ORIGINAL PAPER

Silver birch telomeres shorten in tissue culture

Tuija Aronen & Leena Ryynänen

Received: 7 June 2013 /Revised: 9 September 2013 /Accepted: 16 September 2013 /Published online: 29 September 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Shortening of telomeres has been connected withageing and loss of cell replication or regeneration capacity.The aim of the present study was to examine potential varia-tion in the length of telomeric repeats in silver birch (Betulapendula Roth) using clonal materials consisting of different-aged outdoor trees and tissue cultures of seven genotypes. Theoverall average length of telomeres was 13.6 kb (±0.3), theminimum length of repeats in the different genotypes variedfrom 5.9 kb (±0.5) to 9.6 kb (±0.6), and the maximum lengthvaried from 15.3 kb (±1.1) to 22.8 kb (±0.4). When germinat-ed seeds and leaf and cambium samples from 15- and 80-year-old trees were compared, no correlation of ageing and thelength of telomeric repeats was found. Positional variation inthe telomere length was, however, observed in the cambium ofmature trees, the stem base having longer repeats than theupper parts of the tree. Tissue cultures were found to haveshorter telomeres than outdoor trees: prolonged culture, callusculture and stressful conditions were all observed to shortentelomeric repeats and should thus be avoided in birchmicropropagation. There were significant differences amongthe studied silver birch genotypes in their telomere length, andthese differences were consistent over the sample types. Thisis the first report on variation of telomeric repeats in a long-living organism studied with clonal materials.

Keywords Betula pendula Roth . Duration of tissue culture .

Genotypic variation .Micropropagation . Physiologicalageing . Telomeric repeats

Introduction

Eukaryotic chromosomes are formed of a single DNA mole-cule, which terminates in specialised heterochromatin calledtelomeres. Telomeres are complex nucleoprotein structuresconsisting of repeated DNA sequence and specific set ofDNA-binding proteins. The function of telomeres is to protectchromosomes from degradation and fusion during DNA rep-lication, and therefore, they are especially important for chro-mosome organisation in cell division (McKnight et al. 2002).Without telomeres, the natural ends of chromosomes could berecognised as damaged DNA by cell's own DNA repair ma-chinery. The DNA damage response (DDR) can further triggereither programmed cell death or cellular senescence, in whichcells irreversibly arrest proliferation (Watson and Riha 2011).In all eukaryotes, telomeres are maintained by the specificenzyme, telomerase, because the conventional DNA replica-tion machinery cannot duplicate the ends of the linear chro-mosomes. Without telomerase activity, telomeres shorten ateach cell division, and when they reach a critical length, thecells enter a senescence phase (Vleck et al. 2003).

In plants, the telomere repeat, a heptanucleotide(TTTAGGG)n, has been found to be conserved, with someexceptions (McKnight and Shippen 2004), and the length oftelomeres is known to vary from 2 up to 150 kb in annualspecies (McKnight et al. 2002). The results of telomere studiesin animals have shown that the limit for cellular proliferationis genetically encoded in the lengths of cell's telomeres. Telo-mere shortening is accelerated by external factors, such asstressful conditions (Bauch et al. 2012). The life cycle ofplants differs from that of animals, e.g. in possessing moreplasticity and cell totipotency, but plants also age. The infor-mation on the role of telomeres in ageing of plants is limited(Watson and Riha 2011), and telomeres of long-living treeshave only been studied in a few species (Flanary andKletetschka 2005; Liu et al. 2007; Moriguchi et al. 2007;Song et al. 2010; Aronen and Ryynänen 2012).

