The origin and development of somatic embryos following cryopreservation of an embryogenic suspension culture ofPicea sitchensis

Summary The development of somatic embryos in an embryogenic suspension culture ofPicea sitchensis was followed every day for two weeks after thawing from liquid nitrogen (LN₂). Only a few cells, primarily located at the periphery of the embryonic region of the embryos, survived cryopreservation in LN₂. Surviving cells were classified into two groups: embryogenic cells (EC) and non-embryogenic cells (NEC), based on their morphology and embryogenic competence. The dense cytoplasmic EC underwent organized growth and differentiation with first divisions occurring after 24 h, and embryo formation 6–8 days after thawing from LN₂. No evidence of asymmetrical divisions or free-nuclear stages was found during somatic embryo formation. NEC had less dense cytoplasm with numerous small vacuoles. One to five days after thawing the NEC became progressively more vacuolated and elongated. Histological examination revealed no mitotic activity in NEC, and six days after thawing NECs were seen as single cells or unorganized cell aggregates. Two weeks after thawing the appearance of the cryopreserved cultures was comparable to that of the untreated cultures.Abbreviations EC embryogenic cells - ECC embryogenic cell clusters - FDA fluorescein diacetate - GMA glycol methacrylate - LN₂ liquid nitrogen (–196°C) - NEC non-embryogenic cells     

Expression of the JIM8 cell wall epitope in carrot somatic embryogenesis

Certain single cells in carrot (Daucus carota L.) suspension cultures react with the monoclonal antibody JIM8, and it has been proposed that these cells represent a transitional stage in somatic embryo formation. Shortly after isolation of the single cells by sieving, up to 80% of the cells react with JIM8. Within 4 d, JIM8 labelling becomes restricted to 1% of the single cells. To obtain evidence for the proposed correlation between expression of the JIM8 cell wall epitope and somatic embryo formation the developmental fate of carrot single cells labelled with JIM8 was determined by cell tracking. The results, obtained by recording 43 000 cells, show that only few JIM8-labelled cells give rise to embryos, and most somatic embryos develop from cells devoid of the JIM8 cell wall epitope. We therefore conclude that the presence of the JIM8 cell wall epitope does not coincide with the ability of single suspension cells to form embryos.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - AGP arabino galactan protein - B5-0 Gamborg's B5 medium - B5-0.2 Gamborg's B5 medium supplemented with 0.2 M 2,4-D - FITC fluoresceïn isothiocyanate - PBS phosphate-buffered saline     

Developmental anatomy and ultrastructure of early somatic embryos in European black pine (Pinus nigra Arn.)

Summary Embryogenic callus cultures of European black pine (Pinus nigra Arn.) were established on megagametophytes containing zygotic embryos in early developmental stage. In addition to many elongated cells and disorganized growing clumps they contained early somatic embryos at various stages of development. At all stages of embryogenesis the embryos were organized as bipolar structures. Cell pairs composed of one isodiametric cell with dense cytoplasm and a second large vacuolated cell were the simplest bipolar system. The vacuolated cell underwent senescence. The cytoplasm-rich cell and its derivates divided transversally, resulting in several cytoplasmic cells arranged in row. An early embryonal cylindrical mass was formed by longitudinal division of the cells in a filament. Proximally localized cells in the early embryonal mass became vacuolized and elongated gradually giving rise to the secondary suspensor. Distal cells remained cytoplasmic in character and formed an embryonal mass along the axis of long early somatic embryos. Differences in the proportion of organelles and heterochromatin clumps, thickness of cell walls and number of plasmodesmata between cells at various stages of early somatic embryogenesis were described.     

The effect of auxin type and concentration on pecan (Carya illinoinensis) somatic embryo morphology and subsequent conversion into plants

Summary Embryogenic cultures were initiated from immature pecan zygotic embryos. Explants were induced for one week on Woody Plant Medium with either -naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid at 2, 6 or 12 mg/l, then subcultured monthly to fresh basal medium. Observations were made on callus production, embryo formation, and embryo morphology. Somatic embryo morphology and overall callus proliferation were affected by auxin type. Callus proliferation was less extensive and more somatic embryos resembling zygotic embryos were obtained from cultures initiated with -naphthaleneacetic acid than with 2,4-dichlorophenoxyacetic acid. Repetitive somatic embryogenesis was obtained in all auxin treatments. Conversion into plantlets was affected by somatic embryo morphology in that embryos with poorly developed apices exhibited lower percentages of conversion than those with well developed single or multiple apices. Consequently, although more embryos were obtained with 2,4-dichlorophenoxyacetic acid, naphthaleneacetic acid was the superior auxin for production of somatic embryos more likely to convert into plants.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - WPM Woody Plant Medium (Lloyd & McCown 1980)     

