Traits of transgenic Atropa belladonna doubly transformed with different Agrobacterium rhizogenes strains

Hairy root cultures of Atropa belladonna L. were established by infection either with Agrobacterium rhizogenes ATCC 15834 or MAFF 03-01724, and transgenic plants were obtained from both hairy root cultures. Doubly transformed roots were induced by re-infection of the leaf segments of transgenic Atropa belladonna plants (A. rhizogenes 15834) with MAFF 03-01724. Shoots and viviparous leaves were regenerated from the doubly transformed roots. The genetic transformation was determined by the opine assay (agropine, mannopine and/or mikimopine) and polymerase chain reaction. Physiological changes and tropane alkaloid biosynthesis in the hairy roots (singly and doubly transformed) were investigated. The alkaloid content in the doubly transformed root strain was intermediate as compared to the root strains which were singly transformed. On the other hand endogenous IAA levels in doubly transformed roots were significantly decreased compared to both singly transformed roots.Abbreviations BA benzyladenine - IAA indoleacetic acid - NAA naphthaleneacetic acid - PCR polymerase chain reaction - t-ZR trans-zeatin     

Repetitive somatic embryogenesis in peanut cotyledon cultures by continual exposure to 2,4-d

Somatic embryos from immature cotyledons in peanut (Arachis hypogaea) were initiated on media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-d). Over 90% primary embryogenesis and 41–46% repetitive embryogenesis were obtained 12 weeks after initiation by maintaining embryogenic cultures on medium containing 20 mg 1^-1 2,4-d. Maintenance of cultures on medium with 30 or 40 mg I^-1 2,4-d resulted in lower primary and secondary embryogenesis, and proliferation of nonembryogenic callus. Transfer of embryogenic cultures to a secondary medium with 10 or 20 mg I^-1 2,4-d significantly enhanced secondary embryogenesis compared to basal medium without the growth regulator. The use of Murashige & Skoog versus Finer's media had no significant effect on embryogenesis (85–95%), repetitive embryogenesis (11–37%) or mean embryo number. Secondary embryogenesis was also maintained for over one year by repeated subculture of isolated somatic embryos on medium with 20 mg I^-1 2,4-d.Abbreviations B5 Gamborg et al. medium (Gamborg et al. 1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - FN Finer & Nagasawa medium (Finer & Nagasawa 1968) - MS Murashige & Skoog medium (Murashige & Skoog 1962)     

Trends in accumulation of ephedrine in callus cultures of <Emphasis Type="Italic">Ephedra major</Emphasis> Host

Ephedra major Host, a medicinal plant, belongs to the family of Ephedraceae. Ephedrine is the main alkaloid in Ephedra, which has different medicinal properties. However, the amount of ephedrine in plant material is low and callus culture can be a way to increase the alkaloid content. The aim of this research was to compare Murashige and Skoog (MS) and Gamborg’s B5 culture media for callus induction and ephedrine production. For this purpose, stem explants were cultured on MS or B5 media containing 0.0, 0.5, 1.0, 2.0, or 3.0 mg L^?1 of kinetin (Kin) either alone or in combination with 0.0, 0.5, 1.0, or 2.0 mg L^?1 2,4-dichlorophenoxyacetic acid (2,4-D) and/or naphthalenacetic acid (NAA), in five replicates. MS medium containing 1.0 or 2.0 NAA and 0.5 mg L^?1 Kin were the most effective for callus induction. The highest percentage of callus induction (100%) on B5 culture medium was obtained with 2.0 2,4-D and 0.5 mg L^?1 Kin treatments. The results showed that there was no significant difference between MS and B5 media for callus induction, and fresh and dry weight production. High-performance liquid chromatography was conducted for the identification and quantification of ephedrine in the obtained callus. The highest level of ephedrine (7.38 mg g^?1 DW) was found in callus grown on MS medium containing 0.5 mg L^?1 of 2,4-D. The results revealed that ephedrine can accumulate in callus cultures to levels much higher than in E. major wild plants.     

