J.PlantPhysiol. Vol. 139.pp.

155-160(1991}

Strategies for Plant Regeneration from Mesophyll Protoplasts of

the Recalcitrant Fruit and Farmwoodland Species
Prunus avium L. (sweet/wild cherry), Rosaceae
SERGIO

J. OCHATT

l.N.R.A., Station d'Amelioration des Especes Fruitieres et Ornementales, Route Georges Morel, Beaucouze 49000
Angers, France
Received February 4, 1991 . Accepted July 17, 1991

Summary
Large yields of viable protoplasts were isolated from mesophyll tissues of the sweet cherry (Prunus
avium L.) scion varieties Early Rivers and Van 2D and the rootstock genotype Pontavium 8574 V1813,
provided the antioxidant, PVP-10, was added to the enzyme mixture. Following culture, using modified
MS-based media (for Early Rivers and Pontavium) or K8P medium (for Van 2D), protoplasts entered division to give at first microcallus (for all genotypes), then callus (for Early Rivers and Pontavium), with
shoots being regenerated from the protoplast-derived calli of the rootstock genotype Pontavium. Addition of the antioxidants, glycine and/or PVP-10, to the culture media was a requisite for negation of
phenolic browning of protoplasts and protoplast-derived tissues at all culture stages. The regenerated
shoots of clone Pontavium were successfully rooted when co-cultured with shoots of another, easy-toroot cherry genotype. This is the first example of a protoplast-to-tree system for the recalcitrant species

Prunus avium L.

Key words: Prunus avium L., protoplast culture, plant regneration, antioxidants.
Abbreviations: BAP = 6-benzylaminopurine; CEH = casein enzymatic hydrolysate; CPW = see Power
et al. (1989); 2,4-D = 2,4-dichlorophenoxyacetic acid; FPE = final plating efficiency; f.wt = fresh weight;
GA3 = gibberellic acid; IBA = 4-indole-3yl-butyric acid; IPE = initial plating efficiency; K8P = see
Power et al. (1989); MES = 2-N-morpholinoethane sulphonic acid; MPE = intermediate plating efficiency; MS = Murashige and Skoog (1962); NAA = 1-naphthalene acetic acid; PVP-10 = polyvinylpirrolidone (Av. M.W. 10,000).

Introduction
Among Prunus species, callus has been produced from protoplasts of peach, P. persica (Matsuta et al., 1986), almond,
P. amygdalus (Wu and Kuniyuki, 1985) and P. lannesiana
(Matsuta et aI., 1984), but plants were recovered only from
the protoplasts of sour cherry (P. cerasus) (Ochatt and
Power, 1988 a; Ochatt, 1990 b) and the cherry rootstock
Colt, P. avium x pseudocerasus (Ochatt et al., 1987, 1988). In
contrast to this, the sweet/wild cherry, P. avium L., has
lagged well behind in this field, with protoplast studies being
1991 by Gustav Fischer Verlag, Stuttgart

limited in number and including assessments of antigenantibody reactions (Raff et al., 1980), a description of
conditions for the isolation of viable mesophyll protoplasts for the scion cultivar Early Rivers (Revilla et aI.,
1987) and, more recently, initial attempts at protoplast
culture Oorgensen and Binding, 1988; Ochatt et al.,
1990).
This article reports on the results obtained during protoplast culture studies with various scion and rootstock genotypes of the sweet cherry, Prunus avium L. The difficulties
encountered and strategies developed to resolve them are de-

156

SERGIO

J. OCHATI

tailed. In addition, for the first time, the recovery of protoplast-derived plants is reported.

Material and Methods

Plant Materials
The source of protoplasts were in vitro produced leaves taken
from the uppermost third of axenic shoots of the sweet cherry
(P. avium L.) scions Early Rivers and Van 2D, and the clonal
rootstock genotype Pontavium 8574 V1813 (hereafter denoted Pontavium). Shoots were propagated in a MS-based medium (Murashige
and Skoog, 1962), supplemented with 0.1 mglL IBA, 1.0 mglL BAP
and 0.1 mglL GA3 for Early Rivers, but with 0.05 mglL IBA,
1.5 mglL BAP and 0.3 mglL GA3 for Van 2D and Pontavium. For
all genotypes, shoots were subcultured monthly and kept at 25C
under a 16/8 h (Iightldark) photoperiod of 2000 lux from cool
white fluorescent tubes.

