A quantitative nature of somatic embryogenesis in soybean

Summary The objectives of this research were to investigate the genetic parameters associated with the in vitro formation of somatic embryos in soybean and to determine the effect of light intensity on the embryogenic capability of F₁, F₂, and backcross (RC₁P₁ and RC₁P₂) progenies derived from crosses between embryogenic (IAS-5 and Embrapa-1) and non-embryogenic (Paraná) cultivars. Immature cotyledons (4–6 mm in length) derived from the parental lines, F₁, F₂, RC₁P₁, and RC₁P₂ were grown for 90 d on the inductive N10 medium, after which the number of somatic embryos was recorded. Chi-square tests for goodness of fit showed that the genetic component of the somatic embryogenesis trait is controlled in a quantitative manner by approximately 10 genes. A normal distribution for somatic embryo formation in the F₂ generations was observed reinforcing the quantitative nature of the trait. Variation in light intensity (8–12 and 27–33 μmol m⁻²s⁻¹) had no effect on somatic embryo formation in the parental material tested.     

Somatic embryogenesis in soybean via somatic embryo cycling

Summary The objectives of the present research were: a) to develop an efficient soybean embryogenic regeneration system characterized by a high frequency of explant response and a large number of somatic embryos per explant; b) to evaluate the factors affecting somatic embryogenesis via somatic embryo cycling; and c) to identify the origin of somatic embryos in the system. A highly improved and efficient system for soybean somatic embryogenesis was established using somatic embryo cotyledons and somatic embryo hypocotyl/radicle explants plated on α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented MS basal media. The system included somatic embryo cycling between liquid and solid medium and it consistently gave rise to a much higher frequency of explant response and a larger number of embryos per responding explant than those obtained from zygotic cotyledon explant tissues. Genotype, differences were observed for response in some of the treatments with cv “Fayette” being more responsive than “J103”. Histological studies revealed that somatic embryos induced in the somatic embryo cycling system originated almost exclusively from epidermal cells on both 2,4-D and NAA inductive media. The cells of the epidermis proliferated to produce somatic embryos directly without an intervening callus phase. A single-cell origin of somatic embryos was observed in cultures on a 40 mg/liter 2,4-D treatment. A large number of responding cells in the epidermis was also observed in the 10 mg/liter NAA treatment. The single-cell origin of somatic embryos from epidermal layers of the explant tissues should facilitate development of an efficient transformation system for soybean.     

Optimization of somatic embryogenesis and embryo germination in soybean

Summary Somatic embryos of soybean [Glycine max (L.) Merr.] are induced on immature cotyledons explanted onto a medium containing moderately high levels of auxin. Germinability of embryos is related to morphologic normality, and both are reduced by excessive exposure to auxin during the induction process. Shoot meristem development was improved by reducing exposure of cotyledonary explants from 30 to 10 to 14 d on 10 mg/liter α-naphthaleneacetic acid (NAA). A 3-d exposure was sufficient to induce embryos, and embryo frequency was not significantly increased by exposures to NAA for more than 1 wk. Embryo frequency was enhanced, however, by transfer after 9 d to fresh medium containing 10 mg/liter NAA. Germination of morphologically normal embryos was achieved without growth regulators, after maturation for 1 mo. on hormone-free medium and desiccation for 1 wk in a sealed, dry container. This research was funded by Lubrizol Genetics, Inc., Madison, WI.     

Changes in protein profiles associated with somatic embryogenesis in peanut

The somatic embryogenesis potential of zygotic embryo axes of peanut (Arachis hypogaea L. cv. DRG-12) at different stages of development was evaluated by culturing on MS medium with 18.1 μM 2,4-dichlorophenoxyacetic acid (2,4-D). A 100 % frequency with 18.3 somatic embryos per explant was observed from 4 mm long immature zygotic embryo axes collected 31 – 40 d after pollination. Medium supplemented with 16.6 μM picloram resulted in slow development of somatic embryos whereas in the presence of 21.5 μM α-naphthaleneacetic acid (NAA), the explants underwent maturation with induction of roots after 30 d. The changes in protein profiles in zygotic embryo axes at different stages of development correlated with their potential to form somatic embryos. Immature zygotic embryo axes exhibited high frequency somatic embryogenesis in the stage preceding abundant accumulation of 22 and 65 kDa proteins. The content of 22 and 65 kDa proteins decreased immediately after culture on medium fortified with 18.1 μM 2,4-D and increased again after 12 d of culture coinciding with the development of somatic embryos on the explants. The content of 22 and 65 kDa proteins was low at 15 d of culture on medium supplemented with 16.6 μM picloram possibly due to slow development of the somatic embryos on the explant. On maturation medium containing 21.5 μM NAA, a marked increase in the content of 22 and 65 kDa proteins in 15 d-old cultures was observed.     

