In Vitro Propagation and Secondary Metabolite Production of Medicinal Plant of Euchresta horsfieldii (Lesch) Benn.

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Received January 30, 2023; Revised February 15, 2023; Accepted February 20, 2023; Published March 1, 2023
*Co rrespo nding author Andriyana Setyawati, andriyanasetyawati@staff.uns.ac.id, Tel: +62-852-9394-2727, Fax: +62-0271-637457
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Plant Breed. Biotech. 2023 (March) 11(1):34~48
https://doi.org/10.9787/PBB.2023.11.1.34
Online ISSN: 2287-9366
Print ISSN: 2287-9358
RESEARCH ARTICLE
In Vitro Propagation and Secondary Metabolite Production of
Medicinal Plant of Euchresta horsfieldii (Lesch) Benn.
Andriy ana S etyaw ati1*, S amanhudi S amanhudi1,2, Wuri Pra me s w ari 3, Daimon Syukri4, De frita Fitri Ramadhani1, Okky Tali tha1
1Department of Agrotechnology, Faculty of Agriculture, Universitas Sebelas Maret (UNS), Surakarta 57126, Indonesia
2Center for Research and Development of Biotechnology and Biodiversity, Universitas Sebelas Maret (UNS), Surakarta 57126,
Indonesia
3Department of Agriculture, University of Bengkulu, Bengkulu 38371, Indonesia
4Department of Agricultural Technology, Andalas University, Padang 25175, Indonesia
ABSTRA CT Euchresta horsfieldii (Lesch) Benn. is a highly demanded medicinal plant with many benefits. In vitro
p
ropagation
through callus induction is an effective method for rapid multiplication in a short time. This research aimed to evaluate the effective
concentration of Benzyl Amino Purine and 2,4-Dichlorophenoxiacetic acid on callus induction and organogenesis for in vitro
propagation and secondary metabolite production of E. horsfieldii. The design used in this research was a Complete Randomized
Design in 2 factors: Benzyl Amino Purine and 2,4-Dichlorophenoxiacetic acid with 5 levels for each concentration. Parameters
observed are the percentage of inducted calluses, callus appearance time, the weight of fresh calluses, the weight of dry calluses, the
texture of callus, the color of callus, percentage of the formed shoot, shoot appearance time, the height of shoots, the number of leaves,
flavonoid compounds of callus, and bioactive compounds. The result showed that the single treatment of Benzyl Amino Purine and
2,4-Dichlorophenoxiacetic was significantly affecting the shoots forming. The concentration of 2,4-Dichlorophenoxiacetic 0 ppm gave
the highest average plantlet height by 2.67 cm, increasing the number of shoots by 2.00, and the number of leaves by 5.60. E. horsfieldii
cultured in vitro without additional growth regulators had a higher flavonoid content.
Key words Euchresta horsfieldii, Gas chromatography-mass spectroscopy, Flavonoids, Tissue culture
INTROD UCTION
Euchresta horsfieldii is an Indonesian medicinal plant.
E. horsfieldii is a shrubs plant in the family Fabaceae with
a 45 cm average height. Branches and branchlets are glab-
rous and striate lengthwise. It has thickly papery leaves
with a length of 3-5 cm. Caly x 5-6 m m in diameter, 5-lobed
at the tip, obliquely nearly truncated, with very tiny ap-
pressed hairs (Wu and Raven, 2010). E. horsfieldii has
many medicinal benefits in each of its parts. The fruit of E.
horsfieldii is commonly used as an aphrodisiac and as a
cure to snake poison by Bali’s native villagers (Sutomo and
Mukaromah, 2010). Its seeds and fruits also can be used to
treat lung diseases such as asthma, and TBC (Heyne 1987;
Kloppenburgh 2006; Tirta et al. 2010; Kim et al. 2011).
With those huge potentials and high demand, in vitro
propagation is urgently needed to preserve the population
of E. horsfieldii.
In vitro propagation, is a biotechnological technique that
aids humans in the production of plants. Because of its
ability to generate material in a short amount of time, this
technology is extremely beneficial to plant breeding. Many
types of plants, including decorative plants and forest trees,
have been propagated by this method. In vitro propagation
has also aided in the conservation of rare and endangered
plants that are found in endemic environments or under

In Vitro Propagation and Secondary Metabolite of Pronojiwo (Euchresta horsfieldii (Lesch) Benn.) ∙35
other unique conditions (Chokeli et al. 2020). This appro-
ach, however, has never been used to increase E. horsfieldii
population. As a result, we investigated an effective callus
induction approach using the shoot of E. horsfieldii as an
explant for bulk replication of this medicinal plant. The in
vitro technique entails some procedures in order to produce
the desired output, which in this case is optimum E.
horsfieldii calluses and shoots. The outcome of an in vitro
procedure is impacted by some factors, including media
composition and plant growth regulators (Bidabadi and
Jain, 2020). A particular amount of auxin and cytokinin can
be combined to produce optimal calluses. BAP and 2,4-D
are cytokinins and auxins that have been successfully used
as plant growth regulators in optimum conditions (Gul et
al. 2020). This research aimed to evaluate the effective
concentration of BAP and 2,4-D on callus induction and
organogenesis for in vitro propagation of E. horsfieldii,
which could lead to a future investigation of in vitro
regeneration of this medicinal plant.