Protaplast isolation
For protoplast isolation, 1.0 g f.wt. of leaf tissue was plasmolysed
for at least 1 h in 10 mL of CPW salts medium (Power et al., 1989)
with 13 % (w/v) mannitol, pH 5.6 (CPWI3M medium). Following
plasmolysis, the abaxial epidermis of the leaves was peeled off with a
forceps and some were also finely chopped (1 mm width) and digested in 10 rnL of filter-sterilized (0.2/Lm pore size) enzyme solution (Table 1). Incubation (18-20h) was at 25C under a continuous illumination of 500 lux and on a rotary shaker (40 rpm).
The digested tissues were handled basically as described in Revilla
et al. (1987), with protoplasts being purified either by serial sieving
or by floatation on CPW21S medium (inorganic salts plus 21 % [wi
v] sucrose) or on a Percoll (Sigma, France) gradient (of 20, 25 and
30% Percoll) made up with CPW13M medium, followed by two
rinses with resuspension and centrifugation at 100 x g (for 8, 10 or
12 min for Van 2D, Early Rivers and Pontavium, respectively) to
wash away the Percoll. Purified protoplasts were finally resuspended in 5.0mL of CPW9M medium (inorganic salts plus 9%
[w/v] mannitol) for further determination of protoplast size, yield
and viability (Ochatt et aI., 1990).

Protoplast culture
Protoplast were diluted to 0.5, 0.75, 1.0, 2.5, 5.0 or 10.0 x 10!!I mL
with either MS or ammonium-free MS-based media, or with the Kao
and Michayluk-based (1975) media K8P or Km8P, as described in
Power et al. (1989). Both ammonium-free MS and MS-based media
were supplemented with 2,4-D, NAA, BAP and/or zeatin (0.0, 0.05,
0.1,0.5, 1.0 or 2.0 mglL), and included 9 % (w/v) mannitol as the osmoticum. The effects on protoplast growth of supplementing these
media with the complex mixture of organic compounds as previously used for wild pear mesophyll protoplasts (Ochatt and Caso,
1986) or with 50mglL CEH was also assessed. In these experiments
with sweet cherry, a species with large endogenous contents of
phenolic compounds (Feucht et al., 1986, 1988), a great tendency to
precocious browning of protoplasts and protoplast-derived cells
that hindered the maintenance of cell viability and sustained division was observed (see Results). Therefore, all protoplast culture
media tested were further supplemented (either from day 0 or by
day 15 of culture) with malic, citric or ascorbic acid (0, 100 or
150 mglL), activated charcoal (1.25 or 2.5 giL), PVP-I0 (0.5, 1.0 or
2.0% [w/v]) or L-glycine (0.1, 0.2 or 0.3M). For all media, the pH
was adjusted to 5.8.

The protoplast-medium suspensions were dispensed (as 5.0 mL

aliquots) into 50 mm Petri dishes as liquid or semisolid (0.625 %
[w/v] Seaplaque agarose) layers, or as semisolid l00~ droplets (10
per dish) submerged with 4.0 mL of the same, osmoticum-free liquid
medium (i.e. mannitol-free medium for MS and ammonium-free MS
media, and K8 and Km8 media for K8P and Km8P media, respectively). For cultures in liquid layers, the osmotic pressure of all
media was gradually reduced by adding osmoticum-free medium in
a 3: 1 ratio (protoplast: osmoticumfree medium) at intervals that
were optimized for each genotype (see Results), from 15 to 60 days
of culture. For protoplasts in semisolid layers, small agarose
blocks were progressively transferred to fresh medium with reduced osmoticum. Finally, protoplasts in the semisolid droplets
were surrounded by osmoticum-free medium from day 0 and were
thereafter left undisturbed. All cultures were kept at 25C, and
either in the dark, under the 16/8 h (light/dark) photoperiod as
used for the maintenance of the shoot cultures that provided protoplasts, or with a constant cool white fluorescent illumination of
200 lux.
Cultures were examined twice weekly up to day 15, and once a
week thereafter. Cell wall regeneration was determined with Calcofluor White (0.1 % [w/v] in culture medium), and growth responses
were assessed in terms of the initial (day 15, IPE), intermediate (day
30, MPE) and final (day 60, FPE) plating efficiency (Ochatt et al.,
1991). Each medium and culture condition tested included at least 3
replicated dishes per genotype and all experiments were repeated a
minimum of 3 times.

