Plant Cell Reports

Plant Cell Reports (1996) 16:22-25

© Springer-Verlag1996

Plant regeneration from protoplasts of Vicia narbonensis via somatic embryogenesis and shoot organogenesis Mechthild Tegeder 1, Hubertus Kohn 2, Matthias Nibbe 1, Otto Schieder a, and Thomas Pickardt 1 1 Freie Universit/it Berlin, Institut ffir Angewandte Genetik, Albrecht-Thaer-Weg 6, 14195 Berlin, Germany 2 Western Washington University, Department of Biology, Bellingham, WA 98225, USA Received 16 October 1995/Revised version received 8 March 1996 - Communicated by H. L6rz

Abstract

Protoplasts of Vicia narbonensis isolated from epicotyls and shoot tips of etiolated seedlings were embedded in 1.4% sodium-alginate at a final density of 2.5x1@ protoplasts/ml and cultivated in Kao and Michayluk-medium containing 0.5 mg/I of each of 2,4- dichlorophenoxyacetic acid, naphthylacetic acid and 6 -benzylaminopurine. A division frequency of 36% and a plating efficiency of 0.40.5% were obtained. Six weeks after embedding, protoplast-derived calluses were transferred onto gelritesolidified Murashige and Skoog-media containing various growth regulators. Regeneration of plants was achieved via two morphologically distinguishable pathways. A two step protocol (initially on medium with a high auxin concentration followed by a culture phase with lowered auxin amount) was used to regenerate somatic embryos, whereas cultivation on medium containing thidiazuron and naphthylacetic acid resulted in shoot morphogenesis. Mature plants were recovered from both somatic embryos as well as from thidiazuron-induced shoots. Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; BA, 6 -benzylaminopurine, CH, casein hydrolysate; FDA, fluorescein diacetate; IBA, indole-3-butyric acid; KM, Kao and Michayluk's medium (1975); MS, Murashige and Skoog's medium (1962); NAA, naphthylacetic acid; TDZ, thidiazuron

Introduction

Within the genus Vicia, the species V. narbonensis was placed together with V. serratifolia, V. galilaea, V. johannis and V. hyaeniscyarnus in the narbonensis-group (Sch~fer 1973); previously this narbonensis-complex had been combined with V. faba in the faba-section (Ball 1968). While the degree of taxonomic relationship between these species is still under discussion (e.g. van de Ven et al. 1993), it has been reported that conventional approaches to hybridisation, including embryo rescue, between Vicia faba Correspondence to: T. Pickardt

and other Vicia species resulted in failure (Cubero 1982, Roupakias and Tai 1986, Ramsay and Pickersgill 1986). Thus, in order to improve Vicia faba genetically, e.g. disease resistance or drought tolerance, non-conventional methods like protoplast fusion should be attempted. This method has been shown to successfully produce asymmetric hybrids between different genera and species (e.g. Gilissen et al. 1992, Bauer-Weston et al. 1993, Gerdemann-Kn6rck et al. 1995). However, this technology demands reliable protocols for protoplast isolation, culture, and regeneration of plants from protoplast-derived calluses. Until recently, protocalluses had been obtained through tissue culture from all the Vicia species investigated, yet plant regeneration had not been achieved (see Hammatt et al. 1986). Latest results from our laboratory have shown that shoot regeneration from protocalluses of Vicia faba can be achieved on medium containing thidiazuron as a growth regulator (Tegeder et al. 1995). In order to demonstrate the applicability of this experimental approach to other Vicia species, a protocallus culture was developed and investigated in V. narbonensis. Previous studies in our laboratory have shown that V. narbonensis is generally less recalcitrant to tissue culture conditions compared to other grain legumes. Protocols for the induction of somatic embryogenesis and the transformation via Agrobacterium from V. narbonensis epicotyl-explants have already been established (Pickardt et al. 1995). The present paper describes the conditions for isolation and culture of V. narbonensis protoplasts and different investigative approaches to protocallus culture which led to the recognition of two morphogenetic pathways that resulted in regeneration of plants from these protoplasts.

