Large numbers of viable protoplasts were isolated from leaves of apple rootstocks M.9, MM. 106 and the scion variety Spartan and also from leaf tissues, callus ...
J. PlantPhysiol. Vol. 133. pp. 460-465 (1988)
Plant Regeneration from Protoplasts of Apple Rootstocks and Scion Varieties (Malus X domestica Borkh.) E. M.
PATAT-OCHATT,
S. J.
OCHATT,
and J. B.
POWER
Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, Nottingham NG7 2RD, U.K. Received June 9, 1988· Accepted July 15, 1988
Summary Large numbers of viable protoplasts were isolated from leaves of apple rootstocks M.9, MM. 106 and the scion variety Spartan and also from leaf tissues, callus and cell suspensions of Bramley's Seedling apple. Upon culture, using modified MS-based media (for rootstocks M.9 and MM.106) or modified KM media (for the scions Spartan and Bramley's Seedling), protoplasts entered division to give callus. Shoot buds were regenerated, from the protoplast-derived calli of M.9, MM.106 and Spartan with the production of rooted, autotrophic plants, for M.9 and Spartan apple.
Key words: Fruit trees, Malus X domestica, protoplast culture, plant regeneration. Abbreviations: BAP - 6-benzylaminopurine; 2,4-D - 2,4-dichlorophenoxyacetic acid; f.wt. - fresh weight; GA3 - gibberellic acid; IBA - 4-indole-3yl-butyric acid; KM - Kao and Michayluk (1975); M.9 apple - clone M.9/35; MM.106 apple - clone B.C.221 MM. 106/88; MES - 2-N-morpholinoethane sulphonic acid; MS - Murashige and Skoog (1962); NAA - I-naphthalene-acetic acid; P.E. - plating efficiency; Z - zeatin.
Introduction Somatic hybridisation offers the potential to produce composite fruit trees since rootstock and scion genomes and their cytoplasms could be combined in a single polyploid hybrid with the clear advantage of the retention of heterozygocity, that would normally be reduced in artificially doubled polyploids. However, reproducible protoplast-to-tree systems are a prerequisite for this goal and, in this respect, several reports have described the isolation and culture of apple protoplasts (Doughty and Power, 1988; Hurwitz and Agrios, 1984; James et al., 1984; Kouider et al., 1984; Niizeki et al., 1983; Revilla et al., 1987; Wallin and Welander, 1985) and those of other fruit tree species (Matsuta et al., 1986; Ochatt and Power, 1988 b; Wu and Kuniyuki, 1985). Plant regeneration from protoplasts of woody fruit trees was only achieved for a few species (Ochatt and Caso, 1986; Ochatt et al., 1987; Ochatt and Power, 1988 a; Oka and Ohyama, 1985; Vardi et al., 1982) and, to date, not for apple. © 1988 by Gustav Fischer Verlag, Stuttgart
This paper reports on the isolation, culture and successful regeneration of plants from apple protoplasts (Malus X domes· tica Borkh.) and the differential cultural requirements for several rootstock and scion genotypes.
Materials and Methods Plant material
Apple mesophyll protoplasts were isolated from axenic shoot cui· tures of the rootstocks M.9 and MM.l06 and the scion varieties Spartan and Bramley's Seedling. Shoot cultures were maintained at 25°e, with a constant illumination (1000 lux, daylight fluorescent tubes), by monthly subcultures on MS·based media as shown in Table1. Protoplasts were also isolated from shoot tip callus and cell suspension cultures of Bramley's Seedling apple, the latter initiated and maintained on MS medium with 2.0 mg!l NAA and 0.5 mg!l BAP and grown under the same conditions as for axenic shoot cul-
Plant regeneration from apple protoplasts Table 1: Hormonal composition of MS media used for axenic shoot cultures. The multiplication medium, for M.9, was further supplemented with 1.0 mM phloroglucinol Games and Thurbon, 1981). Apple Variety
IBA (mg/I)
BAP (mg/I)
GA3 (mg/I)
Spartan (scion) Bramley's Seedling (scion) M.9 (rootstock) MM.106 (rootstock)
0.1 0.1 0.0 0.3
1.0 1.0 2.0 1.5
0.1 0.0 0.0 0.0
tures. Subculturing was every 2 and 4 weeks respectively for the cell suspension and callus cultures.
