Electronic Journal of Biotechnology 27 (2017) 80–83

Contents lists available at ScienceDirect

Electronic Journal of Biotechnology

Short communication

In vitro propagation of Vitis vinifera L. cv. ‘Monastrell’

Tània San Pedro a, Rosa Peiró b, Joan Villanova b, Antonio Olmos a, Carmina Gisbert b,⁎
a
Instituto Valenciano de Investigaciones Agrarias, Centro de Protección Vegetal y Biotecnología, Carretera de Moncada a Náquera km 4.5, 46113 Moncada, España
b
Instituto de Conservación y Mejora de la Agrodiversidad Valenciana, Universitat Politècnica de València, Edificio 8E: Escalera J, Camino de Vera s/n, 46022 Valencia, España

a r t i c l e i n f o a b s t r a c t

Article history: Background: A protocol for the micropropagation of the grape (Vitis vinifera L.) cultivar ‘Monastrell’ was
Received 20 October 2016 developed. Initial plant material was obtained from the sanitary selection of grapevine plants performed by
Accepted 22 March 2017 real-time RT-PCR to confirm the absence of Grapevine fanleaf virus, Arabis mosaic virus, Grapevine
Available online 29 March 2017 leafroll-associated virus 1, Grapevine leafroll-associated virus 3, and Grapevine fleck virus.
Results: The effects of the salt composition (comparing Lloyd and McCown woody plant medium and Murashige
Keywords:
and Skoog medium 1/2 macronutrients) and the growth regulator benzylaminopurine (BAP), at 0 and 8.9 μM, on
Mourvedre
Bud induction
plant propagation were evaluated using nodes as explants. The most efficient procedure consisted of bud
Grapevine induction in the medium with Lloyd and McCown woody plant salts and 8.9 μM BAP for 30 d along with
Micropropagation elongation in cytokinin-free medium for 60 d, which gave 22 nodes/explant (174 plants/initial plant). A
Mineral salts second cycle of propagation in a medium without BAP for another 60 d could give approximately 10,000
Node explants nodes, which can be obtained after an additional 2 months of culture. All plants acclimatized after the second
Quality wines cycle of multiplication were successfully transferred to soil.
Real-time RT-PCR Conclusion: We developed an optimal protocol for V. vinifera cv. ‘Monastrell’ micropropagation, the first described
Virus-free grapevine
for this cultivar.
Vitis vinifera micropropagation
Wineries
© 2017 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction it is crucial to use the best available planting material. In this context,
propagation from virus-free materials by micropropagation is of great
Grapevine (Vitis vinifera L.) is one of the most important edible interest because currently propagation of grapevine is performed
fruit crops cultivated worldwide and is mainly used in wineries [1,2]. by wood cuttings. Moreover, multiplication or culture by in vitro
The vast majority of quality wines around the world are made from procedures is of value in the application of techniques such as induced
cultivars resulting from natural or deliberate crosses between different mutation and selection, in vitro screening, and germplasm exchange
varieties belonging to V. vinifera subsp. vinifera [3]. To maintain the [11]. Despite the usefulness of this technique, micropropagation
resulting combination of the distinct genotypes involved in the crosses, attempts using grapevine have had limited success [12,13]. Recently,
which leads to their distinctive characteristics, vegetative propagation micropropagation of several V. vinifera cultivars has been described:
is the common method of grape multiplication. The use of in vitro ‘Malagouzia’ and ‘Xinomavro’ by Skiada et al. [14]; ‘Brasil,’ ‘Sun Red,’
culture for vegetative multiplication, termed micropropagation, offers ‘Pinotage,’ and ‘Zinfandel’ by De Carvalho-Silva et al. [15]; and ‘Pusa
an important alternative to conventional methods of plant propagation Navrang,’ ‘Pearl of Csaba,’ and ‘Julesky Muscat’ by Dev et al. [16].
[4,5,6] and is an important tool to initiate breeding programs [7]. The The work conducted using V. vinifera, interspecific hybrids, or
use of efficient micropropagation protocols will result in the production grape-related species has illustrated the influence of the genotypes and
of numerous plants that can be maintained under controlled conditions the salt composition of the culture medium on the micropropagation
in a reduced space until their transfer to the field for growing or grafting. procedure [14,16,17,18]. Therefore, this work aimed to develop a
In grapevine, virus infection is common and affects the yield and micropropagation protocol for a selected clone of ‘Monastrell,’
fruit quality and therefore may affect wine quality [8,9]. In addition, confirmed as virus free, and compare the most common salt
incompatibility problems can be acute in infected vines when compositions used for grapevine: MS 1/2 [17,19] versus Woody (W)
grafting [10]. Considering the high cost of establishing a vineyard, plant salts [13,20]. ‘Monastrell’ is a grapevine cultivar that originated in
the Valencian region of Spain, and it is very important in the Alicante
⁎ Corresponding author.
designation of origin (DO), Spain. This cultivar is also commonly used in
E-mail address: [email protected] (C. Gisbert). seven DOs in Eastern Spain (Valencia, Bullas, Almansa, Jumilla,
Peer review under responsibility of Pontificia Universidad Católica de Valparaíso. Yecla, Benisalem-Mallorca, and Pla i Llevant) and in Southern France

