Plant Cell, Tissue and Organ Culture59: 1–7, 1999.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

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In vitro shoot regeneration from leaves of wild pear

E. Caboni1,∗, M.G. Tonelli2, P. Lauri1, S. D’Angeli1 & C. Damiano11Istituto Sperimentale per la Frutticoltura, Ciampino Aeroporto 00040 Roma, Italy;2Present address: Uni-versita di Milano, Dipartimento di Biotecnologie (∗requests for offprints. Tel.: +39-06-79348131; Fax:+39-06-79340158; E-mail: isf.propag@mclink.it)

Received 31 May 1999; accepted in revised form 23 November 1999

Key words:cefotaxime, histology, leaf explant, organogenesis, pear,Pyrus communisvarpyrasterL., tissue culture

Abstract

Shoot regeneration was obtained from leaves ofin vitro cultures of wild pear genotypes. The highest regenerationrates, ranging from 40% to 64%, depending on the genotype, were obtained using leaves wounded by threecuts transversely to the mid-rib, a Quoirin and Lepoivre macro-salt composition, 250 mg l−1 cefotaxime andmaintaining the explants in darkness for the first 30 days (induction phase), then transferring them to an auxin-freemedium in light (expression phase). A concentration of 8.8µM BA induced the highest number of explants toproduce adventitious shoots. TDZ was less effective than BA and induced hyperhydricity in regenerated shoots.The histological studies revealed that the regenerated shoots originated mainly from callus formed by epidermaland sub-epidermal cells and by cells of the vascular tissue. The regenerated shoots were micropropagated, rootedand transplanted to the soil.

Abbreviations: AFM – auxin free medium; BA – 6-benzyladenine; CL – Chevreau and Leblay (1993); CX –cefotaxime; IBA – indole-3-butyric acid; LP – Quoirin and Lepoivre (1977); NAA –α-naphthalene-acetic acid;TDZ – 1-phenyl-3-(-1,2,3-thiadiazol-5-yl)urea (thidiazuron)

Introduction

Breeding programmes for perennial fruit species bytraditional methods require several years, particularlywhen interspecific crosses with wild species are usedfor transferring resistance to insects or diseases andseveral generations of backcrossing are required toeliminate undesirable characters. In the case of pearthe high level of heterozygosity and the long juvenileperiod makes genetic improvement through conven-tional breeding a difficult and long-time requiringprocess. In this context,in vitro techniques, and in par-ticular those concerning regeneration of whole plantsfrom adult tissues, permitting the alteration of a fewcharacters through exploitation of somaclonal vari-ation or application of gene transfer, can accelerate therecovery of improved genotypes.

In vitro adventitious shoot formation from ma-ture tissues has been reported for woody fruit species

(James et al., 1988; Fasolo et al., 1989; Stamp et al.,1990; Swartz et al., 1990; Miguel et al., 1996) and his-tological studies have been also performed with apple(Welander, 1988; Welander and Maheswaran, 1992).In pear, shoot regeneration from leaf explants has beenachieved in several cultivars and various factors, suchas growth regulator combinations and basal salt com-position, have been reported to influence adventitiousshoot formation in this species (Chevreau et al., 1989;Predieri et al., 1989; Abu-Qaoud et al., 1991; Leblay etal., 1991). Among the other factors, the cephalosporinantibiotic cefotaxime has been also reported to posit-ively influence adventitious shoot formation in someapple and pear cultivars (Predieri et al., 1989; Yepesand Aldwinckle, 1994a,b). Protocols for regenerationof adventitious shoots from leaves of Japanese pear(P. pyrifolia) (Lane et al., 1998) and Chinese pear (P.bretschneiderii) (Chevreau et al., 1989; Predieri et al.,1989) have been also reported.

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The wild pear (P. communisvar pyrasterL.) is aninteresting rootstock for pear cultivars but only a fewstudies regardingin vitro culture have been reported(Ochatt and Caso, 1986; Damiano et al., 1996) andthere are no reports on adventitious shoot formation.The aim of this work was to establish an efficient andreproducible protocol for induction of shootsin vitrofrom wild pear leaves.