Scale-up of somatic embryogenesis in alfalfa (Medicago sativa L.) I subculture and indirect secondary somatic embryogenisis

Three methods of increasing the productivity of somatic embryogenesis in Medicago sativa L. were investigated. In the basic procedure, somatic embryos were initiated from young petioles and carried through several phases: callus formation, suspension culture, selection of the embryogenic fraction by sieving, development, maturation, desiccation and storage. The suspensions were normally separated into three fractions by sieving. Fraction I (<200 m) containing nonembryogenic cells or cell clusters was discarded. Fraction II (200–500 m) consisting of embryogenic cell clusters was collected for embryo development and maturation. Fraction III (over 500 M) containing the mixture of petiole residues with large pieces of calli and globular somatic embryos was usually discarded. Several methods to scale-up the suspension phase were unsuccessful. Direct subculture of the entire suspension by the addition of fresh liquid medium resulted in the loss of embryogenic capacity by the third subculture. Subculture of fraction II decreased embryogenic cell mass, and hence reduced total productivity. The recycling of fraction III back to fresh B₅g liquid medium resulted in high productivity in the first culture but further subculture of this fraction resulted in a rapid decline in the embryogenic capacity.As an alternative, somatic embryos from the first tissue culture cycle were also used as explants for the initiation of secondary embryogenic callus. The embryogenic capacity of these somatic embryo explants declined rapidly as they matured. More than 100 secondary somatic embryos could be induced from embryo explants removed from development medium at 10 days after sieving the suspension, but only 40 somatic embryos were produced from each mature somatic embryo explant, and 13 from desiccated embryos. The secondary somatic embryos were comparable to the primary embryos in quality according to germination tests. The implications of the results to the efficiency of somatic embryo production of Medicago are discussed.Abbreviations ABA abscisic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - DAS days after sieving - PPF photosynthetic photon flux density - SE somatic embryo     

Rapid propagation of Eleutherococcus senticosus via direct somatic embryogenesis from explants of seedlings

Explants from three different parts (cotyledon, hypocotyl or root) of one week-old seedlings of Eleutherococcus senticosus were cultured on Murashige and Skoog (MS) medium with 1.0 mg l^-1 2,4-D. Somatic embryos were formed directly from the surfaces of explants. The frequency of direct somatic embryo formation was the highest in the hypocotyl segments (75%) as compared to cotyledon (56%) or root segments (12%). When hypocotyl explants from 3 different stages of seedlings (zero, one or three week-old) were cultured on MS medium with 1.0 mg l^-1 2,4-D, the frequency of somatic embryo formation rapidly declined as the zygotic embryos germinated. However most somatic embryos (93%) from explants of zygotic embryos developed as fused state (multiple embryo), whereas somatic embryos (over 89%) from more developed seedlings developed into single state (single embryo). Single embryos germinated and regenerated into plantlets with both shoots and roots, while multiple embryos only regenerated into only multiple shoots. Plantlets that regenerated from single embryos of E. senticosus were acclimatized in a greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

The ultrastructure of somatic embryo development in pearl millet (Pennisetum glaucum; Poaceae)

The ultrastructure, morphology, and histology of somatic embryogenesis in pearl millet (Pennisetum glaucum) were examined using light and electron microscopic techniques. Somatic embryogenesis was initiated from zygotic embryo explants cultured 8 d after pollination. Formation of a ridge of tissue began 3–4 d after culture (DAC) by divisions in the epidermal and subepidermal cells of the scutellum. Ridge formation was accompanied by a decrease in vacuoles, lipid bodies, and cell size, and an increase in endoplasmic reticulum (ER). Proembryonic cell masses (proembryoids) formed from the scutellar ridge by 10 DAC. Proembryoid cells had abundant Golgi bodies and ER while the amounts of lipids and starch varied. Somatic embryos developed from the proembryonic masses 13 DAC and by 21 DAC had all the parts of mature zygotic embryos. Although shoot and root primordia of somatic embryos were always less differentiated than those of zygotic embryos, scutellar cells of somatic and zygotic embryos had similar amounts of lipids, vacuoles, and starch. Somatic scutellar epidermal cells were more vacuolated than their zygotic counterparts. In contrast, somatic scutellar nodal cells were smaller and not as vacuolated as in zygotic embryos. Somatic embryogenesis was characterized by three phases of cell development: first, scutellar cell dedifferentiation with a reduction in lipids and cell and vacuole size; second, proembryoid formation with high levels of ER; and third, the development of somatic embryos that were functionally and morphologically similar to zygotic embryos.     

