Despite major advances in forest biotechnology, clonal regeneration by somatic embryogenesis or organogenesis is still difficult for many woody species and is often limited to the use of juvenile explants. Adventitious regeneration of plants from gymnosperms older than zygotic embryos, and frequently even from highly immature zygotic embryos, is often difficult or has not yet been achieved. A number of experimental approaches that could eventually lead to overcoming recalcitrance are suggested in this review. When cloning trees of various ages, it is important to determine first which part of the individual contains the most responsive cells and at what time of the year these cells are in the most responsive state. This allows selection of the most useful explants. In hardwood trees and a few gymnosperms, responsive tissues are found in root or stump sprouts and in tissues near the site of meiosis at about the time that meiosis takes place. Another potentially active area is the shoot apex with most or all of its leaf or needle primordia removed. Apomixis is a natural form of clonal regeneration but occurs naturally in only one gymnosperm species. As the genetic mechanism of apomixis has been in part elucidated, the induction of apomixis by experimental means may soon be possible. The cytoplasm plays a major role in the expression or repression of nuclear genes that control embryogenesis. Expression of nuclear genes can be manipulated by nuclear transfer into de-nucleated cells (e.g., the cytoplasm of egg cells). Cytoplasmic control also plays a role in regeneration by androgenesis, asymmetric cell division and cell isolation. A short overview is presented of the genetic mechanisms involved in embryo initiation, maturation and germination and how manipulation of these mechanisms by genetic transformation could help in overcoming recalcitrance. It is expected that rapid development in the fields of research areas discussed in this review will over time eliminate the problem of recalcitrance in many instances where it is currently prevalent.

Recalcitrance in clonal propagation, in particular of conifers

Cloning of trees using somatic embryogenesis (SE) could have a major impact on tree breeding and commercial plantation forestry. In conjunction with cryopreservation, SE offers an opportunity to develop highly valuable clone lines. Commercial deployment of such geneti- cally tested clone lines in forestry will dramatically increase forest productivity over any available conventional tree improvement techniques. However, sufficient technical advances must be made to use SE in clonal forestry. Progress in SE techniques and genetic stability of clones is re- viewed, using white spruce (Picea glauca) and eastern white pine (Pinus strobus) as model species. There are several other issues in implemen- ting clonal forestry. The main concern is managing clonal plantations for optimal genetic gain and diversity. The issues and considerations for selecting appropriate numbers of clones and deployment strategies are discussed. A clonal deployment strategy using a "mixture of clones and seedlings" is proposed for eastern Canada. somatic embryogenesis / cryopreservation / genetic stability / clonal forestry / tree improvement

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Implementation of conifer somatic embryogenesis in clonal forestry: technical requirements and deployment considerations

In this study, several improvements and simplifications of SE protocols in Pinus pinaster (Ait.), a species of economic importance in the regions of Western Europe, are described. These improvements pertained to all stages of SE including high initiation frequencies in eight control pollinated seed families, relatively high somatic embryo maturation yield when cells were coated with particles of activated charcoal and a rapid production of plants directly in a shade house. The SE initiation frequency from isolated zygotic embryos was high (up to 100%) and plants were produced from 11 embryogenic lines representing all crosses. Based on these results, the estimated number of somatic embryos required to produce 1,000 plants varied from slightly more than the required number of plants to more than double this number depending on the line. Such an estimate is critical in developing plant production strategy when a number of embryogenic lines are considered for production of clonal plants.

Simplified and improved somatic embryogenesis for clonal propagation of Pinus pinaster (Ait.).

The productivity of plantation forests is essential to meet the future world demand for wood and wood products in a sustainable fashion and in a manner that preserves natural stands and biodiversity. Plantation forestry has enormously benefited from development and implementation of improved silvicultural and forest management practices during the past century. A second wave of improvements has been brought about by the introduction of new germplasm developed through genetics and breeding efforts for both hardwood and conifer tree species. Coupled with the genetic gains achieved through tree breeding, the emergence of new biotechnological approaches that span the fields of plant developmental biology, genetic transformation, and discovery of genes associated with complex multigenic traits have added a new dimension to forest tree improvement programs. Significant progress has been made during the past five years in the area of plant regeneration via organogenesis and somatic embryogenesis (SE) for economically important tree species. These advances have not only helped the development of efficient gene transfer techniques, but also have opened up avenues for deployment of new high-performance clonally replicated planting stocks in forest plantations. One of the greatest challenges today is the ability to extend this technology to the most elite germplasm, such that it becomes an economically feasible means for large-scale production and delivery of improved planting stock. Another challenge will be the ability of the forestry research community to capitalize rapidly on current and future genomics-based elucidation of the underlying mechanisms for important but complex phenotypes. Advancements in gene cloning and genomics technology in forest trees have enabled the discovery and introduction of value-added traits for wood quality and resistance to biotic and abiotic stresses into improved genotypes. With these technical advancements, it will be necessary for reliable regulatory infrastructures and processes to be in place worldwide for testing and release of trees improved through biotechnology. Commercialization of planting stocks, as new varieties generated through clonal propagation and advanced breeding programs or as transgenic trees with high-value traits, is expected in the near future, and these trees will enhance the quality and productivity of our plantation forests.

