Kinetics of Douglas-fir (Pseudotsuga menziesii)

somatic embryo development

Ryan P. Taber, Chun Zhang, and Wei-Shou Hu

Abstract: Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) is one of the most economically important softwood species inthe Pacific Northwest region. Somatic embryogenesis is a potential mass propagation technology for increasing theproductivity of existing forest acreage. Combined with traditional breeding methods and recent advances of geneticengineering in plant species, somatic embryos can shorten the elite clone selection process significantly. Somatic embryoculture of Douglas-fir involves three stages: maintenance, abscisic acid (ABA) singulation, and maturation. At the beginningof all stages of culture, the population of cells with embryogenic potential is increased through the weekly subculturedmaintenance stage; transfer into the ABA singulation stage initiates embryo development, while cotyledonary embryos areformed in the maturation state. The first two stages were carried out in submerged suspension culture, while during thematuration stage the developing embryos were placed on a polyester pad in a Petri dish. The growth kinetics in these stageswere investigated. Fresh and dry weights were observed to double in the maintenance stage, while a smaller increase occurredin the ABA singulation stage. NH4

+ was consumed preferentially to NO3– in all culture stages. Sucrose, the primary carbon

source, was hydrolyzed to glucose and fructose rapidly. During cultivation, glucose and fructose were consumedsimultaneously. The hydrolysis of sucrose resulted in a slight osmolarity increase at the beginning of all culture stages, whilethe subsequent consumption of glucose and fructose coincided with a gradual decrease in osmolarity. This dynamicosmolarity pressure profile is most profound in the maturation stage, in which the initial high osmotic pressure of600 ± 20 mOsm/kg (mean ± SD) increased to 700 ± 27 mOsm/kg after sucrose was hydrolyzed but decreased to350 ± 14 mOsm/kg after the depletion of sugars at the end of cultivation. The complete process of embryo development,from the week-long maintenance culture, through the weekly subcultured ABA singulation culture, to the maturation ofembryos took between 70 and 80 days. Each millilitre of culture present at the onset of maintenance culture gave rise toapproximately 420 mature embryos. During that same time period, the biomass increased approximately 100 times.Prolonging the cultivation time failed to increase the yield of mature embryos. These results give a more complete view of thekinetic behavior of developing Douglas-fir embryos and will aid in the optimization and scale-up of this important process.

Key words: somatic embyrogenesis, Douglas-fir, Pseudotsuga menziesii, growth kinetics, ABA, osmolarity, development.

Résumé: Le sapin Douglas (Pseudotsuga menziezii (Mirb.) Franco) est une des espèces à bois mou des plus économiquementimportantes de la région du Pacific Nord-Ouest. L’embryogénèse somatique est une technologie de production en masseprometteuse pour augmenter la productivité des surfaces forestières existantes. Combinés avec les méthodes d’améliorationgénétique existantes et les progrès récents en génie génétique des espèces végétales, les embryons somatiques peuventraccourcir le processus de sélection des clones élites de façon significative. La culture des embryons somatiques du sapinDouglas implique trois étapes : maintien, individualisation par l’acide abscissique (ABA) et maturation. Au début de cesétapes de la culture, on augmente la population de cellules à potentiel embryogène par repiquage hebdomadaire sur milieugélosé, puis on transfert dans le milieu avec ABA pour l’initiation et l’individualisation des embryons et finalement lesembryons cotylédonaires se développent au cours de la maturation. Les deux premières étapes s’effectuent par culture ensuspension submergée, alors qu’au stade de la maturation, le développement des embryons se poursuit sur un coussin depolyester en plaque de Pétri. Les auteurs ont examiné la cinétique de croissance à ce stade. On observe un doublement despoids frais et secs au stade de maintien, alors qu’une augmentation plus faible se manifeste au stade d’individualisation avecl’ABA. À tous les stades de cultures le NH4

+ est préféré au NO3–. Le sucrose, source de carbone primaire, est rapidement

hydrolysé en glucose et fructose. Au cours de la culture le glucose et le fructose sont consommés simultanément. L’hydrolysedu sucrose augmente légèrement l’osmolarité en début de culture, alors que la consommation subséquente du glucose et dufructose s’accompagne d’une diminution graduelle de l’osmolarité. Ce profile dynamique de pression osmotique est plusmarqué au stade de maturation au cours duquel la haute pression osmotique initiale de 600 ± 20 mOsm/kg (moyen ± SD)atteint 700 ± 7 mOsm/kg après l’hydrolyse du sucrose, mais diminue à 350 ± 14 mOsm/kg après l’épuisement des sucres enfin de culture. Le processus complet de développement des embryons, en commençant par le maintien pour une semaine, puisles sous cultures hebdomadaires d’individualisation à l’ABA et enfin la maturation des embryons, requiert 70 à 80 jours. Pour

Can. J. Bot. 76: 863–871 (1998)

Received November 11, 1997.

