Embryogenic Cell Lines Research Articles

Gene expression during heat-shock in embryogenic carrot cell lines

We have isolated an hsp90 gene from carrot (Daucus carota). The deduced amino acid sequence from this cDNA revealed its similarity to the organelle-type HSP90 protein. It has high homology to other plant organelle-isoforms and shows similar homology to both cytoplasmic and prokaryotic HSP90s. To study the regulation of gene expression during heat-shock, two embryo-specific DC8 and DC59 genes, a tubulin gene and an hsp90 gene were monitored in two embryogenic heat-stressed carrot cell lines. The expression of DC8, a LEA and DC59, an oleosin gene, decreased in both cell lines. In addition, there was a progressive degradation of the accumulated messages with time. The expression of hsp90 gene was induced in both cell lines but with a different pattern of transcript accumulation. These results indicate that the haploid cell line responds differently from the diploid cell line and there is differential transcriptional activity in both cell lines during heat-stress.

Somatic embryogenesis and plantlet regeneration from cotyledon explants isolated from emblings and seedlings of hybrid firs

Summary Embryogenic tissues have been initiated on cotyledon explants dissected from seedlings or emblings of hybrid firs. Cotyledons of seedling origin ( Abies alba x A. cephalonica ) gave a relatively low initiation frequency (1.94 percnt;). In embling-derived cotyledons ( Abies alba x A. cephalonica, Abies alba x A. numidica ), the initiation was cell-line dependent and reached values between 1.25 and 24.28 percnt;. The established embryogenic cell lines are being maintained in long-term cultures. The origin and development of the somatic embryos have been traced histologically. The early stages of somatic embryo development have been characterised by cell division activity (predominantly periclinal) in the epidermal and subepidermal layers of cotyledons and subsequently by development of nodular structures. Further differentiation led to the formation and emergence of somatic embryos on the surface of cotyledons. Somatic embryo development and plantlet regeneration occurred from proliferating tissues initiated from cotyledons of embling as well as seedling origin.

Haploid plants regenerated from androgenic cell cultures of Digitalis lanata.

Androgenic callus was obtained from cold treated anthers and pollen of Digitalis lanata. The callus was mixoploid and contained haploid, diploid and tetraploid cells as shown by impulse cytophotometry. Haploid cell lines were selected by colony cloning. They were unstable and selection had to be repeated every 1-2 months. Mixoploid shoot cultures were derived from embryogenic haploid cell lines via somatic embryos. Haploid shoots were selected by explanting shoot tips. The shoots showed wide variability in cardenolide content and profile. Rooting of the haploid shoots resulted in haploid plants. These plants were smaller in size than diploid plants. Often the flowers were morphologically abnormal and showed male sterility due to crippled anthers.

Somatic embryogenesis in oak (Quercus spp.)

The present review summarizes the factors involved in controlling the process of oak somatic embryogenesis as a method for vegetative plant propagation and includes also data on artificial seed production, cryopreservation and transformation. One major limitation, the inability to initiate embryogenic cultures from mature trees, has been recently overcome. Leaves from selected cork oak trees with an age of 50 yr and more have been used to initiale somatic embryogenesis (SE) with a frequency of up to 20%. These findings offer encouraging prospects for cloning proven superior plant material and to integrate this propagation system into tree improvement programs. Once the process of SE has been initiated, the multiplication cycle proceeds via secondary embryogenesis, which can be maintained indefinitely. Problems are reported by the formation of anomalous embryos. The mutability of somatic embryogenic cell lines of various oak species has been monitored by flow cytometry and molecular markers. No somaclonal variation was detected applying random amplified polymorphic DNA (RAPD) or amplified fragment length polymorphism (AFLP) markers, whereas DNA-content measurements via flow cytometry revealed tetraploidy in some cell lines after several years of continuous subculture. Maturation and low germination frequencies are the main bottlenecks for a broader use of this technique. Recently attention has been on embryo quality and parameters for conversion capacity such as high endogenous cytokinin level and low abscisic acid (ABA) level. Although oak is probably the species that is the most well-developed system for a broadleaved forest tree, data on growth performances of somatic embryo-derived plants are rare.

Auxin metabolism and transport in an embryogenic cell line of Daucus carota L.

