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Plantlet Regeneration through Somatic Embryogenesis in Picea abies (Norway Spruce)
Plantlet Regeneration through Somatic Embryogenesis in Picea abies (Norway Spruce) INGER HAKMAN and SARA VON ARNOLD Institute of Physiological Botany, University of Uppsala, Box 540, 5-75121 Uppsala, Sweden Received April 17, 1985 . Accepted July 4, 1985
Summary Embryogenic callus was produced from immature zygotic embryos of Picea abies cultured on a defined medium supplemented with 2,4-dichlorophenoxyacetic acid (10 - 5 M) and a cytokinin (10- 6 -10- 5 M). Subsequently numerous somatic embryos developed from the callus. Upon subculture the somatic embryos could be stimulated to develop further into plantlets with cotyledons, a hypocotyl and a root. Under suitable conditions 24% of the calli produced plantlets, and up to 25 plantlets were formed in a single callus, in addition to numerous somatic embryos of smaller sizes. Plantlet formation was followed both in living and sectioned materials and showed close similarity to zygotic embryogeny.
Key words: Gymnosperm - Norway spruce - Picea abies - Plantlet regeneration - Somatic embryogenesis - Tissue culture.
Introduction Tissue culture may play an important role in bringing forest yields toward their theoretical maximum (Farnum et ai., 1983). Therefore, much effort has been made during the last decade to apply these culture techniques to conifers in the same way as has been possible for horticultural and agricultural plants. Several promising results have been obtained, for example, it is now possible to multiply juvenile plant material of several coniferous species via adventitious buds in vitro (see e.g. David, 1982). However, the long term goal of forest tissue culture is to induce single cells or cell aggregates to form somatic embryos (Farnum et al., 1983). Somatic embryogenesis in non-conifer gymnosperm tissue culture was reported nearly two decades ago by Norstog and Rhamstine (1967). Cultured immature embryos of lamia integrifolia gave rise to calli in which small somatic embryos were observed. Induction of somatic embryogenesis in suspension cultures derived from cotyledonary calli of Pseudotsuga menziesii has been claimed by u.S. Patent 4217730 (1980). Furthermore, somatic embryogenesis in suspension cultures from P. menziesii Correspondence address: Dr. Inger Hakman, Institute of Physiological Botany, University of Uppsala, Box 540, 5-75121 Uppsala, Sweden Abbreviations: ABA, abscisic acid; BA, 6-benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid; GA, gibberellin; IAA, indole-3-acetic acid; 2iP, (isopentenyl)adenine; KIN, kinetin; NAA, I-naphthaleneacetic acid; ZEA, zeatin.
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and Picea glauca was reported by Durzan (1982), but this author has so far only demonstrated partially formed somatic embryos. It has previously been shown that immature embryos are suitable sources for initiation of embryogenic calli in angiosperms (Vasil, 1983). This seems also to be the case for gymnosperms since the best somatic embryogenesis results have so far been obtained from calli initiated from immature embryos of Z. integrifolia (Norstog and Rhamstine, 1967) and Picea abies (Hakman et al., 1985). As plantlets were formed from the somatic embryos of P. abies only sporadically (Hakman et al., 1985), culture conditions required for embryo initiation and their further development have now been investigated more carefully. Furthermore, the development of regenerated plantlets has been followed by light microscopy to complement the earlier study. In addition, comparison has been made between the development of zygotic and somatic embryos.