Ryan P. Taber, Chun Zhang, and Wei-Shou Hu.1 Department of Chemical Engineering and Materials Science, University of Minnesota,421 Washington Avenue SE, Minneapolis, MN 55455-0132, U.S.A.

1 Author to whom all correspondence should be addressed. e-mail: wshu@cems.umn.edu

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chaque millilitre de culture au début du stade de maintien, on obtient environ 420 embryons matures. Au cours de la mêmepériode, la biomasse augmente environ de 100 fois. Une prolongation de la période de culture ne permet pas d’obtenir plusd’embryons. Ces résultats donnent une meilleure vue d’ensemble du comportement cinétique des embryons de sapin Douglasen développement et aidera à optimiser et mettre à l’échelle cet important procédé.

Mots clés : embryogénèse somatique, sapin Douglas, Pseudotsuga menziezii, cinétique de croissance, ABA, osmolarité,développement.

[Traduit par la rédaction]

Introduction

Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) is one ofthe most economically important timber species, supplyingabout 70% of the demand for softwoods in the United States.Given the increasing demand for forest products and the lim-ited land available for forest production, there is an increasingneed for genetically superior trees with desirable traits such asdisease resistance and rapid growth. Selection of these elitetrees through a conventional breeding program usually takesyears or even decades and only captures partial genetic vari-ance, resulting in lower genetic gains. With the advances ingene transformation techniques, gene(s) responsible for elitetraits can also be incorporated into the plant genome. Somaticembryos can be recovered from these transformed cells andused to generate elite plants. This will significantly shorten thetime span for introducing genetically engineered plants. Forplants with a long life cycle, such as conifers, the prospect ofshortening the time span needed for elite clone selection hasgenerated a great deal of excitement.

Somatic embryos have been recovered from white spruce(Picea glauca (Moench) Voss) protoplasts that were trans-formed using a gene gun (Ellis et al. 1993). Furthermore,propagation of forest trees through somatic embryogenesis of-fers the promise of more uniform progenies and ease of processautomation. The high-quality mature somatic embryos pro-duced can be encapsulated in hydrogels for easy handling andprolonged storage. Recently, the successful germination of en-capsulated somatic embryos of spruce has been reported(Lulsdorf et al. 1993). This clearly demonstrates the potentialimpact of this technology in future forest management.

Somatic embryogenesis was first reported in carrot (Daucuscarota L.) by Steward et al. (1958). However, only in the lastdecade was somatic embryogenesis established in woody gen-era including Abies, Larix, Picea, Pinus, Pseudotsuga, andSequoia (Tautorus et al. 1991). Much effort has been devotedto studying the effects of explants, media composition, andplant growth regulators on somatic embryo cultures in conifers(Tautorus et al. 1991). Immature embryos from fertilizedcones are the most frequently used source of explants and givea higher initiation frequency. Recently, the initiation of em-bryonal suspensor masses (ESMs) from young seedlings wasalso reported (Attree et al. 1990). This renews the prospect ofpreparing explants from tissues of mature trees and potentiallywill allow the desired traits of elite individuals to be cloned.

Environmental factors affect somatic embryo developmentprofoundly. However, even embryos of the same genotyperespond to environmental treatments very differently, resultingin variable quality and inconsistent yield of mature embryos.To date, little work has been done on the kinetics of somaticembryo development of conifers except for studies of themaintenance stage for interior spruce (P. glauca × Picea engel-

mannii Parry) and black spruce (Picea mariana (Mill.) BSP)(Lulsdorf et al. 1992; Tremblay and Tremblay 1991).

