PlantCell Reports
Plant Cell Reports (1991) 10:384-387
9 Springer-Verlag 1991
Characterization of embryogenic cell lines of Picea abies in relation to their competence for maturation Paula Jalonen and Sara von Arnold The Swedish University of Agricultural Sciences, Department of Forest Genetics, Box 7027, S-75007 Uppsala, Sweden Received June 13, 1991/Revised version received July 10, 1991 - Communicated by J. K. Vasil
Summary. Embryogenic ceil lines of Picea abies are categorized into three groups (polar, solar, and undeveloped) based on the organization of the somatic embryos within the tissue and the ability of the somatic embryos to proceed through a maturation process when treated with ABA. The polar and the solar types consist of somatic embryos with densely packed embryonic regions subtended by vacuolated suspensors. Both types of tissue regenerate mature somatic embryos when treated with ABA. Almost all mature somatic embryos develop further into shoots or plantlets. The undeveloped type consists of somatic embryos comprised of only a few loosely aggregated cells in their embryonic regions. Mature somatic embryos were not observed with this tissue type. Keywords: Embryogenic ceil lines - - Maturation - - Picea abies ~ Somatic embryos
Abbreviations: ABA:cis-tmns abscisic acid, Al:polar type, A2:solar type, B:undeveloped type, BA:benzyladenine, 2,4D:2,4 di-chlorophenoxyacetic acid, LP: von Amolds medium (1987).
Introduction Somatic embryogencsis can be achieved in various coniferous species. The appearance of embryogenic cultures seems to be similar in all conifers, i. e. they are white to translucent, mucilaginous and consist of many small, undeveloped somatic embryos. Initiation frequency of embryogenic cultures is relatively high for Picea abies, up to 100% from immature zygotic embryos (von Arnold and Hakman 1988), and about 40% from seedlings (Mo and von Arnold 1991), irrespective of the genotype used. However, the level of embryogenesis varies significantly among embryogenic cell lines derived from different genotypes (Becwar et al. 1987). Embryogenic cultures retain their embryogenic potential and their capacity to develop into plants for several years (Mo and von Arnold 1989). Somatic embryos of P. abies can be converted to phenotypicaily normal plants which overwinter and renew vegetative Offprint requests to: S. von Arnold
growth in a way similar to zygotic embryos (Becwar et al. 1989). Somatic embryos from several coniferous species can attain the germination stage (see for example Krogstrup 1990; Webster et al. 1990). However, the regeneration frequency is generally very low and unpredictable. This may be due to the fact that somatic embryos germinate precociously and do not adequately mature for subsequent development (Roberts et al. 1990 a). Conifer somatic embryos, like those of angiosperms, require ABA for maturation. Conifer zygotic embryos accumulate massive amounts of storage lipids during maturation (Konar 1958). Ultmslructural studies have shown that ABA stimulates somatic embryos of Picea glauca to accumulate storage material, especially lipids but also proteins and carbohydrates (Hakman and von Arnold 1988). These results are confirmed by quantitative and qualitative measurements of ABA-induced increases in storage lipids (Feirer et al. 1989) and proteins (Hakman et al. 1990; Roberts et al. 1990 b) in somatic embryos of P. abies. Although the general appearance of conifer embryogenic cultures are very similar to the unaided eye, significant cell line differences can be observed microscopically. This applies particularly to somatic embryo organization and development. Pronounced differences have also been reported among different cell lines of the same species (Laine and David 1990; Roberts et al. 1990 a). The aim of this study was to characterize embryogenic cell lines ofP. abies of varying genetic origin and to correlate their growth habit to the ability of the somatic embryos to proceed through a maturation process and to develop into
