Plant Cell, Tissue and Organ Culture 63: 81–84, 2000. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
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Research note
Plant regeneration from embryogenic cultures of Phragmites australis (Cav.) Trin. Ex Steud. Csaba M´ath´e1 , M´arta M. Hamvas1 , Istv´an Grigorszky1 , G´abor Vasas1 , Erika Moln´ar1 , J. Brian Power2 , Michael R. Davey2∗ & George Borb´ely1 1 Department
of Botany, Kossuth University of Debrecen, 4010-Debrecen, P.O. Box 14, Hungary; 2 Plant Science Division, School of Biological Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK (∗ requests for offprints; Fax: 15-951-3298; E-mail:
[email protected]) Received 23 November 1999; accepted in revised form 23 October 2000
Key words: common reed, Phragmites australis, plant regeneration, somatic embryogenesis
Abstract Embryogenic cultures of the common reed [Phragmites australis (Cav.) Trin. Ex. Steud.] were induced on Murashige and Skoog (1962)-based medium with 2% (w/v) sucrose, B5 vitamins and 4.5 µM 2,4-dichlorphenoxyacetic acid. Four independent culture lines, two initiated from stem nodes and two from roots, were established. These cultures underwent somatic embryogenesis. In one line of stem node origin, the somatic embryos germinated and developed into plants, following transfer of embryogenic cultures to Murashige and Skoog (1962)-based medium lacking growth regulators, with 108 ± 17 plants being recovered per 100 mg fresh weight of culture. In other lines, the somatic embryos developed roots, but not shoots. Shoot regeneration via somatic embryogenesis offers potential as an in vitro system for physiological studies, including assessments of the response of common reed to environmental pollutants. Abbreviations: 2,4-D – 2,4-dichlorophenoxyacetic acid; IAA – indoleacetic acid; MS – Murashige and Skoog (1962); NAA – α-naphthaleneacetic acid Phragmites australis (common reed) is a widely distributed Gramineaceous species, occurring from tropical regions to latitudes higher than 70◦ , typically in low-lying wetlands (McKee and Richards, 1996). It is often used in wastewater treatment because of its tolerance to environmental pollutants, including toxic heavy metals such as lead and cadmium (Ye et al., 1998). The plant is also exploited for paper and fibre production in Europe (Straub et al., 1988). However, populations of P. australis are declining in this region (Armstrong et al., 1996). Consequently, an efficient micropropagation system would be useful in germplasm preservation. The in vitro response of P. australis has been little studied. Sangwan and Gorenflot (1975) induced callus from stem explants, while Straub et al. (1988) initiated callus from seedlings. Plants were regenerated from
both stem- and seed-derived tissues. The present study was conducted in an attempt to improve the efficiency of previously published culture systems for P. australis and to compare explants with respect to embryogenic culture induction and plant regeneration. Shoots of P. australis were collected from two different locations (3 shoots per location) in the region of Debrecen, Hungary. Ten mm stem segments, containing the shoot apices, were surface sterilized by treatment with 70% (v/v) ethanol for 3 min, followed by 5% (v/v) hydrogen peroxide for 10 min. Explants were washed 3 times in sterile distilled water (5 min each wash). Axenic plants with well developed root systems were produced within 28 days on MS-based medium supplemented with 2% (w/v) sucrose and B5 vitamins (Gamborg et al., 1968), but lacking growth regulators.
