Generation of recombinant antibodies against orchardgrass Acidic nsLTP-like proteins

Embryogenic and non-embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.) secreted into the culture medium a set of proteins, among which low molecular mass (11/12 kDa) proteins were found. However, only the 11/12 kDa proteins from the embryogenic suspension cultures reacted specifically with an antiserum raised against the carrot EP2 non-specific lipid transfer protein (nsLTP). Two-dimensional (2-D) electrophoretic analysis revealed that the extracellular nsLTP-like proteins from the embryogenic lines were acidic proteins, with pI values ranging between 4.3 and 6.4, and the 11/12 kDa proteins of the non-embryogenic lines were basic ones (pI 8-9.3). This is only the second case to report on the accumulation of extracellular acidic nsLTP-like proteins in the culture medium during somatic embryogenesis. A naive phage display Griffin1. library was used to select single-chain phage antibodies, which specifically bind to acidic nsLTP-like proteins. Nine phage clones were selected after four rounds of biopanning of the target proteins blotted on a nitrocellulose membrane. Three soluble monoclonal single-chain phage antibodies, expressed in the non-suppressor E. coli strain HB2151, were purified by metal affinity chromatography and found to be highly specific for the acidic nsLTP-like proteins from the embryogenic suspension cultures. The application of the selected monoclonal antibodies for localization and elucidation of the role of the acidic nsLTP-like proteins in vivo is discussed.     

Three major somatic embryogenesis related proteins in Cichorium identified as PR proteins

In Cichorium hybrid clone '474' (C. intybus L., var. sativum x C. endivia L., var. latifolia), the direct somatic embryogenesis process in leaf tissues is accompanied by an overall increase in the amount of proteins secreted into the culture medium. Amongst these, three major protein bands of 38 kDa, 32 kDa and 25 kDa were found in the conditioned media. These extracellular protein bands accumulated in the medium of the embryogenic Cichorium hybrid up to 8-fold compared with those in the medium of a nonembryogenic variety. 32 and 25 kDa proteins were purified from the medium and their identities were determined as already described for 38 kDa beta-1,3-glucanases. To investigate their possible function in somatic embryogenesis, peptide sequences, serological relationships or biochemical properties revealed that there were at least two acidic chitinases of 32 kDa and one glycosylated osmotin-like protein of 25 kDa in the embryogenic culture medium. Comparing the amounts of the 38 kDa glucanases, the 32 kDa chitinases, and the 25 kDa osmotin-like protein present in the conditioned media of the embryogenic '474' hybrid and of a non-embryogenic variety, a 2-8-fold higher accumulation of these proteins was observed in the embryogenic hybrid culture medium. This may suggest that part of the accumulation of these three pathogenesis-related (PR) proteins could be correlated with the somatic embryogenesis process. Their possible involvement in this developmental process is discussed.     

Effects of glycerol on somatic embryogenesis in Cichorium leaves

 Direct somatic embryogenesis was induced in leaf cells of a Cichorium hybrid (Cichorium intybus L var. sativum×Cichorium endivia L. var. latifolia) through a two-step procedure. Leaf tissue explants were cultured for 5 days in M17 liquid medium supplemented with 30 mM sucrose and 330 mM glycerol (M17S30Gly330 medium). Synchronised divisions of embryogenic cells occurred after transfer for 7 days onto glycerol free-medium (M17S30). By doubling the sucrose concentration (60 mM) in the presence of glycerol (M17S60Gly330) during the induction step, embryogenesis increased and the length of the induction step was reduced from 5 to 4 days. Compared to sucrose, glycerol as carbon source during the induction and the expression steps had an inhibitory effect on the embryogenic response. During culture, glycerol was not detected in M17S60 medium and was at a low level in leaf fragments incubated in this medium. Initially supplied as an osmoticum, glycerol disappeared from M17S60Gly330 medium during the 4-day induction period and penetrated into the tissues where most of was metabolised. Furthermore, glycerol modified it carbohydrate metabolism, particularly during the induction period of embryogenesis. Sucrose hydrolysis was affected in the medium and sucrose and hexose contents in tissues were higher than in glycerol-free medium. The effects of glycerol as osmoticum and as a molecule itself are discussed. Received: 30 January 1998 / Accepted after revision 25 February 1999     

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.     

