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Methyl jasmonate stimulates growth and upregulates the expression of

Phenylalanine Ammonia-Lyase (PAL) gene in Gynura pseudochina in vitro
micropropagation

Article in Biodiversitas Journal of Biological Diversity · May 2024

DOI: 10.13057/biodiv/d250512

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B IOD I V E R S I TA S ISSN: 1412-033X
Volume 25, Number 5, May 2024 E-ISSN: 2085-4722
Pages: 1955-1964 DOI: 10.13057/biodiv/d250512

Methyl jasmonate stimulates growth and upregulates the expression of

Phenylalanine Ammonia-Lyase (PAL) gene in Gynura pseudochina in
vitro micropropagation

TITAH RIGEL ANJALANI1, SALMA ARGYA RASMI1, ANISA ESTI RAHAYU1,

MUHAMMAD RIFQI NUR RAMADHANI1, MAY FIATUS SHOLIHAH1, IRA PUSPANINGTYAS1, IRO
DATUS SOLEHA1,2, SEPTI ANITA SARI1,3, MAULIDIA RAHMAWATI1, NASORI NASORI4, NURUL JADID1,
1Departmentof Biology, Faculty of Science and Data Analytics, Institut Teknologi Sepuluh Nopember. Jl. Teknik Mesin No. 173, Keputih, Sukolilo,
Surabaya 60115, East Java, Indonesia. Tel./fax.: +62-315-963857, email: [email protected]
2Graduated Program of Botany, Department of Botany, Faculty of Science, Chulalongkorn University. 254 Phaya Thai Road, Bangkok 10330, Thailand
3Department of Animal Science, Institute of Agriculture and Technology, College of Agriculture and Life Science, Chonnam National University.

Gwangju 61186, Republic of Korea

4Department of Physics, Institut Teknologi Sepuluh Nopember. Jl. Teknik Mesin No. 173, Keputih, Sukolilo, Surabaya 60115, East Java, Indonesia

Manuscript received: 25 February 2024. Revision accepted: 7 May 2024.

Abstract. Anjalani TR, Rasmi SA, Rahayu AE, Ramadhani MRN, Sholihah MF, Puspaningtyas I, Soleha ID, Sari SA, Rahmawati M,
Nasori N, Jadid N. 2024. Methyl jasmonate stimulates growth and upregulates the expression of Phenylalanine Ammonia-Lyase (PAL)
gene in Gynura pseudochina in vitro micropropagation. Biodiversitas 25: 1955-1964. The use of medicinal plants as primary resources
for traditional medicine is rising. Gynura pseudochina (L) DC. is a well-recognized traditional medicinal plant from Southeast Asia. It is
rich in bioactive compounds like flavonoids, alkaloids, saponins, and tannins. Various techniques and methods have been developed to
ensure plant quality and enhance phytomedicines' production, one of which involves plant in vitro culture. Elicitor-based in vitro culture,
particularly using Methyl Jasmonate (MeJA), has been shown to boost the production of secondary metabolites in plants through the
overexpression of Phenylalanine Ammonia-Lyase (PAL) genes, known to play a role in the excessive production of flavonoid
compounds. This research aims to investigate the impact of MeJA on the growth and PAL gene expression in G. pseudochina in vitro
culture. Several MeJA concentrations of MeJA (0, 75, 150, and 300 µM) were tested. Our findings revealed that the application of
MeJA to G. pseudochina in vitro had a noticeable impact on callogenesis and organogenesis. The optimal condition for callogenesis was
achieved with MeJA at a concentration of 150 µM. On the other hand, the highest frequency of root induction was observed at 75 µM of
MeJA treatment, while the highest shoot frequency occurred at 150 µM of MeJA. Interestingly, the plant height, fresh weight, leaf, and
shoot numbers were significantly affected after treatment with 150 µM of MeJA. Additionally, 75 µM of MeJA was found to promote
optimal root growth compared to other MeJA treatments. Finally, 150 µM of MeJA treatment resulted in higher expression of the PAL
gene (1.29 folds) than the non-treated plants. Our findings suggested that MeJA influenced the G. pseudochina growth and potentially
increased the production of flavonoid compounds in vitro.

