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The Effect of Growth Concentration on In Vitro Shoot Multiplication of Crown Flower (Calotropis gigantea)
Plant Breed. Biotech. 2022;10:244-256
Published online December 1, 2022
© 2022 Korean Society of Breeding Science.

Okky Talitha1, Samanhudi Samanhudi1,2, Andriyana Setyawati1, Muji Rahayu1, Amalia T. Sakya1*

1Department of Agrotechnology, Faculty of Agriculture, Universitas Sebelas Maret, Surakarta 57126, Indonesia
2Center for Research and Development of Biotechnology and Biodiversity, Universitas Sebelas Maret, Surakarta 57126, Indonesia
Corresponding author: Amalia T. Sakya, amaliatetrani@staff.uns.ac.id, Tel: +62-0271-637457, Fax: +62-0271-637457
Received October 17, 2022; Revised November 21, 2022; Accepted November 22, 2022.
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Crown flower (Calotropis gigantea) as an herbaceous plant, is wildly recognized for its benefits as a medicinal plant. Phytochemical compounds which are contained in leaves, roots, and flowers of crown flower can be used as medicines. However, crown flower in Indonesia is still not optimal in their utilization. The study aimed to obtain the right concentration of growth regulators Benzyl Amino Purine and Indole Butyric Acid to stimulate optimal growth and secondary metabolites of crown flower. The study used a Completely Randomized Design arranged in a factorial design. The first factor was Benzyl Amino Purine and the second was Indole Butyric Acid. The data obtained were analyzed using analysis of variance with a 5% level test. The interaction of Benzyl Amino Purine and Indole Butyric Acid significantly increased the number of leaves and shoot emergence time. However, medium without Benzyl Amino Purine improved the percentage of root emergence by 100%, the number of roots by 17.2 roots, and plantlet height by 7.45 cm. Optimal concentration was obtained in 3 ppm Benzyl Amino Purine, which produced the high shoot and emergence time, as well as the number of shoots and leaves. Higher flavonoid contents were contained in cultured plants than in conventional cultivation. Optimal growth and phytochemical content give an opportunity to increase the production and the use of crown flower as medicinal plant.
Keywords : Auxin, Cytokinin, Gas Chromatography-Mass Spectroscopy, Flavonoids, Tissue culture
INTRODUCTION

Crown flower (Calotropis gigantea) is a herbaceous plant widely known to be useful as a medicine. This comes from Southeast Asia which is found in Indonesia, Malaysia, China, Sri Lanka, India, the Philippines, and Cambodia. C. gigantea is a perennial plant that can grow in tropical and subtropical areas (Kumar et al. 2013). This plant belongs to the Asclepiadaceae family with a plant height of up to 3 meters and has an opposite, fleshy, thick, and broad strand of leaves (Chan et al. 2017). This is a wild bush plant that can grow and reproduce rapidly. Besides, this plant lives in marginal land areas, dry grasslands, and coastal areas. This tall and waxy flower plant can adapt to extreme, dry, and hot environments. Crown flowers are perennial plants that are primarily spread in tropical and subtropical regions, Asia, and Africa (Lee et al. 2019).

Bioactive secondary metabolites were produced in crown flowers and antiproliferative activities were used to damage cancer cells (Hasballah et al. 2021). Bioactive compounds are detected widespread in some parts of the plant, such as flavonoids, alkaloids, phenols, saponins, tannins, and steroids which are beneficial as medicine materials. Their synthesis is affected by biotic and abiotic factors such as plant physiology, temperature, light inten-sity, humidity, and culture media composition (Chandran et al. 2020). Flavonoids are the largest group of secondary metabolites that can be found in plant tissues. Flavonoids are a group of phenolic compounds that can prevent cell damage by reactive free radicals and have antioxidant properties (Adamczak et al. 2020). Flavonoids contain antioxidants, namely in cereals, vegetables, and fruit. Secondary metabolite compounds such as flavonoids found in crown flower plants can be helpful as drugs for specific diseases if further identification is carried out.