http://dx.doi.org/10.1016/j.ejbt.2017.03.006
0717-3458/© 2017 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
 T.S. Pedro et al. / Electronic Journal of Biotechnology 27 (2017) 80–83 81

(Provence), where it is known as Mourvedre. This cultivar is also used 0.1 mg/L indolebutyric acid (IBA). Afterward, clones of virus-free
to a lesser degree in five other Spanish DOs [21]. To the best of our plants were obtained and cultured in tubes. The pH of the medium
knowledge, there are no micropropagation protocols for this cultivar. was adjusted to 5.8 before sterilization at 121°C for 20 min. The
cultures were incubated in a growth chamber at 26 ± 2°C under a
2. Materials and methods 16-h photoperiod with cool white light.

2.1. Plant material, virus analysis, and in vitro culture

2.2. Shoot multiplication: effects of mineral salts and benzylaminopurine on
The sanitary status of a single asymptomatic plant of cv. growth and proliferation
‘Monastrell’ was evaluated as described by López-Fabuel et al. [22]
to test for Grapevine fanleaf virus (GFLV), Grapevine fleck virus Four ‘Monastrell’ plants (7–9 cm tall), grown in in vitro culture for 45 d
(GFkV), Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine and obtained from the initial virus-free plant, were used as the source of
leafroll-associated virus 3 (GLRaV-3), and Arabis mosaic virus (ArMV). nodes. Eight nodes per plant (each bearing a single axillary dormant bud)
Viral isolates of each of these virus species, maintained in a screened were obtained and cultured (one node per tube; Fig. 1a) in tubes
greenhouse at the Instituto Valenciano de Investigaciones Agrarias, containing 16 mL of medium B or W [similar to B but with Lloyd and
were used as positive controls. Data acquisition and analysis were McCown woody plant salts (DUCHEFA, The Netherlands): 0.25 mg/L
performed using StepOne Plus 2.0 software. The cv. ‘Monastrell’ was CuSO4·5H2O, 36.7 mg/L FeNaEDTA, 6.2 mg/L H3BO3, 22.30 mg/L
cultured in vitro in basal medium B [Murashige and Skoog salts MnSO4·H2O, 0.25 mg/L Na2MoO4·2H2O, and 8.6 mg/L ZnSO4·7H2O as
(1/2 macronutrients) plus vitamins (DUCHEFA, The Netherlands)] micronutrients; 72.5 mg/L CaCl2, 471.26 mg/L Ca(NO3)2·4H2O,
that contains 0.025 mg/L CoCl 2 ·6H 2 O, 0.025 mg/L CuSO 4 ·5H 2 O, 170 mg/L KH2PO4, 990 mg/L K2SO4, 180.54 mg/L MgSO4, and 400 mg/L
36.7 mg/L FeNaEDTA, 6.20 mg/L H3BO3, 0.83 mg/L KI, 16.90 mg/L NH4NO3 as macronutrients; 2 mg/L glycine; 100 mg/L myo-inositol;
MnSO4·H2O, 0.25 mg/L Na2MoO4·2H2O, and 8.60 mg/L ZnSO4·7H2O 0.5 mg/L nicotinic acid; 0.5 mg/L pyridoxine HCl; and 1 mg/L thiamine
as micronutrients; 166 mg/L CaCl2, 85 mg/L KH2PO4, 950 mg/L KNO3, HCl as vitamins] supplemented with 0 or 8.9 μM benzylaminopurine
87.86 mg/L MgSO4, and 825 mg/L NH4NO3 as macronutrients; 2 mg/L (BAP) (Fig. 1b). On day 30 of culture, explants cultured on media
glycine, 100 mg/L myo-inositol, 0.5 mg/L nicotinic acid, 0.5 mg/L containing BAP were transferred to baby food jars containing medium B
pyridoxine HCl, and 0.1 mg/L thiamine HCl as vitamins; 20 g/L or W (depending on their initial medium) (Fig. 1c). The number of
sucrose; 7.5 g/L plant agar; polyvinylpyrrolidone (0.1 g/L); and sproutings and yield (number of nodes obtained/initial plant, from the

