Growth regulators influence the fatty acid profiles of in vitro induced jojoba somatic embryos

Inducing somatic embryogensis from jojoba [Simmondsia chinensis (Link) Schneider] explants to produce artificial seeds in the laboratory (in vitro) may prove highly profitable, as the seeds contain a characteristic liquid wax of economic importance in industry, nutrition and medicine. Thus, there is a need to examine the effect of the factors involved in the in vitro process on the quality and quantity of the synthesized fatty acids in comparison with those naturally produced in vivo. Immature zygotic embryos and mature leaf explants were cultured on Murashige and Skoog basal medium (MS) supplemented with various levels of 2,4-D, BA and sucrose. Embryogenic calluses developed from the zygotic embryos and leaf explants over a period of 2–4 weeks with the highest response at 0.4 μM 2,4-D, 2.2/4.4 μM BA and 117 mM sucrose (4%). Following induction, the zygotic embryo derived somatic embryos developed to the globular, heart, torpedo, and cotyledon stages. Direct somatic embryogenesis was observed with some of the zygotic embryo explants. Leaf-derived embryogenic calluses did not mature on any of the maturation/germination media examined up to 4 weeks of culture. Analysis of fatty acids indicated that the mature seeds are characterized with long chain saturated fatty acids C22:0 behenic Acid. The zygotic embryo-derived somatic embryos (SE-Z) and leaf-derived somatic embryos (SE-L) are characterized with the induction of the essential polyunsaturated fatty acid C18:2 (omega-6) linoleic acid, (omega-3) alpha-linolenic acid (ALA), with higher values of long chain saturated fatty acids C16:0 palmitic acid and monounsaturated fatty acid C18:1 oleic acid. These results indicate that manipulating the growth regulators in the induction media influenced the fatty acids synthesis and hence the fatty acids profile in jojoba somatic embryos.     

Development of white spruce somatic embryos: II. Continual shoot meristem development during germination

Summary This study compares the development of shoot apical meristems of white spruce somatic and zygotic embryos during germination. In mature somatic embryos, the functional part of the shoot apical meristem was bi-layered. After partial drying, a normal shoot meristem was formed from these two cell layers during germination. Other cells within the meristem were vacuolated and separated by intercellular air spaces. In the absence of the partial drying treatment, somatic embryos enlarged in size primarily due to vacuolation of cells and the formation of large intercellular air spaces. A majority of these somatic embryos failed to form a functional shoot apical meristem. Compared with somatic embryos, the shoot apical meristem of a mature zygotic embryo was well organized with a densely cytoplasmic apical layer. The cells within the meristem were tightly packed. Judging from the cell profiles during germination, all cells within the meristem of the zygotic embryo took part in the formation of the vegetative shoot apical meristem.     

Imbibition of white spruce seeds and somatic embryos: A study of morphological changes in an environmental scanning electron microscope and potassium leakage

Summary Potassium leakage and morphological changes during imbibition of white spruce [Picea glauca (Moench) Voss] seeds and somatic embryos were investigated. A single desiccated somatic embryo, a single somatic embryo exposed to a high relative humidity environment for 2 d, and a single dry zygotic embryo leaked similar amounts of potassium over a 120-min period of imbibition in liquid germination medium. A seed without a seed coat leaked two and eight times more potassium than a single whole seed and a single zygotic embryo, respectively. Nearly 50% of the potassium leaked for all tissues was leaked within the first 20 min of imbibition. Exposure of somatic embryos to an environment with high relative humidity resulted in a reduction in the percentage of potassium leaked after 80 and min to levels equivalent to those for zygotic embryos. Using an environmental scanning electron microscope, we found that desiccated somatic embryos and dry zygotic embryos had wrinkled surface cells, with cells in the surface of zygotic embryos being more shrunken in appearance. Imbibition of both types of embryos in water resulted in turgid surface cells after 2 h. Imbibition in liquid germination medium did not cause much hydration of surface cells, which still had wrinkled appearances after 2 h. Finally, imbibition on filter paper on semisolidified germination medium resulted in slower hydration of somatic and zygotic embryos. Cells near the medium appeared hydrated while cotyledon surface cells furthest from the medium resembled cells in desiccated embryos.     

