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.     

Lauric acid improves the growth of zygotic coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.) embryos in vitro

Coconuts (Cocos nucifera L.) germinating in situ make use of both sucrose and fatty acids, notably lauric acid, as sources of energy and carbon, but current tissue culture methods routinely include only sucrose in the culture medium. The aim of the experiments was to establish whether lauric acid could improve the growth and development of zygotic coconut embryos in culture. The culture medium of zygotic embryos was adjusted to give various concentrations of sucrose and lauric acid. The concentration of free lauric acid was increased at specific times of the culture. At the end of the experiments, plantlet growth was measured. Added at day zero, lauric acid inhibited germination. Added at 60 or 75 days, lauric acid (75 μM, unbound concentration) showed a marked stimulation of plantlet growth and development. When ¹⁴C-labelled lauric acid was used, radioactivity was incorporated mainly into longer chain fatty acids of complex lipids, notably of the phospholipid fraction. Supplementation at these times may mimic conditions in situ and suggests that the supply of fatty acids may represent a physiological requirement for continued growth. The experiments with radioactive lauric acid confirm that it provides carbon for the synthesis of new structural lipids. The method may provide a means of improving the development of coconut somatic embryos in future.     

Storage Lipid Accumulation by Zygotic and Somatic Embryos in Culture

In vitro accumulation of storage lipids occurs in zygotic andsomatic embryos of Brassicu napus L. The concentration of sucrosein the medium modified the pattern of storage lipid accumulationin zygotic and somatic embryos. The sucrose concentration atwhich the maximum amount of lipid is accumulated by the twotypes of embryos is different Analysis of fatty acid compositionshowed that the same fatty acids are present in embryos in vivoand those cultured in vitro although there are quantitativedifferences. The possibility of using this type of system forin vitro production of valuable plant metabolites is discussed Embryo cloning, somatic embryogenesis, in cilro culture, storage lipids, Brussica napus, oilseed rape     

Sucrose requirements and lipid utilization during germination of interior spruce (Picea glauca engelmannii complex) somatic embryos

Both somatic and excised zygotic embryos of interior spruce (Picea glauca engelmannii complex) required exogenous sucrose in the medium for germination in vitro. Over a period of 29 days on sucrose-containing medium germinants with roots and epicotyls developed from both kinds of embryo, and their content of linolenic acid (9,12,15-18:3) increased about six- to eightfold. Without added sucrose, embryos showed retarded growth or were necrotic, and the content of linolenic acid was barely detectable in their fatty acid profiles. Through¹⁴C-sucrose uptake studies, it was determined that germinants consumed only 25% of the sucrose available in a 1% (wt/vol) sucrose-containing medium. Since no radiolabelled fatty acids were detected, it appears that externally supplied sucrose was not used in the synthesis of lipids. Although sucrose was present during plantlet development, 72% of the initial lipids were consumed. To some extent, the plantlets appeared to be obligate storage lipid utilizers.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - FAMEs Fatty acid methyl esters - HPLC High-performance liquid chromatography     

Fatty acid changes during development of zygotic and microspore-derived embryos of Brassica napus

Cultured microspores of Brassica napus L. cvs Topas and Reston initiated cell divisions within 3 to 4 days, and globular, heart and torpedo shaped embryos were prevalent after approximately 6, 8, and 10 days, respectively. Embryos with rudimentary cotyledons were evident within 2 weeks, but those that reached this stage of development represented only 1–5% of the original microspore population. The fresh weight of microspore-derived embryos at all stages of development was significantly greater than that for zygotic embryos, but the pattern of change in fresh weight and fatty acid accumulation was similar in developing zygotic and microspore embryos. In freshly isolated microspores of both Topas (low erucic acid) and Reston (high erucic acid), the predominant fatty acid was 18:3, while 18:1 comprised less than 15% of total fatty acids. During development in both zygotic and microspore embryos, the level of 18:3 declined markedly while 18:1 rapidly increased. Erucic acid (22:1) was not detected in the early stages of embryogenesis in Reston. However, small amounts of 22:1 appeared by early cotyledonary stage and the level gradually increased in both zygotic and microspore embryos through the later stages of development. The fatty acid compositions of mature embryos was nearly identical to that of dry seed, except the level of 22:1 in Reston embryos was consistently less than in the seed. Triacylglycerols comprised only 15% of total lipids in freshly isolated microspores, but increased to more than 90% by 4 weeks. The fatty acid composition of the triacylglycerol fraction was generally similar to that of total lipids at all stages of development of microspore-derived embryos.     

