Flow cytometric and morphological analyses of Pinus pinaster somatic embryogenesis

An approach combining morphological profiling and flow cytometric analysis was used to assess genetic stability during the several steps of somatic embryogenesis in Pinus pinaster. Embryogenic cell lines of P. pinaster were established from immature zygotic embryos excised from seeds obtained from open-pollinated trees. During the maturation stage, phenotype of somatic embryos was characterized as being either normal or abnormal. Based upon the prevalent morphological traits, different types of abnormal embryos underwent further classification and quantification. Nuclear DNA content of maritime pine using the zygotic embryos was estimated to be 57.04 pg/2C, using propidium iodide flow cytometry. According to the same methodology, no significant differences (P ≤ 0.01) in DNA ploidy were detected among the most frequently observed abnormal phenotypes, embryogenic cell lines, zygotic and normal somatic embryos, and somatic embryogenesis-derived plantlets. Although the differences in DNA ploidy level do not exclude the occurrence of a low level of aneuploidy, the results obtained point to the absence of major changes in ploidy level during the somatic embryogenesis process of this economically important species. Therefore, our primary goal of true-to-typeness was assured at this level.     

Genetic variability analyses of the somatic embryogenesis induction process in Olea spp. using nuclear microsatellites

The crop species Olea europaea L. (olive tree) is of great economic importance in the Mediterranean region. Hence, many efforts have been done in the last decades to propagate this commercially valuable species by in vitro methods. On the other hand, the lesser known Olea maderensis (Lowe) Rivas Mart. & Del Arco which is a native species of the Madeira Archipelago has only been the subject of micropropagation from nodal stem cuttings. Therefore, in this work we analysed the stability of ten nuclear simple sequence repeat (SSR) markers at successive stages of the somatic embryogenesis process in two adult trees belonging to these two species from the Madeira Archipelago. For the induction of somatic embryogenesis, petiole and leaf explants were cultivated on solid Murashige and Skoog medium (MS) with 12.25 μM indole-3-butyric acid (IBA) and 4.56 μM of zeatin, in the dark. After 3 months, different callus tissues (non-embryogenic, pre-embryogenic and embryogenic) developed from leaf explants and petioles were later transferred to MS medium without growth regulators in the dark. All ten SSR markers were able to distinguish between species. However, no mutations were found at the SSR loci at any of the successive developmental stages from PEMs (pre-embryogenic masses) to somatic embryos. This genetic uniformity was observed within material derived from each genotype/species and its respective donor plant. Therefore, we conclude that the genomic integrities of both O. europaea and O. maderensis were maintained throughout the stages of the embryogenic processes in study suggesting the absence of somaclonal variation.     

Somatic embryogenesis and two embryo specific proteins (38 and 33 kD) in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

In the present study, the regeneration pathway, especially the different events of somatic embryogenesis (SE) have been studied morphologically and biochemically in Catharanthus roseus. Firstly, the calluses were induced from different explant sources (hypocotyl, epicotyl and root) by using various auxins. Embryogenic and non-embryogenic calluses were identified based on their morphology, colour and dry weight. Embryogenic callus was later cultivated on MS added with 0.45 μM 2,4-D, 6.62 μM BAP and 1.44 μM GA3 for obtaining various developmental stages of embryos. Different stages of embryos have been assayed for the establishment of marker based embryogenesis, particularly on embryo specific proteins whose presence or absence will ensure a rapid and efficient production of embryos that has a special application to clonal biotechnology. Two embryo specific proteins (38 and 33 kD) have been identified for the first time in C. roseus during torpedo stage of embryogenesis. Besides, multiple shoot formation from in vitro raised emblings was also attempted to examine the role of BAP and kinetin for shoot proliferation. The shoots were rooted with 5.37 μM NAA and 5.71 μM IAA before transplantation.     

Genetic control of somatic embryogenesis induction in <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> Labill.

A reproducible protocol for somatic embryogenesis (SE) induction in Eucalyptus globulus from mature zygotic embryos is available since 2002. However, for the use of SE in tree breeding programs, the frequency of SE initiation needs to be improved and controlled, and this was investigated in 13 open-pollinated (OP) families over three consecutive years. A diallel mating design with five parent trees was used to study genetic control of SE induction. Results showed that SE induction varies across E. globulus families and over the years of seed production tested. Somatic embryogenesis was initiated on explants from 84% of the OP families tested in 2002 and 100% of the families tested in 2003 and 2004. The year 2003 gave best results for percentage of induction and total number of somatic embryos produced. Results concerning genetic control showed that SE induction is under the control of additive genetic effects, as 22.0% of variation in SE initiation was due to general combining ability (GCA) effect, whereas 6.4% was due to maternal effects. Neither specific combining ability (SCA) nor reciprocal effects were significant.     