Materials and Methods Plant material Seed cones of Picea abies (L.) Karst. were collected from different parts of Sweden during the maturation period of the embryos Guly and August). The cones were collected from trees either in natural stands or in seed orchards and thereafter were stored in paper bags at 4 °C for a few days up to a couple of months. The plant material was prepared for culture as described earlier (Hakman et aI., 1985). Culture procedure If not stated otherwise the basal medium was that described by von Arnold and Eriksson in 1982 (LP), which is a modified Murashige and Skoog's (1962) medium. Other media tested were based on White's solution (medium 56) and modified Murashige-Skoog medium (medium 59) as described by Norstog and Rhamstine (1967). For callus initiation the media were supplemented with 2,4-D (10 - 5 M) together with various concentrations (10- 6 , 5x 10- 6 , lO- S M) of a cytokinin (BA, 2iP, KIN, ZEA). In a series of experiments different auxins alone (2,4-D, IAA, NAA) of various concentrations (10- 6,5 x 10- 6 , 10 - 5, 5 x 10 - 5, 10 - 4 M) were also tested. In addition, growth regulators originally included in media 56 (5x 10- 7 M 2,4-D and 2.5 x 10- 6 M KIN) and 59 (5x 10- 6 M 2,4-D and 5x 10- 6 M KIN) (Norstog and Rhamstine, 1967) were used. For further development of somatic embryos the calli were transferred after two months either to basal medium or to media supplemented with various concentrations and combinations of growth regulators. The substances tested were BA, 2iP, KIN, ZEA (5 x 10- 6 M); ABA (10- 7 , 5 x 10- 7, 1O- 6 M); IAA (10- 7, 5 x 10- 7, 10- 6 , 5 x 10- 6 M); GA3 and G~/7 (0.05, 0.5, 5.0, 50 mg ,1- 1). The effect of activated charcoal (1 % w/v) was also tested. At least 30 calli were used in each of the differentiation experiments. All media, except those containing GA, were solidified with Gelrite gellan gum (Kelco, Merck) at a concentration of 0.3 % (w/v). The media were autoclaved for 15 min at 121°C before being poured into 50 mm petri dishes. When the GA's were tested these were filter sterilized and added to cooled sterile medium gelled with agarose (Sigma, 0.3 % w/v). Culture conditions The cultures were incubated in the dark at 25°C for the first two months. Thereafter the cultures were placed in a growth chamber with a 16 h per day photoperiod. Irradiance at the level
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of the cultures was 10-40,£ m- 2 s- 1, given by warm white fluorescent tubes, Osram L 36W/ 30. Subculture was carried out monthly to fresh medium. Microscopy
The plant material was processed for microscopy as described previously (Hakman et al., 1985).
Results and Discussion Plant material
The ability to produce callus varied among plant material collected from different localities (Tab. 1). The developmental stages of the seeds were not identical, and the quality of the plant material varied considerably. Therefore, the reason for the varying ability to produce callus cannot easily be given; the physiological condition of the source material seems the most plausible. Table 1: Callus induction on immature embryos of Picea abies collected from different localities and grown for 2 months at 25°C in the dark on various media supplemented with 10 - 5 M 2,4-D and 5x 1O- 6 M BA. Number of explants in brackets. Locality
% of explants forming callus on:
LP medium
59 medium
56 medium
1 2 3 3a )
34 (56) 23 (52) 63 (239) 95 (339)
21 (57) 30 (60)
9 (35) 13 (61)
42 (149)
a) embryos from cold stored cones.
The frequency of callus formation increased when the cones had been stored in the cold for about two months (Tab. 1). Furthermore, the calli produced from these cold stored embryos showed a more uniform growth and a larger number of embryogenic calli. The optimal time for cold treatment has not yet been evaluated. Only when seeds have been collected from the same locality and pretreated in the same way is it possible to compare different experiments; otherwise the results should only be compared within each individual experiment. Callus induction
Explants started to form callus within 1 month of culture. A detailed description of initiation and morphology of embryogenic callus has been made previously (Hakman et al., 1985). The efficiency of callus induction varied among the different media tested (Tab. 1). Best results were obtained with LP medium and medium 59. Although only 42 % of embryos from cold stored cones gave rise to callus on medium 59 compared to 95 % on LP medium, those induced on medium 59 grew very well. ]. PlantPhysiol. Vol. 121.pp.149-158{1985}
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Table2: Effect of different auxins on percentage of immature embryos of Picea abies forming embryogenic-like callus. The calli were cultured for about 2 months at 25°C in the dark on LP medium containing the tested auxin. Number of explants in brackets. Cone. (M)
Auxins
10- 6 5x 10- 6 10- 5 5x 10- 5 10- 4
2,4-D
IAA
NAA
3 (80) 0(80) 3 (80) 1 (80) 3 (80)
0(70) 0(80) 0(80) 0(80) 6 (80)
7 (90) 13 (80) 4 (80) 0(80) 1 (80)
Table 3: Effect of different cytokinins on percentage of immature embryos of Picea abies forming embryogenic-like callus. The calli were cultured for about 2 months at 25°C in the dark on LP medium containing 10 - 5 M 2,4-D and the tested cytokinin. Number of explants in brackets. Cone. (M) 10- 6 5x 10- 6 10- 5
Cytokinins BA
2iP
KIN
ZEA
48 (58) 55 (64) 66 (65)
2 (41) 1 (54) 27 (55)
28 (69) 53 (59) 59 (56)
25 (52) 41 (54) 57 (46)
Embryos explanted on medium 59 sometimes started to develop shoots and roots before they became necrotic and died. On medium 56 many of the immature embryos turned yellow without further growth. Different auxins (2,4-D, IAA, NAA) of various concentrations (10- 6 -10- 4 M) were tested alone for their ability to induce callus growth. None of them was as effective as when combined with a cytokinin, and embryogenic callus was only sporadically produced (Tab. 2). The kind of cytokinin added to the medium had no pronounced effect on callus induction in the range of 1O-6M to 10- 5 M. However, fewer embryogenic calli were found on media with the lower concentrations of added cytokinins and on media containing 2iP (Tab. 3).
