Bakos et al Plant Cell Rep

Plant Cell Reports (2000) 19 : 525–528 Q Springer-Verlag 2000 Á. Bakos 7 T. Borsics 7 O. Toldi 7 K. Babos 7 M. Lados Evidence for somatic embryogenesis during plant regeneration from seedling-derived callus of dodder (Cuscuta trifolii Bab. et Gibs) Received: 9 November 1998 / Revision received: 22 April 1999 / Accepted: 29 June 1999 Abstract In this paper comparative histological studies of embryo-like structures originating from callus cultures, and zygotic embryos originating from sexual seeds of Cuscuta trifolii are reported. The embryos of somatic cell and zygote origin showed similar morphological and anatomical features, such as a complete lack of cotyledon development and the differentiation of a developmentally unique root primordium specialised for water storage. Based on these findings, the regeneration of C. trifolii from callus cultures is shown to proceed along the pathway of somatic embryogenesis. Key words Cuscuta trifolii 7 Somatic embryogenesis 7 Plant regeneration 7 Parasitic weed Abbreviations 2,4-D: 2,4-Dichlorophenoxyacetic acid 7 NAA: Naphthaleneacetic acid 7 MSB5 : Murashige and Skoog (1962) medium supplemented with B5 vitamins Introduction Over 3000 species of flowering plants utilise a parasitic mode of nutrition, yet basic knowledge about the physiology, biochemistry and genetic background of their Communicated by D. Dudits Á. Bakos (Y) National Center for Epidemiology, H-1529 Budapest, Hungary e-mail: bakos6microbi.hu Fax: c36-1-3945409 parasitic interactions is limited (Stewart and Press 1990). Dodder (Cuscuta), a devastating annual holoparasitic flowering plant of legume crops, is widely distributed both in North America and Europe (Graham et al. 1979). One representative of dodder is Cuscuta trifolii. In Hungary it commonly occurs mainly on alfalfa and is fully adapted to parasitic life. The parasite has no root or leaf and is attached to the host by a special shoot-originated organ called a haustorium (Szatala and Gimesi 1965). The entire plant is yellow and although it contains a small amount of chlorophyll it cannot support itself autotrophically. To obtain nutrients the parasite relies solely on its host. Only a few papers are concerned with the in vitro culture of dodders, a precondition of controlled experimentation in molecular biology. Maheshwari and Baldev (1961) reported the initiation and maintenance of the Cuscuta reflexa callus derived from zygotic embryos. According to their observations, somatic embryos differentiated and subsequently formed normal shoots from the callus tissue of C. reflexa. However, somatic embryogenesis was not demonstrated in their paper, either by histological or anatomical examination. We have previously described an in vitro plant regeneration system from seedling-derived embryogenic callus of C. trifolii (Bakos et al. 1995) in which differentiation of embryo-like structures was reported. However, since dodder shows a developmentally unusual zygotic embryogenesis, further morphological characterisation of the in vitro regeneration process would provide new data about its special ontogenesis. Therefore, the aim of our experiments was to determine whether the plant regeneration proceeds through organogenesis or somatic embryogenesis. T. Borsics 7 O. Toldi 7 M. Lados (Y) Agricultural Biotechnology Center, H-2101 Gödöllő, P.O. Box 411, Hungary e-mail: lados6abc.hu Fax: c36-28-430416 Materials and methods K. Babos ELTE Department of Plant Anatomy, H-1088 Budapest, Puskin u. 11–13, Hungary Plant material The seeds of dodder (Cuscuta trifolii Bab. et Gibs) were kindly provided by Prof. I. Bócsa (Rudolf Fleischmann Research Insti- 526 tute of Gödöllő University of Agricultural Sciences, Kompolt, Hungary). All the seeds were kept at 4–6 7C in the dark until used. Surface sterilisation of dodder seeds For the surface disinfection of the Cuscuta seeds Furuhashi’s method (1991) was used with slight modifications. The seeds were treated with 95% (w/w) H2SO4 for 20 min, washed three times in sterile distilled water and then rinsed in 2% (w/v) CaOCl solution supplemented with Tween-20 detergent for 1 h. After this treatment, the seeds were washed five times and finally soaked in sterile distilled water at 4 7C for 1 h. Seed germination was carried out in Petri dishes on growth regulator-free GM medium (Toldi et al. 1994). Establishment of plant or callus cultures In all the experiments for the establishment of plant or callus cultures and plant regeneration, the method described by Bakos et al. (1995) was applied. The basal medium consisted of MS salts, B5 vitamins (Gamborg 1975). For carbon sources both 1.5% maltose (w/v) and 1.5% sorbitol (w/v) were used. Callus formation was initiated on liquid medium in the presense of 5.0 mg/l kinetin and 0.75 mg/l 2,4-D and then later put on agar-solidified (0.7% w/v) media with the same compounds. Germinating seeds were illuminated by cool white fluorescent lights of 20 W/m 2 with a 16 h light photoperiod. Callus cultures were kept in darkness. Plant regeneration experiments were also carried out in darkness or under a 16 h light/8 h darkness photoperiod at 23B2 7C on solidified MSB5 medium containing maltose, sorbitol as above, 2 mg/l kinetin and 7 g/l agar. Histological studies of developing dodder The histological examinations of plant tissues were done by a slightly modified version of the method of Sárkány and Szalai (1957). The plant material was stored in 30% ethanol for at least 24 h. In the following dehydration steps samples were soaked for 30 min in 50%, 75%, 96%, 100% ethanol, ethanol:benzol 2 : 1, 1 : 1, 1 : 2 and finally 100% benzol solutions, respectively. The prepared material was embedded in paraffin containing 5% beeswax. Thin cuttings (13 mm) were made by microtome and Fig. 1A The different stages of somatic embryogenesis of Cuscuta trifolii (1 heart phase, 2 torpedo phase). B The