Synchronization of somatic embryogenesis in a carrot cell suspension culture

Synchronization of somatic embryogenesis was achieved in a carrot (Daucus carota L. cv. “Kurodagosun”) suspension culture by sieving the initial heterogeneous cell population, by density gradient centrifugation in Ficoll solutions, and by subsequent repeated centrifugations at a low speed (50g) for a short time (5 seconds), followed by transferring the cell clusters obtained, which were composed of 3 to 10 cells, to a medium containing zeatin (0.1 micromolar) but no auxin. The frequency of embryo formation reached more than 90%, and synchrony of the embryogenetic process was observed at least in the early stages of the process. The system established in the present work provides a useful system for biochemical research into the mechanisms of somatic embryogenesis.     

Temperature-sensitive carrot variants impaired in somatic embryogenesis

Cultured cells of many plant species are capable of regenerating back to plants. In carrots, plant regeneration takes place via embryogenesis. Because vast numbers of somatic embryos are produced from haploid carrot cultures, it is possible to design a selection procedure for recovering temperature-sensitive variants impaired in early embryogenesis (ts-emb^?). The ts-emb^? lines fall into three classes. The first class is blocked before any growth in embryogenic medium can take place; the second class is blocked in organized growth: cells can grow as callus, but cannot form embryos; the third is blocked at the first stage of embryo development, the globular stage. These classes are useful in delineating the developmental steps during early somatic embryogenesis. The ts-emb^? phenotype of most variant lines is stable in culture and is maintained through plant regeneration. Hypotheses are made concerning the origin of the ts-emb^? phenotype. In addition to the ts-emb^? lines, other classes of variants have been recovered, i.e., ts-growth^?, habituated lines, and lines with altered responses to growth regulators.     

Relationship between auxin-induced cell proliferation and somatic embryogenesis in culture of carrot cotyledons

We elucidated the relationship between cell proliferation and somatic embryogenesis in the culture of carrot cotyledons. Fresh weights of the cotyledon expiants were determined every five days while being cultured on a medium containing 2,4-D. Callus production increased exponentially from Day 20 to Day 25, showing a two-fold rate of proliferation. To examine the embryogenic potential of the callus, we pre-cultured cotyledon explants on an MS medium with 2,4-D, then transferred them to an MS basal medium at five-day intervals. Somatic embryos formed most frequently when the cotyledons were pre-cultured for 20 days on an MS medium that contained 5 μ2,4-D. The frequency of somatic embryo formation was 81%, while that of normal embryos with two cotyledons was 51% among those formed on a hormone-free medium. We used FACScan analysis to relate the embryogenic potential of the callus to the S phase in the cell cycle of cultured cells. The S phase was high after 25 days of culture on the medium with 5 μM 2,4-D. In contrast, the frequency of normal embryogenesis was higher at Day 20 of the pre-culture period. Culturing embryogenic calli on a medium with 5 μM 2,4-D was most favorable for producing somatic embryos with two cotyledons. We verified that active somatic embryogenesis was apparently related to cell division activity; somatic embryos induced from actively dividing cells were apt to accompany cotyledonary abnormality.     

Enhancer-trapping system for somatic embryogenesis in carrot

Somatic embryogenesis in the carrot was used to model zygotic embryogenesis because the spatial and temporal changes in somatic and zygotic embryogenesis are quite similar. To establish an enhancer-trapping system for somatic embryogenesis in the carrot, we constructed 2 enhancer-trap vectors (pETVs) using the GUS reporter gene with a minimal promoter. We also constructed several positive control vectors (pPCVs) using the CaMV 35S promoter. These are models in which pETVs are inserted near a native enhancer region in correct or reverse orientation. First, we tested whether these vectors could be used as enhancer-trap vectors using transgenic hairy root of tobacco. Histochemical GUS assays revealed that pETVs could be used as enhancer-trap vectors, even when the reporter gene in the pETVs was inserted near the native enhancer. Subsequently, we examined the availability of pETVs in somatic embryogenesis in the carrot. The constructed vector was activated in transgenic carrot embryogenic cells at high frequency (64%). This suggests that the enhancer-trapping vector is suitable as a carrot somatic embryogenesis system.     

