EVALUATION OF THE in vitro ESTABLISHMENT OF UVA (Vitis vinifera) IN

THE LABORATORY OF VEGETAL BIOTECHNOLOGY OF THE UFPS,

CAMPOS ELISEOS HEADQUARTERS.

Margarita del Carmen Rosas Rivera 1610344

Email: [email protected]

Biotechnology Engineering

Faculty of Agricultural Sciences and the Environment

Francisco de Paula University Santander, Cúcuta - Norte de Santander

SUMMARY

The purpose of this report is to determine the disinfection protocol and the most appropriate
culture media for the establishment of grape explants (Vitis vinifera); Likewise, monitoring
formats for "in vitro" culture are designed and applied. It is important to perform the
procedures to obtain positive results in the micropropagation of the species. In order to
carry out in vitro sowing, very important characteristics must be taken into account, such as
the asepsis of the sowing room, utensils, equipment and the disinfection protocol that must
be carried out on the initial explants.

Keywords: Micropropagation, "in vitro", "ex vitro", explant, grape.

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 asepsis, growth and development of these
different tissues must be controlled.
1. INTRODUCTION
Microorganisms such as bacteria and
fungi should not grow, and tissues or
plants should show an optimal
        Micropropagation is a biotechnology
development. (Garciglia, 2014)
that is applied to plant species in order to
obtain a population of elite plants selected          To achieve an in vitro culture of
for their agronomic performance plants, tissues and organs (including
characteristics, free of viruses and in the seeds) must be surface sterilized (asepsis)
shortest possible time. It is known as a and cultivated in special nutrient
"rapid response" biotechnology, since solutions, often in agar-solidified media.
results are achieved ranging from 3 to 6 Suitable combinations of auxins and
months to 18 months (depending on the cytokinins, two of the main
species). It can be said that it is also phytohormones of plant growth, are
versatile since it adapts to the incorporated into these culture media.
requirements of each species under study, With the application of these and
by taking maximum advantage of the controlled cultivation such as pH, light
cellular power, to channel it towards mass and temperature, it is possible to
propagation, eradication of pathogens, reproduce all the factors that may affect
propagation of mother material, and the growth and development of tissues or
production of parental lines, among plants in vitro. (Garciglia, 2014)
others. (HortiCultivos, 2016)
The grape, cultivated from the very
          The term "in vitro tissue culture" beginning of history, is especially
means growing some parts of plants also nutritious. Economically it has an
called "explants", such as segments of extraordinary importance. It has been
leaf, stem and roots, in addition to other cultivated for its nutritional value, for its
tissues or plant organs, inside a glass jar healing properties and especially for the
in an artificial environment, in which production of wines. Can be consumed

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fresh in the form of juicy bunches, but vegetative period, when the plant lowers
there is a powerful industry that has its activity, the vine needs an
grown around this fruit. (Frías, 2012) accumulation of daily heat so that its
clusters ripen correctly. (Frías, 2012)
The vines belong to the family of the
ampelidáceas. The grapes have a The vines can be propagated by seeds,
spherical shape, are fleshy, juicy, and are stakes, layering or by grafting of barb or
grouped in clusters. They reproduce by yolk. The seeds are used mainly for the
stakes or grafts. In the commercial scale, production of new varieties. On the
the most used are the stakes, which are commercial scale, the stakes are the most
fragments of stems that are separated to used. In the case of those cultivars of
obtain new plants. They are sown at a difficult rooting, layers are used. (vitis
distance of two to three meters. Later all vinifera, 2019)
buds are pruned except the most vigorous,
             The graft of barb or yolk on
which is trimmed so that only two or
patterns, is used occasionally to increase
three buds remain. The plant that is
the life of the strains, the vigor of the
obtained forms a strong main stem,
plants and the yields. Where there are
similar to a small trunk. When it grows,
harmful soil organisms such as vine
the vine is tied to a tutor or guide of two
phylloxera, root gill nematodes and
meters high. When the fruit appears, it is
susceptible varieties such as Vitis vinifera
carefully pruned to reduce the number of
should be grown, it is necessary to
buds, so that the remaining shoots form
perform barbed or yolk grafts with the
grapes of better quality. (Frías, 2012)
desired varieties on a resistant pattern.
These plants are very resistant to winter (vitis vinifera, 2019)
frosts, but this resistance is reduced after
sprouting, when the first leaves appear.
Therefore, some vineyards are equipped
with frost fighting devices, which are
efficient but expensive. During the

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2. MATERIALS AND METHODS Medio de
Experiment # 1 Disinfection test. cultivo
Componente