Materials and methods

Plant material

Shoot cultures of 3 wild pear (P. communisvar pyras-ter L.) genotypes (ISF50, ISF54 and ISF61), spon-taneous seedlings under evaluation in the Fruit TreesResearch Institute of Rome as putative rootstocks forpear cultivars, were established and propagatedinvitro. The plant growth medium contained Quoirin andLepoivre’s (1977) (LP) macro and micro-elements,1.8µM benzyladenine (BA), 0.2µM indole-3-butyricacid (IBA), 20 g l−1 sucrose and 6 g l−1 agar (B andV, Italy) according to Damiano et al. (1996) and withthe organics (DL organics) modified as following: 0.5mg l−1 nicotinic acid, 0.5 mg l−1 pyridoxine, 2.0 mgl−1 glycine, 0.5 mg l−1 thiamine-HCl, 150 mg l−1

myo-inositol, 1.0 mg l−1 Ca-pantothenate, 0.1 mg l−1

biotin and 0.5 mg l−1 riboflavin. The pH was adjustedto 5.7 and the medium was autoclaved for 20 min at1.1 Kg cm−2 and 120◦C. The cultures were grownat 24±1 ◦C in a 16-h photoperiod at light intensityof 40 µmol m−2 s−1 provided by cool white fluor-escent tubes (Philips TLD, France) and sub-culturedonto fresh medium every 3 weeks.

Adventitious shoot induction

Expanding leaves of the apical internode were ex-cised from three-week-old shoots of each genotype,wounded by three cuts transversely to the mid-rib andplated with the adaxial surface in contact with the me-dium (10 ml/dish) in 6 cm diameter Petri dishes. In allthe experiments the explants were maintained for 30days in darkness, then transferred to light conditionsfor 60 days (see above).

The effect of growth regulator combinations wasevaluated by supplementing a medium containing1/2MS (Murashige and Skoog, 1962) macro-salts, LPmicro-salts and DL organics by BA (2.2, 4.4, 8.8 or17.6µM) or thidiazuron (TDZ, 2.5, 5µM) and 1.0 or

4.0µM IBA or α-naphtaleneacetic acid (NAA). Theexplants were maintained on the same medium.

In a second experiment, three different macro-saltcombinations,12MS, CL (Chevreau and Leblay, 1993)or LP, were compared with LP micro-salts, DL organ-ics, 8.8µM BA and 1.0µM NAA. Half of the explantswere transferred to auxin-free medium (AFM) 30 daysafter the beginning of the experiment and for 60 days,while the others were maintained on the old medium.

The effects of cefotaxime (CX) and of the typeof explant on shoot induction were also evaluatedby supplementing a medium (LP1), containing LPmacro-salts and micro-salts, DL organics, 8.8µM BAand 1.0µM NAA, with 250 mg l−1 CX (HoechstMarion Roussel, Italy) and using the following ex-plants: leaves wounded as reported above, base por-tions of leaf with petiole, petioles alone or leaf por-tions prepared removing the margins and cutting theremaining part into two portions. All the explants weretransferred to AFM as reported above.

Statistical analysis

Each treatment was performed in 8 Petri dishes con-taining 6 explants and each experiment was replicated3 times. Number of explants forming shoots and num-ber of shoots per explants were collected 90 days afterthe beginning of the experiments. Data were subjec-ted to analysis of variance by Fisher’s protected LSD,test at the 5% level of significance (p=0.05) (Fisher,1966). Percentage data were transformed to arcsinbefore analysis.

Histological analysis

Samples from the experiment consisting of leaveswounded by three cuts transversely to the mid-rib fromthe genotype ISF61 were collected 0, 3, 6, 9, 12, 15, 20and 40 days of culture from the most effective regen-eration treatment (LP1 medium+250 mg l−1 CX for30 days in darkness, then transfer to light on AFM).Plant material was fixed in 70% ethanol and dehyd-rated by the tertiary butyl alcohol method (Johansen,1940); subsequently, it was embedded in paraffin wax(melting point: 58◦C, Merck) and sectioned at 10µmwith a rotary microtome (Jung Biocut, Leica). Sec-tions were stained with eosin and Carazzi’s haemalum(Mazzi, 1977).

