Plant Cell Reports
Plant Cell Reports (1993) 12:339-342
9 Springer-Verlag 1993
Improvement of protoplast culture protocols for Beta vulgaris L. (sugar beet) Robert D. Hall, Charlotte Pedersen, and Frans A. Krens Department of Cell Biology, DLO-Centre for Plant Breeding and Reproduction Research (CPRO-DLO), Postbus 16, 6700 AA Wageningen, The Netherlands Received September 10, 1992/Revised version received January 28, 1993 - Communicated by M. R. Davey
Summary. The effects of NaCl, feeder cells and the embedding of protoplasts in calcium alginate have been investigated in an attempt to improve culture conditions of recalcitrant sugar beet (Beta vulgaris L.) mesophyU protoplasts. While the use of NaCl in all instances proved detrimental to protoplast development, the other two treatments had clear beneficial effects. Minimum plating densities, necessary to sustain cell division, could be reduced to < 5 % (< 4000 protopIasts / ml) of the control levels and plating ej~ciencies couM be significantly enhanced by approx. 10 fold. Plants couM still be regenerated from soft calli derived from mesophyll protoplasts cultured under the modified conditions at a frequency of 20 - 30 %. In particular, the use of alginate is considered of potentiaUy great importance for the further application of beet protoplasts for other aims e.g. asymmetric hybridization. Key words: Alginate embedding - Beta vulgaris -feeders protoplasts - regeneration
Introduction In sugarbeet, cytoplasmic male sterility (CMS) is of fundamental importance both in breeding and for the production of F] hybrid seed [Kaul 1988]. In previous publications we outlined how beet breeding programmes were, on a world scale, rather perilously centred around a very narrow cytoplasmic base [Krens et al. 1990; Krens and Hall 1992]. The rapid broadening of this genetic base through the employment of in vitro techniques (e.g. asymmetric hybridization), to incorporate newly-identified mitochondrial genetic variants, is of potentially great importance. Surprisingly, despite being a dicotyledonous crop and despite the considerable attention which it has received, Beta vulgaris remains reealeitrant with respect to many biotechnologieal techniques. However, previously we were able to report, for the first time, the successful regeneration of plants from beet mesophyll protoplasts isolated from two distinct aecessions [Krens et al. 1990]. These regenerants were predominantly diploid and phenotypieally normal. To date we have now obtained plant regeneration from mesophyll protoplasts from 5 of the 7 lines tested (unpublished observations). These results are eneouraging Correspondence to: R. D. Hall
and augur well for future bioteehnologieal application. However, one factor which remains problematic is the disappointingly low plating efficiencies observed even at high plating densities. Generally, plating efficieneies are < 1% in regenerable systems. This represents a potentially serious limitation to success, especially concerning asymmetric somatic hybridizations where few cells remain viable in culture. Consequently, in order to improve the low frequency of division in beet protoplast eultures, we have carried out a series of experiments to help identify valuable modifications to the culture conditions. The effect of the inclusion of NaC1 at a range of concentrations in the protoplast isolation/eulture media was tested following the previously-reported advantageous effects found with beet callus cultures [Hagbge et al 1990]. Additionally, the potential value of using a range of feeder (nurse) systems, already used to great advantage for low density culture [e.g. Waiters and Earle 1990] has herebeen assessed regarding their stimulatory effect on cell division in this recalcitrant system. Finally, the effects of embedding beet protoplasts in agarose and alginate have been compared. Embedding in the latter proved to be greatly beneficial. Materials and Methods P/aM mager/a/: A male fertile B.vulgaris (sugar beet) line SVP31-188 (Bv-NF) [see Kmns et a L 1990] and two male sterile wild B.maritima (sea beet) accessions, Bm-F and Bin-64 were used. Protoplasts were isolated from leaf material from either aseptieally-germlnated seedlings [Krens and Jamar, 1989] or established shoot cultures. Bm-F and Bm-64 shoot cultures were grown on half-strength Murashige & Skoog (MS) medium [Mura~4aige and Skoog 1962] supplemented with 3% (w/v) vaerose, 0.25 nag / I benzylaminopurine and 0.8% (w/v) agar (Daiehin, Brunschwig chemle, Amsterdam, NL). Bv-NF shoot vultures were grown on half-strength MS medium supplemented with 3 % (w/v) sucrose and 0.3 % (w / v) gelrite (Sigma, St Louis, USA). All material was grown at 22 ~ in low light (ca. 3000 lux, photoperiod: 16 h light / 8 h dark). Protoplast isolalion: The isolation and purification of protoplasts was as described previously [Krens et al., 1990] with the exceptions that the preplasmolysis period was reduced to 2 h and, for the lines Bm-F and Bin64, only one quarter of the usual enzyme concentration was used. Protoplast culture: Protoplasts were cultured at several plating densities in a modified KSP medium as described previously [Ka'enset al. 1990]. NaCI experiments: NaCI (0 - 4 g/l) was included in the protoplast isolation and / or culture media. An appropriate amount of marmitol/ glucose was omitted to maintain the usual osmolalities of the solutions [see Krens et al. 1990]. After adjusting the pH to 5.8, all solutions were filter
340 Table 1. Effect of the presence of NaCI in the isolation and / or culture medium on colony formation from Beta mesophyll protoplastsin liquid medium. Isolation Culture Bm-F Bin-64 Bv-NF
0% NaCI
0.4 % NaCI
0 %
0.2 %
0.4 %
0 %
0.2 %
0.4
NaCl
HaCl
NaCl
NaCI
NaCl
NaCl
I00 100 100
58 25 7
74 5 0
34 64 317
23 25
1 3 4
Plating densities 500000 protoplasts / dish. All results are the means of two replicates and are presented as a percentage of the corresponding control value. Analysis of variance analysis of these data has revealed that, in all cases, salt had a significant negative effect upon plating efficiency at P__< 0.05. sterilized.
