In VitroCell.Dev.Biol.29P:109-112. July 1993 © 1993TissueCultureAssociation 1054-5476/93 $01.5040.00

SOMATIC EMBRYO MATURATION FROM LONG-TERM SUSPENSION CULTURES OF

WHITE

SPRUCE

(PICEA GLAUCA)

DAVID I. DUNSTAN, TERRY D. BETHUNE, AND CHERYL A. HOCK

Plant BiotechnologyInstitute, National Research Council Canada, 110 Gymnasium Place, Saskatoon, Saskatchewan, S7N owg, Canada (Received 17 December 1992; accepted 26 February 1993; editor T. A. Thorpe)

SUMMARY The production of cotyledonary somatic embryos of white spruce from cultures grown long-term as suspensions was investigated. We report the effects of removal of 2,4-dichlorophenoxyacetic acid (2,4-D) from the maintenance medium (ordinarily containing both 2,4-D and benzyl adenine) before (+_)-ABA-stimulated maturation. In particular the use of a 1-wk culture period without 2,4-D was found to improve the production of normal-looking cotyledonary somatic embryos. Using high performance liquid chromatography analyses of culture supernatants, it was determined that this affect was not related to altered ABA metabolism. Germination of cotyledonary somatic embryos from cultures pretreated by the 1-wk culture period without 2,4-D was improved compared with similar embryos from cultures that had not been pretreated.

Key words: long-term suspension cultures; white spruce; somatic embryo. tography (HPLC) analyses of ABA metabolism, and by comparison of germination frequencies among the treatments.

INTRODUCTION Embryogenic suspension cultures of Picea glauca have considerable potential in the large-scale automated production of somatic embryos. They also provide an experimental system which facilitates the analysis of, for example, carbohydrate metabolism (Lulsdorf et al., 1992; Tautorus et al., 1992). However, embryogenic suspension cultures of Picea glauca show within-clone differences in their growth rate and morphologic characteristics over the longterm (e.g., after several months of weekly subculture). Differences in number and morphology of somatic embryos, and in the proportion of other cell types which constitute the heterogeneous spruce culture, have been observed (unpublished results). Periodic changes in nutrient medium have been used as one method to reduce such variation (Tautorus et al., 1991). The variability is most evident when aliquots from long-term suspension cultures containing immature spruce somatic embryos are periodically placed on maturation medium with (___)-ABA. Maturation frequencies from such material can fluctuate widely, and the population of aberrant cotyledonary embryos increases relative to normal-appearing cotyledonary embryos (unpublished results). Such differences may result from an alteration in culture sensitivity to phytohormones, or to the carry-over of phytohormones from the maintenance medium to the maturation medium. Either possibility may be due to the longevity of exogenously applied phytohormones, in the medium or in plant tissue (Jacobsen, 1983). Our experiments were designed principally to determine the effects of phytohormone removal from the suspension culture maintenance media used with somatic embryo cultures. We evaluated these effects by monitoring somatic embryo maturation during subsequent culture with (_+)-ABA, by high performance liquid chroma-

MATERIALSANDMETHODS

Plant material. The suspension culture of embryogenic white spruce [Picea glauca (Moench) Voss] used here has been described previously (Dunstan et al., 1988, 1991) and was obtained from an ampoule of cryopreserved material, following the method of Kartha et al. (1988). The suspension culture was incubated in darkness and was routinely maintained over 15 too. by weekly subculture either in autoclaved liquid LP (yon Arnold and Eriksson, 1981) with 1% sucrose, or in autoclaved Liquid 0.5 × LM (Litvay et al., 1981) with 1.5% sucrose, 0.8 g- liter -l casein hydrolysate and 3 mM glutamine, each medium containing 9 #M 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 #M 6-benzylaminopurine. At the time of the experiments, the 15-mo.-old culture consisted of populations of free cells, clusters of isodiametric cells, and immature (stage 1, von Arnold and Hakman, 1988) somatic embryos. Pretreatment. Suspension cultures were subcultured into the above maintenance media (0 wk treatment), or to the equivalent media lacking 2,4-D over periods of 1, 2, 3, or 4 wk before plating under maturation conditions. In each ease, 2 g fresh weight of suspension culture was inoculated into 50 ml of the appropriate medium in 250-ml-capacity DeLong flasks at the start of each weekly culture period. Filter disc cultures. Filter disc cultures (FDC) were prepared and incubated as previously described (Dunstan et al., 1988, 1991). Briefly, filter discs (no. AABG04750; Millipore, Bedford, MA) were placed on agar-solidified (0.5%, wt/vol; no. A-7002, Sigma Chemical Co., St. Louis, MO), autoclaved 0.5 )< LP medium with 1% sucrose, pH 5.6, to which had been added an appropriate filter-steriLizedaliquot of a 1 mM (+)-ABA (A-lO12, Sigma Chemical Co.) stock solution (in acetone/water, 1:9 wt/vol) to give a final concentration of 48 #M. This medium was dispensed into sterile disposable petri dishes (100 × 15 ram), 25 ml per dish. Suspension cultures were harvested, rinsed with phytohormone-free 0.5 X LP medium, and resuspended at 20% (wt/vol) in the same medium. Aliquots of 0.75 ml (equivalent to 0.15 g fresh weight of tissue) were then dispensed onto each filter disc. FDCs were routinely incubated on maturation medium for 63 109