Monoclonal antibody against a cell wall marker protein for embryogenic potential of <Emphasis Type="Italic">Dactylis glomerata</Emphasis> L. suspension cultures

We identified and isolated a monoclonal antibody (MAb 3G2) raised against extracellular proteins from microcluster cells of orchard grass (Dactylis glomerata L.) embryogenic suspension culture. MAb 3G2 recognized with high specificity an antigen ionically bound within the primary cell wall and in the culture medium of microcluster cells. Two-dimensional polyacrylamide gel analysis and blotting of proteins on PVDF membrane showed that MAb 3G2 detected a single polypeptide of apparent molecular mass of 48 kDa and an isoelectric point (pI) of 5.2, designated EP48. A transient expression during somatic embryogenesis was observed for EP48. Indirect immunofluorescence showed that this protein highly accumulated in the cell walls of some single cells, microclusters and partly in proembryogenic masses (PEMs), but not in globular embryos of the embryogenic cell line and microclusters from the non-embryogenic cell line. Signal intensity varied between individual cells of the same population and in successive stages of somatic embryo development. Screening of several D. glomerata L. embryogenic and non-embryogenic cell lines with MAb 3G2 indicated the presence of ECP48 in only embryogenic suspension cultures at early stages of embryo development long before morphological changes have taken place and thus it could serve as an early marker for embryogenic potential in D. glomerata L. suspension cultures.     

Proliferation and maintenance of embryogenic capacity in elm embryogenic cultures

Summary This paper investigates maintenance and proliferation of somatic embryogenesis systems for Ulmus minor and U. glabra. Proliferation occurred with subculture of embryogenic calluses. The calluses were mainly formed by friable nodules composed of meristematic cells organized into proembryogenic cell masses (PEMs) and thin-walled vacuolated parenchymatic cells. Cotyledonary embryos, with procambial strands and differentiation of their vascular tissues as well as visible root meristems, were identifiable after 18d of culture on a proliferation medium with 0.44 μM benzyladenine (BA). The shoot meristem was only occasionally well developed. Somatic embryo multiplication from elm embryogenic calluses is a clearly asynchronic system, and PEMs as well as embryos at all stages of development are observed simultaneously at the end of subculture period. Factors affecting the proliferation of elm embryogenic callus, such as culture medium, carbon source and genotype, were studied. Basal medium (MS) or medium supplemented with 0.44 μM BA produced the highest number of somatic embryos. Somatic embryo production was higher with sucrose or glucose than with maltose, and significant differences were also found among the four embryogenic lines tested. The use of liquid medium with filter paper support is an essential step for the survival of isolated somatic embryos during the germination stage. The addition of 0.22 μM BA′ to liquid MS medium was the best treatment for germination and plantlet conversion of elm somatic embryos.     

Analysis of morphological changes in carrot somatic embryogenesis by serial observation

Carrot somatic embryogenesis was serially observed using a cell cluster immobilizing system with Phytagel. Embryogenic cell clusters ranging in size from 32 to 63 μm were collected by filtration and used for somatic embryo induction. Of the 432 cell clusters, 253 grew, i.e., the size of these cell clusters increased, and 192 developed into globular embryos. Through serial observation, the number of somatic embryos produced from each cell cluster was identified. Cell clusters which developed into two or more embryos grew and developed slowly as compared with cell clusters which developed into single embryos. Serial observation also revealed that some cell clusters consisted of several parts, each of which independently grew as separate units. In cases where two growing parts fused into one embryo, morphological abnormalities such as curvature or lumps in their bodies were occasionally observed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of Gamma Irradiation on Embryogenic Avocado Cultures and Somatic Embryo Development

