Plant Cell Reports
P l a n t Cell R e p o r t s (1996) 1 5 : 8 6 5 - 8 6 8
9 Springer-Verlag 1996
Direct somatic embryogenesis and plant regeneration from single cell suspension cultures of Elymus giganteus Vahl Li Wang, Xiaoguang Wang, and Baiqu Huang Institute o f Genetics a n d Cytology, N o r t h e a s t N o r m a l University, C h a n g c h u n 130024, T h e People's Republic o f C h i n a Received 14 A u g u s t 1995/Revised v e r s i o n received 12 J a n u a r y 1996 - C o m m u n i c a t e d by E. D. Earle
Summary, A yellowish, nodular callus was induced from mature embryos of Elymus giganteus Vahl on MS medium containing 2.0 rag/1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/1 kinetin, from which a cell suspension culture was initiated in liquid MS medium supplemented with 0.5 mg/1 2,4-D, 1.0 mg/1 kinetin and 0.2 rag/1 naphthaleneacetic acid (NAA). By filtering through a series of sieves with decreasing mesh sizes and collecting the resultant filtrate, a suspension culture composed mainly of single embryogenic cells was established. In a medium containing 0.3 rag/1 2,4-D, 1.0 mg/1 6-benzylaminopurine (6-BAP) and 500 mg/1 casein hydrolysate (CH), the single cells underwent direct somatic embryogenesis resulting in the formation of proembryos. These proembryos developed into mature embryos when placed in a double-layer liquid overlay culture. Intact plants were developed from somatic embryos when they were transferred onto solidified MS medium without added growth regulators.
certain useful traits such as tolerance to drought and high salinity, resistance to diseases and insect pests, etc., and therefore is a source of genes that are valuable in genetic improvement of plants and cereals. We have previously reported the regeneration of plants from protoplasts derived from cell suspension cultures in E.giganteus (Zhang et al., 1993). This paper describes the procedure for establishing an embry,ogenic single cell suspension culture from embryo-derived callus, as well as induction of direct somatic embryogenesis from single cells and subsequent plant regeneration in E. giganteus.
Materials and methods Seeds of Elymus giganteus Vahl
were provided by the Chinese
Academy of Agricultural Sciences, Beijing. Mature embryos were used as the source material for the tissue culture, qqae seeds were surface sterilized by immersing in 0.1% mercuric chloride for 30 sec and rinsed in sterile distilled water four times. The embryos were dissected from the
Introduction
seeds under aseptic conditions. The embryos were cut longitudinally
Previous efforts to establish somatic embryogenesis in cell suspension cultures and to use such a system in studies of biochemical and molecular mechanisms of in vitro morphogenesis have been fruitflfl in dicotyledonous plants, particularly in carrot (e.g. Fujimura and Komamine, 1979; Nomura and Komamine, 1985; Sung and Okimoto, 1981, 1983; Choi and Sung, 1984; Choi et al., 1987). Meanwhile, induction of somatic embryogenesis in cell suspension cultures was also reported in a number of monocotyledonous plants including sugarcane (Ho and Vasil, 1983), wheat (Redway et al. 1990) and barley (Jahne et al., 1991). Elymus giganteus Vahl is well known as a superior forage species belonging to the grass family. It bears
medium solidified with 0.7% agar and supplemented with different
into two halves and placed on MS (Murashige and Skoog, 1962)
Correspondence to: B. H u a n g
combinations of auxins and cytokinins. The cultures were maintained at 23-25~ and under a photoperiod of 10 h/day illumination. Calluses were induced two weeks after the inoculation. Yellowish, friable and fast-growing nodular calluses were selected and transferred into liquid MS medium containing 2,4-D and kinetin, and cultured at 25~ in dark on a horizontal rotary shaker operating at 120 rpm. The cultures were subcultured at 7-day intervals by sampling the upper portion of the liquid and transferring into fresh liquid medium. A suspension culture containing free cells and small aggregates was established after 4 subcultures. The suspensions were filtered through a series of nylon sieves (950, 300, 108, 77 and 54 ~tm meshes), and the final filtrate was centrifuged for 5 min at 500g. The pellet of cells was resuspended in fresh liquid medium (MS + 0.3mg/1 2,4-D + 1.0mg/1
866 6-BAP + 500 mg/1 CH) for the induction of somatic embryogenesis. The cell number was estimated by counting cells using a hemocytometer. The single cell suspension thus established was maintained by culturing
Table 1 Influence of exogenous growth regulators on the callus formation in embryo segments of Elymus giganteus cultured on MS medium
in liquid medium at an initial density of 10 s cells/ml on a shaker. When small cell aggregates were formed the culture was filtered through a 77 g m mesh nylon sieve, and the cell aggregates on the sieve were h'ansferred into double-layer cultttre dishes in which a thin layer of
Hormones (mg/L) Medium ............................ Mophology of callus 2,4-D NAA Kinetin
liquid medium of the same composition was laid on the top of solidified agar medium. When the somatic embryos were formed, they were
0.5
--
0.5
2.0
--
0.5
4.0
--
0.5
--
2.0
0.5
transferred onto solidified medium without growth regulators and cultured under the 10 hr/day photoperiod. For histological examination,
materials were fixed in formalin-
acetic acid-alcohol (FAA) for 24hr, dehydrated in a graded ethanol series and embedded in paraffin wax. Sections were cut at a thickness of %10 Ixm, stained with safranin-fast green and finally examined and photographed under a light microscope.