Effects of tissue culture conditions and explant characteristics on direct somatic embryogenesis in Oncidium `Gower Ramsey'

The effects of tissue culture conditions and explant characteristics on direct somatic embryogenesis were studied on Oncidium `Gower Ramsey'. Embryo formation was significantly affected by explant position. Leaf tip segments had a significantly higher embryogenic response than other segments of leaves. Adaxial-side-up orientation significantly promoted embryogenesis in comparison with abaxial-side-up orientation. There was no significant effect of sucrose in a range of concentrations (10–60 g l^–1). Modified 1/2-MS medium (containing 85 mg l^–1 KH₂PO₄) supplemented with 170 mg l^–1 NaH₂PO₄ significantly promoted direct somatic embryogenesis. Peptone at 0.5 mg l^–1 gave significantly higher emrbyogenic response (80%) on leaf tips than control treatment (50%). The best response on direct embryo formation was obtained on the modified 1/2-MS medium supplemented with 10–20 g l^–1 sucrose, 170 mg l^–1 NaH₂PO₄ and 0.5 g l^–1 peptone.     

Isozyme patterns in zygotic and somatic embryogenesis of carrot

Isozyme patterns of carrot (Daucus carota L.) zygotic embryos between the torpedo stage up to 5-day-old seedlings have been compared with those of the similar stages from the embryogenic cell suspension culture to the late somatic plantlet. Somatic embryos blocked at the torpedo stage by -cyclodextrine have also been analyzed. All these stages have been analyzed with respect to seven different enzyme systems: arylesterase, glucosephosphate isomerase, phosphogluconate dehydrogenase, alcohol dehydrogenase, isocitrate dehydrogenase, aspartate aminotransferase and phosphoglucomutase (EC 2.7.5.1, PGM). The relationships between the different stages of both types of embryogenesis have been visualized using an unrooted tree. Generally, profiles of somatic embryos were different from those of zygotic embryos. Interestingly however, a typical zygotic embryo pattern was found in the cyclodextrine-blocked somatic embryos. Only aspartate aminotransferase patterns revealed a similarity between zygotic and somatic torpedo embryos. Both plantlet types showed close patterns with common isozymes. Moreover, similarities were evident between somatic plantlets and cell suspensions. A few isozymes appeared to be stage specific markers: esterase 10–11 were specific to achenes and early germination, phosphogluconate dehydrogenase 8 was specific to 4–5 day-old seedlings and phosphoglucomutase 1 and 7 and alcohol dehydrogenase 4 were markers for zygotic embryos. No somatic embryogenesis specific isozyme could be found. We show that patterns can be associated with particular tissue formation: mainly, aspartate aminotransferase 2 and 1, phosphoglucomutase 8 and 9 and phosphogluconate dehydrogenase 7 coincided with apical meristem initiation and phosphoglucomutase 4 and 5, zones b and d of esterase and zone b of phosphogluconate dehydrogenase coincided with vascular bundle formation.Abbreviations ADH alcohol dehydrogenase - CD -cyclodextrine - CS cell suspension culture - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediaminetetraaeetie acid - LiBo lithium hydroxide/boric acid - PEG polyethylene glycol - PVP polyvinylpyrrolidone - SEg somatic embryo at the globular stage - SEh heart stage - SEte early torpedo stage - SEtl late torpedo stage - SEce early cotyledonary stage - SEcl late cotyledonary stage - SECD somatic embryo blocked at the torpedo stage with -cyclodextrine - EST esterase - GOT aspartate aminotransferase - IDH isocitrate dehydrogenase - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) - PMS phenazonium methosulfate - PGD phosphogluconate dehydrogenase - PGI glucosephosphate isomerase - PGM phosphoglucomutase - SO dry seed - S1–3 seed after 1–3 days of germination - SP1–2 young and old somatic plantlets - ZE zygotic embryo - ZP4–5 4–5 day-old seedlings     

Role of permanent dicentric systems in carrot somatic embryogenesis

Summary A permanent dicentric chromosome system was studied on carrot cultures and regenerated somatic embryos at different stages of development. The large chromosomal variability of the cultures and the presence of the breakage-fusion bridge cycle did not interfere with the initial developmental process up to the seedling stage but subsequent growth proceeded only if healing of the broken ends or dicentric loss had occurred. The behaviour of the dicentric chromosome in culture and during somatic embryogenesis is discussed in relation to chromosomal variability, abnormal development and the somaclonal variation that such mechanisms may generate in regenerated plants.     

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.     