Application of somatic embryogenesis in high-value clonal forestry: Deployment, genetic control, and stability of cryopreserved clones

Summary The most important advantage of cloning conifers by somatic embryogenesis (SE) is that the embryogenic tissue can be cryopreserved without changing its genetic make-up and without loss of juvenility. This offers an opportunity to develop high-value clonal varieties by defrosting and repropagating cryopreserved clones after genetic testing has shown which clones are the best performers. In the current absence of cost-effective automated embling handling systems or artificial seed technology, the deployment of the high-value clones in clonal forestry can be achieved inexpensively by mass serial rooting of cuttings from juvenile donor plants produced from cryopreserved embryogenic cultures. In a genetic analysis of the SE process in white sprucePicea glauca, we found that induction of SE was under strong genetic control. Although the dominance variance diminished rapidly as the zygotic embryos matured, the additive variance remained relatively large during the induction phase. The genetic effects in the subsequent maturation and germination phases were less strong. Furthermore, genetic variation at the different phases of SE was not correlated. Thus, it is the induction phase of SE that can be manipulated by breeding. Most of the embryogenic clones were cryopreserved easily, i.e., there was no apparent genotype effect. To determine stability of cryopreserved clones, a set of 12 clones was retrieved after 3 and 4 years, respectively, from cryopreservation and repropagated by SE. An assessment of morphologicalin vitro development andex vitro survival and growth characters demonstrated general stability of the cryopreserved clones of white spruce. Based on a presentation at the Joint Meeting of the IUFRO Working Parties on Somatic Cell Genetics and Molecular Genetics of Trees held in Quebec City, Canada, August 12–16, 1997.     

Biological characterization of young and aged embryogenic cultures of <Emphasis Type="Italic">Pinus pinaster</Emphasis> (Ait.)

Pinus pinaster (Ait.) somatic embryogenesis (SE) has been developed during the last decade, and its application in tree improvement programs is underway. Nevertheless, a few more or less important problems still exist, which have an impact on the efficiency of specific SE stages. One phenomenon, which had been observed in embryogenic tissue (embryonal mass, EM) initiated from immature seed, has been the loss of the ability to produce mature somatic embryos after the tissue had been cultured for several months. In an attempt to get insight into the differences between young cultures of EM (3-mo-old since the first subculture) of P. pinaster that produced mature somatic embryos and the same lines of significantly increased age (18-mo-old, aged EM) that stopped producing mature somatic embryos, we analyzed in both types of materials the levels of endogenous hormones, polyamines, the global DNA methylation, and associated methylation patterns. In addition, we included in the analysis secondary EM induced from mature somatic embryos. The analysis showed that the two tested genotypes displayed inconsistent hormonal and polyamine profiles in EM cultures of a similar phenotype and that it might be difficult to attribute one specific profile to a specific culture phenotype among genotypes. Experiments were also undertaken to determine if the global DNA methylation and/or the resulting methylation pattern could be manipulated by treatment of the cultures with a hypomethylating drug 5-azacytidine (5-azaC). An aged EM was exposed to different concentrations and durations of 5-azaC, and its response in culture was established by fresh mass increases and somatic embryo maturation potential. All of the analyses are new in maritime pine, and thus, they provide the first data on the biochemistry of EM in this species related to embryogenic potential.     

Somatic Embryogenesis from 20 Open-Pollinated Families of Portuguese Plus Trees of Maritime Pine

Immature zygotic embryos from 20 open-pollinated (OP) families of maritime pine (Pinus pinaster) plus trees were screened for their somatic embryogenic capacity. The best time for zygotic embryo collection was between 30th June and 16th July 1999 when most embryos were at a pre-cotyledonary stage of development. The somatic embryogenesis (SE) initiation frequency was highest on DCR basal medium with 13.6 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 µM 6-benzylaminopurine (BAP) supplemented with L-glutamine and casein hydrolysate. On this medium, initiation frequencies among OP families ranged from 4.6 to 49.1%. Initiation of embryogenic cell lines from all 20 OP families was possible only on DCR based medium, but the addition of L-glutamine and casein hydrolysate significantly increased the number of zygotic embryos producing SE. Most families showed a similar behaviour on different initiation media; however, a few exceptions were observed. Further development of somatic embryos on maturation medium, consisting of DCR with 120 µM abscisic acid (ABA), 100 g l^–1 polyethylene glycol (PEG) and 10 g l^–1 gellan gum, occurred in 29% of 896 embryogenic lines representing all 20 OP families. However, development into cotyledonary somatic embryos was observed in only 11% of the cell lines, but this still represented 18 OP families.     

