PlantCell Reports
Plant Cell Reports (1995) 15:186-191
9 Springer-Verlag1995
In vitro development of globular zygotic wheat embryos Christiane Fischer and Gunther Neuhaus Institute for Plant Sciences, Swiss Federal Institute of Technology (ETH), Universitfitstr. 2, CH-8092 Ziirich, Switzerland Received 14 March 1995/Revised version received 2 May 1995 - Communicated by H. L6rz
Summary. We have established in vitro culture conditions for globular zygotic wheat embryos (Triticum aestivum L.). Their nutritional requirements have been systematically investigated. The initial sucrose concentration, as well as the sucrose concentration during the culture, a 6benzylaminopurine supplement, the use of nitrates and ammonium as nitrogen source have a major influence on the embryo development. Proline has an inhibitory effect on the germination. A double layer system with different media was used to give a continuous variation of the medium composition with time. These culture conditions allowed normal direct embryogenesis in up to 47% of the globular embryos.
symmetry has to be established and the two primary organ systems, the embryonic axis which contains the shoot and root meristems, and the scuteUum have to be properly formed. Subsequent organogenesis has to give properly formed coleoptilar ring and leaf primordia differentiation. The time to achieve complete embryo development must also be in the same range as the in planta development. The medium composition should be defined. Finally, the culture system should be simple to handle and be accessible for manipulations. The influence of different medium components, like sucrose concentration, hormonal requirement, nitrogen source and proline supply have been evaluated in order to set up optimal culture condilions for these globular embryos.
Abbreviations: BAP: 6-benzylaminopurine, MES: 2-Nmorpholinoethane-sulfonic acid, MS: Murashige and Skoog (1962).
Introduction
Our aim is to gain information on the signals influencing early monocot embryogenesis. For this purpose, we have chosen wheat (Triticum aestivum L.) as a model plant. One of the main prerequisites was to establish in vitro culture conditions for very young zygotic embryos (globular to early transition stages). Until now, few publications have described normal in vitro development of the globular proembryo to the mature embryo (e.g. Norstog 1965; Liu et al. 1993). In fact, studies have focused on the growth of very early stages and less on the quality of embryo development (Kranz and LOrz 1993; Holm et al. 1994). Other studies have used older embryos (e.g. Triplett and Quatrano 1982; Iglesias etal. 1994). Several requirements have to be fulfilled in order to establish such a culture system for young embryos. One of the most important is that the embryogenesis in vitro has to be direct and parallel to the in planta development. In particular, a normal embryonic Correspondence to: G. Neuhaus
Material and Methods Wheat plants (Triticum aestivum L. cv Sonora, TDS seeds, Basel, Switzerland) were grown in a greenhouse under 17 h light/7 h dark at 18*C day/15"C night. The daylight was supplemented with 400 W fluorescent lamps (HPL-N 400W mercury lamp, Philips), when a minimum of 5000 lux was not reached during the day. The ears, collected 3 days after anthesis, were surface sterilized with 70% (v/v) ethanol for 1 minute. Immature embryos, with an embryo proper diameter of 100 to 160 grn, were excised aseptically under a dissecting microscope, keeping the tissues in a droplet of isolation medium (Table 1). The excised embryos were then plated on the surface of a double layer media system (Table 1) under a 16/8 h light/dark photoperiod and at a 26/22"C temperature regime. The double layer system consists of two different modified N6 media superposed in a 3.5 cm diameter Petri dish (Table 1). As soon as the first leaf primordium covered the meristem, the embryos were transferred to a dish containing the bottom medium only (Table 1). For germination, the embryos were cultured on solidified MS medium CMurashige and Skoog 1962) without hormones containing 2% sucrose. In order to establish optimal culture conditions for these embryos, the effect of different parameters was tested. These parameters were the sucrose concentration, the hormonal
187 requirements, the effect of proline on the cultures and the optimal nitrogen sources (see results).
Culture media Isolation medium
Double layer s~cstem Top medium
]~ottom medium
Composition N6 inorganic salts (Chu et ai. 1975) N6 vitamins (Chu et al. 1975) 2 mg/1 glyeine 100 rag/1 Casein hydrolysate 16% sucrose 0.6 mg/1 BAP 0.6 g/1MES pH 5.6 1 ml top medium 2 ml bouom medium Isolation medium solidified with 8 g/1 Sea Plaque Agarose (Sea Plaque, FMC Bioproduets, Rockland, USA) Top medium without hormones containing only 5% sucrose
Table 11 Composition of optimized culture media for embryo isolation and culture.
