Plant Cell, Tissue and Organ Culture 66: 89–95, 2001.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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In vitro regeneration of Sophora toromiro from seedling explants

Miguel Jordan∗, Monica Larrain, Andrea Tapia & Carlos RoveraroDepartamento de Ecologia, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile(∗requests for offprints: E-mail: mjordan@genes.bio.puc.cl)

Received 15 December 2000; accepted in revised form 5 January 2001

Key words: endangered tree, Leguminosae, organogenesis, rhizobia, tissue culture, toromiro

Abstract

Embryonic axes with cotyledons, shoot-tips of embryonic axes, isolated cotyledons, as well as axillary buds andleaves from 20-year-old trees of Sophora toromiro, were evaluated for their capacity to trigger organogenesis and toregenerate plantlets under in vitro conditions. Embryonic shoot-tips were the only explants capable of regeneratingplants. They developed rapidly in vitro in the presence of NAA and BA while in subculture roots were induced at theproximal end in the presence of 0.49 µM IBA within 40–60 days. Development was completed with a subculturephase under non-sterile conditions using a mixture of equal parts of sterilized vermiculite/sand/soil in growthchambers, before final acclimation in the greenhouse. In the presence of NAA, BA and GA3, whole embryonicaxes formed multiple shoots that branched when grown in 2.27 or 11.35 µM TDZ in subculture. Similarly, calluswas initiated at the embryo axis base, developing into several new shoots in the presence of TDZ. Because of therelatively high shoot induction rate along the embryonic axis, this axis presents a valuable source of new juvenileexplants. Growth and rhizogenesis was satisfactory only when organs from seed pods of the year or from theprevious season were used. Experiments with isolated cotyledons produced callus only, while axillary buds andleaves did not show any responses in the presence of several growth regulators assayed. Inoculation of seedlingswith various strains of rhizobia under in vitro conditions resulted in root outgrowths, but not in nodules that aretypical of rhizobia infection.

Abbreviations: BA – 6-benzylaminopurine; NAA – α-naphthaleneacetic acid; IBA – indole-3-butyric acid; GA3−– gibberellic acid; MS – Murashige and Skoog (1962) medium; NN – Nitsch and Nitsch (1969) medium; TDZ –1-phenyl-3-(1,2,3,-thidiazol-5-yl)urea (thidiazuron); WPM – Woody Plant Medium (Lloyd and McCown, 1981);Z – zeatin

Introduction

Sophora toromiro (Phil.) Skottsb. (Leguminosae) isan endemic endangered species originally describedfor Easter Island (Rapa Nui or Isla de Pascua) butnow extinct in its natural habitat (Rodriguez et al.,1983). A few individuals remain in botanical gardensin Chile but these are affected by genetic drift and in-breeding depression (autogamic reproduction). Othershave been maintained and propagated in Europeangreenhouses (Lobin and Barthlott, 1988) and haverecently been replanted in apparently good naturalconditions in Easter Island. However, 2-year reintro-duction results were not encouraging with only a few

individuals surviving under very careful nurturing (Y.Erazo, pers. comm.). Toromiro plants, either in nurser-ies or in private hands, are very slow growers in com-parison to the fast development shown by other closelyassociated Sophora spp., such as S. microphylla whichis also from the Section Edwardsia (Peña et al., 2000).Unfortunately very little genetic variation exists withinthe available individuals examined (Maunder et al.,1999). It is assumed that all living trees are self pol-linated offspring of the last individual which grewin Rapa-Nui (Ricci and Eaton, 1997). Many factorsmay affect this species i.e., a serious spider mite at-tack and nutritional deficiencies due to the failure innodulation because of a lack of proper nitrogen-fixing

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Table 1. In vitro germination of S. toromiro seeds and embryo development in sub-culture in presence of different nutrient media and growthregulators

Germination conditions Seed age Scarification Time to first Germination Subculture ∗∗ No. of Embryo size

Medium NAA BA GA3 (year) & treatment germination (%) Medium NAA BA GA3 embryos (cm) after 23

(µM) no. seeds (days) (µM) in culture days

tested χ S.D.

