Plant Physiol. (1984) 76, 1023-10280032-0889/84/76/1023/05/$0 1.00/0

Precocious Germination during In Vitro Growth of SoybeanSeeds'

Received for publication January 9, 1984 and in revised form August 20, 1984

RALPH L. OBENDORF* AND Scorr H. WETTLAUFERDepartment ofAgronomy, New York State College ofAgriculture & Life Sciences, Cornell University,Ithaca, New York 14853-0144

ABSTRACT

Immature Glycine max (L.) Merrill seeds were grown and matured inliquid medium at 25C under fluorescent light. In standard mediumcontaining minerals, 146 millimolar sucrose and 62.5 millimolar gluta-mine (osmolality 0.24), precocious germination seldom occurred with astarting seed size of less than 300 milligrams fresh weight. Frequency ofprecocious germination increased with increased starting seed size. Su-crose concentration strongly affected precocious germination while glu-tamine concentration had no effect. Starting with 300 to 350 milligramsfresh weight seeds, treatments which reduced the sucrose concentrationor lowered the osmolality of the culture medium stimulated precociousgermination, and increased the fresh weight growth but not the dry weightgrowth of seeds. Increasing the osmolality to 0.38 with sucrose ormannitol prevented precocious germination without reducing dry weightaccumulation in seeds. In medium with initially low osmolality, precociousgermination was inhibited by addition of 1 to 100 micromolar abscisicacid to the medium without a reduction in seed growth. During growthand maturation of large soybean seeds in vitro, precocious germinationand other abnormal tissue growth can be prevented by high sucrose ormannitol concentrations in the medium or by addition of abscisic acid.

We recently described the successful growth and maturationof soybean seeds in vitro with ( 15) and without ( 16) pods. Seedsmatured in vitro are similar to naturally matured seeds in com-position (10), are tolerant to desiccation, and are capable ofgermination upon rehydration (10, 15, 16). During 16 to 21 d inculture, seeds grew to a mature dry weight of 100 to 600 mgseed-' at an average rate of 5 to 25 mg dry weight seed-' d-'depending on initial size (16). On field grown plants, dry matteraccumulation continued at a constant rate of about 5 mg seed-'d-' to near maximum dry weight of about 160 mg seed-' (14).When cultured in liquid medium, isolated seeds starting at 50 to70 mg fresh weight accumulated dry matter at an average rate of5 mg seed-' d-'. The average growth rate increased to five timesthe field rate when starting seed size was 400 to 450 mg freshweight (16). A major limitation to experimentation with thesefast-growing, super-sized seeds in vitro is an increase in thefrequency of precocious germination. During studies of seedgrowth and maturation in vitro, control of precocious germina-tion is imperative.Our objective was to evaluate seed growth and precocious

germination after various changes to the medium, includingperiodic addition of glutamine and/or sucrose during culture,

' Funding by Allied Corporation. Agronomy Department Series PaperNo. 1510.

altered initial concentrations of glutamine and sucrose in ninecombinations, and addition of mannitol or ABA to the startingmedium.

MATERIALS AND METHODS

Plant Growth. Soybean (Glycine max [L.] Merrill cv Chippewa64) plants were grown in a greenhouse as previously described(15). Nodulated plants were watered thoroughly as needed andsupplemented with 1 g/pot of complete fertilizer (20-10-20, percent as N-P205-K20 equivalent) in water at weekly intervalsbeginning at 4 weeks after seeding.Seed Culture. Seeds were selected and cultured as previously