Communicated by D. Neale

T. Aronen (*) : L. RyynänenFinnish Forest Research Institute, Finlandiantie 18,58450 Punkaharju, Finlande-mail: tuija.aronen@metla.fi

Tree Genetics & Genomes (2014) 10:67–74DOI 10.1007/s11295-013-0662-4

Flanary and Kletetschka (2005) suggested that an increasedtelomere length and telomerase activity contribute to an in-creased life-span in long-living pines. Studies of ginkgo trees(Ginkgo biloba L.) of up to 1,400 years of age (Liu et al. 2007;Song et al. 2010) showed that telomere length can evenincrease with age. When studying leaf samples of the apple(1–7 years of age) and cherry trees (up to 20 years of age),Moriguchi et al. (2007) observed no difference in the length oftelomeres due to ageing, or between juvenile, non-floweringand mature parts of a tree. In Scots pine (Pinus sylvestris L.),Aronen and Ryynänen (2012) found that after germinationageing per se had no effect on telomere length in cambium,bud or needle tissues of the 1–200-year-old trees. They ob-served, however, the telomeres of the meristematic stem cam-bium shorten towards the tree top in the older trees. Telomeresof Scots pine were also shown to shorten with increasing levelof tissue differentiation, embryo samples having the longestrepeats (Aronen and Ryynänen 2012). Differences in thelength of telomeric repeats among tissue types have beenfound in ginkgo trees too (Liu et al. 2007). In ginkgo leaves,dramatic shortening of telomeres in autumn has been ob-served, and it is suggested to be a part of apoptotic processesat the end of the growing season (Song et al. 2010).

In long-living trees, ageing can be examined also in relationto growth, since increased age and size tend to cause slowergrowth. This can be partly explained by increased physiolog-ical burden, i.e. higher demand of water and nutrients, but it issuggested that meristematic activity in the ageing plants is alsoaffected by accumulation of genome damage including telo-mere dysfunction (Watson and Riha 2011). Vegetative meri-stems of trees can regenerate into a new organism, but theability for clonal propagation reduces with ageing(Greenwood 1995). This may reflect DNA-related meristemarrest, independently of organism size. Shortening of telo-meres is connected with inactivity of telomerase, but can alsoact as sensor for accumulation of overall DNA damage, be-cause the function of general DNA repair system is limitedwithin the telomeric sequence (Watson and Riha 2011). Moreinformation is, however, needed on telomeres of the treemeristems in order to clarify their role in tree ageing.

So far, all the telomere studies in tree species have beenperformed with genetically diverse materials. Significant dif-ferences in the telomere length among genotypes have, how-ever, been reported both in apple (Moriguchi et al. 2007) andScots pine (Aronen and Ryynänen 2012) and should be takeninto account. Thus, in order to obtain unbiased view of telo-mere–ageing connection in trees, studies with different-agedclonal materials would be necessary, especially when only asmall number of tree individuals per age class can be includedinto molecular analyses.

In addition, biotechnological methods are nowadays ap-plied more and more also in tree species. Transgenic trees areactively studied (McDonnell et al. 2010), while clonal

propagation by tissue culture is already a standard practicein many species, and clones can be maintained under tissueculture conditions for long times (Walter and Menzies 2010).There is, however, no data existing on the effects of tissueculture and its duration or stress factors involved in biotech-nologies on telomere length in long-living trees.

The aim of the work presented here was to find answers tothe open questions presented above by studying the variationin telomeric repeats of silver birch (Betula pendula Roth).Silver birch is a wide-ranging species and an economicallyimportant tree in the boreal zone of Eurasia.Micropropagationtechniques for the species were developed in the 1970s–1980s(Huhtinen and Yahyaoglu 1974; Simola 1985; Ryynänen andRyynänen 1986). Therefore, regenerated clonal trees as wellas organogenic tissue cultures were available for examiningthe telomeres of the species. We examined (1) the potentialvariation caused by tree ageing by comparing germinatedseeds with tissues from the 15- and 80-year-old trees, (2) theeffect of tissue culture by comparing outdoor trees with shootand callus cultures, (3) the effect of short and extended tissueculture period and (4) the effect of stress under in vitro con-ditions. We used the same set of seven different genotypesacross all comparisons to test the effect of genotype.