A study reported an anti-inflammatory activity occurred in C. gigantea (Ahmad 2020). The sap on the stem of this plant is commonly used as an herbal medicine for bronchitis, asthma, dyspepsia, leprosy, tumors, and various digestive disorders. The roots are also useful as collagen, diaphoretic, trigger enzyme work, and diuretic. The leaves have also been used as a medicine for wounds, measles, fever, and coughs (Faradilla and Maysarah 2019). Thus, secondary metabolites in plants hold huge prospects to be used as medicine. Therefore, there is an urge to optimize the number of secondary metabolites of C. gigantea through plant tissue culture.

Plant tissue culture is a technique for growing parts of plants that can be cells, tissues, or organs under aseptic conditions. Tissue culture has successfully produced the desired crop yields using appropriate vitamins in media compositions and plant growth regulators (Kazemiani et al. 2018). Plant growth regulators can significantly affect the development of shoots, roots, and calluses. The combination of the right ratio of cytokinin and auxin concentrations can produce plantlets of crown flower plants with optimal secondary metabolites. BAP and IBA are growth regulators of cytokinin and auxin types which are commonly used to induce shoots in tissue culture. This study aimed to assess the effectiveness of BAP and IBA concentrations in inducing shoots, organogenesis, and phytochemical compounds for in vitro propagation of crown flower which could lead to further research of in vitro regeneration.

MATERIALS AND METHODS

Plant material and seed nursery

The stems of C. gigantea were collected from Screen House, Faculty of Agriculture, Universitas Sebelas Maret, Indonesia, through a seed nursery. Crown flower seeds were obtained from the Ministry of Industry, Textile Center, Bandung. Seed selection was done prior to seedling in order to separate seeds based on their quality. A compost planting media was used as a planting mixture. Each polybag contained 4-5 seeds planted. Seedlings were carried out for 1.5 months or until the stems were ready to be used. The abnormal seeds were replanted into new seeds. The plants were irrigated regularly using tap water every day. The pest control was done by spraying the solution of contact pesticides directly to the attacked plant parts. The 1.5-month - old of crown flower plants were picked out of polybags. Roots and leaves were cut off because the explant used was only plant stem parts. Then the stem segments of C. gigantea were cultured in vitro to optimize the production of secondary metabolites of crown flower plants.

Tools and explants sterilization

Sterilization was carried out on tools and explants. Several tools such as culture bottles and dissection equipment (tweezers and scalpel knives) were washed thoroughly and then sterilized using an autoclave at 121℃ and 1 atm pressure for 30 minutes before being stored in the oven or used for initiation. Explant of C. gigantea stems was washed under running tap water to remove the dripping sap from the edges of the leaves cut. After that, explants were immersed using a solution of detergent and aquadest (20 mL detergent for 100 mL aquadest) for one minute and rinsed using distilled water. This was followed by soaking explants in a solution of fungicide and bactericide (1 g fungicide and 1 g bactericide for 1000 mL aquadest) for 45 minutes, then rinsed using distilled water until clean. Sterilization of explants was then continued in a Laminar Air Flow Cabinet (LAF) by rinsing them using sterile distilled water twice, immersing them in 100% Clorox solution for 1.5 minutes, followed by rinsing them with sterile distilled water. The explants were also sterilized by immersing them in 70% alcohol solution for 1.5 minutes and then rinsing with sterile distilled water twice.

Culture medium

Media was prepared by adding various concentrations of BAP and IBA to Murashige and Skoog (MS) base medium. The preparation of the media started by mixing 30 grams of sugar, 50 mL of MS macronutrients, 10 mL of MS micronutrients, 50 mL of Fe-EDTA, 50 mL of vitamins, BAP (0 ppm (A0); 1 ppm (A1); 2 ppm (A2); 3 ppm (A3); 4 ppm (A4)) and IBA (0 ppm (B0); 1 ppm (B1); 2 ppm (B2); 3 ppm (B3); 4 ppm (B4)), followed by adding 1000 mL of distilled water. The medium was adjusted to pH 6.2 before the addition of agar. To control the pH, NaOH is used to lower the pH, while HCl is used to increase it. After that, 8 grams of agar powder were added, then heated with a magnetic stirrer until it boiled. The boiled medium was transferred to a culture bottle and autoclaved. The best treatment for callus induction and organogenesis was determined using control media without the addition of plant growth regulators.

Explant initiation

The initiation was carried out after sterilization under sterile environmental conditions in a Laminar Air Flow Cabinet (LAF). After the sterilization process, the explants were cut along approximately 2 cm in each internode using a scalpel knife. Then, the explants were dried on sterile tissue. Using tweezers, the excised stems were planted into a culture bottle containing Murashige and Skoog medium. Each culture bottle contained one explant. Initiation activities were done nearby the bunsen lamp to minimize contamination.

Culture maintenance

All culture bottles were sealed with plastic wrap and incubated in a growth room at a temperature of 25℃ with a photoperiod of 24 hours under a white fluorescent lamp. Cultured explants were maintained to prevent contamination in the culture bottles by spraying 70% alcohol into the culture bottles every 2 days and removing the contaminated culture bottles. The growth of explants was observed for 65 days, starting from the day after planting.

Data collection and analysis

The data analysis used was Analysis of Variance (ANOVA) with the F test at 5% level, and if there was a significant difference, it was proceeded with Duncan's Multiple Range Test (DMRT) at 5% level. In order to conduct this study, a Completely Randomized Design (CRD) was used and arranged in a factorial design consisting of 2 treatment factors. Each treatment was replicated three times, with 25 treatment combinations per replication. The data will be presented in the form of graphs, pictures, and conclusions will be drawn.

RESULTS

Shoot emergence time

The analysis revealed that the interaction between BAP and IBA had a significant effect on the time of emergence of crown flower shoots (Table 1). The formation of crown flower shoots appeared on average between 2.67-8.33 DAP. The fastest shoot emergence time was found in the combination of 3 ppm BAP + 1 ppm IBA with an average shoot emergence time of 2.67 DAP. Significantly different results were shown in the combination of 0 ppm BAP + 1 ppm IBA which formed shoots in the longest time of 8.33 DAP. The combined effect of BAP and IBA treatments in one medium can trigger shoot growth because the two growth regulators have a synergistic effect. The treatment of 3 ppm BAP + 0 ppm IBA was thought to be the optimal concentration which had a positive impact on the acceleration of the growth of crown flower shoots with the emergence of 3 DAP.

Table 1 . The effect of BAP and IBA interaction on the shoot emergence time in crown flower explants aged 65 DAP.

IBA (ppm)BAP (ppm)BAP Average
01234
04.00a-c4.00a-c4.67a-c3.00a3.67ab3.87
18.33d3.00a4.33a-c2.67a4.00a-c4.46
24.00a-c6.33b-d4.00a-c4.00a-c3.67ab4.40
34.33a-c4.67a-c4.67a-c4.67a-c5.00a-c4.67
44.67a-c7.00cd4.00a-c4.00a-c3.33ab4.59
IBA average5.075.004.333.673.93+

The + sign indicates an interaction.



Number of shoots

The results of the analysis showed that the application of BAP on MS media had a significant effect on the number of shoots. While the interaction between BAP and IBA had no significant effect. Increasing the concentration of BAP up to 4 ppm increased the number of shoots formed on crown flower explants. The highest number of shoots was found in the treatment with 4 ppm BAP concentration, namely 5.87 shoots. Treatment of 4 ppm BAP was significantly different from BAP of 0 and 1 ppm but not substantially different from BAP 2 and 3 ppm because the values were followed by the same notation (Table 2).

Table 2 . The results of number of shoots in crown flower explant aged 65 DAP.

IBA (ppm)BAP (ppm)BAP Average
01234
02.03.05.77.05.04.5
13.75.74.09.34.35.4
21.02.73.75.010.04.5
34.32.03.30.74.73.0
42.32.32.02.35.32.9
BAP average2.67a3.13a3.73ab4.87ab5.87b-

The - sign indicates there is no interaction.



Plantlet height

The results of the analysis revealed that the application of BAP significantly affected the height of the crown flower plantlets. While the interaction between BAP and IBA had no significant effect. The concentration of BAP 0 ppm was significantly different from BAP 1; 2; 3; and 4 ppm. The concentration of 0 ppm BAP produced the highest plantlet which was 7.45 cm, while the treatment which produced the shortest plantlet was the treatment of BAP 3 ppm which was 3.97 cm (Table 3).

