Synthetic seed technology requires the inexpensive production of large numbers of high-quality somatic embryos. Proliferating embryogenic cultures from conifers consist of immature embryos, which undergo synchronous maturation in the presence of abscisic acid and elevated osmoticum. Improvements in conifer somatic embryo quality have been achieved by identifying the conditions in vitro that resemble the conditions during in ovulo development of zygotic embryos. One normal aspect of zygotic embryo development for conifers is maturation drying, which allows seeds to be stored and promotes normal germination. Conditions of culture are described that yield mature conifer somatic embryos that possess normal storage proteins and fatty acids and which survive either partial drying, or full drying to moisture contents similar to those achieved by mature dehydrated zygotic embryos. Large numbers of quiescent somatic embryos can be produced throughout the year and stored for germination in the spring, which simplifies production and provides plants of uniform size. This review focuses on recent advances in conifer somatic embryogenesis and synthetic seed technology, particularly in areas of embryo development, maturation drying, encapsulation and germination. Comparisons of conifer embryogeny are made with other gymnosperms and angiosperms.

Embryogeny of gymnosperms: advances in synthetic seed technology of conifers

Many plants deposit TAG in seeds and fruits as the major form of storage lipid. TAG production is of tremendous socioeconomic value in food, nutraceutical, and industrial applications, and thus numerous conventional and molecular genetic strategies have been explored in attempts to increase TAG content and modify the FA composition of plant seed oils. Much research has focused on the acyl-CoA-dependent reaction catalyzed by diacylglycerol acyltransferase (DGAT), which is an integral endoplasmic reticulum protein and has also been shown to be present in oil bodies and plastids. DGAT enzymes exhibit diverse biochemical properties among different plant species, many of which are summarized here. In addition to catalyzing a critical step in TAG biosynthesis, there is evidence that DGAT has roles in lipid metabolism associated with germination and leaf senescence. TAG can also be formed in plants via two different acyl-CoA-independent pathways, catalyzed by phospholipid: diacylglycerol acyltransferase and diacylglycerol transacylase. The current understanding of the terminal step in TAG formation in plants and the development of molecular genetic approaches aimed at altering TAG yield and FA composition of TAG are discussed.

Diacylglycerol acyltransferase: a key mediator of plant triacylglycerol synthesis.

Somatic embryogenesis is the formation of an embryo from a cell other than a gamete or the product of gametic fusion This is not an artificial phenomenon, and is known in nature as a form of apomixis called adventitious embryony, first described by Strasburger in 1878 [1] (see Sharma and Thorpe, this volume) Although Steward [2] and Reinert [3] are generally given credit for the first descriptions of somatic embryogenesis in 1958, such credit might more properly belong to Levine [4], who in 1947 reported the recovery of carrot “seedlings” from tissues exposed to low levels of α-naphthaleneacetic acid, via a process whose description sounds very much like somatic embryogenesis

Morphogenic Aspects of Somatic Embryogenesis

Somatic embryogenesis of coniferous species was first reported more than 20 years ago. Since then, there has been an explosion of research aimed at developing and optimizing protocols for efficient regeneration of plantlets. Although routinely used both as a means of propagation, as well as a valuable model system for investigating the structural, physiological, and molecular events occurring during embryo development, in vitro embryogenesis is still problematic for some coniferous species. Major problems include: low number of embryos generated; and low frequency of mature embryos able to convert into viable plantlets. Until recent years, despite the fact that embryogenesis is comprised of a sequence of defined steps which include proliferation of embryogenic tissue, embryo maturation, and germination, attempts at improving the whole procedure have been made almost exclusively during the maturation stage. This strategy was based on the assumption that successful regeneration is related to treatments provided during the development of the embryos. Major optimizations of the maturation medium have involved judicious selections of type and concentration of growth regulators, namely abscisic acid, and adjustments of the osmoticum of the culture medium. Extensive work has been conducted in defining the effects of plasmolysing and non-plasmolysing osmoticum agents during maturation, as well as in improving desiccation techniques required for the completion of the maturation program. In the last 2 years, however, work on spruce has clearly demonstrated that the early events in embryogenesis are crucial for the successful completion of the overall embryogenic program. The use of cell tracking techniques, implemented by physiological and molecular studies, has revealed that manipulations of the culture conditions early in the process can increase both number and quality of embryos produced in culture. Additional manipulations of the germination medium can also enhance germination and conversion frequency of somatic embryos matured in a sub-optimal environment. These new findings, together with the unraveling of molecular mechanisms involved in the control/regulation of embryo development hold considerable promise for clonal propagation in conifers.