Keywords: Growth, Gynura pseudochina, in vitro culture, methyl jasmonate, phenylalanine ammonia-lyase

INTRODUCTION al. 2021). All these pharmaceutical activities are tightly

correlated with their phytochemical content. Many reports
Gynura pseudochina, locally named "daun dewa" is a showed that G. pseudochina contains alkaloids, flavonoids,
part of around 46 Gynura species. It is widely distributed in saponins, and tannins. This contributes to Indonesia's wide
Africa, China, and Southeast Asia. This species is part of range of phytochemical-based medicinal plant resources
an Asteraceae plant. Gynura pseudochina, among around that require further development (Jadid et al. 2018). In
30,000 medicinal plants in Indonesia, is recognized for its recent years, there has been an increase in the market
healing properties. Its vegetative organs, including leaves, demand for medicinal herbs (Picking 2024). Therefore,
rhizome, and root, possess inflammatory-reducing activity, preserving natural germplasm is paramount (Bisht et al.
beneficial for treating herpes diseases and pain relievers as 2023). On the other hand, an effective method for
well as for fever ailment treatment, respectively (Saralamp accelerating medicinal plant propagation is necessarily
et al. 2000; Lemmen and Bunyapraphatsara 2003; required. However, the quantity of medicinal plants is not
Siriwatanametanon and Heinrich 2011). In addition, the only factor that should be considered. The quality of the
combined G. pseudochina leaves and Curcuma aeruginosa plant, including the species traits, plant performance, and
rhizome are also used for dengue fever treatment phytochemical content, should also be considered (Luo et
(Moektiwardoyo et al. 2014). Moreover, it has been stated al. 2023).
that G. pseudochina possesses anti-cancer and anti- A traditional method of propagation through stem
allergenic activities and is used for treating ulcers, cutting faces several limitations, including longer
bleeding, rashes, and diabetes mellitus ailments (Meng et establishment time, high risk of disease transmission, and
1956 B I OD I V E R S ITA S 25 (5): 1955-1964, May 2024

