Plant Cell Rep (2004) 23:492–496DOI 10.1007/s00299-004-0870-3

G E N E T I C S A N D G E N O M I C S

M. Martins · D. Sarmento · M. M. Oliveira

Genetic stability of micropropagated almond plantlets,as assessed by RAPD and ISSR markers

Received: 3 February 2004 / Revised: 29 July 2004 / Accepted: 31 July 2004 / Published online: 15 September 2004� Springer-Verlag 2004

Abstract Almond shoots produced by axillary branchingfrom clone VII derived from a seedling of cultivar BoaCasta were evaluated for somaclonal variation usingrandomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) analysis. To verify ge-netic stability we compared RAPD and ISSR patterns ofplantlets obtained after 4 and 6 years of in vitro multi-plication. A total of 64 RAPD and 10 ISSR primers gave326 distinct and reproducible band classes, monomor-phic across all 22 plantlets analysed. Thus, a total of 7,172bands were generated, exhibiting homogeneous RAPDand ISSR patterns for the plantlets tested. These resultssuggest that the culture conditions used for axillarybranching proliferation are appropriate for clonal propa-gation of almond clone VII, as they do not seem to in-terfere with the integrity of the regenerated plantlets.These results allowed us to establish the use of axillarybranching plantlets (mother-plants) as internal controlsfor the analysis of somaclonal variation of shoots regen-erated from other in vitro culture processes performedwith clone VII (adventitious regeneration, regenerationfrom meristem culture, virus sanitation programs andgenetic engineering).

Keywords Somaclonal variation · Randomly amplifiedpolymorphic DNA · Inter-simple sequence repeats ·Axillary branching · Prunus dulcis Mill

Introduction

Almond (Prunus dulcis [Mill.] D. A. Webb.) is an agro-nomically important crop, cultivated mainly for its kerneland oil. For this crop, as for many others, our future needsrequire the adoption of new agricultural technologies.Almond, like other woody plants, is difficult to propagateusing conventional techniques. Nevertheless, in vitro cul-ture has the potential to increase the multiplication rate ofelite genotypes, as well as the ability to produce new andimproved cultivars when combined with other tools ofmodern biotechnology.

The tissue culture of almond has been the subject ofseveral studies focusing on establishment of in vitropropagation (Rugini and Verma 1983; Antonelli 1992;Miguel et al. 1996; Ainsley et al. 2001; Lauri et al. 2001)and transformation (Damiano et al. 1995; Miguel andOliveira 1999). Among the methods developed for plantmicropropagation, the axillary branching system is themost used, and is also considered the most suitable toguarantee genetic stability of the plants obtained. Tissueculture of Portuguese almond cultivars has been estab-lished, and high multiplication rates obtained, using eitheraxillary branching from almond seedlings or adventitiousregeneration from leaves (Miguel et al. 1996). Because ofits simplicity and reliability for clonal propagation, axil-lary branching was the method adopted for almond mi-cropropagation in our laboratory (Miguel 1998). Havingaxillary plantlets as the initial material (mother-plants),other biotechnological studies were performed with theaim of producing improved plants. The strategies em-ployed included Agrobacterium-mediated genetic trans-formation (Miguel and Oliveira 1999), meristem culture(Santos, unpublished), modified adventitious regenera-tion protocols (M. Costa, unpublished) and viral-diseasesanitation work, using chemotherapy and thermotherapy(Gon�alves 1998).

A major problem associated with in vitro culture is theoccurrence of somaclonal variation amongst sub-clones ofone parental line, arising as a direct consequence of invitro culture of plant cells, tissues or organs (Larkin and

M. Martins and D. Sarmento contributed equally to this paper

Communicated by P. Puigdom�nech

M. Martins · D. Sarmento · M. M. Oliveira ())IBET/ITQB,Quinta do MarquÞs, 2784-505 Oeiras, Portugale-mail: mmolive@itqb.unl.ptTel.: +351-21-4469647Fax: +351-21-4421161

M. Martins · M. M. OliveiraFaculdade de CiÞncias, Dep. Biologia Vegetal,Universidade de Lisboa,Campo Grande, 1749-016 Lisboa, Portugal

Scowcroft 1981; Gould 1986). Molecular techniques areat present powerful and valuable tools used in analysis ofgenetic fidelity of in vitro propagated plants, and are thesubject of many publications and reviews.