described (16). Single excised seeds without pods were transferredaseptically into presterilized 1 25-ml Erlenmeyer flasks containing20 ml of liquid medium. Standard medium was prepared aspreviously described ( 15), except that Na2MoO4 was omitted andthe complete medium was sterilized by ultrafiltration. Alterationsto the standard medium included 5-ml additions of 62.5 mmglutamine and/or 146 mm sucrose solution at 4, 8, and 12 dduring culture; adjusting initial medium to 73, 146 (standard),and 296 mM sucrose and 31.25, 62.5 (standard), and 125 mmglutamine (in all combinations); addition of 100 to 300 mmmannitol and adjustment to 73 mM sucrose in otherwise standardmedium; and addition of 0.01 to 100 ,gM ABA and adjustmentto 73 mm sucrose in otherwise standard medium. ABA wasdissolved and diluted in 95% ethanol, and 5 ul ofethanol solutionwas added to 20 ml ofmedium to obtain the final concentrationsindicated. Osmolality was measured by a freezing point osmo-meter. Harvested seeds were air dried to 12% water contentbefore dry weight determinations. Starting dry weight was esti-mated from starting fresh weight by a regression equation (16).Seeds which germinated precociously were excluded from seedweight and seed growth rate calculations (16). Air dried seedswere germinated on rolled paper towels at 25°C for 3 or 5 dunless otherwise indicated. Experimental results from two to fourreplicate experiments are expressed as frequency, percentage, ornumerical mean ± SE.

Postharvest Germination. To evaluate the effect of growthmedium on postharvest germination, seeds were harvested after14 d in culture to optimize germination potential (16). At thistime axis tissues were yellow and the edges of cotyledons werestarting to yellow. Delayed harvest resulted in declining posthar-vest germination potential (16). After excluding seeds whichprecociously germinated or failed to grow, cultured seeds wereharvested aseptically and air dried to 12% water content during2 weeks at 25C and 60 to 70% RH. Dry seeds (embryos) werehumidified overnight to about 17% water content before germi-nation on paper towels saturated with glass distilled H20 at 25°Cfor 3 or 5 d unless otherwise indicated.

Control seeds (matured on plants) were air dried and germi-1024

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nated on paper towels saturated with the respective seed growthmedia to evaluate the potential toxicity to germination.

RESULTS AND DISCUSSIONSeed Size. Precocious germination during seed growth in

standard medium was seldom a problem when starting seed sizewas below 300 mg fresh weight (Table I). However, as startingseed size increased to 500 mg fresh weight, up to 50% or moreof the seeds germinated precociously. Precocious germinationwas observed primarily as root growth during in vitro culture, incontrast to both hypocotyl and root growth during normal ger-mination. In some cases, distal hypocotyl tissues became swollenwith lateral roots growing from the swollen tissues.

Precocious germination was observed approximately 10 d afterthe start of cultures with seeds starting at 300 to 350 mg freshweight. Precocious germination occurred sooner than 10 d withlarger seeds and later with smaller seeds. When seeds greater than300 mg fresh weight were placed in medium containing GA3, allseeds germinated precociously within 5 (2 mM GA3) to 14 d (0.2Mm GA3) (16). Control seeds initially weighing 100 to 200 mg

Table I. Relationship ofStarting Seed Size to Frequency ofPrecociousGermination during Seed Growth In Vitro

Seeds were cultured 21 d in standard medium. Data were pooled fromseveral experiments. Control seeds at the same starting size were germi-nated as fresh seeds without desiccation or air dried to 12% water contentin detached pods before germination. Control seeds were germinated onpaper towels saturated with distilled H20 at 25°C.

Germination of ControlStarting Seed Size Frequency of SeedsPrecocious

Fresh Dry Germination Dried inwt wt Fresh pod

mg n' % na % na %0-49 5 0/56 0 0/19 050-99 13 0/182 0 0/9 0 18/65 28100-149 27 0/62 0 2/16 12 56/78 72150-199 43 0/48 0 11/25 55 45/64 70200-249 61 1/51 2 12/15 80 48/65 74250-299 80 5/68 7 34/41 83 46/54 85300-349 101 17/94 18 17/17 100 41/46 89350-399 123 33/119 28 17/17 100 46/56 82400-449 148 28/82 34 30/31 97450-499 173 21/40 53 27/28 96500-549 201 2/2 100 5/5 100a Number germinating/total seeds.

