JXB Advance Access originally published online on January 19, 2007
Journal of Experimental Botany 2007 58(3):687-698; doi:10.1093/jxb/erl241
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RESEARCH PAPER |
Developmental and genetic variation in nuclear microsatellite stability during somatic embryogenesis in pine
1Austrian Research Center Seibersdorf GmbH, Department of Bioresources, A-2444 Seibersdorf, Austria
2Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Box 7080, SE-750 07 Uppsala, Sweden
* To whom correspondence should be addressed. E-mail: kornel.burg{at}arcs.ac.at
Received 28 June 2006; Revised 23 October 2006 Accepted 24 October 2006
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Abstract |
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Genotypic instability is commonly observed in plants derived from tissue culture and is at least partly due to in vitro-induced stress. In this work, the issues of whether genetic instability induced by in vitro stress varies among families and if genetic instability influences the adaptation to in vitro conditions and embryo development have been addressed. By comparing the stability of four variable nuclear microsatellite loci in embryogenic cultures and zygotic embryos of Pinus sylvestris, a significant difference in genetic stability among families was found. In six out of 10 families analysed, the level of genetic stability was similar between somatic and zygotic embryos. However, for the rest of the families, the mutation rate was significantly higher during somatic embryogenesis. Families showing a low genetic stability during establishment of embryogenic cultures had a higher embryogenic potential than those which were genetically more stable. In contrast, embryo development was suppressed in genetically unstable families. The relatively high mutation rates found for some families might reflect the plasticity of the families to adapt to stress, which is important for widely distributed species such as Pinus sylvestris.
Key words: Embryogenesis, genetic stability, microsatellite mutation, Pinus sylvestris
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Introduction |
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Despite the advantages of in vitro propagation, there are reports showing that plants derived from in vitro cultures are genotypically unstable. Larkin and Scowcroft (1981) coined the term ‘somaclonal variation’ to describe the occurrence of genetic variants derived from in vitro procedures.
Polymorphism in repetitive DNA sequences has for a long time been observed during plant propagation in tissue culture (Smulders et al., 1995). Incorporation of incorrect bases by the DNA replicative polymerases into the DNA occurs at every 106–107 bases. In nucleotide repeat regions such as microsatellites, where slip-mispairing can occur, this frequency is higher (Kunkel and Bebenek, 2000). Microsatellite sequences have been detected within the genomes of every organism so far analysed and are often found at frequencies much higher than would be predicted purely on the basis of nucleotide composition (Behe, 1995). Microsatellites show a more or less even distribution within the genome (Dib et al., 1996). However, a characteristic genomic distribution and motif-dependent dispersion has also been reported in conifers (Schmidt et al., 2000), Oryza sativa (rice), and Arabidopsis (Carninci et al., 2003).
Mutation frequencies for microsatellites are reported in the range of 10–2 in bacteria (Lewinson and Guttman, 1987), 10–4–10–5 in yeast (Strand et al., 1993), and 
10–3 in humans (Weber and Wong, 1993) per generation. The mutation frequency by direct test on the DNA polymerase-generated mutations in microsatellite versus single copy regions was reported to be 4–72 times higher in the microsatellite sequences in an in vitro test system (Eckert et al., 2002). This elevated level of mutation renders microsatellite regions appropriate targets for monitoring putative mutation events in cultured cells. Microsatellite DNA variation was detected in micropropagated Populus tremuloides (trembling aspen) (Rahman and Rajora, 2001) and in Picea abies (Norway spruce) somatic embryos (Burg et al., 1999).
