Plant Cell Reports (1984) 3:88-90
Plant Cell Reports © Springer-Verlag 1984
Somatic embryogenesis in Echinochloa crusgalli Da-yuan Wang and Kong Yan China National Research Institute, Hongzhou, China Received January 9, 1984/Revised version received April 2, 1984 - Communicated by I. K.Vasil
ABSTRACT A white, compact embryogenic tissue was obtained from young inflorescence segments of Echinochloa crusgalli (barnyard grass) cultured on Murashige and Skoog's medium containing various concentrations and combinations of 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. Ilistological and scanning electron microscopic studies revealed that the white compact callus contained embryoids in various stages of development. Typical embryoids were bipolar and possessed scutella, coleoptiles and coleorhizae. The embryogenic nature of the callus was maintained throughout eight to ten subcultures spanning more than six months. A high frequency of plant regeneration was obtained when the 2,4-D concentration was reduced or 2,4-D was removed from the medium. ABBREVIATIONS BA = 6-Benzylaminopurine; CH = Casein IIydrolysate; 2,4-D = 2,4-Dichlorophenoxyacetic acid; MS = Murashige and Skoog's Medium; NAA = Naphthaleneacetic acid. INTRODUCTION Rapid advances are being made in the application of cell and tissue culture techniques for the purpose of genetic manipulation in higher plants. A prerequisite for this application is to regenerate efficiently complete plants from tissues, cells and protoplasts. Only a few years ago, plant regeneration from tissue cultures of the Gramineae was generally considered to be very difficult (Cocking, 1978). llowever, research in recent years has shown that plant regeneration via tissue culture is possible for most of the important species of cereals and grasses (Vasil, 1982, 1983). What is even more interesting is the increasing evidence of plant regeneration in these crops through somatic embryogenesis rather than organogenesis. Somatic embryogenesis is a preferred method of in vitro propagation because the embryoids are unicellular in origin (Vasil and Vasil, 1982b; He and Vasil, 1983), resulting in the uniformity of regenerated plants. This paper reports histological and scanning electron microscopic studies of somatic embryogenesis from cultured immature inflorescences of Echinochloa crusgalli (barnyard grass).
Offprint requests to: D. Wang
MATERIALS AND METIIODS Young, unemerged inflorescences of Echinochloa crusgalli (L.) Beauvois were obtained from field grown plants. After the outermost leaves were removed the material was rinsed in 70% ethanol for 30 seconds, sterilized with 0.1% mercuric chloride for 4 minutes, and washed three times in sterile distilled water. The inflorescences were dissected out, cut into segments 1-3 mm long, and cultured in glass dishes. The basal medium (Murashige and Skoog, 1962) was supplemented with 3% sucrose and different concentrations and combinations of 2,4-D (i.0-i0.0 mg/l), BA (0.1-5.0 mg/l), NAA (0-2.0 mg/l), and CH (500 mg/l). The pH of the medium was adjusted to 5.8 before autoclaving. A l l media were gelled with 0.7% agar. Cultures were incubated at 28 C in a growth room under 16 hours diffused light. Tissues for histological observation were fixed in formalin-acetic-alcohol, embedded in paraplast, sectioned at 8-10 ~m and stained with hematoxylinsafranin. Specimens for scanning electron microscopy were fixed in 2% glutaraldehyde in 0.i M phosphate buffer (pI! 7.0) for 4 hours at room temperature, post-fixed in 1.0% osmium tetroxide for 90 minutes, critical point dried and coated with gold. A JEOL JSH-T20 Scanning Electron Microscope operated at 20 KV was used for the examination and photography of specimens. RESULTS AND DISCUSSION At the time of excision and culture, the inflorescence contained many spikelets and spikelet primordia (Fig. i). Individual floral primordia were recognizable, but no stamen or carpel primordia were evident. A week after culture, the spikelets turned green, and the cut ends of the rachises swelled and produced a small amount of callus. Two weeks after culture initiation, a great deal of callus was formed on the inflorescence segments. There were two distinct types of callus: One was soft, friable, transluscent and non-morphogenic (non-embryogenic callus), the other was white, compact, and organized (Fig. 2). The white and compact embr¥ogenic callus was induced on all the media containing 2,4-D (0.5-5.0 mg/l). This type of callus also was induced from 90% of the
89 through eight to ten subcultures on MS medium containing 4.0 mg/l 2,4-D and 0.2 mg/l BA.