bipolar structure of a callusoriginated somatic embryo at torpedo phase. The upper part (sp shoot pole) consists of small dividing cells, on the lower part (rp root pole) big vacuolate cells are clearly visible. These cells may have role in water storage during the early stages of development (scale bar: 65 mm) stained by malachite green and vesuvin. Specimens were then examined microscopically at magnifications of 50!, 120! and 300!. Results and discussion Regeneration from plant cell or tissue culture can start by organogenesis or by somatic embryogenesis. Somatic embryogenesis, when bipolar embryos develop in the culture, is morphologically and anatomically more or less similar to zygotic embryogenesis. That is, different stages of the differentiation process from the zygote can also be seen in developing somatic embryos. In earlier experiments we observed that in callus cultures, induced on MSB5 medium containing 5 mg/l kinetin and 0.75 mg/l 2,4-D, embryo-like structures were developed (Bakos et el. 1995). We could clearly recognise their morphologically unique way of differentiating (Fig. 1A,B) typical for C. trifolii. However, the somatic embryogenesis of dodder is unique not only from the point of view of morphological development, but also in respect of the mode of in vitro induction. In nearly 60% of tissue-cultured plant species, where plant regeneration was achieved through somatic embryogenesis, a synthetic auxin, 2,4-D, was the only inducer growth regulator (Evans et al. 1981; Finer 1994). Additionally, in a further 15% of tissue-cultured plants, another synthetic auxin, NAA, was used alone (without application of cytokinins) for the same purpose. In only a few percent of tissue-cultured plants were cytokinins also used, either in combination with synthetic auxins or alone in high concentrations to initiate somatic embryogenesis, as with dodder (Evans et al. 1981; Finer 1994). We have found very similar morphological development in the case of putative somatic embryo-originating 527 Fig. 2 Comparison of somatic and zygotic embryogenesis in C. trifolii. Macroscopic picture (A; scale bar: 200 mm) and cross-sectional view of shoot (B; scale bar: 50 mm) and root pole (C; scale bar: 50 mm) of a somatic embryo originated seedling. Macroscopic picture (D; scale bar: 200 mm) and cross-sectional view of shoot (E; scale bar: 10 mm) and root pole (F; scale bar: 50 mm) of a zygotic embryo originated seedling. rp root pole, sp shoot pole, ep epidermis, pc parenchymal cell layer, cc central cylinder, p procambium dodder seedlings (Fig. 2A) and in the case of seedlings originating from zygotes (Fig. 2D). Neither of these had cotyledons, they were thread-like in form, but showed a longitudinal bipolarity (Figs. 1A,B, 2A,D). In the case of the somatic cell-originated embryo-like structures, this longitudinal bipolarity is the first proof of somatic embryogenesis, because its existence is an essential precondition of the simultaneous differentiation of primary root and shoot primordia. However, in contrast to the bipolar development of both zygotic and somatic embryos, a unipolar development is peculiar to plant regeneration through organogenesis. Additional proof of the embryogenic pathway of development is the anatomically classical development of heart-phase (Fig. 1A1) and torpedo-phase somatic embryos (Figs. 1A2,B), on callus induction media. On the crosssectional pictures of a somatic cell and a zygote-originated torpedo-stage embryo (Figs. 2B,E) very primitive and undifferentiated tissue structures were obtained which seem to be similar to bryum stem (Sárkány 1960). The epidermis (ep) and parenchymal cell layers (pc) are clearly distinguishable, as well as the central 528 cylinder (cc) in which the phloem, xylem and procambial bundles are localised along the longitudinal axes of the embryos. Both zygotic and somatic embryos were characterised by two typical kinds of tissue. One was localised in the basal part (root pole: rp) of the embryo, and was less differentiated and filled with parenchymatous cells containing large central vacuoles, underlining the importance of this specialised root primordium for water storage of the developing dodder plantlets (Szatala and Gimesi 1965). The other tissue type consisted of small-celled meristematic centres localised in the apical part of the embryo, characteristic of the shoot primordium (sp). This bipolar structure became clearly expressed during the further development of both somatic and zygotic embryo-originated seedlings of C. trifolii. As a result of somatic embryogenesis, plantlets have been developed (Fig. 2A) which are similar to plants originating from seeds (Fig. 2D). An additional common characteristic of both the somatic and zygotic embryo-originated plantlets was the complete lack of a root system (Fig. 2A,D) due to its necrosis at an early stage of embryogenesis. As explained above, in dodder the root primordium of the embryo is specialised for water accumulation, because the water and nutrient supplier is the parasitic shoot called the haustorium (Szatala and Gimesi 1965). On the basis of these results, we can state that the regeneration of this plant from dedifferentiated calli proceeds through somatic embryogenesis. Acknowledgements The authors are grateful to G. Takács for photography and I. Bárány for technical assistance. This work was supported by an OTKA (F5047) and an OMFB (92-97-160122) research grant awarded to M. Lados and a scholarship from the Foundation for Hungarian Science of the Hungarian Credit Bank (N 3/92/11) granted to Á. Bakos. References Bakos Á, Fári M, Toldi O, Lados M (1995) Plant regeneration from seedling derived callus of dodder (Cuscuta trifolii, Bab. et Gibs.) Plant Sci 109 : 95–101 Evans DA, Sharp WR, Flick CE (1981) Growth and behavior of cell cultures: Embryogenesis and organogenesis. 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