Comparative study on the in vitro multiplication potential of Magnolia ...

Volume 16(2), 39-44, 2012JOURNAL of Horticulture, Forestry and Biotechnologywww.journal-hfb.usab-tm.ro<strong>Comparative</strong> <strong>study</strong> on the in vitro multiplication potential ofMagnolia stellata and Magnolia x soulangiana speciesRadomir Ana-Maria 1*1 National Research & Development Institute for Biotechnology in HorticultureStefanesti, Arges*Corresponding author. Email: radomir.anamaria@yahoo.comAbstract Magnolia genus includes a group of about 80 species withpersistent or falling leaves and with bloom before or after coming into leaf.The goal of this work was to develop a protocol for inducing a highregeneration rate by testing the response of different explants of two magnoliaspecies to different culture media. The aspects of in vitro morphogenesisthrough all the stages from inoculation, multiplication to rooting have beenstudied. Results showed that explants of apical buds represent optimal sourceof inoculums. The period most indicated for sampling and inoculating explantsis November - December when vegetative buds are in the dormant stage.Research in the evaluation of morphogenetic capacity of explants on differentnutrient regeneration formulas have shown that the best results in terms ofpercentage of explants started the trend was obtained on the versionsupplemented with 0.7 mg/l BAP, 1 mg/l NAA, 0.1 mg/l GA 3 , 5 mg/l ascorbicacid and vitamins LS. The greatest number of shoot (10.7microshoots/explant in the case of Magnolia stellata species and 10microshoots/explant at Magnolia x soulangiana) was produced withMurashige-Skoog mineral salts, Miller vitamins and 0,5 mg/l BAP. It took only17 days for root initiation of magnolia shoots. The best concentration ofsupplemented IBA for root initiation was 4 mg/l.Key wordsMagnolia stellata, Magnoliaxsoulangiana,micropropagation, explants,microshoots, growthregulatorsThe magnolias are the most glamorous ofornamental plants in the landscape [2,5]. Magnolias areeffective on the grand scale or in the small gardens. Inlarger garden tree magnolias are in particular moreeffective than evergreen plants, when they dominatethe landscape. They are distiquished by beautifulflowers and decorative leaves. Colours of magnoliasare varied from white to violet ones. They arewidespread in the parks, botanical gardens andarboreta.Magnolias are propagated sexually from seedsand asexually from vegetative tissue. Propagation ofmagnolias by tissue culture is difficult and it needsmodification of culture medium, growth regulators andplanting conditions [8,4]. In vitro propagation is adesired method for multiplication of valuable plants atfaster rates than conventional procedures.Micropropagation of magnolias has been reported fromshoot tip explants and axillary buds [1,3] and viasomatic embriogenesis [7,6].The aim of present <strong>study</strong> was to compare thein vitro multiplication potential of two magnoliaspecies (Magnolia stellata and Magnolia xsoulangiana).Material and MethodBiological material used for cultureestablishment was represented by meristems with 1-2foliar primordiums taken from apical or axillary budsin April-May (during active growth of shoots) or inNovember-December (during the dormant). Theexplants were inoculated on various culture media. Thereactivity of explants was evaluated after a month ofculture.The shoots obtained in the initiation phase aretransfering on the multiplication culture media, toculture proliferation. The plantlets obtained wererooted and then acclimated in order to be readapted totheir natural environment.Nutritive medium used for initiation,multiplication and rooting stage of in vitro culture ofexplants was complex contenting mineral salts,vitamins, auxin, citokinine, NaFeEDTA, dextrose andagar (table 1, 2, 3).To prevent the contamination of culture wasdone a lot of operations: biologic material disinfection,sterilization of nutritive media, sterilization ofinstruments and dishes, transfer of explants to thelaminar air flow hood.The disinfection of biological material wasmade with ethanol 94% for 10 minutes, after which the39