Many globular and organized structures could be seen using scanning electron microscopy. The typical embryoid with a scutellum, coleoptile and coleorhiza was often recognizable (Figs. 3-5). There were intermediate types of embryoids between the globular structures and the typical embryoid. Some types possessed only coleoptiles, others only coleorhizae. In some cases only a secondary scutellum was produced. At times more than two shoots in a scutellum could be seen. From the notch of the folded embryoids arose coleoptiles and coleorhizae. Inflorescences greater than 20 mm in length produced mainly non-embryogenic callus. When the spikelet and rachis were cultured separately, embryogenic callus was induced from both. If one compares the in vitro embryoid with an in vivo embryo using SEM and paraplast sections, it is clear that the somatic embryo is very similar to an in vivo embryo. The wide variety of structures ranging from globular to mature embryoids is possible because of an uneven distribution of hormones under culture conditions. The phenomenon of multiple shoots in somatic embryos has been reported in Pennisetum purpureum (Wang and Vasil, 1982), P. americanum (Vasil and Vasil, 1982a,b) and Triticum aestivum (Ozias-Akins and Vasil, i982). The age of the inflorescence was found to be critical in inducing somatic embryogenesis in E. crusgalli. This is similar to the observations in P_. purpureum (Wang and Vasil, 1982) and P_. americanum (Botti and Vasil, 1984). The authors wish to express their appreciation to Professor I. K. Vasil and Ms. Charlene Boyes for their valuable suggestions in the preparation of this manuscript. Fig. I. Inflorescence at the time of culture (x 19). Fig. 2. White and compact embryogenic callus (ec) and friable nonembryogenic callus (he) 3 weeks after the initial culture (x 8). Fig. 3. A longitudinal section of an embryoid with scutellum, coleoptile and coleorhiza (x85). Fig. 4, 5. Embryoids with defined scutelum and co]eorhiza and a developing coleoptile (note the coleoptile pore in Fig. 5) (x 62). Fig. 6. Plantlet from embryoid after transfer to MS medium with 0.5 mg/l 2,4-D and 0.5 mg/l BA (x 0.8).
inflorescence segments on the medium containing 2 m g / l 2,4-D, 0.5 mg/l BA and 500 m g / l CII. On the media containing BA or NAA without 2,4-D, normally only non-embryogenic calli could be induced. After three to four weeks of culture many embryoids had formed (Figs. 3-5). At this time the calli were transferred to MS medium with no 2,4-D or with reduced concentrations (less than 0.5 mg/l) of 2,4-D. The embryoids germinated and produced complete plantlets in 20 days (Fig. 6). Adding BA (0.5 mg/l) to the medium increased the frequency of plant formation. The embryogenic calli maintained their embryogenic potential for at least six months,
REFERENCES ~otti, C. and I.K. Vasil. (1984)o The o ~ t e g e n y of somatic embryos of Pennisetum americanum (L.) K. Schum. II. In c u l t u r e d immature inflorescences. Canad. J. Bot. In Press. Cocking, E. C. (1978). Protoplast culture and somatic hybridization. In: Proceedings of Symposium on Plant Tissue Culture, pp. 255-263. Scince Press, ]?eking. Ho, W. and I.K. Vasil. (1983). Somatic embryogenesis in sugarcane (Saccharum officinarum L.) I. The morphology and physiology of callus formation and ontogeny of somatic embryos. Protoplasma 118:169-180. Murashige, T. and F. Skoog. (1962): A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. PI. 15, 473-497. Ozias-Akins, P. and I.K. Vasil. (1982). Plant regeneration from cultured immature embryos and inflorescences of Triticum aestivum L. (wheat): evidence for somatic embryogenesis. Protoplasma 110:95-105.
9O Vasil, I.K. (1982). Somatic embryogenesis and plant regeneration in cereals and grasses. Plant Tissue Culture 1982, ed. A. Fujiwara, 101-104. Maruzen, Tokyo.
In: pp.
Vasil, V. and I.K. Vasil. 1982a. Characterization of an embryogenic cell suspension culture derived from inflorescences of Pennisetum americanum (pearl millet, Gramineae). Amer. J. Bot. 69: 1441-1449. Vasil, V. and I. K. Vasil. (1982b). The ontogeny of somatic embryos of Pennisetum americanum
(L.) K. Schum. I. In c u l t u r e d Bot. Gaz. 143: 454-465.
immature
embryos.
Vasil, I.K. (1983). Regeneration of plants from single cells of cereals and grasses. In: Genetic Engineering of Eukaryotes, eds. P. Lurquin and A. Kleinhofs, pp. 233-252. Plenum, New York. Wang, D. and I. K. Vasil. (1982). S o m a t i c embryogenesis and plant regeneration from infloresence segments of Pennisetum purpureum Schum. (Napier or Elephant grass). PI. Sci. Let. 25: 147-154.