Results and discussion
Table 1 summarizes the effects of different combinations of auxins and cytokinins in the culture medium on the induction and morphology of calluses initiated from mature embryos of E. giganteus. It is evident from the table that 2,4-D exerted a critical impact on the efficiency and nature of callus formation in this plant. In general, low 2,4-D level (0.5 mg/1, Medium 1, Table 1) gave rise to slow callusing, whereas high 2,4-D concentration (4.0 rag/l, Medium 3, Table 1) caused rapid callus formation resulting in massive translucent, soft, watery callus. A combination of 2.0 mg/1 2,4-D and 0.5 mg/1 kinetin (Medium 2, Tablel) induced a yellowish nodular callus. This type of callus was friable and easy to disperse in the liquid culture, and possessed a good emblyogenic potential as proved in the later experiments. It was found that NAA was n o t able to replace 2,4-D for induction of desirable callus, because 2.0 rag/1 NAA (Medium 4, Table 1) resulted in a low callusing rate and in partial browning of the callus. To initiate a cell suspension culture, friable nodular callus formed on Medium 2 was selected and placed in a liquid MS medium supplemented with 0.5 mg/1 2,4-D, 1.0 mg/1 kinetin and 0.2 mg/1 NAA. After culturing on a shaker for about 5 weeks with 3-4 subcultures, a suspension culture containing small cell dumps and mnnerous singIe cells was obtained. This culture was filtered through a series of nylon sieves of decreasing mesh size as described. The resultant filtrate was a high-quality single cell suspension culture in which the majority of cells were round, densely cytoplasmic and contained no apparent vacuoles (Fig. 1, A, B). The cell suspension culture thus established was similar in cell morphology and dividing ability to the "embryogenically competent single cells" of carrot reported by Komamine et al. (1991). When the single cells ofE. giganteus were
Moderate amount of white callus. Massive translucent yellowish nodular callus. Massive translucent soft watery callus. Moderate amount of translucent, browning callus.
a O b s e r v a t i o n s r e c o r d e d 25 d a y s after cul~xre.
