Embryogeny of gymnosperms: advances in synthetic seed technology of conifers

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.Abbreviations ABA abscisic acid - LEA late embryogenesis abundant - PEG polyethylene glycol - PGR plant growth regulator - RH relative humidity - TAG triacylglycerol     

Enhanced production of recombinant human gastric lipase in turnip hairy roots

Somatic embryogenesis is a reliable and important tool, and the relevant genes controlling this process act as vital roles through the whole development of somatic embryos. However, regeneration via somatic embryogenesis in Chinese chestnut has been impeded and its molecular mechanism is not known. Therefore, firstly we described a protocol for somatic embryo initiation, development, maturation and germination. Embryogenic calli were obtained in embryo initiation medium containing 1.8 μM 2,4-D and 1.1 μM 6-BA, and then were transferred to embryo development medium without any hormones for at least 4 weeks, until cotyledonary embryos appeared. Next, the somatic embryos were transferred to embryo maturation medium containing Gamborg’s B-5 Basal Salt Mixture with 0.5 μM NAA and 0.5 μM 6-BA for 3 weeks. Finally, these mature embryos were germinated in embryo germination medium consisting of WPM with 0.5 μM NAA and 0.5 μM 6-BA, resulting in shoot regeneration with a 2.1% conversion rate. Additionally, eight embryogenesis-related genes were identified, and the expression profiles of these genes during embryogenesis were analyzed via quantitative real-time RT-PCR (qRT-PCR). The CmSERK, CmLEC1, CmWUS and CmAGL15 genes exhibited high expression in the initial embryo stages, which inferred that these genes played key roles during the initiation of embryogenesis. Studies on embryogenesis-related genes will provide an insight for further elucidating molecular mechanism during somatic embryogenesis of Chinese chestnut. Furthermore, the successful establishment of a somatic embryo regeneration system for Chinese chestnut will lay a significant foundation for a stable genetic transformation system and genetic improvement.     

Altered HBK3 expression affects glutathione and ascorbate metabolism during the early phases of Norway spruce (Picea abies) somatic embryogenesis

Plant homeobox genes play an important role in plant development, including embryogenesis. Recently, the function of a class I homeobox of knox 3 gene, HBK3, has been characterized in the conifer Picea abies (L.) Karst (Norway spruce) [8]. During somatic embryogenesis, expression of HBK3 is required for the proper differentiation of proembryogenic masses into somatic embryos. This transition, fundamental for the overall embryogenic process, is accelerated in sense lines over-expressing HBK3 (HBK3-S) but precluded in antisense lines (HBK3-AS) where the expression of this gene is experimentally reduced. Altered HBK3 expression resulted in major changes of ascorbate and glutathione metabolism. During the initial phases of embryogeny the level of reduced GSH was higher in the HBK3-S lines compared to their control counterpart. An opposite profile was observed for the HBK3-AS lines where the glutathione redox state, i.e. GSH/GSH + GSSG, switched towards its oxidized form, i.e. GSSG. Very similar metabolic fluctuations were also measured for ascorbate, especially during the transition of proembryogenic masses into somatic embryos (7 days into hormone-free medium). At this stage the level of reduced ascorbate (ASC) in the HBK3-AS lines was about 75% lower compare to the untransformed line causing a switch of the ascorbate redox state, i.e. ASC/ASC + DHA + AFR, towards its oxidized forms, i.e. DHA + AFR. Changes in activities of several ascorbate and glutathione redox enzymes, including dehydroascorbate reductase (EC 1.8.5.1), ascorbate free radical reductase (EC 1.6.5.4) and glutathione reductase (GR; EC 1.6.4.2) were responsible for these metabolic differences. Data presented here suggest that HBK3 expression might regulate somatic embryo yield through alterations in glutathione and ascorbate metabolism, which have been previously implicated in controlling embryo development and maturation both in vivo and in vitro.     