rooting difficulties (Qin et al. 2023). Thus, some techniques (Duchefa Biochemie, Netherland) and supplemented by
including genetic engineering and marker-assisted selection 0.25 ppm IAA and 3 ppm BAP. A series of MeJA (Sigma-
could help facing these fundamental problematiques in Aldrich) concentrations (0, 75, 150, and 300 µM) were also
agriculture. However, the previously mentioned techniques, added. Finally, the culture media was solidified using 8.2 g/L
in some cases, have limitations. It includes food safety, gelrite powder (PhytoTech Lab®). The pH of the medium
toxicology, allergenic issues, and time consuming (Hasan was also set from 5.7 to 5.8. The measurement used in
et al. 2021). Other biotechnology-based technique such as autoclaving is at 121°C for 20 minutes (Huang et al. 2023).
in vitro plant cell culture offers an alternative method that The inoculation stage is performed aseptic or sterile in
can be used for accelerating plant propagation (Jadid et al. Laminar Air Flow (LAF) by placing the axenic nodal
2024) and phytochemical isolation (Abbasi et al. 2019). A segments of the G. pseudochina in the MS medium
few attempts have been made to boost the G. pseudochina supplemented with MeJA. All the inoculated explants were
in vitro multiplication. The use of Murashige and Skoog placed in a grow room at 25 ± 2°C under 40W of cool
(MS) medium combined with 3 ppm of 6-benzylaminopurine white fluorescent, 16/8h (light/dark) photoperiod for 75
(BAP) has successfully produced 34.8 shoots and 57.5 days. Each treatment consisted of eight replications.
leaves (Nirwan and Aziz 2006). In addition, a combination
of BAP (3 ppm) and Indole Acetic Acid (IAA) (0.5 ppm) Growth response measurement
illuminated with 1,156 lux of light intensity was also We evaluated the frequency of callogenesis and
reported to increase the number of G. pseudochina shoots, organogenesis of the explants after 75 days of MeJA
height, and leaves (Ghulamahdi et al. 2008). However, no treatment in vitro according to the equation below:
report has been made for G. pseudochina in vitro culture
using an elicitor, a chemical compound that stimulates the
plant secondary metabolites (Malik et al. 2020). Some
elicitors include light (Murthy et al. 2024), nano-elicitors
(Rahmawati et al. 2022), salicylic acid and Methyl Jasmonate
(MeJA) (Mahendran and Rahman 2024). MeJA is a
common abiotic elicitor widely applied through in vitro Additionally, we also recorded the number of leaves per
culture (Jeyasri et al. 2023). MeJA, a jasmonic acid explant. Furthermore, root and shoot proliferation was also
derivative, is a plant protective mechanism that triggers a calculated. Finally, the productivity of the plants formed
defense response. It promotes the control of gene expression, from the in vitro system was also evaluated based on their
resulting in the accumulation of biologically active fresh weight.
compounds like terpenoids, flavonoids, and alkaloids (Ho
et al. 2020; Pisitpaibool et al. 2021). In addition, MeJA was RNA extraction and gene expression measurement
also reported to affect the formation of organs in plants by The G. pseudochina leaves were prepared for total
stimulating an increase in metabolic-related gene expression RNA isolation using Total RNA Mini Kit Plant Geneaid),
levels, including MsPAL, MsC4H, and Ms4CL. They are following the manufacturer's guidelines (Jadid et al. 2016).
recognized as pivotal genes participating in the The total RNA extracted was then quantified using NanoDrop
phenylpropanoid pathway in Mentha x piperita 2000 Spectrophotometers (Thermo Fisher Scientific™).
(Krzyzanowska et al. 2012). In line with this research, Liu For PAL gene expression analysis, we used 25 ng of the
et al. (2022) also proved that the expression of the PAL extracted RNA to be then subjected to RT-qPCR using
(Phenylalanine Ammonia-Lyase) gene in Isatis indigotica SensiFAST SYBR No-ROX One Step Kit (Meridian
increased under MeJA treatment. Nonetheless, the regulation Bioscience). The primers used in this study include PAL1F
of these genes is generally specific or species-dependent. (5'-CGGAACAACACAACCAAGATG-3') and PAL2-R
No prior studies have documented the impact of MeJA (5’-CTGATTGGCATAGAGCGACTAA-3') and the Actin
treatment on the expression of genes associated with the gene as a reference gene with ACT1-F (5’-
biosynthesis of phenol compounds in G. pseudochina. CCAAGGCTAACAGAGAGAAGATG-3') and ACT2-R
Therefore, this work aims to investigate the effect of (5’-CGACCACTGGCATATAGAGAAAG-3'). The RT-
exogenous MeJA treatment in G. pseudochina in vitro qPCR procedure began with a 10 min reverse transcription
culture. phase at 45°C, succeeded by a 2 min activation of the
polymerase at 95°C. Then, the PCR cycling consisted of
approximately 39 cycles at 95°C for 5 seconds
MATERIALS AND METHODS (denaturation), 61.3°C for 10 seconds (annealing), and
72°C for 20 seconds (extension). Finally, the gene
Plant materials, medium preparation, and inoculation expression was calculated using the Livak 2 -ΔΔCT
G. pseudochina plantlet was obtained from the collection method.
of the Laboratory of Bioscience and Plant Biotechnology,
Biology Department, Institut Teknologi Sepuluh Statistical analysis
Nopember, Surabaya. In this in vitro culture study, we This study was performed utilizing a completely
employed an axenic nodal segment of the G. pseudochina randomized design. The G. pseudochina growth data and
as the primary explant. The solid MS basal medium GpPAL gene expression were analyzed using one-way
(PhytoTech Lab®) was prepared with 30 g/L of sucrose ANOVA followed by post-hoc Tukey test (Minitab 19).
 ANJALANI et al. – Methyl jasmonate stimulates of PAL gene in Gynura pseudochin 1957