plants. Material and Methods Embryogenie cultures of Picea abies (L) Karst were established as previously described (yon Arnold 1987). The cultures were initiated in 1986 and 1988 from mature zygotic embryos collected from two different seed orchards in central Sweden. Briefly, the cultures were grown in 9-cm petri dishes containing half-strength LP medium containing 15 mM NH4NO3, 30 mM sucrose, 9 }aM 2,4-D, 4.4 ~tM BA and gelled with 0.4% Gelrite gellan
385 gum. The petri dishes were incubated in darkness at 20~ The cultures were transferred to fresh culture medium every month. Each cell line originated from one zygotic embryo. The following cell lines were used in this study: 86 (1, 2, 3, 19, 29, 36, 55, 58, 59, 66, 70, 71, 72), 88 (6, 12, 14, 21, 29, 30, 33, 36, 37, 46). The somatic embryos were stimulated to mature according to methods described previously (you Arnold 1987). Briefly, the embryogenie cultures were transferred to full-strength LP medium containing 90/aM sucrose and 15/aM ABA, gelled with 0.4% Gelrite gellan gum. When the somatic embryos had matured after 1 to 3 months on ABA containing medium, the cultures were transferred to full-strength LP medium containing 60 mM sucrose and gelled with 0.4% Gellite gellan gum. After one week in darkness the petri dishes were incubated in light at 116 Ixrnol m-Zs-1. After one week in light the embryos were isolated and cultured individually on medium with the same composition as used before. Cultures were examined microscopically both before and during the ABA-treatment.
Results Classification of embryogenic cell lines Based on the appearance of the somatic embryos and their ability to mature and develop into plants, the embryogenic cell lines used in this study were classified into three different groups (Table 1). These three tissue types were easily distinguished in the microscope (Fig. la, 2a, 3a). T a b l e 1. Classification of embryogenic ceil lines ofPicea abies.
tic embryos tend to be more polar, and when fast growing cell suspensions are established the somatic embryos develop the solar configuration (data not shown). Furthermore both (A1) and (A2) tissue types can give rise to planflets after ABA treatment (see below).
Response to ABA treatment The ability of the somatic embryos, from each of the three tissue types, to respond to ABA treatment is shown in Table 2. A positive ABA response was defined as changes in the tissues' growth habit. The following positive responses were observed: differentiation of mature somatic embryos (Fig. lb), precociously germinating somatic embryos (Fig. 2b), nodules, (Fig. 3b) and abnormal somatic embryos (Fig. 3c). Some cell lines showed negative ABA response, i. e. there was no visible change in tissue a ~ c e after ABA treatment. T a b l e 2. Response of eight embryogenic cell lines of Picea abies to ABA
treatment. The cultures were transferred to LP-medium containing 15 pM ABA, 3% sucrose and 0.4% gellan for 8 to 12 weeks. Average weight of each callus was 0.5 g. Numbers of mature somatic embryos, precocious somatic embryos, abnormal somatic embryos and nodules were estimated after 6, 8 and 12 weeks cultivation on ABA containing media. When a cell line had produced an average of I0 somarie embryos per callus the response was regarded as completed. Cell line
Tissue type
No of calli observed
Average no of differentiated structures per callus weeks 6 8 12
Group
Tissue type
Characteristics
A1
polar type
Somatic embryos that are polarized with distinct embryo-heads and well developed suspensors (Fig. la)
86:2
A1
90
5a
10a
A2
solar type
Somatic embryos with a radial symmetry (Fig. 2a)
86:19 86:55
A2 A1
8 18
lb 7a
lb 10a
9b
B
undeveloped type
Somatic embryos that are less developed (Fig. 3a). Embryogenic cell clusters are surrounded by vaeuolated cells. Frequently the embryogenic cells are also intermingled with clusters of vacuolated cells
86:59 86:66
A1 A1
30 47
2a 60a
2a
la
88:12 88:14
B B
24 16
0 0
10e 2c
lc
88:30
B
6
ld
5d
5d