Isozyme patterns in zygotic and somatic embryogenesis of carrot

Isozyme patterns of carrot (Daucus carota L.) zygotic embryos between the torpedo stage up to 5-day-old seedlings have been compared with those of the similar stages from the embryogenic cell suspension culture to the late somatic plantlet. Somatic embryos blocked at the torpedo stage by -cyclodextrine have also been analyzed. All these stages have been analyzed with respect to seven different enzyme systems: arylesterase, glucosephosphate isomerase, phosphogluconate dehydrogenase, alcohol dehydrogenase, isocitrate dehydrogenase, aspartate aminotransferase and phosphoglucomutase (EC 2.7.5.1, PGM). The relationships between the different stages of both types of embryogenesis have been visualized using an unrooted tree. Generally, profiles of somatic embryos were different from those of zygotic embryos. Interestingly however, a typical zygotic embryo pattern was found in the cyclodextrine-blocked somatic embryos. Only aspartate aminotransferase patterns revealed a similarity between zygotic and somatic torpedo embryos. Both plantlet types showed close patterns with common isozymes. Moreover, similarities were evident between somatic plantlets and cell suspensions. A few isozymes appeared to be stage specific markers: esterase 10–11 were specific to achenes and early germination, phosphogluconate dehydrogenase 8 was specific to 4–5 day-old seedlings and phosphoglucomutase 1 and 7 and alcohol dehydrogenase 4 were markers for zygotic embryos. No somatic embryogenesis specific isozyme could be found. We show that patterns can be associated with particular tissue formation: mainly, aspartate aminotransferase 2 and 1, phosphoglucomutase 8 and 9 and phosphogluconate dehydrogenase 7 coincided with apical meristem initiation and phosphoglucomutase 4 and 5, zones b and d of esterase and zone b of phosphogluconate dehydrogenase coincided with vascular bundle formation.Abbreviations ADH alcohol dehydrogenase - CD -cyclodextrine - CS cell suspension culture - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediaminetetraaeetie acid - LiBo lithium hydroxide/boric acid - PEG polyethylene glycol - PVP polyvinylpyrrolidone - SEg somatic embryo at the globular stage - SEh heart stage - SEte early torpedo stage - SEtl late torpedo stage - SEce early cotyledonary stage - SEcl late cotyledonary stage - SECD somatic embryo blocked at the torpedo stage with -cyclodextrine - EST esterase - GOT aspartate aminotransferase - IDH isocitrate dehydrogenase - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) - PMS phenazonium methosulfate - PGD phosphogluconate dehydrogenase - PGI glucosephosphate isomerase - PGM phosphoglucomutase - SO dry seed - S1–3 seed after 1–3 days of germination - SP1–2 young and old somatic plantlets - ZE zygotic embryo - ZP4–5 4–5 day-old seedlings     

Auxin-orientation effects on somatic embryogenesis from immature soybean cotyledons

Summary Development of somatic embryos of soybeanGlycine max (L.) Merr. has been studied using cultivars J103 and McCall and five auxin-sucrose treatment: naphthalene acetic acid at 10 mg/liter with 1.5% sucrose (N10); 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.25, 0.5 or 1.0 mg/liter with 1.5% sucrose (D.25, D.5 D1); and 2,4-D at 25 mg/liter with 3% sucrose (D25). Cotyledons were excised aseptically from immature embryos 3 to 5 mm in length, and placed on the media with either the adaxial or abaxial side down (adaxial or abaxial orientiation, respectively). After 30 d, numbers of normal or total somatic embryos, or both, were counted. For J103 explants on D25 or N10 media, the greatest number of embryos was produced from the central region of abaxially oriented explants on D25, but only 5% of these embryos were normal. The greatest number of normal embryos was produced from the marginal region of adaxially oriented explants on D25. For McCall explants on D.25, D.5, D1, or N10 media, the greatest numbers of normal embryos were produced from the marginal regions of abaxially oriented explants on D1 or N10. Embryo induction was examined histologically for the N10, D25, and D.5 treatment, using serial section of paraffin-embedded cotyledons stained with hematoxylin and safranin. On N10 medium, embryos were produced unicellularly, or more frauently, multicellularly, from homogeneous embryogenic tissue arising from epidermal and subepidermal cells at the distal periphery of the cotyledon. On D25 medium, embryos were produced multicellularly from a geterogeneous embryogenic tissue formed in the central region of the cytoledon, and consisting of embryogenic cells interspersed with large, vacuolate cells. The D.5 treatment showed an intermediate response. Embryo initiation in this soybean system is predominantly multicellular. This work was supported in part by a grant from Lubrizol Genetics, Inc., and by the Agricultural Experiment Station, University of Kentucky. Paper number 88-3-13 from the Kentucky Agricultural Experiment Station.     