fresh weight, and then dried to 12% water content in pods,germinated about 70% after 5 d on wet paper towels. Withoutdesiccation seed of this size germinated 12 to 55% during 17 don wet paper towels (Table I). Seeds that weighed less than 40mg dry weight after in vitro seed growth with or without podsseldom germinated while germination was 64 to 94% for seedsthat weighed more than 40 mg (15, 16). Germination was highestif seeds were removed from culture after axis tissues becameyellow but before complete loss ofgreen color in cotyledon tissues(87 to 94% of maximum dry weight) (16). The germinationpotential of small soybean seeds and embryos has been demon-strated by other workers (2, 3, 14). In concurrence with ourresults, Ackerson (2) also was unable to induce precocious ger-mination in 'Wye' soybean embryos less than 3 weeks old(approximately 100 to 150 mg fresh weight per seed).Glutamine and Sucrose Additions. Addition of glutamine so-

lution during growth of large seeds in vitro markedly stimulatedthe frequency of precocious germination (Table II). Addition ofsucrose solution alone did not stimulate precocious germination,and sucrose reversed the stimulation by glutamine solution. Seedsof this size normally have 5 to 15% precocious germination instandard medium (Table I). The fact that additions of wateralone were sufficient to stimulate precocious germination (TableII), indicated that solute concentration was important.When adjusted for slight differences in starting seed size,

harvested dry weight of seeds which did not precociously germi-nate varied little among treatments in contrast to larger changesin the frequency of precocious germination (Table II). Thus, theadditions appeared to affect precocious germination primarily.The dry weight growth patterns suggested a possible influence ofthe relative concentrations of glutamine and sucrose in themedium on seed dry weight accumulation in vitro (Table II).Postharvest germination of air-dried seeds was 90% for seedsmatured in standard medium, but germination was low for seedswhich were matured in medium receiving additions ofglutamine,sucrose, or water (Table II). Since the increase in volume of themedium may have contributed to a decline in postharvest ger-mination potential, the volume ofthe medium was kept constantin subsequent experiments.

Sucrose-Glutamine Concentrations. Sucrose concentrationstrongly affected precocious germination (Table III). Precociousgermination was very high in 73 mm sucrose medium with astarting osmolality of 167 mOs or less. Increasing the sucroseconcentration to 146 mM (5%) decreased precocious germinationto less than 10%. Precocious germination was not observed in296 mm sucrose medium with a starting osmolality of 375 mOsor greater. Increasing glutamine from 62.5 to 125 mm in 73 mMsucrose medium resulted in reducing precocious germination

Table II. Gluitamine and Sucrose Additions to Large Seeds in Single Seed CultureStarting with 20 ml of standard medium, additions were made after 4, 8, and 12 d in culture. Seeds which

did not germinate precociously were air dried and subsequently germinated on wet paper towels.

Starting Seed After 21 d in Seed CultureAddition to Standard Size Precocious 5 d 25°C

Medium D, Germination Seeds Seed Growth GerminationFresh wt wt harvested dry wt rate

mg na % n mg mg/seed na %None 307 ± I lb 93 0/20 0 20 438 ± 16b 16.4 18/20 905 ml 62.5 mm glutamine 285 ± 24 84 13/18 72 5 396 ± 34 14.9 1/5 20S ml 146mMsucrose 296± 12 88 1/17 6 16 333± 14 11.7 6/16 385 ml glutamine (62.5 mM)+sucrose(146mM) 335 ± 11 105 1/18 6 17 479± 18 17.8 6/17 35

5 ml H20 298 ± 6 86 6/9 67 3 414 ± 23 15.6 0/3 0a Number germinating/total seeds.b Mean ± SE.

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Table III. Influence ofGlutamine and Sucrose Concentrations in the Medium on Seed Growth, Maturation, and Precocious Germination In VitroSeeds were cultured until completely yellow.

Starting Seed Seed Weight at HarvestTreat- Su- Gluta- C:N Starting Size Precocious Days in Seeds See________H0 drywment crose mine Ratio osMityi Fresh Dry Germination Culture Harvested Fresh Dry H20 gro wthrt