Material & Methods

Plant material: Seeds of Vicia narbonensis L. var. narbonensis were surface sterilized by immersion for 1 min in 70% ethanol and 5 min in 5% (w~) sodium hypochlorite solution with a few drops of Tween 80, After several washes seedswere soaked in sterile tap water for 12 h,

2.3 transferred to half strength MS-basal medium solidified with 0.8% agar (Merck) and kept for germination in the dark at 24°C. Shoot-tips from 10-15 d old etiolated seedlings served as the protoplast source. Protoplast isolation: Shoot tips were cut into 1 mm segments and incubated in an enzyme solution (approx. 1.5 g tissue per 50 ml) containing 5% cellulase/Fluka (no. 22177), 1% pectinase/Serva, 1% macerozyme R10/Yakult, 1% hemicellulase/Sigma, 8 mM CaCl2, pH 5.5, and adjusted with mannitol to 500 mOsm.kg~. Digestion was performed in 250 ml-flasks at 25°C in the dark for 14 h under continuous rotation (4 rpm) in a Cell Production Roller Apparatus (BelIco/USA). After enzyme treatment, the protoplast solution was filtered through two layers of steel sieves (125 and 63 IJm mesh size) followed by centrifugation for 8 min at 150 x g. Protoplasts were resuspended in mannitol (500 mOsrn/kg), adjusted to a density of 5x105 protoplasts/ml and kept for 12 h at 4°C in the dark. Protoplast culture: To embed the protoplasts in sodium alginate, equal volumes of the protoplast suspension and a 0.4 M mannitol solution containing 2.8% sodium alginate (Roth-alginate E401) were mixed and then layered in 0.5 ml aliquots on agar-plates (containing 0.4 M mannitol and 0.02 M CaCl2) for polymerisation. The alginate disks were subsequently transferred into 60 x 15 mm plastic Petri dishes containing 4 ml KM-medium (Kao and Michayluk 1975) supplemented with 0.5 mg/I of each of BA, NAA, and 2,4-D adjusted to 500 mOsrn/kg with mannitol. The osmolality was subsequently reduced by diluting the original medium with osmoticum-free medium at a ratio of 1:1 on day 10, 20, 30 and 40, maintaining a constant volume of 4 ml. Protoplasts were cultured at 25°C in the dark. Cell division frequency, defined as the percentage of cells that have divided at least once, was calculated 15 d after protoplast isolation. Initiation of morphogenesis: Protocalluses were released from alginate 6-8 weeks after protoplast isolation. Incubation for 30 rain in a 20 mM sodium-citrate solution caused depolymerisation of the alginate matrix. After centrifugation (100 x g, 3 rain), calluses were washed twice in distilled water and transferred to MS-medium (Murashige and Skoog 1962) solidified with 0.25% Gelrite/Roth (pH adjusted to 5.7 prior to autoclaving) containing different combinations of growth regulators: (i) for the induction of somatic embryogenesis (according to the protocol of Pickardt et al. 1989), protoplast-derived calluses were transferred to MS-medium (3% sucrose) supplemented with 10 mg/I picloram (defined as MS/picloram). Cultures were kept at 25°C in the dark. After 28 d calluses were transferred to MS-medium (1.5% sucrose) containing 1 mg/I NAA (defined as MS/NAA) and incubated at 25°C under a 16 h photoperiod (79 pmol m2 s~). Calluses were subcultured every 28 d on this medium. Developed somatic embryos were transferred to MS-medium supplemented with 0.5 mg/I BA, resulting in continuous proliferation of shoots; (ii) for shoot regeneration protocalluses were placed on MS-medium (3% sucrose) supplemented with 2.5 mg/I thidiazuron, 0.1 mg/I NAA and 50 mg/I casein-hydrolysate and were subcultured every four weeks. Regenerating shoot buds were transferred on MS-medium containing 0.5 mg/I BA. Cultures were incubated at 25°C, 16 h photoperiod, and 79 pmol m2 s-~light intensity. Recovery of plants: Due to the lack of a rooting system, mature plants from both somatic embryos or direct-regenerated shoots were recovered by grafting shoots of 0.5 - 2 cm onto 5-6 d old etiolated seedlings (for a detailed description of the procedure see Pickardt et al. 1995).

