Developmental and genetic variation in nuclear microsatellite stability during somatic embryogenesis in pine

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.     

RAPDs as an aid to evaluate the genetic integrity of somatic embryogenesis-derived populations of Picea mariana (Mill.) B.S.P.

Summary The usefulness of random amplified polymorphic DNA (RAPD) in assessing the genetic stability of somatic embryogenesis-derived populations of black spruce [Picea mariana (Mill.) B.S.P.] was evaluated. Three arbitrary 11-mer primers were successfully used to amplify DNA from both in-vivo and in-vitro material. Twenty-five embryogenic cell lines, additional zygotic embryos and megagametophytes from three controlled crosses involving four selected genotypes of black spruce were used for the segregation analysis of RAPD variants. Ten markers were genetically characterized and used to evaluate the genetic stability of somatic embryos derived from three embryogenic cell lines (one cell line per cross, 30 somatic embryos per cell line). No variation was detected within clones. The utilization of RAPD markers both for the assessment of genetic stability of clonal materials and to certify genetic stability throughout the process of somatic embryogenesis is discussed.     

Long-term cryopreservation of Greek fir embryogenic cell lines: recovery, maturation and genetic fidelity

In coniferous species, including Greek fir (Abies cephalonica Loud), the involvement of somatic embryo plants in breeding and reforestation programs is dependent on the success of long-term cryostorage of embryogenic cultures during clonal field testing. In the present study on Greek fir, we assayed the recovery, morphological characteristics and genetic fidelity of embryogenic cell lines 6 and 8 during proliferation and maturation after long-term cryostorage. Our results indicate successful recovery of both cell lines after 6 years in cryostorage. In the maturation phase, both cell lines were capable of producing somatic embryos although some differences were detected among experiments. However, these changes were more dependent on the differences in the components of the maturation media or in the experimental set-up than on the long-term cryostorage. During both proliferation and maturation phases, the morphological fidelity of the embryogenic cultures as well as of the somatic embryos were alike before and after cryopreservation. The genetic fidelity of the cryopreserved cell line 6 that was assayed by random amplified polymorphic DNA (i.e. RAPD) markers demonstrated some changes in the RAPD profiles. The results indicate possible genetic aberrations caused by long-term cryopreservation or somaclonal variation during the proliferation stage. However, in spite of these changes the embryogenic cultures did not lose their proliferation or maturation abilities.     

Genetic variation in somatic embryogenic response in open-pollinated families of black spruce

Summary Zygotic embryos from open-pollinated seeds of 20 black spruce (Picea mariana) families were used to investigate the proportion of genotypes that would give rise to embryogenic tissue (ET) and mature somatic embryos. Eighty-five percent of the maternal genotypes gave rise to embryogenic tissue. Within-family rates of ET induction ranged from 0 to 17%, with an average of 8%. The largest proportion of variation was among families, indicating the additive nature of the genetic variation. On a medium with 6% sucrose and 3.7 M ABA, 90% of the embryogenic lines gave rise to abundant (>100/100 mg of ET), well-formed, mature somatic embryos. A medium with 2% sucrose, without 2,4-D, was used to germinate the mature somatic embryos. These were grown in the greenhouse and have now been established in field trials.     

Evidence for in vitro induced mutation which improves somatic embryogenesis in Asparagus officinalis L.

Summary Somatic embryogenesis from different genotypes of Asparagus officinalis L. could be obtained by in vitro culture of shoot apices. Apices were first cultured on an auxin-rich inducing medium and then transferred onto a hormone-free development medium. All genotypes tested in this way produced a few somatic embryos. In some experiments, during the development phase, a new kind of friable highly embryogenic tissue appeared in a random manner. These tissues could be continuously subcultured on a hormone-free medium and were named embryogenic lines. Five of these embryogenic lines regenerated plants from somatic embryos. These regenerated plants exhibited an increased embryogenic response compared to the parent plants; e.g. apex culture produced somatic embryos without any auxin treatments. For one of the embryogenic lines, a genetic analysis showed that the improved embryogenic response of regenerated plants was controlled by a mendelian dominant monogenic mutation.Abbreviations LSEA low somatic embryogenesis ability - HSEA high somatic embryogenesis ability - NAA 1-naphthaleneacetic acid     