Plantlet formation The embryogenic calli contained numerous small somatic embryos that developed into plantlets (Fig. 1), and under suitable conditions up to 25 plantlets could be derived from one single callus, which also contained several somatic embryos of smaller sizes (Fig.2). The developmental sequence of somatic embryos isolated from callus cultures is shown in Figure 3. Initially the somatic embryos terminated with long suspensor-like cells. Subsequently they became more seedling-like with cotyledons, a hypocotyl and a root. However, shoots or roots alone developed concomitantly with plantlets. When these were isolated it was observed that either the shoot or the root had failed to develop properly (Fig. 4). We have often observed the same growth dis-
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Figs. 1-4: Plantlet development through somatic embryogenesis in callus cultures initiated from immature zygotic embryos of Picea abies. Figs. 1 a-c. Somatic embryos (1 a and 1 b) and a plantlet (1 c) derived from callus after two to three months in culture. Fig. 2. Plantlets (arrows) developing from somatic embryos in a callus after approximately three months. Fig. 3. Sequence showing plantlet development via somatic embryogenesis. Figs. 4 a- b. «Plantlets» isolated from callus cultures, illustrating inhibition of root (4 a) or shoot (4 b) development. The horizontal bars represent 1 mm.
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turbances in mature zygotic embryos of P. abies cultured in vitro. Similar abnormalities have also been reported for cultured zygotic embryos of P. strobus (Minocha, 1978) and are also frequently seen among somatic embryos of several angiosperm species (Vasil and Vasil, 1981; Ho and Vasil, 1983). Hereafter, the phrase «development of somatic embryos» includes both normal development into plantlets and partly supressed development, while the phrase «plantlet development» stresses a normal development. Calli initiated on LP medium containing 10- 5 M 2,4-D and a cytokinin (BA, 2iP, KIN, ZEA) of various concentrations (10- 6 , 5 x 10- 6 , 10- 5 M) were after 2 months transferred either to basal medium, to cytokinin containing media (5 x 10 - 6 M) or to media of the same composition as they already were growing on. Within 3 months somatic embryos had sporadically developed further in calli cultured on LP media supplemented with 2,4-D and a cytokinin. In cultures that had been subcultured to LP medium lacking growth regulators about 10 % of the calli had developing somatic embryos, while if transferred to cytokinin containing media the frequency was nearly doubled. The most efficient treatment for plantlet development was initiation on medium containing either BA, KIN or ZEA as cytokinin (10- 6 M and 5 x 1O- 6 M), and subculture to medium containing only the cytokinin. Various substances (ABA, IAA, GA's and activated charcoal) were tested for their ability to stimulate a normal development of somatic embryos in P. abies. In these experiments the calli were initiated and maintained on LP medium containing 1O- 5 M 2,4-D and 5 x 10 - 6 M BA for about 2 months before being subcultured to media with the above mentioned additions (for concentrations see Materials and Methods). None of the substances proved to be effective in promoting normal development of the somatic embryos under the culture conditions tested. Plantlets were found to develop sooner (within 2 months) and at a higher frequency if calli produced from cold stored embryos were grown on medium 59 containing 5 x 10- 6 M 2,4-D and 5 x 10- 6 M KIN. In experiments based on 150 immature embryos 24 % of the established calli (62) had produced well developed plantlets after 3 months and another 7 % of the calli had produced either shoots or roots. These figures could be compared to those for calli initiated and grown on LP medium containing 1O- 5 M 2,4-D and 5x 1O- 6 M BA for same time. In that case less than 1 % (2) of Table4: Effect of different media on plantlet formation in callus of Picea abies cultured for 2 months at 25°C in the dark. The calli were initiated on immature embryos, isolated from cold stored cones, and grown on LP medium containing 1O- 5 M 2,4-D and 5x10- 6 M BA for 3 months before being transferred to the tested media. Medium (10- 5 M
No. of calli inoculated 2,4-D+5x10- 6 M
LP BA) 59 (5x10- 6 M 2,4-D+5x lO-6M KIN)
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78 41
% explants showing:
plantlets
o
12
shoots or roots 3
10
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Figs. 5 - 9: Microscopic examinations of somatic embryos of Picea abies. Fig. 5. Somatic embryo at an early stage of development. Figs. 6-8. Sections of developing somatic embryos embedded in plastic and stained with toluidine blue. Figs. 9a-b. Sections of a plantlet embedded in paraffin and stained with safranin-fast green showing the shoot surrounded by cotyledons (9 a) and the root (9 b). The horizontal bars represent 0.1 mm.
the calli (321) had produced well developed plantlets and 5 % of the calli either shoots or roots. However, if these calli were subcultured to medium 59, plantlets developed from 12 % of the calli within 2 months after the transfer, while calli given a continued growth on LP medium did not produce any more plantlets (Tab.4).