82 The medium was semi-solidified with 0.8% (w/v) Difco Bacto agar at pH 5.8 (40 ml aliquots of medium in 175 ml capacity screw capped glass jars). Shoots were assessed for the possible presence of contaminating micro-organisms by macerating 1.0 g portions of material and incubating the macerate in 50 ml aliquots of Luria Broth (Sambrook et al., 1989) at 28 ± 2 ◦ C on a horizontal rotary shaker in the dark for 7 days. Contaminating micro-organisms were not detected following microscopic examination of Luria Broth samples. The second, third and fourth nodes (each 10 mm in length) from the apex were excised from these axenic plants and used for embryogenic culture induction on the same basal medium, but containing 4.5 µM 2,4-D. Ten mm explants were also taken from roots 7 days after their emergence from axenic shoots; root explants were cultured on the same medium as used for stem explants. A 16-h photoperiod (white fluorescent illumination; 30 µmol m−2 s−1 ) at 24 ± 2 ◦ C was used when culturing plants as a source of explants. Stem and root explants were incubated under continuous white fluorescent illumination (20 µmol m−2 s−1 ) at 26 ± 2 ◦ C for the initiation and maintenance of embryogenic cultures. Tissues regenerating shoots were incubated under the same conditions as the plants used as a source of explants. Four culture lines, each composed of tissue 10–13 mm in diameter and obtained from different shoots, were selected 28 days after their induction on medium containing 4.5 µM 2,4-D. Two lines (A, B) were from stem nodes; lines C and D were from roots. Light microscopy confirmed that both stem- and root-derived cultures originated from cells of the pericycle (data not shown). Actively proliferating tissues were detached from the original explants and subcultured every 21 days on medium with 4.5 µM 2,4-D. Embryogenic cultures were compact, with small isodiametric cells and produced green somatic embryos (each 1–2 mm diameter) at their surface (Figure 1). All stages of monocotyledonous type somatic embryos, from globular (Figure 2) to fully developed somatic embryos (Figure 3), were observed by preparing sections of embedded material. In this respect, 2–3 mm portions of embryogenic tissues were embedded in low melting point (40–50 ◦ C) paraffin wax; embedded material was incubated for 3 h in glycerol:glacial acetic acid:70% (v/v) ethanol; 10:15:75, by volume) prior to sectioning to 60–70 µm. Somatic embryos were as described by Emons and Kieft (1991), each with shoot and root primordia, coleorhiza, coleoptile
and scutellum. The number of somatic embryos per 100 mg fresh weight of embryogenic culture reached 96.1 ± 27.8 (± S.D. of mean), 119.8 ± 16.2, 131.4 ± 32.4 and 55.9 ± 12.3 for lines A, B, C and D, respectively, after 28 days of culture. Callus line D produced significantly fewer somatic embryos than the other 3 lines, according to one-way ANOVA. Following detachment from the original explants and at least 3 subcultures (each of 21 days), 5–8 mm portions of the 4 embryogenic culture lines were transferred from medium with 4.5 µM 2,4-D to medium lacking growth regulators, to promote plant regeneration from somatic embryos. Growth regulators were omitted at this stage, since cytokinins, such as zeatin and benzyladenine, inhibited callus growth even at low concentrations (data not shown), while 2,4-D, even at a relatively low concentration of 0.5 µM, stimulated callus formation. After 28 days, the somatic embryos of embryogenic lines A, C and D developed only roots. However, somatic embryos of line B became bipolar with the differentiation of coleoptiles and roots (Figure 4), giving shoots 8–10 mm in height after a further 28 days of growth. Such shoots developed into plants 60–100 mm in height with root systems within 56 days of transfer of embryogenic cultures to growth regulator-free medium. Typically, 108 ± 17 plants were recovered per 100 mg initial fresh weight of embryogenic tissue (9–12 plants per callus; Figure 5). All cultures of line B regenerated plants (Figure 6) after transfer to MS-based medium lacking growth regulators, with plant regeneration being sustained for at least 10 months after culture initiation. Nodal stem segments excised from axenic plants were also transferred to MS-based medium with 2% (w/v) sucrose and B5 vitamins, but with 2,4-D replaced by 1–10 µM NAA. After 28 days of culture, 67.7 ± 4.7% of nodal segments each produced 2–3 shoots from axillary buds of the parent explants in the presence of 1 µM NAA. However, NAA concentrations exceeding 5 µm inhibited shoot formation. Other workers also employed MS-based medium in studies with Phragmites. For example, Sangwan and Gorenflot (1975) initiated callus from stem explants on a medium consisting of MS macroelements, microelements and vitamins of Nitsch and Nitsch (1969) and 4% (w/v) sucrose, with 4.5 µM 2,4-D, or 54 µM NAA or 85.5 µM IAA. Straub et al. (1988) also initiated callus on Phragmites seedlings using a medium containing MS salts, 1000 mg l−1 inositol, 0.1 mg l−1 thiamine-HCl, 3% (w/v) sucrose, 4.5 µM 2,4-
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Figure 1. Embryogenic callus of line B with developing somatic embryos (arrowed) following culture of the callus for 56 days on MS-based medium with 4.5 µM 2,4-D. Bar = 1 mm. (Figures 2–3) Sections through a globular (Figure 2) and a mature somatic embryo (Figure 3) following culture of embryogenic tissue as in Figure 1. co = coleorhiza; et = embryogenic tissue; ge = globular embryo; rt = root tip; sc = scutellum; st = shoot tip. Bars = 500 µm. (Figure 4) Elongation of coleoptiles during development of somatic embryos, following transfer of embryogenic callus to MS-based medium lacking growth regulators for 16 days. Bar = 1 mm. (Figure 5) A 9-cm Petri dish showing the extent of plant regeneration from embryogenic tissue. Bar = 1 cm. (Figure 6) A cluster of regenerated plants, 28 days after their excision from the parent embryogenic tissue. Bar = 1 cm.