Proteomic analyses of somatic and zygotic embryos of Cyclamen persicum Mill. reveal new insights into seed and germination physiology

In the horticulturally important ornamental species Cyclamen persicum Mill., somatic embryogenesis is an efficient vegetative propagation method and the development of artificial seeds is an ultimate aim. This study aims at a systematic comparison of the proteomes of zygotic embryos, somatic embryos grown in liquid medium containing 30 or 60 g l⁻¹ sucrose, germinating embryos of both types and endosperm in order to obtain novel insights into seed and germination physiology. Using high resolution two-dimensional isoelectric focussing/sodium dodecylsulfate polyacrylamide gel electrophoresis (2D IEF/SDS PAGE), 74% of the proteins expressed in zygotic embryos were found in similar abundance in somatic embryos grown in 60 g l⁻¹ sucrose. Somatic embryos grown in 30 g l⁻¹ sucrose accumulated fewer protein species than those grown in 60 g l⁻¹. Selected proteins were identified following mass spectrometry (nano-LC-MS/MS). Four enzymes involved in glycolysis (UDP-glucose pyrophosphorylase, fructose bisphosphate aldolase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase GAPDH) were specifically induced in somatic embryos. 11S globulin proteins identified by MS were present in high levels in somatic embryos, zygotic embryos and endosperm, whereas 7S globulins were detected mainly in endosperm and zygotic embryos. These are the first storage proteins identified in C. persicum. Xyloglucans are known to be another group of seed storage compounds in C. persicum. Interestingly, xyloglucan endotransglycosylases were found to be highly expressed in endosperm tissue. We discuss the physiological implications of these observations.     

Synthetic seed production from somatic embryos of Pinus radiata

Pinus radiata is one of the most important forestry species in the southern hemisphere. This work describes the regeneration of this plant via somatic embryogenesis from immature zygotic embryos. To improve this process, somatic embryogenic cell suspensions were established in liquid media for the generation of material for embryo maturation. Each developmental stage of these suspensions was characterized by microscopy and their growth phases quantified. An alginate-containing medium was used as an encapsulation method for the somatic embryos that were then germinated as artificial seeds in vitro. The protocols described in this work are both useful and reliable for industrial purposes.     

Anatomical study of zygotic and somatic embryos of Tilia cordata

A comparative anatomical study was carried out on zygotic and somatic embryos of Tilia cordata Mill. to evaluate the effect of growth conditions on their development. Zygotic embryos (heart-shaped, torpedo, cotyledonary), collected during two autumn periods, were examined to investigate the effect of growing season on embryo development. In comparison, the influence of growth conditions on the development of somatic embryos in vitro was also studied. Treatment with abscisic acid (ABA) and polyethylene glycol-4000 induced the development of somatic cotyledonary embryos similar to zygotic embryos with respect to morphology and anatomy, as illustrated by the differentiation of the apical meristems and of the procambium. The pattern of accumulation of starch and protein was also similar in these embryos. Somatic cotyledonary embryos that developed spontaneously without ABA showed defective accumulation of storage material and a general failure to form the shoot apical meristem, leading to very low germination rates. Vacuolar phenolic deposits were observed along the procambium of both zygotic and somatic embryos regardless of the maturation stage. Tracheid formation was observed only in somatic embryos formed without ABA in the medium and in precociously germinated somatic embryos. Phenolic vacuolar inclusions were frequently observed in epidermal cells of these embryos. This revised version was published online in June 2006 with corrections to the Cover Date.     

Artificial seeds of alfalfa (Medicago sativa L.). Induction of desiccation tolerance in somatic embryos

Summary The use of somatic embryos from cell culture systems in the clonal propagation of plants would be greatly facilitated if the somatic embryos could be dried and stored in a dormant state similar to true seeds. A cell culture system was developed for alfalfa (Medicago sativa L.) line RL34 which gave high yields of somatic embryos in an approximately synchronized pattern. These somatic embryos were treated with abscisic acid (ABA) at the cotyledonary stage of development to induce desiccation tolerance. With no visual preselection, approximately 60% of the dried embryos converted into plants upon reimbibition. When high quality embryos were selected prior to drying, 90 to 100% conversion rates were observed. The timing of the application of ABA in terms of embryo development was critical with an optimum being at cotyledonary stage spanning approximately 4 days; thus, synchronized embryo development is required for optimal expression in bulk samples. The vigor of the seedlings from dried somatic embryos was greater than those from embryos which had not been dried, but remained substantially lower than those from true seeds.     

