SURAT PERNYATAAN KONTRIBUTORSHIP

Yang bertanda tangan dibawah ini:

1. Nama : Endang Gati Lestari
Instansi : Pusat Riset Tanaman Pangan , Badan Riset dan Inovasi Nasional
2. Nama : Media Fitri Isma Nugraha
Instansi : Pusat Riset bahan baku obat dan obat tradisional. Badan Riset dan Inovasi
Nasional
3. Nama : Rossa Yunita
Instansi : Pusat Riset Hortikultura dan Perkebunan. Badan Riset dan Inovasi Nasional

Menyatakan bahwa makalah dengan judul “The Formula media in vitro Propagation and
Conservation of Ludwigia sp ” yang terbit di Journal of Tropical Biodiversity and
Biotechnology Volume 08, Issue 01 (2023): jtbb75947 DOI: 10.22146/jtbb.75947 Semua
penulis adalah Kontributor Utama.

Demikian surat penyataan ini kami buat untuk dapat dipergunakan sebagaimana mestinya.
Bogor, 13 Januari 2023
Penulis,

Endang Gati Lestari

Media Fitri Isma Nugraha

Rossa Yunita
 Journal of Tropical Biodiversity and Biotechnology
Volume 08, Issue 01 (2023): jtbb75947
DOI: 10.22146/jtbb.75947

Research Article

The Formula media in vitro Propagation and Conservation

of Ludwigia sp.
Endang Gati Lestari1*, Media Fitri Isma Nugraha1, Rossa Yunita1
1) National Research and Innovation Agency (BRIN), Jln Raya Jakarta Bogor Km 46, Cibinong Science
*Corresponding author, email: [email protected]

Keywords: ABSTRACT
conservation The aquatic plant “Red Malang” (Ludwigia sp.) has a fairly high economic value as
Ludwigia sp. an ornamental aquatic plant, so it has the potential to be developed. The growth of
propagation in vitro cultures in culture bottles is high-speed, so it is necessary to find a formula
Submitted: media to inhibit growth so that the frequency of subcultures is reduced. The
02 July 2022 current research aims to produce a formula media for shoot multiplication and in
Accepted: vitro culture conservation. The research was carried out at the ICABIOGRAD
12 December 2022 tissue culture laboratory from April 2020 to June 2021. Research activities included
Published: plant propagation, conservation, and regeneration after conservation. Plant
xxxx material was using in the form of a culture collection in the ICABIOGRAD tissue
Editor: culture laboratory, treatment media for propagation were BA (0; 0.1; 0.3; 0.5; 0.7
Furzani Binti Pa’ee and 0.9 mg/L) + thidiazuron (TDZ) (0 and 0.1mg/L). For conservation were MS
+ BA medium (0 and 0.1 mg/L) + paclobutrazol (0; 0.1; 0.3; 0.5; 0.7 mg/L) and for
shoot regeneration after conservation using MS medium without Plant Growth
Regulator (PGR). Data analysis using the Anova SAS version 9.0 test program.
Further test using DMRT test with alpha level 5%. There was no difference in the
mean value between levels of TDZ treatment on the number of shoots and leaves.
The difference in the mean value between levels of TDZ treatment was very
significant on shoot height, the number of roots, and root length. BA treatment
with a concentration of 0.7 mg/L is better because it gives higher results for each
observation variable. For conservation, treatment with paclobutrazol 0.5 mg/L
inhibited shoot and leaf count, and 0.3 mg/L inhibited shoot formation. Cultures
stored for six months grew normally after being regenerated. The highest shoots
and the highest number of leaves were obtained from the treatment of
paclobutrazol without BA. This study indicated that the propagation media of
aquatic plants Ludwigia sp. did not require high concentrations of BA. Cultures
could be stored for over six months using paclobutrazol with 0.3-0.6 mg/L.

Copyright: © 2023, J. Tropical Biodiversity Biotechnology (CC BY-SA 4.0)

INTRODUCTION
Red Malang (Ludwigia sp.) is an aquatic water plant that has been widely
developed, and its uses include beautifying aquariums. Ludwigia sp.
aquatic plants are attractive for commercialization because they have eco-
nomic value and are unique because of their bright red leaves. The bene-
fits of aquatic ornamental plants are an addition to beautifying aquariums
or fish ponds. It also protects fish from sunlight and improves water
quality because they can produce oxygen and absorb toxins such as am-
monia in the water and as a place for fish to lay eggs (Nugraha et al.
2018). Types of ornamental plants currently being developed include Ba-
copa genus, B. australensis, B. caroliniana, B. ianigera. B. myriophylloides. B.

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monnieri, B. rotundifolia. B. chamaedryoides (Kunth) Wettst (Syn. Herpestis

chamaedryoides Kunth). B. australis (Nugraha et al. 2017).
Aquatic plants' micro-propagation is still conventional (Yunita et al.
2018). Constraints from conventional propagation for mass production of
aquatic ornamental plants include non-uniform and non-sterile seeds pro-
duced. Developing tissue culture techniques to obtain significant and
sterile seedlings is necessary to solve the problem. Tissue culture tech-
nology currently being applied has many benefits, including mass propa-
gation of high-yield seedlings to assemble new varieties and preserve
germplasm (Siddique et al. 2015). The formula media for the propagation
of aquatic plants B. australis has been produced by Yunita et al. (2018),
Nugraha et al. (2017), and Nugraha et al. (2018), using MS media + 0.5
mg/L Benzyl Adenine (BA)+ 0.5 mg/L kinetin and 0.5 mg/L BA + 0.1
mg/L TDZ.
BA, a growth regulator from the cytokinin group, has been widely
used for shoot induction and propagation in vitro culture because BA has
a vital activity for cell division and is more stable than kinetin and zeatin
(Lestari 2011; M. et al. 2017). PGR, such as BA has a significant role in
cell division during plant metabolism in shoot induction and multiplica-
tion (Ashraf et al. 2014). The exact concentration of BA for each plant is
not the same depending on the type of plant, physiological conditions of
the explant, type of primary media, environmental conditions of growth,
and genetic factors (Lestari 2015). Propagation of ginger using ZPT 4.5
mg/L BA is the best concentration to stimulate shoot multiplication
(Abbas et al. 2011). Besides PGR, minerals are essential elements in me-
dia culture (Kumar & Reddy 2011). TDZ is a compound belonging to di-
phenyl urea and has almost the same activity as cytokinins, often used to
stimulate cell division in shoot proliferation, especially in woody plants
(Lestari 2015). Using a combination of TDZ with BA has succeeded in
increasing the ability of cell proliferation in various plants, for example,
Plumbago zeylanica (Syahid & Kristina 2008; Lestari et al. 2013).
In vitro culture conservation for active collection usually uses a for-
mula media for propagation to only last for 2-3 months because the nutri-
ents have run out (Dewi et al. 2016). For culture growth to be extended,
it needs to be inhibited to prolong the time of sub-culture. Reducing the
frequency of sub-cultures will lower maintenance costs and reduce the
risk of contamination (Dewi et al. 2016; Mendes et al. 2021). Conserva-
tion of aquatic ornamental plants B. australis and Alternatia reinecki using
MS media + 0.7 mg/L paclobutrazol showed inhibition until the 6th on
shoot height, number of shoots, number of roots, root length, and num-
ber of leaves. In this conservation medium, the culture remains green and
looks fresh (Lestari et al. 2021). In culture conservation, through in vitro
culture, several techniques can be applied, including cryopreservation,
simple conservation, and slow growth conservation (Dewi et al. 2016).
Conservation media techniques with slow growth techniques gen-
erally use chemical compounds to inhibit the growth of cultures, includ-
ing paclobutrazol, cycocel (CCC), and osmotic compounds such as sorbi-
tol and mannitol (Huang et al. 2014; Silva et al. 2019). Paclobutrazol is
an active compound inhibiting the oxidation of kaurene to ent-kaurene as
a precursor of the growth regulator of gibberellic acid in the apical meri-
stem, causing inhibition of cell division at the growth point (Negi et al.
2017; Bisht et al. 2018). In slow-growth conservation, growth and cell
division are conditioned to occur very slowly, or metabolism is stopped
so that culture development stops and does not change the genetic nature
of the plant (Lestari et al. 2021). The benefits of in vitro culture conserva-
tion include keeping germplasm accessions/collections from becoming
extinct (Huang et al. 2014) by inhibiting cell growth and division from
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becoming very slow (Indrayanti et al. 2018).