Summary In this paper we analysed the endogenous IAA levels in carrot cell cultures and in cell culture medium. Free IAA and its conjugates (esters and amides) were measured in cell cultures exposed to different concentrations of 2,4-D (nil, 2,3 and- 92 μmol/L). The three culture conditions determine different physiological conditions of the cell cultures: somatic embryogenesis, unorganised growth and growth under stress, respectively. The results indicate that these two types of IAA conjugates account for 60–70 % of total IAA and buffer the variations of free IAA in a sequential manner. Free IAA is in a steady state relationship with the ester conjugates and the increased synthesis of IAA is initially buffered by IAA-amide. When a certain threshold (750 ng/g) is reached inside the cell by the amide, further increments are buffered by ester IAA. Another result coming from this analysis is that the presence of tryptophan alone in the culture medium is sufficient to sustain, metabolically as well as physiologically, the conditions of proliferation, shifting the state of the cell culture from somatic embryogenesis to unorganised growth.

Transforming embryogenic cell lines of Gladiolus with either a bar-uidA fusion gene or cobombardment

Embryogenic cell lines of Gladiolus were bombarded with the bar-uidA fusion gene under the cauliflower mosaic virus (CaMV) 35S promoter (pDM327) or cobombarded with uidA under the CaMV 35S promoter (pBCG) and bar under the CaMV 35S promoter (pDM307). Over 500 cell lines were isolated for either the fusion gene or cobombarded cells following selection on Murashige and Skoog's medium supplemented with 2 mg 1−1 (9 μM) 2,4-dichlorophenoxyacetic acid and 6 mg 1−1 phosphinothricin. The optimum DNA concentration for, isolating stable transformants was one-tenth that for optimal isolation of lines with gus expression, and three times as many cell lines were isolated following cobombardment as compared to bombardment with the bar-uidA fusion gene. Three times as many cell lines (72% of the cell lines) containing the bar-uidA fusion gene expressed gus as compared to cobombarded cell lines (23%) following histological staining. Gus expression ceased after 1 yr in culture for 5% of the cell lines containing the fusion gene and 3% of the cobombarded cell lines. The bifunctionality and utility of the bar-uidA fusion gene were demonstrated, accompanied by enhanced gus expression.

Somatic embryogenesis and indole alkaloid production in Catharanthus roseus

ABSTRACT A new protocol to obtain an embryogenic cell line from cultured seedling explants of Catharanthus roseus is described. In order to assess the relationship between tissue differentiation and secondary metabolite biosynthesis, the biosynthetic capabilities (alkaloid production) of an embryogenic cell line and two non-embryogenic C. roseus strains were comparatively examined. Faster cell growth rate was associated with higher alkaloid production in the embryogenic cell line. The kinetics of ajmalicine and serpentine production by the three cell lines is also reported.

Embryogenic Cell Culture, Protoplast Regeneration, Cryopreservation, Biolistic Gene Transfer and Plant Regeneration in Japanese Cedar (Cryptomeria japonica D. Don).

Somatic embryogenesis in Cryptomeria japonica was initiated at a relatively high frequency from immature seeds collected from the end of June to mid-July. Induction of embryogenic cultures was possible on media with or without plant growth regulators, and the initiation frequency varied from 5 to 16%. Embryogenic cell lines have been maintained and proliferated for more than 2 years in solid and liquid media. For long-term storage embryogenic cells were cryopreserved using a simple freezing method. Cotyledonary embryos were obtained mostly on maturation media containing abscisic acid (ABA) and polyethylene glycol (PEG) as osmotic agent, however, the plant conversion rate was still low. Plants regenerated from somatic embryos continued growing in a greenhouse. Furthermore, a procedure for the individual culture of protoplasts isolates from embryonal masses, and an approach for microprojectile bombardment-mediated transformation using pIPT and pMAT vectors was also described.

A Basic Chitinase-like Protein Secreted by Embryogenic Tissues of Pinus caribaea acts on Arabinogalactan Proteins Extracted from the same Cell Lines

Embryogenic cell lines of Pinus caribaea consist of a high number of somatic embryos. We have previously characterized the embryogenic state by studying the proteins and glycoproteins ionically bound to the cell surfaces of preglobular somatic embryos. The embryogenic tissues and nonembryogenic calli produce proteins of 48 kDa or 56 and 25 kDa, respectively. All of these proteins cross-react with several classes of tobacco chitinases (CHs). These CH-like proteins express a potential chitinase activity on SDS-PAGE gels overlaid with glycol chitin as a synthetic substrate. When an arabinogalactan protein (AGP) fraction from embryogenic tissues substitutes for glycol chitin on gels, only the 48 kDa embryo-specific CH-like protein acts on this substrate, indicating that an interaction between this protein and a specific set of AGPs might exist within embryogenic tissues of Carribean pine.

