Plant Cell Reports (1997) 16:550-554

© Springer-Verlag 1997

D. J. Carrier. J. E. C u n n i n g h a m • D. C. Taylor D. I. Dunstan

Sucrose requirements and lipid utilization during germination of interior spruce (Picea glauca engelmannii complex) somatic embryos

Received: 10 July 1996 / Revision received: 6 December 1996 / Accepted 31 December 1996

Abstract Both somatic and excised zygotic embryos of interior spruce (Picea glauca engelmannii complex) required exogenous sucrose in the medium for germination in vitro. Over a period of 29 days on sucrose-containing medium germinants with roots and epicotyls developed from both kinds of embryo, and their content of linolenic acid (9,12,15-18:3) increased about six- to eightfold. Without added sucrose, embryos showed retarded growth or were necrotic, and the content of linolenic acid was barely detectable in their fatty acid profiles. Through ~4C-sucrose uptake studies, it was determined that germinants consumed only 25% of the sucrose available in a 1% (wt/vol) sucrose-containing medium. Since no radiolabelled fatty acids were detected, it appears that externally supplied sucrose was not used in the synthesis of lipids. Although sucrose was present during plantlet development, 72% of the initial lipids were consumed. To some extent, the plantlets appeared to be obligate storage lipid utilizers. Key words Carbohydrates • Fatty acid • Germination Somatic embryogenesis Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid • FAMEs Fatty acid methyl esters. HPLC High-performance liquid chromatography

Communicated by F. Constabel D. J. Carrier 1 • J. E. Cunningham D. C. Taylor. D. I. Dunstan ([]) Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, Saskatchewan, S7N OW9, Canada Fax: +1-306-975-4839

Present address: 1 Agricultural and Bioresource Engineering Department, University of Saskatchewan, 57 Campus Drive, Saskatoon, Saskatchewan, S7N 5A9, Canada

Introduction Spruce somatic embryogenesis has been well documented in recent years, and its use for the establishment of trial field plantings has been realized (Attree et al. 1992, 1995; Dunstan et al. 1991, 1993; Roberts et al. 1993; Webster et al. 1990). Synthetic conifer seeds (synseed, artificial seed, encapsulated embryos) have the potential to be an integral step in the transition from in vitro culture to greenhouse or field planting. To date there are two principal methods of synthetic seed production which have been described for use with conifer somatic embryos. Lulsdorf et al. (1993) developed a procedure involving the use of a hydrated alginate capsule surrounding the somatic embryo and containing added nutrients, and Attree et al. (1995) suggest the direct use of desiccated somatic embryos with high lipid content. Whichever type of synthetic seed is used, it will be essential that the germinant has a sufficient energy supply to support its development. In spruce plantlet development, the accumulation of adequate lipid reserves and their subsequent utilization is apparently of key importance. Cyr et al. (1991) and Attree et al. (1992) have reported a decrease in total lipid content during germinant development after mature excised zygotic or somatic spruce embryos were placed on germination medium with sucrose. Because lipids are an integral component of the embryo and are degraded during germinant development even in the presence of exogenous sucrose, we investigated the relationship between sucrose requirement and lipid degradation. Understanding the carbon requirements of the germinants until photoautotrophy will provide useful information for the development of an artificial seed. This report presents our findings on sucrose requirements during germinant and plantlet development from interior spruce somatic and excised zygotic embryos.

