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Centre de Recherche en Biologie Forestière, Pavillon Charles-Eugène Marchand, Local 2210, Université Laval,Québec, Canada G1K 7P4
Received for publication September 22, 1998. Accepted for publication March 25, 1999.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Key Words: aneuploidy • chromosome counts • conifers • morphological variations • Picea • Pinaceae • plagiotropism
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMaterials and MethodsResultsDiscussionLITERATURE CITED |
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). In the last decade, somatic embryogenesis has been the most promising multiplication technique available for conifers. Indeed, this technique allows clonal plant production for field tests, and clone preservation by cryopreservation, as well as regrowth of the selected clones for reforestation. We are at the point where field tests are being conducted with techniques scaled-up to meet industrial production levels. The production of true-to-type plants is a prerequisite for mass clonal propagation, but we do not know to what extent the embryogenic process itself might result in somaclonal variation in the regenerated plants. Therefore, there is a need to characterize deviant phenotypes and to determine the level of clonal fidelity (true-to-type plants) among somatic embryo-derived plants of conifers.
Somaclonal variation is the variation observed among plants regenerated from in vitro culture (Larkin and Scowcroft, 1981
). These variations are heritable, i.e., transmitted through meiosis and are usually irreversible. Somaclonal variation can be assessed by analysis of phenotype, chromosome number and structure, proteins, or direct DNA evaluation of plants (De Klerk, 1990
). Somaclonal variation in embryogenic and nonembryogenic cultures has been studied for many species including crop species (reviewed by Bajaj, 1990
) as well as angiosperm and gymnosperm species (reviewed by Deverno, 1995
). More recently for the latter, nuclear DNA variation was reported in embryogenic tissues of Abies alba (Roth, Ebert, and Schmidt, 1997
). Somaclonal variation was also reported in somatic embryo-derived plants of conifers. Four P. glauca plants with variegata phenotypes were found among 2270 regenerated plants (Isabel et al., 1996
). In this study, one out of 250 RAPD markers was correlated with the white needles of the variegated plants. In P. abies, an abnormal chromosome number was found in one of two plants presenting a dwarf morphology (Fourré et al., 1997
). For conifers, these studies suggested that phenotypic variations in regenerated plants from somatic origin can be caused by genetic instability.
Besides somaclonal variation, another source of variations observed in plants regenerated from tissue culture concerns variations from epigenetic origin. Epigenetics refer to modifications in gene expression brought about by heritable, but potentially reversible, changes in chromatin structure and/or DNA methylation (reviewed by Henikoff and Matzke, 1997
). Several epigenetic systems that have been studied were surveyed by Russo, Martienssen, and Riggs (1996)
.
Morphological variations resulting from somaclonal variation have been extensively studied for several crop and fruit tree species. For instance, plants showed considerable variation for morphological traits such as flower color and shape, leaf morphology and color, plant height, resistance to disease, and maturity date (reviewed by Bajaj, 1990
; Hammerschlag, 1992
). For conifers, phenotypic evaluation might also represent a valuable means for assessing somaclonal variation.
In our laboratory, evaluation of 
7000 black spruce (P. mariana) and 4000 white spruce (P. glauca) plants regenerated from somatic embryogenesis has been carried out at the phenotypic level within a 5-yr period. For the first time, this work describes in detail several variant phenotypes found in acclimatized spruce plants derived from somatic embryogenesis. Chromosome counts were performed on these phenotypes to see whether, at least at the chromosomic level, genetic instability could be confirmed. Moreover, even if their frequency was low, we were able to correlate statistically the appearance of variant phenotypes with two factors, clone and time in maintenance.
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Materials and Methods |
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TOPABSTRACTINTRODUCTIONMaterials and MethodsResultsDiscussionLITERATURE CITED |
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), or from bulk seeds. Seeds had been stored for 2–3 yr before use. All embryogenic tissues were induced from mature zygotic embryos according to Tremblay (1990)
. A total of 11 042 plants was produced through somatic embryogenesis from 65 black spruce clones and 22 white spruce clones (Table 1).