Polar somatic embryos (A1) have a distinct embryo-head consisting of small closely packed cells and attached suspensor cells which are highly vacuolated (Fig la). Frequently, the suspensors from many embryos develop in bundles. The solar somatic embryos (A2) consist of the same ceil types as in the polar type, e.g. small closely packed cells and long vacuolates suspensor cells (Fig. 2a). However, the small, densely packed cells of the (A2) are completely surrounded by the elongated, vacuolated suspensor ceils. In the third type of tissue the undeveloped somatic embryos (B) are polarized, but the embryo-heads are comprised of only a few loosely aggregated clusters of ceils, which are not separated from the vacuolated suspensor cells (Fig. 3a). In addition to the embryos, the B-type contains long, highly vacuolated cells and rounded, vacuolated cells, both single and in clusters. These vacuolated cells frequently contain starch grains. Since cell line A1 can switch over to A2 and vice versa it appears that the polar type (A1) and the solar type (A2) belong to the same main group. We also observed that when the growth rate of a fast growing cell line slows down, the soma-
a = normal mature somatic embryos b = precociously germinating embryos c = nodules d = abnormal embryos
Normal mature somatic embryos (Fig. lb) were only obtained from cell lines classified as type A1. The optimal treatment time with ABA varied from 6 to 12 weeks. The best cell line (86:66) gave an average of 120 mature embryos per gram fresh weight. In contrast only a few mature somatic embryos were formed from the cell lines classified as type A2. These cell lines regenerated somatic embryos which germinated precociously (Fig. 2b), i.e. the embryos turned green much earlier than that was observed with the A1 tissue type. No mature somatic embryos were obtained from the cell lines classified as B. These cell lines either did not respond to ABA treatment or they differentiated nodules (Fig. 3b) or abnormal embryos. Upon prolonged culture on medium containing ABA the nodules either regenerated new callus, turned brown or developed roots. By sectioning it was found, that the abnormal embryos had several bud apices.
386
Figs. 1--3: Development of somatic embryos from different types of embryogenic tissues. Figures 1, 2 and 3 show the development of somatic embryos from tissue type A1, A2 and B respectively. a) Proliferating embryogenic tissue on medium containing 2,4-D and BA. Note that the somatic embryos in la are polarized, in 2a are solar shaped and in 3a are less differentiated. Scale bars = 0.1 ram. b) Embryogenic tissue cultured on medium containing ABA for 4, 6 and 12 weeks respectively in figs 1, 2 and 3. Note that mature somatic embryos are formed in lb; that the somatic embryo in 2b has started to develop cotyledons; that nodules are formed in 3b. Scale bars in lb and 2b = 1 mm, in 3b = 5 mm. c) Embryogenic tissue first treated with ABA and then cultured on medium lacking growth regulators for two weeks. Note that normal somatic embryos have developed in Fig. Ic; that the embryo in 2c retains a suspensor, that the somatic embryo in 3c has an abnormal number of cotyledons. Scale bars in Ic and 3c = 5 ram, in 2c = 1 mm. d) Isolated somatic embryos cultured on medium lacking growth regulators. (ld) Normal mature somatic embryo after one week. (3d) Abnormal embryo after three weeks. Scale bars = 0.5 ram.
The experiments were repeated twice with the same and new cell lines. In all cases the three types of embryogenic tissues (A1, A2 and B) responded to ABA in a manner typical for each tissue type (Table 3). Table 3. The ability of different cell lines to differentiate mature somatic embryos. The calli (as described in Table 2) were cultured on ABA containing medium for 2 to 3 months. Tissue type
A1 A2 B
Total number of cell lines 6 6 11
Total number of calli 192 30 90
Maturation on ABA + + -
Plant development Mature somatic embryos derived from type A tissue developed further when transferred to medium lacking ABA (Figs. lc and d). Almost all of the embryos developed into shoots or plantlets (Table 4). However, some variation in the developmental pattern could be observed between embryos from different cell lines. The majority of the cell lines with type A tissue gave rise to plantlets with a single tap root, which usually was covered with root hairs. Plantlets from some cell lines also formed a large number of secondary roots. Shoots from some cell lines developed roots only after prolonged culture in vitro. In general, shoots either with or without roots, had a tendency to form a resting bud. However, in some cell lines the epicotyl continued to develop without a rest period.
