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Improvement of Sugar Beet (Beta vulgaris L.) Protoplast Culture: Leaf Petioles as a Protoplast Source
f. Plant Pbysiol. Vol. 143. pp. 227-233 {1994)
Improvement of Sugar Beet (Beta vulgaris L.) Protoplast Culture: Leaf Petioles as a Protoplast Source MARION ScHLANGSTEDT 1• 2, KuRT ZoGLAUER 1• MICHEL jACOBS3
*, STEFFEN LENZNER1, BoB HERMANS3, and
1
Humboldt-Universitat zu Berlin, FB Biologie, Institut fiir Pflanzenphysiologie und Zellbiologie, InvalidenstraBe 43, D-10115 Berlin, BRD
2
Present address: Planta Angewandte Pflanzengenetik und Biotechnologie GmbH, Grimsehlstr. 31, D-37574 Einbeck, BRD
3
Instituut voor Moleculaire Biologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium
* To whom correspondence should be addressed Received May 13, 1993 · Accepted September 22, 1993
Summary
Petioles of in vitro shoots were tested as potential sources for sugar beet (Beta vulgaris L.) protoplasts. Regardless of the genotype used, petiole protoplasts showed much higher plating efficiencies (average P.E.: 4.6 %) than mesophyll protoplasts (average P.E.: 1.0%) when cultured under identical conditions in a Schenk and Hildebrandt (SH) as well as in a Kao and Michayluk (K8p) medium. Additionally, DNA histograms indicate that petiole protoplast populations have a higher proportion with doubled DNA content, i.e. nuclei in the G2/M stage of the cell cycle.
Key words: Beta vulgaris, alginate-embedding, flow cytometry, mesophyll protoplasts, nurse culture, petiole pro toplasts. Abbreviations: BAP = 6-benzylaminopurine; C.I. = confidence interval; 2,4-D = 2,4-dichlorophenoxyacetic acid; fw = fresh weight; illA = indole-3-butyric acid; Kin = kinetin; NAA = 1-naphthaleneacetic acid; ppl = protoplasts; TillA = tri-iodobenzoic acid; P.E. = plating efficiency. Introduction
Suspension cells and leaf mesophyll tissue are the sources most commonly used for isolation of sugar beet protoplasts (review see Table 1). Because suspension cell protoplasts can be easily isolated and cultured, they were often used in manipulation experiments such as fusion (Eady et al., 1988; Pedersen et al., 1988, 1991), electroporation (Eady et al., 1988; Lindsey and Jones, 1987 a, b) and gene transfer (Lindsey and Jones, 1987, 1989; Joersbo and Brunstedt, 1990). While stable yields and high plating efficiencies can be achieved from this type of protoplasts, plant regeneration was never observed. As long as protoplasts derived from non-embryogenic and © 1994 by Gustav Fischer Verlag, Stuttgart
long-time cultured suspensions are involved, plant regeneration is not to be expected. On the other hand only Hermansen et al. (1990) have presented a method for initiation of morphogenic suspensions in sugar beet potentially capable of plant regeneration. In order to apply protoplast technology in sugar beet genetics and breeding, high yields and plating efficiency as well as plant regeneration are absolutely required. Methods for plant regeneration in more than one genotype are rare and to our knowledge only described by Krens et al. (Krens et al., 1990; Vander Maas et al., 1990) and mentioned by Pedersen et al. (pers. commun.). Krens et al. used leaves of in vitro grown seedlings, which indicates that at least this type of donor material is suited. However, the
228
MAiuoN ScHLANGSTEDT, KuRT ZoGLAUER, STEFFEN LENZNER, BoB HERMANs, and MicHEL jACOBS
Table 1: Overview ot the type of protoplasts used in sugar beet research and their response upon culturing. Origin of protoplasts
Response
References
Outdoor and greenhouse plants (leaves, apex)
P.E.: 0%
Binding et a!., 1981
Suspension cells (late log phase)
colonies after 7 to 10 days callus after 30 to 40 days
Smolenskaya and Raldugina, 1981
Suspension cells (4 to 5 days old)
colonies after 21 days P.E.: 7.3%
Bhat eta!., 1985
Suspension cells (2 days old)
colonies after 30 days P.E.: 35 %, cell suspension
Szabados and Gaggero, 1985
In vitro shoot culture (4 weeks old, leaves)
microcolonies after 3 weeks P .E.: 0.3 to 13 %, callus formation and root organogenesis
Bhat et a!., 1986
Suspension cells (4 days old)
colonies after 3 weeks P.E.: 60%
Senanayake, 1986
Suspension cells (3 days old)
P.E.: 7%
Miiller and Ryschka, 1987
In vitro grown seedlings (4 to 6 weeks old, leaves)
occasionally cell division
Krens and Jamar, 1989
Suspension cells
routinely calli without any regeneration
Krens and Jamar, 1989
Suspension cells (5 to 7 days old)
P.E.: 14.8% (liquid medium) 64.7% (agarose embedding)
Lindsey and Jones, 1989
In vitro grown seedlings (6 to 8 weeks old, leaves)
P.E.: 0.005 to 1.0% shoot regeneration, 10 to 20%
Krens et a!., 1990
In vitro grown seedlings
P.E.: O.Ql to 7% shoot regeneration, 3 to 20%
In vitro shoot culture and in vitro grown seedlings
P.E.: 0.5 to 12% callus formation
In vitro shoot culture (2 to 3 weeks old, leaves)
microcolonies after 3 weeks P.E.: 3.7% callus formation and root organogenesis
Schlangstedt et a!., 1992
Suspension cells (2 days old)
P.E.: 5% (alginate embedding), 15% (liquid medium)
Schlangstedt (unpublished)
microcolonies after 3 weeks In vitro shoot culture (2 to 4 weeks old, petioles) P.E.: up to 40% callus formation and root organogenesis
Vander Maas
et a!., 1990 Weyens et a!., 1991
Schlangstedt
et a!.,
(present paper)
plating efficiencies obtained with those protoplasts, i.e. prepared from fully differentiated mesophyll tissue, are often low and show substantial variance (see Table 1}. Petioles from in vitro sugar beet shoots are known to have a high morphogenic capacity (Saunders and Shin, 1986; T etu et al., 1987; Detrez et al., 1988, 1989; Freytag et al., 1988}. Therefore, it has already been proposed that sub-epidermal petiole parenchyma protoplasts may prove for the development of a system capable of shoot regeneration (Ritchie et al., 1989}, but until now, no results have been published. In order to profit from the morphogenic capacity of leaf petioles in protoplast technology we developed a method for using petiole tissue of in vitro shoots as a potential protoplast source in sugar beet.
