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.     

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.     

Changes in cell wall polysaccharides during elongation in a 2, 4-D free medium in a carrot suspension culture

Cell elongation occurred when carrot (Daucus carota L. ev. Kurodagosun) cells subcultured through sieving (Y. Ozeki and A. Komamine, Physiol. Plant. 53: 570-577. 1981) were transferred to a medium lacking auxin, while the cells showed no elongation in a medium containing 2, 4-D. Changes in polysaccharides of the cell walls and in their sugar composition during elongation were investigated. All wall components, EDTA-soluble pectic substance, 5 and 24%, KOH-soluble hemicelluloses and cellulose increased markedly during elongation. The increase of hemicelluloses correlated especially with elongation. In the 5% KOH-soluble hemicellulose, galactose and arabinose contents in the walls increased significantly both in amounts (per fresh weight) and relative contents (% in total neutral sugars) during elongation, while the relative contents of glucose and xylose decreased rapidly in the 5 and 24% KOH-soluble hemicelluloses. The methylation analysis tentatively indicated that larger amounts of galactan and/or arabinogalactan and lower amount of xyloglucan were found as components of the two hemicelluloses of elongating cells than those of non-elongating cells. The amounts of total carbohydrate and of uronic acid of extracellular polysaccharides secreted into the medium increased to a larger extent in the elongation culture than in the non-elongation culture. The contents of galactose and arabinose in extracellular polysaccharides increased rapidly in the elongation culture. The biochemical aspects of cell elongation in the absence of auxin were discussed from the viewpoint of the results obtained here.     

DNA RNA and protein content of tissue during growth and embryogenesis in wild-carrot suspension cultures

Summary A highly selected population of cells (clumps from 63 to 125 μm in diameter), obtained by screening 14-day-old stock suspension cultures of wild carrot (Daucus carota L.), was used to initiate cultures in this study. Time-course changes in DNA, RNA and protein were followed when these cultures were grown in the presence or absence of 2.25 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The data show that growth of these cultures, particularly in the early part of the growth curve, is different from that in most other studies reported on suspension cultures initiated without screening. The gross compositional analysis shows that this difference stems from the very high RNA:DNA and protein:DNA ratios of the cellular material used as the inoculum in this study. The presence of 2,4-D in the medium promoted total RNA and protein levels. Correlations were sought between the appearance of embryos in the absence of exogenous 2,4-D and gross compositional differences developing in cultures grown in the presence and absence of 2,4-D. The handling of cultures during inoculation appeared to have led to a substantial loss of DNA. This had, however, little effect on dry weight or protein content of the tissue. This research was supported by the W. Alton Jones Foundation.     

Evidence for phosphorylation of tubulin in carrot suspension cells

Two-dimensional gels of phosphoproteins from carrot ( Daucus carota L. var. Juwarot) suspension cells labeled in vivo or in vitro revealed phosphoproteins that comigrate with carrot tubulin. A polyclonal antiserum to hibiscus tubulin immunoprecipitated an in vivo labeled phosphoprotein of 50 kDa. Cell-free extracts of carrot suspension cells phosphorylated both purified carrot and bovine brain tubulins in the presence of gamma-labeled adenosine triphosphate. This tubulin phosphorylating activity was reduced 2-fold in extracts from globular stage embryos and approximately 10-fold in extracts from heart/torpedo stage embryos. These data suggest that carrot cells phosphorylate tubulin, and that tubulin phosphorylating activity may be developmentally regulated     

An evaluation of gene expression during somatic embryogenesis of two temperature-sensitive carrot variants unable to complete embryo development