Ms 100% 4,43 g/l

Myo- Inositol 100 mg/l

Sacarosa 30 g/l

Ácido ascórbico 50 mg/l

6- Benzylaminopurine 1,15 mg/l

(6BAP)

Agar 8,5

Evaluations every 7 days for 30 days. Ph 6,2

Variables to be evaluated: endogenous,
exogenous contamination, phenolization, Table 1. Composition of the
bud formation, shoot length. The plant establishment of grape

material for the establishment of grape

(Vitis vinifera) variety Uva Isabella was After 40 days of planting of grape (vitis
collected from the patio of the house of a vinifera) variety Isabella was made a pass
relative in the city of Cúcuta. The plant to plant material in the means of
material was in good phytosanitary treatments influence of hormones.
condition. The explants used in this
investigation were axillary buds.

In this phase, the following variables

were evaluated at 20 days:

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• Endogenous contamination and • Death: the death of the explant, was
exogenous contamination: the presence of evaluated by observation since the
bacterial or fungal colonies was observed explant is completely necrotic.
in the culture medium and / or explant.
Experiment # 2 Influence of hormones.
• Number of outbreaks
Preparatory phase
• Bud length: by measuring with ruler in
In this phase, the collection of plant
units of mm
material was carried out. The plant
• Callus formation material used were axillary buds of grape,
variety Uva Isabella collected in the patio
• Root training
of the house of a relative of the city of
• Bud formation: is the ability of the Cúcuta. Once the material was collected,
explant to break the latency phase and it was moved to the plant biotechnology
produce new shoots. laboratory.

• Phenolization: the phenolization of the Establishment phase. It consisted of two

explant, was evaluated by observation and stages: disinfection of the explant and
classified with values of grade 1 to grade establishment in vitro.
4.
        Disinfection of the explant
 Grade 1: light phenolization in the
explant

 Grade 2: medium phenolization in the

explant

 Grade 3: advanced phenolization in the

explant

 Grade 4: death occurs by phenolization Next, in the sterile area, the disinfection
in the explant. protocol was developed based on the

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addition of three solutions: A) soap laminar flow chamber. tools to be used
solution, B) 70% alcohol and C) 3% (scalpel, forceps, napkins and Kraft paper
sodium hypochlorite, each separately, sterilized previously).
during a given time of exposure as shown
Once the sowing area was adequate, with
in table 1. Once the time of exposure to
the sterile forceps, the explant was cut by
each disinfectant is complete, three rinses
cutting the axillary bud and then planted
are made with sterile distilled water with
in the culture medium.
antioxidant to eliminate waste, and then
axillary buds were extracted and sown. A 100% MS basal medium supplemented
Table 2. Disinfection protocol with growth regulators was used as
shown in the following table 3.
Componente Tiempo (mn)

Solución jabonosa 5 Componente Medio de

cultivo
(detergente
Ms 100% 4,43 g/l
comercial + agua)
Myo- Inositol 100 mg/l
Alcohol al 70% 3
Sacarosa 30 g/l
Hipoclorito de 20
Ácido ascórbico 50 mg/l
sodio al 3% +
tween 80 (2 gotas) Agar 8,5

Ph 6,2
* In laminar flow cabinet
Table 3

Planting of explants: sowing was carried

Add hormones
out in a laminar flow cabin previously
sterilized with ultraviolet light for 30 6BA 0 5µm 15
minutes, laminar flow for 30 minutes and P µm
booth cleaning with 70% alcohol, and all
ANA 0 2 µm 4 µm
the instruments were placed in the

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 Tratamiento Hormona Hormona
s 6BAP ANA
In the previous photograph the sowing of
1 (6 tubos) 0 0
the establishment of this plant species is
2 (6 tubos) 5 0 observed.
3 (6 tubos) 15 0
In this phase, the following variables will
4 (6 tubos) 5 2 be evaluated after 20 days:
5 (6 tubos) 5 4
• Endogenous contamination and
6 (6 tubos) 15 2
exogenous contamination: the presence of
7 (6 tubos) 15 4 bacterial or fungal colonies was observed
8 (6 tubos) 0 2 in the culture medium and / or explant.
9 (6 tubos) 0 4
• Number of outbreaks

• Bud length: by measuring with ruler in

units of mm

• Callus formation
After the seeding process, the tubes are
• Root training
sealed with polyethylene to wrap
(Envoplast), labeled and taken to the • Bud formation: is the ability of the
growing room, at a temperature of 20 ° C explant to break the latency phase and
± 2 ° C and photoperiod of 16 light produce new shoots.
hours / 8 dark. After 20 days of planting,
• Phenolization: the phenolization of the
the weekly evaluation of the explants
explant, was evaluated by observation and
begins.
classified with values of grade 1 to grade
4.

