Embryogenic Cell Clusters Research Articles

Analysis of morphological changes in carrot somatic embryogenesis by serial observation

Carrot somatic embryogenesis was serially observed using a cell cluster immobilizing system with Phytagel. Embryogenic cell clusters ranging in size from 32 to 63 μm were collected by filtration and used for somatic embryo induction. Of the 432 cell clusters, 253 grew, i.e., the size of these cell clusters increased, and 192 developed into globular embryos. Through serial observation, the number of somatic embryos produced from each cell cluster was identified. Cell clusters which developed into two or more embryos grew and developed slowly as compared with cell clusters which developed into single embryos. Serial observation also revealed that some cell clusters consisted of several parts, each of which independently grew as separate units. In cases where two growing parts fused into one embryo, morphological abnormalities such as curvature or lumps in their bodies were occasionally observed.

Selection and microculture of single embryogenic cell clusters in japanese conifers: Picea jezoensis, Larix leptolepis and Cryptomeria japonica

A microculture method for single embryogenic cell clusters was established for several Japanese conifer species, namely Picea jezoensis, Larix leptolepis and Cryptomeria japonica. Individual small, dense cell clusters were identified and picked up by a micromanipulator and cultured in 50 µl liquid medium in a 96-well culture plate. In all three species studied, a majority of the cell clusters actively proliferated within the wells. Maturation of somatic embryos was successful when the newly proliferated cell clusters were transferred to solid abscisic acid-containing medium. Thus, the small, dense cell clusters could be a useful morphological marker for cells capable of proliferation and differentiation.

Immunogold localization of plant surface arabinogalactan-proteins using glycerol liquid substitution and scanning electron microscopy.

We have studied the spatial distributions of arabinogalactan-protein (AGP) epitopes on the surface of maize embryogenic calli and roots using monoclonal antibodies JIM4 and MAC207. For this purpose, a new immunogold-scanning electron microscopy (SEM) method was employed which is based on liquid substitution of samples with glycerol. Using this method, we were able to show that the AGP epitopes are distributed along callus and root surfaces and they decorate filamentous structures. In callus cells, the JIM4 epitope was specifically enriched in the outer extracellular layers covering compact clusters of embryogenic meristematic callus cells. In roots, the MAC207 epitope was abundant on the root epidermal surface corresponding to the outer root pellicle, but was only occasionally found on the mucilage layer covering the root cap cells. Silver-enhanced gold particles, indicating AGP epitopes, were often linearly arranged suggesting that AGPs associate with filamentous structures both on the surface of embryogenic calli and root epidermal cells. These results indicate that AGPs are components of the outer extracellular layers and networks that cover the surface of roots and cells undergoing somatic embryogenesis.

Synchronization of somatic embryogenesis at high frequency using carrot suspension cultures: model systems and application in plant development.

Materials and methods for the high frequency induction and synchronous somatic embryogenesis from cultured cells of higher plants are described, using carrot suspension cultures as a model system of higher plants. The following four synchronous systems of somatic embryogenesis, which were established in our laboratories, are reported: (1) Somatic embryogenesis from single cells. a) Small spherical single cells, obtained from suspension cultures in the presence of 2,4-D, zeatin and mannitol by sieving, density gradient centrifugation in Percoll solutions and manual picking up, form embryogenic cell clusters, which differentiate to embryos at high frequency, when embryogenic cell clusters are transferred to a medium lacking 2,4-D. b) Explants of hypocotyls of regenerated plantlets from somatic embryos were cultured after treatment with 2,4-D for 12-24 h, and then transferred into a fresh medium lacking 2,4-D. Single cells are released from hypocotyl explants and differentiated into embryos at high frequency. In this system, a large number of single cells and embryogenic cells can be collected. (2) Somatic embryogenesis from embryogenic cell clusters, which are obtained from suspension cultures by sieving, density gradient centrifugation in Ficoll solutions, and subsequent centrifugation at a low speed, differentiate synchronously to globular embryos at high frequency. Plantlets are formed from globular embryos. (3) Embryogenic cell clusters obtained according to the procedure described in (2) are cultured at cell densities of 2x10(3) cell clusters ml(-1). Globular embryos differentiate to torpedo-shaped embryos and subsequently to plantlets at high frequency when they are cultured at densities below 150 globular embryos ml(-1).