LECs go crazy in embryo development

We have reviewed studies in which LEC TFs have been used to explore totipotency via SE and regulation of the maturation phase during zygotic embryogenesis. LEC TFs are master regulators of the maturation phase, activating genes encoding seed proteins that define this phase of embryo development. Regulation of the maturation phase seems to involve a feedback loop between the LEC TFs and hormones. LEC TFs stimulate ABA levels and activate genes that repress GA levels, contributing to the high ABA to GA ratio characteristic of the maturation phase. High ABA levels in turn stimulate LEC TFs to activate seed protein genes, and the reduction in GA levels might facilitate LEC TF activity. Although the LEC TFs are master regulators of the maturation phase, LEC genes are initially expressed before the onset of the maturation phase. The cellular process that initiates the maturation phase is not known. Nor is it known how LEC TFs interact with ABA and GA at the molecular level.SE is an outstanding example of totipotency in plants. Ectopic expression of LEC genes causes vegetative or reproductive cells to change their fate and undergo somatic embryo development. LEC TFs, via LEC2, activate auxin biosynthetic enzymes, and we propose that an increase in endogenous auxin levels serves to induce SE (Figure 3). How exogenous or endogenous auxin acts as the induction signal remains to be determined. We suggest that LEC TFs enable cells to become competent to respond to the induction signal by inactivating GA and, perhaps, by increasing ABA levels (Figure 3). Thus, a potential thread between the roles of LEC TFs in the maturation phase and SE might be their involvement in controlling the ABA to GA balance. It remains to be determined whether and how ABA and GA influence embryogenic competence. Although many questions remain, substantial progress has been made in determining how the LEC TFs ‘go crazy’ during embryo development.     

Expression of the viviparous 1 (Pavp1) and p34cdc2 protein kinase (cdc2Pa) genes during somatic embryogenesis in Norway spruce (Picea abies [L.] Karst)

Detailed expression analysis of the Norway spruce (Picea abies [L.] Karst) Viviparous 1 (Pavp1) and p34cdc2 (cdc2Pa) genes was carried out during somatic embryogenesis. Pavp1, a gene associated with embryo development, was expressed in proliferating embryogenic suspension cultures in the absence of exogenous ABA. When somatic embryo formation was promoting by blocking proliferation, Pavp1 expression was reduced. During maturation, exogenous ABA induced increased Pavp1 expression, which peaked at the early cotyledonary stage of somatic embryogenesis. Following partial desiccation of mature somatic embryos at high relative humidity, Pavp1 expression persisted under germination conditions. Pavp1 expression was also detected in non-dormant immature male strobili and dormant terminal buds. These data confirm the functional conservation of Pavp1 during the evolution of seed plants and extend its function beyond the embryo. Cdc2Pa, a gene associated with the cell cycle, was up-regulated when the proliferation of embryogenic cells was blocked. Expression was again up-regulated in early embryogeny and again during germination. The implications of this up-regulation of cdc2Pa are discussed.     

<Emphasis Type="Italic">LEAFY COTYLEDON1</Emphasis>, <Emphasis Type="Italic">FUSCA3</Emphasis> expression and auxin treatment in relation to somatic embryogenesis induction in Arabidopsis

The expression pattern of the LEC1 and FUS3 genes during somatic embryogenesis in Arabidopsis explants (immature zygotic embryos) induced in vitro was analysed, using Real-time quantitative PCR (qRT-PCR). The analysis revealed differential expression of LEC1 but not FUS3 within a 30 day time course of somatic embryo development, and a significant auxin-dependent upregulation of LEC1 was found over the time course. In contrast to embryogenic culture, the level of LEC1 and FUS3 expression was noticeably lower in non-embryogenic callus of Col-0 and hormonal mutants (cbp20 and axr4-1) with low SE-efficiency. In addition, the expression profile of LEC1 and FUS3 was followed in the embryogenic culture derived from 35S::LEC2-GR explants. A significant increase of LEC1 but not FUS3 activity was observed under LEC2 overexpression induced in auxin-treated explants. The work provides further experimental evidence on LEC gene involvement in the embryogenic response in Arabidopsis somatic cells, and also implicates LEC1 function in more advanced stages of SE culture in relation to somatic embryo differentiation and development.     

Gene Expression Patterns During Somatic Embryo Development and Germination in Maize Hi II Callus Cultures

