Non-embryogenic Lines Research Articles

The WOX Genes from the Intermediate Clade: Influence on the Somatic Embryogenesis in Medicago truncatula.

Transcription factors from the WOX family are well-known regulators of cell proliferation and differentiation in plants. Herein, we focused on several WOX genes from the intermediate clade and checked their impact on somatic embryogenesis using the model legume object Medicago truncatula. As a result, we show that MtWOX9-1 overexpression not only stimulates somatic embryogenesis in the embryogenic M. truncatula line, as it was shown previously, but can also induce somatic embryogenesis in the non-embryogenic line. Other intermediate clade WOX, including the close paralog of MtWOX9-1, as well as WOX11 homologs, did not have any significant impact on somatic embryogenesis in our in vitro cultivation system. Together, our results give new information about the diversity of the WOX family proteins and their specific functions. These data can be used for the search of new regeneration stimulators.

Comparative analysis of the composition of sterols in embryogenic and nonembryogenic cell lines of <i>Larix sibirica</i> Ledeb.

The cell lines of Siberian larch (Larix sibirica Ledeb.), possessing different embryogenic potential, were studied. Qualitative and quantitative content of sterol components was comparatively analyzed in their tissues with the help of gas chromatography-mass spectrometry. Significant differences in these parameters were found between both groups of the lines in fractions of free sterols and their esters. In addition to sterol compounds, squalen, which is a triterpenic intermediate in sterol biosynthesis, was revealed. Of free sterols, -sitosterol, campesterol, isofucosterol, and stigmasterol dominated in the embryogenic lines, while -sitosterol, campesterol, and stigmasterol in nonembryogenic ones. The campesterol concentration was 1.31.9 times higher in the embryogenic than nonembryogenic lines. Since campesterol is a precursor of brassinosteroids, it is likely that its level is high due to an embryogenic state of these cell lines. Only the embryogenic lines contained appreciable amounts of isofucosterol. The fraction of sterol esters exhibited wider component diversity in the nonembryogenic than the embryogenic lines. In all the cell lines, among the identified sterol esters, the substances without double bonds, bearing, as a structural fragment, a sterane coreperhydrocyclopenta[a]phenanthrenewere the most abundant. Their content ranged from 52 to 71% of the total amount of sterol esters. The revealed differences in the compositions of sterols and their esters, between the L. sibirica cell lines with different embryogenic potential, witness to the considerable rearrangements in sterol metabolism in the course of embryogenesis. The line-specific differences may indicate the involvement of these metabolites in formation of embryos.

Differences in DNA methylation, DNA structure and embryogenesis-related gene expression between embryogenic and non embryogenic lines of Pinus radiata D. don

Pinus radiata is the most important conifer species for commercial forestry in countries such as Australia, New Zeeland and Chile. Nowadays, SE (somatic embryogenesis) is considered the most promising in vitro method for large scale vegetative propagation of woody plants. The understanding of the molecular basis of SE is in its very beginning and a number of embryogenesis-related genes have been identified in conifers. Among the molecular mechanisms involved in regulation of SE, DNA methylation, which is an epigenetic modification associated with transcriptional silencing, has shown to be a pivotal factor controlling gene expression. In this work, we studied the morphological and molecular differences between cell lines previously characterized in terms of their embryogenic potential as embryogenic (E) and non embryogenic (NE), obtained from immature zygotic embryos of P. radiata. In contrast to E lines, NE lines were composed of multicellular aggregates lacking polarity, and they were characterized by the presence of significantly lower transcript levels of embryogenesis-related genes and higher global DNA methylation. Furthermore, the detection of vibrational markers of DNA conformation indicated that DNA samples obtained from E lines presented the common B-DNA conformation, while NE samples presented Z-conformation. Taken together, our results highlight the role of epigenetic mechanisms such as DNA methylation in regulating the expression of embryogenesis- related genes, having impact on the embryo patterning and cell differentiation.