Maturation and Plant Recovery from Embryogenic Cells of Japanese Larch: Effect of Abscisic Acid in Relation to Their Morphology

Two embryogenic cell lines characterized by different morphology and color, white and red, were obtained from an immature zygotic embryo of Japanese larch (Larix leptolepis Gord.). Mature somatic embryos with cotyledons and regenerated plants were obtained from both cell lines. However optimal concentration of abscisic acid (ABA) for maturation varied depending on morphology of ECs. From the white ECs which intermingled with somatic embryos of very early stage, mature somatic embryos were induced on maturation medium containing 50 μM of ABA. On the other hand, mature somatic embryos with cotyledons were observed from the red ECs which consisted of somatic embryos of more developed stage on hormone-free medium or 0.1 μM ABA containing-medium.

Maturation Capacity of Somatic Embryos of Picea Abies After Prolonged Proliferation Culture

Matured somatic embryos of different embryogenic cell lines of Picea abies formed as a response to abscisic acid and polyethylene glycol were categorised into three main groups: well-developed, less-developed and undeveloped type. Only the well-developed embryos were able to convert into normal plants. They were positively influenced by prolonged proliferation up to 4 months in contrast to the less-developed ones. Seed extract positively affected both well-developed and less-developed embryos, but only in the beginning of tested period. Intermediate cell lines after a peak of acquisition of maximum maturation competence decrease the maturation frequency with prolonged culture.

Biolistic transformation, expression, and inheritance of bovine β-casein in soybean (Glycine max)

A 630-bp DNA fragment encoding a bovine milk protein, β-casein, was cloned into a seed-specific lectin promoter expression cassette and introduced into soybean somatic embryos via particle bombardment. Four hygromycin-resistant embryogenic cell lines were selected. All four lines were positive for the β-casein gene via Southern blot analysis; however, only one line did not show significant rearrangement of the β-casein gene and/or lectin promoter. The presence of the β-casein gene in the genome of the four plants regenerated from this culture was confirmed via Southern blot hybridization analysis. DNA blot experiments and progeny inheritance analysis indicated that the plants contain four to eight copies of the β-casein gene and that the insertion occurred at a single genetic locus. Bovine β-casein mRNA was highly expressed in developing cotyledons. A very low level of β-casein mRNA was found in leaf tissues although endogenous lectin transcripts were not found in the same samples. Bovine β-casein protein was expressed in cotyledons of transformed plants but was not detectable in leaf tissues of transformed plants by immunoblotting. Bovine β-casein produced in the individual developing seed of the first generation of transgenic plants migrated as two closely spaced bands very similar to those of pure bovine β-casein. This experiment represents the first report of the expression of a milk protein in soybean and opens the way for the general improvement of protein quality in soybean directed by seed-specific promoters.

Somatic embryogenesis and plantlet development in Pinus sylvestris and Pinus pinaster on medium with and without growth regulators

To initiate somatic embryogenesis in Pinus sylvestris and Pinus pinaster, immature seeds were collected from June to August and the developmental stage of the zygotic embryos was determined. Four developmental stages were distinguished and the response of the zygotic embryos at each of the four developmental stages was compared intra‐ and inter‐species. For this study, modified Litvay's medium (LM), with or without growth regulators, was chosen. Somatic embryogenesis was initiated and maintained on both media but the two species displayed different propensities. In P. sylvestris, the highest initiation frequency was obtained with intact megagametophytes containing embryos at the four‐cell stage to the stage of cleavage polyembryony (up to 22 and 9%, respectively). The culture medium had no significant effect on the initiation and proliferation of embryogenic cultures. In P. pinaster, however, the best response occurred from excised zygotic embryos at the stage prior to elongation of cotyledon primordia (up to 40% explants responded), on medium with growth regulators. Another characteristic distinguishing the two species in culture was that in some embryogenic cell lines of P. sylvestris, somatic embryos matured spontaneously when initiated and maintained on medium without growth regulators. Some of these embryos developed into plantlets on the same medium at the frequency of 40%. Therefore, in P. sylvestris all the stages of somatic embryogenesis were achieved on the medium without growth regulators. However, in both species, maturation of a large number of somatic embryos was greatly improved on medium containing high concentration of gellan gum (Gelrite 10 g l−1) and abscisic acid (60 μM). Cotyledonary somatic embryos subsequently germinated (72 and 80% for P. sylvestris and P. pinaster, respectively) and developed into plantlets (48 and 29%, for P. sylvestris and P. pinaster, respectively). This represents a significant improvement in plantlet recovery from somatic embryos of both species.