MATERIALS AND METHODS
Plant material and seed germinatio n
This research was conducted from August to November
2021 in the Laboratory of Plant Physiology and Biotechno-
logy Faculty of Agriculture, Sebelas Maret University. The
study began with the sterile germination of E. horsfieldii
seeds, followed by medium preparation, initiation, and
observation. This study used a completely randomized
design (CRD) with 25 treatment combinations that were
duplicated three times. Explants of a month-old E.
horsfieldii shoots, fungicide, Murashige and Skoog (MS)
medium with B AP (0; 0.5; 1; 1.5; 2 ppm) and 2,4-D (0; 0. 5;
1; 1.5; 2 ppm), aquadest, 70% alcohol, spirtus, tween 80
percent, and chlorox were employed. Parameters observed
are the percentage of inducted calluses, callus appearing
time, the weight of fresh calluses, the color of callus, the
texture of callus, percentage of formed shoots, shoots
appearing time, the height of shoots, and the number of
leaves.
Tools and explants sterilization
Sterilization was done for the tools and seeds. For the
tools sterilization, tweezers, culture knife (scalpel), and
petri dish are among the dissection equipment that is
cleaned. The instruments were then disinfected in an
autoclave for 30 minutes at 121℃ and 1 atm pressure
before being stored in the oven to retain their sterility. The
seeds were sterilized twice. E. horsfieldii seeds were
removed from their seed coat. The peeled seeds were
properly washed under running tap water, steeped in a
detergent and aquadest solution (20 mL detergent for 100
mL aquadest) for 1 minute, cleaned on aquadest, then
soaked in a fungicide (0.1 g for 100 mL aquadest) for 30
minutes, then cleaned on aquadest for four times. Further
sterilization was carried out in an aseptic environment
under a laminar air flow cabinet by soaking the seeds in a
chlorox 2.5% solution for 30 seconds, followed by soaking
in alcohol 35% for 30 seconds, then washing twice with
sterile aquadest.
Culture medium
The medium was produced by adding varying concen-
trations of BAP and 2,4-D to Murashige & Skoog (MS)
based medium. Medium making starts with 30 g of sugar,
50 mL of MS macronutrients, 10 mL of MS micro-
nutrients, 50 mL of Fe-EDTA, 50 mL of vitamins, 6.25 mL
of BAP (0 ppm (B0); 0.5 ppm (B1); 1 ppm (B2); 1.5 ppm
(B3); 2 ppm (B4)) and 6.5 mL of 2,4-D (0 ppm (D0); 0.5
ppm (D1); 1 ppm (D2); 1.5 ppm (D3); 2 ppm (D4)),
followed by 1,000 mL of distilled water and pH control up
until 6.2. As a controller, NaOH was used to lower pH
while HCl was used to raise it. Furthermore, 8 grams of
agar powder was added, heated with a magnetic stirrer until
it boiled, then transferred to a culture bottle and autoclaved.
The best treatment for callus induction and organogenesis
was determined using a controlled media without the
addition of plant growth regulators.
Expl ant ini tiation and mai ntenance
Explants were made by growing E. horsfieldii seeds in a
sterile environment. The seeds were planted in a cotton
medium and germinated in an aseptic environment. After

36 ∙Plant Breed. Biotech. 2023 (March) 11(1):34~48
Table 1 . The average percentage of E. horsfieldii callus induction at single treatment of BAP and 2,4-D concentration.
2,4-D (ppm) BAP (ppm) 2,4-D Average
00.511.52
0 33.3 33.3 100 100 100 73.33a
0.5 66.6 100 100 100 100 93.33
b
1 100 100 100 100 100 100
b
1.5 100 100 100 100 100 100
b
2 100 100 100 100 100 100
b
BAP average 80a80.67ab 100
b
100
b
100
b
-
In Table 1, a, b are the significant concentration levels, with arepresents the least and brepresents the highest of callus
induction percentage. Numbers followed by the same letter in the same rows or column are not significantly different
at 5% level DMRT. The (-) sign indicates there is no interaction.
one month, the explants were harvested. The second shoot
of the germinated seeds was used as an explant in this
study. Scissors were used to separate the shoot from the
main plant. The shoot’s leaves were trimmed to reduce
their weight and allow it to stand in the medium. Tweezers
were used to plant the shoots in the medium, one for each
culture bottle. The explants in this study were kept in a
growing environment with a temperature of 25℃ for 24
hours under white fluorescent light. To keep the bottles
aseptic and prevent contamination, they were sprayed with
70% alcohol regularly.
Data col lectio n and analysi s
The color and texture of calluses data are described and
the rest parameters were analyzed by ANOVA (Analysis of
Variance) 5% then continued by DMRT (Duncan Multiple
Range Test) 5% if the data showed a significant result.
The design used in this research was a Complete Ran-
domized Design in 2 factors: Benzyl Amino Purine and
2,4-Dichlorophenoxiacetic acid with 5 levels for each
concentration: 0; 0.5; 1; 1.5; and 2 ppm. 25 combinations
were obtained and repeated 3 times producing 75 experi-
ment units.
RESU LTS
Inducti on of callus
The percentage of inducted calluses demonstrates that
BAP and 2,4-D had a considerable impact on E. horsfieldii
callus growth. Table 1 demonstrates that practically all
2,4-D treatments were able to generate calluses 100% of
the time. It reveals that the treatment with 2,4-D 0 ppm
concentration was considerably different from the other
treatments, while 2,4-D treatments of 0.5; 1; 1.5; and 2 ppm
had similar results. However, treatment with a 0 ppm
concentration of BAP was completely different from
treatment with a 1; 1.5; and 2 ppm concentration of BAP.