387 Table 4. Plant regeneration from mature somatic embryos, cultured on hormone-free LP medium containing 2% sucrose and solidified with 0.4% gellan in light for two weeks. Ceil line 86:2 86:55 86:59 86:66
No. of mature somatic embryos 357 286 24 441
Shoots only (fro) 89 95 92 8
Rooted shoots (%) 11 5 8 92
Normal plants were never observed with the type B tissue. The abnormal embryos developed several bud-meristems which produced a cluster of shoots (Figs. 3c and 3d). The nodules, irrespective of whether they were within the associated tissue or isolated, either degenerated or developed into roots. Discussion This study has shown that embryogenic tissues ofPicea abies can be categorised into three groups. These groups are based on the following criteria: (1) the presence of somatic embryos and (2) the manner in which the somatic embryos proceed through a maturation process and subsequently develop into plants. Since each cell line represents one genotype it is reasonable to assume that the regeneration ability, under the same culture conditions, varies among different genotypes. Similar genotypic differences have been reported for several angiosperm species (Tomes and Smith 1985; Hedges et al. 1986). Both the polar and solar tissue types have several characteristics in common. Their immature somatic embryos consist of a densely packed embryonic region subtended by vacuolated suspensor cells. Furthermore, these somatic embryos can be stimulated to go through a maturation process when treated with ABA. We have ample evidence to conclude that the differences between the solar and polar tissue types are mainly due to the growth rate of the cell line. The rationale for separating the types into two groups are (1) anatomical differences observed microscopically and (2) differences in the maturation process of their somatic embryos. In interior spruce, precocious germination of somatic embryos occurred at 10---20 ~tM ABA (Roberts et al. 1990 a). In our study, the tendency to precocious germination was only observed in cell lines with A2 tissue. It is reasonable to assume that the fast growing cell lines with solar type embryos would require a higher ABA concentration for complete inhibition of precocious germination.
The undeveloped tissue type consists of less organized somatic embryos. It is characterised by cells in the embryo head that are loosely aggregated and frequently intermingled with vacuolated cells. These somatic embryos cannot proceed through a normal maturation process. An embryogenic cell line of Pinus caribaea consisting of poorly organized somatic embryos was also unable to regenerate plants (Laine and David 1990). Several experiments (not shown) have been performed attempting to stimulate undeveloped embryogenic tissue type of P. abies to differentiate into mature somatic embryos. Different solvents for ABA (Dunstan et al. 1990), increased ABA concentration and prolonged ABA treatment time, were not sufficient to induce maturation. The reason why undeveloped tissue does not regenerate mature somatic embryos is unknown. One hypothesis is that the somatic embryos are blocked in their development and have not reached the stage where they can respond to ABA and mature.
Acknowledgements.This study was supported by the National Board of Forestry, The Scandinavian Contact Agency for Agricultural Research and The Troedsson Fund.