osmot walreitDr H20 growth ratewt wt wt wt

mM mM mg:mg mOs mg na % n mg mg/seed % mg/dA 73 31.25 14.1 135 331 ± 25b 103 17/26 65 27± lb 9 1114 ± 49b 314± 11b 800 71 ± 1b 7.8 0.4bB 73 62.5 8.1 167 321 ± 15 99 16/25 64 30 ± 1 9 1385 ± 93 401 ± 15 984 70 ± 1 10.2 ± 0.5C 73 125 5.1 222 333 ± 14 104 4/26 15 27 ± 1 22 1583 ± 52 513 ± 9 1070 67 ± 1 15.3 ± 0.4D 146 31.25 26.2 205 348 ± 16 111 1/25 4 28 ± 1 24 1124 ± 34 375 ± 7 749 67 ± 1 9.7 ± 0.5EC 146 62.5 14.1 245 325 ± 8 101 1/28 4 29 ± 1 27 1262±43 461 ±9 801 63 ± 1 12.7 ±0.5F 146 125 8.1 297 308 ± 13 93 1/29 4 26 ± 1 26 1325 ± 57 506 ± 21 819 62 ± 1 15.8 ± 0.7G 296 31.25 50.2 375 324 ± 13 100 0/26 0 28 ± 1 26 997 ± 42 396 ± 13 601 61 ± 1 10.6 ± 0.5H 296 62.5 26.2 407 304 ± 10 92 0/28 0 29 ± 1 28 1029 ± 39 438 ± 15 591 57 ± 1 11.9 ±0.4I 296 125 14.1 465 323 ± 8 100 0/28 0 25 ± 1 29 1085 ± 47 444 ± 19 641 59 ± 1 14.0 ± 0.7a Number germinating/total seeds.b Mean ± SE.I Standard medium.

from 64 to 15% (Table III). These results suggested that preco-cious germination may be regulated by sucrose concentration(perhaps an osmotic effect) and that glutamine concentrationwas less important in the regulation of precocious germinationduring in vitro growth of large soybean seeds.Although related to sucrose concentration, the frequency of

precocious germination appeared to be regulated by factors otherthan the C:N ratio of the seed growth medium (Table III). At aC:N ratio of 14.1 mgC:mgN, the frequency of precocious ger-mination ranged from 0 to 65%. Likewise, at a C:N ratio of 8.1,the range was 4 to 64%. Comparing the highest and lowest C:Nratios, the frequency of precocious germination was 0 and 15%,respectively. The high C:N ratios were not evaluated at lowosmolality.Days in culture were not markedly altered by sucrose concen-

tration, glutamine concentration or C:N ratio, although an in-crease in glutamine from 62.5 to 125 mm resulted in seedsyellowing 3 to 4 d earlier (Table III). Expansion of seed tissuesas judged by seed fresh weight and mg H20 per seed at harvest,was enhanced by high glutamine and low sucrose concentrations.Cotyledons were visibly more expanded when grown in 73 mmsucrose, and the seeds retained a higher per cent water contentat harvest. With 73 mm sucrose medium, distal hypocotyl tissueshad considerable cell proliferation and occasional proliferationof cotyledon tissues, but tissue proliferation was markedly lesswith 146 or 296 mm sucrose medium. Seed weight at harvestand seed growth rate were influenced more by glutamine con-centration than by sucrose concentration (Table III). Averagegrowth rates ranged from 8 to 16 mg dry weight seed-' d-' forthe period of culture. Highest growth rates occurred in 125 mmglutamine medium, but these seeds yellowed sooner, signaling ashortened duration of the filling period.

Seed dry weight at harvest after culture in standard medium(treatment E) was exceeded only by treatments C and F (TableIII). Tissue proliferation and enhanced precocious germinationevents preclude the use of 73 mm sucrose (treatment C, TableIII) as a reliable culture treatment. Some increased seed weightmay be achieved by increasing the glutamine concentration, buta doubling of glutamine to 125 mm resulted in no significantincrease in weight (treatment F, Table III). In addition to yellow-ing sooner, many cotyledons in 125 mM glutamine medium wereunusually 'cupped' in shape and formed deep transverse crackson the adaxial side. Occurrence of these abnormalities does notfavor use of 125 mm glutamine medium. Large seeds grown andmatured in the 296 mm sucrose, 62.5 mM glutamine medium

(treatment H, Table III) accumulated 95% as much dry weightas seeds in standard medium without the occurrence of preco-cious germination and abnormal growth of axis and cotyledontissues.Although the C:N ratio was not correlated (r = -0.38) with

seed growth in vitro, the C:N ratio of the 296-mM sucrose, 62.5-mM glutamine medium (treatment H, Table III) was comparableto that of leaf petiole exudate (13) and seed coat exudate (20),while the C:N ratio of the standard medium was comparable tothat of pod phloem exudate (13).

