PlantCeU Reports
Plant Cell Reports (1992) 11:299-303
9 Springer-Verlag 1992
Somatic embryogenesis and plant regeneration from zygotic embryo culture in blue spruce (Picea pungens Engelman.) John C. Afele 1, Tissa Senaratna 2, Bryan D. McKersie 23 and Praveen K. Saxena 1 1 Horticultural Science Department, University of Guelph, Guelph, Ontario, Canada N 1 G 2Wl 2 Crop Science Department, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Received December 9, 1991/Revised version received March 15, 1992 - Communicated by E Constabel
Summary. Somatic embryogenesis and plantlet formation have been achieved from cultured mature zygotic embryos of blue spruce (Picea pungens Engelman.). The effect of three basal media LP, LM, and BLG, all used at half-strength, was tested at the induction phase. LM medium induced somatic embryogenesis to a higher extent than LP whereas BLG did not produce any embryonal-suspensor mass representing stage 1 somatic embryos. The embryonalsuspensor mass was induced on a wide range of auxin/cytokinin ratios. However, media containing either 2 #M NAA and 10 #M BA, or 10 #M NAA and 5 #M BA produced somatic embryos that gave the highest frequency of planflets. The level of ABA required in the maturation medium for somatic embryos to mature properly varied with the auxin/cytokinin levels in the induction medium on which the somatic embryos were derived. Inclusion of AgNO3 (10 - 100 #M) in the induction medium reduced somatic embryogenesis and embryo conversion.
Key words: Blue spruce - Somatic embryogenesis Auxin and cytokinin ratio - Abseisic acid Abbreviations: naphthalene-acetic acid (NAA), N 6benzylaminopurine (BA), abscisic acid (ABA)
Introduction Induction of somatic embryogenesis and plantlet recovery in tree species is now possible in a wide range of species. Several conifer species have been successfully regenerated through somatic embryogenesis, including black spruce (Picea mariana) (Hakman and Fowke 1987; Tautorus et al. 1990), white spruce (P. glauca) (Dunstan Correspondence to: J.C. Afele
et al. 1988; Hakman and von Arnold 1988), Norway spruce (P. abies) lyon Arnold and Hakman 1988; Mo and von Arnold 1991), and interior spruce (mixture of P. glauca and P. engelmanni0 (Webb et al. 1989; Roberts et al. 1990; Webster et al. 1990; Flinn et al. 1991). Blue spruce is an important ornamental plant in North America. A procedure for somatic embryogenesis in this species is desirable to facilitate improvement through large-scale clonal multiplication and genetic manipulation. However, in vitro differentiation and development of blue spruce has not been described except the induction of non-embryogenic cultures from stem explant cultures (Bychenkova 1963; Harvey and Grasham 1969). This report, to our knowledge, is the first to describe a method for inducing somatic embryogenesis and plant recovery in blue spruce.
Materials and method Induction of embryonal-suspensor mass. Mature blue spruce s~ds were supplied by F.W. Sehumacher Co., Sandwich, Massachussets. Seeds were surface sterilized by sequentially immersing in 70% (v/v) ethanol for 2 rain, in 30% (v/v) Sanitizing Sodium Hypochlorite (Lilo Products, Hamilton, Ont.) for 20 rain, and rinsed 3 times in sterile distilled water for 5 rain each. The zygotic embryos were then isolated and cultured on different induction media, LM (Litvay 9 el. 1981), LP (yon Arnold and Erleksson 1981), and BLG (Verhagen and Warm 1989), all used at half strength, to test for the ability to support induction of embryonal-suspensor mass. Induction media were modified by incorporating filter-sterilized glutsmine (750 mg.L') into cooled autoclaved media. Other supplements in the induction medium were 60 mM sucrose, 0.8% (w/v) agar (Difco), and 10 gM NAA and 5 gM BA as growth regulators. The pH of the media was adjusted to 5.8 before autoclaving at 0.122 MPa for 20 rain. In the second experiment, the effect of different ratios of auxin and cytokinin (NAA and BA) contained in the induction medium on the production of embryonal-suspensor masses, and conversion of somatic embryos to plantlets was investigated. The basal medium for this experiment was a half-strength LM (HLM). Similarly, the influence of AgNO3, an ethylene inhibitor, on embryogenesiswas evaluated. Silver
300 nitrate at 10, 50, and 100 ~M was added to HLM containing 2 NAA and 10 ~ BA. In all treatments, 6 zygotic embryos were cultured on 10 ml of induction medium contained in a 60 x 15 mm Petri dish. Each replicate consisted of 3 Petri dishes (or 18 embryos). Experiments were repeated between 3 and 15 times. The total number of embryos cultured for each treatment is shown in the respective tables of results. Cultured zygotic embryos were incubated in the dark at 25 + 20C for induction of embryonal-suspensor masses. Embryogenic cultures were subcultured every 2 weeks. The frequency of embryonal-suspensor mass production was computed as a percentage of number of embryos cultured. A typical embryogenic tissue possessed small, densely cytoplasmic cells subtended by a long suspensor of highly vacuolated cells, corresponding to stage 1 embryos as described by yon Arnold and Hakman (1988). Treatment means and standard error (s.c.) were determined as described in Snedecor and Cochran (1980).