Embryogenic avocado cultures were exposed to ionizing irradiation in order to determine its effect on proliferation and subsequent somatic embryo development. The approximate PD₅₀ as determined by linear regression is 35 Gy 2 weeks after irradiation for Fuerte 2.11.1 and 4 weeks after irradiation for T362 2.11.1. Irradiation of embryogenic cultures did not significantly affect the number of early stage Fuerte 2.11.1 somatic embryos that developed directly from irradiated cultures; however, 10–50 Gy inhibited somatic embryo development. Irradiation of T362 2.11.1 embryogenic cultures at 25–50 Gy inhibited the number of intermediate and mature stages of somatic embryos that developed directly from irradiated cultures, and 50 Gy inhibited somatic embryo maturation. Inhibition of somatic embryo development could be partially offset by proliferation of irradiated embryogenic cultures as suspensions. Irradiation up to 10 Gy significantly increased the number of mature Fuerte 2.11.1 somatic embryos that developed from suspension cultures. Irradiation with doses up to 25 Gy stimulated development of heart stage T362 2.11.1 somatic embryos; however, mature somatic embryo development was suppressed at dosages of 10 Gy and greater.     

Characterization of embryogenic cell lines of Picea abies in relation to their competence for maturation

Summary Embryogenic cell lines of Picea abies are categorized into three groups (polar, solar, and undeveloped) based on the organization of the somatic embryos within the tissue and the ability of the somatic embryos to proceed through a maturation process when treated with ABA. The polar and the solar types consist of somatic embryos with densely packed embryonic regions subtended by vacuolated suspensors. Both types of tissue regenerate mature somatic embryos when treated with ABA. Almost all mature somatic embryos develop further into shoots or plantlets. The undeveloped type consists of somatic embryos comprised of only a few loosely aggregated cells in their embryonic regions. Mature somatic embryos were not observed with this tissue type.Abbreviations ABA cis-trans abscisic acid - A1 polar type - A2 solar type - B undeveloped type - BA benzyladenine - 2,4-D 2,4 di-chlorophenoxyacetic acid - LP von Arnolds medium (1987)     

虎眼万年青的直接体细胞胚胎发生和植株再生

以虎眼万年青(Ornithogalum caudatum Jacq.)子鳞茎的鳞叶为外植体,不经过愈伤组织阶段,直接诱导体细胞胚胎发生并再生植株。组织切片和扫描电镜的观察结果表明:体细胞胚来自鳞叶表皮最外层的单个细胞。这个细胞经第一次平周分裂,成为2-细胞原胚,然后依次经过4-细胞原胚、球形胚、香蕉形胚等阶段发育成小植株,其形态学发育过程与合子胚相似。小植株移入土中,1周后即可成活。此外,还观察到直接体细胞胚只产生于鳞叶的近轴面,而远轴面无体细胞胚产生,这两个部位在细胞结构和形态上有显著差异。     

Comparative anatomy of embryogenic and non-embryogenic calli from<Emphasis Type="Italic">pimpinella brachycarpa</Emphasis>

Anatomical differences between embryogenic and non-embryogenic calli ofPimpinella brachycarpa were investigated by light microscopy and electron microscopy. Initial callus tissue emerged from expiants after 14 d of culturing. The embryogenie calli (EC) were firm, rather opaque, and light yellow in color. The cells usually formed small, compact clusters. Nonembryogenic calli (NEC), however, were friable, semitransparent, and yellow or gray. These formed relatively larger and loosely held clusters. Scanning electron microscopy showed that EC were composed of individual compact and spherical cells that were rather regular in size and approximately 20 μm long. All were tightly held together and appeared to organize globular embryos. In contrast, the NEC comprised elongated and loosely held cells that were approximately 50 μm long. Tubular and u-shaped NEC cells protruded irregularly, and were of varying heights along the cell aggregates. Transmission electron microscopy of the EC revealed typical eukaryotic cytoplasmic components, including nuclei, mitochondria, and vacuoles in the cytoplasm enclosed by an electron-transparent cell wall. Based on the numerous ribosomes within the cytoplasm, these cells appeared to be well-organized and metabolically active. The NEC cells were much larger and more highly vacuolated than those of the EC. In ultrathin sections, the former seemed to be almost devoid of other cellular contents except for plastids and nuclei. Furthermore, EC and NEC showed different regeneration capacities in their somatic embryo formation. Most EC produced hyperhydric somatic embryos, followed by normal somatic embryos; whereas only a few shooted or rooted somatic embryos arose from the NEC.     

