InVitroCell.Dev.Biol.30P:156-159,July1994 © 1994SocietyforIn VitroBiology 1054-5476/94 $01.50+0.00

(PICEA ABIES) AND WHITE SPRUCE

ABA C O N S U M P T I O N I N NORWAY SPRUCE

(PICEA GLAUCA) SOMATIC E M B R Y O C U L T U R E S 1 DAVID I. DUNSTAN, STEPHANIE BERRY, AND CHERYL A. BOCK

Plant Biotechnology Institute, National Research Council Canada, 1t0 Gymnasium Place, Saskatoon, Saskatchewan, S7N OIV9, (Received 12 January 1994; accepted 9 April 1994; editor T. A. Thorpe)

SUMMARY W e investigated abscisie acid (ABA) metabolism among Norway and white spruce somatic embryo cultures which exhibited differencesin maturation response when placed on racemic abscisicacid [(_+)-ABA]. Differences in metabolic rate among the spruce genotypes could affectthe A B A pool availablefor the maturation process, and might therefore be responsible for the differences in maturation response. The production of cotylcdonary (stage 3) somatic embryos in cultures (gcnotypes) of Norway spruce (PA86:26A and PA88:25B) and of white spruce (WSIFcryoD and WS46) was compared. In each species pair one of the two genotypes failed to show stage 3 embryo development (respectively, PA88:25B and WS46). The investigation of ABA metabolism of each species pair showed that no substantial differences in ABA consumption or in the production of metabolites occurred. In each case ABA was metabolized to phaseic acid and dihydrophaseic acid over the 42-day culture period, metabolites were recoverable from the agar-solidified medium, and the sum of residual ABA and metabolites were equivalent to the ABA initially supplied. The results indicate that the process of ABA metabolism occurs essentially independently of somatic embryo maturation. Key words: ABA; somatic embryo; maturation; Picea abies; Picea glauca; metabolism; photoisomerism. 1990; Dunstan et al., 1988, 1991, 1993; Hakman and van Arnold, 1988; Roberts et al., 1990a,b; yon Arnold and Hakman, 1988) and that natural [(+)-ABA] is metabolized to phaseic acid (PA) and dihydrophaseic acid (DPA) (Dunstan et al., 1992, 1993). These metabolites were recoverable from the culture filtrates of suspension cultures; the sum of the residual ABA and metabolites were essentially equal to the ABA initially supplied. It seems that the unnatural enantiomer [(-)-ABA] in the racemic mixture was not metabolized (Dunstan et al., 1992) and that PA does not promote somatic embryo maturation equivalent to that obtained with (+)ABA (D. I. Dunstan and C, A. Bock, unpublished results). One question that arises is whether differences in the maturation of the Norway and white spruce cultures could be attributed to variations in specific ABA-related events; for example, ABA metabolism, gene induction, and expression. In this report we present the results from an investigation of ABA metabolism, in cultures of Norway and white spruces that differed in their abilities to undergo somatic embryo maturation after placement on medium containing ABA. We decided to investigate ABA metabolism first because the rate of metabolism in liquid suspension cultures of white spruce suggested that fed ABA could be depleted within 8 days. Different rates of metabolism among the spruce genotypes could affect the ABA pool available for the maturation process, and might therefore be responsible for the differences in maturation response.

INTRODUCTION Somatic embryogenesis of white and Norway spruces and the maturation of somatic embryos has been extensively studied, and the main characteristics of somatic embryo maturation have been described, e.g., morphogenesis (Hakman and van Arnold, 1988; von Arnold and Hakman, 1988; Joy et al., 1991), requirement for exogenous abscisic acid [(+)-ABA] (Attree et al., 1990; Dunstan et al., 1988, 1991; Hakman and von Arnold, 1988; van Arnold and Hakman, 1988), and storage product deposition (Attree et at., 1992; Flinn et al., 1991; Hakman, 1993; Joy et al., 1991). However, maturation is not obtainable with all somatic embryo cultures of these spruces, and may be genotype dependent. For example, Jalonen and van Arnold (1991) classified Norway spruce embryogenic cultures into either of two classes, based on the abilities of the embryos of independent genotypes to mature; type "A" were able to mature, type "B "were unable to mature. These two classes also differed morphologically. With white spruce, we have found similar differences among genotypes in their abilities to mature, although morphologically the white spruce cultures have seemed to be relatively more uniform than the Norway spruce cultures. The focus for our research is on understanding the requirements for effecting conifer somatic embryo maturation, particularly the effects of ABA on the process. It is known that exogenous ABA in the form of (+)-ABA will promote spruce somatic embryo maturation, most often used between 8 and 60 #M (_+)-ABA(Attree et at.,