Modulation of berberine bridge enzyme levels in transgenic root cultures of California poppy alters the accumulation of benzophenanthridine alkaloids

California poppy (Eschscholzia californica Cham.) root cultures produce a variety of benzophenanthridine alkaloids, such as sanguinarine, chelirubine and macarpine, with potent biological activity. Sense and antisense constructs of genes encoding the berberine bridge enzyme (BBE) were introduced into California poppy root cultures. Transgenic roots expressing BBE from opium poppy (Papaver somniferum L.) displayed higher levels of BBE mRNA, protein and enzyme activity, and increased accumulation of benzophenanthridine alkaloids compared to control roots transformed with a -glucuronidase gene. In contrast, roots transformed with an antisense-BBE construct from California poppy had lower levels of BBE mRNA and enzyme activity, and reduced benzophenanthridine alkaloid accumulation, relative to controls. Pathway intermediates were not detected in any transgenic root lines. Suppression of benzophenanthridine alkaloid biosynthesis using antisense-BBE also reduced the growth rate of the root cultures. Two-dimensional ¹H-NMR spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic roots lines. However, transformed roots with low levels of benzophenanthridine alkaloids contained larger cellular pools of certain amino acids compared to controls. In contrast, cellular pools of several amino acids were reduced in transgenic roots with elevated benzophenanthridine alkaloid levels relative to controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was only marginally altered in all transgenic root lines; thus, altering metabolic flux through benzophenanthridine alkaloid pathways can affect cellular pools of specific amino acids. Consideration of such interactions is important for the design of metabolic engineering strategies that target benzophenanthridine alkaloid biosynthesis.     

Somatic embryogenesis from leaf protoplasts of Rauvolfia vomitoria shoot cultures

Rauvolfia vomitoria mesophyll protoplasts have been isolated from axenic shoot cultures and cultured (10⁵-10⁶ protoplasts per ml) in Murashige and Tucker liquid medium containing growth regulators. Within 6–8 weeks, a mixed population of calli and proembryos were obtained and transferred on solid media. Calli produced shoots; however, rooting did not occur. Somatic embryos achieved different patterns of development. In particular, whole plantlets have been obtained either directly through germination of primary embryos or via embryogenic calli.Abbreviations B5 Gamborg et al. (1968) medium - BA N⁶ (benzyl) adenine - 2,4-D 2,4 dichlorophenoxyacetic acid - MT Murashige and Tucker (1969) medium - NAA naphthalene acetic acid - Z zeatin - K kinetin     

Putative sites of cytokinin action during their enhancing effect on indole alkaloid accumulation in periwinkle cell suspensions

Summary The effects of cytokinins on the different branches of the indole alkaloid pathway were investigated in Catharanthus roseus cell cultures. Addition of zeatin to a 2,4-dichlorophenoxyacetic acid-containing medium decreased tryptamine levels and increased the bioconversion of secologanin to ajmalicine. Zeatin also enhanced the geraniol-10 hydroxylase activities and modified the indole alkaloid pattern. The results are discussed in the light of previous works showing that cytokinins have a positive effect on indole alkaloid accumulation in some lines of C. roseus.Abbreviations BSTFA bis(trimethylsilyl) trifluoroacetamide - CK cytokinin - 2,4-D 2,4-dichlorophenoxyacetic acid - dw dry weight - G-10H geraniol-10 hydroxylase - NAA naphthaleneacetic acid - SE standard error - TDC tryptophan decarboxylase - Z zeatin     

Single-cell cloning of a crown gall from protoplasts regenerated in hormone-free medium. Establishment of pure transformed cell-lines of Catharanthus roseus

Protoplasts enzymatically isolated from cell line of Catharanthus roseus G. Don crown gall, were cultured at high density (10⁵ P ml^-1) in modified B5 liquid medium (Gamborg et al. 1976). In the absence of growth regulators C. roseus protoplasts were able to regenerate a cell-wall, divide and, subsequently, yield very numerous clones in the absence of growth regulators. After two weeks, the cultures were greatly diluted in order to obtain clones of single-cell origin. Most of the clones individually transferred onto solid medium can proliferate indefinitely, without growth regulators. Among analyzed clones, 90% were nopaline positive. Their ajmalicine and serpentine content was compared with that of the parental crown gall line, and was found to be low. The CR10 protoplasts were very easy to grow, they were an interesting model for the development of pure tumorous lines. Moreover, we found that the tumorous protoplasts were useful for cell fusion experiments or for the delicate culture of tree protoplasts.Abbreviations B5 Gamborg et al. (1976) medium - 2,4-d 2,4-dichlorophenoxyacetic acid - Kin Kinetin - NAA naphthalene acetic acid - BA N⁶ (benzyl) adenine     

