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. Received: 6 June 2000 / Revision received: 17 July 2000 / Accepted: 17 July 2000     

Acquisition of embryogenic potential in carrot cell-suspension cultures

Embryogenic suspension cultures of domesticated carrot (Daucus carota L.) are characterized by the presence of proembryogenic masses (PEMs) from which somatic embryos develop under conditions of low cell density in the absence of phytohormones. A culture system, referred to as starting cultures, was developed that allowed analysis of the emergence of PEMs in newly initiated hypocotyl-derived suspension cultures. Embryogenic potential, reflected by the number of FEMs present, slowly increased in starting cultures over a period of six weeks. Addition of excreted, high-molecular-weight, heat-labile cell factors from an established embryogenic culture considerably accelerated the acquisition of embryogenic potential in starting cultures. Analysis of [³⁵S]methionine-labeled proteins excreted into the medium revealed distinct changes concomitant with the acquisition of embryogenic potential in these cultures. Analysis of the pattern of gene expression by in-vitro translation of total cellular mRNA from starting cultures with different embryogenic potential and subsequent separation of the [³⁵S]methionine-labeled products by two-dimensional polyacrylamide gel electrophoresis demonstrated a small number of abundant in-vitro-translation products to be present in somatic embryos and in embryogenic cells but absent in nonembryogenic cells. Several other in-vitro-translation products were present in explants, non-embryogenic and embryogenic cells but were absent in somatic embryos. Hybridization of an embryoregulated complementary-DNA sequence, Dc3, to RNA extracted from starting cultures showed that the corresponding gene is expressed in somatic embryos and PEMs but not in non-embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - cDNA complementary DNA - PAGE polyacrylamide gel electrophoresis - PEM proembryogenic mass     

The origin and development of somatic embryos following cryopreservation of an embryogenic suspension culture ofPicea sitchensis

Summary The development of somatic embryos in an embryogenic suspension culture ofPicea sitchensis was followed every day for two weeks after thawing from liquid nitrogen (LN₂). Only a few cells, primarily located at the periphery of the embryonic region of the embryos, survived cryopreservation in LN₂. Surviving cells were classified into two groups: embryogenic cells (EC) and non-embryogenic cells (NEC), based on their morphology and embryogenic competence. The dense cytoplasmic EC underwent organized growth and differentiation with first divisions occurring after 24 h, and embryo formation 6–8 days after thawing from LN₂. No evidence of asymmetrical divisions or free-nuclear stages was found during somatic embryo formation. NEC had less dense cytoplasm with numerous small vacuoles. One to five days after thawing the NEC became progressively more vacuolated and elongated. Histological examination revealed no mitotic activity in NEC, and six days after thawing NECs were seen as single cells or unorganized cell aggregates. Two weeks after thawing the appearance of the cryopreserved cultures was comparable to that of the untreated cultures.Abbreviations EC embryogenic cells - ECC embryogenic cell clusters - FDA fluorescein diacetate - GMA glycol methacrylate - LN₂ liquid nitrogen (–196°C) - NEC non-embryogenic cells     

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.     

Callus concentration regulates somatic embryo production in orchardgrass suspension cultures

Summary The effects of callus inoculation concentration and culture duration on somatic embryogenesis of orchardgrass,Dactylis glomerata L., were evaluated in suspension cultures of an embryogenic genotype Embryogen-P. Somatic embryo formation was induced in liquid SH medium containing 30 μM dicamba (SH-30 and 1.5% casein hydrolysate; embryo development was in liquid SH medium without plant growth regulators (SH-0); and embryo maturation and germination occurred on solid SH-0 medium. Callus proliferation in SH-30 suspension cultures was greatest when callus was inoculated into the liquid medium at a relatively high concentration of 4% (4 g callus/100 ml medium), but the induction of somatic embryos was highest in this medium if the callus was inoculated at a lower concentration (<2%). In a second experiment, somatic embryo yield was highest when SH-0 development medium was inoculated with suspension culture callus at 0.1% concentration and declined markedly as inoculation concentration increased. Cell concentration is a critical factor in regulating the somatic embryogenesis response in orchardgrass suspension cultures.     