Materials and Methods 1. Plant material . The plant material for protoplast isolation was taken from in vitro cloned shoots 2 to 4weeks after subculture. In all basic experi-
ments the following sugar beet lines were used: the diploid E-line (Institut fiir Riibenforschung, Klein Wanzleben, Germany) and the triploid Prot-line (Instituut voor Moleculaire Biologie, Vrije Universiteit Brussel, Belgium). In comparative studies the lines VRB (diploid, Faculty of Biology, University of Sofia, Bulgaria), 3915 (haploid, lnstitut fiir Riibenforschung, Klein Wanzleben, Germany) and NF (diploid, Centre for Plant Breeding and Reproduction Research (CPRO-DLO) Wageningen, The Netherlands) were included too. The shoots were propagated as described by Schlangstedt et al. (1992).
2. Protoplast isolation In principle, protoplasts were isolated according to the method described in an earlier paper on mesophyll protoplasts (Schlangstedt et al., 1992). The following modification was made: for petiole protoplasts, the enzymes Cellulase TC (1 %, Serva), Pectinase (1.5 %, Serva), Cellulysin (0.2 %, Calbiochem), Cellulase Onozuka R10 (0.1 %, Serva), Macerozyme R10 (0.2 %, Serva) and Driselase (0.075 %, Fluka) were used. For mesophyll protoplasts, the enzyme mixture used was as described by Schlangstedt et al. (1992). Enzymatically isolated protoplasts were separated from debris by centrifugation in 0.4 M sucrose solution overlayered with WG solution (WG solution: 30.03g glycine, 7.50g NaCl, 0.67g KCl and 0.91 g glucose monohydrate per liter, pH 5.8). The protoplast band at the interface of the sucrose and WG layer was removed and added to fresh WG solution. After a 20-min cold treatment at 8 °C, protoplasts were pelleted by centrifugation (100 x g, 5 min). For culturing purposes, the protoplasts were then diluted with 0.5 M sorbitol to various densities (see Fig. 1). Protoplast densities were determined by counting the cells in a Neubauer chamber.
3. Protoplast culture For culturing, protoplasts were embedded in alginate discs and transferred on a SH or K8p liquid medium to which nurse cells were added (see Schlangstedt et al., 1992). Plating efficiency is expressed as the percentage of protoplasts that form colonies after 3 weeks of culture by considering only colonies larger than 0.5 mm in diameter. Callus development and shoot/root organogenesis were microscopically estimated 4 to 6weeks after transfer of protoplast derived microcolonies onto different hormone-containing media. In some experiments conditioned medium was tested for nursing purposes. This medium was produced by filtration (0.21J.m fl.lter, Minisart/Sartorius) of 7-day-old sugar beet cell suspension in SH (Schenk and Hildebrandt, 1972) medium containing 0.02mg/L kinetin (Kin) and 0.5 mg/mL 2,4-dichlorophenoxyacetic acid (2,4-D). The freshly prepared flltrate was added in different concentrations (2, 5, 10, 20 or 50%) to SH based culture medium.
4. Flow cytometry {FCM) For FCM, the CA-ll Cell Analyzer (Partee AG) was used. Before measuring, purified protoplasts were suspended in W solution (Schlangstedt et al., 1992) and stored about 24 h at 5 °C in the dark. Samples for FCM analysis were prepared by combining the protoplast suspension (approx. 2 x 105 ppl/mL) with a 5-fold volume of commercial Partee staining solution and passing through a nylon sieve of 40 11m mesh size. The DNA histograms were automatically analyzed and plotted by a LX-400 plotter (EPSON).
------
Results 1. Protoplast culture
229
Sugar beet protoplast culture
The culturing method for petiole protoplasts is an adaption of the standard culture method described for mesophyll protoplasts, which is based on cell immobilization in Ca-alginate {Schlangstedt et al., 1992). Several parameters, such as alginate type, plating density, 2,4-D concentration of the culture medium and the nursing system, have been tested.
Alginate type Petiole protoplasts of the E- as well as of the Prot-line consistently showed high plating efficiencies, which repeatedly reached 20%. However, over the entire long experimental period, absolute values ranged from 0 to 40 %. The specific type of alginate used was of major importance for achieving high division frequencies. On the average, 2.8 times higher plating efficiencies could be obtained if Sigma alginate of low viscosity was used in comparison to embedding petiole protoplasts in the Roth alginate. Sigma alginate was also found to be the most suited alginate-type for mesophyll protoplast cultivation (Schlangstedt et al., 1992).
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Initial protoplast density Plating densities between 1 x 104 and 5 x 105 ppllmL alginate were tested for highest division frequency. For the Protline, maximum plating efficiency was obtained when a plating density of 1 x 105 ppllmL alginate was used. For the E-line protoplasts, the maximum was reached at a concentration of 2.5 x 105 ppl/ mL alginate. Within each genotype the optimum plating densities for both petiole and mesophyll protoplasts appeared to coincide (Fig. 1, Fig. 2A-D).
Plating efficiency (%)
•
..
ft
Fig. 2: Microcalli obtained by culturing of sugar beet petiole protoplasts in alginate of different initial densities: A = 10\ B = 5 x 10\ C = 105, D = 5x105 ppl/mL alginate {3weeks old) and E 5 x 105 ppl/mL alginate {5 weeks old), bar = 3 mm.
Table 2: Effect of the culture medium {2,4-D concentration) on plating efficiency' of sugar beet petiole protoplasts (means of 10 independent experiments ± 95 %-C.I.)
12,--------------------10 8
6
2,4-D {mg/L)
Genotype
0.5
1.0
Prot E
4.8±0.7% 6.0 ± 1.0%
8.1±1.0% 10.3 ± 1.4%
1.5
2.0
7.0±0.7% 6.8 ± 1.0%
6.6±0.8% 7.1 ± 1.0%
• Sigma alginate {1 %) of low viscosity was used.
4
r
mesophyll
2
3
4
Initial density (ppl/ml alginate)
Fig.1: Effect of initial protoplast density (ppl/mL alginate) on the plating efficiency of sugar beet petiole and mesophyll protoplasts (E-line and Prot-line) (mean of 4 repetitions with 3 parallels at a time ± 95 %-C.I.).
