Access to this full-text is provided by EDP Sciences.
Content available from BIO Web of Conferences
This content is subject to copyright. Terms and conditions apply.
In vitro shoot response of Rauvolfia serpentina
to the type and concentration of cytokinin
Rossa Yunita1
*
, Endang Gati Lestari1 Sitti Fatimah Syahid1, Media Fitri Isma Nugraha2,
Sustiprijatno3, Deden Sukmadjaja1, Diana Widiastuti4 , Evan Maulana3
1Research Organization for Agriculture and Food, National Research and Innovation Agency of
Indonesia, Cibinong Science Center, Jl. Raya Jakarta-Bogor, Cibinong Km 46, Bogor, West Java
16915, Indonesia.
2Organisation Research of Health. National Research and innovation Agency of Indonesia. Jl. Raya
Jakarta-Bogor, Cibinong Km 46, Bogor, West Java 16915, Indonesia
3 Research Organization for Life Sciences and Environment. National Research and Innovation
Agency of Indonesia, Jl. Raya Jakarta-Bogor, Cibinong Km 46, Bogor, West Java 16915, Indonesia
4Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Pakuan, Bogor
16144, Indonesia
Abstract.. Rauvolfia serpentina is widely recognized for its use as a raw
material in hypertension and antihypertensive medications, including
reserpine. Since this plant is used directly from the natural world, cultivation
activities are necessary. Seeds for cultivation must be consistent, high-
quality, and free of pests and diseases. Thus, a suitable propagation
technique is required. In vitro propagation is one method that can produce
homogeneous plants with a relatively high rate of multiplication. Cytokinin-
family regulatory molecules are crucial for in vitro proliferation techniques.
The aim of this research was to determine the optimal type and concentration
of cytokinin for the in vitro induction of R. serpentina shoots. This study
employed a completely randomized factorial design. The first factor was the
type of cytokinin (Benzylaminopurine (BA), Zeatin, Kinetin, and 2iP), and
the second factor was the cytokinin concentration (0, 0.5, 1.0, 1.5 mg/l).
Each treatment was replicated 10 times. The results showed that the best
cytokinin for R. serpentina shoot induction in vitro was BA at a
concentration of 0.5 mg/l. This treatment produced a greater number of
shoots and leaves, taller shoots compared to other treatments, and resulted
in more well-developed plant visualization.
1 Introduction
Rauvolfia serpentina belongs to the family Apocynaceae and is a potential medicinal plant
for development because it is used as raw material for pharmaceuticals. This is due to the fact
that R. serpentina contains 21 types of alkaloids, including reserpine, rescinnamine,
deserpidine, serpentine, yohimbine, and ajmaline, which can be used as treatments for high
blood pressure, as tranquilizers, and for circulatory system disorders [1].
*
Corresponding author: rossa_yunita@yahoo.com
© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons
Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/).
BIO Web of Conferences 127, 01004 (2024)
ICW Biotech 2024
https://doi.org/10.1051/bioconf/202412701004
R. serpentina is utilized in traditional medicine for treating conditions such as shortness
of breath, stomach pain, dysentery, headaches, and snake bites. It can also be used to reduce
fever, lower high blood pressure, and treat dysentery, cholera, loss of appetite, intestinal
inflammation, and more [2][3][4].
The amount of R. serpentina simplicia used domestically was 6,898 kg in 2000,
representing a 25.89% yearly growth [5]. Because R. serpentina is taken straight from the
wild, it is regarded as rare. The species is included in Appendix II of CITES (the Convention
on International Trade in Endangered Species of Wild Fauna and Flora), which lists species
that are not currently threatened with extinction but may do so if trade is not regulated. The
species is listed as threatened by the International Union for Conservation of Nature (IUCN).
The fact that roots of R. serpentina are employed as medicinal materials adds to its rarity by
making ordinary propagation difficult and restricting its range [6].
Conventional propagation of R. serpentina is very limited, with seed germination and
stem cutting percentages both below 15%. The stiff seed shell is the cause of the low growth
percentage and extremely limited capacity for seed germination.. This makes seed
propagation of R. serpentina suboptimal. To balance the demand for R. serpentina simplicia
and prevent its extinction, conservation and cultivation efforts are necessary. Therefore, mass
production of seedlings is needed. In vitro culture is one potential technique that can generate
a lot of seedlings in a short amount of time [ [7].
In vitro culture is a technique for growing plant parts such as protoplasts, cells, tissues,
or organs in a suitable medium under aseptic conditions [8]. Growth regulators are one of the
factors that affect the success of tissue culture. Plant tissues' biological processes are largely
regulated by growth regulators [9]. The chemical molecules known as growth regulators are
non-nutritive substances that, when present in small amounts, can promote the growth and
development of plants and are crucial in improving the metabolism of explants [9].