Invited review: Genetic regulation of somatic embryogenesis with particular reference to arabidopsis thaliana and Medicago truncatula

Summary Investigations into the mechanisms of somatic embryogenesis (SE) have largely focused on the hormonal regulation of the process and a repertoire of strategies has been developed to regenerate many species via SE. However, the genes that regulate the induction and development of somatic embryos have not been defined. In the recent times, regeneration via overexpression of genes, such as WUSCHEL or LEAFY COTYLEDON, in Arabidopsis has started to provide a basis for understanding the genes involved in SE. This has gone hand in hand with the availability of genome sequence information and the availability of mutants in model plants such as Arabidopsis and Medicago. An improved understanding of zygotic embryogenesis and the maintenance and differentiation of stem cells in the shoot meristem also helps to provide novel insights into the mechanisms of SE. This review examines the current understanding of the genetic regulation of SE in the context of current molecular understanding of plant development.     

Factors affecting maintenance, proliferation, and germination of secondary somatic embryos of Eucalyptus globulus Labill

The described protocol for repetitive somatic embryogenesis (SE) in Eucalyptus globulus produced more somatic embryos than the primary SE protocol. Primary somatic embryos (induced on MS_3NAA) were transferred to the same medium, leading to new cycles of somatic embryos, for at least 2 years. The influence of medium (MS and B5), plant growth regulators (auxins and cytokinins), and light on secondary SE was tested. The MS medium without growth regulators (MS_WH) was more efficient for cotyledonary embryo formation and germination than the B5 medium. Reducing auxin (NAA) levels increased the proliferation of globular somatic embryos and allowed SE competence to be maintained on medium free of plant growth regulators. The addition of two cytokinins (BAP and KIN) to the MS medium did not improve proliferation of globular secondary embryos, but was crucial during later stages of the SE process (germination and conversion). Data also show that light may influence the quality of the process, depending on its stage. Darkness should be maintained until the cotyledonary stage is reached, after which exposure to light is recommended.     

Plant regeneration via somatic embryogenesis in cotton

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and stem explants of abnormal seedling as an explant have been developed in Gossypium hirsutum L. Embryogenic callus and somatic embryos have been obtained directly from the explants of cotton abnormal seedlings. Plant growth regulators influenced the induction of cotton somatic embryogenesis. The optimal medium for direct somatic embryogenesis was modified MS medium supplemented with 0.1 mg l^-1 ZT and 2 g l^-1 activated carbon. On this medium, an average of 28.0 and 28.1 matured somatic embryos formed from per leaf and stem explants respectively. The highest frequency of somatic embryogenesis was 100%. The somatic embryos were converted into normal plantlets when cultured on modified MS medium supplemented with 0.1 mg l^-1 ZT. Upon transfer to soil, plants grew well and appeared normal. Plants could be regenerated within 60–80 days. The system of cotton somatic embryogenesis and plant regeneration described here will facilitate the application of plant tissue culture and genetic engineering on cotton genetic improvement. This revised version was published online in June 2006 with corrections to the Cover Date.     

Histocytological changes and reserve accumulation during somatic embryogenesis in Eucalyptus globulus