Thus, it can be seen that treatments 1 to 3 ppm in both BAP
and 2,4-D were the more optimal concentration because the
percentage of inducted callus at that concentration was
100% (Table 1).
Callus developments are marked by the appearance of a
bulge or swelling at the base of the explant. However, the
result of the ANOVA analysis of callus appearance time
showed an insignificant number (P ＞ 0.05). Therefore, the
DMRT test is unlikely to be done. Fig. 1 displayed the
complete data of the callus appearance time for each
treatment. Treatment B2D2 or the combination of BAP 1
ppm + 2,4-D 1 ppm had the quickest average callus appear-
ance time, along with BAP 1.5 ppm + 2,4-D 1 ppm (B3D2),
1.5 ppm + 2,4-D 1.5 ppm (B3D3), and 1.5 ppm + 2,4-D 2
ppm (B3D4), with a 5-day appearance time. Treatment
B4D1 consists of a combination of BAP 2 ppm + 2,4-D 0.5
ppm, with a 9-day appearing seed, which had the slowest
average appearing time. It can be concluded that B2D2 is
the best treatment to induce callus fast. The time it takes for
a callus to form is mostly controlled by the amount of
exogenous auxin present.
Table 2 displayed the complete data of callus texture that

In Vitro Propagation and Secondary Metabolite of Pronojiwo (Euchresta horsfieldii (Lesch) Benn.) ∙37
Fig. 1. Histogram of callus appearance time of E. horsfieldii at 8 WAP. B0D0: 0 ppm BAP + 0 ppm 2,4-D, B0D1: 0
ppm BAP + 0.5 ppm 2,4-D, B0D2: 0 ppm BAP + 1 ppm 2,4-D, B0D3: 0 ppm BAP + 1.5 ppm 2,4-D, B0D4:
0 ppm BAP + 2 ppm 2,4-D, B1D0: 0.5 ppm BAP + 0 ppm 2,4-D, B1D1: 0.5 ppm BAP + 0.5 ppm 2,4-D, B1D2:
0.5 ppm BAP + 1 ppm 2,4-D, B1D3: 0.5 ppm BAP + 1.5 ppm 2,4-D, B1D4: 0.5 ppm BAP + 2 ppm 2,4-D,
B2D0: 1 ppm BAP + 0 ppm 2,4-D, B2D1: 1 ppm BAP + 0.5 ppm 2,4-D, B2D2: 1 ppm BAP + 1 ppm 2,4-D,
B2D3: 1 ppm BAP + 1.5 ppm 2,4-D, B2D4: 1 ppm BAP + 2 ppm 2,4-D, B3D0: 1.5 ppm BAP + 0 ppm 2,4-D,
B3D1: 1.5 ppm BAP + 0.5 ppm 2,4-D, B3D2: 1.5 ppm BAP + 1 ppm 2,4-D, B3D3: 1.5 ppm BAP + 1.5 ppm
2,4-D, B3D4: 1.5 ppm BAP + 2 ppm 2,4-D, B4D0: 2 ppm BAP + 0 ppm 2,4-D, B4D1: 2 ppm BAP + 0.5 ppm
2,4-D, B4D2: 2 ppm BAP + 1 ppm 2,4-D, B4D3: 2 ppm BAP + 1.5 ppm 2,4-D, B4D4: 2 ppm BAP + 2 ppm
2,4-D.
Table 2 . E. horsfieldii callus texture at 8 WAP.
2,4-D (ppm) BAP (ppm)
00.511.52
0 - Compact Compact Compact Compact
0.5 Friable Compact Friable Compact Compact
1 Friable Friable Friable Compact Compact
1.5 Friable Compact Friable Compact Compact
2 Friable Compact Compact Compact Compact
ppm: part per million (mg/L), WAP: week after plantation.
Fig. 2. Texture of E. horsfieldii calluses, (a) compact callus
on treatment BAP 1 ppm + 2,4-D 1 ppm, (b) fri-
able callus on treatment BAP 0 ppm + 2,4-D 2 ppm.
is divided into 2 groups: friable and compact calluses.
Compact calluses predominated among inducted calluses.
When a callus was exposed to lignification, the callus
consolidated, forming compact calluses. The cytokine that
operates as nutrition transporters has an impact on this
occurrence. Castro et al. (2016) also found that on a
medium with a greater BAP concentration, compact
calluses can be formed. Fig. 2 displayed the visualization of
friable and compact calluses. Friable calluses were
obtained on treatment 0 ppm + 2,4-D 0.5 ppm (B0D1), 0
ppm + 2,4-D 1 ppm (B0D2), 0 ppm + 2,4-D 1.5 ppm

38 ∙Plant Breed. Biotech. 2023 (March) 11(1):34~48
Fig. 3. Color of E. horsfieldii calluses, (a) whitish green callus, (b) white callus, (c) yellowish green callus, (d) green
callus, (e) brownish green callus.
Table 3 . E. horsfieldii callus colors at 8 WAP.
2,4-D (ppm) BAP (ppm)
00.511.52
0 - Brownish green Whitish Green White White
0.5 Green Green Green Green Yellowish green
1 Green Green Brownish green Brownish green Brownish green
1.5 Green Brownish green Brownish green Brownish green Brownish green
2 Green Green Green Yellowish green Brownish green
ppm: part per million (mg/L), WAP: week after plantation.
(B0D3), 0 ppm + 2,4-D 2 ppm (B0D4), 0.5 ppm + 2,4-D 1
ppm (B1D2), 1 ppm + 2,4-D 0.5 ppm (B2D1), 1 ppm +
2,4-D 1.5 ppm (B2D3), and 1 ppm + 2,4-D 1 ppm (B2D2).