Soluble carbohydrate content of soybean [Glycine max (L.) merr.] somatic and zygotic embryos during development

Summary Somatic and zygotic embryos of soybean cv. Jack were analyzed for soluble carbohydrate, total lipids, and protein during development. Zygotic embryos accumulated trace amounts of fructose, galactose, and galactinol., whereas somatic embryos contained only trace amounts of galactose. Somatic embryos accumulated much higher glucose levels than zygotic embryos. Both somatic and zygotic embryos contain low levels of sucrose, myoinositol, and pinitol. Raffinose and stachyose accumulated in the late developmental stages of zygotic embryos, but only stachyose was found to accumulate in the late stage somatic embryos. Zygotic embryos contained low total lipid levels up to 50 d after flowering (DAF) and then the levels increased to 16% by 55 DAF and 21% at 65 DAF. Somatic embryos had low levels of total lipids throughout development with the maximum of only 4.7%. Soybean zygotic embryos contained about 40% protein throughout development, while the protein concentration of somatic embryos decreased from 44% to 25% as maturation approached. These studies demonstrate that the composition of Jack zygotic embryos is similar to that described for other cultivars during development while the somatic embryo composition and size is markedly different. The low somatic embryo germination often noted might be due to the abnormal development as shown by a composition different from that of mature zygotic embryos. The low concentration of the raffinose series sugars might be especially important factors.     

Significance of the zygotic seed coat on quiescence and desiccation tolerance of Medicago sativa L. somatic embryos

Summary The influence of the zygotic seed coat on precocious germination and desiccation tolerance of somatic embryos has been studied using alfalfa (Medicago sativa L.). When cultured in contact with somatic embryos, seed coats at certain developmental stages inhibited precocious germination and induced desiccation tolerance in the somatic embryos. Germination of somatic embryos was inhibited by seed coats at the age of 16–26 days after pollination (DAP) and desiccation tolerance was induced after 20–26 DAP. Both phenomena were related to the synthesis of abscisic acid in the seed coat. The absence of a quiescent phase and desiccation tolerance in alfalfa somatic embryos may be related to the lack of developmental control by the seed coat.Abbreviations ABA Abscisic acid - DAP Days after pollination     

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

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 M 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 M 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.Abbreviations ABA abscisic acid - PEG polyethylene glycol - TAG triacylglycerol - TL total lipid - TEM transmission electron microscopy Plant Research Centre contribution No. 1383We are grateful to Dawn Moore and Ken Stanley for technical assistance, and thank Pat Rennie for the electron microscopy. We acknowledge financial support through an NSERC/Forestry Canada/Weyerhaeuser Canada Ltd (Prince Albert, Sask.) research partnership programme.     

Quantitative analysis of ultrastructural changes during zygotic and somatic embryogenesis in pearl millet (Pennisetum glaucum [L.] R. Br.)

Ultrastructural changes during zygotic and somatic embryogenesis in pearl millet (Pennisetum glaucum [L.] R. Br.) were quantified using morphometric techniques. The total area per cell profile and the cell volume percentage of the whole cell, endoplasmic reticulum (ER), Golgi bodies, mitochondria, nuclei, lipids, plastids, starch grains and vacuoles were measured and comparisons made between three zygotic and three somatic embryo developmental stages. All measurements were taken from scutellar or scutellar-derived cells. Zygotic embryogenesis was characterized by increases in cell size, lipids, plastids, starch, Golgi bodies, mitochondria and ER. Somatic embryogenesis was characterized by two phases of cell development: (1) the dedifferentiation of scutellar cells involving a reduction in cell and vacuole size and an increase in cell activity during somatic proembryoid formation and (2) the development of somatic embryos in which most cell organelle quantities returned to values found in late coleoptile or mature predesiccation zygotic stages. In summary, although their developmental pathways differed, the scutella of somatic embryos displayed cellular variations which were within the ranges observed for later stages of zygotic embryogenesis.     

Plant regeneration from encapsulated somatic embryos of pedunculate oak (Quercus robur L.)

Summary Cotyledonary Quercus robur L. somatic embryos from two cell lines were encapsulated in 4% (w/v) sodium alginate. An artificial endosperm was provided by the addition of P₂₄ medium plus 3% (w/v) sucrose. Oak somatic embryos and oak synthetic seeds were germinated on P₂₄ medium plus 0.1 μM indole-3-butyric acid and 0.9 μM 6-benzylaminopurine or were dehydrated prior to germination. The highest conversion rates (26%) were obtained with encapsulated somatic embryos as well as artificial endosperm-coated somatic embryos. Encapsulation improved the regeneration into oak plantlets in one of the two cell lines tested. The artificial endosperm had no additional beneficial effect on conversion frequency, but increased germination rate in one cell line tested. Significant higher conversion could be attributed to slow desiccation compared to the non-encapsulated control. Cold storage as a post-maturation treatment had no influence on the germination ability of oak synthetic seeds. Differences in the response of the cell lines with respect to conversion frequencies and timing of germination were observed. Fifty-six well-developed plantlets regenerated 12 wk after germination, and 29 plants were transferred to the greenhouse, where they have been successfully established in substrate.     