Embryogenic and non-embryogenic cell lines of Daucus carota cloned from meristematic cell clusters: relation with cell ploidy determined by flow cytometry

The presence of totipotent and non-totipotent cells in embryogenic carrot cell suspension cultures was examined by cloning of cell microclusters. Forty clones were isolated and the distribution of their embryogenic potential was studied. Nonembryogenic, weakly and highly embryogenic cell lines were selected. After one year of subculture a second cloning round showed that the highly embryogenic and the non-embryogenic cell lines were homogenous and stable. A measurement of ploidy levels of clones by flow cytometry showed that the embryogenic clones were all diploid whereas the non-embryogenic were diploid or tetraploid. Hence, for our strain, there was a strict relationship between the tetraploid state and the inability to produce somatic embryos.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume - MS Murashige and Skoog medium - MES 2(N-morpholino) ethanesulfonic acid - a.u. arbitrary units     

Acetylsalicylic acid and ammonium-induced somatic embryogenesis and enhanced plumbagin production in suspension cultures of <Emphasis Type="Italic">Plumbago rosea</Emphasis> L.

Summary Somatic embryogenesis was induced from suspension cultures (derived from leaf callus) of an important medicinal plant, Plumbago rosea L. While acetylsalicylic acid (ASA) alone induced embryogenesis, indole-3-acetic acid (IAA) failed to elicit a similar response. This is the first time that ASA-induced somatic embryogenesis has been reported in cultured cells. Optimal embryogenic response per culture was observed in Murashige and Skoog’s medium containing a combination of ASA (8.32 μM) and IAA (5.06 μM). but 1-naphthaleneacetic acid and indole-3-butyric acid individually did not induce somatic embryogenesis. Increase in the concentration of ammonium enhanced the number of embryos formed per culture. Accumulation of plumbagin, an important naphthoquinone and a medicinal compound, was three times higher in embryogenic compared to non-embryogenic suspensions.     

Proteins related with embryogenic potential in callus and cell suspensions of sugarcane (Saccharum sp.)

Summary Previous results have shown that some proteins secreted in the culture medium are involved with the formation of embryogenic cells and can modify somatic embryo differentiation. Undifferentiated cell suspensions grown in the presence of 13 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and obtained from embryogenic and non-embryogenic callus were used to study these events in sugarcane plants (cv.PR-62258). The cell suspension growth curves were determined and soluble proteins were extracted from embryogenic and non-embryogenic callus and culture medium from cell suspensions. In embryogenic callus we detected 1.43 times more protein than in non-embryogenic callus and the electrophoretic protein patterns show specific polypeptides for both callus types. In embryogenic callus we detected a cluster of four polypeptides in the range of 38–44 kDa and another polypeptide of 23 kDa that were not observed in non-embryogenic callus. In nonembryogenic callus there is a 35-kDa polypeptide that was not detected in embryogenic callus. In the case of extracellular proteins, the medium from embryogenic cell suspensions contained four polypeptides of 41, 38, 34 and 28 kDa that were slightly detected in the medium from non-embryogenic cell cultures; we also detected a band at 15 kDa that could not be observed in the medium from non-embryogenic cell suspensions. These results suggest that the development of embryogenic callus and cell suspensions is related to the type and amount of intracellular proteins in the callus cells and to the secreted proteins from these cells into the medium.     

Large impact of the apoplast on somatic embryogenesis in <Emphasis Type="Italic">Cyclamen persicum</Emphasis> offers possibilities for improved developmental control <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Clonal propagation is highly desired especially for valuable horticultural crops. The method with the potentially highest multiplication rate is regeneration via somatic embryogenesis. However, this mode of propagation is often hampered by the occurrence of developmental aberrations and non-embryogenic callus. Therefore, the developmental process of somatic embryogenesis was analysed in the ornamental crop Cyclamen persicum by expression profiling, comparing different developmental stages of embryogenic cell cultures, zygotic vs. somatic embryos and embryogenic vs. non-embryogenic cell cultures.     