The microspore-derived embryo ofBrassica napus L. as a tool for studying embryo-specific lipid biogenesis and regulation of oil quality

Summary A time-course study of lipid accumulation in microspore-derived embryos and developing zygotic embryos of rapeseed (Brassica napus L. ssp.oleifera) is presented. Rapid storage fat (triacylglycerol) biosynthesis was induced in microspore-derived embryos of oilseed rape (cv Topas) when the embryos were transferred from standing cultures (10 ml) to fresh medium (75 ml) and shake cultured. Triacylglycerols accumulated, after a lag period of 7 days, at a linear rate of approximately twice that of the developing zygotic embryo. The fatty acid composition of triacylglycerols in microspore-derived embryos closely parallelled that of the developing zygotic embryos. In the microspore-derived embryos, the amount of phosphatidylcholine, the major substrate for the production of polyunsaturated fatty acids in oilseeds, remained constant during the linear phase of triacylglycerol production, whereas it increased steadily in the zygotic embryos. The fatty acid composition of individual cotyledons from microspore embryos shake cultured for 15 days was compared with that of individual mature seeds. Relative amounts of the major fatty acids, i.e. palmitic, oleic and linoleic acids, were essentially the same, whereas the microspore-derived embryos had about 35% less stearic acid and 35% more linolenic acid than the mature seeds. Variation in the amounts of oleic, linoleic and linolenic acids between seeds was similar to that found between cotyledons of microspore-derived embryos, whereas variation in palmitic and stearic acid levels was significantly lower between microsporederived cotyledons than between the seeds. The results indicate that microspore-derived embryos from shake cultures should be convenient for use in studying the regulation of oil biosynthesis and for rapidly screening for oil quality in genetically altered rapeseed.     

Water and sucrose regulate canola embryo development

The effect of water and sucrose on the growth and development of zygotic, 30-day-old canola ( Brassica napus L. cv. Bounty) embryos was examined in vitro by manipulating the levels of sucrose and/or sorbitol present in the culture medium. In some experiments, the medium water potential was allowed to vary with sucrose concentration, while in other experiments, the medium water potential was held constant by adding sorbitol to varying amounts of sucrose. Our results showed that embryos cultured on sorbitol alone exhibited two developmental patterns: embryos germinated precociously on media containing up to 0.70 M sorbitol, whereas embryos became yellow and quiescent on media with higher concentrations of sorbitol. For embryos cultured on media containing sucrose alone, three distinct developmental patterns were noted: at low sucrose concentrations, embryos germinated precociously; at intermediate concentrations, embryos continued to grow in an embryonic mode; and, at high concentrations, embryos became yellow and quiescent. Continued embryonic growth was never observed in embryos cultured on media containing sorbitol alone. Embryos never germinated precociously when cultured on media maintained at a constant water potential of -1.4 MPa, rather dry weight increased in these embryos with an increase in sucrose concentration. We envision the effect of sucrose on embryo growth and development to be nested within the effect of water availability. When water availability is restricted, embryos become quiescent. When water is available, embryos have the potential to grow, but the developmental growth pattern depends on the availability of sucrose. In the absence of sucrose, embryos germinate and initiate the transition to autotrophy. If sufficient sucrose is available, embryos remain photohet-erotrophic and continue to grow in an embryonic mode.     