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). After different times in maintenance, calculated as date of maturation minus date of induction, mature somatic embryos were obtained as described previously (Isabel et al., 1993
) except that abscisic acid (ABA) concentrations varied from 15 to 60 µmol/L. After 4–6 wk in maturation, somatic embryos were germinated and acclimatized according to previously published procedures (Khlifi and Tremblay, 1995
). Some plants were regenerated from experiments conducted to test different media components (growth regulators, basal medium composition, vitamin and amino acid, and carbohydrate) during either maintenance or maturation. Maturation could be achieved using embryogenic tissues maintained on solid or in liquid medium.
Plants in the greenhouse were treated with a standard high-fertilization regime (Plantek, Ministry of Natural Resources, Quebec) under a 18/6 h day/night photoperiod. When necessary, additional illumination was provided by High Pressure Sodium light beams. The light intensity was 
800 µmol·m-2·s-1. Dormancy was induced by short days (10–12 h) and cold treatments (10°–12°C) before plants were transplanted to the field.
Since 1992, each plant produced has been individually tagged according to clone and lot number. The lot number refers to any treatment (ABA concentration, incubation conditions, etc.) used to produce a particular set of plants, from the induction to the transfer in soil. For example, plants from the same clone produced with the same protocol but with different time intervals in maintenance had different lot numbers. Similarly, plants produced in a particular experiment but with different treatments were tagged by treatment. The lot numbers were linked to a computerized data bank that allows for tracing the complete tissue culture history of every plant under observation in relation to the phenotype observed.
Data collection
During the growing period in the greenhouse and later on in the field, each lot of plants was regularly checked for the presence of variant phenotype plants. Deviant phenotypes were closely monitored to confirm phenotype stability.
Light microscopy
Some of the regenerated plants had fasciated needles. Light microscopy was performed on these needles and on normal needles from the same plant. Needles (10 mm long) were cut into 5-mm sections and immediately placed in a solution of 3% glutaraldehyde in 0.1 mol/L cacodylate (Sigma, St. Louis, Missouri) buffer, pH 7.4, for 3 h at room temperature. They were then washed in 0.1 mol/L cacodylate buffer, pH 7.4, and postfixed in 1% osmium tetroxyde in the same buffer for 2 h at room temperature. After washing in cacodylate buffer, the material was dehydrated in an ethanol series, passed through propylene oxide, and embedded in Epon 812 resin (J. B. EM Services, Montreal). Sections for light microscopy (2 µm thick) were stained with toluidine blue at 0.1%.
Chromosome squashes
Metaphase chromosome spreads were obtained from both root tips and flushing buds of acclimatized plants. Excised roots were pretreated for 6 h with 0.3% colchicine (Fisher Scientific, Montreal, Quebec), fixed for 24 h at room temperature in a 3 : 1ethanol : glacial acetic acid solution and hydrolyzed for 9 min at room temperature in a 5 mol/L HCl solution. The samples were stained in acetocarmine for at least 6 h, and after heating at 60°C for few seconds, they were squashed under a cover slip in a drop of acetocarmine, i.e., 0.5% (w/v) carmin in 45% (v/v) acetic acid. Metaphase chromosomes were counted in 2–4 cells from each sample. The same protocol was used for flushing buds except that after staining, the samples were dissected under a microscope to take only the meristematic area of the apex.
Statistical analysis
All the variant phenotypes were used for the statistical analysis except the type I plants (plagiotropism) since they were associated with a physiological rather than a genetical disorder.
Due to the high number of lots of plants without variant phenotypes it was not possible to perform a logistic regression analysis on all the plants from the computerized data bank. Therefore, it was necessary to eliminate the families (and all the clones within the family) for which no variant was observed. As a result the effect of the family on variant appearance had to be analyzed separately. This was done by an analysis of frequencies (SAS Institute Inc., Cary, North Carolina) with the P level calculated with the Fisher's exact test. This analysis included all the plants produced from the 19 families of black spruce. White spruce was not analyzed since only one family out of eight produced variant phenotypes. For the factors "clone" and "time in maintenance," a logistic regression analysis (Hosmer and Lemeshow, 1989
) was performed on each species with the CATMOD procedure from SAS using only families in which variant phenotypes were observed. In this way, the analysis was restricted. The logistic regression model was: g(x) = µ + b1x1 + ß2x2+ ß3x3, where g(x) = estimated logit, µ = intercept, ß = estimated coefficients, x1 = family, x2 = clones inside family, and x3= time in maintenance. Data were transformed by the addition of 0.05 to avoid an undefined point estimate. This was the least transformation possible that did not change our data in the analysis. The likelihood ratio chi-square test determined the P level and served to assess the goodness of fit of the final model. Other factors (medium, growth regulators, etc.) could not be analyzed because they were associated with a statistically insignificant number of variant plants.