Gene expression patterns were profiled during somatic embryogenesis in a regeneration-proficient maize hybrid line, Hi II, in an effort to identify genes that might be used as developmental markers or targets to optimize regeneration steps for recovering maize plants from tissue culture. Gene expression profiles were generated from embryogenic calli induced to undergo embryo maturation and germination. Over 1,000 genes in the 12,060 element arrays showed significant time variation during somatic embryo development. A substantial number of genes were downregulated during embryo maturation, largely histone and ribosomal protein genes, which may result from a slowdown in cell proliferation and growth during embryo maturation. The expression of these genes dramatically recovered at germination. Other genes up-regulated during embryo maturation included genes encoding hydrolytic enzymes (nucleases, glucosidases and proteases) and a few storage genes (an α-zein and caleosin), which are good candidates for developmental marker genes. Germination is accompanied by the up-regulation of a number of stress response and membrane transporter genes, and, as expected, greening is associated with the up-regulation of many genes encoding photosynthetic and chloroplast components. Thus, some, but not all genes typically associated with zygotic embryogenesis are significantly up or down-regulated during somatic embryogenesis in Hi II maize line regeneration. Although many genes varied in expression throughout somatic embryo development in this study, no statistically significant gene expression changes were detected between total embryogenic callus and callus enriched for transition stage somatic embryos.Supplementary material is available for this article at     

松柏类植物体细胞胚胎发生的研究进展

松柏类植物的体细胞胚胎发生既是繁育的一种手段,又是研究胚胎发育过程中结构、生理和分子事件的一种重要的模式系统。整个体细胞胚胎发生过程主要包括3个步骤：胚性组织的诱导和增殖、体细胞胚的成熟以及体细胞胚的萌发和转换。过去为了提高胚胎发育过程所做的努力主要都集中在胚的成熟阶段,这是因为一直认为能否成功再生的关键在于胚发育成熟阶段的处理。然而,在过去几年里,结合生理生化以及分子生物学的研究发现,胚胎发生的早期阶段对于完成整个发育过程也是至关重要的,早期阶段培养条件的优化可以显著提高培养过程中体细胞胚的数量和质量。此外,萌发过程培养条件的调节对于提高成熟体细胞胚的萌发率和转换率也很重要。因此,这些新的研究成果对于改善松柏类植物体细胞胚胎发生中的胚的诱导率和转换率低的现象具有重要的意义。     

Precociously germinating rapeseed embryos retain characteristics of embryogeny

We compared the germination of Brassica napus L. embryos at three stages of development-mid-cotyledon, maturation and mature dry-to determine at which stage they acquired the capacity for normal germination and seedling development. Embryos were removed from the seed and cultured on hormone-free medium, allowing them to germinate. The transition from embryogeny to germination was monitored both morphologically and biochemically, using synthesis of 12 S storage protein as a marker of embryogeny. The mature embryos (dry seeds) set the standard for normal seedling development: radicle emergence, hypocotyl extension and cotyledon expansion occurred within 2 d and true leaves were formed within a week of germination. Rocket immunoelectrophoresis indicated that the storage proteins in seedlings from mature dry embryos were completely degraded within a week. In contrast, the midcotyledon-stage embryos appeared to germinate abnormally, retaining many embryonic characteristics. Although the roots emerged, the hypocotyls did not elongate and secondary cotyledons instead of leaves were formed at the shoot apex. Also, the seedlings continued to synthesize and accumulate storage proteins. The maturation-stage embryos did develop into normal-looking seedlings, but complete degradation of storage proteins required several weeks, presumably reflecting continued synthesis and turnover. We conclude that embryogenic and germination-specific processes can occur concurrently and that the capacity to develop as normal seedlings is acquired gradually during the maturation process.Abbreviations dpa days post anthesis - EDTA ethylenedi-aminetetraacetic acid - FW fresh weight     

Factors Affecting Somatic Embryogenesis from Cotyledonary Explants of Safflower

Frequency of safflower (Carthamus tinctorius L.) somatic embryogenesis, number of somatic embryos per responding explant and somatic embryo maturation and germination were affected by genotype, explant age, carbon source, and ethylene. Among 8 cultivars tested, 7 were embryogenic with varying frequencies. The best response was obtained with cv. Girna. Whole cotyledonary explant from 10-d-old plants was best responding compared to 5- or 15-d-old ones. Among different carbon sources, sucrose at 87.6 mM concentration was most suitable for embryo induction, maturation and germination. Of the different ethylene inhibitors, silver nitrate at 50 [micro ]M concentration significantly increased the embryogenic frequency and also the number of embryos per responding explant. Silver nitrate has pronounced effect on embryo maturation but had no effect on germination.     

Influence of freezing and non-freezing temperature on somatic embryogenesis and vinblastine production in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don.

