Food Structure

Volume 6 Number 2 Article 4

1987

Cellular Rupture and Release of Protoplasm and Protein Bodies From Bean and Pea Cotyledons During Imbibition

Stephen C. Spaeth

Joe S. Hughes

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Recommended Citation Spaeth, Stephen C. and Hughes, Joe S. (1987) "Cellular Rupture and Release of Protoplasm and Protein Bodies From Bean and Pea Cotyledons During Imbibition," Food Structure: Vol. 6 : No. 2 , Article 4. Available at: https://digitalcommons.usu.edu/foodmicrostructure/vol6/iss2/4

This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Food Structure by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. FOOD MICROSTRUCTURE, Vol. 6 (1987 ) , pp. 127-134 Scanning Mi croscopy International, Chicago (AMF O'Hare), IL 60666 USA

CELLULAR RUPTURE AND RELEASE OF PROTOPLASM AND PROTEIN BODIES FROH BEAN AND PEA COTYLEDONS DURING IMBIBITION

Stephen C, Spaeth* and Joe S. Hughes**

Grain Legume Genetics and Physiology Research Unit Agricult.ural Research Servi;e 0 0 8 Depa r~~e~t· o ;p;oror:dme;ctie nfceAgarnidc~l~uar: Nu t ;idtion** Washington State Univers i ty Pullman, Wa shington, 99164

Introductio n

Imbibition is a critical phase in germina­ Legwne seeds and excised cotyledons which tion and processing of legume seeds because cell­ are soaked i n water or sown in wet soil release ular disruption during imbibition may influence intracellular substances into the surrounding seedling vigor and processing quality Cellular water (Schroth and Cook, 1964; Duke and Kakefuda, disruption of cotyledonary surfaces of beans 1981, Duke et al., 1983, Spaeth, 1987), Substan­ (Phaseolus vulgaris L.) and peas (Pisum sacivum tial quantities of nutrients can be lost from L.) and the cellular contents released during seeds during soaking, e.g., 60% of K content in imbibition were examined with scanning electron 24 h (Simon, 1984). In crop production, losses microscopy. Two types of c e llular disruption of intracell ular materials are associated with we r e observed during imbibition: ruptures and poor seedl ing vigor and stand (Larson, 1968, fractures. Individual cells and small groups of Powell and Matthews, 1978) because cells are cells on t he surfnces of cotyledons ruptured ruptured and storage reserves are not nvailable after immersion in water. Ruptured cells had for growth of seedlings Loss of intracellular flaps of cell walls which remained attached to substances is also associated with increased intact portions of cell walls. Fractured cells pathogen growth (Sch roth and Cook, 1964) because spl it in half, and remnant portions of cell walls nutrient sources become available to soil-borne were completely separated from each other. pathogens. Disrupted cells on the interior surfaces of The loss of int racellular substances has blister cavities were of the fractured type. been attributed to large scale disruption of Materials released from cotyledonary tissues manifested as transverse cracks (Morris tissues consisted of both dense aggregates of et. al., 1970), displacement of tissue strips protein bodies and a dispersed phase of proto­ (Duke and Kakefuda, 1981), and blistering of plasm. In some cases, protoplasm and protein embryonic axes (Dunn et al., 1980) and cotyle­ bodies on cotyledonary surfaces were found donary surfaces (Spaeth, 1987). Multicellular adjacent to single cell ruptures and, in others, blist:ers on surfaces of bean cotyledons ranged the sites of losses were not found. The presence from less than 0. 4 to more than 0. 8 mm in of protoplasm and protein bodies and absence of diameter (Spaeth, 1987). One mechanism for sites for their release indicate an additions 1 release of intracellular materials from blisters mechanism other than fracture or rupture may is pressure-driven extrusion (Spaeth , 1987). contribute co losses of intracellular substances Partially hydrated protoplasm, protein bodies during imbibition. and, in some cases , starch grains (granules) exhibit characteristics of a viscous fluid which is extrud e d as streams of material t h rough irreg­ ular orifices in blisters. Solute leakage from cotyledonary tissue is Initial paper rec eived April 6, 1987 not always accompanied by visible tissue disrup­ Manuscript rec eived October 16, 1987 tion. While cotyledons of one bean cultivar Direc t inquiries to S.C . Spaeth produced surface blisters, a second bean cultivar Telephone number: 509 335-9521 and two pea cult i vars extruded small diameter streams of intracellular material but did not form visible blisters (Spaeth, 1987). Seeds with intact seed coats may leak substantial amounts of intracellular substances, yet not exhibit visible Key wo rds: beans, Phaseolus vulgaris (L), peas, tissue disrup tion (Simon and Raja Harun, 1972, Pisum sacivum (L), cellular rupture, protoplasm Duke and Kake f uda, 1981, Simon, 1984). The extrusion , nutrient loss, solute leakage, soaking existence of cellular ruptures has been inferred injury, imbibitional stress, imbibitional injury, from the appearance of intracellul ar substances, protein bodies. e. g. ot·ganelle specific enzymatic marke·cs (Duke