Culture medium (2,4-D concentration) Among the various 2,4-D doses added to the SH based culture medium, the best one was found to be 1.0 mg/L, regardless of the genotype used. On the average, at this concentration a plating efficiency of about 8% {Prot-line) or 10% (Eline) was observed {Table 2). For mesophyll protoplasts, the
MARioN ScHLANGSTEDT, KuRT ZoGLAUER, STEFFEN LENZNER, BoB HERMANs, and MicHEL jAcoBs
230
Plating efficiency (%) 5,---------------------------------------, +E-line -B- Prot-line
4
one-fourth of the plating efficiency that can be achieved by adding nurse cells instead of a mere filtrate. As further culture experiments pointed out, it proved to be impossible to induce divisions in protoplast-derived cells in SH medium without any type of nursing.
2. Comparison ofpetiole and mesophyll protoplasts Petiole protoplasts are much more heterogenous in size and structure than mesophyll protoplasts (see Fig. 4). This
10
20
30
40
50
Conditioned medium (%)
Fig. 3: Influence of conditioned medium on the plating efficiency of petiole protoplasts. SH culture medium was supplemented with different proportions(%) of a filtrate of a 7-day-old sugar beet suspension (mean of 3 independent repetitions ± 95 %-C.I.).
•-.
.
: ~
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optimum 2,4-D concentration appeared to be 1.5 mg/L (Schlangstedt et al., 1992). Embedded protoplasts lost their spherical shape within 48 h. The first divisions occurred after 5 or 6 days. The number of cells starting to divide increased constantly until day 14. Subsequent divisions led to the development of microcalli within 3 more weeks (Fig. 2 E).
Conditioning Apart from the routinely used nurse cells (see Schlangstedt et al., 1992), we also tested conditioned medium for promoting the induction of cell division. Both E-line and Prot-line derived petiole protoplasts showed a maximum plating efficiency of about 2 to 3 % if the culturing solution contained 10% conditioned medium (Fig. 3). This is, however, only
-
Fig. 5: Paraffin section of a leaf petiole, Haematoxylin-Eosin stained, bar= 100 J.Lm.
r~; • . .i
~
t..
1), I
Fig. 4: Freshly prepared mesophyll (A) and petiole protoplasts (B), bar = 50 J.Lm.
Sugar beet protoplast culture
goes together with the different cell types that make up the sub-organs and that become visible in a cross-sectional view of a petiole (Fig. 5). Large cells correspond to parenchymatic tissue. Small cells can be found in the meristems of vascular bundles but also in certain epidermal and sub-epidermal areas. The sub-epidermal cell layers at the concave adaxial side of the petiole containing these areas are known to be meristematically active and a site of de novo bud formation (Detrez et al., 1988). In general, dividing cells were found in petiole protoplast cultures derived from the small protoplasts. On the average, petiole protoplasts showed a 4-fold higher plating efficiency under identical culture conditions in comparison to protoplasts isolated from differentiated leaf mesophyll tissue (3- to 6-fold higher, see Table 3). However, in protoplast cultures derived from young, not fully expanded leaves relatively high division frequencies were obtained (P.E.: 3.8 to 5.0%, Table 3). In contrast to mature leaves these young leaves consisted mainly of midrib/petiole tissue. Flow cytometric analyses of mesophyll and petiole protoplasts showed differences in their DNA histograms, which indicate a higher division activity in petioles compared with mesophyll tissue (Fig. 6). Two distinct peaks could be observed in all histograms. The prominent one corresponds to the nuclear DNA content of (non-dividing) cells of the GO/ G 1 stage of the cell cycle. This peak reflects the ploidy state of the shoots used for protoplast isolation. The minor peak
IC
231
Table 3: Plating efficiency (P.E. in%) in function of the genotype and the protoplast source (in vitro shoot culture) (E, Prot: means of 20 repetitions with 3 parallels at a time ± 95 %-C.I.; 3915, VRB, NF: means of 4 repetitions with 2 parallels at a time ± 95 %-C.I.) Genotype
Culture medium
Mesophyll (mature leaves)
Mesophyll (young leaves)
Petiole
E
SH SH K8p KSr K8p K8p2 K8p
1.30 ± 0.50 2.40 ± 0.70 0.04 ± 0.01 0.16 ± 0.04 3.89 ± 2.28 0.05 ± O.o2 0.28 ± 0.07
3.80 ± 0.50 5.00 ± 0.50
8.30±0.60 8.80±0.60 0.21 ±0.07 0.45±0.44
Prot
3915 VRB VRB NF NF1 1 2 3
n.d.3 n.d. n.d. n.d. n.d.
n.d.
0.40±0.18 0.96±0.04
In vitro grown seedlings, 6 weeks old. K8p medium (Kao and Michayluk, 1975} without nursing. Not determined.
corresponds to the doubled-DNA phase of the cells involved in divisions (G21M stage). The G2/M-peaks of petiole protoplasts were always higher than those of mesophyll protoplasts. 3. Callus formation and root organogenesis
We tested and compared agar-solidified SH basic media supplemented with different doses of BAP (0.1 to 5.0 mg/L)
A
IC
B
2C
2C
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uc
j
0
...8.
c
2C
__
..l.lC
D
1'o
4C
.8 ~
4C
-~
..j.
~
JC
Fig. 6: Flow cytometric analysis of nuclei released from protoplasts derived from mesophyll (A, C, E) or petioles (B, D, F) of haploid (A, B), diploid (C, D) and triploid (E, F) in vitro shoots.
E
JC
F
6C
fluorescence intensities (I>WI>ortionalto relative DNA content)
232
MAiuoN ScHLANGSTEDT, KullT ZOGLAUEll, STEFFEN LENZNER, BoB HERMANs, and MicHEL jACOBS
Table 4: Callus and root formation from sugar beet petiole protoplasts on SH medium in light or dark (5 repetitions). Supplements (mg/L) none BAP 0.1
0.2 0.3 0.4 0.5 1.0 3.0 5.0
Darkness
Light
Prot-line Rb +"
E-line
+ + + ++ +++ (R) +++ (R) ++ +
+ + + + (R) + ++ +++ ++
R
E-line n.d.c ++ R ++ ++ ++ ++ +++ +++ +
• Signs -, +, + + and + + + show the level of callus formation after 4 weeks of incubation no further development of microcolonies, + formation of small, brown to dark brown calli, + + growing, yellow to brown calli, + + + well growing, white to yellow calli. b R indicates root formation (R) rare root formation, R little root formation with high frequency (1 to 3 roots per alginate disc). c Not determined. for their ability to induce callus formation and subsequent shoot/root organogenesis from petiole-derived protoplasts. Callus growth was maximal when microcolonies were cultured on media supplemented with 0.5 to 3.0 mg/L BAP. This was true under dark as well as under light conditions (Table 4). However, calli were compact and yellowish-white in darkness while they turned green and grew better when cultured in the light. White and more friable callus that may be capable of shoot development (Saunders and Daub, 1984; Ritchie et al., 1989; Krens et al., 1990) was obtained only from Prot-line protoplasts cultured on hormone-free medium. These white loose calli arose after the initial microcalli had turned dark brown. Likewise, calli grew well when the medium contained 0.4 mg/L BAP and 0.1 mg/L indole-3-butyric acid (ffiA) or 1.0 mg/L 1-naphthaleneacetic acid (NAA), but without further development. Media containing 2,4-D or tri-iodobenzoic acid (TmA) were not suited for the development of callus from microcolonies (results not shown). Calli grown from petiole protoplasts from different genetic origin did not differ in their rhizogenic capacity. They all showed root formation on hormone-free medium only (Table 4) or on medium supplemented with 1.0 mg/L NAA. When kept in the dark on BAP containing media, the calli barely produced any roots (see Table 4). In light, however, rhizogenesis occurred regularly on medium containing 0.1mg/LBAP. Up until now, shoot formation could not be induced from petiole protoplast derived calli.