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Figs. 10-11: Zygotic embryos of Picea abies. Fig. 10. A very young embryo isolated shortly after fertilization. Fig. 11: Sectioned zygotic embryo embedded in plastic and stained with toluidine blue. The horizontal bars represent 0.1 mm.
Callus growth was more uniform in cultures obtained from cold treated material compared to unstored material and plantlets also started to develop much faster. In addition, in these experiments plantlets were formed in calli incubated in the dark. This reflects a difference in plant regeneration through somatic embryogenesis and via adventitious bud development in P. abies, since light is a prerequisite in the latter case (von Arnold and Eriksson, 1979). However, light regimes for optimal development of plantlets through somatic embryogenesis still has to be investigated. Plantlets regenerated from somatic embryos were isolated and cultured individually on different media. One problem with these plantlets was that they often produced a dormant bud and stopped growing. Therefore, we are now trying to improve the culture conditions in order to keep the plantlets in a continuous state of growth.
Microscopy In our previous report we described the initiation of somatic embryos and their further development into green nodules, from which plantlets sporadically were pro-
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duced (Hakman et al., 1985). In the present study, plantlets have been obtained at a much higher frequency and this has enabled us to more precisely follow their development both in living and sectioned materials. During their early stages, the somatic embryos consisted of long vacuolated cells at one end (suspensor) and a small group of meristematic cells at the other (embryonal end) (Figs. 5 and 6). Subsequently, when they grew further, they became more organized (Fig. 7). Later, cotyledons were formed (Figs. 8 and 9 a) and a root developed (Fig. 9 b). The internal anatomy of immature somatic embryos was very similar to immature zygotic embryos (d. Fig. 5 with Fig. 10 and Figs. 7 and 8 with Fig. 11).
Conclusion This study has shown that embryogenic callus can be induced on immature embryos of Picea abies cultured on LP medium supplemented with 2,4-D and cytokinin. For regeneration of plantlets, transfer of the embryogenic callus to medium 59 was favourable. Both callus initiation and plantlet regeneration was most frequent in cultures derived from immature embryos isolated from female cones that first had been stored in cold for about 2 months. Under suitable conditions 24 % of the established calli produced plantlets and in some instances up to 25 plantlets were formed in a single callus. Acknowledgements We thank Prof. Tage Eriksson for his support in this research, Ms. Ann-Charlott Johansson and Ms. Agneta Ottosson for technical assistance and Ms. Anette Axen for preparing the sections. Research was supported by a grant from Commission of the European Communities, contract BOS.140.S(H).
References DAVID, A.: In vitro propagation of gymnosperms. In: J. M. BONGA and D. J. DURZAN (eds.). Tissue Culture in Forestry, p. 72-108. Martinus Nijhoff, Dr. W. Junk Publishers, The Hague, Boston, London. ISBN 90-247-2660-3 (1982). DURZAN, D. J.: Somatic embryogenesis and sphaeroblasts in conifer suspensions. In: A. FUJIWARA (ed.). Plant tissue culture, p. 113-114. Tokyo: Jap. Assoc. for Plant Tissue Culture (1982). FARNUM, P., R. TIMMIS, and J. L. KuLP: Biotechnology of forest yield. Science 219, 694-702 (1983). lliKMAN, I., L. C. FOWKE, S. VON ARNOLD, and T. ERIKSSON: The development of somatic embryos in tissue cultures initiated from immature embryos of Picea abies (Norway spruce). Plant Science 38,53-59 (1985). Ho, W. J. and I. K. VASIL: Somatic embryogenesis in sugarcane (Saccharum officinarum L.) I. The morphology and physiology of callus formation and the ontogeny of somatic embryos. Protoplasma 118, 169-180 (1983). MINOCHA, S. c.: Callus and adventitious shoot formation in excised embryos of white pine (Pinus strobus). Can. J. Bot. 58, 366-370 (1978). MURASHIGE, T. and F. SKOOG: A revised medium for the rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15, 473-497 (1962).
