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Isolation of a floating cell strain from submerged cell cultures of Euphorbia marginata pursh
Experimenfal
290 ISOLATION
OF A FLOATING
SUBMERGED
CELL
CELL
CULTURES
MARGINATA
Cell Research 26, 290-295 (1962)
STRAIN
FROM
OF EUPHORBIA
PURSH.l
P. G. MAHLBERG Biological
Sciences, University
of Pittsburgh,
Pittsburgh,
Pa., U.S.A.
Received June 29, 1961
THE continuous
propagation of plant cells and cell aggregates under submerged conditions in agitated liquid cultures has been reported by several investigators [l-4, s-121. Growth of such cultures has been dependent upon some type of agitation, either very slow (1 rpm) as performed by Steward et al. [lo] or more rapid (230 ‘pm), with forced aeration as reported by Nickel1 et al. [7]. Agitation is functional in providing aeration to the suspended cell cultures and physically aiding in the separation of cells from each other. Requirement for agitation may well become a limiting factor in the versatility of the cultural technique if it remains the only method whereby single cells or cell aggregates can be readily secured in liquid culture. The physical environment in which a culture is maintained can be a potent factor in selection of cells from the population which are able to survive and multiply. This was evidenced in a chance isolation in this laboratory of a strain of cells which grows upon the surface of the culture medium without agitation or forced aeration. MATERIALS
AND
METHODS
The callus material employed during these observations was derived from proliferated embryos of Euphorbia marginata Pursh. [5]. Comparably sized calli were cultured in liquid and on agar media for comparative purposes. Composition of the basic medium, reported elsewhere [5] was slightly modified. Nicotinic acid and thiamin hydrochloride were removed from the medium. Sucrose level was altered to 30 g/l. The following additions were made (mg/l): CoCl, -6 H,O, 0.005; AlCl, 0.003; NiCl, .6H,O, 0.003; Kl, 0.001. For agar cultures, 0.5 per cent agar was employed for solidification of the medium. The inoculum was cultured in 50 ml of liquid medium in 125 ml Erlenmeyer flasks on a gyroshaker at 175 rpm. Calli on agar were maintained on 30 ml of medium 1 This investigation Foundation. Experimental
was supported
Cell Research 26
by a research
grant (G-8698) from the National
Science
Isolation of floating strain from cultures of E. marginata
291
in baby food bottles. Cultures were maintained in diffuse fluorescent lighting on a 12 hr light-12 hr dark cycle at 25°C. Liquid cultures of single cells and cell aggregates were serially propagated by pipetting 1-3 ml aliquots of the cell suspensions to fresh quantities of liquid media at approximately four week, occasionally eight week, intervals. Calli on agar were transferred to fresh media at similar intervals. Both flasks and bottles were capped with 1.5 mil polyethylene sheeting to prevent desiccation. RESULTS When mature embryos of E. marginata are cultured on an agar medium in the presence of 2,4-dichlorophenoxyacetic acid and u-naphthaleneacetic acid, induced meristematic activity of the provascular tissues of the hypocotyl region results in the formation of a callus mass [S]. The callus is friable in character and is readily separated into small calli during subsequent transfers. This friable character indicated that the calli when cultured in a liquid medium may be suitable for establishment of cell suspension cultures. Origin of suspended cells.-Callus masses (about 3 mm3) transferred to a liquid medium were observed to proliferate rapidly and after incubation for 30-60 days yielded suspension cultures of single cells and cell aggregates of varving size. A culture established in this may was subsequently maintained by introducing either a pipetted quantity of suspension or a large cell aggregate into a new flask of medium (Fig. 1). In liquid culture dissociation of cells from the callus resulted in the formation of a suspension culture consisting of numerous single cells (Fig. 2) and aggregates of varying cell numbers. Morphological variations became apparent in approximately 30 different flasks established after several subcultures. Cultures can be characterized as developing calli of comparatively large size (3 mm or more in diameter) with little dissociation of cells from the calli, in contrast to cultures developing calli of small size (3 mm and less in diameter) \\-ith cell suspensions of varying density. These variations are maintained in culture by subculturing of calli or pipetted quantities of the supernatant containing single cells and cell aggregates. Isolation of floating strain.--4 stable strain of floating cells was detected in one of six liquid cultures placed in the dark and not subjected to agitation. The origin of the floating culture, whether from single cell or multiple cell origin, could not be ascertained. Either a buoyant cell(s) from the suspension or those adherent to the side of the flask gave rise to the initial floating culture. The floating strain was multiplied by transferring small portions of the original culture to the surface of ne\v media. Proliferation occurs in complete darkness Experimental
Cell Research 26
P. G. Mlrhlberg
Fig. l.-Dense suspension of isolated tured for 60 days. x 1.1.
