Plant Physiol. (1989) 89, 768-7750032-0889/89/89/0768/08/$01 .00/0

Received for publication July 7, 1988and in revised form September 7, 1988

Maturation of Soybean Somatic Embryos and the Transitionto Plantlet Growth

Julie A. Buchheim1, Susan M. Colbum, and Jerome P. Ranch*2United AgriSeeds, P.O. Box 4011, Champaign, Illinois 61820

ABSTRACT MATERIALS AND METHODS

The maturation of soybean (Glycine max L. Merr.) somaticembryos was characterized. Maturation was assayed by evalu-ating the ability of somatic embryos to make the transition to aplantlet through a germination-like process. Somatic embryoswere organized from cotyledons of immature soybean embryos.Maturation of somatic embryos occurred on a Murashige-Skoogbasal medium supplemented with activated charcoal and 0.28molar sucrose. After 8 weeks on this medium, somatfc embryosexhibited vigorous, high frequency development to plantdets. The"germination" frequency (conversion) of somatic embryos, andplantlet recovery frequency varied concurrently with maturationperiod. Conversion and plant recovery required no exogenousgrowth regulators. Desiccation of immature somatic embryosunder controlled humidity regimes resulted in increased fre-quency of conversion of immature somatic embryos. Morpholog-ical abnormalities appeared in the somatic embryos, but few weredetrimental to conversion velocity. There was little effect ofgenotype on conversion velocity or frequency.

There have been several contemporary reports concerningthe production of somatic embryos from cotyledons of im-mature soybean embryos (5, 6, 15, 23, 24). Although consid-erable effort has been expended in the characterization ofsomatic embryo induction, there is sparse quantitative infor-mation on the subsequent performance of the somatic em-bryos, especially with regard to their morphology, anatomy,development, physiology, and biochemistry. Although so-matic embryogeny recapitulates zygotic embryology in manyways (7, 22, 28), this correlation has yet to be developed insoybean. Plant cell and tissue culture provides an excellentopportunity to study developmental processes in a controlledenvironment. However, cell and tissue cultures typically arenot well-synchronized in development or differentiation.Therefore, the goals of this study are to understand theprocesses of soybean somatic embryo development, and tocontrol expression of this process in a reproducible and con-trolled fashion. Our long-term goal is to determine if therequirements for somatic embryo maturation and develop-ment are comparable to that demanded by zygotic embryos.In this way, somatic embryos may function as a model forzygotic embryogenesis.

' Present address: Department of Entomology, 402 Life SciencesBldg., Louisiana State University, Baton Rouge LA 70803.

2 Present address: American Cyanamid, P.O. Box 400, Princeton,NJ 08540.

Induction and Maturation of Somatic Embryos

Somatic embryos were generated on cotyledons of imma-ture soybean (Glycine max L. Merr.) embryos as described byRanch et al. (23, 24). The genotypes used in these studieswere cv 'Gem,' 'Star,' 'Shawnee,' 'A3127,' 'Williams 82,' and'Union.' Immature seed were harvested from greenhouse orfield-grown plants. At the appropriate stage for explant, theimmature embryo possessed cotyledons 3 to 5 mm long andwere 15 to 21 DAF in field-grown plants. Cotyledons werecultured abaxial surface down on solidified induction mediumcomposed of Murashige and Skoog (17) medium (MS) + 87,uM 2,4-D + 0.8% (w/v) TC agar (KC Biological) + 0.0875 Msucrose (20MX) adjusted to pH 5.8 prior to autoclaving. Thecotyledons were cultured 10 per 60 x 20 mm Petri dish andsealed with Parafilm. The cotyledons were cultured at 28°Cunder continuous illumination at 20 lux (0.3 ,uE).

After 30 d incubation, somatic embryos were removed fromthe cotyledon and transferred individually, or in clumps of 2to 8 embryos, to a solidified maturation medium. This me-dium was formulated with MS + 0.28 M sucrose + 0.5% (w/v) activated charcoal + 0.8% TC agar (medium MM). Coty-ledonary tissue was removed carefully to avoid injury to thesomatic embryos. Approximately 50 somatic embryos werecultured per 60 x 20 mm Petri dish. The Petri dishes wereleft unsealed in a plastic box (Flambeau A801) at 28°C undera 16 h light diurnal photoperiod and 1000 lux (15.5 AE) fromcool-white fluorescent lamps.