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NORSTOG, K. and E. RHAMSTINE: Isolation and culture of haploid and diploid cycad tissues. Phytomorphology 17, 374-381 (1967). United States Patent Abo EI-Nil No. 4217730, 1980. Embryogenesis of gymnosperm forest trees. VASIL, 1. K.: Regeneration of plants from single cells in cereals and grasses. In: P. F. LURQuIN and A. KLEmHOFS (eds.). Genetic Engineering in Eukaryotes, p. 233-252. Plenum Publishing Corporation (1983). VASIL, V. and I. K. VASIL: Somatic embryogenesis and plant regeneration from suspension cultures of Pearl millet (Pennisetum americanum). Ann. Bot. 47, 669-678 (1981). VON ARNOLD, S. and T. ERIKSSON: Bud induction on isolated needles of Norway spruce (Picea abies [L.] Karst.) grown in vitro. Plant Sci. Lett. 15, 363-372 (1979). - - In vitro studies of adventitious shoot formation in Pinus contorta. Can. J. Bot. 59, 870874 (1982).
J Plant Physiol.
Vol. 121. pp. 149-158 (1985)
Summary Embryogenic callus was produced from immature zygotic embryos of Picea abies cultured on a defined medium supplemented with 2,4-dichlorophenoxyacetic acid (10 - 5 M) and a cytokinin (10- 6 -10- 5 M). Subsequently numerous somatic embryos developed from the callus. Upon subculture the somatic embryos could be stimulated to develop further into plantlets with cotyledons, a hypocotyl and a root. Under suitable conditions 24% of the calli produced plantlets, and up to 25 plantlets were formed in a single callus, in addition to numerous somatic embryos of smaller sizes. Plantlet formation was followed both in living and sectioned materials and showed close similarity to zygotic embryogeny.
Key words: Gymnosperm - Norway spruce - Picea abies - Plantlet regeneration - Somatic embryogenesis - Tissue culture.
Introduction Tissue culture may play an important role in bringing forest yields toward their theoretical maximum (Farnum et ai., 1983). Therefore, much effort has been made during the last decade to apply these culture techniques to conifers in the same way as has been possible for horticultural and agricultural plants. Several promising results have been obtained, for example, it is now possible to multiply juvenile plant material of several coniferous species via adventitious buds in vitro (see e.g. David, 1982). However, the long term goal of forest tissue culture is to induce single cells or cell aggregates to form somatic embryos (Farnum et al., 1983). Somatic embryogenesis in non-conifer gymnosperm tissue culture was reported nearly two decades ago by Norstog and Rhamstine (1967). Cultured immature embryos of lamia integrifolia gave rise to calli in which small somatic embryos were observed. Induction of somatic embryogenesis in suspension cultures derived from cotyledonary calli of Pseudotsuga menziesii has been claimed by u.S. Patent 4217730 (1980). Furthermore, somatic embryogenesis in suspension cultures from P. menziesii Correspondence address: Dr. Inger Hakman, Institute of Physiological Botany, University of Uppsala, Box 540, 5-75121 Uppsala, Sweden Abbreviations: ABA, abscisic acid; BA, 6-benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid; GA, gibberellin; IAA, indole-3-acetic acid; 2iP, (isopentenyl)adenine; KIN, kinetin; NAA, I-naphthaleneacetic acid; ZEA, zeatin.
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and Picea glauca was reported by Durzan (1982), but this author has so far only demonstrated partially formed somatic embryos. It has previously been shown that immature embryos are suitable sources for initiation of embryogenic calli in angiosperms (Vasil, 1983). This seems also to be the case for gymnosperms since the best somatic embryogenesis results have so far been obtained from calli initiated from immature embryos of Z. integrifolia (Norstog and Rhamstine, 1967) and Picea abies (Hakman et al., 1985). As plantlets were formed from the somatic embryos of P. abies only sporadically (Hakman et al., 1985), culture conditions required for embryo initiation and their further development have now been investigated more carefully. Furthermore, the development of regenerated plantlets has been followed by light microscopy to complement the earlier study. In addition, comparison has been made between the development of zygotic and somatic embryos.