The synthesis of putative stage-specific polypeptides during somatic embryogenesis of the carrot ( Daucus carota L. cv. Danvers) was investigated in the temperature-sensitive variants OB-2 and OB-3. These variants undergo normal embryo development to produce mature plantlets at the permissive temperature (24°C), but are arrested at the oblong stage to form elongated embryos without cotyledons at the restrictive temperature (33°C). Using two-dimensional polyacrylamide gel electrophoresis of in vivo labelled polypeptides, the patterns of stage-specific polypeptides in both lines were compared in: (1) oblong embryos grown at continuous 24°C vs oblong embryos exposed to 33°C during their temperature-sensitive period (i.e. embryos of identical morphology but different developmental fates); and (2) heartshaped embryos grown at constant 24°C vs enlarged oblong embryos exposed to 33°C during their temperature-sensitive period (i.e. embryos of the same age but different morphologies). The 22 putative stage-specific, polypeptides observed in this study fall into four classes: (1) line-specific, (2) age-specific, (3) unsynchronized, and (4) synchronized polypeptides. Only the last class, which consists of 4 polypeptides, exhibits synthesis patterns which are consistent with the polypeptides being causally involved in somatic embryo development. It is concluded that stage-specific behavior as assayed by PAGE analyses of simple 'present or absent' comparisons is insufficient to identify most of the polypeptides that may be relevant for somatic embryogenesis.     

Effect of nalidixic acid and novobiocine on the metabolism of suspension cultured carrot cells

Suspension cells of Daucus carota L. cv. Lunga di Amsterdam are extremely sensitive to both nalidixic acid and novobiocine. The synthesis of DNA, RNA and protein are inhibited within minutes of exposure even at concentrations less than 0.1 mM of both drugs. Moreover, uptake of deoxythymidine by cells is impaired more severely than by sodium azide. Reduction of oxygen consumption caused by the two drugs is slower than in the case of NaN₃ and occurs a few minutes after the inhibition of macromolecular biosynthesis. This excludes the possibility that the block in oxidation is the primary cause of the observed inhibition of cellular functions and suggests that the inhibition of ATP synthesis, which is immediately affected in both cases, may be responsible for these effects. A possible interaction with other cellular targets may be secondary to the uncoupling effect. Cell growth is completely inhibited by 0.3 mM concentration of both drugs, but this inhibition is irreversible only in the case of novobiocine. The irreversibility of cell growth inhibition by novobiocine can be explained by the total disappearance of ATP from the cell.     

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.     

Initial identification of a phosphoprotein that appears to be involved in the induction of somatic embryogenesis in carrot

We examined changes in the absence versus presence patterns of phosphoproteins with respect to the acquisition of embryogenic competence during somatic embryogenensis in carrot (Daucus carota L.). To characterize a possible correlation between the induction of embryogenic competence and protein phosphorylation, we examined the patterns of protein phosphorylation in embryogenic cells (EC) and non-embryogenic cells (NC) that had lost the ability to form somatic embryos. Two-dimensional polyacrylamide gel electrophoresis and subsequent autoradiography revealed the presence of 31 phosphoproteins in EC but not in NC. Furthermore, when we examined the induction of somatic embryogenesis by certain stress compounds in the absence of phytohormones, we identified one specific phosphoprotein (ECPP-44). ECPP-44 was found to be induced in all treatments that resulted in embryogenic competence. The partial amino acid and nucleotide sequence of ECPP-44 shows partial homology to two dehydrins (ERD10 and ERD14) from Arabidopsis. Received: 1 October 1999 · Accepted: 3 November 1999     

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.     

Transient reduction in secreted 32 kD chitinase prevents somatic embryogenesis in the carrot (Daucus carota L.) variant ts11

At the nonpermissive temperature, somatic embryos of the temperature-sensitive (ts) carrot (Daucus carota L.) cell variant ts11 only proceed beyond the globular embryo stage in the presence of medium conditioned by wild-type cells. The causative component in the conditioned medium has been identified as an acidic 32 kD endochitinase. An antiserum raised against the 32 kD chitinase detected this protein in culture medium from ts11 embryo cultures grown at the permissive temperature as well as at the nonpermissive temperature. No difference in biochemical characteristics or in effect on ts11 embryo development could be detected between the 32 kD chitinase purified from wild-type cultures and the chitinase from ts11 cultures grown at the permissive or at the nonpermissive temperature. Compared to the amount present in a ts11 embryo culture at the permissive temperature, a reduction in the amount of 32 kD chitinase was observed during the temperature-sensitive period at the nonpermissive temperature. These results imply that the arrested embryo phenotype of ts11 is not the result of a structural difference in its 32 kD chitinase, but is the result of a transient decrease in the amount of 32 kD chitinase present. Morphological observations indicate that the ts11 phenotype is pleiotropic and also affects the cell wall of nonembryogenic cells. © 1995 Wiley-Liss, Inc.     

