Analysis of the genetic stability of <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> Labill. somatic embryos by flow cytometry

Flow cytometry was used to measure the nuclear DNA content of Eucalyptus globulus Labill. somatic and zygotic embryos and leaves in order to determine if somatic embryogenesis induces DNA content and ploidy changes in this species. Mature zygotic embryos derived from open-pollination orchard families were collected from a location in the centre of Portugal. One group was kept for nuclear DNA content and ploidy analysis, and the other group was used for establishing embryogenic cultures. Mature zygotic embryos were grown on Murashige and Skoog (MS) medium supplemented with 3% (w/v) sucrose and 3 mg l^–1 -naphthaleneacetic acid (NAA) for 3 weeks and then transferred to MS medium without growth regulators. Globular somatic embryos from approximately 8-month-old embryogenic cultures were used in the assay. DNA ploidy levels and the nuclear DNA content of mature zygotic embryos, somatic embryos and leaves from the mother field tree were determined using flow cytometry combined with propidium iodide staining. Zygotic embryos had a nuclear DNA content of 1.32 pg/2C, somatic embryos had a nuclear DNA content of 1.39 pg/2C and leaves from the field tree had a nuclear DNA content of 1.40 pg/2C. The values estimated for the somatic embryos and mother plant did not differ statistically from each other (P0.05), but both differed from those of the zygotic embryos (P0.05). These results clearly indicate that no changes were induced during the embryogenic process. However, the differences found between the field plants and zygotic embryos did suggest that some aspects must be evaluated carefully, as propidium iodide fluorescence may potentially be influenced by the presence of secondary compounds (e.g. anthocyanins, tannins) in E. globulus somatic embryos and mature leaves. Therefore we believe that the somatic embryogenesis methodology used did not induce major genetic changes in the somatic embryos and that our primary goal of true-to-type propagation was assured.     

Assessment of ploidy stability of the somatic embryogenesis process in <Emphasis Type="Italic">Quercus suber</Emphasis> L. using flow cytometry

Flow cytometry analyses were used to verify the ploidy stability of Quercus suber L. somatic embryogenesis process. Leaf explants of two adult cork oak trees (QsG0 and QsG5) of the North of Portugal were inoculated on MS medium with 2,4-D and zeatin. After 3 months, calluses with embryogenic structures were isolated and transferred to fresh MS medium without growth regulators and somatic embryo evolution was followed. Morphologically normal somatic embryos (with two cotyledons) and abnormal somatic embryos (with one or three cotyledons) were used in this assay. Flow cytometry combined with propidium iodide staining was employed to estimate DNA ploidy levels and nuclear DNA content of somatic embryos and leaves from mother plants. No significant differences (P0.05) were detected among embryos, and between the embryos and the mother plants. Also, after conversion of these embryos, no significant morphological differences were observed among the somatic embryo-derived plants. These results and further studies using converted plantlet leaves and embryogenic callus tissue indicate that embryo cultures and converted plantlets were stable with regard to ploidy level. As no major somaclonal variation was detected our primary goal of true-to-type propagation of cork oak using somatic embryogenesis was assured at this level. The estimation of the 2C nuclear DNA content for this species is similar to the previously obtained value.     

Flow cytometric and morphological analyses of Pinus pinaster somatic embryogenesis

An approach combining morphological profiling and flow cytometric analysis was used to assess genetic stability during the several steps of somatic embryogenesis in Pinus pinaster. Embryogenic cell lines of P. pinaster were established from immature zygotic embryos excised from seeds obtained from open-pollinated trees. During the maturation stage, phenotype of somatic embryos was characterized as being either normal or abnormal. Based upon the prevalent morphological traits, different types of abnormal embryos underwent further classification and quantification. Nuclear DNA content of maritime pine using the zygotic embryos was estimated to be 57.04 pg/2C, using propidium iodide flow cytometry. According to the same methodology, no significant differences (P ≤ 0.01) in DNA ploidy were detected among the most frequently observed abnormal phenotypes, embryogenic cell lines, zygotic and normal somatic embryos, and somatic embryogenesis-derived plantlets. Although the differences in DNA ploidy level do not exclude the occurrence of a low level of aneuploidy, the results obtained point to the absence of major changes in ploidy level during the somatic embryogenesis process of this economically important species. Therefore, our primary goal of true-to-typeness was assured at this level.     