cultured in a liquid medium supplemeuted with 0.3 rag/1 2,4-D, 1.0 rag/1 6-BAP and 500 rag/1 CH, first cell divisions were observed on day 3 of culture. The two daughter cells of the first division appeared to undergo a certain extent of differentiation, as one of them behaved like the apical cell in zygotic embryogenesis (Fig.lC, solid arrow), which was destined to develop into the embryo proper later on. The other daughter cell of the first division appeared like the basal cell of zygotic embryos (Fig.lC, hollow arrow). The apical-like cell of the 2-celled proembryo entered the second cell division earlier than the basal-like cell. Two types of division of the apical-like cell were observed, i.e., it may undergo either a longitudinal division (vertical to the first division) to give rise to a "T"-shaped 3-celled proembryo (Fig. 1C, 1D), or a transverse division (parallel to the first division) resulting in a filamentous proembryo (Fig. 1E). Successive cell divisions of the 3-celled proembryos gave rise to either an ovoid-shaped (Fig.IF) or filamentshaped (FigAG) multi-celled proembryos. It is clear from Fig.lF that the basal-like cell in the ovoid proembryo remained undivided at this stage (arrow). By day 10 of culture, small globular proembryos were formed (Fig. 1H). At this stage, the culture was filtered through a 77 gm mesh nylon sieve. The cultures retained on the sieve were mainly globular embryos and cell aggregates (Fig.lI, J). When they were transferred into the double-layer dishes (see Materials and Methods), mature somatic embryos were formed. Fig.lK is the histological section of a 25-day mature embryo, from which it can be seen that the vascular system had been developed in the embryo (solid arrow) and the plumular
867
Fig.1. Direct somatic embryogenesis in cell suspension culture of Elymus giganteus. A. Embryogenic suspension cells obtained from the filtration through a series of nylon sieves (950, 300, 108, 77, 54 lain meshes), x105. B. Densely cytoplasmic embryogenic cells in liquid culture, x210. C. The first division of an embryogenic cell gave rise to a 2-celled proembryo. The apical-like cell (solid arrow) entered the second division earlier than the basal-like cell (hollow arrow), x210. D. A "T"-shaped 3-celled proembry~resulted from the longitidinal division of the apical-like cell, x290. E. A filamentous 3-celled proembryo resulted from the transverse division of the apical-like cell, x210. F. An early ovoid-shaped embryo originated from divisions of a single embryogenic cell. Note the basal-like cell remained undivided at this stage (arrow), x210. G. An early filamentous embryo formed from the successive transverse divisions of the apical-like cell of a 2-celled proembryo, x210. I-I. A globular proembryo formed from a single embryogenic cell at day 10 of culture, x184. I, J. Globular proembryos and cell aggregates resulted from the filtration of embryogenic suspension culture through a 77~tm mesh sieve, x120. K. Longitudinal section of a mature somatic embryo showing the vascular system in the embryo (solid arrow). The plumular and radical apices are visible (hollow arrows), x120. L. A well-developed intact somatic embryo with a scutellum (Sc), a coleoptile (Cp) and a coleorhiza (Cr), x132. M. Regenerated plants transplanted to soil after rooting on hormone-free MS medium.
868 and radical apices were visible at the opposite poles of the embryo (hollow arrows). An intact mature somatic embryo possessed a scutellum, a coleoplile and a coleorhiza similar to a zygotic embryo (Fig. 1L). Based on four repeats of induction, it was estimated that nearly 30% of the globular cell aggregates placed in doublelayer medium formed mature somatic embryos. On average, about 100-150 mature embryos can be picked up from each 5-cm Petri dish after 3 weeks in double-layer culture. Therefore, this culture system provides a procedure by which harvest of hundreds or even thousands of somatic embryos is possible. This is useful in biochemical and molecular studies of somatic embryogenesis, for instance, the identification and isolation of embryo-associated proteins and their genes. When the mature somatic embryos were placed on solidified MS medium without added growth regulators, over 90% of them germinated and developed into plants which can be transplanted into soil (Fig. 1M). The histological study showed that the cleavage pattern of embryogenic cells and the development of somatic embryogenesis in E. giganteus as presented in this paper are similar to those described for other monocotyledonous plants, for example, Freesia refracta. However, in direct somatic embryogenesis of F. refraeta, the basal-like cell of the proembryo continued to divide to form a suspensor-like structure (Wang et aI., 1994). The suspensor-like structure of F. refraeta somatic embryos may have two functions. Firstly, it helped to push the somatic embryo out of the maternal tissue (immature inflorescence segments), and secondly it may have served as an absorption organ for transportation of mttrients from maternal tissue to the embryo (Wang et aL, 1994). Suspensor-like structures were not developed in somatic embryos of E. giganteus presumably because they were suspended in a liquid medium and such functions played by a suspensor were apparently not essential. A similar description of the existence and function of suspensor-like structures in other plants can be found in the literature (Williams and Maheswaran, 1986). Numerous investigations have provided evidence that somatic embryos originate from divisions of single individual embryogenic cells (e.g., Haccius, 1978; Street and Withers, 1974; Trigiano et aL, 1989; Wang et al., 1990, 1994), while some others suggested that somatic embryos may be organized from aggregates of cells (e.g., Raghavan, 1976; Tisserat et al., 1979). In our study with E. giganteus cell cultures, the process from the cleavage of single embryogenic cells to the formation of globular proembryos was observed and followed, and this has led us to the conclusion that the direct somatic embryogenesis in suspension cultures ofE. giganteus has a single-cell origin.