84 D and 5.7 µM IAA, although the origin of the callus was not confirmed. The efficiency of plant regeneration from callus line B was superior to that reported previously from cultured tissues of Phragmites. In the study by Sangwan and Gorenflot (1975), 66% of calli gave 1 shoot per tissue, when the tissues were transferred from medium with 54 µM NAA or 85.5 µM IAA to medium containing 0.45 µM 2,4-D and 5% (v/v) coconut milk. Straub et al. (1988) reported shoot and plant regeneration from seedling-derived tissues, on a medium lacking growth regulators, although the number of shoots regenerated (20 plants per 100 mg fresh weight of callus) was considerably less than in the present study. However, in the studies of Sangwan and Gorenflot (1975) and Straub et al. (1988), there was no discussion as to whether or not regenerated plants were derived from somatic embryos. Phytotoxins, such as sulphide or acetic acid, induce histological changes in Phragmites stems and rhizomes, leading to decreased viability of the plants (Armstrong et al., 1996). A micropropagation system will be useful in providing a supply of material with which to study the mechanisms of die-back in Phragmites, including the effects of phytotoxins. Embryogenic line B should be useful in this respect. In addition, the production of plants, either through somatic embryogenesis from line B or by shoot regeneration from axillary buds of stem nodal explants, should ensure an indefinite supply of material for such long-term studies. Thus, the system described in this report may be exploited to micropropagate plants of common reed for transfer to field conditions for conservation purposes, and for use in establishing biofilter ponds.
Acknowledgements The authors thank P. Anthony for assistance with statistical analysis and Mrs Katalin Havelant for excellent technical assistance. This work was supported by Grants OTKA N◦ . T 5235, 22988 and U 28679.
References Armstrong J, Armstrong W & van der Putten WH (1996) Phragmites die-back: bud and root death, blockages within the aeration and vascular systems and the possible role of phytotoxins. New Phytol. 133: 399–414 Emons AMC & Kieft H (1991) Histological comparison of single somatic embryos of maize from suspension culture with somatic embryos attached to callus cells. Plant Cell Rep. 10: 485–488 Gamborg OL, Miller RA & Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 151–158 McKee J & Richards AJ (1996) Variation in seed production and germinability in common reed (Phragmites australis) in Britain and France with respect to climate. New Phytol. 133: 233–243 Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473–479 Nitsch JP & Nitsch C (1969) Haploid plants from pollen grains. Science 163: 85–87 Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning: A Laboratory Manual. 2nd edition. Cold Spring Harbor Laboratory Press, New York Sangwan RS & Gorenflot R (1975) In vitro culture of Phragmites tissues. Callus formation, organ differentiation and cell suspension culture. Z. Pflanzenphysiol. 75 S: 256–269 Straub PF, Decker DM & Gallagher JL (1988) Tissue culture and long-term regeneration of Phragmites australis (Cav.) Trin. Ex. Steud. Plant Cell Tiss. Org. Cult. 15: 73–78 Ye ZH, Wong MH, Baker JM & Willis AJ (1998) Comparison of biomass and metal uptake between two populations of Phragmites australis grown in flooded and dry conditions. Ann. Bot. 82: 83–87
