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Crown gall protoplasts—isolation, culture and ultrastructure
Plant Science Letters, 1 (1973) 451--456 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
CROWN GALL PROTOPLASTS -- ISOLATION, CULTURE AND U L T R A S T R U C T U R E *
W.R. SCOWCROFT**, M.R. DAVEY*** and J.B. POWER*.
Department of Botany, University of Nottingham, University Park, Nottingham NG7 2RD (Great Britain) (Received April l l t h , 1973)
SUMMARY
Large quantities of protoplasts were enzymatically isolated from cell suspensions of Parthenocissus tricuspidata crown gall. Their behaviour under fully defined cultural conditions was followed at t h e light- and electron-microscope level. Some protoplasts regenerated a new cell wall and divided to form small cell colonies; others underwent nuclear division without concomitant cytokinesis to form multinucleate, wall-less structures. In each case there was an absolute requirement for exogenous growth regulators.
INTRODUCTION
Many plant species from diverse families form neoplasms following infection with Agrobacterium tumefaciens '. The persistence of the inciting bacterium is not essential to maintain the non self-limiting growth o f crown gall neoplasms, which, when cultured in vitro, are independent of exogenous growth regulators 2 Power and Frearson 3 and Benbadis and Bauman 4 isolated protoplasts from crown gall of Parthenocissus tricuspidata and Vitis vinifera respectively, by the enzymatic degradation of in vitro cultured material. This report is concerned with the isolation, colony formation and ultrastructure of protoplasts of P. tricuspidata. The successful culture of protoplasts required an exogenous supply of growth regulators which is contrary to the independence of normal crown gall cultures. * Supported by a grant from the Agricultural Research Council. ** On leave from Division of Plant Industry, CSIRO, Canberra, Australia and supported in part by a Royal Society Nuffield F o u n d a t i o n Bursary. *** Supported by a Royal Society Pickering Research Fellowship. Abbreviation: NAA, naphthalene acetic acid.
451
MATERIALS AND METHODS
Crown gall of P. tricuspidata was maintained on Heller's saltsm e d i u m s with the addition of 2 % sucrose and 0.4 mg/l thiamine HCI, as either callus or a suspension culture in 500-ml flasks (150 ml media) on a rotary shaker (90 cycles/min) in the dark at 26 ° . Actively dividing cultures with large numbers of small cell colonieswere obtained 2--4 weeks after initiation of the cell suspension. In order to isolate protoplasts, the cellswere allowed to sediment and were transferred to a 100-ml flask together with 40 ml of a filter-sterilizedenzyme solution comprising 2 % Cellulase "Onozuka" R-10, 0.01% Macerozyme R-10 in 1 3 % mannitol at p H 5.6. The mixture was incubated in the dark at 22 ° on a rotary shaker (70 cycles/rain) for 16 h. Following centrifugation (90 g; 5 min), the supernatant was discarded and the pellet resuspended in 1 5 % sucrose and centrifuged (200 g; 5 rain).The sucrose flotation yielded a clean preparation of protoplasts which were washed twice in the final culture m e d i u m and embedded in agar according to the method of Nagata and Takebe 6 at a minim u m density of I- 10 s/ml and maintained in the dark at 25 °. Material for electron microscopy was fixed in 3 % glutaraldehyde (in 0.1 M sodium phosphate buffer, p H 7.1) for 16 h at 22 °. The solution contained 1 5 % sucrose during fixation of freshly isolated protoplasts and 1 3 % mannitol for fixation of cultured protoplasts. The latter were fixed in situ in agar medium. Preparations were washed in 0.1 M phosphate buffer (pH 7.1 ), postfixed in 0.1 M phosphate buffered 1 % OsO4 (pH 7.1) (3 h at 2°), and subsequently embedded and sectioned as described by Davey and Short 7 RESULTS
The efficiency of release from cell suspensions was 21%, thus yielding approximately 1.107 protoplasts (Fig. 1). Several protoplast culture media and auxin/cytokinin levels were employed; the most successful was that of Nagata and Takebe 6 with 0.1 mg/1 NAA and 0.2 rag/1 kinetin replacing the original growth regulators. Plate I.