The aim of this work was to assess the impact of theprotocol used for almond axillary branching on the in-duction of somaclonal variation. As almond has a longgeneration cycle, possible variations may be detected onlyin late developmental stages, or even in the offspring.Consequently, early detection of somaclonal variationbecomes an issue of great importance. For this study, wehave therefore selected for analysis one clone derivedfrom a seedling of ‘Boa Casta’ (clone VII) and tested twomolecular methods, randomly amplified polymorphicDNA (RAPD; Williams et al. 1990) and inter-simple se-quence repeats (ISSR; Zietkiewicz et al. 1994).

Materials and methods

Plant material

Seeds obtained from open pollination of the Portuguese almondcultivar Boa Casta, maintained in a collection at Centro de Ex-perimenta�¼o Agr�ria de Tavira, in Algarve, Portugal, were pre-viously established in culture and analysed for their regenerationpotential (Miguel et al. 1996). Plantlets obtained by axillarybranching from one selected seedling were identified as clone VIIand used in the present study.

Briefly, shoots obtained from clone VII were excised andtransferred to micropropagation medium (MJ), which is composedof Murashige and Skoog (1962) basal medium (MS), supplement-ed with 1.33 mM N6-benzyladenine, 0.049 mM indole-3-butyricacid, 20 g/l sucrose and solidified with 7 g/l agar (Miguel et al.1996). Thereafter, axillary branching shoots were separated andtransferred to fresh MJ medium every 21 days and kept in culturein a growth chamber at 23€2�C under a 16-h photoperiod of35 mEm�2s�1 (Miguel et al. 1996).

Twenty plantlets obtained by axillary branching, randomlychosen from 150 plantlets of clone VII (AB01–AB20), monthlysub-cultured in vitro for a period of 6 years, were used for analysisof genetic stability. Two plantlets of the same clone, but sub-cul-tured for just 4 years, were used as a control and a comparison ofRAPD and ISSR patterns of the 6- and 4-year plantlets was per-formed.

DNA extraction and PCR amplification conditions

Total DNA was extracted from plantlets grown in vitro followingthe method described by Doyle and Doyle (1987), modified byWeising et al. (1995) and adapted for almond (Martins et al. 2003).

Purified total DNA was quantified and its quality verifiedby spectrophotometry (GeneQuant spectrophotometer; Pharmacia,Uppsala, Sweden), and each sample was diluted to 12.5 ng/ml in TEand stored at 4�C.

Axillary branching plantlets were tested for somaclonal varia-tion using 64 RAPD primers (Table 1) and 10 ISSR primers (Ta-

ble 2), previously selected by Martins (2003) for their unambiguousand reproducible band patterns.

The protocol for RAPD analysis was adapted from that ofWilliams et al. (1990). PCR was performed in a volume of 25 mlcontaining 25 ng total DNA, 1� PCR buffer (Gibco-BRL, Paisley,UK), 2.0 mM MgCl2, 200 mM dNTPs, 1 mM 10-mer oligode-oxynucleotide primer (Operon Technologies, Alameda, Calif.) and1 unit (U) Taq DNA polymerase (Gibco-BRL). The amplificationreaction consisted of an initial denaturation step at 94�C for 3 min,followed by 40 cycles of 1 min at 94�C, 1 min at 36�C and 2 min at72�C.

ISSR amplifications were performed in a volume of 40 mlcontaining 40 ng total DNA, 1� PCR buffer (Gibco-BRL), 2.0 mMMgCl2, 200 mM dNTPs, 1 mM oligodeoxynucleotide primer and 2 UTaq DNA polymerase (Gibco-BRL).

Optimised PCR conditions for ISSR primers (Martins 2003)were used for DNA amplifications. For primers IS01, IS03, IS10,IS17, IS18 and IS19, a touchdown PCR was used: initial denatur-ation step at 94�C for 3 min; 36 cycles of 45 s at 94�C, 45 s at aspecific annealing temperature and 1 min at 72�C; one last exten-sion step of 7 min at 72�C. The annealing was performed withtouchdown decrements of 0.5�C, starting at 65�C (for primers IS01and IS03) or 60�C (for primers IS10, IS17, IS18 and IS19), for thefirst 11 cycles, followed by 25 cycles at 60�C (for primers IS01 andIS03) or 57�C (for primers IS10, IS17, IS18 and IS19). For primersIS06, IS07, IS12 and IS16, the amplification consisted of an initialdenaturation step at 94�C for 3 min, 36 cycles of 30 s at 94�C, 45 sat 60�C and 90 s at 72�C, followed by one last extension step of5 min at 72�C.

