Vorgabe neu

Journal of Applied Botany and Food Quality 81, 1 - 6 (2007)

Palacký University in Olomouc, Faculty of Science, Department of Botany, Olomouc, Czech Republic

Factors affecting protoplast isolation and cultivation of Cucumis spp.
Gajdová, J., Navrátilová, B., Smolná, J., Lebeda, A.*

(Received October 19, 2006)

Summary

Protoplasts of Cucumis anguria, Cucumis melo (3 accessions),
Cucumis metuliferus and Cucumis sativus were isolated from leaves,
growing apices, hypocotyls and calluses. Plants were cultured on
2 concentrations of sucrose. The effect of plant culture medium, explant
age and explant type on protoplast viability were investigated. The
protoplasts were cultured in 3 types of culture medium and at two
temperatures. Optimal age range for protoplast isolation was 1-5 weeks
depending on explant type and genotype. Viabilities ranging between
50 % and 80 % were obtained from all explants and genotypes.
Increased concentration of sucrose had negative impact on viability
of protoplasts. The highest level of regeneration achieved was callus,
regenerated from leaf protoplasts of C. melo cv. ‘Charentais’ and C.
melo ‘MR-1’. The lowest regeneration capability was observed in
hypocotyls. Liquid LCM medium (B5 macro and microelements
(1 g · l-1 CaCl2), B5 vitamins with 1g · l

-1 myo-inositol, 2 mg · l-1 ascorbic
acid, 0.8 mg · l-1 glycine, 20 mg · l-1 glutamine, 100 mg · l-1 casein hydro-
lysate, 70 g · l-1 mannitol, 10 g · l-1 sucrose, 5 g · l-1 glucose, 585 mg · l-1

MES, 5.37 µmol · l-1 NAA, 2.26 µmol · l-1 2,4-D, 2.22 µmol · l-1 BA)
was optimal for protoplast regeneration. Agarose-solidified medium
caused a decrease in the number of cell divisions (used in C. melo ‘PI
124112’). Cultivation at 25ºC resulted in a higher frequency of cell
divisions (tested in C. metuliferus).

Abbreviations

2,4-D – 2,4-dichlorphenoxyacetic acid; BA – benzylaminopurine;
IBA – indole-3-butyric acid; IPA – indolylpropionic acid; MES – 2-
N-morfolin-etansulfonic acid; NAA – α-naphthaleneacetic acid

Introduction

Cucurbit downy mildew (Pseudoperonospora cubensis) and cucurbit
powdery mildews (Podosphaera xanthii, Golovinomyces cichora-
cearum) are responsible for significant loss of cucurbit crops
worldwide annually (JAHN et al., 2002; LEBEDA and WIDRLECHNER,
2003). There are different methods used to control outbreaks of such
diseases but these are all chemical based treatments which are not
only sometimes inefficient but also have significant economic and
environmental costs (HOLLOMON and WHEELER, 2002; LEBEDA and
WIDRLECHNER, 2003; MCGRATH, 2001; URBAN and LEBEDA, 2006).
Classical methods of cucumber breeding like intraspecific and
interspecific hybridisation have not been reported to confer viable
resistance (CHEN and ADELBERG, 2000; LEBEDA et al., 2007). Recent
studies have shown that by using protoplast fusion hybrid plants may
be obtained, however these did not produce viable seeds (GAJDOVÁ
et al., 2004). In order to address the problems with somatic hybridi-
sation we continued on work with previously reported accessions of
wild and cultivated species of Cucumis (Gajdová et al., 2007).
Various protocols were developed and some successfully used in
protoplast cultures of Cucurbitaceae. Many of them led to regeneration
of normal plants able to form roots in soil and produce fruits (GAJDOVÁ