In vitro culture conditions appear to affect the stability of the plant genomes, with various plant species and genotypes responding differently under various conditions. In this work, the following questions have been raised. (i) Does the genetic instability induced by in vitro stress vary among families? (ii) Does the genetic instability influence the adaptation to in vitro conditions? (iii) Does the genetic instability affect the competence for plant regeneration? To address these questions, somatic embryogenesis of Pinus sylvestris (Scots pine) was used as a biological system. The whole procedure of conifer plant regeneration through somatic embryogenesis comprises a sequence of steps including establishment and proliferation of embryogenic cultures, maturation and germination of somatic embryos, and, finally, ex vitro acclimatization of somatic embryo plants (von Arnold et al., 2002). The genetic stability of four nuclear microsatellite loci has been studied during establishment and proliferation of embryogenic cultures in 10 half-sib families of Scots pine and in cotyledonary somatic embryos and zygotic embryos. A high mutation rate in microsatellites was observed during establishment of embryogenic cultures. It is shown that the mutation rate varies significantly among families. Furthermore, the genetic instability of the families correlates positively with their embryogenic potential, but negatively with the frequency of cotyledonary embryo formation. It is also shown that a relatively high mutation rate takes place during zygotic embryogenesis.
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Materials and methods |
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Plant material
Immature, open pollinated seed cones were collected from 15 clones (W1005, W1006, W2015, W2017, W3006, W3011, S3051, S3144, S3244, S3246, S3256, S3259, W4009, W4011, and S6201) corresponding to half-sib seed families designated F01–F15 of P. sylvestris L. from the Swedish breeding programme growing in a seed orchard in central Sweden (Brunsberg). Three harvests were performed; harvest I on 19 June estimated as 5–7 days after fertilization (DAF), harvest II on 27 June (13–15 DAF), and harvest III on 3 July (19–21 DAF). At harvest I, the ovules predominantly contained few-celled proembryos. At harvest II, the ovules mainly contained four or more early cleaving embryos of equal size, whereas at the last harvest, a single dominant embryo could be clearly distinguished among several monozygotic embryos (Filonova et al., 2002).
The procedure for cone sterilization and isolation of ovules was as described by Keinonen-Mettälä et al. (1996). At each of the three harvests, 450 ovules of each family were cultured on the DCR proliferation medium of Gupta and Durzan (1985) modified by replacing Ca(NO3)2 by 8.1 mM KNO3 and 1.16 mM CaCl2, containing the auxin 2, 4-dichlorophenoxyacetic acid (2, 4-D) and the cytokinin benzyladenine (BA) at 9.0 µM and 4.4 µM, respectively, as plant growth regulators (PGRs), 3% sucrose, and 0.35% gelrite. The cultures were incubated in darkness at 22 °C and subcultured every 3 weeks. Establishment of embryogenic cell lines was recorded after 4–6 months of culture. An embryogenic cell line was classified as established when the tissue increased in size by at least 2-fold within the subculture interval. In total, 325 embryogenic cell lines were established. Samples for microsatellite analyses were taken from each established line (Fig. 1).
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The newly established cell lines were cryopreserved 10 months after starting the cultures following the protocol developed by Nörgaard et al. (1993) (Fig. 1). The cell lines were thawed and proliferated on modified DCR medium of the same composition as during establishment. After 3 months, samples were taken for microsatellite analyses from 48 proliferating cell lines representing four families. At the same time, five cell lines representing two families were chosen for determining whether the cultures were mosaics (Fig. 1). In this case, large culture aggregates (200 mg fresh weight) were separated into at least eight sectors and each sector was analysed separately.
To stimulate embryo maturation, the cell lines were first pre-treated for 4 weeks on PGR-free DCR medium and then transferred to DCR maturation medium containing 7.5% polyethylene glycol (PEG-4000), 60 µM abscisic acid (ABA), 3% maltose, and 0.35% gelrite. The cultures were incubated in darkness at 22 °C. Embryogenic tissues were subcultured every third week by 50 mg fresh weight pieces. Four pieces were incubated in each Petri dish (50 mm in diameter). Initially 130 cell lines were screened for maturation; out of these 88 developed cotyledonary embryos (data not shown). The experiment was repeated with 16 cell lines (10–40 Petri dishes per cell line) representing six families. In order to genotype the mother trees, needles were collected from the 15 mother trees. For genotyping zygotic embryos, seeds were extracted from mature cones collected in October from the open pollinated mother trees corresponding to families F02, F03, F05, F07, F08, F11, F12, F13, F14, and F15. Zygotic embryos from at least 45 seeds for each family were isolated and analysed for microsatellite stability.