Materials and Methods Plant material Seed cones of Picea abies (L.) Karst. were collected from different parts of Sweden during the maturation period of the embryos Guly and August). The cones were collected from trees either in natural stands or in seed orchards and thereafter were stored in paper bags at 4 °C for a few days up to a couple of months. The plant material was prepared for culture as described earlier (Hakman et aI., 1985). Culture procedure If not stated otherwise the basal medium was that described by von Arnold and Eriksson in 1982 (LP), which is a modified Murashige and Skoog's (1962) medium. Other media tested were based on White's solution (medium 56) and modified Murashige-Skoog medium (medium 59) as described by Norstog and Rhamstine (1967). For callus initiation the media were supplemented with 2,4-D (10 - 5 M) together with various concentrations (10- 6 , 5x 10- 6 , lO- S M) of a cytokinin (BA, 2iP, KIN, ZEA). In a series of experiments different auxins alone (2,4-D, IAA, NAA) of various concentrations (10- 6,5 x 10- 6 , 10 - 5, 5 x 10 - 5, 10 - 4 M) were also tested. In addition, growth regulators originally included in media 56 (5x 10- 7 M 2,4-D and 2.5 x 10- 6 M KIN) and 59 (5x 10- 6 M 2,4-D and 5x 10- 6 M KIN) (Norstog and Rhamstine, 1967) were used. For further development of somatic embryos the calli were transferred after two months either to basal medium or to media supplemented with various concentrations and combinations of growth regulators. The substances tested were BA, 2iP, KIN, ZEA (5 x 10- 6 M); ABA (10- 7 , 5 x 10- 7, 1O- 6 M); IAA (10- 7, 5 x 10- 7, 10- 6 , 5 x 10- 6 M); GA3 and G~/7 (0.05, 0.5, 5.0, 50 mg ,1- 1). The effect of activated charcoal (1 % w/v) was also tested. At least 30 calli were used in each of the differentiation experiments. All media, except those containing GA, were solidified with Gelrite gellan gum (Kelco, Merck) at a concentration of 0.3 % (w/v). The media were autoclaved for 15 min at 121°C before being poured into 50 mm petri dishes. When the GA's were tested these were filter sterilized and added to cooled sterile medium gelled with agarose (Sigma, 0.3 % w/v). Culture conditions The cultures were incubated in the dark at 25°C for the first two months. Thereafter the cultures were placed in a growth chamber with a 16 h per day photoperiod. Irradiance at the level
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of the cultures was 10-40,£ m- 2 s- 1, given by warm white fluorescent tubes, Osram L 36W/ 30. Subculture was carried out monthly to fresh medium. Microscopy
The plant material was processed for microscopy as described previously (Hakman et al., 1985).
Results and Discussion Plant material
The ability to produce callus varied among plant material collected from different localities (Tab. 1). The developmental stages of the seeds were not identical, and the quality of the plant material varied considerably. Therefore, the reason for the varying ability to produce callus cannot easily be given; the physiological condition of the source material seems the most plausible. Table 1: Callus induction on immature embryos of Picea abies collected from different localities and grown for 2 months at 25°C in the dark on various media supplemented with 10 - 5 M 2,4-D and 5x 1O- 6 M BA. Number of explants in brackets. Locality
% of explants forming callus on:
LP medium
59 medium
56 medium
1 2 3 3a )
34 (56) 23 (52) 63 (239) 95 (339)
21 (57) 30 (60)
9 (35) 13 (61)
42 (149)
a) embryos from cold stored cones.
The frequency of callus formation increased when the cones had been stored in the cold for about two months (Tab. 1). Furthermore, the calli produced from these cold stored embryos showed a more uniform growth and a larger number of embryogenic calli. The optimal time for cold treatment has not yet been evaluated. Only when seeds have been collected from the same locality and pretreated in the same way is it possible to compare different experiments; otherwise the results should only be compared within each individual experiment. Callus induction
Explants started to form callus within 1 month of culture. A detailed description of initiation and morphology of embryogenic callus has been made previously (Hakman et al., 1985). The efficiency of callus induction varied among the different media tested (Tab. 1). Best results were obtained with LP medium and medium 59. Although only 42 % of embryos from cold stored cones gave rise to callus on medium 59 compared to 95 % on LP medium, those induced on medium 59 grew very well. ]. PlantPhysiol. Vol. 121.pp.149-158{1985}
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Table2: Effect of different auxins on percentage of immature embryos of Picea abies forming embryogenic-like callus. The calli were cultured for about 2 months at 25°C in the dark on LP medium containing the tested auxin. Number of explants in brackets. Cone. (M)
Auxins
10- 6 5x 10- 6 10- 5 5x 10- 5 10- 4
2,4-D
IAA
NAA
3 (80) 0(80) 3 (80) 1 (80) 3 (80)
0(70) 0(80) 0(80) 0(80) 6 (80)
7 (90) 13 (80) 4 (80) 0(80) 1 (80)
Table 3: Effect of different cytokinins on percentage of immature embryos of Picea abies forming embryogenic-like callus. The calli were cultured for about 2 months at 25°C in the dark on LP medium containing 10 - 5 M 2,4-D and the tested cytokinin. Number of explants in brackets. Cone. (M) 10- 6 5x 10- 6 10- 5
Cytokinins BA
2iP
KIN
ZEA
48 (58) 55 (64) 66 (65)
2 (41) 1 (54) 27 (55)
28 (69) 53 (59) 59 (56)
25 (52) 41 (54) 57 (46)
Embryos explanted on medium 59 sometimes started to develop shoots and roots before they became necrotic and died. On medium 56 many of the immature embryos turned yellow without further growth. Different auxins (2,4-D, IAA, NAA) of various concentrations (10- 6 -10- 4 M) were tested alone for their ability to induce callus growth. None of them was as effective as when combined with a cytokinin, and embryogenic callus was only sporadically produced (Tab. 2). The kind of cytokinin added to the medium had no pronounced effect on callus induction in the range of 1O-6M to 10- 5 M. However, fewer embryogenic calli were found on media with the lower concentrations of added cytokinins and on media containing 2iP (Tab. 3).