Somatic embryogenesis from seed explants of Abies alba

Megagametophytes of Abies alba containing the immature embryos were dissected from the seed coats and divided by longitudinal and transverse sections. They were placed with the cut surface down on modified Schenk & Hildebrandt medium containing 50 mgl^-1 myo-inositol and 2% sucrose, supplemented with 1 mgl^-1 N⁶-benzyladenine (BAP). An embryogenic type of callus proliferated after one month of culture. Closer examination revealed the presence of structures resembling early stages of embryogenesis as well as of single elongated, vacuolated cells and clusters of cells with dense cytoplasm. Under appropriate conditions, some of the somatic embryos elongated and formed cotyledons.     

Origin and development of secondary somatic embryos in transformed embryogenic cultures of <Emphasis Type="Italic">Medicago sativa</Emphasis>

Non-transformed and transformed embryogenic cultures of alfalfa (Medicago sativa L. cv. Zaječarska 83), long-term maintained on growth regulator-free medium, were histologically analyzed. In all examined cultures, somatic embryos at various stages of development were observed and secondary embryos were formed in the cotyledonary, hypocotylary and radicular region of the primary embryos. Detailed histological analysis of the torpedo shape somatic embryo revealed that secondary somatic embryos arose directly from single epidermal cells of hypocotylary axis after an unequal periclinal division. Bipolar proembryos were composed of one smaller cytoplasm rich cell and one larger more vacuolated cell. Further cell division pattern was similar for both non-transformed and transformed embryos. However, multicellular origin of secondary embryos in a direct process and even from callus can not be excluded.     

SV40 Large T Antigen Disrupts Embryogenesis of Canine and Porcine Somatic Cell Nuclear Transfer Embryo

Background

Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for transgenic animal production using genetically modified somatic cells (GMSCs). However, there are several limitations preventing successful transgenic animal generation by SCNT, such as obtaining proper somatic donor cells with a sufficiently long life span and proliferative capacity for generating GMSCs. Here, we established simian virus 40 large T antigen (SV40LT)-mediated lifespan-extended canine fibroblast cells (SV40LT-K9 cells) and evaluated their potential as nuclei donors for SCNT, based on cellular integrity and SCNT embryo development.

Results

SV40LT did not cause canine cell transformation, based on cell morphology and proliferation rate. No anchorage-independent growth in vitro and tumorigenicity in vivo were observed. After SCNT with SV40LT-K9 cells, embryos were transferred into surrogate dogs. All dogs failed to become pregnant. Most embryos did not proceed past the 8-cell stage and only one surrogate showed an implantation trace in its oviduct, indicating that the cells rarely developed into blastocysts. Because of the absence of an in vitro maturation method for canine embryos, we performed identical experiments using porcine fibroblast cells. Similarly, SV40LT did not transform porcine fibroblast cells (SV40LT-Pig cells). During in vitro development of SV40LT-Pig cell-driven SCNT embryos, their blastocyst formation rate was clearly lower than those of normal cells. Karyotyping analysis revealed that both SV40LT-K9 and SV40LT-Pig cells had aberrant chromosomal statuses.

Conclusions

Although lifespan-extended canine and porcine cells via SV40LT exhibit no apparent transforming changes, they are inappropriate for use as nuclei donors for SCNT because of their aneuploidy.

     

Asparagus somatic embryos: Production in suspension culture and conversion to plantlets on solidified medium as influenced by carbohydrate regime