Use of paromomycin as a selective agent for oat transformation

Summary Friable, embryogenic oat (Avena sativa L.) tissue cultures were stably transformed with two different plasmids containing the E. coli tn5 neomycin phosphotransferase II gene (npt II). Selection was accomplished using the antibiotic paromomycin sulfate following microprojectile bombardment. From two independent experiments, 88 paromomycin-resistant tissue cultures were shown to be transgenic based on Southern blot analysis and detection of the neomycin phosphotransferase (NPT II) protein using ELISA. Copy numbers of the npt II gene ranged from one to eight copies per haploid oat genome integrated into high molecular weight DNA of the paromomycin-resistant cultures. Plants were regenerated from 32 of the 88 transgenic tissue cultures. Plants from 17 of the 32 regenerable cultures exhibited fertility. Stable transformation was shown by segregation patterns of the NPT II protein in R₁ seedlings produced from 16 fertile culture lines that were tested. The overall results demonstrate that the combination of the npt II gene and paromomycin provides efficient selection of transgenic oat tissue cultures. Oat plants transformed with the npt II gene present reduced ecological risk compared to the previously used herbicide-resistance selection system.Abbreviations GUS beta-glucuronidase - uid A E. coli gene coding for GUS - NPT II neomycin phosphotransferase II of Tn 5 - npt II gene for NPT II - 2,4-D 2,4-dichlorophenoxy acid - X-gluc 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid cyclohexyl-ammonium salt - NOS nopaline synthase - NAA naphthalene acetic acid - BAP benzylaminopurine - ELISA enzyme-linked immunosorbant assay     

Antisense RNA-mediated suppression of benzophenanthridine alkaloid biosynthesis in transgenic cell cultures of California poppy

California poppy (Eschscholzia californica Cham.) cell cultures produce several benzophenanthridine alkaloids, such as sanguinarine, chelirubine, and macarpine, with potent pharmacological activity. Antisense constructs of genes encoding two enzymes involved in benzophenanthridine alkaloid biosynthesis, the berberine bridge enzyme (BBE) and N-methylcoclaurine 3'-hydroxylase (CYP80B1), were introduced separately into California poppy cell cultures. Transformed cell lines expressing antisense BBE or antisense CYP80B1 constructs and displaying low levels of BBE or CYP80B1 mRNAs, respectively, showed reduced accumulation of benzophenanthridine alkaloids compared with control cultures transformed with a beta-glucuronidase gene. Pathway intermediates were not detected in any of the transformed cell lines. The suppression of benzophenanthridine alkaloid biosynthesis using BBE or CYP80B1 antisense RNA constructs also reduced the growth rate of the cultures. Two-dimensional (1)H-nuclear magnetic resonance and in vivo (15)N-nuclear magnetic resonance spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic cell lines. However, transformed cells with reduced benzophenanthridine alkaloid levels contained larger cellular pools of several amino acids including alanine, leucine, phenylalanine, threonine, and valine compared with controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was less than 2-fold higher in antisense-suppressed cells relative to controls. These results show that alterations in the metabolic flux through benzophenanthridine alkaloid biosynthesis can affect the regulation of amino acid pools. These data provide new insight into the metabolic engineering of benzophenanthridine alkaloid pathways.     