Cryopreservation of immature spring wheat zygotic embryos using an abscisic acid pretreatment

Spring wheat (Triticum aestivum L.) zygotic embryos were successfully cryopreserved, without the addition of exogenous cryoprotectants, using only an abscisic acid (ABA) pretreatment. Optimum survival was obtained when embryos were cultured in vitro for 10 days on semisolid Murashige and Skoog (MS) nutrient medium supplemented with 0.5 mg/L (±) ABA prior to cryopreservation. The embryos resumed growth within three days when returned to MS medium devoid of ABA but containing 2mg/L 2,4-dichlorophenoxyacetic acid. The embryogenic calli produced from these embryos exhibited normal plant regeneration on auxin-free media. Changes in dw/fw ratio, as well as the esterified fatty acid and sucrose concentrations correlated positively with the development of tolerance to cryopreservation.NRCC Publication No. 33519     

Enhancement of somatic embryogenesis in camphor tree (Cinnamomum camphora L.): osmotic stress and other factors affecting somatic embryo formation on hormone-free medium

The aim of this study was to improve the direct somatic embryogenesis and initiate embryogenic callus formation in camphor tree (Cinnamomum camphora L.) on hormone-free medium. The influence of osmotic stress pretreatment of immature zygotic embryos (0.5 and 1.0 M solution of sucrose for 12, 24, 48, 72, 96, 120, and 144 h at 4 or 25°C) before cultured on hormone-free medium, on embryogenesis efficiency was assessed. The embryogenesis frequency was improved from 16.29 to 93.27%, while the average number of somatic embryos per explant increased from 3 to 12.57. Activated charcoal (AC), medium renewal, basal medium, light conditions and sucrose concentration in culture medium were also evaluated for their effect on somatic embryogenesis. AC addition and 10-day medium renewal did not increase embryogenesis efficiency significantly, and Murashige and Skoog (MS) medium proved to be more beneficial for somatic embryo formation than others. No differences were found between embryogenesis frequencies when cultured in darkness or under light, but culturing under light yielded more embryos. After the sucrose solution pretreatment, high level concentration of sucrose in induction medium was not needed for somatic embryogenesis, for it had a negative effect on somatic embryo formation when the concentration of sucrose was higher than 50 g l⁻¹. The derived embryogenic lines were maintained via repetitive embryogenesis on hormone-free medium. Low ratio formation of embryogenic callus was observed on the surface of somatic embryos both on induction and proliferation medium. Plantlets derived from somatic embryos grew vigorously with normal appearance similar to germinated zygotic embryos.     

Induction of adventitious shoots or somatic embryos on in vitro cultured zygotic embryos of Helianthus annuus: Variation of endogenous hormone levels

The induction of both shoots and somatic embryos from the same cells of immature zygotic embryos (IZE) of sunflower (Helianthus annuus L.) is dependent on the sugar concentration of the induction medium. With the same concentration of benzyladenine (BAP), shoots are induced at a sucrose concentration of 3 %, while somatic embryos will develop at 12 % sucrose. To understand the role played by endogenous hormones, we have investigated the temporal variation of their levels during culture of IZE on caulogenic or embryogenic medium. The BAP taken up from the medium reaches a maximum concentration in the explants at 6–15 h of culture. When the IZE were cultured under caulogenic conditions, the endogenous concentration of BAP was 2-fold higher than in an IZE cultured on embryogenic medium. Inversely, the concentration of indolyl acetic acid (IAA) induced by the exogenously added BAP was 4-fold higher in explants cultured on embryogenic than in those cultured on caulogenic medium. The IAA concentration peaked in the sample taken at 24 h, the time when the responding cells become determined towards one or the other morphogenic reaction. The IAA/BAP ratio was higher in the IZE induced to embryogenesis compared to those that gave rise to shoots. The effect of abscisic acid (ABA) on the morphogenic process appears to be indirect and can be explained by a modification of the IAA content.     