DNA preparation and microsatellite analysis
DNA was isolated from the sampled material as described previously (Helmersson et al., 2004) with the aid of a DNeasy Plant Mini Kit (Qiagen, Cologne, Germany).
The PCR amplifications for the microsatellite regions were made in an MJ Research PCR machine (PTC100), in 96-well plates. Samples used for capillary electrophoresis were PCR amplified without oil, using a heated lid. The reactions were performed in 96-well plates (Corning). A single reaction of 25 µl contained the following components: 10x buffer (supplied with the enzyme), 2.5 µl of MgCl2 (4.0 mM), primers (0.25 µM), dNTPs (Amersham) (200 µM), HotStarTaq DNA Polymerase (Qiagen) 0.625 U, and 5–50 ng of DNA. The sequence information of the four nuclear microsatellite loci (see accession numbers below) analysed in this study and the primer pairs for their amplification were published by Soranzo et al. (1998): SPAC11.4 (accession number AJ223766) Forw. 5'-TCA CAA AAC ACG TGA TTC ACA-3'/Rev. 5'-GAA AAT AGC CCT GTG TGA GAC A-3'; SPAC11.6 (accession number AJ223767) Forw. 5'-CTT CAC AGG ACT GAT GTT CA-3'/Rev. 5'-TTA CAG CGG TTG GTA AAT G-3'; SPAG11.8 (accession numberAJ223770) Forw. 5'-AGG GAG ATC AAT AGA TCA TGG-3'/Rev. 5'-CAG CCA AGA CAT CAA AAA TG-3'; and SPAC12.5 (accession number AJ223772) Forw. 5'-CTT CTT CAC TAG TTT CCT TTG G-3'/Rev. 5'-TTG GTT ATA GGC ATA GAT TGC-3'. All the four loci were amplified by the following protocol: 95 °C for 15 min followed by three cycles of 94 °C for 15 s, 60 °C for 1 min, 70 °C for 35 s, then 12 cycles [93 °C 45 s, 55 °C (–0.5 °C per cycle), 70 °C 45 s] followed by 20 cycles of 92 °C for 30 s, 50 °C for 30 s, 70 °C for 45 s and a final extension at 70 °C for 5 min.
Determination of the simple sequence repeat (SSR) fragment length was performed on either an ABI373XL or an ABI3100 fragment analyser (Applied Biosystems). Slab gels (22 cm) were used for the ABI373XL, using 6% Rotiphorese Gel 30 (Roth catalogue no. 3029.1). The runs on the ABI3100 sequence analyser were performed using a 22 cm capillary array with POP4 polymer. The samples were analysed in duplicate using independent PCR amplifications. At the stage of culture establishment, the genotype was considered as mutant if none of the two alleles for a given microsatellite locus had a length comparable with any of the two parental alleles. A 2 bp mismatch was set as a detection limit. The allelic sizes determined at the stage of culture establishment served as reference for monitoring mutations at subsequent stages of embryogenesis. Putative mutants were confirmed by additional independent PCR amplifications. Performing multiple PCRs substantially decreased the possibility of misdetecting alleles.
None of the four analysed microsatellite regions contained null alleles in the families studied as judged by analysing the segregation of the maternal loci in zygotic embryos and established embryogenic cell lines, for example, in case a mother tree was heterozygous for a given locus, the segregation of both alleles was observed (Fig. 2A–C). Homozygote status of the maternal loci may hide null alleles which would result in the absence of the maternal allele in about half of the established cell lines as well as in the zygotic embryos within a family. None of the loci analysed yielded this type of segregation (data not shown). However, in this study, ‘loss’ (not PCR amplified) of alleles was occasionally observed (Fig. 2E). During the course of the present study, both loss of alleles and alteration in the fragment length were considered as mutations of the locus.