et al., 2004). Despite this progress, fusion experiments are still
problematic, because even when regeneration of hybrid plants was
achieved, the genes of one of the parents may gradually become
silenced (YAMAGUCHI and SHIGA, 1993).
Recently several studies have reported on factors influencing proto-
plast yield, viability and regeneration capability, such as genotype,
explant type, explant pre-treatment, presence of glycine in the enzyme
solution, and culture medium composition (e.g. growth regulator
concentration) (FELLNER and LEBEDA, 1998; SUTIOJONO et al., 1998,
2002). MCCARTHY et al. (2001b) studied the effect of light on proto-
plast division, alongside temperature and agarose effects. They also
demonstrated the influence protoplast density has on division rates
and microcallus production (MCCARTHY et al., 2001b). Despite
improving the culture technique and increasing of the culture efficiency
of protoplasts, the regeneration of plants was not always achieved
(GAJDOVÁ et al., 2004; MCCARTHY et al., 2001a, b; SUTIOJONO et al.,
1998, 2002).
To improve the protocol for protoplast isolation and cultivation it is
first necessary to increase the protoplast viability (the percentage of
protoplasts surviving the isolation and purification procedure). Second
is to increase the regeneration efficiency (the percentage of protoplasts
regenerating into dividing cells and subsequently into calluses or
plants).
The aim of this investigation was: a) to contribute towards a better
understanding of regeneration from protoplasts which could then be
used in subsequent somatic hybridisation; b) to find out optimal plant
or callus age, plant culture media, donor plant organs, temperature
and composition of culture media for cultivation and regeneration of
plants.

Material and methods
Plant material and explants

Cucumis spp. genotypes that in previous experiments indicated good
regenerative capabilities (calluses obtained) (GAJDOVÁ et al., 2007)
were used for this study. The following four accessions were selected
for good regenerative capabilities: Cucumis meloa ‘600’ (MR-1, CZ
09-H40-0600), Cucumis meloa ‘16’ (cv. Charentais, CZ 09-H40-1116),
Cucumis metuliferusa (CZ 09-H41-0587), Cucumis sativusa line SM
6514 (CZ 09-H39-0768). Both Cucumis anguria var. anguriab (Ames
23536) and Cucumis melob ‘12’ (PI 124112) were selected because
they have increased yield and viability of protoplasts. The plant
material originated from the vegetable germplasm collection of the
Research Institute of Crop Production (Prague) a, Department of Gene
Bank (Olomouc, Czech Republic), and Regional Plant Introduction
Stationb (Ames, Iowa, USA).
Seeds were surface sterilised with 8 % Chloramin B for 30 minutes
and rinsed 3 times with sterile distilled water. The seat coat was excised
and they were germinated on half strength MS (MURASHIGE and
SKOOG, 1962) medium in Petri dishes (60 mm) in the dark at 25°C.
After germination, hypocotyls were detached and seedlings planted
on OK medium (MS medium supplemented with 20 g · l-1 sucrose,

20 mg · l-1 ascorbic acid, 0.8 % agar, 0.049 µmol · 1-1 IBA and
0.044 µmol · 1-1 BA) in plastic boxes (volume 800 ml) or Erlenmeyer’s
flasks (100 ml). Plants were cultivated in culture room with a 16 hour
day (light intensity 32 - 36 µmol · m-2 · s-1) and the temperature at
22 ± 2°C. They were subcultured every 3 weeks. Hypocotyls were
used either for protoplast isolation or for callus derivation. Leaves
and growing apices served as sources of protoplasts. Calluses were
derived from hypocotyls of in vitro plants and in the case of C. melo
‘12’ and C. sativus from leaves. Leaves for callus derivation were cut
into quarters, hypocotyls were cut in to several pieces and then halved
and placed on MSC medium (MS with 30 g · l-1 sucrose, 13.43 µmol · l-1

NAA, 4.4 µmol · l-1 BA and 0.8 % agar) to induce callus growth and
growing calluses were subcultured every 2 weeks. Calluses were
cultivated in the dark at 25°C.

Protoplasts isolation and culture

Protoplast isolation enzyme solutions for leaves, growing apices and
hypocotyls contained 1% (w/v) Cellulase Onozuka R-10 (Duchefa)
and 0.25 % (w/v) Macerozyme R-10 (Duchefa) dissolved in PGly
washing solution (DEBEAUJON and BRANCHARD, 1992; composition:
27.2 mg · l -1 KH2PO4, 101 mg · l