We recently described a protocol for Eucalyptus globulus somatic embryogenesis (SE). For its immediate use at industrial levels, some stages of the process require better control. In particular, SE germination rates are variable, decreasing SE efficacy. As reserves may play a central role in embryogenic processes, we followed histocytological changes and reserve fluctuations, during SE. For SE induction, explants of mature zygotic embryos were grown on Murashige and Skoog (MS) medium with 3 mg l⁻¹ α-naphthalene acetic acid and later transferred to MS without growth regulators (MS_WH). Samples of zygotic embryo cotyledons (explants), of globular and dicotyledonar somatic embryos, and of embling leaves were analysed for reserve accumulation and histocytological profiles. Cotyledon cells of zygotic embryos were rich in lipid and protein bodies, having almost no starch. After 3 weeks of induction, starch grain density increased in differentiated mesophyll regions, while in meristematic regions their occurrence was diffuse. In globular somatic embryos, starch accumulation increased with time (in amyloplasts), but protein bodies were absent. Cotyledonary somatic embryos had lower density of starch grains and absence of lipid and protein bodies. Embling leaves showed typical histological organisation. This is the first comprehensive study on histological and cytological changes during Eucalyptus SE with emphasis in reserve accumulation. With this work we demonstrate that the presently available SE protocol for E. globulus leads to reserve fluctuations during the process. Moreover, the reserves of somatic embryo cotyledons differ from those of their zygotic embryo counterparts, which reinforce the importance of reserves in the embryogenic process and suggests that manipulating external conditions, SE may be optimised giving suitable emblings production for industrial purposes.     

Validating a QTL region characterized by multiple haplotypes

Validation of a quantitative trait locus (QTL) for outcrossing perennial plants is rarely reported due to complexity of plausible genetic models and reliance on field designs already available. Here, a particular marker-QTL haplotype exerted a large, positive effect on height for Pinus taeda and its origin could be traced to a founder, GP₃, in a three-generation QTL pedigree. To validate this QTL effect, we used an extended GP₃-based pedigree. In the validation cross, each of the 46 offspring was clonally propagated from developing seeds using somatic embryogenesis technology. Subsequent analyses were conducted separately for seedlings and for other somatic emblings. For seedlings, the original QTL effect could not be fully validated. For somatic emblings, a strong negative QTL effect was detected in the validation cross; some evidence from another cross-supported the original positive QTL effect. From this part of the analysis, three distinct marker-QTL haplotypes at a single locus could be inferred. Validating QTL haplotypes in readily available field tests was feasible despite the genetic model complexity inherent to outcrossing long-lived perennials.     

Somatic embryogenesis induction system for cloning an adult Cyphomandra betacea (Cav.) Sendt. (tamarillo)

Somatic embryogenesis is a valuable tool for plant breeding. In recent years, different aspects related to somatic embryogenesis (SE) induction in tamarillo have been studied at our laboratory. In this work, results concerning the establishment of a protocol for cloning an adult tamarillo tree through SE are presented. Attempts to induce SE in tamarillo from various explants directly taken from an adult tree were unsuccessful and only calli with no embryogenic potential were initiated. To overcome the lack of potential of adult tissues for SE, an indirect approach was attempted in which shoots from an adult tree were first established in vitro and then wounded leaves were used for SE induction. A low rate of embryogenic tissue formation was obtained (19.4%), but it was in the range of initiation rates from leaf explants of in vitro cloned plantlets of different tamarillo cultivars (red, orange and yellow) that originated from a single seedling (13.3–54.4%). High variation in SE initiation among juvenile controls could not be explained by different organogenetic potential, as no significant differences in shoot proliferation or rooting ability during micropropagation could be detected. Subcultures of embryogenic lines from the adult tree allowed us to obtain a large amount of embryogenic tissue that, after 8 weeks on a PGR-free medium, gave an average of 111 plants per gram of fresh mass of embryogenic tissue. A RAPD comparative analysis of somatic embryo-derived plantlets and the donor tree confirmed that the plantlets had no variation in the DNA regions amplified by 12 primers. These results open the way for large-scale cloning of elite tamarillo trees through SE.     

Somatic embryogenesis in oak (<Emphasis Type="Italic">Quercus</Emphasis> spp.)

Summary The present review summarizes the factors involved in controlling the process of oak somatic embryogenesis as a method for vegetative plant propagation and includes also data on artificial seed production, cryopreservation and transformation. One major limitation, the inability to initiate embryogenic cultures from mature trees, has been recently overcome. Leaves from selected cork oak trees with an age of 50 yr and more have been used to initiale somatic embryogenesis (SE) with a frequency of up to 20%. These findings offer encouraging prospects for cloning proven superior plant material and to integrate this propagation system into tree improvement programs. Once the process of SE has been initiated, the multiplication cycle proceeds via secondary embryogenesis, which can be maintained indefinitely. Problems are reported by the formation of anomalous embryos. The mutability of somatic embryogenic cell lines of various oak species has been monitored by flow cytometry and molecular markers. No somaclonal variation was detected applying random amplified polymorphic DNA (RAPD) or amplified fragment length polymorphism (AFLP) markers, whereas DNA-content measurements via flow cytometry revealed tetraploidy in some cell lines after several years of continuous subculture. Maturation and low germination frequencies are the main bottlenecks for a broader use of this technique. Recently attention has been on embryo quality and parameters for conversion capacity such as high endogenous cytokinin level and low abscisic acid (ABA) level. Although oak is probably the species that is the most well-developed system for a broadleaved forest tree, data on growth performances of somatic embryo-derived plants are rare.     