Maturation and germination of somatic embryos. Embryonal-suspensor masses, about 0.5 cm in diameter, containing stage 1 embryos were transfert'ed onto maturation medium which consisted of HLM supplemented with 90 mM sucrose and either 7.6 tam or 25 p2d ABA (Sigma Chemical Co., St Louis, M e ; Cataloq No. A-1049) for 4 weeks in the dark. ABA was prepared freshly, filter-sterilized using a 0.22 /tam nylon membrane filter system (Coming Glass Works, Coming, NY), and the required volume dispensed into cooled autoclaved medium. During the ABA treatment, cultures were transferred every 2 weeks onto fresh medium, without division into smaller pieces. Following maturation, stage 2 embryos (yon Arnold and Hakman 1988) were divided into smaller clumps and placed on HLM lacking growth regulators for plantlet formation under light intensity of 60 #reel ma.s-', 16/8 h photoperiod supplied by 40W cool white fluorescent lamps (Philips Canada, Scat'borough, Ontario), at 25*(2 and relative humidity of about 8 5 + 5%. Root elongation and further plant development was achieved on a half-strength GD medium (Gresshoff and Doy 1972), supplemented with 30 mM sucrose, and 0.6% (w/v) agar (pH 5.5).
Results
Induction of somatic embryogenesis Stage 1 embryos (Fig. 1) arising from the hypocotylcotyledonary region of the zygotic embryos were visible within one month of culturing. The frequency of embryonal-suspensor mass varied significantly among the three basal media (Table 1). More embryonal-suspensor masses were produced on LM medium than LP whereas BLG was found to be totally ineffective in inducing embryogenesis. Calli produced on BLG were small, brittle and turned brown faster than in other treatments. Embryonal-suspensor masses were produced on HLM containing a wide range of auxin/cytokinin combinations. The treatment means did not vary significantly from each other (P > 0.05). However, the best combination for embryogenesis was 2/10 which resulted in about 16 % of the cultured zygotic embryos producing embryonalsuspensor masses (Table 2). This was closely followed by NAA/BA ratio of 5/10 (14%), 10/5 (13%), 7.5/10
(11%), and 10/10 (10%). When the ratio of NAA/BA was 1/10 or lower, 0.1/10, the frequency of embryonalsuspensor mass decreased to 9 % and 6 %, respectively. Although the frequency of embryonal-susponsor mass did not vary significantly among most of the auxin/cytokinin treatments, the ability of the somatic embryos to differentiate into plantlets differed markedly (Table 2). Thus, NAA/BA ratio of 2/10 and 10/5 produced 10.83 and 9.71 plantlets/embryonal-suspensor mass transferred onto regeneration medium, respectively, which were more than five-fold higher than that obtained with other growth regulator treatments. Silver nitrate did not appear to be a requirement for embryogenesis in blue spruce. There was a significant decline in embryogenic response when AgN03 was included in the induction medium (Table 3). Plant regeneration was similarly inhibited.
Maturation and germination of somatic embryos Stage 1 embryos matured into stage 2 embryos (Fig. 2) within 4 weeks when transferred onto medium containing ABA. Stage 2 embryos displayed a more dense head region but were still subtended by the long suspensor. Transfer of stage 2 embryos onto HLM lacking growth regulators yielded cotyledouary or stage 3 embryos (Fig. 3) within 2 weeks. Plant regeneration or germination of the somatic embryos was achieved subsequently (Fig. 4). However, not all stage 3 embryos were able to differentiate into plantlets, which probably was a reflection of the quality of the somatic embryos produced by preceding treatments. Some factors involved in germination of the somatic embryos include the interaction of the growth regulator treatment during the induction phase and the level of ABA in the maturation medium. For example, stage 1 embryos derived from induction medium containing 10 pM NAA and 5 #M BA required a lower level of ABA (7.6 pM) to mature into stage 2 embryos, and proceed to stage 3 embryos, followed by plant differentiation. Plant regeneration frequency was about 9.7 planflets/embryonal-suspensor mass for such treatment, and was one of the highest (Fig. 5). For this induction medium treatment, plant regeneration was completely inhibited when the ABA level was raised to 25 #M. On the other hand, embryonal-suspensor mass induced on induction medium containing 2/xM NAA and 10/~M BA required a higher ABA level to yield stage 2 embryos capable of forming stage 3 embryos that later differentiated plantlets. Plant regeneration for these embryos when matured on 7.6 #M ABA was 4.3 plantlets/embryonal-suspensor mass but this frequency was increased to.9.7 plantlets/embryonal-suspensor mass when embryos were matured on 25/tM ABA.