When placed in suitable initiation media, ESMs ofDouglas-fir form on the surface of explants. They can be fur-ther maintained either on solid medium or in suspension cul-ture for an extended time period without losing theirtotipotency. Removal of growth hormones and the use of ahigh level of abscisic acid (ABA) prevent cleavage polyem-bryony and result in more robust proembryos (Durzan andGupta 1987). This stage has been termed ABA singulation andis unique to Douglas-fir. In the following maturation, or coty-ledonary embryo development stage, medium osmolarity isincreased to about 600 mOsm/kg and the cell cultivation isswitched from suspension culture to being on a polyester padsoaked with maturation medium (Gupta et al. 1995). In thismaturation stage, somatic embryos undergo a series of mor-phological changes similar to their zygotic counterparts.

The development of somatic embryos of conifer is oftendescribed in three distinct stages (von Arnold and Hakman1988). An S1 embryo consists of dense embryonic cellsflanked by elongated suspensor cells. It has a dome-shapedhead region. With further development, the shoot apex startsto protrude. At this stage, the head of the somatic embryobecomes cone shaped. This stage is called S2. With the devel-opment of cotyledons, the embryo gradually becomes a fullymature cotyledonary embryo (S3 embryo).

In this study, the development of Douglas-fir somatic em-bryos in batch cultures was investigated. Parameters such asnutrient consumption, growth rates, and embryo yields will becritical for the bioreactor design and developing a large-scaleprocess.

Materials and methods

Cell line and suspension cultureSuspension cultures of Douglas-fir were kindly provided by Weyer-haeuser Corp, Tacoma, Wash. Cells were grown in liquid culturesduring the maintenance and ABA singulation stage. The basal me-dium components and their concentration are listed in Table 1. Thisbasal medium was modified for each culture stage according to Guptaet al. (1995). The pH was adjusted to 5.7 with 1.0 M KOH prior toautoclaving. Cultures were subcultured weekly by transferring settledcells to fresh medium at a volumetric ratio of one part cell volume innine parts of fresh medium. The suspension cultures were maintainedin the dark at 24 ± 1°C on a rotatory shaker at 80 rpm with a radius ofrotation of 2 cm at 80 rpm (New Brunswick Scientific, Edison, N.J.).

Maintenance stageFor maintenance culture, the basal medium was supplementedwith the following (in mg/L): KNO3, 1250; myo-inositol, 5000;L-glutamine, 1000; sucrose, 30 000; 2,4-dichlorophenoxyacetic acid(2,4-D), 1.1; kinetin, 0.22; and N6-benzylaminopurine (BAP), 0.22.

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Cell suspensions (100 mL) were maintained in 500-mL Erlenmeyerflasks.

ABA singulation stageIn the ABA singulation stage, the medium contained only ABA as agrowth regulator. The basal medium was supplemented with the fol-lowing (in mg/L): KNO3, 1050; Ca(NO3)2⋅4H2O, 200; myo-inositol,100; sucrose, 20 000; and L-glutamine, 1000. The ABA singulationstage lasted 3 weeks. Cells from the maintenance stage were used asinoculum for week 1 ABA singulation cultures. Cells were subcul-tured to fresh medium every week with the same dilution ratio as thatfor the maintenance stage. The ABA concentrations in the mediumwere as follows: 10 mg/mL in week 1 and 5 mg/mL in weeks 2 and 3.

Embryo developmentThe maturation medium was the basal medium supplemented with thefollowing (in mg/L): KNO3, 2500; myo-inositol, 100; L-glutamine,750; L-proline, 100; L-asparagine, 100; L-arginine, 50; L-alanine, 20;L-serine, 20; sucrose, 60 000; polyethylene glycol of molecularweight 8000, 190 000; activated charcoal, 1000; and ABA, 10.

Maturation culture was carried out in 60 × 15 mm Petri dishes. Acellulose membrane with 0.45 µm pore size (Millipore, Bedford,Mass.) was laid on a 35 × 35 × 5 mm polyester pad placed in the Petridish. Six millilitres maturation medium was added to the dish andabsorbed into the polyester pad. This allowed the surface of the mem-brane to be moistened but not inundated by medium. Cells fromweek 3 ABA singulation culture were allowed to settle. The cellswere washed with fresh maturation medium twice and then used asinoculum of maturation cultures. Aliquots (1.2 mL) of the cell suspen-sion were transferred to the Petri dish and evenly laid on the surfaceof the membrane. The Petri dishes were sealed with parafilm andplaced in the dark at 24 ± 1°C.