Fig. 1. Freshly isolated protoplasts maintained in agar medium (× 470). Fig. 2. First division of a protoplast detected after 5 days (× 470). Fig. 3. Small cell colony produced as a result o f sustained division of an individual protoplast (14 days) (× 60). Fig. 4. Predominantly wall-less structure produced after 24 days. Such structures are frequently multinucle ate and contain protoplasmic subunits and arise as a result o f nuclear division without associated cytokinesis (x 30). Fig. 5. Suspension culture cell in thin section showing peripheral cytoplasm and large central vacuole (v). Plastids (arrow) contain starch, n, nucleus iX 1500). Fig. 6. Section through a suspension cell wall. The fibrils are closely compacted (middle lamella, arrow) (X 9000). Fig. 7. Freshly isolated protoplast. Organelles have increased in number and are aggregated around the nucleus (n). Plastids have lost their starch (× 1650). Fig. 8. Plastids (arrow) and mitochondria in the cytoplasm near the nucleus of the protoplast in Fig. 7. Mitochondria are elongated. The mitochondrial matrix and plastid stroma are electron opaque as a result o f plasmolysis (x 9750).
453
Both suspension cells and freshly isolated protoplasts were highly vacuolated (Figs. 1, 5, 7). Accumulated starch was apparent l y utilised during protoplast isolation and initial culture (cf. Figs. 5, 8). During cul t ure the protoplasts became less vacuolated and highly cytoplasmic (Fig. 9) with the nucleus and associated organelles assuming a central position (Figs. 9, 10). The d e v e l o p m e n t of cultured protoplasts was variable. A p p r o x i m a t e l y 30% of protoplasts progressively u n d e r w e n t cell wall regeneration (Figs. 11--13} and subsequent division after five days (Fig. 2) which was o f t e n sustained, resulting in small cell colonies (Fig. 3). It is clear t hat the regenerated cell walls were n o t as substantial as those o f nor mal cultured crown gall cells (Fig. 6). Alternatively, cultured protoplasts f r e q u e n t l y gave rise to near spherical structures after 3 weeks (Fig. 4), which were o f t e n multinucleate (Fig. 14) as a result of nuclear division w i t h o u t associated cytokinesis. Although pred o m i n a n t l y wall-less, these structures appeared to contain nucleated protoplasmic subunits separated by p o o r l y developed walls (Fig. 15). Often there was no detectable cell wall material separating these subunits (Fig. 16). During early culture, as a possible result o f a fission-like process, clusters of small protoplasts were also de t e c t e d. These protoplasts, normally o f equal size (20 #), were non-vacuolated, nucleated, probably wall-less, and a b o u t one-third o f the normal protoplast diameter. Browning o f protoplasts and regenerating cells towards t he end o f the culture period was possibly due t o accum ul a t i o n of phenolic substances. T he e x t e n t o f browning could be minimised b y restricting atmospheric exposure during handling.
Plate II.
Fig. 9. Protoplast cultured for 10 days. The cytoplasm has increased in extent compared with Fig. 7. The nucleus now occupies a central position surrounded by plastids which have accumulated starch (× 1650). Fig. 10. Amyloplasts and mitochondria in the cytoplasm of a 10-day-old protoplast. The mitochondrial matrix and plastid stroma have now recovered from the effect of plasmolysis and have lost their electron opacity. Mitochondria are mainly oval in shape at this stage, n, nucleus (× 11000). Fig. 11. The initial stage in wall formation of a cultured protoplast (10 days). Electron opaque material (arrow) has accumulated immediately external to the plasmalemma (x 26000). Fig. 12. Completed cell wall (arrow) of a 14-day-old cultured protoplast (x 4200). Fig. 13. Cell wall of Fig. 12. The fibrils are loosely compacted as compared with those in the walls of suspension cells (Fig. 6). The first formed wall material of Fig. 11 is now integrated with the fibrillar structure (× 21000). Fig. 14. Multinucleate structure containing 5 nuclei after 24 days in culture (× 3600). Fig. 15. Section through part of a spherical structure of Fig. 4. Most protoplasts within the structure are cytoplasmicaUy active (left hand protoplast of Fig. 15); others show degenerating cytoplasm. Sparse wall material may be present between some protoplasts (arrows), but individual protoplasts of these spherical structures are mainly naked (x 3600). Fig. 16. Portion of two adjacent naked protoplasts from a spherical structure. The plasmalemmae (arrows) are nearly touching and there is a complete absence of wall material (X 15000).