RESULTS AND DISCUSSION to increased anthocyanin production from the MeJA

treatment.
Impact of Methyl Jasmonate (MeJa) on morphogenic In addition to the callogenesis responses, we observed
responses of Gynura pseudochina different shoot and root formations responses of the
The morphogenic responses of G. pseudochina after explants planted in the MeJA-containing medium (Table
MeJA treatments were determined by the frequency of 1). The percentage of shoot formation ranged from 0-
explant forming callus, shoots and roots. Variations of 100%. The maximum shoot formation percentage was
callus, shoot and root formationswere observed in all MeJA attained with a MeJA treatment concentration of 150 µM
treatments of G. pseudochina in vitro culture after 75 days (100%), followed by 0 µM (87.5%) and 75 µM (50%)
of culture. We observed a significant delay in callus (Table 1). Interestingly, we did not observe any shoot
formation in explants treated with 300 µM of MeJA (47.5 ± formation in the explants treated with the highest
6.8 days) compared to other MeJA treatments. Meanwhile, concentration of MeJA treatment (300 µM).
explants treated with 150 µM of MeJA demonstrated the
lowest values (14.4 ± 1.1 days), followed by the control
and 75 µM of MeJA, which took 13.8 ± 3.4 days and 22.3 A B
± 5.1 days, respectively. All explants treated with zero
MeJA formed callus (100%). At the same time, treatment
of MeJA in all concentrations tested seemed to lower the
ability of the explant to form callus. Even though the
highest frequency of explant forming callus was obtained
in G. pseudochina treated with 150 µM of MeJA (87.5%).
Meanwhile, the other MeJA treatment-induced callus is
only 75% of the explants (Table 1). C D
The reduction of callus formation frequency as a result
of the MeJA treatment, follows a previous study by Sun et
al. (2017). Another report also demonstrated that MeJA
lower callus formation and growth in Taxus x media var.
Hatfieldii (Furmanowa et al. 1997). However, some
different results were also observed in other plants in vitro.
This suggests that MeJA effect was species-dependent and
concentration-dependent (Rodríguez-Sánchez et al. 2020).
Overall, our results suggested that the optimal Figure 1. Callus formation in Gynura pseudochina in vitro
concentration of MeJA in inducing callogenesis and shoot culture after 75 days of culture. Note: A. Control, B. 75 µM, C.
formation is at 150 µM. Even though further studies are still 150, D. 300 µM; (a) Callus (Scale bar: 1 cm)
needed to obtain an optimal MeJA concentration for root
induction.
We also observed that MeJA treatments also resulted in Table 1. Morphogenic responses of Gynura pseudochina post-75
different characteristics of callus (Table 2). Callus texture days of MeJA treatment
varies at various levels of MeJA concentration. Callus
derived from the MS medium's explants with no more than Morphogenic responses (%)
75 µM and 150 µM MeJA showed a compact (non-friable) Frequency of Frequency of Frequency of
callus texture. Furthermore, the 300 µM of MeJA treatment MeJA
explant shoot root
produced a friable callus texture. These results are similar Treatment
forming formation formation
(µM)
to those obtained in Salacia chinensis in vitro culture after callus per explants per explants
treatment with 50 and 100 µM of MeJA; the latter 0 100 87.5 62.5
produced a compact and hard callus texture (Chavan et al. 75 75 50 75
2021). In addition, the callus color was also different 150 87.5 100 0
among MeJA treatments (Table 2). 300 75 0 0
The callus obtained from zero MeJA treatment
possesses a green callus. Meanwhile, 75 µM induced the
Table 2. Texture and color of Gynura pseudochina callus after 75
formation of a green-yellowish callus. In contrast, 150 µM
days of MeJA treatment
and 300 µM of MeJA produced green-purplish callus
(Figure 1). The alteration of callus color indicates callus
cells are actively dividing, while darker or browner callus MeJA treatments (µM) Texture Color
color generally indicates a decrease in cell growth 0 Compact Green
(Pisitpaibool et al. 2021). The green color in the control 75 Compact Green yellowish
callus can occur due to the influence of Plant Growth 150 Compact Green purplish
300 Friable Green purplish
Regulators (PGRs) supplemented with the medium. In
comparison, the purple color in the callus is probably due
1958 B I OD I V E R S ITA S 25 (5): 1955-1964, May 2024