Recent advances in conifer somatic embryogenesis: improving somatic embryo quality

Vegetative propagation of forest trees offers advantages to both tree breeders and the forest industry. This review will describe benefits, type of vegetative propagation, and its integration into breeding programmes. Of all of the different methods for vegetative propagation, only rooted cuttings and somatic embryogenesis (and the combined use of both) offer any practical methods for large-scale commercial use. However, it is very difficult to fully appreciate the overall level of activity of the research and application of somatic embryogenesis of forest trees. Publications and reports only highlight a small fraction of the ongoing work. To this end, a survey was conducted across Europe (under EU Research Infrastructure Concerted Action “Treebreedex”) to document the species involved, the state-of-the-art of somatic embryogenesis, its stage of development and its application in tree improvement programmes and to commercial forestry. The results of this survey are presented and discussed. In addition, this review presents the challenges (biological, economic, public acceptance and regulatory) and their relationships to European forestry. Finally, a strategy to promote the use of this technology is proposed.

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Somatic embryogenesis in forestry with a focus on Europe: state-of-the-art, benefits, challenges and future direction

In order to enhance post-germinative vigour, somatic embryos of Picea glauca (Moench) Voss. were matured under in-vitro conditions that stimulated triacylglycerol (TAG) biosynthesis. In P. glauca seeds over 90% of the TAG was stored within the megagametophyte, and isolated zygotic embryos contained twice the amount of TAG of somatic embryos cultured for four weeks on basal medium containing 16 μM abscisic acid (ABA). Polyethylene glycol-4000 (PEG) as a non-permeating osmoticum with ABA promoted TAG biosynthesis by somatic embryos and sustained maturation throughout an eight-week culture period. Treatments that promoted TAG biosynthesis also prevented precocious germination and promoted desiccation tolerance. Thus, the optimal culture conditions for maturation, desiccation survival, and plantlet regeneration were 16–24 μM ABA and 7.5% PEG for eight weeks, followed by desiccation. Under these conditions the levels of TAG per somatic embryo were raised ninefold to about five times the zygotic-embryo level, and the TAG fatty-acid composition became similar to that of zygotic embryos. A study of sectioned material, using light and transmission electron microscopy, showed that the structure and distribution of lipid bodies within these somatic embryos and the degree of embryo development were similar to mature zygotic embryos. Up to 81% of the desiccated somatic embryos regenerated to plantlets during which time the TAG was utilised in a manner similar to zygotic seedlings.

Manipulation of conditions for the culture of somatic embryos of white spruce for improved triacylglycerol biosynthesis and desiccation tolerance.

The limit of permeability of white spruce (Picea glauca [Moench.] Voss) somatic embryo cell walls to molecules was in the order of 30 A. Polyethylene glycols (PEGs) and dextrans of molecular weights greater than 1000 and 6000, respectively, produced a nonpermeating (non-plasmolysing) water stress which improved embryo development. Somatic embryos converted to plantlets at frequencies of 76-84% following slow drying and storage at − 20°C for 1 year, which was similar to the 77% recorded for control somatic embryos slowly dried then germinated without freezing or storage. Culture for 7-8 weeks with medium containing abscisic acid, 3% sucrose, and 7.5% PEG 4000 yielded somatic embryos with five times the embryo storage lipid content recorded for zygotic embryos. During culture with PEG the moisture content of the somatic embryos decreased from 96% for immature suspension-cultured somatic embryos, to 47% for mature embryos. Somatic embryos cultured for 7-8 weeks survived rapid drying to 5% moisture content, and converted to plantlets at frequencies of 60-70%, but no somatic embryos survived rapid drying when cultured for only 4 weeks; however, slow drying did induce desiccation tolerance in 3-week cultured somatic embryos. Abscisic acid was important to maintain embryos in a developmental state, but ABA alone did not induce desiccation tolerance. In order to induce desiccation tolerance a water stress treatment was required. Tolerance of rapid drying coincided with moisture contents below 55%, which occurred after 5 weeks of culture in the presence of PEG 4000 and abscisic acid

Development of white spruce (Picea glauca (Moench.) Voss) somatic embryos during culture with abscisic acid and osmoticum, and their tolerance to drying and frozen storage

S. M. Attree

Papers

Manipulation of conditions for the culture of somatic embryos of white spruce for improved triacylglycerol biosynthesis and desiccation tolerance.

Development of white spruce (Picea glauca (Moench.) Voss) somatic embryos during culture with abscisic acid and osmoticum, and their tolerance to drying and frozen storage