Scientia Horticulturae 256 (2019) 108552

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Isolation and culture of strawberry protoplasts and field evaluation of T

regenerated plants
Marta Barcelóa, Anita Wallinb, Juan J. Medinac, David J. Gil-Arizaa, Gloria López-Casadod,
José Juareze, José F. Sánchez-Sevillaa, Carlos López-Encinaf, José M. López-Arandaa,
⁎
José A. Mercadod, , Fernando Pliego-Alfarod
a
IFAPA Centro de Málaga, Cortijo de la Cruz s/n, 29140, Málaga, Spain
b
Department of Physiological Botany, Uppsala University, Villavägen 6, S-752 36, Uppsala, Sweden
c
IFAPA Centro Las Torres-Tomejíl, 41200, Sevilla, Spain
d
Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora” (IHSM-UMA-CSIC), Departamento de Biología Vegetal, Universidad de Málaga, 29071, Málaga,
Spain
e
Instituto Valenciano de Investigaciones Agrarias-IVIA, Ctra. Moncada-Náquera, Km 4.5, 46113, Moncada, Valencia, Spain
f
Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora” (IHSM-CSIC-UMA), 29750, Algarrobo-Costa, Málaga, Spain

A R T I C LE I N FO A B S T R A C T

Keywords: Protoplasts are an useful biotechnological tool for plant improvement. In strawberry, very few studies on pro-
Agronomic evaluation toplast technology have been carried out. In this investigation, a procedure for the isolation and culture of
Fragaria × ananassa strawberry protoplasts, cv. ‘Chandler’, has been developed. The effect of several factors affecting the successful
Protoplast isolation isolation of protoplasts and formation of microcalli, e.g. explant source, washing procedure, hormonal compo-
Protoplast regeneration
sition of the culture medium and protoplast density, were evaluated. For shoot regeneration, microcalli derived
Somaclonal variation
from isolated protoplasts were transferred to MS medium supplemented with 0.2 mg l−1 NAA and either
Strawberry
5 mg l−1 BA or 3 mg l−1 TDZ, obtaining a similar regeneration rate, 17%, in both media. Twenty-one in-
dependent protoclones were transferred to field conditions for agronomic evaluation. Significant alterations in
the growth habit, density of foliage, leaf color and leaf morphology were detected in some lines. Fruit yield was
significantly reduced in 15 out of the 21 protoclones evaluated due to a reduction in fruit weight and/or the
number of fruits. Ploidy level was unaffected in a sample of 6 lines selected at random; however, a study of
genetic stability by using 10 EST-SSR markers showed genetic alterations in all the lines analyzed. Despite the
high rate of somaclonal variation detected in the protoclones, some of the lines displayed an agronomical be-
havior similar to control plants, indicating that this protocol could be useful for genetic improvement in this
species.

1. Introduction cytoplasmic organelles that are unique, being the obtainment of

Protoplasts are single cells devoid of their walls, with potential to
divide, proliferate and dedifferentiate forming a callus prior to re-
generate whole plants. Protoplasts represent a versatile experimental
system to perform functional genetic analysis and to study a wide range
of cellular processes, e.g. cellular structure, membrane function, hor-
monal signaling (Davey et al., 2005). Furthermore, protoplasts might
also be an important biotechnological tool in breeding programs,
especially in vegetatively propagated species of high heterozygosity and
ploidy level, such as strawberry (Nyman and Wallin, 1988). Through
protoplasts, it is possible to create gene combinations of nucleus and/or
asymmetric hybrids and cybrids one the main applications for proto-
plast fusion (Geerts et al., 2009; Li et al., 1999). As the variation ob-
tained in plants regenerated from protoplasts is much greater than that
in plants coming from cultures with organized growth (Karp, 1993),
protoplast culture could have an enormous value as a source of soma-
clonal variation, so-called protoclonal variation, expanding the existing
germplasm by creating variations that may be used within breeding
programs (Davey et al., 2005).
The use of protoplasts for genetic transformation has received minor
attention mainly due to the simplicity and higher transformation rates
of Agrobacterium tumefaciens and biolistic-based technologies. The

⁎
Corresponding author.
E-mail address: mercado@uma.es (J.A. Mercado).