Acknowledgements'. This work was supported by a grant from the National Natural Science Foundation of China to BH.
References Choi JH, Sung ZR (1984) Plant/viol. Biol. Report 2 : 19-25. Choi JH, Liu L, Borkird C, Sung ZR (1987) Proc. Natl. Acad. Sci. USA 84 : 1906-1910. Fujimura T, Komamine A (1979) Plant Physiol. 64 : 1162-1164. Haccius B (i978) Phytomorphol. 28 : 74-8I. Ho W, Vasil IK (1983) Ann. Bot. 51: 719-720. Jahne A, Lazzeri PA, Jager-gussen M, Lorz H (1991) Theor. AppI. Gen. 82:74-80. Kommnine A, Matsumoto M, Tsukahara M, Fujimara A, Kawahara R, Ito M, Smith J, Nomura K, Fujimara T (1991) In: Nijkamp HJJ, Van Der Pas LHW, Van Aartrijk J (ed) Progress in Plant Cellular & Molecular Biology, Kluwer Academic Publishers, the Netherlands, pp 307-313. Murashige T, Skoog F (1962) Physiol. Plant. 15 : 473-497. Nomura K, Komamine A (1985) Plant Physiol. 79 : 988-991. Raghavan V (1976) In: Experimental Embryoganesis in Vascular Plants, chapter 14, Academic Press, London, pp 349-381. Redway FA, Vasil V, Vasil IK (1990) Plant Cell Reports 8 : 714-717. Street HE, Withers LA (1974) In: Street HE (ed) Tissue Culture and Plant Science, Proceeding 3rd International Congress of Plant Tissue and Cell Culture. U.K. Academic Press London, pp 7-100. Sung ZR, Okimoto R (1981) Proc. Natl. Acad. Sci. USA 80 3683-3687. Sung ZR, Okimoto R (1983) Proc. Natl. Acad. Sci. USA 80
2661-2665. Tisserat B, Esan EE, Murashige T (1979) Horticul. Rev. 1: 1-78. Trigiano RN, Gray DJ, Conger BV, McDaniel JK (1989)13ot. Gaz. 150 (1): 72-77. Wang L, Huang B, He M, Hao S (1990) Ann. Bot. 65 : 271-276. Wang L, Bao X, Liu Y, Hao S (1994) Internatl. J. Plant Sci. 155 : 672-676. Williams EG, Maheswaran G (1986) Ann. Bot. 57 : 443-462. Zhang G, Huang B, Wang L, Luo X, Zheng X, He M, Hao S (1993) Acta Bot. Sinica 35 : 422-428.
P l a n t Cell R e p o r t s (1996) 1 5 : 8 6 5 - 8 6 8
9 Springer-Verlag 1996
Direct somatic embryogenesis and plant regeneration from single cell suspension cultures of Elymus giganteus Vahl Li Wang, Xiaoguang Wang, and Baiqu Huang Institute o f Genetics a n d Cytology, N o r t h e a s t N o r m a l University, C h a n g c h u n 130024, T h e People's Republic o f C h i n a Received 14 A u g u s t 1995/Revised v e r s i o n received 12 J a n u a r y 1996 - C o m m u n i c a t e d by E. D. Earle
Summary, A yellowish, nodular callus was induced from mature embryos of Elymus giganteus Vahl on MS medium containing 2.0 rag/1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/1 kinetin, from which a cell suspension culture was initiated in liquid MS medium supplemented with 0.5 mg/1 2,4-D, 1.0 mg/1 kinetin and 0.2 rag/1 naphthaleneacetic acid (NAA). By filtering through a series of sieves with decreasing mesh sizes and collecting the resultant filtrate, a suspension culture composed mainly of single embryogenic cells was established. In a medium containing 0.3 rag/1 2,4-D, 1.0 mg/1 6-benzylaminopurine (6-BAP) and 500 mg/1 casein hydrolysate (CH), the single cells underwent direct somatic embryogenesis resulting in the formation of proembryos. These proembryos developed into mature embryos when placed in a double-layer liquid overlay culture. Intact plants were developed from somatic embryos when they were transferred onto solidified MS medium without added growth regulators.
certain useful traits such as tolerance to drought and high salinity, resistance to diseases and insect pests, etc., and therefore is a source of genes that are valuable in genetic improvement of plants and cereals. We have previously reported the regeneration of plants from protoplasts derived from cell suspension cultures in E.giganteus (Zhang et al., 1993). This paper describes the procedure for establishing an embry,ogenic single cell suspension culture from embryo-derived callus, as well as induction of direct somatic embryogenesis from single cells and subsequent plant regeneration in E. giganteus.
