Non-embryogenic Suspension Cultures Research Articles

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.

Arabinogalactan-proteins stimulate the organogenesis of guard cell protoplasts-derived callus in sugar beet

Arabinogalactan proteins (AGPs) represent a class of proteoglycans implicated in the development and differentiation of cells and tissues both in planta and in vitro. Here we report that AGP-rich extracts isolated from media of embryogenic and non-embryogenic suspension cultures of sugar beet (Beta vulgaris L.) are able to enhance the organogenesis of guard protoplast-derived callus and to increase the number of shoots formed, in comparison to control cultures. Immunocytochemical detection of carbohydrate antigens in the extracts revealed the presence of epitopes that typify both AGP and pectin, the latter being frequently bound to AGPs or, in some cases, even contributing to the polysaccharide structure of proteoglycan molecules. The most abundant epitopes proved to be those recognized by the JIM13, LM2, and MAC207 antibodies, whereas some others could be found only in relatively small or trace amounts--these included epitopes recognized by JIM16, JIM5, and LM6. Surprisingly, the JIM4- and JIM8-binding epitopes that are expressed in the course of in vitro morphogenetic processes of many species could not be detected at all in sugar beet AGPs. This is the first report of the improvement of sugar beet protoplast-derived callus organogenesis by exogenous AGP-rich extracts, an achievement that will have great impact on the biotechnological applications of protoplast technology in this species.

Secretion of a chitinase-like protein in embryogenic suspension cultures of Dactylis glomerata L.

A chitinase-like 32 kDa acidic protein with a potential chitinase activity has been identified in the medium of embryogenic suspension cultures of Dactylis glomerata L. using an antiserum raised against endochitinase EP3 from Daucus carota L. The presence of this protein discriminates between Dactylis glomerata L. embryogenic and nonembryogenic suspension cultures and thus could be possibly used as a marker for embryogenic potential.

Two-Dimensional Protein Pattern Analysis of Extracellular Proteins Secreted by Embryogenic and Non-Embryogenic Suspension Cultures of Dactylis Glomerata L.

ABSTRACTProteins from the medium of orchardgrass (Dactylis glomerata L.) embryogenic suspension culture during defined stages of somatic embryogenesis were compared with those of non-embryogenic suspension culture during unorganized cell proliferation. After separation on two-dimensional polyacrylamide gel electrophoresis, we were able to classify 40 proteins in each of 6 groups: a group common to both embryogenic and non-embryogenic suspension cultures, a group specific only for embryogenic, respectively non-embryogenic suspension cultures and 3 groups of microcluster-, proembryogenic masses- and somatic embryo-specific proteins. One of the groups is particularly interesting as it corresponds to polypeptides that are related to the earliest stages of somatic embryo-genesis-the transition of microclusters into proembryogenic masses when no morphological changes are visible yet. Using this experimental approach, we identified the major extracellular proteins involved in somatic embryo development and we discuss on the possibility for their use as early markers for somatic embryogenesis.

Induction of a class III acidic chitinase in foliar explants of Coffea arabica L. during somatic embryogenesis and wounding

A gene fragment, termed AR-52, was cloned by differential display analysis during the induction of somatic embryogenesis in foliar explants of Coffea arabica. It is homologous to several class III chitinases and Northern blot shows that it is up regulated during somatic embryogenesis and scarcely expressed in embryos at any developmental stage. AR-52 is also induced by wounding the leaves of in vitro cultured plantlets but not induced in a non-embryogenic suspension culture. Chitinase zymography revealed the existence of at least three-chitinolitic bands that are also regulated during somatic embryogenesis and wounding.

The maize MADS box gene ZmMADS3 affects node number and spikelet development and is co-expressed with ZmMADS1 during flower development, in egg cells, and early embryogenesis.

MADS box genes represent a large gene family of transcription factors with essential functions during flower development and organ differentiation processes in plants. Addressing the question of whether MADS box genes are involved in the regulation of the fertilization process and early embryo development, we have isolated two novel MADS box cDNAs, ZmMADS1 and ZmMADS3, from cDNA libraries of maize (Zea mays) pollen and egg cells, respectively. The latter gene is allelic to ZAP1. Transcripts of both genes are detectable in egg cells and in in vivo zygotes of maize. ZmMADS1 is additionally expressed in synergids and in central and antipodal cells. During early somatic embryogenesis, ZmMADS1 expression is restricted to cells with the capacity to form somatic embryos, and to globular embryos at later stages. ZmMADS3 is detectable only by more sensitive reverse transcriptase-PCR analyses, but is likewise expressed in embryogenic cultures. Both genes are not expressed in nonembryogenic suspension cultures and in isolated immature and mature zygotic embryos. During flower development, ZmMADS1 and ZmMADS3 are co-expressed in all ear spikelet organ primordia at intermediate stages. Among vegetative tissues, ZmMADS3 is expressed in stem nodes and displays a gradient with highest expression in the uppermost node. Transgenic maize plants ectopically expressing ZmMADS3 are reduced in height due to a reduced number of nodes. Reduction of seed set and male sterility were observed in the plants. The latter was due to absence of anthers. Putative functions of the genes during reproductive and vegetative developmental processes are discussed.