Protoplast isolation Leafprotoplasts {all varieties} The most recent, fully expanded leaves of the axenic shoot cultures were chopped into strips (1- 2 mm wide) and plasmolized (1 h) in CPW 13M medium (Power et aI., 1984). Leaf tissues (500mg f.wt.) were incubated in 10.0 ml aliquots of an enzyme mixture which consisted of 1.0% (w/v) Cellulase Onozuka R-10 (Yakult Honsha Co., Nishinomiya, Japan), 1.0 % (w/v) Hemicellulase (Sigma, U.K.), 0.1 % (w/v) Pectolyase Y-23 (Seishim Pharmaceutical, Tokyo, Japan), 5.0 mM MES and 1.0% (w/v) polyvinylpyrrolidone (PVP-10, average MW 10,000) in CPW 13M medium (pH 5.6) (Revilla et aI., 1987). Incubation was overnight (17 h), at 25°C, with a continuous illumination of 100 lux (cool white fluorescent tubes) plus agitation (40 rev/min).
Callus/cell suspension protoplasts (Bramley's Seedling) Callus slices (1 mm thick) or cells of suspension cultures were plasmolized and incubated as for the leaf material with aliquots (1.0 g f.wt. or 1.0 ml packed cell volume respectively) in 10.0 ml of an enzyme solution which contained 2.0% (w/v) Meicelase (Meiji Seika Kaisho, Tokyo, Japan), 2.0% (w/v) Rhozyme HP-150 (Rohm and Hass, Philadelphia, U.S.A.) and 0.03 % (w/v) Macerozyme R-10 (Yakult Honsha Co., Nishinomiya, Japan) in CPW 13M solution with 5.0 mM MES (pH 5.6). After incubation, tissues (both sources) were filtered through a nylon sieve (64~m) and the filtrate centrifuged (10 min; 100 xg for leaf protoplasts; 120 x g for callus and cell suspension protoplasts) and the supernatant discarded. Protoplasts were resuspended in a small volume (2 ml) of CPW 13M medium and layered on top of CPW 21S medium (10ml) (Power et al., 1984) and centrifuged (100 xg, 5-7 min for callus and cell suspension protoplasts; 10 min for leaf protoplasts). Protoplasts free of cell debris were collected at the interphase, resuspended in 10.0 ml of CPW 13M medium and the yield and viability assessed, the latter using fluorescein diacetate (Power et aI., 1984).