MATERIALSANDMETHODS

Plant material. Two liquid suspensioncultures of embryogenicwhite spruce [Piceaglauca (Moench)Voss] were compared, WS1FcryoD(Dunstart et al., 1988, 1991, 1993) and WS46 (initiatedfrom a mature zygotic

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ABA CONSUMPTION IN SPRUCE CULTURES embryo in 1990). Also compared were two liquid suspension cultures of Norway spruce [Picea abies (L.) Karst], PA86:26A, and PA88:25B which had been obtained as tissue cultures growing on solidified medium from Dr. Sara van Arnold (Swedish University of Agricultural Sciences, Uppsala, Sweden). These four spruce cultures had been maintained as liquid suspensions by weekly subculture for at least 26 wk before the start of the following experiments. Subculture of the spruce suspension cultures was in autoclaved liquid 0.5 X LM (Litvay et al., 1981) with 1.5% sucrose, 0.8 g • liter-1 casein hydrolysate and 3 mM glutamine, containing 9 #M 2,4dichlorophenoxy acetic acid and 4.4 ttM benzyl adenine (BA). The white spruce and Norway spruce embryogenic suspension cultures differed in appearance. At the time of the experiments the two white spruce cultures each consisted of populations of free cells, clusters of isodiametric cells, and stage 1 immature somatic embryos, as previously described (Hakman and Fowke, 1987; Dunstan et al., 1988, 1993). Stage 1 white spruce somatic embryos resemble the polar-type Norway spruce somatic embryos described by Jalonen and yon Arnold (1991). The two Norway spruce cultures also contained populations of free cells and clusters of isodiametric cells, although the somatic embryos were either of the solar type "A" (PA86:26A) or undeveloped type "B" (PA88:25B), as described by Jalonen and van Arnold (1991). Filter disc cultures. Filter disc cultures (FDCs) were prepared and incubated as previously described (Dunstan et al., 1988, 1991, 1993). Presterilized gridded black filter discs (no. AABG047S0; Miflipore, Bedford, MA) were placed on 25 ml agar-solidified (0.5%, wt/vol; no. A-7002, Sigma Chemical Co., St. Louis, MO) 0.5 × LP medium (van Arnold and Eriksson, 1981) containing 15 ttM (+)-ABA (A-1012, Sigma Chemical), 1% (wt/vol) sucrose, pH 5.6, contained in sterile disposable Petri dishes (100 X 15 mm). The (+)-ABA was added to medium as a filter-sterilized aliquot from a 1 mM stock solution (in acetone/water, 1:9 wt/vol) before dispensing into the Petri dishes. A 0.75-ml aliquot of a 20% wt/vol suspension (0.15 g fresh weight culture) of each culture line was then dispensed onto the center of each disc. FDCs were routinely incubated on maturation medium for 42 days, under low light (16 h, 40 W cool white fluorescent lights) providing a photosynthetically active radiation (Krizek, 1982) of 1 #E" m -2- s -1 in the dish. The FDCs were observed throughout the culture period, and the numbers of stage 3 cotyledonary somatic embryos were counted on Days 35 and 42. Maturation treatments were replicated for each culture line with 20 dishes on each of two separate occasions. Of these, eight dishes were used to count stage 3 embryos on Days 35 and 42, of each of the two repeat experiments. Data (Table 1) are presented as mean numbers of stage 3 somatic embryos + the standard deviation of the mean number (SD). FDCs were also harvested for analyses of metabolism as described next. Abscisic acid metabolism. The experiments were designed to compare the metabolism of 15 #M (_+)-ABAin the FDCs for each spruce line. Of the 20 dishes available per culture line in each experiment, 3 were taken for analysis on Days 3, 7, 14, 28, and 42. Equivalent analyses of non-inoculated dishes were made throughout the course of the experiment, beginning at Day 0. After removal of the filters with embryo tissue, each dish was placed at - 2 0 ° C to disrupt the agar matrix, allowing removal of liquified medium when thawed. Abscisic acid and its metabolites in the culture medium were quantified by direct-injection high performance liquid chromatography (HPLC), following a modification (Dunstan et al., 1993) to the procedure of Dunstan et al., 1992. A 1-ml aliquot of medium was passed through a 0.2-#m filter and a 10-#1 portion of the filtrate was then injected into a Gilson modular HPLC operated at ambient temperature and equipped with a Supelco Hiscp 15 cm × 4.6 mm 5-#m column preceded by a Supelco Hisep 2 cm × 4.6 mm guard. The column was eluted at 1.50 ml • min-1 with 1% aq HOAc-MeCN (3:1, vol/vol), and the eluent was monitored by detection at 262 rim. Typical elution times were: DPA 2.2 min, PA 2.5 min, ABA 3.7 min. For quantitation, calibration curves were constructed using solutions having an appropriate range of concentrations of known compound, as previously described (Dunstan et al., 1992). Data (Figs. 1-4) are presented as means _+ the SD of each compound vs. time. RESULTS AND DISCUSSION