-1 KNO3, 1117.6 mg · l
-1 CaCl2,

246 mg · l-1 MgSO4 · 7H2O, 0.16 mg · l
-1 KI, 0.025 mg · l-1 CuSO4 · 5 H2O,

11.5 g · l-1 glycine, 18.016 g · l-1 glucose, 0.58572 g · l-1 MES and
65.58 g · l-1 mannitol). Enzyme solution containing 2% Cellulase
Onozuka R-10, 1% Macerozyme R-10 and 0.3 % Driselase (Fluka)
was used for callus protoplast isolation. Hypocotyls, growing apices
and the youngest fully developed leaves were cut into fine strips or
pieces and incubated in the enzyme solution for 16-17 hours in the
dark at 25°C (approximately 2 ml of enzyme solution for 100-
200 mg of tissue). Approximately 5 ml enzyme solution was used for
1 g of callus. Calluses were cut into pieces and placed on a shaker
(80 rpm) for 30 minutes, then in an incubator (25°C, dark) for
17 hours and finally on the shaker for 30 minutes again. The proto-
plast suspension was filtered through nylon mesh (72 µm), mixed with
PGly solution (approximately 5 ml) and centrifuged at 100 × g for
5 minutes. After pouring off the supernatant the pellet was resuspended
in 20 % (w/v) sucrose (4 ml) and overlaid with the PGly washing
solution (2 ml) ensuring they did not mix. They were centrifuged at
100 × g for 10 minutes, protoplasts were isolated from the layer
between sucrose and PGly using Pasteur pipette. Protoplasts were
mixed with approximately 3 ml of PGly solution and centrifuged at
100 × g for 5 minutes.
Viability of protoplasts was established after purification using an
Olympus fluorescent microscope ‘BX60’ and fluorescein diacetate
stain (LARKIN, 1976) and a BW filter. Results were calculated by using
the percentage of the protoplasts which were living; ten readings
were made for each sample. Protoplasts were cultured in modified
liquid LCM medium (DEBEAUJON and BRANCHARD, 1992) containing:
B5 macro and microelements (1 g · l-1 CaCl2), B5 vitamins with 1g · l

-1

myo-inositol, 2 mg · l-1 ascorbic acid, 0.8 mg · l-1 glycine, 20 mg · l-1

glutamine, 100 mg · l-1 casein hydrolysate, 70 g · l-1 (0.38 mol · l-1)
mannitol, 10 g · l-1 sucrose, 5 g · l-1 glucose, 585 mg · l-1 MES, 5.37
µmol · l-1 NAA, 2.26 µmol · l-1 2,4-D, 2.22 µmol · l-1 BA, in Petri dishes
(diameter 40mm), approximately 1.5 ml of medium per dish, at density
105 protoplasts · ml-1. Protoplast cultures were placed in the dark
at 25°C for 14 days and then transferred into a culture room with
16/8 hours day/night cycles with light intensity 32-36 µmol · m-2 · s-1

at 22 ± 2°C. At this time LCM2 medium containing 3.3 µmol · l-1 BA
and no mannitol (DEBEAUJON and BRANCHARD, 1992) was added to
dishes (approximately 1 ml) and protoplasts from each dish were
subdivided between two dishes (using a pipette). Growing micro-
calluses (after 4 weeks of culture) were transferred onto a solid
F medium (MS macro and microelements, B5 vitamins, 10g · l-1

sucrose, 0.537 µmol · l-1 NAA, 2.2 µmol · l-1 BA, 0.8% agar) (PELLETIER

et al., 1983) and the resultant calluses were subcultured after 2-3 weeks
onto a fresh medium.

Effect of plant culture media on viability of isolated protoplasts

Plants were cultured on OK medium with 30 g · l-1 of sucrose. Leaves
and growing apices were detached between 15- 63 days. Protoplast
isolation was carried out according to the protocol in the previous
section. Protoplasts were cultured in liquid LCM medium. The viability
of isolated protoplasts was compared to those derived from plants
grown in standard OK medium.

Optimal age of plants, seedlings and calluses
for protoplast isolation

Leaves were detached from plants between 8 -107 days old, growing
apices from 7- 56 days old, hypocotyls from seedlings 4 -17 days after
sowing and calluses were used for protoplast isolation 14 - 96 days
after starting their cultivation. The viabilities of isolated protoplasts
were used as criteria to find the optimal age of in vitro materials for
protoplast isolation. The yield of protoplasts was usually sufficient if
the viability was high.