AtLTP1 luciferase expression during carrot somatic embryogenesis

The carrot (Daucus carota L.) EP2 gene encodes a Lipid Transfer Protein (LTP) which is expressed during protoderm formation in developing embryos. To develop a vital reporter system for gene expression during somatic embryo development a 1.1 kB fragment of the Arabidopsis thaliana LTP1 promoter was fused to the firefly luciferase (LUC) coding sequence. The AtLTP1 luciferase expression pattern in transformed carrot suspension cultures was identical to the expression pattern of the endogenous carrot EP2 gene. Cell tracking experiments revealed that all somatic embryos were derived from AtLTP1 luciferase expressing cell clusters. However, not all cell clusters that expressed the AtLTP1 luciferase reporter gene developed into a somatic embryo, suggesting that initiation of an embryogenic pathway in tissue culture does not always lead to development of a somatic embryo.     

Embryogenic cells in Dactylis glomerata L. (Poaceae) explants identified by cell tracking and by SERK expression

 Single mesophyll cells in leaf explants of Dactylis glomerata L. (Dactylis) that were competent to form somatic embryos directly or through callus were identified by semi-automatic cell tracking. These competent cells were a subpopulation of small, isodiametric, cytoplasm-rich cells located close to the vascular bundles. Using whole mount in situ hybridization, we showed that a similar subpopulation of cells expressed the Somatic Embryogenesis Receptor-like Kinase (SERK) gene during the induction of embryogenic cell formation. In both leaf explants and suspension cultures, a transient pattern of SERK gene expression was found during early embryo development, up to the globular stage. In later embryo stages, SERK mRNA was present in the shoot apical meristem, scutellum, coleoptile and coleorhiza. Received: 14 May 1999 / Revision received: 27 August 1999 / Accepted: 8 September 1999     

Identification of a transitional cell state in the developmental pathway to carrot somatic embryogenesis

We have located a novel carbohydrate epitope in the cell walls of certain single cells in embryogenic, but not in non-embryogenic, suspension cultures of carrot. Expression of this epitope, recognized by the mAb JIM8, is regulated during initiation, proliferation, and prolonged growth of suspension cultures such that changes in the abundance of JIM8-reactive cells always precede equivalent changes in embryogenic potential. Therefore, a direct correlation exists between the presence of the JIM8-reactive cell wall epitope and somatic embryo formation. The JIM8-reactive cell wall epitope is expressed in the cell walls of three types of single cells and one type of cell cluster. One of the single cell types seems able to follow one of two phytohormone-controlled developmental pathways, either a cell elongation pathway that eventually leads to cell death, or a cell division pathway that gives rise to proembryogenic masses. We demonstrate that all JIM8-reactive cell types in embryogenic carrot suspension cultures are developmentally related, and that the switch by one of them to somatic embryogenesis is accompanied by the immediate dissipation of the JIM8-reactive cell wall epitope. The cell wall carbohydrate epitope recognized by JIM8 therefore represents a cell wall marker for a very early transitional cell state in the developmental pathway to carrot somatic embryogenesis.     

4-Hydroxybenzyl alcohol accumulates in suspension-cell cultures and inhibits somatic embryogenesis in carrot

Somatic embryogenesis in carrot ( Daucus carota L.) is strongly inhibited by certain factors that accumulate in culture medium of high-density cultures of embryogenic cells. We previously identified 4-hydroxybenzyl alcohol (4HBA) as one of the inhibitory factors. In this study, we analyzed the accumulation pattern of 4HBA in the cultures of carrot suspension cells. When somatic embryogenesis was induced by culturing embryogenic cells in phytohormone-free Murashige and Skoog medium at various initial cell densities, 4HBA accumulated in the culture medium. The concentration of 4HBA in high cell density cultures was higher than in low cell density cultures. The accumulation of 4HBA in high cell density cultures was rapid during the early days of culture. This rapid accumulation of 4HBA in high cell density cultures might result in the strong inhibition of somatic embryogenesis. The production of 4HBA decreased as the somatic embryos developed. In addition, embryogenic cells released larger amount of 4HBA into the culture medium compared with non-embryogenic cells. These results suggest that the production of 4HBA is both related to embryogenic competence and developmentally regulated during somatic embryogenesis.     