One commonly used group of growth regulators in tissue culture is cytokinins. The
cytokinins commonly used in tissue culture include 2-IP (2-Isopentenyl adenine), zeatin, BA
(benzil adenin), and kinetin (6-furfurylaminopurine). Generally, cytokinins function in cell
division, cell enlargement, delaying flower and fruit aging, and root and shoot differentiation
[10]. This description serves as the basis for the study's investigation of the impact of various
cytokinin formulations on the in vitro growth and provision of seedlings for R. serpentina
(L.) Benth. ex Kurz explants. Finding the right cytokinin type and concentration for R.
serpentina in vitro shoot induction is the aim of this study.
2 Methodology
The plant material used in this study was in vitro shoots of R. serpentina that were cultivated
for 14 days on MS (Murashige and Skoog) medium without the use of growth regulators. A
factorial totally randomized design with two components was employed in this investigation.
Two factors were identified: the kind of cytokinin (BA, Zeatin, Kinetin, and 2iP) and its
concentration (0, 0.5, 1, and 1.5 mg/l). There were ten duplicates of each therapy. The pH of
each treatment medium was brought down to 5.8 and added 30 g/l sucrose and 2.5 g/l gelrite
as supplements. Subculture on the same media was carried out after the plants were 2 weeks
old.
The number of roots, number of leaves, shoot height (cm), and number of shoots were the
variables that were observed. After the second subculture or during the fourth week,
observations were made. An F-test was used at a 5% significance level to statistically
examine the data gathered from observations of R. serpentina explants.
2
BIO Web of Conferences 127, 01004 (2024)
ICW Biotech 2024
https://doi.org/10.1051/bioconf/202412701004
3 Results and Discussion
Generally, the plant growth response to cytokinin treatment was observed through changes
in plant height and the emergence of new shoots. Each treatment produced different responses
across the various observed parameters.
3.1 Number of Shoots
The number of shoots was observed in the fourth week after culture. This measure is crucial
because the number of shoots produced indicates the success of the multiplication activity.
According to the ANOVA results, the interaction between the type and concentration of
cytokinin had a significant effect on the number of shoots. Table 1 shows that increasing the
concentration of cytokinin resulted in an increased number of shoots. The best treatment was
BA 0.5 mg/l, which produced 4.3 shoots, more than any other treatment. Table 1 also
indicates that BA produced more shoots than Kinetin, Zeatin, and 2iP. A synthetic cytokinin
made from adenine, BA is very effective at encouraging the growth of new shoots.
Chlorophyll production, tissue and organ differentiation, cell division, and other
physiological responses are all influenced by BA [11]. Stronger than other cytokinins like
kinetin or 2-iP, the BA is a cytokinin that plays a major role in shoot development and
multiplication [12].
Table 1. The effect of cytokinin type and concentration on the number of shoots in R. serpentina
cultur
Concentration of growth regulators
(mg/l)
Types of growth regulators
BA
Zeatin
Kinetin
2IP
0
1.3a
1.1a
1.1a
1.1a
0.5
4.3d
3.5c
1.4a
1.2a
1
3.5c
2.5b
1.9ab
2.1b
1.5
2.5b
2.5b
2.9bc
2.4b
There were 3.5 and 2.5 fewer shoots when the concentration of BA was increased to 1
mg/l and up to 1.5 mg/l, respectively. This also happened when zeatin was administered; at
0.5 mg/l, zeatin produced 3.5 shoots, but as the concentration of zeatin increased, fewer
shoots were produced. Because high quantities of cytokinins can interfere with food
absorption and decrease explant growth, it appears that employing growth regulators at
higher concentrations inhibits shoot growth (Kusmianto, 2008).
3.2 Plant Height
Because of the nutrients in the medium, the shoots are growing and developing, as seen by
the increase in shoot height. The measurement of shoot height serves as a gauge for the
growth that the administered treatments have produced. During the in vitro culture phase, the
growth rate pattern can be explained by the increase in shoot height.
3
BIO Web of Conferences 127, 01004 (2024)
ICW Biotech 2024
https://doi.org/10.1051/bioconf/202412701004
Fig. 1. The effect of cytokinin type and concentration on the shoot height of R. serpentina Culture
When compared to other treatments, the BA therapy often had the most positive
outcomes. Because cytokinins and auxins work synergistically as growth regulators, using
them in the same treatment media can promote shoot proliferation [14] [15].