Materials and methods

Plant material

Seven silver birch genotypes named E1987, E5201, E5382,E5387, E5389, E5396 and E5398 were used in the study. Theoriginal trees are growing in a silver birch stand at Punkaharju,in southeastern Finland (61°49′N; 29°18′E; 90 m above sealevel). The stand had been planted with seedlings of localorigin in 1926. The material consisted of samples taken fromthe original trees, from the 15-year-old micropropagated treesof the same genotypes growing at the plastic house atPunkaharju and from in vitro cultures of the same genotypes(Table 1).

From the outdoor trees, fresh and healthy, full-sized leavesand cambium samples were taken in spring (15-year-old treesin the plastic house) or early summer (original, 80-year-oldtrees) in 2008–2009. The open-pollinated seeds were collect-ed in years 2001–2003 and stored at −5 °C. Prior to sampling,the seeds were germinated on moist filter paper in sterile Petridishes under constant light with a light intensity of 16–20 μEm−2 s−1) at 20 °C in 2008. Pooled samples of thegerminated seeds (tiny seedlings with cotyledons) were col-lected per donor tree for DNA extraction (Table 1).

The in vitro shoot cultures were initiated from dormantvegetative buds of the donor trees. The cultures wereestablished as described by Ryynänen (1996) and subculturedevery third week on woody plant medium (WPM) (Lloyd and

68 Tree Genetics & Genomes (2014) 10:67–74

McCown 1980) containing 4.4μM6-benzyladenine (BA) and0.09 M sucrose (w /v ) solidified with 1 % (w /v ) agar. Thegrowth conditions for the shoot cultures were a 16:8-h light/dark photoperiod with a light intensity of 95–115 μEm−2 s−1,cool white lights F25W/30′′/133-T8, Sylvania, Germany, at22°C. During micropropagation, the new shoots regeneratedfrom adventitious buds on the basal callus of the mothershoots. The young cultures were subcultured from 10 to 20times, and the old ones over 80 times before sampling. Sam-ples for DNA extraction were taken separately from in vitroshoots and underlying callus tissue (Table 1).

In addition to the in vitro shoot cultures, undifferentiatedcallus cultures were studied both under normal and stressconditions caused by a selective agent, kanamycin, usedwidely in plant biotechnology (Table 1). Phytotoxicity ofkanamycin is based on its effect on chloroplasts and mito-chondria in which it inhibits protein synthesis in ribosomes(Nap et al. 1992). The cultures were subjected to kanamycin atconcentration considered detrimental to normal, i.e. non-transformed, birch tissues (Valjakka et al. 2000; Aronenet al. 2003). For the experiment, new callus cultures wereestablished by cutting the in vitro shoots into 0.5–1.0-cmpieces that were cultivated on WPM containing 2.3 μMthidiazuron (TDZ) and 1.1 μM naphthaleneacetic acid(NAA) for 2 days, after which half of the explants weretransferred onto the same medium with 100 mg−l kanamycin(K4000, Sigma-Aldrich, Finland). Three Petri dishes, eachcontaining ten explants were used per tissue culture age/genotype combination. The explants were transferred onto

fresh medium every second week during the 12 weeks ofcultivation. The explants on each dish were weighed prior toand after the cultivation period, and their growth was calcu-lated. The growth conditions during the kanamycin stresstreatment were the same as for the in vitro shoot cultures.For DNA extraction, samples both from the stressed andcontrol calli were taken after 12-week treatment.

DNA extraction and Southern blot analysis of telomericrepeats

Immediately after collection, the 300–500-mg samples werefrozen in plastic bags in liquid nitrogen and stored at −80 °C.Total genomic DNA was isolated from the samples by themodified method of Lodhi et al. (1994), as described inValjakka et al. (2000) and Aronen et al. (2003). DNA analysiswas performed using Southern blot hybridisation, as describedby Kilian et al. (1995), with the modifications described byAronen and Ryynänen (2012). For the positive control, asynthetic telomere sequence was generated by PCR accordingto Cox et al. (1993), using oligomer primers T1 (5′-TTTAGGG-3′) and T2 (5′-CCCTAAA-3′). Chemilumines-cence detection of the hybridisation products was performedaccording to the manufacturer's (Roche Diagnostics GmbH)instructions. The output was then scanned with theAlphaImager Imaging System (Alpha Innotech Co./ProteinSimple, CA, USA), and the size of the signals wasanalysed using AlphaEase®FC software and digoxigenin-labeled marker for molecular weight (MW).