Endogenous auxin hormones found in the explant stimulate
the formation of friable calluses.
Callus color is a parameter that can be an indicator of the
compounds contained in the callus. The color of the callus
ranged from green to whitish green, white, yellowish
green, green, and brownish green (Fig. 3). According to
Rasud and Bustaman (2018), callus color change is an
indicator of the growth of cells in callus tissue. Based on the
data in Table 3, it can be seen that the most common callus
color found was green. Furthermore, white callus was
found in B3D0 and B4D0 treatments. The white color of
the callus indicates the lack of pigment in the callus cells.
Yellow ish green call us found in the 1.5 ppm + 2,4-D 2 ppm
(B3D4) and 2 ppm + 2,4-D 0.5 ppm (B4D1) treatments
were suspected as a potential embryogenic callus. Wu et al.
(2020) stated that proper handling such as the application
of the subculture process was able to induce embryogenic
callus that had the potential to form buds.
The weight of fresh calluses was observed on a
month-old callus. Fig. 4 shows that the highest average of
callus fresh weight was obtained on treatment B1D4 (0.5
ppm BAP and 2 ppm 2,4-D) with an average weight of
3,610 g. This demonstrated that this combination had the
best balance of auxin (2,4D) and cytokinin (BAP),
resulting in the production of optimal callus masses in E.
horsfieldii. However, the lowest average resulted at 0.086
g on treatment B0D1 (0 ppm BAP + 0.5 ppm 2,4-D). The
low-growth regulators are directly proportional to low
callus quality. Treatment B0D0 was discluded from
observation for not developing any calluses.
The weight of dry calluses was calculated on some
samples (Fig. 5). The selected treatments are chosen as
representatives for the combinations of high and low auxin
and cytokinin. The harvested callus was dried in the oven at
60℃ temperature. The highest callus dry weight average
was observed on treatment 0.5 ppm + 2,4-D 2 ppm (B1D4)
with 0.111 g. Treatment BAP 0.5 ppm + 2,4-D 0 ppm
(B1D0) has the lowest callus dry weight average of 0.027 g.
However, the result of the ANOVA analysis of callus dry
weight showed an insignificant number (P ＞ 0.05).
Therefore, the DMRT test was unlikely to be done.

In Vitro Propagation and Secondary Metabolite of Pronojiwo (Euchresta horsfieldii (Lesch) Benn.) ∙39
Fig. 4. Average of E. horsfieldii fresh callus weight.
Fig. 5. Average of E. horsfieldii dry callus weight.
Shoot development
When a shoot reaches a length of 0.5 cm, the criteria of
shoots are calculated. The ANOVA revealed that every
single treatment of BAP and 2,4-D had a significant effect
on the percentage of developed shoots on E. horsfieldii.
Nonetheless, the interaction of BAP and 2,4-D did not
influence the formation of shoots. Table 4 showed that a
single treatment of BAP by 0; 0.5; 1; and 1.5 ppm con-
centration has no significantly different effect on the
percentage of E. horsfieldii shoots forming. However,
those treatment is significantly different from the treatment
BAP by 2 ppm. The application of 2,4-D can influence the
percentage of E. horsfieldii shoots forming. The average
percentage of E. horsfieldii planet shoots showed that a
single treatment of 2,4-D with concentrations of 0.5 ppm
and 1 ppm gave no noticeable different results from
treatment with 2,4-D 2 ppm and 1.5 ppm. 2,4-D 0 ppm
differs noticeably from 2,4-D with any other treatments.
Fig. 6 displayed the complete data of the percentage of E.
horsfieldii formed shoots. Treatment B0D1, B0D2, B2D1,
B2D2, and B3D2 was showing a 33.33% percentage of E.
horsfieldii shoots forming which means 1 of 3 explants was
able to produce a shoot. Treatment B3D1, B4D2, and
B4D3 reached 66.66% percentage of E. horsfieldii shoots
forming, this means 2 of 3 planted explant was able to
successfully form shoots. Treatment B0D0, B1D0, B2D0,
B3D0, B4D0, and B4D1 was able to reach the 100%

40 ∙Plant Breed. Biotech. 2023 (March) 11(1):34~48
Table 4 . Average percentage of E. horsfieldii shoots forming at single treatment of BAP and 2,4-D concentration.
2,4-D (ppm) BAP (ppm) 2,4-D Average
0 0.5 1 1.5 2
0 100 100 100 100 100 100.00c
0.5 0 0 33.3 66.6 100 40.00
b
1 0 0 33.3 33.3 66.6 26.67ab
1.5 33.3 0 0 0 66.6 20.00ab
2 33.30000 6.67
a
BAP average 33.33a20.00a33.33a40.00a66.67
b
-
In Table 4, a, b, c are the significant concentration levels, with arepresents the least, brepresents mild, and crepresents the
highest of shoot forming percentage. Numbers followed by the same letter in the same rows or column are not
significantly different at 5% level DMRT. The (-) sign indicates there is no interaction.
Fig. 6. Percentage of E. horsfieldii formed shoots.
percentage of E. horsfieldii shoots forming. It can be seen
that the addition of auxin in the form 2,4-D decreased the
percentage of formed shoots.
The shoot appearance time is observed when a shoot
reaches a length of 0.5 cm. The ANOVA analysis results
revealed that each BAP and 2,4-D treatment has no
significant effect on the shoot appearance time, as does the
interaction of BAP and 2,4-D, which has no significant
effect on the shoot appearance time of E. horsfieldii shoots.