Forty Petri dishes were inoculated in each experiment, and fivewere randomly sacrificed every week for analysis. The morphologicaldevelopment of embryos was monitored using a stereo microscope(Olympus Optical Co. Ltd., Tokyo, Japan). Only S2 and S3 embryoswere counted by an operator. S2 embryos were defined as those hav-ing smooth cone-shaped heads and opaque appearance, whereas S3embryos showed distinct development of cotyledons around the shootapex.

AnalysisCulture samples were first centrifuged at 100 × g to separate cells andsupernatant. The supernatant was then collected and stored at –20°Cuntil analysis.

For the maintenance and ABA singulation stages, fresh weightwas determined by placing the cells on a preweighed Whatman No. 1filter paper, washing with 10 mL distilled water under vacuum, thenweighing the sample. At the maturation stage, fresh weight was deter-mined by weighing the membrane plus cell mass first and then weigh-ing the membrane with the cells removed. Samples used for freshweight measurement were then dried at 65°C and used to obtain dryweight. All experiments were repeated at least twice, and each hadtwo replicates.

Sucrose concentration was determined enzymatically. It was firsthydrolyzed into glucose and fructose in 0.1 M acetate buffer solution(pH 4.6) with 10 g/L invertase (Sigma Chemical Co., St. Louis, Mo.)at 55°C (Bergmeyer and Bernt 1974). The glucose and fructose con-centrations were then measured using the enzyme assay kits for glu-

Fig. 1. Growth kinetics of Douglas-fir somatic embryos inmaintenance culture: (A) fresh weight (h) and dry weight (j);(B) sucrose (h), glucose (n), and fructose (×) concentrations;(C) NO3

– (s) and NH4+ (d) concentrations. Error bars are ± 1 SD.

ComponentConcentration

(mg/L)

Ca(NO3)2⋅4H2O 200.0KH2PO4 340.0MgSO4⋅7H2O 400.0MnSO4⋅H2O 20.8ZnSO4⋅7H2O 8.0CuSO4⋅5H2O 0.02FeSO4⋅7H2O 27.85Na2EDTA 37.25H3BO3 5.0NaMoO4⋅2H2O 0.20CoCl2⋅6H2O 0.02KI 1.0Thiamine⋅HCl 1.0Nicotinic acid 0.50Pyridoxine⋅HCl 0.50Glycine 2.0

Table 1.Composition of Douglas-fir culturebasal medium.

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cose and fructose, respectively (Boehringer Mannheim, Indianapolis,Ind.). Nitrate and ammonium concentrations were determined spec-trophotometrically using different enzymatic assay kits (BoehringerMannheim, Indianapolis, Ind., and Sigma Chemical Co., respec-tively). Osmolarity measurements were made with a vapor pressureosmometer (Wescor Inc., Logan, Utah).

Results

A somatic embryo culture of Douglas-fir involves three con-secutive stages: maintenance, ABA singulation, and matura-tion. Embryonic cells were maintained in suspension culturesfor the first two stages, then transferred to a polyester padsoaked with liquid maturation medium for further cotyledon-

ary embryo development. To improve the efficiency ofDouglas-fir somatic embryo cultures, the kinetics of embryogrowth and development were studied for all three stages ofculture.

In the maintenance culture, numerous white, mucilaginouscell aggregates or embryonal suspensor masses (ESMs), wereformed. These ESMs consisted of an embryonal region of mer-istematic cells with large suspensor cells connected at one end.The culture doubled, as measured by packed cell volume,every 7 days and was subcultured in the same time interval. Atday 7, cells consisted of approximately 12% of the total work-ing volume.

Figure 1 shows the kinetics of the cell growth and nutrientconcentration profiles at the maintenance stage. Fresh weightincreased approximately twofold over 7 days and reached48.6 ± 5.4 g/L at day 7 (Fig. 1A). A similar trend was observedfor the dry weight. The ratio of dry weight to fresh weight wasabout 0.11 during the cultivation period. Nearly all (95%) ofthe initial sucrose was hydrolyzed to glucose and fructosein 2 days (Fig. 1B). Subsequently, glucose and fructose wereconsumed by cells simultaneously. By day seven, 3.65 ±0.60 g/L of glucose and 7.20 ± 1.40 g/L of fructose had beenmetabolized giving a molar ratio of glucose to fructose con-sumption of 0.5. The yield coefficient was calculated to be0.22 g dry weight/g sugar.