Callus growth and plant regeneration

Protoplast-derived microcalli (1-2 mm diam., day 75) were transferred to a range of agar-solidified (0.7% w/v) media, all based on
MS medium with 2.0 mglL NAA plus 0.1, 0.25 or 0.5 mglL BAP
and with or without the addition of antioxidants (see above) to alleviate browning. After 2 weeks, microcalli were transferred to and
subcultured (twice, every 3 weeks) on MS medium with 2.0mglL
NAA, 0.5mg/L BAP, 50mg/L CEH and antioxidants, for proliferation.
By day 130, plant regeneration from the protoplast-derived callus
(approx. 200 mg f.wt. [125 mm)] portions) was first attempted.
Thus, callus pieces were transferred (10 per dish with 10 mL medium) to a range of regeneration media based on MS medium supplemented with IBA or NAA (Om, 0.025, 0.05 or 0.1 mg/L), BAP
(1.0,1.5,3.0 or 5.0mglL) and/or zeatin (0.1, 0.25, 0.5 or 1.0mglL).
The effect on regeneration ability of CEH (50, 100 or 200 mglL), of
doubling the concentration of group B vitamins as included in the
original MS formulation (i.e. to 0.2 mglL thiamine-HCI, 1.0 mglL
pyridoxine-HCl and 1.0 mg/L nicotinic acid), and/or the addition
of antioxidants to the medium as used in the preceding culture
stages (see above) were also assessed. All cultures, for both the callus
proliferation and shoot bud regeneration stages, were kept at 25C
with a 16/8 h (light/dark) photoperiod of 2000 lux from cool white
fluorescent tubes. Each medium was replicated at least ten times and
the experiments were repeated at least three times.
Any shoot buds regenerated from the protoplast-derived callus
(day 160) were detached and transferred to the medium as used for
the original axenic shoot cultures that provided protoplasts. Following two (4-week) cycles of multiplication, individual shoots (> 2 em
tall) were separated and rooted in half-strength MS medium with
3.0 mglL IBA and 0.5 mglL NAA (1 week) plus 3 weeks in
hormone-free half-strength MS medium (Patat-Ochatt and Power,
1990), either on their own or when co-cultured with similar, axenic
shoots of the easy-to-root cherry rootstock Colt (Ranjit and Kester,
1988). The protoplast-derived plants of sweet cherry thus obtained
were then transferred to pots in a glasshouse and acclimated following standard procedures (Ochatt et al" 1990).

Plant regeneration from protoplasts of the Prunus avium L.

157

Table 1: Results for protoplast isolation with sweet cherry genotypes (mean data from 3 successive experiments each with 3 replicated dishes).
Enzyme
Mixture'

Genotype

Peeled leaves
0% PVP-10
1.0% PVP-10
YieldY
Viabz
Yield
Viab
19
1.46
55
1.14
76
25
0.55
0.20
36
84
1.35
1.55

Peeled + chopped leaves

0% PVP-10
1.0% PVP-10
Yield
Viab
Yield
Viab
1.88
2.36
E. Rivers
5
31
0.75
Van 2D.
13
0.97
44
2.05
2.50
Pontavium
45
68
6,45
27
3.24
73
5.25
17
E. Rivers
2.90
56
II
0.67
97
Van2D
40
0.85
33
1.08
71
0.50
1,45
42
0.96
81
1.20
Pontavium
0.76
9
47
, I - 1.0% Cellulase Onozuka R-10 + 1.0% Hemicellulase + 0.1 % Pectolyase Y-23; II = 1.0% Cellulase Onozuka R-10 + 0.1 %
Driselase+0.2 % Macerozyme R-10. Both enzyme mixtures were dissolved in CPW13M medium (Power et aI., 1989) with 5 mM MES, at
pH 5.6.
6
Y Yield is expressed as x 10 protoplasts/g f.wt. of digested tissue.
Z Viability is expressed as a percentage.