Regenerative capability of protoplasts isolated
from different types of explants

The level of regeneration from protoplasts was investigated for all
tissue types. Levels of regeneration were categorised as the following:
cell division (< 0.2 mm), microcallus (colonies of cells 0.2- 2 mm which
were visible by the eye) and callus (> 2 mm in diameter). Results were
recorded by the percentage of samples showing each level (category)
of regeneration.

Protoplast culture technique

Cucumis melo ‘12’ leaf protoplasts were cultivated both in filter
sterilised liquid medium and in solidified (0.6% agarose) LCM
medium. The number of divisions observed after 14 days represented
the regeneration efficiency when expressed as a percentage of total
sample number. In Cucumis anguria modified CML medium (without
edamin) (COLIJN-HOOYMANS et al., 1988) was compared to LCM.
CML medium contained MS macro and micro elements, 25 µmol · l-1

NAA and 15 µmol · l-1 IPA, 2 g · l-1 sucrose, 0.25 mol · l-1 mannitol.
Cucumis metuliferus leaf protoplasts were used to compare effect of
culture temperature during first 14 days of cultivation. Temperatures
of 25°C and 27°C were compared. Due to constraints of time it was
not possible to carry replicates of each variable with each species
within this study, future work will focus on the most responsive
candidates.

Results and discussion

Effect of plant culture media on viability of isolated protoplasts

It is evident that using of the culture medium with a higher concen-
tration of sucrose (30 g · l-1) resulted in decreased protoplast viability
(Fig. 1). Protoplasts were obtained in normal yields, and being normal
in size and shape. They were however, not able to survive digestion
and purification procedure. In C. melo ‘16’, C. metuliferus and C.
sativus a big decrease in viability was recorded, particularly in leaf
protoplasts. However, in C. melo ‘600’ an increase of viability was
seen in protoplasts from growing apices. This suggests that concen-
tration of sugar, namely sucrose in culture medium can have strong
effect on subsequent isolation of protoplasts from Cucumis tissue
dependent on genotype.

2 Gajdová, J., Navrátilová, B., Smolná, J., Lebeda, A.

There are reports about positive effect of a high concentration of
sucrose (85.6 g · l-1) on somatic embryogenesis in cotyledon tissue (LOU
and KAKO, 1995) but no studies on sucrose concentration in donor
plant culture medium have been reported. MORENO et al. (1984) have
reported increasing protoplast divisions when donor plants were
cultivated on medium with yeast extract, but this medium also caused
strong decrease of protoplast yield. This suggests that composition of
culture medium can greatly influence protoplast physiological
properties.

Optimal age of plants, seedlings and calluses
for protoplast isolation

The optimal age for protoplast isolation varies both among genotypes
and explants (Tab. 1). For leaves, the highest viabilities were obtained
from 3-5 weeks old plants (2-3 weeks after subculture), but in some
genotypes the suitable age was up to 10 weeks. Plants of C. anguria
and C. metuliferus grew rapidly and exhibited increased vitality in
vitro for prolonged intervals (up to several months) but the optimal
age for C. metuliferus was only 3-5 weeks. Flowers of the C. sativus

started budding after 4 weeks and plant growth decreased rapidly
thereafter. Plants of C. melo grew slowly from the beginning with
poor rooting and leaves were rather tough in texture. Despite this,
these plants provided good results. COLIJN-HOOYMANS et al. (1988)
and DEBEAUJON and BRANCHARD (1992) reported plant regeneration
from leaf protoplasts isolated from 3 week old plants of C. melo and
C. sativus. Growing apices generated good protoplast viability from
1-8 weeks, depending on genotype. Soft and fine grained calluses
only were suitable for protoplast isolation, optimally up to 5 weeks
for most genotypes, 7-14 days after last subculture. BURZA and
MALEPSZY (1995) reported plant regeneration from callus protoplasts
used 7-10 days after subculture, and after 4-8 subcultures. Hypocotyls
need to be prepared within 8 days after sowing seeds. This is in line
with published data from C. melo ‘Green Delica’ and ‘Fastoso’
(SUTIOJONO et al., 2002) where it was reported that hypocotyls older
than 8 days had lignified cell walls which prevented successful iso-
lation of protoplasts.