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GeneScan 3.7 (Applied Biosystems) software was used for the size calibration of the raw data, while the genotypes were established with the Genotyper 3.7 (Applied Biosystems) software. Genotypes identified after establishment of a cell line served as reference for the evaluation of mutation events both at proliferation phase and at embryo maturation. Popgene (Yeh et al., 1997) software was used for estimating the allele frequencies, while the statistical analysis was done using MS Excel functions.
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Results |
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The process of somatic embryogenesis
Embryogenic cultures were established from immature seeds. The frequency of established cell lines varied in the range of 0.2–11% depending on seed family and harvest time (Fig. 3). In general, based on all families, the highest frequency of cell lines was detected at the first harvest, for example, 1 week after fertilization, which coincides with the period when cleavage polyembryony has just started (Filonova et al., 2002). The established cell lines were sampled for genotyping and then cryopreserved (Fig. 1). The regrowth after thawing was in the range of 75–100% for different families. Cotyledonary somatic embryos developed after the withdrawal of PGRs followed by the addition of ABA and PEG. The ability to form cotyledonary somatic embryos varied significantly among and within families (see below).
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Single paternal genotypes in embryogenic cell lines
Polyembryony is common in gymnosperms (Singh, 1978). In polyzygotic polyembryony, the embryos have identical maternal but different paternal haplotypes, while in monozygotic polyembryony the embryos have identical maternal and paternal haplotypes. Most of the excess embryos in a seed are eliminated by programmed cell death (Filonova et al., 2002). The optimal stage of zygotic embryo development for initiation of embryogenic cultures of Scots pine is 1 week after fertilization (Fig. 3). At this stage, the zygotic embryos cannot be isolated, rather the whole ovule is used as the explant. The ovules may contain multiple living embryos derived from both monozygotic and polyzygotic polyembryony. Consequently, the embryogenic cultures established from a single ovule could originate from more than one embryo derived from different zygotes and could therefore represent a mixture of genotypes. Three out of 36 ovules of loblolly pine produced cell lines with multiple genotypes as shown by isozyme polymorphism (Becwar et al., 1991). Newly established embryogenic cell lines from 15 mother trees of Scots pine were analysed using four nuclear SSR markers. The mother trees as well as all 325 embryogenic cell lines had normal diploid genotypes yielding one or two amplified fragments in all the four SSRs tested. These results indicate the absence of mixed cultures at the initiation of somatic embryogenesis.
Natural and induced variability of four nuclear SSR loci
Microsatellites are among the most rapidly evolving DNA sequences with high mutation rate, which leads to their high polymorphism in terms of repeat units. In the present study, the genetic stability/mutability of four hypervariable nuclear microsatellite regions SPAC11.4, SPAC11.6, SPAG11.8, and SPAC12.5 (Soranzo et al., 1998) was analysed in newly established embryogenic cell lines, in proliferating cell lines, in cotyledonary somatic embryos (Fig. 1), as well as in zygotic embryos. Previously the same four loci were used in analysing Austrian Scots pine populations, and yielded 23, 17, 25, and 29 alleles based on the analysis of 635, 322, 561, and 321 loci, respectively (data not shown). In the 15 mother trees included in this study, nine, 14, six, and 17 alleles were detected for loci SPAC11.4, SPAC11.6, SPAG11.8, and SPAC12.5, respectively (Table 1). Paternal contribution of alleles and in vitro conditions had a strong effect on the allelic diversity in the 325 newly established embryogenic cell lines, resulting in 12, 31, 19, and 27 alleles for the corresponding loci in the 15 families analysed. Likewise, high diversity was observed in 481 zygotic embryos representing 10 families yielding 11, 35, 15, and 28 alleles for the corresponding loci (Table 1). Taken together, the lowest allelic diversity of 12 alleles was observed in the SPAC11.4 locus, while 20, 31, and 39 alleles were found for the SPAG11.8, SPAC12.5, and SPAC11.6 loci, respectively.
