1. INTRODUCTION
Curculigo latifolia, often known as Lemba or Marasi, is a member of the family Hypoxidaceae. It grows in the Indonesian highlands of Kalimantan Java, Papua New Guinea, Bangka, and Sumatra on well-drained, fertile soils that are rich in organic matter. According to reports, the plant is found in the Philippines, West Africa, India, Myanmar, and Thailand [1,2]. Because of its many applications, C. latifolia is a plant with significant economic worth and promising future growth. Fishing nets are made in Kalimantan, Indonesia, and Malaysia using the fibers from C. latifolia leaves. The Dayak tribe in East Kalimantan uses fibers from C. latifolia leaves to make their traditional clothes, Ulap doyo. C. latifolia is a significant medicinal plant in Malaysia [1,2]. Furthermore, the plant is well-known for two unique sweet proteins: Curculin which has been demonstrated to be 500–9,000 times sweeter than sucrose by weight [3,4], and Neoculin, a taste-modifying protein that changes sour taste to sweet [5].
Curculigo latifolia is only propagated spontaneously in the wild in Kalimantan, Indonessia. Curculigo grows on well-drained, organic matter-rich soils. Curculigo grows in rain forests in Java up to an elevation of 1,100 m; it has not yet been regularly domesticated [2]. This plant develops slowly, which makes challenging standard propagation. According to Ismail et al. [6], seeds are difficult to germinate. The seeds need certain media and attention because they are resistant. An alternate option is the in vitro method. Babaei et al. [7] reported on the micropropagation of C. latifolia using C. latifolia shoot tips from Malaysia as explants on Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ: 0, 0.5, 1, 1.5, and 2 mg l−1) and indole-3-butyric acid (IBA: 0, 0.25, and 0.5 mg l−1) for shoot regeneration, Farzinebrahimi et al. [8], other researchers, successfully produced callus from the leaves and tuber of Malaysian-origin C. latifolia explants. They used MS base medium, which was supplemented with different concentrations of IBA, either alone or in combination with BAP (0.5–4 mg l−1) and IBA (0–4 mg l−1) and contained 3% sucrose that had been solidified using 2.5 g l−1 Gelrite. On the other hand, leaf explants with IBA and BAP (4.0 mg l−1) produced the most callus. Furthermore, callus and shoot induction on medium supplemented with IBA and BAP were also observed by Umar et al. [9]. Hormones called auxins and cytokinins are frequently utilized alone or in combination on in vitro culture for callus initiation, shoot induction or regeneration, and root induction. Typically, auxin is utilized to promote elongation and cell division as well as the development of callus, roots, and culture suspension. Cytokines are substances that can promote leaf growth, development, and cell division. Combining auxin with cytokines will promote cell division and growth, boost protein synthesis, and have an impact on callus growth [10]. This study, however, provides a more efficient stage for the micropropagation of C. latifolia on employing growth regulators.
DNA-based molecular markers have proven to be adaptable instruments and have established themselves in a number of domains, including genetic engineering, plant breeding, and taxonomy. Because they are the most affordable among all the molecular markers available, polymerase chain reaction (PCR)-based RAPD and ISSRs have become the most widely employed markers for assessing genetic fidelity in micropropagated plants [11]. In addition, the RAPD and ISSR markers have the benefit of being fast, easy to use, and requiring very little DNA and require only small amounts of DNA [12].
Many challenges have been well defined on in vitro plant research, namely physiological, epigenetic, and genetic quality problems associated with cells, tissues, and organs of in vitro plants, absence or loss of organogenic potential (re-calcitrance), hyperhydricity (vitrification) and somaclonal variation. Somaclonal variation is one of the very important problems on in vitro culture. It is imperative to determine the genetic stability of in vitro regenerated plants [13]. Molecular markers especially RAPD and ISSR have been widely applied to monitor somaclonal variation and verify the genetic stability of micropropagated plants [11–19]. However, genetic stability tests on in vitro propagation of C. latifolia have never been reported. The objectives of this study were to describe an efficient protocol for micropropagation of C. latifolia along with the RAPD and ISSR analysis to determine the genetic fidelity of the regenerated plants.
2. MATERIALS AND METHODS
2.1. Micropropagatin of C. latifolia
2.1.1. Callus induction and culture condition
The plant material or explants used in this study were sterile leaves from seedlings grown in vitro. Leaves were cut about 1.5 × 2 cm and inoculated on MS solid medium with the addition of 6 g l−1 agar and 3% sucrose. Medium pH was adjusted to 5.8. MS medium was enriched with 5 mg l−1 IBA dan 3 mg l−1 BA according to Umar et al. [9]. The cultures were incubated at 23°C ± 2°C without lighting (dark room) for 30 days.
