclimatized plantlets didnot show symptoms of nutrient deficiency or excess.Furthermore, the plantlets in the greenhouse grew in heightand were vigorous with dark green leaves and shoots ofsufficient thickness akin to good growth and development ofthe root system. Additionally, the induction of multiple shoots

Figure 3. In vitro callus induction, shoot differentiation, and rooting ofMxM 14 explants in medium containing 17.6 μM ΒΑ and 2.68 μMNAA. (a) Leaf callus and root formation in the control treatment (0 μMSNP), (b) and in the 50 μM SNP treatment, (c) shoot differentiation and

rooting from leaf callus with 30 μM SNP, (d , e) shoot induction with20 μM SNP, (f , g) 30 μM SNP, (h) 50 μM SNP, and (i) leaf callusformation with 40 μM SNP.

ORGANOGENESIS OF CHERRY ROOTSTOCKS WITH SNP 231

and the development of numerous lateral roots that weresubstantially smaller in both length and thickness than theprimary roots, were also observed.

Discussion

SNP, which is an NO donor, affected in vitro callus induction,shoot proliferation, and rooting of CAB-6P, Gisela 6, andMxM 14 cherry explants in a dose-dependent manner. In otherwords, the in vitro callus induction, shoot proliferation, androoting characteristics appear to be affected to a lesser orgreater extent by the SNP concentrations. Furthermore, thethree cherry rootstocks responded differently to SNP concen-trations due to their different genotypes. In order to study theeffects of NO, its donors, such as SNP, are generally appliedexogenously rather than direct gas application, since directexposure to NO is technically difficult and cumbersome.

SNP promoted the in vitro shoot proliferation of CAB-6Pand MxM 14 explants. However, in the Gisela 6 explants, onlythe shoot induction rate was enhanced, reaching its maximumvalue with 40 μMSNP. Our results are in agreement with thosereported by others including Han et al. (2009) for Malushupehensis Rehd. var. pinyiensis Jiang; Kalra and Babbar(2012) for Albizzia lebbeck ; and Xu et al. (2009) forDioscorea opposita Thunb. In the Gisela 6 rootstock, on theother hand, better results concerning the number of shoots perexplant and shoot length were recorded in the absence of SNPfrom the culture medium. Our findings are in disagreementwith those of Kalra and Babbar (2010) for Linum usitatissimumL, where SNP (1–6 μM) augmented the caulogenic responseby increasing the average number of shoots per explant withsignificant increase being only at 4 and 5 μM.

Having a direct effect on cell wall components, SNP (NO)may cause cell wall relaxation, thus inducing cell enlargementand stimulating plant growth (Lamattina et al. 2003).Similarly, Leshem and Haramaty (1996) purport that NO actson the lipid bilayer of the cell membrane and wall compo-nents, loosening cell walls and so permitting their expansion.In the present study, although SNP stimulated shoot length inthe CAB-6P and MxM 14 rootstocks, it seems to have had aninhibitory effect on Gisela 6. Regarding rooting, SNP promot-ed growth of longer roots in all the three cherry rootstocks.The different responses among the three cherry rootstocksconcerning shoot and root length are most probably genotypedependent. Results that were inconsistent with both CAB-6Pand MxM 14, but in accordance with Gisela 6 rootstock, werealso exhibited in wheat plants (Tian and Lei 2007),Kosteletzkya virginica shoots (Guo et al. 2009), andBrassica napus plants (Jhanji et al. 2012).

SNP at 50 μΜ had a considerable effect in promoting thein vitro rooting of CAB-6P explants. A possible mechanism

could be that SNP induces indole-3-acetic acid (IAA) synthe-sis, which shortens the lag period for the first transversedivision and hastens the formation of root initials(Mitsuhashi-Kato et al. 1978). Furthermore, SNP and IAAhasten the cells’ entrance into the longitudinal division stagein the rooting zone, thereby increasing root length (Blazichand Heuser 1979). Most SNP effects might be mediated byethylene since its biosynthesis is stimulated by auxin. Similarresults were reported for M. hupehensis Rehd. var. pinyiensisJiang (Han et al. 2009) and mung bean (She and Huang2004). For the MxM 14 rootstock in our study, it appears that10 μM of SNP completely inhibited rooting. In their study onPhalaenopsis and Doritaenopsis orchids, HsuanHsuan et al.(2009) also found similar results as for MxM 14, but were indisagreement with our findings for CAB-6P. Our findingsindicated that SNP promoted rhizogenesis of Gisela 6 leavesat 40 μMwhich are in agreement with those reported by Liaoet al. (2012) for marigold (Tagetes erecta L.) and Kalra andBabbar (2010) for L. usitatissimum L. Additionally, we foundthat SNP enhanced root length in both the CAB-6P and MxM14 cherry rootstocks and similar results have been reported inB. napus (Jhanji et al. 2012) and B . rapa (Sung and Hong2010). A possible explanation is that in these genotypes, SNPdecreases cell wall lignification and accelerates cell expan-sion. In Gisela 6, SNP had an inhibitory effect on root length,which could be attributed to the different genotype of thisrootstock. A high level of SNP, however, may have actuallyenhanced membrane leakage due to oxidative stress and im-paired cell wall growth, thus inhibiting plant growth (Wanget al. 2010).