Transgenic Central American, West African and Asian Elite Rice Varieties Resulting from Particle Bombardment of Foreign DNA into Mature Seed-derived Explants Utilizing Three Different Bombardment Devices

We developed a novel system based on particle bombardment for the transfer of foreign genes into eight important rice varieties grown in Central America, West Africa and Asia. Novel features of this improved protocol, which we believe will replace alternative systems currently utilizing immature embryos, embryogenic cell lines or suspension cultures include: (a) bombardment of target tissue 5 d after seed germination; (b) no requirement to select for embryogenic callus prior to bombardment; (c) high transformation frequencies (up to 14%); (d) ability to recover transgenic material at similar frequencies irrespective of bombardment devices used. Southern blot analysis and enzymatic assays in primary transformants and progeny confirmed the stable integration and expression of introduced transgenes in all engineered cultivars.

Screening of large numbers of seed families of Pinus strobus L. for somatic embryogenesis from immature and mature zygotic embryos

The somatic embryogenic process was investigated using immature and mature zygotic embryos originating from 13 open-pollinated seed families selected for their embryogenic capacity from over 128 seed families of Pinus strobus. In a first step, intact megagametophytes with precotyledonary embryos from these families were placed on modified Litvay medium. Embryogenic tissues (ETs) were obtained for 12 of them, with initiation rates varying from 2.6% to 23%. On average, 14% of the ETs (36/258) formed stable embryogenic cell lines (ECLs) after 4–6 months of subculture. Mature somatic embryos were produced for 30 out of 52 cell lines, and plants were regenerated. Later, initiation of ETs from mature zygotic embryos was also tested for the selected families. ECLs were obtained for five of them, with a maximum initiation rate of 2.7%, and plants were produced for four ECLs.

Somatic cell cryopreservation and protoplast regeneration of important disease-resistant wild riceOryza meyeriana Baill

Oryza meyeriana Baill is one of the three wild rice species found in Chiia.O. mcyeriana possesses valuable characteristics but is reluctant in cell culturein vitro. In a series of experiments, callus with no regeneration ability was induced from young panicle ofO. meyeriana. The callus was subcultured and propagated. Embryogenic cell clones were obtained after cryopreswation. Suspension cultures were established and protoplasts were isolated and regenerated into plants. Results of artificial inoculation ofXanthomonas campestris pv.Oryzae showed that the strong resistance did not change in the regenerated plants. The development of protoplast-to-plant system is an important progress towards utilization ofO. meyeriana via cellular engineering. The experiments demonstrated that cryopreservation of plant calli was a new way to obtain embryogenic cell line.

Somatic embryogenesis in Arabidopsis thaliana is facilitated by mutations in genes repressing meristematic cell divisions.

Embryogenesis in plants can commence from cells other than the fertilized egg cell. Embryogenesis initiated from somatic cells in vitro is an attractive system for studying early embryonic stages when they are accessible to experimental manipulation. Somatic embryogenesis in Arabidopsis offers the additional advantage that many zygotic embryo mutants can be studied under in vitro conditions. Two systems are available. The first employs immature zygotic embryos as starting material, yielding continuously growing embryogenic cultures in liquid medium. This is possible in at least 11 ecotypes. A second, more efficient and reproducible system, employing the primordia timing mutant (pt allelic to hpt, cop2, and amp1), was established. A significant advantage of the pt mutant is that intact seeds, germinated in 2,4-dichlorophenoxyacetic acid (2, 4-D) containing liquid medium, give rise to stable embryonic cell cultures, circumventing tedious hand dissection of immature zygotic embryos. pt zygotic embryos are first distinguishable from wild type at early heart stage by a broader embryonic shoot apical meristem (SAM). In culture, embryogenic clusters originate from the enlarged SAMs. pt somatic embryos had all characteristic embryo pattern elements seen in zygotic embryos, but with higher and more variable numbers of cells. Embryogenic cell cultures were also established from seedling, of other mutants with enlarged SAMs, such as clavata (clv). pt clv double mutants showed additive effects on SAM size and an even higher frequency of seedlings producing embryogenic cell lines. pt clv double mutant plants had very short fasciated inflorescence stems and additive effects on the number of rosette leaves. This suggests that the PT and CLV genes act in independent pathways that control SAM size. An increased population of noncommitted SAM cells may be responsible for facilitated establishment of somatic embryogenesis in Arabidopsis.