Mannitol Additions. Addition of mannitol to 73 mM sucrosemedium reduced the frequency of precocious germination with-out decreasing seed growth rate and dry weight accumulation(Table IV). No precocious germination was observed when start-ing osmolality was 374 mOs or higher (Table IV). Although somevariability occurred in individual flasks, the average osmolalityof the medium in flasks containing a precociously germinatedseed was not significantly different than the osmolality of thestarting medium (data not shown). Our results confirm theimportance of sucrose or mannitol concentrations (osmolality)of the medium in control of precocious germination duringsoybean seed growth in vitro. Sucrose concentration and osmo-lality of the medium appear to be important for the preventionof precocious germination during embryo culture of severalspecies (4, 8, 18). When making experimental additions to themedium during seed culture, care must be taken to avoid aninadvertant reduction in the sucrose concentration (or osmolal-ity) of the medium and a resultant stimulation of precociousgermination in cultured seeds.During growth and maturation of Phaseolus seeds, values for

water potential and solute potential became more negative forboth axis tissues and cotyledons. From 16 to 34 d after anthesis,the solute potential of axis tissues changed from -1.0 to -2.0MPa (22). A similar but slightly delayed change occurred incotyledons. If comparable changes occur in soybean, one wouldexpect an increase in precocious germination as solute potentialof the axis tissues surpassed that of the culture medium at aboutmidseed fill corresponding to our observations (Table I). Like-wise, increased osmolality of the medium would be required toprevent precocious germination with increased starting seedweight during the last half of seed fill. A differential in solutepotential between the axis tissues and cotyledon tissues (as ob-served by Yeung and Brown [22]), would be consistent with anosmotic prevention ofprecocious germination without inhibitionof seed growth (Tables III, IV).

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Table IV. Etlect ofMannibol on Precocious Germination during Seed Growth In VitroMannitol was added to 73 mm sucrose and 62.5 mm glutamine in otherwise standard medium.

Starting Seed After 21 d in CultureStarting Size Precocious ___________________

Mannitol Milli- . .osmolality Fresh Dry Germination Seeds Fresh H20 Dry Dry wt

wt wt harvested wt wt growth rate

mM mOs mg na % n mg % mg mg/d0 170 354 ± 8b 114 13/18 72 5 740 ± 105b 61 283 ± 33b 8.1 1.7b

107.6 266 373 ± 11 122 5/14 36 9 808 ± 49 61 312 ± 17 9.2 ± 0.8206.2 374 346± 11 110 0/16 0 16 771 ±28 60 309± 10 9.4±0.5305.6 485 322±15 100 0/13 0 13 601±24 58 251±10 7.2±0.6a Number germinating/total seeds.b Mean ± SE.

Table V. Effect ofABA on Precocious Germination during Seed Growth In VitroABA was added in 5 Al of 95% ethanol to 20 ml of medium containing 73 mm sucrose and otherwise

standard medium.

Starting Seed After 21 d in CultureABAe Precocious Days to Seeds _________ Dry wtSize GPrecoctious Precocious Harvested Fresh Driy Growth RateFresh Dry Germinatio Germination H20

wt wt wt wt

AM mg na % n mg % mg mg/d0 357 ± 15b 115 7/11 64 10 2b 4 1113± 33b 64 402 ± 2lb 14.0 ± o.9b0.01 354± 12 114 7/14 50 9±2 7 1189±65 65 413± 13 14.5±0.40.1 321 ±9 99 6/12 50 10±3 6 1132± 140 65 390±37 14.3± 1.71 330 ± 10 103 1/12 8 14 11 1238 ± 42 65 427 ± 12 15.4 ± 0.5

10 317±5 97 2/16 13 21 ±0 14 1141 ±50 64 403±8 14.5±0.8100 321 ± 8 99 0/18 0 18 1040 ± 38 62 397 ± 10 14.2 ± 0.4a Number germinating/total seeds.b Mean ± SE.