Feederexperiments: Two basic nurse systems were tested. Firstly, an agarose ring system in which suspension cell protoplasta were embedded in a 0.5 em wide ring of 0.8% (w/v) agarose (Seaplaque agarose; Duchefa, Haarlem, NL) around the circumference of the Petri dish (Fig 1A). In an attempt to prevent feeder cell "escapes', this was then covered with an approx. 1 mm layer of 1% (w/v) agarose. Mesophyll cells were then cultured in the central well. In the second system, Millipore miniwells (3 era, Millicell-CM; Millipore, Etten Leur, NL) were used. By placing the feeder cells in 1 ml medium within the well these could be physically isolated from the mesophyll cells in the Petri dish but nevertheless remained in chemical contact via the basal membrane (Fig 1A). As feeder, 125000 protoplasts or cells from a finely-dispersed suspension culture of the sugar beet variety Nemee could be used interehangably with equal effectiveness. This suspension had been selected for growth in a medium identical to that used for protoplast culture. Alginate embedding: The method used was modified from that of Datum etal. (1989). Protoplasts were resnspended in 9% (w/v) marmitol containing 1 mM CaCIz at twice the desired final density. This was then mixed thoroughly with an equal volume of 2% (w/v) Na alginste (made up in 9% (w/v) mannitol containing 1 mM CaCl2 and autoclaved). Aliquots (l ml) were spread onto caleiunffagarose plates (6 cm Petrl dishes containing 10 ml 50 mM CaC12, 7.25 % (w/v) mannitol and 0.8% (w/v) agarose). After 1 h the solidified alginste discs were transferred to 5 mi 50 mM CaCl2 containing 7.25 % (w/v) mannitol and were maintained for 1 h at 4~ Thereafter, the bathing solution was removed and was replaced with culture medium. Dishes were incubated at 25*(2 in darkness. Analysis: (i) Liquid cultures - Plating efficieneies, defined as the percentage of origlnally-plated protoplasts which gave rise to viable colonies, were determined after 28 - 35 d by averaging the number of visible microcalli in 3 randomly-chosen sectors (9~ or 36", i.e. 2.5 or 10 % of the total area) of each Petri dish and then multiplying by the appropriate conversion factor. (ii) Alginate eulturas - The numbers of mieroealli in entire alginate discs were determined after 18 d. R ~
The influence of NaCI on protoplast culture. Preliminary experiments revealed that even after compensating for the negative effects o f the salt-induced increase in osmolality o f the culture m e d i u m b y slightly reducing the concentration o f the osmoticum, an inhibition o f colony formation was still observed (results not presented). This inhibition was both genotype and concentration dependent, with Bm-64 and B v - N F proving particularly sensitive (Table 1). T h e further inclusion o f NaC1 in the isolation m e d i a had an additional, significant negative effect (Table 1). The single positive effect observed for B.vulgaris (a 3 fold increase in plating efficiency in standard culture m e d i u m following isolation in the presence o f salt) could not b e reproduced and furthermore, the inclusion o f NaCI in the enzyme mix had a distinct inhibitory effect on protoplast yield for all lines tested (results not presented).
The influence of feeder cells on the plating efficiency of B. maritima protoplasts. B.maritima mesophyll protoplasts were used, as division o f these cells was found to be greatly dependent upon plating density. F o r example, while protoplasts o f B m - F have m a x i m u m plating efficieneies at a density o f 500000 / dish, halving this density totally eliminates cell division and further cell development (unpublished observations). W h i l e the agarose system was able to sustain mesophyll protoplast divisions at sub-critical densities it nevertheless p r o v e d unusable. Before the mesophyll-derived colonies had reached a size suitable for transfer, the feeder colonies derived from the m o r e rapidly-dividing suspension cell protoplasts had b e g u n to escape and mix with the cells in the central well (Fig 1B). Thickening the agarose isolation layer o r substituting the suspension protoplasts for mesophyll protoplasts b o t h resulted in the failure o f the nurse cells to develop and thus the feeder effect was lost (results not presented). Table 2. Influence of plating density and minlwell feeders on plating efficieneies of Beta ma~timamesophyll protoplasts.
Protoplast density/dish
250000 125000 125000 62500 62500 31300 15600 62500
Feedera
% Plating
Efficiency
Bm-F
Bin-64
0.0 0.0 1.3
0.0
+ + + + meso
1.7 0.5
0.0
0.0
1.2 0.3 0.3 0.3
0.6 0.2 0.2
a(+) 125000 suspension protoplasts / miniwell were used as a feeder. a(.) Miniwell contained only 1 ml medium, meso - Bm-F, 125000 mesophyll protoplasts. Results are the means of two replicates. The miniwells p r o v e d a simple and excellent nurse system (Table 2). These permitted mesophyll protoplast division d o w n to approx. 15000 protoplasts / dish ( < 4 0 0 0 / ml) which, for BIn-F, is equivalent to ea. 3 % o f the usual critical m i n i m u m density (125000 / ml) ( F i g 1C). Again, mesophyll protoplasts proved to be p o o r nurse cells. The optimal density for suspension protoplast nurse cultures was 125000 protoplasts / dish. H i g h e r densities resulted in premature culture senescence and lower densities had a reduced feeder effect (results not presented).