Effect of ploidy increase on transgene expression: example from <Emphasis Type="Italic">Citrus</Emphasis> diploid cybrid and allotetraploid somatic hybrid expressing the EGFP gene

Polyploidization is an important speciation mechanism for all eukaryotes, and it has profound impacts on biodiversity dynamics and ecosystem functioning. Green fluorescent protein (GFP) has been used as an effective marker to visually screen somatic hybrids at an early stage in protoplast fusion. We have previously reported that the intensity of GFP fluorescence of regenerated embryoids was also an early indicator of ploidy level. However, little is known concerning the effects of ploidy increase on the GFP expression in citrus somatic hybrids at the plant level. Herein, allotetraploid and diploid cybrid plants with enhanced GFP (EGFP) expression were regenerated from the fusion of embryogenic callus protoplasts from ‘Murcott’ tangor (Citrus reticulata Blanco × Citrus sinensis (L.) Osbeck) and mesophyll protoplasts from transgenic ‘Valencia’ orange (C. sinensis (L.) Osbeck) expressing the EGFP gene, via electrofusion. Subsequent simple sequence repeat (SSR), chloroplast simple sequence repeat and cleaved amplified polymorphic sequence analysis revealed that the two regenerated tetraploid plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from the callus parent, while the five regenerated diploid plants were cybrids containing nuclear DNA of the leaf parent and with complex segregation of cytoplasmic DNA. Furthermore, EGFP expression was compared in cells and protoplasts from mature leaves of these diploid cybrids and allotetraploid somatic hybrids. Results showed that the intensity of GFP fluorescence per cell or protoplast in diploid was generally brighter than in allotetraploid. Moreover, same hybridization signal was detected on allotetraploid and diploid plants by Southern blot analysis. By real-time RT-PCR and Western blot analysis, GFP expression level of the diploid cybrid was revealed significantly higher than that of the allotetraploid somatic hybrid. These results suggest that ploidy level conversion can affect transgene expression and citrus diploid cybrid and allotetraploid somatic hybrid represents another example of gene regulation coupled to ploidy.     

Morphohistological and flow cytometric analyses of somatic embryogenesis in <Emphasis Type="Italic">Trifolium nigrescens</Emphasis> Viv.

Microscopy and flow cytometry (FCM) were used to study somatic embryogenesis (SE) from zygotic embryos of Trifolium nigrescens Viv. to determine if there were any relationships between characteristics of somatic embryos (morphology, anatomy, genome size stability) and their regenerability. Embryoids were induced on Murashige and Skoog (MS) medium containing 4 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg l⁻¹ N⁶-[2-isopentenyl]-adenine (2iP) either directly from hypocotyls or via an intervening callus, depending on the duration of culture. The morphology of somatic embryos varied from zygotic-like structures to abnormal structures including horn-shaped, polycotyledonary, and fused embryoids. The incidence of abnormalities was higher in callus cultures than in direct regeneration. Horn-shaped embryoids were the most frequent type of abnormal embryos. Only embryoids having zygotic-like morphology regenerated into plantlets. Histological observations revealed that the absence of shoot and root apical meristems along with parenchymatization of embryos were major obstacles to conversion of horn-shaped embryoids. The estimated 2C value for T. nigrescens was 0.9 pg. FCM analysis revealed differences in DNA content between embryoids induced via an intervening callus and those produced directly from explants. Individuals with species-specific as well as increased DNA content were detected among those zygotic-like embryos derived from callus, but all horn-shaped embryoids had increased genome sizes. The observed lack of differences in DNA content between zygotic-like and horn-shaped embryoids, from direct SE, indicated that these phenotypic abnormalities were of physiological origin. The mean DNA content of regenerants was species-specific, suggesting that only diploid embryoids were capable for regeneration into plantlets.     

Nuclear DNA content of somatic and zygotic embryos ofVitis vinifera cv. Grenache noir at the torpedo stage

Summary A flow cytometric analysis and an in situ DNA microspectrophotometric study were made concomitantly to establish why somatic grapevine (Vitis viniferacv. Grenache noir) embryos showed a low level of conversion into plantlets. In somatic embryos at the torpedo stage and in zygotic embryos at the same stage of development, ploidy level, DNA content per 2 C nucleus, and the cell-cycle state of the shoot apical meristem were examined. The frequency distribution histograms of nuclear DNA values were similar in the two types of embryos. At the torpedo stage both types of embryos had a majority of nuclei with 2 C DNA content equal to 1.6pg. In the shoot apices of somatic and zygotic embryos, DNA microspectrophotometry showed preferential blockage of the cell cycle at the G_0–1 stage; however, 20% of somatic embryo shoot apices were blocked at the G_0–2 stage. Analogies between somatic embryos and their zygotic homologues were shown. The genetical and environmental causes of the low level of conversion of grapevine somatic embryos into plantlets are discussed. Our work suggests that the in vitro culture conditions which were used could be incompatible with normal morphogenesis from the torpedo stage.     