The concentration of BAP and 2,4-D is thought to be less
efficient against the emergence of E. horsfieldii shoots.
The average E. horsfieldii shoots appearance time is
ranged from 4.67 days to 25 days after the initiation. Fig. 7
displayed the complete data of average shoot appearance
time in E. horsfieldii. Treatment B2D0 which consists of 1
ppm BAP and 0 ppm 2,4-D was the fastest shoot average
appearing time with 4.67 days after the initiation. The
slowest treatment was B0D3 which consist of 0 ppm BAP
and 1.5 ppm 2,4-D with 25 days after the initiation. From
this result, it seems likely that BAP affects shoot formation
by accelerating the process and 2,4-D slows it down. BAP
affected shoot formation by accelerating the process and
2,4-D slows it down. For this situation, Dinesh et al. (2019)
research on Punica granatum assumes that the influ ence of
BAP on shoot formation might be related to the faster
metabolism of BAP by plant tissues compared with other
synthetic growth regulators.
The ANOVA analysis result for the 2,4-D effect on E.
horsfieldii shoots height was also significant. Table 5
showed that the effect of treatment with 0 ppm has a
different effect from any other treatments and resulted in
the highest average height of E. horsfieldii. The treatment

In Vitro Propagation and Secondary Metabolite of Pronojiwo (Euchresta horsfieldii (Lesch) Benn.) ∙41
Fig. 7. Shoot appearance time of E. horsfieldii.
Table 5 . Average height of E. horsfieldii at single treatment of BAP and 2,4-D concentration.
2,4-D (ppm) BAP (ppm) 2,4-D Average
00.511.52
0 2.00 2.17 3.33 2.50 2.83 2.67c
0.5 0.00 0.00 0.67 1.17 2.00 0.77
b
1 0.00 0.00 0.33 0.33 1.17 0.37ab
1.5 0.33 0.00 0.00 0.00 1.00 0.27a
2 0.33 0.00 0.00 0.00 0.00 0.07a
BAP average 0.53a0.43a0.87a0.80a1.40
b
-
In Table 5, a, b, c are the significant concentration levels, with arepresents the least, brepresents mild, and crepresents the
highest of average height. Numbers followed by the same letter in the same rows or column are not significantly different
at 5% level DMRT. The (-) sign indicates there is no interaction.
Table 6 . Average number of E. horsfieldii leaves at single treatment of BAP and 2,4-D concentration.
2,4-D (ppm) BAP (ppm) 2,4-D Average
0 0.5 1 1.5 2
0 2.67 4.00 6.67 6.00 8.67 5.60c
0.5 0.00 0.00 0.67 3.33 5.33 1.87
b
1 0.00 0.00 1.33 0.67 1.33 0.67a
1.5 0.67 0.00 0.00 0.00 2.67 0.67a
2 0.67 0.00 0.00 0.00 0.00 0.13a
BAP average 0.80a0.80a1.73ab 2.00
b
3.60c-
In Table 6, a, b, c are the significant concentration levels, with arepresents the least, brepresents mild, and crepresents the
highest of leaves number. Numbers followed by the same letter in the same rows or column are not significantly different
at 5% level DMRT. The (-) sign indicates there is no interaction.
with 1 ppm of BAP has no significant difference from
treatments 0.5; 1.5; and 2 ppm.
The interaction of BAP and 2,4-D did not have a
significant influence on the number of E. horsfieldii
plantlet leaf growth. However, treatment with BAP and
2,4-D concentration alone has significantly influenced the
number of E. horsfieldii plantlet leaves growth. Table 6
showed that 0 ppm of BAP concentration has a similar

42 ∙Plant Breed. Biotech. 2023 (March) 11(1):34~48
Table 7 . Flavonoid compound on field and callus samples
of E. horsfieldii.
Sample Flavonoid compound (% b/b)
Field 3.27
B0D0 4.35
B0D4 1.61
B1D0 2.10
B1D4 1.94
B2D0 2.87
B2D4 1.79
B3D0 2.88
B3D4 1.87
B4D0 2.98
B4D4 1.97
B0D0: Treatment of BAP 0 ppm + 2,4-D 0 ppm, B0D4:
Treatment of BAP 0 ppm + 2,4-D 2 ppm, B1D0: Treat-
ment of BAP 0.5 ppm + 2,4-D 0 ppm, B1D4: Treatment
of BAP 0.5 ppm + 2,4-D 2 ppm, B2D0: Treatment of
BAP 1 ppm + 2,4-D 0 ppm, B2D4: Treatment of BAP
1 ppm + 2,4-D 2 ppm, B3D0: Treatment of BAP 1.5
ppm + 2,4-D 0 ppm, B3D4: Treatment of BAP 1.5 ppm
+ 2,4-D 2 ppm, B4D0: Treatment of BAP 2 ppm + 2,4-D
0 ppm, B4D4: Treatment of BAP 2 ppm + 2,4-D 2 ppm.
effect with BAP 0.5 and 1 ppm on the number of leaves of
E. horsfieldii. The treatment of BAP 2 ppm gave the highest
number of leaves. On the contrary, 2,4-D 2 ppm has the
lowest number of leaves while 2,4-D 0 ppm treatment
showed the highest number of leaves of E. horsfieldii
plantlet. That treatment showed significantly different
effects with 1; 1.5; and 2 ppm concentrations of 2,4-D.