Plant Cell Reports © Springer-Verlag 1984
Somatic embryogenesis in Echinochloa crusgalli Da-yuan Wang and Kong Yan China National Research Institute, Hongzhou, China Received January 9, 1984/Revised version received April 2, 1984 - Communicated by I. K.Vasil
ABSTRACT A white, compact embryogenic tissue was obtained from young inflorescence segments of Echinochloa crusgalli (barnyard grass) cultured on Murashige and Skoog's medium containing various concentrations and combinations of 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. Ilistological and scanning electron microscopic studies revealed that the white compact callus contained embryoids in various stages of development. Typical embryoids were bipolar and possessed scutella, coleoptiles and coleorhizae. The embryogenic nature of the callus was maintained throughout eight to ten subcultures spanning more than six months. A high frequency of plant regeneration was obtained when the 2,4-D concentration was reduced or 2,4-D was removed from the medium. ABBREVIATIONS BA = 6-Benzylaminopurine; CH = Casein IIydrolysate; 2,4-D = 2,4-Dichlorophenoxyacetic acid; MS = Murashige and Skoog's Medium; NAA = Naphthaleneacetic acid. INTRODUCTION Rapid advances are being made in the application of cell and tissue culture techniques for the purpose of genetic manipulation in higher plants. A prerequisite for this application is to regenerate efficiently complete plants from tissues, cells and protoplasts. Only a few years ago, plant regeneration from tissue cultures of the Gramineae was generally considered to be very difficult (Cocking, 1978). llowever, research in recent years has shown that plant regeneration via tissue culture is possible for most of the important species of cereals and grasses (Vasil, 1982, 1983). What is even more interesting is the increasing evidence of plant regeneration in these crops through somatic embryogenesis rather than organogenesis. Somatic embryogenesis is a preferred method of in vitro propagation because the embryoids are unicellular in origin (Vasil and Vasil, 1982b; He and Vasil, 1983), resulting in the uniformity of regenerated plants. This paper reports histological and scanning electron microscopic studies of somatic embryogenesis from cultured immature inflorescences of Echinochloa crusgalli (barnyard grass).
Offprint requests to: D. Wang
MATERIALS AND METIIODS Young, unemerged inflorescences of Echinochloa crusgalli (L.) Beauvois were obtained from field grown plants. After the outermost leaves were removed the material was rinsed in 70% ethanol for 30 seconds, sterilized with 0.1% mercuric chloride for 4 minutes, and washed three times in sterile distilled water. The inflorescences were dissected out, cut into segments 1-3 mm long, and cultured in glass dishes. The basal medium (Murashige and Skoog, 1962) was supplemented with 3% sucrose and different concentrations and combinations of 2,4-D (i.0-i0.0 mg/l), BA (0.1-5.0 mg/l), NAA (0-2.0 mg/l), and CH (500 mg/l). The pH of the medium was adjusted to 5.8 before autoclaving. A l l media were gelled with 0.7% agar. Cultures were incubated at 28 C in a growth room under 16 hours diffused light. Tissues for histological observation were fixed in formalin-acetic-alcohol, embedded in paraplast, sectioned at 8-10 ~m and stained with hematoxylinsafranin. Specimens for scanning electron microscopy were fixed in 2% glutaraldehyde in 0.i M phosphate buffer (pI! 7.0) for 4 hours at room temperature, post-fixed in 1.0% osmium tetroxide for 90 minutes, critical point dried and coated with gold. A JEOL JSH-T20 Scanning Electron Microscope operated at 20 KV was used for the examination and photography of specimens. RESULTS AND DISCUSSION At the time of excision and culture, the inflorescence contained many spikelets and spikelet primordia (Fig. i). Individual floral primordia were recognizable, but no stamen or carpel primordia were evident. A week after culture, the spikelets turned green, and the cut ends of the rachises swelled and produced a small amount of callus. Two weeks after culture initiation, a great deal of callus was formed on the inflorescence segments. There were two distinct types of callus: One was soft, friable, transluscent and non-morphogenic (non-embryogenic callus), the other was white, compact, and organized (Fig. 2). The white and compact embr¥ogenic callus was induced on all the media containing 2,4-D (0.5-5.0 mg/l). This type of callus also was induced from 90% of the
89 through eight to ten subcultures on MS medium containing 4.0 mg/l 2,4-D and 0.2 mg/l BA.