Inhibitory conditioning for carrot somatic embryogenesis in high-cell-density cultures

Carrot somatic embryogenesis was strongly inhibited in high-cell-density cultures. This inhibition was not caused by depletion of nutrients or physical damage but by factor(s) released into the culture medium from cells during culture. A conditioned medium prepared by eliminating cells after high-cell-density culture inhibited somatic embryogenesis. The degree of inhibition increased with the amount of conditioned medium. A dialysis experiment revealed that the molecular weight of the inhibiting factor(s) was below 3,500. We also found that the conditioned medium contained a high-molecular-weight factor(s), which stimulated somatic embryogenesis. Received: 13 March 1998 / Revision received: 19 May 1998 / Accepted: 1 June 1998     

Stimulation of carrot somatic embryogenesis by proline and serine

Serine and proline, when added in a wide range of concentrations to Daucus carota cultures during pre-growth in the presence of 2,4-D(2,4-dichlorophenoxyacetic acid), extended in time and quantity the mitotic divisions and stimulated significantly the number of embryos regenerated when the hormone subsequently was omitted from the medium. A specific effect of these amino acids on regeneration is suggested, since proline (as hydroxyproline) and serine are the two major constituents of the cell wall glycoprotein, extensin, which thus may have a morphoregulatory function. The significant role of the conditions during pregrowth of the cultures in the presence of hormone is underlined by the effect of hydroxyproline and D-proline which also stimulate embryogenesis, but alter markedly the development of the embryos.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylaminopurine - Pro proline - Ser serine - Ala alanine - Glu glutamic acid - Leu leucine - Hyp Hydroxyproline - FC fusicoccin     

The effect of genotype and explant age on somatic embryogenesis of coffee

We demonstrate that embryogenic capacity of coffee (Coffea arabica) depends on the genotype, and that it is a character fixed at the early generations F3 or F4. Therefore, the embryogenic capacity F5 lines can be predicted in a breeding program if it is possible to evaluate the F3 of F4 ancestors, or lines with the same parents on those generations.Regeneration via somatic embryogenesis of genotypes from a C. arabica cv. Caturra by Timor Hybrid cross were evaluated, to determine the effect of the pedigree, month of leaf collection, as well as its interaction with the genotype. Large variations in embryogenic capacity among genotypes were detected with rates ranging from 4.8 to 72.7. There was association between embryogenic response of the progenies and the progenitor from which they proceeded. Significant differences were also found in embryogenic responses depending on the month of leaf explant collection, as well as a significant interaction of genotype by planting month.The results are relevant for breeding and tissue culture purposes of coffee because they will allow speed up of the breeding program and save effort by being able to predict embryogenic capacity of a line, based on the response of either its progenitors or the F3 or F4 lines of the same origin. In addition, high embryogenic capacity lines can be considered directly for genetic transformation or for massive multiplication (bioreactors).     

The effect of explant material on somatic embryogenesis of Cyclamen persicum Mill

Somatic embryos of Cyclamen persicum Mill. could be produced through a callus phase from juvenile explant material including anthers, ovaries and zygotic embryos. The auxin 2,4-D (1.0–1.5 mg l^-1) and coconut milk (10% v/v) in MS medium were important factors for the induction of somatic embryogenesis. Somatic embryos germinated into plantlets in MS medium without growth regulators. The plants grew well in the greenhouse and flowered normally. The plants were phenotypically identical to the mother plants with a few exceptions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthylacetic acid - IAA 3-indoleacetic acid - BA 6-benzyladenine - ABA abscisic acid - CM coconut milk     

Aspects of DNA, RNA and protein synthesis during somatic embryogenesis in a carrot cell suspension culture

Changes in DNA, RNA and protein content, incorporation of ³H-thymidine, ¹⁴C-uridine and ³H-leucine and template activity of chromatin were investigated in the early process of somatic embryogenesis in a carrot (Daucus carota L. cv. Kurodagosun) cell suspension culture using a synchronous system. An embryogenetic culture in a medium containing 10^-7M zeatin was compared with a non-embryogenetic culture in a medium containing 10^-7M zeatin and 5 x 10^-7M 2,4-D. DNA was synthesized very actively prior to and during the formation of globular embryos in the embryogenetic culture. The RNA and protein content per tube increased at an almost constant rate in both cultures, while the rate of incorporation of labelled precursors of RNA and protein rose much more prior to active DNA synthesis in the embryogenetic culture than in the non-embryogenetic culture. Template activity of chromatin was high in the early stage of embryogenesis in the embryogenetic culture. The results obtained here showed that synthesis and turnover of RNA and protein became active prior to active DNA synthesis in the early stage of embryogenesis, and that these changes at macromolecular levels may play important roles in embryogenesis.     

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. This revised version was published online in June 2006 with corrections to the Cover Date.     

Tunicamycin affects somatic embryogenesis but not cell proliferation of carrot

The antibiotic tunicamycin which specifically blocks the first step in the lipid-linked oligosaccharide pathway is capable of arresting somatic embryogenesis in a reversible way. At the same drug concentration cell proliferation is not affected. The quantitative and qualitative changes induced by tunicamycin in glycolipids and glycoproteins are the same in embryogenic and non-embryogenic conditions and this might therefore indicate some proteins whose glycosylation is essential for development.     