Secondary me taboli te producti on
In this study, the flavonoid content test was applied to 10
samples of callus and plantlets that had formed stems,
leaves, and roots and 1 field sample of E.horsfieldii. Those
10 callus samples were selected based on the high and low
concentrations of BAP and 2,4-D used to represent all
existing treatments. The flavonoid content test was also
carried out on field samples to be used as a comparison
against callus and plantlets from tissue culture. The data
obtained showed that the value of the flavonoid content of
the field samples was 3.27%. The treatment B0D4 had the
lowest flavonoid concentration, with a value of 1.61%. The
highest content of flavonoid compounds can be found in
sample B0D0 or the treatment without additional growth
regulators with a value of 4.35% (Table 7).
Bioactive compound analysis was conducted using
GCMS (Gas Chromatography Mass Spectroscopy). Analysis
was applied to the field sample and callus of treatment BAP
1 ppm + 2,4-D 1.5 ppm (A2B3) as the best treatment
observed. The results of the GCMS test on field samples
and BAP 0.5 ppm + 2,4-D 1 ppm (B1D2) plantlet samples
showed a difference. Some of the major compounds
detected were found in both the field sample and B1D2
plantlet samples, some of which could only be found in one
of the samples. Table 8 displays a list of major compounds
in both samples. The presence of compounds that cannot be
found in other samples is thought to be caused by differ-
ences in nutrients obtained during the planting process. In
vitro cultivation with the addition of BAP and 2,4-D was
thought to trigger the formation of compounds that could
not be found in field samples.
Based on the data in Table 8, Benzene ethyl and Benzene
1,3 dimethyl are 2 types of alkene compounds found in field
samples and plantlets. However, Benzene 1,2 dimethyl can
only be found in plantlet samples. Another compound
found in both types of samples is pentadecanoic acid and
9-octadecenamide. Siloxane compounds are one of the
major compound groups in this study and were commonly
found in B1D2 plantlet samples. Apart from the major
compound, there are several other components identified.
These components are impurity components and minor
components. Compounds detected in small amounts in one
of the samples are thought to be impurity components.
Compounds suspected as impurity components identified
include (4R,5R)-2,2-Dimethyl-4-pentadecenamide,
2-Hydroxycyclopentadecamethyl, and (Z,Z)-6,9-cis-3,4-
epoxy-nonadecadiene. Other compounds that are not in-
cluded in the major compound group but are present in both
types of samples are called minor compounds. The cause of
the presence of minor compounds is thought to be related to
the age of the plants when processed to be a sample. This is
because some compounds are only formed at certain
periods during plant growth. One of the minor compounds
from E. horsfieldii detected in this study was 2-pentanol, 4
methyl. 2-pentanol, 4 methyl is a compound used as a raw
material for flavored products. According to Api et al.

In Vitro Propagation and Secondary Metabolite of Pronojiwo (Euchresta horsfieldii (Lesch) Benn.) ∙43
Table 8 . List of major compounds in field samples and B1D2 plantlets using the GC-MS analysis method.
No. Compounds Structure Field
sample
B1D2
Plantlet sample
1 Benzene ethyl
CH3
++
2 Benzene, 1-3-Dimethyl CH3
H3C
++
3 Pentadecanoic acid
O
OH
++
4 9-Octadecenamide
CH
3
O
NH2
++
5 Octadecamethyl cyclononasiloxane
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
‒+
6 Benzene, 1,2-dimethyl
CH3
CH3
‒+
7 Hexadecamethyl cyclooctasiloxane
Si O
Si
O
Si
O
Si
O
SiO
Si
O
Si
O
Si
O
‒+
8 Eicosamethyl-cyclodecasiloxane
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
‒+
9 Tetracosamethyl cyclododecasiloxane
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
+‒
The + sign indicates the presence of chemical compounds in the test sample with GC-MS.
(2019) 2-pentanol, 4 methyl is an aromatic compound that
does not have the potential for toxicity when used in
humans. This compound is often used in detergents, per-
fumes, clothing fragrances, and several types of cosmetics.

44 ∙Plant Breed. Biotech. 2023 (March) 11(1):34~48
Table 9 . Average of E. horsfieldii callus fresh weight at single treatment of BAP and 2,4-D concentration.
2,4-D (ppm) BAP (ppm) 2,4-D Average
00.511.52
0 0.07 0.37 0.77 0.70 2.01 0.78a
0.5 0.09 0.12 0.66 0.69 0.70 0.45a
1 0.13 0.33 0.98 0.77 1.63 0.77a
1.5 0.85 1.61 2.17 0.97 3.47 1.81
b
2 0.81 3.61 2.16 1.96 2.97 2.30
b
BAP average 0.40a1.20
b
1.35
b
1.02ab 2.16c-
In Table 9, a, b, c are the significant concentration levels, with arepresents the least, brepresents mild, and crepresents the
highest of callus fresh weight. Numbers followed by the same letter in the same rows or column are not significantly
different at 5% level DMRT. The (-) sign indicates there is no interaction.
DISCUSSION
Inducti on of callus
The explants’ meristematic tissue contains endogenous
hormones that aid in the formation of calluses. When
endogenous hormones were coupled with BAP and 2,4-D,
cells that would be actively differentiated began to pro-
liferate. This finding was consistent with Fadhilasari et al
(2018), who found that juvenile explants were faster and
more likely to generate callus than older explants. However,
there was no callus development in treatment B0D0, which
was a pure MS media. This is thought to be caused by a lack
of supportive hormones that induce callus development.