References Becwar M R, Noland T L, Wann S.R (1987) Plant Cell Reports 6:35-38 Becwar M tL Nagmani R, Warm S R (1990) Can. L Bot. 20:810-817 Becwar M R, Noland T L, Wyckoff J (1989), In Vitro Cellular and Developmental Biology 25(6): 575-580 Dunstan D J, Boch C A, Abrams G D, Abrams R (1990) Abstract B5-15 at VII th ~APTC Congress in Amsterdam Feirer R P, Conkey J H, Verhagen S A (1989) Plant Cell Reports.8:207209 Hakman I, yon Arnold S (1988) Physiologia Plantamm 72:579-587 Hakman I, Stabel P, Engstrtm P, Eriksson T (1990) Physiologia Plantamm 80:1-5 Hedges T K, Kamo K K, Imbrie C W, Becwar M R (1986) Bio/Teehnology 4:219-223 Konar R N (1958) Phytomorphology 8:174-176 Krogstmp P (1990) Plant Science 72:115-123 Laine E, David A (1990) Plant Science 69:215-224 Mo H, yon Arnold S, Lagercranz U. (1989) Plant Cell Reports 8:375-378 Mo H, yon Arnold S (1991) J. Plant Physiology (in press) Roberts D S, Flinn B S, Webb D T, Webster F B, Sutton B C S (1990 a) Physiologia Plantarum 78:355-360 Roberts D S, Sutton B C S, Flinn B S (1990 b) Can J. Bot 67:1086-1090 Tomes D T, Smith O S (1985) Theor. App1. Genet. 70:505-509
Plant Cell Reports (1991) 10:384-387
9 Springer-Verlag 1991
Characterization of embryogenic cell lines of Picea abies in relation to their competence for maturation Paula Jalonen and Sara von Arnold The Swedish University of Agricultural Sciences, Department of Forest Genetics, Box 7027, S-75007 Uppsala, Sweden Received June 13, 1991/Revised version received July 10, 1991 - Communicated by J. K. Vasil
Summary. Embryogenic ceil lines of Picea abies are categorized into three groups (polar, solar, and undeveloped) based on the organization of the somatic embryos within the tissue and the ability of the somatic embryos to proceed through a maturation process when treated with ABA. The polar and the solar types consist of somatic embryos with densely packed embryonic regions subtended by vacuolated suspensors. Both types of tissue regenerate mature somatic embryos when treated with ABA. Almost all mature somatic embryos develop further into shoots or plantlets. The undeveloped type consists of somatic embryos comprised of only a few loosely aggregated cells in their embryonic regions. Mature somatic embryos were not observed with this tissue type. Keywords: Embryogenic ceil lines - - Maturation - - Picea abies ~ Somatic embryos
Abbreviations: ABA:cis-tmns abscisic acid, Al:polar type, A2:solar type, B:undeveloped type, BA:benzyladenine, 2,4D:2,4 di-chlorophenoxyacetic acid, LP: von Amolds medium (1987).
Introduction Somatic embryogencsis can be achieved in various coniferous species. The appearance of embryogenic cultures seems to be similar in all conifers, i. e. they are white to translucent, mucilaginous and consist of many small, undeveloped somatic embryos. Initiation frequency of embryogenic cultures is relatively high for Picea abies, up to 100% from immature zygotic embryos (von Arnold and Hakman 1988), and about 40% from seedlings (Mo and von Arnold 1991), irrespective of the genotype used. However, the level of embryogenesis varies significantly among embryogenic cell lines derived from different genotypes (Becwar et al. 1987). Embryogenic cultures retain their embryogenic potential and their capacity to develop into plants for several years (Mo and von Arnold 1989). Somatic embryos of P. abies can be converted to phenotypicaily normal plants which overwinter and renew vegetative Offprint requests to: S. von Arnold
growth in a way similar to zygotic embryos (Becwar et al. 1989). Somatic embryos from several coniferous species can attain the germination stage (see for example Krogstrup 1990; Webster et al. 1990). However, the regeneration frequency is generally very low and unpredictable. This may be due to the fact that somatic embryos germinate precociously and do not adequately mature for subsequent development (Roberts et al. 1990 a). Conifer somatic embryos, like those of angiosperms, require ABA for maturation. Conifer zygotic embryos accumulate massive amounts of storage lipids during maturation (Konar 1958). Ultmslructural studies have shown that ABA stimulates somatic embryos of Picea glauca to