2.1.2. Shoot induction and culture conditions
The callus obtained was subcultured on a shoot induction medium. The medium was MS with the addition of a growth regulator with different compositions. They are MS free growth regulator (MS0); 5 mg l−1 IBA and 3 mg l−1 BA; 1 mg l−1 2,4D and 3 mg l−1 BA; 0.5 mg l−1 NAA and 5 mg l−1 BA. The medium was added 6 g l−1 agar and 3% sucrose. The pH of the media was adjusted to 5.8. The cultures were incubated at 23°C ± 2°C with 1,500 lux LED lighting (PhilipTM TL) on a 16 hour/8 hour light/dark photoperiod.
2.1.3. Growth medium and culture conditions
The shoots were separated from the callus and subcultured onto MS-free hormone medium with the addition of 6 g l−1 agar and 3% sucrose. The cultures were incubated at 23°C ± 2°C under 1,500 lux white LED (PhilipTM TL) on a 16 hour/8 hour light/dark photoperiod. The cultures were incubated for 14 weeks.
2.2. Assessment of Genetic Stability using RAPD and ISSR Markers
2.2.1. Plant material
The materials used for genetic stability testing were callus with 3-level subcultured, leaves of 14-week plantlets from callus and mother plant.
2.2.2. DNA extraction and PCR amplification conditions
Genomic DNA of all material was isolated using Genomic DNA Mini Kit (Plant) (Geneiaid Biotech Ltd, Taiwan). Genetic stability tests were conducted using RAPD and ISSR molecular markers. This study used 5 RAPD primer, OPA1, OPA2, OPA3, OPF 4, OPG17 [20] and 5 ISSR primers, BC815, BC823, BC825, BC835, and BC868 [21]. Quantitative assessment of genomic DNA was carried out by NanoDrop™ 2000 (Thermo Fisher Scientific, USA). The final concentration was set to 20 µg µl−1 for PCR containing 12,5 µl My Taq HS Red Mix 2X (Meredian Bioscience, USA), 1 µl primer RAPD/ISSR (IDT, Singapore), 3 µl template DNA, and 3,5 µl Nuclease free water. RAPD and ISSR reactions were performed in Select Cycler II Thermal Cycler (Select BioProduct, Taiwan). The amplification reaction for RAPD consisted of an initial denaturation step at 95°C for 3 minutes, followed by 40 cycles of 94°C for 45 seconds (denaturation), 45 seconds at specific annealing temperatures (Table 2), and 72°C for 1 minute (extension) followed by a final extension step at 72°C for 5 minutes. The amplification reaction for ISSR consisted of an initial denaturation step at 94°C for 5 minutes, followed by 40 cycles of 94°C for 45 seconds (denaturation), 45 seconds at specific annealing temperatures (Table 2), and 72°C for 30 seconds (extension) followed by a final extension step at 72°C for 5 minutes.
2.2.3. DNA visualization
RedSafe (iNtRON Biotechnology, Korea) was used to dye the amplified products after they were electrophoresed in 2% agarose gels using Tris-Borate-Ethylene diamine tetra acetic acid buffer. The amplicons’ sizes were measured using a comparison with a 1 kb DNA ladder from Thermoscientific, USA. Using a gel imaging device, the number of amplified products was counted (Digibox 7000, Mbiotech, Korea). To ensure the reproducibility of all PCR reactions for RAPD and ISSRs, significant bands were graded. The presence of a band was indicated by a “1” in the data, and its absence by a “0”. Plant samples that included every band were classified as monomorphic, while DNA samples lacking some bands were classified as polymorphic.
3. RESULT
3.1. Efficient Micropropagation of C. latifolia
The characteristics of callus that appeared on the medium of 5 mg l−1 IBA and 3 mg l−1 BA had a yellowish–white color with a compact texture (Fig. 1).
Furthermore, the callus was subcultured into the medium with different compositions for medium selection for shoot induction. However, the media with an additional growth regulator did not produce shoots but shoots appeared on media without the addition of a growth regulator (MS0) (80%) (Table 1).
Shoots developed from callus 30–35 days after subculture on MS0 medium. Callus on medium with the addition of auxin and cytokinin did not show a response. In the next step, the shoots that appear are separated and subcultured into MS0 to produce plantlets with roots. Shoot growth on MS0 medium is an efficient technique because it reduces production costs by not using growth regulators. Our micropropagation protocol produced morphologically normal plantlets or seedlings.