Protoplast culture Protoplasts were plated in a range of media including K8P, Km8P media (Kao and Michayluk, 1975) or MS medium with a range of concentrations of NAA, BAP and/or Z (0.0, 0.1, 0.25, 0.5, 1.0 or 2.0 mg/I) and 9 % (w/v) mannitol (pH 5.8). In addition, the MSbased protoplast media were modified either without ammonium ions or by the inclusion, with the inorganic constituents, of a filter sterilized organic mixture which consisted of (mg/I): glycine (2.0), thiamine-HCI (2.0), pyridoxine-HCI (1.0), nicotinic acid (1.0), riboflavine (0.5), biotin (0.05), cyanocobalamin (0.01), folic acid (0.1), calcium panthotenate (1.0), caseine hydrolysate (enzymatic) (50) and
461
myoinositol (500). Protoplasts were maintained statically, at 25°C, either in the dark or with a continuous illumination of 1000 lux (cool white fluorescent tubes), as liquid cultures or embedded in Seaplaque agarose-containing media (0.625 %), as semisolid layers or bead cultures (Shillito et al., 1983). The initial plating density was 0.5, 0.75, 1.0, 2.5 or 5.0 x 105 protoplasts/ ml. For liquid cultures, the concentration of the osmoticum was reduced by adding an osmoticum-free media counterpart, in a 3: 1 (v/v) ratio (protoplast: mannitol-free medium), after 14 and 21 days. Protoplast-derived cell colonies, in all liquid media, were subsequently dispensed over semisolid (agarose) MS-based medium for further proliferation. For protoplasts in agarose layers, small blocks, containing dividing protoplasts, were progressively transferred to fresh media of a reduced mannitol concentration whilst those plated as bead cultures were surrounded by osmoticum-free liquid medium immediately after plating and left undisturbed until cell colonies were visible. All cultures were examined twice weekly and cell wall regeneration confirmed with Calcofluor White (Nagata and Takebe, 1970). Protoplast P.E., defined as the percentage of protoplasts that had regenerated a cell wall and divided mitotically at least once, was calculated at day 15 (initial plating efficiency - l.P.E.) and at day 60 (final plating efficiency - F.P.E.) with F.P.E. being expressed as the percentage of the originally plated protoplasts that had developed to the microcallus stage.
Plant regeneration Protoplast-derived cell colonies (60 days old; 1-2 mm diam.) were transferred to MS-based semisolid media (0.8 % agar) supplemented with NAA or 2,4-D and BAP or Z for two 3-week subculture periods and finally to agar-solidified (0.8 %) regeneration media. These regeneration media, all based on MS, contained NAA (0.0 to 0.5 mg/I), BAP (0.0 to 2.0 mg/I) and Z (0.0 to 1.0 mg/I) (PH 5.5). Caseine hydrolysate (250 or 500 mg/I), phloroglucinol (0.1 or 1.0 mM), thiamine-HCI (0.2 or 0.4 mg/I), pyridoxine-HCI (1.0 mg/I) and nicotinic acid (1.0 mg/I), individually or in combination, were also assessed for their influence on plant regeneration. All cultures were kept at 25±2 °C, with a constant illumination (1000 lux, daylight fluorescent tubes) and subcultured every 3 weeks. All media were assessed twice with at least 20 replicates (for callus proliferation) or 10 replicates (for plant regeneration). Shoot buds, which had regenerated from the protoplast-derived calli, were detached and propagated using the same multiplication media as used for the original axenic shoot cultures (Table 1). Shoots (2.0 em in height) were rooted as a two-step sequence, root initiation being achieved by placing shoots, for 7 days, in half-strength MS medium with 3.0 mg/I IBA, followed by root elongation for 3 weeks in half-strength hormone-free MS medium. Rooted plants were finally transferred to pots which contained soil-less compost (Professional Levington M3, Fisons, U.K.) with each pot being enclosed in a polyethylene bag, after watering, under glasshouse conditions (23 ± 5°C; 16h light photoperiod, 10,000 lux from cool white fluorescent tubes). Bags were progressively opened over a 3 week period and by 4 weeks ex vitro transfer and acclimatization was considered to be complete. The protoplast-derived apple trees were thereafter maintained in the open glasshouse.
Results
Protoplast isolation Data relating to protoplast yield and viability is given in Table 2. Protoplasts isolated from callus/cell suspension cultures had a higher viability (Bramley's Seedling) than those
462
E. M. PATAT-OCHATT, S. J. OCHATI, and J. B. POWER
Table 2: Protoplast yield and viability for apple genotypes and tissue sources (mean±S.D. from 3 isolations with 3 replicates each). Apple Protoplast Source Yield Variety (xl0 6 /g f.wt.) M.9 leaves of axenic shoot cultures 2.27±0.55 MM. 106 leaves of axenic shoot cultures 1.45±0.60 leaves of axenic shoot cultures 7.75±2.70 Spartan Bramley's leaves of axenic shoot cultures 16.31±3.08 3.56± 1.01 Seedling { callus 23.23±3.77 cell suspension
Viability (%)
64±4 76±6 71±5 78±3 92±7 97±3
isolated from leaf tissues whilst for mesophyll protoplasts there was a clear effect of genotype on both yield and viability. Apple scion varieties gave larger yields of protoplasts than rootstocks (Fig. 1).
