1 Introduction

Medicinal plants play a vital role in enhancing human health, serving as key sources for herbal therapies, anti-infective treatments, therapeutic medications, and dietary supplements [1]. Particularly in developing nations, these plants are fundamental for disease treatment and healthcare [2]. However, in Egypt, numerous medicinal plant species face endangerment due to factors like over-grazing, excessive harvesting for fuel and medicines through cutting and uprooting, tourism, and urbanization [3].

Cleome droserifolia (Forssk.) Delile Descr. (Syn. Roridula droserifolia Forssk. or Rorida droserifolia (Forssk.) Thulin & Roalson), is a wild aromatic shrub. It is found in Arabian countries like Syria, Palestine, Libya and Sinai in Egypt [4,5,6,7,8]. It belongs to the Cleomaceae family, it is called “spider flower” and “mountain bee plant” [3, 9]. In Egypt, it is known as Afein, Samwah, or Reeh El-Bard [3, 6]. This species is endangered due to ecological challenges, over-collection for various uses, and over-grazing in the Saint Katherine Protectorate. So, it is classified as endangered plant [3, 10].

Cleome genus exhibits anticancer, hepatoprotective [6, 11, 12], antiparasitic, antimicrobial [6], antioxidant, and robust antidiabetic properties [1, 13]. Various studies on Cleome droserifolia highlight the presence of phytochemicals like tannins, alkaloids, coumarins, amino acids, and terpenoids [3, 14,15,16,17]. The plant extract has bioactive compounds including terpenoids, flavonoids, glycosides, carbohydrates, sterols, and sesquiterpenes like daucosterol, teucladiol, and buchariol, contributing to its anticancer and antibacterial effects [16, 18,19,20]. It has anti-hyperglycemic properties, making it used by herbalists and Bedouins in Sinai for diabetes treatment [21]. Additionally, the plant is employed in treating various ailments such as liver diseases, skin issues, inflammation, scabies, and abdominal pain [22]. Rare and endangered species require conservation through in situ or ex situ methods to prevent their extinction. conservation involves safeguarding species within their natural habitats, while ex situ conservation entails preserving genetic resources outside their original environments. This is often crucial when species are on the brink of extinction. Ex situ strategies encompass techniques like seed banking, field gene banks, living collections, cryopreservation, DNA barcoding and in vitro propagation. [23, 24].

DNA barcoding is an innovative and valuable tool for the ex situ conservation of medicinal and rare species. By utilizing short DNA sequences, or barcodes, this method compares individual samples with a reference library to identify species and address taxonomic and ecological issues. It aids in species discovery, phylogenetic diversity assessment, and traditional medicine identification. Moreover, it effectively differentiates species, resolves uncertainties, and contributes to the protection of endangered species. [3, 25,26,27,28]. El-Atroush et al. [29] utilized DNA barcoding (ITS and rbcL) for Cleome droserifolia identification, highlighting the need for updated primers and protocols to enhance plant identification efficiency.

In vitro propagation is a pivotal technology for conserving endangered or medicinally significant species. Plant tissue culture, employing vegetative explants, yields numerous plants quickly and in limited space [30]. Even a small sample generates hundreds of genetically identical plants regardless of seasons. It’s crucial for non-seed-forming or seed-trouble species [31], and those challenging conventional propagation methods [32]. Micropropagation of Cleome species is scarcely documented. Hassan [33] found that 30 mg/l adenine sulphate with 1 mg/l 6-benzylaminopurine (BA) in Murashige and Skoog (MS) [34] medium resulted in the highest axillary shoots/plantlet count of 3.22 in Cleome droserifolia. Another protocol for in vitro organogenesis of Cleome viscosa utilized leaf explants, yielding 95% callusing and 7.6 shoots using 2 mg/l BAP in MS medium [35].

This research aimed to conserve the Cleome droserifolia plant by adding more barcodes to the universal plant databases to enhance species and landrace differentiation, boosting accurate identification for biodiversity conservation. Also, it aimed to develop a protocol to improve the in vitro propagation of the plant for germplasm conservation using different kinds of growth regulators to increase the number of multiplied shoots.