The effect of alginate embedding. B v - N F mesophyll protoplasts derived from shoot cultures,
341 have a consistently low plating efficiency in liquid medium (Table 3). In stark contrast, plating effieieneies following embedding in Ca alginate were, in all cases, significantly enhanced. Furthermore, this enhancement in colony formation was the end result of culture development which was dearly greatly improved in comparison to that in liquid culture. From the first day onwards, substantial morphological and physiological differences were observed in the immobilized cells. In alginate cultures, a greater proportion of the protoplasts remained viable, resynthesized a cell wall, retained well distributed chloroplasts and an active cytoplasm (Fig 1D, 1E). Furthermore, cell growth was more extensive than in liquid culture, while budding and cell browning were totally eliminated. The greatest differences were however observed in cell division. This began much earlier in alginate. After 6 days, in the alginate treatments, colonies of up to 20 cells were already present whereas in the control dishes the first cell division was just occurring. Consequently, mierocalli in alginate cultures reached the size for subculture after 14 - 18 days, in contrast to the usual 35 days in liquid medium (Fig. IF). Attempts to further enhance the plating efficiency through the use of feeder cells or conditioned medium (harvested from 4 day old suspensions; adjusted for pH and osmolality) had a clear stimulatory effect particularly at the lower plating densities (Table 3). Clearly suspension culture cells, as protoplasts, can be successfully used as feeders. Statistical analysis of the data in Table 3 has indicated that embedding significantly enhances plating efficieneies and that conditioned medium and feeder ceils both significantly enhance plating effieiencies in comparison to embedding alone. In alginate cultures, both compact and loose cell colonies were observed, in comparison to only the latter type in liquid medium (Fig 1G). Preliminary experiments have shown that plant regeneration from the latter type (Fig 1H) was however, 20 - 30% in alginate treatments in comparison to just 1 - 2% in the liquid medium controls. Table 3. The influence of different culture conditions on the plating efficiency (%) of Beta vulgaris mesophyll protoplasts. Density pps/dish
KSp liquid
KSp +ALG
K8p +ALG + FEED
KSp +ALG + COND
500000 250000 125000 62500 31250
0.01 0.02 0.03 0.02 0.00
n.t. 0.20 0.21 0.12 0.10
n.t. n.t. 0.34 0.27 0.20
n.t. n.t. 0.37 0.28 0.30
ALG, alginate embedding; FEED, Feeder cells (approx 125000 susp. cells); COND, 25% (v/v) conditioned medium. Results are the means of between 3 to 7 experiments with two replicates per experiment, n.t. = not tested.
In an attempt to identify which features of the immobilization process gave rise to the effects observed, a further experiment was performed where different components were tested separately (Table 4). While the addition of an equivalent amount of extra calcium chloride had no effect on colony formation, ungelled alginate or embedding in agarose totally eliminated cell division. Cells in liquid culture, to which a solidified alginate disc was
added, divided at a reduced frequency in comparison to algiuate-embedded cells. Table 4. The influence of modified culture conditions on the subsequent plating efficiencies of Bv-HF mesophyll protoplasts. Treatment
Liquid medium Embedded in alginate Embedded in 0.8 % aga~se Liquid medium + lml 50 mM CaCIz Liquid medium + ungelled alginate Liquid medium + algiuate disc*
Plating efficiency
(%)
0.05 a
0.22b 0.00a 0.05 a 0.00 ~
0.02 a
Plating density 125000/dish. *Solidified in the usual way but cells located only in the liquid phase. Results are the means of two replicates. Different superscripts indicate statistical differences at P < 0.05 (Multiple Range Test: Student Newman Keuls procedure).