455
DISCUSSION Although cultured crown gall t u m o u r tissues are independent of an exogenous supply of growth regulators, protoplasts isolated from such tissue are, at least initially, d e p e n d e n t on their presence in the medium, for the initiation of division. It was possible to transfer colonies and the wall-less structures to a medium devoid of growth substances, and still maintain division. Once a certain degree of structural organisation or physiological stability was achieved in these small colonies, growth regulator dependence apparently ceased. The general interest in crown gall disease stems, not only from its patholological effects on economic species, but also from the fact that an understanding of neoplastic transformation in plants may shed some light on the basic causes of tumourigenesis 2. The salient feature of crown gall tissues is growth regulator a u t o n o m y , in spite of their different origins (bacterial, viral or genetic). Our findings suggest that the derepression of growth regulator biosynthesis in crown gall cells is intimately related to the presence of a functional cell wall. The removal of the cell wall leads to a transient reversion to growth regulator dependence during the early culture of the protoplasts. Following wall regeneration and division, the derepressed state is apparently re-established. Some confirmation of this comes from other crown gall systems which are being investigated at the protoplast level (Daucus carota, Vinca rosea, N i c o t i a n a tabacum and Petunia hybrida) in which protoplast division has not been observed in culture media devoid of growth regulators. REFERENCES
1 C. Elliot, Manual of Bacterial Plant Pathogens, 2nd ed., Chronica Botanica, Waltham, 1951. 2 A.C. Braun (Ed. ), Plant Tumor Research (Progr. Exptl. Tumor Res., 15), Karger, B~el,
1972. 3 J.B. Power and E.M. Frearson, in Protoplastes et Fusion de Cellules Somatiques VJgdtales, Coll. Intern., C.N.R.S., No. 212 (1973) 409. 4 A. Benbadis and F. Bauman, in Protoplastes et Fusion de Cellules Somatiques V~ge~tales, Coll. Intern., C.N.R.S., No. 212 (1973) 189.
5 R. Heller, Ann. Sci. Natl. Bot. Biol. Veg., 14 (1953) 1. 6 T. Nagata and I. Takebe, Planta, 99 (1971) 12. 7 M.R. Davey and K.C. Short, Protoplastes et Fusion de Cellules Somatiques Vdgdtales, Coll. Intern., C.N.R.S., No. 212 (1973) 437.
456
CROWN GALL PROTOPLASTS -- ISOLATION, CULTURE AND U L T R A S T R U C T U R E *
W.R. SCOWCROFT**, M.R. DAVEY*** and J.B. POWER*.
Department of Botany, University of Nottingham, University Park, Nottingham NG7 2RD (Great Britain) (Received April l l t h , 1973)
SUMMARY
Large quantities of protoplasts were enzymatically isolated from cell suspensions of Parthenocissus tricuspidata crown gall. Their behaviour under fully defined cultural conditions was followed at t h e light- and electron-microscope level. Some protoplasts regenerated a new cell wall and divided to form small cell colonies; others underwent nuclear division without concomitant cytokinesis to form multinucleate, wall-less structures. In each case there was an absolute requirement for exogenous growth regulators.
INTRODUCTION
Many plant species from diverse families form neoplasms following infection with Agrobacterium tumefaciens '. The persistence of the inciting bacterium is not essential to maintain the non self-limiting growth o f crown gall neoplasms, which, when cultured in vitro, are independent of exogenous growth regulators 2 Power and Frearson 3 and Benbadis and Bauman 4 isolated protoplasts from crown gall of Parthenocissus tricuspidata and Vitis vinifera respectively, by the enzymatic degradation of in vitro cultured material. This report is concerned with the isolation, colony formation and ultrastructure of protoplasts of P. tricuspidata. The successful culture of protoplasts required an exogenous supply of growth regulators which is contrary to the independence of normal crown gall cultures. * Supported by a grant from the Agricultural Research Council. ** On leave from Division of Plant Industry, CSIRO, Canberra, Australia and supported in part by a Royal Society Nuffield F o u n d a t i o n Bursary. *** Supported by a Royal Society Pickering Research Fellowship. Abbreviation: NAA, naphthalene acetic acid.