Protoplast culture and plant regeneration Rootstocks (M9, MMI06) The optimum protoplast cultural requirements for the two rootstocks differed as did their requirement for plant regeneration, as shown in Table 3. Media based on KM salts failed to sustain protoplast growth for both rootstocks, whereas MS-based media supplemented with any hormonal combination would support initial division of protoplasts (Fig. 1) but this could only be maintained using the growth regulators shown in Table3. Microcalli obtained after 60 days from protoplasts of both genotypes could be transferred and subcultured every 3 weeks in a semisolid (0.8 % agar) MS medium (Table 3) with a continuous illumination of 1000 lux. Twelve weeks from isolation protoplast-derived callus pieces (approx. 200 mg f.wt.) were transferred to the regeneration medium (Table 3) and by week 18 shoot buds were evident. As shown in Table 3 the rootstocks exhibited different media requirements for regeneration. For M.9, 40% of the protoplast-derived calli gave two or more buds (Fig. 1). During the first three weeks of culture on regeneration medium, calli that were originally white-greenish and friable became hard and browned. Following subculture smooth nodules appeared on the callus surface after 10 days resulting in shoot buds. Regeneration capacity was sustained over two successive subcultures and then lost. The
regenerated shoots could be micropropagated (see Table 1) if necessary and to date a sample of 100 plants have been rooted and established in soil (Fig. 1). For protoplast-derived calli of rootstock MM.106, 10 % of calli gave shoot buds (Table 3) but would not regenerate on the medium as used for M.9. Regeneration capacity was lost after one subculture. Moreover, the shoots were slow growing even on the medium used for the maintenance of the appropriate parental shoot cultures (Table 1). All attempts at rooting have, so far, been unsuccessful.
Scions (Spartan, Bramley's Seedling) Like the rootstocks, mesophyll protoplasts of Spartan and Bramley's Seedling exhibited a different nutritional requirement for culture (Table 4), not only between each other but also when compared to the rootstocks. Further differences were noted, for protoplasts of Bramley's Seedling, dependant on the tissue source (Table 4). For Spartan mesophyll protoplasts K8P medium supported growth but with a reduced plating efficiency when compared to Km8P medium. All MS-based media failed to sustain growth beyond first division. Spartan protoplast microcalli (1-2 mm diam.) were transferred (day 60) to the proliferation medium (for 6 weeks) and then to a regeneration medium (Table 4) which by the 21st week of culture had stimulated shoot bud formation, with as many as 60 % of calli giving an average of 8 buds. Regeneration capacity was retained for up to 6 subcultures and by the addition of 0.005 mg/l GA3 shoot-forming ability could be extended for a further three (3 weekly) subculture periods. Regeneration media as used for the rootstocks M.9 and MM.I06 were unsuccessful. Regenerated shoots of Spartan were readily propagated on the axenic shoot culture medium (Table 1) with up to 30 % of shoots ultimately being successfully rooted (Fig. 1). Leaf mesophyll protoplasts of Bramley's Seedling apple exhibited the best plating efficiency on K8P medium as shown in Table 4. All MS-based media were unsuccessful, since growth ceased after second division. Microcalli could be proliferated (Table 4) but to date have not regenerated. Conversely, protoplasts from Bramley's Seedling callus required an MS-based medium (Table 4) for growth and, in the dark, exhibited the highest LP.E. and F.P.E. of all the rootstock and scion protoplast systems. Cell suspension proto-
Table 3: Optimum conditions for culture and plant regeneration for apple mesophyll protoplasts (rootstocks). Protoplast Culture Apple LP.E. Medium Culture Method Plating F.P.E. Rootstock (mg/I) and Conditions Density (%) (%) (x lOs/ml) (day 15) (day 60) Genotype M.9 MS+organic agarose beads 5.0 10.5 0.8 mixture*; or layers; NAA (2.0); dark; 25°C BAP (0.25); 9% mannitol MM.I06 as above; liquid; 2.5 1904 1.1 Z (0.1) dark; 25°C * See Materials and Methods. Media pH 5.8 unless otherwise stated.