Growth and Different during the Maturation Regime

At the time of initial transfer to MM, and at intervals for70 d, the length, fresh weight, dry weight, and moisturecontent of somatic embryos of 'Gem' were determined.Length measurements were determined nondestructively with20 somatic embryos. Fresh weight and dry weight values werethe mean of 5 somatic embryos. Dry weight was determinedby drying somatic embryos in tared plastic boats at 56°C. Thesamples were weighed daily for 72 h. When the weight stabi-lized, that value was used in dry weight computation. Mois-ture content was computed from the fresh and dry weightdeterminations.

Also at intervals during the maturation process, somaticembryos were removed from MM and transferred to a "ger-mination" and plantlet recovery medium. This conversionmedium (SHGG) was composed of Schenk and Hildebrandt(27) basal formulation + 0.028 M sucrose + 0.2% (w/v) Gelrite

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SOYBEAN SOMATIC EMBRYO MATURATION

(Kelco Co.). The term, conversion, has been used to denotethe recovery of plantlets or plants from somatic embryos (25)and therefore was used to describe the activation of soybeansomatic embryos. The somatic embryos were cultured 10 per100 x 25 mm Petri dish. A total of 378 somatic embryos wereevaluated for conversion using an average of 34 embryos permaturation interval. During conversion evaluation, the disheswere unsealed in a plastic box, and maintained at 28°C undera 16 h light diurnal photoperiod with 3000 lux (42 ,uE) fromcool-white fluorescent lamps. Conversion was recorded as thedevelopment of the primary root, greening of the cotyledonsand hypocotyl, and formation of the first set of pubescenttrifoliolate leaves when initially observed at a young stage.Conversion data were recorded at 7 d intervals for 10 weeks.At the termination of a conversion evaluation, the finalpercentage conversion was recorded, and conversion days wascomputed using the expression,

Conversion daysNIT1 + N22 +. -*NxTx

total normalized number of conversion events

where Nx is the normalized number of somatic embryosconverted within consecutive intervals of time Tx (13). So-matic embryos for all experimental conversion studies wereofmonocotyledonous, dicotyledonous, and polycotyledonoustypes unless otherwise stated.During the 10 week conversion assay period on SHGG,

converted embryos continued development. The frequencyof converted somatic embryos that became established asplantlets with 2 to 3 nodes was subsequently recorded. Whenthe plantlets attained this stage, they were transferred to 10mL solidified SHGG in 125 x 25 mm culture tubes. Afterthe plantlets grew to the top of the culture tube, they weretransferred to a 5 gallon pot containing standard greenhousepotting mix. The plantlets were covered with transparent 20ounce plastic cups for acclimatization. Although the vastmajority of regenerated plants were fertile, a quantitativedetermination of fertile plant recovery was not recorded aspart of this study.

Effect of Genotype and Physical Manipulation ofEmbryos on Development

Somatic embryos of 'Gem' and 'Star' were matured on

MM for 50 d. After removal of the cotyledons, twenty dico-tyledonous somatic embryos of each genotype were trans-ferred to SHGG. For cotyledon removal, the embryonic axiswas held carefully with a tweezers. Gentle basipetal pressure

was then applied to the distal edge of the cotyledon with a

blunt instrument. The cotyledons were cleanly detached fromthe embryonic axis at the cotyledonary node with this tech-nique. Somatic embryos with the cotyledon intact were thecontrol. Conversion was recorded at 4 d intervals for 20 d.

In other experiments, after 40 d on MM, 'Gem' somaticembryos were placed into a sterile, dry 60 x 20 mm Petridish. The embryos were immediately transferred to a sealedplexiglass vessel containing H2SO4 in concentrations whichprovided 75% or 93% relative humidity (26). Proper H2SO4concentration for each humidity was determined by measur-