Catharanthus roseus (L.) G. Don. (Apocynaceae) is an important dicotyledonous medicinal plant. It produces vinblastine and vincristine, two alkaloids that are being used against a variety of cancers. In the present study, the freezing (−196, 4, 15°C) and non-freezing (25°C) temperature was imposed on embryogenic cultures, and later in vitro embryogeny and vinblastine production in C. roseus was studied. Somatic embryo (SE) production was maximum at 15°C, but the SE maturation was high at 4°C. The SEs, grown at 25°C, showed highest germination and plantlet conversion. Quantitative estimation of vinblastine was carried out using high-performance liquid chromatography in various in vitro raised tissues (embryogenic callus), embryo stages (proliferated, matured and germinated embryos)], and SE-derived plantlets (leaf, shoot, root and whole plant) after various freezing- and non-freezing temperature treatments. Vinblastine synthesis was temperature dependent in C. roseus that has been discussed in this present article.     

Improved somatic embryo maturation in loblolly pine by monitoring ABA-responsive gene expression

During loblolly pine zygotic embryo development, increases in mRNAs for three ABA-responsive LEA-like genes coincided with the two developmental stage-specific peaks of endogenous ABA accumulation (Kapik et al. 1995). These ABA concentration profiles from zygotic embryo development were used to develop several tissue culture approaches that altered the exposure of somatic embryos to exogenous ABA. Elevating exogenous ABA at a time corresponding to mid-maturation improved the germination and resulted in more zygotic-like expression of selected genes in somatic embryos. Extending the time on maturation medium for a fourth month increased embryo yield, dry weight, and germination in high-and low-yield genotypes. Optimizing the amounts of embryogenic suspension, plated and exogenous ABA concentration increased from 22 to 66% in the early-stage bipolar embryos that developed to the cotyledonary stage.     

Effects of cadmium and lead stress on somatic embryogenesis of coniferous species. Part I: Evaluation of the genotype-dependent response

Somatic embryogenesis is an important biotechnological tool that has significant potential to be used in studies related to environmental stress. In this study, embryogenic cell masses of Abies alba and Picea abies were grown on media enriched with 50–500 µM cadmium (Cd²⁺) or lead (Pb²⁺). The effects of cadmium and lead were evaluated during the subsequent stages of somatic embryogenesis: proliferation, maturation, and germination. The following characteristics were evaluated: proliferation potential, cell viability, average number of somatic embryos obtained per 1 g of fresh weight, and morphology of the developed somatic embryos. The tested heavy metals significantly reduced the proliferation rate of A. alba and P. abies embryogenic cell masses. The highest tested cadmium concentration markedly slowed or stopped the growth of embryogenic cell masses in both species. Unexpectedly, the proliferation ratio remained fairly high for the P. abies cell lines treated with lead at all concentrations tested. During the maturation stage, the total number of somatic embryos declined under cadmium exposure. The formation of early precotyledonary and cotyledonary somatic embryos in both species was similarly reduced, although cadmium caused a higher death rate and was more toxic than lead. To the best of our knowledge, this is the first report to study the effects of heavy metals on A. alba embryogenic cell masses during the proliferation stage as well as on the maturation and germination processes of both species. This in vitro system can be used for testing the response of large sets of genotypes, and the best performing lines can be used in the future for in vivo performance tests of heavy metal-polluted soils.     

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.     

Long-term subculture randomly affects morphology and subsequent maturation of early somatic embryos in maritime pine

Somatic embryogenesis is affected by highly variable maturation yields in Pinus pinaster. Origins of this variability were investigated by testing effects of spatial and temporal division of initial embryogenic tissue into independently cultivated pieces. One embryogenic cell-line was proliferated to obtain six embryonal-suspensor masses (ESM) treated as six sub-lines within a single dish. After proliferation to reach 2, 4 and 8 dishes (12, 24, 48 ESM), the 48 ESM (six sub-lines) were maintained by weekly subculture (34 weeks). ESM observations and maturation tests were regularly performed during the amplification and maintenance periods. Relations between maturation yields as well as cotylfedonary embryo length and immature embryo morphology were analyzed. ESM yielded variable results, independently from culture spatial (dish) and temporal (sub-line) subdivision. Maturation yields and length of regenerated embryos globally decreased as a function of subculture number. Concomitantly, morphological degradation of immature embryos occurred, indicating a global loss of embryogenic ability. Maturation variability probably results from immature embryos heterogeneity, which could be increased by manipulations during subculture. This is the first time that this evolution along time in culture is precisely described in conifers somatic embryos.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .