In vitro Propagation of Singgalang Cabbage (Brassica oleracea var. capitata L.) on Murashige and Skoog Modification Media for Preservation Purpose

Abstract

Singgalang Cabbage is one of the local cabbage cultivated on the foothills of Mount Singgalang, West Sumatra. Recently, the existence of this cabbage has decreased, so conservation and preservation efforts need to be carried out. This research was conducted to develop method for in vitro propagation and preservation of singgalang cabbage. The Murashige and Skoog (MS) were used as basal media with two experimental stages, i.e., shoot initiation with 6-Benzylaminopurine (BAP), and root induction and plantlet preservation with modification of MS media. The nodal and shoots were used as explants. The results showed that increment of BAP concentrations gave a significant effect on shoot initiation after 60 days of treatment. MS media-enriched with BAP 2 mg/L gave significant increment of shoots (4 shoots/nodus) and leaves (11.67) numbers when compared to other treatments. For root induction and plantlet preservation, it was found that the earlier of root formation was observed in modification of MS media at ½ and ¼ strength. Meanwhile, MS media at ⅛ strength was observed to be better media for plant height increment (4.75 cm) when compared to other treatments. It was found that the plantlets survived and grew well after 120 days under in vitro condition.

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Original Research Paper
In vitro Propagation of Singgalang Cabbage (Brassica oleracea var.
capitata L.) on Murashige and Skoog Modification Media for Preservation
Purpose
Maya Sari1, & Muhammad Idris1*
1Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Andalas,
Padang, Indonesia;
Article History
Received : Maret 25th, 2024
Revised : April 01th, 2024
Accepted : April 27th, 2024
*Corresponding Author:
Muhammad Idris,
Department of Biology ,
Faculty of Mathematics and
Natural Sciences, Universitas
Andalas, Padang, Indonesia;
Email: midris@sci.unand.ac.id
Abstract: Singgalang Cabbage is one of the local cabbage cultivated on the
foothills of Mount Singgalang, West Sumatra. Recently, the existence of this
cabbage has decreased, so conservation and preservation efforts need to be
carried out. This research was conducted to develop method for in vitro
propagation and preservation of singgalang cabbage. The Murashige and
Skoog (MS) were used as basal media with two experimental stages, i.e.,
shoot initiation with 6-Benzylaminopurine (BAP), and root induction and
plantlet preservation with modification of MS media. The nodal and shoots
were used as explants. The results showed that increment of BAP
concentrations gave a significant effect on shoot initiation after 60 days of
treatment. MS media-enriched with BAP 2 mg/L gave significant increment
of shoots (4 shoots/nodus) and leaves (11.67) numbers when compared to
other treatments. For root induction and plantlet preservation, it was found
that the earlier of root formation was observed in modification of MS media
at ½ and ¼ strength. Meanwhile, MS media at ⅛ strength was observed to be
better media for plant height increment (4.75 cm) when compared to other
treatments. It was found that the plantlets survived and grew well after 120
days under in vitro condition.
Keywords: 6-Benzylaminopurine, MS modification media, root induction,
shoot initiation, singgalang cabbage.
Introduction
Cabbage (Brassica spp.) is a widely
cultivated vegetable crop in Indonesia that is
divided into two main types, namely annuals
and biennial types. Annual cabbage is widely
cultivated because it can flower in the tropics
and produce seeds. This is in contrast to biennial
cabbage, which cannot flower in the tropics, so
the provision of seedlings only comes from
shoot cuttings, which causes the quality of
seedlings to decrease (Singh et al., 2010).
Singgalang Cabbage (Brassica oleracea var.
capitata L.), one of the local cabbages of West
Sumatra, has been cultivated for a long time by
farmers in the area around the slopes of Mount
Singgalang. The three variants are Biaso,
Batang Hitam, and Senggan (Afdi et al., 2005).
Singgalang cabbage has a distinctive
flavor widely used in West Sumatra's culinary
specialties, but its existence is decreasing due to
declining of farmers to cultivate it. It was
caused by the reason of less productivity of this
vegetable when compared to introduced
cultivars. On the other hand, according to
(Rincon-Sanchez & Ruiz-Torres, 2018) local
cultivars have higher potential than introduced
cultivars, including being more adaptive to the
local climate and tolerant of environmental
stresses. In addition, the lack of farmers to
cultivate singgalang cabbage is also caused by
the retention of pests and diseases and the
conversion of land functions for annual cabbage
planting. Therefore, efforts to maintain the
existence of the singgalang cabbage variant to
exist as a typical vegetable of West Sumatra are

Idris et al., (2024). Jurnal Biologi Tropis, 24 (2): 128 – 138
DOI: http://dx.doi.org/10.29303/jbt.v24i2.6651
129
significant to be attempted, one of which is
through tissue culture techniques and in vitro
storage so that its existence will be maintained
in the Future.
Tissue culture techniques for mass
propagation and preservation purposes have
been widely developed, including in cabbage
species (Alawadi et al., 2019; Gambhir et al.,
2017; Gerszberg et al., 2015; Pavlović et al.,
2010; Srikanth et al., 2016). Gerszberg et al.
(2015) used Murashige-Skoog (MS) basal
medium with the addition of 6-
Benzylaminopurin (BAP) in shoot propagation
of eight B. oleracea var. capitata cultivars.
Rahman et al. (2021) also used BAP to
regenerate shoots in vitro on MS media for B.
oleracea var. italica plants. The range of BAP
used in the above research commonly from 1-5
mg/L.
In germplasm storage or preservation,
tissue culture techniques play a pivotal role in
the conservation of genetic diversity through the
use of growth inhibition technique (Chauhan et
al., 2019; Gianní & Sottile, 2015; Trejgell et al.,
2015). The preservation of genetic diversity is
further facilitated by minimal growth strategies.
In the context of in vitro preservation utilizing
minimal growth techniques, this entails the
reduction of incubation temperatures and the
modification or manipulation of the culture
medium, thereby altering the availability of
nutrients (Chen & Dribnenki, 2004).
Explant with roots is important in
preservation of plant material genetics. Research
conducted by Ghanbar et al. (2016) and Islam et
al. (2017), found that the use of MS ½ is better
for root induction. Before, Azad et al. (2005)
found that increasing the MS strength was better
for root formation, while Dhavala & Rathore
(2010) found that MS ½ is the most effective
medium for root induction.
Based on the description above, research
on in vitro propagation of singgalang cabbage
(B. oleracea var. capitata) needs to be done.
Given the limited planting area and the
declining farmers interest in cultivation of this
vegetable, the existence of this cabbage will be
reduced or even lost if there is no effort to
propagate and preserve it. The tissue culture
technique is an alternative way to maintain its
existence as a local vegetable with high cultural
value for the Minangkabau tribe in West
Sumatra.
Materials and Methods
Time and place
This research was conducted in the
Laboratory of Plant Physiology, Department of
Biology, Faculty of Mathematics and Natural
Sciences, Universitas Andalas. It was held from
February to May 2023.
Plant material
The singgalang cabbage variant used in
this experiment was Biaso variant. The samples
were collected in the foothills of mount
Singgalang. The shoots were cultured in MS
media to get nodal free contaminated explants
(Figure 1).
Figure 1. Source of singgalang cabbage explants
used in this experiment. The explants were grown in
MS media without growth regulator for
approximately 4-6 weeks in each sub-culture cycle.
Experiment design
This research was conducted in an
experimental method using a completely
randomized design (CRD) consisting of four
treatments and six replications. The experiment
was conducted in two stages, i.e.:
1. In vitro shoot initiation from nodal explants.
In this stage, the nodal were planted in MS
media supplemented with 6-
Benzileaminopurine (BAP) as follows:
A= MS as a control (without BAP)
B= MS + 1 mg/L BAP
C= MS + 2 mg/L BAP
D= MS + 3 mg/L BAP
2. In vitro root induction from shoot explants.
In this stage, the shoots were planted in
modification of MS media as follows:
A= MS full strength (MS)
B= MS half strength (MS ½)
C= MS a quarter strength (MS ¼)

Idris et al., (2024). Jurnal Biologi Tropis, 24 (2): 128 – 138
DOI: http://dx.doi.org/10.29303/jbt.v24i2.6651
130
D= MS one-eighth strength (MS ⅛)
Research procedure
Sterilization of equipment
Glass bottles, tweezers, scalpel handles,
and a Petri dish were soaked in commercial
bleaching solution for one day. All equipment
was washed and rinsed with running tap water
then dried in the oven before sterilized using
autoclave (17.5 psi and 121°C for 15 min).
Preparation of MS media stock solution
In this experiment, five stock of MS
media solutions were prepared containing
macronutrient, micronutrient, iron sources,
vitamins, and myo-inositol. Their solubilities
were 10x concentration for macronutrients, 50
times concentration for micronutrient, iron
sources, and vitamins, and 20x concentration for
myo-inositol based on MS media recipe by
Murashige and Skoog (1962).
Preparation of treatment media
a. Shoot initiation media using BAP.
The treatment media for shoot initiation
were MS media supplemented with BAP at the
concentration of 0-3 mg/L. For each treatment
media, BAP stock solution (100 mg/L) was
pipetted based on concentration mentioned
above. The pH of media was adjusted at 5.8-6
using pH meter. For one liter of MS media, 30 g
sucrose and 7 g agar were added before boiled
and poured into culture bottles. The bottles were
tightly closed with a sterilized bottle cap. All
treatment media were sterilized using an
autoclave (17.5 psi 121°C for 15 min). The
sterilized media were kept in the culture room
for 3-7 days before being used to check for
contamination.
b. Root induction media using modification of
MS concentration.
The modification of MS concentration
was used for root induction media. The
concentration of MS media was modified from
full strength to one-eighth strength (MS ⅛).
After preparation of each stock solution based
on concentration treatments, the procedure was
similar to preparation of shoot initiation media,
except for using of BAP stock solution.
Planting explants into treatment media and
growth maintenance in growth room
The explants used for treatment consist of
nodal and shoots. Nodal was used as explants
for shoot initiation, while shoots for root
induction. Nodal explants were planted into MS
media supplemented with BAP, while shoot
explants were planted into modification of MS
media. All explants were cultured in treatment
media, sealed with transparent parafilm before
kept in growth room. The culture bottles were
placed in the growth room for maintaining the
explants growth. The growth room was set
under photoperiodism 12HL/12HD with
temperature 25±1oC. The cultured bottles were
kept under this condition until 60 days for shoot
initiation and 30 to 120 days for root induction
and plantlet preservation.
Parameter of observation and data analysis
The parameters were observed in this
experiment including 1) shoot initiation - the
first day of shoot emergence, number of leaves,
number of shoots, shoot length, and root
formation, 2) root induction and plantlet
preservation - plantlet height, first day of root
emergence, number of primary roots, root
length, and plantlet morphology. All data were
collected and categorized into qualitative and
quantitative data. Qualitative data were analyzed
descriptively, while quantitative data were
analyzed using Analysis of variant (ANOVA) at
p < 0.05. If the ANOVA was significantly
difference between each treatment, the data
were then analyzed using Duncan’s New
Multiple Range Test (DNMRT) at p<0.05.
Result and Discussion
In vitro Shoot Initiation
Average of first day shoot emergence
Table 1 shows that the average of first day
shoot emergence is not significantly different
between MS and MS media supplemented with
BAP. It was shown that using MS media or the
addition of BAP (plant growth regulator) did not
affect the rapid emergence of a shoot on nodal
explants. It was showed that BAP does not
affect the time of shoot emergence. BAP is a
synthetic cytokinin that induces shoot in the
explant (i.e., leaves cutting, nodal, and petiole)

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DOI: http://dx.doi.org/10.29303/jbt.v24i2.6651
131
and stimulates the growth of axillary and
adventitious buds. In can be concluded that
addition of BAP in MS media did not accelerate
the emergence of shoot in this experiment by
using nodal explants.
Table 1. The average of first day shoot emergence in
nodal explants of singgalang cabbage on MS media
supplemented with BAP (mg/L)
Treatment
First-day of shoot
emergence (days after
planting)
MS
6,83 ± 0,12 a
MS + 1 BAP
7,17 ± 0,03 a
MS + 2 BAP
7,50 ± 0,06 a
MS + 3 BAP
7,67 ± 0,07 a
Note: The values are means ± SE of 6 explants (n =
24). Numbers followed by the same letter indicate no
significant difference on DNMRT at p<0.05.
Number and length of shoot
Based on the results in Table 2, the
average number of shoots in MS media
supplemented with BAP 2 mg/L has the highest
shoots numbers when compared to other
treatments. The high average number of shoots
shows that BAP has an effect on initiation of
new shoots in the meristematic area of nodus.
Number of leaves
Table 3 shows that the highest average
number of leaves was found in the MS treatment
with the addition of 2 mg. L-1 BAP, significantly
different from the control treatment and other
treatments. The high number of leaves is due to
the large number of new shoots formed. Plant
leaf growth is influenced by the number of
axillary buds formed due to the interaction of
exogenous growth regulators and endogenous
growth regulators in the explants.
Table 2. The average of shoots numbers and shoot
length in nodal explants of singgalang cabbage on
MS media supplemented with BAP (mg/L).
Treatment
Number of
shoots*
Length of
shoot (mm)
MS
1,13 ± 0,08 a
3,82 ± 0,63 a
MS + 1 BAP
1,27 ± 0,08 a
3,64 ± 0,90 a
MS + 2 BAP
1,87 ± 0,30 b
3,76 ± 0,25 a
MS + 3 BAP
1,12 ± 0,12 a
2,37 ± 0,31 a
Note: The values are means ± SE of 6 explants (n =
24). Numbers followed by the same letter indicate no
significant difference on DNMRT at p<0.05. *Data
were transformed with √𝑥 before statistically
analysed.
Table 3. The average of leaves number in nodal
explants of singgalang cabbage on MS media with
the addition of BAP (mg/L)
Note: The values are means ± SE of 6 explants (n =
24). Numbers followed by the same letter indicate no
significant difference on DNMRT at p<0.05.
Observation of root formation
Based on Figure 2, the morphology of
shoots produced from nodal explants shows that
treatment B (MS+1 BAP) is the explant with the
most root formations compared to other
treatments. This treatment shows that the
addition of BAP is able to form roots. Table 4
shows that the treatment media with the addition
of BAP affects the formation of roots in
singgalang cabbage node explants. The roots
that appeared in the MS treatment were longer
than those with the addition of BAP.
In vitro Root Induction and Plantlet
Preservation
Height of plantlet
Based on table 5 shows that the ⅛ MS
treatment has a high growth of (4.74 cm) which
is significantly different compared to other
treatments. In this study, the lower the
concentration in the media showed the higher
the plants formed. seen in MS media as a
control has a plantlet height (1.99 cm) is the
lowest plantlet height growth compared to other
explants.
Treatment
Number of leaves
MS
6,83 ± 0,94 a
MS + 1 BAP
6,83 ± 0,94 a
MS + 2 BAP
11,67 ± 0,98 b
MS + 3 BAP
5,33 ± 0,42 a

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DOI: http://dx.doi.org/10.29303/jbt.v24i2.6651
132
Figure 1. Morphology of shoot produced from nodal
explants in (a) MS media, and MS media
supplemented with (b) 1 mg/L BAP, (c) 2 mg/L
BAP, and (d) 3 mg/L BAP.
Table 4. The average of root formation in nodal
explants of singgalang cabbage on MS media with
the addition of BAP (mg/L)
Table 5. The average of plantlet height on
modification of MS media 30 days after planting
Treatment
Height of plantlet (cm)
MS
1,99 ± 0,30 a
MS ½
2,72 ± 0,82 a
MS ¼
2,63 ± 0,60 a
MS ⅛
4,74 ± 0,80 b
Note: The values are means ± SE of 6 explants (n =
24). Numbers followed by the same letter indicate no
significant difference on DNMRT at p<0.05.
Average of first day root emergence
Based on table 6 shows that the average
days to appear roots on ⅛ MS is significantly
different from the other treatments. In the
treatment it can be seen that the lower the
concentration of media given, the longer it takes
to appear roots on singgalang cabbage. This
difference can be caused by low nutrients in the
tissue and endogenous cytokinin so that plants
are not optimal enough in root formation.
Table 6. The average of first day root emergence on
modification of MS media 30 days after planting.
Treatment
First day of root emergence
MS
7,50 ± 0,19 a
MS ½
8,17 ± 0,03 ab
MS ¼
8,17 ± 0,03 ab
MS ⅛
10,50 ± 0,21 b
Note: The values are means ± SE of 6 explants (n =
24). Numbers followed by the same letter indicate no
significant difference on DNMRT at p<0.05.