Identification of LEC1, L1L and Polycomb Repressive Complex 2 genes and their expression during the induction phase of Medicago truncatula Gaertn. somatic embryogenesis

Although somatic embryogenesis (SE), a multi-step process starting from somatic tissues and ending with somatic embryos, has been applied to numerous plants including Medicago sp., the molecular basis of development reprograming in somatic cells toward the embryogenic pathway is still incompletely known. Though recent analysis of the proteome and transcriptome has led to the identification and characterization of new genes involved in SE, lot of these genes are up-regulated only in the late developmental stages. Consequently, this work was aimed at finding out if and when the genetic program changed during the SE induction phase in both the highly embryogenic line M9-10a of Medicago truncatula cv. Jemalong and its non-embryogenic predecessor line, M9. Based on multipoint (day 0, 2, 7, 14 and 21) gene expression qPCR analysis of two embryogenesis marker genes LEC1 and L1L and selected genes encoding proteins of PRC2 complex (CLF, SWN, FIE, MSI1, VRN2) it was possible to distinguish two periods during the induction phase. The first week was related to dedifferentiation with no visible changes in explant morphology and lack of transcripts of LEC1 and L1L; the expression of PRC2 members, however, increased in the embryogenic line. The next two weeks were regarded as the expression phase involving the beginning of a rapid callus growth and the appearance of products for LEC genes, which was observed in the embryogenic line only. However, the callus formation was observed in the non-embryogenic line as well, but the LEC1 and L1L genes were not expressed and the transcription of PRC2 genes was at stable level. It was only the SWN expression that decreased at the beginning of induction and did not change in the subsequent days in both lines. Our results indicate that LEC1 and L1L, known as marker genes of the late developmental stages, may be associated with the acquisition of embryogenic competency by somatic cells (prime events in the SE induction). The PRC2 complex genes are also expressed during embryogenic callus tissue formation on Medicago tuncatula leaf explants.

Wczesne etapy somatycznej embriogenezy w kalusie korzeniowym lędźwianu siewnego (Lathyrus sativus L.)

Callus cultures from root explants of Lathyrus sativus L. ‘Derek’ were tested for their morphogenic capacity. Primary explants (fragments of roots) were cultivated on three induction media. We obtained three lines of callus tissue among which we identified two non-embryogenic lines and one embryogenic line. Callus originally cultivated on modified MS medium supplemented with 0.05 mg*L-1 picloram, formed embryo-like structures upon transfer to media containing 0.1 mg*L-1 picloram or 0.9 mg*L-12,4-D. Histological examinations confirmed embryogenity of obtained structures. Previous studies had revealed that, notwithstanding efficient callus induction and proliferation, its capacity to differentiate shoots or somatic embryos is limited. Consequently, rhizogenesis was only form of complete organogenesis obtained in our experiments. However attempts to develop the methods for indirect plant regeneration in L. sativus would allow creation of new genetic variations required to improvement of this species.

DNA demethylation and decrease on free polyamines is associated with the embryogenic capacity of Pinus nigra Arn. cell culture

Embryogenic cell lines initiated from immature zygotic embryos of Pinus nigra Arn. ssp. Austriaca were characterized in terms of macromorphological traits (colour, bipolar structures formation, germination ability) and their embryogenic potential was defined as high, medium or null. Quantification of global genomic DNA methylation revealed the existence of specific DNA methylation levels for the determinated embryogenic potentials. The line considered as effectively embryogenic, i.e., with the ability of develop the whole embryogenic program and producing plants, showed the lowest methylation levels. There was also proved the existence of an inverse relationship between total contents of free PAs and embryogenic potential, being the highest contents of free putrescine and spermidine in the non-embryogenic line and the lowest in the effectively embryogenic one. Relationships among DNA methylation levels, profiles of free individual polyamine contents and embryogenic potentials based on the ability to produce well-formed somatic embryos with effective plant conversion are discussed.

Identification and characterization of PgHZ1, a novel homeodomain leucine-zipper gene isolated from white spruce (Picea glauca) tissue

A member of the homeodomain-leucine zipper (HD-Zip) family was isolated from white spruce (Picea glauca) and designated as PgHZ1 (Gene Bank Accession No. DQ201170). The gene has an open reading frame of 1268bp and encodes a protein of 309 amino acid residues. PgHZ1 has all the features of a HD-ZIP protein: a homeodomain composed by three alpha-helices involved in DNA binding and an adjacent leucine zipper motif for protein-protein interaction. Phylogenetic analyses and sequence allignments with several Arabidopsis HD-ZIP members reveal that PgHZ1 belongs to the same monophyletic group of ATHB3, 13, 20, and 23 with which it shares a respective amino acid similarities of 74%, 71%, 68%, and 61%. Expression studies during spruce somatic embryogenesis reveal that the transcript levels of PgHZ1 increase during the late phases of proliferation and remain high during the subsequent embryo growth on the ABA-containing maturation medium. Such an increase does not occur in a non-embryogenic line characterized by a developmental block. Arabidopsis plants with ectopic PgHZ1 expression show an increase sensitivity to ABA, as estimated in seed germination and root growth tests. Compared to wild type plants, plants over-expressing PgHZ1 driven by the CAMV 35S promoter show a variety of phenotypic deviations, including a reduced inflorescence growth, increased branching, small rosette leaves and a delay in flowering. Somatic embryos produced from 35S:PgHZ1 Arabidopsis plants display a heavy accumulation of storage products and remain in a developmental program even if subjected to prolonged culture. This is in contrast to wild type somatic embryos in which storage products are quickly mobilized and the germination program is initiated after only 15 days in maturation. Overall these data support the notion that PgHZ1 confers hypersensitivity to ABA and that proper expression of this gene may be required for proper embryonic growth.

Generation of Recombinant Antibodies against Orchardgrass Acidic nsLTP-Like Proteins

Embryogenic and non-embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.) secreted into the culture medium a set of proteins, among which low molecular mass (11/12 kDa) proteins were found. However, only the 11/12 kDa proteins from the embryogenic suspension cultures reacted specifically with an antiserum raised against the carrot EP2 non-specific lipid transfer protein (nsLTP). Two-dimensional (2-D) electrophoretic analysis revealed that the extracellular nsLTP-like proteins from the embryogenic lines were acidic proteins, with pI values ranging between 4.3 and 6.4, and the 11/12 kDa proteins of the non-embryogenic lines were basic ones (pI 8-9.3). This is only the second case to report on the accumulation of extracellular acidic nsLTP-like proteins in the culture medium during somatic embryogenesis. A naive phage display Griffin1. library was used to select single-chain phage antibodies, which specifically bind to acidic nsLTP-like proteins. Nine phage clones were selected after four rounds of biopanning of the target proteins blotted on a nitrocellulose membrane. Three soluble monoclonal single-chain phage antibodies, expressed in the non-suppressor E. coli strain HB2151, were purified by metal affinity chromatography and found to be highly specific for the acidic nsLTP-like proteins from the embryogenic suspension cultures. The application of the selected monoclonal antibodies for localization and elucidation of the role of the acidic nsLTP-like proteins in vivo is discussed.

Expression of the gene encoding transcription factor PaVP1 differs in Picea abies embryogenic lines depending on their ability to develop somatic embryos

Maturation of Norway spruce (Picea abies L.) somatic embryos is induced by abscisic acid (ABA). Several proteins were proven to be involved in ABA sensing including ABI3/VP1 transcription factors and their orthologue PaVP1 was characterized in spruce. To evaluate the role of PaVP1 both in embryogenic potential and in the process of embryo maturation, we studied PaVP1 expression in lines with contrast embryogenic capacities in parallel with detailed anatomical characterization. PaVP1 expression was determined by northern blot hybridisation, which revealed presence of two differentially regulated VP1-like B3-domain transcripts. Full-length PaVP1 transcript level was negligible in all lines on the proliferation media, but it differed strongly on the maturation media containing ABA. In non-embryogenic line, lacking any differentiated structures, the transcript remained undetectable. In contrast, in embryogenic lines with meristematic centres attached to suspensor cells, PaVP1 expression increased strongly after transition onto the maturation media. In highly embryogenic lines, it kept on a high level until the embryos reached cotyledonary stage, while in developmentally arrested line incapable to form mature embryos, the expression dropped down in connection with advanced disintegration of the meristematic centres. Removal of ABA from the maturation media after 2 weeks of maturation resulted in aberrant embryo development and rapid decrease in PaVP1 expression, indicating the impact of exogenously supplemented ABA on both initiation and maintenance of PaVP1 expression and proper embryo development. Since permanently high or increasing PaVP1 transcript levels accompanied proper embryo development in all experiments, it could be regarded as a good marker of this process.

GhSEM-1 Marker Potentially Associated with Regeneration Ability in Cotton

A marker protein for embryogénie potential could be useful in determining if target tissue for Agrobacterium tumefaciens or microprojectile bombardment has the ability to regenerate plants. Certain varieties of cotton, especially Coker 312, are known to form somatic embryos readily, while others are more recalcitrant. Callus induced from Coker 312–17wa s found to be more embryogénie than that from TM-1. To investigate the molecular basis for this difference in somatic embryo potential, total RNA was isolated from globular stage callus embryos, calli with heart-stage embryos, and non-embryogenic calli. Northern analyses were performed using a developmentally regulated cotton fiber gene as a probe. This probe hybridized to a single band, GhSEM-1 (Gossypium hirsutum somatic embryogénie marker 1), only in globularstage embryos. Genomic DNA was also isolated from young leaf tissue of both embryogénie (Coker 312–17) and non-embryogenic (TM-1) lines and a Southern analysis using the same probe was performed. No differences were found between the genomic DNA of both embryogenie and non-embryogenic lines, indicating that this gene was structurally present in both lines but only expressed in Coker 312–17 at the globular stage. Antibodies against GhSEM-1 were developed and used in immunoblots after SDS-PAGE separation of proteins from embryogenie and non-embryogenic calli. The antibody recognized a -15 kDa protein in the globular and heart-stage somatic embryos from Coker 312–17 and was designated as GEP15 (15 kDa Gossypium embryogénie protein). This protein could be used as a marker for determining if tissue has embryogénie potential. Our results represent the first reported potential association of a marker gene (GhSEM-1) and protein (GEP15) with somatic embryogenesis in cotton.

Strategies to obtain stable transgenic plants from non-embryogenic lines: complementation of the nn 1 mutation of the NORK gene in Medicago sativa MN1008

Strategies to introduce genes into non-embryogenic plants for complementation of a mutation are described and tested on tetraploid alfalfa (Medicago sativa). Genes conditioning embryogenic potential, a mutant phenotype, and a gene to complement the mutation can be combined using several different crossing and selection steps. In the successful strategy used here, the M. sativa genotype MnNC-1008(NN) carrying the recessive non-nodulating mutant allele nn ( 1 ) was crossed with the highly embryogenic alfalfa line Regen S and embryogenic hybrid individuals were identified from the F1 progeny. After transformation of these hybrids with the wild-type gene (NORK), an F2 generation segregating for the mutation and transgene were produced. Plants homozygous for the mutant allele and carrying the wild-type NORK transgene could form root nodules after inoculation with Sinorhizobium meliloti demonstrating successful complementation of the nn ( 1 ) mutation.

Identification of differentially expressed cDNA sequences and histological characteristics of Hevea brasiliensis calli in relation to their embryogenic and regenerative capacities

Different friable Hevea callus lines from the same genotype can display different embryogenic and regenerative potentials. These lines can not be distinguished on the basis of macroscopic criteria. A histological and molecular study was undertaken to characterize the differences existing between five callus lines with different potentials. The genes differentially expressed during induction were analyzed using the differential display (DD-RT) technique. Twenty-eight cDNAs were found to be differentially expressed during induction in the embryogenic regenerating line (ER). Embryogenic nodules were formed earlier in the ER lines than in the embryogenic non-regenerating lines and were completely absent in the non-embryogenic line. Of these 28 cDNAs, five could be used to distinguish between calli prior to induction, thereby enabling an early diagnosis of friable Hevea callus embryogenic potential.

Ascorbic acid metabolism during white spruce somatic embryo maturation and germination

Changes in ascorbic acid (AA) metabolism were investigated in 3 different cell lines of white spruce; a non‐embryogenic line (NE)WS and two embryogenic lines with low (E)WS1 and high (E)WS2 percentage of embryo germination. During embryo maturation, a high AA/dehydroascorbate (DHA) ratio was observed in the two embryogenic cultures. The increment of this ratio was mainly ascribed to the high ability of the (E)WS1 and (E)WS2 lines to generate AA from its last precursor, l‐galactono‐γ‐lactone, since no increase in the activities of the AA‐recycling enzymes, i.e., AA free‐radical reductase (AFRR) and DHA reductase (DHAR), was observed as the embryos developed. During the partial drying treatment needed for successful embryo germination, the ability to de novo generate AA continued to increase in the embryos of the two embryogenic lines, although the total ascorbate pool (AA+DHA) sharply decreased. A reactivation of the overall AA metabolism was observed at the inception of germination, especially in the (E)WS2 embryos. These results suggest that (1) the ability to metabolically alter the AA/DHA ratio may be a fundamental requirement for somatic embryo maturation and (2) the restoration of the AA metabolism during the first days of germination may be necessary for successful embryo germination.