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Results |
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TOPABSTRACTINTRODUCTIONMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Type D corresponds to plants with a reduced height compared to the control (7 vs. 70 cm after 4 yr) and additional abnormalities, including a large-diameter stem and thick and short needles. The number of vegetative buds is higher than on control plants (Fig. 4). Plants were difficult to grow. After 4 yr, only ten were alive. This variation was found in seven black spruce clones for a frequency of 0.5% (33/7047) (Table 3). Chromosome counts were performed on two variant plants and one control plant. One variant was aneuploid with cells at 2n = 38, while the second was a mosaic of aneuploid cells with 2n = 30, 2n = 39, 2n = 40, and 2n = 55 in the same tissue (Fig. 12). The control plant was 2n = 24.
Type E corresponds to plants similar in height and shape to controls, but with fasciated needles (Fig. 5a). Needle number per plant never surpassed the number of normal needles, and the fasciation did not affect the growth of the plants even after 5 yr in soil. A frequency of 0.2% (7/3995) was observed with one white spruce clone (Table 2) and 0.3% (18/7047) with three black spruce clones (Table 3). Fasciated and normal needles from the same plant were characterized by light microscopy. Transverse sections of fasciated needles showed tissue continuity between the needle units, each unit being otherwise normally constituted (Fig. 5b). A chromosome count was not done on this type.
Type F corresponds to plants with an abnormal architecture, i.e., branches at more acute angles to the main shoot compared to the control (Fig. 6). Furthermore, the position of the branches is not according to the normal phyllotaxy of the species. Only one white spruce plant out of 3995 plants had this morphology (Table 2), and none was found in black spruce (Table 3). Chromosomes were not counted.
Type G corresponds to plants with different variegata patterns previously characterized by Isabel et al. (1996)
. These plants are partly achlorophyllous, displaying different distributions and proportions of white and green tissues (Fig. 7). The achlorophyllous areas are sensitive to sunlight, resulting in drying and shedding of the white needles under high light intensity (800 µmol·m-2·s-1). This problem was overcome by lowering light intensity in the greenhouse while maintaining the same vapor pressure deficit. A frequency of 0.3% (10/3995) was found from two white spruce clones (Table 2) and 0.1% (9/7047) from seven black spruce clones (Table 3). Chromosome counts on both variegata and normal plants were 2n = 24.
Type H refers to variation in plant height. Plants with this phenotype have a normal morphology but remain smaller, from one-third to one-quarter of the height of the control plants after 4 yr (Fig. 8). A frequency of 0.4% (15/3995) was found from one white spruce clone (Table 2) and 0.4% (26/7047) from nine black spruce clones (Table 3). The chromosome numbers, determined on three variant plants and two controls, were normal (2n = 24).
The type I phenotype describes plants with a plagiotropic growth compared to orthotropic control plants (Figs. 9, 10). The plagiotropic growth was reversible and, within 5 yr in the field, all the plagiotropic plants reverted to an orthotrophic growth comparable to normal plants. Plagiotropic plants were regenerated from tissues that had been maintained for long periods of time (Fig. 13). They were found at a frequency of 8.5% (341/3995) for white spruce (Table 2) and 0.4% (30/7047) for black spruce but in a limited number of clones (Table 3). Within the clones with plagiotropic plants, the frequency varied from 1.1 up to 42.6%.