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 Grade 1: light phenolization in the
explant

 Grade 2: medium phenolization in the

explant
In the initial phase of the establishment
 Grade 3: advanced phenolization in the
10% of endogenous contamination was
explant
observed, in the disinfection protocol of 5

 Grade 4: death occurs by phenolization minutes which shows us that it was not as

in the explant. effective as compared to the disinfection

protocol of 15 and 20 minutes that
• Death: the death of the explant, was
reached 100%. In the phenolization
evaluated by observation since the
degrees 0 - 1 predominate. Zero
explant is completely necrotic.
indicating that there is no phenolization
and 1 light phenolization.

        In the establishment "in vitro" of

grape there was no death of the explants
due to phenolization or oxidation. It is
generally considered that intact and
3. RESULTS AND DISCUSSIONS healthy plant tissues are internally aseptic
Establishment "in vitro" of Grape and that the main task of cleaning
(Vitis vinifera) variety Isabella. material for explants is limited to
Experiment # 1 superficial sterilization (Roca &
Mroginski, 1993), which allows to deduce
that the protocol used was effective
Disinfection test

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 After 20 days, the reading was made
where a degree of phenolization 1-2 was
observed, with grade 2 predominating,
with an average phenolization of the
explant, the growth of the explants always
After 20 days of planting, it was possible
being advanced in treatment 3 (T3 20
to evaluate the percentage of sprouting of
MIN). after 60 days of planting the
the explants in each treatment, observing
appearance of the leaves, no exogenous
that the treatment T2, and T3 reach 100%
contamination was present and shows that
and the average length of the shoots in the
the average length of the plant was
treatment T3 shows a greater growth
greater in the treatment of 20 minutes of
compared to the T1 and T2 that is
the disinfection protocol. The success of
observed lower growth and slower. After
plant propagation systems by
40 days of planting in the medium 100%
biotechnology depends to a large extent
Ms and the addition of cytokinins (6-
on the control and prevention of
benzylaminopurine promoted a good
microbial contamination. There are
development of the explant, the passage
several strategies to control and manage
of the plant material was made to the
pollution, which include prevention
means carried out in the experiment # 2
through the selection and treatment of the
influence of the hormones After 20 days
mother plant, the surface disinfection of
of the sowing pass, the reading was made.
the explant and the identification of
contaminating microorganisms, the
control of contamination through the use
of antimicrobial substances. and the
cultivation of meristems. (Niedz &
Bausher, 2002). The physiological state
of the mother plant influences its
morphogenic capacity, since the

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totipontenciality decreases with age, 8 (6 tubos) 0 2
therefore, while the tissue is younger and 9 (6 tubos) 0 4
less differentiated (meristems, young non-
Influence of hormones.
woody explants in vegetative state and
larger) , the in vitro response will be The respective reading was carried out 20
improved, other factors must also be days after planting, as it can be seen, its
taken into account, such as that the degree of phenolization is in the range of
experimental material is healthy, since 2-3 of the explants where the grade 3 is
this affects the percentage of infection present, being an advanced phenolization
after isolation. (Coba, 2009) of the explant. The exogenous
contamination in the treatment T4 and T8
Establishment "in vitro" of Grape
was of a percentage of 33.3; and T6,
(Vitis vinifera) variety Isabella.
16.6% of contamination was presented,
Experiment # 2 Influence of hormones
which is within the permitted range. The
percentage of sprouting of the explants in
the treatments T3, T5, T7 exceeds 50
percent, demonstrating that the addition
of the growth regulating hormones (6BAP
and ANA) to the MS medium at 100%
Tratamiento Hormona Hormona
helps the development of the explant.
s 6BAP ANA

1 (6 tubos) 0 0         As for the budding of the explants,

we can clearly see the influence of plant
2 (6 tubos) 5 0
hormones in the sprouting process. It is
3 (6 tubos) 15 0
observed that the treatments that do not
4 (6 tubos) 5 2 incorporate the cytokinin 6 BAP have the
5 (6 tubos) 5 4 lowest results (T1, T8 and T9) with
6 (6 tubos) 15 2 values between 0 and 16.6%, while those
that incorporate it, either alone or
7 (6 tubos) 15 4
combined with the auxin ANA , they give
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the best results with values of 50 to reported by different authors [14], [16].
66.6%. (Pedro, 2016)

        Cavazos et al, 2018, found that the            [14], According to Fotini G.