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Microsatellite stability during zygotic embryogenesis
In the present study, the interest was to monitor the genetic stability during somatic embryogenesis of Scots pine. For comparison, the stability of the four selected microsatellite regions was also analysed in 481 zygotic embryos representing the 10 mother trees/families used for establishing embryogenic cell lines. Since the mother trees were open pollinated, the fate of the maternal alleles, but not the paternal ones, could be traced in the zygotic embryos. The highest frequency of mutated maternal alleles was observed in the SPAC11.6 locus (9%), while the SPAG11.8 and SPAC12.5 loci yielded fewer mutations, 3.5% and 1%, respectively, and no mutations were observed in the SPAC11.4 locus (Table 2).
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In order to characterize the genetic stability of a family, two values were calculated; (i) the frequency (as a percentage) of genotypes in the family carrying perfect maternal alleles (FPG; frequency of perfect genotypes); and (ii) the mean of the mutation frequencies (in decimals) observed in the four microsatellites in each family (MMF; mean mutation frequency). The FPG value was, on average, 84% for the zygotic embryos. However, the FPG value varied considerably among families, ranging from 66% (F05) to 98% (F02) (Table 2). The MMF value of the analysed zygotic embryos ranged between 0.01 (F03) and 0.083 (F07) (Table 2).
Microsatellite stability during somatic embryogenesis
Newly established cell lines: At establishment of embryogenic cell lines, the genetic integrity of the maternal but not the paternal contribution to the offspring genome could be analysed since the ovules were taken from open pollinated trees. However, values obtained for the paternal alleles at this stage served as reference for later stages to trace putative mutations in them. A total of 325 newly established cell lines representing 15 families were tested for the stability of the four nuclear microsatellite loci. For estimating genotype stability, only families represented by >10 established cell lines with complete genotypes (all four loci successfully identified) have been included. In the following calculations, 268 cell lines representing 10 families have been included (Table 2). The four loci showed different genetic stability (Table 2). Loci SPAC11.4 and SPAC12.5 yielded 2% and 6% mutated maternal alleles, respectively, while the loci SPAC11.6 and SPAG11.8 yielded >12% mutated maternal alleles.
On average, 40% of the established embryogenic cell lines carried mutated maternal alleles (Table 2). However, there was a significant difference in mutation frequency among families (Table 2). The FPG values ranged from 40% (F15) to 100% (F02). The MMF values varied between 0 (F02) and 0.165 (F05) (Table 2). Based on the FPG and MMF values for newly established cell lines, the families could be divided into two groups. Families with an FPG value >70% and an MMF value <0.1 were considered as genetically stable, while families with FPG <70% and MMF >0.1 were considered as genetically unstable. On this basis, families F02, F03, F07, F08, F12, and F14 were classified as group 1 (stable families) and families F05, F11, F13, and F15 as group 2 (unstable families). Group 1 families were represented by 104 cell lines and group 2 families by 164 cell lines; however, from the latter, 94 (57%) represented F05. Comparing the FPG and MMF values of the established cell lines with those of zygotic embryos showed no significant differences for group 1 families; however, both values showed a significant difference for group 2 families (P=0.01 and P=0.00008, respectively). Out of 268 cell lines, 160 (60%) carried the perfect maternal genotype (Table 2). Eighty-two per cent of the cell lines in group 1 and 46% of the cell lines in group 2 carried perfect genotypes (Table 2). Accumulation of mutations within cell lines could be observed in group 2 families only. Nine cell lines were found with two mutated maternal alleles (six from F05, two from F13, and one from F15) and three cell lines with three mutated maternal alleles (two from F05 and one from F11). These results clearly show that microsatellite instability varies significantly among families both during somatic embryogenesis and in zygotic embryos. However, for some families, the instability is significantly higher during somatic embryogenesis compared with zygotic embryos.