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For both species, statistical analyses showed that the main factors involved in somaclonal variation were clone and time in maintenance. The family did not significantly affect the appearance of variants (P = 0.921). The clone was a more important source of genetic instability (
2 = 230.42, df = 44, P = 0.00001 and 2 = 30.44, df = 4, P = 0.00001 for black and white spruce, respectively) than the time in maintenance (2 = 15.99, P = 0.0001 and 2 = 17.99, P = 0.00001 for black and white spruce, respectively). Variant plants from types A–H were found in two out of 22 clones for white spruce (Table 2) and in 22 out of 65 clones for black spruce (Table 3). For the most unstable clones, the frequency of variant plants reached 57.6% for black spruce and 3.9% for white spruce. For both species, the number of variant phenotypes increased significantly with increasing time in maintenance, but was independent from the number of plants in a clone. The assessment of the goodness of fit of the predictive model was good for black spruce (P = 0.9580) but not for white spruce (P = 0.00001), indicating that for white spruce, other factors not included in the statistical analysis might have influenced the appearance of variant plants.
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Discussion |
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TOPABSTRACTINTRODUCTIONMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Our work emphasizes the fact that phenotypic evaluation should not be neglected as a tool to assess the genetic integrity of the somatic embryogenesis process. In the absence of reliable genetic markers of somaclonal variation and considering the long life cycle of coniferous species to confirm the inheritability of abnormal phenotypic traits, phenotype still represents the easiest and fastest way to identify putative mutants.
The variations found in this study were similar to those reported for crop and tree species: dwarfism, fasciation, variegata patterns, height alteration, modified branch angle, and bushy shape (reviewed by Bajaj, 1990
; Hammerschlag, 1992
). In coniferous species regenerated through somatic embryogenesis, cases of dwarfism and variagation have already been documented. In P. abies, two dwarf plants were characterized: one was a chimera with trisomic buds and diploid roots, while the second had a normal chromosome complement (Fourré et al., 1997
). In our study, two phenotypes could be related to reduced height: one dwarf plant (type A) with a chromosomic anomaly and several plants with reduced height (type H) but normal chromosome numbers. In a previous work, we characterized four P. glauca plants with variegata phenotypes in terms of ultrastructure, chromosome number, and genetics using RAPD markers (Isabel et al., 1996
). Although these plants had normal chromosome numbers, the genetic origin of the phenotype was proven by the presence of an additional RAPD fragment in the variegata plants. In this study, variegata plants (type G) were observed in both P. glauca and P. mariana with frequencies of 0.3 and 0.1%, respectively.
In addition to the previous cases, other phenotypic variations were also observed. Two were lethal, i.e., type C (hooked stem) and to a lesser extent type D (aneuploid). Type C has not been described in other species and could not be related in this study to a chromosome anomaly. A supplementary B chromosome was found in these plants, as well as in plants with a normal phenotype within the same clone. Type-D plants represent the first plants constituted of aneuploid cells (2n = 38) or a mosaic of aneuploid cells (2n = 30–55) to be regenerated from black spruce through somatic embryogenesis. The only other cases of aneuploid conifer plants regenerated through somatic embryogenesis were three trisomic (2n = 25) Norway spruce plants, two of which exhibited a normal phenotype (Fourré et al., 1997
). Our results together with Fourré's results show that conifer plants can survive and grow in soil in spite of chromosome anomalies. Our aneuploid plants showed heavier morphological disorders than Fourré's plants probably because of their higher number of chromosomes. For some clones, the high frequency of the type-D phenotype suggests that variation in chromosome number had occurred in the tissue before maturation. This is supported by the fact that, except for the clone 50 with only two aneuploid/859 plants, this mutation was particularly frequent (3.4–50%) in clones with low numbers of plants (e.g., clones 10, 51, 56, 92, and 100). Plant production was particularly difficult with these clones, which might have been due to a high proportion of aneuploid cells in the tissue. Although no chromosome counts were performed in our embryogenic tissues prior their maturation, different ploidy levels were found in embryogenic tissues of different coniferous species (Lelu, 1987
; von Aderkas and Anderson, 1993
; Fourré et al., 1997
; Roth, Ebert, and Schmidt, 1997
).