accumulate storage material, especially lipids but also proteins and carbohydrates (Hakman and von Arnold 1988). These results are confirmed by quantitative and qualitative measurements of ABA-induced increases in storage lipids (Feirer et al. 1989) and proteins (Hakman et al. 1990; Roberts et al. 1990 b) in somatic embryos of P. abies. Although the general appearance of conifer embryogenic cultures are very similar to the unaided eye, significant cell line differences can be observed microscopically. This applies particularly to somatic embryo organization and development. Pronounced differences have also been reported among different cell lines of the same species (Laine and David 1990; Roberts et al. 1990 a). The aim of this study was to characterize embryogenic cell lines ofP. abies of varying genetic origin and to correlate their growth habit to the ability of the somatic embryos to proceed through a maturation process and to develop into
plants. Material and Methods Embryogenie cultures of Picea abies (L) Karst were established as previously described (yon Arnold 1987). The cultures were initiated in 1986 and 1988 from mature zygotic embryos collected from two different seed orchards in central Sweden. Briefly, the cultures were grown in 9-cm petri dishes containing half-strength LP medium containing 15 mM NH4NO3, 30 mM sucrose, 9 }aM 2,4-D, 4.4 ~tM BA and gelled with 0.4% Gelrite gellan
385 gum. The petri dishes were incubated in darkness at 20~ The cultures were transferred to fresh culture medium every month. Each cell line originated from one zygotic embryo. The following cell lines were used in this study: 86 (1, 2, 3, 19, 29, 36, 55, 58, 59, 66, 70, 71, 72), 88 (6, 12, 14, 21, 29, 30, 33, 36, 37, 46). The somatic embryos were stimulated to mature according to methods described previously (you Arnold 1987). Briefly, the embryogenie cultures were transferred to full-strength LP medium containing 90/aM sucrose and 15/aM ABA, gelled with 0.4% Gelrite gellan gum. When the somatic embryos had matured after 1 to 3 months on ABA containing medium, the cultures were transferred to full-strength LP medium containing 60 mM sucrose and gelled with 0.4% Gellite gellan gum. After one week in darkness the petri dishes were incubated in light at 116 Ixrnol m-Zs-1. After one week in light the embryos were isolated and cultured individually on medium with the same composition as used before. Cultures were examined microscopically both before and during the ABA-treatment.
Results Classification of embryogenic cell lines Based on the appearance of the somatic embryos and their ability to mature and develop into plants, the embryogenic cell lines used in this study were classified into three different groups (Table 1). These three tissue types were easily distinguished in the microscope (Fig. la, 2a, 3a). T a b l e 1. Classification of embryogenic ceil lines ofPicea abies.
tic embryos tend to be more polar, and when fast growing cell suspensions are established the somatic embryos develop the solar configuration (data not shown). Furthermore both (A1) and (A2) tissue types can give rise to planflets after ABA treatment (see below).
Response to ABA treatment The ability of the somatic embryos, from each of the three tissue types, to respond to ABA treatment is shown in Table 2. A positive ABA response was defined as changes in the tissues' growth habit. The following positive responses were observed: differentiation of mature somatic embryos (Fig. lb), precociously germinating somatic embryos (Fig. 2b), nodules, (Fig. 3b) and abnormal somatic embryos (Fig. 3c). Some cell lines showed negative ABA response, i. e. there was no visible change in tissue a ~ c e after ABA treatment. T a b l e 2. Response of eight embryogenic cell lines of Picea abies to ABA
treatment. The cultures were transferred to LP-medium containing 15 pM ABA, 3% sucrose and 0.4% gellan for 8 to 12 weeks. Average weight of each callus was 0.5 g. Numbers of mature somatic embryos, precocious somatic embryos, abnormal somatic embryos and nodules were estimated after 6, 8 and 12 weeks cultivation on ABA containing media. When a cell line had produced an average of I0 somarie embryos per callus the response was regarded as completed. Cell line