Many globular and organized structures could be seen using scanning electron microscopy. The typical embryoid with a scutellum, coleoptile and coleorhiza was often recognizable (Figs. 3-5). There were intermediate types of embryoids between the globular structures and the typical embryoid. Some types possessed only coleoptiles, others only coleorhizae. In some cases only a secondary scutellum was produced. At times more than two shoots in a scutellum could be seen. From the notch of the folded embryoids arose coleoptiles and coleorhizae. Inflorescences greater than 20 mm in length produced mainly non-embryogenic callus. When the spikelet and rachis were cultured separately, embryogenic callus was induced from both. If one compares the in vitro embryoid with an in vivo embryo using SEM and paraplast sections, it is clear that the somatic embryo is very similar to an in vivo embryo. The wide variety of structures ranging from globular to mature embryoids is possible because of an uneven distribution of hormones under culture conditions. The phenomenon of multiple shoots in somatic embryos has been reported in Pennisetum purpureum (Wang and Vasil, 1982), P. americanum (Vasil and Vasil, 1982a,b) and Triticum aestivum (Ozias-Akins and Vasil, i982). The age of the inflorescence was found to be critical in inducing somatic embryogenesis in E. crusgalli. This is similar to the observations in P_. purpureum (Wang and Vasil, 1982) and P_. americanum (Botti and Vasil, 1984). The authors wish to express their appreciation to Professor I. K. Vasil and Ms. Charlene Boyes for their valuable suggestions in the preparation of this manuscript. Fig. I. Inflorescence at the time of culture (x 19). Fig. 2. White and compact embryogenic callus (ec) and friable nonembryogenic callus (he) 3 weeks after the initial culture (x 8). Fig. 3. A longitudinal section of an embryoid with scutellum, coleoptile and coleorhiza (x85). Fig. 4, 5. Embryoids with defined scutelum and co]eorhiza and a developing coleoptile (note the coleoptile pore in Fig. 5) (x 62). Fig. 6. Plantlet from embryoid after transfer to MS medium with 0.5 mg/l 2,4-D and 0.5 mg/l BA (x 0.8).
inflorescence segments on the medium containing 2 m g / l 2,4-D, 0.5 mg/l BA and 500 m g / l CII. On the media containing BA or NAA without 2,4-D, normally only non-embryogenic calli could be induced. After three to four weeks of culture many embryoids had formed (Figs. 3-5). At this time the calli were transferred to MS medium with no 2,4-D or with reduced concentrations (less than 0.5 mg/l) of 2,4-D. The embryoids germinated and produced complete plantlets in 20 days (Fig. 6). Adding BA (0.5 mg/l) to the medium increased the frequency of plant formation. The embryogenic calli maintained their embryogenic potential for at least six months,
REFERENCES ~otti, C. and I.K. Vasil. (1984)o The o ~ t e g e n y of somatic embryos of Pennisetum americanum (L.) K. Schum. II. In c u l t u r e d immature inflorescences. Canad. J. Bot. In Press. Cocking, E. C. (1978). Protoplast culture and somatic hybridization. In: Proceedings of Symposium on Plant Tissue Culture, pp. 255-263. Scince Press, ]?eking. Ho, W. and I.K. Vasil. (1983). Somatic embryogenesis in sugarcane (Saccharum officinarum L.) I. The morphology and physiology of callus formation and ontogeny of somatic embryos. Protoplasma 118:169-180. Murashige, T. and F. Skoog. (1962): A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. PI. 15, 473-497. Ozias-Akins, P. and I.K. Vasil. (1982). Plant regeneration from cultured immature embryos and inflorescences of Triticum aestivum L. (wheat): evidence for somatic embryogenesis. Protoplasma 110:95-105.
9O Vasil, I.K. (1982). Somatic embryogenesis and plant regeneration in cereals and grasses. Plant Tissue Culture 1982, ed. A. Fujiwara, 101-104. Maruzen, Tokyo.
In: pp.
Vasil, V. and I.K. Vasil. 1982a. Characterization of an embryogenic cell suspension culture derived from inflorescences of Pennisetum americanum (pearl millet, Gramineae). Amer. J. Bot. 69: 1441-1449. Vasil, V. and I. K. Vasil. (1982b). The ontogeny of somatic embryos of Pennisetum americanum
(L.) K. Schum. I. In c u l t u r e d Bot. Gaz. 143: 454-465.
immature
embryos.
Vasil, I.K. (1983). Regeneration of plants from single cells of cereals and grasses. In: Genetic Engineering of Eukaryotes, eds. P. Lurquin and A. Kleinhofs, pp. 233-252. Plenum, New York. Wang, D. and I. K. Vasil. (1982). S o m a t i c embryogenesis and plant regeneration from infloresence segments of Pennisetum purpureum Schum. (Napier or Elephant grass). PI. Sci. Let. 25: 147-154.