Efficient plant regeneration from embryogenic suspension cultures of sweetpotato

Summary Using 15 Chinese and Japanese cultivars of sweetpotato, Ipomoea batatas (L.) Lam., we succeeded in developing an efficient plant regeneration system from embryogenic suspension cultures. The embryogenic callus derived from shoot apices of the 15 cultivars was used to initiate embryogenic suspension cultures in Murashige and Skoog (MS) medium containing 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Rapidly proliferating and well-dispersed embryogenic suspension cultures were established. Cell aggregates 0.7–1.1 mm in size from embryogenic suspension cultures were transferred to solid MS medium supplemented with 9.05 μM of 2,4-D and formed embryogenic callus with somatic embryos. The embryogenic callus with somatic embryos was further transferred to MS medium supplemented with 3.78 μM of abscisic acid, resulting in the germination of somatic embryos. Within 20 wk after the initiation, the frequencies of cell aggregates forming plantlets reached approximately 100% for the 15 tested cultivars. These plantlets, when transferred to soil, showed 100% survival. No morphological variations were observed.     

Synthesis of extracellular proteins in embryogenic and non-embryogenic cell cultures of alfalfa

Extracellular proteins, released into the culture medium from alfalfa cells grown in embryogenic and non-embryogenic conditions, were ³⁵S-methionine labelled at different days of culture. SDS-PAGE analysis showed significant differences between the patterns of extracellular proteins secreted into the medium devoid of 2,4-d, in which cells formed somatic embryos, or in presence of 2,4-d, in which undifferentiated cell proliferation took place. Some proteins, evident in 2,4-d-supplied cultures, disappeared when cells were subcultured in the embryogenic conditions. Western analysis with antibodies against the carrot extracellular proteins EP1 and EP2 showed the presence of homologous alfalfa proteins. In 2,4-d depleted alfalfa cells, an EP1-like protein disappeared and another one was reduced, while the presence of the EP2-like protein was, in the same conditions, strongly enhanced.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - EP extracellular proteins - ns-LTP non specific lipid transfer protein - SDS-PAGE Sodium dodecyl sulphate polyacrilamide gel electrophoresis     

Synchronized somatic embryo development in embryogenic suspensions of Fraxinus Angustifolia

Summary The synchronization of somatic embryo development in embryogenic suspension cultures is a crucial step in taking advantage of somatic embryogenesis for high production potential and reduction of unit cost through automation. In the present study, a synchronous somatic embryogenic system was developed for Fraxinus angustifolia suspension cultures. High cell density, 6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid proved essential for the establishment and maintenance of suspension cultures. Low cell density, BA and 1-naphthaleneacetic acid enhanced somatic embryo development. Cell and cell cluster fractionation by density gradient centrifugation in Ficoll solution proved useful for separation of subpopulations with differing potentials for embryo development. A synchronous development of somatic embryos at high frequency was achieved only from the heaviest cell population.     

Phenolic compounds and somatic embryogenesis in cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

Studies of phenolic compounds were performed during cell suspension cultures in relation with the induction of embryogenic structures in two cultivars of cotton. Coker 312 produced embryogenic structures, unlike R405-2000 which was found to be a non-embryogenic cultivar. Embryogenesis induction in Coker 312 was strongly linked to a higher content of caffeic, ferulic and salicylic acids and to the appearance of p-coumaric acid, benzoic acid, trans-resveratrol, catechin and naringenin.     

Embryo induction and regeneration from root explants of Medicago truncatula after osmotic pre-treatment

Embryo induction and regeneration from suspension culture of two Medicago truncatula cvs. (cv. R 108 1 and cv. Jemalong) have been studied. The influence of osmotic pre-treatment (1 M solution of sucrose for 48 h and 72 h) of roots as an initial explant, on embryogenic efficiency of the suspension culture was assessed. In comparison to the control, the level of abscisic acid (ABA) increased significantly after osmotic stress. The increased ABA level did not correlate with the induction of embryogenesis neither with the improved embryogenic potential of cv. R 108 1. The shortest regeneration period and the highest percent of conversion to plants were found in cv. R 108 1 after 72-h pre-treatment of roots. The efficiency of somatic embryo conversion was less after 48-h pre-treatment and much less for the untreated control. Osmotic stress did not positively affect the process of embryogenesis from root explants of cv. Jemalong, confirming its cultivar dependence. A single cell suspension fraction was produced in both Medicago trunacatula cvs. during the somatic embryo maturation stage. A higher embryogenic potential than the initial suspension culture was established only for the cell suspension originating from 72-h pre-treated roots of cv. R 108 1. The data confirms that the process of somatic embryo induction and embryo conversion from root explants of cv. R 108 1 could be promoted by osmotic stress pre-treatment.