Results

Protoplast isolation and culture The use of epicotyls and shoot tips from etiolated seedlings as a protoplast source yielded approximately 2.5x106 protoplasts/g fresh weight. A clean preparation of protoplasts varying in size from 20-40 pm diameter was obtained by filtration and several mannitol washings. Staining with FDA revealed that 90% of the protoplasts survived the isolation procedure. After overnight incubation in mannitol at 4°C, protoplasts were embedded in 1.4% sodium alginate and cultivated in KM-medium supplemented with 0.5 mg/I of each of BA, NAA, and 2,4-D. Protoplasts started division after 6-10 d. The frequency of cell division on the 15th day of culture varied from 25-60% with a mean of 36%. During the following weeks, the protoplast-derived microcalluses grew into a contiguous aggregation of colonies in and on the alginate disk (Fig. l a). Due to the high frequency of colony formation, it was difficult to distinguish individual colonies. It was roughly estimated that each alginate disk contained approximately 400-700 protocalluses. After 40-55 d of culture, the alginate matrix was depolymerised by incubation in sodium citrate. The released protocalluses were transferred to the solidified media mentioned below for further development.

Plant regeneration Regeneration of plants was achieved via two morphologically distinguishable pathways: (i) In order to induce somatic embryogenesis a protocol was used which had been developed for the initiation of embryogenic callus from epicotyl explants (Pickardt et al. 1989). Protoplast-derived calluses were exposed for a period of four weeks to MS+10 mg/I picloram followed by several subcultures on MS+I mg/I NAA. During the first culture on MS/picloram, a smooth, whitish callus developed without morphogenic activity. Upon transfer to MS/NAA, calluses formed yellowish, nodular structures from which somatic embryos arose singly or in clusters (Fig. 1b-d). The first embryos appeared after 4-5 weeks on MS/NAA. During the following months, the number of somatic embryos increased continuously with a maximum at week 10-12 (10-20 embryos per callus). After 12 weeks, embryo formation declined rapidly and from week 16 embryos were no longer produced. The remaining calluses showed only poor growth with occasional root formation. This pattern of regeneration resembles the results we have previously encountered studying embryogenic cultures of V.narbonensis derived from epicotyl segments (Pickardt et al. 1989). Attempts to achieve direct development from these somatic embryos to fully differentiated root- and shoot-systems (i.e. plantlets) were unsuccessful. Embryos, therefore, were transferred to MS-medium containing 0.5 mg/l BA. Under these conditions, shoot development was promoted. Due to the lack of an efficient rooting system, mature plants were generated by grafting shoots of >0.5 cm onto young seedlings (see Pickardt et al. 1995).

24

Fig. 1a-h: Plant regeneration via somatic embryogenesis (b-d) and shoot organogenesis (e-h) in Vicia narbonensis. (a) protoplast-derived

microcalluses growing as a contiguous aggregation of colonies in and on the alginate disk, (b-d) somatic embryos emerging singly and in clusters from catlus on MS/NAA, (e) development of green and compact caIluses on Ms,rFDZ, (f) formation of dark green, nodular structures, (g) development of shoot buds, (h) stem eIongation on mediumwith 0.5 mg/l BA. (ii) Protocalluses were transferred onto MS-medium supplemented with 2.5 mg/I thidiazuron and 0.1 mg/t NAA, a medium which was successfully used for shoot regeneration from V. faba protocalluses (Tegeder et al. 1995). Within the first 6-8 weeks of culture, the initially yellowish-coloured protocalluses developed into green, compact calluses (Fig. l e). During the following weeks small, dark green nodular structures appeared on the surface (Fig. l f) which occasionally differentiated into shoot buds (Fig. l g). The time until redifferentiation started varied in individual calluses between 3 and more than 7 months. A prolonged cultivation of calluses which had started to regenerate shoot buds on TDZ-containing medium resulted in continuous proliferation of buds that were, however, incapable of stem elongation and normal leaf development. Only upon transfer to MS-medium supplemented with 0.5 mg/I BA did several buds exhibit normal shoot development (Fig.lh). As mentioned above, rooting of V. narbonensis shoots occurs with only limited success. Mature plants were, therefore, generated by grafting. Efficiency of regeneration Due to the fact that embryogenic protocalluses realize their entire regeneration potential within 5 months after being transferred to solidified medium, the number of calluses which formed somatic embryos (on picloram/NAA) or shoots (on TDZ) within this period were considered for a comparison between both systems (Table 1). Regeneration via somatic embryogenesis occurred with higher frequency. Moreover, somatic embryos appeared earlier and more synchronously, whereas development of shoot-initials on MS/TDZ proved to be a slow process with high variability between individual calluses in respect to the time of appearance (see above). Therefore, somatic embryogenesis

required less time to produce flowering plants (6-7 months) compared to TDZ-induced shoot morphogenesis (8-10 months).