from a 1 ml inoculum
cul-
Fig. S.-Habit of floating strain on surface of medium. Both small calli are evident (center) the very friable cells (horizontal growth pattern) to the left and right of center. x 1.1.
and
Fig. Z.-Single
Fig. 4.--I~oose
spherical
cell derived
cells and cell aggregates from a suspension culture.
cluster of three cells from the floating
Fig. 5.-Two-celled cluster from a suspension culture, lignified (phloroglucinol positive) element. x 490. Experimental
Cell Research 26
derived
strain.
x 490.
x 490.
one cell of which has differentiated
into a
Isolation of floating strain from cultures of E. marginata
293
or under the light-dark regime. The strain has been under continuous cultivation for over one year (Fig. 3). The floating strain differs from the suspension cultures in several respects. Numerous observations upon the submerged cells of the floating strain indicated no survival of cells after 30 days. Floating cells upon subjection to agitation, or forced submersion, did not survive the treatment. The floating strain, originally consisting of very friable calli, displayed a Initially the calli formed three-dimensomewhat variable growth pattern. sional masses attaining variable diameters most frequently 4-3 mm in size. The weight of such masses resulted in the gradual submersion and subsequent death of the component cells. Varying numbers of small calli dissociated from such masses prior to complete submersion and continued to grow. Over a period of several months some changes became evident in the growth pattern. Various of the small calli in different cultures developed a horizontal or tjvo-dimensional grolvth pattern. This was evidenced by the development of a very thin ant1 delicate layer of loosely aggregated cells on the surface of the medium. The surface growth pattern of the floating cultures appeared to be a result of separation of neighboring cells along much of the cell wall surface. The cells often form loose chains or groups of elements anti tlo not develop into compact cell aggregates (Fig. 4). Isolated cells can be secured by gently tapping the floating culture with the transferring loop or small spatula. Individual cells or small aggregates then float free of the surface film of cells. Alicroscopical observations upon the floating cultures indicate greater uniformity of morphological features for these cells than is apparent for the suspended cultures. Cells of the floating strain are more uniform in shape being more or less spherical in form in contrast to the variably shaped cells in the suspended culture condition. Both elongated cells and lignified cells occur under the two cultural conditions (Fig. 3). 1)uplication of a floating culture has not been successful. In contrast, suspended cell cultures can be initiated readily \vith callus inocula or intact embryos introduced into the liquid medium. Further attempts will be made to develop floating cultures of other genera presently being maintained in addition to Rr~phorbirr. DISCUSSION 1’ariabilit.v in the character of cultures of common origin indicates that considerable morphogenetical and physiological plasticity exists within a population of dissociated cell groupings and possibly single cells. Selection SC) - 62173269
Experimental
Cell Xeseurch 26
P. G. Mahlberg of cells capable of surviving under particular environmental conditions is emphasized in particular upon the isolation in standing flasks of a floating cell strain which does not survive when cultured under conditions of agitation. A dii’i’erential 0,/W, requirement of the floating versus suspended cultures is indicated by the inability of the floating strain to survive when submerged in both standing and agitated cultures. The floating ccl1 strain was originally derived from a supension culture; no other cells in the culture were observed to survive the standing culture conditions. The importance of supplemental aeration, especially forced aeration, for increasing the growth rate of cultured cells has been reported for cultures grown in large volumes of liquid medium [7]. It should be pointed out, however, that the cells in the agitated suspension cultures will not survive prolonged periods without agitation. Evidence for deleterious ef‘fects