Discussion Petioles are well suited for the in vitro clonal propagation of sugar beet (Rogozinska and Goska, 1978; Hussey and
Hepher, 1978; Detrez et al., 1988; Freytag et al., 1988; Ritchie et al., 1989). Direct adventitious shoot formation from petiole explants could be initiated by treatment with BAP (Hussey and Hepher, 1978; Ritchie et al., 1989) or with BAP and auxin (Detrez et al., 1988; Freytag et al., 1988). The newly obtained shoots proliferated along the length of the petiole and emerged generally along the adaxial surface. Several authors noticed that the origin of these shoots is localized in the adaxial sub-epidermal tissue of the leaf petioles (Detrez et al., 1988; Freytag et al., 1988; Ritchie et al., 1989). Concerning the differentiation state of these cell layers, opinions conflict. Whereas Freytag et al. (1988) assumed that a high proportion of adventitious shoots arise from pre-existing meristems in the petiole explants, Detrez et al. (1988) pointed out that it is precisely the preceding hormone treatment (BAP) of the tissue that is responsible for the formation of meristematic zones within the petioles as no pre-existing buds were observed in the petiole explants before culture. Irrespective of the controversy it seems to be the case that precisely these non- or little-differentiated cell layers right below the epidermis and the cambium cells are responsible for the high plating efficiencies observed with petiole-derived protoplasts, because only small protoplasts underwent cell divisions and subsequent callus formation. This also explains the high plating efficiency in protoplast cultures derived from young, not fully expanded leaves (Table 3), which consist mainly of midrib/petiole tissue. The generally higher plating efficiencies in petiole protoplast cultures might be a consequence of their much higher proportion of cells in the G 2/M stage of the division cycle, as can be seen in the corresponding DNA-histograms. Despite of the not yet solved plant regeneration from petiole protoplasts the considerable improvement of the plating efficiencies with those protoplasts in all tested genotypes is a further important step towards application of protoplast technology in sugar beet breeding, e.g. with regard to fusion and transformation experiments. It remains to be seen, however, if the progress in plating efficiency and callus formation will result in an improvement of complete plant regeneration.
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DETREZ, C., T. TETU, R. S. SANGWAN, and B. S. SANGWAN-NOllllEEL: Direct organogenesis from petiole and thin cell layer explants in sugar beet cultured in vitro. J. Exp. Bot. 39, 917-926 (1988). DETREZ, C., R. S. SANGWAN, and B. S. SANGWAN-NOllllEEL: Phenotypic and karyotypic status of Beta vulgaris plants regenerated
Sugar beet protoplast culture from direct organogenesis in petiole culture. Theor. Appl. Genet. 77, 462-468 (1989). EADY, C., G. WARREN, K. LINDSEY, and M.G. K. joNES: Electrofusion and electroporation of sugar beet (Beta vulgaris L.) protoplasts. In: PmTE, K. J., J. J. M. DoNs, H. J. HmZING, A. J. KooL, M. KooRNNEEF, and F. A. KRENs (eds.): Progress in Plant Protoplast Research, pp. 261-262. Kluwer Academic Publishers (1988). FREYTAG, A. H., S. C. ANAND, A. P. RAo-ARELLI, and L. D. OwENs: An improved medium for adventitious shoot formation and callus induction in Beta vulgaris L. in vitro. Plant Cell Rep. 7, 3034 (1988). HERMANSEN, P. R., B. KEIMER, and H. C. PEDERSEN: Induction of morphogenetic callus as a source for regenerating cells suspensions of sugarbeet: Influence of silver nitrate and gelling agents. In: Abstracts 7th International Congress on Plant Tissue and Cell Culture, p. 296. Amsterdam {1990). HussEY, G. and A. HEPHER: Clonal propagation of sugar beet plants and the formation of polyploids by tissue culture. Ann. Bot. 42, 477-479 {1978). JoERSBO, M. and J. BRUNSTEDT: Direct gene transfer to plant protoplasts by mild sonication. Plant Cell Rep. 9, 207-210 (1990). KAo, K. N. and M. R. MICHAYLUK: Nutritional requirements for growth of Vicia hajastana cells and protoplasts at a very low population density in liquid media. Planta (Berl.) 126, 105-110 (1975). KRENs, F. A. and D. JAMAR: The role of explant source and culture conditions on callus induction and shoot regeneration in sugarbeet (Beta vulgaris L.). J. Plant Physiol. 134, 651-655 (1989). KRENs, F. A., D. JAMAR, G. J. A. RouwENDAL, and R. D. HALL: Transfer of cytoplasm from new Beta CMS sources to sugar beet by asymmetric fusion. 1. Shoot regeneration from mesophyll protoplasts and characterization of regenerated plants. Theor. Appl. Genet. 79, 390-396 {1990). LINDSEY, K. and M. G. K. JoNEs: The permeability of electroporated cells and protoplasts of sugar beet. Planta 172, 346-355 (1987 a). - - Transient gene expression in electroporated protoplasts and intact cells of sugar beet. Plant Mol. Bioi. 10, 43-53 (1987 b). - - Stable transformation of sugar beet by electroporation. Plant Cell Rep. 8, 71-74 (1989). MOLLER, B. and U. RYSCHKA: Effect of various osmotic substances on protoplast isolation by enzymes. Biochem. Physiol. Pflanzen 182, 183-186 (1987). PEDERSEN, H. C., A. BUCHTER-LARSEN, and B. KEIMER: Inactivation of sugar beet protoplasts using acridin orange, an agent for late selection of fusion products. In: PuiTE, K. J., J. J. M. DoNs, H. J. HUIZING, A. J. KooL, M. KooRNEEF, and F. A. KRENs (eds.): Pro-
233
gress in Plant Protoplast Research, pp. 265-266. Kluwer Academic Publishers {1988). PEDERSEN, C., R. D. HALL, F. A. KRENs, and L. BucH: Transfer of mitochondria in Beta vulgaris via asymmetric protoplast fusion. In: Abstracts 8th International Protoplast Symposium, Sweden. Physiol. Plant. 82, A 27 (1991). RITcHIE, G. A., K. C. SHORT, and M. R. DAVEY: In vitro shoot regeneration from callus, leaf axils and petioles of sugar beet (Beta vul· garis L.). J. Exp. Bot. 40, 277-283 {1989). RoGOZINSKA, J. H. and M. GosKA: Induction of differentiation and plant formation in isolated sugar beet leaves. Bull. Acad. Pol. Sci., Cl. II, Vol. XXVI, 343-345 {1978). SAUNDERS, J. W. and K. SHIN: Germplasm and physiologic effects on induction of high-frequency hormone autonomous callus and subsequent shoot regeneration in sugar beet. Crop Sci. 26, 1240-1245 (1986). SAUNDERS, J. W. and M. E. DAUB: Shoot regeneration from hormone-autonomous callus from shoot cultures of several sugarbeet (Beta vulgaris L.) genotypes. Plant Sci. Lett. 34, 219-223 {1984). SCHENK, R. U. and A. C. HILDEBRANDT: Medium and techniques for induction and growth of monocotyledonus plant cell cultures. Can. J. Bot. 50, 199-204 {1972). ScHLANGSTEDT, M., B. HERMANS, K. ZoGLAUER, and 0. ScHIEDER: Culture of sugar beet (Beta vulgaris L.) mesophyll protoplasts in alginate - callus formation and root organogenesis. J. Plant Physiol. 140, 345-349 {1992). SENANAYAKE, A.M. S.: In vitro tissue culture of sugar beet and selection of cell lines resistant to herbicides. Thesis, Vrije U niversiteit Brussel, Belgium (1986). SMOLENSKAYA, I. N. and G. N. RALDUGINA: Protoplast cultivation from sugar beet cell suspensions. Soviet Plant Physiol. 28, 10221029 {1981). SZABADOS, L. and C. GAGGERO: Callus formation from protoplasts of a sugar beet cells suspension culture. Plant Cell Rep. 4, 195198 (1985). T:Eru, T., R. S. SANGWAN, and B. S. SANGWAN-NORREEL: Hormonal control of organogenesis and somatic embryogenesis in Beta vul· garis callus. J. Exp. Bot. 38, 506- 517 (1987). VANDER MAAs, H. M., F. A. KRENs, and D. JAMAR: A reproductive technique for the regeneration of shoots from mesophyll protoplasts of Beta vulgaris L. In: Abstracts 7th International Congress on Plant Tissue and Organ Culture, p. 22. Amsterdam (1990). WEYENS, G., J. LATHoUWERS, J. VAN GEYT, and M. jACOBS: Steps toward regeneration from leaf mesophyll protoplasts of Beta vulga· ris. In: Abstracts 8th International Protoplast Symposium, Sweden. Physiol. Plant. 82, A 38 (1991).
Improvement of Sugar Beet (Beta vulgaris L.) Protoplast Culture: Leaf Petioles as a Protoplast Source MARION ScHLANGSTEDT 1• 2, KuRT ZoGLAUER 1• MICHEL jACOBS3
*, STEFFEN LENZNER1, BoB HERMANS3, and
1
Humboldt-Universitat zu Berlin, FB Biologie, Institut fiir Pflanzenphysiologie und Zellbiologie, InvalidenstraBe 43, D-10115 Berlin, BRD
2
Present address: Planta Angewandte Pflanzengenetik und Biotechnologie GmbH, Grimsehlstr. 31, D-37574 Einbeck, BRD
3
Instituut voor Moleculaire Biologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium
* To whom correspondence should be addressed Received May 13, 1993 · Accepted September 22, 1993
Summary
Petioles of in vitro shoots were tested as potential sources for sugar beet (Beta vulgaris L.) protoplasts. Regardless of the genotype used, petiole protoplasts showed much higher plating efficiencies (average P.E.: 4.6 %) than mesophyll protoplasts (average P.E.: 1.0%) when cultured under identical conditions in a Schenk and Hildebrandt (SH) as well as in a Kao and Michayluk (K8p) medium. Additionally, DNA histograms indicate that petiole protoplast populations have a higher proportion with doubled DNA content, i.e. nuclei in the G2/M stage of the cell cycle.
Key words: Beta vulgaris, alginate-embedding, flow cytometry, mesophyll protoplasts, nurse culture, petiole pro toplasts. Abbreviations: BAP = 6-benzylaminopurine; C.I. = confidence interval; 2,4-D = 2,4-dichlorophenoxyacetic acid; fw = fresh weight; illA = indole-3-butyric acid; Kin = kinetin; NAA = 1-naphthaleneacetic acid; ppl = protoplasts; TillA = tri-iodobenzoic acid; P.E. = plating efficiency. Introduction
Suspension cells and leaf mesophyll tissue are the sources most commonly used for isolation of sugar beet protoplasts (review see Table 1). Because suspension cell protoplasts can be easily isolated and cultured, they were often used in manipulation experiments such as fusion (Eady et al., 1988; Pedersen et al., 1988, 1991), electroporation (Eady et al., 1988; Lindsey and Jones, 1987 a, b) and gene transfer (Lindsey and Jones, 1987, 1989; Joersbo and Brunstedt, 1990). While stable yields and high plating efficiencies can be achieved from this type of protoplasts, plant regeneration was never observed. As long as protoplasts derived from non-embryogenic and © 1994 by Gustav Fischer Verlag, Stuttgart
long-time cultured suspensions are involved, plant regeneration is not to be expected. On the other hand only Hermansen et al. (1990) have presented a method for initiation of morphogenic suspensions in sugar beet potentially capable of plant regeneration. In order to apply protoplast technology in sugar beet genetics and breeding, high yields and plating efficiency as well as plant regeneration are absolutely required. Methods for plant regeneration in more than one genotype are rare and to our knowledge only described by Krens et al. (Krens et al., 1990; Vander Maas et al., 1990) and mentioned by Pedersen et al. (pers. commun.). Krens et al. used leaves of in vitro grown seedlings, which indicates that at least this type of donor material is suited. However, the
228
MAiuoN ScHLANGSTEDT, KuRT ZoGLAUER, STEFFEN LENZNER, BoB HERMANs, and MicHEL jACOBS
Table 1: Overview ot the type of protoplasts used in sugar beet research and their response upon culturing. Origin of protoplasts
Response
References
Outdoor and greenhouse plants (leaves, apex)
P.E.: 0%
Binding et a!., 1981
Suspension cells (late log phase)
colonies after 7 to 10 days callus after 30 to 40 days
Smolenskaya and Raldugina, 1981
Suspension cells (4 to 5 days old)
colonies after 21 days P.E.: 7.3%
Bhat eta!., 1985
Suspension cells (2 days old)
colonies after 30 days P.E.: 35 %, cell suspension
Szabados and Gaggero, 1985
In vitro shoot culture (4 weeks old, leaves)
microcolonies after 3 weeks P .E.: 0.3 to 13 %, callus formation and root organogenesis
Bhat et a!., 1986
Suspension cells (4 days old)
colonies after 3 weeks P.E.: 60%
Senanayake, 1986
Suspension cells (3 days old)
P.E.: 7%
Miiller and Ryschka, 1987
In vitro grown seedlings (4 to 6 weeks old, leaves)
occasionally cell division
Krens and Jamar, 1989
Suspension cells
routinely calli without any regeneration
Krens and Jamar, 1989
Suspension cells (5 to 7 days old)
P.E.: 14.8% (liquid medium) 64.7% (agarose embedding)
Lindsey and Jones, 1989
In vitro grown seedlings (6 to 8 weeks old, leaves)
P.E.: 0.005 to 1.0% shoot regeneration, 10 to 20%
Krens et a!., 1990
In vitro grown seedlings
P.E.: O.Ql to 7% shoot regeneration, 3 to 20%
In vitro shoot culture and in vitro grown seedlings
P.E.: 0.5 to 12% callus formation
In vitro shoot culture (2 to 3 weeks old, leaves)
microcolonies after 3 weeks P.E.: 3.7% callus formation and root organogenesis
Schlangstedt et a!., 1992
Suspension cells (2 days old)
P.E.: 5% (alginate embedding), 15% (liquid medium)
Schlangstedt (unpublished)
microcolonies after 3 weeks In vitro shoot culture (2 to 4 weeks old, petioles) P.E.: up to 40% callus formation and root organogenesis
Vander Maas
et a!., 1990 Weyens et a!., 1991
Schlangstedt
et a!.,
(present paper)
plating efficiencies obtained with those protoplasts, i.e. prepared from fully differentiated mesophyll tissue, are often low and show substantial variance (see Table 1}. Petioles from in vitro sugar beet shoots are known to have a high morphogenic capacity (Saunders and Shin, 1986; T etu et al., 1987; Detrez et al., 1988, 1989; Freytag et al., 1988}. Therefore, it has already been proposed that sub-epidermal petiole parenchyma protoplasts may prove for the development of a system capable of shoot regeneration (Ritchie et al., 1989}, but until now, no results have been published. In order to profit from the morphogenic capacity of leaf petioles in protoplast technology we developed a method for using petiole tissue of in vitro shoots as a potential protoplast source in sugar beet.
Materials and Methods 1. Plant material . The plant material for protoplast isolation was taken from in vitro cloned shoots 2 to 4weeks after subculture. In all basic experi-
ments the following sugar beet lines were used: the diploid E-line (Institut fiir Riibenforschung, Klein Wanzleben, Germany) and the triploid Prot-line (Instituut voor Moleculaire Biologie, Vrije Universiteit Brussel, Belgium). In comparative studies the lines VRB (diploid, Faculty of Biology, University of Sofia, Bulgaria), 3915 (haploid, lnstitut fiir Riibenforschung, Klein Wanzleben, Germany) and NF (diploid, Centre for Plant Breeding and Reproduction Research (CPRO-DLO) Wageningen, The Netherlands) were included too. The shoots were propagated as described by Schlangstedt et al. (1992).
2. Protoplast isolation In principle, protoplasts were isolated according to the method described in an earlier paper on mesophyll protoplasts (Schlangstedt et al., 1992). The following modification was made: for petiole protoplasts, the enzymes Cellulase TC (1 %, Serva), Pectinase (1.5 %, Serva), Cellulysin (0.2 %, Calbiochem), Cellulase Onozuka R10 (0.1 %, Serva), Macerozyme R10 (0.2 %, Serva) and Driselase (0.075 %, Fluka) were used. For mesophyll protoplasts, the enzyme mixture used was as described by Schlangstedt et al. (1992). Enzymatically isolated protoplasts were separated from debris by centrifugation in 0.4 M sucrose solution overlayered with WG solution (WG solution: 30.03g glycine, 7.50g NaCl, 0.67g KCl and 0.91 g glucose monohydrate per liter, pH 5.8). The protoplast band at the interface of the sucrose and WG layer was removed and added to fresh WG solution. After a 20-min cold treatment at 8 °C, protoplasts were pelleted by centrifugation (100 x g, 5 min). For culturing purposes, the protoplasts were then diluted with 0.5 M sorbitol to various densities (see Fig. 1). Protoplast densities were determined by counting the cells in a Neubauer chamber.
3. Protoplast culture For culturing, protoplasts were embedded in alginate discs and transferred on a SH or K8p liquid medium to which nurse cells were added (see Schlangstedt et al., 1992). Plating efficiency is expressed as the percentage of protoplasts that form colonies after 3 weeks of culture by considering only colonies larger than 0.5 mm in diameter. Callus development and shoot/root organogenesis were microscopically estimated 4 to 6weeks after transfer of protoplast derived microcolonies onto different hormone-containing media. In some experiments conditioned medium was tested for nursing purposes. This medium was produced by filtration (0.21J.m fl.lter, Minisart/Sartorius) of 7-day-old sugar beet cell suspension in SH (Schenk and Hildebrandt, 1972) medium containing 0.02mg/L kinetin (Kin) and 0.5 mg/mL 2,4-dichlorophenoxyacetic acid (2,4-D). The freshly prepared flltrate was added in different concentrations (2, 5, 10, 20 or 50%) to SH based culture medium.