Abscisic acid and proline improve desiccation tolerance and increase fatty acid content of celery somatic embryos

Desiccation tolerance of celery (Apium graveolens L.) somatic embryos was increased by supplementation of embryo-production medium with 1 M abscisic acid (ABA) or 1 mM proline, with highest survival obtained with a combination of 1 M ABA and 1 mM proline. Addition of ABA and proline increased fatty acid accumulation by somatic embryos; the effect on fatty acid composition was inconsistent. Somatic embryos capable of germination differed from mature zygotic embryos by greater size, lower fatty acid level, and substantially lower proportion of oleic acid (18:1) as compared to linoleic acid (18:2).     

Developmental plasticity of glandular trichomes into somatic embryogenesis in Tilia amurensis

BACKGROUND AND AIMS: In Tilia amurensis, two types of trichomes (hairy and glandular) develop from epidermal surfaces of cotyledons and hypocotyls of zygotic embryos soon after germination. Here, it is demonstrated that glandular trichome initials develop directly into somatic embryos when treated in vitro with 2,4-dichlorophenoxyacetic acid (2,4-D). METHODS: Zygotic embryos of Tilia amurensis were cultured on Murashige and Skoog medium with 3 % sucrose and various concentrations (0, 2.2, 4.4 and 8.8 microm) of 2,4-D. Morphological development of trichomes and somatic embryos was analysed by scanning electron microscope and light microscope after histological sectioning. KEY RESULTS: In zygotic embryos cultured on medium with 4.4 microM 2,4-D, formation of hairy trichomes was completely suppressed but formation of glandular trichome initials increased. That some filamentous trichome initials developed directly into somatic embryos was confirmed by histological and scanning electron microscope observation. When explants with different stages of trichome initials (two-, four- and eight-celled filamentous and fully mature trichomes) were temporally pre-treated with 4.4 microM 2,4-D for 24 h and transferred into hormone-free medium, two-celled and four-celled filamentous trichome initials were the effective stage of trichomes for somatic embryo induction. CONCLUSIONS: It is suggested that early developing filamentous trichome initials have developmental plasticity and that with 2,4-D treatment these trichome initials develop directly into somatic embryos.     

Lipid composition of somatic and zygotic embryos from Prunus avium. Effect of a cold treatment on somatic embryo quality

In order to evaluate the quality of Prunus avium somatic embryos, a comparison of lipid composition between somatic and zygotic embryos was undertaken. In both zygotic and somatic embryos, neutral glycerolipids (NL) and phosphatidylcholine (PC) were the 2 major lipid classes. The content of NL increased over the course of development in zygotic embryos and reached 490 μg per embryo, while the PC content reached 100 μg per embryo. However, the contents of NL and PC in somatic embryos were similar to immature zygotic embryos at stage 3. Fatty acid composition of NL from both zygotic and somatic embryos revealed more unsaturated than saturated fatty acids. In somatic embryos, the saturated/unsaturated fatty acid ratios of NL and phosphatidylinositol (PI) were similar to those observed in immature zygotic embryos up to stage 6. Conversely, in phosphatidylethanolamine (PE) the ratio was similar to the ratio observed in mature zygotic embryos, at stage 7. Histological studies confirmed the immaturity of somatic embryos: no protein or lipid reserves were observed in the vacuolated cotyledonary cells. Maturation of somatic embryos was improved by a 2-month cold period. In cold-treated somatic embryos, both NL and PC increased to levels comparable to those observed in mature zygotic embryos, and the PE content reached 10 times the level of that in mature zygotic embryos. The cold treatment induced a large increase in the saturated/unsaturated fatty acid ratio in phospholipids but only a slight increase in that of neutral glycerolipids. Histological studies revealed a lipid accumulation at cellular level. Lipid bodies surrounded by protein bodies were observed in cotyledonary cells of cold-treated somatic embryos. Furthermore, the cold-treated somatic embryos developed into plantlets with a frequency of 14%, whereas no development was obtained with the non-treated somatic embryos.     