Plantlet formation The embryogenic calli contained numerous small somatic embryos that developed into plantlets (Fig. 1), and under suitable conditions up to 25 plantlets could be derived from one single callus, which also contained several somatic embryos of smaller sizes (Fig.2). The developmental sequence of somatic embryos isolated from callus cultures is shown in Figure 3. Initially the somatic embryos terminated with long suspensor-like cells. Subsequently they became more seedling-like with cotyledons, a hypocotyl and a root. However, shoots or roots alone developed concomitantly with plantlets. When these were isolated it was observed that either the shoot or the root had failed to develop properly (Fig. 4). We have often observed the same growth dis-
J Plant Physiol. Vol.
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Figs. 1-4: Plantlet development through somatic embryogenesis in callus cultures initiated from immature zygotic embryos of Picea abies. Figs. 1 a-c. Somatic embryos (1 a and 1 b) and a plantlet (1 c) derived from callus after two to three months in culture. Fig. 2. Plantlets (arrows) developing from somatic embryos in a callus after approximately three months. Fig. 3. Sequence showing plantlet development via somatic embryogenesis. Figs. 4 a- b. «Plantlets» isolated from callus cultures, illustrating inhibition of root (4 a) or shoot (4 b) development. The horizontal bars represent 1 mm.
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turbances in mature zygotic embryos of P. abies cultured in vitro. Similar abnormalities have also been reported for cultured zygotic embryos of P. strobus (Minocha, 1978) and are also frequently seen among somatic embryos of several angiosperm species (Vasil and Vasil, 1981; Ho and Vasil, 1983). Hereafter, the phrase «development of somatic embryos» includes both normal development into plantlets and partly supressed development, while the phrase «plantlet development» stresses a normal development. Calli initiated on LP medium containing 10- 5 M 2,4-D and a cytokinin (BA, 2iP, KIN, ZEA) of various concentrations (10- 6 , 5 x 10- 6 , 10- 5 M) were after 2 months transferred either to basal medium, to cytokinin containing media (5 x 10 - 6 M) or to media of the same composition as they already were growing on. Within 3 months somatic embryos had sporadically developed further in calli cultured on LP media supplemented with 2,4-D and a cytokinin. In cultures that had been subcultured to LP medium lacking growth regulators about 10 % of the calli had developing somatic embryos, while if transferred to cytokinin containing media the frequency was nearly doubled. The most efficient treatment for plantlet development was initiation on medium containing either BA, KIN or ZEA as cytokinin (10- 6 M and 5 x 1O- 6 M), and subculture to medium containing only the cytokinin. Various substances (ABA, IAA, GA's and activated charcoal) were tested for their ability to stimulate a normal development of somatic embryos in P. abies. In these experiments the calli were initiated and maintained on LP medium containing 1O- 5 M 2,4-D and 5 x 10 - 6 M BA for about 2 months before being subcultured to media with the above mentioned additions (for concentrations see Materials and Methods). None of the substances proved to be effective in promoting normal development of the somatic embryos under the culture conditions tested. Plantlets were found to develop sooner (within 2 months) and at a higher frequency if calli produced from cold stored embryos were grown on medium 59 containing 5 x 10- 6 M 2,4-D and 5 x 10- 6 M KIN. In experiments based on 150 immature embryos 24 % of the established calli (62) had produced well developed plantlets after 3 months and another 7 % of the calli had produced either shoots or roots. These figures could be compared to those for calli initiated and grown on LP medium containing 1O- 5 M 2,4-D and 5x 1O- 6 M BA for same time. In that case less than 1 % (2) of Table4: Effect of different media on plantlet formation in callus of Picea abies cultured for 2 months at 25°C in the dark. The calli were initiated on immature embryos, isolated from cold stored cones, and grown on LP medium containing 1O- 5 M 2,4-D and 5x10- 6 M BA for 3 months before being transferred to the tested media. Medium (10- 5 M
No. of calli inoculated 2,4-D+5x10- 6 M
LP BA) 59 (5x10- 6 M 2,4-D+5x lO-6M KIN)
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78 41
% explants showing:
plantlets
o
12
shoots or roots 3
10
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Figs. 5 - 9: Microscopic examinations of somatic embryos of Picea abies. Fig. 5. Somatic embryo at an early stage of development. Figs. 6-8. Sections of developing somatic embryos embedded in plastic and stained with toluidine blue. Figs. 9a-b. Sections of a plantlet embedded in paraffin and stained with safranin-fast green showing the shoot surrounded by cotyledons (9 a) and the root (9 b). The horizontal bars represent 0.1 mm.