301 Discussion Table 1. Effect of basal media on induction o f embryonal-suspensor masses containing stage 1 embryos, and embryo conversion in cultures of blue spruce BM" (x 0.5)
Number o f zygotic embryos cultured
Embryonal-suspensor mass (%) (mean + s.e)
Number of plantlets / e m b r y o n a 1suspensor mass (mean . 6 s.e)
LP BLG LM
120 60 264
1.79 . 6 1.78 0.00 13.00 + 2.40
0.04 . 6 0.01 0.00 9.71 + 1.97
9 Basal media were used at half-strength, and supplemented with 10/aM N A A and 5 p.M BA
Table 2. Influence of N A A and BA combinations on the frequency of embryonal-suspensor mass and plant recovery from cultures o f blue spruce Treatment
Number of Embryonal-suspensor zygotic mass (%) embryos (mean + s.e) cultured
Number o f plantlets / 9m b r y o n a Isuspensor mass (mean + s.e)
264 78 78 78 198 198 48
9.71 -6 1.97 4.67 -6 0.93 2.33 -6 0.30 1.50 -6 0.24 10.83 -6 1.76 0.27 -6 0.07 0.00
NAA / BA l0 / 5 10 / 10 7.5/10 5 / 10 2 / 10 1 / 10 0.1/10
13.00 -6 2.40 9.47 -6 4.10 10.65 -6 5.48 13.89 + 7.03 15.64 + 2.88 9.13 -6 1.75 6.25 -6 3.98
Table 3. Effect of silver nitrate on the frequency of embryonalsuspensor masses and plant recovery from cultures o f blue spruce Treatment~ Number o f AgNO~ zygotic (j~M) embryos cultured
Embryonal-suspensor mass (%) (mean . 6 s.e)
Number of plantlets / e m b r y o n a 1suspensor mass (mean + s.e)
0 10 50 100
15.64 7.87 2.78 1.39
10.83 + 1.76 0.17 + 0.04 0.00 0.00
198 78 78 78
+ + + +
2.88 2.72 2.78 1.39
~ The basal medium was a half-strength LM containing 2/tM N A A and 10 p,M BA
The four stages of somatic embryogenesis described by yon Arnold and Hakman (1988) in Norway spruce have been observed in blue spruce as well. Stage 1 embryos were initiated on basal medium supplemented with an auxin and a cytoldnin. LM medium proved to be more inductive than LP while BLG medium produced only non-embryogenic cultures. LP medium has been used extensively for inducing somatic embryogenesis in many conifers, and BLG has also been shown to be more inductive than other media investigated for Norway spruce (Verhagen and Warm 1989). The superiority of LM medium over LP and BLG in the present study suggests blue spruce cultures might require different nutrients, compared to other spruces, for optimal expression of emhryogenlc potential. The three basal media compared in this report differed markedly in a number of nutrient elements, hence it is difficult to attribute the superiority of LM to any particular nutrient component. Stage 1 embryos of spruces have usually been obtained on basal medium supplemented with 10 #M NAA (or 2,4-D) combined with 5 #M BA. However, it appears that a wide range of auxin to cytokinin ratios might be effective as well. The quality of embryos would vary though, as reflected in their ability to mature and differentiate into plantlets. Several reports have established a role for ABA in the maturation of spruce and other conifer somatic embryos (Durzan and Gupta 1987; Boulay et al. 1988; Dunstan et al. 1988; Roberts et al. 1990), but the optimum level of ABA has not been resolved unequivocally. For example, yon Arnold and Hakman (1988) observed that 7.6 #M ABA was optimal in promoting embryo maturation in Norway spruce cultures, while Roberts et al. (1990) reported that higher ABA levels (30-40 /~M) were required. One plausible explanation of the variation among different groups could be the incorporation of ABA in the medium before, or after autoclaving. In the present study, filter-sterilized ABA was incorporated into cooled medium after autoclaving. The age of the cultures might also affect the ABA requirement for nmturation. For example, yon Arnold and Hakman (1988) observed that younger embryogenlc cultures growing more rapidly required longer durations of ABA treatment than older cultures growing slowly. Results in the present investigation demonstrated that ABA requirement for maturation depended on growth regulator treatment in the induction medium. Stage 1 embryos that developed on induction medium containing 10 #M NAA and 5 #M BA matured into stage 2 embryos when treated with 7.6 #IV[ABA. A higher ABA level (25 #M) was required by embryos that were
302
Figures 1 - 4. Somatic embryogenesis in blue spruce (Picea pungens Engelman.) Fig. 1. Embryonal-suspensor mass containing stage 1 embryos from mature zygotic embryo culture. Stage 1 embryos appeared within 4 weeks of culture of the zygotic embryos on a halfstrength LM medium supplemented with various levels of NAA and BA. Bar = 2 ram. F'~g. 2. Stage 2 embryos (arrow) developed when stage 1 embryos were transferred onto basal medium supplemented with 7.6 or 25 pm ABA. Bar = 2 roan. Fig. 3. Cotyledonary or stage 3 embryos were formed upon the transfer of stage 2 embryos from ABA-containing medium onto basal medium without growth regulators. Bar = 1.3 mm. Fig. 4. Germination of a cotyledonary embryo and development of a plantlet. Bar = 2 mm.