Materials and methods Seeds of Elymus giganteus Vahl
were provided by the Chinese
Academy of Agricultural Sciences, Beijing. Mature embryos were used as the source material for the tissue culture, qqae seeds were surface sterilized by immersing in 0.1% mercuric chloride for 30 sec and rinsed in sterile distilled water four times. The embryos were dissected from the
Introduction
seeds under aseptic conditions. The embryos were cut longitudinally
Previous efforts to establish somatic embryogenesis in cell suspension cultures and to use such a system in studies of biochemical and molecular mechanisms of in vitro morphogenesis have been fruitflfl in dicotyledonous plants, particularly in carrot (e.g. Fujimura and Komamine, 1979; Nomura and Komamine, 1985; Sung and Okimoto, 1981, 1983; Choi and Sung, 1984; Choi et al., 1987). Meanwhile, induction of somatic embryogenesis in cell suspension cultures was also reported in a number of monocotyledonous plants including sugarcane (Ho and Vasil, 1983), wheat (Redway et al. 1990) and barley (Jahne et al., 1991). Elymus giganteus Vahl is well known as a superior forage species belonging to the grass family. It bears
medium solidified with 0.7% agar and supplemented with different
into two halves and placed on MS (Murashige and Skoog, 1962)
Correspondence to: B. H u a n g
combinations of auxins and cytokinins. The cultures were maintained at 23-25~ and under a photoperiod of 10 h/day illumination. Calluses were induced two weeks after the inoculation. Yellowish, friable and fast-growing nodular calluses were selected and transferred into liquid MS medium containing 2,4-D and kinetin, and cultured at 25~ in dark on a horizontal rotary shaker operating at 120 rpm. The cultures were subcultured at 7-day intervals by sampling the upper portion of the liquid and transferring into fresh liquid medium. A suspension culture containing free cells and small aggregates was established after 4 subcultures. The suspensions were filtered through a series of nylon sieves (950, 300, 108, 77 and 54 ~tm meshes), and the final filtrate was centrifuged for 5 min at 500g. The pellet of cells was resuspended in fresh liquid medium (MS + 0.3mg/1 2,4-D + 1.0mg/1
866 6-BAP + 500 mg/1 CH) for the induction of somatic embryogenesis. The cell number was estimated by counting cells using a hemocytometer. The single cell suspension thus established was maintained by culturing
Table 1 Influence of exogenous growth regulators on the callus formation in embryo segments of Elymus giganteus cultured on MS medium
in liquid medium at an initial density of 10 s cells/ml on a shaker. When small cell aggregates were formed the culture was filtered through a 77 g m mesh nylon sieve, and the cell aggregates on the sieve were h'ansferred into double-layer cultttre dishes in which a thin layer of
Hormones (mg/L) Medium ............................ Mophology of callus 2,4-D NAA Kinetin
liquid medium of the same composition was laid on the top of solidified agar medium. When the somatic embryos were formed, they were
0.5
--
0.5
2.0
--
0.5
4.0
--
0.5
--
2.0
0.5
transferred onto solidified medium without growth regulators and cultured under the 10 hr/day photoperiod. For histological examination,
materials were fixed in formalin-
acetic acid-alcohol (FAA) for 24hr, dehydrated in a graded ethanol series and embedded in paraffin wax. Sections were cut at a thickness of %10 Ixm, stained with safranin-fast green and finally examined and photographed under a light microscope.