Effect of donor explant variation on protoplast viability

Fig. 2 shows viabilities of protoplasts which were isolated within their
optimal explant age ranges for respective genotypes. Differences
among species and varieties and especially among explant types within
one genotype are visible in the graph. In C. melo ‘12’ and C. melo
‘600’ leaf protoplasts were the most viable. All explant types of
C. melo ‘16’ were suitable for protoplast isolation, in C. metuliferus
growing apex and callus gave the best results using our isolation
protocol. In C. sativus callus protoplasts were the most viable. Different
species have different requirements for cultivation and isolation, for
example in C. melo ‘600’ better results were achieved for growing
apices by growing plants on OK medium with higher concentration
of sucrose (Fig. 1). To also increase the protoplasts viability in other
explant types it is necessary to optimise the culture protocols and
subsequent isolation techniques for each individual genotype. For
example hypocotyls of C. metuliferus were much softer than in other
species so they may need a lower concentration of digesting enzymes
or cutting into bigger pieces then other studied species. The isolation
technique has been developed and optimised for C. melo which is
why its protoplast viabilities are increased over that of the other species.
The yield also differed among explant types being approximately ten
times higher in leaves and growing apices than in calluses. Hypocotyls
gave lower yields than leaves and growing apices (unpublished data).
Other authors have reported differences among Cucumis species and
tissue types as well (GAJDOVÁ et al., 2007; MCCARTHY et al., 2001b;
SUTIOJONO et al., 2002).

Fig. 1: Effect of sucrose concentration on protoplast viability.
Data points comprised of a minimum of 16 individual explants. Y-
error bars represent standard deviation.

100

C. melo '16'
C. melo '600'

C. metuliferus
C. sativus

Cu cumis specie s

V
ia

b
le

p
ro

to
p

la
st

s
(%

)

Leaf on OK medium with 20g·l¯¹ sucrose
Leaf on OK medium with 30g·l¯¹ sucrose
Growing apex on OK medium with 20g·l¯¹ sucrose
Growing apex on OK medium with 30g·l¯¹ sucrose

Tab. 1: Optimal age of plants, cultivated calluses and seedlings used for protoplast isolation

Leaf Growing apex Callus Hypocotyls
(days after planting) (days after planting) (days of cultivation) (days after sowing)

Genotype Tested Range Optimal Tested Range Optimal Tested Range Optimal Tested Range Optimal

C. anguria 15-74 (22) 21-74 — — — — — —

C. melo ‘12’ 12-40 (51) 20-27 — — 17-96 (22) 33-34 — —

C. melo ‘16’ 20-35 (4) 20-35 7-35 (7) 7-22 14-94 (10) 14-28 — —

C. melo ‘600’ 8-107 (18) 30-64 8-34 (3) 8-34 18-42 (13) 35-42 4-15 (4) 4-8

C. metuliferus 22-91 (8) 22-35 — — 18-42 (12) 32-35 — —

C. sativus 11-75 (16) 22-34 11-39 (6) 11-39 — — 6-17 (5) 6-8

The sample number is represented in brackets, each sample comprises of 2-4 explants for leaves, 8 explants for growing apices, 4 dishes for callus and 10 explants
for hypocotyls.

Factors affecting protoplast isolation 3

Regenerative capability of protoplasts isolated from different types
of explants

The highest level of regeneration was achieved using leaf proto-
plasts in all tested genotypes (Tab. 2) despite that leaf protoplasts
were not as viable as other types of protoplasts in most genotypes.
Calluses were regenerated from leaf protoplasts in C. melo ‘16’ and
C. melo ‘600’. Microcalluses were obtained in C. metuliferus and
C. sativus leaf protoplasts. Using the same technique calluses are also
possible using C. metuliferus and C. sativus (GAJDOVÁ et al., 2007).
Protoplasts from growing apices did not achieve this level of re-
generation but had higher regeneration efficiency than hypocotyl and
callus protoplasts. Using growing apices as source of protoplasts has
not been reported yet. Hypocotyl and callus derived protoplasts
exhibited very low regenerative capability. It has been reported that
callus protoplasts require a different medium for regeneration (with
0.045 µmol · l-1 2,4-D and 0.913 µmol · l-1 zeatin) whereas leaf
protoplasts of the same C. sativus variety had the highest regeneration
rate using 25 µmol · l-1 NAA and 14.8 µmol · l-1 2iP (BURZA and
MALEPSZY, 1995). For this study the same regeneration protocol was
used for all types of protoplasts. Hypocotyl protoplasts have not been

Fig. 2: Effect of donor explant variation on viability of protoplasts. Each value
in the graph is based on at least 16 leaf explants, 24 growing apices,
20 hypocotyls and 12 dishes of callus. Y-error bars represent standard
deviation.