Fig.5. Interaction o f growth regulators in induction and maturation media, on plant regeneration in blue spruce.
{~
1/10 2/10 5/10 7.5/10 10/10 10/5 Growth regulator in induction medium (NAA/BA uM)
[ ] 7.6 uM ABA [ ] 25 uM ABA No. of embryogenic mass used: NAA/BA 1/10 (10), z / t o Os), 5/70 (e), 7.5/70 (7), to/70 (5.1, to~5 (72)
303 initiated on induction medium containing 2/~M NAn, and 10 #M BA. All embryonal-suspensor masses were reduced to about the same size (0.5 cm in diameter) before explanting on maturation medium. However, cultures initiated on 2 #M NAA and 10 #M BA grew slightly bigger than other treatments on the maturation medium. This increased size was a reflection of the faster rate of growth of these cultures; hence, it is likely that a higher ABA level was required to mature these embryos. Ethylene accumulates during embryogenesis in white spruce, and high levels of the gas are known to be inhibitory to embryogenesis CKumar et al. 1989). Silver nitrate has been shown to stimulate embryogenesis in species in which the endogeneous ethylene level is generally high, and inhibited embryogenesis in those species with lower ethylene concentrations (Biddington et al. 1988; Cho and Kasha 1989); AgNO3 is thought to act as an ethylene inhibitor. The inclusion of various concentrations of AgNOs in our induction medium reduced embryogenesis and plant recovery in the blue spruce cultures. It is possible that the endogeneous ethylene level in the blue spruce cultures was below the optimum for embryogenesis, and AgNO3 further decreased its availability in the culture vessels. Another possibility is that AgNO3 itself was toxic or inhibitory to the cultures. The frequency of embryonal-suspensor masses observed in the present study was generally low. Mo and von Arnold (1991) reported a high frequency of embryonal-suspensor masses (39 %) in Norway spruce, however, the frequency of embryonal-suspensor masses that actually reached a stage to be transferred onto maturation medium was 10-15%. The frequency of embryonal-suspensor masses reported in our study was that which could be subcultured or transferred onto regeneration medium, hence our results did not differ much from other reports in which the explants were mature zygotic embryos. Efforts are continuing to improve the efficiency of plant regeneration. Our goal was to clonally propagate blue spruce using tissue culture methods. The choice of zygotic embryos as explants is not ideal in view of clonal propagation. However, most of the plantlets recovered in our experiments exhibited blue needles. Thus the protocol, apart from its application in genetic manipulation, could still be an effective tool in multiplication of propagules.
Acknowledgement. This research was supported by White Rose Nurseries Ltd., and the Province of Ontario's University Research Incentive Fund. JA acknowledges the generous and valuable discussions with Dr. R.H. Ho, and Dr. A.Y. Raj of the Ontario Ministry of Natural Resources.