Table 1 Silver birch materialssampled (√) for estimating thelength of telomeric repeats

Origin and age of material Birch genotype

E1987 E5201 E5382 E5387 E5389 E5396 E5398

Original donor trees (>80-year-old)

Leaves √ √ √ √ √ √ √Branch cambium √ √ √ √ √ √ √Stem base cambium √ √ √ √ √ √ √

Micropropagated trees (15-year-old)

Germinated seeds from the tree √ √ √ √ √Branch cambium √ √ √ √ √

Tissue cultures after 10–20 subcultures

In vitro shoots √ √ √ √ √ √ √Basal callus of the in vitro shoots √ √ √ √ √ √Callus culture √ √ √ √ √ √ √Callus on kanamycin medium √ √ √ √ √ √ √

Tissue cultures after >80 subcultures

In vitro shoots √ √ √ √ √ √ √Basal callus of the in vitro shoots √ √ √ √ √ √Callus culture √ √ √ √ √ √ √Callus on kanamycin medium √ √ √ √ √ √ √

Tree Genetics & Genomes (2014) 10:67–74 69

Chromosomal position of the telomeric sequences detectedby Southern blot hybridisation—if they truly are located at theends of the chromosomes—was studied using BAL-31 exo-nuclease treatment, as described by Aronen and Ryynänen(2012).

Experimental design and statistical analyses

The experimental design and statistical analyses were asfollows:

1. The influence of tree ageing was studied in the samplescollected from the outdoor trees: germinated seeds werecompared with the leaf and cambium tissues of the 15-and 80-year-old trees.

2. The effect of tissue culture was examined by comparingthe outdoor and in vitro materials, either using all the dataor excluding the callus cultures. Within in vitro materials,shoot and callus cultures were compared with each other.In addition, the samples from the young (10–20 subcul-tures) and old (>80 subcultures) tissue cultures werecompared.

3. The influence of stress was studied under in vitro condi-tions by comparing callus cultures subjected to antibiotickanamycin with the callus cultures grown on the controlmedium.

4. Genotypic differences among tree individuals were inves-tigated in all the above-mentioned data sets, excluding,however, the germinated seed samples that consisted ofnumerous, pooled tiny seedlings.

The effect of tree age, tissue culture, tissue type, stress andtree genotype on the maximum, mean and minimum lengthsof the telomeric repeats was studied by analysis of variance.Post hoc comparisons among the group means, if necessary,were performed using the Student–Newman–Keuls multiplerange test. All the statistical analyses were performed usingthe PASW Statistics 17.0 software.

Results

The overall average length of telomeric repeats in the studied89 silver birch samples was 13.6 kb (±0.3 SE), the minimumlength in the different genotypes varying from 5.9 kb (±0.5) to9.6 kb (±0.6) and the maximum length varying from 15.3 kb(±1.1) to 22.8 kb (±0.4). The BAL-31 exonuclease treatmentconfirmed the detected sequences being true telomeres at theend of the chromosomes (Fig. 1).

The effect of tree ageing

When the three age classes of the outdoor trees, i.e. germinat-ed seeds, 15-year-old micropropagated trees and the original

80-year-old trees were compared, no significant difference wasfound in the average or maximum length of the telomericrepeats (ANOVA p =0.382 and 0.981, respectively). For theminimum length, a significant (p =0.017) difference was ob-served, the germinated seeds having shorter telomeres (6,540±356 kb) than the 15-year-old (9,103±708) or 80-year-old trees(9,579±485 kb).When examining different sample typeswith-in the 80-year-old trees, the stem base cambium was found tohave significantly longer repeats than the samples representingthe upper parts of the tree, i.e. branch cambium and leaves(Fig. 2).