Isoperoxidases as markers of somatic embryogenesis in carrot cell suspension cultures

Growth, peroxidase activity and isoperoxidase pattern were studied during the growth cycle of 3 cell suspension lines of carrot ( Daucus carota L.), an embryogenic, a non-embryogenic and a habituated cell line. Isoelectric focusing of extracted proteins on agarose gels revealed the isoperoxidase pattern of the embryogenic line to include, among other differences, an isoperoxidase with a pl of pH 7.0 when grown under conditions stimulating embryogenesis. This isoperoxidase (P7.0: EC 1.11.1.7) was present between days 2 and 6 after subculturing, and this period correlates well with the early stages of somatic embryogenesis. This isoenzyme showed very low activity in the non-embryogenic and habituated cell suspension lines as well as in the embryogenic cell line in the presence of Daucus carota , 2,4–dichlorophenoxyacetic acid. P7.0 could probably be used as a biochemical marker of somatic embryogenesis.     

Gene expression during induction of somatic embryogenesis in carrot cell suspensions

We report the identification, via their cDNAs, of genes which are temporarily transcribed during the initiation of somatic embryogenesis in carrot (Daucus carota L.) cells cultured in an auxin-free medium. Their expression is roughly associated with the first morphogenetic, or globular, stage. A cDNA library ( gt 10) was established using poly(A)⁺ -rich RNAs from cells deprived of auxin for 8 d. By differential screening a number of clones corresponding to early-induced embryogenic genes were identified. For several a temporary accumulation of the specific mRNA between 6 and 16 d after induction was observed. With regard to the nucleotide sequence and the respective deduced amino-acid sequence, two glycine-rich proteins and a polypeptide with a proline-rich domain were among the products of genes activated at the onset of somatic embryogenesis.Abbreviations b, bp bases, basepairs - 2,4-D 2,4-dichlorophenoxyacetic acid Sequence data reported here will appear in the EMBL Genbank and DDBJ Nucleotide Sequence Databases under the following accession numbers: X 15436 for clone DC 2.15 (proline-rich protein), X 15706 for clone DC 7.1 (glycine-rich protein, DCGRP) and X 14067 for clone DC 9.1 (glycine-rich protein, DCGRP)This research was supported by the Deutsche Forschungsgemeinschaft. We thank Mrs. I. Liebscher for her competent assistance.     

Genetic analysis of somatic embryogenesis in carrot cell culture: Initial characterization of six classes of temperature-sensitive variants

Cell cultures of the carrot Daucus carota are a useful experimental system for studying the genetic regulation of plant embryogenesis. A modified filtration-enrichment procedure was used to isolate 21 temperature-sensitive variants in somatic embryogenesis; the variants display normal embryo development at the permissive temperature (24°C) and altered development at the restrictive temperature (33°C). Temperature-shift experiments were performed on these variants to determine the timing of gene action for the putative temperature-sensitive alleles. According to their phenotypes at the restrictive temperature, these variants can be divided into six classes: No Growth, Callus Proliferation, Globularstage Block, Oblong-stage Block, Lateral Growth, and Root Formation. Although many variants exhibit lengthy temperature-sensitive periods, the temperature sensitivity of some variants is restricted to one or two embryonic stages. These results plus those in the literature are incorporated into a preliminary model concerning the genetic regulation of carrot embryogenesis.     

Possible involvement of abscisic acid in the induction of secondary somatic embryogenesis on seed-coat-derived carrot somatic embryos

When seed coats (pericarps) were picked from 14-day-old carrot (Daucus carota) seedlings and cultured on agar plates, embryogenic cell clusters were produced very rapidly at a high frequency on the open side edge. Embryo induction progressed without auxin treatment; indeed treatment caused the formation of non-embryogenic callus. The embryogenic tissues (primary embryos) developed normally until the torpedo stage; however, after this a number of secondary somatic embryos were produced in the hypocotyl and root regions. Tertiary embryos were formed on some of the secondary embryos, but many developed into normal plantlets. The primary embryos contained significantly higher levels of abscisic acid (ABA) than the hypocotyl-derived normal and seed-coat-derived secondary embryos. Fluridone inhibited the induction of secondary embryogenesis, while exogenously supplied ABA induced not only tertiary embryogenesis on the seed-coat-derived secondary embryos, but also secondary embryos on the hypocotyl-derived normal somatic embryos. These results indicate that ABA is one of the important endogenous factors for the induction of secondary embryogenesis on carrot somatic embryos. Higher levels of indole-3-acetic acid (IAA) in primary embryos also suggest the presence of some concerted effect of ABA and IAA on the induction of secondary embryogenesis in primary embryos.