References Biddington NL, Suthedand RA, Robinson FIT (1988) Ann. Bot. 62:
181-185. Boulay MP, Gupte PK, Krogstrup P, Durzan DJ (1988) Plant Cell Rep. 7: 134-137. Bychenkova EA (1963) l~fiol.Plant. 5: 302-309. Cho U-H, Kasha KI (1989) Plant Cell Rep. 8: 415-417. Dunstan DI, Bekkaoui F, Pilon M, Fowke LC, Abrams SR (1988) Plant Sci. 58: 77-84. Durzan DJ, Gupta PK (1987) Piaa~ Sci. 52: 229-235. Flinn BS, Roberts DR, Taylor IEP (1991) Physiol. Plant. 82: 624-632. GresshoffPM, Doy CH (1972)Ptente 107: 161-170. Hakman IC, Fowke LC (1987) Plant Cell Rep. 6: 20-22. Hakman IC, yon Arnold (1988) Physiol. Plant. 72: 579-587. Harvey AE, Grasham JL (1969) Can. J. Pot. 47: 547-549. Kumar PP, Richard WJ, 'thorpe TA (1989) J. Plant Physiol. 135: 592596. Litvay JD, Johnson MA, Verma DC, Einspahr D, Kaustinen H (1981) Inst. Paper Chem., Tech. Paper 114, Appleton, WI. Mo LH, yon Arnold S (1991) I. Plant Physiol. 138: 223-230. Roberts DR, Fllnn BS, Webb DT, Webster FB, and Sutton BCS (1990) Physiol. Plant. 78: 355-360. Snedecor GW, Cochrsn WG (1980) Statistical Methods. Seventh edition. Iowa State University Press, Ames, Iowa. Tautorus TE, Attree SM Fowke LC, Dunstan DI (1990) Plant Sci. 67:115-124. Verhagen SA, Wann SR (1989) Plant Cell Tissue Organ Cult. 16: 103111. yon Arnold S, Hakman l[ (1988) J. Plant Physiol. 132: 164-169. yon Arnold S, Eriksson T (1981) Can. J. Pot. 59: 870-874. Webb DT, Webster FB, Flinn BS, Roberts DR, Ellis DD (1989) Can I. For. Res. 19: 1303-1308. Webster FB, Roberts DR, McInnis SM, Sutton BCS (1990) Can J. For. Res. 20: 1759-1765.
Plant Cell Reports (1992) 11:299-303
9 Springer-Verlag 1992
Somatic embryogenesis and plant regeneration from zygotic embryo culture in blue spruce (Picea pungens Engelman.) John C. Afele 1, Tissa Senaratna 2, Bryan D. McKersie 23 and Praveen K. Saxena 1 1 Horticultural Science Department, University of Guelph, Guelph, Ontario, Canada N 1 G 2Wl 2 Crop Science Department, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Received December 9, 1991/Revised version received March 15, 1992 - Communicated by E Constabel
Summary. Somatic embryogenesis and plantlet formation have been achieved from cultured mature zygotic embryos of blue spruce (Picea pungens Engelman.). The effect of three basal media LP, LM, and BLG, all used at half-strength, was tested at the induction phase. LM medium induced somatic embryogenesis to a higher extent than LP whereas BLG did not produce any embryonal-suspensor mass representing stage 1 somatic embryos. The embryonalsuspensor mass was induced on a wide range of auxin/cytokinin ratios. However, media containing either 2 #M NAA and 10 #M BA, or 10 #M NAA and 5 #M BA produced somatic embryos that gave the highest frequency of planflets. The level of ABA required in the maturation medium for somatic embryos to mature properly varied with the auxin/cytokinin levels in the induction medium on which the somatic embryos were derived. Inclusion of AgNO3 (10 - 100 #M) in the induction medium reduced somatic embryogenesis and embryo conversion.
Key words: Blue spruce - Somatic embryogenesis Auxin and cytokinin ratio - Abseisic acid Abbreviations: naphthalene-acetic acid (NAA), N 6benzylaminopurine (BA), abscisic acid (ABA)
Introduction Induction of somatic embryogenesis and plantlet recovery in tree species is now possible in a wide range of species. Several conifer species have been successfully regenerated through somatic embryogenesis, including black spruce (Picea mariana) (Hakman and Fowke 1987; Tautorus et al. 1990), white spruce (P. glauca) (Dunstan Correspondence to: J.C. Afele
et al. 1988; Hakman and von Arnold 1988), Norway spruce (P. abies) lyon Arnold and Hakman 1988; Mo and von Arnold 1991), and interior spruce (mixture of P. glauca and P. engelmanni0 (Webb et al. 1989; Roberts et al. 1990; Webster et al. 1990; Flinn et al. 1991). Blue spruce is an important ornamental plant in North America. A procedure for somatic embryogenesis in this species is desirable to facilitate improvement through large-scale clonal multiplication and genetic manipulation. However, in vitro differentiation and development of blue spruce has not been described except the induction of non-embryogenic cultures from stem explant cultures (Bychenkova 1963; Harvey and Grasham 1969). This report, to our knowledge, is the first to describe a method for inducing somatic embryogenesis and plant recovery in blue spruce.