The BA 0.5 mg/l treatment provided the best response in terms of plant height. The shoot
height produced with BA 0.5 mg/l treatment was 3.32 cm (Figure 1). Treatments with Zeatin,
Kinetin, and 2iP showed similar plant heights across various cytokinin concentrations.
However, compared to the treatment without cytokinin, the addition of cytokinin resulted in
taller shoots. By encouraging cell division and preventing elongation, cytokinins can promote
shoot growth, resulting in comparable shoot lengths for the three cytokinin types [16][17] .
3.3 Number of Leaves
The development of leaves is crucial because the leaf axils are where new shoots will sprout.
After four weeks of culture, leaves may grow on all treatments. Table 2 demonstrates that the
number of leaves was significantly impacted by the interaction between the type and
concentration of cytokinin. Out of all the treatments, the BA 0.5 mg/l treatment produced the
highest yield of 11.0 leaves and was found to have a noteworthy impact. Other treatments
with cytokinin produced less leaves at the same concentration. This suggests that BA offers
a good leaf-formation-inducing reaction. The BA treatment produced more leaves than the
kinetin treatment did in Photos tener, demonstrating another instance of this [18].
Table 2. The effect of cytokinin type and concentration on the number of leaves in R. serpentina
culture
concentration of growth
regulators (mg/l)
Types of growth regulators
BA
Zeatin
Kinetin
2IP
0
4.0a
4.17a
4.11a
4.15a
0.5
11.10f
6.80c
6.11c
5.11b
1
9.10e
5.80b
5.50b
4.70a
1.5
8.11d
5.20b
5.80b
6.40c
Compared to other treatments, the BA 0.3 mg/l treatment produced more leaves. There
was less leaf formation when the BA concentration was raised to 0.5 mg/l. Table 2
demonstrates that 8.11 leaves were produced as a result of the BA 1.5 mg/l treatment. The
response to other cytokinin therapies was comparable. For instance, there were nine leaves
2,01 2,03 2,1 2,09
3,32
2,47 2,27 2,304
2.74 2,23 2,46 2,13
2,32 2,23 2,25 2,12
0
0,5
1
1,5
2
2,5
3
3,5
BA Zeatin Kinetin 2iP
Shoot height increase(cm)
Type of growth regulator
0 mg/l
0.5 mg/l
1 mg/l
1.5 mg/l
4
BIO Web of Conferences 127, 01004 (2024)
ICW Biotech 2024
https://doi.org/10.1051/bioconf/202412701004
when the concentration of zeatin was 1.5 mg/l; but, when the concentration was raised to 1.5
mg/l, there were only 5.2 leaves. Plant growth is inhibited by high growth regulator
concentrations.
3.4 Number of Roots
For in vitro plant micropropagation to be successful during the acclimation stage, high-
quality root formation is essential. Figure 2 illustrates that fewer roots are seen at greater
cytokinin concentrations. The treatment without cytokinin had the greatest number of roots.
Fig. 2. The effect of cytokinin type and concentration on the number of roots of R. serpentina Culture
The number of roots significantly decreases as the concentration of BA increases, as seen in Figure
2. Treatments with zeatin, kinetikin, and 2iP have the same result. The number of roots decreases as
Zeatin, Kinetin, and 2iP concentrations are increased. The correct ratio of auxin to nutrients determines
the production of roots [19][20] [21]. Apart from the impact of exogenous auxin, genetic variations
resulting from the utilized explants and their native cytokinin concentration also have an influence.
4 Conclusion
Equations From the research conducted, it is clear that BA, at a concentration of 0.5 mg/l, is
the ideal cytokinin for shoot induction. Compared to previous treatments, this one produced
more leaves and shoots that were both taller and more numerous. Increasing the cytokinin
concentration can prevent leaves and shoots from developing. The application of cytokinin
can also inhibit root growth, with the highest number of roots forming in treatments without
cytokinin.
References
1. D. Lobay, Rauwolfia in the treatment of hypertension. Integrative Medicine. 14(3):40-
46 (2015).
2. J. Kaur, S. Gulati, Therapeutic potential of Rauwolfia serpentina. Indian Journal of
Advanced Research in Society. 2(1):99-104 (2017).
1,71 1,7 1,68 1,69
0,8
1,2
0,8 1
0,84
1,2
0,6
0,9
0,82
0,5 0,4
0,8
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
2
BA Zeatin Kinetin 2iP
Number of roots
Type of growth regulator
0 mg/l
0.5 mg/l
1 mg/l
1.5 mg/l
5
BIO Web of Conferences 127, 01004 (2024)
ICW Biotech 2024
https://doi.org/10.1051/bioconf/202412701004
3. A. Malviya, R. Sason, The phytochemical and pharmacological properties of
sarpagandha: Rauwolfia serpentina. Ayushdhara. 3(1):473-478 (2016).