Endoreduplication in <Emphasis Type="Italic">Phalaenopsis</Emphasis> is affected by light quality from light-emitting diodes during somatic embryogenesis

Endoreduplication is a developmental process that is unique to plants and occurs in all plants. The present study aimed to assess endoreduplication in various explant tissues and regenerated somatic embryos of Doritaenopsis. We further investigated the effects of light quality on endoreduplication and somatic embryo proliferation. To this end, we studied endoreduplication in leaves and root tips from regenerated plantlets and somatic embryos and in developing somatic embryos under 4 types of lighting conditions: red light, red + far-red light, red + blue light, and white light. We found that the degree of endoreduplication varied in different explants, and that the choice of explants used also influenced the ploidy levels of the newly regenerated somatic embryos. The DNA content of the leaf (2C–8C) was less than that of the root tip (2C–16C) and somatic embryo (2C–64C). In terms of light quality, the combination of red and far-red light produced the highest number of somatic embryos, while maintaining a low degree of endoreduplication. The data obtained indicate that this light combination stimulates somatic embryogenesis in Doritaenopsis and may exert some control on endoreduplication during cell division. These findings can be applied to achieve a reduction in somaclonal variations for the purpose of mass proliferation and genetic improvement.     

Nuclear DNA content of Vitis vinifera cultivars and ploidy level analyses of somatic embryo-derived plants obtained from anther culture

Flow cytometry was employed to determine the ploidy level of Vitis vinifera L. somatic embryo-derived plants obtained from anther culture. Only one among the 41 analysed plants (2.4%) presented somaclonal variation (tetraploidy); the other plants were diploid. No significant differences (P≤0.05) were detected between diploid and parental field plants. No haploid or aneuploid plants were observed. The nuclear DNA content of nine V. vinifera cultivars was also estimated using flow cytometry. A non-significant variation was found among the cultivars, with DNA content ranging from 1.17 pg/2C (cv. ‘Tinta Barroca’ and ‘Viosinho’) to 1.26 pg/2C (cv. ‘Cabernet Sauvignon’). These results and previous studies on other Vitis species suggest that Vitis genome is stable with regard to nuclear DNA content.     

Effect of anti-microtubular drug amiprophos-methyl on somatic embryogenesis and DNA ploidy levels in alfalfa and carrot cell suspension cultures

A short treatment with the anti-microtubular drug amiprophos-methyl (APM) blocked somatic embryogenesis in alfalfa(Medicago sativa L.) and carrot (Daucus carota L.). The interruption was temporary and restoration of somatic embryogenesis was observed in long-term cultures. In addition to the effect on somatic embryogenesis, APM treatment induced polyploidization the extent of which was concentration dependent. In long-term alfalfa cultures, APM-induced loss of somatic embryogenesis led to ploidy instability and to a shift to DNA aneuploidy. Critical stages of somatic embryogenesis sensitive to disruption of microtubule-mediated processes were determined in carrot cell cultures. Complete embryogenic arrest occurred when APM was added within the first 5 d of embryogenesis from single cells. The role of the cytoskeleton in the first events of somatic embryogenesis and the relation between totipotency and ploidy stabilityin vitro is discussed.     

Flow cytometric analysis of somatic embryos, shoots, and calli of the cactus Copiapoa tenuissima Ritt. forma monstruosa

Flow cytometry was used for analyzing DNA contents of nuclei isolated from in vitro grown somatic embryos, shoots and calli, as well as mammillae of in vivo grown shoots of cactus, Copiapoa tenuissima Ritt. forma monstruosa. Endoreduplication was detected in both in vitro grown somatic embryos, shoots and calli and in mammillae derived from in vivo grown shoots. However, the lowest ploidy levels ranged from 2C to 4C in somatic embryos, and reaching up to 32C for in vitro grown shoots and calli from mammillae. Whereas, ploidy levels of in vivo-derived mammillae ranged from 2C to 16C. The presence of 2,4-dichlorophenoxyacetic acid (2,4-D) in the culture medium had no influence on levels of ploidy of regenerated tissues. The mean genome size of cactus was calculated as 2C = 2.87 ± 0.05 picogram (pg).     