Table 3 . The results of plantlet height in crown flower explant aged 65 DAP.

IBA (ppm)BAP (ppm)BAP Average
01234
08.64.64.93.83.55.1
18.94.04.93.75.25.3
26.03.54.23.83.54.2
38.05.93.94.33.75.2
45.83.74.04.44.04.4
BAP average7.45b4.35a4.37a3.97a3.99a-

The - sign indicates there is no interaction.



Root emergence time

The results of the analysis showed that the application of BAP had a significant effect on root emergence time. However, the interaction between BAP and IBA treatments and single IBA treatments had no significant effect. Treatment with 0 ppm BAP concentration had the longest root emergence time of 10.73 DAP, while 3 ppm BAP showed the fastest root emergence time of 1.33 DAP. Roots did not appear at a BAP concentration of 4 ppm until the end of the observation. Although the 3 ppm BAP treatment resulted in the fastest shoot emergence time, the result was not significantly different from 1 ppm BAP. Therefore, the BAP treatment of 1 to 3 ppm was the more optimal concentration because the root emergence time at that concentration was relatively fast but not significantly different (Table 4).

Table 4 . The results of root emergence time in crown flower explant aged 65 DAP.

IBA (ppm)BAP (ppm)BAP Average
01234
07.82.55.50.00.03.2
110.04.50.00.00.02.9
24.30.00.00.00.00.9
39.80.08.05.00.04.6
48.54.09.00.00.04.3
BAP average10.73c2.93ab6.00b1.33ab0a-

The - sign indicates there is no interaction.



Number of roots

The results of the analysis showed that the application of BAP on MS media had a significant effect on the number of roots. While the interaction between BAP and IBA had no significant effect. The highest number of roots was found at 0 ppm BAP concentration treatment, namely 17.2 roots. The treatment of BAP 0 ppm was significantly different from other treatments, so it can be seen that 0 ppm BAP is the most optimal treatment to increase the number of roots (Table 5).

Table 5 . The results of number of roots in crown flower explant aged 65 DAP.

IBA (ppm)BAP (ppm)BAP Average
01234
09.73.00.30.00.02.6
125.32.70.00.00.05.6
22.00.00.01.00.30.7
329.00.01.30.70.06.2
420.03.75.00.00.05.7
BAP average17.20b1.87a1.33a0.33a0.07a-

The - sign indicates there is no interaction.



Number of leaves

For all levels of BAP, the treatment of IBA that produced the highest average number of leaves was at a concen-tration of 1 ppm IBA, which was 19.4 leaves. This result is better than the average at IBA concentrations 0; 2; 3; and 4 ppm. Treatment of 0 ppm BAP + 2 ppm IBA resulted in the least number of leaves, namely 1.33 leaves. In contrast, the highest number of leaves was found in the treatment of 2 ppm BAP + 1 ppm IBA, which was 23.67 leaves. However, the treatment was not significantly different from all treatments of 1 ppm IBA with the addition of BAP 0, 1, 3, and 4 ppm.

Observational data (Table 6) showed that at all BAP levels, the IBA treatment that produced the highest average number of leaves was at a concentration of 1 ppm IBA, which was 19.4 leaves. This result is better than the average at IBA concentrations 0; 2; 3; and 4 ppm. Treatment of BAP 0 ppm + IBA 2 ppm resulted in the least number of leaves, namely 1.33 leaves. While the highest number of leaves was found in the treatment of 2 ppm BAP + 1 ppm IBA, which was 23.67 strands. However, the treatment was not significantly different from all treatments of 1 ppm IBA with the addition of BAP 0, 1, 3, and 4 ppm.

Table 6 . The results of the interaction of BAP and IBA on the number of leaves in crown flower explants aged 65 DAP.

