In spite of the importance attained by somatic embryogenesis and of the many studies that have been conducted on this developmental process, there are still many aspects that are not fully understood. Among those features, the involvement of plant hormones and plant growth regulators on deTermining the conversion of somatic onto embryogenic tissues, and on allowing progression and maturation of somatic embryos, are far away from being completely comprehended. Part of these difficulties relies on the frequent appearance of contradictory results when studying the effect of a particular stimulus over a specific stage in somatic embryogenesis. Recent progress achieved on understanding the interaction between exogenously added plant growth regulators over the concentration of endogenous hormones, together with the involvement of sensitivity of the tissues to particular hormone groups, might help clarifying the occurrence of divergent patterns in somatic embryogenesis, and in tissue culture in general. The aspects described above, emphasizing on the effect of the concentration of plant hormones and of the addition of plant growth regulators during the different phases of somatic embryogenesis, will be reviewed in this paper. Citations will be limited to review articles as much as possible and to individual articles only in those cases in which very specific or recent information is presented.

Involvement of plant hormones and plant growth regulators on in vitro somatic embryogenesis

Recent advances in the genetic transformation of trees

Trees are an integral part of human life, and a vital component of biodiversity. Forest trees in particular are renewable sources of food, fodder, fuel wood, timber and other valuable non-timber products. Due to the rapid growth of population and the human desire to progress, there has been a tremendous reduction in forest cover from the earth’s surface. To maintain and sustain forest vegetation, conventional approaches have been exploited in the past for propagation and improvement. However, such efforts are confronted with several inherent bottlenecks. Biotechnological interventions for in vitro regeneration, mass micropropagation and gene transfer methods in forest tree species have been practised with success, especially in the last decade. Against the background of the limitations of long juvenile phases and life span, development of plant regeneration protocols and genetic engineering of tree species are gaining importance. Genetic engineering assumes additional significance, because of the possibility of introducing a desired gene in a single step for precision breeding of forest trees. There are no comprehensive and detailed reviews available combining research developments with major emphases on tissue culture and basic genetic transformation in tree species. The present communication attempts to overview the progress in tissue culture, genetic transformation and biotechnological applications in the last decade and future implications.

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Progress in tissue culture, genetic transformation and applications of biotechnology to trees: an overview

A simple, rapid and effective system to regenerate Arabidopsis plants via direct somatic embryogenesis has been established. Somatic embryogenesis was induced directly during culture of immature zygotic embryos. The frequency of somatic embryogenesis was strongly influenced by the stage of development of the explants. Explants in different developmental stages were cultured on B5 agar medium containing 5 μM 2,4-dichlorophenoxyacetic acid and the highest frequency (up to 90%) of somatic embryogenesis was observed in zygotic embryos with fully-developed cotyledons. The first somatic embryos developing directly from explant tissue were noticed after 8 days of culture. Somatic embryogenesis of a high frequency (87–96%) was observed in cultures of the all six genotypes tested (Columbia, C-24, RLD, Wassilewskaja, Landsberg erecta and Wilna). Subculture of somatic embryos onto auxin-free medium resulted in their conversion into plants with an average frequency of 79.5%. The regenerates showed normal morphological characteristics and were fertile. All 56 analysed plants displayed a diploid number of chromosomes and two out of 96 (2.1%) tested plants carried a chlorophyll or embryo-lethal mutation.

Direct somatic embryogenesis as a rapid and efficient system for in vitro regeneration of Arabidopsis thaliana

Somatic embryogenesis is a developmental process where a plant somatic cell can dedifferentiate to a totipotent embryonic stem cell that has the ability to give rise to an embryo under appropriate conditions. This new embryo can further develop into a whole plant. In woody plants, somatic embryogenesis plays a critical role in clonal propagation and is a powerful tool for synthetic seed production, germplasm conservation, and cryopreservation. A key step in somatic embryogenesis is the transition of cell fate from a somatic cell to embryo cell. Although somatic embryogenesis has already been widely used in a number of woody species, propagating adult woody plants remains difficult. In this review, we focus on molecular mechanisms of somatic embryogenesis and its practical applications in economic woody plants. Furthermore, we propose a strategy to improve the process of somatic embryogenesis using molecular means.

