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

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

Woody plants may be defined as trees and shrubs “whose stems live for a number of years and increase in diameter each year by addition of woody tissue” [1]. This contrasts with the description for herbaceous plants “with a relatively short-lived aboveground stem that is comparatively thin, soft, and nonwoody” [1]. Included in this definition of woody plants are those vines which are woody, i.e. “plants whose stems are not self-supporting, but either trail on the ground or climb by attaching to other plants or supports” [1]. In this context species such as Manihot esculenta and Carica papaya, included as woody by others [2,3] are regarded as herbaceous here.

Somatic Embryogenesis in Woody Plants

Several factors affecting somatic embryogenesis (SE) in Pinus sylvestris from self- and cross-pollinated seed families were studied with the aim of producing large quantities of clonal plants. Somatic embryogenesis initiation from zygotic embryos was improved on a medium with lower than standard concentrations of 2,4-dichlorophenoxyacetic acid (2.2 vs. 9.5 μM) and 6-benzyladenine (2.2 vs. 4.5 μM). On this medium, initiation rates of four controlled crosses, including one self-cross, varied from 3% to 25%. Among the maturation factors tested, the concentration of abscisic acid (ABA 80, 120 μM) had no significant effect on the production of mature somatic embryos when the medium contained 0.1 M sucrose. When sucrose concentration was 0.2 M, however, 1.4 times more mature somatic embryos were produced on medium with 80 μM compared with 120 μM ABA. Under our best maturation conditions, mature somatic embryos accumulated amounts of storage proteins that were similar to the amounts in mature zygotic embryos. Activated charcoal exerted a beneficial effect on mature somatic embryo production of 24-week-old cultures; there was no evidence of such an effect in 8-week-old cultures. Thirty-seven embryogenic lines from a self-cross and an out-cross were chosen for clonal plant production. Highly embryogenic lines produced mature somatic embryos that were more likely to convert to plants than those from less embryogenic lines. After 4 months of growth in a shade house, plantlet survival rates exceeded 70% for 31 lines out of 35. This report describes an improved method for accelerated production of large quantities of Scots pine for clonal tests.

Clonal plant production from self- and cross-pollinated seed families of Pinus sylvestris (L.) through somatic embryogenesis

The development of techniques for somatic embryogenesis in conifers has led to rapid advances in the ability to culture conifer tissue in vitro. Somatic embryogenesis now provides a means to clonally propagate commercially valuable conifers of several species and offers an in vitro experimental system for studying embryogenesis. As a result of these developments, the conifer zygotic and somatic embryo system has recently attracted the attention not only of the cell biologist but also of biochemists and molecular biologists. In this review I have attempted to consolidate the recent information on ultrastructure, biochemical and molecular aspects of conifer zygotic and somatic embryogenesis. In the first section, salient features of zygotic embryogenesis are highlighted followed by biochemical and ultrastructural characterization of development with reference to storage reserve deposition. In the second section, detailed characterization of seed storage proteins, changes in gene expression with emphasis on storage protein genes and Lea genes during zygotic and somatic embryogenesis are described. Effect of culture treatments such as abscisic acid, osmoticum and desiccation on storage protein gene expression in somatic embryos is discussed. Based on these studies, comparisons are presented between angiosperm and gymnosperm embryogeny regarding the unique morphological and developmental patterns of conifer embryogenesis. Finally, a brief discussion of recent progress in biochemical and molecular characterization of conifer seed germination is presented.

https://www.cambridge.org/core/services/aop-cambridge-core/content/view/S0960258500002439

Conifer zygotic embryogenesis, somatic embryogenesis, and seed germination: Biochemical and molecular advances

Utilization of lipid and protein storage reserves was examined during germination and early growth of somatic and excised seed embryos in vitro, and seed of interior spruce. High germination frequencies were recorded for somatic and excised seed embryos, and elongation of radicles and hypocotyls observed for both embryo types. Although elongation of the embryo types differed (i.e. somatic ≤ excised seed < seed), fresh weight gain was similar. Utilization of triglycerides in somatic embryos was more rapid than in excised seed and seed embryos, thus it appeared to be under embryo infuence. By contrast, depletion of storage proteins appeared to be influenced by the megagametophyte, with hydrolysis in somatic and excised seed embryos preceding that of the seed embryo by 2 days. Differential utilization of the storage reserves was temporally associated with differences in growth patterns of somatic and seed embryos. The evidence presented indicates that the in vitro germination medium does not entirely supplant the role of the megagametophyte during germination and early growth. The relevance of these results to artificial seed technology is discussed.

D. R. Cyr

Papers

Biochemical events during germination and early growth of somatic embryos and seed of interior spruce ( Picea glauca engelmanni complex)