Callus Proliferation Plant Regeneration Medium Shoot Bud Regeneration Medium (mg/I) (mg/I) MS+NAA (2.0); BAP (0.5)
as above
MS+thiamine/HCI (0.2); pyridoxine-HCI (1.0); nicotinic acid (1.0); caseine hydrolysate (500); phloroglucinol (1.0 mM); NAA (0.01); BAP (2.0); pH 5.5 as above but thiamine-HCI (004); phloroglucinol (0.1 mM); NAA (0.05)
a
d
b
e
-
Fig. 1: Plant regeneration from apple protoplasts a. Isolated cell suspension protoplasts of Bramley's Seedling; b. Dividing callus protoplast-derived cells of Bramley's Seedling after 15 days of culture; c. Callus protoplast-derived cell colonies after 30 days of culture; d. Shoots emerging from protoplast-callus of M.9 apple after 20 weeks from isolation; e. Rooted plants (left - M.9; right - Spartan) at 28 weeks from isolation; f. Sample of plants in soil (left - M.9; right - Spartan) after 1 month from ex vitro transfer. For media composition see Tables 3 and 4. Scale bars represent 40/Lm (1 a), 125/Lm (1 b), 175/Lm (1 c), 1 em (1 d; 1 e), 5 em (1£).
464
E. M. PATAT-OCHATT, S. J. OCHATT, and J. B. POWER
Table 4: Optimum conditions for culture and plant regeneration for protoplasts of apple scion varieties. Apple Source Scion Genotype Spartan leaf
leaf
Medium (mg/I) Km8P
I.P.E.
F.P.E.
(%)
(%)
Callus Proliferation Plant Regeneration Medium Shoot Bud Regeneration Medium (mg/I) (mg/I)
(day 15) (day 60) 9.2 0.65 MS+2,4-D (0.1); BAP (0.1)
liquid; light; 25°C
1.0
7.5
0.09
MS + 2,4-D (0.1); BAP (0.1)
MS + NAA liquid; dark; (2.0); 25°C BAP (0.5) 9% mannitol cell Km8P liquid; dark; suspensIOn 25°C
1.0
31.1
3.2
MS + NAA (2.0); BAP (0.5)
1.0
16.9
0.2
MS + 2,4-D (0.1); BAP (0.1)
Bramley's callus Seedling
K8P
Protoplast Culture Culture Method Plating and Conditions Density (x10 5 /ml) agarose layers; 1.0 dark; 25°C
plasts grew best on Km8P medium (Table 4) with all MSbased permutations failing to support division. Protoplastderived calli (of callus or cell suspension origin) did not exhibit plant regeneration.