ing the specific gravity of each dilution with a hydrometer.The specific gravities to generate these relative humiditieswere, 75%, p = 1.230, and 93%, p = 1.102. The ratio of thevolume of acid to total container volume was 1:2. Somaticembryos were treated for 4 or 7 d under these humidityregimes. Twenty somatic embryos were used in each treat-ment. The humidity chambers were kept at 25°C underambient laboratory lighting conditions. For ambient desicca-tion treatment, somatic embryos were transferred to a dryPetri dish, sealed with one layer of Parafilm, and kept in alaminar flow hood at 25°C for 4 d. After desiccation treat-ments, the somatic embryos were rehydrated with sterile,distilled water for 1 h before transfer to SHGG. Desiccatedsomatic embryos were evaluated for conversion on SHGG at2 d intervals for 21 d. Controls were somatic embryos derivedfrom the same batch of induced cotyledons and cultured onSHGG without desiccation treatment. Loss of viability wasgenerally associated with the embryo becoming pure whiteand vitrified. When an embryo had this appearance anddid not develop a radicle or apex, it was considered to benonviable.

Finally, to determine if maturation was genotype specific,30 somatic embryos each of 'A3 127,' 'Harper,' 'Williams 82,'and 'Union' were incubated on MM for 53 d. These maturedsomatic embryos were transferred to SHGG, and conversionwas recorded daily for 12 d.

Embryo Morphology and Its Effect on Conversion

For morphometric studies, 411 'Gem' somatic embryosand 449 'Shawnee' somatic embryos were classified into mor-phological classes after 8 weeks maturation on MM. Twentyembryos of each 'Gem' morphology, and from 11 to 42embryos of each 'Shawnee' morphology were cultured 10 to15 per plate ofSHGG in 100 x 25 mm Petri dishes. Conver-sion days and % conversion were determined at 2 d intervalsfor 10 weeks. The frequency ofthe classifications was recordedas the percentage of all the somatic embryos observed.

Data Analyses

Enumeration data (% conversion and % viability), wereevaluated using the x2 statistic. Counts were entered intocontingency tables using the classes, converted versus noncon-verted, or, viable versus nonviable. We assumed all treatmentswithin an experiment had a similar effect on the measuredresponse. If this hypothesis was rejected based on the x2statistic, 95% confidence intervals were computed. The meanof conversion days were computed from replicated experi-ments and tested for similarity using an analysis of variance(ANOVA). Least significant differences (LSD) for means werecomputed from ANOVA tables.

RESULTS

After incubation for 3 to 4 weeks on 20MX, somaticembryos had organized upon the adaxial surface and marginsof the explanted cotyledons. Somatic embryos resembled aglobular stage zygotic embryo (Fig. IA). No further embryo-logical development was observed on this medium regardless

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Plant Physiol. Vol. 89, 1989

Figure 1. A, Induction of somatic embryos on 20MX (x8). B, Somatic embryo freshly excised from the cotyledon, and after 4 d on MM (toprow); somatic embryos after 17, 28, and 56 d on MM (bottom row) (x8.8). C, 'Germinating' somatic embryos on SHGG (x4.3). D, In vitroplantlets derived from converted dicotyledonous somatic embryos (right); trumpet-shaped somatic embryos with extensive root growth but noapical development (left) (xO.37).

ofthe length of culture. Somatic embryos were approximately1 mm in length, largely translucent, and were pale yellow.Occasionally, the apical area ofthe immature somatic embryowas slightly green. Embryos at this developmental stage weretransferred to MM for maturation. After 4 to 7 d on MM, thesomatic embryos developed a pale-green translucent appear-ance, particularly at the apical area, and resembled a torpedostage zygotic embryo (Fig. 1B). During culture on MM, thesomatic embryos passed through morphological stages asso-ciated with zygotic embryogeny. The green color associatedwith the somatic embryos during early maturation graduallybecame a creamy-white color (Fig. 1B).

Somatic embryos increased in embryo length, fresh weight,and dry weight while maturing on MM (Fig. 2). Fresh weightand dry weight reached a maximum after 40 to 50 d matu-ration and varied concurrently. After 40 to 50 d maturation,both fresh weight and dry weight decreased. At 10 d matura-tion, the moisture content was approximately 80%. Moisturecontent at 0 d could not be determined due to the fragility ofthe embryos and excessive variation. The moisture content ofthe embryos decreased gradually throughout the maturationregime. Somatic embryos were reduced to 52% moisture after70 d maturation. Fresh weight, dry weight, and moisturecontent data were fitted with the NONL module of PlotIt.