Number and length of roots
The results in Table 7 show that the
average number of roots is not significantly
different between the control treatment and
other treatments. The roots produced in both
media treatments did not affect the average
number of roots. Judging from the results of the
study, the lower the concentration of media
given, the lower the number of roots produced
by the explants. This is because the endogenous
hormones in the explants are more focused on
the formation and elongation of shoot height
rather than root formation.
Table 7. The average of root numbers and length on
modification of MS media 30 days after planting
Treatment
Number of
roots
Root length
(mm)
MS
5,16 ± 1,35 a
72,50 ± 0,63 a
MS ½
5,83 ± 1,07 a
86,33 ± 0,90 a
MS ¼
5,00 ± 0,77 a
70,83 ± 0,25 a
MS ⅛
4,00 ± 0,51 a
61,67 ± 0,31 a
Note: The values are means ± SE of 6 explants (n =
24). Numbers followed by the same letter indicate no
significant difference on DNMRT at p<0.05.
Growth of plantlet after preservation in rooting
media
Figure 3. shows the appearance of
singgalang cabbage plantlets after being stored
in MS modification media for 120 days. It can
be seen that reducing the strength of MS media
(from ½ to ⅛ did not affect the growth of the
plantlets. The growth of plantlet in MS
modification media is better when compared to
MS full strength. It can be concluded that
reducing the concentration of MS media is still
able to support the growth of plantlet in the
media for preservation purpose.
Treatment
Presence/absence of
root formation (%)
MS
( 50.00 )
MS + 1 BAP
( 66.67 )
MS + 2 BAP
( 16.67 )
MS + 3 BAP
( 33.33 )

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133
Figure 3. Growth of singgalang cabbage 120 days
after planting in (a) MS (b) MS ½ (c) MS ¼, and (d)
MS ⅛.
Discussion
Micropropagation of cabbage varieties for
conservation and preservation purposes
The plant tissue culture system allows the
aseptic propagation of plant material
environments with high multiplication rates
(Sharma et al., 2018). Plant tissue culture
technique has been reported as an effective tool
to conserve many plant species, especially of
tropical origin (Engelmann, 2011). For the
short- and mid-term conservations, various
techniques have been developed, which not only
results in slow growth of the cultures but also
prolongs the time interval between two
subcultures (Cordeiro et al., 2014). In vitro
culture under slow-growth conditions is
supposed to be the most effective method of
plant germplasm conservation. The use of this
approach is aimed at slowing down the growth
of cultures and prolonging the interval between
two successive transfers (Cordeiro et al., 2014).
Tissue culture techniques for mass
propagation purposes have been widely
developed, including in cabbage species
(Pavlovic et al., 2010; Gerszberg et al., 2015).
In most Brassica species, the success of in vitro
regeneration is mostly dependent on the
genotype and the influence of plant growth
regulators (Ravanfar et al., 2009). The addition
of cytokinin and auxins would enhance shoot
multiplication in many species (Pierik, 1997;
Razdan, 2003; Thorpe, 2007; George et al.,
2008).
It was explained before that the use of MS
media for shoot propagation and root induction
have been widely performed in Brassica species
(Gerszberg et al., 2015; Pavlovic et al., 2010;
Rahman et al., 2021; Farooq et al., 2023;
Kaminska & Sliwinska, 2023). The use of
growth regulator is common in the tissue culture
of Brassica species (for review, see Ravandar et
al., 2017; Gerszberg, 2018). Conservation of
genetic sources of Brassica species has been an
important part of the research to preserve and
produce new varieties or clones of the species
for improving their ability to cope with climate
change. Preservation by tissue culture technique
is now developed by researcher beside the use
of seeds in Brassica species (Hammer et al.,
2018; Subramanian et al., 2023).