Protease activities in non-embryogenic and embryogenic carrot cell strains during callus growth and embryo formation

Embryogenic and non-embryogenic cell strains of Daucus carota L. were examined for their protease activity using a wide range of chromogenic synthetic peptides as substrates. High arginine-specific activity was present in all strains, but no protease activity "specific" for embryogenic or non-embryogenic strains could be detected with the substrates tested. The specific protease activity was 5–10 times higher in the non-embryogenic as compared to the embryogenic strain for most tested substrates, and this difference was not due to release of proteases in the latter. All strains showed a decrease in protease activity when cultured in media without 2,4-dichlorophenoxyacetic acid, but the embryos had high protease activity in comparison with the nondifferentiated cell aggregates. In the latter aggregates, hydrolyzing activity towards three of the substrates (H-D-Phe-Pip-Arg- p -nitroanilide, Suc-Ala-Pro-Phe- p -nitro-anilide and Bz-Phe-Val-Arg- p -nitroanilide) was absent, whereas the embryos were able to hydrolyze them.     

Somatic embryo development in carrot is associated with an increase in levels of S-adenosylmethionine,S-adenosylhomocysteine and DNA methylation

Almost homogeneous populations representing different developmental stages of somatic embryos (globular, torpedo-shaped, plantlets) and vacuolated cells were obtained from a cell suspension culture of carrot. The concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and methylated DNA were determined in embryos at different developmental stages and were found to increase during somatic embryogenesis. The highest increase during embryogenesis was a 5-fold increase in the level of SAM. A considerable increase in the methylation index (SAM/SAH ratio) was also found. We propose that the levels of SAM and SAH may be involved in the control of somatic embryogenesis by affecting the level of DNA methylation, which in turn might cause differential changes in gene activation. An increase in the level of SAM may be a prerequisite for progression of embryogenesis and the development of complete embryos.     

Calcium increases the yield of somatic embryos in carrot embryogenic suspension cultures

An upward shift in the concentration of calcium present in the medium during somatic embryogenesis increased the number of embryos produced approximately two-fold. This was observed when embryogenic suspension cells grown in 2,4-D medium with the normal calcium concentration of 10^–3 M were transferred to hormone-free medium containing 10^–2 M calcium and when embryogenic suspension cells grown in 2,4-D medium containing 10^–4 M calcium were transferred to hormone-free medium with 10^–3 M calcium. At calcium concentrations between 6·10^–3 and 10^–2 M globular stage somatic embryos were found in cultures supplemented with 2·10^–6 M of 2,4-D indicating that elevated calcium counteracts the inhibitory effect of 2,4-D on somatic embryogenesis. No qualitative changes were found in the pattern of extracellular polypeptides as a result of growth and embryogenesis in media with different calcium concentrations.     

The rates of deceleration of nuclear and organellar DNA syntheses differ in the progenitor cells of the apical meristems during carrot somatic embryogenesis

 The synthesis of DNA in nuclei and organellar nucleoids at the various stages of somatic embryogenesis in carrot (Daucus carota L. cv. Kurodagosun) was analyzed using anti-5-bromo-2′-deoxyuridine (BrdU) immunofluorescence microscopy. The active syntheses of both nuclear and organellar DNA started in the cells forming the embryo proper 3 d after the initiation of embryogenesis, but not in cells forming suspensor-like cell aggregates. In the early globular embryo, active DNA syntheses were continuously observed in the whole embryo proper, except for the progenitor cells of the root apical meristem (RAM) and shoot apical meristem (SAM). These were recognized as slowly cycling cells with a non-BrdU-labelled nucleus and strongly BrdU-labelled organellar nucleoids. At the heart- and torpedo-shaped embryo stages, both nuclear and organellar DNA syntheses were inactive in the presumptive RAM and SAM. Thus, slowing down of organellar DNA synthesis is not coupled with, but is later than, that of nuclear DNA synthesis in the progenitor cells of the embryonic RAM and SAM. These findings clearly indicate that the timing of DNA synthesis is similar in the progenitor cells of both the RAM and SAM in the early stages of somatic embryogenesis. Received: 18 January 2000 / Accepted: 2 March 2000     