Some explants suffered mortality as a result of the 75 days of culture (Figure 4). The highest plant height was
elevated concentration of methyl jasmonate (Figure 2.D). demonstrated by 150 µM of MeJA (3.64 cm), followed by
We noticed that shoot formation appeared from the callus the control plant (0 µM of MeJA). Meanwhile, we noted no
(Figure 1.C) and the nodal part (Figure 2.B). These data significant difference between plant height after treatment
might suggest that MeJA treatment might induce the 75 and 300 µM. They showed the lowest plant height of
formation of embryogenic callus. Nevertheless, additional 1.32 and 1.06 cm, respectively. However, they significantly
microscopic assessment should be carried to identify the differ from the treatment at the control and 150 µM dose
presence of embryogenic callus (Zhang et al. 2021). Our (Figure 4).
results were similar to those observed in Centella asiatica Gynura pseudochina plants cultured in vitro without
in vitro culture, demonstrating that higher jasmonic acid MeJA (0 µM) exhibited greater growth than those
concentrations might inhibit shoot formation (Krishnan et subjected to 75 µM and 300 µM MeJA treatments. This
al. 2019). Machado et al. (2017) demonstrated that MeJA could be attributed to the presence of IAA and BAP in the
acts antagonistically with Gibberellic Acid (GA) to medium. The concurrent application of auxin and cytokinin
decrease shoot growth. can potentially enhance plant growth and development,
Another organogenesis response is the percentage of influencing height, branching, and biomass (Talukdar et al.
root formation. We noticed a variation in root formation 2022).
frequency after MeJA treatments (Table 1, Figure 3). The
percentage of root formation ranged from 0 to 75%. The
highest percentage of root formation was obtained in 75
µM MeJA (75%), followed by 0 µM of MeJA (62.5%).
Explants placed in the 150 and 300 µM of MeJA resulted
in no root formation response (0%) (Table 1). This aligns
with the results reported by Mangas et al. (2006), who
observed that elevated concentrations of externally applied
MeJA hinder root growth in the in vitro culture of Ruscus
aculeatus.

Effect of MeJA on plant height of Gynura pseudochina

in vitro culture
The increase in plant height resulted from the sequential
progression involving cell division, elongation, and Figure 4. Effect of MeJA on plant height of Gynura pseudochina
enlargement (Zhou et al. 2023). In this study, MeJA in vitro culture after 75 days of culture. Note: All data represented
application in G. pseudochina in vitro culture significantly by mean ± standard deviation. Different letters represented a
affects plant height (P<0.05). The G. pseudochina height statistically significant effect by Tukey's test (P<0.05)
ranged from 1.06 to 3.64 cm after MeJA treatment during

A B
A B

C D C D

Figure 2. Shoot formation in Gynura pseudochina in vitro culture Figure 3. Root formation in Gynura pseudochina in vitro culture
after 75 days of MeJA treatment. Note: A. Control, B. 75 µM, C. after 75 days of MeJA treatment. Note: A. Control, B. 75 µM, C.
150 µM, D. 300 µM (No shoot formation). A. Shoots (Scale bar: 1 150 µM (No roots detected), D. 300 µM (No roots formed); (a)
cm) Root (Scale bar: 1 cm)
 ANJALANI et al. – Methyl jasmonate stimulates of PAL gene in Gynura pseudochin 1959