Alkaloid formation by habituated and tumorous cell suspension cultures of Catharanthus roseus

Habituated and tumorous Catharanthus roseus cells grown in the absence of hormones accumulated indole alkaloids. Total alkaloids and alkaloid pattern were the same when cells were cultured in medium without hormones or in alkaloid production medium with and without indole acetic acid. Treatment of cells with Pythium homogenate as elicitor did not increase total alkaloids or change the pattern of alkaloids produced. When either habituated or tumorous cells were grown in 1B5 medium after Gamborg et al (1968) containing 2,4-dichlorophenoxyacetic acid (2,4-D), their capacity to accumulate alkaloids decreased with time. The levels of tryptophan decarboxylase (TDC) and strictosidine synthase (SS) specific activities were constant throughout growth except when cells were exposed to 2,4-D in 1B5 medium, where enzyme activities declined in step with the decrease in alkaloid accumulation. Neither habituated nor tumorous cell suspension cultures accumulated vindoline, nor could they be induced to produce this alkaloid by any of the given treatments.NRCC No. 27514     

Transformation of protoplast-derived cell colonies and suspension cultures by Agrobacterium tumefaciens

In this paper we describe procedures for transforming micro colonies derived from mesophyll protoplasts of Petunia hybrida with Agrobacterium tumefaciens. The method is efficient, up to 70% of the colonies were transformed, and we used a similar method to transform cells from a suspension culture of haploid Nicotiana plumbaginifolia.Abbreviations RH Relative humidity - MES 2 (N - Morpholino) ethane sulphonic acid - NAA 1 - Naphthylacetic acid - BAP 6 - Benzyl amino purine - B5 Gambourg's B5 medium (Gambourg 1970) - MS Murashige and Skoog Medium (1962) - PZ saline 0.9% NaCl pH 7.0 - 2, 4-D 2,4, Dichlorophenoxyacetic acid - Ty tryptone yeast     

The production of callus capable of plant regeneration from immature embryos of numerous Zea mays genotypes

In the summer of 1983, immature embryos from 101 selfed inbred lines and germplasm stocks of Zea mays L. were examined for their ability to produce callus cultures capable of plant regeneration (regenerable cultures) using a medium with which some limited success had previously been obtained. Forty-nine of the genotypes (49%) produced callus which visually appeared similar to callus previously cultured and shown to be capable of plant regeneration. After five months, 38 of these genotypes were alive in culture and plants were subsequently regenerated from 35 (92%) of them. No correlation was observed between plant regeneration and callus growth rate, the vivipary mutation (genes vp1, 2, 5, 7, 8 and 9), or published vigor ratings based on K⁺ uptake by roots. When F₁ hybrid embryos were cultured, 97% of the hybrids having at least one regenerable parent also produced callus capable of plant regeneration. No regenerable cultures were obtained from any hybrid lacking a parent capable of producing a regenerable callus culture.In the summer of 1984, immature embryos from 218 additional inbred lines and germplasm stocks were plated and examined for their ability to produce regenerable callus cultures on media containing altered micronutrient concentrations, 3,6-dichloro-o-anisic acid (dicamba), glucose, and elevated levels of vitamin-free casamino acids and thiamine. Of these genotypes 199 (91%) produced callus that was regenerable in appearance. In the 1984 study, plant regeneration was noted in many commercially important inbreds, including B73, Mo17, B84, A632, A634, Ms71, W117, H99³H95 and Cm105. Thus tissue-culture techniques are now available to obtain callus cultures capable of plant regeneration from immature embryos of most maize genotypes.Abbreviations trade names 2,4-D 2,4-dichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid     

Production of tropane alkaloids in diploid and tetraploid plants and in vitro hairy root cultures of Egyptian henbane (Hyoscyamus muticus L.)

In this study, the effects of ploidy level and culture medium were studied on the production of tropane alkaloids. We have successfully produced stable tetraploid hairy root lines of Hyoscyamus muticus and their ploidy stability was confirmed 30?months after transformation. Tetraploidy affected the growth rate and alkaloid accumulation in plants and transformed root cultures of Egyptian henbane. Although tetraploid plants could produce 200% higher scopolamine than their diploid counterparts, this result was not observed for corresponding induced hairy root cultures. Culture conditions did not only play an important role for biomass production, but also significantly affected tropane alkaloid accumulation in hairy root cultures. In spite of its lower biomass production, tetraploid clone could produce more scopolamine than the diploid counterpart under similar growth conditions. The highest yields of scopolamine (13.87?mg?l^?1) and hyoscyamine (107.7?mg 1^?1) were obtained when diploid clones were grown on medium consisting of either Murashige and Skoog with 60?g/l sucrose or Gamborg??s B5 with 40?g/l sucrose, respectively. Although the hyoscyamine is the main alkaloid in the H. muticus plants, manipulation of ploidy level and culture conditions successfully changed the scopolamine/hyoscyamine ratio towards scopolamine. The fact that hyoscyamine is converted to scopolamine is very important due to the higher market value of scopolamine.     