Significance of preprophase bands of microtubules in the induction of microspore embryogenesis of Brassica napus

Microspores of Brassica napus L. cv. Topas, undergo embryogenesis when cultured at 32.5 °C for the first 18–24 h and then at 25 °C. The first division in heat-treated microspores is a symmetric division in contrast to the asymmetric division found after the first pollen mitosis in-planta or in microspores cultured continuously at 25 °C. This asymmetric division is unique in higher plants as it results in daughter cells separated by a non-consolidated wall. The cytoskeleton has an important role in such morphological changes. We examined microtubule (MT) organization during the first 24 h of heat induction in the embryogenic B. napus cv. Topas and the non-embryogenic B. napus breeding line 0025. Preprophase bands (PPBs) of MTs appeared in cv. Topas microspores in late uninucleate microspores and in prophase figures after 4–8 h of heat treatment. However, more than 60% of the PPBs were not continuous bands. In contrast, PPBs were never observed in pollen mitosis; MT strands radiated from the surface of the nuclear envelope throughout microspore maturation to the end of prophase of pollen mitosis I, during in-planta development and in microspores cultured at 25 °C. Following 24 h of heat treatment, over 95% of the microspores appeared to have divided symmetrically as indicated by the similar size of the daughter nuclei, but only 7–16% of the microspores eventually formed embryos. Discontinuous walls were observed in more than 50% of the divisions and it is probable that the discontinuous PPBs gave rise to such wall abnormalities which may then obstruct embryo development. Preprophase bands were not formed in heat-treated microspores of the non-embryogenic line 0025 and the ensuing divisions showed discontinuous walls. It is concluded that the appearance of PPBs in heat-induced microspores marks sporophytic development and that continuous PPBs are required for cell wall consolidation and embryogenesis. It follows that induced structures with two equally condensed nuclei, do not necessarily denote symmetric divisions.

Extracellular beta-1,3-glucanases are induced during early somatic embryogenesis in Cichorium.

In leaf tissues of the Cichorium hybrid clone '474' (C. intybus L. var. sativum x C. endivia L. var. latifolia), the acquisition and expression of embryogenic competence was characterised by the appearance of 15 polypeptides (Boyer et al., 1993, Plant Sci 93: 41-53). The 38-kDa proteins were found to be abundantly present in conditioned embryogenic medium after the first division of the induced cells. These proteins seemed to be glycosylated as indicated by general carbohydrate detection methods. Internal amino-acid sequences obtained after microsequencing tryptic peptides appeared to be 36-57% homologous with plant beta-1,3-endoglucanases. In addition, these 38-kDa proteins were recognised by antibodies raised against the pathogenesis-related tobacco glucanase PR2a and their beta-1,3-glucanase activity was demonstrated by direct detection in polyacrylamide gels after electrophoresis. These results strongly suggested that the 38-kDa somatic-embryogenesis-related (SER) polypeptides are beta-1,3-glucanases. Moreover, the level of glucanase activity was nearly three times higher in the medium of the embryogenic '474' line than in the medium of a non-embryogenic line. The possible involvement of the extracellular 38-kDa proteins in callose degradation during somatic embryogenesis is discussed.

Characterization of a germin-like protein gene expressed in somatic and zygotic embryos of pine (Pinus caribaea Morelet).

Germin-like proteins (GLPs) ionically bound to the walls of preglobular somatic embryos of Pinus caribaea Morelet are markers of this early developmental stage. In order to reveal the physiological implications of such markers during early embryo development, we isolated a cDNA clone from somatic embryos predicted to encode a protein with sequence similarity to GLPs. PcGER1 has an open reading frame corresponding to a 220 amino acid polypeptide with a putative N-glycosylation site on Asn-69. The presence of a 24 amino acid putative signal peptide supports the hypothesis of an apoplastic location. The N-terminal 20 amino acid sequence of the predicted mature protein is identical to the amino terminal sequence of GP111, one of the extracellular pine GLPs previously identified. Southern blot hybridizations indicate that PcGER1 is probably unique in the pine genome. Transcripts homologous to PcGER1 are abundant in all embryogenic lines, absent from nonembryogenic lines, and present in quiescent zygotic embryos but not in the female gametophyte, the haploid storage tissue of conifers. Their abundance sharply decreases during germination. Isolation of gf-0.8, a genomic fragment identical to PcGER1 cDNA sequence, confirms that no introns disrupt the coding region as it has been already described for wheat gf-2.8 and gf-3.8 genomic clones. Recombinant PcGER1, produced in Escherichia coli, is recognized by antibodies raised against the GP111 N-terminal nonapeptide and the unglycosylated wheat germin monomer. The implications of GLPs in pine embryogenesis are discussed.