Comparative histology of microprojectile-mediated gene transfer to embryogenic calli in japonica rice ( Oryza sativa L.): influence of the structural organization of target tissues on genotype transformation ability

The histology and time course of hygromycin-resistant callus formation from bombarded, seed embryo scutellum-derived embryogenic nodular units (ENUs) were investigated in four japonica rice cultivars (Taipei309, IDSA6, IR47686, IRAT361) exhibiting contrasting amenabilies in a microprojectile-mediated transformation system. ENUs from the above genotypes collected at a stage considered suitable for bombardment, exhibited contrasted organization in their peripheral regions. This organization was found to influence the hygromycin concentration requested to stop ENU growth, along with the amenability of transformed cells to evolve into isolated, clonal, transgenic resistant calli, from the rest of the ENU. Organized globular proembryos located at the periphery of ENUs proved to be quite tolerant to antibiotic selection. They also developed in selective induction medium, irrespective of whether they had been sectorially transformed or not, and produced chimaeric or clonal, non-transgenic proliferations. In contrast, the presence of a single layer or small clusters of embryogenic cells at the periphery of the bombarded ENU, whose growth was fully inhibited under a selective regime, generally led to the formation of clonal transgenic calli. An average of 2.5, 4.3, and 3.2 hygromycin resistant calli were produced per responding ENU in Taipei 309, IDSA6 and IR47686, respectively. We obtained histological and molecular evidence that the hygromycin resistant calli arising from a single bombarded ENU may have been formed through independent transformation events.

Transformation system of rice suspension-cultured microcolonies by Electroporation

For establishing a transformation system of rice (Oryza sativa), after three days of culture embryogenic suspension-cultured cell clusters were enzymatically macerated for 2 hours in electroporation buffer containing 2% cellulase and filtered through 550, 400, 250 and 100 μm stainless mesh. Filtered embryogenic microcolonies of 100–250 μm with pBI121 were electroporated at 400 V/cm for 1.2 ms. Four weeks after the electroporation, stable transformed calli were obtained at a frequency of 72% on the selection medium containing 100 mg/L kanamycin. GUS gene in the genomic DNA among 20 out of 22 putative transformed calli lines were detected by PCR analysis. The expression of GUS gene into the kanamycin-resistance calli was confirmed by spectrophotometric assay and histochemical assay of GUS activity. In a histochemical study of the transgenic rice regenerants, it was shown that the GUS activity directed by the CaMV 35S promoter was localized mainly in leaf vein and root apex.

A chromo box gene from carrot ( Daucus carota L.): its cDNA structure and expression during somatic and zygotic embryogenesis

A cDNA clone, designated DcDB1, was isolated from a cDNA library prepared from embryogenic cell clusters of carrot ( Daucus carota L.) and characterized. The cDNA (1416 bp) encoded for a protein of 392 amino acid residues that contained a conserved chromo domain. The chromo domain is a 37 aa region found in both the Polycomo gene product, which is a repressor of homeotic genes, and a heterochromatin protein 1 of Drosophila. This domain is postulated to function in the binding of proteins to chromatin. Genomic blot hybridization experiments suggested that the number of DcCB1 genes in the carrot genome is low. The level of DcCB1 mRNAs was high in somatic embryos at globular and heart-shaped stages but low in torpedo-shaped somatic embryos. The level of DcCB1 transcripts decreased during the formation of seeds. The existence of both homeo and chromo box genes in plants suggests that regulatory mechanisms of developmental genes in plants may resemble those in Drosophila.

A routine system for generation of transgenic rice ( Oryza sativa L.) plants by microprojectile bombardment of embryogenic cell clusters

We have developed a routine system, based on microprojectile bombardment of embryogenic cell clusters for recovery of fertile transgenic rice ( Oryza sativa L.) plants. Embryogenic cell clusters were established within 3–4 months from calli derived from mature seeds of nine diverse cultivars. The cell clusters were bombarded with plasmids carrying genes encoding hygromycin phosphotransferase ( hph, conferring resistance to hygromycin B) and β-glucuronidase ( gus A), both driven by the cauliflower mosaic virus (CaMV)35S promoter. Resistant calli were readily recovered from hygromycin-containing medium. On average 4.6 resistant calli were recovered from each bombarded plate. Transgenic plants were routinely regenerated from such resistant calli. In this system, where the transformation procedure was based on bombardment of embryogenic cell clusters, there were no escapes. Data from Southern blot analysis of T0 and T1 plants confirmed that the transgene was stably integrated into the host genome and inherited in a Mendelian fashion.