of agitation upon cultured cells remains incomplete. The cells from either culture do not appear physically distorted from constant movement \\-ithin the medium. Employing a slow rate of rotation [lo] for submerged cells does reduce the traumata to \\-hich cells are subjected during continuous culture. It is possible that the cells from standing cultures could not survive the traumata \vhen reintroduced into the agitated culture condition. Nevertheless the physical effects associated with agitation can be entirely eliminated with floating cultures. Selection of cell populations forming a delicate two-dimensional grokvth pattern from the three-dimensional form, has made it possible to enhance the friable character of the floating strain. The surface growth pattern in the floating cultures appears to be a result of separation of neighboring cells along much of the cell wall circumference. The cells often form loose chains or clusters of elements with intercellular pockets of air contributing to the buoyancy of the culture. The change in the growth pattern from a three- to two-dimensional form with resultant increased friability of associated cells suggests that an alteration in the metabolism of the middle lamellar calcium pectate may have occurred. Torrey and Shigemura [13] suggested that friability within suspension cell cultures may be a result of the breakdown of calcium pectate either by enzymic processes or by chelation of calcium. The progressive change occurring in the floating strain suggests, in addition to the above, that a progressive decrease in synthesis of calcium pectate associated with the middle lamella during and after cell divisions could illicit the friable character. A progressive development and selection toward the completely free-form or dissociated character of cells may be occurring in the floating cell strain. Experimental
Cell Research 26
Isolation of floating strain from cultures of E. marginata
2%
The standing flask technique can be used to advantage in conjunction with agitated liquid or agar culture techniques. Standing flasks provide an en\Gronment in which cultured cells are subjected to little physical trauma. Natural selection lvithin the surface cultures will select for mitotically active ccl1 groupings possessing the character of friability. Compact anti enlarged cell groupings submerge with resultant death of the cells. In liquid, therefore, a two-dimensional growth pattern can be developed more readily than on agar \vith the advantage of culturing cells and cell groupings LIIIOII a liquid surface. SUMMARY
A strain
of floating cells, isolated from cell suspensions of Euz@orbirr is being maintained in standing flask culture without requirements of agitation or supplemental aeration for growth. The floating strain exhibits two-dimensional anti three-dimensional growth patterns both of which are being maintained as subcultures. The strain consists of cells more uniform in morphology than the cells in suspension culture. Paths of cellular differentiation are similar in both cultures \vith resultant development of elongated and lignified cells. Isolated single cells or small cell groupings can be readily secured from both the floating cultures and the suspension cultures. Standing flask cultures as a technique can aid in the selection and maintenance of friable cell cultures on a liquid surface to supplement studies on agitated suspension cultures anti agar base cultures.
mcrr~ginntcr,
REFERENCES 1. 2. 3. 4.
5. 6. 7.
8. 9. 10. 11. 12. 13.
RERGMAXN, L., J. Gen. Physiol. 43, 841 (1960). -~ Nature 184, 648 (1959). 1)~ Row, R. S., Proc. Roy. Sot. (London) B 144, 86 (1955). .JONES, L. E., HILDEBRANDT, A. C., RIKER, A. J. and Wu, J. H., Am. J. Botany 47, 468 (1960). MAHL~~G; P. G., Phytomorphot. 9, 156 (1959). MELCHERS, G. and ENGELMAKN, U., Naturluissenschaften 20, 564 (1955). KICKELL, L. G. and TULECKE, W., J. Biochem. Microbiot. Technol. Eng. 2, 287 (1960). ~ Proc. Natl. Acad. Sci. 42, 848 (1956). REINERT, J., Science 123, 457 (1956). STEXI’ARD, F. C., MAPES, M. 0. and SMITH, J., Am. J. Botany 45, 693 (1958). ~ Am. .J. Botany 48, 351 (1961). TORREY, .J. G., Proc. Natl. Acad. Sci. 43, 887 (1957). TORREY, .J. G. and SRIGEMURA, Y., Am. J. Botany 44, 334 (1957).