application of the MS culture medium Skiada1, in the Micropropagation study
added with the growth regulator BAP, in of Vitis vinifera L.cv. "Malagouzia" and
different concentrations, allowed to "Xinomavro" performed the experiments
obtain better results in the stage of shoot on the effect of BA, the number of new
induction and multiplication to conserve shoots, as well as the proliferation rate of
in vitro the cultivars of Cabernet both cultivars increased in parallel with
Sauvignon vine. and Merlot. (Cavazos- the growth regulator, while the length of
Galindo, 2018) the seedlings, roots and the internodes
decrease in relation to the means of
          (6BAP) 6-Benzylaminopurine is a
control.
broad spectrum plant growth regulator. It
can accelerate the growth of cell. When            The combinations of BA and NAA
used with gibberellins, the shape of the revealed that in "Malagouzia" two media
fruit can be improved. 6- seemed to be more appropriate, those of
benzylaminopurine stimulates the 0.5 μM BA combined with 0.1 or 0.3 μM
following effects: cell division; NAA. Of these, the second was
Emergency side shoot (apples, oranges); considered to be more suitable because
Formation of basal shoots (roses, the first combination caused large
orchids); flowering (cyclamen, cactus); calluses to develop in the basal stem.
Set of fruits (grapes, oranges, melons). Hyperhydricity appeared in seedlings
(Delong chemistry CO., 2009) grown in other combinations, especially
when the high concentration of cytokinin
            The effect of the mineral
was used for 'Xinomavro', media
composition of the culture medium in the
supplementation with 0.1 μM BA and
in vitro culture of the vine has been
0.03 μM NAA seemed to favor
proliferation producing vigorous

11
seedlings with green leaves in complete presence of BA in the culture medium
form without Callus. (Skiada1, 2010) was a decisive factor in the growth of
axillary buds, elongation. In media
            [16], In the research conducted by
containing 1 or 2 mg / l of BA, the mean
Dev Rahul; which was entitled in vitro
number of shoots and axillary buds
comparative multiplication of some grape
developed by explants.
genotypes (Vitis vinifera) the application
of an MS medium containing growth            They were significantly greater
regulators BAP (2 mg / l) + NAA (0.2 mg than in his absence. Benzyladenine (BA)
/ l) and IBA (2 mg / l) ) + AC (200 mg / l) has been reported to be effective in
best found for the start of cultivation and improving axillary bud proliferation in
rapid multiplication, respectively. several species of Euvitis (Harris and
Stevenson 1982, Gray and Fisher 1985,
Heloir et al., 1997, Dzazio et al., 2002,
To improve the survival of the explant, Ayman et al. 2011). Heloir et al. (1997)
two cytokinins (BAP and Kinetin) and and Mhatre et al. (2000) found that higher
auxin (NAA) were tested in different BA concentrations stimulated axillary
combinations in grape genotypes. The bud growth without much mortality
beneficial role of BAP in the initiation of effect. (DEV, 2015)
the culture was More remarkable when it
was used together with a low level of
NAA. Among the growth regulator in the
treatments, 2.0 mg / l BAP + 0.2 mg / l
NAA gave the first sprouting of shoots
(11.6 days). Previously Mhatre et al.
(2000) reported that NAA 0.09 mg / l was
essential in the medium initiation culture
for three cultivars of V. vinifera. Ibáñez In the previous photograph we show that
and Alabama. (2005) found that the the treatments T3, T5, T7 there is an
advanced growth of the explant; The
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following table shows how many
hormones each treatment contains.
5. BIBLIOGRAPHICAL
4. CONCLUSIONS REFERENCES

 The disinfection protocol of 20 Cavazos-Galindo, J. (Enero de 2018).

minutes was chosen for the PROPAGACIÓN CLONAL DE
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and for the optimal development DE VID (Vitis vinifera) PARA
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 The 6BAP hormone is essential in file:///C:/Users/ufps/Downloads/1
the addition of 100% MS medium, 405-2768-polib-45-181.pdf
to obtain high sprouting rates of
Delong química CO., L. (2009). D
the buds of the grape crop (Vitis
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vinifera).
de
 The hormone ANA (naphthalene
http://www.bestplanthormones.co
acetic acid) at 4 micro molar must
m/plant-growth-regulator/cell-
be combined with a cytokinin
division-plant-hormones/6-
6BAP in the MS medium at
benzylaminopurine-plant-growth-
100%, otherwise this auxin does
regulator.html
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 The in vitro establishment of Vitis DEV, R. (11 de julio de 2015).

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contamination, was achieved with multiplication of some grape

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Universidad Michoacana de San
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