127 S. C. Spaeth and J. S. Hughes and Kakefuda, 1981), which are too large for dehydrated in a graded ethanol series (30 to diffusion through intact membranes. Evans Blue 100%) (Hughes and Swanson, 1985). Fixed samples stain has been used to demonstrate plasma were dried in carbon dioxide with a critical membrane rupture during imbibition (Duke and point drier (Bomar SPC·l500) 1 , sputter coated Kakefuda, 1981; Schoetcle and Leopold, 1984). with gold (Hummer·Technics) 1 • All samples were Detailed microscopic studies of cell rupture viewed and photographed at: 20 kV with an ETEC U ·1 would help to elucidate mechanisms of solute Scanning Electron Hicroscope 1 • leakage and its role in reduced legume seed viability. The first objective of this research was to examine cellular disruption on a scale smaller In addition to the large scale cracking and than transverse cracks, crescent cracks, and multicellular blisters previously reported, blisters. The second and third objectives were epidermal cells of legume cotyledons were to identify various types of cellular disruption, ruptured during imbibition. Areas of Apollo bean and to identify some materials lost from cotyle· cotyledons which were not blistered exhibited dons during imbibition Scanning electron ruptured cells and the contents from the ruptured microscopy (SEM) was used co examine excised cells on adjacent unblistered tissue (Fig 1). cotyledons a nd protoplasm lost during imbibition. Only a few of the total number of cells on the surface were ruptured (Fig. l). Holes consisted Materials and Methods of individual or small groups of ruptured cells. Flaps of cell walls, which were formed by the Seeds of two bean cult:ivars (Phaseolus rupture process, were attached to remaining walls vulgaris L. cv Apollo, a snap bean, and cv Royal of ruptured cells. Cell contents released from Red Mexican, a dry bean) and two pea cultivars cells did not contain recognizable constituents (Plsum sativum L. cv OSU 605, a wrinkled pea, and (Fig. 1). Cell contents from ruptures did not cv Carfield 81, a round pea) were obtained from form extrusion streams as did cell contents from local commercial or research sources. Seeds were blisters (Spaeth, 1987). equilibrated to constant water content over a The scale of individual cell rupture was saturated solution of potassium carbonate. Water too small to be seen without SEM, therefore, it content of seeds was determined by oven drying at was not possible to use light microscopy to 10s•c for 48 h. Initial water contents of bean directly confirm that individual cells or small and pea cotyledons were 11 and 10% of seed dry groups of cells ruptured during soaking To weight, respectively. Coats of dry seeds were carefully removed with a scalpel. Individual cotyledons were £.i&...... l. Ruptured cells (R) with wall flaps (F) immersed in 6 ml of distilled, deionized water at and cell contents (C) on t h e surface of Apollo 24•c. Cotyl edons and extrusion streams from snap·bean cotyledons soaked prior to fixation cotyledons were observed with a dissecting micro · Bar - 10 pm. scope during imbibition. Cotyledons for SEH were prepared in several .E.i&...,___,f. Surface cells of Apollo bean cotyledons different ways. To prepare unfixed samples for which had single drops of water applied to SEH, some cotyledons were simply excised from dry cotyledonary surfaces. Low magnification (2a) seeds. Other cotyledons were excised, soaked in shows dry (untreated) tissue (D), perimeter of water for 30 min, blotted to remove excess water, the treated area (P), ruptured cells (R), unrup· and then allowed to dry at ambient temperature tured cells (U), cell contents (C), smooth and humidity. In one set of Apollo bean cotyle· surface (S) with fissures (f). Bar - 100 ~m. dons, a droplet of water was applied to abaxial Same seed at higher magnification (2b) shows surfaces. After the water was absorbed into the ruptured cells (R), cell contents (C), and cotyledon or evaporated, the sample was reequili· fissures (F) in the epidermal surface covered brated at ambient relative humidity. Another set with a film of cell contents. Bar - 10 pm. of bean cotyledons was fractured dry. Fracture surfaces were washed free of protoplasm a nd .Eig,_,_l. Ruptured cells (R) in OSU 605 pea cotyle­ cellular inclusions as described by Wo lf and dons which were soaked but not fixed. Wall flaps Baker (1980), Dry tissues of all unfixed samples (F) were attached to undisturbed portions of were sputter coated with gold (Hummer·Technics) 1 • ruptured cell walls. Cotyledon surface was free To prepare fixed samples for SEM, cotyle· of cellular contents. Bar- 10 pm. dons were immersed in 6 ml distilled, deionized water unt:il blisters extrusion ~· Fracture surfaces of transverse cracks appeared (20 min). To preserve the structure of through bean cotyledons. Dry fracture without extruded protoplasm during fixation, a washing surface (4a) shows intercellular spaces glutaraldehyde :water solution (25%) was gently (1), cell walls (\.l), starch grains (S) imbedded added to Petri plates containing t:he soaking in protoplasm and protein bodies (PB). seed, and mixed with the soak water to create a Bar - 50 pm. Surface, after washing to remove 5% glutaraldehyde solution for fixing tissue and protoplasm and cellular inclusions (4b), shows extruded protoplasm. Thirty min after addition primarily empty cell walls (E), with few starch of the glutaraldehyde solut i on, cotyledons were grains (S) on the surface, and some starch grains rinsed and transferred to a 2% solution of osmium trapped (T) in a cell with a small hole in it. tetroxide in water for 30 min. Samples were Bar - 100 pm