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Material and methods Embryogenic cultures Embryogenic tissue cultures of interior spruce (Picea glauca engelmannii complex) were initiated by British Columbia Research Inc., Forest Biotechnology Centre (BCRI/FBC,Vancouver, British Columbia, Canada), from a 1990 full-sib cross of tree PG2 (F, growthrank 5/173) xtree PG96 (M, growth-rank 84/173) (Prince George Selection Unit, Kalamalka Research Station, British Columbia Ministry of Forests and Lands, British Columbia, Canada). Isolated mature zygotic embryos were placed on 0.5xLM medium (Litvay et al. 1981), with 1.5% (wt/vol) sucrose, 0.8 g 1-1 casein hydrolysate, 3 mM glutamine, 9 #M 2,4-dichlorophenoxyacetic acid (2,4-D), 4.4 pM N6-benzyladenine, and 8 g 1-~ agar, at pH 5.6. Embryogenic cultures obtained from BCRI/FBC were maintained on agar-solidified medium as above and then transferred into suspension cultures. This involved placing 4-6xl-cm-diameter portions of tissue into 50 ml liquid 0.5xLM medium in 250-ml baffle flasks agitated at 150 rpm in the dark at 25°C. Cultures were allowed to equilibrate for 1-2 weeks, after which they were subcultured each week by transfer of 10 ml suspension culture into 50 ml fresh nutrient medium in 250 ml capacity non-baffle flasks. Cultures had been maintained as liquid suspensions for 6 months before the start of these experiments. Somatic embryo maturation Prior to somatic embryo maturation, suspension cultures were grown for 1 week in nutrient medium without 2,4-D (Dunstan et al. 1993). Immediately following this, the tissue was placed under maturation conditions after Dunstan et al. (1988, 1993). Briefly, suspension cultures were harvested, weighed, washed and resuspended to 20% (wt/vol) with phytohormone-free 0.5×LP medium (yon Arnold and Eriksson 1981). Aliquots of 0.75 ml (150 mg fresh mass) were dispensed onto filter disks (AABG047SO, Millipore, Bedford, MA) placed over maturation medium [0.5×LP medium, 0.5% (wt/vol) agar, 3% (wt/vol) sucrose (unless otherwise specified) with 48 pM filter-sterilized (_+)-abscisic acid (A-1012, Sigma, St. Louis, MO), pH 5.8] in Petri dishes (100x15 mm, 25 ml/dish). The cultures were then incubated for 42 days at 25°C and 25 cd sr-~ m -2 for 16 h day -1 (40 W cool white fluorescent lamps). After 42 days, morphologically normal cotyledonary embryos with a symmetric arrangement of cotyledons, defined as stage three embryos, were harvested. Zygotic embryos Interior spruce seeds obtained from BCRI/FBC were soaked overnight in running tap water, and surface sterilized for 2 min in a 3% (vol/vol) sodium hypochlorite solution, to which a few drops of dishwashing detergent were added. After thorough rinsing with sterile water, the embryos were removed and placed on 0.5xGMD (Mohammed et al. 1986) germination medium [0.8% (wt/vol) agar, 0.1% (wt/vol) sucrose, pH 5.8]. Twenty excised zygotic embryos were placed into each Petri dish. The experiment had at least three replicates. Germination of somatic embryos, and use of 14C-sucrose Twenty morphologically normal stage-three somatic embryos were placed onto germination medium as described, with or without 1.0% (wt/vol) sucrose. Cultures were incubated for 42 days at 25°C and 2092 cd sr- 1 m- 2 for 16 h day- 1 (40 W cool white fluorescent lamps). For the radiolabelling experiment, the 0.5xGMD germination medium contained 1% (wt/vol) uniformly labelled a4C-sucrose (2.2x106 dpm, NEN-Dupont Biotechnology, Wilmington, DE; specific activity 0.11 MBq/mmol). The experiment had at least three replicates.

Lipid and 14C-label analysis Margaric acid (17:0) was added to all samples as an internal standard. Total lipid extracts of lyophilized embryos were prepared as described in Carrier et al. (1995). It should be noted that germinants grown in the presence of labelled 14C-sucrose were extracted without lyophilization. In our hands, lyophilization decreased the efficiency of lipid recovery by up to 70%, the data shown in Figures 1 and 2 are therefore presented as percentage values. Fatty acid methyl esters (FAMEs) were prepared, and analyzed by gas chromotography as described by Holbrook et al. (1992). The fatty acids monitored were palmitic acid (16:0), oleic (9-18:1), linoleic (9,12-18:2) and linolenic (9,12,15-18:3). The fatty acid 5,9,12-18:3 (Carrier et al. 1995) was also monitored. The prepared FAMEs were also analysed by radio-high-performance liquid chromatography (HPLC) as described by Taylor et al. (1992). Samples from the extraction procedure were diluted to a 1:4 proportion of fraction: scintillation cocktail (Aquasol-2/DuPont, Biotechnology Systems, Boston, MA), and counted (LKB 1219 Rack-Beta instrument, LKB-Wallac Oy, Turku, Finland). Data are presented as means _+SD.