Experimental
Cell Research 26
290 ISOLATION
OF A FLOATING
SUBMERGED
CELL
CELL
CULTURES
MARGINATA
Cell Research 26, 290-295 (1962)
STRAIN
FROM
OF EUPHORBIA
PURSH.l
P. G. MAHLBERG Biological
Sciences, University
of Pittsburgh,
Pittsburgh,
Pa., U.S.A.
Received June 29, 1961
THE continuous
propagation of plant cells and cell aggregates under submerged conditions in agitated liquid cultures has been reported by several investigators [l-4, s-121. Growth of such cultures has been dependent upon some type of agitation, either very slow (1 rpm) as performed by Steward et al. [lo] or more rapid (230 ‘pm), with forced aeration as reported by Nickel1 et al. [7]. Agitation is functional in providing aeration to the suspended cell cultures and physically aiding in the separation of cells from each other. Requirement for agitation may well become a limiting factor in the versatility of the cultural technique if it remains the only method whereby single cells or cell aggregates can be readily secured in liquid culture. The physical environment in which a culture is maintained can be a potent factor in selection of cells from the population which are able to survive and multiply. This was evidenced in a chance isolation in this laboratory of a strain of cells which grows upon the surface of the culture medium without agitation or forced aeration. MATERIALS
AND
METHODS
The callus material employed during these observations was derived from proliferated embryos of Euphorbia marginata Pursh. [5]. Comparably sized calli were cultured in liquid and on agar media for comparative purposes. Composition of the basic medium, reported elsewhere [5] was slightly modified. Nicotinic acid and thiamin hydrochloride were removed from the medium. Sucrose level was altered to 30 g/l. The following additions were made (mg/l): CoCl, -6 H,O, 0.005; AlCl, 0.003; NiCl, .6H,O, 0.003; Kl, 0.001. For agar cultures, 0.5 per cent agar was employed for solidification of the medium. The inoculum was cultured in 50 ml of liquid medium in 125 ml Erlenmeyer flasks on a gyroshaker at 175 rpm. Calli on agar were maintained on 30 ml of medium 1 This investigation Foundation. Experimental
was supported
Cell Research 26
by a research
grant (G-8698) from the National
Science
Isolation of floating strain from cultures of E. marginata
291
in baby food bottles. Cultures were maintained in diffuse fluorescent lighting on a 12 hr light-12 hr dark cycle at 25°C. Liquid cultures of single cells and cell aggregates were serially propagated by pipetting 1-3 ml aliquots of the cell suspensions to fresh quantities of liquid media at approximately four week, occasionally eight week, intervals. Calli on agar were transferred to fresh media at similar intervals. Both flasks and bottles were capped with 1.5 mil polyethylene sheeting to prevent desiccation. RESULTS When mature embryos of E. marginata are cultured on an agar medium in the presence of 2,4-dichlorophenoxyacetic acid and u-naphthaleneacetic acid, induced meristematic activity of the provascular tissues of the hypocotyl region results in the formation of a callus mass [S]. The callus is friable in character and is readily separated into small calli during subsequent transfers. This friable character indicated that the calli when cultured in a liquid medium may be suitable for establishment of cell suspension cultures. Origin of suspended cells.-Callus masses (about 3 mm3) transferred to a liquid medium were observed to proliferate rapidly and after incubation for 30-60 days yielded suspension cultures of single cells and cell aggregates of varving size. A culture established in this may was subsequently maintained by introducing either a pipetted quantity of suspension or a large cell aggregate into a new flask of medium (Fig. 1). In liquid culture dissociation of cells from the callus resulted in the formation of a suspension culture consisting of numerous single cells (Fig. 2) and aggregates of varying cell numbers. Morphological variations became apparent in approximately 30 different flasks established after several subcultures. Cultures can be characterized as developing calli of comparatively large size (3 mm or more in diameter) with little dissociation of cells from the calli, in contrast to cultures developing calli of small size (3 mm and less in diameter) \\-ith cell suspensions of varying density. These variations are maintained in culture by subculturing of calli or pipetted quantities of the supernatant containing single cells and cell aggregates. Isolation of floating strain.--4 stable strain of floating cells was detected in one of six liquid cultures placed in the dark and not subjected to agitation. The origin of the floating culture, whether from single cell or multiple cell origin, could not be ascertained. Either a buoyant cell(s) from the suspension or those adherent to the side of the flask gave rise to the initial floating culture. The floating strain was multiplied by transferring small portions of the original culture to the surface of ne\v media. Proliferation occurs in complete darkness Experimental
Cell Research 26
P. G. Mlrhlberg
Fig. l.-Dense suspension of isolated tured for 60 days. x 1.1.