4. S. Paul, S. Thilagar, G. Nambirajan, A. ·Elangovan, D. K. Lakshmanan, G.
Ravichandran, A. Arunachalam, S.· Murugesan, Rauwolfia serpentina: a potential
plant to treat insomnia disorder. Sleep and Vigilance. 6:31-40 (2011).
5. A. W. Karyaningtyas, A. Lestari, E. Sandra, The effect of several cytokinin formulations
on the provision of seeds and explant growth of pule pandak (Rauvolfia serpentina (L.)
Benth.ex Kurz) plants in vitro. Jurnal Agroplasma. 10(1):237-251 (2023).
6. B. B. Kunwar, Establishing in situ gene bank of Rauvolfia serpentina (L.) Benth ex Kurtz
in western Nepal with a focus on conservation and sustainability. Biodiversity
International Journal. 3(4) :134–143 (2019).
7. R. Yunita, E.G. Lestari, Propagation of Rauwolfia serpentina L. plants using tissue
culture techniques. Jurnal Natur Indonesi. 14(1):68–72 (2011).
8. C. A. Espinosa‑Leal, C. A. Puente‑Garza, S. García‑Lara, In vitro plant tissue culture:
means for production of biological active compounds. Planta. 248:1–18 (2018).
9. O. G. Ogunyale, O.O. Fawibe, A. A. Ajiboye, D. A. Agboola, A review of plant growth
substances: their forms, structures, synthesis and functions journal of advanced
laboratory. Research in Biology. 4(5):152-168. (2014).
10. J. Sosnowski, M. Truba, V. Vasileva, The Impact of auxin and cytokinin on the growth
and development of selected crops agriculture, 13,724 (2023).
11. M. Hönig, L. Plíhalová, A. Husiˇcková, J. Nisler, K. Doležal, Role of cytokinins in
senescence, antioxidant defence and photosynthesi. Int. J. Mol. Sci. 19,404 (2018)
12. I. W. WereJeżyna, I. Kuźma, A. K. Kiss, I. G. Karolak. Effect of cytokinins on shoots
proliferation and rosmarinic and salvianolic acid B production in shoot culture of
Dracocephalum forrestii W. W. Smith. Acta Physiologiae Plantarum, 40:189 (2018)
13. T. P. Pasternak , D. Steinmacher, Plant growth regulation in cell and tissue culture in
vitro. Plants. 13(2), 327 (2024).
14. B. S. Stefanowska, M. K. Baranowska, R. Hałasa, Influence of plant growth regulators
on the shoot culture of Phyllanthus glaucus and accumulation of indolizidine alkaloids
with evaluation of antimicrobial activity. Acta Physiologiae Plantarum. 41,6 (2019).
15. D. Raj, A, Kokotkiewicz, A. Drys, M. Luczkiewicz, Effect of plant growth regulators
on the accumulation of indolizidine alkaloids in Securinega suffruticosa callus cultures.
Plant Cell Tissue Organ Cult. 123:39–45(2015).
16. W. Wu, K. Du, X. Kang, H. Wei, The diverse roles of cytokinins in regulating leaf
development. Hortic. Res. 8,118 (2021)
17. G.E Schaller, I.H. Street, J. J. Kieber, Cytokinin and the cell cycle. Curr. Opin. Plant
Biol. 21:7–15 (2014).
18. R. Yunita, M. F. I. Nugraha, L. Sari, M. A. L. Rajamuddin, In vitro multiplication of
Pothos tener shoots. IOP Conf. Series: Earth and Environmental Science
1255:012045.(2013)
19. H. Jing, C. Strader, Interplay of auxin and cytokinin in lateral root development. Int. J.
Mol. Sci. 20:486 (2019).
20. H. Takatsuka, M. Umeda, Hormonal control of cell division and elongation along
differentiation trajectories in roots. J. Exp. Bot. 65,2633–2643 (2014)
21. C. H. Sun, J. Q. Yu, D. G. Hu, Nitrate: A crucial signal during lateral roots development.
Front. Plant Sci. 8,485 (2017)
6
BIO Web of Conferences 127, 01004 (2024)
ICW Biotech 2024
https://doi.org/10.1051/bioconf/202412701004
Content uploaded by Evan Maulana
Author content
All content in this area was uploaded by Evan Maulana on Sep 19, 2024
Content may be subject to copyright.