Methods were developed for the production of somatic embryos of asparagus (Asparagus officinalis L.) in suspension culture and subsequent conversion to plantlets on solidified medium. Stem-derived callus that was subcultured twice on Murashige and Skoog (MS) medium + 0.54 M naphthaleneacetic acid (NAA) and 1.4 M 2-isopentenyladenine (2iP) was used to initiate suspension cultures. Six out of 15 such cell suspensions (MS medium with 54 to 107 M NAA) had a high embryogenic capacity. These cell suspensions consisted primarily of single elongated cells (about 90% of all single cells), embryogenic cell clusters (2571/ml), and globular translucent embryos (32/ml). The latter converted to plantlets within four weeks on embryo development medium (EDM), which was solidified MS medium containing 0.54 M NAA and 0.98 M 2iP. Suspension-derived embryos formed secondary globular embryos at high frequencies (251 to 258/g callus) when placed on EDM with a low carbohydrate (sucrose, glucose or fructose) level (2%). In contrast, EDM with a high carbohydrate level (10%) caused a reduction in the frequency of secondary embryos (30 to 85/g callus), while resulting in the promotion of embryo growth and conversion, 3.6 to 8.5 times higher than 2% carbohydrates. Transfer of globular somatic embryos from cell suspension to EDM with high carbohydrate levels (4 to 10%) for two weeks followed by transfer to EDM with a low carbohydrate level (2%) resulted in a 2 to 4 times higher conversion rate to plantlets than those that remained at the 4 to 10% levels.Abbreviations ANOVA analysis of variance - EDM embryo development medium - NAA naphthaleneacetic acid - MS Murashige and Skoog - RCBD randomized complete block design - 2iP 2-isopentenyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid     

Possible involvement of abscisic acid in the induction of secondary somatic embryogenesis on seed-coat-derived carrot somatic embryos

When seed coats (pericarps) were picked from 14-day-old carrot (Daucus carota) seedlings and cultured on agar plates, embryogenic cell clusters were produced very rapidly at a high frequency on the open side edge. Embryo induction progressed without auxin treatment; indeed treatment caused the formation of non-embryogenic callus. The embryogenic tissues (primary embryos) developed normally until the torpedo stage; however, after this a number of secondary somatic embryos were produced in the hypocotyl and root regions. Tertiary embryos were formed on some of the secondary embryos, but many developed into normal plantlets. The primary embryos contained significantly higher levels of abscisic acid (ABA) than the hypocotyl-derived normal and seed-coat-derived secondary embryos. Fluridone inhibited the induction of secondary embryogenesis, while exogenously supplied ABA induced not only tertiary embryogenesis on the seed-coat-derived secondary embryos, but also secondary embryos on the hypocotyl-derived normal somatic embryos. These results indicate that ABA is one of the important endogenous factors for the induction of secondary embryogenesis on carrot somatic embryos. Higher levels of indole-3-acetic acid (IAA) in primary embryos also suggest the presence of some concerted effect of ABA and IAA on the induction of secondary embryogenesis in primary embryos.     

Tissue-specific expression of Pa18, a putative lipid transfer protein gene,during embryo development in Norway spruce (Picea abies)

A full-length Picea abies cDNA clone Pa18, encoding a protein with the characteristics of plant lipid transfer proteins, has been isolated and characterized. The size of the deduced 173 amino acid (aa) long protein is around 18 kDa. The first 100–120 aa show similarity to angiosperm lipid transfer proteins in amino acid sequence as well as in predicted secondary structure. The Pa18 gene is constitutively expressed in embryogenic cultures of Picea abies representing different stages of development as well as in non-embryogenic callus and seedlings. The Pa18 gene product has an antimicrobial activity. In situ hybridization showed that the Pa18 gene is equally expressed in all embryonic cells of proliferating embryogenic cultures but during embryo maturation the expression of the gene in maturing and mature somatic as well as in mature zygotic embryos is stronger in the outer cell layer than in other tissues. Southern blot analysis at different stringencies was consistent with a single gene with one or two copies rather than a gene family. Twenty independent transgenic sublines over- and under-expressing the Pa18 gene under the Zea mays ubiquitin promoter were established. There was a high yield of mature somatic embryos with a smooth surface only in untransformed, control cultures. Irrespective of the expression level of Pa18, the somatic embryos started to mature when given a maturation treatment. However, in the transgenic sublines, the outer cells in the maturing embryos frequently became elongated and vacuolated instead of remaining small and uniform. One explanation for this was that the expression of Pa18 was not restricted to the outer cell layer in transformed sublines. Angiosperms and gymnosperms separated about 300 million years ago and the embryo genesis is different in the two groups. The outer cell layer (protoderm), the first tissue to differentiate, is less clearly delineated in gymnosperms. For normal embryo development in angiosperms, expression of the LTP gene must be restricted to the protodermal cells. In this work we show that the expression of the Pa18 gene must be restricted to the putative protodermal cells of the gymnosperm.