Discussion

In the present study protoplasts of Vicia narbonensis were regenerated to mature plants via two different developmental pathways. Through macroscopical observations we found a difference in the morphology of differentiation dependent on the growth regulators to which protoplast-derived calluses were exposed: (i) induction of somatic embryogenesis by the auxins picloram and NAA in two succeeding culture steps and (ii) initiation of shoot morphogenesis by thidiazuron. Since an identical protocol was used to generate somatic embryos from epicotyl segments in V. narbonensis (Pickardt et al. 1989), the isolated protoplasts derived from the same explant obviously retain their embryogenic potential under the given conditions. Table 1: Frequency of somatic embryogenesis (A) and direct shoot

organogenesis (B) in V. narbonensis within 5 months of subculture after transfer of protoplast-derived colonies to solidified medium. A

B

No. of callusestested 517 1049 No. of regenerating calluses 151 140 Regeneration rate (%) 29.2 13.3 A: Somatic embryogenesis on MS-medium containing 10 mg/I picloram (4 weeks) and subsequent culture on MS-medium with 1 mg/I NAA B: Shoot regeneration on MS-medium containing 2.5 mg/I TDZ, 0.1 mg/I NAA and 50 mg/I CH

25 The high efficacy of thidiazuron in tissue culture has already been demonstrated in several studies (see Huetteman and Preece 1993). The recent success in plant regeneration from protoplasts in Pisum sativum (BOhmer et al. 1995) and Vicb faba (Tegeder et al. 1995) together with our results in Vicia narbonensis clearly indicate that thidiazuron will play an important role in protoplast technology with previously recalcitrant grain legumes. Our observations regarding the phytohormonedependent mode of in vitro-morphogenesis are in accordance with the standard practices of initiating embryogenic cultures in the presence of auxins (Ammirato 1983) and shoot differentiation using a high cytokinin/auxinratio (grain legumes reviewed by Parrott et al. 1992). However, in contrast to the clear distinction between pidoram-induced somatic embryogenesis and thidiazuroninduced shoot morphogenesis in V. narbonensis, it has been shown that thidiazuron also induces the formation of somatic embryos in Arachis hypogaea (Saxena et al. t992), Geranium (Visser et al. 1992), tobacco (Gill and Saxena 1993), and Cayratia japonica (Zhou et al. 1994). In addition, a simultaneous formation of somatic embryos and shoots in Phaseolus coccineus has also been reported (Malik and Saxena 1992). It is important to state that the basic processes underlying both morphogenetic pathways are not necessarily as distinct from each other as the macroscopical observations described in this study would suggest. A histological based analysis of shoot regeneration and somatic embryogenesis in V. narbonensis is currently being carried out in our laboratory. The development of effective protocols for plant regeneration from V. narbonensis protoplasts through this study, together with the successful plant regeneration from V. faba protoplasts recently achieved in our laboratory establishes the prerequisites for attempting somatic hybridisations between these two species. These protocols may also prove valid for other Vicb species, in which case these species could be included in somatic hybridisation as well. In addition to the primary task of establishing the prerequisites for somatic hybridisation between Vicia species, the two regeneration systems also provide exciting opportunities for further studies regarding the effects of morphoregulatory compounds as well as the physiological events during in vitro-morphogenesis.

Acknowledgements The authors thank Verena Schade for excellent technical assistance. The work was supported by a grant of the Friedrich-Naumann-Stiftung/ Germany to M.T. and by the Bundesminister for Forschung und Technologie/Germany to O.S.