Identification of a RAPD marker associated with somatic embryogenesis in alfalfa

The current study was conducted to identify random amplified polymorphic DNA (RAPD) markers linked to genes controlling somatic embryogenesis in alfalfa. Segregation analyses of the somatic embryogenesis trait and the RAPD markers in an F₁ population of 83 plants, derived from a cross between embryogenic A70-34 and non-embryogenic Arrow36 alfalfa plants, identified a polymorphic band that is associated with somatic embryogenesis. Based on the assumptions that somatic embryogenesis in alfalfa is controlled by two dominant genes with complementary effects and that the genotypes of A70-34 and Arrow36 are AAaaBbbb and aaaabbbb, respectively, the segregation data for the marker and the somatic embryogenesis trait in the F₁s indicate that the marker is linked to the A locus. The maximum recombination fraction estimated for the linkage between the marker and the gene is 36.3%.     

Comparison of somatic embryogenesis and embryo conversion in commercial soybean cultivars

Six commercially important soybean cultivars and one control cultivar were compared for differences in induction-efficiency of somatic embryogenesis, primary embryo yield, and embryo conversion. Cotyledons from immature seeds of similar developmental stage for all soybean cultivars were used for embryo induction. The experiments utilized a Latin square design to exclude the effect of differential lighting and position due to plate location in the growth chamber on the embryogenesis process. Results indicated that the efficiency of embryo induction and yield of primary somatic embryos were genotype-dependent. In contrast, no dependence on genotype was observed for the conversion of embryos to form roots and shoots. The percentage of cotyledons that gave a positive embryogenic response ranged from 26 to 89% for the soybean cultivars tested. The average number of primary globular-stage embryos per responding cotyledon after one month on induction medium ranged from 6 to 13 among the seven cultivars. Conversion frequencies for all genotypes ranged from 27 to 45%.     

Effect of genotype on somatic embryogenesis from immature cotyledons of soybean

Genotype has a large effect on the ability of immature soybean cotyledons to undergo auxin-stimulated somatic embryogenesis. Among 33 soybean lines, all those showing good regeneration were found to have in their pedigrees one or both of the highly regenerative ancestral lines, ‘Manchu’ or ‘A.K. Harrow’. When ‘Manchu’ was crossed with ‘Shiro’, a genotype showing extremely poor regeneration, F₁ hybrid cotyledons showed intermediate regeneration capacity. This paper (No. 88-3-128) is published with the approval of the director of the Kentucky Agricultural Experiment Station.     

Clustering methods for microarray gene expression data

Within the field of genomics, microarray technologies have become a powerful technique for simultaneously monitoring the expression patterns of thousands of genes under different sets of conditions. A main task now is to propose analytical methods to identify groups of genes that manifest similar expression patterns and are activated by similar conditions. The corresponding analysis problem is to cluster multi-condition gene expression data. The purpose of this paper is to present a general view of clustering techniques used in microarray gene expression data analysis.     

Cellular and molecular changes associated with somatic embryogenesis induction in Agave tequilana

In spite of the importance of somatic embryogenesis for basic research in plant embryology as well as for crop improvement and plant propagation, it is still unclear which mechanisms and cell signals are involved in acquiring embryogenic competence by a somatic cell. The aim of this work was to study cellular and molecular changes involved in the induction stage in calli of Agave tequilana Weber cultivar azul in order to gain more information on the initial stages of somatic embryogenesis in this species. Cytochemical and immunocytochemical techniques were used to identify differences between embryogenic and non-embryogenic cells from several genotypes. Presence of granular structures was detected after somatic embryogenesis induction in embryogenic cells; composition of these structures as well as changes in protein and polysaccharide distribution was studied using Coomassie brilliant blue and Periodic Acid-Schiff stains. Distribution of arabinogalactan proteins (AGPs) and pectins was investigated in embryogenic and non-embryogenic cells by immunolabelling using anti-AGP monoclonal antibodies (JIM4, JIM8 and JIM13) as well as an anti-methyl-esterified pectin-antibody (JIM7), in order to evaluate major modifications in cell wall composition in the initial stages of somatic embryogenesis. Our observations pointed out that induction of somatic embryogenesis produced accumulation of proteins and polysaccharides in embryogenic cells. Presence of JIM8, JIM13 and JIM7 epitopes were detected exclusively in embryogenic cells, which supports the idea that specific changes in cell wall are involved in the acquisition of embryogenic competence of A. tequilana.     