from a 1 ml inoculum
cul-
Fig. S.-Habit of floating strain on surface of medium. Both small calli are evident (center) the very friable cells (horizontal growth pattern) to the left and right of center. x 1.1.
and
Fig. Z.-Single
Fig. 4.--I~oose
spherical
cell derived
cells and cell aggregates from a suspension culture.
cluster of three cells from the floating
Fig. 5.-Two-celled cluster from a suspension culture, lignified (phloroglucinol positive) element. x 490. Experimental
Cell Research 26
derived
strain.
x 490.
x 490.
one cell of which has differentiated
into a
Isolation of floating strain from cultures of E. marginata
293
or under the light-dark regime. The strain has been under continuous cultivation for over one year (Fig. 3). The floating strain differs from the suspension cultures in several respects. Numerous observations upon the submerged cells of the floating strain indicated no survival of cells after 30 days. Floating cells upon subjection to agitation, or forced submersion, did not survive the treatment. The floating strain, originally consisting of very friable calli, displayed a Initially the calli formed three-dimensomewhat variable growth pattern. sional masses attaining variable diameters most frequently 4-3 mm in size. The weight of such masses resulted in the gradual submersion and subsequent death of the component cells. Varying numbers of small calli dissociated from such masses prior to complete submersion and continued to grow. Over a period of several months some changes became evident in the growth pattern. Various of the small calli in different cultures developed a horizontal or tjvo-dimensional grolvth pattern. This was evidenced by the development of a very thin ant1 delicate layer of loosely aggregated cells on the surface of the medium. The surface growth pattern of the floating cultures appeared to be a result of separation of neighboring cells along much of the cell wall surface. The cells often form loose chains or groups of elements anti tlo not develop into compact cell aggregates (Fig. 4). Isolated cells can be secured by gently tapping the floating culture with the transferring loop or small spatula. Individual cells or small aggregates then float free of the surface film of cells. Alicroscopical observations upon the floating cultures indicate greater uniformity of morphological features for these cells than is apparent for the suspended cultures. Cells of the floating strain are more uniform in shape being more or less spherical in form in contrast to the variably shaped cells in the suspended culture condition. Both elongated cells and lignified cells occur under the two cultural conditions (Fig. 3). 1)uplication of a floating culture has not been successful. In contrast, suspended cell cultures can be initiated readily \vith callus inocula or intact embryos introduced into the liquid medium. Further attempts will be made to develop floating cultures of other genera presently being maintained in addition to Rr~phorbirr. DISCUSSION 1’ariabilit.v in the character of cultures of common origin indicates that considerable morphogenetical and physiological plasticity exists within a population of dissociated cell groupings and possibly single cells. Selection SC) - 62173269
Experimental
Cell Xeseurch 26
P. G. Mahlberg of cells capable of surviving under particular environmental conditions is emphasized in particular upon the isolation in standing flasks of a floating cell strain which does not survive when cultured under conditions of agitation. A dii’i’erential 0,/W, requirement of the floating versus suspended cultures is indicated by the inability of the floating strain to survive when submerged in both standing and agitated cultures. The floating ccl1 strain was originally derived from a supension culture; no other cells in the culture were observed to survive the standing culture conditions. The importance of supplemental aeration, especially forced aeration, for increasing the growth rate of cultured cells has been reported for cultures grown in large volumes of liquid medium [7]. It should be pointed out, however, that the cells in the agitated suspension cultures will not survive prolonged periods without agitation. Evidence for deleterious ef‘fects of agitation