“FACULTATEA DE HORTICULTURĂ ŞI SILVICULTURĂ”material was transferred in a solution of calciumhypochlorite concentration of 6% for another 20minutes – treatment applied on vegetating buds. Forthe buds in the foliation stage as well as for the apicalbuds during full growth, the treatment was reduced tohalf of the initial time.The culture media were sterilized byautoclaving at 120°C temperature for 20 minutes.Before autoclaving, the pH registered in a culturemedium was adjusted to 5.6-5.8.The surgical type instruments used weresterilized in the drying stove, at 120 o C temperature for2 hours.For the growing, multiplication and rooting ofexplants we ensured in the growing room controlledconditions of temperature (22-24 o C), photoperiod (16hours) and light intensity.Composition of nutritive media used for culture initiationGrowth regulators used and theirVariconcentration in culture medium (mg/l)antsBasic mediumexperimentalascorbicBAP IAA NAA GA 3acidV.1 Macroelements MS, 1 0,2 - - -V.2 microelements MS, vitamins JQ 0,5 - 1 0,1 5V.3 Macroelements MS, 1 0,2 - - -V.4 microelements MS, vitamins MS 0,5 - 1 0,1 5V.51 0,2 - - -Macroelements MS,V.6 0,5 - 1 0,1 5microelements MS, vitamins LSV.7 0,7 - 1 0,1 5Legend: MS = Murashige - Skoog (1962); LS = Linsmaier – Skoog (1965); JQ = Jaquoit (1956)VariantsexperimentalComposition of nutritive media used for micropropagationBasic mediumGrowth regulators used and theirconcentration in culture medium (mg/l)BAP 2iP TDZ Kin0,25 - - -V.1V.2 0,5 - - -V.3V.4V.5Macroelements MS,microelements MS,vitamins M---0,250,5---0,25---V.6 - - 0,5 -V.7 - - - 0,25V.8 - - - 0,5Legend: MS = Murashige - Skoog (1962); M = Miller şi colab. (1982)AComposition of nutritive media used for in vitro rooting of shootsGrowth regulators used and theirVariantsBasic medium concentration in culture medium (mg/l)experimentalIBA GA 3V.1- 0,1V.2 2 0,1V.3 Macroelements MS½ n,4 0,1V.4 microelements MS½ n,6 0,1V.5 vitamins LS n8 0,1V.6 10 0,1V.7 12 0,1V.8 14 0,1Legend: MS = Murashige - Skoog (1962); LS = Linsmaier – Skoog (1965)Table 1Table 2Table 3The achieved results was registered asmultiplication rate being express inmicroshoots/explants and was statistic interpretation byDuncan Test. Growing of explants and rooting ofmicrocuttings was express in percents.40

Results ObtainedThe initiation phaseThe reactivity of different types of explants onvarious culture media was tested in order to establishexperimental protocol for in vitro multiplication ofmagnolia species.In the initiation stage of in vitro culture, thegrowing of explants was influenced by the type ofexplants and composition of nutritive media.Results obtained have shown that explants ofapical buds represent optimal source of inoculums.The period most indicated for sampling andinoculating explants is November-December whenvegetative buds are in the dormant stage. Research inthe evaluation of morphogenetic capacity of explantsshowed that nutrient media composition influencedreactivity of explants taken during active growth ofbuds similar with explants taken from dormant stage,differences consisting of percentages of explants turnedevolving, which were lower.It was found that the best results in terms ofpercentage of explants started the trend was obtainedon the version supplemented with 0.7 mg/l BAP, 1mg/l NAA, 0.1 mg/l GA 3 , 5 mg/l ascorbic acid andvitamins LS (V.7), followed the versions V6, V3, V5,V4, V1 and V2.In general, nutrient media compositioninfluenced the reactivity of explants taken of axillarybuds similar with explants taken from apical buds,differences consisting of percentage of explants startedevolving, that were smaller (fig. 1).apical budaxillary budFig. 1. The in vitro reactivity of magnolia species considering the explant type, genotype and culture mediumcompositionFig. 2. Morphogenetic response of explants, 4 weeks after initiation of in vitro cultureThe multiplication phaseIn multiplication phase, the analysis of theeffect of type and concentration of citokinine could beseen the clearly influence of citokinine type on theregeneration rate.In the case of Magnolia stellata species,referring to the effect of citokinine depending on its41