AFLP analysis of variation in pecan somatic embryos

Before somatic embryogenesis can be applied, the genetic fidelity of cultures needs to be determined. Problematic of tissue-cultured woody species is the extensive evaluation time needed for assessments. The development of methods whereby plants could be rapidly screened for potential tissue culture-derived genetic changes would be very valuable. We evaluated the applicability of AFLP (amplified fragment length polymorphism) analysis for the assessment of genetic variability in somatic embryos of pecan [Carya illinoinensis (Wangenh.) C. Koch] and made comparisons between and within embryogenic culture lines. AFLP readily detected differences between culture lines, with 368 polymorphic loci identified. Individual culture lines generally produced somatic embryos with similar overall banding patterns. Embryos derived from the same culture line generally grouped together in a phenogram generated by UPGMA (unweighted pair-group method, arithmetic average) analysis. However, a few somatic embryos exhibited higher levels of polymorphism and failed to group with others regenerated from the same line. The relation between the detected within-line differences and their contribution to phenotypic variation is yet to be determined. Received: 9 September 1998 / Revision received: 12 November 1998 / Accepted: 8 December 1998     

Plant regeneration through somatic embryogenesis from tissues of mature oak trees: true-to-type conformity of plantlets by RAPD analysis

Somatic embryogenesis was induced in expanding leaf explants excised from epicormic shoots forced from branch segments taken at four different times of year from a mature oak (Quercus robur L.). Branch segments 2–4 cm in diameter produced most shoots when collected in March. Somatic embryos were induced on explants derived from branches of all collection dates, although collection in November seemed to afford the best results. Germination and conversion ability of embryos of embryogenic lines derived from six oak trees depended heavily on genotype, conversion rates ranging from 0 to 70%. RAPD analyses found no evidence of genetic variation either within or between the embryogenic lines established from three of these trees, or between these lines and the trees of origin, or between somatic embryo derived plantlets and the trees of origin. The embryogenic system used in this study appears to be suitable for true-to-type clonal propagation of mature oak genotypes.     

Instability of (GATA)<Subscript> n</Subscript> microsatellite loci in the parthenogenetic Caucasian rock lizard<Emphasis Type="Italic"> Darevskia unisexualis</Emphasis> (Lacertidae)

Mini- and microsatellites, comprising tandemly repeated short nucleotide sequences, are abundant dispersed repetitive elements that are ubiquitous in eukaryotic genomes. In humans and other bisexual species hypervariable mini- and microsatellite loci provide highly informative systems for monitoring of germline and somatic instability. However, little is known about the mechanisms by which these loci mutate in species that lack effective genetic recombination. Here, multilocus DNA fingerprinting was used to study M13 minisatellite and (GATA) _n microsatellite instability in the parthenogenetic Caucasian rock lizard Darevskia unisexualis (Lacertidae). DNA fingerprinting of 25 parthenogenetic families, from six isolated populations in Armenia (comprising a total of 84 siblings), using the oligonucleotide (GATA)₄ as a hybridization probe, revealed mutant fingerprinting phenotypes in 13 siblings that differed from their mothers in several restriction DNA fragments. In three families (8 siblings), the mutations were present in the germline. Moreover, the mutant fingerprint phenotypes detected in siblings were also present in population DNA samples. No intrafamily variations in DNA fingerprint patterns were observed with the M13 minisatellite probe. Estimates of the mutation rate for (GATA) _n microsatellite loci in D. unisexualis showed that it was as high as that seen in some bisexual species, reaching 15% per sibling or 0.95% per microsatellite band. Furthermore, in one case, a somatic (GATA) _n microsatellite mutation was observed in an adult lizard. These findings directly demonstrate that mutations in (GATA) _n microsatellite loci comprise an important source of genetic variation in parthenogenetic populations of D. unisexualis.Communicated by G. P. Georgiev     

A key to totipotency: Wuschel-like homeobox 2a unlocks embryogenic culture response in maize (Zea mays L.)