128 Cell Rupture In Cotyledons During Imbibition

129 S. C. Spaeth and J. S. Hughes ensure that ruptures of individual cells were not dons differed from those of bean and round pea caused by glutaraldehyde induced modification of Before imbibition, small, isolated particles of cell walls, cotyledons were prepared for SEM by intracellular material were observed on the applying isolated drops of water to cotyledonary wrinkled surfaces of OSU 605 cotyledons (Fig. surfaces and allowing the drops to be absorbed or 6a). Epidermal cells gave the surface a rough evaporate. This treatment also partially texture During imbibition and fixation, t h e simulated the wetting and drying cycles employed wrinkles changed to creases, cotyledonary sur­ by Powell and Matthews (1981) faces became smoother, and large quantities of Cells on the surfaces of Apollo cotyledons flecks appeared on the central portion of the ruptured and lost intracellular materials when cotyledon, near the creases (Fig. 6b) Blisters drops of water were applied to surfaces (Fig were not found on the surfaces of pea cotyledons 2a). Spots with irregular outlines were formed as in earlier results (Spaeth, 1987) where water drops lay on cotyledon surfaces (Fig Creases in surfaces of OSU 605 cotyledons 2a). The rough-textured areas around spots did were filled with dense particles and surfaces not come into contact with water. Three zones adjacent to the creases were covered with dense were observed where water was applied, a rough particles and patches of less dense material center with ruptured cells and cellular contents, (Fig. 6c). Large numbers of single cell ruptures a bumpy surface without ruptures, and a perimeter or small tissue ruptures were not found which was smooth and cracked (Fig. 2a). Isolated epidermal cells on cotyledonary Cellular ruptures and intracellular materi­ surfaces of OSU 605 (Fig. 6d) were ruptured als were clearly visible at higher magnification Flaps of cell walls remained attached to the main (Fig. 2b). The intracellular materials on portion of the wall Protoplasm released from unfixed cotyledons were more consolidated than ruptured cells was in two forms Dense particles that on fixed cotyledons, but constituents were were roughly spherical or irregular in shape and no more recognizable. Air-drying of soaked ranged from l to 10 J-Im in effective diameter tissues without fixation resulted in formation of (Fig. 6d). The disperse phase of cellular cracks and other predictable artifacts. Thin contents often covered large areas of cotyle­ layers or films of material covered entire donary surfaces and consisted of small particles cotyledonary surfaces (Fig . 