Somatic embryogenesis in elm

We show that isolated zygotic embryos of Ulmus minor and U. glabra can produce embryogenic cultures provided they are isolated from immature seeds before storage proteins begin to accumulate. Rates of somatic embryogenesis were highest among zygotic embryos collected 6 weeks post-anthesis when they were at the midcotyledonary stage, were about 5 mm long and had a fresh weight of approx. 10 mg. At this time, induction was even possible in Murashige and Skoog basal medium with no plant growth regulators, but addition of 2,4-dichlorophenoxyacetic acid was necessary at earlier stages of zygotic development. In medium supplemented with benzyladenine (BA) only, no embryogenic induction was observed. The formation of callus was an essential step not only for the induction of embryogenic masses, but also for the maintenance of embryogenic competence through successive subculture of callus on induction media supplemented with 0.1 mg l(-1) BA. Nine embryogenic U. minor lines and 24 U. glabra lines have been maintained in this way for 3 years. However, conversion into plantlets has occurred only rarely.     

Extracellular β-1,3-glucanases are induced during early somatic embryogenesis in Cichorium

In leaf tissues of the Cichorium hybrid clone `474' (C. intybus L. var. sativum × C. endivia L. var. latifolia), the acquisition and expression of embryogenic competence was characterised by the appearance of 15 polypeptides (Boyer et al., 1993, Plant Sci 93: 41–53). The 38-kDa proteins were found to be abundantly present in conditioned embryogenic medium after the first division of the induced cells. These proteins seemed to be glycosylated as indicated by general carbohydrate detection methods. Internal amino-acid sequences obtained after microsequencing tryptic peptides appeared to be 36–57% homologous with plant β-1,3-endoglucanases. In addition, these 38-kDa proteins were recognised by antibodies raised against the pathogenesis-related tobacco glucanase PR2a and their β-1,3-glucanase activity was demonstrated by direct detection in polyacrylamide gels after electrophoresis. These results strongly suggested that the 38-kDa somatic-embryogenesis-related (SER) polypeptides are β-1,3-glucanases. Moreover, the level of glucanase activity was nearly three times higher in the medium of the embryogenic `474' line than in the medium of a non-embryogenic line. The possible involvement of the extracellular 38-kDa proteins in callose degradation during somatic embryogenesis is discussed. Received: 5 May 1997 / Accepted: 25 September 1997     

Morphogenetic effects of Brefeldin A on embryogenic cell cultures of Daucus carota L.

Brefeldin A, an inhibitor of protein secretion, caused typical alterations to the endomembrane system with limited effects on viability when given to unorganized carrot cells growing in suspension. When given to the same cells during particular stages of embryogenesis, it caused similar endomembrane lesions and an almost complete arrest of the embryogenic process. Addition of conditioned medium containing extracellular secreted proteins to the embryos during treatment with Brefeldin A allowed acquisition of polarity and the continuation of a quasi-normal embryogenic process. Received: 4 December 1996 / Accepted: 4 March 1997     

Histology,histochemistry and ultrastructure of pre-embryogenic cells determined for direct somatic embryogenesis in the palm tree Syagrus oleracea

Somatic embryogenesis in palm trees is, in general, a slow and highly complex process, with a predominance of the indirect route and, consequently, a lack of knowledge about the direct route. We present new knowledge related to the morphological, histochemical and ultrastructural aspects of the transition from somatic to embryogenic cells and direct formation of somatic embryos from mature zygotic embryos of Syagrus oleracea, a palm tree. The results support the general concept that 2,4-dichlorophenoxyacetic acid plays a critical role for the formation of somatic embryos of direct and multicellular origin. Seven days in medium with auxin were enough for the identification of embryogenic cells. These cells had a set of characteristics corresponding to totipotent stem cells. At 14 days on induction medium, nodular formations were observed in the distal region of inoculated embryos, which evolved into globular somatic embryos. At 120 days on induction medium, the quality of the somatic embryos was compromised. The dynamics of the mobilization of reserve compounds was also demonstrated, with emphasis on starch and protein as energy sources required for the embryogenic process. This study shows for the first time the anatomical and ultrastructural events involved in direct somatic embryogenesis in a palm tree and incites the scientific community to return to the discussion of classical concepts related to direct somatic embryogenesis, especially regarding the characteristics and location of determined pre-embryogenic cells.     

The effect of auxins, time exposure to auxin and genotypes on somatic embryogenesis from mature embryos of wheat