Similar to the previous parameters observed, the effect did not undergo treatment (Sirhindi et al. 2020). The
of MeJA on plant height also depends on the concentration enhancement of the plant's fresh weight was also reported
given. Alam and Albalawi (2020) demonstrated that 2 and to be followed by an increase in the production of plant
5 µM of MeJA treatment increased plant height of secondary metabolites. However, MeJA is also known to
Artemisia annua in vitro culture. This effect is due to cause a decrease in biomass. MeJA treatment at 100 µM
positive plant growth regulation due to exogenous MeJA has been reported to decrease the biomass of Polygonum
application. In line with this study, Lalnunzawma et al. multiflorum and Echinacea purpurea by 5.8 and 22.97%,
(2020) also showed that giving lower MeJA concentrations respectively. MeJA is proposed to potentially stimulate the
(0.3 and 0.6 mM) to strawberry plants (Fragaria x production of Reactive Oxygen Species (ROS),
ananassa) increased plant height. However, it decreased subsequently negatively impacting plant growth (Ho et al.
plant growth at higher concentrations (0.9 mM). In 2020). This is similar to those in other plants under
contrast, other studies demonstrated different results, for environmental stress conditions. The plants tend to produce
instance, the application of MeJA (Helianthus annuus) and significant amounts of antioxidants to counter the existence
tomatoes (Solanum lycopersicum) (Li et al. 2018). It of detrimental radicals (Jadid et al. 2017).
strongly supports the previous results, which explained that
the effect of MeJA seems species-dependent. Moreover, Impact of MeJA on the number of leaves of Gynura
plant height decreases because MeJA also plays an pseudochina in vitro culture
important role in plant senescence. Additionally, an Leaves are plants' primary site for photosynthesis,
unfavorable concentration of MeJA inhibits plant height by making them vitally significant in plant development and
blocking the G1/S cell cycle transition, thereby reducing biomass accumulation (Sari et al. 2018). In addition, leaves
cell number and suppressing endoreduplication (which are considered the most frequently used plant organ for
supports cell differentiation) (Heinrich et al. 2013). These traditional ailment treatment. The leaves of the G.
consequently affect cell size and further generate a dwarf pseudochina plant are also used in traditional medicine.
phenotype (Larrieu and Vernoux 2016). The leaf extract of this plant is utilized for treating
inflammation and herpes infections (Siriwatanametanon
Effect of MeJA on fresh weight of Gynura pseudochina and Heinrich 2011). Therefore, leave formation resulting
in vitro culture from the MeJA treatment should be well-evaluated. Our
Fresh weight is a commonly employed quantitative study demonstrated that MeJA significantly affects the
measurement of plant growth and development. The number of leaves in G. pseudochina treatment.
augmentation of fresh weight primarily results from cell Our data showed that exogenous MeJA significantly
enlargement due to high water absorption rate and cell wall affected the number of plant leaves with an average range
expansion regulated by turgor pressure within the cell of 0-80.6 (P<0.05) (Figure 6.A). The highest average
(Dale 1988). In addition, fresh weight is also related to number of leaves was found when MeJA was given at 150
plant productivity, indicating good photosynthate transport µM (80.6). We also observed a purple leaf on explants
(Mehmandar et al. 2023). In this investigation, the treated with µM of MeJA. This might indicate the
application of external MeJA had a notable impact on the production of flavonoid-based compounds compared to
fresh weight of the plants (P<0.05). The fresh weight plants possessing 40.75 leaves per explant (Figure 6).
ranged from 0.07-6.95 g per plant (Figure 5).
The highest average of G. pseudochina fresh weight
was found at 150 µM of MeJA treatment (6.95 g), followed
by control treatment (3.44 g). Meanwhile, the lowest fresh
weight was at a MeJA concentration of 300 µM (0.072 g).
However, the latter was not significantly different from
those observed at 75 µM of MeJA treatment (0.354 gr).
Our results suggested that 150 µM of MeJA was the
optimal concentration; in contrast, the other concentration
seemed to inhibit plant growth and development. This
result was also supported by data obtained from the control
plant. It indicates that PGRs added to the medium affected
plant growth (Amoanimaa-Dede et al. 2022).
We noticed that the alteration of fresh weight of the G.
pseudochina obtained from the MeJA treatment varies
depending on the MeJA concentration. Other studies also
exhibited similar results. For instance, administering Figure 5. Effect of MeJA on fresh weight of Gynura pseudochina
exogenous MeJA at 50 and 200 µM to Talinum in vitro culture after 75 days of treatment. Note: All data
paniculatum callus increased biomass compared to control represented by mean ± standard deviation. Different letters
(Restiani et al. 2022). The application of MeJA at a represented a statistically significant effect by Tukey's test
concentration of 1 µM led to a 10.89% increase in the fresh (P<0.05)
weight of Brassica oleracea plants compared to those that
1960 B I OD I V E R S ITA S 25 (5): 1955-1964, May 2024