2a, 2b). Films were and a network of interconnecting material (Fig probably mixtures of soluble and insoluble compo­ 6d). Particle size distribution of the dispersed nents of cell contents which dried in a thin phase was similar to the granular cytoplasm found coating Cracks in the film and cell walls in un-aged dry-bean cells (Varriano-Marston and probably formed during dehydration of the tissue . Jackson, 1981). Dry films were similar to films found on parti­ Dense part icles from cotyledonary t issues cles of soy- flour which were moistened with were aggregates of spheroidal particles which buffer and dried (Wolf and Baker, 1972) ranged from l to 2 Jlm in effective diameters When cotyledons of OSU 605, a wrinkled pea, (Fig 6d, 7). Spheroidal particles included were soaked in water, blotted dry, and allowed to protein bodies which were a major component of dry in air, isolated cells on the surface also materials from ruptured cells Although some ruptured ( Fig. 3) Remnant flaps of cell walls protoplasm and protein bodies released from hung over the empty cells Cotyledon surfaces cotyledonary tissues clearly came from single immersed in large quantities of water and not subsequently fixed lacked protoplasm and inclu­ sions (Fig. 3). Protoplasm and inclusions, which .Ei...g_.______Fracture surface of blister top after in other cases were fixed to cotyledonary removal from Apollo bean cotyledon showing empty surfaces or restricted to small volwnes of water cell walls (E) and starch grains (S). in drops, apparently were washed away in the Bar - 100 jJm. l arger quantity of soak water. Ruptured bean and pea cells contrasted with f.i.g_,____Q. Release of cell contents from OSU 605, a another form of damage to cotyledonary tissues, wrinkled pea. Unsoaked and unfi xed cotyledon which also contributed to losses of intracellular (6a) shows wrinkles (W) and material (M) on the materials When cotyledonary tissues of beans surface. Bar - 1 mm Soaked and fixed cotyledon fracture as a result of tensile stress, e.g., (6b) shows creases (Cr) and large quantities of crack transversely, cells in the fracture plane material (M) on the surface. Bar - l mm Higher split completely apart (Morris et al., 1970, magnification of same cotyledon soaked and fixed Spaeth, 1987) (Fig. 4a) and parts of cell walls (6c) with a crease (Cr) in surface and large separated completely. When protoplasm, protein quantities of cell contents (C) on the surface bodies and starch grains were washed out of and large quantities of dense material (M) in the fractured cells , incomplete cell wall fragments crease Bar - 100 Jlm. Highest magnification of comprised most of the surface (Fig. 4b) When soaked and fixed cotyledonary surface (6d) show­ holes in cell walls were small, starch grains ing ruptured cells (R) with cell wall flaps (F) were retained in fractured cells while protein and cell contents including a disperse phase of bodies and protoplasm were washed out (Fig. 4b). protoplasm (Pr) and protein bodies (PB). Cells at fracture surfaces did not exhibit Bar - 100 Jlm. remnant flaps characteristic of ruptured cells. Blister separation surfaces (Fig. 5), were E.ig_,__2. Protein bodies (PB) and disperse phase of similar to tensile fracture surfaces (Morris et protoplasm ( Pr) on surfaces of soaked and fixed al., 1970, Spaeth, 1987). samples of Royal Red Mexican bean with adjacent The shape of OSU 605 (wrinkled pea) cotyle· cells not ruptured. Bar = 10 t-tm