Results and discussion Development of somatic embryos on germination media with or without sucrose The utilization of the total lipid pool and the formation of linolenic acid (9,12,15-18:3) were monitored in cultures developing either on sucrose-free germination medium or

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on medium with 1% (wt/vol) sucrose. Embryos placed on germination medium with sucrose developed roots and epicotyls, and increased their fresh mass by about 13-fold. Figure 1 shows the utilization of the total lipid pool (Fig. 1A) and the appearance of linolenic acid (Fig. 1B) for somatic embryos placed on either type of germination medium. With 1% (wt/vol) sucrose, utilization of lipid reserves occurred between approximately days 0 and 11. A small increase in endogenous total lipids was observed between days 11 and 29. The concentration of linolenic acid was low in embryos at day 0, and increased when they were placed on germination medium with sucrose. On sucrose-free germination medium, embryos failed to develop. The total lipid content in such material declined throughout the monitored period, and linolenic acid was not formed.

Development of excised zygotic embryos on germination media with or without sucrose To determine if the initial lipid content of zygotic embryos was sufficient to allow germination without any external source of carbohydrates, embryos were excised from seeds and placed either on sucrose-free germination medium or on medium with 0.1% (wt/vol) sucrose. Embryos placed on medium with sucrose developed into germinants with

roots and epicotyls, while those placed on sucrose-free medium either became necrotic or showed stunted growth. At 29 days, the dry mass of excised zygotic embryos on germination media, with or without sucrose, was 797+ 118 gg embryo -1 and 290_+72 ~tg embryo -1, respectively. Figure 2 shows the utilization of the total lipid pool (Fig. 2A) and the appearance of linolenic acid (Fig. 2B) in excised zygotic embryos on each type of medium. When developing on medium with sucrose, excised embryos utilized 62% of their total lipid pool between days 0 and 11. The content of linolenic acid increased between days 5 and 29. When excised zygotic embryos were placed on sucrose-free medium, 76% of the total lipid reserve was utilized over the 29-day observation period, and no synthesis of linolenic acid occurred. These results suggest that the lipid reserves of the excised zygotic embryo are not sufficient to support growth and development. Our results show that germination medium supplemented with sucrose can support somatic embryo and excised zygotic embryo development and subsequent germination. During normal seed germination, the lipids contained in the megagametophyte are metabolized to malate via the glyoxylate cycle, leading ultimately to sucrose synthesis (Ching 1970; see Mullen and Gifford 1995, and references therein). The sucrose is made available to the developing embryo until photoautotrophy is reached. The carbon skeletons provided by the megagametophyte are essential for seedling development.

Development of somatic embryos on ]4C-sucrose-supplemented germination medium To further understand the role of sucrose as a supplement in the germination medium, somatic embryos were incubated on germination medium with14C-sucrose. Substrate uptake from the medium and the fresh mass increases of somatic embryos are shown in Fig. 3. Within the 24-day time course, an increase of 25 mg in somatic embryo fresh mass was recorded. Embryos consumed 25% of the avail-