Tissue type
No of calli observed
Average no of differentiated structures per callus weeks 6 8 12
Group
Tissue type
Characteristics
A1
polar type
Somatic embryos that are polarized with distinct embryo-heads and well developed suspensors (Fig. la)
86:2
A1
90
5a
10a
A2
solar type
Somatic embryos with a radial symmetry (Fig. 2a)
86:19 86:55
A2 A1
8 18
lb 7a
lb 10a
9b
B
undeveloped type
Somatic embryos that are less developed (Fig. 3a). Embryogenic cell clusters are surrounded by vaeuolated cells. Frequently the embryogenic cells are also intermingled with clusters of vacuolated cells
86:59 86:66
A1 A1
30 47
2a 60a
2a
la
88:12 88:14
B B
24 16
0 0
10e 2c
lc
88:30
B
6
ld
5d
5d
Polar somatic embryos (A1) have a distinct embryo-head consisting of small closely packed cells and attached suspensor cells which are highly vacuolated (Fig la). Frequently, the suspensors from many embryos develop in bundles. The solar somatic embryos (A2) consist of the same ceil types as in the polar type, e.g. small closely packed cells and long vacuolates suspensor cells (Fig. 2a). However, the small, densely packed cells of the (A2) are completely surrounded by the elongated, vacuolated suspensor ceils. In the third type of tissue the undeveloped somatic embryos (B) are polarized, but the embryo-heads are comprised of only a few loosely aggregated clusters of ceils, which are not separated from the vacuolated suspensor cells (Fig. 3a). In addition to the embryos, the B-type contains long, highly vacuolated cells and rounded, vacuolated cells, both single and in clusters. These vacuolated cells frequently contain starch grains. Since cell line A1 can switch over to A2 and vice versa it appears that the polar type (A1) and the solar type (A2) belong to the same main group. We also observed that when the growth rate of a fast growing cell line slows down, the soma-
a = normal mature somatic embryos b = precociously germinating embryos c = nodules d = abnormal embryos
Normal mature somatic embryos (Fig. lb) were only obtained from cell lines classified as type A1. The optimal treatment time with ABA varied from 6 to 12 weeks. The best cell line (86:66) gave an average of 120 mature embryos per gram fresh weight. In contrast only a few mature somatic embryos were formed from the cell lines classified as type A2. These cell lines regenerated somatic embryos which germinated precociously (Fig. 2b), i.e. the embryos turned green much earlier than that was observed with the A1 tissue type. No mature somatic embryos were obtained from the cell lines classified as B. These cell lines either did not respond to ABA treatment or they differentiated nodules (Fig. 3b) or abnormal embryos. Upon prolonged culture on medium containing ABA the nodules either regenerated new callus, turned brown or developed roots. By sectioning it was found, that the abnormal embryos had several bud apices.
386
Figs. 1--3: Development of somatic embryos from different types of embryogenic tissues. Figures 1, 2 and 3 show the development of somatic embryos from tissue type A1, A2 and B respectively. a) Proliferating embryogenic tissue on medium containing 2,4-D and BA. Note that the somatic embryos in la are polarized, in 2a are solar shaped and in 3a are less differentiated. Scale bars = 0.1 ram. b) Embryogenic tissue cultured on medium containing ABA for 4, 6 and 12 weeks respectively in figs 1, 2 and 3. Note that mature somatic embryos are formed in lb; that the somatic embryo in 2b has started to develop cotyledons; that nodules are formed in 3b. Scale bars in lb and 2b = 1 mm, in 3b = 5 mm. c) Embryogenic tissue first treated with ABA and then cultured on medium lacking growth regulators for two weeks. Note that normal somatic embryos have developed in Fig. Ic; that the embryo in 2c retains a suspensor, that the somatic embryo in 3c has an abnormal number of cotyledons. Scale bars in Ic and 3c = 5 ram, in 2c = 1 mm. d) Isolated somatic embryos cultured on medium lacking growth regulators. (ld) Normal mature somatic embryo after one week. (3d) Abnormal embryo after three weeks. Scale bars = 0.5 ram.