https://doi.org/10.1016/j.scienta.2019.108552
Received 21 March 2019; Received in revised form 30 May 2019; Accepted 31 May 2019
0304-4238/ © 2019 Elsevier B.V. All rights reserved.
M. Barceló, et al.
elevated frequency of protoclonal variation is also an additional han-
dicap to the use of protoplasts for stable transformation. In recent years,
the development of the CRISPR/Cas9 system for gene editing has in-
creased the interest in protoplasts technology. In some species, proto-
plasts are used to validate the efficiency of the CRISPR/Cas9 plasmids
prior to stable transformation (Lin et al., 2018; Zhang et al., 2011; Zhou
et al., 2018). Additionally, transient expression of Cas9 in protoplasts
would allow the recovery of non-transgenic plants, free from any for-
eign genetic material (Malnoy et al., 2016; Woo et al., 2015). Never-
theless, isolation of high-quality protoplasts and plant regeneration is
far from being a routine technique in many species.
In strawberry, very few investigations on protoplast culture have
been carried out. Nyman and Wallin (1988) described a procedure for
the isolation and culture of protoplasts from in vitro leaves of cvs.
‘Sengana’ and ‘Canoga’. Isolated protoplasts were cultured in a modified
8p-liquid medium (Glimelius et al., 1986) for 3–4 weeks, until micro-
colonies had formed, and later transferred to semi-solid medium. Plant
regeneration was achieved in MS medium supplemented with 1.07 μM
NAA and either 22.2 μM BA or 22.7 μM TDZ (Nyman and Wallin, 1992).
Almost half of the plants recovered contained anomalous ploidy levels
(Nyman and Wallin, 1992). Using a similar protocol, Infante and Rosati
(1993) regenerated plants from protoplasts of wild strawberry, F. vesca,
clon ‘Alpine’. In this case, regenerated plants did not show any ab-
normality in the greenhouse compared to the original micropropagated
clone (Infante and Rosati, 1993). In order to develop biotechnological
tools for the genomic editing of strawberry, the aim of this research was
to develop an efficient protocol for strawberry protoplast isolation and
culture, as well as regeneration of plants from resulting microcalli. In
addition, a study of the agronomic performance of the obtained mate-
rial was carried out.
2. Material and methods
2.1. Plant material
In vitro grown plants of Fragaria × ananassa, cv. ‘Chandler’, were
used as source of material. Shoots, derived from 3 mm long meriste-
matic buds, were maintained in a medium containing N30K macroele-
ments (Margara, 1984), MS micronutrients and vitamins (Murashige
and Skoog, 1962), 20 g l−1 sucrose and 0.47 mg l−1 kinetin with sub-
cultures at four week intervals. Prior to protoplasts obtainment, shoots
were transferred to MS medium supplemented with 10 g l−1 sucrose,
2 mg l−1 BA and 0.3% Gelrite for 3–4 subcultures, to induce hyperhy-
dricity. Culture conditions were 16 h photoperiod under 40 μmol
m−2 s−1 and 25 ± 2 °C temperature.
2.2. Protoplast isolation and purification
Initially, the protocol described by Nyman and Wallin (1988) for
strawberry protoplasts isolation was employed. Strawberry shoots in a
hyperhydric state were chopped into a solution of preplasmolysis with
W5 medium (Menczel et al., 1981; Supplementary Table 1), containing
0.3 M sorbitol and 0.05 M CaCl2·H2O (Carlberg et al., 1983), in darkness
over 30–60 min. Afterwards, W5 medium was replaced by a sterile
enzymatic solution containing 1% cellulysin and 0.1% macerase dis-
solved in K3 medium (Menczel et al., 1981; Supplementary Table 1)
and 0.4 M sucrose. The material was incubated in darkness at 25 °C
under slow stirring (20 rpm) for 18 h. Purification of released proto-
plasts was carried out by filtration through a 53 μm nylon sieve. The
enzymatic solution containing the protoplasts was diluted at 1:1 ratio
with CPW16 (CPW salts (Banks and Evans, 1976; Supplementary
Table 1) and 16% sucrose (w/v)), and centrifuged for 5 min at 50g.
Then, floating protoplasts were carefully removed, poured on top of
fresh CPW 16 medium at a 2:8 ratio and centrifuged as above. This
process was repeated in W5 medium (Menczel et al., 1981) to pellet the
protoplasts.
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Scientia Horticulturae 256 (2019) 108552
To improve the recovery of viable protoplasts, different washing
media were evaluated. Enzymatic solutions containing protoplasts were
washed in three different ways: a) two consecutive rinses in CPW16
medium, b) a first wash in CPW16 medium followed by a rinse in
CPW21 medium (21% sucrose), and c) a rinse in CPW21 followed by a
second rinse in a solution of 0.8 M sucrose and percoll (1:1).
2.3. Protoplast culture and plant regeneration
Viable protoplasts at different densities (104, 2.5 × 104, 5 × 104,
10 , 2 × 105 protoplastos ml−1) were cultured in a modified bead
5
culture system (Shillito et al., 1983). Agarose at 0.6% was added to the
protoplast solution the same day or one day after protoplast isolation.
Thereafter, the medium containing the protoplasts was separated into
pieces that were located in Petri dishes. Three ml of modified 8p liquid
medium (Glimelius et al., 1986; Supplementary Table 1), supplemented
with 0.4 M glucose, 1 mg l−1 NAA and either 0.5 mg l−1 BA (8 pm BA)
or 0.25 mg l−1 TDZ (8 pm TDZ) were added to each Petri dish, to pro-
mote microcalli formation. The osmolarity was progressively reduced
through weekly changes to fresh medium, decreasing the glucose con-
centration from 0.4 to 0.2 M. Frequency of divisions was estimated by
counting the protoplasts which made up group of cells after the first
week of protoplast isolation.
After 3–4 weeks, the medium was replaced by a modified 8p liquid
medium containing 2% sucrose, 0.1 mg l−1 NAA and 1.0 mg l−1 BA.
Once the microcalli were visible, segments of the agarose beads were
placed on the same modified 8p medium described above solidified
with agarose. Then, calli of 2–3 mm, usually obtained after 4 weeks of
culture, were transferred to the regeneration medium.
Three different regeneration media were evaluated: MS medium
containing 0.2 mg l−1 NAA and either 5 mg l−1 BA (MS-BA) or 3 mg l−1
TDZ (MS-TDZ), both supplemented with 2% sucrose and 0.3% agarose
(Nyman and Wallin, 1992), and a modified MS medium with N30K
macroelements (Margara, 1984) supplemented with 0.5 mg l−1 IBA,
2 mg l−1 BA and solidified with 6 g l−1 agar (N30K-BA) (Barceló et al.,
1998). Regenerated plants were micropropagated in the medium of
López-Aranda et al. (1994) containing N30K macroelements, MS mi-
croelements and vitamins and 0.47 mg l−1 kinetin.
For acclimatization, rooted plants from 27 independent protoclones,
and a micropropagated control were transferred to trays with substrate
1:perlite:sand (70:10:20) and kept into a tunnel for 6–8 weeks, reducing
progressively the humidity during the hardening process.
2.4. Field evaluation of protoclones
After acclimatization, mother plants were established in a nursery at
IFAPA-Centro de Málaga (Spain) for 6 months in order to get the ve-
getative progeny to be used in the fruiting field. Plants were planted
onto ridges in 4 lines distributed in a field within an area of 400 m2.
Mineral nutrients were periodically incorporated to the drip irrigation
system. The agronomic study of the F1 vegetative progeny was carried
out in the production fields of “El Cebollar” (IFAPA-Las Torres, Huelva,
Spain), under macrotunnel conditions. Plants were planted in three
completely randomized plots, each with 10 plants for protoclon, on
ridges covered with black plastic. Later on, plants were covered with
macrotunnel thermal plastic 600G. Twenty one protoclones and two
controls, plants derived from micropropagated shoots (CM) and plants
conventionally propagated by runners (CR), were evaluated.
Seven months after plants had been established in the field, mor-
phological data were taken. Growth habit, density of foliage and leaf
color were estimated with arbitrary scales accordingly to the guidelines
described by The International Union for the Protection of New
Varieties of Plants for strawberry (UPOV, 2012); growth habit: (3)
upright, (5) semi-upright, (7) spreading; density of foliage: (3) sparse,