Flow cytometric analyses in embryogenic and non-embryogenic callus lines of Cyclamen persicum Mill.: relation between ploidy level and competence for somatic embryogenesis.

Embryogenic and non-embryogenic callus lines derived from the same diploid Cyclamen persicum genotype (`Purple Flamed') were analyzed by flow cytometry and compared to the initial plant material. The DNA content of the diploid plant in the greenhouse was 1.12 pg DNA/2C as estimated in relation to the internal standards tomato nuclei and chicken erythrocytes. In both callus lines the majority of cells contained the same amount of DNA as the initial plant, indicating that no polyploidization has taken place after 5 years of culture on medium containing 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.8 mg/l 6-(γ-γ-dimethylallylamino)purine(zip). Thus, our data suggest that in Cyclamen callus lines there was no strict correlation between the ploidy level and the ability to produce somatic embryos. Furthermore, following the proportion of cells in the three phases of the cell cycle (G0/G1, S, G2/M) during one subculture period of 4 weeks revealed high division activity within the first 2 weeks for both callus lines cultured on the 2,4-D-containing medium. However, when transferred to hormone-free medium, the division activity of the embryogenic cell line decreased markedly, corresponding to the differentiation of somatic embryos. In contrast, for the non-embryogenic callus an increase in cells in the G2/M phase was observed.

Development of somatic embryos in Norway spruce

Egertsdotter et al., 1993). The group B embryos are considered to be developmentally blocked in the sense Embryogenic cell lines of Norway spruce consist of a that they cannot reach the developmental stage required large number of somatic embryos. The cell lines have for response to abscisic acid (ABA) and formation of been divided into two groups, A and B. The group B mature somatic embryos. However, addition of extracts embryos are developmentally blocked. Extracts of from Norway spruce seeds to the culture medium can mature spruce seeds stimulate group B embryos to release group B embryos from their developmental arrest develop a morphology comparable to group A embryos. ( Egertsdotter and von Arnold, 1995). However, seed extract inhibits early embryo develop- When trying to understand the diVerences between ment. The active components in seed extract were group A and B cell lines, it was found that diVerent shown probably to be proteins. Extracts of mature extracellular proteins were secreted to the culture medium seeds contain chitinase-like proteins as recognized by in group A and B cell lines and that concentrated extracelan antibody towards chitinase 4 in sugar beet. Proteins lular proteins (CEPs) from group A cell lines aVected of similar sizes were detected by the same antibody embryo morphology in B cell lines in a positive way in the conditioned medium of group A, but not in group ( Egertsdotter et al., 1993). The content and composition B. A chitinase 4-related chitinase and a nod factor had of extracellular arabinogalactan proteins (AGPs) also a stimulating effect on early embryo development, but diVered between A and B cell lines. Furthermore, the did not influence the later stages of embryo AGP-fraction from group A CEP promoted the formation development.

Xylose-rich polysaccharides from the primary walls of embryogenic cell line of Pinus caribaea

Embryogenic cell lines of Pinus caribaea were isolated from somatic embryogenesis from zygotic embryos. Previous studies showed that the proteins and glycoproteins were characteristic of the embryogenic state. In the present work we were seeking typical feature in the polysaccharide from the cell walls of embryogenic calli at nine days of culture. Sequential extraction with water, ammonium oxalate, dimethyl sulfoxide, sodium borohydride and 4.3 M potassium hydroxide revealed that the extracted polysaccharides contained high proportions of arabinose and significant amounts of xylose. Fractionation of the hydrosoluble polymers on DEAE cellulose afforded a xylose-rich fraction (80% xylose, 24% glucose and lower properties of fucose and mannose). Methylation analysis and 13C-NMR spectra showed that the glycan backbone consisted of β 1→4 linked xylosyl residues Similar study of the fractions extracted respectively with DMSO and 4.3 M KOH showed the presence of polydisperse glycoxylans but excluded the presence of xyloglucan in significant amount. This could be a characteristic feature of embryogenic cells walls of Pinus caribaea or could be typical of cells grown as calluses. In the various fractions obtained from DEAE cellulose chromatography of the alkaline extract the infrequent occurrence of fucoxylans beside an arabinogalactan showed again the unusual nature of the cell wall polymers of this embryogenic lines, which seems to differ greatly from those found in the primary wall of cells from suspension cultures.