Somatic embryo cultures and maturation. Immature somatic embryos from type A Norway spruce cultures were a characteristic solar shape, in which elongate suspensorlike cells radiated out from

TABLE 1 MEAN NUMBERS OF COTYLEDONARY SOMATIC EMBRYOS 42 DAYS AFTER PLACEMENT ON FDCs WITH 15 ttM (+)-ABA" Genotype

Cotyledonary Somatic Embryos

SD (+-)

WSiFcryoD WS46 PA86:26A PA88:25B

21.7 0.0 11.0 0.0

10.6 0 6.8 0

a SD is the standard deviation of the mean number shown. Inoculum in each case was 0.15 g fresh weight per FDC.

a central embryo; immature embryos from type B cultures were less developed, having a more diffuse organization of embryo and suspensor tissue as described by Jalonen and von Arnold, 1991. The white spruce immature somatic embryos most closely resemble a third type of Norway spruce culture described by Jalonen and van Arnold, 1991, the polar type. Suspensors or embryos in the white spruce cultures showed varying degrees of lateral fusion with similar tissues. The polar-type embryo typical of the white spruce cultures is termed a stage 1 (immature) somatic embryo as previously described (Hakman and Fowke, 1987; Dunstan et al., 1988). Norway and white spruce maturation commences when immature somatic embryos are placed on medium containing exogenous ABA, and typically leads to the development of an increasingly differentiated polar structure possessing at one pole a series of small cotyledons surrounding a central shoot meristem, and at the other pole a radicle associated with suspensor tissue (stage 3, von Arnold and Hakman, 1988; Hakman and von Arnold, 1988; Dunstan et al., 1988). The embryogenic cultures WS1FcryoD and PA86:26A have been reported previously to be capable of somatic embryo maturation, respectively, by Dunstan et al. (1993) and Jalonen and von Arnold (1991, "A" type) following the general pattern described above. Conversely no maturation has previously been obtained with the embryogenic cultures WS46 (Dunstan et al. unpublished) and PA88:25B (Jalonen and von Arnold, 1991, "B" type). These results were reproduced here (Table 1). Although achievement of a maximum number of stage 3 somatic embryos was not a goal of this research, it should be noted that the numbers of such somatic embryos of white spruce was maximum at 42 days, whereas Norway spruce cultures achieved their maximum at 49 days (data not shown). Abscisic acid metabolism. The analysis of ABA metabolism showed that all genotypes were capable of metabolizing ABA to PA and DPA in very similar ways (Figs. 1-4). In each case, metabolites were recoverable from the agar-solidified medium; the sum of the metabolites and residual ABA essentially equalled the ABA originally supplied. With PA86:26A, and the two white spruce genotypes, about 5 0 % of the ABA remained in the medium after 42 days of culture; previous data indicated that this amount represents the unnatural [(R)-] enantiomer from (+)-ABA (Dunstan et al., 1992). ABA consumption with PA88:25B was slightly less than for its counterpart PA86:26A. Data for each of the two repeat experiments were similar. At the (+)-ABA concentrations used and the low-light intensity under which the Petri dishes were incubated there was no photoisomerism to trans-ABA (Figs. 1-4). Under higher (+)-ABA concentrations there can be measurable photoiso-