Materials and method Induction of embryonal-suspensor mass. Mature blue spruce s~ds were supplied by F.W. Sehumacher Co., Sandwich, Massachussets. Seeds were surface sterilized by sequentially immersing in 70% (v/v) ethanol for 2 rain, in 30% (v/v) Sanitizing Sodium Hypochlorite (Lilo Products, Hamilton, Ont.) for 20 rain, and rinsed 3 times in sterile distilled water for 5 rain each. The zygotic embryos were then isolated and cultured on different induction media, LM (Litvay 9 el. 1981), LP (yon Arnold and Erleksson 1981), and BLG (Verhagen and Warm 1989), all used at half strength, to test for the ability to support induction of embryonal-suspensor mass. Induction media were modified by incorporating filter-sterilized glutsmine (750 mg.L') into cooled autoclaved media. Other supplements in the induction medium were 60 mM sucrose, 0.8% (w/v) agar (Difco), and 10 gM NAA and 5 gM BA as growth regulators. The pH of the media was adjusted to 5.8 before autoclaving at 0.122 MPa for 20 rain. In the second experiment, the effect of different ratios of auxin and cytokinin (NAA and BA) contained in the induction medium on the production of embryonal-suspensor masses, and conversion of somatic embryos to plantlets was investigated. The basal medium for this experiment was a half-strength LM (HLM). Similarly, the influence of AgNO3, an ethylene inhibitor, on embryogenesiswas evaluated. Silver
300 nitrate at 10, 50, and 100 ~M was added to HLM containing 2 NAA and 10 ~ BA. In all treatments, 6 zygotic embryos were cultured on 10 ml of induction medium contained in a 60 x 15 mm Petri dish. Each replicate consisted of 3 Petri dishes (or 18 embryos). Experiments were repeated between 3 and 15 times. The total number of embryos cultured for each treatment is shown in the respective tables of results. Cultured zygotic embryos were incubated in the dark at 25 + 20C for induction of embryonal-suspensor masses. Embryogenic cultures were subcultured every 2 weeks. The frequency of embryonal-suspensor mass production was computed as a percentage of number of embryos cultured. A typical embryogenic tissue possessed small, densely cytoplasmic cells subtended by a long suspensor of highly vacuolated cells, corresponding to stage 1 embryos as described by yon Arnold and Hakman (1988). Treatment means and standard error (s.c.) were determined as described in Snedecor and Cochran (1980).
Maturation and germination of somatic embryos. Embryonal-suspensor masses, about 0.5 cm in diameter, containing stage 1 embryos were transfert'ed onto maturation medium which consisted of HLM supplemented with 90 mM sucrose and either 7.6 tam or 25 p2d ABA (Sigma Chemical Co., St Louis, M e ; Cataloq No. A-1049) for 4 weeks in the dark. ABA was prepared freshly, filter-sterilized using a 0.22 /tam nylon membrane filter system (Coming Glass Works, Coming, NY), and the required volume dispensed into cooled autoclaved medium. During the ABA treatment, cultures were transferred every 2 weeks onto fresh medium, without division into smaller pieces. Following maturation, stage 2 embryos (yon Arnold and Hakman 1988) were divided into smaller clumps and placed on HLM lacking growth regulators for plantlet formation under light intensity of 60 #reel ma.s-', 16/8 h photoperiod supplied by 40W cool white fluorescent lamps (Philips Canada, Scat'borough, Ontario), at 25*(2 and relative humidity of about 8 5 + 5%. Root elongation and further plant development was achieved on a half-strength GD medium (Gresshoff and Doy 1972), supplemented with 30 mM sucrose, and 0.6% (w/v) agar (pH 5.5).
Results
Induction of somatic embryogenesis Stage 1 embryos (Fig. 1) arising from the hypocotylcotyledonary region of the zygotic embryos were visible within one month of culturing. The frequency of embryonal-suspensor mass varied significantly among the three basal media (Table 1). More embryonal-suspensor masses were produced on LM medium than LP whereas BLG was found to be totally ineffective in inducing embryogenesis. Calli produced on BLG were small, brittle and turned brown faster than in other treatments. Embryonal-suspensor masses were produced on HLM containing a wide range of auxin/cytokinin combinations. The treatment means did not vary significantly from each other (P > 0.05). However, the best combination for embryogenesis was 2/10 which resulted in about 16 % of the cultured zygotic embryos producing embryonalsuspensor masses (Table 2). This was closely followed by NAA/BA ratio of 5/10 (14%), 10/5 (13%), 7.5/10
(11%), and 10/10 (10%). When the ratio of NAA/BA was 1/10 or lower, 0.1/10, the frequency of embryonalsuspensor mass decreased to 9 % and 6 %, respectively. Although the frequency of embryonal-susponsor mass did not vary significantly among most of the auxin/cytokinin treatments, the ability of the somatic embryos to differentiate into plantlets differed markedly (Table 2). Thus, NAA/BA ratio of 2/10 and 10/5 produced 10.83 and 9.71 plantlets/embryonal-suspensor mass transferred onto regeneration medium, respectively, which were more than five-fold higher than that obtained with other growth regulator treatments. Silver nitrate did not appear to be a requirement for embryogenesis in blue spruce. There was a significant decline in embryogenic response when AgN03 was included in the induction medium (Table 3). Plant regeneration was similarly inhibited.