(5) medium, (7) dense; leaf color: (1) yellow green, (3) light green, (5)
medium green, (7) dark green. Plant area was estimated assuming that
M. Barceló, et al.
the plant acquired an ellipse-like form by measuring long (a) and short
(b) axis (A = (a × b/4) π) (Mercado et al., 2015). The number of
leaflets per leaf in a sample of 10 leaves per plant was also evaluated.
Fruits were harvested at the stage of full ripeness and the fruit yield was
calculated as the g of fruits produced per plant. Fruit firmness was
measured using a hand-penetrometer with a 3.5 mm diameter needle.
2.5. Genetic stability of protoclones
Leaf sections of approximately 1 × 1 cm from ‘Chandler’ control and
each evaluated protoclon were used for the analysis of the ploidy level
in a Partec (Germany) flow cytometer. The plant high resolution DNA
kit type P (Partec, Germany) was employed for DNA extraction and
staining. The measurements in the flow cytometer were carried out
with a minimum of 1000 nuclei per sample. The fluorescence intensity
histograms obtained were analyzed with the program CAPD v 2.0
(Partec).
Additionally, genetic stability of some of the protoclones obtained
was analyzed by using 10 single sequence repeat markers derived from
expressed sequence tags (EST-SSR markers) as described by Gil-Ariza
et al. (2009). DNA from young leaves was extracted with the DNeasy
Plant Mini Kit (Qiagen, Valencia, CA) and amplified in a iCycler
thermal cycler (BioRad Laboratories, Hercules, CA) in a total volumen
of 15 μL containing 1 × PCR buffer, 2 mM MgCl2, 200 μM of each
dNTPs, 200 nM of each specific primer, 0.5 U of Taq DNA polymerase
and 25 ng of genomic DNA. The PCR program was 94 °C for 5 min, 35
cycles of denaturation at 94 °C for 1 min, annealing at 58–60 °C for
1 min and extension at 72 °C for 1 min, and a final extension step at
72 °C for 5 min. The PCR fragments were separated in polyacrylamide
gels for analysis of polymorphisms in a Sequi-Gen GT (BioRad, Munich)
sequencer. From the similarity values based on these markers, a den-
drogram was generated using the UPGMA (Unweighted Pair Group
Method Average) to determine the relationships between protoclones
analyzed.
2.6. Statistical analysis
Data were subjected to analysis of variance (ANOVA) using SPSS
software. The Levene test for homogeneity of variance was performed
prior to ANOVA. Tukey test was used for post hoc multiple compar-
isons. Mean comparisons in pairs were performed with Student t-test or
Mann-Whitney U test, both with sequential Bonferroni correction, in
case of homogeneous or non-homogenous variances, respectively. For
principal component analysis, the psych package of R was employed,
with Varimax rotation of factors.
3. Results
3.1. Protoplasts isolation and plant regeneration
Preliminary experiments showed that hyperhydric leaves from in
vitro strawberry shoots were adequate explants for protoplast isolation.
Hence, proliferating shoots were transferred to a medium supplemented
with lower sucrose concentration, 10 g l−1, and a greater BA con-
centration, 2 mg l−1, to induce hyperhydricity. Plants in this medium
formed smaller shoot colonies, with folded sheets, shorter petioles and a
generally stumpy and hyperhydric appearance (Fig. 1A). Optimal
duration of last subculture in the hyperhydricity inducing medium prior
to protoplast isolation was found to be 3 weeks. An increase of the
culture period caused a deterioration in the quality of the explant
material that began to get brown, especially at the callus of shoot base.
Strawberry protoplasts were isolated following the protocol de-
scribed by Nyman and Wallin (1988) with some modifications. In-
creasing the proportion of sucrose in the CPW washing medium from 16
to 21% significantly increased the percentage of viable protoplasts from
58% to 73% (Fig. 2). The recovery of viable protoplasts was further
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Scientia Horticulturae 256 (2019) 108552
enhanced when replacing the second CPW rinse by a 0.8 M sucrose and
Percoll solution at 1:1 ratio, reaching a value of viable protoplasts close
to 90% (Fig. 2). Moreover, the density gradient generated by using a
sucrose-percoll solution, allowed the isolation of protoplasts of different
sizes. Fig. 1B shows the appearance of protoplasts obtained after
washing and purification with this optimized protocol. In general, two
cell populations were visible, one with large protoplasts containing
many green chloroplasts, and the other one formed by smaller proto-
plasts with a lower amount of chloroplasts.
Isolated protoplasts were cultured in a modified bead culture system
using agarose for embedding the cells. It is important to notice that
regeneration of the cell wall was visible one day after protoplast iso-
lation. The effect of protoplasts density and hormonal composition of
the culture medium, 0.5 mg l−1 BA or 0.25 mg l−1 TDZ, in the forma-
tion of microcalli were evaluated (Table 1). At low protoplast densities
(1–2.5 × 104 protoplasts ml−1) no cell divisions were observed. In ad-
dition, with the lowest population density, few alive protoplasts could
be distinguished. At higher densities, cluster of cells were visible after 7
days of culture (Fig. 1C). After two weeks of culture, best results were
obtained when using the highest protoplast density (2 × 105 proto-
plasts ml−1) in the medium supplemented with TDZ (Fig. 1D).
Microcalli were transferred to three different regeneration media,
MS supplemented with 0.2 mg l−1 NAA and either 5 mg l−1 BA or
3 mg l−1 TDZ, and N30K supplemented with 0.5 mg l−1 IBA and
2 mg l−1 BA. MS media induced an earlier response than N30K medium;
microcalli started to regenerate shoots after 4 weeks of culture in MS
while shoots, at lower frequency, were visible in N30K medium after 8
weeks. At the end of the experiment, 16 weeks, the percentage of ex-
plants regenerating shoots was similar in MS supplemented with BA or
TDZ, ca. 17%, and significantly higher than N30K (2.3%) (Fig. 3).
Fig. 1E shows the aspect of a shoot regenerated from protoplasts in MS
medium supplemented with BA. Subsequently, 25 independent proto-
clones were obtained. These plants were micropropagated in a N30K
medium supplemented with 0.47 mg l−1 kinetin and later acclimated to
ex vitro conditions (Fig. 1F). A workflow diagram of the main steps of
the developed protocol for protoplast isolation and plant regeneration
in strawberry is displayed in Fig. 4.
3.2. Behaviour of protoclones at the nursery
Control micropropagated plants and the different clones derived
from protoplasts were planted in a nursery to obtain the vegetative
progeny for field evaluation. A reduction in plant vigour and leaf
chlorosis was observed in some clones. Additionally, a 36% of the
clones produced leaves with 4–5 leaflets, being 3 the standard value of
this parameter. This variation was also observed in the runner progeny
of these lines. In relation to the average number of first-order runners,
significant differences were found among different protoclones. The
highest number of runners per plant was found in clone 21
(29.7 ± 2.7) followed by control micropropagated plants
(23.0 ± 1.0). A 44% of the clones also showed a high capacity for
runner production, ranging the number of stolons per plant from 20.7
to 11.7. The rest of clones produced less than 5 runners per plant and
some of them also showed a delay in runner production. Finally, en-
ough daughter plants for field evaluation were obtained from 21 out of
the 25 clones obtained after the acclimatization phase. Generally, these
daughter plants showed normal appearance and green colour, except
those from clones 6, 8, 19 and 22, where a 33.3% of the daughter plants
were slightly chlorotic, and clones 4, 24 and 26, with a 66.7% of
chlorotic plants.
3.3. Behaviour of protoclones at the fruiting field
Plants from the first vegetative generation of the 21 protoclones and
two controls (CM, micropropagated control plants obtained through
axillary branching, and CR, plants conventionally propagated by
M. Barceló, et al. Scientia Horticulturae 256 (2019) 108552