4. Flow cytometry {FCM) For FCM, the CA-ll Cell Analyzer (Partee AG) was used. Before measuring, purified protoplasts were suspended in W solution (Schlangstedt et al., 1992) and stored about 24 h at 5 °C in the dark. Samples for FCM analysis were prepared by combining the protoplast suspension (approx. 2 x 105 ppl/mL) with a 5-fold volume of commercial Partee staining solution and passing through a nylon sieve of 40 11m mesh size. The DNA histograms were automatically analyzed and plotted by a LX-400 plotter (EPSON).
------
Results 1. Protoplast culture
229
Sugar beet protoplast culture
The culturing method for petiole protoplasts is an adaption of the standard culture method described for mesophyll protoplasts, which is based on cell immobilization in Ca-alginate {Schlangstedt et al., 1992). Several parameters, such as alginate type, plating density, 2,4-D concentration of the culture medium and the nursing system, have been tested.
Alginate type Petiole protoplasts of the E- as well as of the Prot-line consistently showed high plating efficiencies, which repeatedly reached 20%. However, over the entire long experimental period, absolute values ranged from 0 to 40 %. The specific type of alginate used was of major importance for achieving high division frequencies. On the average, 2.8 times higher plating efficiencies could be obtained if Sigma alginate of low viscosity was used in comparison to embedding petiole protoplasts in the Roth alginate. Sigma alginate was also found to be the most suited alginate-type for mesophyll protoplast cultivation (Schlangstedt et al., 1992).
.··~
,,
•
.·'(·· . ~~
Initial protoplast density Plating densities between 1 x 104 and 5 x 105 ppllmL alginate were tested for highest division frequency. For the Protline, maximum plating efficiency was obtained when a plating density of 1 x 105 ppllmL alginate was used. For the E-line protoplasts, the maximum was reached at a concentration of 2.5 x 105 ppl/ mL alginate. Within each genotype the optimum plating densities for both petiole and mesophyll protoplasts appeared to coincide (Fig. 1, Fig. 2A-D).
Plating efficiency (%)
•
..
ft
Fig. 2: Microcalli obtained by culturing of sugar beet petiole protoplasts in alginate of different initial densities: A = 10\ B = 5 x 10\ C = 105, D = 5x105 ppl/mL alginate {3weeks old) and E 5 x 105 ppl/mL alginate {5 weeks old), bar = 3 mm.
Table 2: Effect of the culture medium {2,4-D concentration) on plating efficiency' of sugar beet petiole protoplasts (means of 10 independent experiments ± 95 %-C.I.)
12,--------------------10 8
6
2,4-D {mg/L)
Genotype
0.5
1.0
Prot E
4.8±0.7% 6.0 ± 1.0%
8.1±1.0% 10.3 ± 1.4%
1.5
2.0
7.0±0.7% 6.8 ± 1.0%
6.6±0.8% 7.1 ± 1.0%
• Sigma alginate {1 %) of low viscosity was used.
4
r
mesophyll
2
3
4
Initial density (ppl/ml alginate)
Fig.1: Effect of initial protoplast density (ppl/mL alginate) on the plating efficiency of sugar beet petiole and mesophyll protoplasts (E-line and Prot-line) (mean of 4 repetitions with 3 parallels at a time ± 95 %-C.I.).
Culture medium (2,4-D concentration) Among the various 2,4-D doses added to the SH based culture medium, the best one was found to be 1.0 mg/L, regardless of the genotype used. On the average, at this concentration a plating efficiency of about 8% {Prot-line) or 10% (Eline) was observed {Table 2). For mesophyll protoplasts, the
MARioN ScHLANGSTEDT, KuRT ZoGLAUER, STEFFEN LENZNER, BoB HERMANs, and MicHEL jAcoBs
230
Plating efficiency (%) 5,---------------------------------------, +E-line -B- Prot-line
4
one-fourth of the plating efficiency that can be achieved by adding nurse cells instead of a mere filtrate. As further culture experiments pointed out, it proved to be impossible to induce divisions in protoplast-derived cells in SH medium without any type of nursing.
2. Comparison ofpetiole and mesophyll protoplasts Petiole protoplasts are much more heterogenous in size and structure than mesophyll protoplasts (see Fig. 4). This
10
20
30
40
50
Conditioned medium (%)
Fig. 3: Influence of conditioned medium on the plating efficiency of petiole protoplasts. SH culture medium was supplemented with different proportions(%) of a filtrate of a 7-day-old sugar beet suspension (mean of 3 independent repetitions ± 95 %-C.I.).
•-.
.
: ~
.•
'
~-/
'
'f
~'
optimum 2,4-D concentration appeared to be 1.5 mg/L (Schlangstedt et al., 1992). Embedded protoplasts lost their spherical shape within 48 h. The first divisions occurred after 5 or 6 days. The number of cells starting to divide increased constantly until day 14. Subsequent divisions led to the development of microcalli within 3 more weeks (Fig. 2 E).
Conditioning Apart from the routinely used nurse cells (see Schlangstedt et al., 1992), we also tested conditioned medium for promoting the induction of cell division. Both E-line and Prot-line derived petiole protoplasts showed a maximum plating efficiency of about 2 to 3 % if the culturing solution contained 10% conditioned medium (Fig. 3). This is, however, only
-
Fig. 5: Paraffin section of a leaf petiole, Haematoxylin-Eosin stained, bar= 100 J.Lm.
r~; • . .i
~
t..
1), I
Fig. 4: Freshly prepared mesophyll (A) and petiole protoplasts (B), bar = 50 J.Lm.
Sugar beet protoplast culture
goes together with the different cell types that make up the sub-organs and that become visible in a cross-sectional view of a petiole (Fig. 5). Large cells correspond to parenchymatic tissue. Small cells can be found in the meristems of vascular bundles but also in certain epidermal and sub-epidermal areas. The sub-epidermal cell layers at the concave adaxial side of the petiole containing these areas are known to be meristematically active and a site of de novo bud formation (Detrez et al., 1988). In general, dividing cells were found in petiole protoplast cultures derived from the small protoplasts. On the average, petiole protoplasts showed a 4-fold higher plating efficiency under identical culture conditions in comparison to protoplasts isolated from differentiated leaf mesophyll tissue (3- to 6-fold higher, see Table 3). However, in protoplast cultures derived from young, not fully expanded leaves relatively high division frequencies were obtained (P.E.: 3.8 to 5.0%, Table 3). In contrast to mature leaves these young leaves consisted mainly of midrib/petiole tissue. Flow cytometric analyses of mesophyll and petiole protoplasts showed differences in their DNA histograms, which indicate a higher division activity in petioles compared with mesophyll tissue (Fig. 6). Two distinct peaks could be observed in all histograms. The prominent one corresponds to the nuclear DNA content of (non-dividing) cells of the GO/ G 1 stage of the cell cycle. This peak reflects the ploidy state of the shoots used for protoplast isolation. The minor peak
IC
231
Table 3: Plating efficiency (P.E. in%) in function of the genotype and the protoplast source (in vitro shoot culture) (E, Prot: means of 20 repetitions with 3 parallels at a time ± 95 %-C.I.; 3915, VRB, NF: means of 4 repetitions with 2 parallels at a time ± 95 %-C.I.) Genotype
Culture medium
Mesophyll (mature leaves)
Mesophyll (young leaves)
Petiole
E
SH SH K8p KSr K8p K8p2 K8p
1.30 ± 0.50 2.40 ± 0.70 0.04 ± 0.01 0.16 ± 0.04 3.89 ± 2.28 0.05 ± O.o2 0.28 ± 0.07
3.80 ± 0.50 5.00 ± 0.50
8.30±0.60 8.80±0.60 0.21 ±0.07 0.45±0.44
Prot
3915 VRB VRB NF NF1 1 2 3
n.d.3 n.d. n.d. n.d. n.d.