The advantages of in vitro culture for conservation include that it
does not require a large/wide place/container, accessions, or germplasm
stored in thousands and can be stored for more than five years (Dewi et
al. 2016). To maintain accession, which is very valuable germplasm has a
high economic value. In contrast, if it is stored conventionally, there is a
risk of damage due to natural disasters or drought (Silva et al. 2019). The
collection of germplasm accessions is also beneficial as genetic material in
the assembly of new varieties (Arrigoni-Blank et al. 2014). Formulas me-
dia for the propagation of aquatic ornamental plants are still limited,
namely B. australis (Nugraha et al. 2017; Yunita et al. 2018), as well as
formula media for in vitro culture conservation.
Lestari et al. (2021) conducted a conservation study through in
vitro culture of B. australis and A. reinecki using MS + paclobutrazol me-
dia. So many aquatic plants are currently being developed that research is
needed to obtain formula media for in vitro propagation and conservation.
The study aimed to produce the best formula media for the propagation
and conservation of ornamental plant cultures.

MATERIALS AND METHODS

Materials
Plant material is a sterile culture of aquatic plants aged two months and
collected of the Tissue Culture Laboratory.

Methods
The research was carried out at the tissue Culture Laboratory, the Indo-
nesian Centre for Agricultural Biotechnology and Genetic Resources Re-
search and Development (ICABIOGRAD), at Cimanggu street No. 3 Bo-
gor from April 2020 to July 2021.
The research consisted of 3 activities, namely (1) Testing the for-
mula media for propagation, (2) Testing the formula media for conserva-
tion (3) Testing the regeneration ability of the culture after conservation.
The media used were MS basic media plus macro salt, micro salt, vita-
mins from group B (thiamine, pyridoxine, nicotinamide acid), and myo
inositol, two grams of agar gel were added as a compactor, and 30 g of
sucrose as a carbon source.
The media's pH was 5.7 by adding 0.1 N HCl or NaOH solution.
The media was sterilized using an autoclave with a pressure of 121 Psi
for 15 minutes. Sterile culture bottles filled with 25 ml of treatment me-
dia

Test the media formula for propagation.

The ex-plant was used as a single node with a size of ± 1 cm. The experi-
mental design was factorial and completely randomized. consisting of
two factors. the first factor was the concentration of BA (0; 0.1; 0.3; 0.5;
0.7 and 0.9 mg/L). Moreover, the second factor was the concentration of
TDZ (0 and 0.1 mg/L). Each treatment was repeated in ten bottles. The
observed variables were shoot height, number of shoots, number of
leaves, number of roots, and root length. The basic medium commonly
used for shoot induction and shoot multiplication is MS base media
(Murashige & Skoog 1962).

Test the formula media for conservation.

Ex-plants are stem from sterile single-node culture ± 1 cm. The experi-
mental design was factorial and completely randomized, consisting of
two factors: two levels of BA concentration (0 and 0.1 mg/L), and the
second factor was five levels of paclobutrazol concentration (0; 1; 0.3; 0.5;
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0.7 and 0.9 mg/L). Bottles planted with ex-plants are placed on the cul-
ture rack under irradiation using a TL lamp. Illumination intensity 1500
lux for 16 hours in 1 day. The variables observed were shoot height,
number of shoots, number of leaves, root length, and number of roots.

Culture regeneration after conservation.