110

DUNSTAN ET AL.

days and observed for their content of cotyledonary (stage 3, von Arnold and Hakman, 1988) somatic embryos. Maturation frequency (MF, %) is described as: the number of stage 3 embryos per FDC )< 100. number of stage 1 embryos plated per FDC

Data collection and analysis. Each treatment regimen was replicated with 10 dishes on each of two separate occasions. Data for the production of cotyledonary somatic embryos maturing on FDCs, relative to prior culture on each maintenance medium, were collected and analyzed independently by one-way analysis of variance. Statistical differences were evaluated using Duncan's multiple range test. Statistical analyses were calculated using Slatgraphics Version 5 (Statistical Graphics Corp., Rockville, MD). Somatic embryo germination. The effect of culture pretreatment was further compared by recording root, hypocotyl, and cotyledon development from the stage 3 somatic embryos placed under germination permissive conditions. To do this, 100 cotyledonary somatic embryos from non-pretreated and 100 from 1 wk pretreated cultures, grown on 0.5 X LM-based medium during maintenance, were placed on phytohormone-free conditions, as previously described (Dunstan et al., 1991). Radicle emergence was scored after 3 wk, and hypoeotyl and cotyledon development were monitored throughout this period. The comparison was made on two separate occasions. ABA metabolism. The metabolism of (---)-ABAin suspension cultures of white spruce pretreated for 1 wk by culture in the absence of 2,4-D was compared with that in non-pretreated cultures, using 0.5 X LM-based medium during maintenance. For the metabolism study, appropriate filtersterilized aliquots of a l mM (-+)-ABAstock solution (in acetone/water, 1:9 wt/vol) were added to previously autoclaved 0.5 × LP medium containing 1% sucrose, to give final concentrations of 0 or 30 #M (-+)-ABA. The medium was dispensed as 25-ml aliquots in 125-ml Erlenmeyer flasks, as previously described (Dunstan et at., 1992). The appropriate 7-day-old liquid suspension cultures were harvested by filtration on two layers of Miracloth, rinsed with phytohormone-free medium, and then resuspended as a 7.5% (wt/vol) solution. Aliquots of 10 ml resuspended culture were then added to each 25 ml (---)-ABA-containingmedium described. These suspensions were then incubated for 8 days. Three replicate flasks of each treatment were inoculated for each of 5 sample days. Each replicate flask was sampled, and extracts were prepared as previously described (Dunstan et al., 1992). ABA and metabolites of ABA in the culture medium were quantified by direct injection HPLC. A 1-ml aliquot of medium was passed through a 0.2-gm filter and a 10-,ul portion of the filtrate was then injected into a Gilson modular HPLC. This was operated at ambient temperature and equipped with a Supelco Hisep 15 cm X 4.6 mm 5-gm column preceded by a Supelco Hisep 2 cm X 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 at 262 nm, as previously described (Dunstan et al., 1992). RESULTSANDDISCUSSION