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Table 1. Level of ions (mM) of the macro-salt compositions usedin the regeneration experiments

LP CL 12MS

NO−3 29.6 20.1 19.7

PO3−4 19.8 0.6 0.6

SO2−4 14.6 0.7 0.7

Cl− – – 3.0

K+ 37.6 12.0 10.7

Ca2+ 0.9 1.3 1.5

Mg2+ 14.6 0.7 0.7

NH+4 5.0 7.6 10.0

NH+4 :NO−3 1:6 1:3 1:2

Table 2. Effect of hormonal combinations on adventitious shootformation (%) and mean number of shoots (N) per explant inleaves of wild pear (P. communisvar pyraster L.) genotypes(ISF50, ISF54 and ISF61)

Genotypes

Growth ISF50 ISF54 ISF61

regulators (µM) % Na % Na % Na

BA NAA

2.2–1.0 0b – 6cd 1.4 0c –

2.2–4.0 2b 2.1 7cd 1.3 0c –

4.4–1.0 4b 1.0 10c 1.5 0c –

4.4–4.0 6b 1.4 9cd 1.2 0c –

8.8–1.0 15a 2.1 23a 2.4 14a 1.9

8.8–4.0 14a 2.0 18b 2.8 10b 2.1

17.6–1.0 5b 1.2 13c 1.4 7bc 0.9

17.6–4.0 4b 1.0 8cd 1.6 6bc 1.1

TDZ NAA

2.5–1.0 0b – 5d 1.7 0c –

2.5–4.0 0b – 4d 1.4 2c 1.9

5.0–1.0 2b 1.0 15c 1.2 12ab 1.8

5.0–4.0 4b 1.1 14c 1.4 9ab 1.5

aSE was less than 10% in all the values. Macro-salt combinationwas 1

2MS. Data were collected 90 days after the beginning of theexperiment. Means within a column followed by the same letterare not significantly different from each other atp=0.05 accordingto Fisher’s least significant difference test. Percentages data weretransformed to arcsin before analyses.

Results and discussion

Regeneration studies

The effect of growth regulator combination on adven-titious shoot formation is shown in Table 2. A concen-tration of 8.8µM BA induced the highest number of

explants to produce adventitious shoots in all the gen-otypes. A higher BA concentration was detrimental foradventitious shoot regeneration in these genotypes, incontrast to what was reported by Predieri et al. (1989)in pear cultivars. TDZ, which was reported to be veryefficient for the induction of organogenesis in pear byLeblay et al. (1991) and Lane et al. (1998), was lesseffective than BA in wild pear genotypes and inducedhyperhydricity in regenerated shoots. Similar resultshave been reported in some apple genotypes, decreas-ing the regenerated shoot survival (Van Nieuwkerk etal., 1986; Pawlicki et al., 1994; Caboni et al., 1996).The lowest NAA concentration (1.0µM) slightly en-hanced the regeneration rate when used together with8.8µM BA with genotypes ISF54 and ISF61, while nodifferences were detected in the other combinations.A low effect of NAA concentration compared to theeffect of cytokinins was also reported by other authors(Leblay et al., 1991), but IBA had no effect on wildpear regeneration, and caused slight callus in 80–90%of the explants of all the genotypes.

The effects of macro-salt composition and of trans-fer to an auxin-free medium after the induction phaseon shoot regeneration response is detailed in Table 3.Despite the differences due to genotype, it is evidentthat the LP macro-salts were most effective for in-ducing shoot formation from leaf explants in all thegenotypes. Differences in the morphogenic effect ofthe other macro-salt compositions were not detectedin the other treatments, except for the genotype ISF50,without the change to an AFM,12MS was more ef-fective than CL. It was reported that12MS and CLmacro-salts can be used to induce adventitious shootregeneration in pear cultivars (Leblay et al., 1991;Chevreau and Leblay, 1993). According to Leblayet al. (1991), the effect may be related to the NH+4concentration and they suggested that concentrationsranging from 7.5 and 10 mM of this ion are the mostsuitable for regeneration of some pear cultivars. In ourexperiments, the LP macro-salt formulation, whichhas lower NH+4 concentration than12MS and CL for-mulations (Table 1), induced higher regeneration ratesin wild pear and this is in agreement with Welander(1998) and Lane et al., (1998) who reported that amedium with reduced ammonium is more effective forinducing regeneration in apple and in Japanese pear,respectively. The lower NH+4 : NO−3 ratio (1:6) of theLP formulation than those of CL (1:3) and12MS (1:2)could also positively affect the regeneration ability ofwild pear as already proposed for apple (Fasolo et al.,

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Table 3. Effect of three macro-salt combinations and of transferring of explant to fresh auxin free medium (AFM) on adventitiousshoot formation rate (%) and mean number of shoots per explant (N) in leaves of wild pear (P. communisvar pyrasterL.) genotypes