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Application of Somatic Embryogenesis in Woody Plants.

Internodal and leaf segments from pedunculate oak (Quercus robur L.) seedlings were used as explant source to induce somatic embryogenesis. Auxin treatment influenced embryogenic response, which only occurred in explants initially cultured on media containing 4 mg/l naphthaleneacetic acid (NAA) and different benzyladenine (BA) concentrations. After 6 weeks of culture on induction medium, the explants were transferred to medium supplemented with 0.1 mg/l BA and 0.1 mg/l NAA, and 4 weeks later, they were subcultured in a growth-regulator-free medium, in which somatic embryos arose through indirect regeneration on the surface of a nodular callus. Somatic embryos were induced in explants of two out of four seedling provenances. The induction frequency ranged from 16% in leaf explants to 4% in internodal explants. Somatic embryos developed two cotyledons, which were translucent or opaque-white in appearance, but anomalous morphologies were also observed. Different embryogenic lines were established and maintained by repetitive embryogenesis in multiplication medium containing 0.1 mg/l BA plus 0.05 mg/l NAA. These results indicate that tissues from explants other than Q. robur zygotic embryos are able to produce embryogenic cultures.

Somatic embryogenesis from stem and leaf explants of Quercus robur L.

Somatic embryo induction and plant regeneration have been obtained in tissues from mature Quercus robur L. trees. Epicormic shoots were forced to flush in branch segments collected from the crown of trees growing in selected stands on different collection dates. Expanding leaves from five genotypes, cultured following a multistage treatment procedure, produced somatic embryos at frequencies ranging from 0.3 to 3.6% of leaf explants, depending on genotype and collection date. After being induced, somatic embryos started a recurrent process by secondary embryogenesis which amplified the 15 embryogenic lines established. Plant recovery was achieved in 60% and 17% of matured embryos from two genotypes.

Somatic Embryogenesis in Mature Quercus Robur Trees

Cryopreservation of embryogenic cultures of Quercus robur using desiccation and vitrification procedures

A procedure for inducing somatic embryos in shoot apex explants (2 mm) excised from shoot proliferation cultures established from adult oak trees (Quercus robur) was investigated. Embryogenesis was induced in shoot tip as well as leaf explants in three out of the five genotypes evaluated. Somatic embryos were formed by culture in induction medium supplemented with 21.48 μM naphthalene acetic acid and 2.22 μM benzyladenine for 8 weeks, and successive transfer of explants to expression media with a low concentration of growth regulators and without them. Both types of explants formed callus tissue from which somatic embryos developed, indicating indirect embryogenesis. Although the embryogenic frequencies were lower than 12%, it did not prevent the establishment of clonal embryogenic lines maintained by repetitive embryogenesis. Histological study confirmed an indirect somatic embryogenesis process from shoot tip explants, in which leaf primordia and the corresponding axial zones were involved in generating callus, whereas the apical meristem itself did not proliferate. The origin of embryogenic cells appeared to be associated with dedifferentiation of certain parenchymal cells in callus regions after transfer of explants to expression media without auxin. Division of embryogenic cells gave rise to proembryo aggregates of unicellular origin, although a multicellular origin from bulging embryogenic areas would also seem possible. Further development led to the formation of cotyledonary-stage somatic embryos and nodular embryogenic structures that may be considered as anomalous embryos with no clear bipolarity. Inducement of somatic embryos from explants isolated from shoot cultures ensures plant material all year round, thus providing a significant advantage over the use of leaf explants from field-grown trees.

Shoot apex explants for induction of somatic embryogenesis in mature Quercus robur L. trees

Hardwood species are valuable biological resources that have an important role in the economy and ecology of ecosystems worldwide. Non-zygotic or somatic embryogenesis (SE) is a powerful tool in pl...

M. T. Martínez

Papers

Somatic embryogenesis from stem and leaf explants of Quercus robur L.

Somatic Embryogenesis in Mature Quercus Robur Trees

Cryopreservation of embryogenic cultures of Quercus robur using desiccation and vitrification procedures

Shoot apex explants for induction of somatic embryogenesis in mature Quercus robur L. trees

Non-Zygotic Embryogenesis in Hardwood Species