The effects of different factors on the embryogenesis and plant regeneration from mature embryos of Russian spring and winter genotypes were studied. Embryogenic callus induction was achieved on MS medium supplemented with different concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) or Dicamba (3,6-dichloro-o-anisic acid). Although all auxins were able to induce callus from explants with high frequency (98–100%), Dicamba was more effective for the induction of embryogenic callus (21.8–38.3%). Maximum embryogenic callus formation and high number of regenerated plants were observed at 12 mg l⁻¹ of Dicamba. The time exposure to Dicamba (7, 14, 21 and 28 days) had a significant effect on efficiency of somatic embryogenesis. When contact of explants with callus induction medium was increased from 7 to 21 days the rate of somatic embryogenesis and number of regenerated plants per embryogenic callus gradually increased from 13.0 to 38.4% and 3.6 to 8.0%, respectively. Supplement of additional auxins (indoleacetic acid (IAA), indolebutyric acid (IBA), and naphthaleneacetic acid (NAA)) to callus induction medium with Dicamba had a positive effect on the rate of embryogenic callus formation, while the average number of regenerated shoots was not affected. The best rate of somatic embryogenesis was observed at the addition of 0.5 mg l⁻¹ IAA with Dicamba (61.0%). The optimum combination of Dicamba and IAA increased the efficiency of somatic embryogenesis and plant regeneration from seven spring and winter wheat genotypes, thought overall morphogenic capacity was still genotype dependent.     

Genotype effects on proliferative embryogenesis and plant regeneration of soybean

Summary Proliferative somatic embryogenesis is a regeneration system suitable for mass propagation and genetic transformation of soybean [Glycine max (L.) Merr.]. The objective of this study was to examine genotypic effects on induction and maintenance of proliferative embryogenic cultures, and on yield, germination, and conversion of mature somatic embryos. Somatic embryos were induced from eight genotypes by explanting 100 immature cotyledons per genotype on induction medium. Differences in frequency of induction were observed among genotypes. However, this step was not limiting for plant regeneration because induction frequency in the least responding genotype was sufficient to initiate and maintain proliferative embryogenic cultures. Six genotypes selected for further study were used to initiate embryogenic cultures in liquid medium. Cultures were evaluated for propagation of globular-stage tissue in liquid medium, yield of cotyledon-stage somatic embryos on differentiation medium, and plant recovery of cotyledon-stage embryos. Genotypes also differed for weight and volume increase of embryogenic tissue in liquid cultures, for yield of cotyledon-stage embryos on differentiation medium, and for plant recovery from cotyledon-stage embryos. Rigorous selection for a proliferative culture phenotype consisting of nodular, compact, green spheres increased embryo yield over that of unselected cultures, but did not affect the relative ranking of genotypes. In summary, the genotypes used in this study differed at each stage of plant regeneration from proliferative embryogenic cultures, but genotypic effects were partially overcome by protocol modifications.     

Expression of auxin and light-regulated arrestin-like proteins,G proteins and nucleoside diphosphate kinase during induction and development of wheat somatic embryos

The modulation of three signal transduction elements: arrestin-like proteins, G proteins and NDPK was assessed during the induction of wheat (Triticum aestivum L.) somatic embryogenesis under different auxin (2,4-D) and light conditions. Immunological approaches using specific antibodies, kinase activity measurement and [α-³²P]-GTP-binding assay were performed. The induction of embryogenic capacity by 2,4-D was characterised both by the increased expression of the classical 40-kDa arrestin-like form and by the appearance of an additional arrestin-like protein of 29 kDa. The 40-kDa arrestin-like soluble form was unaffected by light stimuli. On the other hand, the 29-kDa arrestin-like form, specific of the embryogenic tissue culture, was found to be light regulated. From embryogenic cultures grown under light or dark, different soluble G proteins from 22 to 48 kDa were detected by probing polyvinylidene fluoride (PVDF) blots with [α-³²P]-GTP. In addition, in the microsomal fraction from light-grown cultures, a polypeptide of 20 kDa was heavily labelled. Under light conditions, cell proliferation induced by 2,4-D stimulated the appearance of a 32-kDa nucleoside diphosphate kinase (NDPK) form in addition to the classical 16–18-kDa protein, without a significant change in the NDPK activity. The modulated expression of plant arrestin-like proteins, G proteins and NDPK molecules in response to auxin and light support the view that they play key roles in signalling cascades participating in plant development.     

Repetitive somatic embryogenesis in Medicago truncatula ssp. Narbonensis and M. truncatula Gaertn cv. Jemalong

Medicago truncatula ssp Narbonensis and four genotypes of M. truncatula Gaertn cv. Jemalong were tested for their somatic embryogenesis potential using a two-step protocol. In the first step, embryogenic callus was induced in folioles isolated from shoots grown in vitro and cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid and zeatin. In the second step, somatic embryos were allowed to develop from the induced callus in MS growth-regulator-free medium. Individual somatic embryos were then isolated and transferred again to growth regulator free medium where they formed secondary somatic embryos in repetitive cycles. Conversion of somatic embryos into plantlets was achieved by isolating late-torpedo-phase somatic embryos with distinct cotyledons and reculturing them onto MS growth regulator free medium. The system of repetitive somatic embryogenesis in M. truncatula described here represents a permanent source of embryogenic material that can be used for the genetic modification of this species. Received: 7 August 1997 / Revision received: 22 December 1997 / Accepted: 20 January 1998     