The ability of plant somatic cells to dedifferentiate, form somatic embryos and regenerate whole plants in vitro has been harnessed for both clonal propagation and as a key component of plant genetic engineering systems. Embryogenic culture response is significantly limited, however, by plant genotype in most species. This impedes advancements in both plant transformation-based functional genomics research and crop improvement efforts. We utilized natural variation among maize inbred lines to genetically map somatic embryo generation potential in tissue culture and identify candidate genes underlying totipotency. Using a series of maize lines derived from crosses involving the culturable parent A188 and the non-responsive parent B73, we identified a region on chromosome 3 associated with embryogenic culture response and focused on three candidate genes within the region based on genetic position and expression pattern. Two candidate genes showed no effect when ectopically expressed in B73, but the gene Wox2a was found to induce somatic embryogenesis and embryogenic callus proliferation. Transgenic B73 cells with strong constitutive expression of the B73 and A188 coding sequences of Wox2a were found to produce somatic embryos at similar frequencies, demonstrating that sufficient expression of either allele could rescue the embryogenic culture phenotype. Transgenic B73 plants were regenerated from the somatic embryos without chemical selection and no pleiotropic effects were observed in the Wox2a overexpression lines in the regenerated T0 plants or in the two independent events which produced T1 progeny. In addition to linking natural variation in tissue culture response to Wox2a, our data support the utility of Wox2a in enabling transformation of recalcitrant genotypes.     

大豆体细胞胚系培养及其微卫星DNA稳定性研究(简报)

植物离体培养是植物基因操作中的重要一环,也是植物个体发育研究中基因表达研究的有益参考体系。在继代培养过程中发生的遗传变异有时会使再生植株丧失优良的性状而需加以控制和避免。为此,首先需要了解培养过程中的遗传变异情况。     

Biochemical and molecular analysis of plants derived from embryogenic tissue cultures of napier grass (Pennisetum purpureum K. Schum)

Summary We have investigated the extent of biochemical and molecular variation in 63 plants of napier grass (Pennisetum purpureum K. Schum.) regenerated from 3- to 24-week-old embryogenic callus cultures. The calli were derived from cultured basal segments of young leaves and immature inflorescences obtained from a single fieldgrown donor plant. The entire population was analyzed for the activity of 14 isozyme systems, but no qualitative variation was found at any of the loci examined. Similarly, no restriction fragment length polymorphisms (RFLPs) were detected in the mitochondrial, plastid and nuclear genomes in a representative sample of regenerated plants. Our results confirm earlier reports of the genetic uniformity of plants derived from somatic embryos and highlight their value both for clonal propagation and for genetic transformation.     

Improving genetic transformation of European chestnut and cryopreservation of transgenic lines

The aim of the present work was to study the effect of the developmental stage of the somatic embryos and of the genotype on the genetic transformation of embryogenic lines of European chestnut (Castanea sativa Mill.) and the cryopreservation of the embryogenic lines that are generated. As an initial source of explants in the transformation experiments, it was found that the use of somatic embryos isolated in the globular stage or clumps of 2–3 embryos in globular/heart-shaped stages was more effective (30%) than when embryos at the cotyledonary stage were used (6.7%). All of the seven genotypes tested were transformed, and transformation efficiency was clearly genotype dependent. Three transgenic lines were successfully cryopreserved using the vitrification procedure, and the stable integration of the uidA gene into the transgenic chestnut plants that were regenerated subsequent to cryopreservation was demonstrated.     

Analysis of genetic stability at SSR loci during somatic embryogenesis in maritime pine (Pinus pinaster)

Somatic embryogenesis (SE) is a propagation tool of particular interest for accelerating the deployment of new high-performance planting stock in multivarietal forestry. However, genetic conformity in in vitro propagated plants should be assessed as early as possible, especially in long-living trees such as conifers. The main objective of this work was to study such conformity based on genetic stability at simple sequence repeat (SSR) loci during somatic embryogenesis in maritime pine (Pinus pinaster Ait.). Embryogenic cell lines (ECLs) subjected to tissue proliferation during 6, 14 or 22 months, as well as emblings regenerated from several ECLs, were analyzed. Genetic variation at seven SSR loci was detected in ECLs under proliferation conditions for all time points, and in 5 out of 52 emblings recovered from somatic embryos. Three of these five emblings showed an abnormal phenotype consisting mainly of plagiotropism and loss of apical dominance. Despite the variation found in somatic embryogenesis-derived plant material, no correlation was established between genetic stability at the analyzed loci and abnormal embling phenotype, present in 64% of the emblings. The use of microsatellites in this work was efficient for monitoring mutation events during the somatic embryogenesis in P. pinaster. These molecular markers should be useful in the implementation of new breeding and deployment strategies for improved trees using SE.     