the calli (321) had produced well developed plantlets and 5 % of the calli either shoots or roots. However, if these calli were subcultured to medium 59, plantlets developed from 12 % of the calli within 2 months after the transfer, while calli given a continued growth on LP medium did not produce any more plantlets (Tab.4).
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Figs. 10-11: Zygotic embryos of Picea abies. Fig. 10. A very young embryo isolated shortly after fertilization. Fig. 11: Sectioned zygotic embryo embedded in plastic and stained with toluidine blue. The horizontal bars represent 0.1 mm.
Callus growth was more uniform in cultures obtained from cold treated material compared to unstored material and plantlets also started to develop much faster. In addition, in these experiments plantlets were formed in calli incubated in the dark. This reflects a difference in plant regeneration through somatic embryogenesis and via adventitious bud development in P. abies, since light is a prerequisite in the latter case (von Arnold and Eriksson, 1979). However, light regimes for optimal development of plantlets through somatic embryogenesis still has to be investigated. Plantlets regenerated from somatic embryos were isolated and cultured individually on different media. One problem with these plantlets was that they often produced a dormant bud and stopped growing. Therefore, we are now trying to improve the culture conditions in order to keep the plantlets in a continuous state of growth.
Microscopy In our previous report we described the initiation of somatic embryos and their further development into green nodules, from which plantlets sporadically were pro-
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duced (Hakman et al., 1985). In the present study, plantlets have been obtained at a much higher frequency and this has enabled us to more precisely follow their development both in living and sectioned materials. During their early stages, the somatic embryos consisted of long vacuolated cells at one end (suspensor) and a small group of meristematic cells at the other (embryonal end) (Figs. 5 and 6). Subsequently, when they grew further, they became more organized (Fig. 7). Later, cotyledons were formed (Figs. 8 and 9 a) and a root developed (Fig. 9 b). The internal anatomy of immature somatic embryos was very similar to immature zygotic embryos (d. Fig. 5 with Fig. 10 and Figs. 7 and 8 with Fig. 11).
Conclusion This study has shown that embryogenic callus can be induced on immature embryos of Picea abies cultured on LP medium supplemented with 2,4-D and cytokinin. For regeneration of plantlets, transfer of the embryogenic callus to medium 59 was favourable. Both callus initiation and plantlet regeneration was most frequent in cultures derived from immature embryos isolated from female cones that first had been stored in cold for about 2 months. Under suitable conditions 24 % of the established calli produced plantlets and in some instances up to 25 plantlets were formed in a single callus. Acknowledgements We thank Prof. Tage Eriksson for his support in this research, Ms. Ann-Charlott Johansson and Ms. Agneta Ottosson for technical assistance and Ms. Anette Axen for preparing the sections. Research was supported by a grant from Commission of the European Communities, contract BOS.140.S(H).
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NORSTOG, K. and E. RHAMSTINE: Isolation and culture of haploid and diploid cycad tissues. Phytomorphology 17, 374-381 (1967). United States Patent Abo EI-Nil No. 4217730, 1980. Embryogenesis of gymnosperm forest trees. VASIL, 1. K.: Regeneration of plants from single cells in cereals and grasses. In: P. F. LURQuIN and A. KLEmHOFS (eds.). Genetic Engineering in Eukaryotes, p. 233-252. Plenum Publishing Corporation (1983). VASIL, V. and I. K. VASIL: Somatic embryogenesis and plant regeneration from suspension cultures of Pearl millet (Pennisetum americanum). Ann. Bot. 47, 669-678 (1981). VON ARNOLD, S. and T. ERIKSSON: Bud induction on isolated needles of Norway spruce (Picea abies [L.] Karst.) grown in vitro. Plant Sci. Lett. 15, 363-372 (1979). - - In vitro studies of adventitious shoot formation in Pinus contorta. Can. J. Bot. 59, 870874 (1982).
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