According to Robles et al, (2016) as an auxin, 2,4-D
increased osmotic pressure and cell permeability, lowering
cell wall pressure and increasing synthetic protein, plas-
ticity, and cell wall growth. The development of calluses
appears to be influenced by cytokinin concentrations. After
9 days of induction, treatment with a combination of higher
cytokine and lower auxin are slow to develop calluses. This
suggests that increased cytokinin concentrations influenced
callus growth, which could be related to cytokinin’s role in
creating shoots and growing plantlets. These findings
support Ahmad et al. (2020) findings that the kind and
concentration of PGRs used had a substantial impact on
callus development.
The color and texture of the callus were observed in this
study as callus morphology. The texture of a callus is used
to determine its quality. Friable and compact callus were
the two types of callus texture identified. According to
Karimi et al. (2018), callus texture can be categorized as
friable to compact depending on the type of explant, media,
growth regulators, and biotic and abiotic nutrients used.
The endogenous auxin hormone found in explants also
stimulates the formation of friable callus. According to El
Aouad et al. (2019), friable callus generally has nucleates
in its tissue. Furthermore, Salim et al. (2019) explained that
friable callus formed faster than compact callus, this was
due to the friable callus cell division being faster than
compact callus.
According to Li et al. (2021), the green color of the callus
is an indication of the presence of chloroplasts in the callus
tissue formed from the development of proplastids due to
exposure to light. The brownish-green color is also common.
This was allegedly caused by observations made when the
callus was 8 WAP so that browning events had occurred in
several callus. Cai et al (2020) explained that a callus with
brown color was a green callus that had undergone a
browning process, this indicated the presence of toxic
phenolic chemicals in the callus.
A certain concentration of cytokinin combined with
auxin proved to be able to help explants produce calluses.
However, the interactions between BAP and 2,4-D in this
research do not affect the weight of fresh callus (Table 9).
Cytokinin itself has an important role to develop a good
callus quality. Mahadi et al. (2016) in Castro et al. (2016)
stated that the cytokinin transport system, which runs from
the base to the top of the plant, carries water and nutrients
through the transport vessel, affecting the osmotic potential
of cells. This activity causes cell walls to turn rigid and

In Vitro Propagation and Secondary Metabolite of Pronojiwo (Euchresta horsfieldii (Lesch) Benn.) ∙45
Table 10. Average number of E. horsfieldii shoots formed
at single treatment of 2,4-D concentration.
2,4-D concentration (ppm) Number of shoots
02.00
b
0.5 0.53a
10.20
a
1.5 0.27a
20.07
a
In Table 10, a, b are the significant concentration levels,
with arepresents the least and brepresents the highest of
number of shoots formed. Numbers followed by the same
letter in the same column are not significantly different at
5% level DMRT.
eventually develop calluses. This was shown in Table 9,
where 0.5 ppm concentration of BAP was different from 0
ppm and 2 ppm but similar to 1 ppm and 1.5 ppm.
Treatment with 2 ppm concentration of BAP was signi-
ficantly different from any other treatment and can be
concluded as the best treatment for increasing the fresh
weight of callus. Acid secretion causes cell membrane
relaxation by activating the enzyme at a certain pH. This
enzyme is responsible for breaking the links between
cellulose molecules in the cell wall. Turgor pressure occurs
when cells absorb water molecules in response to an
increase in soluble material concentration in the vacuole,
allowing them to expand. Table 9 shows that the existence
of 2,4-D alone has a significant effect on the weight of fresh
calluses. Treatments with 0; 0.5; and 1 ppm were observed
to have a similar effect to the fresh weight of calluses.
However, the effect of those treatments was significantly
different from treatment 1.5 ppm and 2 ppm concentration
of 2,4-D.
The result also showed that higher concentrations of
2,4-D were able to produce higher callus dry weight. This
indicates the significant effect of 2,4-D to callus weight.
Another research by Abdelmaksood (2017) showed that
auxin concentration plays a big role in callus quality, a
combination of auxin and cytokinin was also a better
treatment for the high frequency of callus. The drying
process of calluses will reduce 60% to 90% its weight as in
the research conducted by Osman et al. (2016) where they
recorded the fresh weight [(1.681 ± 0.770) g], and the dry
weight [(0.239 ± 0.239) g] of Barringtonia racemosa L.
Shoot development
Those number was calculated based on the results of
three replications of the calculation per treatment, and the
MS base media with growth regulator concentration
provided the best response. This statement is in accordance
with the research of Paramartha et al. (2012) that the
addition of high concentrations of auxin has the effect of
inhibiting tissue growth because there is competition with
endogenous auxin to get a position for receiving cell
membrane signals so that the addition of auxin from the
outside does not have a major effect on cell growth and
development. BAP affected shoot formation by accelera-
ting the process and 2,4-D slows it down. For this situation,
Dinesh et al. (2019) research on Punica granatum assumes
that the influence of BAP on shoot formation might be
related to the faster metabolism of BAP by plant tissues
compared with other synthetic growth regulators.
The long circumstances of each explant’s shoots vary.
This can happen because the absorption of nutrients for
regeneration varies depending on the explant. Shoots
explant with no new shoot growth and a length of buds are
conceivable because there is still sufficient nutritional
content from the previous culture on the body of the
explant. The appropriateness of the regulatory compounds
administered during multiplication can also influence the
length of the shoots. According to Buko and Trine (2020),
the number of internodes indicates a potential mass
increase because each node usually has at least one axillary
shoot.