130 Cell Rupture In Cotyledons During Imbibition

131 S. C. Spaeth and J . S. Hughes cell ruptures (Fig. 6d), substantial quantities Table l. Frequency of various features of injury of protoplasm and protein bodies appeared on and losses of intracellular substances from bean cotyledonary surfaces where cell ruptures were (Apol lo and Royal Red Mexican) and pea (OSU 60S no t observed (Fig. 6c,d, 7). and Garfield 81) cultivars during imbibit ion. A summary indicates the absence or pres· ence, and frequency of various features observed in each of the cultivars (Table 1). Note that Crop Beans Peas Royal Red Mexican and Garfield 81 had relatively Cultivar: Apollo RRM OSU 605 Garfield few ruptures and substantial quantities of proto· Type: ~ Qu Wrinkled Round plasm and protein bodies on the surfaces of cotyledons after imbibition. Feature: Transverse Cracks Blisters Micrographs of cell ruptures (Figs. 1·3,6d) Cell Ruptures corroborated histochemical evidence for single Extrusion streams cell ruptures (Duke and Kakefuda, 1981). Hypo· In soak water (LM) +++ theses about cellular rupture with complete loss On cotyledons (SEM) +++ of cell contents (Simon, 1984) were partially Protoplasm confirmed; however, cellular rupture demonstrated Protein bodies here was more deleterious than the membrane rupture that Simon ( 1984) and Powell and Matthews (1981) discuss. Cell walls as well as plasma t + - presence and frequency, - - absence. membranes were ruptured during imbibition. Duke et al. (1983) suggested that cells might be able to repair mild cases of membrane rupture. of soybean cotyledons and interior surfaces of Rupture of bean and pea cell walls during imbibi­ seed coats observed by Yaklich et al. (1984) tion (Figs. 1-3,6d) was sufficient to preclude apparently were formed by a different (unidenti­ recovery or repair. However, it must be remem­ fied) process. bered that seed coats were not present to modify In contrast to the extrusion streams water uptake observed on fixed Apollo bean cotyledons and in Cellular ruptures were observed in cotyle­ water surrounding unfixed OSU 605 pea cotyledons dons which were imbibed but not fixed, and to a (Spaeth, 1987), protoplasm and protein bodies lesser extent in cotyledons which were fixed in from fixed OSU 605 cotyledons did not form extru­ aqueous solutions Therefore, ruptures in fixed sion streams. The disperse phase released from cotyledons were caused by imbibition of water ruptures of OSU 605 cells was protoplasm fixed by from the fixation solution and were not caused by the glutaraldehyde solution. Protoplasm quickly glutaraldehyde in the fixation processes. Fixa­ dispersed into soak water which did not contain tion with glutaraldehyde was useful because glutaraldehyde. Rapid dispersion of protoplasm protoplasm and protein bodies were retained on will make it difficult to determine to what cotyledonary surfaces which otherwise would have extent fracture, rupture, extrusion, and diffu­ been lost in the soak water. sion each contribute to the aggregate quantities Large holes were produced in walls of of material released from cotyledons. ruptured cells, but flaps of cell walls w(!re The intracellular material observed on the attached to the undisturbed portions of cell surfaces of Royal Red Mexican, OSU 605, and walls (Fig. 1,3,6d). Ruptures appeared to be Garfield 81 but not associated with cellular cases of plasmoptysis, osmotically induced ruptures was also not in the form of extrusion rupture of cells. The presence of cell wall streams. The dense particles from cotyledons flaps opening to the surface was characteristic were individual or aggregates of protein bodies of wall rupture and explosive release of the (Fig . 6d, 7). The disperse phases on the surfaces contents. of Royal Red Mexican, OSU 605, and Garfield 81 Rupture of single cells contrasted wi th differed slightly in form. Differences in proto­ another process by which intracellular materials plasm might reflect differences in composition of were completely lost from cotyledonary cells , protoplasm from different genera and cultivars. cell fracture in the plane of transverse cracks. Protoplasm and protein bodies may be one of the Fractures of cells in transverse cracks are primary constituents of 'deposits' which have caused by tensile stresses in dry tissue (Spaeth , been observed on surfaces of excised cotyledons 1986, 1987) which split cells through the middle (Wolf and Baker, 1972). and leave empty cell walls after protoplasm and The low frequency of cellular rupture in inclusion s wash out (Morris et al., 1970, Wolf Royal Red Mexican, OSU 605, and Garfield 81 and Saker, 1980) Walls of cells in the fracture cotyledons did not appear to account for the plane are split in half and no longer attached to quantities of protoplasm and protein bodies lost one another. Fractured cel ls lacked flap from cotyledons of these cultivars. Some remnants of cell wall (Fig. 4b). Separation ruptures may have been hidden by t he protoplasm surfaces in blister cavities were similar to released from ruptured cells, or protoplasm and fracture surfaces associated with transverse protein bodies may have been fixed to surfaces at cracks (Fig. 5). The similarity indicates that some distance from ruptured cells. However, the tensil e stresses were probably important in the low frequency of single cell ruptures on cotyle· formation of blisters. Holes in epidermal cells donary surfaces in which protoplasm was not fixed