Results

Protoplast isolation and purification

The data for protoplast isolation for all three genotypes is
given in Table 1. The use of chopped leaves resulted in an almost doubled yield of protoplasts compared with peeled
leaves, but was coupled with a significant reduction of protoplast viability. The use of peeled leaves and the addition of
1.0% PVP-I0 to the enzyme mixture greatly enhanced the
viability of protoplasts. On the average, protoplasts had a
diameter of 15/Lm and preparations were very difficult to rid
of debris. Floatation of CPW21S medium had to be adopted
as the purification method, as serial sieving resulted in preparations that were highly contaminated with debris and undigested cells; Percoll gradients could not be used due to protoplast fragility, which impeded additional centrifugation to
wash out Percoll.

The effects ofantioxidants on protoplast proliferation

For all genotypes, extensive browning of isolated mesophyll protoplasts and protoplast-derived cells was a problem
in all media tested. Protoplasts regenerated a cell wall
(10-20% depending on the hormonal composition of the
medium) after 7 days, but only small cell colonies (4-6 cells
each) were obtained by day 20. These quickly browned and
subsequently died. Protoplast cell viability assessments
revealed that, after 10 days, viability was reduced by 90 %.
Therefore, antioxidants were added to negate phenolic
browning.
Citric, malic and ascorbic acids all failed to control
browning of cells, irrespective of the concentration used. Activated charcoal negated browning, but cell wall regeneration was never observed when this was added at day 0, while,
if added at day 15, the growth of protoplast-derived cell
colonies was arrested. Since a similar result was obtained in
media with low hormone contents, it is likely that activated
charcoal may have adsorbed the growth regulators in the optimum medium for each genotype so that their concentration fell below the minimum critical level needed to support
mitotic cycles.

Table 2: Effects of the addition of glycine or PVP-10 to the optimum

culture medium on percentage FPE (day 60) for mesophyll protoplasts of sweet cherry cv. Early Rivers. Data are the mean S.D.
from 3 successive experiments each with 3 replicated dishes. FPE for
control protoplasts (medium without antioxidants)-O.OO%.
Antioxidant Concentration % Plating efficiency (FPE) when added at
Day 0
Day 15
PVP-10

0.5 %
1.0%
2.0%

0.SHO.17
0.SHO.1S
0.470.14

0.090.03
0.160.07
0.070.02

Glycine

O.lM

2.020.lS
1.6S0.10
1.170.20

0.910.11

0.2M
0.3M

1.260.lS

0.390.10

For Early Rivers, L-glycine or PVP-I0 reduced browning

without any associated negative effects on subsequent
growth and proliferation of the protoplast-clerived cell
colonies. High plating efficiencies were maintained when
either of these two antioxidants were added at day 0, where
0.1 M glycine consistently gave the best results, in terms of
FPE (at day 60), when microcalli had reached the 100-cell
stage (Table 2).
For Van 2D, too, growth was arrested at the lO-cell colony
stage due to extensive cell browning. Addition to the protoplast culture medium of 0.1 M glycine, as successfully used
for Early Rivers, failed to negate browning, as did all the
other antioxidants tested. Only culturing protoplasts as
semisolid droplets in the dark during the first 15 days permitted the sustained division of cells. At day 15, the liquid
medium surrounding the droplets was replaced by fresh, osmoticum-free medium of the same composition, but with
0.2M glycine and 0.5% PVP-I0, and dishes were moved
under the photoperiodic light regime.
Finally, for the cultured protoplasts and protoplast-derived cells of clone Pontavium, several approaches were similarly successful in negating the adverse effects of browning
on growth, e.g. either by adding PVP-I0 (1.0%) or glycine
(0.1 M) to the protoplast culture medium, from day O. For
consistency with studies for the other genotypes, however,
0.1 M glycine was routinely used with protoplast cultures of
Pontavium.