Amplifications were performed in a UNO-Thermoblock ther-mocycler (Biometra, G�ttingen, Germany) for both RAPD andISSR. At least two independent PCR amplifications were per-formed for each sample with RAPD and ISSR primers. DNA am-plification fragments were separated in a 2% agarose gel using1� TAE buffer, and stained with ethidium bromide. Gels wereanalysed with Gel Doc 2000 software (Bio-Rad, Hercules, Calif.).

Data analysis

Only consistently reproducible, well-resolved fragments, in the sizerange of 200 bp to 2.8 kb were scored as present or absent forRAPD and ISSR markers in each micropropagated plantlet. We

Table 1 Randomly amplifiedpolymorphic DNA (RAPD)primers used to screen 22 axil-lary branching almond plantlets

Primers

Kit OPA A01, A03, A04, A05, A06, A07, A08, A10, A11, A12, A13, A16, A17, A18, A20Kit OPB B01, B03, B06, B07, B08, B10, B11, B14, B15, B17, B18, B20Kit OPC C02, C03, C04, C05, C07, C08, C11, C12, C14, C15, C16, C18, C20Kit OPD D01, D02, D03, D04, D05, D08, D09, D10, D11, D15, D16, D18, D19, D20Kit OPE E01, E02, E03, E05, E06, E13, E14, E16, E18, E20

Table 2 Inter-simple sequence repeats (ISSR) primers used toscreen 22 axillary branching almond plantlets. Y C and T residues;B C, G and T residues; D A, G and T residues; H A, C and Tresidues

Primer Sequence

IS01 GTG(5)IS03 GACA(4)IS06 GTGC(4)IS07 GAC(5)IS10 GGAT(4)IS12 AGC(6)TYIS16 DHB CGA(5)IS17 BDB ACA(5)IS18 DDB CCA(5)IS19 YHY GT(5)G

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compared RAPD and ISSR profiles obtained for the 22 plantletswith each primer.

Results and discussion

In order to confirm whether or not axillary branching is areliable propagation method to maintain genetic integrityof almond plantlets, a comparison of RAPD and ISSRpatterns of 22 regenerants from clone VII was carried out.This study is of high significance since axillary branchingof clone VII is currently being used as a source of plantmaterial for other biotechnological procedures.

The 64 RAPD primers used in this analysis gave rise to266 scorable band classes, ranging from 200 bp to 2.8 kbin size. The number of bands for each primer varied from1 to 13, with an average of 4.16 bands per RAPD primer.The screening with the 10 ISSR primers, generated 60scorable band classes, ranging in size from 240 bp to2.8 kb. The number of bands for each primer varied from3 to 9, with an average of 6 bands per ISSR primer. Atotal of 7,172 bands (number of plantlets analysed � num-ber of band classes with all the primers) were generatedby the RAPD and ISSR techniques, giving rise to mono-morphic patterns across all 22 plantlets analysed. Anexample of the monomorphic band classes obtained isshown in Fig. 1 for RAPD markers and in Fig. 2 for ISSRmarkers.

Among the various methods for plant micropropaga-tion, axillary branching has become one of the most im-portant and widely used in in vitro regeneration systems.Rani et al. (1995) described this method as being partic-ularly useful due to its simplicity and the relatively highpropagation rates obtained. In the particular case of al-mond clone VII, the propagated shoots showed a multi-plication rate of 3.6€1.4 (Miguel 1998). Additionally, thisin vitro culture system is generally considered as havinglow risk of genetic instability due to the existence of or-ganised meristems, which are less susceptible to geneticvariations that might occur during cell division or dif-ferentiation under in vitro conditions (Shenoy and Vasil1992). Moreover, the culture conditions used to achieveplant regeneration from tissues where organised meris-tems are already present are less aggressive than thoseusually needed to induce shoots from differentiated cellsvia a disorganised callus.

Numerous studies on somaclonal variation analysishave been developed using PCR-based techniques such asRAPD, SSR and AFLP, RAPD being one of the mostused. Genetic stability has been described in severalcases, namely, Picea mariana plants regenerated fromsomatic embryogenesis (Isabel et al. 1993), micropropa-gated shoots of Pinus thunbergii Parl. (Goto et al. 1998),adventitious shoots of Pinus taeda (Tang 2001), and ax-illary bud proliferation of chestnut rootstock hybrids(Carvalho et al. 2004). However, somaclonal variationwas also reported for a number of species, such as Pop-

Fig. 1 Randomly amplifiedpolymorphic DNA (RAPD)amplification pattern obtainedfor axillary branching 6th-yearplantlets (a; 1–20), and 4th-yearplantlets used as control (b; c1,c2); using primer OPB-15. M aGeneRuler ladder, b 1 Kb PlusDNA ladder

Fig. 2 Inter-simple sequencerepeats (ISSR) amplificationpattern obtained for axillarybranching 6th-year plantlets (a;1–20), and 4th-year plantletsused as control (b; c1, c2); us-ing primer IS06. M a,b Gen-eRuler ladder

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ulus deltoides (Rani et al. 1995), Populus tremuloides(Rahman and Rajora 2001), Prunus persica (Hashmi et al.1997) or Actinidia deliciosa (Palombi and Damiano2002).