451
MATERIALS AND METHODS
Crown gall of P. tricuspidata was maintained on Heller's saltsm e d i u m s with the addition of 2 % sucrose and 0.4 mg/l thiamine HCI, as either callus or a suspension culture in 500-ml flasks (150 ml media) on a rotary shaker (90 cycles/min) in the dark at 26 ° . Actively dividing cultures with large numbers of small cell colonieswere obtained 2--4 weeks after initiation of the cell suspension. In order to isolate protoplasts, the cellswere allowed to sediment and were transferred to a 100-ml flask together with 40 ml of a filter-sterilizedenzyme solution comprising 2 % Cellulase "Onozuka" R-10, 0.01% Macerozyme R-10 in 1 3 % mannitol at p H 5.6. The mixture was incubated in the dark at 22 ° on a rotary shaker (70 cycles/rain) for 16 h. Following centrifugation (90 g; 5 min), the supernatant was discarded and the pellet resuspended in 1 5 % sucrose and centrifuged (200 g; 5 rain).The sucrose flotation yielded a clean preparation of protoplasts which were washed twice in the final culture m e d i u m and embedded in agar according to the method of Nagata and Takebe 6 at a minim u m density of I- 10 s/ml and maintained in the dark at 25 °. Material for electron microscopy was fixed in 3 % glutaraldehyde (in 0.1 M sodium phosphate buffer, p H 7.1) for 16 h at 22 °. The solution contained 1 5 % sucrose during fixation of freshly isolated protoplasts and 1 3 % mannitol for fixation of cultured protoplasts. The latter were fixed in situ in agar medium. Preparations were washed in 0.1 M phosphate buffer (pH 7.1 ), postfixed in 0.1 M phosphate buffered 1 % OsO4 (pH 7.1) (3 h at 2°), and subsequently embedded and sectioned as described by Davey and Short 7 RESULTS
The efficiency of release from cell suspensions was 21%, thus yielding approximately 1.107 protoplasts (Fig. 1). Several protoplast culture media and auxin/cytokinin levels were employed; the most successful was that of Nagata and Takebe 6 with 0.1 mg/1 NAA and 0.2 rag/1 kinetin replacing the original growth regulators. Plate I.
Fig. 1. Freshly isolated protoplasts maintained in agar medium (× 470). Fig. 2. First division of a protoplast detected after 5 days (× 470). Fig. 3. Small cell colony produced as a result o f sustained division of an individual protoplast (14 days) (× 60). Fig. 4. Predominantly wall-less structure produced after 24 days. Such structures are frequently multinucle ate and contain protoplasmic subunits and arise as a result o f nuclear division without associated cytokinesis (x 30). Fig. 5. Suspension culture cell in thin section showing peripheral cytoplasm and large central vacuole (v). Plastids (arrow) contain starch, n, nucleus iX 1500). Fig. 6. Section through a suspension cell wall. The fibrils are closely compacted (middle lamella, arrow) (X 9000). Fig. 7. Freshly isolated protoplast. Organelles have increased in number and are aggregated around the nucleus (n). Plastids have lost their starch (× 1650). Fig. 8. Plastids (arrow) and mitochondria in the cytoplasm near the nucleus of the protoplast in Fig. 7. Mitochondria are elongated. The mitochondrial matrix and plastid stroma are electron opaque as a result o f plasmolysis (x 9750).
453
Both suspension cells and freshly isolated protoplasts were highly vacuolated (Figs. 1, 5, 7). Accumulated starch was apparent l y utilised during protoplast isolation and initial culture (cf. Figs. 5, 8). During cul t ure the protoplasts became less vacuolated and highly cytoplasmic (Fig. 9) with the nucleus and associated organelles assuming a central position (Figs. 9, 10). The d e v e l o p m e n t of cultured protoplasts was variable. A p p r o x i m a t e l y 30% of protoplasts progressively u n d e r w e n t cell wall regeneration (Figs. 11--13} and subsequent division after five days (Fig. 2) which was o f t e n sustained, resulting in small cell colonies (Fig. 3). It is clear t hat the regenerated cell walls were n o t as substantial as those o f nor mal cultured crown gall cells (Fig. 6). Alternatively, cultured protoplasts f r e q u e n t l y gave rise to near spherical structures after 3 weeks (Fig. 4), which were o f t e n multinucleate (Fig. 14) as a result of nuclear division w i t h o u t associated cytokinesis. Although pred o m i n a n t l y wall-less, these structures appeared to contain nucleated protoplasmic subunits separated by p o o r l y developed walls (Fig. 15). Often there was no detectable cell wall material separating these subunits (Fig. 16). During early culture, as a possible result o f a fission-like process, clusters of small protoplasts were also de t e c t e d. These protoplasts, normally o f equal size (20 #), were non-vacuolated, nucleated, probably wall-less, and a b o u t one-third o f the normal protoplast diameter. Browning o f protoplasts and regenerating cells towards t he end o f the culture period was possibly due t o accum ul a t i o n of phenolic substances. T he e x t e n t o f browning could be minimised b y restricting atmospheric exposure during handling.