132 Cell Rupture l n Cotyl edons During Imb ibition

( Fig . 3; indicated that protoplasm and protein Dunn BL, Obendorf RL, Paolillo OJ Jr. bodies nay h,ave been released from cotyledonary (1980). Imbibitional surface damage i n isolated cells bf some mechanism other than complete hypocotyl-root axes of soybean {Glycine max (L) rupture :lf ce 11 walls. Merr, cv. Chippewa 64]. Plant Physiol. U , S-139. Powel 1 and Matthews (1981) demonstrated Hughes JS, Swanson BG. (1985). Microstruc­ that substantial quantities of intracellular t u ral changes i n maturing seeds of the common substances are lost from cotyledonary tissue bean (Phaseolus vulgaris L.). Food Mi crostruc. !!., dur ing : mbibition and that cells are a ble to 183- 189. recover from s uch losses. A process by whic h Larson LA. (1968). The effect soaking pea cells cculd lose protoplasm and protein bodies seeds with or without seed coats h as on seedling and not rupt:ure and complete ly discharge cell growt h . Plant Physiol. !!}, 255-269. contents h as yet to be identified. The distinc­ Morris JI, Campbell WF, Pollard I. (1970). tions bet ween cellular rupture and fracture, and Re lation of imbibition and drying on cotyledon among the for ms in wh ich intracellular substances cracking in snap beans, Phaseolus vulgaris L. J. were released , will help efforts to deve lop Amer. Soc. Hort. Sci. 22., 541 - 543. breeding. cultura l, and processing strategies Powell AA, Matthews S. ( 1978). The damaging which mi nimize the problems of tissue disrupt ion effect of water on dry pea embryos during imbibi­ during bbibition . tion. J. Exp. Bot. 1.2, 1215-1229. Powell AA, Ma tthews S. ( 1981). A physical Conclusions explanation for solute leakage from dry pea em­ bryos during imbibition . J. Exp. Bot. ,ll, 1045- Di s rupt ion of cotyledonary cells during 1050. imbibition occurs on both large and small dimen­ Schoettle AW, Leopold AC. (1984). Solute sions. Large disrupti ons include transverse lea kage from artificially aged seed s after cracking and blistering. Small disruptions were imbibition . Crop Sci. .2.!!.. 835-838. cell wall r uptures. Ce llular fracture is caused Schroth MN , Cook RJ. (1964). Seed exudation by tensile stresses induced during imbibition b y and its influence on pre-emergence damping-off of surrounding tissue. Fractur e d cells split open bean . Phytopathology 2!!. 670-673. and most of t hei r contents were released i n to Simon E\1. (1984). Early even ts in germina­ soak water . In con trast, individual and small tion. In: Seed Ph ysiology. Vol 2. Germination and groups of cells d ischarged cellular conten ts reserve mobilization, DR Murray (Ed.), Academic under pressure during imbibition. Cellular Pr ess, Orlando, FL, pp 77-115. contents d ischarged from ruptured cells included Simon EW, Raja Harun RM . (1972), Leakage pro toplas m, and protein bodies in dispersed and during seed imbibition. J. Exp. Bot. 21. 1076- dense phases, respectively. Efforts to measure 1085. and control leakage from seeds should take into Spaeth SC. (1986). !mbibitional stress and account variation among process es by whic h intra ­ t r ansverse cracking of bean, pea , and c h ickpea cellular sub stances are released from cotyl edons cotyledons. HortScience 11. 110-111. during imb i bi cion. Spaeth SC. (1987). Pressure-driven extrusion of protoplasm from cotyledons of b ean Ac kno wledgements and pea during imbibition. Plant Physiol. !l2,, 217- 223. A contribution from the Grain Legume Varriano- Ma rston E, Jackson GM. (1981) Genetics and Physiology Research Unit, U. S. Hard-to-cook phenomenon in beans: Structural Department of Agriculture, Agricultural Research changes during storage and imbibition. J. Food Service in cooperation with the Agricultural Sci. ~. 1379-1385. Research CEnter , Washington State University. The Wolf WJ , Baker FL . ( 1980). Scanning Electron Microscope Center , Washington State e l ectron microscopy of soybeans and soybean Universi ty provided facilities and technical produc ts . Scanning Electron Hic rosc. 1980; Ill: assistance Dr. M. J. Silbernagel and Crites­ 621-634. Moscow Co, supplied seeds. Jerry Coker provided Wolf WJ, Baker FL. (1972). Scanning technical ass i stance. electron microscopy of soybeans. Cereal Sci. Today 11., 124-126,128-130,147. 1 Mention o : a trademark, proprietary product, or Yaklich RW, Vigil EL, Wergin WP . (1984). vendor does not constitute guarantee o r warranty Scanning electron microscopy of soybean seed of t he product by the U _ S. Department of Agri ­ Scanning Electron Microsc. 1984; II: 991- culture, a nd does not i mply its approval to the !000. exclusion of other products or vendors that may also be s u i table. Discussion with Reviewers