“FACULTATEA DE HORTICULTURĂ ŞI SILVICULTURĂ”concentration was found that the best results in termsof multiplication rate (10.7 microshoots/explant,respectively 8.9 microshoots/explant) was recorded onthe versions that contain a higher BAP (0.5 mg/l - V.2)and K (0.5 mg/l - V.8). On the nutritive media in witchBAP and K were found in lower concentrations (0.25mg/l - V.1 and V.7), the number of shoots/explantshowed values lower than the alternatives discussedabove (8.2 microshoots/explant and 4.3microshoots/explant). On versions supplemented with2iP (V.3 and V.4) and TDZ (V.5 and V.6) regenerationrate was 0, in both concentrations used.In general, the type and concentration ofcitokinine had a similar influence on the multiplicationrate of Magnolia x soulangiana species differencesconsisting in the multiplication rate values were lower(fig. 3).Magnolia stellataMagnolia x soulangianaFig. 3. Influence of type and concentration of citokinine for the multiplication rateFig. 4. Multiple shoot proliferation on MS medium supplemented with 0.5 mg/l BAPThe rooting phaseObservations made after 32 days of culturerevealed that magnolia has a good behaviour in vitrorooting process, the differences registered at therooting percentages were influenced by thecomposition of culture media, especially by theconcentration of indolilbutiric acid.Observations in the first 14 days of incubationrevealed no initiation process rootedness. Appearanceof the first primary root was recorded after 17 days ofculture, regardless of the culture medium used.In the case of Magnolia stellata species, onthe control variant (without IBA) has identified thelowest number of microshoots rooted respectively inthe proportion of 10-15% at each interval ofincubation, by the end of 32 days of experiment.The highest values of the rooting rate (90%)occurred after 24 days of culture at a concentration of 4mg/l IBA (fig. 5).42

Fig. 5. The influence of indolilbutiric acid over rooting of Magnolia stellata microshootsIn general, IBA concentration had a similar influenceon the percentage of rooted microshoots in the case ofMagnolia x soulangiana species, but their values arelower (fig. 6).Fig. 6. The influence of indolilbutiric acid over rooting of Magnolia x soulangiana microshootsFig. 7. In vitro rooting of shoots on ½ MS medium supplemented with 4 mg/l IBA43

“FACULTATEA DE HORTICULTURĂ ŞI SILVICULTURĂ”Rooted plantlets had been acclimatized in the greenhouse and transferring to the soil substrate (fig. 8).Fig. 8. Magnolia plants after transfer from greenhouse conditions to soilConclusions1. The results presented in this paper indicatethat the percentage of regenerating, shoot number perexplant, percentage of rooting were affected bygenotype and the interaction of genotype with theculture media. Between Magnolia stellata andMagnolia x soulangiana species tested, the best resultsas well as percent of regeneration, number of shootsper explant and rooting rate were recorded forMagnolia stellata species.2. Explants of apical buds represent optimalsource of inoculums. The period most indicated forsampling and inoculating explants is November-December when vegetative buds are in the dormantstage.3. Research in the evaluation ofmorphogenetic capacity of explants on differentnutrient regeneration formulas and results obtainedhave shown that the best results in terms of percentageof explants started the trend was obtained on theversion supplemented with 0.7 mg/l BAP, 1 mg/lNAA, 0.1 mg/l GA 3 , 5 mg/l ascorbic acid and vitaminsLS.4. Our results demonstrate the clearlyinfluence of citokinine type on the regeneration rate.The best shoot multiplication rate (10.7microshoots/explant in the case of Magnolia stellataspecies and 10 microshoots/explant at Magnolia xsoulangiana) was established with Murashige-Skoogmineral salts, Miller vitamins and 0,5 mg/l BAP.5. It took only 17 days for root initiation ofmagnolia shoots. The best concentration ofsupplemented IBA for root initiation was 4 mg/l.6. The results obtained in our investigationsproved that in vitro culture could be used as anefficient alternative method for multiplication ofMagnolia stellata and Magnolia x soulangiana species.Bibliography1. Biederman I. E. G. - 1987 - Factors affectingestablishment and development of Magnolia hybrids invitro, Acta Horticulturae, 212, s. 625-629.2. Callaway D.J. – 1994 - The world of Magnolias,Timber Press, Portland, 260 pp.3. Kamenicka Aurelia, Lanakova Maria – 2000 -Effect of medium composition and type of vesselclosure on axillary shoot production of magnolia invitro, Acta Physiologiae Plantarum, vol: 22, number: 2,pages: 129-134.4. Kamenicka Aurelia, Takats J. – 1997 - Magnolia, 1:1-6.5. Liu Y.H. – 2004 - Magnolia of China. BeijingScience&Technology Press, 391 pp.6. Merkle S.A. - 1995 - Somatic embryogenesis inMagnoliaceae (Liliodendron and Magnolias). In: BajajY.P.S. (ed.) Somatic embryogenesis and SyntheticSeed I. Springer-Verlag, Berlin, Heidelberg: 388-403.7. Merkle S.A., Watson-Pauley B.A. – 1994 - Ex vitroconversion of pyramid magnolia somatic embryos,HortScience, v. 29(10) p. 1186-1188.8. Tobe J.D. – 1990 - Journal of Magnolia Soc., 26 : 4844