Pretreatment. Fresh weight gains were obtained under all treatment regimens, and were an indication of culture viability. Two maintenance medium nutrient formulations were compared because long-term maintenance of somatic embryo suspension cultures in hquid LP has proven to be difficult (unpublished observations). The growth of suspension cultures and yield of stage 1 somatic embryos under the treatment regimens were generally greater when cultured on 0.5 × LM compared to LP (Table 1). By Week 2 on media without 2,4-D, suspension cultures had turned brown and had visible aggregates of culture material. The MF for cultures previously grown for 1 wk on LP and 0.5 × LM in the absence of 2,4-D was 4 and 2%, respectively (Table 1). These values, obtained with a suspension culture maintained for 15 mo. before the start of experiments, are much lower than those obtained by Dunstan et at. (1991) for a 6-mo.-old suspension culture, but compara-

ble to those obtained by Dunstan et al. (1988) for an 18-mo.-old suspension culture, in each case using the same clonal suspension line. The better maturation frequency obtained with the younger suspension culture may be a consequence of culture age. Analyses of data for yield of stage 3 embryos after 42 days on FDCs (Table 2) showed significant differences among phytohormone treatment regimens (P < 0.05) with each maintenance medium. Data for the production of stage 3 embryos (Table 2) show that there was a beneficial affect of the prior 1 wk removal of 2,4-D from each maintenance medium. Longer pretreatment periods resuited in a reduction in number of stage 3 embryos. Analyses of data for yield of aberrant stage 3 embryos (i.e. those embryos with an asymmetry of their cotyledons or with malformed cotyledons) after 42 days on FDCs (Table 2) also showed significant differences among phytohormone treatment regimens (P < 0.05). Data for the production of aberrant stage 3 embryos (Table 2) show that the greatest amount, approximately 71% of the total, was produced with LP as the maintenance medium containing 2,4-D immediately before maturation. The beneficial affect of the 1-wk culture period without 2,4-D during maintenance on this medium can be seen in the consequent reduction in aberrant embryo yield to 23% of the total yield. Somatic embryo germination. Cotyledonary somatic embryos from 1-wk pretreated cultures showed an average 86% germination after 3 wk, compared with 59% for non-pretreated somatic embryos. The development of cotyledons and hypocotyls was equivalent with each group. ABA metabolism. Ana/ysis of suspension culture fi/trates during 8 days of culture in the presence of (+)-ABA showed that essentially similar patterns of ABA metabolism had occurred for the nonpretreated cultures, and for those cultures pretreated for 1 wk by culture in the absence of 2,4-D. The (+)-ABA was partially metabohzed during the culture period leading to the production of phaseic acid (PA) and dihydrophaseic acid (DPA) (Fig. 1 a,b). The level of ABA at the conclusion of the experiment was approximately half the amount initially supplied, representing the non-metabolized ( - ) ABA enantiomer. This pattern of metabolism resembles that reported previously (Dunstan et at., 1992). The similarity between the non-pretreated and the prctreated cultures indicates that the effect of the pretreatment was not due to altered ABA metabolic activity. These results indicate that the conditions of suspension culture maintenance affect subsequent culture response to maturation conditions. Improved maturation and germination responses were obtained when 2,4-D was removed from the maintenance medium for 1 wk before inoculation onto maturation conditions. Preliminary experiments had indicated that the greatest impact on somatic embryo maturation was the total removal of 2,4-D from maintenance medium, in comparison with the use of various BA and 2,4-D combinations (unpubhshed results). 2,4-D has been reported to be retained in pea tissue as free auxin, a result of its high mobility and low rate of oxidation and conjugation (Jacobsen, 1983). The consequence of this in the long-term suspension cultures used here could be retention, of 2,4-D between consecutive culture passages. This could include carry-over of 2,4-D into the (+)-ABA-containing maturation media, even though cultures were previously rinsed with phytohormone-free medium (Dunstan et at., 1988). Other authors have used charcoal before maturation treatments with conifer so-

111

SOMATIC EMBRYO MATURATION IN WHITE SPRUCE TABLE 1 GROWTH PARAMETER (F. WT. AND STAGE 1 EMBRYOS) MEAN VALUES OF SUSPENSION CULTURES WHEN GROWN UNDER DIFFERENT TREATMENTS GIVEN BEFORE MATURATION, AND CORRESPONDING MEAN VALUES FOR INOCULUM DENSITY AND NORMAL STAGE 3 EMBRYO PRODUCTION (MF~) Treatment, b week