Fig. 1. (A) In vitro strawberry shoots, cv. ‘Chandler’, cultured in the medium for inducing hyperhydricity, used as the source of explants. (B) Isolated protoplasts after
washing and purification. (C) Cluster of protoplast derived cells after 7 days of culture in 8 pm medium containing 1 mg l−1 NAA and 0.25 mg l−1 TDZ. (D) Microcalli
formed in the same medium after 14 days of culture. (E) Shoot regenerated in MS medium supplemented with 0.2 mg l−1 NAA and 5 mg l−1 BA. (F) Acclimated
plants. (G) and (H) Protoclones growing in the production field.

Fig. 3. Effect of the culture medium on the percentage of adventitious shoot

Fig. 2. Effect of washing solution in the recovery of viable protoplasts. WS1: regeneration in calli derived from strawberry protoplasts, cultivar ‘Chandler’.
two washes in CPW medium with 16% sucrose; WS2: an initial wash in CPW16 Data were taken after 16 weeks of culture. Bars represent mean ± SD. Mean
medium followed by CPW21 medium (21% sucrose); WS3: a rinse in CPW21 separation was performed by Tukey test at P < 0.05.
followed by a rinse in a solution of 0.8 M sucrose and percoll (1:1). Bars re-
present mean ± SD. Mean separation was performed by Tukey test at runners), were cultivated under macrotunnel cover for agronomical
P < 0.05. evaluation (Fig. 1G, H). Growth characteristics of plants are shown in
Table 2. Controls showed an upright growth habit and a slightly sparse
Table 1 density of foliage. Plant area was slightly higher in CM plants than in
Effect of protoplasts density and culture medium in the induction of cell divi- CR. However, leaf color was greener in CR. The number of leaflets per
sions in strawberry protoplasts. Data were taken after two weeks of culture in leaf was always three in both cases. Growth habit in most protoplasts
8 pm medium supplemented with 1 mg l−1 NAA and either 0.5 mg l−1 BA or derived clones was between upright and semi-upright; however, clones
0.25 mg l−1 TDZ. Frequency of cell proliferation: (−) absence of divisions, (+) 12, 18, and 23 showed a spreading growth habit. The density of foliage
low, (++) intermediate, (+++) high. was higher than control in 52.4% of the lines. In general, the variations
Protoplasts density (protoplasts ml−1) Culture medium found in plant area in the mother plants were maintained in the first
vegetative generation; a 71.4% of the clones showed similar values of
BA (0.5 mg l−1) TDZ (0.25 mg l−1)
plant area than controls, while 6 lines (lines 4, 10, 12, 18, 23, and 27)
1 × 104 − − displayed a significant reduced growth. Leaf color varied from yellow
2.5 × 104 − − green in some of the lines (lines 1, 3, 4, 10, 18, 19, 26) to dark green
5 × 104 ++ ++ (lines 7, 21, 24, 27). As regards the number of leaflet per leaf, clones
1 × 105 ++ ++ with all their leaves trifoliated, clones with a number of leaflets dif-
2 × 105 ++ +++
ferent from three in all leaves (clone 11), as well as others showing a

4
M. Barceló, et al. Scientia Horticulturae 256 (2019) 108552

Fig. 4. Workflow chart describing the main steps of the protocol for protoplasts
isolation and plant regeneration in strawberry.
Fig. 5. Fruit yield and fruit weight in plants obtained from strawberry proto-
plasts. CM: control micropropagated plants. CR: control plants obtained by
conventional runner propagation. Asterisks indicate significant differences with
the control CM by Student t-test (fruit yield) or Mann-Withney U test (fruit
weight) with Bonferroni correction at P < 0.05.