Production of solamargine by in vitro cultures of Solanum paludosum

Callus cultures of Solanum paludosum were established from roots, hypocotyles, cotyledons and leaf limbs of plantlets cultivated in sterile conditions on a Murashige and Skoog's modified medium. Non organogenous calluses were obtained with addition of BA or kinetin (10^-5M to 10^-6M) as the cytokinin and 2,4-d or NAA (10^-5M to 10^-6M) as the auxin. These calluses permitted the establishment of a cell suspension culture with BA (10-6M) and 2,4-d (10^-6M). Zeatin (10^-6M) with IAA (10^-6M) gave rise to organogenous calluses. These organogenous callus cultures developed multiple shoots which either proliferated if they were cultivated on a medium containing zeatin with IAA or IBA or were able to regenerate into whole plants when zeatin was used as the only hormone. The different plant material produced solamargine, the main steroidal glycoalkaloid present in the unripe fruits. The best production was obtained with the fruits of regenerated plants from organogenous callus cultures after reintroduction of these plants in their brasilian biotope. The solamargine content of the two types of plant materials was about 0.06% and 2.5% (dry weight) respectively for the callus cultures and the fruits from in vitro plants. The fruits were harvested a year after the beginning of the plantlet regeneration step.Abbreviations HPTLC high performance thin layer chromatography - HPLC high performance liquid chromatography - 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - IAA 3-indolebutyric acid - NAA -naphthaleneacetic acid - IBA 3-indolebutyric acid - IPA isopentenyladenine     

Regulation of product synthesis in cell cultures of Catharanthus roseus. V. Long-term maintenance of cells on a production medium

Three unselected cell lines of C. roseus maintained on a growth-associated alkaloid production medium were studied over a period of 2 to 5.5 years for the stability of alkaloid production (serpentine and ajmalicine). Large fluctuations in the total alkaloid content of 20-day-old cells were found for all three cell lines at each subculture over a two-year period. Growth rates increased during prolonged subculture and one cell line became unproductive after five years culture. By selection of small autofluorescent aggregates, high alkaloid production was restored in this cell line, while the parent line was found to be unresponsive to alkaloid induction treatments. The instability in both alkaloid production and spectrum and the loss of alkaloid productivity are discussed in relation to the selection pressures present during long-term maintenance of cell suspension cultures.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - nHS n-heptane sulphonate     

The effect of plant growth regulators on growth, morphology and condensed tannin accumulation in transformed root cultures of Lotus corniculatus

This study concerns the effects of four different classes of plant growth regulators on root morphology, patterns of growth and condensed tannin accumulation in transgenic root cultures of Lotus corniculatus L. (Bird's-foot trefoil). Growth of transformed roots in 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in decreased tannin levels relative to controls at concentrations of 10^-6 M and above, while gibberellic acid (GA₃) inhibited tannin accumulation at concentrations of 10^-7 M and above. Benzyladenine (BA) had little effect at low concentrations (10^-7 M and below) but resulted in an increase in tannin levels at 10^-6 M. Abscisic acid had little effect on levels of condensed tannins at any of the concentrations used. Experiments involving growth regulator addition and medium transfer demonstrated that 2,4-D inhibition of tannin accumulation could be reversed by GA₃ and BA, while GA₃ downregulation could only be reversed by the addition of 2,4-D. Although 2,4-D inhibited tannin accumulation, addition of 2,4-D to root cultures grown for 14 or 28 days in the absence of plant growth regulators stimulated both growth and tannin biosynthesis. Characteristic alterations in root morphologies accompanied growth regulator-mediated modulation of tannin biosynthesis. Growth in 2,4-D resulted in partially de-differentiated root cultures while growth in GA₃ produced roots with an elongated phenotype. Restoration of tannin biosynthesis in 2,4-D-treated roots was accompanied by root re-differentiation and the production of new lateral roots.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid 3 - FW fresh weight     

Studies on a drought resistant legume: The moth bean,Vigna aconitifolia (Jacq) marechal. II. morphogenetic studies

