Further evidence of a role for abscisic acid in conversion of somatic embryos ofDaucus carota

Summary Somatic embryogenesis has been shown to be an imperfect recapitulation of stages involved to form embryos from vegetative tissues. Although abscisic acid has been implicated in normalizing development, studies that specifically investigate conversion (vegetative leaf initiation) in somatic embryos are lacking. This report documents a follow-up of a study that implicated abscisic acid as a vital factor in allowing embryos ofDaucus carota to progress to the plantlet stage. Abscisic acid was determined to enhance conversion at doses ranging from 1 to 50 µM. Younger embryo stages were more responsive to abscisic acid application with regards to plantlet recovery. Pulses of abscisic acid were shown to elicit more rapid response with younger embryo stages, indicating more plastic development. Fluridone, an abscisic acid synthesis inhibitor, was shown to dramatically reduce conversion, even at low doses (<5µM). When abscisic acid was applied concurrently with fluridone, partial restoration of conversion was observed. Histologically, fluridone was seen to cause pronounced vacuolation in the shoot apical notch which resulted in the loss of meristematic cells, negating conversion capacity. Quantitation of total cytoplasmic area showed that abscisic acid reduced vacuolar intrusion into the apical notch, while fluridone caused a significant increase in vacuolation of cells in this region. This report documents further evidence of a role for abscisic acid in plantlet establishment from somatic embryos ofDaucus carota.     

Somatic embryogenesis and organogenesis inGuizotia Abyssinica

Summary Induction of somatic embryogenesis, shoot organogenesis, and subsequent plant regeneration in niger seem to be dependent on genotype, choice of explant, and composition of media growth regulators. Two distinct regeneration protocols have been developed for somatic embryogenesis and shoot organogenesis. Somatic embryogenesis was induced from epicotyls and cotyledonary explants (9 to 35%) (but not from hypocotyls and roots) in presence of 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, and 2,4,5-trichlorophenoxypropionic acid. These embryos matured in MS medium containing Kinetin plus naphthalene acetic acid (NAA), Kinetin plus Zeatin, and Kinetin plus abscisic acid (ABA). Matured embryos could be germinated on LS and MS basal media without hormones. Non-embryogenic callus initiated on Linsmaier and Skoog’s (LS) medium from cotyledons of six different genotypes produced shoots (9 to 32%) on Murashige and Skoog’s (MS) medium fortified with 6-benzylaminopurine (BAP, 0.5 mg · liter^−1), and BAP (1 mg · liter^−1) plus NAA (0.1 mg · liter^−1). These shoots were rooted with 100% frequency by using indole-3-acetic acid or NAA and transferred successfully to the soil.     

Differential molecular responses to abscisic acid and osmotic stress in viviparous maize embryos

Substantial quantities of mRNA encoding the abundant Em polypeptide accumulate, in planta, in developing embryos of maize (Zea mays L.). By contrast, accumulation of Em mRNA is only barely detectable in embryos with the vp-5/vp-5 genotype [an abscisic acid (ABA)-deficient viviparous phenotype]. Em mRNA is not detectable within viviparous embryos of the vp-1/vp-1 genotype that are non-responsive to ABA. Culture of immature wild-type and vp-5/vp-5 embryos in the presence of exogenous ABA or of an osmotically active agent prevents precocious germination and results in expression of the Em genes. When vp-1/vp-1 embryos are cultured under similar conditions, only the application of osmotic stress prevents precocious germination. However, Em mRNA does not accumulate either in ABA-treated or stressed, arrested embryos, indicating a requirement for ABA perception through a VP-1-mediated mechanism for Em gene expression. Nevertheless, vp-1/vp-1 embryos do show both ABA and stress responses at the molecular level. Treatment with ABA causes the accumulation of mRNA encoding a polypeptide of approx. 30 kDa, whilst osmotic stress induces the accumulation both of a 30-kDa polypeptide and a set of approx. 20-kDa polypeptides. This indicates the existence of discrete, parallel ABA and stress response pathways in developing maize embryos.Abbreviations ABA abscisic acid - cDNA copy-DNA - DAP days after pollination - kDa kilodaltons - MS Murashige and Skoog medium - LEA late embryogenesis abundant - NEpHGE non-equilibrium pH gradient gel electrophoresis - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Plant regeneration via somatic embryogenesis in many cultivars of cotton (Gossypium hirsutum L.)

Summary Embryogenic callus was formed from several cultivars of cotton (Gossypium hirsutum L.) when sections of hypocotyl and cotyledon were cultured on medium supplemented with 5 mg/liter 6-(γ, γ-dimethylallyl-amino)-purine (2iP) and 0.1 mg/liter α-naphthaleneacetic acid (NAA) for callus initiation and proliferation, and subcultured on medium supplemented with 5 mg/liter NAA and 0.1 to 1 mg/liter 2iP for embryogenic callus induction. It seems that a high 2iP:auxin ratio is preferred for callus initiation and proliferation, but should be exchanged with a higher NAA:cytokinin ratio before differentiation will occur. Embryogenic calluses were recovered at a frequency of 2 to 85% depending on the cultivar used. Coker cultivars produced embryogenic callus faster and at higher frequencies than other cultivars. Embryogenic callus produced somatic embryos on phytohormone-free medium. This medium was used to maintain and proliferate embryogenic callus for a perid of 18 to 24 mo. Somatic embryos were converted to plants on a lower ionic strength medium supplemented with 0.1 mg/liter gibberellic acid (GA₃) and 0.01 mg/liter NAA. Glucose was the only carbohydrate used through all phases of tissue culture and was much better than sucrose, on which phenolic production was very high. High temperature (30° C) and low light intensity (9 μE · m⁻² · s⁻¹) were optimal conditions for callus initiation, embryogenic callus induction, and maintenance, whereas lower temperature (25° C) and high light intensity (90 μE · m⁻² s⁻¹) were the optimal conditions for somatic embryo maturation, germination, and plantlet development. Plants could be regenerated within 10 to 12 wk in Cokers or 7 to 8 mo. in others.     