Discussion Two of the three protocols reported here proved greatly beneficial to the culture of Beta mesophyll protoplasts. While the inclusion of NaCI in protoplast media had, in general, a detrimental effect, the use of feeder systems proved a very effective means of significantly reducing the critical cell density, and alginate embedding greatly enhanced plating effieiencies. Both should prove of considerable value in asymmetric fusion experiments. Previously, it was reported that the inclusion of NaCI in the medium ofB. vulgaris callus cultures stimulated growth rates (I-Iag~ge et al. 1990). Furthermore, this was correlated with significant reductions in ethylene production and peroxidase activity. As both of these are stressassociated and protoplast isolation is considered a stressful process, an attempt was made to improve our culture conditions by supplementing protoplast media with NaCI. Previous experiments along these lines using n-propyl gallate, another compound which works against the consequences of stress, had already proved greatly beneficial (Krens et al. 1990). However, even with maritime genotypes, the lowest concentration of salt used still had detrimental effects on cell development. Protoplasts, in contrast to callus cells, through their exposed nature appear to be over-sensitive to the presence of NaC1. However, preliminary experiments in which salt was included, not in the protoplast media, but in the medium on which the source tissue was grown and/or the regeneration medium, have revealed a possible stimulatory effect on plant regeneration. This is under further investigation. Nurse cultures have been widely used for the culture of single cells or protoplasts at low densities (e.g. Larkin et al. 1988). It is considered that such cultures stimulate the development ofprotoplasts either through the production of physiologically-active compounds or through the prevention of leaching of vital components out of the ceils [Dix 1986]. To determine if such a system might also promote cell division at relatively high plating densities with a recalcitrant species (B.maritima), protoplasts from a rapidly-dividing suspension culture of a related species (B. vulgaris) was chosen as the feeder. Cell division could be greatly stimulated in this way and colonies could be
342
Figure 1. [A] Two different nurse culture systems using (a) agarose embedding and (b) miniwells; AG, agarose ring; LQ, liquid medium; IL, agamse isolation layer; MP, mesophyll protoplasts; SP, suspension cell protoplasts. [B] Feeder cell (FC) colonies bursting through the IL (see [A]) after 2t d; MC, B.maritima mesophyll protoplast colonies. [C] Colony formation from B.maritima mesophyll protoplasts after 21 d. Let~, 6 cm dish contained 125000 protoplasts + an empty miniweU (Control); Right, ditto + 125000 suspension cell protoplasts in the mlniweU. [D] B.vulgaris mesophyll protoplast culture after 3 d in liquid medium (Bar = 100/tin). [E] As [13] but embedded in Ca alginate. [F] Development ofB.vulgaris mesophyll protoplast colonies after 18 d in liquid medium (left) or embedded in a Ca alginate disc (right). [G] Sot~ callus (SC) and hard callus (HC) types growing out of Ca alginate (CA) after transfer to solid culture medium (Bar = l nun). [H] Plantlet from sof[ callus (Bar = 100/tm).
obtained at plating densities reduced to just a few percent of the standard critical value. This also complements the findings of Schlangstedt et al., (1992) who were notably only able to obtain cell division in beet mesophyll protoplasts in the presence of feeder cells. The greatest effects on cell division were however, observed when mesophyll protoplasts were embedded in alginate. Dramatic differences in cell development were evident even after the first day. Stimulatory effects of such a treatment have also been reported for other recalcitrant systems such as apple [Huanearuna Perales and Schieder 1991] and wheat mesophyll protoplasts [Ha/me et al. 1991]. In Crocus sativus, while liquid culture could not support any call division, alginate embedding resulted in successful plant regeneration from protoplasts [Isa et al. 1990]. The precise mode of action of alginate remains unclear [Schnabl et al. 1983] although it is evident from the results presented here, that it is not due to one of the individual components. Alone, these were found to have no, or in some cases, even a toxic effect. It would appear that calcium alginate in some way provides a unique environment which protects and/or stimulates the cells to develop further. Schlangstedt et al. (1992), using a somewhat more elaborate culture protocol, have also reported on the great advantage of embedding beet protoplasts in calcium alginate although in their system no plants could be obtained. An interesting point in this regard is to consider which cells are actually involved. Are two cell populations involved, or is it simply that, in alginate, more cells of the type which give rise to colonies in liquid medium proceed to division? It was evident that the majority of the cell colonies developing in alginate had a harder, more compact nature in comparison to the very loose type characteristic of liquid medium. This point is of particular importance as, to date, it has only proven possible to regenerate plants from the latter type [Krens et al. 1990]. Whether these more compact colonies are the
result of the different (physically, more-restricting) culture conditions or are indeed derived from another subpopulation of cells, is presently under investigation. Resulting from the advantageous effects reported here, both the use of feeder systems and the embedding of protoplasts in alginate are presently being applied in asymmetric somatic hybridization experiments with these beet genotypes. The latter technique warrants further attention where other recalcitrant protoplast systems are concerned. Acknowledgemeats: Discussions with Dr K. Zoglauer and S. Lenzner (Humboldt Univ, Berlin)were especially useful regarding the application of the alginate techniques. Dr C. Kik is thanked for carrying out the statistical analysis and Dr. J. Creemers-Molenaar and W.M. Mattheij are thanked for their critical reading of the manuscript. This work wusfunded by Danish Plant Breeding Ltd. References Datum B, Schmidt R, WUlmitzer L (1989) Mol Gen Genet 217:6-12 Dix PH (1986) Cell line selection. In Yeoman MM (ed) Plant cell culture technology; Blackwell Sci Publ London:143-201 Hag~ge D, Kevers C, Le Dily F, Gaspar T, Boucaud J (1990) CRAcad Sci Paris 310:259-264 Hahne B, Fleck J, Hahne, G (1991) Physiol Plant 82:A7 Huancaruna Perales E, Schieder O (1991) Physiol Plant 82:A15 Isa T, Ogasawara T, Kaneko H (1990) Jap J Breed 40:153-157 Kaul MLH (1988) Male sterility in higher plants. Springer Vedag, Bedin Krens FA, Hall RD (1992) Prophyta 1:12-16 Krens FA, Jamar D (1989) J Plant Physiol 134:651-655 Krens FA, Jamar D, Rouwendal GJA, Hall RD (1990) Theordppl Genet 79:390-396 Larkin PJ, Davies, PA, Tanner, GJ (1988) Pl Sci 58, 203 - 210. Mursshige T, Skoog F (1962) Physiol Plant 15:473-497 Waiters TW, Earle ED (1990) Plant Cell Rep 9:316-319 Sehlangstedt M, Hermans B, Zoglauer K, Schieder O (1992) J Plant Physiol 140:339 -344 Schnabl H, Youngman RI, ZimmermannU (1983) Planta 158:392-397