100

C. anguria C. melo '12' C. melo '16' C. melo '600' C. metuliferus C. sativus

Cucum i s species

V
ia

b
le

p
ro

to
p
la

s
ts

(
%

)

leaf growing apex hypocotyl callus

Tab. 2: Effect of plant tissue on regeneration of protoplasts

Genotype Level of regeneration Regeneration efficiency¹ (%) Number of cultivated samples2

C. anguria

Leaf Cell division 58 12

C. melo ‘12’

Leaf Cell division 25 28

Callus No regeneration 100 15

C. melo ‘16’

Leaf Callus 50 2

Growing apex Microcallus and cell division 33 and 33 3

Callus Microcallus and cell division 14 and 7 14

C. melo ‘600’

Leaf Callus and cell division 4 and 38 26

Growing apex No regeneration 100 3

Hypocotyl No regeneration 100 1

Callus Cell division 14 14

C. metuliferus

Leaf Microcallus and cell division 5 and 53 19

Growing Apex Cell division 100 3

Hypocotyl Cell division 25 4

Callus No regeneration 100 12

C. sativus

Leaf Microcallus and cell division 25 and 25 8

Growing apex Microcallus 50 2

Hypocotyl No regeneration 100 1

Callus No regeneration 100 8

Only samples with viabilities of over 40% were used for regenerative capability assessment. ¹Regeneration efficiency is calculated as percentage of regenerating
samples given for each level of regeneration. 2One sample consists of at least 2-4 explants for leaves, 8 explants for growing apices, 4 dishes for callus and
10 explants for hypocotyls. In cases, where two levels of regeneration were observed, the regeneration efficiencies are given respectively.

4 Gajdová, J., Navrátilová, B., Smolná, J., Lebeda, A.

regenerated into plants yet in Cucumis spp. (GAJDOVÁ et al., 2004).
SUTIOJONO et al. (1998) have reported that leaf protoplasts had the
highest regeneration potential, however cotyledon protoplasts di-
vided slower and hypocotyl protoplasts did not divide at all. Plants
were regenerated from protoplasts isolated from leaves (DEBEAUJON
and BRANCHARD, 1992; ORCZYK and MALEPSZY, 1985), cotyledons
(ROIG et al., 1986; COLIJN-HOOYMANS et al., 1988) and calluses (BURZA
and MALEPSZY, 1995) in C. melo and C. sativus. Cotyledon protoplasts
were mostly tetraploid and regenerated shoots were not normal
however leaf protoplasts were diploid and produced normal plants
(COLIJN-HOOYMANS et al., 1988). Regarding this, we did not use
cotyledons in our study.

In C. melo ‘12’ the highest regeneration efficiency was achieved with
protoplasts from plants at 20-27 days old (which was also optimal
age for protoplast isolation as shown in Tab. 1). In all other genotypes
protoplasts from explants of various age were regenerating. Calluses
from C. melo ‘16’ and C. melo ‘600’ were regenerated from 20-28
days old plant-derived protoplasts. Regenerated calluses grew several
months but organogenesis did not occur on the media used in this
study. Although no plant growth was observed in this study, further
investigation into the callus regeneration media would likely yield
plants (DEBEAUJON and BRANCHARD, 1992; ORCZYK and MALEPSZY,
1985).

Protoplast culture technique

The number of cell divisions decreased using agarose-solidified
medium in our experiment (Tab. 3). Interestingly, several studies
showed increased plating efficiency using this technique compared
to liquid culture (COLIJN-HOOYMANS et al., 1988; MCCARTHY et al.,
2001b; SUTIOJONO et al., 1998). The decrease in number of cell
divisions in our study may have been caused by heat stress when