Enhancing initiation and proliferation in radiata pine (<Emphasis Type="Italic">Pinus radiata</Emphasis> D. Don) somatic embryogenesis through seed family screening,zygotic embryo staging and media adjustments

In Pinus spp., initiation of somatic embryogenesis (SE) is influenced by the developmental stage of immature embryos, the genotype of the parent trees and the formulation of tissue culture medium. Optimizing all these factors can lead to improved initiation and proliferation response; however, few studies have focused on improving these stages. For this reason, the objectives of this research were to determine the best immature zygotic embryo developmental stage for initiation and to test the effect of different sources of organic nitrogen in the initiation and proliferation steps in Pinus radiata SE. We have determined and verified the optimum zygotic embryo developmental stages 2–4 for embryogenic tissue (ET) initiation and proliferation and identified the most responsive seed families in two consecutive years. Besides EDM (Walter et al. 1998), medium with high gellan gum content during ET proliferation maintained the embryogenic tissue in a better micro-morphological arrangement for a longer time.     

Novel avenues for passion fruit in vitro regeneration from endosperm culture,and morpho-agronomic and physiological traits of triploid Passiflora cincinnata Mast. emblings

The present study describes a new regeneration system based on somatic embryogenesis from mature endosperm Passiflora cincinnata Mast. cultures. Moreover, the morpho-agronomic and phenological traits, as well as enzymatic activity of regenerated triploid emblings are compared to those of diploids. Mature endosperms were cultured on Murashige and Skoog medium supplemented with various concentrations (4.5–45.2 µM) of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 μM 6-benzylaminopurine (BA). No plant growth regulators were included in the control group. Embryogenic calli were observed only in treatments supplemented with 13.6 and 18.1 µM 2,4-D?+?4.5 µM BA, with the highest number of somatic embryos per explant and regenerated plants (emblings) obtained with 18.1 µM 2,4-D. Most emblings (70%) were triploid (2n?=?3x?=?27), with a DNA amount (4.38 pg) similar to that of endosperm and 1.5 times greater than in diploid P. cincinnata seedlings (2n?=?2x?=?18), that contained 2.98 pg of DNA. While the number of organs and/or structures was akin to that in diploids, triploid emblings generally exhibited larger and longer vegetative and floral structures. The flower lifespan was also slightly altered by triploidy, nectar concentration was 27% higher, and the activity of plant defense enzymes β-1,3-glucanase and polyphenol oxidase was 29.8% and 22.1% higher. This study describes a new regeneration system for the production of phenotypic variants of this ornamental passion fruit species, opening new perspectives for future studies on genetic passion fruit breeding.

     

Detection of somaclonal variation of cotton (Gossypium hirsutum) using cytogenetics, flow cytometry and molecular markers

Two protocols of plant regeneration for cotton were adopted in this study, namely, 2, 4-D and kinetin hormone combination and IBA and kinetin hormone combination. Twenty-eight embryogenic cell lines via somatic embryogenesis and 67 regenerated plants from these embryogenic calli were selected and used for random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), chromosomal number counting, and flow cytometric analysis. The roles of RAPD and SSR markers in detecting somaclonal variation of cotton (Gossypium hirsutum L.) were evaluated. Two cluster analyses were performed to express, in the form of dendrograms, the relationships among the hormone combinations and the genetic variability. Both DNA-based techniques were able to amplify all of the cell clones and regenerated plantlets genomes and relative higher genetic variation could be detected in the culture type with 2, 4-D and kinetin hormone combination. The result suggested that 2, 4-D and kinetin hormone combination could induce relative high somaclonal variation and RAPD and SSR markers are useful in detecting somaclonal variation of regenerated cotton plants via somatic embryogenesis. Chromosome number counting and flow cytometry analysis revealed that the number of chromosomes and ploidy levels were nearly stable in all regenerated plants except two regenerated plantlets (lost 4 and 5 chromosomes, respectively) which meant that cytological changes were not correlated with the frequency of RAPD and SSR polymorphisms. This result also might mean that the cell lines with variation of chromosome numbers were difficult to regenerate plants.     