high (clones 2, 8, 10, 22) or low frequency (clones 1, 4, 6, 18 and 20) of

Table 2 this abnormality could be found.
Growth characteristics of daughter plants obtained from strawberry protoplasts,
Fruit yield and average fruit weight are shown in Fig. 5. Both
growing in the production field. CM: control micropropagated plants. CR:
parameters were slightly higher in the micropropagated control when
control plants obtained by conventional runner propagation. Growth habit: (3)
upright, (5) semi-upright, (7) spreading. Density of foliage: (3) sparse, (5) compared with macropropagated plants, although the differences were
medium, (7) dense. Leaf color, (1) yellow green, (3) light green, (5) medium not significant. Fruit yield was reduced in most of the plants derived
green, (7) dark green. Number of leaflet abnormalities: (−) absence, (+) low from protoplasts. Three out of 21 clones, 14.2% of the lines, did not
frequency, (++) medium, (+++) high. Asterisks indicate differences with produce fruits (lines 15, 18 and 23) and 12 clones, 57.1%, displayed
CM plants by Mann-Whitney U test with sequential Bonferroni correction. lower yield values than CM. Average fruit weight in 8 out of the 18
Line Growth Density of Plant area Leaf Number of leaflet
protoclones that produced fruits was lower than in CM plants. By
habit foliage (cm2) color abnormalities contrast, three lines, protoclones 2, 10 and 22, produced fruits bigger
than control although the differences were not significant. Globally,
CM 3.5 4.1 629.9 3.5 − only protoclones 2, 9 and 20 displayed fruit yield and fruit weight
CR 3.7 3.7 488.1 5.4* −
values similar to CM plants. As regard as fruit quality, all protoclones
1 5.0* 5.7* 467.7 1.0* +
2 4.4 6.2* 672.4 4.5 ++ produced fruits with similar color, internal cavity and firmness than
3 5.0* 5.8* 610.2 1.5* − control plants. However, fruit shape was highly variable. Suction cup
4 6.4* 3.4 399.9* 1.0* + (lines 1, 6, 8, 9 and 24), short cuneiform or cordiform (4, 10 and 26),
6 5.6* 5.3 748.1 2.5 +
conical (7, 10, 20 and 21), multilobular (2, 3, 7, 9, 22, 24 and 26) and
7 4.4 6.5* 663.2 6.4* −
8 3.8 6.9* 645.6 4.1 ++ bell fruit shape (8, 12, 19, 22 and 27) were observed in different pro-
9 3.7 4.2 511.9 2.9 − toclones whereas control fruits had a biconical shape. Interestingly,
10 6.0* 4.2 377.3* 1.1* ++ some lines produced anomalous fruits with presence of phyllody; e.g.
11 4.6 5.5* 754.8 1.7 +++ clones 6, 7, 11 and 22. In the case of protoclone 22, phyllody gave rise
12 7.0* 5.1 385.5* 2.4 −
to a complete plant. Protoclones that produced fruits with a mor-
15 5.0* 6.5* 625.8 4.8 −
18 7.0* 4.2 374.1* 1.0* + phology more similar to the controls were 7, 10, 19, 20 and 21.
19 6.0* 3.3 372.3 1.0* − Principal component analysis (PCA) was applied to characterize the
20 3.0 6.3* 697.7 2.7 + independent clones with respect to the vegetative and fruiting para-
21 3.8 6.8* 708.9 7.0* −
meters measured. Two principal components with eigenvalues higher
22 3.9 6.2* 845.8 2.5 ++
23 7.0* 3.3 243.1* 1.6 −
than 1 were obtained. Factor loadings of the two PCs, explaining 72.8%
24 5.0* 5.7* 656.5 6.8* − of total variance, are shown in Supplementary Table 2. PC1 was mainly
26 5.0* 4.7 544.7 1.6* − related to the vegetative growth whereas fruit parameters and growth
27 6.4* 3.8 280.9* 6.2* − habit were correlated with PC2. The factor score plot is shown in Fig. 6.
Line 9 was the clone that had factor scores more similar to controls.
Among the rest of clones, line 2 stood out for its positive values on the
first component and similar score values than control in the second PC,