Characterization of genes associated with polyembryony and in vitro somatic embryogenesis in Citrus

Apomixis is a common reproduction system in the genus Citrus. To investigate the molecular mechanism of apomictic embryogenesis in Citrus, genes expressed specifically in an apomictic genotype were isolated by PCR-selected suppression subtractive hybridization with total RNAs obtained from the ovule at anthesis. Several genes showing conspicuously different expressions between polyembryonic (apomictic) and monoembryonic (nonapomictic) genotypes were selected, and their expression profiles during ovule development were analyzed in detail. This analysis identified two apomictic and three nonapomictic genotype-specific genes. Among the latter, msg-2 was highly expressed in the late stage of somatic embryogenesis. Specific expression during ovule development in monoembryonic cultivars and in the late stage of somatic embryogenesis indicated that msg-2 is not expressed in the initiation stage of polyembryogenesis and somatic embryogenesis, suggesting its role in suppressing initial cell formation of somatic embryos. The full-length complementary DNA of msg-2 contained small open reading frames in its sequence but showed no homology to functionally known genes in the public databases. As sequences similar to msg-2 were frequently found among Citrus expressed sequence tags, msg-2 may be associated with polyembryogenesis and somatic embryogenesis in a Citrus-specific manner.     

Extracting gene expression patterns and identifying co-expressed genes from microarray data reveals biologically responsive processes

Background  

A common observation in the analysis of gene expression data is that many genes display similarity in their expression patterns and therefore appear to be co-regulated. However, the variation associated with microarray data and the complexity of the experimental designs make the acquisition of co-expressed genes a challenge. We developed a novel method for Extracting microarray gene expression Patterns and Identifying co-expressed Genes, designated as EPIG. The approach utilizes the underlying structure of gene expression data to extract patterns and identify co-expressed genes that are responsive to experimental conditions.     

大豆主栽品种体细胞胚胎发生的影响因素及再生植株

Factors on in vitro somatic embryogenesis of soybean (three elite cultivars) were studied using cotyledons of 3.0-6.0 mm immature seed as explants. Not only the kinds, concentrations and combinations of plant growth regulatory substances but also immature embryo length and inoculum density have main effects on the approaches of embryogenesis. The results of two-factors analysis of variance experiments showed that immature embryo length, plant growth substance concentration and basic medium type have very significant effects on the frequency of embryogenic response, furthermore, interactions exist between the former two factors and are just very significant(at 1% level). The best combinations between 2,4-D concentration and cotyledon length are 10 mg/L 2,4-D & 4.0 mm immature embryos, 20-40 mg/L 2,4-D & 5.0 mm immature embryo. Under these combinations, the salt composition of E1 are very significantly better than that of MS. In conclusion, in the regeneration system established by us the frequency of somatic embryogenesis from the soybean immature cotyledons is greater than 50% and the frequency of conversion of normal (not fused) somatic embryos is about 52.9%-62.6%.     

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.     

Protein patterns associated with <Emphasis Type="Italic">Pisum sativum</Emphasis> somatic embryogenesis

Total protein patterns were studied in the course of development of pea somatic embryos using simple protocol of direct regeneration from shoot apical meristems on auxin supplemented medium. Protein content and total protein spectra (SDS-PAGE) of somatic embryos in particular developmental stages were analysed in Pisum sativum, P. arvense, P. elatius and P. jomardi. Expression of seed storage proteins in somatic embryos was compared with their accumulation in zygotic embryos of selected developmental stages. Pea vegetative tissues, namely leaf and root, were used as a negative control not expressing typical seed storage proteins. The biosynthesis and accumulation of seed storage proteins was observed during somatic embryo development (since globular stage), despite of the fact that no special maturation treatment was applied. Major storage proteins typical for pea seed (globulins legumin, vicilin, convicilin and their subunits) were detected in somatic embryos. In general, the biosynthesis of storage proteins in somatic embryos was lower as compared to mature dry seed. However, in some cases the cotyledonary somatic embryos exhibited comparatively high expression of vicilin, convicilin and pea seed lectin, which was even higher than those in immature but morphologically fully developed zygotic embryos. Desiccation treatments did not affect the protein content of somatic embryos. The transfer of desiccated somatic embryos on hormone-free germination medium led to progressive storage protein degradation. The expression of true seed storage proteins may serve as an explicit marker of somatic embryogenesis pathway of regeneration as well as a measure of maturation degree of somatic embryos in pea.     

Genotypes of high competence for somatic embryogenesis and plant regeneration in soybean Glycine max

Summary Soybean somatic embryos were induced from cultured immature embryos in the presence of a highlevel concentration of Naphthalenacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Embryogenetic capacity was strongly influenced by genotypes of explants. Induced somatic embryos could be sorted into normal or abnormal types according to the morphological shapes of cotyledons and hypocotyls. Somatic embryos were transferred sequentially to three different media until germination. Germination capacity from the somatic embryos was also influenced by the genotype. Following germination, these regenerated plantlets were transferred to soil in the greenhouse and were stably matured to set seeds.