Ploidy stability of somatic embryogenesis-derived <Emphasis Type="Italic">Passiflora cincinnata</Emphasis> Mast. plants as assessed by flow cytometry

In this study, flow cytometric analysis was used to evaluate the genetic stability of Passiflora cincinnata Mast. plants regenerated via primary and secondary somatic embryogenesis. Embryogenic calli obtained from culturing zygotic embryos on Murashige and Skoog (MS) medium containing 18.1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 μM benzyladenine (BA) were transferred to differentiation medium. Torpedo and cotyledonary embryos were obtained. These primary embryos were maintained on differentiation medium to generate secondary embryos. Conversion of primary and secondary embryos yielded 305 and 138 normal plants, respectively. Almost 90% of plantlets survived following acclimatization. Flow cytometric analysis revealed that seed-derived plants had on average 3.01 pg nuclear DNA (2C), and all plants, except for a single plant regenerated via primary embryogenesis, maintained their ploidy. This single plant contained more than twice the average DNA content: 6.21 pg (4C). Epidermal stomata of leaves of the tetraploid plant were larger but lower in density than those of diploid plants, indicating that stomatal characteristics are useful in distinguishing between diploid and tetraploid plants of passion fruit. In summary, the procedure we employed to regenerated P. cincinnata plants via somatic embryogenesis generated mostly genetically true-to-type plants.     

Somatic embryogenesis from immature zygotic embryos and monitoring the genetic fidelity of regenerated plants in grapevine

Somatic embryogenesis and plant regeneration were successfully established on Nitsch and Nitsch (NN) medium from immature zygotic embryos of six genotypes of grapevine (Vitis vinifera). The optimum hormone combinations were 1.0 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction and 1.0 mg dm⁻³ α-naphthalene acetic acid (NAA) + 0.5 mg dm⁻³ 6-benzyladenine (BA) for embryos production and 0.03 mg dm⁻³ NAA + 0.5 mg dm⁻³ BA for embryos conversion and plant regeneration. The frequency of somatic embryogenesis varied from 10.5 to 37.5 % among six genotypes and 15.5–42.1 % of somatic embryos converted into normal plantlets. The analysis of DNA content determined by flow cytometry and chromosome counting of the regenerated plantlets clearly indicated that no ploidy changes were induced during somatic embryogenesis and plant regeneration, the nuclear DNA content and ploidy levels of the regenerated plants were stable and homogeneous to those of the donor plants. RAPD markers were also used to evaluate the genetic fidelity of plants regenerated from somatic embryos. All RAPD profiles from regenerated plants were monomorphic and similar to those of the field grown donor plants. We conclude that somaclonal variation is almost absent in our grapevine plant regeneration system.     

黄藤染色体核型及基因组大小分析

为探究黄藤(Daemonoropsjenkinsiana)染色体核型和基因组的大小,采用体细胞染色体常规制片法与显微摄影技术相结合的方法,对黄藤染色体进行了核型分析,同时以番茄(Lycopersicon esculentum)为内标,应用流式细胞术对黄藤叶片基因组大小、DNA含量和DNA倍性进行了测定。结果表明,黄藤茎尖是理想的染色体制片材料;黄藤的染色体数为2n=24,核型公式为K(2n)=1M+17m+5sm+1st,核型类型为2C;核型不对称系数61.20%;黄藤的DNA含量为1.57 pg,基因组大小为1 539.53 Mb,黄藤的DNA倍性为二倍体(2n)。这是首次报道黄藤的核型和基因组大小,为深入开展黄藤属及其近缘属植物的核型和基因组比较分析提供了参考依据。     

Induction of streptomycin-resistant plantlets in <Emphasis Type="Italic">Solanum surattense</Emphasis> through <Emphasis Type="Italic">in vitro</Emphasis> mutagenesis

The species Solanum surattense Burm.f. has importance in ayurvedic medicine and also as vegetable. Streptomycin-resistant plantlets were induced showing chloroplast encoded mutants in S. surattense from mutagenised (ethyl methane sulphonate and gamma-rays) cotyledon explants. Chloroplast encoded – streptomycin resistant – shoots were developed from green (unbleached) sectors of the cotyledons. The streptomycin-resistant plants were similar to parental plants in morphology and ploidy level (2n=2x=24). Reciprocal crosses between streptomycin-resistant and the original streptomycin sensitive plants have shown the non-Mendelian transmission under the control of chloroplast – DNA. These antibiotic resistant plants are useful in designing biochemical selection schemes aimed at somatic hybrid/cybrid recovery in S. surattense.     