Microsatellite Variation in Two Populations of Free-Ranging Yellow Baboons (Papio hamadryas cynocephalus)

We investigated genetic variation at six microsatellite (simple sequence repeat) loci in yellow baboons (Papio hamadryas cynocephalus) at two localities: the Tana River Primate Reserve in eastern Kenya and Mikumi National Park, central Tanzania. The six loci (D1S158, D2S144, D4S243, D5S1466, D16S508, and D17S804) were all originally cloned from and characterized in the human genome. These microsatellites are polymorphic in both baboon populations, with the average heterozygosity across loci equal to 0.731 in the Tana River sample and 0.787 in the Mikumi sample. The genetic differentiation between the two populations is substantial. Kolmogornov–Smirnov tests indicate that five of the six loci are significantly different in allele frequencies in the two populations. The mean F _ST across loci is 0.069, and Shriver's measure of genetic distance, which was developed for microsatellite loci (Shriver et al., 1995), is 0.255. This genetic distance is larger than corresponding distances among human populations residing in different continents. We conclude that (a) the arrays of alleles present at these six microsatellite loci in two geographically separated populations of yellow baboons are quite similar, but (b) the two populations exhibit significant differences in allele frequencies. This study illustrates the potential value of human microsatellite loci for analyses of population genetic structure in baboons and suggests that this approach will be useful in studies of other Old World monkeys.     

Embryogenic cell lines of <Emphasis Type="Italic">Juniperus communis</Emphasis>; easy establishment and embryo maturation,limited germination

Several coniferous species belonging to the Pinaceae family can be propagated via somatic embryogenesis, while species belonging to the Cupressaceae family cannot. The aim of this study was to identify possibilities and limitations with somatic embryogenesis in Cupressaceae. Juniperus communis was chosen as model species. We show that a high initiation frequency of embryogenic cell lines can be established from intact megagametophytes at the time when intensive cleavage polyembryogeny takes place. The embryogenic cell lines proliferate fast on medium lacking plant growth regulators. Early somatic embryos develop after transfer to medium with decreased content of nitrogen and calcium. The early embryos mature after exposure to abscisic acid. Mature cotyledonary embryos germinate after partial desiccation. A high proportion, over 40%, of the germinating embryos retain the embryogenic potential in the basal part, resulting in development of new embryogenic tissue.     

Morphological and microsatellite diversity associated with ecological factors in natural populations of Medicago laciniata Mill. (Fabaceae)

Genetic variability in 10 natural Tunisian populations of Medicago laciniata were analysed using 19 quantitative traits and 12 polymorphic microsatellite loci. A large degree of genetic variability within-populations and among-populations was detected for both quantitative characters and molecular markers. High genetic differentiation among populations for quantitative traits was seen, with Q _ST = 0.47, and F _ST = 0.47 for microsatellite markers. Several quantitative traits displayed no statistical difference in the levels of Q _ST and F _ST . Further, significant correlations between quantitative traits and eco-geographical factors suggest that divergence in the traits among populations may track environmental differences. There was no significant correlation between genetic variability at quantitative traits and microsatellite markers within populations. The site-of-origin of eco-geographical factors explain between 18.13% and 23.40% of genetic variance among populations at quantitative traits and microsatellite markers, respectively. The environmental factors that most influence variation in measured traits among populations are assimilated phosphorus (P₂0₅) and mean annual rainfall, followed by climate and soil texture, altitude and organic matter. Significant associations between eco-geographical factors and gene diversity, H _e , were established in five-microsatellite loci suggesting that these simple sequence repeats (SSRs) are not necessarily biologically neutral.     