Discussion Large yields of viable protoplasts were obtained, for all the genotypes and source tissues with an enhancement of viability via the plasmolysis stage prior to enzyme incubation. This concurs with earlier findings for protoplasts of apple (Doughty and Power, 1988; Wallin and Welander, 1985) and other fruit and nut tree species (Ochatt et aI., 1987; Ochatt and Power, 1988 a, b; Revilla et aI., 1987). Media based on those of Kao and Michayluk (1975) supported the growth of Spartan mesophyll protoplasts and mesophyll and cell suspension protoplasts of Bramley's Seedling apple. This was in line with earlier observations for the scion variety Greensleeves (Doughty and Power, 1988), cell suspension protoplasts of the variety Jonathan (Kouider et aI., 1984) and callus protoplasts of the variety Orei (Niizeki et aI., 1983). However, maintenance of Spartan or Bramley's Seedling protoplast-derived cultures on these media, beyond the microcallus stage, always resulted in a lack of differentiation. Several reports on protoplasts of woody species, have shown that a reduction or elimination of ammonium ions from the culture medium has a beneficial effect on protoplast growth (Ochatt and Caso, 1986; Ochatt and Power, 1988 b; Oka and Ohyama, 1985). However, for the apple protoplast systems described here this only applied to callus protoplasts of Bramley's Seedling apple. The concentration of organic components in protoplast culture media has an important effect on the growth of protoplasts for apple (Doughty and Power, 1988; Kouider et aI., 1984; Niizeki et aI., 1983) and wild pear (Ochatt and Caso, 1986). But in this study this only applied to the rootstocks. Such a difference between scion and rootstock was also found for Pyrus species protoplasts (Ochatt and Caso, 1986; Ochatt and Power, 1988 b).
MS+thiamine-HCI (0.2); pyridoxine-HCI (1.0); nicotinic acid (1.0); caseine hydrolysate (50); NAA (0.5); BAP (1.5); Z (0.1); pH 5.5
Protoplast-to-protoplast relationships seem to be particularly relevant for apple, since a relatively high initial plating density was required for all the systems, in common with the situation for most woody fruit tree species (Doughty and Power, 1988; James et aI., 1984; Kouider et aI., 1984; Matsuta et al., 1986; Niizeki et al., 1983; Ochatt et aI., 1987; Vardi et al., 1982; Wu and Kuniyuki, 1986). Differences in protoplast cultural requirements were also found within genotypes particularly in relation to source. This too seems to be common for many rosaceous fruit tree species (Ochatt et aI., 1987; Ochatt and Power, 1988 a) where high auxin to low cytokinin ratios favoured growth in general, with NAA the best for rootstocks and 2,4-0 generally preferable for scions. As might be expected there were also differences in terms of media requisites for plant regeneration and in this respect, group B vitamins and caseine hydrolysate were always needed for successful regeneration. Furthermore, protoplastderived calli of both the rootstocks required phloroglucinol for shoot bud regeneration. Phloroglucinol has proven to be beneficial for shoot growth of M.9 (James and Thurbon, 1981) and apple micropropagation in general (Jones, 1976). However, an enhanced rooting effect of phloroglucinol on shoots (James and Thurbon, 1981; Jones, 1976) was not evident for regenerated shoots of rootstock MM.106. In apple, breeding programmes based on sexual hybridisation are, in general, hampered by a segregation of useful traits, following crossing. An alternative, for example somatic hybridisation between superior rootstocks and elite scion varieties, could avoid such problems. The establishment now of protoplast-to-tree systems clearly lays a foundation for the develpoment of such an approach to apple improvement.
Acknowledgements E.M.P-O was supported by a grant from the University of Nottingham/Allied-Lyons Research Fund and S.J.O. was in receipt of a fellowship from the Consejo Nacional de Investigaciones Cientificas y Tecnicas de la Republica Argentina. The authors wish to thank
Plant regeneration from apple protoplasts Mr. B. V. Case for photographic assistance and Dr. D. J. James, East Mailing Research Station, for the provision of rootstocks.