Asymmetric somatic hybridization between UV-irradiated <Emphasis Type="Italic">Citrus unshiu</Emphasis> and <Emphasis Type="Italic">C</Emphasis>. <Emphasis Type="Italic">sinensis</Emphasis>: regeneration and characterization of hybrid shoots

In the present paper, attempts were made to explore the possibility of employing ultraviolet (UV) irradiation in citrus asymmetric fusion for transfer of limited amount of favorable traits from a desirable cultivar to a target one. Exposure of Satsuma mandarin (Citrus unshiu Marc.) embryogenic protoplasts to UV at an intensity of 300 μW cm⁻² led to reduced viability, especially under long irradiation duration. The protoplasts could not grow during culture when they were irradiated for over 30 s. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay revealed extensive DNA fragmentation in the UV-irradiated protoplasts compared with those without UV treatment. Electrofusion between UV-irradiated protoplasts of Satsuma mandarin (donor) with those of Jincheng (C. sinensis Osbeck, recipient), a local cultivar of superior quality, gave rise to regeneration of several lines of shoots, which failed to root despite enormous endeavors. Ploidy analysis via flow cytometry and chromosome counting showed that four selected shoots were either diploid, triploid or tetraploid. Random amplified polymorphism DNA (RAPD) and amplified fragment length polymorphism (AFLP) confirmed the shoots, irrespective of their ploidy level, as putative somatic hybrids. Cleaved amplified polymorphism sequences (CAPS) demonstrated that the shoots predominantly got their cytoplasmic components, in terms of chloroplast (cp) and mitochondrion DNA, from Jincheng, along with possible recombination of cpDNA in some shoot lines. The current data indicated that UV-based asymmetric fusion could also be employed in citrus somatic hybridization with the intention of creating novel germplasms, which may provide an alternative approach for cultivar improvement.     

DNA hypomethylation as Achilles’ heel of tumorigenesis: A working hypothesis

There are at least two findings that show DNA hypomethylation plays a key role in carcinogenesis. The first major evidence is that DNA hypomethylation induces target chromosomal and genomic instability with cancer manifestations. The second reason that cancer progression is associated with deepening DNA hypomethylation. Nevertheless, the evolution of this crucial epigenomic alteration in the somatic cellular malignant transformation remains unclear.From some of the experimental data to be present, a key role of DNA hypomethylation in early development of epigenetic somatic cancer biology is proposed. We have observed the significant increasing of genome ploidy at the level of peripheral blood lymphocytes taken from the patients with different solid carcinomas. Similarly, 5-azacytidine demethylating DNA treatment of cultured healthy lymphocytes induces increased nuclear DNA content. We argue that somatic lymphocyte ploidy induced by genomic DNA hypomethylation during carcinogenesis is related to global demethylation and decondensation of mitotic constitutive pericentromeric heterochromatin. This results in disturbances of pericentromeric heterochromatin that are expressed in nuclear heterochromatinization on the basis of extrachromosomal chromomerization.On the basis of literature searches and experimental findings, it is proposed that DNA hypomethylation plays the role of an initiator in epigenetic somatic cancer biology.     

Decreased DNA content of birch (Betula) chromosomes at high ploidy as determined by cytophotometry

Relative amounts of DNA were determined on telophase nuclei by Feulgen cytophotometry for euploid taxa of birch (Betula) with somatic chromosome numbers of 28, 42, 56, 70, and 84. A direct correlation was found between observed DNA absorbance and chromosome number except for plants of B. papyrifera with 84 somatic chromosomes. The DNA density value for nuclei of the 84-chromosome plants fitted a 12.25 ratio instead of the expected 13.0 ratio. The DNA density value for these plants was calculated to be approximately equivalent to plants which would possess 63 somatic chromosomes. The average DNA value per chromosome was 2.73 for the 84-chromosome plants in contrast to 3.50 per chromosome in each of the lower euploids. Nuclear diameters of the 84-chromosome plants were directly related to chromosome number and not to DNA density value. The genomic number of Betula was considered to be x=7, rather than x=14, since a DNA value equivalent to 63 chromosomes is a multiple of 7 and not 14. Diploid birch species (2n=2x=28), therefore, would actually be tetraploids (2n=4x=28). The reduction in DNA content may be an adaptation for the establishment of higher ploidy in birches.     