The genetic stability in different families was compared with the ability to establish embryogenic cell lines and to develop cotyledonary somatic embryos. A negative correlation was found between embryogenic potential and genetic stability (Fig. 4). In contrast, the yield of cotyledonary somatic embryos positively correlated with genetic stability (Fig. 5).
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Proliferation of embryogenic cell lines: The stability of the four microsatellite regions was analysed in proliferating embryogenic cell lines (Fig. 1). Families F03 and F08 were selected as representatives of group 1 and families F05 and F11 as representatives of group 2 (Table 2). Cell lines with both perfect and altered genotypes (identified at the stage of establishment) were chosen. In total, 31 cell lines with perfect genotypes and 17 cell lines with altered genotypes at establishment were analysed during the proliferation phase (7/4, 12/2, 7/7, and 5/4 perfect/altered genotypes for family F03, F05, F08, and F11, respectively). Out of the 48 analysed cell lines, 13 showed altered genotypes during proliferation compared with after establishment (FPG=73%). About 30% of the cell lines with altered genotypes after establishment incurred further mutations during proliferation, and 26% of the cell lines with perfect genotypes after establishment carried new mutations during proliferation. Data obtained with the 13 cell lines, which showed altered genotypes during proliferation, are shown in Table 3. In family F05, two out of 14 cell lines analysed carried mutated alleles. In cell line F05:53, a shortening of the paternal allele was observed in the SPAC11.4 locus, a loss of the paternal allele was seen in the SPAC11.6 locus, and both alleles were mutated in the SPAC12.5 locus. In some cases, for example for cell lines F08:19 and 21, the maternal or paternal origin of the locus could not be followed since the reference genotypes were homozygotes for the corresponding loci.
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If mutations occur during proliferation, these should result in a mosaic nature of the cell aggregate. In order to analyse the appearance of new mutations, five cell lines representing both group 1 (F08:05 and F08:10) and group 2 (F05:31, F05:53, and F05:58) were selected. Cell lines F05:58 and F08:05 kept their perfect genotypes both after establishment and during proliferation, cell line F05:53 had perfect genotype after establishment but accumulated mutations during proliferation, cell line F05:31 had an altered genotype after establishment and kept that genotype during proliferation, and cell line F08:10 accumulated mutations both after establishment and during proliferation. Large aggregates of proliferating embryogenic tissue were split into several sectors, which were genotyped individually (Fig. 1). None of the sectors from cell line F05:58 showed any mutation. Mutations observed in the other cell lines are shown in Table 4. In cell lines F05:31 and F08:05, only one sector out of eight and 10, respectively, showed one mutated allele. In cell line F05:53, five adjacent sectors carried the same mutant paternal allele of the locus SPAC11.6. In cell line F08:10, consecutive mutational events could be detected. These data clearly show that mutations take place continuously when cells are cultured in vitro.
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Establishment of embryogenic cell lines took up to 6 months and the proliferation time after establishment was 4 months before cryopreservation and 3 months after thawing (Fig. 1). It is not possible to estimate the number of cell divisions during the two periods (establishment and proliferation). Since the length of the proliferation phase was longer than the phase of establishment, it is assumed that the average mutation rate was lower during proliferation compared with during establishment of embryogenic cell lines, resulting in 27% altered genotypes at proliferation versus 40% observed after establishment.