W J Wo1 f: One would expect to be able to discern starch grains and p rotein bodies as Duke SH, Kakefuda G. (1981). Role of the constituents of cellular contents on the surfaces testa i n preventing cellular rupture dur i ng of cotyledons. Were the cell con tents shown in imbibition of l egume seeds. Plant Physiol , 21. Figs. 1 and 2b examine d at higher mag nification? 449-456. &!th2.n: We did not find starch grains among the Duke SH , Kakefuda G, Harvey TM. (1983). contents of ruptured cells in any of the seeds Differential l eakage of intracellular s ubstances examined. Viewing the cell contents s hown i n from i mbibl ng soybean seeds. Plant Physiol. 21. Fig. 1 at high magnification revealed protein 919-924.

133 S. C. Spaeth and J. S. Hughes bodies similar to those of OSU 605 and Royal Red K Saio: Losses of sol id and chemical components Mexican (Figs. 6d and 7). !-l owever, we could not during inunersion in water clearl y increase discern protein bodies in contents from ruptured depending on time and conditions during storage cells of air-dried samples, including Fig. 2b. (Saio et al. , 1980, Cereal Chern. ll. 77-82). Are such losses related to the frequency of cellular E Varriano-Marston: Structures in Fig. 3 bear r upture? some similarity to stomates. How did you distin­ E Varriano-Marston: Cultivars differ i n leakage guish between cellular ruptures and stomates on and seedling vigor. Do differences in cellular the surfaces of cotyledons? rupture cause differences in leakage and vigor? Authors: Stomates are generally bilaterally Authors: Given the variety of p rocesses which synunetric. Cellular ruptures ""ere often asymme­ can contribute to losses of intracellular s u b ­ tric. The large flaps of cell wall which often stances, we would not necessarlly expect to find remained attached to rupture d cell s also served strong corre lations between any one form of to distinguish them from stomates. imbibitional injury and total losses of i ntr a­ cellular ma terials. Differences in protein leak­ K...... fu!i: You have shown that some cells rupture age among cul tivars were consisten t with t h e while others do not. What differences exist extent of cellular fracture in excised cotyledons between ruptured and unruptured cells? of susceptible and resistant cul tivars (Spaeth, Authop:: Rupture of an individual cell is prob­ 1987) ably influenced by numerous factors including the mo vement of water, characteristics of the cell R W. Yaklich · Are the results obtained in this itself and characteristics of adjacent tissue. research applicable to seeds with intact coats? Epidermal t i ssues influence water movement into Authors: Seed coats certainl y can influence subepidermal cells and may dimi nish cellular water moveme n t into cotyledonary cel l s. However, r upture t here. Variation in strength of indivi· fractured ce lls associated with transverse cracks dual cell walls might predispose some cells to and multicellular blisters are found in some rupture. Finally, the tendency of a cell to cultivars even when seed coats are intact ( Morris rupture may be influenced by surrounding ce lis. et al. 1970, Spaeth , 1987). Rupture of indivi­ If compressive stresses in hydrating tissue dual cell s seems to be a less severe case of contribute significantly to pressures which cause imbibitional injury so we expect it will be found cellular rupture, then rupture of some cells may in seeds with intact coats of some cultivars . partially relieve t he stresses and , thereby, diminis h t he number of neighboring cells which rupture.

~: Hard beans, which cannot absorb water, can be made to absorb water if the seed coat is scarified. The rupture of cells on the cotyle­ donary surface may act in a similar manner to promote rapid absorption of water while also resulting in the loss of nutrients from the seeds. Pl ease conunent . Authors: The ce llular ruptures which result from imbibiti.onal stresses should be of interest to othe r areas of legume seed research including the serious problems assoc i ated with hard beans. Hardness in beans is caused by different factors including the i nability of water to penetrate seed coats and failure of cotyledons to soften even when seeds imbibe water. Cotyledonary cell rupture would not appear to play a role in hard­ ness resulting from seed coat impermeab i lity wh ere water does not enter cotyl edons, but may be involved in forms of hardness wh ere bean seeds r e main hard even after cotyledons imbibe wa ter

134