Polyploidization in embryogenic microspore cultures ofBrassica napus L. cv. Topas enables the generation of doubled haploid clones by somatic embryogenesis

Summary Embryogenic microspore and pollen culture followed by subculture of microspore-derived plantlets enabled the production of clones ofBrassica napus cv. Topas. Flow-cytometric analysis revealed that most microspore- and pollen-derived embryos (pEMs) were haploid initially. Spontaneous diploidization occurred at the globular stage of the pEMs, and was expressed as the relative increase of the 2C and 4C nuclear DNA content. Diploidization occurred throughout various organs of the pEMs and resulted in the formation of haploid and doubled haploid chimerics. In some embryos, nearly all cells were doubled haploid. From early cotyledon stage onward, pure haploid embryos were not observed anymore. At late cotyledon and germination stages, pure doubled haploid embryos and plantlets increased in number. Tetraploid pEMs were found occasionally. A culture regime was established to induce somatic embryos on the pEM-derived young plantlets. The ploidy of the somatic embryos varied highly and tended to be the same as that of the tissue at the initiation site on the pEM-plant. The results show that during the embryogenic development ofB. napus microspores, spontaneous diploidization occurs at globular stage, and increases progressively, resulting in the formation of chimerical haploid and doubled haploid plants as well as pure doubled haploid plants; ploidy neither affects pEM development at embryo developmental stages nor somatic embryogenesis, that starts on young pEM-derived plantlets; doubled haploid somatic embryos can be cloned from single pEM-derived plantlets; and doubled haploid embryos develop to fertile plants.     

Determination of ploidy level and nuclear DNA content in blueberry by flow cytometry

The technique of DNA flow cytometry was used to study variation in DNA content among different ploidy levels, as well as among diploid species, of Vaccinium section Cyanococcus. In a sample of plants of varying ploidy level, the relative fluorescence intensity (RFI) of nuclei stained with propidium iodide was a function of the number of chromosome sets (x), as represented by the linear equation RFI=3.7x-2.3 (r²=95%). The data indicated that DNA flow cytometry could be useful for the determination of ploidy level at the seedling stage in blueberry. They also suggest that conventional polyploid evolution has occurred in this section of the genus Vaccinium with an increase in nuclear DNA content concurrent with the increase in chromosome number. The nuclear DNA content of diploid species of Vaccinium section Cyanococcus was estimated from the relationship of the observed RFI to an internal known DNA standard (trout red blood cells). A nested analysis of variance indicated significant variation among species, as well as among populations within species, in nuclear DNA content, although this variation was small compared to the variation among ploidy levels. The variation in nuclear DNA content corresponded to the phylogenetic relationships among species determined from previous studies.     

Somatic embryogenesis and plant regeneration from leaves of<Emphasis Type="Italic"> Ulmus minor</Emphasis> Mill.

Somatic embryogenesis from mature elm (Ulmus minor Mill.) in vitro-cloned material is possible. Embryogenic callus was obtained from leaves inoculated on two different MS-based media—one supplemented with 2.3 M 2,4-dichlorophenoxyacetic acid (I2) and the other supplemented with 1.1 M kinetin (I6). However, only leaves cultured on medium I6 produced somatic embryos, at the globular stage, when embryogenic callus was maintained in induction media. When embryogenic callus from medium I6 was transferred to basal medium, somatic embryos with green cotyledons were obtained. An average of 35.9% of these embryos converted easily into normal plants in conversion medium with 1% sucrose. Acclimatisation reached 39.7%, and this was not significantly different from a control group consisting of plants propagated by axillary buds. No morphological differences were observed between plants derived from somatic embryos and control plants. Also, no differences in ploidy were detected between the somatic embryo-derived plants and the mother plants.Abbreviations BA: Benzyladenine - C1, C2: Conversion media - 2,4-D: 2,4-Dichlorophenoxyacetic acid - Kn: Kinetin - NAA: -Naphthaleneacetic acid - PI: Propidium iodide - I2, I6: Induction media Communicated by D. Bartels