Stability of the nuclear SSR loci in somatic embryos
One group 1 (F08) and one group 2 (F05) family was used to test the appearance of mutations in cotyledonary somatic embryos. In somatic embryos, accumulation of mutations may take place during culture establishment and proliferation as well as during embryo maturation. After establishment, the fate of maternal alleles only could be analysed, while during proliferation and maturation the fate of both the maternal and the paternal alleles could be followed. During proliferation and maturation, the genotypes obtained at culture establishment were taken as the reference to monitor putative mutations. When analysing the genotype of 39 cotyledonary somatic embryos derived from family F05, no mutations were observed in the SPAC11.4 locus. SPAC11.6 yielded 11.5%, while the SPAG11.8 locus yielded 29.5% mutated alleles. In the SPAC12.5 locus, the frequency of altered alleles was only 2.6%. Altogether 15 embryos with perfect genotypes were observed, yielding 38% of perfect genotypes, which coincides well with the 45% FPG observed in established embryogenic cell lines (Table 2). Of the 46 cotyledonary somatic embryos genotyped for family F08, similarly to F05, no mutations were observed in the locus SPAC11.4. The other three loci (SPAC11.6, SPAG11.8, and SPAC12.5) yielded 1.1, 8.7, and 6.5% mutated alleles, respectively. In this family, the frequency of perfect genotypes was as high as 83%, a value similar to the genetic stability found in established embryogenic cell lines (82% FPG).
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Discussion |
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Embryogenic cultures were established from immature seeds of Scots pine collected from 15 half-sib families. The entire period of in vitro embryogenic competence of Scots pine zygotic embryos is rather narrow, comprising 2–3 weeks (Keinonen-Mettälä et al., 1996). The peak of in vitro embryogenic activity occurs 1 week after fertilization (Fig. 3) which coincides with the period when cleavage polyembryony has just started (Filonova et al., 2002). Initiation frequency varied from 0.2% to 11% depending on the collection date and family. Established embryogenic cell lines were cryopreserved and new cultures were established after thawing. Cotyledonary somatic embryos were obtained after maturation treatment. However, the yield of cotyledonary somatic embryos varied significantly among families. In this work, it is shown that the genetic instability induced by in vitro stress varies among families and that the embryogenic potential correlates with the genetic instability. It is also shown that the microsatellite loci used can detect genetic stability in zygotic embryos.
All ‘above ground’ parts of the plant are ultimately derived from the shoot apical meristem. As the plant enters the flowering phase, the vegetative shoot meristems are transformed into floral meristems. Consequently, the gametes in plants arise from cell lineages that have undergone many mitotic divisions. The number of mitotic divisions is not known because the cell division activity within meristems may have evolved to minimize errors during somatic DNA replication from being passed on from one generation to the next (Cloutier et al., 2003). In the present study, up to 34% of the zygotic embryos carried mutated maternal alleles with a significant difference among families. In contrast, no evidence of mutations in the analysed microsatellite loci was observed during somatic growth and/or gamete formation in Pinus strobus (Eastern white pine) (Cloutier et al., 2003), Pinus taeda (loblolly pine) (Elsik et al., 2000), or Pinus radiata (radiata pine) (Smith and Devey, 1994). However, in another study of radiata pine, 6% of the progeny and megagametophytes carried non-parental alleles (Fisher et al., 1998). In addition to the fact that the mutability differs both among species and between different loci within the same species, the data show that there are differences among families within species.
In order to determine if the genetic stability changes during in vitro culture of Scots pine, embryogenic cell lines from 10 half-sib families were analysed. The frequency of mutated cell lines varied significantly among families (Table 2). New mutations occurred during proliferation although at a lower rate than during establishment (Table 3). Since the embryogenic cell lines were cryopreserved after establishment and proliferating cell lines were established after thawing, the possibility cannot be excluded that the cryopreservation procedure as such caused genetic aberrations. However, it is also shown that new mutations successively take place during proliferation, resulting in mosaic cultures consisting of mutated and non-mutated cells. Previously it was shown that when analysing a locus displaying hypermutability, it is possible to detect a high mutation rate also within an individual plant (Lian et al., 2004). Additionally, phenotypic and genotypic alterations after prolonged proliferation in vitro have also been shown in several species including conifers (De Verno et al., 1994, 1999; Isabel et al., 1996; Fourré et al., 1997). In the present case, the mutation rate during proliferation was higher for cell lines belonging to families that showed a high mutation rate during establishment. It is well known that the use of PGRs, and especially 2, 4-D, can cause genetic changes. In the present culture system, treatment with PGRs does not increase the genetic instability since similar results were obtained with cultures grown in the absence of PGRs (data not shown). The results indicate that in vitro stress triggers mutations in the microsatellite regions in Scots pine. The mutation rate for the microsatellites studied was higher in embryogenic cultures than in zygotic embryos; however, a considerable mutation rate was also detected in zygotic embryos (Table 2).