Somatic embryogenesis of two new <Emphasis Type="Italic">Panax ginseng</Emphasis> cultivars,Yun-Poong and Chun-Poong

Somatic embryogenesis from single cells is important for normal plant regeneration of ginseng. Cotyledon explants from zygotic embryos of two new ginseng cultivars, Chun-Poong and Yun-Poong, produced somatic embryos on Murashige and Skoog (MS) basal medium and MS medium containing growth regulators. The highest frequency of single somatic embryo formation was obtained when cotyledon explants were excised from premature (cultured for 1 day) zygotic embryos (about 6 mm in length) of both cvs. Chun-Poong and Yun-Poong and then cultured on MS medium supplemented with 7% sucrose. The frequency of single somatic embryo formation was strongly enhanced when Chun-Poong cotyledons were subjected to plasmolysis with 0.1–0.5 M sucrose for 24 h and Yun-Poong cotyledons to plasmolysis with 1.0 M sucrose for 24 h and then cultured on MS medium with 2,4-D.     

Changes of lipid composition in non-cultured and cultured porcine embryos

The objective of the present study was to evaluate the lipid composition of cultured and non-cultured pig embryos during cleavage using histochemical methods. The authors studied pig zygotes as well as 2-to 4-cell embryos, morulae, and blastocysts that were either non-cultured or cultured in NCSU-23 medium. To detect different types of lipids, the authors used the Churukian method with Oil red O, the Sudan black B method, the Cain method with Nile blue sulfate, and the modified osmium tetroxide-ethanol treatment. In the zygotic lipid droplets, diverse classes of unsaturated and saturated lipids were found, with particularly high levels of unsaturated hydrophobic lipids, mainly triglycerides and other esters, free fatty acids, and phospholipids. In the zygotic cytoplasm, the authors observed high levels of fatty acids and phospholipids. The total lipid content remained constant up to the morula stage, decreasing later at the blastocyst stage, but the overall amount of unsaturated lipids declined earlier, at the 2-to 4-cell stage. The amount of free fatty acids and phospholipids decreased during cleavage in both non-cultured and cultured porcine embryos. The main differences between the non-cultured and cultured embryos were the more pronounced reduction in the amount of unsaturated hydrophobic lipids in droplets and the cytoplasmic free fatty acids observed in the cultured morula and the lower content of phospholipids in the cytoplasm of the cultured 2-to 4-cell embryos relative to the non-cultured embryos. The decrease in the unsaturated lipid, free fatty acid, and phospholipid content during in vivo development and the differences in the amount of these types of lipids between developmentally matched cultured and non-cultured pig embryos correlate well with modifications of lipid and carbohydrate metabolism.     

Fatty acid composition of membranes of L-forms ofStreptococcus faecalis andProteus mirabilis cultured at different osmolalities

The fatty acid composition of membranes of L-forms ofStreptococcus faecalis andProteus mirabilis cultured at different osmolalities and in different osmotic stabilizers was examined.S. faecalis L-forms cultured with sucrose in the medium showed a decrease in the unsaturated fatty acid C₁₈₁ and an increase in the C₁₈ fatty acid and C₁₉ cyclopropane fatty acid. Fatty acid composition ofS. faecalis L-forms cultured in medium containing 1.8% NaCl was similar to the fatty acid composition of L-forms cultured in brain-heart infusion broth (BHI) without osmotic stabilizer and was between the composition of fatty acids of L-forms in BHI with sucrose and that in BHI without 0.5 M sucrose. InProteus mirabilis L-forms, there were differences between L-forms cultured with and without sucrose, but these differences were not comparable to the changes observed inS. faecalis L-forms.P. mirabilis L-forms cultured with and without NaCl in the medium had similar fatty acid compositions.     

Changes in fatty acid composition during development of tissues of coconut (Cocos nucifera L.) embryos in the intact nut and in vitro.