5
M. Barceló, et al.
Fig. 6. PCA analysis of vegetative and fruiting variables evaluated in the
strawberry protoclones. Factor score plot of the different protoclones on the two
PC. CM: control micropropagated plants; CR: control plants obtained by con-
ventional runner propagation.
indicating an excellent vigor and fruit production.
3.4. Genetic stability of protoclones
Initially, the ploidy level of 6 protoclones selected at random was
analyzed. No differences in the ploidy level were found between the
clones analyzed and the ‘Chandler’ octoploid control (results not
shown). Subsequently, a genetic variability analysis was carried out by
means of microsatelite markers in 10 out of the 21 lines evaluated in the
field. The dendogram obtained is shown in Fig. 7. Similarity coefficients
ranged between 0.94 and 0.85. The first group, formed solely by clone
21, showed a high similarity with control. A second group was formed
by clones 3, 9 and 26, with a coefficient of similarity of 0.90. Finally,
the third group obtained included a high fruit yield clone (line 2), four
of intermediate production (clones 4, 8, 11, and 20), and one of ex-
tremely low production (clone 18). Interestingly, all clones of the latter
group had leaves with 4 or 5 leaflets.
4. Discussion
There is a wide agreement regarding the difficulties of isolating and
culturing protoplasts (Eriksson, 1988; Debnath and Texeira da Silva,
2007). In this investigation, using as reference the protocol developed
by Nyman and Wallin (1988), an improved procedure for plant re-
generation from protoplasts of cv. ‘Chandler’ has been developed.
Fig. 7. Genetic relationships among the different protoclones and the control line analyzed with EST-SSR markers. The motifs and characteristics of the 10 markers
used can be found in Gil-Ariza et al. (2009). The dendogram was generated using the unweighted pair group method with arithmetic mean.
6
Scientia Horticulturae 256 (2019) 108552
4.1. Protoplast isolation
Besides genotype, the type and physiological state of the donor
material greatly influences the success in the isolation of viable proto-
plasts (Eeckhaut et al., 2013; Ortín-Parraga and Burgos, 2003). Along
this line, we were unable to obtain protoplasts when using young
strawberry leaves from greenhouse grown plants. Nyman and Wallin
(1988) indicated that the in vitro culture of the strawberry plants used
as source of explants in a low-sucrose high-cytokinin medium stimu-
lated the vegetative growth, although it induces an hyperhydric state in
the shoots. Moreover, a lower carbohydrate content in the medium
decreases the intracellular sugar available for the synthesis of the cell
wall, and this might reduce its thickness facilitating protoplasts se-
paration (Warren, 1991). However, Boulay (1985) indicated that if the
hyperhydric state was irreversible it would lead to cell death. On the
other hand, it is known the negative influence of the presence of phe-
nolics to obtain viable protoplasts (Warren, 1991). In the present work,
a culture of shoots during three weeks in the hyperhydric inducing
medium was found to be optimum, since both the appearance of phe-
nolic compounds and irreversible damage in the plant material were
avoided. As high BA concentrations in the hyperhydric inducing
medium could cause somaclonal variation, the effect of this treatment
in genetic variability should be examined.
Protoplast isolation is a stress-inducing process (Papadakis et al.,
2001; Papadakis and Roubelakis-Angelakis, 2002), in which waste
products accumulation could induce cell disruption. Sucrose and glu-
cose are usually used in the protoplast isolation and purification media
(Davey et al., 2005). In the strawberry cv. ‘Chandler’, an increase in the
concentration of sucrose in the CPW washing medium from 16 to 21%
improved protoplasts viability; however, Nyman and Wallin (1988)
recommended CPW 16 in ‘Sengana’ and ‘Canoga’ strawberry cultivars,
while Infante and Rosati (1993) recommended CPW with 18% sucrose
for Fragaria vesca cv. ‘Alpine’. These results corroborate the need to
adjust the isolation protocol for each genotype.
Protoplasts from cells containing chloroplasts have high densities,
whereas those from cells with large vacuoles and small plastids, show
lower densities. Gradients of osmotically inactive substances, such as
Percoll, in combination with a low density osmotic agent, e.g. sucrose,
allow the recovery of clean populations (Potrykus and Shillito, 1986).
Protoplasts obtained in cv. ‘Chandler’ showed differences in size and
density, and the use of a density gradient with a Percoll-sucrose 0.8 M
solution increased viability by 10%. These results clearly differ from the
observations of Nyman and Wallin (1992) in the breeding line 77,101;
these authors observed a significant increase in the number of proto-
plasts that precipitated with the cellular debris after using a density
M. Barceló, et al.
gradient.
4.2. Protoplast culture and plant regeneration
The use of gelling agents for protoplasts culture prevent cell ag-
gregation, stabilize membranes and reduce the diffusion of wall pre-
cursors outside protoplasts (Chen et al., 1988; Eeckhaut et al., 2013;
Shillito et al., 1983). In cv. ‘Chandler’, the culture of protoplasts in
agarose beads showed to be superior compared with liquid medium, as
previously reported by Nyman and Wallin (1992). Cell density is a key
factor for protoplasts regeneration, being the optimum density in the
range 2 × 104–2 × 105 protoplasts. ml−1 (Potrykus and Shillito, 1986;
Bhojwani and Dantu, 2013). Nyman and Wallin (1988, 1992) suggested
a density of 5 × 104 protoplasts. ml−1 for strawberry to prevent ag-
gregation; however, in cv. ‘Chandler’, best results were obtained at a
higher culture density (2 × 105 protoplasts. ml−1), in accordance with
previous observations of Ortín-Parraga and Burgos (2003) in peach.
High cell density facilitates cellular contact and stimulates the growth
of adjacent cells by releasing growth factors, a phenomenon known as
medium conditioning (Morgan, 1999; Davey et al., 2005).
Strawberry tissues require a medium with cytokinin and auxin for
successful adventitious shoot regeneration. A combination of BA and
IBA is the most commonly used, although TDZ instead of BA has also
been successfully employed in some cultivars (Palomo-Ríos et al.,
2018). Nyman and Wallin (1992) obtained higher shoot regeneration
rates in protoplasts cultured in a medium supplemented with TDZ in
comparison with BA; however, in cv. ‘Chandler’, both cytokinins
yielded the same result, 17% of callus regenerating shoots, a value
much lower than that obtained by Nyman and Wallin (1992) in the
breeding line 77,101. On the other hand, Barceló et al. (1998) found