The nitrogen source plays important roles in the growth anddevelopment of plant cells. Both the chemical form of thenitrogen source and its concentration have significant effectson growth rate, cell morphology, and cell totipotency (Thorpe1980). Ammonia and nitrate are the two most commonly usednitrogen sources. Organic nitrogen sources, such as glutamine,arginine, and casamino acids are also frequently used to stimu-late cell growth in conifer tissue culture. Glutamine, casaminoacids, and nitrate were used in our culture media. Glutamineis thermally unstable and decomposes during autoclaving.No NH4

+ was included in the medium. However, because ofthe decomposition of glutamine at an initial concentration of6.8 mM, the ammonia concentration was 6.0 ± 0.3 mM afterautoclaving. NO3

– and NH4+ were consumed simultaneously

with NH4+ being utilized at a slightly faster rate. By day 7,

NH4+ concentration was reduced to the low level of 1.0 ±

0.2 mM. There were still detectable amounts of NO3– present

in the medium at that time. However, a total of 4.8 ± 0.5 mMNH4

+ and 3.3 ±.8mM NO3– were consumed over the 7-day

culture period. Because the packed cell volume increased sig-nificantly during the 7 days, the volume of liquid at the begin-ning of the culture was different from that at the end of theculture. The nutrient consumption values reported here werecorrected for this volume change.

The initial osmolarity of the maintenance medium was200 ± 7 mOsm/kg. It increased to 220 ± 21 mOsm/kg at day 2and then decreased to 160 ± 15 mOsm/kg at day 7 as a resultof nutrient consumption. The pH of the medium decreasedfrom 5.20 ± 0.07 to 4.10 ± 0.08 by day 2 and then remainedconstant for the rest of the culture period.

The proembryos of Douglas-fir formed in the maintenancestage tend to form tight clumps. In the subsequent ABA sin-gulation stage, a high ABA concentration was used to separatethese proembryos into small clusters. The singulation stageinvolves three weekly subcultures into medium containingABA. Cells were transferred to fresh medium with designated

Fig. 2. Growth kinetics of Douglas-fir somatic embryos in ABAsingulation culture: (A) fresh weight (j) and dry weight (∆)expansion over 3 weeks; (B) sucrose (h), glucose (n), and fructose(×) concentration; (C), NO3

– (s) and NH4+ (d) concentration. Cells

were subcultured at days 7 and 14. Error bars are ± 1 SD.

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ABA concentration every 7 days. In the first week, the con-centration of ABA was 10 mg/L, which was reduced to 5 mg/Lfor the two successive subcultures. The kinetic behavior of the

three ABA singulation cultures is presented in Fig. 2. The totalcell mass expansion in fresh weight and dry weight is shownin Fig. 2A. During the 3-week period, total biomass increased

Fig. 3. Micrographs of early proembryos at the end of the maintenance culture (A) and morphology of late proembryos at the end of week 3 ofthe ABA singulation stage (B).

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about 4.5-fold as indicated by fresh weight. The increase intotal biomass was smaller in the last 2 weeks. Carbohydrateconcentration profiles again indicated a rapid hydrolysis ofsucrose followed by simultaneous consumption of glucose andfructose except during week 3 (Fig. 2B). Trends in NO3

– andNH4

+ profiles were similar to those in the maintenance stagewith NH4

+ being consumed faster than NO3– (Fig. 2C).

By the third week of the ABA singulation stage, the proem-bryo’s morphology was distinctly different from that in themaintenance culture. Shown in Fig. 3 are two micrographs ofproembryos from a maintenance culture and from the thirdweek of the ABA singulation stage, respectively. In the main-tenance culture, proembryos had a smaller embryonic regionwith four or five long and highly vacuolated suspensor cells(Fig. 3A). The head region consisted of small cytoplasm-richcells that are embryonic. By week 3 of the ABA singulationstage, these proembryos have further developed a more de-fined embryo head region with a mass of elongated suspensorcells connected at one end (Fig. 3B). The 3-week cultivationof cells in the ABA singulation medium is thought to producesingulated proembryos with increased robustness, as illus-trated in the micrographs. These late proembryos were thentransferred to the maturation stage for further cotyledonaryembryo development. Cells were overlaid on a polyester padsoaked with maturation medium and sealed in Petri dishes.Mature embryos began to emerge in 2–3 weeks.