Somatic embryos freshly removed from the cotyledon andcultured on SHGG did not convert with high frequency orgreat vigor (Fig. 3). However, somatic embryos converted

more rapidly and with higher frequency with increased mat-uration time. After 56 d maturation, the maximum conver-sion frequency of 98.2%, and the minimum conversion daysof 7.1 was achieved. Culture on MM for periods longer than56 d led to a deterioration of embryo vigor as judged by bothconversion days and conversion frequency. Conversion ofsomatic embryos cultured on MM for an optimal or near-optimal period included development of the root, greening ofthe cotyledons and hypocotyl, and formation of the apicalarea (Fig. IC). Root development and greening of the cotyle-dons and hypocotyl usually occurred first. The first true leaveswere simple and alternate to the cotyledonary node. Subse-quent nodes produced trifoliolate leaves. While most somaticembryos produced a root, shoot development was generallythe limiting factor in conversion.The frequency of plantlet development from converted

somatic embryos paralleled that ofconversion frequency (Fig.3). In vitro plantlets from converted somatic embryos ap-peared normal in all regards (Fig. ID). In this study, we didnot evaluate whole plants for heritable variation which mayhave been generated by passage through culture. Althoughmost quantitative measurements used somatic embryos of'Gem,' embryos of other genotypes also converted rapidlyand with high frequency after maturation on MM (Table I).There was no significant effect of genotype on conversiondays or conversion frequency.

After 40 d on MM, somatic embryos were desiccated to

BUCHHEIM ET AL.770

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SOYBEAN SOMATIC EMBRYO MATURATION

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MATURATION PERIOD (days)Figure 2. Change in somatic embryo length, fresh weight, dry weight,and moisture content as a function of interval on maturation medium,MM. Bars indicate standard error of observations.

determine if this treatment could reduce the requirement forextended maturation on MM. The conversion of desiccatedembryos proceeded more vigorously than that of controlsomatic embryos (Table II). There were significant differencesin both viability and conversion frequency between the treat-ments. Of the desiccation treatments, embryos exposed to75% RH for 4 d had the greatest viability and vigor. Incontrast, untreated somatic embryos, although having highviability, did not convert within the 21 d conversion testperiod. Treatment with higher RH, or for longer periods at75% RH, resulted in the reduction of viability and embryovigor. The mortality ofsomatic embryos desiccated in a sealedPetri dish under ambient conditions was 100%. Ambient RHwas 40%. This ambient RH was likely translated into RHwithin the sealed dish. Somatic embryos were green beforedesiccation, and remained green throughout the treatment.

Somatic embryo morphologies were classified into 9 types.Mono-, di-, poly-, and fused cotyledon, as well as trumpet,and long hypocotyl-vestigial cotyledon, were morphologies ofsolitary somatic embryos. Diaxial fusion, moderate fasciation,and gross fasciation were dactyloid types based on the severityof axis fasciation of the somatic embryos. The nine morpho-types varied in their conversion days and percent conversion(Fig. 4, Table III). x2 analyses resolved significant effects of

MATURATION PERIOD (days)Figure 3. Change in conversion days, conversion frequency, andfrequency of plantlet development as a function of interval on matu-ration medium, MM. Data points which represented fewer than fiveobservations were not included.

Table I. Conversion Days and Percent Conversion of SomaticEmbryos from Genotypes 'A3127,' 'Williams 82,' 'Union,' and'Harper' after Maturation on MM for 53 da

Conversion was measured on SHGG.

Genotype Conversion Conversion

d %

Union 3.3 54.5A3127 3.6 96.3Williams 82 3.8 71.4Harper 4.0 68.0

Fe = 0.57Nsa x2= 4.6 NSa Conversion velocities were averaged and analyzed using a one-

way ANOVA. Conversion frequency data were entered into a contin-gency table and tested for significance using the x2 statistic (NS =not significant).

morphology on conversion frequency in both genotypes.There was poorer evidence for differences in conversion daysbetween the different morphologies if stringent requirementsfor significance were used (P c 0.05). The most morphologi-cally abnormal somatic embryos of 'Gem' (trumpet, fused

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Plant Physiol. Vol. 89,1989

Table II. Effect of Desiccation on Conversion of Immature SoybeanSomatic Embryosa

Somatic embryos were matured for 40 d on MM, subjected tovarious desiccation treatments, and tested for conversion on SHGG.