upon cultured cells remains incomplete. The cells from either culture do not appear physically distorted from constant movement \\-ithin the medium. Employing a slow rate of rotation [lo] for submerged cells does reduce the traumata to \\-hich cells are subjected during continuous culture. It is possible that the cells from standing cultures could not survive the traumata \vhen reintroduced into the agitated culture condition. Nevertheless the physical effects associated with agitation can be entirely eliminated with floating cultures. Selection of cell populations forming a delicate two-dimensional grokvth pattern from the three-dimensional form, has made it possible to enhance the friable character of the floating strain. The surface growth pattern in the floating cultures appears to be a result of separation of neighboring cells along much of the cell wall circumference. The cells often form loose chains or clusters of elements with intercellular pockets of air contributing to the buoyancy of the culture. The change in the growth pattern from a three- to two-dimensional form with resultant increased friability of associated cells suggests that an alteration in the metabolism of the middle lamellar calcium pectate may have occurred. Torrey and Shigemura [13] suggested that friability within suspension cell cultures may be a result of the breakdown of calcium pectate either by enzymic processes or by chelation of calcium. The progressive change occurring in the floating strain suggests, in addition to the above, that a progressive decrease in synthesis of calcium pectate associated with the middle lamella during and after cell divisions could illicit the friable character. A progressive development and selection toward the completely free-form or dissociated character of cells may be occurring in the floating cell strain. Experimental
Cell Research 26
Isolation of floating strain from cultures of E. marginata
2%
The standing flask technique can be used to advantage in conjunction with agitated liquid or agar culture techniques. Standing flasks provide an en\Gronment in which cultured cells are subjected to little physical trauma. Natural selection lvithin the surface cultures will select for mitotically active ccl1 groupings possessing the character of friability. Compact anti enlarged cell groupings submerge with resultant death of the cells. In liquid, therefore, a two-dimensional growth pattern can be developed more readily than on agar \vith the advantage of culturing cells and cell groupings LIIIOII a liquid surface. SUMMARY
A strain
of floating cells, isolated from cell suspensions of Euz@orbirr is being maintained in standing flask culture without requirements of agitation or supplemental aeration for growth. The floating strain exhibits two-dimensional anti three-dimensional growth patterns both of which are being maintained as subcultures. The strain consists of cells more uniform in morphology than the cells in suspension culture. Paths of cellular differentiation are similar in both cultures \vith resultant development of elongated and lignified cells. Isolated single cells or small cell groupings can be readily secured from both the floating cultures and the suspension cultures. Standing flask cultures as a technique can aid in the selection and maintenance of friable cell cultures on a liquid surface to supplement studies on agitated suspension cultures anti agar base cultures.
mcrr~ginntcr,
REFERENCES 1. 2. 3. 4.
5. 6. 7.
8. 9. 10. 11. 12. 13.
RERGMAXN, L., J. Gen. Physiol. 43, 841 (1960). -~ Nature 184, 648 (1959). 1)~ Row, R. S., Proc. Roy. Sot. (London) B 144, 86 (1955). .JONES, L. E., HILDEBRANDT, A. C., RIKER, A. J. and Wu, J. H., Am. J. Botany 47, 468 (1960). MAHL~~G; P. G., Phytomorphot. 9, 156 (1959). MELCHERS, G. and ENGELMAKN, U., Naturluissenschaften 20, 564 (1955). KICKELL, L. G. and TULECKE, W., J. Biochem. Microbiot. Technol. Eng. 2, 287 (1960). ~ Proc. Natl. Acad. Sci. 42, 848 (1956). REINERT, J., Science 123, 457 (1956). STEXI’ARD, F. C., MAPES, M. 0. and SMITH, J., Am. J. Botany 45, 693 (1958). ~ Am. .J. Botany 48, 351 (1961). TORREY, .J. G., Proc. Natl. Acad. Sci. 43, 887 (1957). TORREY, .J. G. and SRIGEMURA, Y., Am. J. Botany 44, 334 (1957).
Experimental
Cell Research 26