553 4000-

counting; the results are presented in Fig. 4. Up to day 11, the aqueous phase, composed primarily of hydrophilic proteins and soluble carbohydrates, showed constant counts, -~ 3000whereas on days 18 and 24, increased counts were re~- 2500o corded. The insoluble phase, composed mainly of starch ~" 2000and cellulose products, showed an increase in counts up to E 1500day 18. A conversion of 14C-starch to ~4C-sugar could pos1000sibly explain the drop in counts observed on day 24. How:~ 500Q.. ever, this remains to be verified. The ~4C counts in the orO" ganic phase remained constant throughout the monitored 3 5 11 18 24 period. To determine if the organic phase contained laTime (days) belled fatty acids, samples were transmethylated and anaFig. 4 Fresh somatic embryos: amount of 14C label in the organic lysed by radio-HPLC. No labelled fatty acids were detected (left bar) aqueous (right bar) and insoluble (middle bar) phases of during the monitored period, indicating that within the limtotal lipid extracts during 24 days of development on germination • " medmm with I°Ao (wt/vol) 1 4 C-sucrose. Twenty embryos per Petn its of detection, labelled sucrose was not utilized for lipid dish, with at least three dishes per experiment. Values are means _+SD biosynthesis. This suggests that the label detected in the organic phase was labelled carbon incorporated into nonfatty acyl organic molecules such as photosynthetic pig40 ments. Utilization and synthesis of major fatty acids during ger35 minant development on medium with sucrose were moni3O o tored during the time period, as presented in Fig. 5. In ad_~ 25 A dition to the relative abundance of total lipids (Fig. 5B) E 180and of linolenic acid (Fig. 5A), as noted earlier, depletion -B 20 160of linoleic acid (9,12-18:2) ended after 11 days and re=15 140::zk o mained constant for the remainder of the period (Fig. 5B). 10. .B 120Palmitic acid (16:0) and oleic acid (9-18:1) were consumed E100until day 11, and some accumulation was observed until .z2_ 80o_ day 24 (Fig. 5A). On the other hand, the fatty acid 5,9,1260 3 5 11 18 24 18:3 was consumed, and re-accumulation was not subse=L Time (days) 40 quently observed (Fig. 5B). The presence of this fatty acid 20 in interior spruce somatic embryos is consistent with other reports in conifers (Jamieson and Reid 1972; Ekman 1980; 1 3 5 11 18 24 Nilsson and Liljenberg 1991; Olsson et a1.1994; Carrier et Time (days) al. 1995). The amount of linolenic acid appears to be inB 180 versely proportional to the amount of 5,9,12-18:3. Olsson 160 et al. (1994) attributed the increase of linolenic acid to the 140 presence of chloroplasts dominated by membrane galactoO ~'120 lipids rich in this fatty acid. The appearance of linolenic 10o acid could be a potential marker for successful germinant -if3 -~ 8 0 development and assessment of artificial seed formula60 tions. :::L 40 In conclusion, our data show that the addition of sucrose 20 was necessary for the viability of somatic and zygotic embryos, and for subsequent development during germina3 5 11 18 24 tion. Embryos showed an apparent obligate requirement Time (days) for the carbohydrate. Based on the amount of label detected Fig. 5 A, B Fatty acid utilization and synthesis in fresh somatic em- in the sampled germination medium, developing somatic bryos during 24 days of development on germination medium with embryos consumed sucrose; however, there was a decrease 1% (wt/vol) sucrose. Twenty embryos per Petri dish, with at least three dishes per experiment. Values are menas _+SD. A oleic acid of only 25% of the initial carbohydrate. The external su(9-18:1; left bar), palmitic acid (16:0; middle bar) and linolenic crose was not used for the synthesis of new lipids. Although acid (9,12,15-18:3; right bar). B Total lipids (left bar), linoleic ac- somatic embryos were cultured in the presence of excess id (9,12-18:2; middle bar), and 5,9,12-18:3 (right bar) sucrose, they still consumed 72% of their internal total lipid reserves during development (Fig. 5B). To some extent, able sucrose, showing that substrate is not limiting in ger- the developing spruce embryos also appeared to be oblimination medium with 1% (wt/vol) sucrose. Embryos were gate storage lipid utilizers. Lipid degradation is most likely harvested and analysed for their total lipids throughout the an inherent part of the germination program (Mullen and extraction procedure, and ~4C present in the aqueous, or- Gifford 1995, and references therein). From a synthetic ganic and insoluble phases was monitored by scintillation seed perspective, addition of sucrose to germination me3500-

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554 d i u m is a r e q u i r e m e n t for spruce somatic e m b r y o d e v e l o p ment. H o w e v e r , the role of the external sucrose supply, and its contribution to e m b r y o d e v e l o p m e n t require further clarification. It is p o s s i b l e that it has m o r e than one role, for e x a m p l e acting as a carbon s u p p l y m i m i c k i n g translocared photosynthates, and having an osmotic effect w h i c h influences e m b r y o d e v e l o p m e n t . M o n i t o r i n g the d e v e l o p m e n t of somatic e m b r y o s e n r i c h e d in lipid content, such as those p r o d u c e d by A t t r e e et al. (1992, 1995), in the presence or absence of an external sucrose s u p p l y c o u l d provide further i n f o r m a t i o n on the a p p a r e n t l y o b l i g a t e cons u m p t i o n o f lipid stores and c a r b o h y d r a t e as r e p o r t e d here.