Maturation and germination of somatic embryos Stage 1 embryos matured into stage 2 embryos (Fig. 2) within 4 weeks when transferred onto medium containing ABA. Stage 2 embryos displayed a more dense head region but were still subtended by the long suspensor. Transfer of stage 2 embryos onto HLM lacking growth regulators yielded cotyledouary or stage 3 embryos (Fig. 3) within 2 weeks. Plant regeneration or germination of the somatic embryos was achieved subsequently (Fig. 4). However, not all stage 3 embryos were able to differentiate into plantlets, which probably was a reflection of the quality of the somatic embryos produced by preceding treatments. Some factors involved in germination of the somatic embryos include the interaction of the growth regulator treatment during the induction phase and the level of ABA in the maturation medium. For example, stage 1 embryos derived from induction medium containing 10 pM NAA and 5 #M BA required a lower level of ABA (7.6 pM) to mature into stage 2 embryos, and proceed to stage 3 embryos, followed by plant differentiation. Plant regeneration frequency was about 9.7 planflets/embryonal-suspensor mass for such treatment, and was one of the highest (Fig. 5). For this induction medium treatment, plant regeneration was completely inhibited when the ABA level was raised to 25 #M. On the other hand, embryonal-suspensor mass induced on induction medium containing 2/xM NAA and 10/~M BA required a higher ABA level to yield stage 2 embryos capable of forming stage 3 embryos that later differentiated plantlets. Plant regeneration for these embryos when matured on 7.6 #M ABA was 4.3 plantlets/embryonal-suspensor mass but this frequency was increased to.9.7 plantlets/embryonal-suspensor mass when embryos were matured on 25/tM ABA.
301 Discussion Table 1. Effect of basal media on induction o f embryonal-suspensor masses containing stage 1 embryos, and embryo conversion in cultures of blue spruce BM" (x 0.5)
Number o f zygotic embryos cultured
Embryonal-suspensor mass (%) (mean + s.e)
Number of plantlets / e m b r y o n a 1suspensor mass (mean . 6 s.e)
LP BLG LM
120 60 264
1.79 . 6 1.78 0.00 13.00 + 2.40
0.04 . 6 0.01 0.00 9.71 + 1.97
9 Basal media were used at half-strength, and supplemented with 10/aM N A A and 5 p.M BA
Table 2. Influence of N A A and BA combinations on the frequency of embryonal-suspensor mass and plant recovery from cultures o f blue spruce Treatment
Number of Embryonal-suspensor zygotic mass (%) embryos (mean + s.e) cultured
Number o f plantlets / 9m b r y o n a Isuspensor mass (mean + s.e)
264 78 78 78 198 198 48
9.71 -6 1.97 4.67 -6 0.93 2.33 -6 0.30 1.50 -6 0.24 10.83 -6 1.76 0.27 -6 0.07 0.00
NAA / BA l0 / 5 10 / 10 7.5/10 5 / 10 2 / 10 1 / 10 0.1/10
13.00 -6 2.40 9.47 -6 4.10 10.65 -6 5.48 13.89 + 7.03 15.64 + 2.88 9.13 -6 1.75 6.25 -6 3.98
Table 3. Effect of silver nitrate on the frequency of embryonalsuspensor masses and plant recovery from cultures o f blue spruce Treatment~ Number o f AgNO~ zygotic (j~M) embryos cultured
Embryonal-suspensor mass (%) (mean . 6 s.e)
Number of plantlets / e m b r y o n a 1suspensor mass (mean + s.e)
0 10 50 100
15.64 7.87 2.78 1.39
10.83 + 1.76 0.17 + 0.04 0.00 0.00
198 78 78 78
+ + + +
2.88 2.72 2.78 1.39
~ The basal medium was a half-strength LM containing 2/tM N A A and 10 p,M BA
The four stages of somatic embryogenesis described by yon Arnold and Hakman (1988) in Norway spruce have been observed in blue spruce as well. Stage 1 embryos were initiated on basal medium supplemented with an auxin and a cytoldnin. LM medium proved to be more inductive than LP while BLG medium produced only non-embryogenic cultures. LP medium has been used extensively for inducing somatic embryogenesis in many conifers, and BLG has also been shown to be more inductive than other media investigated for Norway spruce (Verhagen and Warm 1989). The superiority of LM medium over LP and BLG in the present study suggests blue spruce cultures might require different nutrients, compared to other spruces, for optimal expression of emhryogenlc potential. The three basal media compared in this report differed markedly in a number of nutrient elements, hence it is difficult to attribute the superiority of LM to any particular nutrient component. Stage 1 embryos of spruces have usually been obtained on basal medium supplemented with 10 #M NAA (or 2,4-D) combined with 5 #M BA. However, it appears that a wide range of auxin to cytokinin ratios might be effective as well. The quality of embryos would vary though, as reflected in their ability to mature and differentiate into plantlets. Several reports have established a role for ABA in the maturation of spruce and other conifer somatic embryos (Durzan and Gupta 1987; Boulay et al. 1988; Dunstan et al. 1988; Roberts et al. 1990), but the optimum level of ABA has not been resolved unequivocally. For