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Forest biotechnology : Innovative methods, emerging opportunities

Advances in conifer biotechnology offer new opportunities in the field of vegetative propagation and genetic engineering. Development of clonal propagation methods, especially somatic embryogenesis (SE), has numerous potential applications. Owing to its efficiency in plant regeneration, coupled with the ease of long-term storage in liquid nitrogen, SE became an indispensable tool for accelerating the development of tree varieties and deployment. Early SE protocols were developed for Picea species, however, when applied to Pinus species, especially at the somatic embryo maturation stage, they were unsuccessful. It became apparent that pines required more research and development to bring the SE biotechnology to its potential. This review emphasizes the most recent progress made in SE and cryopreservation in the genus Pinus, focusing on major plantation and forest species in Europe (Pinus pinaster), New Zealand, Australia, and South America (P. radiata), as well as North America (P. taeda and P. strobus). Much consideration is also given to applying SE in pine improvement and deployment strategies in multivarietal forestry.

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Recent Progress in Somatic Embryogenesis of Four Pinus spp.

Somatic embryogenesis (SE) initiation in Pinus strobus was optimized by the manipulation of plant growth regulator (PGR) concentrations in the culture medium. Modified Litvay medium (MLV) of Litvay et al. (1985) supplemented with lower than routinely used PGR concentration increased initiation of established embryogenic cultures from approximately 20 to 53%. The original developmental stage of zygotic embryos had a pronounced effect on the SE response. The optimum stage was the pre- to shortly post-cleavage stage. A substantial genetic influence on initiation of SE was indicated by a significant variance component due to families. Genotype X collection date and genotype X media interactions had large effects on initiation of SE. The PGR levels in the culture medium prior to maturation had a significant effect on subsequent production of mature somatic embryos. Embryogenic tissue initiated and proliferated on medium with a low level of PGR consistently produced a high number of somatic embryos, indicating that optimized initiation protocol also enhanced somatic embryo production. Somatic embryos of 93 embryogenic lines (representing five families) that were initiated on media with different PGR concentrations were converted to plants at an overall frequency of 76%, and grown in the greenhouse. With these improved protocols, application of P. strobus SE in commercial clonal forestry is feasible as an alternative to traditional breeding and reforestation.

Optimized somatic embryogenesis in Pinus strobus L.

Among the many commercial opportunities afforded by somatic embryogenesis (SE), it is the ability to clonally propagate individual plants with rare or elite traits that has some of the most significant implications. This is particularly true for many long-lived species, such as conifers, but whose long generation times pose substantive challenges, including increased recalcitrance for SE as plants age. Identification of a clonal line of somatic embryo-derived trees whose shoot primordia have remained responsive to SE induction for over a decade, provided a unique opportunity to examine the molecular aspects underpinning SE within shoot tissues of adult white spruce trees. Microarray analysis was used to conduct transcriptome-wide expression profiling of shoot explants taken from this responsive genotype following one week of SE induction, which when compared with that of a nonresponsive genotype, led to the identification of four of the most differentially expressed genes within each genotype. Using absolute qPCR to expand the analysis to three weeks of induction revealed that differential expression of all eight candidate genes was maintained to the end of the induction treatment, albeit to differing degrees. Most striking was that both the magnitude and duration of candidate gene expression within the nonresponsive genotype was indicative of an intense physiological response. Examining their putative identities further revealed that all four encoded for proteins with similarity to angiosperm proteins known to play prominent roles in biotic defense, and that their high-level induction over an extended period is consistent with activation of a biotic defense response. In contrast, the more temperate response within the responsive genotype, including induction of a conifer-specific dehydrin, is more consistent with elicitation of an adaptive stress response. While additional evidence is required to definitively establish an association between SE responsiveness and a specific physiological response, these results suggest that biotic defense activation may be antagonistic, likely related to the massive transcriptional and metabolic reprogramming that it elicits. A major issue for future work will be to determine how and if suppressing biotic defense activation could be used to promote a physiological state more conducive to SE induction.

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Potential link between biotic defense activation and recalcitrance to induction of somatic embryogenesis in shoot primordia from adult trees of white spruce (Picea glauca)

Within a plantation of clonal somatic embryo-derived white spruce trees that belonged to four genotypes, one genotype (G6) has consistently responded for the last 16 years, to the induction of somatic embryogenesis within primordial shoot explants. Analysis of fourteen individuals within this genotype subsequently revealed a group of clonal trees that were nonresponsive. This in turn provided a unique opportunity to conduct differential gene expression analysis in the absence of genotype-specific factors. Absolute qPCR was first used to expand the analysis of several genes previously identified via microarray analysis to be differentially expressed during SE induction, along with the inclusion of two nonresponsive genotypes. While this demonstrated a high level of repeatability within, and between, responsive and nonresponsive genotypes, it did not support our previous contention that an adaptive stress response plays a role in SE induction responsiveness, at least with respect to the candidate genes we analyzed. RNAseq analysis was then used to compare responsive and nonresponsive G6 primordial shoots during the somatic embryogenesis induction treatment. Although not analyzed in this study, this included samples of callus and embryonal masses previously generated from G6 explants. In addition to revealing a large number of differentially expressed genes, de novo assembly of unmapped reads was used to generate over 25,000 contigs that potentially represent previously unidentified transcripts. This included a MADS-domain gene that was found to be the most highly differentially expressed gene within responsive shoot explants during the first seven days of the induction treatment.

Gene expression analysis of primordial shoot explants collected from mature white spruce (Picea glauca) trees that differ in their responsiveness to somatic embryogenesis induction.

Cathy Overton

Papers

Optimized somatic embryogenesis in Pinus strobus L.

Potential link between biotic defense activation and recalcitrance to induction of somatic embryogenesis in shoot primordia from adult trees of white spruce (Picea glauca)

Gene expression analysis of primordial shoot explants collected from mature white spruce (Picea glauca) trees that differ in their responsiveness to somatic embryogenesis induction.