The combination of IAA and BAP at the control Impact of MeJA on root number in Gynura pseudochina
medium successfully induced the formation of leaves. BAP tissue culture
is known as cytokinin, which contains nitrogen Plant roots absorb nutrients to support developmental
compounds, involved in protein synthesis; the latter is used processes (Lopez et al. 2023). A large number of roots can
for leaf growth (Pantin et al. 2011). Also, IAA increases optimize the absorption of nutrients in the culture media
the number of leaves and leaf area (Jin et al. 2023). No (Sathyanarayan et al. 2023). In this study, the addition of
explants treated with 300 µM of MeJA exhibited zero leaf MeJA significantly affected the number of roots of G.
formation. This data suggests that MeJA application acts pseudochina, with an average range of root numbers of
concentration-dependent, where the optimal concentration 1.25-1.60 (P<0.05) (Figure 7). Our data showed that the
was obtained at 150 µM. highest average number of roots was found when MeJA
Previous studies demonstrated that leaves can generally was added at 75 µM (1.6), followed by 0 µM of MeJA
induce callus (Rakesh et al. 2022). Another study explained (1.25). We observed no root formation at 150 and 300 µM
that MeJA, in some cases, might increase leaf growth and of MeJA treatment. Our results suggest that 75 µM of
senescence (Yamashita et al. 2021). In addition, 5 µM of MeJA might help the plant to initiate root formation (Jásik
MeJA has been reported to increase the number of leaves in and de Klerk 2006; Li et al. 2018).
wheat (Triticum aestivum L) (Islam et al. 2019). However,
in other cases, MeJA can also cause a decrease in the
number of leaves. For instance, adding 100 µM of MeJA
decreased the number of Celosia argentea L. leaves (El-
Sayed et al. 2023). The form of jasmonoyl-isoleucine (JA-
Ile), which is catalyzed from the jasmonate compound,
binds to the COI1 (coronatine insensitive-1) complex,
which then recruits the transcription repressor JAZ
(Jasmonate Zim-Domain) to degrade the COI-JAZ bond
via the 26S proteasome. The JAZ repressor will ultimately
inhibit the MYC transcription factor, increasing leaf
growth. However, when the JA-Ile accumulation is low, the
JAZ protein will be stably bound with MYC, which inhibits
plant growth during defense responses (Kamińska 2021;
Delgado et al. 2021). In addition, it is known that MeJA
might delay the transition from the mitotic cell cycle to the Figure 7. The Effect of MeJA on the root number of Gynura
endoreduplication cycle, which consequently reduces cell pseudochina in vitro culture after 75 days of culture. Note: All
number and size (Noir et al. 2013). data represented by mean ± standard deviation. Different letters
represent statistically significant differences by Tukey's test
(P<0.05)

A B

Figure 6. A. Effect of MeJA on the number of leaves of Gynura pseudochina in vitro culture after 75 days of treatment. Note: All data
represented by mean ± standard deviation. Different letters represented a significant effect by Tukey's test (P<0.05). B. Morphological
responses of G. pseudochina in vitro culture, showing leaf proliferation after MeJA treatment for 75 days. (1) Control (2) 75 µM (3) 150
µM (4) 300 µM (No shoot formed); (a) leaf (Scale bar: 1 cm)
 ANJALANI et al. – Methyl jasmonate stimulates of PAL gene in Gynura pseudochin 1961