Figure 4 illustrates the growth kinetics in the maturationstage. Fresh weight increased about 5.5-fold in 6 weeks, whiledry weight increased about 11-fold in the same time period(Fig. 4A). The ratio of dry weight to fresh weight increased

from 0.11 to 0.29 during the first week, then decreased slightlyto 0.23 at the end of cultivation. In maintenance and ABAsingulation cultures, this ratio was typically in the range of 0.1to 0.13, indicating that a significant decrease in water content,probably also a concurrent increase in the content of storagecompounds, occurred in the maturing embryos.

The concentrations of S2 and S3 embryos in maturationculture are shown in Fig. 4B. Both the S2 and S3 embryoconcentrations increased in the first 4 weeks. At day 28 theconcentration of S2 embryos reached a maximum of 22 ± 7per mL of packed cell volume inoculated initially. After thattime point, the number of S2 embryos started to decrease. Incontrast, the concentration of S3 embryos continued to in-crease to a maximum of 42 ± 16 at day 35 before it began todecrease. This decrease of S2 embryo concentration aroundday 28 is likely to be the results of continuous conversion ofS2 embryos to S3 embryos at a time when few new S2 embryoswere produced.

Corresponding nutrient data for the maturation stage aregiven in Fig. 5. Carbohydrates were nearly depleted by day 45(Fig. 5A). There was a significant amount of NH4

+ consump-tion at the beginning of the maturation culture. NH4

+ was de-pleted by day 20, followed by an increased consumption ofNO3

– (Fig. 5B). In addition to sucrose, glutamine, and nitrate,the medium for maturation culture included casamino acids.HPLC analysis showed that only small amounts of amino acidswere present by day 35 (data not shown). Over the 6-weekmaturation period, a total of 4.1 ± 0.3 mM NH4

+ and 6.1 ±1.2 mM NO3

– were consumed.The osmolarity profiles for all three culture stages are

Fig. 4. Growth kinetics of Douglas-fir somatic embryos at thematuration stage: (A) fresh weight (j) and dry weight (n);(B) concentrations of S2 (s) and S3 (d) somatic embryos.Error bars are ± 1 SD.

Fig. 5. Nutrient concentration profiles at the maturation stage:(A) concentrations of sucrose (u), glucose (n), and fructose (×);(B) concentrations of NO3

– (s) and NH4+ (d). Error bars

are ± 1 SD.

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given in Fig. 6. The initial osmolarity was approximately200 mOsm/kg for the maintenance stage and 150 mOsm/kgfor the three subcultures of the ABA singulation stage. Thisdifference in osmolarity is mainly due to the different concen-tration of sucrose and myo-inositol used in the medium ofthese two stages. For the maintenance stage, 30 g/L of sucroseand 5 g/L myo-inositol were used compared with 20 g/L ofsucrose and 0.1 g/L of myo-inositol for ABA singulation culture.In both the maintenance and ABA singulation stages, su-crose was quickly hydrolyzed to glucose and fructose. Theincrease in molarity was reflected in the increase of the me-dium osmolarity during the early stage of cultivation. Sub-sequently the osmolarity decreased gradually to about160 mOsm/kg in maintenance cultures and 75 mOsm/kg inABA singulation cultures at the end of cultivation, probablybecause of significant nutrient consumption. The initial osmo-larity in the maturation stage was 600 mOsm/kg. By transfer-ring the cells from week 3 ABA singulation culture to thematuration culture, the proembryos were exposed to a suddenincrease in osmolarity. It was thought that this increase inosmolarity is at least partially responsible for triggering theembryo maturation process. The osmolarity remained above600 mOsm/kg during the first 4 weeks and decreased rapidlyto 350 mOsm/kg at the end of the cultivation. It was noticedthat a decrease in embryo quality was coincident with thisosmolarity drop.