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Figs. 1-4. Graphs showing the metabolism of 15 #M racemic abscisic acid by two somatic embryo cultures of white spruce (Fig. 1: WS46, Fig. 4: WS1FeryoD) and two somatic embryo cultures of Norway spruce (Fig. 2: PA88:25B, Fig. 3: PA86:26A), over a 42-day culture period on agar-solidified medium, under low light intensity. Data show compound concentration as means + the SD vs. time. Open circles ABA control, solid circles mean ABA, open squares mean PA, solid squares mean DPA.

merism under various lighting conditions, which effectively reduces the availability of (+)-ABA for the maturation process (Fig. 5). For example, using 30 #M (+)-ABA, after 35 days culture at 85 #E. m-2. s-1, approximately 30% of the initial (_)-ABA was isomerized to trans-ABA; the ABA remaining at 35 days is attributable to non-isomerized (-)-ABA. Figure 5 also shows that (+)-ABA and its metabolites diffused freely through the medium. The results presented indicate that the process of ABA metabolism occurs independentlyof the process of somatic embryo maturation in the material compared. This could be due to the metabolic enzymes being constitutively expressed, or induced independently of maturation-related gene expression. The literature cites evidence in support of constitutive (in corn suspension cultures; Balsevich et

al., 1994) and induced expression (in barley aleurone layers; Uknes and Ho, 1984) of ABA metabolic enzymes. There are several other possibilities why some spruce genotypes show maturation while others do not. For example, there may be some differences in ABA entry into the cells. Balsevich et al. (1994) have shown that ABA enters corn suspension culture cells principally by passive diffusion, although there is a small component attributable to carrier-saturable uptake. Carrier-saturable uptake of ABA has also been reported for carrot suspension cells (Windsor et al., 1994). More likely, there may be repression of inducible ABArelated gene expression associated with somatic embryo maturation but unrelated to ABA metabolism. Our current investigations therefore aim at characterizing patterns of gene expression in different

ABA CONSUMPTION IN SPRUCE CULTURES 120 5.

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a

b

c

FIG. 5. Shows the presence of ABA, trans-ABA, and metabolites of ABA in agar-solidified medium, following 35 days of culture after WS1FcryoD was inoc~ated onto 30/zM (+)-ABA, under high light intensity (85 #E • m-2 • s-l). Sectors show the occurrence of these compounds in a, the region of medium flanking the acetate disc; b, the region beneath the acetate disc but not directly beneath the somatic embryo culture; and c, the region beneath the disc that was immediately below the somatic embryo culture. [] % ABA, [] % trans ABA [] % metabolites.

spruce genotypes under conditions stimulatory to maturation, using ABA.