Results and Discussion
In planta development of wheat immature embryos The globular proembryo has a hemispherical embryo proper, and a cylindrical suspensor (Figure 1, A). One of the major steps in further embryo development is the transition phase, in which a shift from the radial symmetry of the proembryo to the bilateral symmetry of the embryo takes place. This transition phase starts with the initiation of the scutellum and the shoot apical meristem. The scutellum grows in both the axial and the lateral directions resulting in a prominent but well characterized structure on one side of the embryo proper (Figure 1, B). Almost in parallel, the swelling on the other side of the embryo proper indicates the initiation of the shoot apical meristem. (Figure 1, B). During subsequent development, the shoot meristem will be surrounded by the coleoptilar ring (Figure 1, C). Then, the first leaf primordium is initiated at the apical meristem (Figure 1, 13). This leaf primordium progressively overgrow the shoot meristem. As a result of this development, the apical meristem is entirely covered by the first leaf primordium which is itself, except for a small pore, enclosed by the coleoptile (Figure 1, E). At the same time, the scutellum develops into an oval shield-like organ (Figure 1, E). It is attached to one side of the embryonic axis structure and makes up most of the bulk of the embryo. In contrast to the shoot meristem, the root meristem is not located on the exterior of the embryo and hence cannot be detected macroscopically. Thus, the mature embryo contains two primary organ systems : (1) the embryonic axis which contains the shoot and root meristems, and (2) the scutelium.
Required culture conditions for normal in vitro development The embryos were excised from the kernels at the globular to late globular stage (embryo proper diameter of 100 to 150 lxm). Embryos which might be in early transition phase (embryo proper diameter of 160 lun) were also isolated. The initial solidified medium used for the culture of these embryos was based on N6 medium containing the inorganic salts and vitamins of N6, supplemented with 2 mg/l glycine, 100 mg/l casein hydrolysate, 8% sucrose, 25 mM proline, and 0.6 mg/i BAP. The development of globular embryos cultured on this medium was abnormal. Therefore, the influence of different medium components was evaluated. These components were optimized separately. For each new factor evaluated, the optimal conditions determined for the previous factors were used. The osmotic pressure of the medium during isolation and subsequent culture of tile embryos plays a critical role. We observed that the younger the excised embryos, the higher the medium osmolarity had to be. The sucrose concentration for freshly excised globular embryos was found to be optimal at 16% (Table 2). Below this concentration, the embryo was either not able to grow or developed into an abnormal structure. The embryos were excised in a droplet of isolation medium. This medium has the same composition as the subsequent solid culture medium. Thus, dehydratation of the embryos and osmotic shocks during the transfer of the embryos from the endosperm to the culture medium were minimized. Sucrose concentration of the isolation and initial solid culture medium (%)
Normal embryogenesis
(%)
abnormal development
16 14 12
45 7 0
55 93 100
No or
Table 2: Influence of the initial sucrose concentration on the development of young wheat embryos. The criteria for "normal embryogenesis" are the initiation of a~ embryonic symmetry, initiation of the scutellum and shoot apicad meristem. The embryos are observed after 5 days in culture. I~ each condition, an average of 10 embryos have been cultured.
As pointed out by Smith (1973), the chen-dcal composition of the medium for in vitro embryo culture should adequately reflect the nutritional requirements of the developing embryo in vivo. Monnier (1976) suggested that this nutritional requirement varied during the development. Therefore a double layer system has been developed which ensures a continuous variation of the medium composition with time. The double layer system is composed of the superposition of two different modified N6 media (Table 1). Monnier (1976) was the first to establish this kind of system
188 Differentiation degree reached by 9the shoot apical meristem :
Control: ~ccession of single layer media a 16%=~12%=:,10% ~8%=~5%
Sucrose concentration in the upper / lower layer of the different double layer systems used b
(I) 16%/10% (2) 10o/o/5%
0)16%/8% (2)8%/5%
(3) 5%
(3) 5~o
5
8
0
5
23
27
Early abnormal or stop of meristeandifferentiation Mefistem differentiation
29
0)16%15% (2)5%
First leaf primordium differentiation Table 3: Influence of double layer systems on the development of young wheat embryos The calculated numbers illustrate the percentages of embryos blocked at the different degree of shoot apical meristem differentiation considering the total amount of embryos cultured. The frequencies of embryos which do not differentiate a meristem have not been indicated but can be deducted from the indicated frequencies. For each culture condition, a total of 13 to 19 embryos were used in two independent experiments. a The embryos were initially cultured on a single layer medium containing 16% sucrose. They were then transferred on a medium containing 12% sucrose. Several transfers were performed in order to reduce the sucrose concentration down to 5%. b (1),(2),(3) refers to the succession of double layer systems used. 25 mM proline was added to all media. BAP was added only to the upper layer of the first system at a concentration of 0.6 rag/1. Ia-tthe control experiment, BAP has been added only to the first medium.