Discussion

This report presents the kinetics of Douglas-fir somatic em-bryo development. Success in generating embryos from cul-tured somatic cells of Douglas-fir is rather recent (Durzan andGupta 1987). Field tests of trees regenerated from somaticembryos are underway in industry. For future widespread ap-plications, the process of somatic embryo culture will need tobe optimized to improve the efficiency and to be highly auto-mated. Acquiring kinetic information on nutrient consumptionand embryo development is the first step toward the optimiza-tion process.

The three cultivation stages in Douglas-fir somatic embryodevelopment serve to expand the embryogenic population, toinitiate the embryo development, and finally to allow them tomature. The overall objective is certainly to generate a large

number of mature embryos in a short cultivation period. Ofconcern to the manufacturing process is the rate of cell propa-gation. Frequent sampling is necessary for growth rate mea-surement. In practice, frequent sampling for fresh weight ordry weight measurements is not convenient as samples willhave to be sacrificed after measurements. Alternatively,packed cell volume measurement, if operated aseptically, en-ables biomass to be returned for continued cultivation. Thepacked cell volume profiles for the maintenance and ABAsingulation stages are shown in Fig. 7. In the four subculturesof both stages, the time period needed for the packed cell vol-ume to double was about 4 days. The growth appeared to slowdown toward the latter part of each subculture. The decreasein growth was more profound at the ABA singulation stage.Although in two of these subcultures, fructose and ammoniaappeared to have depleted at the end of subculture, it is notclear whether the depletion of these nutrients is the direct causeof the diminished growth rate. Supplementary media with 5 mMammonia did not result in improved embryo growth (data notshown). Other possibilities include potential inhibitory meta-bolite accumulation.

During the 4-week suspension cultures, the packed cell vol-ume increased approximately 10-fold. This estimated extent ofincrease in biomass was similar to that determined by dry weightor fresh weight measurements. At the end of the maturationstage, a maximum of 42 ± 16 cotyledonary embryos were gen-erated from each millilitre of packed cell inoculum obtainedfrom ABA singulation culture. This is roughly equivalent to0.1 mL of packed cells in the first subculture (maintenancestage) of embryo propagation. The packed cell volume at thatstage was about 6% or 60 mL/L. Thus, each initial litre ofculture volume at the maintenance stage would generate about25 000 mature embryos. The frequency of mature embryo for-mation has been reported to be higher for other species (Tautoruset al. 1992; Attree et al. 1993). Many factors affect the fre-quency of mature embryo formation, including the explant andgenotypes. The frequency of mature embryo formation mightalso be improved by manipulation of cultivation conditions.Recently, we employed a perfusion reactor for the maturationstage and observed a substantially increased frequency of ma-ture embryo formation, indicating the possibility of furtherprocess improvements through environmental manipulations.

Fig. 6. Osmolarity (e) and macronutrient concentration (m)profiles for the entire propagation process of Douglas-fir somaticembryo culture. Error bars are ± 1 SD.

Fig. 7. Packed cell volume in at maintenance and ABA singulationstages. Error bars are ± 1 SD.

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At least three aspects of the physical and chemical environ-ments warrant further investigation in optimizing the embryopropagation process. They are carbon and nitrogen metabo-lism, the dynamics of osmolarity, and the role of activatedcharcoal in the maturation stage.

In plant tissue cultures, carbohydrates serve as both carbonand energy sources. Sucrose is the most commonly used sugar.In conifer somatic embryo cultures, sucrose is also used as anosmoticum in the medium. It was noticed that sucrose in themedium was rapidly hydrolyzed to glucose and fructose at allculture stages. Such hydrolysis of sucrose resulted in an initialincrease in medium osmolarity (Fig. 6). Glucose and fructosewere metabolized by Douglas-fir cells simultaneously withmaybe a slight preference of fructose over glucose. This pref-erence among carbohydrates has been shown to be speciesspecific (Tautorus et al. 1992, 1994; Tremblay and Tremblay1991). We substituted sucrose for an equivalent amount ofglucose, or an equivalent amount of glucose and fructose, andobserved no difference in cell growth at the maintenance stage(data not shown). However, replacing sucrose with an equiva-lent amount of glucose, or glucose and fructose, in the matu-ration medium yielded many fewer mature somatic embryos(data not shown). Sucrose, despite its rapid hydrolysis to glu-cose and fructose, appears to have certain stimulatory effectson embryo development that are not replaceable by glucose orfructose.