The effect of tissue culture

Tissue culture affected telomere length in silver birch. Thein vitro tissues had shorter telomeres than outdoor trees, andthis effect was seen similarly in maximum, average and min-imum lengths of telomeric repeats (ANOVA p =0.009, 0.006and 0.003, respectively). According to tissue type and age, thein vitro shoots do not differ significantly from most of theoutdoor tree samples, but the callus cultures do (Fig. 2).

Within the in vitro materials, the shoots differed signifi-cantly from the callus cultures when examining the maximumand average lengths of telomeric repeats (ANOVA p =0.001and 0.003, respectively) and had longer repeats, but not in thecase of minimum length (p =0.064). When the effect of ex-tended tissue culture period on telomere length was analysed,a significant difference between samples taken after 10–20subcultures or over 80 subcultures was seen in the minimumlength of telomeres, the old tissue cultures having shorterrepeats (Figs. 3 and 4).

The effect of kanamycin stress on callus cultures

The inclusion of 100 mg/l of kanamycin into the mediumreduced the growth of silver birch calli significantly (ANOVAp <0.001): The average growth of the control cultures was608±47 mg under the 12-week experiment, and that of thecalli subjected to kanamycin was only 20±1.1 mg. On thekanamycin medium, shortening of the telomeres could beobserved (Fig. 2), but this effect was not significant (p =0.386, 0.493 and 0.837 for the maximum, average and mini-mum lengths, respectively).

Genotypic differences

There were significant differences among the studied silverbirch genotypes in their telomere length (ANOVA p <0.001for the maximum, average and minimum lengths), and thesedifferences were consistent over the sample types of theoutdoor trees (Fig. 5) and the in vitro culture periods (Fig. 4).

70 Tree Genetics & Genomes (2014) 10:67–74

MW 1 2 3 4 5 1 2 3 4 5 MW T

BAL31-treated control samples

21.2 kb -

5.1 kb - 4.3 kb - 3.5 kb -

2.0/1.9 kb -

Fig. 1 Southern hybridization of a telomere probe to the birch DNAsamples following BAL-31 exonuclease treatment and without it. TheBAL-31 degrades termini of duplex DNA but does not generate internalscissions. Thus, dramatic shortening of the signals following the BAL-31treatment suggest their position in the genome being at the ends ofchromosomes, i.e. truly telomeric. T = positive telomere control

0

5

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30MW, kb

Outdoor trees In vitro tissues Means

max mean min

Germ

inate

d se

eds

Branc

h ca

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5 y)

Leav

es (8

0 y)

Branc

h ca

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0 y)

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bas

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Stress

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Fig. 2 Length of telomeric repeats in the studied silver birch mate-rials, n =89, together with the mean values for the samples of theoutdoor trees and the in vitro tissues. Both the tissue cultures after 10–20 subcultures and >80 subcultures were included. Significant differ-ences in the maximum, average or minimum length of telomericrepeats among the sample types according to the S–N–K test (p <0.05) are marked with different letters

Fig. 3 Southern hybridisation showing within-clone variation in thelength of silver birch telomeric repeats. Samples of the outdoor treesand the in vitro tissues of the genotypes E5396 and E5398

0

5

10

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25

30MW, kb

10-20 subcultures > 80 subcultures Means

E1987

E5201

E5382

E5387

E5389

E5396

E5398

E1987

E5201

E5382

E5387

E5389

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E5398

10-2

0 su

bcult

ures

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ultur

es

max mean min

a

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Fig. 4 The effect of tissue culture period on the length of telomericrepeats in the studied silver birch genotypes E1987, E5201, E5382,E5387, E5389, E5396 and E5398, n =58. Significant differences in themaximum, average or minimum length of telomeric repeats among thesample types according to the S–N–K test (p<0.05) are marked withdifferent letters