Plant Cell Reports (1993) 12:339-342
9 Springer-Verlag 1993
Improvement of protoplast culture protocols for Beta vulgaris L. (sugar beet) Robert D. Hall, Charlotte Pedersen, and Frans A. Krens Department of Cell Biology, DLO-Centre for Plant Breeding and Reproduction Research (CPRO-DLO), Postbus 16, 6700 AA Wageningen, The Netherlands Received September 10, 1992/Revised version received January 28, 1993 - Communicated by M. R. Davey
Summary. The effects of NaCl, feeder cells and the embedding of protoplasts in calcium alginate have been investigated in an attempt to improve culture conditions of recalcitrant sugar beet (Beta vulgaris L.) mesophyU protoplasts. While the use of NaCl in all instances proved detrimental to protoplast development, the other two treatments had clear beneficial effects. Minimum plating densities, necessary to sustain cell division, could be reduced to < 5 % (< 4000 protopIasts / ml) of the control levels and plating ej~ciencies couM be significantly enhanced by approx. 10 fold. Plants couM still be regenerated from soft calli derived from mesophyll protoplasts cultured under the modified conditions at a frequency of 20 - 30 %. In particular, the use of alginate is considered of potentiaUy great importance for the further application of beet protoplasts for other aims e.g. asymmetric hybridization. Key words: Alginate embedding - Beta vulgaris -feeders protoplasts - regeneration
Introduction In sugarbeet, cytoplasmic male sterility (CMS) is of fundamental importance both in breeding and for the production of F] hybrid seed [Kaul 1988]. In previous publications we outlined how beet breeding programmes were, on a world scale, rather perilously centred around a very narrow cytoplasmic base [Krens et al. 1990; Krens and Hall 1992]. The rapid broadening of this genetic base through the employment of in vitro techniques (e.g. asymmetric hybridization), to incorporate newly-identified mitochondrial genetic variants, is of potentially great importance. Surprisingly, despite being a dicotyledonous crop and despite the considerable attention which it has received, Beta vulgaris remains reealeitrant with respect to many biotechnologieal techniques. However, previously we were able to report, for the first time, the successful regeneration of plants from beet mesophyll protoplasts isolated from two distinct aecessions [Krens et al. 1990]. These regenerants were predominantly diploid and phenotypieally normal. To date we have now obtained plant regeneration from mesophyll protoplasts from 5 of the 7 lines tested (unpublished observations). These results are eneouraging Correspondence to: R. D. Hall
and augur well for future bioteehnologieal application. However, one factor which remains problematic is the disappointingly low plating efficiencies observed even at high plating densities. Generally, plating efficieneies are < 1% in regenerable systems. This represents a potentially serious limitation to success, especially concerning asymmetric somatic hybridizations where few cells remain viable in culture. Consequently, in order to improve the low frequency of division in beet protoplast eultures, we have carried out a series of experiments to help identify valuable modifications to the culture conditions. The effect of the inclusion of NaC1 at a range of concentrations in the protoplast isolation/eulture media was tested following the previously-reported advantageous effects found with beet callus cultures [Hagbge et al 1990]. Additionally, the potential value of using a range of feeder (nurse) systems, already used to great advantage for low density culture [e.g. Waiters and Earle 1990] has herebeen assessed regarding their stimulatory effect on cell division in this recalcitrant system. Finally, the effects of embedding beet protoplasts in agarose and alginate have been compared. Embedding in the latter proved to be greatly beneficial. Materials and Methods P/aM mager/a/: A male fertile B.vulgaris (sugar beet) line SVP31-188 (Bv-NF) [see Kmns et a L 1990] and two male sterile wild B.maritima (sea beet) accessions, Bm-F and Bin-64 were used. Protoplasts were isolated from leaf material from either aseptieally-germlnated seedlings [Krens and Jamar, 1989] or established shoot cultures. Bm-F and Bm-64 shoot cultures were grown on half-strength Murashige & Skoog (MS) medium [Mura~4aige and Skoog 1962] supplemented with 3% (w/v) vaerose, 0.25 nag / I benzylaminopurine and 0.8% (w/v) agar (Daiehin, Brunschwig chemle, Amsterdam, NL). Bv-NF shoot vultures were grown on half-strength MS medium supplemented with 3 % (w/v) sucrose and 0.3 % (w / v) gelrite (Sigma, St Louis, USA). All material was grown at 22 ~ in low light (ca. 3000 lux, photoperiod: 16 h light / 8 h dark). Protoplast isolalion: The isolation and purification of protoplasts was as described previously [Krens et al., 1990] with the exceptions that the preplasmolysis period was reduced to 2 h and, for the lines Bm-F and Bin64, only one quarter of the usual enzyme concentration was used. Protoplast culture: Protoplasts were cultured at several plating densities in a modified KSP medium as described previously [Ka'enset al. 1990]. NaCI experiments: NaCI (0 - 4 g/l) was included in the protoplast isolation and / or culture media. An appropriate amount of marmitol/ glucose was omitted to maintain the usual osmolalities of the solutions [see Krens et al. 1990]. After adjusting the pH to 5.8, all solutions were filter
340 Table 1. Effect of the presence of NaCI in the isolation and / or culture medium on colony formation from Beta mesophyll protoplastsin liquid medium. Isolation Culture Bm-F Bin-64 Bv-NF
0% NaCI
0.4 % NaCI
0 %
0.2 %
0.4 %
0 %
0.2 %
0.4
NaCl
HaCl
NaCl
NaCI
NaCl
NaCl
I00 100 100
58 25 7
74 5 0
34 64 317
23 25
1 3 4
Plating densities 500000 protoplasts / dish. All results are the means of two replicates and are presented as a percentage of the corresponding control value. Analysis of variance analysis of these data has revealed that, in all cases, salt had a significant negative effect upon plating efficiency at P__< 0.05. sterilized.