Treatment Viability Conversion Conversion% % d

None 100 ± 0 0 >2175%RH,4d 100±0 70±26 11.4(0.91)75% RH, 7 d 60 ± 18.9 60 ± 27 8.6 (1.1)93% RH, 4 d 50 ± 19.3 20 ± 23 10.0 (0.73)93% RH, 7 d 60 ± 18.9 40 ± 27 16.0 (1.1)40%RH,4d 0

(ambient)

X2 = 58.2** X2=27.8**

aStandard deviations of conversion velocities are enclosed inparentheses. Conversion frequency data were entered into a contin-gency table and tested for significance using the x2 statistic. Ninety-five percent confidence intervals are indicated by ± values (** signifi-cant at <0.01% level).

A

G

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B

H

Figure 4. Line drawings of the nine classifications of soybean so-matic embryos based on morphology. A, monocotyledonous; B,dicotyledonous; C, polycotyledonous; D, fused cotyledon; E, longhypocotyl, vestigial cotyledon; F, trumpet; G, moderately fasciated;H, grossly fasciated, and 1, proximal diaxial fusion (x5.6).

cotyledon, and grossly fasciated) accounted for 16.3% of thesomatic embryos. The conversion frequency of this popula-tion was less than 35%. All other somatic embryo morphol-ogies converted with a frequency of 70% or greater. Uncon-verted somatic embryos, particularly abnormal ones, pro-duced an even more extensive root system than those embryoswhich converted (Fig. 1D). Although 'Shawnee' had nearly10 times fewer trumpet-shaped embryos, there were only

minor differences in the frequency of other embryo morphol-ogies between the two genotypes.Two to 4 somatic embryos composed any fasciated mor-

phology. Except for diaxial fusions, data were not recordedon the various solitary types comprising the fasciated mor-phologies. Data for diaxial fusion reflected conversion ofeither one or both of the somatic embryos. In the genotypesevaluated, both embryos generally convert. More severe fas-ciation prevented classification of individual embryo types.However, only rarely did more than one conversion eventoccur in severely fasciated morphotypes. Data on the fre-quency of plantlet recovery from the different morphologicalclassifications were not recorded.There was a direct relationship between the amount of

cotyledonary tissue and conversion days. For example, so-matic embryos which possessed little cotyledonary tissue fre-quently organized a pubescent apical area on MM medium,and embryos with fewer cotyledons or with vestigial cotyle-dons converted more rapidly (Table IV). Removal of cotyle-dons from 50-d matured somatic embryos also promoted amore rapid conversion. With intact cotyledons, 'Gem' and'Star' somatic embryos converted in 10.2 (sd = 1.2) and 14.2(sd = 0.93) d, respectively. With the cotyledons removed,conversion days were reduced to 8.8 (sd = 0.91) and 8.8 (Sd =0.95) days, respectively. The conversion frequency was 90%in all these treatments.

DISCUSSION

Our observations demonstrated that soybean somatic em-bryos required a period of maturation before acquiring thecapacity for vigorous conversion and plantlet development.Zygotic soybean embryos also require a certain period ofmaturation in situ before they germinate vigorously and arecapable of development into a seedling (3, 18). Whether thebasis of the maturation period and the acquisition of physio-logical maturity has similar physiological and biochemicalbasis in zygotic and somatic embryos has yet to be determined.

It has been suggested that the use of 2,4-D in the inductionof soybean somatic embryogenesis inhibits rapid and produc-tive embryo conversion and plantlet recovery (5, 15). Soy-beans sequester much of the available 2,4-D as amino acidconjugates (9). This biochemistry has been assessed as apotential basis for the lack of morphogenic competence his-torically observed in soybeans (16). Somatic embryos inducedby 2,4-D, after transfer to 2,4-D-free medium, may havereleased 2,4-D from the conjugated state (8). Since com-pounds with high auxin activity effectively inhibit somaticembryo development, the reported lack of rapid and highfrequency conversion might be due to the continued presenceof 2,4-D. Developmental and morphological abnormality ofsoybean somatic embryos have been related to 2,4-D exposure(5, 15, 23, 24). Activated charcoal was used in maturation todecrease the effects of residual 2,4-D. Activated charcoal hasoften been used to adsorb 2,4-D and aromatic compoundsproduced by plant tissues which are inhibitory to growth ordevelopment (10).The inability of immature zygotic soybean embryos to

make the transition to a seedling with great vigor has beencorrelated with a requirement for a period of desiccation (2,