Separation and analysis of cell types involved in early stages of carrot somatic embryogenesis

The occurrence of the polarized synthesis of DNA in embryogenic cell clusters of carrot on the third and fourth days after transfer to an embryogenesis-inducing medium was observed by labeling with [3H]thymidine and autoradiography. The cells that were actively synthesizing DNA were separated from cells that were not synthesizing DNA by maceration of cell clusters into individual protoplasts and centrifugation in a Percoll density gradient. [35S]Methionine-labeled proteins extracted from the two types of cell were analyzed by SDS-PAGE and fluorography. Three polypeptides (of 69, 98 and 108 kD, respectively) were found only in cells that were actively synthesizing DNA and could be candidates for markers of the polarity of DNA synthesis that is specific to embryogenesis.

Effect of microprojectile bombardment parameters and osmotic treatment on particle penetration and tissue damage in transiently transformed cultured immature maize ( Zea mays L.) embryos

Immature maize ( Zea mays L.) embryos of the tropical inbred line Cat-100-6, plated in callus induction medium for short periods, were used to study the effect of microprojectile bombardment parameters and osmotic treatment on the transformation of embryogenic competent cells. Somatic embryogenesis in this material arose from sub-epidermal cell clusters located at the third or deeper cell layer. Experiments were carried out focusing on the transformation of these cell clusters following microprojectile bombardment. Physical conditions such as particle delivering method, particle size, helium pressure and target distance were analyzed in factorial experiments. The best condition which permitted the transformation of embryogenic cell clusters at relatively high frequency was found to cause deleterious tissue damage because of the bombardment at high pressure and short target distance. Tissue damage as successfully prevented by the addition of mannitol in the culture medium before and after bombardment.

Ontogeny of Grape Somatic Embryos

The development of grape somatic embryos (Vitis vinifera cv. Thompson Seedless) was studied using high-resolution light microscopy and scanning electron microscopy. Somatic embryos develop either from discrete embryogenic cell clusters (indirect somatic embryogenesis) or from previously formed somatic embryos (direct somatic embryogenesis). In both instances, embryo development begins when a small, isodiametric, densely cytoplasmic cell undergoes a series of organized divisions, which are identical to those observed during zygotic embryogenesis. Developing embryos pass through recognizable embryonic stages, remaining white and opaque through maturity. Upon germination, embryos begin to enlarge, become yellow, then green, and develop into morphologically correct plants. The cells of somatic embryos contain little starch, but abundant storage proteins. However, lipids comprise the primary storage compound. Some developmental abnormalities occur during embryogenesis, including overly enlarged hypocotyls and fewer or more than two cotyledons. In addition, relatively few somatic embryos grow into plants primarily due to inadequate shoot apical meristem development. These abnormalities are best attributed to inadequacies of the in vitro environment of medium in a culture vessel when compared to the in vivo environment of a seed.

Production of somatic embryos and plantlets from root cells of the Greater Yam

Morphogenically competent cell clusters were induced from 3–4 mm-long root segments excised from in vitro shoot cultures of the greater yam (Dioscorea alata L. ‘Oriental Lisbon’)plated in a liquid modified Murashige and Skoog (1962) medium (PMS) supplemented with 1 mg l−1 2,4-dichlorophenoxy-acetic acid and 3% sucrose at 25°C in the light. Embryogenic cell clusters released from subcortical regions of the root explants proliferated further into proembryonic cell masses which, when transferred to auxin-free medium, differentiated into somatic embryos. A proportion of the somatic embryos (as high as 80% in some experiments) converted to plantlets within two months after initial root segment culture. A description of the different stages of yam somatic embryogenesis is presented. Cultural factors which affected somatic embryo maturation in liquid medium to the greatest extent were embryogenic cell cluster density and gibberellic acid (GA3) level present in the medium. Optimal somatic embryo maturation occurred when the density of embryogenic clusters was adjusted to 2–4 per 5 ml PMS and when GA3 was present in conditioned PMS medium at a concentration of 1.5 mg l−1

Isolation of a carrot gene expressed specifically during early-stage somatic embryogenesis

We report the first successful isolation by subtractive hybridization of a gene expressed specifically during somatic embryogenesis. Embryogenic cell clusters, 32-50 microns in diameter, were isolated by sieving and density-gradient centrifugation. The cDNA library was constructed from proglobulars which were formed from embryogenic cell clusters 3 days after transfer to auxin-free modified Lin and Staba's medium. For use as probe in screening, the same cDNA used for library construction was enriched for specific sequences using subtractive hybridization. The cDNA used for subtraction was prepared from suspension cultures 5 days after subculturing in auxin-containing medium. Nine independent differentially expressed cDNA clones were obtained from a screen of 150,000 recombinant phages. Northern analysis indicated one of these, CEM6, to be expressed specifically during somatic embryogenesis. In addition, one hybridizing transcript was detected in plantlet cotyledons, and two transcripts were detected in hypocotyls. Two separate and distinct hybridizing transcripts are expressed specifically in hypocotyl tissue. The amino acid sequence deduced from the nucleotide sequence of the CEM6 cDNA indicates that it encodes a glycine-rich protein containing a hydrophobic signal-sequence like domain. Its early embryo-specific expression and sequence characteristics suggest an important role as a cell wall protein in embryogenesis.