Genotypes

Macro-salt ISF50 ISF54 ISF61

composition AFM % Na % Na % Na

12 Murashige + 20b 1.8 28c 2.3 24b 2.1

and Skoog – 15b 2.0 23d 1.7 14c 1.9

Chevreau + 18b 1.9 27c 2.3 26b 1.7

and Leblay – 10c 2.0 21d 1.0 16c 2.1

Quoirin + 28a 1.9 52a 3.4 42a 2.6

and Lepoivre – 18b 2.1 40b 1.8 25b 2.1

aSE was less than 10% in all the values. Growth regulator combination was 8.8µM BA and 1µM NAA. Means within a columnfollowed by the same letter are not significantly different from each other atp=0.05 according to Fisher’s least significant differencetest. Percentages data were transformed to arcsin before analyses.

Table 4. Effect of explant source and of 250 mg l−1 cefo-taxime (CX) on adventitious shoot formation rate (%) andmean number of shoots per explant (N) in leaves of wild pear(P. communisvar pyrasterL.) genotypes

Genotypes

Explant ISF50 ISF54 ISF61

sourcea CX % Nb % Nb % Nb

WL + 40a 1.7 64a 4.9 58a 4.2

– 28b 1.9 52b 3.4 42b 2.6

LC + 0d – 12c 2.1 7d 1.8

– 0d – 10c 1.5 4d 1.8

PL + 31b 1.3 48b 4.4 36c 3.9

– 24c 1.3 46b 4.1 34c 3.6

P + 2d 1.2 16c 1.9 10d 1.7

– 0d – 14c 1.6 8d 1.8

aWL=leaves wounded by three cuts transversely to the mid-rib; LC=leaf portions prepared removing the margins andcutting the remaining part into two portions; PL=petioles withbase portion of leaf; P=petioles.bSE was less than 10% in all the values. Growth regulatorcombination was 8.8µM BA and 1µM NAA and macro-saltcomposition was LP. Data were collected 90 days after the be-ginning of the experiment. Means within a column followed bythe same letter are not significantly different from each otheratp=0.05 according to Fisher’s least significant difference test.Percentages data were transformed to arcsin before analyses.

1989) and pear leaves (Leblay et al., 1991; Predieri etal., 1989).

Transfer of explants to auxin-free medium, 30 daysafter the beginning of the experiments, strongly in-creased morphogenic ability, inducing regenerationrates of 28, 52 and 42% in genotypes ISF50, ISF54and ISF61, respectively, and the highest number ofshoots per explant in genotype ISF54 (Table 3). Sub-dividing the regeneration process into two phases, an

Figure 1. Various stages of plant regeneration from leaf of wild pear(P. communisvar pyrasterL.). The explants were cultured on LP1medium plus 250 mg l−1 CX for 30 days and then transferred on theAFM medium and to the light. (A) Genotype ISF61. Adventitiousshoot formation 90 days after the beginning of the experiment.(B)Genotypes ISF54 (left) and ISF61 (right). Multiplied shoots.(C)Genotypes ISF50, ISF54 and ISF61 (from the left). Acclimatisedplantlets.

induction period of 30 days in darkness, then an ex-pression phase in the light, has already been shownto improve regeneration in pear cultivars (Leblay etal., 1991). Regeneration rates and the mean numberof shoots per explants from different explant sourcesand with CX are reported in Table 4.

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Figure 2. Histology of the morphogenic response of the leaf cut transversally to the mid-rib of wild pear (P. communisvarpyrasterL.), genotypeISF61, cultured on LP1 medium supplemented with 250 mg l−1 CX. (A) Day 0, cross-section of leaf lamina showing single layer epidermis,spongy and palisade parenchyma (arrows on bundle sheath cells); bar=50µ. (B) Day 3, bundle sheath enlarged cells and first divisions (arrow)in vascular tissue; bar=50µ. (C) Day 6, callus formation from mesophyll; bar=50µ. (D) Day 9, vegetative dome on the callus surface ofleaf margin (leaf thickness is increased by callus formation in the mesophyll cells); bar=100µ. (E) Day 9, meristemoid formation on callusoriginated from mid-rib; bar=100µ. (F) Day 12, vegetative bud from callus of the leaf margin in the base part of the leaf, bar=200µ.