In our study we adopted the use of two PCR-basedtechniques, RAPD and ISSR, for somaclonal evaluationof almond plantlets. These methods were chosen becauseof their simplicity and cost-effectiveness. The use of twotypes of markers, which amplify different regions of thegenome, allows better analysis of genetic stability/varia-tion of the plantlets. When analysing micropropagatedplants of kiwifruit, Palombi and Damiano (2002) alsosuggested the use of more than one DNA amplificationtechnique as advantageous in evaluating somaclonalvariation.

The number of RAPD and ISSR primers (64+10) usedin this study, as well as the total number of band classesgenerated (266+60), together with the observed morpho-logic normality and homogeneity of the plantlets, aftereither 4 years or 6 years in culture, strongly suggest thatthe axillary branching strategy ensures maintenance ofgenomic integrity in almond shoot propagation. Theseresults have thus allowed the selection of axillary branch-ing shoots to be used as an internal control when ana-lysing somaclonal variation associated with adventitiousregeneration and culture of isolated meristems of almondclone VII (Martins 2003). Also in this latter study, theauthor found genetic homogeneity among the six differentregenerants analysed and the control plantlets obtained byaxillary branching, using the same RAPD and ISSR pri-mers.

In almond, the time plantlets were kept in culture(4 years and 6 years, with regular sub-culturing) did notseem to affect their genetic integrity. Angel et al. (1996)also found no variation in cassava plantlets regeneratedfrom isolated meristems and kept in in vitro culture for10 years. Some authors, however, have reported that thetime in in vitro culture could promote somaclonal varia-tion (Orton 1985; Hartmann et al. 1989). Channuntapipatet al. (2003), studying somaclonal variation in almondcultivars ‘Ne Plus Ultra’ and ‘Nonpareil’, found a fewchanges in genomic DNA methylation from in vitro cul-ture and cryopreserved plantlets, suggesting that the pe-riod of time in in vitro culture influenced this result.However, the authors do not clearly specify the treatmentsapplied to each sample subjected to molecular analysis,nor the length of the culture period. According to Gould(1986), culture time does not seem to be the only pa-rameter affecting genetic stability. Vendrame et al. (1999)analysed somaclonal variation in somatic embryos ofpecan (Carya illinoinensis) using AFLP markers andconcluded that, in this case, genetic variation in a cultureline could be affected more by the genotype than by theperiod in culture. In studies performed with micropropa-gated peach plantlets, it was suggested that the genotypeand the nature of the explant could influence the pheno-typic stability of the plants obtained (Hammerschlag et al.1987). This was verified when improved peach cultivarswere obtained through the use of somaclonal variation;

the results obtained were clearly dependent on the geno-type used (Hammerschlag 2000).

In the case of almond, it is possible that the absence ofsomaclonal variation among regenerants of differentprotocols, namely axillary branching (as studied here), butalso adventitious regeneration and culture of isolatedmeristems reported elsewhere (Martins 2003), may haveresulted from an influence of the genotype used, derivedfrom a “Boa Casta” seedling. However, further researchusing other almond genotypes is necessary to confirm thishypothesis.

In the study reported here, RAPD and ISSR analysiswere used to evaluate the genetic homogeneity (or vari-ation) of almond plantlets regenerated through axillarybranching after 4 years and 6 years of in vitro multipli-cation. The results obtained suggest that axillary branch-ing regeneration is an in vitro process that may be usedfor clonal propagation with a low risk of generatingsomaclonal variants, particularly for almond clone VII.These results were also important to ascertain the validityof using axillary branching plantlets of clone VII (mother-plants) as internal controls in studies of somaclonalvariation of regenerants obtained from other in vitroculture systems developed for this clone (adventitiousregeneration protocols, virus sanitation programs and ge-netic improvement).

Acknowledgements Part of this research was supported by PraxisXXI through the PhD grant BD/11323/97 provided to M.C. Martinsand the research projects PRAXIS 3/3.2/HORT/2143/95. We alsogratefully acknowledge Dr. C�lia Miguel and Milene Costa, forproviding the in vitro plant material.

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