MS∗ 1 26 Pricked 7 100 WPM 1.61 0.44 – 14 0.7 ± 0.26

WPM 1.61, 0.44, 0.03 1 29 4% H2SO4 10 72.4 WPM 1.61 0.44 0.03 21 3.3 ± 0.24

(3 min)

D.W 1 22 Pricked 5 100 WPM 1.61 0.44 – 22 1.6 ± 0.83

D.W 2 20 None – 0.0 – – –

D.W 2 20 Pricked – 0.0 – – –

D.W 2 20 4% H2SO4 8 16.6 WPM 1.61 0.44 0.03 5 1.7 ± 0.92

(3 min)

WPM 1.61, 0.44, 0.03 >2 35 4% H2SO4 – 0.0 – – –

(3 min)

∗Medium without sucrose nor growth regulators.∗∗All embryos selected for subculture were approx. 2 mm sized. D.W. distilled water.

Rhizobium bacteria can lead to low survival. Althoughit has been reported that within the subfamily Papilion-oideae nodulation can reach 97% (van Berkum andEardly, 1998; Polhill, 1981), no nodules have beenfound on roots of toromiro trees or S. microphylla.Conventional propagation methods are not suitable forthis species (Alden and Zizka, 1989) and few in vitrostudies have been carried out in related plants, i.e.Pterocarpus indicus (Rao and Lee, 1982). S. angus-tifolia and S. flavescens were investigated to obtainsecondary metabolites (Furuya and Ikuta, 1968; Yagiet al., 1992). Previously we reported (Iturriaga et al.,1994) the morphogenic potential in some explants oftoromiro under in vitro conditions. This paper presentsadditional results on the enhancement of regenerationusing organs of juvenile explants. Seedlings are a goodexplant source in various woody leguminous speciesof the related sub-family Mimosoideae (Nandwani andRamawat, 1992a,b; Jordan, 1987, 1996, 1998). Pre-liminary results on nodulation of seedling roots withspecific strains of rhizobia are presented.

Material and methods

Seed germination, explant preparation andestablishment

Because of the rarity of mature Sophora toromirotrees, the number of seeds available was too smallfor a fully balanced experiment with several replic-ations. Only two trees, each with only a few seeds,

were available to us for seed collection. One was a 20-year old tree in Santiago, (INACAP, Tabancura) andthe other was in a nursery (Las Brujas de Talagante)near Santiago. Seeds were mechanically or chemic-ally (4% H2SO4, 3 min) scarified and disinfected withCaptan/Benlate (0.02% each) for 30 min under con-stant agitation, then washed in sterile distilled water.Seeds were germinated in vitro under various growthconditions (Table 1).

In vitro germination and growth

After germination, 2-mm long embryos attached to aportion of cotyledons, were disinfected for 3 min in4% commercial NaHClO, rinsed in sterile distilled wa-ter and cultured in tubes containing 14 ml autoclavedliquid WPM (Lloyd and McCown, 1981) contain-ing 2% sucrose (final pH 5.6) and growth regulators(Table 1) on Whatman Nr.1 paper-bridges.

Culture of various explants

Various explants were obtained from seedlings ger-minated in distilled water. They consisted of em-bryonic axes (2 cm long) attached to cotyledons, em-bryonic shoot-tips (0.5 cm) excised from approx. 4 cmlong embryonic axes and isolated cotyledons. Also,shoot-tips and leaves from adult donor trees were as-sayed. Cultures were maintained at 22◦C in a growthchamber under a 16/24-h light regime at light intens-ities of 40 or 80 µmol m−2 s−1 provided by GeneralElectric F40T10-SS fluorescent lamps.

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Figure 1. (a) S. toromiro embryo axis in presence of TDZ showing new shoots, axillary sprouting and branching. (b) An embryonic axisshowing profuse shoot formation from callus initiated at both sides of the embryonic axis beneath the attached cotyledons in presence of TDZ.(c–e) Embryonic shoot-tips developing one or several roots after 60 days in presence of 0.49 µM IBA.