Developmental requirements for the ecdysoneless (ecd) locus in Drosophila melanogaster

The ecdysoneless locus in Drosophila melanogaster has been defined previously by a single conditional mutation, I(3)ecd¹, that causes an ecdysteroid deficit and larval death at the restrictive temperature, 29°C, although the primary role of the mutation in developmental processes has been unclear. Gene dosage and complementation studies reported here for ecd¹ and five nonconditional lethal alleles indicate that the ecd locus plays prezygotic and postzygotic roles essential for normal embryonic development, the successful completion of each larval molt, adult eclosion, and female fertility. The ecd locus is also required for normal macrochaete differentiation. For each observed phenotype, the severity of mutational effects was correlated with ecd mutant genotypes. In all cases, ecd¹ homozygotes were least affected. Mutants heteroallelic for ecd¹ and any one of four nonconditional recessive mutations were more severely affected than ecd¹ homozy-gotes, revealing these as hypomorphic alleles. For all phenotypic effects, mutants heteroallelic for ecd¹ and a dominant mutation (ecd^3D) were most severely affected. These individuals died during embryogenesis at 29°C and developed no macrochaetes on the dorsal thorax when transferred to 29°C during the white prepupal stage. The ecd^3D mutation also caused female semisterility in heterozygotes. Ecdysteroid regulation has been implicated previously in all the developmental processes disrupted by these ecd mutations except for macrochaete differentiation. © 1993 Wiley-Liss, Inc.     

Agrobacterium-mediated transformation of Asparagus officinalis L. long-term embryogenic callus and regeneration of transgenic plants

Summary Twenty-three independent kanamycin resistant lines were obtained after cocultivation of longterm embryogenic cultures of three Asparagus officinalis L. genotypes with an Agrobacterium tumefaciens strain harboring ß-glucuronidase and neomycin phosphotransferase II genes. All the lines showed ß-glucuronidase activity by histological staining. DNA analysis by Southern blots of the kanamycin resistant embryogenic lines and of a plant regenerated from one of them confirmed the integration of the T-DNA.Abbreviations GUS ß-glucuronidase - X-Gluc 5-bromo-4-chloro-3indolyl ß-D-glucuronic acid - NPT II neomycin phosphotransferase II     

Embryogenesis and plant regeneration of spinach (Spinacia oleracea L.) from hypocotyl segments

A system for somatic embryogenesis and plant regeneration of spinach from hypocotyl segments has been established. Callus was induced on solid media supplemented with 8.5–15.0 mg.l⁻¹ of indole-3-acetic acid and 3.46–34.64 mg.l⁻¹ gibberellic acid. Callus was then subcultured on different media (solid or liquid) with or without IAA, or continuously maintained on the initiating media. Somatic embryos were obtained in subcultures on IAA-containing media as well as in long-term cultures on initiating media. The best results were achieved in liquid subcultures. About 60% of plantlets survived after transplanting in pots.     

鼠兔核质杂交胚胎早期发育的研究

细胞核移植技术已被证明是研究发育中核质相互关系的非常重要的手段之一,电融合技术也是近十年发展起来的新型细胞融合技术。本实验运用这两项技术,进行了鼠、兔目间核质杂交实验,小鼠8-细胞核在激活的兔去核卵母细胞中,发生了染色体超前凝聚及核膨胀,融合卵移植到小鼠输卵管4.5天后,冲洗出,有5.4%的重构卵发育到囊胚期,通过染色体检查,囊胚细胞中均为小鼠染色体,其中一个囊胚为正常小鼠核型(2 n=40,XX)。通过本实验,我们认为:鼠兔远缘核质杂交胚胎的早期发育是可能的。     

Evaluation of somatic embryos of interior spruce. Characterization and developmental regulation of storage proteins

Storage proteins of interior spruce ( Picea glauca engelmanii complex) somatic embryos were compared to those of zygotic embryos by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Somatic embryos contain the same storage proteins as zygotic embryos based on similarities of molecular weight, isoelectric variants, solubility characteristics and disulfide linkages. Storage protein levels varied among different somatic embryo genotypes; however, all genotypes tested accumulated significant amounts of storage proteins. Zygotic and somatic embryos display a similar developmental accumulation of storage proteins. The 22, 24, 33 and 35 kDa proteins appear in early stage embryos, while the 41 kDa protein begins to accumulate during mid cotyledon development. The 22, 24 and 41 kDa proteins accumulate continuously during cotyledon development in somatic embryos cultured on abscisic acid. In contrast, zygotic embryos display a more rapid and transient accumulation of these proteins.