158

SERGIO

J. OCHATT

Table 3: Optimum protoplast culture responses for each genotype.

Genotypes

Culture Parameters
and Responses

Early Rivers

Van2D

Pontavium

Protoplast culture
medium

MS + complex organic mixture plus

(mg/L) NAA(2.0), 2(0.1), 9 % mannitol, 5 mM MES, 0.1 M glycine

KSP plus antioxidants (see Text)

MS plus (mg/L)2,4-D(0.5),
NAA(1.0), BAP(1.0), 2(0.1),
CEH(50), 9% mannitol, O.lM
glycine

Plating conditions

5 x lOs I mL, as shaken liquid layers,

under continuous 500 lux

1 X 105I mL, as droplets, in the dark

0.75 x lOS I mL, as liquid layers,

2000 lux photoperiod

%IPE(day 15)
%MPE (day 30)
%FPE(day60)

19.6
S.9
2.0

1.1

29.2
19.9

<0.1

1.1

Callusing medium

MS+2.0mg/L NAA, 0.1 mg/L

BAP, 0.1 M L-glycine

MS+2.0mg/L NAA, 0.1-0.5mgl

L BAP plus antioxidants

MS+2.0 mg/L NAA, 0.25 mg/L

BAP, 1.0% PVP-l0

% Microcallus survival
Medium for shoot bud
regeneration

25

15
MS plus (mg/L) IBA(O.l),
BAP(1.0), 2(0.25), CEH(200)

9.S

% Regenerating calli
Nb. shootsl callus

12
5

* shoot bud regeneration not obtained.

Protoplast culture and plant regeneration from protoplastderived callus
The media and conditions supporting optimum responses
with Early Rivers protoplasts are detailed in Table 3. The
osmotic pressure of the medium was reduced every 2 weeks,
beginning at day 15. Noteworthy, replacing the organic mixture of Ochatt and Caso (1986) by the vitamins of MS medium (at a single or double strength) had little effect on responses of Early Rivers protoplasts. Protoplast-derived
microcalli (1- 2 mm diam, day 60) were transferred for further proliferation, but browning of tissues still being apparent at this stage, glycine (0.1 M) had to be added to the
medium in order to maintain such calli in culture. Plant
regeneration from protoplast-derived tissues of Early Rivers
was not established.
Optimum responses for Van 2D protoplasts were on K8P
medium (Table 3), where microcalli of about 100 cells each
were occasionally obtained when the osmotic pressure of the
medium was reduced once weekly from day 15. To date,
however, all attempts at maintaining these microcalli and
subsequently assessing their regeneration competence have
been unsuccessful due to persistent cell browning and
ultimate callus death.
Optimum culture responses for Pontavium protoplasts are
also described in Table 3. The osmotic pressure of the medium was reduced every week, beginning at day 15 (after
protoplasts had divided once), whereby lO-cell colonies proliferated by day 30 when the first significant drop in cell
viability occurred due to phenolic browning. By day 60,
1-2 mm in diameter protoplast-derived microcalli were recovered and were transferred for further proliferation. At
this stage, PVP-10 (1.0%) was added to the medium as a preventive measure to reduce browning. Nevertheless, many of
the protoplast-derived microcalli obtained failed to sustain
growth (Table 3). Addition of alternative antioxidants (e.g.