In summary, the stages of efficient micropropagation of C. latifolia are, callus induction on media containing IBA and BA, then callus that appears is subcultured on shoot induction medium, MS without growth regulator. Shoots that appear are incubated on the same media until they have good performance, namely the appearance of leaves, and sturdy. Furthermore, small plantlets without roots that grow in clusters are separated and subcultured to the MS-free growth regulator. On MS free growth regulator, plantlets grown into good performance, sturdier plantlets, with many leaves and roots (Fig. 2). The development of callus to plantlets in a closed system of in vitro culture can be a cycle of development. The resulting plantlets can be used as a supply of seedlings ready for acclimatization or the leaves can be used as sterile explants to produce callus again which will eventually produce seedlings or green stock in larger quantities.
 | Figure 1. Morphology of callus. (A). Callus emerged from the leaf of C. latifolia; (B). Shoot emerged from callus. [Click here to view] |
 | Table 1. Effect of growth regulator on percentage of callus emmerged. [Click here to view] |
3.2. Genetic Stability
All of the tested primers produced scorable bands although some of the resulting bands are thin under the optimized PCR conditions (Table 2). This study produced a total of 35 bands on RAPD primers and 38 bands on ISSR with an average of 7 bands from each RAPD and ISSR primer.
 | Figure 2. Efficient micropropagation of C. latifolia. (A). Callus formed from leaf explants on medium containing IBA and BA; (B). The callus swells and potential shoots begin to appear on MS free growth regulator medium; (C). Shoots on MS free growth regulator medium; (D). The shoots grow into small plantlets on MS free growth regulator medium; (E). Plantlet after being separated from the callus, subculture on MS free growth regulator medium; (F). Plantlets that are ready to be acclimatized or can be used as sterile explants to obtain new callus. [Click here to view] |
 | Table 2. List of RAPD and ISSR primers used for assessment of genetic fidelity of C. latifolia. [Click here to view] |
 | Figure 3. PCR amplification obtained with RAPD primers (A) OPA1; (B) OPA2; (C) OPA3; (D) OPF4 (E) OPG17. Lane L, represents 1 kb ladder (red arrows indicate ladder sizes of 250, 500, 750, and 1,000 bp in all electrophoresis results in A, B, C, D, and E), lane M represents the mother plant, and lanes K1–K3 represent callus and P1–P3 represents in vitro-raised clones. [Click here to view] |
 | Figure 4. PCR amplification obtained with ISSR primers. (A) UBC815; (B) UBC823; (C) UBC825; (D) UBC835 (E) UBC868. Lane L, represents a 1 kb ladder (t red arrows indicate ladder sizes of 250, 500, 750, and 1,000 bp in all electrophoresis results in A, B, C, D, and E), lane M represents the mother plant, and lanes K1–K3 represent callus and P1–P3 represents in vitro-raised clones. [Click here to view] |
The results of DNA amplification using RAPD showed no polymorphism. The size of the band on each primer shows a size that is not much different, namely OPA1 produces 7 bands with range sizes 350–1,500 bp, OPA2 formed 6 bands measuring 400–1,000 bp, OPA3 produces 6 bands with 350–1,000 bp, OPAF4 produces the least number of bands, namely 5 bands with 250–700 bp and OPG17 produces the most number of bands, 11 bands with 300–1,500 bp (Fig. 3).
Amplification using ISSR primers obtained the same results as RAPD, namely no polymorphism, except for primer UBC815 which showed polymorphism of 33.33%. Polymorphism is shown from the results of DNA amplification in plantlets that only detected 4 bands out of 6 bands in the mother plant and callus with a size of 400–900 bp. This indicates a reduction or deletion in plantlet DNA amplification. Primer UBC823 detected 7 bands of monomorphysm of 400–8,900 bp, UBC825 detected 7 bands of 300–900 bp, UBC835 detected 10 bands of 100–800 bp and UBC868 detected 8 bands of 300–750 bp (Fig. 4).
4. DISCUSSION
Not all C. latifolia callus are able to multiply cells and develop into shoots because they experience tissue damage due to browning. A serious problem faced in in vitro culture of C. latifolia is browning as has been reported in according research to Babaei et al. [7]. Browning in plant tissue culture refers to the phenomenon in which explants release phenolic compounds into the medium from their own tissues in the process of dedifferentiation and/or redifferentiation. Phenolic compounds are commonly present in healthy tissues and can accumulate in cells and be released as a defense response, especially after tissue injury or stress. Consequently, this natural defense response can lead to the accumulation of phenolic compounds that ultimately damage or kill plant cells and tissues. The process of cutting explants causes the release and accumulation of phenols in the tissue and media. Browning can be seen in almost all explants, especially on the wounded side and the color of the media turns brown. This caused some callus to break down and turn brown.
In some species, it is reported that MS free growth regulator produces better growth compared to growth on medium with the addition of growth regulator. In a previous study Sulaiman et al. [22] reported pineapples were cultured on MS medium without a plant growth regulator produced the highest shoot development. In addition, Nepeta rtanjensis shoots spontaneously rooted in vitro on a hormone-free medium [23]. In addition, Haddadi et al. [24] reported that the stunted shoots lengthened and took on a normal look after being subculturing on hormone-free MS media and on the medium MS free growth regulator obtained the longest root.