a b

c d e f

g h i

Fig. 1. (a) Nodes on culture media at day 0. (b) Explants grown on medium W or B supplemented or not with BAP (8.9 μM) after 30 d of culture. (c) Shoots induced in BAP-containing media
and developing in media without growth regulators. (d, e, f) Shoots developed after 90 d of culture. (g) Plants, 15 d after acclimatization. (h, i) Micropropagated plants grown under
greenhouse (h) and field conditions (i).
82 T.S. Pedro et al. / Electronic Journal of Biotechnology 27 (2017) 80–83

eight initial nodes) were measured 30 and 90 d after the start of the initial observed during the protocol developed here, possibly because of the
culture (Fig. 1d). This assay was performed twice. use of another genotype, differences in the composition of the culture
Chi-square test was used to analyze the percentage of sprouting at media, or the transfer of shoots induced in a BAP-containing medium
30 and 90 d of culture. The effect of the media on yield (number of to a medium without growth regulation for elongation.
nodes per initial plant after 60 and 90 d of culture) was analyzed Yield, measured as the number of nodes obtained from an initial
using ANOVA. As significant differences were found, the means were plant after a period of culture, was calculated after 60 and 90 d of
separated by a post-hoc Tukey HSD test (P b 0.05). The Statgraphics initial culture. Statistical differences were found between the media at
program was used for all the analyses. both times of initial culture (P-value = 0.0022 at 60 d and 0.0001
For a second multiplication cycle, four plants (10 nodes per plant) at 90 d). The yield observed from the explants cultured on medium W
obtained from the best procedures (media with W + BAP) were was approximately double that of explants cultured on medium B, in
cultured on medium W without cytokinin for another 60 d (Fig. 1e). the absence or presence of BAP, on both days of scoring (Fig. 2).
The percentage of sprouted buds and the number of nodes of the Therefore, it was concluded that medium W is better than B for
developed plants were noted at the end of this period. the in vitro growth of the grape cv. ‘Monastrell.’ The most efficient
multiplication was obtained from nodes cultured on medium
2.3. Acclimatization and growth in greenhouse conditions W supplemented with BAP and elongated in medium W; 174
shoots—5–15 cm tall—were obtained from each initial plant (8 nodes)
Twenty plants obtained after the second cycle of multiplication were at day 90 of the initial culture, averaging 21.75 nodes/explant. This
acclimatized in pots containing soil and vermiculite (1:1). The plants result is better than that obtained by De Carvalho-Silva et al. [15]
were covered with a plastic vessel for 1 week and were grown in a using a lower BAP concentration and similar time of culture for four
chamber with 70–80% humidity, 26 ± 2°C, and 1160 lx luminance cultivars of V. vinifera (ranging from 1.9 to 2.8 nodes/explant).
for 20 d. Then they were transplanted to pots and grown under Medium W has an auxin, indole-butyric acid (IBA), that favors rooting
hydroponic conditions in a greenhouse. A sample of these plants was and also contains polyvinylpyrrolidone that may favor rooting
transferred to the field. induction [26]. Concerning the mineral composition of the media, the
main differences were the higher levels of SO2- 3-
4 , PO4 , and Ca
2+
and
3. Results and discussion lower NO-3 in medium W than in medium B. Moreover, the thiamine
HCl concentration was 10 times higher in medium W.
The analysis of the mother plant, a clone of cv. ‘Monastrell,’ was After the initial propagation step, the number of clones can
performed to confirm the absence of GFLV, ArMV, GFkV, GLRaV-1, and be increased by using a second cycle of multiplication. Of the 40
GLRaV-3 in the starting plant material. Only positive controls gave a nodes (extracted from four plants) obtained in the first cycle of
successful amplification by real-time RT-PCR, while the mother plant propagation and cultured on medium W without cytokinin for 60 d,
tested negative for all five viruses. Subsequently, four plants were 38 shoots sprouted and grew (each with 6.53 ± 0.21 nodes/shoot).
obtained from this initial virus-free mother plant to use as sources of Therefore, in this second cycle of multiplication, approximately 62
nodes and to determine the effects of the mineral salt composition nodes were obtained per plant (6.53 nodes/shoot × 38 shoots/4
and/or BAP addition on ‘Monastrell’ in vitro plant growth and bud plants). Considering that we obtained 174 plants in the first cycle,
induction. The effect of culture medium mineral composition on overall approximately 10,000 nodes (174 plants × 62 nodes/plant)
the in vitro culture of grapevine has been reported by different could have been produced to start a third multiplication step.
authors [14,16,17,18]. With regard to the addition of cytokinin to Finally, the acclimatized plants were 8.4 ± 0.40 cm tall, on average,
the culture medium, which is essential to increase multiplication in 20 d after transplanting. All the plants transferred for growing under
micropropagation procedures, the BAP concentration chosen in this greenhouse and field conditions were adapted (Fig. 1g–i).
study was similar to that used by Alizadeh et al. [23] for the In conclusion, the salt composition of medium W doubled the yield
micropropagation of four grape rootstocks (8.9 μM) and by Abido with respect to medium B, with and without the addition of BAP. By
et al. [24] for the grapevine cv. ‘Muscat de Alexandria.’ In addition, following the most efficient micropropagation procedure of those
this dose of BAP was reported as adequate with regard to inducing tested (nodes of the mother plant cultured on medium W containing
new buds with good development in other species. For instance,
Bhatt et al. [25] considered this concentration optimal for five
200
Alocasia species; higher concentrations (22.2 or 44.4 μM) induced a
pale and stunted shoots. 180
Yield (multiplication rate)