Plantlets regenerated from shoot apices, cotyledons and callus cultures in Moth bean, Vigna aconitifolia (JACQ) Marechal, a drought resistant legume and pulse crop, were rooted and transferred to soil. Explants for these studies were derived from seedlings pre-conditioned by germination of seeds on B5BA and WMB (control).Abbreviations MS Murashige and Skoog (1962) - B5 B5 basal medium (Gamborg et al 1968) - B5BA B5 basal medium containing BA (2.25 mg/l) - WMB Modified White's medium (Mascarenhas et al 1976) - BA 6-benzyladenine - IAA indole-3-acetic acid - NAA 1-napthaleneaceticacid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - 2iP N(^–2 isopentyl) adenine - CM coconut milk NCL Communication No. 3375     

Influence of auxin type and concentration on peanut somatic embryogenesis

Somatic embryogenesis in peanut (Arachis hypogaea L.) using immature cotyledonary explants was induced on a wide range of 2,4-dichlorophenoxyacetic acid (2,4-D) (5 to 60mg l^–1) and naphthaleneacetic acid (NAA) (20 to 50 mg l^–1) levels. Percent embryogenesis ranged from 31 to 94%. As auxin level increased in induction medium, percent embryogenesis decreased and was associated with browning of explants. However, with higher 2,4-D induction levels (40 mg l^–1 and over), embryogenic explants had dense masses of embryogenic areas and repetitive embryogenesis was enhanced. Higher auxin concentrations during induction decreased precocious germination of embryos, but had no marked effect on somatic embryo morphology. The use of 2,4-D compared to NAA in the induction medium resulted in greater per cent embryogenesis and mean number of embryos. Embryos induced on NAA were harder, less pliant, and less succulent; cultures exhibited more extensive root development and nonembryogenic callus proliferation.Abbreviations B5 Gamborg et al. (1968) - BA benzyladenine - 2,4-D dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige & Skoog (1962) - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Relationship of indole-3-acetic acid and tryptophan concentrations in normal and 5-methyltryptophan-resistant cell lines of wild carrots

A 5-methyltryptophan(5-MT)-resistant cell line of wild carrot (Daucus carota L.), W001, that exhibited auxin-independent callus growth, was found to accumulate indole-3-acetic acid (IAA) and tryptophan (trp). Anthranilate-synthetase activity in W001 cell extract was less sensitive to feedback inhibition by trp than in the original 5-MT-sensitive cell lines. It is hypothesized that the resistant enzyme allowed more trp synthesis and accumulation which, in turn, affected the IAA concentration in the cell. Since carrot cultures cannot regenerate in the presence of exogenous auxin, the elevated IAA concentration in W001 may be responsible for its drastically reduced capacity to regenerate. The relationship between trp and IAA levels was further investigated by examining the effect of 2,4-dichlorophenoxy acetic acid (2,4-D) on the endogenous concentration of trp and IAA. In general, the IAA level was reduced but the trp concentration was elevated when 2,4-D was present in the culture medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 5-MT 5-methyltryptophan - 5-MT^r 5-MT-resistant - 5-MT^s 5-MT-sensitive - trp tryptophan     

Spontaneous somatic embryogenesis and plant regeneration from root cultures of Peucedanum palustre

The regeneration of Peucedanum palustre (L.) Moench (milk parsley) was established for the first time via somatic embryogenesis from primary root cultures. Callus formation occurred on the root cultures and showed spontaneous embryogenic capability on B5 basal medium supplemented with a low concentration of indoleacetic acid (5.5 × 10^–7 M). 2,4-Dichlorophenoxyacetic acid was not needed for the initiation of embryogenesis. The somatic embryos germinated and formed plantlets on hormone-free B5 medium. These plantlets were easily transferable to pots, and are presently passing their second growing season in the greenhouse.Development of the somatic embryos progressed through the globular, heart-shaped, torpedo-shaped, and cotyledonary stages, typical of zygotic embryos. Synchronization performed by sieving the embryos did not affect the development time. The culture has retained its embryogenic capacity for 25 months.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - IAA indoleacetic acid - IBA 3-indolebutyric acid - BAP 6-benzylaminopurine