IBA (ppm)BAP (ppm)BAP Average
01234
08.67a-c11.67a-c20.00bc21.33bc15.00a-c15.33
121.00bc17.33a-c23.67c23.33c11.67a-c19.4
21.33a7.33ab16.00a-c22.67bc21.33bc13.73
320.67bc12.67a-c8.00a-c5.67ab17.00a-c12.80
418.33a-c12.33a-c9.00a-c9.00a-c22.00bc14.13
IBA Average14.0012.2715.3316.4017.40+

The + sign indicates an interaction.



Percentage of root appearance

The results showed that the percentage of explants that appeared roots in each treatment combination differed (Fig. 1). Treatments A0B0, A0B1, and A0B3 showed the percentage of root growth at 100%, which means that roots appeared in all replicates in that treatment. In A0B4, A1B4, and A2B4 treatments, the percentage of root growth was 66.67%, which means roots appeared in 2 replications out of a total of 3 replications in that treatment. Treatments A0B2, A1B0, A1B1, A2B0, A3B2, A3B3, and A4B2 showed a percentage of root growth of 33.34%, which means roots appeared in 1 replication of a total of 3 replications in the treatment. While other treatments did not show any root emergence up to 65 DAP.

Figure 1. Histogram of the effect of BAP and IBA on the percentage of root emergence in crown flower. A0B0 (0 ppm BAP + 0 ppm IBA); A0B1 (0 ppm BAP + 1 ppm IBA); A0B2 (0 ppm BAP + 2 ppm IBA); A0B3 (0 ppm BAP + 3 ppm IBA); A0B4 (0 ppm BAP + 4 ppm IBA); A1B0 (1 ppm BAP + 0 ppm IBA); A1B1 (1 ppm BAP + 1 ppm IBA); A1B2 (1 ppm BAP + 2 ppm IBA); A1B3 (1 ppm BAP + 3 ppm IBA); A1B4 (1 ppm BAP + 4 ppm IBA); A2B0 (2 ppm BAP + 0 ppm IBA); A2B1 (2 ppm BAP + 1 ppm IBA); A2B2 (2 ppm BAP + 2 ppm IBA); A2B3 (2 ppm BAP + 3 ppm IBA); A2B4 (2 ppm BAP + 4 ppm IBA); A3B0 (3 ppm BAP + 0 ppm IBA); A3B1 (3 ppm BAP + 1 ppm IBA); A3B2 (3 ppm BAP + 2 ppm IBA); A3B3 (3 ppm BAP + 3 ppm IBA); A3B4 (3 ppm BAP + 4 ppm IBA); A4B0 (4 ppm BAP + 0 ppm IBA); A4B1 (4 ppm BAP + 1 ppm IBA); A4B2 (4 ppm BAP + 2 ppm IBA); A4B3 (4 ppm BAP + 3 ppm IBA); A4B4 (4 ppm BAP + 4 ppm IBA).

Analysis of flavonoid content

Testing the flavonoid content as secondary metabolites in crown flowers is one of the destructive observations, namely removing samples of crown flower plantlets aged 65 DAP and then drying the explants in an oven for 24 hours. The samples used were 1 field crown flower sample and 5 plantlet samples with varying concentrations of BAP and IBA to represent all treatments. The plantlet samples used in the flavonoid content analysis were A0B0, A1B4, A4B1, A1B1, and A4B4. While the field crown flower sample was used as a comparison.

Table 7 shows the flavonoid content in the field crown flower sample, which is 1.08%. The highest flavonoid content was found in sample A0B0 (BAP 0 ppm + IBA 0 ppm) which was 1.73%. This value is higher than the value of flavonoid content in the field sample. While the lowest flavonoid content was found in sample A1B4 (BAP 1 ppm + IBA 4 ppm) which was only 0.75%. Crown flower plantlet samples without BAP and IBA had a higher percentage of flavonoid-type secondary metabolites when compared to field crown flower samples.

Table 7 . The results of the flavonoid content in several samples of crown flower aged 65 DAP.

SampleFlavonoid content (% w/w)
Field1.08
A0B01.73
A1B11.22
A1B40.75
A4B11.11
A4B40.94

A0B0: Treatment of BAP 0 ppm + IBA 0 ppm, A1B1: Treatment of BAP 1 ppm + IBA 1 ppm, A4B4: Treat-ment of BAP 4 ppm + IBA 4 ppm.