Intact coconuts were germinated in situ and compared with excised zygotic embryos germinated in vitro. The growth of the embryonic tissue and their fatty acid compositions were measured. Haustoria, plumules and radicles of coconuts germinated in situ grew continuously and proportionately throughout the 120 d experiment with haustauria increasing to 45 g x nut(-1) and weighing 4-5-fold more than the other two tissues. The plumules and radicles of the seedlings cultured in vitro also grew continuously but the haustoria grew sporadically between 15 d and 75 d in culture and, at 250 mg x nut(-1) after 75 d, were smaller than the other two tissues. All the tissues of the nuts grown in situ contained significant amounts of lauric acid, the acid characteristic of coconut oil, as well as longer chain saturated and unsaturated fatty acids. The content of medium and long chain fatty acids increased in all growing tissues as the experiment proceeded, especially the haustorium which contained 24-35% of its fatty acid as lauric acid; the fat content of solid endosperm reduced during this period. Seedlings grown in vitro, on the other hand, failed to accumulate lauric acid in any of their tissues (haustorium contained 6-11% of its fatty acid as lauric acid). The results may have implications for the design of growth media for growing zygotic and somatic cultures of coconut and may provide a marker for successful germination.     

In vitro zygotic embryo germination and propagation of an endangered Boswellia serrata Roxb., a source of boswellic acid

This study aims to establish an efficient protocol for development of seedlings of an endangered medicinally important forest tree Boswellia serrata Roxb., for mass plantation and consistent supply of salai guggul. The green mature fruits served as source of seeds. The excised green zygotic embryos were cultured on Gamborg (B5), McCown and Loyd (WPM) and Schenk and Hildebrandt (SH) media fortified with different concentration of sucrose and on Murashige and Skoog (MS) medium containing 3 % sucrose, polyvinylpyrrolidone (PVP) (0–300 mg l^−l), Gibberellic acid (GA₃), Indoleacetic acid (IAA), Naphthaleneacetic acid (NAA), Indole-3-Butyric acid (IBA) or 2,4-dichlorophenoxyacetic acid (2,4 D) and 6-benzylaminopurine (BA) or kinetin (Kin) individually. The highest frequency of embryo germination (96 %) and conversion into seedling was obtained on MS medium containing 3 % sucrose together with 200 mg l^−l PVP; other media were either inferior or induced abnormalities in the seedlings including callus formation from the zygotic embryos. Fully developed seedlings could be successfully established in soil with about 94 % survival. The embryos from mature dry seeds did not respond for germination in any of the experiments. In conclusion, selection of zygotic embryo from green mature seeds and their in vitro germination is important for propagation of B. serrata.     

Effects of cholesterol on viability and (n - 6) fatty acid metabolism in cultured human monocyte-like cells (U937).

Effects of supplementation of growth-promoting cholesterol on metabolism of the cytotoxic (n - 6) polyunsaturated fatty acids in cultured human monocyte-like cells (U937) have been examined. U937 cells were incubated in 5% delipidated fetal bovine serum containing 0 or 38.7 microM cholesterol. The rate of uptake and the distribution of metabolites of (n - 6) fatty acids (such as 18:2(n - 6), 18:3(n - 6), and 20:3(n - 6), and 20:4(n - 6)) were examined by adding radiolabelled fatty acid at a level of 1 microgram/mL (3.3 microM for 20-carbon fatty acids and 3.6 microM for 18-carbon-fatty acids). For assessing the cytotoxicity, (n - 6) fatty acids were added to medium at a concentration of 5 micrograms/mL (16.4 microM for 20-carbon fatty acids and 17.9 microM for 18-carbon fatty acids). Cholesterol supplementation suppressed the uptake of all (n - 6) fatty acids and reduced the cytotoxic effects of 18:2(n - 6), 20:3(n - 6), and 20:4(n - 6), but not 18:3(n - 6). In addition, cholesterol supplementation increased peroxide production and metabolism of (n - 6) fatty acids in U937 cells. Thus, the differential suppressive effect of cholesterol on the cytotoxicity of different fatty acids could not be attributed to an inhibitory effect on fatty acid delta 6- and delta 5-desaturation, or to an antioxidant effect on peroxide formation.     

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.