An interesting difference in the offspring of the mother trees was observed by comparing the stability of established embryogenic cell lines versus zygotic embryos. For characterizing the genetic stability of the families, the MMF value was used, which is the mean of the mutation frequencies observed in the four studied microsatellite loci within each family, and the FPG value showing the frequency of perfect genotypes within each family. Based on the MMF and FPG values for established embryogenic cell lines, the families were categorized into two groups, where group 1 includes genetically stable families and group 2 genetically unstable families. By comparing the MMF and FPG values for the established embryogenic cell lines in group 2 with zygotic embryos derived from the same families, it was shown that the mutation frequency was significantly higher in embryogenic cell lines. By contrast, no differences in mutation frequency between established embryogenic cell lines and zygotic embryos were detected in group 1. It is important to stress that no correlation was found between the genomic stability of the families observed in zygotic embryos versus those in established cell lines (Table 2). Consequently, it is not possible to predict the genetic stability of embryogenic cell lines established from different families by analysing the inherent stability of zygotic embryos. However, a correlation of the mutability of a family during establishment of embryogenic cell lines and its embryogenic potential could be observed (Fig. 4). Therefore, the recorded mutation frequencies at establishment of embryogenic cell lines might be somewhat overestimated since only families yielding >10 lines were selected for the analysis. Furthermore, the ability to form cotyledonary somatic embryos was higher for genetically stable families than for those that were unstable (Fig. 5), suggesting that severely mutated cell lines lose the capacity to differentiate cotyledonary somatic embryos. However, further investigations are required for assessing if instability in the studied microsatellite loci reflects alteration in functional genes. In Drosophila melanogaster, it has been shown that mutations in neutral microsatellites can be used for identification of ecologically important mutations (Harr et al., 2002).
Widely distributed plant species such as Scots pine must adapt to a broad range of environmental conditions to maintain their large geographic distribution. Mutations induced by stress can, in some cases, allow adaptation to stress (Rosenberg, 2001). Although microsatellite regions are considered to represent mostly neutral regions of the genome as far as genetic selection is concerned, a high correlation between microsatellite diversity and allozyme gene diversity was reported in Elymus alaskus (Alaskan wheatgrass) (Sun and Salomon, 2003). The authors suggested that the parallelism between microsatellite and allozyme patterns indicates that similar and primarily deterministic (selection) evolutionary forces might be involved in shaping the genomic structure of both microsatellite and allozyme loci, i.e. across non-coding and coding genomic regions. If this is the case for Scots pine, the genetic instability would reflect the plasticity of the family, which also includes adaptation to in vitro conditions.
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Acknowledgements |
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We are grateful to Agnes Burg for carrying out the microsatellite analysis, and Ingrid Schenning for isolating DNA. We thank Torborg Jansson and the Forestry Research Institute of Sweden in Ekebo for their expert care of the cultures. The study has been carried out with financial support from the commission of the European Communities, Community research programme ‘Quality of life and Management of Living Resources’ (Project SEP QLRT1-CT99-0679); however, this publication does not necessarily reflect the European Commission's views and in no way anticipates its future policy in this area.
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Abbreviations |
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ABA, abscisic acid; BA, benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid; DAF, days after flowering; FPG, frequency of perfect genotypes; MMF, mean mutation frequency; PEG, polyethylene glycol; PGR, plant growth regulator; SSR, simple sequence repeat.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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