Phytochemical compounds

GC-MS (Gas Chromatography- Mass Spectroscopy) analysis was performed on samples of field crown flowers and plantlets cultured at 65 DAP to identify different compounds using liquefied gas chromatography and mass spectrometry methods. According to Revanthi et al. (2015), GC-MS analysis is able to provide crucial information on the compound components and relatively low molecular weights. A total of 0.5 g fraction of the dried A3B2 (3 ppm BAP + 2 ppm IBA) crown flower plantlets were used in the GCMS analysis.

The dominant compounds contained in both samples were pentadecanoic acid, benzene ethyl, and benzene 1,3-dimethyl. Cyclononacyloxane octadecamethyl was not found in crown flower plantlets but only in field crown flower samples. Pentadecanoic acid was produced in both samples, namely, field crown flower and plantlet crown flower A3B2, with a high percentage. Pentadecanoic acid, or pentadecanoic acid is a group of saturated fatty acid compounds with a long straight chain structure with 15 carbon atoms (C). Pentadecanoic acid, also known as my-ristic methyl ester, has the molecular formula C15H30O2. However, according to Pfeuffer and Jaudszus (2016), some plants contain high amounts of pentadecanoic acid. This compound has the same long-chain structure as hexade-canoic acid and oleic acid. The function of this compound is as an antibacterial and antifungal.

DISCUSSION

Shoot emergence time

The slow emergence of shoots in explants was caused by the concentration of growth regulators BAP which was too low. The time of emergence of the different shoots was thought to be caused by differences in the response of the cells and tissues of the explants of crown flower to the availability of endogenous and exogenous auxin. This is supported by Phillips and Garda (2019), that shoot growth requires deficient concentrations of auxin with moderate concentrations of cytokinins. The fast shoot emergence time indicated that the cells in 3 ppm BAP + IBA 0 ppm were more actively dividing (Fig. 2). Therefore, if the interaction between cytokinin and auxin used is in accordance with the needs of the explant, the cell division process will be faster. The faster shoots formation showed an increase in the nutrients absorbed by the explants, where all the nutrients came from the agar medium in the culture bottle.

Figure 2. The growth of crown flower explants in vitro (a) root emergence, (b) shoot emergence, (c) leaves of the explant.

Number of shoots

The increase in BAP concentration increased the number of shoots, but the overall growth of crown flower shoots was not optimal. This is because BAP is an artificial cytokinin that inhibits shoot growth if the concentration used is too high. It is supported by Hussain et al. (2021), that plant organ development is determined by possible synergistic and antagonistic interaction of auxin and cytokinin. This is supported by research by Kazemiani et al. (2018), that the increase of BAP increased shoot multiplication. According to Agustina et al. (2020), the advantage of BAP is that it is one of a group of synthetic cytokinin hormones that are active and have a longer duration of stimulation. BAP has high effectiveness in stimulating shoot formation, is more stable, and is resistant to oxidation. This shows the implication that using BAP concentrations of 2 to 4 ppm is more recommended because it produces a high number of shoots with results that are not significantly different or the same. Shoots in each explant had various conditions, such as shoots with long nodes and shoots with tight nodes. Giving BAP at high concentrations can cause plantlets to be smaller and leaf growth not to be optimal and crowded (rosette). So that the use of BAP with high concentrations for an extended period resulted in the number of plantlets with abnormal growth tending to increase.

Plantlet height

The increase in BAP concentration was inversely proportional to the increase in the height of growing crown flower plantlets. Therefore, 0 ppm BAP treatment is the best treatment because, in addition to producing the highest plantlets, it can be concluded that a low BAP concentration can increase the plantlet’s height. Plants can naturally produce phytohormones which in low concentrations can affect plant physiological processes, so the addition of growth regulators can inhibit plant height growth. According to Arnao and Hernández-Ruiz (2014), plant growth regulators have an inhibitory effect at high concentrations. In addition, growth regulators in the form of BAP with a higher concentration than auxin increased the number of shoots and leaves, and plantlet height decreased.