Spatiotemporal genetic differentiation of Cuban natural populations of the pink shrimp <Emphasis Type="Italic">Farfantepenaeus notialis</Emphasis>

We analyzed the spatiotemporal genetic structure of Farfantepenaeus notialis populations using five microsatellites loci in order to understand the influence of natural events such as hurricanes on the genetic drift/migration balance as the main cause for the variation of allele frequencies over time. The results were compared with the previous ones obtained from allozymes and mtDNA. High and stable genetic diversity levels (He=0.879+/-0.0015) were found over eight years for the populations that inhabit the south Cuban platform, however significant changes of allele frequencies were detected over time. The F(ST) estimates, albeit low, revealed significant differences among populations inside the Ana Maria Gulf for 1995 but not for the 1999 and 2003 samples. The F(ST), AMOVA and the genetic distance analysis revealed the instability of the genetic structure over time in accordance with allozymes results. The correspondence of the microsatellite results with those obtained from allozymes confirm the effects of migration enhanced by natural events as the main cause of the temporal variation of allele frequencies. The genetic drift effect was discarded through the evaluation of Ne and the M ratio, while natural selection effects were rejected because of the lowest probability of microsatellite loci being under selective pressures. The microsatellite data are also consistent with the results obtained with mtDNA in detecting significant and persistent genetic differences between the Gulfs of Ana María and Batabanó for the years 1995 and 2003.     

Fine Spatial Structure of Atlantic Hake (<Emphasis Type="Italic">Merluccius merluccius</Emphasis>) Stocks Revealed by Variation at Microsatellite Loci

Genetic variation at 5 microsatellite loci was analyzed for European hake Merluccius merluccius sampled from 9 different regions in the Atlantic Ocean and the Mediterranean Sea. Significant genetic differentiation was found between samples, suggesting a fine subdivision of Atlantic and Mediterranean hake stocks. These results are discussed in the context of the decline of demersal fish species, probably due to overfishing.     

Proliferation and maintenance of embryogenic capacity in elm embryogenic cultures

Summary This paper investigates maintenance and proliferation of somatic embryogenesis systems for Ulmus minor and U. glabra. Proliferation occurred with subculture of embryogenic calluses. The calluses were mainly formed by friable nodules composed of meristematic cells organized into proembryogenic cell masses (PEMs) and thin-walled vacuolated parenchymatic cells. Cotyledonary embryos, with procambial strands and differentiation of their vascular tissues as well as visible root meristems, were identifiable after 18d of culture on a proliferation medium with 0.44 μM benzyladenine (BA). The shoot meristem was only occasionally well developed. Somatic embryo multiplication from elm embryogenic calluses is a clearly asynchronic system, and PEMs as well as embryos at all stages of development are observed simultaneously at the end of subculture period. Factors affecting the proliferation of elm embryogenic callus, such as culture medium, carbon source and genotype, were studied. Basal medium (MS) or medium supplemented with 0.44 μM BA produced the highest number of somatic embryos. Somatic embryo production was higher with sucrose or glucose than with maltose, and significant differences were also found among the four embryogenic lines tested. The use of liquid medium with filter paper support is an essential step for the survival of isolated somatic embryos during the germination stage. The addition of 0.22 μM BA′ to liquid MS medium was the best treatment for germination and plantlet conversion of elm somatic embryos.     

Demarcation of informative chromosomes in tropical sweet corn inbred lines using microsatellite DNA markers

A study of genetic variation among 10 pairs of chromosomes extracted from 13 tropical sweet corn inbred lines, using 99 microsatellite markers, revealed a wide range of genetic diversity. Allelic richness and the number of effective alleles per chromosome ranged from 2.78 to 4.33 and 1.96 to 3.47, respectively, with respective mean values of 3.62 and 2.73. According to the Shannon’s information index (I) and Nei’s gene diversity coefficient (Nei), Chromosome 10 was the most informative chromosome (I = 1.311 and Nei = 0.703), while Chromosome 2 possessed the least (I = 0.762 and Nei = 0.456). Based on linkage disequilibrium (LD) measurements for loci less than 50 cM apart on the same chromosome, all loci on Chromosomes 1, 6 and 7 were in equilibrium. Even so, there was a high proportion of genetic variation in Chromosomes 4, 5, 8, 9 and 10, thereby revealing their appropriateness for use in the genetic diversity investigations among tropical sweet corn lines. Chromosome 4, with the highest number of loci in linkage disequilibrium, was considered the best for marker-phenotype association and QTL mapping, followed by Chromosomes 5, 8, 9 and 10.