Modulation of auxin-binding proteins in cell suspensions

Cultured cell lines from carrot (Daucus carota L.) with little or no embryogenic potential were examined for the auxin-binding capacity of their membranes. The lines belonged to different classes: (a) wild-type lines kept in culture for different periods (the longer the period, the lower being their embryogenic potential); (b) variants, isolated after mutagenesis, showing normal growth but a lack of embryogenic response; (c) auxin-resistant lines, isolated as colonies on solid media containing 45 μM 2,4-D; (d) a previously described tumorous line (E9) isolated because of its resistance to hypomethylating drugs. All of these lines showed alterations in auxin-induced, auxin-binding capacity (modulation), i.e. in the non-embryogenic lines the addition of auxin increased the auxinbinding capacity to a very small degree, or removal of the hormone did not produce the proper decrease in that capacity, or both defects could be simultaneously present. Both types of defects were shown to be correctable: after treatments designed to increase the amplitude of modulation, embryogenic capacity was restored in a number of lines.

Auxin levels at different stages of carrot somatic embryogenesis

The role of auxin in somatic embryogenesis was evaluated by characterizing the changes in the concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), and their conjugates in callus suspension cells and developing embryos of Daucus carota. Both embryogenic and non-embryogenic lines exhibited similar growth rates and levels of IAA and 2,4-D on 2,4-D-supplemented medium. Total endogenous IAA in both lines exposed to 2,4-D reached high levels greater than 600 ng g −1 fresh weight which suggests that IAA levels in carrot callus are not regulated via auxin feedback mechanisms. After being transferred to 2,4-D-free medium, the embryogenic line exhibited a rapid decline in both free and conjugated 2,4-D metabolites within seven days, while IAA levels remained relatively steady for seven days in the preglobular stage after which the levels declined steadily in all subsequent stages of embryo development. Individual analyses of different embryo fractions collected from asynchronous cultures confirmed that each stage in embryo development had lower IAA levels than the preceding stage. The non-embryogenic line maintained similar 2,4-D levels but higher IAA levels than the embryogenic line throughout the experiment. The present results suggest that high IAA levels may be necessary but are not sufficient for the initial events in plant embryogenesis, whereas low IAA levels are associated with the later stages of embryo development.

Changes in enzyme activity and protein patterns during the maturation phase of somatic embryogenesis in hybrid larch (Larix × eurolepis)

Changes were monitored in the activity of 10 enzymes and in the protein patterns revealed by one-dimensional SDS–PAGE gels during the maturation phase of hybrid larch somatic embryos up to the globular stage of the embryogenic cell line L2. For all the enzymes tested, activity increased up to 15 days after transfer of the proembryogenic calli on maturation media and then gradually decreased. In the globular somatic embryos, enzyme activity increased. Analyses of total protein patterns on SDS–PAGE indicated a decrease in the amount and number of detectable protein bands. As expected, mature globular embryos showed a different protein pattern. The decrease in calli metabolism, as demonstrated by enzyme activity and protein patterns, is consistent with the phenotypic observation of calli degeneration during the maturation phase. No differences in the protein pattern were observed between the embryogenic line L2 and another nonembryogenic line, L1. From these results, protein patterns specific to time in the maturation phase have been identified, but further investigation is required to determine if they are causes or effects of the process.

Peroxidase as a developmental marker in plant tissue culture.

Peroxidase was studied as a developmental marker in pumpkin (Cucurbita pepo L.) callus lines and horse-radish (Armoracia lapathifolia Gilib) transformants. Embryogenic callus lines DE grown on MS medium with 2.4-D and NA-3 grown on medium with NAA and adenine sulfate showed about a 20 times higher enzyme activity than the habituated non-embryogenic line Z5b/T grown on medium without hormones. A rise in peroxidase activity indicated that somatic embryogenesis was triggered in a few habituated tissue cultures. Separated globular embryoids had a manifold lower enzyme activity than the callus from which they originated. SDS-electrophoresis showed distinct polypeptide patterns between the horse-radish leaves and crown galls, but the tumor characteristic protein bands failed to be identified. In horse-radish crown galls and short bushy plants regenerated from hairy roots an enhanced peroxidase activity was registered. Due to its high peroxidase level and abundant biomass production horse-radish transformants should facilitate enzyme production.