Root emergence time

Root emergence time is one of the qualitative parameters indicating the growth of cultured explants. The function of the roots is to absorb nutrients and water for plants so that if the roots appear faster, the nutrients will be fulfilled more quickly. According to (Karyanti et al. 2021), the accuracy of the composition of the primary media used supported the success of root induction in tissue culture. Cytokinins can reduce auxin levels in the roots and inhibit root formation at high cytokinin concentrations (Márquez et al. 2019). This implies that using 1 to 3 ppm BAP concentrations is more economically recommended than other BAP concentra-tions (Fig. 2).

Number of roots

If the number of roots formed is increasing, this indicates that the nutrients absorbed are also more optimal. Accord-ing to Agustina et al. (2020), the root growth of explants in tissue culture cultivation depends on the growth regulators’ concentration. A low BAP concentration was able to increase the number of roots formed in crown flower explants. The increase in BAP concentration was inversely proportional to the number of roots formed. The impli-cation is that root initiation has produced a fairly optimal number of roots without being given BAP treatment on culture media. This is supported by previous research by Chen et al. (2019) that cytokinins with high concentration could harm root growth.

Number of leaves

Leaf formation is part of the growth of shoots and leaves that are formed in tissue culture. Leaf formation is influenced by the application of auxin and cytokinin in specific concentrations. Leaves are plant parts that grow on stems and are in the form of sheets and function as a place for photosynthesis to take place. The number of leaves is a growth indicator showing biomass formation in plants. The combination treatment of 0 ppm BAP + 1 ppm IBA is the optimal treatment and is economically recommended to increase the leaves number compared to the treatment of 2 ppm BAP + 1 ppm IBA (Fig. 2). This is because auxin triggers the performance of the gibberellin hormone in elongating the internodes, thereby increasing the number of nodes and the number of leaves. While cytokinins can trigger cell division, one of which is the formation of leaves. According to Agustina et al. (2020), both auxin and cytokinin have long been known to work synergistically and antagonistically in controlling several significant developmental processes.

Percentage of root appearance

According to Hughes (1987) in Widyarso (2010), different plant genotypes will show different morpho-genesis directions, so there is no universal comparison between cytokinins and auxins that can be used as a basic reference in inducing roots. This is the cause of no root formation in several combinations of IBA and BAP treatments. The combination of treatments without BAP tends to be able to produce roots optimally, which is 80%. This is supported by research by Gethami & Sayed (2020), that culture media without any concentrations of cytokinin growth regulators (BAP) stimulates root formation. The addition of cytokinins, in general can inhibit root forma-tion; to stimulate root formation, it is necessary to apply auxin growth regulators.

Flavonoid content

The crown flower plantlets used as samples have varied growths resulting in various organ morphology (Fig. 3). Crown flower plantlets with A0B0 treatment had the widest, thickest, dark green leaf blade morphology compared to other treatments. The stems are erect and sturdy, and the roots formed are numerous and long. Treatments A1B1 and A4B1 produced small leaves, formed small calluses, and there were branches. A1B4 and A4B4 treatments produced larger callus than others, but leaf formation was less than optimal. This proves the statement of Chandana et al. (2018) that the benefits of tissue culture in the development of biotechnology in medicinal plants, among others, for the production of secondary metabolites.

Figure 3. The performance of crown flower plantlets aged 65 DAP at concentrations of (a) 0 ppm BAP + 0 ppm IBA (A0B0); (b) BAP 1 ppm + IBA 1 ppm (A1B1); (c) BAP 1 ppm + IBA 4 ppm (A1B4); (d) BAP 4 ppm + IBA 1 ppm (A4B1); (e) BAP 4 ppm + IBA 4 ppm (A4B4).

Crown flower plantlets treated without BAP and IBA had a percentage of root emergence of 100% and a percentage of callus emergence of 0%. This treatment resulted in long roots in all replicates, had a shoot emergence time of 4 DAP, and the number of leaves was 8 pieces. The morphology of the leaves formed is wide, thick, and dark green. According to Chandrawat & Sharma (2018), crown flower leaves contain secondary metabolites such as alkaloids, phenols, saponin, and flavonoids which are useful as antibacterial. Kumar et al. (2013) and Patel et al. (2014) stated that the results of the phytochemical test showed that flavonoid compounds were contained in the roots of the crown flower plant. A study by Kumar et al. (2021) showed that flavonoid compounds were present in the bark of crown flower stems. Therefore, the accumulation of flavonoid compounds in the treatment without BAP and IBA was higher because the crown flower’s roots, stems, and leaves were formed optimally. It can be concluded that the treatment without BAP and IBA resulted in the highest flavonoid content compared to other samples. The flavo-noid content in crown flower samples was influenced by growth regulators IBA and BAP added to MS media.