that the basal medium N30K, a mineral formulation of lower ionic
strength than MS, was better for leaf regeneration of cv. ‘Chandler’.
However, this medium was not adequate in the case of protoplasts, i.e.
calli cultured in MS regenerated shoots earlier and at higher rates than
those cultured in N30K.
4.3. Field evaluation of protoclones and genetic stability
The regulatory mechanisms of the cellular cycle play a direct role in
the growth and morphogenesis of plants, so that any alteration in the
cellular cycle might produce somaclonal variation (Bairu and Aremu,
2011). The variations found in plants derived from protoplasts are
higher than that of plants derived from explants with a high degree of
organization (Karp, 1993). Protoplast regeneration is normally asso-
ciated with chromosomal instability, being the polyploidization process
the most commonly observed variation. Nyman and Wallin (1992)
found high ploidy levels in strawberry plants obtained from protoplasts;
in addition, they observed a correspondence between certain anomalies
in morphology and chromosome duplication; e.g. leaves and petioles
from 16x and mixoploids plants were thicker and more rigid than those
of the octoploid counterparts, although the latter plants had more
leaves. Changes in leaf colour were also observed. In cv. ‘Chandler’, no
variation in the ploidy level was observed in the 6 protoclones ana-
lyzed. However, significant genetic variations were detected when
using microsatellite markers. The absence of changes in the ploidy level
in ‘Chandler’ protoclones could be due to the genotype, as has been
observed in other species; e.g. in potato, Jones et al. (1988) obtained
protoclones from leaf mesophyll with the same ploidy level than their
parent lines; however, Sadia (2015) observed variability in the chro-
mosome number. Alternatively, the use of 2,4-D in the culture medium
of protoplasts might also be the cause of the high incidence of ploidy
alterations reported by Nyman and Wallin (1992).
Among the vegetative characteristics analyzed, the number of
leaflet and the plant size showed great variability in the different pro-
toclones. Alterations in both parameters were related to genetic
changes, i.e. the two groups closer to the ‘Chandler’ control in the
7
Scientia Horticulturae 256 (2019) 108552
microsatellite analysis (protoclones 21, 3, 9 and 26) displayed similar
plant size than the control and three leaflets per leaf; however, the
group most distant to the control showed a variable number of leaflets
and included protoclones of low vigor. Nyman (1993) also observed
variability in the number of leaflets in plants derived from protoplasts
in the breeding line 77,101. Variants in leaf structure were also fre-
quent in strawberry somaclones derived from somatic embryos (Biswas
et al., 2009). Dwarf variants have been obtained in shoots regenerated
from leaf derived callus cultured in the presence of high BA and 2,4-D
concentrations (Nehra et al., 1992). Regarding fruit yield, a large
variability was observed among the different protoclones. This para-
meter was not related to the genetic changes detected with the micro-
satellite markers, i.e. some protoclones in the group close to the control
showed a reduced yield. A reduction in fruit size was also observed in
the strawberry breeding line 77,101 by Nyman (1993). Interestingly,
some of the vegetative characters analyzed correlated with fruit yield.
Thus, clones showing a lower yield had, in general, a growth pattern
between spreading and semiupright and a smaller plant area, while
clones with a higher production showed upright or semiupright growth
pattern. Actually, fruit yield was negatively correlated with the growth
habit (Pearson correlation coefficient of -0.647, significant at
P < 0.01). However, no significant correlations were found between
fruit yield and plant area, density of foliage, leaf color or fruit size. By
contrast, fruit weight was positively correlated with plant area (Pearson
correlation coefficient of 0.499, significant at P < 0.05).
In conclusion, a reliable protocol for protoplast isolation, culture
and plant regeneration in strawberry cv. ‘Chandler’ has been developed
in this research. This protocol could be applicable to other important
commercial cultivars showing similar in vitro response than ‘Chandler’,
e.g. ‘Camarosa’. Microsatellite markers showed a high incidence of so-
maclonal variation in the regenerated plants, although the ploidy level
was not affected. The protoclones obtained displayed a high variability
in the vegetative growth and fruit yield, and some of these changes
were related to the genetic changes detected with the microsatellites
study. Despite this variability, a few number of clones, 5% of the lines
analyzed, behaved like control plants, suggesting that the protocol es-
tablished could be useful for gene editing of strawberry.
Acknowledgements
This work was supported by INIASC97-009, INIA RTA2010-0027-
00-00 and Ministerio de Ciencia, Innovación y Universidades and
FEDER EU funds (grant number AGL2017-86531-C2-1-R).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.scienta.2019.108552.
References
Bairu, M.W., Aremu, A.O., 2011. Somaclonal variation in plants: causes and detection
methods. Plant Growth Regul. 63, 147–173.
Banks, M.S., Evans, P.K., 1976. A comparison of the isolation and culture of mesophyll
protoplasts from several Nicotiana species and their hybrids. Plant Sci. Lett. 7,
409–416.
Barceló, M., El-Mansouri, I., Mercado, J., Quesada, M.A., Pliego-Alfaro, F., 1998.
Regeneration and transformation via Agrobacterium tumefaciens of the strawberry
cultivar Chandler. Plant Cell Tissue Organ Cult. 54, 29–36.
Bhojwani, S.S., Dantu, P.K., 2013. Plant Tissue Culture: An Introductory Text. Springer,
New Delhi.
Biswas, M.K., Dutt, M., Roy, U.K., Islam, R., Hossain, M., 2009. Development and eva-
luation of in vitro somaclonal variation in strawberry for improved horticultural
traits. Sci. Hortic. 122, 409–416.
Boulay, M., 1985. Aspects pratiques de la multiplication in vitro des essences forestières.
Ann. AFOCEL 1984, 7–43.
Carlberg, I., Glimelius, K., Eriksson, T., 1983. Improved culture ability of potato proto-
plasts by use of activated charcoal. Plant Cell Rep. 2, 223–225.
Chen, W.H., Davey, M.R., Power, J.B., Cocking, E.C., 1988. Sugarcane protoplasts: factors
affecting division and plant regeneration. Plant Cell Rep. 7, 344–347.
M. Barceló, et al.
Davey, M.R., Anthony, P., Power, J.B., Lowe, K.C., 2005. Plant protoplasts: status and
biotechnological perspectives. Biotechnol. Adv. 23, 131–171.
Debnath, S.C., Texeira da Silva, J.A., 2007. Strawberry culture in vitro: applications in