Factors Influencing Somatic Embryogenesis Induction and Plant Regeneration with Particular Reference to Arabidopsis thaliana (L.) Heynh

The broad applications of somatic embryogenesis, both in basic and applied research, have stimulated studies on the determination of in vitro conditions for the induction of somatic embryos and their conversion into plants. As a result, efficient protocols on SE induction and plant regeneration have recently become available for many plant species, including Arabidopsis thaliana (L.) Heynh., a model plant in genetics and embryogenesis.Studies on factors controlling in vitro plant morphogenesis are highly desirable not only for the development of improved regeneration systems, but also for the analysis of molecular mechanisms underlying plant embryogenesis. This review focuses on the conditions influencing the induction of embryogenic potential in in vitro cultured plant cells. The roles of explant type, endo- and exogenous plant growth regulators and stress factors in the induction of somatic embryogenesis are especially emphasized. Possible mechanisms by which different factors induce or modify embryogenic competence in cultured plant cells are also discussed. Since the production of genetically solid and true-to-type plants is desired, especially for transformation and micropropagation practice, the problem of the genetic characteristics of regenerants, in terms of their chimerism and somaclonal variation, is discussed in some detail.Special consideration is given to A. thaliana– a major model plant species for classical genetics and genomics. Recent availability of efficient embryogenic cultures in this organism makes it possible to benefit from advanced genomic research of Arabidopsis to study plant embryogenesis on the molecular level.     

Estimation of population structure in coastal Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] using allozyme and microsatellite markers

Characterizing population structure using neutral markers is an important first step in association genetic studies in order to avoid false associations between phenotypes and genotypes that may arise from non-selective demographic factors. Population structure was studied in a wide sample of ∼1,300 coastal Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] trees from Washington and Oregon. This sample is being used for association mapping between cold hardiness and phenology phenotypes and single-nucleotide polymorphisms in adaptive-trait candidate genes. All trees were genotyped for 25 allozyme and six simple sequence repeat (SSR) markers using individual megagametophytes. Population structure analysis was done separately for allozyme and SSR markers, as well as for both datasets combined. The parameter of genetic differentiation (θ or F _ST) was standardized to take into account high within-population variation in the SSR loci and to allow comparison with allozyme loci. Genetic distance between populations was positively and significantly correlated with geographic distance, and weak but significant clinal variation was found for a few alleles. Although the STRUCTURE simulation analysis inferred the same number of populations as used in this study and as based on previous analysis of quantitative adaptive trait variation, clustering among populations was not significant. In general, results indicated weak differentiation among populations for both allozyme and SSR loci (θ _s = 0.006–0.059). The lack of pronounced population subdivision in the studied area should facilitate association mapping in this experimental population, but we recommend taking the STRUCTURE analysis and population assignments for individual trees (Q-matrix) into account in association mapping.     

The identification of QTLs associated with the <Emphasis Type="Italic">in vitro</Emphasis> response of rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

This study was conducted in order to identify quantitative trait loci (QTLs) for the in vitro culture response of winter rye (Secale cereale L.) immature embryos and immature inflorescences. A genetic linkage map comprising 67 SSRs, 9 ISSRs, 13 SAMPLs, 7 RAPDs, 2 SCARs and one EST marker was created based on the analyses of 102 recombinant inbred lines from the cross between lines L318 (which has a good response in tissue cultures) and L9 (which is unable to regenerate plants from somatic tissues and anthers). The map spans 979.2 cM, and the average distance between markers is 9.9 cM. Two characteristics were evaluated: callus induction (CI) and somatic embryogenesis ability (SE). They were expressed as the percentage of immature embryos/inflorescences producing callus (designated ECI/ICI) and the percentage of explants producing somatic embryos (ESE/ISE). All the analysed traits showed continuous variation in the mapping population but a non-normal frequency distribution. We identified nine putative QTLs controlling the tissue culture response of rye, explaining up to 41.6% of the total phenotypic variation: two QTLs for ECI — eci-1, eci-2; 4 for ESE — ece-1, ese-2, ese-3, ese-4; 2 for ICI — ici-1, ici2; and 1 for ISE — ise-1. They were detected on chromosomes 1R, 4R, 5R, 6R and 7R.