The application of SNP in the MxM 14 rootstock had theeffect of reducing root fresh weight. This can be ascribed, onthe one hand, to the morphology of the plant’s root system, asthe formed roots were very thin, and on the other hand, to adecrease in the volume of the already existing cells, as well asto the reduced cell expansion of recently divided cells. Incontrast, SNP application in the CAB-6P rootstock increasedthe root fresh weight, indicating that the different responsebetween the two rootstocks is genotype dependent.

In the CAB-6P rootstock, 50 μΜ of SNP promoted thepositive effects of BA and NAA on in vitro rooting, exhibitinga synergistic action. The same trend was observed in theMxM14 rootstock with 30 and 40 μΜ SNP. Similar results werereported in rice (Liu et al. 2011), cucumber (Pagnussat et al.2003), and mung bean (Phaseolus radiatus L.; Huang et al.2007). NO is a downstream messenger in the IAA-signalingpathway that promotes adventitious root development.Adventitious roots arise endogenously from dedifferentiatedparenchyma cells around vascular tissues with SNP action.The SNP effects root primordia elongation differently to for-mation of root initials. Pagnussat et al. (2003) found that NOtreatments were able to induce adventitious rooting in cucum-ber explants to the same extent as the plant hormone IAA.

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Furthermore, application of SNP to tomato seedlings(Lycopersicon esculentum Mill.) induced lateral root (LR)emergence in a dose-dependent manner, while primary rootgrowth diminished (Correa-Aragunde et al. 2004). More thanone hypothesis could be postulated to explain which targetmolecules SNP exerts its action through in order to promoteLR development. For example, it can be hypothesized that: (1)SNP via NO produces an antioxidant environment (Beligniand Lamattina 2002), which might protect auxins from oxi-dation, and/or (2) SNP regulates cell-cycle genes or enzymeactivities involved in auxin signal transduction. NO and/orSNP mediate(s) plant hormones and transduction in plantgrowth and development, as well as function(s) as a signalin the auxin-induced signaling cascade leading to adventitiousroot development (Tewari et al. 2008). Furthermore, SNP hasbeen shown to play a central role in lateral root formation(Correa-Aragunde et al. 2006; Tewari et al. 2008).

In the three cherry rootstocks studied, callus inductionfrequency was 100% in all the treatments (Table 2). A highcallus induction frequency has been also reported for tuberexplants of D. opposita Thunb treated with SNP (Xu et al.

2009). Root regeneration occurred only after callus formationin all the treatments for all three rootstocks in the presentstudy. In contrast, it appears that SNP did not promote callusinduction in M. hupehensis Rehd. var. pinyiensis Jiang (Hanet al. 2009).

In sum, it is of note that SNP, a donor of NO, has a directeffect on in vitro callus induction, shoot differentiation, androoting of the CAB-6P, Gisela 6, and MxM 14 explants. SNPmay interact with auxin and cytokinin, linking the regulationof cell division to cell differentiation during the dedifferenti-ation and redifferentiation of plant cells. This has been report-ed in a previous study by Ötvos et al. (2005). Furthermore, itwas found that not only does SNP improve plant regenerationefficiency in the three cherry rootstocks but it also shortens thein vitro propagation cycle by combining the steps of shootproliferation and rooting. Finally, it appears that SNP actssynergistically with BA and NAA, giving better results thanthe combination BA plus NAA.

Acknowledgments We would like to express our sincere gratitude toAngelos Xylogiannis for kindly providing the CAB-6P (P. cerasus L.),Gisela 6 (P. cerasus × P. canescens), and MxM 14 (P. avium × P.mahaleb) plants; also our thanks to Sofia Kuti and Vasiliki Tsakiridoufor technical assistance. The authors gratefully acknowledge the financialsupport of the Aristotle University of Thessaloniki.

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Table 2. Effect of SNP on callus growth from leaf explants of cherryrootstocks CAB-6P, Gisela 6, and MxM 14

SNP (μΜ) Size of callus(mm)

Callus freshweight (g)

Callus inductionfrequency (%)

CAB-6P

0 26.07 b 1.100 b 100 a

10 25.00 a 0.950 a 100 a

20 27.27 d 0.960 a 100 a

30 28.46 f 1.600 d 100 a

40 27.50 e 1.600 d 100 a

50 26.92 c 1.290 c 100 a

Gisela 6

0 26.54 b 1.540 a 100 a

10 28.89 d 2.170 e 100 a

20 29.55 d 1.700 b 100 a

30 31.50 e 1.670 b 100 a

40 27.73 c 1.890 d 100 a

50 24.00 a 1.790 c 100 a

MxM 14

0 36.82 e 1.250 c 100 a

10 29.17 a 1.030 a 100 a

20 32.50 c 1.350 d 100 a

30 35.36 d 1.480 e 100 a

40 37.00 e 1.860 f 100 a

50 30.50 b 1.070 b 100 a

The medium contained 17.6 μM ΒΑ and 2.68 μM NAA plus SNP(0–50 μM). Treatments denoted by the same letter in each columnand in each rootstock, separately, are not significantly differentaccording to Duncan’s multiple range test at P ≤0.05

ORGANOGENESIS OF CHERRY ROOTSTOCKS WITH SNP 233

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