Plagiotropic growth was observed at overall respective frequencies of 0.4 and 8.5% for black spruce and white spruce, but values of 42.6% were observed for the most affected clone. This variation was attributed to an epigenetic rather than to a genetic modification of the trees since it corresponds to a developmental process (aging) that was reversible in all the plants observed. Although in coniferous species, plagiotropic growth and other unusual stem morphologies have been observed in micropropagated trees such as Douglas-fir and loblolly pine (Leach, 1979
; Ritchie and Long, 1986
; Timmis, Ritchie, and Pullman, 1992
), this is the first observation of plagiotropism in plants derived from somatic embryogenesis. One plagiotropic plant was previously observed from a haploid embryogenic culture of Larix decidua (von Aderkas and Bonga, 1993
). The number of plants exhibiting plagiotropic growth seems to increase with increasing time in maintenance, indicating that some loss of juvenility might occur under in vitro conditions. Although affecting only a few clones, this phenomenom will have to be further studied since it negatively affects early growth of trees and therefore may influence results of early selection trials of clonal material.
Spruce variants, types A–H (excluding plagiotropic plants), were obtained at relatively low frequencies (1.0% for white spruce and 1.6% for black spruce) but above the rate of spontaneous mutations (Karp, 1991
). The variation frequencies obtained here are within the range observed for other crop species (Skirvin, McPheeters, and Norton, 1994
). However, they might be underestimated since only plants with abnormal phenotypes were considered. For example, Fourré et al. (1997)
found two trisomic P. abies plants from somatic origin with a normal phenotype. Furthermore, our observations were made with juvenile trees, while somaclonal variations should also be expected to affect mature characteristics such as those related to flowering.
In this study, two factors were identified as directly involved in somaclonal variation: clone and time in maintenance, with the first having a stronger effect than the second. Independently of any other factor, some clones remained invariant, while others could have up to 50% abnormal plants. This confirms previous studies that identified the genotype as an important source of somaclonal variation (reviewed by Karp, 1995
). The genotypic effect can be seen with the most unstable black spruce clone, clone 62, which had 19 variants out of only 33 regenerated plants, while few variations were found with clone 50, which also had the highest number of regenerated plants. Time in maintenance is also known to influence the rate of somaclonal variation (reviewed by Sibi, 1990
; Skirvin, McPheeters, and Norton, 1994
). In our study, the effect of the time in maintenance is probably underestimated since increasing time in maintenance is accompanied by decreased regenerative capacities of the cell lines, and this study considers only regenerated plants growing in soil. It is known that factors such as explant source, duration in culture, and growth regulators influence the frequency of somaclonal variation (reviewed by Karp, 1995
). Because of the low frequency of variants, it was impossible to determine the effect of particular in vitro treatments on somaclonal variation in this study. We therefore do not exclude the fact that other factors related to the in vitro protocols might influence the frequency of somaclonal variation, but we could not quantify them.
For the first time, a phenotypic study was conducted on conifer plants regenerated through somatic embryogenesis. The frequencies of variants are within the range observed in other species and appear acceptable for commercial production. However, to ensure true-to-type plant production, it appears necessary to establish embryogenic cultures from a large number of genotypes and to keep the time in maintenance as short as possible.
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FOOTNOTES |
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2 Author for correspondence (e-mail: francine.tremblay{at}sbf.ulaval.ca
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMaterials and MethodsResultsDiscussionLITERATURE CITED |
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De Klerk, G.-J. 1990 How to measure somaclonal variation. Acta Botanica Neerlandica 39: 129–144.[ISI]
Deverno, L. L. 1995 An evaluation of somaclonal variation during somatic embryogenesis. In S. Jain, P. Gupta, and R. Newton [eds.], Somatic embryogenesis in woody plants, vol. 1, 361–377. Kluwer Academic Publishers, Dordrecht.
Fourré, J.-L., P. Berger, L. Niquet, and P. André. 1997 Somatic embryogenesis and somaclonal variation in Norway spruce: morphogenetic, cytogenetic and molecular approaches. Theoretical and Applied Genetics 94: 159–169.[CrossRef][ISI]
Hakman, I., L. C. Fowke, S. von Arnold, and T. Eriksson. 1985 The development of somatic embryos in tissue cultures initiated from immature embryos of Picea abies (Norway spruce). Plant Science 38: 53–60.