Table VI. Effect ofSucrose, Mannitol, andABA Adjustments to the In Vitro Growth Medium onGermination after Harvest and Desiccation

Seeds starting at 300 to 350 mg fresh weight were grown in vitro for 14 d in standard medium adjusted asindicated. Seeds which germinated precociously were excluded. Seeds were harvested aseptically and air driedto 12% water content at 25°C and 60 to 70% RH. Dry seeds (embryos) were humidified to about 17% watercontent before germination on wet (distilled H20) paper towels for 5 d at 25°C. Control seeds were maturedon plants, air dried, and germinated on paper towels saturated with seed growth medium.

Seed Growth Medium Postharvest Control SeedseGermination of Seeds

Grown In Vitro Hypocotyl RootSucrose Mannitol ABA Germination length length

mM AM nb % mm % mm

73 0 0 6/14 43 38±14c 98 19+±c 8± lc146 0 0 12/15 80 23±3 93 13± 1 5±1296 0 0 19/21 90 49±12 75 10±1 2±173 206 0 11/22 50 86±15 95 14±1 3±173 305 0 12/22 51 73± 10 88 11 ± 1 2± 173 0 1 3/17 18 24 ± 11 99 17 ± 1 8 ± 173 0 100 5/19 26 70 ± 35 93 16 ± 1 8 ± 1

Glass-distilled H20 98 63 ± 2 118 ± 4a Five replications of 20 seeds each, n = 100. Seeds were germinated for 4 d at 25°C.b Number germinating/total seeds.I Mean ± SE.

ABA in Medium. Addition ofABA at 1 gM or higher reducedthe frequency of precocious germination (Table V) to the lowlevels observed in standard medium for seeds at the same startingsize (Table I). ABA concentrations of 0.01 to 100 gM had noeffect on seed growth and dry matter accumulation during cul-ture of initially large seeds (Table V). Since young soybean seeds

contain high levels of ABA (1, 2, 7, 17), it was not surprisingthat in vitro growth of seeds (Table V; [ 16]) or excised cotyledons(1, 19) was not affected by addition of ABA. Ackerson (1)observed a stimulation in in vitro dry weight growth of soybeanembryos by 1 uM ABA only when embryos (20 to 60 mg initialfresh weight) had not yet reached peak ABA concentration in

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vivo. ABA is generally recognized as a potent inhibitor of pre-cocious germination (2, 4-6, 8, 9, 12, 21). The decrease inconcentration of ABA during rapid dry matter accumulation insoybean seeds (1, 2, 7, 17) and a lower ABA concentration inaxis tissues than in cotyledons or seed coats (17) is consistentwith the increase in precocious germination at larger startingseed weights (Table I). Ackerson (2) noted an enhanced frequencyof precocious germination of soybean embryos with a decline inendogenous ABA concentration to less than 4 gg/g fresh weight,but a reduction in ABA did not always result in germination.Inhibition of precocious germination by exogenous applicationof 1 ,AM ABA (Table V) is similar to results with other species (4,6, 8). The onset of dormancy during Arabidopsis seed develop-ment correlated with the presence of embryo controlled ABAbut not with maternally controlled ABA or exogenously appliedABA (1 1). However, Karssen et al. (11) concluded that restrictedaccess to water may be the most important limiting factor forprevention of precocious germination in vivo.

Postharvest Germination. Seeds grown in 146 or 296 mmsucrose medium retained a postharvest germination potential of80 to 90% (Table VI). In contrast, seeds grown in 73 mm sucrosemedium, a treatment with 65% precocious germination (TableIII), had a postharvest germination potential of 40 to 50% (TableVI). Addition of mannitol to 73 mM sucrose medium, to preventprecocious germination, enhanced the growth of postharvestseedlings without improving the frequency of postharvest ger-mination. The addition of ABA to 73 mm sucrose mediumappeared to restrict the potential for postharvest germination.Seed cultured in glass-distilled H20 without an exogenous energysource failed to increase in dry weight and retained green color.After desiccation, these seeds also failed to germinate.