Cultures stored for ± six months were transferred to MS 0 media
(without PGR). Ex-plants in the form of stems of single nodes, one ex-
plant per bottle. The number of ex-plants from each conservation medi-
um was planted in as many as ten bottles. The observed variables includ-
ed shoot height, number of shoots, and number of leaves.

Data Analysis
The data were analysed using the Anova SAS version 9.0 test program,
further testing using DMRT with an alpha level of 5%.

RESULTS AND DISCUSSION

Results
Induction of shoot multiplication
Analysis of variance on the variables of shoot height, number of shoots,
number of leaves, and number of roots and interactions between TDZ
and BA treatments are presented in Tables 1 and 2. Analysis of variance
on all observed variables showed an interaction between TDZ treatment
and BA.

Table 1. The variance of each observation variable on shoot proliferation.

F-Calculate treatment
Variable Thidiazuron Benzil Adenin CV (%)
TDZ*BA
(TDZ) (BA)
Shoot height 107.88** 8.30** 6.39** 27.90
Number of shoots 0.05ns 2.57* 3.51** 46.19
Number of leaves 3.55 ns 1.59 ns 1.81ns 36.82
Number of roots 175.06 ** 3.25 ** 17.89** 68.35
Root length 137.28** 5.16** 10.52** 79.07
Note: ns) not significantly different at = 5% based on F-test results. *) signifi-
cantly different at = 5% based on F-test results. **) very significant difference
at = 1% based on F-test results. tdz*BA Interaction between this treatment and
BA. CV) Coefficient of Diversity.

Test formula media for conservation

Analysis of variance and Interaction between BA treatment and paclobu-
trazol on the variables of shoot height, number of leaves, number of
roots, root length and number of shoots are presented in Tables 3 and 4.

Shoot regeneration after conservation

Analysis of the various effects of BA with paclobutrazol treatments on
conservation media showed an interaction, and the results were signifi-
cantly different for all observed variables (Table 5). The effect of interac-
tion between BA and paclobutrazol during conservation on shoot regen-
eration ability is presented in Table 6.
The Interaction between TDZ and BA for shoot propagation
showed that BA treatment of 0.7 mg/L without TDZ produced the high-
est shoots at 5.93 cm. However, for the number of shoots, the number of
roots and root length were not significantly different (Table 2).
It is suspected that the Ludwigia sp. contains high levels of PGR,
both cytokinins and auxins, so in the media, without BA and TDZ quite a
lot of shoots were produced (Table 1). The results prove that the activity
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Table 2. Interaction of Thidiazuron and Benzyl Adenine. on shoot height, number of shoots, number of roots, and
root length five weeks after planting (MST).
Thidiazuron Benzil Adenin (mg/L) Mean
(mg/L) 0.0 0.1 0.3 0.5 0.7 0.9
shoot height (cm)
0.0 3.91b 3.80b 3.20bc 3.10bc 5.40a 3.80b 3.86
0.1 3.41b 1.83d 1.83d 1.86d 2.06d 2.51cd 2.25
Mean 3.66 2.81 2.51 2.48 3.73 3.15
CV (%) 7.90

Number of shoots
0.0 4.60abc 5.50ab 5.80ab 6.80a 3.80bc 5.40ab 4.35
0.1 7.00a 2.60c 6.20ab 5.70ab 5.70ab 4.10bc 5.21
Mean 5.8 4.05 6 6.25 4.75 4.75
CV (%) 46.19

Number of roots
0.0 7.50a 0.00c 8.02a 7.00a 0.00c 7.90a 5.1
0.1 3.20b 0.30c 0.00c 0.00c 0.00c 0.00c 0.58
Mean 5.35 0.15 4.01 3.5 0 3.95
CV (%) 8.35

Root length (cm)

0.0 0.00c 1.65a 1.05b 1.37ab 1.43ab 1.32ab 0.94
0.1 0.37c 0.00c 0.00c 0.00c 0.00c 0.00c 0.37
Mean 0.18 0.85 0.5 0.68 0.7 0.66
CV (%) 79.07
Note: Data followed by the same letter in the same variable are not significantly different based on DMRT test lev-
el = 5%.