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Table 2. Summarized responses observed in different explants of S. toromiro

Explant n First culture Subculture Responses Remarks

type growth regulators growth regulators

(µM) (µM)

Medium Z TDZ IBA Medium NAA BA Z TDZ

Whole 10 D.W – – – MS – 22.2 – – Thickening of embryo axes Observed in all explants in

embryonic 10 D.W – – – MS – – 4.56 – only presence of BA, Z and TDZ

axis

10 D.W – – – MS – – – 2.27 Thickening, new axillary All explants with thickened

10 D.W – – – MS – – – 11.35 buds and branching. Callus shoots and callus formation

at the base of both sides of after 30 days. New shoots

embryonic axes formed new from callus observed after 70

shoots (> 10/callus) in all days. Callus green.

explants

Embryonic 20 D.W – – – WPM 1.61 0.44 – – Root formation after second Seven explants with one or more

shoot-tips subculture in presence of roots after 60 days in

IBA 0.49 µM subculture. Six plantlets

adapted in greenhouse

Cotyledons 20 D.W – – – WPM – 22.2 – – Callus only (25%) Cot. & callus white

(isolated) 20 D.W – – – WPM – – 4.56 – Callus only (n.e.) Cot & callus green

15 D.W – – – WPM – – – 18.16 Callus only (40%) Cot. & callus intensive green,

some necrotic

Axillary 30 WPM – – 0.49 None No response All brown/necrotic

buds

Leaves 30 WPM 0.45 – None No response Yellow-green after 30 days

30 WPM 4.56 4.54 – None No response All brown

30 WPM 22.7 4.54 – None No response All brown

D.W: distilled water. n.e: not examined.

Seedling inoculation and Rhizobium strains

Infection with R. leguminosarum was evaluated by in-oculating 45-day-old seedlings grown in vitro with theviciae, strains 1399, 1400, D-138, the pisum strainsC-17 and D-58 and the less specific strain 154 forvarious woody leguminous plants such as Vigna andAcacia (P. Carrancá, M. Granger, pers. comm.). TheRhizobium strains were provided by ‘Probical S.A’,Peñaflor. Strains grown on 0.6% agar were suspendedin 3 ml of distilled water and a few drops were placeddirectly on the roots of seedlings grown on 0.6% agarin autoclaved NN-solution (Nitsch and Nitsch, 1969)with or without nitrogen salts. Other attempts werecarried out using a commercial sample containingstrain 1400 which was suspended in 100 ml of distilledwater. Five ml of this suspension were mixed with 15

ml autoclaved NN-solution solidified with 0.6% agar.Inoculation was also performed with the same strainsthat had been exposed separately to UV light-253 nmat a distance of 12 cm from source for 20 min prior toinoculation/infection.

Results and discussion

Germination

Germination and explant responses were affected bythe age of the seeds. About 100% of pricked seedsfrom pods collected during the current or previousgrowing season germinated. Fewer seeds (72.4%) ger-minated after chemical scarification (4% H2SO4) andmany older scarified seeds failed to germinate or

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developed poorly. Germination of pricked seeds oc-curred in vitro within 5–7 days (Table 1). Best embryogrowth was observed using WPM with 1.61 µM NAA,0.44 µM BA and 0.03 µM GA3 which increased thesize of the embryo by 2–3 times after 23 days.