Seeds cultured in glass-distilled H20 without an exogenousenergy source failed to increase in dry weight and retained greencolor. After desiccation, seeds which were cultured in glass-distilled H20 also failed to germinate. Control seeds which werematured on plants exhibited superior germination and seedlinggrowth on paper towels saturated with water. Germination ofcontrol seeds on paper towels saturated with seed growth mediumwas greater than 90% (except in medium with high osmolality),but seedling growth was markedly reduced. Hypocotyl growthwas reduced 70% and root growth was reduced more than 90%in the presence of seed growth medium (Table VI). Hypocotylswere thickened in treatments with severe root growth inhibition.

Conclusion and Recommendations. During growth and matu-ration of large soybean seeds in vitro, precocious germinationand other abnormal tissue growth can be prevented by highconcentrations of sucrose or mannitol in the medium or byaddition of ABA to low osmolality medium. For most experi-ments, 296 mm (10%) sucrose would be recommended for soy-

bean seed growth and maturation in vitro when starting seed sizeis greater than 250 mg/seed fresh weight (weight may vary withcultivar). The high sucrose concentration provided substrate forseed growth, adjusted osmolality of the medium, permitted op-

timum seed dry weight growth without abnormal tissue growth,and prevented precocious germination of large seeds during

culture while preserving postharvest germination potential. Forexperiments requiring a low osmolality medium, addition of 100,M ABA to the medium would prevent precocious germination,but the potential for postharvest germination may be reduced.In all cases of in vitro growth of soybean seeds in liquid mediumwithout pods, we were unable to prevent the rupture of the seedcoat (16). Therefore, care must be taken to avoid mechanicalinjuries to the embryo during in vitro growth and also duringrapid dehydration and/or rehydration.

Acknowledgments-The authors thank L. Vargas, S. Weerasena, and I. Saab forexpert assistance with some of the experiments.

LITERATURE CITED

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2. ACKERSON RC 1984 Abscisic acid and precocious germination in soybeans. JExp Bot 35: 414-421

3. ADAMS CA, RW RINNE 1981 Seed maturation in soybeans (Glycine max L.Merr.) is independent of seed mass and of the parent plant, yet is necessaryfor the production of viable seeds. J Exp Bot 32: 615-620

4. AMMIRATO PV 1983 Embryogenesis. In DA Evans, WR Sharp, PV Ammirato,Y Yamada, ed, Handbook of Plant Cell Culture, Vol I. MacMillian, NewYork, pp 82-123

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soybean seeds. Plant Sci Lett 27: 129-13511. KARSSEN CM, DLC BRINKHORST-VAN DER SWAN, AE BREEKLAND, M KOORN-

NEEF 1983 Induction of dormancy during seed development by endogenousabscisic acid: studies on abscisic acid deficient genotypes of Arabidopsisthaliana (L.) Heynh. Planta 157: 158-165

12. KING RW 1982 Abscisic acid in seed development. In AA Khan, ed, ThePhysiology and Biochemistry of Seed Development, Dormancy and Germi-nation. Elsevier Biomedical, New York, pp 157-181

13. LAYZELL DB, TA LARUE 1982 Modeling C and N transport to developingsoybean fruits. Plant Physiol 70: 1290-1298

14. OBENDORF RL, EN ASHWORTH,GT RYTKO 1980 Influence of seed maturationon germinability in soybean. Crop Sci 20: 483-486

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16. OBENDORF RL, EE TIMPO, MC BYRNE, TV TOAI, GT RYTKO, FC Hsu, BGANDERSON 1984 Soybean seed growth and maturation in vitro without pods.Ann Bot 53: 853-863

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19. THOMPSONJF, JT MADISON, AME MUENSTER 1977 In vitro culture of imma-ture cotyledons of soya bean (Glycine max L. Merr.). Ann Bot 41: 29-39

20. THORNE JH, RM RAINBIRD 1983 An in vivo technique for the study of phloemunloading in seed coats of developing soybean seeds. Plant Physiol 72: 268-271

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22. YEUNG EC, DCW BROWN 1982 The osmotic environment of developingembryos ofPhaseolus vulgaris. Z Pflanzenphysiol 106: 149-156

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