Effects of MethylJasmonate and Concanavalin A on Embryogenesis and the Induction of Secondary Somatic Embryos of Carrot

When embryogenic cell clusters prepared in a novel embryo-forming system were cultured in a standard liquid medium that contained methyljasmonate (MeJ), their development invariably proceeded to the torpedo stage but, thereafter, their morphological development to plantlets was suppressed to a considerable extent. Plantlets that occasionally regenerated in the presence of MeJ were very dwarfed. Secondary somatic embryogenesis was observed to occur at a frequency on the surface of embryos that remained at the torpedo stage and on dwarfed plantlets. Similar results were obtained with concanavalin A (Con A). These secondary embryos became developed normally into plantlets after transfer to a liquid medium without MeJ and Con A.

Effect of abscisic acid and cytokinins on the development of somatic embryos in Hevea brasiliensis

Addition of liquid medium, conditioned by an embryogenic suspension, to MH1 solid medium (3,4-dichlorophenoxyacetic acid 9 μM, 6-benzyladenine 9 μM) permitted the frequent induction of highly embryogenic calli from slices of internal integument of immature seeds of Hevea brasiliensis Mull. Arg. The proliferation of embryogenic cell clusters was achieved in MH1 liquid medium. Abscisic acid (ABA), cytokinins and adenine were tested for their ability to affect development of somatic embryos to plantlets. The transfer of embryogenic cell clusters on auxin-free solid medium with 10-5M ABA for 2 months stimulated embryo development. When torpedo-shaped embryos were transferred to medium with adenine or cytokinins they turned green in 1 month. Green embryos produced secondary embryos when they were collected and placed on medium without growth regulators.

Synchronization of somatic embryogenesis from carrot cells at high frequency as a basis for the mass production of embryos

Synchronization of somatic embryogenesis at high frequency is a useful system for the mass production of embryos. Many attempts have been carried out, however, it was difficult to obtain the system in which most of the initial embryogenic cells or cell clusters synchronously differentiate to embryos. In carrot suspension cultures, high frequency, synchronous embryogenesis systems (following three systems) have been established.

Scale-up of somatic embryogenesis in alfalfa (Medicago sativa L.) I subculture and indirect secondary somatic embryogenisis

Three methods of increasing the productivity of somatic embryogenesis in Medicago sativa L. were investigated. In the basic procedure, somatic embryos were initiated from young petioles and carried through several phases: callus formation, suspension culture, selection of the embryogenic fraction by sieving, development, maturation, desiccation and storage. The suspensions were normally separated into three fractions by sieving. Fraction I (<200 μm) containing nonembryogenic cells or cell clusters was discarded. Fraction II (200–500 μm) consisting of embryogenic cell clusters was collected for embryo development and maturation. Fraction III (over 500 μM) containing the mixture of petiole residues with large pieces of calli and globular somatic embryos was usually discarded. Several methods to scale-up the suspension phase were unsuccessful. Direct subculture of the entire suspension by the addition of fresh liquid medium resulted in the loss of embryogenic capacity by the third subculture. Subculture of fraction II decreased embryogenic cell mass, and hence reduced total productivity. The recycling of fraction III back to fresh B5g liquid medium resulted in high productivity in the first culture but further subculture of this fraction resulted in a rapid decline in the embryogenic capacity.