Shoot and root initiation

Embryonic axes and embryonic shoot-tipsEmbryos excised from seeds germinated in water andsub-cultured on MS medium containing 22.2 µM BA,4.56 µM Z, 2.27 µM or 11.35 µM TDZ showed rapidgrowth in length and diameter of their embryonic axes,followed by a slight elongation of the shoot-tip. Inthe presence of TDZ multiple shoot formation oc-curred after 3 weeks (Figure 1a and Table 2). Severalother responses were observed in these explants, i.e.,profuse branching of elongating shoots in both con-centrations of TDZ tested and callus initiation at thebase of the embryonic axis next to each cotyledon.After 60–70 days in culture a high number of new ad-ventitious green shoots (>10) were formed from thiscallus on each side of the embryonic axis (Figure 1b).Branching and the induction of new shoots are im-portant since they provide an extra source of juvenileexplants for use in subculture. Other explants such asembryonic shoot-tips, when cultivated on WPM for 35days in the presence of 1.61 µM NAA and 0.44 µMBA, initially showed active growth. After sub-culturein 0.49 µM IBA for another 60 days, one or moreroots were formed at their proximal ends in light(Figure 1 c–e and Table 2). Root formation occurredin approx. 30% of subcultured shoot-tips. Plantletsreaching approx. 3.5 cm were transferred to non-sterile conditions using a substrate composed of equalvolumes of sand/vermiculite/soil (soil taken from thesite where mother plants grow) in growth chambersfor 1-month acclimatization followed by transfer tothe greenhouse. From six plants three could adapt tofield conditions reaching 18–25 cm after approx. oneyear (Figure 2). Low light conditions caused some ini-tial etiolation, yellowing and some loss of plantlets.However, this effect was corrected by exposures tohigher light intensity (80 µmol m−2 s−1). The shootinduction pattern as well as rooting of the explantsclosely corresponded to those obtained with Prosopistamarugo embryonic axes (Nandwani and Ramawat,1992a,b) and seedling explants of Acacia caven, A.visco (Jordan, 1998), Leucaena leucocephala (Nag-mani and Venketeswaran, 1983; Jordan, 1998), andL. retusa (Nagmani and Venketeswaran, 1987). How-

Figure 2. A hardened 1-year old plant of Sophora toromiro.

ever, initiation of somatic embryos, as described for L.diversifolia (Nagmani and Venketeswaran, 1983) wasnot observed in any of the toromiro explants examined.

Cotyledons

Callus and shoot formation occurred in embryo axeswith cotyledons attached. Callus but no shoot forma-tion was observed on isolated cotyledons in the pres-ence of 22.2 µM BA, 4.56 µM Z or 4.54 µM TDZ,using each as single growth regulator. The green colorof the cotyledons growing on TDZ intensified duringculture. Callus growth was limited in time; morpho-genesis did not occur under all conditions tested.

Explants from mature donor trees

Several other explants, i.e., axillary buds or leaveswith petioles from terminal twigs of mother plantswere cultivated in a range of growth regulator com-binations. Those cultivated in lower TDZ levels main-tained their green color in vitro but did not undergomorphogenesis.

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Figure 3. (a) Root outgrowth formation at the sub-apical region of a root after rhizobia inoculation for 8 days in vitro. (b) Section of a rootoutgrowth showing cell proliferation initiating close to the central cylinder. No infection signs are visible after 8 or more days.

Inoculation of seedlings with Rhizobia

The roots of several mother plants growing in nurser-ies of seedlings grown in the greenhouse showed noevidence of nodule formation. Seedlings of S. micro-phylla and of S. toromiro when planted together inthe same container exhibited fast growth and a ro-bust state for the former compared to slow growthof the latter. Neither species had nodules after 2–3months. Under in vitro conditions, after inoculatingS. toromiro seedlings with UV-irradiated Rhizobiumstrain 1400, nodule-like outgrowths were observedafter 7–8 days. The effect was not observed using otherUV-irradiated and non-irradiated Rhizobium strains.These structures surrounded the sub-apical region ofthe roots (Figure 3a). Histological observations after8 days indicated that these outgrowths formed fromdividing cells close to the central cylinder (Figure 3b).No infection threads were microscopically visible inthe invasion zone at this time or later. The samenegative response was found when examining cellsfrom outgrowths fixed 40–50 days after inoculation,with no evidence of infection or recognition of bac-teroids inside the cells. The failure to infect toromiroclearly indicates that other proper host-specific rhizo-bia strains must be evaluated to determine the potentialfor nodulation in this species. The role of specific lipo-

polysaccharides involved in the cell-cell recognitionand attachment processes of compatible rhizobia to thelegume host root has to be defined (Kannenberg et al.,1998).

Acknowledgements

We thank Forestry Engineer Mr Patricio Carrancá andMrs Marta Granger (Probical S.A., Peñaflor) whogenerously provided most rhizobia samples. We alsothank nursery ‘Las Brujas’ and INACAP-Tabancurafor plant material and Ms A. Goreux for revision ofthe manuscript.

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