glycine, activated charcoal) to the medium was equally ineffective to resolve the problem, and an average of only 670
fast-growing calli/mL of initially cultured protoplasts were
recovered 3 weeks later (equivalent to an effective P.E. of
about 0.2 %).
Following two successive (3-week) passages on the callusing medium, 130-day-old callus portions were transferred for
assessments of their organogenic competence. Under optimum conditions (Table 3), a total of 51 calli (out of 449 calli
transferred) became caulogenic. The zeatin content in the
regeneration medium played a key role in organogenesis. A
similar medium, but with 0.1 mg/L zeatin, only supported
shoot bud regeneration on 9 out of 438 callus portions
tested, whereas in a zeatin-free medium only one out of the
454 calli transferred regenerated a single shoot bud, which
quickly browned and died. The caulogenic ability of such
calli was lost after the first subculture on the same regeneration medium, but could be prolonged for up to five successive subcultures by doubling the concentration of group B
vitamins of the regeneration medium.
The regenerated shoot buds of sweet cherry clone Pontavium were detached from the callus and were transferred to
MS medium with 0.05 mg/L IBA, 1.5 mg/L BAP and
0.3 mg/L GA 3, as used for the original shoot cultures that
provided protoplasts, but further supplemented with 1.0 %
PVP-I0 to prevent browning. About two thirds of the protoplast-derived shoots obtained were capable of survival and
multiplication under these conditions and were cloned separately for the subsequent assessment of their rootability.
Then, only those shoot buds produced during the first culture passage and on the optimum regeneration medium (i.e.
the one containing 0.25 mg/L zeatin) could be rooted. In
addition, the protoplast-derived shoots of clone Pontavium
were only rarely rooted if transferred to the sequence of
rooting media on their own. Conversely, when they were
co-cultured during rooting with the cherry rootstock Colt,

Plant regeneration from protoplasts of the Prunus avium L.

Table 4: Rooting responses of protoplast-derived Pontavium shoots.
% rooting % ex vitro survival
Rooting conditions
Protoplast-derived Pontavium shoots
15
20
on their own
Protoplast-derived Pontavium shoots
co-cultured with Colt cherry shoots
45
75
Shoots from rooted protoplast-derived
48
80
Pontavium plants on their own

an easy to root genotype (Feucht and Dausend, 1976), the

percentage of rooted shoots was improved significantly
(Table 4). Interestingly, shoots from axenic cultures that
were reinitiated taking apices of the rooted protoplast-derived plants of Pontavium thus produced were rooted on
their own and at a rate similar to those co-cultured with Colt
cherry shoots as above. The rooted plants obtained by either
of these strategies were generally enfeebled and difficult to
acclimate due to dehydration during the first days ex vitro.
Discussion
For Prunus avtum L., a species rich in endogenous
phenolic compounds (Feucht et aI., 1986, 1988), the
browning of protoplasts and protoplast-derived cells has so
far hampered the use of protoplast technology for breeding.
In this article, a compromise between a successful negation
of browning and an acceptable growth of cultures was dependent both on the genotype and on the type and timing of
addition of the antioxidants to the medium. Generally, optimum responses were obtained when the antioxidants were
added from day 0 of culture, with glycine as the most effective antioxidant and PVP-10 as the second best. In this respect, glycine had previously been used to enhance the
viability of protoplasts for herbaceous species (Orczyk and
Malepszy, 1985), but never before with woody plant genotypes. On the other hand, protoplast cultures of the
scion genotypes Early Rivers and Van 2D appeared to be
more prone to browning than those of the clonal rootstock
Pontavium. Similar differences in this respect were observed
between scion and rootstock genotypes in the genus Pyrus
(Ochatt and Caso, 1986; Ochatt and Power, 1988b; Ochatt,
1990 a) but have not been verified with apple (Patat-Ochatt
et al., 1988).
In a recent report with sour cherry protoplasts, Ochatt
(1990 b) suggested that, compared with other rosaceous
woody genotypes, Prunus species might be generally less responsive to the addition of organic compounds, while the
hormonal balance of the medium might be the key factor for
the induction of sustained growth and differentiation from
their protoplasts. For the sweet cherry genotypes studied
here, also, only minor differences in responses were observed (in terms of IPE, MPE and FPE) by changing the organic
composition of the optimum medium for each genotype
(data not shown), while no proliferation at all was detected
when it was the hormonal compotision that was modified.
The same applied, at the regeneration stage, to the protoplast-derived calli of the clone Pontavium. It is noteworthy,
in this respect, that protoplasts of clone Van 2D proliferated