Enhancing carrot somatic embryos survival during slow dehydration, by encapsulation and control of dehydration

In order to obtain dry artificial seeds, carrot somatic embryos were encapsulated and dehydrated. Encapsulation in some hydrogels delayed the dehydration and preserved the water content of carrot somatic embryos. In particular, a matrix made of alginate with gellan gum was found to be the most efficient in maintaining a high water activity (a_w) around somatic embryos. By delaying dehydration, and also rehydration, encapsulation seemed to protect somatic embryos against desiccation and imbibition damages, giving better germination and emergence of cotyledons. Matrices made of alginate mixed with kaolin or gellan gum were particularly adapted to protect the embryos during the dehydration. Apart from the matrix composition, the control of dehydration speed enhanced the survival and regeneration of encapsulated-dehydrated somatic embryos. Using a slow dehydration protocol (95-15% RH—relative humidity into the chamber—in 11.5 days), it was possible to exert different dehydration speeds. Slowing the dehydration between 70 and 45% RH stabilized the water activity (a_w) of the encapsulation matrix, and enhanced the survival and regeneration frequencies of encapsulated-dehydrated embryos. In the absence of any maturing pretreatment, alginate-gellan gum encapsulated carrot somatic embryos, dehydrated to 15% RH, and rehydrated in moistured air (90% RH), germinated up to 72.9%. Therefore, encapsulation in alginate-gellan gum, combined with a slow dehydration, leads to enhance the somatic embryos' desiccation tolerance.     

Isolation of two genes that were induced upon the initiation of somatic embryogenesis on carrot hypocotyls by high concentrations of 2,4-D

 This study describes the formation of somatic embryos directly on the surface of hypocotyl sections of Daucus carota L. after exposure to 450 μM of 2,4-D for 2 h, followed by culturing without 2,4-D for 2 weeks. A search for differentially expressed genes immediately after the 2,4-D treatment resulted in the identification of two genes, Dchsp-1 and Dcarg-1. Dchsp-1 has homology to low-molecular-weight heat-shock proteins, while Dcarg-1 has homology to auxin-regulated genes. Dchsp-1 was expressed upon the exposure to 2,4-D treatment but also responded to general stress responses. Dcarg-1 responded primarily to auxin; however, a parallel relationship between the expression of Dcarg-1 and the formation of somatic embryos on the hypocotyl sections was observed. It seems the exposure to auxin shock could serve as a trigger in inducing cell division in the epidermal cells and promoting their further differentiation to somatic embryos. These results are discussed in relationship to our current understanding of the molecular basis of somatic embryogenesis. Received: 3 May 1999 / Accepted: 9 August 1999     

Stimulation of somatic embryogenesis in carrot by ethylene: Effects of modulators of ethylene biosynthesis and action

Endogenous levels of ethylene appeared to he suhoptimal for somatic embryogenesis in a suspension culture of carrot. Low concentrations of 1-aminocyclopropane-1-carboxylic acid (ACC). 2-chloroethylphosphonic acid (ethephon) and elhylene stimulated embryogenesis whereas higher concentrations were inhibitory. The stimulation by ACC was through its conversion to ethylene. whereas the inhibition by ACC was not. Low concentrations of AgNO₃. an inhibitor of ethylene action, inhibited embryo-genesis but stimulated ethylene production. Aminoethoxyvinylglycine (AVG) and aminooxyacetic acid (AOA). commonly used inhibitors of ACC synthase. inhibited both embryogenesis and ethylene production. However, the inhibition of embryogenesis was not related to the inhibition ote ethylene production. Very low concentrations of AVG stimulated embryo production in a way unrelated to its effect on ethylene production. Salicylic acid and CoCl₂. inhibitors of ACC oxidase in other systems, inhibited embryogenesis but. again, in way(s) unrelated to their inhibition of ethylene production. In fact, low concentrations of salicylic acid stimulated rather than inhibited ethylene production. The results show that in suspension-cultured cells, caution is warranted in the interpretation of results obtained with agents presumed to inhibit ethylene biosynthesis. The stimulation of somatic embryogenesis by ethylene unequivocally shows that the inhibition of embryo development by 2.4-dichlorophenoxyacetic acid (2.4-D) and other auxins cannot be through their stimulatory effect on ethylene production.