Tree Genetics & Genomes (2014) 10:67–74 71

Discussion

The length of telomeric repeats was studied in seven silverbirch genotypes by comparing the 80-year-old donor treeswith the clonally replicated 15-year-old trees and tissue-culturedmaterials. The observed size of silver birch telomeres,varying from 5.9 kb (±0.5 SE) to 22.8 kb (±0.4 SE), is longerthan in some other angiosperm tree species studied, e.g. cherryand apple having telomeres of 2–6 or 2–7 kb, respectively(Moriguchi et al. 2007), but shorter than found in papaya, 25–50 kb (Shakirov et al. 2008).

In the present birch material, there were no age-relateddifferences in the maximum or average length of telomericrepeats among germinated seeds and 15- and 80-year-oldtrees, but the minimum telomere size was bigger in the oldertrees than in the germinated seeds. This difference may also becaused by genotypic effect that was observed to be significantin silver birch. The germinated seeds do not represent theexactly the same genotypes as other materials used in thestudy, although they were collected from the same trees, i.e.share the genotypic background on the maternal side. Inginkgo trees reaching ultimate old ages, the length oftelomeric repeats has been observed to extend with increasingage, the most rapid changes taking place before 200 years ofage (Liu et al. 2007), with the lengthening trend continuingtowards the oldest age studied, 1,400 years (Song et al. 2010).In Scots pine, embryos were found to have the longest telo-meres, while following germination, ageing per se had nosignificant effect on the telomere length in 1- to 200-year-old trees (Aronen and Ryynänen 2012). Ageing had no effecton telomere length in 1- to7-year-old apples or in cherries ofup to 20 years of age (Moriguchi et al. 2007).

The present observation that the telomeric repeats in thecambium of 80-year-old silver birch trees were significantlylonger at the stem base than in the branches is in accordancewith the results obtained for the 50- to 200-year-old Scots pinetrees that showed shortening of telomeres in the stem cambiumtowards the tree top (Aronen and Ryynänen 2012). The in-creasing evidence that the telomere length in the meristematiccambium may vary positionally within a tree, the stem basepositions having longer repeats than the upper parts of the tree,

gives new insight to the well-established idea of cyclophysis.According to this theory, the lower part of the tree stemremains more juvenile than the top of the tree, because thelower stem originates from a juvenile apical meristem, whereasthe upper parts of the tree are formed from the matured apicalmeristem that has undergone numerous cell divisions (Olesen1978; Bonga 1982). In the case of Scots pine (Aronen andRyynänen 2012), hormonal gradients within the stem weresuggested to affect telomere length via telomerase activity thatis known to be increased by auxin and decreased by abscisicacid (Tamura et al. 1999; Yang et al. 2002). It has, however,also been argued that changes in relative concentrations ofhormones, as well as gene expression regulating them, mayalso be only symptoms rather than a primary cause of matura-tion in woody plants (Greenwood 1995).

In the present study, in vitro tissues of silver birch hadshorter telomeric repeats than the outdoor trees. The in vitroshoots derived from vegetative buds in the tree tops did,however, not differ significantly from the most of the outdoorsamples, the stem base cambium with longer telomeres beingan exception. On the other hand, both shoot basal callus andcallus cultures produced from the in vitro shoots had signifi-cantly shorter telomeres than the outdoor trees. The effect oftissue culture on the length of telomeric repeats has previouslybeen investigated only in annual plant species. The size oftelomeres in barley embryos used as explants for initiatingcallus cultures was estimated to be around 60 kb. During thefirst 4 weeks of callus culture, the barley telomeres wereobserved to shorten, but the trend then reversed, and thetelomeres up to 250 kb were seen in long-term cultures(Kilian et al. 1995). An increase in telomere size has also beenreported for Melandrium callus cultures, 3 months after theirinitiation (Riha et al. 1998). In tobacco, propagation throughtwo cycles of callus formation did not change the telomerelength significantly, and it was assumed that any loss oftelomeric DNA during the experiment was compensated byincreased telomerase activity (Fajkus et al. 1998).

