Micropropagation of Ilex glabra (L.) A. Gray

Additional index words. inkberry, microcutting, MS medium, rooting, shoot proliferation,tissue culture protocolAbstract.Nodalsegmentscontainingoneaxillarybud(1to1.5cm)weredisinfectedusing10% bleach and were established on a Murashige and Skoog (MS) medium withouthormones at 27 8C and with a 16-h photoperiod. The sprouted shoots (’’1.0 cm) werecultured on a MS medium supplemented with 6-benzylaminopurine (BAP), kinetin(KIN), or zeatin (ZT) at 2.3, 4.5, 9.1, or 18.2 mM. After 38 d, ZT and BAP signiﬁcantlyinduced multiple shoot formation with multiplication rates of 4 to 6, whereas themultiplicationrateofKINwaslessthan2. ShootsculturedonZTgrewsigniﬁcantlytallerthanthoseonBAPandKIN. TheheightofthelongestshootstreatedwithZTwas4.6cm,whichwas1.6to2. 2timesgreaterthanthosetreatedwithBAPorKIN.Toinducerooting,shoots (’2 cm) were subcultured on one-fourth strength MS (1/4 MS) mediumcontaining either 3-indolebutyric acid (IBA) or 1-naphthylacetic acid (NAA) at 2.6,5.1, or 10.3 mM. Adventitious roots formed in vitro after 2 to 4 weeks. IBA at 10.3 mMproduced the best rooting (100%) compared with other treatments after 38 d of culture.The average number of roots per shoot for IBA was ’’15, which was 1.6 to 3.1 times asmany as that of other treatments. All rooted plantlets were then transplanted into a mixof peatmoss and perlite (1:1 v/v) and acclimatized in a mist system. Average plantletsurvival was 73.6% after 35 d. After acclimatization, they were grown in a pot withMetro-mix under greenhouse conditions for 10 weeks where 95% of plants survived andgrewupto6.8cmhigh. Themicropropagationprocedure,i.e.,nodalsegmentscontainingone axillary bud proliferated on MS with 4.5 mM ZT followed by in vitro rooting on 1/4MS plus 10.3 mM IBA, could be used for commercial mass production of new inkberrycultivars.Ilex, a member of the Aquifoliaceae(holly family), comprises more than 500deciduous and evergreen shrubs or trees witheconomic importance as crops and ornamen-tals in tropical and temperate regions of theworld (Galle, 1997; Hu, 1989). Ilex glabra(L.) A. Gray (inkberry) is a native evergreenshrub with glossy green foliage and blackberry-like drupes. This species grows toa mature height of 1.8 to 2.4 m and a widthof 2.4 to 3.0 m (Dirr, 1998). It is a desirableornamental plant for colder regions. Conven-tional seed germination and vegetative prop-agation are two major procedures for masspropagation. However, similar to other Ilexspecies, seed germination of inkberry is in-efﬁcient as a result of low germination rate,long germination time, and seedling varia-tion. It may take 2 to 3 years to overcome thedouble dormancy caused by hard, imperme-able seedcoat and immature embryos (Dirrand Heuser, 1987). In addition, vegetativepropagation of inkberry by cuttings may besubject to seasonal variations.Tissue culture is a well-known and efﬁ-cient tool for mass propagation of uniformplants. In vitro culture of zygotic embryos forpropagation of various Ilex species plantsfrom rudimentary embryos has been reported(Hu,1975, 1976;Hu et al., 1979;Mattis et al.,1995; Sansberro et al., 1998, 2001a). Theyfocused on the inﬂuences of different factorson overcoming dormancy in rudimentaryembryos. Hu (1989) reported that cell sus-pension cultures and somatic embryos wereobtained from immature Ilex aquifolium L. (English holly) zygotic embryos; however,the conversion rate of embryos into plantswas low. In general, Ilex species have beenpropagated by shoot proliferation using nodalsegments, shoot tips, and/or apical meristems(Bernasconi et al., 1998; Luna et al., 2003;Majadaet al., 2000; Mattis et al., 1995;Morteet al., 1991; Sansberro et al., 1999, 2000,2001b; Zaniolo and Zanette, 2001). Because there is no reported protocol formass propagation of inkberry using tissueculture, our evaluation of this procedureincludes the study of the effects of plantgrowth regulators on shoot proliferation androoting.Materials and MethodsPlant material and culture establishment.A 1-year-old plant of Ilex glabra ‘Pretty Boy’(Rareﬁnd Nursery, Jackson, NJ) maintainedunder greenhouse conditions was used as thesource of explants. On 2 June 2008, young,nonligniﬁed branches were collected, surface-disinfected in 70% ethanol for 10 s, thenimmediatelytransferredinto10%Ultrableach(6.0% sodium hypochlorite; Wal-Mart Stores,Inc., Bentonville, AR) with seven drops ofTween 20 (ACC00528/0030; Agdia Inc.,Elkhart, IN) per 200 mL for 10 min, andwashed with three to ﬁve rinses of steriledistilled water. The branches were dissectedinto 1- to 1.5-cm-long segments of stemcontaining a single bud (Fig. 1A). The nodalsegments were transferred to 60-mL Pyrexglass tubes containing Murashige and Skoog(MS) (1962) basic medium plus 90 m