The medium used in this study largely consists of two cate-gories of chemical species: those consumed by cells to becomebiomass and (or) to generate energy and those that are noncon-sumable or are consumed to a minor extent. The consumablespecies include carbohydrates, nitrogen sources, and some mi-cronutrients. The nonconsumables are PEG and bulk salts.During cultivation, the biovolume increased significantly, as aresult of cells taking up nutrients and water from the medium.Because of the reduced water volume in the medium, or thevolume of the abiotic phase, osmolarity increased. Conversely,the consumption of nutrients reduced the total concentrationof soluble species in the medium and reduced the osmolarity.The percentage of medium volume change caused by waterloss due to biomass expansion was relatively small. Hence, theincreased concentration of nonconsumable species was notlikely to be the major factor in osmolarity change; on the otherhand, the consumption of nutrients changes the concentrationof soluble species significantly and is more likely to be thedominating factor affecting the variation of osmolarity duringcultivation.

The nutrients that were present at high concentration andcontributed significantly to osmolarity change are sucrose,glucose, fructose, ammonium, and nitrate. The sum of the mo-larity of these nutrients over the course of embryo propagationis shown along with the osmolarity profiles (Fig. 6). In themaintenance and ABA singulation stages, no PEG was presentin the medium, and the osmolarity was relatively low, in therange of 75–220 mOsm/kg. Nevertheless, the general agree-ment between the profiles of macronutrients concentration andosmolarity was apparent, especially at the maturation stage.Because of the presence of PEG, the osmolarity in the matu-ration culture was rather high, initially at about 600 mOsm/kg.This increase in osmolarity, sometimes referred to as osmoticshock, is thought to be responsible for the onset of maturationprocess. The initial high value of the osmolarity was sustained

for about 30 days before decreasing gradually to about half ofits peak value. The decrease coincided with the consumptionof macronutrients. It is interesting to note that the increase ofdry weight was slower than the increase of fresh weight in thematuration culture after 30 days (Fig. 4), indicating an in-creased specific cell volume. It is conceivable that the increasein specific cell volume is related to decreased osmolarity.There were also fewer new embryos formed during this periodof decreasing osmolarity. It was reported that an osmolarity ofgreater than 450 mOsm/kg was necessary for optimal embryoformation (Gupta and Pullman 1991). As a result of suddenexposure to high osmolarity, turgor pressure of the cells de-creases. This decreased turgor pressure is thought to act as thesignal for endogenous ABA synthesis and is important to em-bryo development. Activated charcoal was included in the me-dium at the maturation stage. Activated charcoal is thought toabsorb ABA in the medium initially and slowly release it dur-ing the cultivation (Attree and Fowke 1993; Gupta and Pull-man 1991). It may also absorb toxic metabolites produced bycells. The embryos produced using the ABA-activated char-coal combination can be directly stored at low temperature onthe maturation plates for extended period without losing vi-ability (Gupta and Pullman 1991). However, the use of acti-vated charcoal has several limitations. The sources and qualityof activated charcoal may greatly affect the adsorption of ABAand other nutrients. Its presence also makes defined kineticstudies difficult.

If the role of activated charcoal is either to absorb ABAinitially and then slowly release it or to absorb inhibitory sub-stances, it may be possible to replace it with a continuousperfusion culture in which nutrients and growth regulators arecontinuously supplied and metabolites or inhibitors are con-tinuously removed. Indeed, we have successfully eliminatedactivated charcoal in a perfusion culture system. After remov-ing activated charcoal from the medium, the only undefinedcomponent left is casamino acid. We are currently investigat-ing the possibility of replacing casamino acids with a definedmixture of amino acids, the results of which will be presentedin a separate report. Our aim is to develop a completely definedenvironment to further elucidate the kinetic behavior ofDouglas-fir somatic embryos. A better understanding of thekinetic behavior of the developing somatic embryos will cer-tainly facilitate the implementation of somatic embryo culturefor the micropropagation of Douglas-fir.

Acknowledgments

This work was supported in part by grants from the NationalScience Foundation (BCS 9015817 and BES 9321426). R.P.T.was supported by NIH Biotechnology Traineeship (GM08347).We also acknowledge Dr. Roger Timmis of WeyerhaeuserCorp. for kindly providing Douglas-fir cell lines.

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