Callus on medium with the addition of auxin and cytokinin showed no response. In fact, the callus will experience browning when subcultured on media containing auxin and cytokinin. This indicates that the addition of exogenous growth regulators is not efficient or can even inhibit growth. The germinated plants reported by Muslihatin et al. [25] and the plantlets raised from callus have the same character of growing well in medium without growth regulators. The growth of C. latifolia does not require the addition of exogenous growth regulators. A basic medium called MS is typically used to propagate a wide range of plant species. This foundational media contains a lot of minerals that promote organogenesis [26], This means that plant tissue culture for C. latifolia is cost-effective because no growth regulator was required to obtain a mass quantity of in vitro clonal C. latifolia.
The process of plant micropropagation aims to produce clones (true copies of a plant in large numbers). The use of fewer growth regulators is thought to reduce production costs of plant micropropagation. Our method of micropropagation resulted in morphologically regular plantlets or seedlings. Nonetheless, one of the most crucial prerequisites for the micropropagation of crop species is true-to-type clonal fidelity. The potential for cryptic genetic defects to arise in regenerates can significantly restrict the broad application of the technology of micropropagation [12].
In general, a total of 10 primers used in this study produced monomorphism, which means that there is no genetic variation from C. latifolia callus and plantlets compared to the mother plant except for the ISSR primer UBC815 which shows a low level of polymorphism (33.33%). The absence of genetic variation using RAPD has been reported such as in micropropagation of Rosa hybrida [27], Aloe vera [12], Musa sp. cv. Williams [28], Rauwolfia tetraphylla L [16], Neolamarckia cadamba [15], Dendrocalamus strictus (Roxb.) [29], Muntingia calabura [17], and Dendrobium transparens Wall. Ex Lindl. [18]. In addition, Patel et al. [30] reported no genetic variation was observed on micropropagated Curculigo orchioides through RAPD analysis.
Although there is polymorphism in the UBC815 primer, the value is less than 50%, which means that the genetic variation that occurs is low [31]. According to Wibowo et al. [32], polymorphisms with a value of ≤50% are still classified in the category of high genetic profile stability. This study is in line with the assessment of the clonal fidelity of the in vitro regenerated Thunbergia coccinea, Artocarpus altilis, Orthosiphon stamineus using ISSR produced polymorphism 1%, 22.2%, and 7.32% [19,33,34]. In contrast, previous study showed no genetic variation in many species using ISSR marker, Andrographis alata [35], Valeriana jatamansi [36], Momordica cymbalaria [37], Solanum khasianum [38]. Polymorphism was detected in C. latifolia plantlets, DNA bands that appeared less than DNA bands that appeared in control or mother plants and callus. Micropropagated plantlets are frequently exposed to in vitro stress, which induces modifications at specific locations, including repetitive DNA, activating transposable elements and potentially leading to genetic diversity through insertion or deletion [39]. Variations in the RAPD and ISSR patterns can be attributed to the loss or gain of primer annealing sites resulting from transposon insertion/deletion and point mutation [34]. Using distinct marker systems increases the likelihood that variation brought about by genetic and epigenetic mechanisms will be reflected in banding profiles. Molecular markers’ effectiveness and dependability are regularly questioned, nevertheless. Combining two different types of markers that amplify distinct areas of the plant genomes allows for a more thorough investigation of the genetic stability of micropropagated plants. Earlier reports suggested that using more than one type of molecular marker in genetic fidelity studies of in vitro regenerated plants is always useful for reliability [16]. Therefore, it is recommended to use multiple markers or several DNA amplification techniques as useful in evaluating somaclonal variation.
5. CONCLUSION
This work offered a productive technique for C. latifolia in vitro micropropagation. By using RAPD and ISSR analysis, genetic stability between the mother plant, callus, and the in vitro-raised plants was found. Conversely, the five RAPD primers that were evaluated revealed no genetic variation, whereas the five ISSR primers that were tested revealed high genetic profile stability (66.67%). The in vitro micropropagation procedure created in this work can be used commercially to produce C. latifolia on a large scale that is genetically stable and free of pathogens.
6. ACKNOWLEDGMENTS
This research was supported by facilities of the Institute Tropical Diseases Airlangga University and Biotechnology Laboratory Institut Teknologi Sepuluh Nopember, Indonesia.
AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the international committee of medical journal editors (ICMJE) requirements/guidelines.
CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
ETHICAL APPROVALS
This study does not involve experiments on animals or human subjects.
DATA AVAILABILITY
All the data is available with the authors and shall be provided upon request.
PUBLISHER’S NOTE
All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED TECHNOLOGY
The authors declares that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI.
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