Plate II.
Fig. 9. Protoplast cultured for 10 days. The cytoplasm has increased in extent compared with Fig. 7. The nucleus now occupies a central position surrounded by plastids which have accumulated starch (× 1650). Fig. 10. Amyloplasts and mitochondria in the cytoplasm of a 10-day-old protoplast. The mitochondrial matrix and plastid stroma have now recovered from the effect of plasmolysis and have lost their electron opacity. Mitochondria are mainly oval in shape at this stage, n, nucleus (× 11000). Fig. 11. The initial stage in wall formation of a cultured protoplast (10 days). Electron opaque material (arrow) has accumulated immediately external to the plasmalemma (x 26000). Fig. 12. Completed cell wall (arrow) of a 14-day-old cultured protoplast (x 4200). Fig. 13. Cell wall of Fig. 12. The fibrils are loosely compacted as compared with those in the walls of suspension cells (Fig. 6). The first formed wall material of Fig. 11 is now integrated with the fibrillar structure (× 21000). Fig. 14. Multinucleate structure containing 5 nuclei after 24 days in culture (× 3600). Fig. 15. Section through part of a spherical structure of Fig. 4. Most protoplasts within the structure are cytoplasmicaUy active (left hand protoplast of Fig. 15); others show degenerating cytoplasm. Sparse wall material may be present between some protoplasts (arrows), but individual protoplasts of these spherical structures are mainly naked (x 3600). Fig. 16. Portion of two adjacent naked protoplasts from a spherical structure. The plasmalemmae (arrows) are nearly touching and there is a complete absence of wall material (X 15000).
455
DISCUSSION Although cultured crown gall t u m o u r tissues are independent of an exogenous supply of growth regulators, protoplasts isolated from such tissue are, at least initially, d e p e n d e n t on their presence in the medium, for the initiation of division. It was possible to transfer colonies and the wall-less structures to a medium devoid of growth substances, and still maintain division. Once a certain degree of structural organisation or physiological stability was achieved in these small colonies, growth regulator dependence apparently ceased. The general interest in crown gall disease stems, not only from its patholological effects on economic species, but also from the fact that an understanding of neoplastic transformation in plants may shed some light on the basic causes of tumourigenesis 2. The salient feature of crown gall tissues is growth regulator a u t o n o m y , in spite of their different origins (bacterial, viral or genetic). Our findings suggest that the derepression of growth regulator biosynthesis in crown gall cells is intimately related to the presence of a functional cell wall. The removal of the cell wall leads to a transient reversion to growth regulator dependence during the early culture of the protoplasts. Following wall regeneration and division, the derepressed state is apparently re-established. Some confirmation of this comes from other crown gall systems which are being investigated at the protoplast level (Daucus carota, Vinca rosea, N i c o t i a n a tabacum and Petunia hybrida) in which protoplast division has not been observed in culture media devoid of growth regulators. REFERENCES
1 C. Elliot, Manual of Bacterial Plant Pathogens, 2nd ed., Chronica Botanica, Waltham, 1951. 2 A.C. Braun (Ed. ), Plant Tumor Research (Progr. Exptl. Tumor Res., 15), Karger, B~el,
1972. 3 J.B. Power and E.M. Frearson, in Protoplastes et Fusion de Cellules Somatiques VJgdtales, Coll. Intern., C.N.R.S., No. 212 (1973) 409. 4 A. Benbadis and F. Bauman, in Protoplastes et Fusion de Cellules Somatiques V~ge~tales, Coll. Intern., C.N.R.S., No. 212 (1973) 189.
5 R. Heller, Ann. Sci. Natl. Bot. Biol. Veg., 14 (1953) 1. 6 T. Nagata and I. Takebe, Planta, 99 (1971) 12. 7 M.R. Davey and K.C. Short, Protoplastes et Fusion de Cellules Somatiques Vdgdtales, Coll. Intern., C.N.R.S., No. 212 (1973) 437.
456