Phytochemical compounds

The results of the GC-MS test were written as a series of peaks representing one compound in the sample. The analysis results show there are 4 major phytochemical compounds in the field sample and A2B3 plantlets (Table 8). The most formed compounds (major compound) in the field crown flower samples were pentadecanoic acid with a peak height of 17.8%, an area of 9.93%, and a retention time of 31.641 minutes, benzene ethyl with a peak height of 13.54%, an area of 9.43%, in retention time 7.652 minutes, and Cyclononacyloxane octadecamethyl with a peak height of 13.05%, an area of 15.08%, in a retention time of 34.09 minutes.

Table 8 . Major compounds of crown flower field samples and A2B3 plantlets using GCMS analysis.

No.Compound nameCompound structureMolecular formulaField sampleA3B2 plantlets
1Pentadecanoic acidC15H30O2++
2Benzene, 1-3-DimethylC8H10++
3Benzene ethylC8H10++
42,2,4,4,6,6,8,8,10,10,12,12,14,14,16,16,18,18-Cyclononasiloxane octadecamethylC18H54O9SI9+-

The + sign indicates the presence of chemical compounds in the test sample with GC - MS.



Retention time is the time required for the sample from the time of injection or running until it comes out at the peak of the chromatogram (Fig. 4). Each compound has a different retention time depending on the compound’s boiling point, solubility in the liquid phase, and column temperature. According to Ruwindya (2019), the difference in retention time is caused by the stationary phase in the GC-MS tool, which is different in nature, affecting the interaction of compounds at the separation stage in the column.

Figure 4. Representative chromatogram of C. gigantea (a) A3B2 plantlet and (b) in vivo.

The analysis results show that the number of compounds in A3B2 crown flower plantlets is less than in the field sample, which contains 10 chemical compounds. The major compound in the A3B2 crown flower plantlet was benzene ethyl with the molecular formula C8H10, the peak height percentage was 28.71%, and the area percentage was 28.94%, with a retention time of 7.710 minutes. Next is benzene, 1-3-Dimethyl, with the molecular formula C8H10, the peak height percentage is 18.27%, and the area percentage is 18.34% in a retention time of 7.939 minutes. Pentadecanoic acid with molecular formula C15H30O2, peak height percentage 14.43%, area percentage 12.81%, in retention time 31.665 minutes. The highest percentage of peak height was found in the benzene ethyl compound, with a peak height of 28.71%. Benzene ethyl also has the largest percentage of area, which is 28.94%. The fastest retention time is the compound 2-Pentanol, 4-Methyl with a retention time of 5.091 minutes.

This is because biochemical compounds in plants can increase or decrease after propagation through tissue culture with the addition of IBA and BAP. This is supported by Gunawan (1992), the content of secondary metabolites in tissue culture will be different from the original plant qualitatively and quantitatively due to differences in environmental conditions of growth.

CONCLUSION

In the present study, in vitro shoot induction was developed from stem explants of a crown flower. Here-after, treatments of 2 ppm IBA followed by an increase in BAP of 1 ppm to 4 ppm accelerated the time of shoot emergence and increased the number of leaves. The con-centration of BAP 0 ppm gave the highest average per-centage of root emergence, namely 80%, increasing the number of roots by 17.2, the number of shoots by 2.67, and the plantlet height of 7.45 cm. Crown flower cultured in vitro without PGR had a higher flavonoid content than crown flower grown conventionally. Also, there are dif-ferences in the types of bioactive compounds presence between crown flower cultivated in vitro and conventionally.

ACKNOWLEDGEMENTS

This research was funded by a non-APBN UNS grant (PUT-UNS) 2021.

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