Polyamine levels during the development of zygotic and somatic embryos of Pinus radiata

Changes in the cellular content of three polyamines (putrescine, spermidine and spermine) were compared at different stages of development in zygotic and somatic embryos of Pinus radiata D. Don . During embryo development, both the zygotic and the somatic embryos showed a steady increase in spermidine content, with either a small decrease or no significant change in putrescine. This led to a several-fold increase in spermidine/putrescine ratios during development of both types of embryos. Cell cultures of plant-forming and non-plant-forming lines derived from the same clone and growing on proliferation (maintenance) medium differed significantly in their polyamine levels. Mature, cotyledonary stage somatic embryos capable of germination and formation of plants could be distinguished by their higher spermidine/putrescine ratios from abnormal cotyledonary stage somatic embryos which were incapable of forming plants.     

Comparative protein accumulation patterns in soybean somatic and zygotic embryos

Summary The relative maturity and competence of somatic embryos is often estimated on the basis of their morphologic similarity to various stages of immature zygotic embryo development. Morphologic abnormalities noted in soybean [Glycine max (L.) Merr.] somatic embryos are similar to those observed in zygotic embryos maturing in vitro and may reflect common interruptions of normal developmental processes. We provide here a more objective means of assessing the point(s) at which cultured embryos deviate from the normal embryogenical pathway by comparing the accumulation of the embryo-specific marker proteins (11S and 7S storage globulins, soybean agglutinin, and seed lipoxygenase) between somatic and immature zygotic embryos maturing in culture to zygotic embryos maturingin planta. Immature (heart-stage) soybean (cv. ‘McCall’) zygotic embryos were removed from the testa and cultured for 5, 15, or 45 days in nien modified Linsmaer-Skoog salts, 5% sucrose liquid medium. Somatic embryos were induced from immature cotyledon explants on a medium containing either naphthalene acetic acid or 2,4 dichlorophenoxyacetic acid (10 mg·liter⁻¹). The measured level of the marker proteins present in cultured embryos never exceeded those observed in mature soybean seeds. During the culture period, immature zygotic embryos accumulated significant levels of all marker proteins except a 29 kDa soybean agglutinin associated with the final stages of seed maturationin planta. Somatic embryos of all morphologic classes exhibited similar levels of the marker proteins suggesting that morphology may not accurately represent the developmental state of the culture-derived embryos. Somatic embryos induced on naphthalene acetic acid-containing medium accumulated detectable levels of all maturation-specific marker proteins except the 7S β and 29-kD soybean agglutinin antigen and seemed similar in most respects to the cultured zygotic embryos. Embryos induced on 2,4-dichlorophenoxyacetic acid accumulated none of the mature 7S or 11S storage globulin subunits nor any soybean agglutinin antigen, and yet the synthesis of 7S and 11S precursor polypeptides was similar in both naphthalene acetic acid-and 2,4-dichlorophenoxyacetic acid-induced somatic embryos. These observations are consistent with the view that embryos induced on high 2,4-dichlorophenoxyacetic are arrested at a relatively earlier developmental stage than naphthalene acetic acid-induced embryos of similar morphology and may indicate that some external signal (e.g., abscisic acid or desiccation or both) is necessary for the transition to the late maturation stage of seed ontogeny.     

Release of somatic embryogenic potential from excised zygotic embryos of carrot and maintenance of proembryonic cultures in hormone-free medium

Excised zygotic embryos, mericarps ("seeds") and hypocotyls of seedlings of cultivated carrot Daucus carota cv. Scarlet Nantes were evaluated for their ability to generate somatic embryos on a semisolid hormone-free nutrient medium. Neither intact zygotic embryos nor hypocotyls ever produced somatic embryos. However, mericarps and broken zygotic embryos were excellent sources for somatic embryo production (response levels as high as 86%). Somatic embryo formation was highest from cotyledons, but was also observed on isolated hypocotyls and root tips of mature zygotic embryos. On media containing unreduced nitrogen, somatic embryo formation led to the generation of vigorous cultures comprised entirely of somatic embryos at various stages of development which in turn proliferated still other somatic embryos. However, a medium was devised which when 1-5 mM NH4+ was the sole nitrogen source, led only to a proliferation of globular proembryos. Sustained subculturing of these proembryos at 2-3 week intervals enabled establishment of highly uniform cultures in which no further development into more mature stages of embryonic development occurred. These have been maintained, without decline, as morphogenetically competent proembryonic globules for over ten months. A basal medium containing from 1-5 mM NH4+ as the sole nitrogen source appears not to be inductive to somatic proembryo formation. Instead, such a medium is best thought of as permissive to the expression of embryogenically determined cells within zygotic embryos. By excising and breaking or wounding zygotic embryos, constituent cells are probably released from positional or chemical restraints and thus are able to express their innate embryogenic potential. Once a proembryonic culture is established, this medium containing 1-5 mM NH4+ as the sole nitrogen source provides a nonpermissive environment to the development and growth of later embryonic stages, but it does allow the continued formation and multiplication of globular somatic proembryos. The sequence of events leading from excised broken zygotic embryos to the formation of somatic embryos and the maintenance of somatic proembryos are demonstrated by scanning electron microscopy and histological preparations. Germination levels from intact zygotic embryos on media with varying levels and ratios of unreduced vs. reduced inorganic nitrogen were determined as well and provided baseline or control data on the type of response obtained from nonwounded material.     