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Whole leaves wounded transversally to the mid-ribwere more morphogenic in all genotypes than the baseportions of leaf with petiole. Nevertheless, most of theshoots obtained from whole leaves were regeneratedfrom the base part of the leaf (Figure 1A). The othertype of explants were poorly morphogenic (Table 4).Cefotaxime may increase morphogenic ability in sev-eral cereal crops (Mathias and Boyd, 1986; Borrelli etal., 1992; Pius et al., 1993) and some woody speciessuch as pear (Predieri et al., 1989) and apple (Yepesand Aldwinckle, 1994). Regeneration in wild pear wasimproved with 250 mg l−1 CX with all genotypeswhen it was used on leaves cut transversally to themid-rib (Table 4).

The shoots obtained with the most morphogenictreatment (Figure 1A) were excised, multiplied (Fig-ure 1B), rooted and successfully acclimatised (Figure1C).

Histology

Cross-sections of leaf samples showing the develop-mental stages involved in adventitious shoot formationare depicted in Figure 2. At day 0 the explants showeda single layered epidermis, a single layered palisadeparenchyma and some layers of spongy mesophyll(Figure 2A). After 3 days there was enlargement ofbundle-sheath cells and the first divisions of cells ofthe vascular tissue of the main veins (Figure 2B). Celldivisions increased in this tissue during the following6 days (Figure 2C). At the same time, other cells of themesophyll became enlarged, some divided producingcallus and some xylogenesis was also evident. After9 days adventitious meristem formation was observedas well as vegetative domes on the callus surface closeto the leaf margin (Figure 2D) and above the mid-rib (Figure 2E), particularly in the basal part of theleaf. At day 12 the first vegetative buds were evident(Figures 2F). Continued cell divisions, bud differenti-ation and callus-derived shoot formation occurred overthe next 10 days of culture, involving both epidermal,sub-epidermal and vascular callus and vascular cells,mainly in the basal part of the leaf.

The inner cells of the vascular bundles and the epi-dermal and sub-epidermal callus cells located close tothe base of the leaf are responsive sites, as alreadyreported for apple (Welander, 1988; Pawlicki andWelander, 1994; Caboni et al., 1996). The increasinggradient in morphogenic ability from the leaf tip to-ward the base of the leaf has already been observedin apple and explained by the fact that leaves reach

maturity first at the distal part and subsequently ba-sipetaly toward the proximal part (Welander, 1988).Furthermore, an increased density of vascular bundlespresent in the proximal region of the leaf could alsoplay a role in inducing the higher morphogenic re-sponse of this part of thePyrusleaf as already shownin apple cotyledons (Kouider et al., 1984).

Acknowledgements

The authors wish to thank Prof. Maria MaddalenaAltamura (Dipartimento di Biologia Vegetale, Uni-versità “La Sapienza” – Roma, Italy) for the helpfuldiscussions on the results of histological analysis. Theauthors also thank Loreta Marinaccio for the skilfulassistance in sub-culturing thein vitro material. Thework was developed within the framework of the re-search project “Plant Biotechnologies” supported bythe Italian Ministry of Agriculture.

References

Abu-Qaoud H, Skirvin RM & Below FE (1991) Influence of ni-trogen form and NH+4 -N/:NO−3 -N ratios on adventitious shootformation from pear (Pyrus communis) leaf explantsin vitro.Plant Cell Tiss. Org. Cult. 27: 315–319

Borrelli GM, Di Fonzo N & Lupotto E (1992) Effect of cefotaximeon callus regeneration in durum wheat. J. Plant Physiol. 140:372–374

Caboni E, Tonelli M, Falasca G & Damiano C (1996) Factors affect-ing adventitious shoot regenerationin vitro in the apple rootstock‘Jork 9’. Adv. Hort. Sci. 10: 1–5

Chevreau E, Skirvin RM, Abu-Qaoud H, Korban SS & Sullivan JG(1989) Adventitious shoot regeneration from leaf tissue of threepear (Pyrussp.) cultivarsin vitro. Plant Cell Rep. 7: 688–691

Damiano C, Liberali M, Avanzato D & Preka P (1996). Micropro-pagazione diPyrus communisvar.pyraster. Macfrut: Agro. Bio.Frut. Cesena 10–11 May pp. 132–133

Fasolo F, Zimmerman RH & Fordham I (1989) Adventitious shootformation on excised leaves ofin vitro grown shoots of applecultivars. Plant Cell Tiss. Org. Cult. 16: 75–87