Concentration of BAP (mgfl)
Differentiation degree achieved by the shoot apical meristem :
0
0.3
0.6
0.9
Early abnormal or stop of mefistem differentiation
8
13
0
9
Meristem differeraiation
28
38
23
17
First leaf primordium differentiation
4
4
~i~iiiiiii~iiiii!!iiiii
13
Table 4: Influence of the BAP concentration on the wheat apical meristem differentiation For each BAP concentration, the percentages of embryos blocked at the different degrees of shoot apical meristem differentia~on are indicated. These frequencies are calculated considering the total amount of embryos cultured. For each culture condition, a total of 22 to 25 embryos were cultured in 3 independent experiments. 25 mM proline was added to all media.
which has subsequently been used in several other studies (Liu etal. 1993; Iglesias etal. 1994). Smith (1973) showed that tile embryo in vivo is subjected to a high osmotic pressure at early globular stages, which rapidly decreases during development. Based on this observation, different sequences of double layer systems containing different sucrose concentrations in the upper and lower layer have been tested to reduce gradually the osmotic pressure (Table 3). In the first system, the concentration of sucrose is higher in the upper layer than in the lower layer. The embryos were then transferred to a second double layer system. This second system has in the upper layer the same sucrose concentration as in the lower layer of the previous system 1 and in the lower layer, a yet more reduced sucrose concentration. Several transfers were thus performed in order to decrease progressively the osmotic pressure of the medium. A double layer system in which the top layer medium contains 16% sucrose and the bottom layer medium 5% sucrose appeared to be the best for the optimal development of young embryos. During subsequent culture, the embryos were transferred to a single layer medium containing 5% sucrose. With this system up to 20% of
the cultured embryos differentiated the first leaf primordium. As the embryos were blocked at this stage of development and were unable to germinate, further improvement o1' the culture conditions had to be performed. As second parameter, the effect of hormones, in particular the eytokinin requirement was tested. It has been assumed that growth factors are not required in embryo culture media. However, very young embryos might be stimulated by hormones (for review see Hu et Wang 1986). Therefore, different concentrations of B AP were added only to the upper medium (Table 4) thus leading to a gradual reduction of the BAP concentration during culture. We observed that the addition of BAP increases the proportion of embryos able to differentiate the first leaf primordium (Table 4). The optimal concentration of BAP was 0.6 mg/l. In this condition, 18% of the embryos differentiated the first leaf primordium. At lower concentrations or without BAP, meristem and leaf primordium differentiation occured, but the frequency of embryos blocked before the first leaf primordium differentiation, was higher. At higher BAP concentration, the number of embryos able to
189 Developmental stages reached by
Coneentral~on of preline (raM) in the upper / lower medium
the embryos :
25/25
25/0
12.5/12.5
0/0
Early abnormal or stop of mefistem differentiation
5
4
13
9
Mefistem differentiation
20
26
37
14
First leaf pfimordium differentiation
10
22
17
9
Growth of the leaf/leavesblocked early (onlyfew ram)
0
4
0
14
Shoot
0
0
4
5
Plantlet (shoot + root)
0
4
0
iiiiiiiiiiiiiiiiiiiiiiiiiiiiii~iiiiiiiiiiiiiiiiiiiiiiiiiiiii
Table 5: Effect of proline on embryo development. For each proline concentration, the percentages of embryos blocked at different stages of development are indicated. For each culture condition, a total of 20 to 24 embryos were used in 3 independent experiments.
Developmental stages reached by
Concentration of glutamine (rag/l)
the embryos :
400
800
800 without nitrates and arr~onium
Early abnormal or stop of meristem differentiation
0
0
0
22
Meristem differentiation
5
10
4
4
First leaf primordium differentiation
9
3
0
4
Coleolxilar ring and first leaf primordiurn differentiation
9
0
0
17
Growth of the leaf/leavesblocked early (onlyfew ram)
7
8
7
9
Shoot
0
5
0
0
Planflet (shoot + root)
47
45
40
0
Table 6 : Effect of the nitrogen source on embryo development For each glutamine concentration used, the percentages of embryos blocked at the different developmental stages are indicated. For each treatment, 23 to 45 embryos were cultured in 4 independent experiments.