ACKNOWLEDGMENTS The authors thank Dr. Sara von Arnold for her helpful comments during this work, Drs. Garth and Suzanne Abrams for their guidance with ABA metabolism research, and Mr. Terry Bethune for his assistance with spruce somatic embryo cultures in liquid suspension. REFERENCES Attrce, S. M.; Tantorus, T. E.; Dunstan, D. I., et al. Somatic embryo maturation, germination, and soil establishment of plants of black and white spruce (Picea mariana and Picea glauca). Can. J. Bot. 68:25832589; 1990. Attree, S. M.; Pomeroy, M. K.; Fowke, L. C. Manipulation of conditions for the culture of somatic embryos of white spruce for improved triacylglycerol biosynthesis and desiccation tolerance. Planta 187:395-404; 1992. Balsevich, J. J.; Cutler, A. J.; Lamb, N., et al. Response of cultured maize ceils to (+)-ABA, (-)-ABA and their metabolites. Plant Physiol. in press; 1994. Dunstan, D. I.; Bekkaoui, F.; Pilon, M., et al. Effects of abscisic acid and

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analogues on the maturation of white spruce (Picea glauca) somatic embryos. Plant Sci. 58:77-84; 1988. Dunstan, D. I.; Bethune, T. D.; Abrams, S. R. Racemic abscisic acid and abscisyl alcohol promote maturation of white spruce (Piceaglauca) somatic embryos. Plant Sci. 76:219-228; 1991. Dunstan, D. I.; Bock, C. A.; Abrams, G. D., et al. Metabolism of (+)- and (-)-abscisic acid by somatic embryo suspension cultures of white spruce. Phytochemistry 31:1451-1454; 1992. Dunstan, D. I.; Bethune, T. D.; Bock, C. A. Somatic embryo maturation from long-term suspension cultures of white spruce (Picea glauca). In Vitro Cell. Dev. Biol. 26P:109-112; 1993. Flinn, B. S.; Roberts, D. R.; Taylor, I. E. P. Evaluation of somatic embryos of interior spruce. Characterization and developmental regulation of storage proteins. Physiol. Plant. 82:624-632; 1991. Hakman, I. Embryology in Norway spruce (Picea abies). Immunochemical studies on transport of a seed storage protein. Physiol. Plant. 88:427-433; 1993. Hakman, I.; Fowke, L. C. An embryogenic cell suspension culture of Picea glauca (white spruce). Plant Cell Rep. 6:20-22; 1987. Hakman, I.; von Arnold, S. Somatic embryogenesis and plant regeneration from suspension cultures of Picea glauca (white spruce). Physiol. Plant. 72:579-587, 1988. Jalonen, P.; van Arnold, S. Characterization of embryogenic cell lines of Picea abies in relation to their competence for maturation. Plant Cell Rep. 10:384-387; 1991. Joy, R. W., IV; Yeung, E. C.; Kong, L., et al. Development of white spruce somatic embryos: 1. Storage product deposition. In Vitro Cell. Dev. Biol. 27P:32-41; 1991. Krizek, D. T. Guidelines for measuring and reporting environmental conditions in controlled environment studies. Physiol. Plant. 56:231235; 1982. Litvay, J. D.; Johnson, M. A.; Verma, D., et al. Conifer suspension culture medium development using analytical data from developing seeds. Institute of Paper Chemistry technical paper series no. 115. Appleton, Wisconsin. Roberts, D. R.; Flinn, B. S.; Webb, D. T., et al. Abscisic acid and indole-3butyric acid regulation of maturation and accumulation of storage proteins in somatic embryos of interior spruce. Physiol. Plant. 78:355-360; 1990a. Roberts, D. R.; Sutton, B. C. S.; Flinn, B. S. Synchronous and high frequency germination of interior spruce somatic embryos following partial drying at high relative humidity. Can. J. Bot. 68:10861090; 1990b. Uknes, S. J.; Ho, T.-H. Mode of action of abscisic acid in barley aleurone layers: abscisic acid induces its own conversion to phaseic acid. Plant Physiol. 75:1126-1132; 1984. van Arnold, S.; Eriksson, T. In vitro studies of adventitious shoot formation in Pinus contorta. Can. J. Bot. 59:870-874; 1981. van Arnold, S.; Hakman, I. Regulation of somatic embryo development in Picea abies by abscisic acid (ABA). J. Plant. Physiol. 132:164-169; 1988. Windsor, M. L.; Milborrow, B. V.; Abrams, S. R. Stereochemical requirements of the saturable carrier for abscisic acid in carrot suspension culture cells. J. Exp. Bot. in press; 1994.