Maintenance Medium b

0 1 2 3 4

0.5 0.5 0.5 0.5 0.5

LM + LM + LM + LM + LM +

0

LP + BA/2,4-D

1 2 3 4

LP + LP + LP + LP +

F. wt./Flask, g"

Stage ] Embryos, c × l O s

lnoculum per FDC (0.15 g),~ × 1 0 s

MF, %

5.6 8.3 6.3 9.7 5.0

52.9 87.4 43.2 28.7 7.4

1.4 1.6 1.0 0.43 0.23

0.9 2.0 2.4 0.5 2.0

1.8 4.5 4.2 2.9 4.5

18.6 29.0 16.4 11.5 9.5

1.5 0.97 0.58 0.62 0.32

0.6 4.0 0.4 0.5 1.5

BA/2,4-D BA BA BA BA

BA BA BA BA

° (MF, %, the number of stage 3 embryos per FDC/number of stage 1 embryos plated per FDC × 100). b Duration of treatment, and the medium used during the treatment. LP medium (yon Arnold and Eriksson, 1981) with 1% sucrose, or 0.5 X LM (Litvay et al., 1981) with 1.5 % sucrose, 0.8 g. 1-1 casein hydrolysate and 3 mM glutamine, each medium containing 9 ttM 2,4-D and 4.4 #M BA. c Yield of suspension culture grown under each treatment regimen, in grams fresh weight (f.wt.), and mean total number of embryos per culture flask. Inoculation density of stage 1 somatic embryos/0.15 g f.wt. tissue, inoculated in 0.75 ml of a 20% wt/vol dilution of suspension culture for maturation.

matic embryo cultures (Becwar et al., 1987; Hakman et al., 1985; Roberts et al., 1990). Charcoal has been used in situations in which it was desirable to remove inhibitory substances or excess phytohormones (Thorpe and Patel, 1984; Gaspar and Coumans, 1987). When used in preliminary experiments with the white spruce culture, a 1-wk pretreatment on phytohormone-free medium with activated charcoal did not improve subsequent embryo maturation (unpublished observations). One week of 2,4-D removal from the maintenance medium in itself did not have a direct influence on ABA metabolism. This is consistent with our observation that embryogenic cultures are capable of metabolizing exogenous ABA, irrespective of their abilities to mature.

Non-optimal nutrient supply in the maintenance medium used with suspension cultures could also contribute to altered maturation responses, such as those identified by Tautorus et al. (1991), and by Tremblay and Tremblay (1991a,b). In addition, the suspension culture milieu places a selection pressure on culture material, favoring the evolution of a fast-growing culture-type which could have altered responses when the culture is exposed to exogenous ABA to promote maturation. This may be the cause of the differences in maturation frequency obtained with the different ages of this white spruce suspension culture. Such evolution in shake-flask culture may result in periodic variation in response during research evaluations. Our future experiments will directly compare the maturation

TABLE 2 STAGE 3 EMBRYO PRODUCTION PER MATURATION DISH AFTER 42 DAYS, RELATIVE TO MAINTENANCECONDITIONSa Treatment? Week

Maintenance Medium b

0 1 2 3 4

0.5 0.5 0.5 0.5 0.5

LM + LM + LM + LM + LM +

0 1 2 3 4

LP LP LP LP LP

+ + + + +

BA/2,4-D BA BA BA BA

BA/2,4-D BA BA BA BA

Total St. 3 Embryos

Norm St. 3 Embryos

Percent

Aberrant St. 3 Embryos

27.3 43.6 37.3 0.3 9.4

17.1 32.1 25.0 0.15 4.45

+_ 2.9c ~ + 2.8a + 2.8b + 0.08d ± 1.2d

63 74 67 50 47

10.2 11.6 12.3 0.15 4.95

+ + + + ±

1.2a 1.5a 1.1a 0.08c 1.2b

30.7 48.2 3.0 27.7 6.1

8.9 37.1 2.15 22.4 4.75

± 1.4c + 4.2a -4- 0.5e -+ 5.9b -+ 1.1e

29 77 72 81 78

21.8 11.1 0.80 5.32 1.35

+ 2.1a + 1.5b + 0.2d ± 1.0c +-- 0.4d

Percent

37 26 33 50 53 71 23 28 19 22

a Data are mean numbers for total stage 3 embryos, and mean numbers ± standard error of the mean and percentage proportions of each type of embryo; b duration of treatment, and the medium used during the treatment. LP medium (yon Arnold and Eriksson, 1981) with 1% sucrose, or 0.5 X LM (Litvay et al., 1981) with 1.5% sucrose. 0.8 g" liter -l casein hydrolysate and 3 mM glutamine, each medium containing 9/.tM 2,4-D and 4.4 #M BA; c Means with the same letters are not significantly different (P < 0.05).

112

DUNSTAN ET AL.

A 0

B 30

0

2

4

6

8

DAYS IN CULTURE

i

0

.

.

2

.

.