differentiate the first leaf prirnordium was also reduced (13%). Nevertheless, even with an optimal BAP concentration, the embryos were not able to germinate. The N6 modified medium used initially contained a high proline concentration (25 mM, Lupotto and Lusardi 1988), which has been shown to induce highly ernbryogenic callus for maize somatic embryogenesis (Amstrong and Green 1985). We tested whether proline was also required for zygotic embryogenesis. Different proline concentrations were added to the upper and lower medium (Table 5). The embryos were then transferred to a single medium containing the same proline concentration as the lower layer of the previous system. At proline concentrations less than 25 mM, the embryos were able to reach later developmental stages. In fact, they were able to germinate and develop into plantlets. The best results were obtained without any proline in the medium. In this case, 18% of the embryos were able to develop into morphologically normal plants (Table 5). Thus,
although proline allows differentiation and subsequent organogenesis to occur, it has an inhibitory effect on embryo development, reflected in their inability te germinate. Finally, the effect of the nitrogen source on the in vitro development of globular zygotic embryos was tested. Glutamine, at different concentrations, was added to the two layers of the system and to the subsequent single medium. This was done either by supplementing the normal N6 medium, that already contained nitrates and ammonium (Table 1, 6), or by using an N6 medium devoid of nitrates and ammonium (Table 6). Differentiation of the shoot apical meristem and subsequent organogenesis occurred in the presence of nitrates/ammonium and to a certain extent with glutamine as sole nitrogen source, whereas germination required nitrates and ammonium. With glutamine as sole nitrogen source, the latest developmental stage reached by these embryos was a limited growth of leaves. They were unable to germinate into plants
190
191 Fig. 1: Comparison between inplanta and in vitro development of wheat globular zygotic embryos. A: globular embryo; B, C, D, E: in planta development; F, G, H, ]: in vitro development; bars represent 100 p.m (sc: scutellurn, m: shoot apical meristem, or: eoleoptilar ring, lp: first leaf primordium, arrowhead: pore not enclosed by the coleoptilar ring.)
without nitrates and ammonium. Otherwise, we observed that adding glutamine to nitrates and ammonium did not improve the frequency of plantlets obtained, which is in the range of 45%. However, glutamine had no inhibitory effect on the germination. These results were surprising, as glutamine has been described as an ideal nitrogen source for the in vitro culture of embryos for many species (for review see Raghavan 1976; Hu and Wang 1986). It has been suggested that cultured embryos might not develop an efficient system for the uptake and metabolism of nitrates or ammonium (Pdjven 1958; Monnier 1990). However, Mordhorst and L0rz (1993) showed that glutamine as sole nitrogen source inhibited plant regeneration from barley microspore derived embryoids but did not interfere with cell divisions and growth. It has to be noticed that the quality of the donor material, which was better in summer than in winter, has an influence on the quality of the in vitro development. This is reflected by a higher percentage of globular embryos able to develop into mature plants in summer. From the described data, we deduced the following optimal culture conditions for globular wheat embryos as shown in table 1. The embryos were excised in an isolation medium containing 16% sucrose and then cultured on a double layer system containing 16% sucrose in the upper layer and 5% sucrose in the lower layer. This system contained no proline but 0.6 rag/1 BAP in the upper layer. Futhermore, both layers contained 2830 mg/l KNO3 and 463 mg/l (NH4)2SO4. After 8 to 10 days, the embryos were transferred onto a single medium identical to the lower layer of the previous system. Subsequently, the embryos were germinated on MS hormone free medium containing 2% sucrose. Using these conditions, 18 to 47% of the embryos in culture, depending on the quality of the donor material, developed into a plantlet. These plantlets were transferred to the greenhouse and were fertile.
Quality of the in vitro development of wheat globular zygotic embryos In this optimized culture system, the pattern of development of the embryos in vitro (Figure 1, F to I) mimics the one observed in planta (Figure 1, B to E). The shoot apical meristem, the root meristem, as well as the scutellum were well differentiated and normal embryonic symmetry was established. Thus, these embryos were able to undergo direct embryogenesis which resulted in the germination of morphologically normal fertile plants. However, minor differences between the development in vitro and in planta might occur. In vitro, the scutellum had variable shapes (Figurel, I) in contrast to the well defined shield-like structure
(Figure 1, E) observed in planta. In addition, the coleoptilar ring sometimes did not enclose totally the shoot meristem and the leaf primordia and remained open (Figure 1, I). Sometimes, the differentiation of the coleoptile was blocked early. In this case, the first sheath observed during germination is the first leaf and not the coleoptile. Instead of covering the meristem from the base to the top, the first leaf primordiurn formed a sheath surrotmding the meristem. However, these minor differences did not affect the embryonic symmetry.
Conclusion In this study, we have established an in vitro culture system that allows the development through direct embryogenesis of up to 47% of globular wheat embryos in culture. The pattern of development ot these embryos is close to the one observed in planta. Furthermore, this culture system provides all the othe~ important requirements; namely defined composition, easy handling, accessibility, and a comparable time schedule for embryo development in vitro and in planta. This system will be useful for future investigations on cellular and molecular aspects ol embryogenesis in one of the most important monw.ot families, namely the Poacea.
Acknowledgments. We thank ProfessorI. Pot~kus ~TH - Zrarieh)/n whose laboratory these experiments were perforated and V.A. Iglesias, E. Fenner, and C. Sautter for providing us with the plant material. We also acknowledge G. Neuhaus-Urlfor critical reading of the manuscriptand 19I. Saul for language corrections. C. Fischer was supported by the grant (n~ALTF 287-1993)from EMBO.
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