Feederexperiments: Two basic nurse systems were tested. Firstly, an agarose ring system in which suspension cell protoplasta were embedded in a 0.5 em wide ring of 0.8% (w/v) agarose (Seaplaque agarose; Duchefa, Haarlem, NL) around the circumference of the Petri dish (Fig 1A). In an attempt to prevent feeder cell "escapes', this was then covered with an approx. 1 mm layer of 1% (w/v) agarose. Mesophyll cells were then cultured in the central well. In the second system, Millipore miniwells (3 era, Millicell-CM; Millipore, Etten Leur, NL) were used. By placing the feeder cells in 1 ml medium within the well these could be physically isolated from the mesophyll cells in the Petri dish but nevertheless remained in chemical contact via the basal membrane (Fig 1A). As feeder, 125000 protoplasts or cells from a finely-dispersed suspension culture of the sugar beet variety Nemee could be used interehangably with equal effectiveness. This suspension had been selected for growth in a medium identical to that used for protoplast culture. Alginate embedding: The method used was modified from that of Datum etal. (1989). Protoplasts were resnspended in 9% (w/v) marmitol containing 1 mM CaCIz at twice the desired final density. This was then mixed thoroughly with an equal volume of 2% (w/v) Na alginste (made up in 9% (w/v) mannitol containing 1 mM CaCl2 and autoclaved). Aliquots (l ml) were spread onto caleiunffagarose plates (6 cm Petrl dishes containing 10 ml 50 mM CaC12, 7.25 % (w/v) mannitol and 0.8% (w/v) agarose). After 1 h the solidified alginste discs were transferred to 5 mi 50 mM CaCl2 containing 7.25 % (w/v) mannitol and were maintained for 1 h at 4~ Thereafter, the bathing solution was removed and was replaced with culture medium. Dishes were incubated at 25*(2 in darkness. Analysis: (i) Liquid cultures - Plating efficieneies, defined as the percentage of origlnally-plated protoplasts which gave rise to viable colonies, were determined after 28 - 35 d by averaging the number of visible microcalli in 3 randomly-chosen sectors (9~ or 36", i.e. 2.5 or 10 % of the total area) of each Petri dish and then multiplying by the appropriate conversion factor. (ii) Alginate eulturas - The numbers of mieroealli in entire alginate discs were determined after 18 d. R ~
The influence of NaCI on protoplast culture. Preliminary experiments revealed that even after compensating for the negative effects o f the salt-induced increase in osmolality o f the culture m e d i u m b y slightly reducing the concentration o f the osmoticum, an inhibition o f colony formation was still observed (results not presented). This inhibition was both genotype and concentration dependent, with Bm-64 and B v - N F proving particularly sensitive (Table 1). T h e further inclusion o f NaC1 in the isolation m e d i a had an additional, significant negative effect (Table 1). The single positive effect observed for B.vulgaris (a 3 fold increase in plating efficiency in standard culture m e d i u m following isolation in the presence o f salt) could not b e reproduced and furthermore, the inclusion o f NaCI in the enzyme mix had a distinct inhibitory effect on protoplast yield for all lines tested (results not presented).
The influence of feeder cells on the plating efficiency of B. maritima protoplasts. B.maritima mesophyll protoplasts were used, as division o f these cells was found to be greatly dependent upon plating density. F o r example, while protoplasts o f B m - F have m a x i m u m plating efficieneies at a density o f 500000 / dish, halving this density totally eliminates cell division and further cell development (unpublished observations). W h i l e the agarose system was able to sustain mesophyll protoplast divisions at sub-critical densities it nevertheless p r o v e d unusable. Before the mesophyll-derived colonies had reached a size suitable for transfer, the feeder colonies derived from the m o r e rapidly-dividing suspension cell protoplasts had b e g u n to escape and mix with the cells in the central well (Fig 1B). Thickening the agarose isolation layer o r substituting the suspension protoplasts for mesophyll protoplasts b o t h resulted in the failure o f the nurse cells to develop and thus the feeder effect was lost (results not presented). Table 2. Influence of plating density and minlwell feeders on plating efficieneies of Beta ma~timamesophyll protoplasts.
Protoplast density/dish
250000 125000 125000 62500 62500 31300 15600 62500
Feedera
% Plating
Efficiency
Bm-F
Bin-64
0.0 0.0 1.3
0.0
+ + + + meso
1.7 0.5
0.0
0.0
1.2 0.3 0.3 0.3
0.6 0.2 0.2
a(+) 125000 suspension protoplasts / miniwell were used as a feeder. a(.) Miniwell contained only 1 ml medium, meso - Bm-F, 125000 mesophyll protoplasts. Results are the means of two replicates. The miniwells p r o v e d a simple and excellent nurse system (Table 2). These permitted mesophyll protoplast division d o w n to approx. 15000 protoplasts / dish ( < 4 0 0 0 / ml) which, for BIn-F, is equivalent to ea. 3 % o f the usual critical m i n i m u m density (125000 / ml) ( F i g 1C). Again, mesophyll protoplasts proved to be p o o r nurse cells. The optimal density for suspension protoplast nurse cultures was 125000 protoplasts / dish. H i g h e r densities resulted in premature culture senescence and lower densities had a reduced feeder effect (results not presented).