Plant regeneration from cultured protoplasts of the cooking banana cv. Bluggoe (Musa spp., ABB group)

Suspensions of embryogenic cells of a triploid banana (Musa spp., cv. Bluggoe) were initiated from the uppermost part of meristematic buds, and used as protoplast source. After 20 weeks in culture, the suspension contained a mixture of globular structures or globules and embryogenic cell clusters, as well as single cells. Two types of protoplasts were obtained from embryogenic suspension culture: small (20-30 μm) and larger (30-50 μm) protoplasts with a dense cytoplasm and large starch grains respectively. The small protoplasts probably originated from embryogenic cell clusters, and also from pseudocambial cells of globules, while larger protoplasts were probably released from oval starchy cells and those of the globule peripheral area. In co-culture with a suitable feeder, consisting of suspensions of diploid banana cells, the protoplasts of triploid banana reformed the cell wall within 24 h and underwent sustained divisions leading to the formation of small clusters of 2-3 cells within 7 days. The latter developed directly into embryos without passing through an apparent callus phase. 10% of such embryos gave rise to plantlets when subcultured in 2.2 μM 6-benzylaminopurine and 2 μM 4 amino-3,5,6-trichloropicolinic acid for 1 week, before transfer to MS medium containing 10 μM 6-benzylaminopurine. The rest of the embryos underwent intensive direct secondary embryogenesis which could lead to the formation of plantlets with a frequency of up to 50% upon further transfer to hormone-free medium.

Pyrolysis mass spectrometry of developmental stages of maize somatic embryos

SUMMARY The diversity in biopolymers such as polysaccharides, (poly)phenols, nucleic acids and lipids in the developmental stages of two types of histologically different somatic embryos of maize was investigated, using in-source pyrolysis low voltage electron impact mass spectrometry (PyMS) and the data were compared with those obtained for zygotic embryos. The first type of somatic embryo is attached to callus tissue and shows the same histo-differentiation characteristics as zygotic embryos. The second type grows from clusters of cells in liquid culture and its shoot meristem is blocked in development (Emons & Kieft 1991). In-source pyrolysis low voltage electron impact mass spectrometry shows that during callus initiation from young zygotic embryos the relative abundances of di- and triglycerides decrease, but increase again after establishment of the embryogenic callus. During callus initiation mass peaks for ferulic acid appear. The embryogenic cell clusters contain more pentosans and hexosans than the non-embryogenic elongated cells. In the globular stage, the hexosans increase. At the ovoid stage, when the first cells with vascular thickenings are observed in the sections, which are brightly fluorescent and can be stained with phloroglucinol, mass peaks for lignin are observed in the mass spectrum. The mass spectra of the developmental stages of somatic embryos grown attached to callus are quite comparable to those of zygotic embryos. Both structures contain far less phenolics, no lignin-like polyphenols, and a much higher amount of polysaccharides than the single somatic embryos.

CDNA cloning of ECP40, an embryogenic-cell protein in carrot, and its expression during somatic and zygotic embryogenesis

A cDNA of 1.2 kb was isolated from a cDNA library of embryogenic cells of carrot (Daucus carota L.) by use of nucleotide sequences that encode two internal amino-acid sequences of ECP40 (an embryogenic-cell protein with a relative molecular mass of 40,000). A genomic Southern blot using the cDNA as probe suggested that there are at least two genes for ECP40 in the carrot genome. The cDNA encoded an open reading frame of 306 amino acids, and the deduced amino-acid sequence was found to share two motifs, namely SSSSSSEDDGXGGRRKKGXXXKIKEKLXGG and EKKXXXDKIKXKLPG, with rab16 protein from rice and dehydrins from barley and maize. The level of expression of these proteins has been reported to be high during late embryogenesis and to be induced by a plant hormone, ABA. Accumulation of ECP40-specific transcripts started 18 days after flowering and continued until maturation of seeds, but the levels decreased within 24 h after imbibition. ECP40 and its mRNAs were detected in the endosperm and zygotic embryos of mature seeds by immunohistochemistry and in situ hybridization. Exogenous application of 0.1 mM ABA to carrot seedlings did not induce expression of the gene for ECP40, while drought treatment induced the accumulation of low levels of the mRNAs. During somatic embryogenesis, the mRNAs were found at high levels in embryogenic cells and at low levels in somatic embryos at the torpedo stage. Immunohistochemical analysis and in situ hybridization showed that both ECP40 and its transcripts were preferentially localized in the peripheral cells of the clusters of embryogenic cells. In somatic embryos, application of ABA resulted in increases in levels of mRNAs for ECP40 up to the levels in embryogenic cells, but no such increases were observed in ABA-treated embryogenic cells. The pattern of expression of the gene for ECP40 during somatic embryogenesis was basically the same as that of ECP31, another ABA-regulable embryogenic-cell protein of carrot, the presence of which has been correlated with the embryogenic competence of cultured cells (T. Kiyosue, S. Satoh, H. Kamada and H. Harada, Plant Physiol 95 (1991) 1077-1083). The various results together imply that a group of ABA-inducible genes is expressed in these embryogenic cells.