The number of shoots was calculated after 4 weeks of
initiation. The result revealed that the presence of 2,4-D
alone had a substantial effect on the number of shoots
generated. However, the single treatment of BAP and the
interaction of BAP and 2,4-D gave no significant effect on
the number of shoots. Table 10 displays the result of the
DMRT test on a single treatment of 2,4-D concentration.
The treatment with 0.5; 1; 1.5; and 2 ppm showed no
significantly different effect on the number of shoots
formed. However, these treatments gave significantly
different effects with treatment 2,4-D 0 ppm.
The parameter of the number of leaves can be observed
right after observing the parameter of the number of shoots.

46 ∙Plant Breed. Biotech. 2023 (March) 11(1):34~48
Khan et al. (2015) stated that various combinations of
growth regulators influence the number of shoots. Accord-
ing to the result, the treatment of BAP 2 ppm is the highest
number of leaves observed. For this situation, Batti et al.
(2020) stated that the addition of exogenous auxin did not
significantly affect the number of leaves formed because
the plants already had sufficient amounts of endogenous
auxin to trigger leaf formation.
Secondary me taboli tes production
Flavonoids have been found several times in various
parts of E. horsfieldii. Mizuno et al. (1990) stated that a
large number of flavonoid compounds can be found in the
stems and roots of E. horsfieldii. Matsuura (1994) also
added that at least 60 types of flavonoid compounds can be
obtained by extracting the roots and stems of E. horsfieldii.
The treatment without additional growth regulators has the
highest content of flavonoids caused by the hormone stress
conditions in B0D0 treatment which is pure MS media that
can trigger the formation of flavonoid compounds better
than planting in the field which is not under stress con-
ditions. According to Setyorini and Kusnawan (2016),
flavonoid compounds are formed when there is an increase
in the activity of the phenylalanine ammonia-lyase (PAL)
enzyme as a defense action of plant tissue from both biotic
and abiotic stresses.
The results of the analysis indicated that in vitro
cultivation was able to produce more types of active
compounds than cultivation in the field. This is thought to
be caused by differences in the organs used as samples and
the presence of a self-protection system formed in the
plantlets after explant injury. The GCMS method has pre-
viously been applied to E. horsfieldii field plants. Accord-
ing to Tirta et al. (2015), active compounds found in the
leaves of E. horsfieldii include apigenin, palmitic acid,
2-Tridecanone, 4-Ethyloctane, 4-propylheptadecane, and
antioxidants as much as 196.94 ppm. Trujillo-Chacon et al.
(2019) explained that the differences in the active com-
pounds possessed by field plants and plantlets were caused
by the activity of cytokinins and auxins which not only
affected cell differentiation and proliferation but also
affected cell biosynthetic pathways. Benzene ethyl with the
compound formula C8H10 is a single-ringed alkyl aromatic
organic compound that is flammable, colorless, and gene-
rally smells like gasoline. In the industrial sector, according
to Gaurh and Pramanik (2018), Benzene ethyl has u ses as a
basic ingredient in the manufacture of paint, paper, and
plastic.
Another compound found in both types of samples is
pentadecanoic acid. Pentadecanoic acid is a saturated fatty
acid with the compound formula C15H30O2. Saturated fatty
acids concerning human health, have anti-bacterial and
anti-fungal activities. Fatty acids effectively help in the
prevention and management of diseases associated with
pathology. According to To et al. (2020) pentadecanoic
acid is also known to be used in the prevention of coronary
heart disease and breast cancer. 9-octadecenamide com-
pound or also called oleamide is an organic compound with
many benefits that can be found in both animals and plants.
Hachisu et al. (2015) explained that oleamide is a pro-
tective agent against memory loss in Alzheimer’s disease
and can increase drowsiness cause deep sleep, increase
appetite, and has no side effects on blood pressure, heart
rate, and body temperature. According to Ameamsri et al.
(2021), oleamide exhibits anti-inflammatory effects and
has been used in the prevention and treatment of various
diseases such as atherosclerosis, arthritis, and cancer
through its metabolic conversion activity.
According to Falowo et al. (2017), siloxane compounds
are compounds that have high antioxidant activity and play
an active role as anti-microbials. The types of siloxane com-
pounds identified in this study include Cyclooctasiloxane
hexadecamethyl, Octadecamethyl-cyclonasiloxane, and Eico-
samethyl-cyclodecasiloxane. Furthermore, Sahab et al.
(2018) explained that Cyclooctasiloxane hexadecamethyl
is an essential organic compound that can act as an anti-
fungal. Another siloxane compound found in the field
samples is Cyclododecasiloxane tetracosamethyl.
CONCLUSIONS
Callus and shoots of E. horsfieldii can be obtained by
combining BAP and 2,4-D. The callus appearance time of
E. horsfieldii is around 5 to 9 days after initiation. The
callus is mostly compact textured and green-colored.

In Vitro Propagation and Secondary Metabolite of Pronojiwo (Euchresta horsfieldii (Lesch) Benn.) ∙47
Single treatment of BAP and 2,4-D was significantly
affecting the shoots forming. The concentration of 2,4-D 0
ppm gave the highest average plantlet height by 2.67 cm,
increasing the number of shoots by 2.00, and the number of
leaves by 5.60. The average E. horsfieldii shoots appear-
ance time is ranged from 4.67-25 days after the initiation.
E. horsfieldii cultured in vitro without additional growth
regulators had a higher flavonoid content with a value of
4.35%. The in vitro cultivation of E. horsfieldii was able to
produce more types of active compounds than cultivation
in the field.
ACKNOWLEDGEMENTS
The authors would like to thank Sebelas Maret
University for funding the Domestic Universities Colla-
borative Research.
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