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SOYBEAN SOMATIC EMBRYO MATURATION

Table Ill. Effect of Somatic Embryo Morphology on Conversion Days and Final Percent Conversion with Soybean Genotypes 'Gem' and'Shawnee'"

Somatic embryos were matured on MM for 56 d. 'Gem' somatic embryos were evaluated for 21 d, while 'Shawnee' embryos were evaluatedfor 14 d for conversion on SHGG.

Total Conversion Time ConversionMorphology

Gem Shawnee Gem Shawnee Gem Shawnee

% d %

Monocotyledon 12.7 13.9 3.9 5.4 70 ± 18 64 ± 12Dicotyledon 10.5 12.4 4.4 5.6 100 ± 0 55 ± 22Polycotyledon 12.2 10.3 5.9 6.2 75 ± 17 71 ± 11Fused cotyledon 8.3 12.4 6.9 12.4 35 ± 18 50 ± 13Trumpet 7.1 0.9 3.0 13.0 10 ± 11 33 ± 13Long hypocotyl vestigial 5.1 3.2 4.2 4.0 90 ± 11 45 ± 26

cotyledonModerately fasciated 22.6 31.2 4.8 7.7 85 ± 14 72 ± 8Grossly fasciated 10.9 7.9 4.4 13.9 35 ± 18 61 ± 17Diaxial fusion 10.7 7.6one 3.4 4.4 25± 16 15± 12both 7.2 7.6 70 ± 18 73 ± 15

F9= 1.43 NS F93 = 1.8 NS X2= 62.5** x2 = 36.7**1 SDo.30 = 1.8 1 SDo5s1 = 6.5

a Data for diaxial fusion were recorded as one or both of the embryos converting. Conversion velocities were averaged and analyzed using aone-way ANOVA. The LSD at the level of significance indicated by the F-test was computed for conversion days in both genotypes. Conversionfrequency data were entered into a contingency table and tested for significance with the x2 statistic. Ninety-five percent confidence intervalsare indicated by ± values (** significant at <0.01% level, NS = not significant at <0.05% level).

Table IV. Effect of Extent of Cotyledon Development on ConversionDays and Final Percent Conversiona

Morphology Conversion ConversionTime

d %

Long hypocotyl, vestigial 4.1 74 ± 1.1cotyledon

Monocotyledon 4.2 61 ± 0.25Dicotyledon 4.9 63 ± 0.26Polycotyledon 5.8 76 ± 1.1Fused cotyledon 7.4 45 ± 0.61

F17 = 3.5* x2 = 13.8**

Data from genotypes 'Gem' and 'Shawnee' (Table Ill) were pooledand averaged. Conversion velocities were averaged and analyzedusing a one-way ANOVA. Conversion frequency data were enteredinto a contingency table and tested for significance using the x2statistic. Ninety-five percent confidence intervals are indicated by +values. For significance tests, * = significant at <0.05 level, 8* =significant at <0.01 level.

3) and a high endogenous concentration of ABA in theembryo (1, 20, 22). The dormancy of immature soybeanzygotic embryos may be circumvented by experimentallyintroducing an early desiccation (18, 26). Somatic embryosmight recapitulate zygotic embryology to the extent that theytoo required a similar period of maturation and dormancy toachieve rapid and high frequency conversion. This interpre-tation seems reasonable particularly since desiccated imma-ture somatic embryos converted with increased frequency andvelocity. The ambient 40% RH treatment probably providedtoo rapid a dehydration. Desiccation under 75% and 93%