References DOUGHTY, S. andJ. B. POWER: Callus formation from leaf mesophyll protoplasts of Malus X domestica Borkh., cv. Greensleeves. Plant Cell Rep. 7, 200-202 (1988). HURWITZ, C. D. and G. N. AGRIos: Isolation and culture of protoplasts from apple callus and cell suspension cultures. J. Amer. Soc. Hort. Sci. 109, 348-350 (1984). JAMES, D. J. and 1. J. THURBON: Shoot and root initiation in vitro in the apple rootstock M.9 and the promotive effects of phloroglucinol. J. Hort. Sci. 56, 15-20 (1981). JAMES, D. J., A. J. PASSEY, and S. B. MALHOTRA: Isolation and fusion of protoplasts. Rep. E. Mailing Res. Stn. for 1983, pp. 63-65 (1984). JONES, O. P.: Effect of phloridzin and phloroglucinol on apple shoots. Nature 262, 392. Erratum, Id. 262, 724 (1976). KAo, K. N. and M. MICHAYLUK: Nutritional requirements for growth of Vicia hajastana cells and protoplasts at very low population density in liquid media. Planta 126, 105-110 (1975). KOUIDER, M., R. HAUPTMANN, J. M. WIDHOLM, R. M. SKIRVIN, and S. S. KORBAN: Callus formation from Malus X domestica cv. «Jonathan» protoplasts. Plant Cell Rep. 3, 142-145 (1984). MATSUTA, N., T. HIRABAYASHI, T. AKIHAMA, and 1. KOZAKI: Callus formation from protoplasts of peach cell suspension cultures. Sci. Hort. 28, 59-64 (1986). MURASHIGE, T. and F. SKOOG: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497 (1962). NAGATA, T. and I. TAKEBE: Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts. Plant a 92, 301- 308 (1970).
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NUZEKI, M., Y. HIDANO, and K. SAITO: Callus formation from isolated protoplasts of apple, Malus pumila Mill. Japan. J. Breed. 33, 369-374 (1983). OCHATT, S. J. and O. H. CASO: Shoot regeneration from leaf mesophyll protoplasts of wild pear (Pyrus communis var. pyraster L.). J. Plant Physiol. 122,243-249 (1986). OCHATT, S. J., E. C. COCKING, and J. B. POWER: Isolation, culture and plant regeneration of Colt cherry (Prunus avium x pseudoce. rasus) protoplasts. Plant Sci. 50, 139-143 (1987). OCHATT, S. J. and J. B. POWER: An alternative approach to plant regeneration from protoplasts of sour cherry (Prunus cerasus L.). Plant Sci. 56, 75 -79 (1988 a). - - Rhizogenesis in callus from Conference pear (Pyrus communis L.) protoplasts. Plant Cell Tissue Organ Culture 13, 159-164 (1988 b). OKA, S. and K. OHYAMA: Plant regeneration from leaf mesophyll protoplasts of Broussonetia kazinoki Sieb. (Paper mulberry). J. Plant Physiol. 119, 455-460 (1985). POWER, J. B., J. V. CHAPMAN, and D. WILSON: Laboratory Manual: Plant Tissue Culture. Plant Genetic Manipulation Group, Dept. of Botany, Univ. of Nottingham, U.K., 125pp. (1984). REVILLA, M. A., S. J. OCHATT, S. DOUGHTY, and J. B. POWER: A general strategy for the isolation of mesophyll protoplasts from deciduous fruit and nut tree species. Plant Sci. 50, 133-137 (1987). SHILLITO, R. D., J. PASZKOWSKI, and 1. POTRYKUS: Agarose plating and a bead-type culture technique enable and stimulate development of protoplast-derived colonies in a number of plant species. Plant Cell Rep. 2, 244-247 (1983). V AWl, A., P. SPIEGEL-Roy, and E. GALUN: Plant regeneration from Citrus protoplasts, variability in methodological requirements among cultivars and species. Theor. Appl. Genet. 62, 171-176 (1982). WALLIN, A. and M. WELANDER: Improved yield of apple leaf protoplasts from in vitro cultured shoots by using very young leaves and adding L-methionine to the shoot medium. Plant Cell Tissue Organ Culture 5, 69-72 (1985). Wu, S. C. and A. H. KUNIYUKI: Isolation and culture of almond protoplasts. Plant Sci. 41,55-60 (1985).