The experiments were repeated twice with the same and new cell lines. In all cases the three types of embryogenic tissues (A1, A2 and B) responded to ABA in a manner typical for each tissue type (Table 3). Table 3. The ability of different cell lines to differentiate mature somatic embryos. The calli (as described in Table 2) were cultured on ABA containing medium for 2 to 3 months. Tissue type
A1 A2 B
Total number of cell lines 6 6 11
Total number of calli 192 30 90
Maturation on ABA + + -
Plant development Mature somatic embryos derived from type A tissue developed further when transferred to medium lacking ABA (Figs. lc and d). Almost all of the embryos developed into shoots or plantlets (Table 4). However, some variation in the developmental pattern could be observed between embryos from different cell lines. The majority of the cell lines with type A tissue gave rise to plantlets with a single tap root, which usually was covered with root hairs. Plantlets from some cell lines also formed a large number of secondary roots. Shoots from some cell lines developed roots only after prolonged culture in vitro. In general, shoots either with or without roots, had a tendency to form a resting bud. However, in some cell lines the epicotyl continued to develop without a rest period.
387 Table 4. Plant regeneration from mature somatic embryos, cultured on hormone-free LP medium containing 2% sucrose and solidified with 0.4% gellan in light for two weeks. Ceil line 86:2 86:55 86:59 86:66
No. of mature somatic embryos 357 286 24 441
Shoots only (fro) 89 95 92 8
Rooted shoots (%) 11 5 8 92
Normal plants were never observed with the type B tissue. The abnormal embryos developed several bud-meristems which produced a cluster of shoots (Figs. 3c and 3d). The nodules, irrespective of whether they were within the associated tissue or isolated, either degenerated or developed into roots. Discussion This study has shown that embryogenic tissues ofPicea abies can be categorised into three groups. These groups are based on the following criteria: (1) the presence of somatic embryos and (2) the manner in which the somatic embryos proceed through a maturation process and subsequently develop into plants. Since each cell line represents one genotype it is reasonable to assume that the regeneration ability, under the same culture conditions, varies among different genotypes. Similar genotypic differences have been reported for several angiosperm species (Tomes and Smith 1985; Hedges et al. 1986). Both the polar and solar tissue types have several characteristics in common. Their immature somatic embryos consist of a densely packed embryonic region subtended by vacuolated suspensor cells. Furthermore, these somatic embryos can be stimulated to go through a maturation process when treated with ABA. We have ample evidence to conclude that the differences between the solar and polar tissue types are mainly due to the growth rate of the cell line. The rationale for separating the types into two groups are (1) anatomical differences observed microscopically and (2) differences in the maturation process of their somatic embryos. In interior spruce, precocious germination of somatic embryos occurred at 10---20 ~tM ABA (Roberts et al. 1990 a). In our study, the tendency to precocious germination was only observed in cell lines with A2 tissue. It is reasonable to assume that the fast growing cell lines with solar type embryos would require a higher ABA concentration for complete inhibition of precocious germination.
The undeveloped tissue type consists of less organized somatic embryos. It is characterised by cells in the embryo head that are loosely aggregated and frequently intermingled with vacuolated cells. These somatic embryos cannot proceed through a normal maturation process. An embryogenic cell line of Pinus caribaea consisting of poorly organized somatic embryos was also unable to regenerate plants (Laine and David 1990). Several experiments (not shown) have been performed attempting to stimulate undeveloped embryogenic tissue type of P. abies to differentiate into mature somatic embryos. Different solvents for ABA (Dunstan et al. 1990), increased ABA concentration and prolonged ABA treatment time, were not sufficient to induce maturation. The reason why undeveloped tissue does not regenerate mature somatic embryos is unknown. One hypothesis is that the somatic embryos are blocked in their development and have not reached the stage where they can respond to ABA and mature.
Acknowledgements.This study was supported by the National Board of Forestry, The Scandinavian Contact Agency for Agricultural Research and The Troedsson Fund.
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