Expression of the Daucus carota somatic embryogenesis receptor kinase (DcSERK) protein in insect cells

The Daucus carota somatic embryogenesis receptor kinase (DcSERK) gene serves as marker to monitor the transition from somatic into embryogenic plant cells. To determine the intrinsic biochemical properties of the DcSERK protein, a predicted transmembrane receptor, the kinase domain was expressed as a 40-kDa his-tag fusion protein in the baculovirus insect cell system. The kinase domain fusion protein was able to autophosphorylate in vitro. Phosphoamino acid analysis of the autophosphorylated DcSERK protein revealed that it was autophosphorylated on serine and threonine residues. This is the first evidence of the biochemical characterization of a transmembrane receptor kinase from embryogenic plant cell cultures.     

Organogenesis and somatic embryogenesis in Foeniculum vulgare: histological observations of developing embryogenic callus

Different NAA plus kinetin or BA combinations were tested on Francia Pernod fennel seedlings for callus induction and plant regeneration. Callogenesis from hypocotyls was obtained in all auxin/cytokinin-containing media. The organogenic response was observed especially in presence of NAA plus kinetin. The highest frequency of shoot regeneration was found when the auxin and kinetin were used at a 1:1 ratio. Moreover, a prolonged culture period increased shoot formation. Somatic embryogenesis was tested on several fennel populations. The results gave evidence of the genotypic importance. Two different protocols were used for somatic embryo induction. Using the first protocol among the different fennel genotypes tested, only Francia Pernod showed embryogenic capacity. In this case, from a primary non-embryogenic callus cultured for 12 months in presence of 2,4-D, an embryogenic secondary callus was produced. When transferred to the medium without 2,4-D (agarized or liquid), this gave embryogenic plants in high frequency. As far as the second embryogenic method is concerned, secondary embryogenic callus developed only in the presence of 2,4-D plus kinetin in Francia Pernod genotype. Thereafter, the replacement of those growth regulators by GA₃ into the medium greatly increased the somatic embryo development, especially in `Francia Pernod', but also in `Aboca erbe' callus, a population with a very poor embryogenic capacity. In Francia Pernod, the primary and secondary (embryogenic) calli showed different morphological and histological responses, either when the secondary callus was induced by 2,4-D alone or by 2,4-D plus kinetin. Ontogenetic processes leading to somatic embryo formation are described in this context. This revised version was published online in June 2006 with corrections to the Cover Date.     

盐肤木体细胞胚胎发生及植株再生

以盐肤木(Rhus chinensis Mill.)幼胚为外植体,研究不同植物生长调节剂组合对其愈伤组织诱导及体细胞胚胎发生的影响,以建立盐肤木体细胞胚胎发生及植株再生体系。结果表明,最适愈伤组织诱导培养基为MS+6-BA 0.2 mg/L+2,4-D 1.0 mg/L,诱导率为84.57%,诱导出的初代愈伤组织白色或淡黄色,质地疏松,表面光滑,为非胚性愈伤。初代愈伤组织转移到1/2 MS+6-BA 2 mg/L+NAA 0.5 mg/L培养基上培养1个月后,长出淡黄色质地紧密的胚性愈伤组织,诱导率高达100%,在此培养基上胚性愈伤组织增殖倍数为854.73%。所获得的胚性愈伤组织转接到1/2 MS+6-BA 2 mg/L+NAA 0.5 mg/L+蔗糖4%的培养基上培养1个月后可诱导体细胞胚胎发生,诱导率可达32.67%。诱导得到的体细胞胚胎经历球形胚、心形胚、鱼雷胚、子叶胚进一步分化发育成苗。无菌苗炼苗后栽种到泥炭土∶蛭石∶珍珠岩为2∶1∶1的生长基质上,能100%稳定成活。经过细胞学观察分析,体细胞胚的发育与合子胚相似。     

Plant regeneration through somatic embryogenesis from callus induced on immature embryos of Alstroemeria spp. L.

Summary The plant regeneration ability of callus obtained from zygotic embryos of the monocot Alstroemeria spp. was studied. The best explants for somatic embryogenesis were immature zygotic embryos in half-ovules when the endosperm was still soft and white. For 2 genotypes embryogenic callus was induced on callus induction medium with a success rate of 54%. The best callus induction period was 10 weeks. The morphology of embryogenic callus was nodular. Somatic embryos were formed after transfer of the callus to regeneration medium. These somatic embryos revealed later on the typical features of zygotic Alstroemeria embryos. The total duration of the plant regeneration protocol, from inoculation till rooted plantlets ready for transfer to the greenhouse, was 28 weeks.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - NAA -naphthaleneacetic acid