Hammerschlag, F. A. 1992 Somaclonal variation. In F. A. Hammerschlag and R. E. Litz [eds.], Biotechnology of perennial fruit crops, 35–55. CAB International, Cambridge.
Henikoff, S., and M. A. Matzke. 1997 Exploring and explaining epigenetic effects. Trends in Genetics 13: 293–295.[CrossRef][ISI][Medline]
Hosmer, D. W., and S. Lemeshow. 1989 Applied logistic regression. Wiley-Interscience Publication, New York, NY.
Isabel, N., R. Boivin, C. Levasseur, P. M. Charest, J. Bousquet, and F. M. Tremblay. 1996 Occurrence of somaclonal variation among somatic embryo-derived white spruces (Picea glauca, Pinaceae). American Journal of Botany 83: 1121–1130.[CrossRef][ISI]
———, L. Tremblay, M. Michaud, F. M. Tremblay, and J. Bousquet. 1993 RAPDs as an aid to certify the genetic integrity of somatic embryogenesis-derived populations of Picea mariana (Mill) B.S.P. Theoretical and Applied Genetics 86: 81–87.[ISI]
Jain, S. M., P. K. Gupta, and R. J. Newton [eds.]. 1995 Somatic embryogenesis in woody plants, Gymnosperms, vol. 3. Kluwer Academic Publishers, Dordrecht.
Karp, A. 1991 On the current understanding of somaclonal variation. Oxford Surveys of Plant Molecular and Cell Biology 7: 1–58.
——— 1995 Somaclonal variation as a tool for crop improvement. Euphytica 85: 295–302.
Khlifi, S., and F. M. Tremblay. 1995 Maturation of black spruce somatic embryos: Part 1. Effect of L-glutamine on the number and germinability of somatic embryos. Plant Cell, Tissue and Organ Culture 41: 23–32.[CrossRef]
Larkin, P. J., and W. R. Scowcroft. 1981 Somaclonal variation—a novel source of variability from cell cultures for plant improvement. Theoretical and Applied Genetics 60: 197–214.[CrossRef][ISI]
Leach, G. N. 1979 Growth in soil of plantlets produced by tissue culture. Tappi Journal 62: 59–61.
Lelu, M. A. 1987 Variations morphologiques et génétiques chez Picea abies obtenues après embryogenèse somatique. Étude préliminaire. In Annales de Recherches Sylvicoles, 35–47. Association Forêt-Cellulose, Paris.
Ritchie, G. A., and A. J. Long. 1986 Field performance of micropropagated Douglas-fir. New Zealand Journal of Forestry Science 16: 343–356.
Roth, R., I. Ebert, and J. Schmidt. 1997 Trisomy associated with loss of maturation capacity in a long-term embryogenic culture of Abies alba. Theoretical and Applied Genetics 95: 353–358.[CrossRef][ISI]
Russo, V. E. A., R. A. Martienssen, and A. D. Riggs. 1996 Epigenetic mechanisms of gene regulation. Cold Spring Harbor Laboratory Press, New York, NY.
Sibi, M. 1990 Genetic bases of variation from in vitro tissue culture. In Y. P. S. Bajaj [ed.], Somaclonal variation in crop improvement. I, vol. 2, 112–133. Springer-Verlag, Berlin.
Skirvin, R. M., K. D. McPheeters, and M. Norton. 1994 Sources and frequency of somaclonal variation. HortScience 29: 1232–1237.
Timmis, R., G. A. Ritchie, and G. S. Pullman. 1992 Age- and position-of-origin and rootstock effects in Douglas-fir plantlet growth and plagiotropism. Plant Cell, Tissue and Organ Culture 29: 179–186.
Tremblay, F. M. 1990 Somatic embryogenesis and plantlet regeneration from embryos isolated from stored seeds of Picea glauca. Canadian Journal of Botany 68: 236–242.
von Aderkas, P., and P. Anderson. 1993 Aneuploidy and polyploidization in haploid tissue cultures of Larix decidua. Physiologia Plantarum 88: 73–77.[CrossRef]
———, and J. M. Bonga. 1993 Plants from haploid tissue culture of Larix decidua. Theoretical and Applied Genetics 87: 225–228.[ISI]
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