After 30 d of culture (Fig. 1b) in media without cytokinin, bud break 160
was observed in approximately 45% of the explants (precisely, 37.5% of 140
those cultured on B and 50% of those cultured on W), whereas in the b
120 b
BAP-containing media, new bud induction was observed in 87.5% of
100 a
the nodes cultured on B + BAP and in all the nodes (100%) cultured
on W + BAP. The Chi-square test comparing the two media without 80 b c
cytokinin showed no significant difference (P-value = 0.78). Similarly, 60
b
no difference was obtained when comparing the two media with c
40
cytokinin (P-value = 0.85). Adventitious buds were transferred to
20
media without growth regulators for elongation (Fig. 1c); the
remaining nodes were maintained in the corresponding tubes for 0
sprouting or elongation. After 90 d of culture, 62.5% sprouting was 60d 60d 60d 60d 90d 90d 90d 90d
achieved on medium B and 87.5% on medium W; no significant B W B+BAP W+BAP B W B+BAP W+BAP
difference between media B and W was found (P-value = 0.84). For
both media with BAP, 100% of the nodes had new shoots. Days of culture on each medium
Adequate elongation of shoots was produced for all treatments
Fig. 2. Mean values of the propagation rate (yield) after 60 and 90 d of culture on media B
(Fig. 1d). In grape, difficulties in shoot elongation [13] or deficiencies and W, supplemented or not with BAP (8.9 μM). Yield: number of nodes obtained per
such as vitrification [23] in BAP-containing media have been initial plant after a period of culture. Mean values separated by different letters are
described. Difficulties in shoot elongation or vitrification were not significantly different (P b 0.05) according to Tukey's test.
 T.S. Pedro et al. / Electronic Journal of Biotechnology 27 (2017) 80–83 83

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