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Ball PW (1968) Vicia. In: Flora Europaea (Tutin TC, Heywood VH, Burges NA, Moore DM, Valentine DH, Waiters SM, Webb DA, eds), Cambridge University Press, pp. 29-136 Bauer-Weston B, Keller W, Webb J, Gleddie S (1993) Production and characterisation of asymmetric somatic hybrids between Arabidopsis thaliana and Brassica napus. Theor Appl Genet 86: 150-158 B6hmer P, Meyer B, Jacobsen HJ (1995) A TDZ-based efficient system for protoplast regeneration in pea. Plant Cell Rep 15:26-29 Cubero Jl (1982) Interspecific hybridisation in Vicia. In: Faba bean improvement (Hawtin G, Webb C, eds), Icarda/Martinus Nijhoff, The Netherlands, pp. 91-108 Gerdemann-Kn6rck M, Nielen S, Tzscheetzsch C, Iglish J, Schieder O (1995) Transfer of disease resistance within the genus Brassica through asymmetric somatic hybridisation. Euphytica 85:247-253 Gilissen LIW, van Taveren M J, Verhoeven HA, Sree Ramulu K (1992) Somatic hybridisation between potato and Nicotiana plumbaginifolia. Theor Appl Genet 84:73-80 Gill R, Saxena PK (1993) Somatic embryogenesis in Nicotiana tabacum L. - Induction by thidiazuron of direct embryo differentiation from cultured leaf discs. Plant Cell Rep 12:154-159 Hammatt N, Ghose TK, Davey MR (1986) Regeneration in legumes. In: Cell culture and somatic cell genetics of plants, Vol. 3 (Vasit IK, ed), Academic Press/New York, pp. 67-95 Huetteman CA, Preece JE (1993) Thidiazuron - a potent cytokinin for woody plant tissue culture. Plant Cell Tiss Org Cult 33:105-119 Kao KN, Michayluk MR (1975) Nutritional requirements for growth of Viola hajastana ceils and protoplasts at a very low population density in liquid media. Planta 126:105-110 Malik KA, Saxena PK (1992) Somatic embryogenesis and shoot regeneration from intact seedlings of Phaseolus acutifolius A., P. aureus (L) Wilczek, P. coccineus L., and P. wrightii L. Plant Cell Rep 11:163-168 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant: 15:473-497 Parrott WA, Bailey MA, Durham RE, Mathews HV (1992)Tissue culture and regeneration in legumes. In: Biotechnology and crop improvement in Asia (Moss JP, ed) Patancheru, A.P. 502324, India, pp. 115-148 Pickardt T, Huancaruna Perales E, Schieder O (1989) Plant regeneration via somatic embryogenesis in Vicia narbonensis. Protoplasma 149: 5-10 Pickardt T, Saalbach I, Waddell D, Meixner M, MOntz K, Schieder O. (1995) Seed specific expression of the 2S albumin gene from Brazil nut (Bertholletia excelsa) in transgenic Vicia narbonensis. Molec Breeding 1:295-301 Ramsay G, Pickersgill B (1986) Interspecific hybridisation between Vicia faba and other species of Vicia: Approaches to delying embryo abortion. Biol Zentralb1105:171-179 Roupakias DG, Tai W (1986) Interspecific hybridisation in the genus Vicia under controlled environment. Z PflanzenzOchtung 96: 177180 Saxena PK, Malik KA, Gill R (1992) Induction by thidiazuron of somatic embryogenesis in intact seedlings of peanut. Planta 187:421-424 Sch~fer HO (1973) Zur Taxonomie der Vicia narbonensis Gruppe. Kulturpflanze 21 : 211-273 Tegeder M, Gebhardt D, Schieder O, Pickardt T (1995)Thidiazuroninduced plant regeneration from protoplasts of Vicia faba cv. Mythos. Plant Cell Rep 15:164-169 Van den Ven WTG, Duncan N, Ramsay G, Philips M, Powell W, Waugh R (1993)Taxonomic relationships between V. faba and ist relatives based on nuclear and mitochondrial RFLPsand PCR analysis. Theor Appl Genet 86:71-80 Visser C, Qureshi JA, Gill R, Saxena PK (1992) Morphoregulatory role of thidiazuron - substitution of auxin and cytokinin requirement for the induction of somatic embryogenesis in Geranium hypocotyl cultures. Plant Physio199:1704-1707 Zhou J, Ma H, Guo F, Luo X (1994) Effect of thidiazuron on somatic embryogenesis of Cayratia japonica. Plant Cell Tiss Org Cult 36: 73-79

Plant regeneration from protoplasts ofVicia narbonensis via somatic embryogenesis and shoot organogenesis.

Protoplasts ofVicia narbonensis isolated from epicotyls and shoot tips of etiolated seedlings were embedded in 1.4% sodium-alginate at a final density...
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