The extended tissue culture period shortened the minimumlength of silver birch telomeric repeats in the present study.The inclusion of a selective chemical stress factor, kanamycin,caused also some shortening of telomeres, although not

Fig. 5 Southern hybridisationshowing genotypic variation inthe length of silver birch telomericrepeats in the outdoor treesamples (germinated seeds, leavesof 80-year-old trees and branchcambium of 15-year-old trees)

72 Tree Genetics & Genomes (2014) 10:67–74

statistically significantly. Kanamycin inhibits protein synthe-sis in the mitochondria and chloroplasts of the plant cells (Napet al. 1992). In animals and humans, various stress factorsincluding harmful chemicals have been shown to acceleratetelomere shortening both in vivo and in vitro, and this effecthas been connected to mitochondrial dysfunction and gener-ation of reactive oxygen species (Liu et al. 2002, 2003; Passosand von Zglinicki 2005; Valdes et al. 2005). It can also beargued that tissue culture conditions per se act as stress envi-ronment (Rani and Raina 2000). Increased telomerase activityhas been found in undifferentiated cell cultures of severalplant species (Fizgerald et al. 1996), but in the present silverbirch tissue culture system based on the maintenance of dif-ferentiated shoots, the minimum length of telomeres wasshown to shorten with time. This is an important observation,because it is now commonly accepted that replicative cellularsenescence can be initiated at only one or a subset of telomeresthat shorten below a critical length, and therefore, mainte-nance of an average telomere length is not so critically impor-tant as maintaining all telomeres above a minimum threshold(Watson and Riha 2011).

Genotypic differences in telomere length were obviousamong the studied birch individuals, and the observeddifferences were found to be consistent over the outdoorand tissue-cultured samples. This is the first time whenvariation in telomeric repeats has been studied indifferent-aged clonal materials of a long-living organism.Genotypic variation in telomere length has been reportedalso in other plants, including barley (Kilian et al. 1995),apple (Moriguchi et al. 2007) and Scots pine (Aronen andRyynänen 2012) and has been studied in more detail inmaize. Burr et al. (1992) reported more than 25-foldgenotype-dependent variation among maize inbred lines,attributed most of this variation to three loci and assumedhigh heritability for telomere length. Also, in apple, thetelomere lengths were found to segregate into various pat-terns in F1 seedlings originating in the crosses of two applecultivars with different telomere lengths, suggesting thatthe telomere length in apple is maintained by multigenes(Moriguchi et al. 2007). The present results obtained fromclonal birch materials underline the importance of takinggenotypic variation into account when interpreting molec-ular results—comparing, e.g. telomere lengths among tis-sues or time points using different individuals may other-wise lead to biased view on telomere dynamics. To circum-vent this problem, clonal materials should preferably beused or number of sampled trees increased enough.

In conclusion, no direct effect of ageing on the length oftelomeric repeats was observed in silver birch. Positional var-iation in the telomere length was, however, observed in thecambium of mature trees, the stem base having longer repeatsthan the upper parts of the tree. The same phenomenon haspreviously been discovered in Scots pine (Aronen and

Ryynänen 2012) and needs to be studied further. Does telomerelength in meristematic cambium reflect different ontogenic ageof tree parts? Another important finding was that tissue culturecan affect telomere length in silver birch: prolonged cultureperiods, callus culture and stressful conditions may all shortentelomeric repeats and should thus be avoided.

Data archiving statement The research performed in the present studydid not produce any data on nucleic acid or protein sequences, geneticmaps, SNPs or gene expression that could be deposited in the publicdatabases. Digitalized data of original Southern blots is stored at FinnishForest Research Institute, Punkaharju (www.metla.fi).

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