embedding protoplasts in warm agarose medium (approximately
40°C). In our experiments the LCM medium was much better for
protoplast regeneration of C. anguria leaf protoplasts than CML
medium (Tab. 4). LCM medium has been successfully used for
plant regeneration from cotyledon and leaf protoplasts in C. melo
(DEBEAUJON and BRANCHARD, 1992). Similar concentrations of
growth regulators (2.69 µmol · l-1 NAA, 4.56 µmol · l-1 2,4-D and
2.22 µmol · l-1 BA) were used in C. sativus and leaf and cotyledon
protoplast of C. melo (DABAUZA et al., 1991; ROIG et al., 1986). CML
medium was successfully used for plant regeneration from leaf
protoplasts of C. sativus (ORCZYK and MALEPSZY, 1985; COLIJN-
HOOYMANS et al., 1988). Both temperatures used in this study had
similar effects on cell division (Tab. 5). No increase was seen when
using 27°C. MCCARTHY et al. (2001a) have obtained increased
rates of cell division and microcalluses in cotyledon protoplasts of
C. metuliferus cultured at 30°C over those cultured at 25°C.

Conclusions

From this study it is evident that the most important factor for ob-
taining the highest viability of Cucumis protoplasts was donor explant
age (optimal age range was mostly 1- 5 weeks, varying according to
explant type and genotype). Using the plant culture medium with
increased level of sucrose resulted in production of protoplasts with
poor viability except with C. melo ‘600’ growing apex protoplasts.
Explant type was also important for viability, but there was no ‘high
viability explant’ common to all genotypes. All explants were able to
produce over 50 % (some over 80 %) viable protoplasts. C. melo
varieties gave slightly higher protoplast viabilities than C. metuliferus
and C. sativus. The regenerative capability of protoplasts was primarily
dependent on source explant type, with the highest regeneration level
(callus) observed in leaf protoplasts. Calluses were obtained only

Tab. 5: Effect of culture temperature on cell division

Tested species Cell divisions in 25°C Cell divisions in 27°C

Cucumis metuliferus Regeneration Number of Regeneration Number of
efficiency¹ (%) cultivated samples2 efficiency¹ (%) cultivated samples2

75 8 63 8
1Regeneration efficiency is the percentage of regenerating samples. 2Each sample consisted of 8-16 leaves.

Tab. 3: Effect of viscosity of the protoplast culture medium on cell division

Tested species Cell divisions in liquid medium Cell divisions in agarose solidified medium

Cucumis melo ‘12’ Regeneration Number of Regeneration Number of
efficiency¹ (%) cultivated samples2 efficiency¹ (%) cultivated samples2

25 28 5 20

1Regeneration efficiency is the percentage of regenerating samples. 2Each sample consisted of 2-4 leaves.

Tab. 4: Effect of the protoplast culture medium composition on cell division

Tested species Cell divisions in LCM medium Cell divisions in CML medium

Cucumis anguria Regeneration Number of Regeneration Number of
efficiency¹ (%) cultivated samples2 efficiency¹ (%) cultivated samples2

58 12 10 10
1Regeneration efficiency is the percentage of regenerating samples. 2Each sample consisted of 4-8 leaves.

Factors affecting protoplast isolation 5

in C. melo ‘16’ and C. melo ‘600’. Temperature and culture medium
composition affected number of cell division but there were no
differences in level of regeneration obtained. This study has highlighted
some of the important factors in promoting healthy regeneration of
protoplasts. Future work will concentrate on further optimising the
growth conditions of regenerating microcalluses specific to the donor
species. Investigation on the effects of protoplast density in culture
medium may help increase the plating efficiency. These may yield the
answer in producing viable plants from protoplast fusions in the future.

Acknowledgements

We would like to thank the Ministry of Agriculture of the Czech
Republic for the co-funding of this project by a grant QF 4108 and the
Ministry of Education of the Czech Republic for grant MSM
6198959215 who made this research possible. Technical assistance
of Anna Zedkova is acknowledged. We would also like to thank John
Mitchels and Alan Scragg, the University of the West of England
(Bristol, UK) for kindly reading the manuscript.

References

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Address of the authors:
Mgr. Jana Gajdová, Prof. Dr. Aleš Lebeda *, RNDr. Bozena Navrátilová,
Ph.D. and Mgr. Jitka Smolná; Department of Botany, Faculty of Science,
Palacký University in Olomouc, Slechtitelu 11, 783 71 Olomouc - Holice, Czech
Republic.

*corresponding author (ales.lebeda@upol.cz)

6 Gajdová, J., Navrátilová, B., Smolná, J., Lebeda, A.

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