n.d.
0.40±0.18 0.96±0.04
In vitro grown seedlings, 6 weeks old. K8p medium (Kao and Michayluk, 1975} without nursing. Not determined.
corresponds to the doubled-DNA phase of the cells involved in divisions (G21M stage). The G2/M-peaks of petiole protoplasts were always higher than those of mesophyll protoplasts. 3. Callus formation and root organogenesis
We tested and compared agar-solidified SH basic media supplemented with different doses of BAP (0.1 to 5.0 mg/L)
A
IC
B
2C
2C
__.__._.
uc
j
0
...8.
c
2C
__
..l.lC
D
1'o
4C
.8 ~
4C
-~
..j.
~
JC
Fig. 6: Flow cytometric analysis of nuclei released from protoplasts derived from mesophyll (A, C, E) or petioles (B, D, F) of haploid (A, B), diploid (C, D) and triploid (E, F) in vitro shoots.
E
JC
F
6C
fluorescence intensities (I>WI>ortionalto relative DNA content)
232
MAiuoN ScHLANGSTEDT, KullT ZOGLAUEll, STEFFEN LENZNER, BoB HERMANs, and MicHEL jACOBS
Table 4: Callus and root formation from sugar beet petiole protoplasts on SH medium in light or dark (5 repetitions). Supplements (mg/L) none BAP 0.1
0.2 0.3 0.4 0.5 1.0 3.0 5.0
Darkness
Light
Prot-line Rb +"
E-line
+ + + ++ +++ (R) +++ (R) ++ +
+ + + + (R) + ++ +++ ++
R
E-line n.d.c ++ R ++ ++ ++ ++ +++ +++ +
• Signs -, +, + + and + + + show the level of callus formation after 4 weeks of incubation no further development of microcolonies, + formation of small, brown to dark brown calli, + + growing, yellow to brown calli, + + + well growing, white to yellow calli. b R indicates root formation (R) rare root formation, R little root formation with high frequency (1 to 3 roots per alginate disc). c Not determined. for their ability to induce callus formation and subsequent shoot/root organogenesis from petiole-derived protoplasts. Callus growth was maximal when microcolonies were cultured on media supplemented with 0.5 to 3.0 mg/L BAP. This was true under dark as well as under light conditions (Table 4). However, calli were compact and yellowish-white in darkness while they turned green and grew better when cultured in the light. White and more friable callus that may be capable of shoot development (Saunders and Daub, 1984; Ritchie et al., 1989; Krens et al., 1990) was obtained only from Prot-line protoplasts cultured on hormone-free medium. These white loose calli arose after the initial microcalli had turned dark brown. Likewise, calli grew well when the medium contained 0.4 mg/L BAP and 0.1 mg/L indole-3-butyric acid (ffiA) or 1.0 mg/L 1-naphthaleneacetic acid (NAA), but without further development. Media containing 2,4-D or tri-iodobenzoic acid (TmA) were not suited for the development of callus from microcolonies (results not shown). Calli grown from petiole protoplasts from different genetic origin did not differ in their rhizogenic capacity. They all showed root formation on hormone-free medium only (Table 4) or on medium supplemented with 1.0 mg/L NAA. When kept in the dark on BAP containing media, the calli barely produced any roots (see Table 4). In light, however, rhizogenesis occurred regularly on medium containing 0.1mg/LBAP. Up until now, shoot formation could not be induced from petiole protoplast derived calli.
Discussion Petioles are well suited for the in vitro clonal propagation of sugar beet (Rogozinska and Goska, 1978; Hussey and
Hepher, 1978; Detrez et al., 1988; Freytag et al., 1988; Ritchie et al., 1989). Direct adventitious shoot formation from petiole explants could be initiated by treatment with BAP (Hussey and Hepher, 1978; Ritchie et al., 1989) or with BAP and auxin (Detrez et al., 1988; Freytag et al., 1988). The newly obtained shoots proliferated along the length of the petiole and emerged generally along the adaxial surface. Several authors noticed that the origin of these shoots is localized in the adaxial sub-epidermal tissue of the leaf petioles (Detrez et al., 1988; Freytag et al., 1988; Ritchie et al., 1989). Concerning the differentiation state of these cell layers, opinions conflict. Whereas Freytag et al. (1988) assumed that a high proportion of adventitious shoots arise from pre-existing meristems in the petiole explants, Detrez et al. (1988) pointed out that it is precisely the preceding hormone treatment (BAP) of the tissue that is responsible for the formation of meristematic zones within the petioles as no pre-existing buds were observed in the petiole explants before culture. Irrespective of the controversy it seems to be the case that precisely these non- or little-differentiated cell layers right below the epidermis and the cambium cells are responsible for the high plating efficiencies observed with petiole-derived protoplasts, because only small protoplasts underwent cell divisions and subsequent callus formation. This also explains the high plating efficiency in protoplast cultures derived from young, not fully expanded leaves (Table 3), which consist mainly of midrib/petiole tissue. The generally higher plating efficiencies in petiole protoplast cultures might be a consequence of their much higher proportion of cells in the G 2/M stage of the division cycle, as can be seen in the corresponding DNA-histograms. Despite of the not yet solved plant regeneration from petiole protoplasts the considerable improvement of the plating efficiencies with those protoplasts in all tested genotypes is a further important step towards application of protoplast technology in sugar beet breeding, e.g. with regard to fusion and transformation experiments. It remains to be seen, however, if the progress in plating efficiency and callus formation will result in an improvement of complete plant regeneration.
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