The role of cytokinin in shoot initiation
Cytokinin represents a class of growth
regulators comprising small quantities of non-
nutritive organic compounds that can either
support, inhibit, or modify various physiological
processes within plants. The overarching
function of growth regulators, broadly speaking,
is to induce and facilitate morphogenesis across
cell, tissue, and organ cultures (Nisak et al.,
2012). The efficacy of a tissue culture technique
is contingent upon the judicious use of growth
regulators. In tissue culture, cytokinin plays a
pivotal role by fostering cell division in the
utilized explants and promoting shoot
development. Within the realm of in vitro plant
propagation, cytokinin is strategically employed
to mitigate apical dormancy and enhance the
branching of lateral shoots emanating from
axillary shoots. Cytokinin serves to stimulate
shoot formation, influence cell metabolism, and
activate dormant cells, with their principal
function being the promotion of cell division.
According to Badriah et al. (1998),
cytokinin affects shoot initiation. The most
commonly used type of cytokinin is BAP
because of its high effectiveness (Yusnita,
2003). BAP is one of the cytokinin containing
adenine, which is active in induction shoot
formation (Sutriana et al., 2014) and can work
effectively in induction cell division and shoot
multiplication in plants (Azis et al., 2017).
Research by Ravantar et al. (2011), showed that
adventitious shoots can be regenerated for plant
propagation in vitro as produced in B. oleracea
sub-sp. Green Marvel. Farzinebrahim et al.
(2012) have regenerated and propagated B.
oleracea var. italica in vitro where MS media
supplemented with 1 mg/L BAP and 1.5 mg/L
indole-3-butyric acid (IBA) gave the highest
number of formations of new shoots.
The role of MS media modification in root
induction and in vitro preservation of plantlet
Media modification is one of the
important steps for rooting induction of the
shoot produced in vitro. Many experiments
conducted by researchers chose to reduce
concentration of basal media composition for
improving root induction and addition some of

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growth regulator from auxin group as also
applied in Brassica species. Alam et al. (2008)
used MS ½ for root induction of five oilseed
Brassica species. This media was supplemented
with low concentration of IBA and NAA. Basak
et al. (2012), also used MS ½ for rooting
process of mustard shoots produced by in vitro
technique. Attaya et al. (2017), using MS and
MS ½ to evaluate the ability of shoot to produce
roots for canola varieties in vitro.
In vitro culture is an effective method for
ex situ conservation of plant genetic diversity,
allowing rapid propagation from minimal plant
material and exerting little impact on wild
populations. Two types of in vitro preservation
methods are employed in tissue culture: a)
growth inhibition and b) cryopreservation. The
first method is utilized for the medium-term
preservation of genetic resources (from several
months to several years), while the second
method is employed for long-term preservation
extending over decades or more (Day & Stacey,
2007). The development of in vitro slow-growth
storage methods has emerged as a viable
alternative for the medium-term preservation of
fruit germplasm (Neveen & Bekheet, 2008). The
goal of medium-term storage is to extend the
duration between subcultures by reducing
growth. This objective can be achieved through
the application of various strategies, such as
modifying environmental conditions, changing
culture media, using growth inhibitors, low
temperature, and osmotic regulators
(Kameswara, 2004; Chauhan et al., 2019).
Slow-growth retention via in vitro culture
has been documented across a wide spectrum of
species (Maqsood & Muhammad, 2010;
Engelmann, 2011). To ensure the integrity of in
vitro cultures, regular subculturing under
standard conditions is essential to reduce the
risk of contamination and safeguard stock
material from potential damage (Niino &
Arizaga, 2015). Modification of media
composition is another way to enhance plant
preservation through tissue culture. This
technique is commonly used by reducing the
basal media concentration without causing
deficiency symptoms in the explants that growth
in vitro (Ashrafi et al., 2009; Vahdati et al.,
2009; (Khas et al., 2020).
Conclusions
Based on the results explained above, it
was concluded that (1) The use of MS media
with the addition of 2 BAP significantly
enhances shoot initiation, resulting in increased
numbers of shoots and leaves in singgalang
cabbage (2) MS modified media did not show a
significant effect on root induction and growth,
which was significantly in MS 1/8 and plantlet
can be preserved for 12 weeks in these
treatments media.
Acknowledgements
This research was funded by
SIMBELMAWA DIKTI with Program
Kreatifitas Mahasiswa- Riset (PKM-RE) 2022.
Thanks to Zainal S.P. for laboratory assistance
during the research. Thanks also to Dr. Zozy
Aneloi Noli, Suwirmen, MS and Dr. Nurainas
for critical discussion of this article.
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