genetic transformation and biotechnology. Fruit Veg. Cereal Sci. Biotechnol. 1, 1–12.
Eeckhaut, T., Lakshmanan, P.S., Deryckere, D., Van Bockstaele, E., Van Huylenbroeck, J.,
2013. Progress in plant protoplast research. Planta 238, 991–1003.
Eriksson, T.R., 1988. Obstacles and perspectives in protoplast research. In: In: Puite, K.J.,
Dons, J.J.M., Huizing, H.J., Kool, A.J., Koornneef, K.M., Krens, F.A. (Eds.), Progress
in Plant Protoplast Research. Current Plant Science and Biotechnology in Agriculture,
vol. 7. Springer, Dordrecht, pp. 7–14.
Geerts, P., Henneguez, A., Druart, P., Watillon, B., 2009. Protoplast electro-fusion tech-
nology as a tool for somatic hybridisation between strawberries and raspberries. Acta
Hortic. 842, 495–498.
Gil-Ariza, D.J., Amaya, I., López-Aranda, J.M., Sánchez Sevilla, J.F., Botella, M.A.,
Valpuesta, V., 2009. Impact of plant breeding on the genetic diversity of cultivated
strawberry as revealed by expressed sequence Tag-derived simple sequence repeat
markers. J. Am. Soc. Hortic. Sci. 134, 1–11.
Glimelius, K., Djupsjobacka, M., Felner-Feldegg, H., 1986. Selection and enrichment of
plant protoplast heterokaryons of Brassicaceae by flow sorting. Plant Sci. 45,
133–141.
Infante, R., Rosati, P., 1993. Fragaria vesca L. Alpine protoplast culture and regeneration.
Acta Hortic. 348, 432–434.
Jones, O.P., Waller, B.J., Beech, M.G., 1988. The production of strawberry plants from
callus cultures. Plant Cell Tissue Organ Cult. 12, 235–241.
Karp, A., 1993. Mechanisms of somaclonal variation. Biotechnol. Biotechnol. Equip. 7,
20–25.
Li, Y.-G., Stoutjestijk, P.A., Larkin, P.J., 1999. Somatic hybridization for plant improve-
ment. Morphogenesis in Plant Tissue Cultures. Springer, Dordrecht, pp. 363–418.
Lin, C.-S., Hsu, C.-T., Yang, L.-H., Lee, L.-Y., Fu, J.-Y., Cheng, Q.-W., Wu, F.-H., Hsiao,
H.C.-W., Zhang, Y., Zhang, R., Chang, W.-J., Yu, C.-T., Wang, W., Liao, L.-J., Gelvin,
S.B., Shih, M.-C., 2018. Application of protoplast technology to CRISPR/Cas9 mu-
tagenesis: from single-cell mutation detection to mutant plant regeneration. Plant
Biotechnol. J. 16, 1295–1310.
López-Aranda, J.M., Pliego-Alfaro, E., López-Navidad, I., Barceló-Muñoz, M., 1994.
Micropropagation of strawberry (Fragaria x ananassa Duch.). Effect of mineral salts,
benzyladenine levels and number of subcultures on the in vitro and field behaviour of
the obtained microplants and the fruiting capacity of their progeny. J. Hortic. Sci. 69,
625–637.
Malnoy, M., Viola, R., Jung, M.-H., Koo, O.-J., Kim, S., Kim, J.-S., Velasco, R.,
Nagamangala Kanchiswamy, C., 2016. DNA-free genetically edited grapevine and
apple protoplast using CRISPR/Cas9 ribonucleoproteins. Front. Plant Sci. 7, 1904.
Margara, J., 1984. Bases de la multiplication vegetative. INRA, Versailles, Paris.
Menczel, L., Nagy, F., Kiss, Z.R., Maliga, P., 1981. Streptomycin resistant and sensitive
somatic hybrids of Nicotiana tabacum + Nicotiana knightiana: correlation of resistance
to N. tabacum plastids. Theor. Appl. Genet. 59, 191–195.
Mercado, J.A., Barceló, M., Pliego, C., Rey, M., Caballero, J.L., Muñoz-Blanco, J., Ruano-
Rosa, D., López-Herrera, C., de los Santos, B., Romero-Muñoz, F., Pliego-Alfaro, F.,
2015. Expression of the β-1,3-glucanase gene bgn13.1 from Trichoderma harzianum in
strawberry increases tolerance to crown rot diseases but interferes with plant growth.
Scientia Horticulturae 256 (2019) 108552
Transgenic Res. 24, 979–989.
Morgan, E.R., 1999. Callus production from protoplasts of Cylamen persicum. Plant Cell
Tissue Organ Cult. 55, 63–65.
Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with
tobacco tissue cultures. Physiol. Plant. 15, 473–497.
Nehra, N.S., Kartha, K.K., Stushnoff, C., Giles, K.L., 1992. The influence of plant-growth
regulator concentrations and callus age on somaclonal variation in callus-culture
regenerants of strawberry. Plant Cell Tissue Organ Cult. 29, 257–268.
Nyman, M., 1993. Protoplast Technology in Strawberries. Ph.D. Thesis. Uppsala
University, Sweden.
Nyman, M., Wallin, A., 1988. Plant regeneration from strawberry (Fragaria x Ananassa)
mesophyll protoplasts. J. Plant Physiol. 133, 375–377.
Nyman, M., Wallin, A., 1992. Improved culture technique for strawberry (Fragaria ×
ananassa Duch.) protoplasts and the determination of DNA content in protoplast
derived plants. Plant Cell Tissue Organ Cult. 30, 127–133.
Ortín-Parraga, F., Burgos, L., 2003. Isolation and culture of mesophyll protoplasts from
apricot. J. Hort. Sci. Biotechnol. 78, 624–628.
Palomo-Ríos, E., Quesada, M.A., Matas, A.J., Pliego-Alfaro, F., Mercado, J.A., 2018. The
history and current status of genetic transformation in berry crops. In: Hytönen, T.,
Graham, J., Harrison, R. (Eds.), The Genomes of Rosaceous Berries and Their Wild
Relatives. Springer International Publishing, Cham, pp. 139–160.
Papadakis, A.K., Roubelakis-Angelakis, K.A., 2002. Oxidative stress could be responsible
for the recalcitrance of plant protoplasts. Plant Physiol. Biochem. 40, 549–559.
Papadakis, A.K., Simis, C.I., Roubelakis-Angelakis, K.A., 2001. Reduced activity of anti-
oxidant machinery is correlated with suppression of totipotency in plant protoplasts.
Plant Physiol. 126, 434–444.
Potrykus, I., Shillito, R.D., 1986. Protoplasts: isolation, culture, plant regeneration.
Methods Enzymol. 118, 549–578.
Sadia, B., 2015. Improved isolation and culture of protoplasts from S. chacoense and
potato: morphological and cytological evaluation of protoplast-derived regenerants
of potato cv. Desiree. Pak. J. Agric. Sci. 52, 51–61.
Shillito, D., Paszkowski, J., Potrykus, I., 1983. 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.
UPOV, 2012. Strawberry. Guidelines for the Conduct of Test for Distinctness, Uniformity
and Stability. http://www.upov.int.
Warren, G., 1991. Protoplast isolation and fusion. In: Stafford, A., Warren, G. (Eds.), Plant
Cell and Tissue Culture. Open University Press, Buckingham, pp. 48–81.
Woo, J.W., Kim, J., Kwon II, S., Corvalán, C., Cho, S.W., Kim, H., Kim, S.-G., Kim, S.-T.,
Choe, S., Kim, J.-S., 2015. DNA-free genome editing in plants with preassembled
CRISPR-Cas9 ribonucleoproteins. Nat. Biotechnol. 10–13.
Zhang, Y., Su, J., Duan, S., Ao, Y., Dai, J., Liu, J., Wang, P., Li, Y., Liu, B., Feng, D., Wang,
J., Wang, H., Zhu, J., Bi, Y., Si, C., Hu, B., Dong, G., Wang, H., Zhou, Z., Li, T., Tan, T.,
Pu, X., Wang, F., Ji, S., Zhou, Q., Huang, X., Ji, W., Sha, J., 2011. A highly efficient
rice green tissue protoplast system for transient gene expression and studying light/
chloroplast-related processes. Plant Methods 7, 30.
Zhou, J., Wang, G., Liu, Z., 2018. Efficient genome editing of wild strawberry genes,
vector development and validation. Plant Biotechnol. J. 16, 1868–1877.