The effect of alginate embedding. B v - N F mesophyll protoplasts derived from shoot cultures,
341 have a consistently low plating efficiency in liquid medium (Table 3). In stark contrast, plating effieieneies following embedding in Ca alginate were, in all cases, significantly enhanced. Furthermore, this enhancement in colony formation was the end result of culture development which was dearly greatly improved in comparison to that in liquid culture. From the first day onwards, substantial morphological and physiological differences were observed in the immobilized cells. In alginate cultures, a greater proportion of the protoplasts remained viable, resynthesized a cell wall, retained well distributed chloroplasts and an active cytoplasm (Fig 1D, 1E). Furthermore, cell growth was more extensive than in liquid culture, while budding and cell browning were totally eliminated. The greatest differences were however observed in cell division. This began much earlier in alginate. After 6 days, in the alginate treatments, colonies of up to 20 cells were already present whereas in the control dishes the first cell division was just occurring. Consequently, mierocalli in alginate cultures reached the size for subculture after 14 - 18 days, in contrast to the usual 35 days in liquid medium (Fig. IF). Attempts to further enhance the plating efficiency through the use of feeder cells or conditioned medium (harvested from 4 day old suspensions; adjusted for pH and osmolality) had a clear stimulatory effect particularly at the lower plating densities (Table 3). Clearly suspension culture cells, as protoplasts, can be successfully used as feeders. Statistical analysis of the data in Table 3 has indicated that embedding significantly enhances plating efficieneies and that conditioned medium and feeder ceils both significantly enhance plating effieiencies in comparison to embedding alone. In alginate cultures, both compact and loose cell colonies were observed, in comparison to only the latter type in liquid medium (Fig 1G). Preliminary experiments have shown that plant regeneration from the latter type (Fig 1H) was however, 20 - 30% in alginate treatments in comparison to just 1 - 2% in the liquid medium controls. Table 3. The influence of different culture conditions on the plating efficiency (%) of Beta vulgaris mesophyll protoplasts. Density pps/dish
KSp liquid
KSp +ALG
K8p +ALG + FEED
KSp +ALG + COND
500000 250000 125000 62500 31250
0.01 0.02 0.03 0.02 0.00
n.t. 0.20 0.21 0.12 0.10
n.t. n.t. 0.34 0.27 0.20
n.t. n.t. 0.37 0.28 0.30
ALG, alginate embedding; FEED, Feeder cells (approx 125000 susp. cells); COND, 25% (v/v) conditioned medium. Results are the means of between 3 to 7 experiments with two replicates per experiment, n.t. = not tested.
In an attempt to identify which features of the immobilization process gave rise to the effects observed, a further experiment was performed where different components were tested separately (Table 4). While the addition of an equivalent amount of extra calcium chloride had no effect on colony formation, ungelled alginate or embedding in agarose totally eliminated cell division. Cells in liquid culture, to which a solidified alginate disc was
added, divided at a reduced frequency in comparison to algiuate-embedded cells. Table 4. The influence of modified culture conditions on the subsequent plating efficiencies of Bv-HF mesophyll protoplasts. Treatment
Liquid medium Embedded in alginate Embedded in 0.8 % aga~se Liquid medium + lml 50 mM CaCIz Liquid medium + ungelled alginate Liquid medium + algiuate disc*
Plating efficiency
(%)
0.05 a
0.22b 0.00a 0.05 a 0.00 ~
0.02 a
Plating density 125000/dish. *Solidified in the usual way but cells located only in the liquid phase. Results are the means of two replicates. Different superscripts indicate statistical differences at P < 0.05 (Multiple Range Test: Student Newman Keuls procedure).