Fisher RA (1966) The Design of Experiments, 8th ed. Hafner, NewYork, Chapter 10

James DJ, Passey AJ & Rugini E (1988) Factors affecting highfrequency plant regeneration from apple leaf tissues culturedinvitro. J. Plant Physiol. 132: 148–154

Johansen DA (1940) Plant Microtechnique. McGraw-Hill and Co.,New York

Kouider M, Korban SS, Skirvin RM & Chu MC (1984) Influence ofembryonic dominance and polarity on adventitious shoot forma-tion from apple cotyledonsin vitro. J. Amer. Soc. Hort. Sci. 109:381–385

Lane DW, Iketani H & Hayashi T (1998) Shoot regeneration fromcultured leaves of Japanese pear (Pyrus pyrifolia). Plant CellTiss. Org. Cult. 54: 9–14

7

Leblay C, Chevrau E & Raboin LM (1991) Adventitious shoot re-generation fromin vitro leaves of several pear cultivars (PyruscommunisL.). Plant Cell Tiss. Org. Cult. 25: 99–105

Mathias RJ & Boyd LA (1986) Cefotaxime stimulates callusgrowth, embryogenesis and regeneration in hexaploid breadwheat (Triticum aestivumL.EM.Thell). Plant Sci. 46: 217–223

Mazzi V (1977) Manuale di Tecniche Istologiche ed Istochimiche.Piccin, Padova, pp 127

Miguel CM, Druart P & Oliveira MM (1996) Shoot regenerationfrom adventitious buds induced on juvenile and adult almond(Prunus dulcisMill.) explants. In Vitro Cell Dev. Biol. Plant. 32:148–153

Murashige T & Skoog F (1962) A revised medium for rapid growthand bioassays with tobacco tissue cultures. Physiol. Plant 15:473–497

Ochatt SG & Caso OH (1986) Shoot regeneration from leaf meso-phyll protoplast of wild pear (Pyrus communisvar. pyraster). J.Plant Physiol. 122: 243–249

Pawlicki G & Welander M (1994) Adventitious shoot regenera-tion from leaf segments ofin vitro cultured shoots of the applerootstock ‘Jork 9’. J. Hort. Sci. 69 (4): 687–696

Pius J, George G, Eapen S & Rao PS (1993) Enhanced plant re-generation in pearl millet (Pennisetum americanum) by ethyleneinhibitors and cefotaxime. Plant Cell Tiss. Org. Cult. 32: 91–96

Predieri S, Fabbri Malavasi F, Passey AJ, Ridout MS & James DJ(1989) Regeneration fromin vitro leaves of “Conference” andother pear cultivars (Pyrus communisL.) J. Hort. Sci. 64: 553–559

Quoirin M & Lepoivre P (1977) Improved media forin vitro cultureof Prunussp. Acta Hort. 78: 437–442

Stamp JA, Colby SM & Meredith CP (1990) Direct shoot organo-genesis and plant regeneration from leaves of grape (Vitis spp.)Plant Cell Tiss. Org. Cult. 22: 127–133

Swartz HJ, Bors R, Mohamed F & Naess SK (1990) The effect ofin vitro pretreatments on subsequent shoot organogenesis fromexcisedRubusandMalus leaves. Plant Cell Tiss. Org. Cult. 21:179–184

Van Nieuwkerk JP, Zimmerman RH & Fordahm I (1986)Thidiazuron stimulation of apple shoot proliferationin vitro.HortSci. 21: 516–518

Welander M (1988) Plant regeneration from leaf and stem segmentsof shoots raisedin vitro from mature apple trees. J. Plant Physiol.132: 738–744

Welander M & Maheswaran G (1992) Shoot regeneration from leafexplants of dwarfing apple rootstocks. J. Plant Physiol. 140: 223–228

Wetzstein HY & Sommer HE (1982) Leaf anatomy of tissue-culturedLiquidambar styraciflua(Hamamelidaceae) during ac-climatization. Amer. J. Bot., 69 1579–1586

Yepes LM & Aldwinckle HS (1994a) Factors that affect leafregeneration efficiency in apple, and effect of antibiotics inmorphogenesis. Plant Cell Tiss. Org. Cult. 37: 257–269

Yepes LM & Aldwinckle HS (1994b) Micropropagation of thir-teenMaluscultivars and rootstocks, and effect of antibiotics onproliferation. Plant Growth Reg. 15: 55–67