Acknowledgements The authors wish to thank British Columbia Research Inc. (Vancouver, British Columbia, Canada) for supplying the embryogenic spruce cultures and interior spruce seeds. We are gratefully to Mr. Darwin Reed for assistance with radio-HPLC analyses. This work was funded by the National Research Council of Canada through technology contribution agreement GC 103-3-2021 between the National Research Council of Canada and British Columbia Research Incorporated. This is NRCC publication no. 40028.

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Dunstan DI, Bekkaoui F, Pilon M, Fowke LC, Abrams SR (1988) Effects of abscisic acid and analogues on the maturation of white spruce (Picea glauca) somatic embryos. Plant Sci 58:77-84 Dunstan DI, Bethune TB, Abrams SR (199I) Racemic absciscic acid and abscisyl alcohol promote maturation of white spruce (Picea glauca) somatic embryos. Plant Sci 76:219-228 Dunstan DI, Bethune TB, Bock CA (1993) Somatic embryo maturation from long-term suspension cultures of white spruce (Picea glauca). In Vitro Cell Dev Biot 29P: 109-112 Ekman R (1980) New polyenoic fatty acids in Norway spruce wood. Phytochemistry 19:147-148 Holbrook L, Magus J, Taylor DC (1992) Abscisic acid induction of elongase activity, biosynthesis and accumulation of very long chain mono-unsaturated fatty acids and oil body proteins in microspore-derived embryos of Brassica napus L. cv Reston. Plant Sci 84:99-115 Jamieson GR, Reid EH (1972) The leaf lipids of some conifer species. Phytochemistry 11:269-275 Litvay JD, Johnson MA,Verma DC, Einspahr D, Weyrauch K (1981 ) Conifer suspension culture medium development using analytical data from developing seeds. Institute of Paper Chemistry Technical Paper Series no 115, Appelton, WI Lulsdorf MM, Tautorus TE, Kikcio SI, Bethune TD, Dunstan DI (1993) Germination of encapsulated embryos of interior spruce (Picea glauca engelmannii complex) and black spruce (Picea mariana Mill.). Plant Cell Rep 12:385-389 Mohammed GH, Dunstan DI, Thorpe TA (1986) Influence of nutrient medium shoot initiation on vegetative explants excised from 15 to 18-year-old Picea gIauca. N Z J For Sci 16:297-305 Mullen RT, Gifford DJ (1995) Isocitrate lyase from germinating loblolly pine megagametophyte enzyme purification and immunocharacterization. Plant Physiol Biochem 33:87-95 Nilsson R, Liljenberg C (1991) The determination of double bond positions in poly-unsaturated fatty acids-GC/MS of the diethylamide derivative. Phytochem Anal 2:253-259 Olsson M, Nilsson R, Norberg R Arnold S von, Liljenberg C (1994) Maturation in Norway spruce: alterations in lipid composition. Plant Physiol Biochem 32:225-232 Roberts DR, Webster FB, Flinn B, Lazaroff W, Cyr DR (1993) Somatic embryogenesis of spruce. In: Redenbaugh K (ed) Synseeds: applications of synthetic seeds to crop improvement. CRC, Boca Raton, pp 427-450 Taylor DC, Barton D, Rioux K, MacKenzie S, Reed D, Underhill E, Pomeroy K, Weber N (1992) Biosynthesis of acyl lipids containing very-long chain fatty acids in microspore derived and zygotic embryos of Brassica napus L. cv Reston. Plant Physiol 99:1609-1618 Webster FB, Roberts DR, McInnis S, Sutton BC (1990) Propagation of interior spruce by somatic embryogenesis. Can J For Res 20:1759-1765