4

6

8

DAYS IN CULTURE

FI¢. 1. Themetab~ism~f3~M(+)-ABAins~maticembry~suspensi~ncu~turesprevi~us~yn~n-pretreated(npt)~rpretreated(pt)~ as described in Materials and Methods. A, solid squares DPA (npt); solid triangles PA (npt); solid circles racemic ABA (npt). B, open squares DPA (pt); open triangles PA (pt); open circles raeemic ABA (pt).

responses of somatic embryos from suspension and tissue cultures differing in age, taking advantage of cryopreserved material. ACKNOWLEDGEME~'S The authors thank Drs. Garth and Suzanne Abrams for their guidance with ABA metabolism research. This manuscript is NRCC no. 35894. REFERENCES Becwar, M. R.; Noland, T. L.; Wann, S. R. Somatic embryo development and plant regeneration from embryogenic Norway spruce callus. TAPPI J. 70:155-160; 1987. Dunstan, D. I.; Bekkaoui, F.; Pilon, M., et al. Effects of abscisic acid and 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 (Picea glauca) somatic embryos. Plant Sci. 76:219-228; 1991. Dunstan, D. I.; Bock, C. A.; Abrams, G. D., et al. Metabolism of (+)- and (-)-abscisic acid hy somatic embryo suspension cultures of white spruce. Phytochemistry 31:1451-1454; 1992. Gaspar, Th.; Coumans, M. Root formation. In: Bonga, J. M.; Durzan, D. J., eds. Cell and tissue culture in forestry, vol 2. Dordrecht, Netherlands: Martinus Nijhoff; 1987:202-217. Hakman, I.; Fowke, L. C.; yon Arnold, S., et al. The development of somatic embryos in tissue cultures initiated from immature embryos of Picea abies (Norway spruce). Plant Sci. 38:53-59; 1985. Jacobsen, H.-J. Biochemical mechanisms of plant hormone activity. In: Evans, D. A.; Sharp, W. R.; Ammirato, P. V., et al., eds. Handbook of plant cell culture, vol 1. New York: MacMillan Publishing; 1983:672-695. Kartha, K. K.; Fowke, L. C.; Leung, N. L., et al. Induction of somatic embryos and plantlets from eryopreserved cell cultures of white spruce (Picea glauca). J. Plant. Physiol. 132:529-539; 1988.

Litvay, J. D.; Johnson, M. A.; Verma, D., et al. Conifer suspension culture medium development using analytical data from developing seeds. Appleton, WI: Institute of Paper Chemistry technical paper series no. 115:1981. Lulsdorf, M. M.; Tautorus, T. E.; Kikcio, S. I., et al. Growth parameters of embryogenic suspension cultures of interior spruce (Picea glauca engelmannii complex) and black spruce (Picea mariana Mill.). Plant Sci. 82:227-234; 1992. Roberts, D. R.; Fhnn, 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; 1990. Tautorus, T. E.; Fowke, L. C.; Dunstan, D. I. Somatic embryogenesis in conifers. Can. J. Bot. 69:1873-1899; 1991. Tautorus, T. E.; Lulsdorf, M. M.; Kikcio, S. I., et al. Bioreactor culture of Picea mariana Mill. (black spruce) and the species complex Picea glauca-engelmannii (interior spruce) somatic embryos. Growth parameters. Appl. Microbial. Biotech. 38:46-51; 1992. Thorpe, T. A.; Patel, K. R. Clonal propagation: adventitious buds. In: Vasil, 1. K., ed. Cell culture and somatic cell genetics of plants, vol 1. Orlando: Academic Press; 1984:49-60. Tremblay, L.; Tremblay, F. M. Carbohydrate requirements for the development of black spruce (Picea mariana (Mill.) B.S.P.) and red spruce (P. rubens Sarg.) somatic embryos. Plant Cell Tissue Organ Cult. 27:95-103; 1991a. Tremblay, L.; Trcmblay, F. M. Effects of gelling agents, ammonium nitrate, and light on the development of Picea mariana (Mill.) B.S.P. (black spruce) and Picea rubens Sarg. (red spruce) somatic embryos. Plant Sci. 77:233-242; 1991b. yon Arnold, S.; Eriksson, T. In vitro studies of adventitious shoot formation in Pinus contorta. Can. J. Bot. 59:870-874; 1981. yon Arnold, S.; Hakman, I. Regulation of somatic embryo development in Picea abies, with emphasis on ABA effects. J. Plant Physiol. 132:164-169; 1988.