RH, however, produced a dramatic increase in the conversionvigor. Perhaps even greater vigor of desiccated somatic em-bryos might be achieved with a sequence of relative humiditytreatments. Slow dehydration of soybean seed as a require-ment for maximum germination vigor has been documented(2, 3). If somatic embryos have a similar requirement, theslow dehydration of somatic embryos under controlled RHconditions may have produced such a required desiccation.Culture of somatic embryos on MM for extended periods alsomay have imposed a slow desiccation as MM contained 10%sucrose. During the maturation of embryos on MM, themedium also became dehydrated as fracture of the agar sur-face was frequently noted. This physical modification likelycaused further elevation of the osmolarity of the culturemedium and may have caused further osmotic conditioningofthe somatic embryos. The maximum conversion frequencyand velocity occurred when the moisture content ofthe wholesomatic embryo decreased to less than 60% (Fig. 2). Germi-nation of soybean seed (26) and growth of the embryonic axis(18) also occurred with increased frequency when the mois-ture content decreased below 60%. A decrease of fresh weightafter 40 d maturation occurred in vivo (18, 26). However, asimultaneous decrease in dry weight was not observed. Thedecrease of dry weight in vitro was probably due to embryomortality (Figs. 2 and 3).

It is unlikely that desiccation of somatic embryos wouldhave inactivated 2,4-D or prevented its suppression ofembry-ogeny. Desiccation of somatic embryos, however, may havecaused a decrease in the endogenous ABA level. The lack ofhigh frequency conversion in immature soybean somaticembryos might therefore be related to an inhibitory endoge-nous ABA titer which was regulated toward lower concentra-

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Plant Physiol. Vol. 89,1989

tions with extended maturation. Finally, besides the effect onABA levels, desiccation may have initiated a cascade of otherphysiological functions associated with physiological maturity(2, 26).

In an earlier report, ABA was used to promote maturationof somatic soybean embryos (23). Without this addition,immature somatic embryos precociously germinated by pro-ducing only a root. This 'rhizogenic structure' never devel-oped an apical shoot area. Although ABA can promote amore normal somatic embryogeny (4) and limits precociousgermination (19), a high sucrose level was substituted in thecurrent studies to inhibit precocious germination (19). Be-cause MM contained activated charcoal, ABA would likelyhave been adsorbed and lost biological availability or activity.Use of culture medium of high osmolarity may have evokedan endogenous ABA biosynthesis, and may have mimickedthe in vivo osmotic environment for a developing embryo.Medium high in osmolarity has often been used for zygoticembryo rescue (30).Matured soybean somatic embryos, like physiologically

mature soybean embryos, required no exogenous growth reg-ulators to promote germination. Ranch et al. (23) described'germination' of soybean somatic embryos as development ofthe apical area followed by root elongation. By the judgementof our current studies, these former somatic embryos wereimmature. Properly matured soybean somatic embryos exhib-ited a more germination-like process in which root elongationgenerally preceded cotyledon greening and shoot develop-ment. After maturation, embryo conversion was also morerapid since in former studies, conversion required 4 to 6weeks (23). Previously, studies on the recovery of plants fromsoybean somatic embryos have recommended the use ofgrowth regulators such as gibberellic acid or a cytokinin. Theseregulators may stimulate or compel development, or causeshoot apex organization (5, 15, 23). Regulators might stimu-late the de novo organization of either apex in an apparentlywell-developed somatic embryo. However, perhaps morelikely, these regulators may activate the embryo or otherwiseaid in overcoming dormancy (29). The differences in somaticembryo behavior among these reports was likely due to thelack of standardization of the ontogenic status of the somaticembryos. There has been little documentation of the embry-ological stage at which somatic embryos were transferred to aconversion or 'germination' medium. Although the ontogenicstages of somatic embryos from various reports cannot beunambiguously equated, the report of Ranch et al. (24) de-scribed the propagation and maintenance ofsomatic embryosat a considerably lower ontogenic stage than that of Barwaleet al. (5) or Lazzeri et al. (15). In the two latter studies,somatic embryos, while still on induction medium, eventuallydisplayed morphological characteristics of size, color, andshape, resembling a cotyledonary stage embryo. In contrast,this embryological stage was not observed until approximately14 d on MM (Fig. 1B). Regulators would likely have differenteffects on somatic embryos or embryogenic tissue dependingon the ontogenic stage of the target tissue (1 1). The concen-tration of2,4-D we used in induction ofembryogenesis main-tained the somatic embryos in a lower stage of ontogeny thandid weaker auxins or 2,4-D at a lower concentration (24).