Results and discussion
Table 1 summarizes the effects of different combinations of auxins and cytokinins in the culture medium on the induction and morphology of calluses initiated from mature embryos of E. giganteus. It is evident from the table that 2,4-D exerted a critical impact on the efficiency and nature of callus formation in this plant. In general, low 2,4-D level (0.5 mg/1, Medium 1, Table 1) gave rise to slow callusing, whereas high 2,4-D concentration (4.0 rag/l, Medium 3, Table 1) caused rapid callus formation resulting in massive translucent, soft, watery callus. A combination of 2.0 mg/1 2,4-D and 0.5 mg/1 kinetin (Medium 2, Tablel) induced a yellowish nodular callus. This type of callus was friable and easy to disperse in the liquid culture, and possessed a good emblyogenic potential as proved in the later experiments. It was found that NAA was n o t able to replace 2,4-D for induction of desirable callus, because 2.0 rag/1 NAA (Medium 4, Table 1) resulted in a low callusing rate and in partial browning of the callus. To initiate a cell suspension culture, friable nodular callus formed on Medium 2 was selected and placed in a liquid MS medium supplemented with 0.5 mg/1 2,4-D, 1.0 mg/1 kinetin and 0.2 mg/1 NAA. After culturing on a shaker for about 5 weeks with 3-4 subcultures, a suspension culture containing small cell dumps and mnnerous singIe cells was obtained. This culture was filtered through a series of nylon sieves of decreasing mesh size as described. The resultant filtrate was a high-quality single cell suspension culture in which the majority of cells were round, densely cytoplasmic and contained no apparent vacuoles (Fig. 1, A, B). The cell suspension culture thus established was similar in cell morphology and dividing ability to the "embryogenically competent single cells" of carrot reported by Komamine et al. (1991). When the single cells ofE. giganteus were
Moderate amount of white callus. Massive translucent yellowish nodular callus. Massive translucent soft watery callus. Moderate amount of translucent, browning callus.
a O b s e r v a t i o n s r e c o r d e d 25 d a y s after cul~xre.
cultured in a liquid medium supplemeuted with 0.3 rag/1 2,4-D, 1.0 rag/1 6-BAP and 500 rag/1 CH, first cell divisions were observed on day 3 of culture. The two daughter cells of the first division appeared to undergo a certain extent of differentiation, as one of them behaved like the apical cell in zygotic embryogenesis (Fig.lC, solid arrow), which was destined to develop into the embryo proper later on. The other daughter cell of the first division appeared like the basal cell of zygotic embryos (Fig.lC, hollow arrow). The apical-like cell of the 2-celled proembryo entered the second cell division earlier than the basal-like cell. Two types of division of the apical-like cell were observed, i.e., it may undergo either a longitudinal division (vertical to the first division) to give rise to a "T"-shaped 3-celled proembryo (Fig. 1C, 1D), or a transverse division (parallel to the first division) resulting in a filamentous proembryo (Fig. 1E). Successive cell divisions of the 3-celled proembryos gave rise to either an ovoid-shaped (Fig.IF) or filamentshaped (FigAG) multi-celled proembryos. It is clear from Fig.lF that the basal-like cell in the ovoid proembryo remained undivided at this stage (arrow). By day 10 of culture, small globular proembryos were formed (Fig. 1H). At this stage, the culture was filtered through a 77 gm mesh nylon sieve. The cultures retained on the sieve were mainly globular embryos and cell aggregates (Fig.lI, J). When they were transferred into the double-layer dishes (see Materials and Methods), mature somatic embryos were formed. Fig.lK is the histological section of a 25-day mature embryo, from which it can be seen that the vascular system had been developed in the embryo (solid arrow) and the plumular
867
Fig.1. Direct somatic embryogenesis in cell suspension culture of Elymus giganteus. A. Embryogenic suspension cells obtained from the filtration through a series of nylon sieves (950, 300, 108, 77, 54 lain meshes), x105. B. Densely cytoplasmic embryogenic cells in liquid culture, x210. C. The first division of an embryogenic cell gave rise to a 2-celled proembryo. The apical-like cell (solid arrow) entered the second division earlier than the basal-like cell (hollow arrow), x210. D. A "T"-shaped 3-celled proembry~resulted from the longitidinal division of the apical-like cell, x290. E. A filamentous 3-celled proembryo resulted from the transverse division of the apical-like cell, x210. F. An early ovoid-shaped embryo originated from divisions of a single embryogenic cell. Note the basal-like cell remained undivided at this stage (arrow), x210. G. An early filamentous embryo formed from the successive transverse divisions of the apical-like cell of a 2-celled proembryo, x210. I-I. A globular proembryo formed from a single embryogenic cell at day 10 of culture, x184. I, J. Globular proembryos and cell aggregates resulted from the filtration of embryogenic suspension culture through a 77~tm mesh sieve, x120. K. Longitudinal section of a mature somatic embryo showing the vascular system in the embryo (solid arrow). The plumular and radical apices are visible (hollow arrows), x120. L. A well-developed intact somatic embryo with a scutellum (Sc), a coleoptile (Cp) and a coleorhiza (Cr), x132. M. Regenerated plants transplanted to soil after rooting on hormone-free MS medium.