Table 3. Analysis of the variance of each variable in the treatment of BA and paclobutrazol Six months after plant-
ing.
F calculate
Variable CV (%)
Benzil Adenin (BA) Paklobutrazol BA*paclobutrazol
Shoot height 17.9** 11.93** 29.8** 26.56
Number of leaves 8.99** 10.74** 6.55** 30.14
Number of roots 3.31ns 3.50* 5.21** 35.23
Root length 22.62** kon11.35 ** 0.52 ns 55.39
Number of shoots 7.12** 3.52* 11.48** 32.92
Note: ns) is not significantly different at = 5% based on the F-test results. *) is significantly different at = 5% based
on the F-test results. **) is significantly different at = 1% based on the F-test results. BA*paclobutrazol) interac-
tion between BA and paclobutrazol treatments. CV) Coefficient of diversity.

of growth regulators depends on the type of chemicals, chemical struc-

ture, and chemical concentration, plant genotype and physiological phase
(Satyavathi et al. 2004).
TDZ can be given together with other growth regulators, such as
cytokinins or auxins. At the initiation of mangosteen shoots, using BA
plus TDZ is the best treatment for shoot formation (Lestari et al. 2015).
TDZ stimulates shoot multiplication in several plants, including bread-
fruit (Supriati et al. 2005) and aromatic ginger, by adding 0.1 mg/L TDZ
to MS + BA 5 mg/L (Lestari & Hutami 2005). However, not all plants
responded to shoot multiplication in starfruit plants, TDZ only increased
shoot height and number of leaves (Supriati et al. 2005).
Paclobutrazol is a growth inhibitory compound that has been ap-
plied to inhibit the growth of cultures in in vitro culture. and in various
plants, including fruit trees (Hasan et al. 2013; Mendes et al. 2021). The
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Table 4. Effect of Interaction between BA and paclobutrazol on the growth of shoot height number of leaves, num-
ber of roots, and number of shoots.
Benzil Adenin Paclobutrazol(mg/L)
Mean
(mg/L) 0.1 0.3 0.5 0.7
Shoot height (cm)
0.0 2.50d 4.67b 2.95 cd 2.88cd 3.25
0.1 6.80a 3.30cd 3.78 c 2.90cd 4.19
Mean 4.65 3.98 3.36 2.89
CV (%) 26.56

Number of leaves
0.0 29.30bc 3.70b 20.10 d 23.10cd 26.3
0.1 47.20a 25.90bcd 25.67 bcd 30.30bc 32.26
Mean 38.25 29.3 282.88 26.7
CV (%) 30.14

Number of roots
0.0 18.30bc 21.40bc 15.00 c 21.50bc 19.05
0.1 29.30a 14.80c 20.56 bc 23.40ab 22.01
Mean 23.8 18.1 17.78 22.45
CV (%) 35.23

Number of shoots
0.0 1.40b 1.60b 1.10 b 1.50b 1.4
0.1 2.20 a 1.10 b 2.22 a 1.30b 1.7
Mean 1.8 1.35 1.66 1.45
CV (%) 32.92
Note: Data followed by the same letter on the same variable are not significantly different based on Duncan's test
with a level of = 5%.

Table 5. The variance of each variable of the effect of BA and paclobutrazol treatment media on shoot regeneration
after conservation.
F-Calculate
Variable
Benzil Adenin (BA) Paclobutrazol BA*Paclobutrazol CV (%)
Shoot height (cm) 6.59 * 1.74 ns 8.37 ** 33.81
Number of shoots 3.32 ns 7.54 ** 14.24 ** 46.77
Number of leaves 9.97 ** 4.76 ** 8.62 ** 38.40
Note: ns) is not significantly different at = 5% based on the results of the F-test. *) is significantly different at = 5%
based on the results of the F-test. **) significantly different at = 1% based on the results of the F-test.
BA*paclobutrazol interaction between the treatment of BA and paclobutrazol. CV=Coefficient of diversity.