Therefore, somatic embryos induced with high 2,4-D concen-trations may have required a longer period away from 2,4-Dto complete development.Embryos which converted at high frequency also made the

transition to plantlet with high frequency. This relationshipindicated that the behavior of regenerated plants was influ-enced by prior somatic embryo manipulation and behavior.Therefore, conversion days might be an excellent predictor ofsubsequent plantlet vigor. As the conversion days approachesthe total period of the evaluation, however, the frequency ofconversion becomes a less reliable estimate of the real con-version frequency. Other issues must be addressed when usingthis measure. For example, the study which tested for theeffect of maturation period on conversion velocity had asensitivity of 7 d since this was the lag before the first meas-urement. At the optimal maturation time, conversion dayswas measured as 7.1 d. Since this value was close to thesensitivity of the measurements, the actual conversion periodmight be less. Conversion periods of 3 to 4 d were obtainedwith daily measurement (Table I). In the desiccation andmorphology experiments, the lag before the first measurementwas 2 d. Since no conversion data approached the value ofthis interval, these data likely reflect the true conversion days.In general, conversion days might be used to quantitativelystandardize this important stage in somatic embryo develop-ment. Different conversion periods between experiments maylead to apparent conflicts. For example, there appears to be aconflict between the percent conversion data of Table II andFigure 3. The conversion frequency data of Figure 3 werederived from a 10-week long conversion evaluation. Fromthese data, embryos matured for 40 d converted in approxi-mately 21 d. Conversion in Table II was only evaluated for21 d. Thus, in this latter experiment, the cause for observing0% conversion is related to the length of the experimentalmeasurement period. Although the data from the two exper-iments were consistent, the length of conversion evaluationcan have an influence on the percent conversion observed.We suggest that percent conversion be accompanied by ameasure ofconversion days. Alternatively, percent conversioncould be determined by measurement over a constant periodof time when making comparisons between experiments.Soybean somatic embryos passed through morphological

stages roughly comparable to zygotic embryology. Since mor-phological markers are only one aspect of comparison be-tween zygotic and somatic embryos, there was a degree ofuncertainty about the classification of somatic embryos intospecific ontogenic stages. Molecular markers would be ofassistance in this regard. The morphological variation ob-served was largely associated with cotyledon development andthe interaction of cotyledon formation with the apical meris-tem. Soybean somatic embryos when fully matured also ex-hibited a much larger embryonic axis than found in seed.This effect may be due to either nutritional factors or lack ofphysical constraint. Most of the morphological abnormalitieswe observed did not prevent normal development. This vari-ation might be used to assess the extent to which morphologymay be disturbed without adversely affecting subsequent de-velopmental events or processes. For example, the relation-ship between the amount of cotyledon tissue and the conver-

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SOYBEAN SOMATIC EMBRYO MATURATION

sion days deserves further attention. At certain stages duringthe development of zygotic soybean embryos, cotyledons dopossess more ABA than the axis (21). The relationship ofamount of cotyledonary material and conversion rate indi-cated that cotyledons have a regulating influence on soybeansomatic embryo dormancy and conversion. This influencemay be due to the ABA content of cotyledons.We have described a method for the maturation and con-

version of soybean somatic embryos. By using a relativelyhigh 2,4-D concentration for induction, it was possible toarrest somatic embryo development at a low ontogenic stage.Somatic embryos were subsequently transferred to a matura-tion medium to produce a relatively synchronized embry-ogeny. Immature somatic embryos converted with high fre-quency after a desiccation treatment. If this in vitro matura-tion process parallels the acquisition of quiescence in vivo,this method may yield somatic embryos well-suited for syn-thetic seed (12, 25). This developmental system could be usedto further study the biochemistry of somatic embryo devel-opment in this important crop on a truly temporal basisrather than on a strictly morphological or post hoc basis. Withrespect to maturation and "germination," somatic embryosbehaved similarly to zygotic embryos. This behavior wasconsistent with the hypothesis that the transition from im-mature embryo to mature embryo was mediated primarily bydesiccation (2). Further experiments are required to documentthis mechanism fully.

ACKNOWLEDGMENTS

Our gratitude is extended to M. Ranch for the technical linedrawings, and to Dr. N. Cowen for his expert statistical assistance.

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