Discussion Two of the three protocols reported here proved greatly beneficial to the culture of Beta mesophyll protoplasts. While the inclusion of NaCI in protoplast media had, in general, a detrimental effect, the use of feeder systems proved a very effective means of significantly reducing the critical cell density, and alginate embedding greatly enhanced plating effieiencies. Both should prove of considerable value in asymmetric fusion experiments. Previously, it was reported that the inclusion of NaCI in the medium ofB. vulgaris callus cultures stimulated growth rates (I-Iag~ge et al. 1990). Furthermore, this was correlated with significant reductions in ethylene production and peroxidase activity. As both of these are stressassociated and protoplast isolation is considered a stressful process, an attempt was made to improve our culture conditions by supplementing protoplast media with NaCI. Previous experiments along these lines using n-propyl gallate, another compound which works against the consequences of stress, had already proved greatly beneficial (Krens et al. 1990). However, even with maritime genotypes, the lowest concentration of salt used still had detrimental effects on cell development. Protoplasts, in contrast to callus cells, through their exposed nature appear to be over-sensitive to the presence of NaC1. However, preliminary experiments in which salt was included, not in the protoplast media, but in the medium on which the source tissue was grown and/or the regeneration medium, have revealed a possible stimulatory effect on plant regeneration. This is under further investigation. Nurse cultures have been widely used for the culture of single cells or protoplasts at low densities (e.g. Larkin et al. 1988). It is considered that such cultures stimulate the development ofprotoplasts either through the production of physiologically-active compounds or through the prevention of leaching of vital components out of the ceils [Dix 1986]. To determine if such a system might also promote cell division at relatively high plating densities with a recalcitrant species (B.maritima), protoplasts from a rapidly-dividing suspension culture of a related species (B. vulgaris) was chosen as the feeder. Cell division could be greatly stimulated in this way and colonies could be
342
Figure 1. [A] Two different nurse culture systems using (a) agarose embedding and (b) miniwells; AG, agarose ring; LQ, liquid medium; IL, agamse isolation layer; MP, mesophyll protoplasts; SP, suspension cell protoplasts. [B] Feeder cell (FC) colonies bursting through the IL (see [A]) after 2t d; MC, B.maritima mesophyll protoplast colonies. [C] Colony formation from B.maritima mesophyll protoplasts after 21 d. Let~, 6 cm dish contained 125000 protoplasts + an empty miniweU (Control); Right, ditto + 125000 suspension cell protoplasts in the mlniweU. [D] B.vulgaris mesophyll protoplast culture after 3 d in liquid medium (Bar = 100/tin). [E] As [13] but embedded in Ca alginate. [F] Development ofB.vulgaris mesophyll protoplast colonies after 18 d in liquid medium (left) or embedded in a Ca alginate disc (right). [G] Sot~ callus (SC) and hard callus (HC) types growing out of Ca alginate (CA) after transfer to solid culture medium (Bar = l nun). [H] Plantlet from sof[ callus (Bar = 100/tm).
obtained at plating densities reduced to just a few percent of the standard critical value. This also complements the findings of Schlangstedt et al., (1992) who were notably only able to obtain cell division in beet mesophyll protoplasts in the presence of feeder cells. The greatest effects on cell division were however, observed when mesophyll protoplasts were embedded in alginate. Dramatic differences in cell development were evident even after the first day. Stimulatory effects of such a treatment have also been reported for other recalcitrant systems such as apple [Huanearuna Perales and Schieder 1991] and wheat mesophyll protoplasts [Ha/me et al. 1991]. In Crocus sativus, while liquid culture could not support any call division, alginate embedding resulted in successful plant regeneration from protoplasts [Isa et al. 1990]. The precise mode of action of alginate remains unclear [Schnabl et al. 1983] although it is evident from the results presented here, that it is not due to one of the individual components. Alone, these were found to have no, or in some cases, even a toxic effect. It would appear that calcium alginate in some way provides a unique environment which protects and/or stimulates the cells to develop further. Schlangstedt et al. (1992), using a somewhat more elaborate culture protocol, have also reported on the great advantage of embedding beet protoplasts in calcium alginate although in their system no plants could be obtained. An interesting point in this regard is to consider which cells are actually involved. Are two cell populations involved, or is it simply that, in alginate, more cells of the type which give rise to colonies in liquid medium proceed to division? It was evident that the majority of the cell colonies developing in alginate had a harder, more compact nature in comparison to the very loose type characteristic of liquid medium. This point is of particular importance as, to date, it has only proven possible to regenerate plants from the latter type [Krens et al. 1990]. Whether these more compact colonies are the
result of the different (physically, more-restricting) culture conditions or are indeed derived from another subpopulation of cells, is presently under investigation. Resulting from the advantageous effects reported here, both the use of feeder systems and the embedding of protoplasts in alginate are presently being applied in asymmetric somatic hybridization experiments with these beet genotypes. The latter technique warrants further attention where other recalcitrant protoplast systems are concerned. Acknowledgemeats: Discussions with Dr K. Zoglauer and S. Lenzner (Humboldt Univ, Berlin)were especially useful regarding the application of the alginate techniques. Dr C. Kik is thanked for carrying out the statistical analysis and Dr. J. Creemers-Molenaar and W.M. Mattheij are thanked for their critical reading of the manuscript. This work wusfunded by Danish Plant Breeding Ltd. References Datum B, Schmidt R, WUlmitzer L (1989) Mol Gen Genet 217:6-12 Dix PH (1986) Cell line selection. In Yeoman MM (ed) Plant cell culture technology; Blackwell Sci Publ London:143-201 Hag~ge D, Kevers C, Le Dily F, Gaspar T, Boucaud J (1990) CRAcad Sci Paris 310:259-264 Hahne B, Fleck J, Hahne, G (1991) Physiol Plant 82:A7 Huancaruna Perales E, Schieder O (1991) Physiol Plant 82:A15 Isa T, Ogasawara T, Kaneko H (1990) Jap J Breed 40:153-157 Kaul MLH (1988) Male sterility in higher plants. Springer Vedag, Bedin Krens FA, Hall RD (1992) Prophyta 1:12-16 Krens FA, Jamar D (1989) J Plant Physiol 134:651-655 Krens FA, Jamar D, Rouwendal GJA, Hall RD (1990) Theordppl Genet 79:390-396 Larkin PJ, Davies, PA, Tanner, GJ (1988) Pl Sci 58, 203 - 210. Mursshige T, Skoog F (1962) Physiol Plant 15:473-497 Waiters TW, Earle ED (1990) Plant Cell Rep 9:316-319 Sehlangstedt M, Hermans B, Zoglauer K, Schieder O (1992) J Plant Physiol 140:339 -344 Schnabl H, Youngman RI, ZimmermannU (1983) Planta 158:392-397