Storage protein dynamics in zygotic and somatic embryos of white fir

Composition and accumulation patterns of storage proteins in female gametophyte and embryos of the white fir (Abies concolor) were investigated during embryogenesis and germination of mature seeds using SDS-PAGE and immunological approach. Altogether 9 major and minor protein components with molecular masses of 14, 16, 22, 24, 27, 30, 35, 38, and 43 kDa were detected in female gametophytes and 9 protein bands in the embryos with the molecular sizes of 14, 16, 22, 24, 25, 27, 34, 38, and 43 kDa. The species seems to deviate in this respect from other representatives of Pinaceae. A conspicuous increase of storage protein synthesis was observed at the stage of fully cellularized female gametophytes and at the cotyledonary stage of embryo development. There exists a high degree of similarity between storage protein profiles of white fir zygotic and somatic embryos. Successive stages of somatic embryogenesis exhibited a high degree of similarity of storage proteins except for cotyledonary stage when a noticeable increase in storage protein synthesis was registered. Conversely, during germination of somatic embryos, an overwhelming majority of storage proteins was depleted.     

Water relations parameters and tissue development in somatic and zygotic embryos of three pinaceous conifers

There is increasing interest in using somatic embryogenesis to meet the demand for high quality seedlings. However, in vitro production of propagules on a large scale depends on the optimization of the maturation and germination steps promoted by desiccation and subsequent imbibition of the embryo, respectively. It is therefore important to characterize zygotic and somatic embryos in terms of their water relations. Bound water, elastic modulus, osmotic potential at full turgor, and relative water content at turgor loss point were determined for somatic and zygotic embryos of western larch and loblolly pine and somatic embryos of white spruce at two developmental stages. These water relations parameters were derived from water-release curves obtained by suspending tissue samples in sealed vials over unsaturated NaCl solutions of known water potential. There was little difference in water relations parameters among species but marked stage dependency for bound water and elastic modulus. The amount of bound water was lowest in western larch somatic embryos (0.02-0.07) and highest in zygotic loblolly pine embryos (0.10-0.18). Elastic modulus ranged from 2.5 to 6.2 MPa in somatic embryos but varied between 1.4 and 1.8 MPa in zygotic embryos. The osmotic potential was lower in somatic embryos than in their zygotic counterparts. Our results show that water relations parameters are remarkably conservative across species but that, within a given species, these parameters are stage specific. It would seem, therefore, that desiccation protocols might be developed for a given developmental stage and applied across a range of species without the confounding effects of differences in water relations parameters.     

Development of white spruce somatic embryos: I. Storage product deposition

Summary The growth and development of white spruce somatic embryos was followed from the filamentous immature to the mature cotyledonary embryo stage. Histochemical examination of the various stages of embryo development showed that lipids, proteins, and polysaccharides were produced to varying degrees during the process. During early stages (1 to 2 wk on ABA), mostly polysaccharide was produced, whereas during later stages, polysaccharides, lipids, and protein accumulated. Electron microscopy indicated that lipid deposition in somatic embryos started during the first week after transfer to ABA-containing medium. Deposition of the storage products began at the basal end of the embryonal mass and within the proximal zone of the suspensors. Accumulation continued to the peripheral regions and then inward toward the cortex of the developing embryo. In all cases, polysaccharide accumulated first, followed by lipid and lastly, protein. Quantitatively, cotyledonary stage somatic embryos had less lipid and protein and more starch when compared to zygotic embryos at the same developmental stage. Total protein profiles elucidated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the majority of proteins were similar in zygotic and somatic embryos. Prominent protein bands were found at 30, 20, 19.5, 15, 14.4, 12, and 10 Kd. However, protein bands at 40, 15, and 12 Kd in total protein from somatic embryos were either absent or highly underexpressed.