MeJA application is known to modulate root and shoot of the PAL gene relative to the housekeeping gene (b-actin)
growth (Yamashita et al. 2021). MeJA has been reported to was evaluated. We detected no significant difference in
increase the number of roots by 37.21% in Cucumis GpPAL expression of the explant after treatment with 75
sativus. However, higher concentrations of MeJA (150 and and 300 µM of MeJA compared to the control (Figure 9).
300 µM) inhibit root formation (Feng et al. 2023). On the However, a significant expression of GpPAL was detected
other hand, 100 µM of MeJA also reduced the number of at MeJA 150 µM (1.29-fold compared to control) (Figure
roots in Triticale (Triticosecale wittmack), as reported by 9). PAL, 4CL, FLS, and F3′H genes, and DFR gene gene
Lahuta et al. (2017). In addition, root inhibition has also are responsible for the biosynthesis of many secondary
been reported in Arabidopsis after MeJA treatment. This metabolites, including flavonoids (Olsen et al. 2008).
might be due to the involvement of COI1 (Kamińska Overexpression of the PAL gene is commonly correlated
2021). Moreover, applying jasmonates (including MeJA) with the accumulation of flavonoid compounds (Xu et al.
can inhibit root apical growth and produce a short root 2020). Kianersi et al. (2020) reported that adding MeJA at
phenotype. The latter is due to Jasmonate-Auxin signaling 150 µM increases the flavonoid production in Capparis
(Jang et al. 2020). spinosa. Similarly, the callus culture of Givotia moluccana
treated with 150 µM of MeJA also accumulated a
Impact of MeJA on shoot multiplication of Gynura significant amount of total phenolic and flavonoid content
pseudochina in vitro culture compared to the control plant (Woch et al. 2023). Kianersi
All MeJA treatment significantly affected shoot number et al. (2020) also presented comparable findings concerning
generated from the nodal segment of G. pseudochina (p- the application of MeJA to the PAL gene in Salvia yangii
value <0.05), except 300 µM of MeJA (Figure 8). Shoot and Salvia abrotanoides plants. Their study indicated that
proliferation is important in plant tissue culture (Wu et al. adding MeJA at 150 µM yielded the highest PAL
2023); the number of shoots varied from 1.50 to 13.0 expression levels.
shoots per explant. The highest number of shoots was
observed at 150 µM of MeJA. Stimulation of shoot
proliferation was also increased when 100 µM of MeJA
concentration was given to the medium in Musa acuminata
in vitro culture (Mahmood et al. 2012). It is suggested that
MeJA modulates the biosynthesis of cytokinin, which is
important for shoot proliferation. Similarly, two-fold
cytokinin accumulation after MeJA treatment has also been
reported in Triticum aestivum (Avalbaev et al. 2016).
Despite the findings of an adversarial relationship between
jasmonic acid and cytokinin during Arabidopsis thaliana
development, Jang et al. (2017) also observed that MeJA
boosted cytokinin accumulation.
Moreover, adding MeJA at 75 µM resulted in the
lowest shoot number (1.5 shoots per explant), which was
not significantly different from those observed in the
control plant. Interestingly, we did not detect any shoot Figure 8. The Effect of MeJA on shoot proliferation of Gynura
formed in the explant treated with a higher amount of pseudochina in vitro culture after 75 days of culture. Note: Data
MeJA (300 µM). Rasouli et al. (2021) also reported a shows mean ± standard deviation. Different letters represent a
decrease in shoot multiplication rate when MeJA statistically significant effect by Tukey's test (P<0.05)
concentration exceeded 200 mg/L. Other studies also
demonstrated similar results, indicating that a higher
amount of MeJA negatively impacts shoot multiplications.
MeJA-mediated growth suppression may result from
reduced ATP production, proteins involved in energy
metabolism, and alteration of the mitochondrial membrane
(Patil et al. 2014).

Exogenous MeJA increases the expression of GpPAL in

the in vitro culture of Gynura pseudochina
Gynura pseudochina contains bioactive compounds like
flavonoids (Siriwatanametanon and Heinrich 2011). MeJA
supplementation in this study increased Phenylalanine
Ammonia-Lyase (GpPAL) gene expression. This gene is
engaged in the initial committed step of phenylpropanoid
Figure 9. The expression of the GpPAL gene in Gynura
synthesis (Yang et al. 2022). PAL functions to catalyze the
pseudochina in vitro culture after 75 days of MeJA treatment
formation of trans-cinnamic acid via phenylalanine
deamination. In this present study, the change in expression
1962 B I OD I V E R S ITA S 25 (5): 1955-1964, May 2024

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