868 and radical apices were visible at the opposite poles of the embryo (hollow arrows). An intact mature somatic embryo possessed a scutellum, a coleoplile and a coleorhiza similar to a zygotic embryo (Fig. 1L). Based on four repeats of induction, it was estimated that nearly 30% of the globular cell aggregates placed in doublelayer medium formed mature somatic embryos. On average, about 100-150 mature embryos can be picked up from each 5-cm Petri dish after 3 weeks in double-layer culture. Therefore, this culture system provides a procedure by which harvest of hundreds or even thousands of somatic embryos is possible. This is useful in biochemical and molecular studies of somatic embryogenesis, for instance, the identification and isolation of embryo-associated proteins and their genes. When the mature somatic embryos were placed on solidified MS medium without added growth regulators, over 90% of them germinated and developed into plants which can be transplanted into soil (Fig. 1M). The histological study showed that the cleavage pattern of embryogenic cells and the development of somatic embryogenesis in E. giganteus as presented in this paper are similar to those described for other monocotyledonous plants, for example, Freesia refracta. However, in direct somatic embryogenesis of F. refraeta, the basal-like cell of the proembryo continued to divide to form a suspensor-like structure (Wang et aI., 1994). The suspensor-like structure of F. refraeta somatic embryos may have two functions. Firstly, it helped to push the somatic embryo out of the maternal tissue (immature inflorescence segments), and secondly it may have served as an absorption organ for transportation of mttrients from maternal tissue to the embryo (Wang et aL, 1994). Suspensor-like structures were not developed in somatic embryos of E. giganteus presumably because they were suspended in a liquid medium and such functions played by a suspensor were apparently not essential. A similar description of the existence and function of suspensor-like structures in other plants can be found in the literature (Williams and Maheswaran, 1986). Numerous investigations have provided evidence that somatic embryos originate from divisions of single individual embryogenic cells (e.g., Haccius, 1978; Street and Withers, 1974; Trigiano et aL, 1989; Wang et al., 1990, 1994), while some others suggested that somatic embryos may be organized from aggregates of cells (e.g., Raghavan, 1976; Tisserat et al., 1979). In our study with E. giganteus cell cultures, the process from the cleavage of single embryogenic cells to the formation of globular proembryos was observed and followed, and this has led us to the conclusion that the direct somatic embryogenesis in suspension cultures ofE. giganteus has a single-cell origin.
Acknowledgements'. This work was supported by a grant from the National Natural Science Foundation of China to BH.
References Choi JH, Sung ZR (1984) Plant/viol. Biol. Report 2 : 19-25. Choi JH, Liu L, Borkird C, Sung ZR (1987) Proc. Natl. Acad. Sci. USA 84 : 1906-1910. Fujimura T, Komamine A (1979) Plant Physiol. 64 : 1162-1164. Haccius B (i978) Phytomorphol. 28 : 74-8I. Ho W, Vasil IK (1983) Ann. Bot. 51: 719-720. Jahne A, Lazzeri PA, Jager-gussen M, Lorz H (1991) Theor. AppI. Gen. 82:74-80. Kommnine A, Matsumoto M, Tsukahara M, Fujimara A, Kawahara R, Ito M, Smith J, Nomura K, Fujimara T (1991) In: Nijkamp HJJ, Van Der Pas LHW, Van Aartrijk J (ed) Progress in Plant Cellular & Molecular Biology, Kluwer Academic Publishers, the Netherlands, pp 307-313. Murashige T, Skoog F (1962) Physiol. Plant. 15 : 473-497. Nomura K, Komamine A (1985) Plant Physiol. 79 : 988-991. Raghavan V (1976) In: Experimental Embryoganesis in Vascular Plants, chapter 14, Academic Press, London, pp 349-381. Redway FA, Vasil V, Vasil IK (1990) Plant Cell Reports 8 : 714-717. Street HE, Withers LA (1974) In: Street HE (ed) Tissue Culture and Plant Science, Proceeding 3rd International Congress of Plant Tissue and Cell Culture. U.K. Academic Press London, pp 7-100. Sung ZR, Okimoto R (1981) Proc. Natl. Acad. Sci. USA 80 3683-3687. Sung ZR, Okimoto R (1983) Proc. Natl. Acad. Sci. USA 80
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