An acidic esterase as a biochemical marker for somatic embryogenesis in orchardgrass (Dactylis glomerata L.) suspension cultures.

The isoenzyme pattern of esterases (EC 3.1.1.2) secreted into the medium of orchardgrass (Dactylis glomerata L.) embryogenic suspension cultures during defined stages of somatic embryogenesis was compared with that of non-embryogenic suspension cultures during unorganised cell proliferation. Isoelectric focusing revealed the presence of 7-14 predominantly acidic isoforms. Comparison with the corresponding cell-wall isoenzyme pattern showed minor, mainly quantitative differences. The pattern of intracellular soluble esterases did not change markedly during somatic embryo development. A unique esterase whose migration in two-dimensional gel electrophoresis corresponds to an apparent molecular mass of 36 kDa and pI=3.8 was detected only in embryogenic cultures at very early stages of development. Since this isoform appeared long before morphological changes had taken place, it could possibly be used as a biochemical marker for embryogenic potential in D. glomerata L. suspension cultures.

Transient expression of a reporter gene changes significantly during somatic embryogenesis in alfalfa

Transient expression of the β -glucuronidase (GUS) gene driven by a 35S constitutive promoter was investigated during somatic embryo development in alfalfa (Medicago sativa L.) following micro-projectile bombardment. The level of gene expression varied with the different developmental stages and changed dramatically within the stages. During embryogenic callus induction (stage I), expression was low in explants freshly transferred to culture, but increased when cells started to divide and show competence for somatic embryogenesis. Gene expression, however, decreased when extensive callus formation began and reached the lowest observed level when callus was fully developed. Reporter gene expression was consistently low during embryogenic cell proliferation (stage II). In contrast, high levels of reporter gene expression were detected in non-embryogenic suspension cultures during cell proliferation. Extremely low reporter gene expression was detected in embryonal tissues and young embryos at the embryo development and maturation stage (stage III). Expression increased as the somatic embryos developed and reached the highest level when the embryos reached maturity. Transient expression levels were similar in excised petiole tissue across 10 alfalfa genotypes, which included both diploid and tetraploid genotypes. In general, reporter gene expression was found to be higher in more organised mature tissues and organs than in less-organised or young tissues. The developmental status of the cells and tissues appears to be an important factor in the degree of transient expression of the introduced gene. Changes of gene expression during embryogenesis and factors affecting gene expression are discussed. Key words: GUS expression, Medicago sativa, bombardment, forage

Expression pattern of the carrot EP3 endochitinase genes in suspension cultures and in developing seeds

Carrot (Daucus carota) extracellular protein 3 (EP3) class IV endochitinases were previously identified based on their ability to rescue somatic embryos of the temperature-sensitive cell line ts11. Whole-mount in situ hybridization revealed that a subset of the morphologically distinguishable cell types in embryogenic and nonembryogenic suspension cultures, including ts11, express EP3 genes. No expression was found in somatic embryos. In carrot plants EP3 genes are expressed in the inner integumentary cells of young fruits and in a specific subset of cells located in the middle of the endosperm of mature seeds. No expression was found in zygotic embryos. These results support the hypothesis that the EP3 endochitinase has a "nursing" function during zygotic embryogenesis and that this function can be mimicked by suspension cells during somatic embryogenesis.

Differences in compounds released by embryogenic and non-embryogenic suspension cultures of Euphorbia pulcherrima

Media from embryogenic and non-embryogenic cell suspension cultures were analysed for protein content, electrophoretic protein patterns, glycoproteins and activity of peroxidases and β-glucosidases in order to characterize the physiological status of the cultures. On a dry mass basis the amount of extracellular proteins per cell was greater in embryogenic suspensions than in non-embryogenic suspensions. Non-embryogenic suspensions contained unidentified slimy compounds which were not present inembryogenic cultures. The extracellular Concanavalin A-specific glycoproteins gave different isoelectric focussing patterns and thus enabled embryogenic and non-embryogenic cultures to be differentiated. The extracellular peroxidase activity per cell dry mass was far greater in embryogenic than in non-embryogenic cultures. The isoenzymes differed in number and composition of the anionic bands. β-glucosidases were found in the same range of activity in both culture types, but the time course of enzyme activity during cultivation was significantly different. In the embryogenic culture the activity was correlated with dry mass increase, whereas in the non-embryogenic suspension the activity reached maximum during the linear growth phase. Polyphenoloxidase which was recently recognized as an intracellular marker for embryogenic stages was not released into culture media.