159

on K8P, as media based on Kao and Michayluk's medium

(1975) have been successfully employed for culturing apple
protoplasts (Patat-Ochatt et aI., 1988, and references therein)
and Pyrus (+) Prunus heterokaryons (Ochatt et al., 1989), but
generally failed to support growth of the Prunus protoplast
systems studied to date (Ochatt et aI., 1991).
The requirements for regeneration proved to be highly
genotype-dependent, with the calli of clone Pontavium
exhibiting a marked dependence on the addition of zeatin to
the regeneration medium whereas those of Early Rivers
failed to regenerate whatever the hormonal composition of
the medium employed. Zeatin, in conjunction with BAP and
an auxin, has previously been used for the successful regeneration of protoplast-derived shoot buds for several Prunus
genotypes (Ochatt et al., 1987; Ochatt and Power, 1988 a;
Ochatt, 1990 b). The loss of regeneration capacity by protoplast-derived callus has been reported for cultures of several
fruit tree genotypes before (Ochatt and Power, 1988 b; PatatOchatt et al., 1988) and, for protoplast-derived cultures of
the apple rootstock MM. 106, was resolved by doubling the
concentration of the group B vitamins of the regeneration
medium (Patat-Ochatt and Power, 1990), the same as in these
studies.
Protoplast-derived shoots of fruit trees have generally
proved to be weaker and more difficult to root than shoots
obtained by other in vitro techniques (Ochatt et aI., 1991).
This was also true for the shoots of sweet cherry clone Pontavium produced in this study, and was probably worsened
by the natural difficulty for the rooting of P. avium shoots,
irrespectively of their origin (Feucht and Dausend, 1976). In
this respect, co-culturing of such shoots with those of a more
amenable genotype, Colt cherry, as described by Ranjit and
Kester (1988), significantly enhanced rooting of the protoplast-derived shoots produced. The improved rooting responses, as observed for shoots from axenic cultures initiated
from apices of protoplast-derived rooted plants of clone Pontavium, is difficult to explain. In this context, it is possible
that diffusible compounds (rooting cofactors?), diffused to
the medium by the rooting shoots of Colt cherry as
suggested by Ranjit and Kester (1988), were in turn translocated and stored in the Pontavium shoots, where they
were still present when portions of these were taken to reinitiate axenic cultures.
These results with Prunus avium L., an important fruit and
farm woodland species that to date did not lend itself to biotechnological breeding approaches based on protoplasts,
now lay a foundation for using such techniques with a view
of producing protoclonal variants, for direct gene transfer
experiments, and also by including the sweet cherry as a
partner in somatic hybridization studies. Equally important
though is the fact that some of the strategies as applied here
to sweet cherry could also serve for the development of protoplast-to-plant systems for hitherto recalcitrant genotypes,
particularly those where phenolic browning of protoplasts
and protoplast-derived tissues is the barrier to success.
Acknowledgements
The author acknowledges Dr. J. Boccon-Gibod for access to the
laboratory facilities at the E.N.I.T.H.P. (Angers, France), and the

160

SERGIO J. OCHATr

G.I.E. Delbard Nurseries (Malicorne, France) for financial support.

The original axenic shoot cultures of sweet cherry clones Van 2D
and Pontavium 8574 V1813 were kindly provided by Dr. F. Dosba,
from the Station d' Arboriculture Fruitiere, INRA, at Bordeaux
(France).

References
FEUCHT, W. and B. DAUSEND: Root induction in vitro of easy-toroot Prunus pseudocerasus and difficult-to-root Prunus avium. Sci.
Hort. 4, 49-54 (1976).
FEUCHT, W., P. P. S. SCHMID, and E. CHRIST: Distribution of lavanols in meristematic and mature tissues of Prunus avium
shoots. J. Plant Physiol. 125, 1-8 (1986).
FEUCHT, W., D. TREUTTER, and P. SCHMID: Inhibition of growth and
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