example, yon Arnold and Hakman (1988) observed that 7.6 #M ABA was optimal in promoting embryo maturation in Norway spruce cultures, while Roberts et al. (1990) reported that higher ABA levels (30-40 /~M) were required. One plausible explanation of the variation among different groups could be the incorporation of ABA in the medium before, or after autoclaving. In the present study, filter-sterilized ABA was incorporated into cooled medium after autoclaving. The age of the cultures might also affect the ABA requirement for nmturation. For example, yon Arnold and Hakman (1988) observed that younger embryogenlc cultures growing more rapidly required longer durations of ABA treatment than older cultures growing slowly. Results in the present investigation demonstrated that ABA requirement for maturation depended on growth regulator treatment in the induction medium. Stage 1 embryos that developed on induction medium containing 10 #M NAA and 5 #M BA matured into stage 2 embryos when treated with 7.6 #IV[ABA. A higher ABA level (25 #M) was required by embryos that were
302
Figures 1 - 4. Somatic embryogenesis in blue spruce (Picea pungens Engelman.) Fig. 1. Embryonal-suspensor mass containing stage 1 embryos from mature zygotic embryo culture. Stage 1 embryos appeared within 4 weeks of culture of the zygotic embryos on a halfstrength LM medium supplemented with various levels of NAA and BA. Bar = 2 ram. F'~g. 2. Stage 2 embryos (arrow) developed when stage 1 embryos were transferred onto basal medium supplemented with 7.6 or 25 pm ABA. Bar = 2 roan. Fig. 3. Cotyledonary or stage 3 embryos were formed upon the transfer of stage 2 embryos from ABA-containing medium onto basal medium without growth regulators. Bar = 1.3 mm. Fig. 4. Germination of a cotyledonary embryo and development of a plantlet. Bar = 2 mm.
Fig.5. Interaction o f growth regulators in induction and maturation media, on plant regeneration in blue spruce.
{~
1/10 2/10 5/10 7.5/10 10/10 10/5 Growth regulator in induction medium (NAA/BA uM)
[ ] 7.6 uM ABA [ ] 25 uM ABA No. of embryogenic mass used: NAA/BA 1/10 (10), z / t o Os), 5/70 (e), 7.5/70 (7), to/70 (5.1, to~5 (72)
303 initiated on induction medium containing 2/~M NAn, and 10 #M BA. All embryonal-suspensor masses were reduced to about the same size (0.5 cm in diameter) before explanting on maturation medium. However, cultures initiated on 2 #M NAA and 10 #M BA grew slightly bigger than other treatments on the maturation medium. This increased size was a reflection of the faster rate of growth of these cultures; hence, it is likely that a higher ABA level was required to mature these embryos. Ethylene accumulates during embryogenesis in white spruce, and high levels of the gas are known to be inhibitory to embryogenesis CKumar et al. 1989). Silver nitrate has been shown to stimulate embryogenesis in species in which the endogeneous ethylene level is generally high, and inhibited embryogenesis in those species with lower ethylene concentrations (Biddington et al. 1988; Cho and Kasha 1989); AgNO3 is thought to act as an ethylene inhibitor. The inclusion of various concentrations of AgNOs in our induction medium reduced embryogenesis and plant recovery in the blue spruce cultures. It is possible that the endogeneous ethylene level in the blue spruce cultures was below the optimum for embryogenesis, and AgNO3 further decreased its availability in the culture vessels. Another possibility is that AgNO3 itself was toxic or inhibitory to the cultures. The frequency of embryonal-suspensor masses observed in the present study was generally low. Mo and von Arnold (1991) reported a high frequency of embryonal-suspensor masses (39 %) in Norway spruce, however, the frequency of embryonal-suspensor masses that actually reached a stage to be transferred onto maturation medium was 10-15%. The frequency of embryonal-suspensor masses reported in our study was that which could be subcultured or transferred onto regeneration medium, hence our results did not differ much from other reports in which the explants were mature zygotic embryos. Efforts are continuing to improve the efficiency of plant regeneration. Our goal was to clonally propagate blue spruce using tissue culture methods. The choice of zygotic embryos as explants is not ideal in view of clonal propagation. However, most of the plantlets recovered in our experiments exhibited blue needles. Thus the protocol, apart from its application in genetic manipulation, could still be an effective tool in multiplication of propagules.
Acknowledgement. This research was supported by White Rose Nurseries Ltd., and the Province of Ontario's University Research Incentive Fund. JA acknowledges the generous and valuable discussions with Dr. R.H. Ho, and Dr. A.Y. Raj of the Ontario Ministry of Natural Resources.
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