Polyphenoloxidase-activity and -activation in embryogenic and non-embryogenic suspension cultures of Euphorbia pulcherrima

The activity and activation potential of polyphenoloxidase (PPO, E.C. 1.10.3.1.) of tissue from shoot tips, adult leaves and embryogenic and non-embryogenic cell suspension cultures of Euphorbia pulcherrima was investigated using an oxygen probe technique. PPO derived from differentiated in vivo plant tissue (shoot tips, leaves) cannot be activated either by storage at 0-4°, freezing and thawing, incubation with CaCl2, sodium dodecyl sulfate or by incubation with trypsin. Embryogenic cells are characterized by high initial PPO activity and strong activation potential of membrane bound enzyme. Non-embryogenic material reveals low phenolase activity and low activation potential. An activation quotient (based on the ratio between "PPO-activity determined after sodium dodecyl sulfate incubation" to "PPO-activity determined after CaCl2-incubation") was calculated. This is independent of absolute enzyme activity and can be used for characterization of the embryogenic status of cells.

Molecular analysis of plants regenerated from embryogenic cultures of wheat (Triticum aestivum L.)

Total DNAs of plants regenerated from immature embryo-derived 2-month-old embryogenic calli of wheat (cultivars Florida 302, Chris, Pavon, RH770019) were probed with six maize mitochondrial genes (atpA, atp6, apt9, coxI, coxII, rrn18-rrn5), three hypervariable wheat mitochondrial clones (K', K3, X2), five random pearl millet mitochondrial clones (4A9, 4D1, 4D12, 4E1, 4E11) and the often-used wheat Nor locus probe (pTA71), in order to assess the molecular changes induced in vitro. In addition, protoplast-derived plants, and 24-month-old embryogenic and non-embryogenic calli and cell suspension cultures of Florida 302 were also analyzed. No variation was revealed by the wheat or millet mitochondrial clones. Qualitative variation was detected in the nonembryogenic suspension culture by three maize mitochondrial genes (coxI, rrn18-rrn5, atp6). A callus-specific 3.8-kb Hind III fragment was detected in all four cultivars after hybridization with the coxI gene. The organization of the Nor locus of the plants regenerated from Florida 302 and Chris was stable when compared to their respective control plants and calli. The Nor locus in regenerants of Pavon and RH, on the other hand, was found to be variable. However, Nor locus variability was not observed in 14 individual seed-derived control plants from either Pavon or RH sources. In Pavon, a 3.6-kb Taq I or a 5.6-kb Bam HI+ Eco RI fragment was lost after regeneration. In one of the RH regenerants, which lost a fragment, an additional fragment was observed.

Somatic hybridization in the Gramineae: Triticum monococcum L. (Einkorn) + Pennisetum americanum (L.) K. Schum. (Pearl Millet)

Summary Somatic hybrid callus lines were recovered following the fusion of protoplasts from a non-embryogenic suspension culture of Triticum monococcum L. (Einkorn) with iodoacetate inactivated protoplasts of an embryogenic suspension of Pennisetum americanum (L.) K. Schum. (Pearl millet) resistant to S-2-aminoethyl-L-cysteine (AEC). More than 100 cell colonies grew on an AEC-supplemented selection medium. Two of these lines were confirmed as somatic hybrids by the electrophoretic pattern of the dimeric enzyme 6-phosphogluconate dehydrogenase and restriction length polymorphism using a Zamia pumila (Cycadaceae) ribosomal DNA probe.

Induction of Nitrogenase Activity in Azospirillum brasilense by Conditioned Medium from Cell Suspension Cultures of Pennisetum americanum (Pearl Millet) and Panicum maximum (Guinea Grass)

Summary Nitrogenase activity ( = acetylene reduction activity) was observed in Azospirillum brasilense cultures grown on conditioned medium obtained from embryogenic pearl millet ( Pennisetum americanum ) and Guinea grass ( Panicum maximum ) cell suspension cultures, but no bacterial growth or activity was observed on unconditioned medium. A peak of maximum activity was obtained with medium harvested mid-way through a 13 d culture period. Non-embryogenic suspension cultures of pearl millet and Guinea grass supported little or no bacterial growth or nitrogenase activity.