effectiveness of paclobutrazol concentrate in inhibiting plant growth de-

pends on the physiological conditions of each plant and environmental
conditions (Mog et al. 2019). Its inhibitory effect is through the regula-
tion of physiological processes such as inhibiting plant size from shorten-
ing, namely the presence of short internodes, and reduction of leaf size
(Muengkaew & Chaiprasart 2016).
The research of Roostika et al. (2009) showed that Pimpinella pruat-
jan cultures conservation in vitro using paclobutrazol inhibitors also
caused robust inhibition, so the conservation period could not be extend-
ed more than four months from the culture during recovery a rosette ap-
pears. In addition to using paclobutrazol inhibitors to inhibit culture, it
can be done by diluting the primary medium to 50 and 75% in combina-
tion with the mannitol osmoregulator 20 g/L, as was done for conserva-
tion on Carica papaya Dieng. The culture can be stored in that media for
16 weeks (Rahayu et al. 2015).
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Table 6. Effect of Interaction of Benzyl Adenine and Paclobutrazol on Culture Growth after.
Benzil Adenin Paclobutrazol (mg/L)
(mg/L) 0.1 0.3 0.5 0.7
Shoot height (cm)
0.0 3.41e 4.97abc 5.23ab 5.93a
0.1 4.73bcd 4.47 bcde 3.85cde 3.78de
Mean 4.07 4.72 4.54 4.85
CV (%) 33.81

Number of shoots
0.0 1.72c 1.80c 1.73c 3.80a
0.1 1.93c 2.67b 1.60c 1.63c
Mean 1.82 2.2 1.66 2.71
CV (%) 46.77

Number of leaves
0.0 21.95cd 22.20cd 25.93bc 35.73a
0.1 19.23 cd 29.20 b 17.20 d 20.13cd
Mean 20.59 25.7 21.56 27.93
CV (%) 38.40
Note: Data followed by the same letter on the same variable are not significantly different based on Duncan's test
with a level of = 5%.

Cultures grown on media with paclobutrazol without BA showed

lower yields on all observed variables other than the number of roots.
Inhibition of shoot height growth and the number of leaves obtained
from treatment with paclobutrazol of 0.5 mg/L and paclobutrazol of 0.3
mg/L. It is inhibited the formation of the number of roots, root length,
and the number of shoots (Table 4).
The results showed that the paclobutrazol concentration up to 0.7
mg/L did not inhibit the culture when regenerated after conservation.
The average shoot height was 4.85 cm, the number of shoots was 2.71,
and the number of leaves was 27.9, but this concentration influenced
growth inhibition during Conservation (Table 4). Shoots regenerated
from the treatment of BA 0 + paclobutrazol 0.7 mg/L produced higher
shoots than those from treatment paclobutrazol 0.1-0.5 mg/L, as well as
for variables number of shoots and number of leaves except in the treat-
ment paclobutrazol of 0.5 mg/L (Table 6). Shoots from paclobutrazol 0.7
mg/L + BA 0.1 mg/L treatment shorter shoots were obtained and fewer
leaves. The study results by Mendes et al. (2021) showed that the citrus
cultures stored for 12 months using a paclobutrazol growth inhibitor did
not undergo genetic changes based on analysis using SSR markers.

CONCLUSIONS
Based on the results of this research, it can be concluded that the best
media for the propagation of Ludwigia sp. is MS primary media without
PGR. The best medium for culture conservation was MS + 0.5-0.7 mg/L
paclobutrazol. Cultures stored in 0.7 mg/L paclobutrazol for six months
produced the best response to growth after conservation.

AUTHOR CONTRIBUTION
All authors in this article have the same contribution as the main contrib-
utors both in research and in the preparation of the paper

ACKNOWLEDGEMENTS
We gratefully acknowledge ICABIOGRAD, who has provided laboratory
facilities and technicians, and Mr. Suparjo and Mr. Sukmawan, who has
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helped prepare planting media.

CONFLICT OF INTEREST
The Authors declare that there is no conflict of interest.

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