2 Materials and methods

2.1 Samples collection

Cleome droserifolia leaf specimens and mature seeds were harvested from shrubs grown in Wadi Zaghra, Saint Katherine, Southern Sinai (N: 28.63501, E: 34.13972) as shown in Figs. 1 and 2. Plant identification was carried out by Dr. Ibrahim Abdelrafee El Gamal, Nature Conservation Sector, Egyptian Environmental Affairs Agency, Southern Sinai, Egypt. Specimens were placed in the Herbarium of Faculty of Science, Cairo University with the voucher number 35.120.335.

Fig. 1
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A map showing the location of Wadi Zaghra, Saint Katherine, Southern Sinai, the collection site of Cleome droserifolia plant

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Fig. 2
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Naturally growing Cleome droserifolia plant in Wadi Zaghra, Saint Katherine, Southern Sinai, with a close-up view of a flower

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2.2 DNA barcoding

2.2.1 DNA Extraction and Purification

Leaf samples from Cleome droserifolia plant were gathered and turned into a fine powder using liquid nitrogen, a mortar and pestle. The DNA within the samples was then extracted and cleaned using the DNeasy Plant Kit from Qiagen, Germany. The ND-1000 System spectrophotometer (Nano-Drop Technologies, Thermo Fisher Scientific Inc., Waltham, MA, USA) gauged extracted DNA’s quantity and purity. A 1% agarose gel, with a DNA size marker (Lambda DNA Hind III digest Phi X 174/HaeIII digest), estimated purified DNA’s concentration and quality.

2.2.2 Polymerase chain reaction (PCR) amplification and sequencing

The reaction of PCR was done as mentioned by Ibrahim et al. [36] in a net volume of 50 µl master mix containing: 1x buffer (Promega, USA), 15mM MgCl2, 0.2 mM dNTPs (Promega, USA), 20 pcoml of each primer (Invitrogen), 1 u of Tag DNA polymerase (GoTaq, Promega, USA), 40 ng DNA and pure water to the final volume. Three chloroplast DNA (cpDNA) genes; ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL), maturase K (matK) and trnH-psbA were used for DNA barcoding analysis. The sequences of the primers are listed in Table 1.

Table 1 List of DNA barcoding loci and the primer pairs used in the PCR reactions
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The PCR amplification of the three genes started with an initial step at 94 °C for 5 min. This was followed by 40 cycles, each consisting of three phases. First, the DNA was denatured at 94 °C for 30 s. Then, it was annealed at 45 °C for 30 s. Lastly, elongation occurred at 72 °C for 30 s. The final cycle included a longer 7-minute extension at 72 °C. The Perkin-Elmer/Gen-eAmp PCR System 9700, provided by PE Applied Biosystems in the USA, was used for these PCR processes.

The PCR products were separated using a 1.5% w/v agarose gel that was stained with ethidium bromide (EtBr) at a concentration of 0.5 ug/ml. The gel was run in 1X TBE buffer at a voltage of 95 volts. To determine the sizes of the DNA fragments, a 100 bp DNA ladder from Promega in the USA was used as a reference standard. UV transilluminator aided visualization, and images were captured. To recover the PCR-amplified fragments from the agarose gel, the Q1A Quik PCR Purification Kit from Qiagen Inc. in Venlo, The Netherlands, with Catalog Number 28,106, was employed. The recovery process followed the instructions provided by the manufacturer. Purified PCR amplicons underwent DNA sequencing using ABIPRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Waltham, MA, USA) as per the supplied protocol.

2.2.3 Data analysis and assigning of species

The terminal noisy regions on the 3′ and 5′ ends of Cleome droserifolia’s rbcL, matK, and trnH-psbA barcode sequences were excised. These purified sequences were then deposited in the GeneBank, each acquiring an accession number. Alignment occurred against previously published sequences utilizing the Basic Local Alignment Search Tool (BLAST) on the National Centre of Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/BLAST). Online tool Clust Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) facilitated the creation of phylogenetic trees.

2.3 In vitro propagation experiments

2.3.1 Seeds sterilization

Mature seeds of Cleome droserifolia were cleaned by washing under continuous running tap water. They were subsequently treated with tween twenty solution for 10 min, followed by immersion in a 15% commercial bleach solution containing 5.25% sodium hypochlorite for 5 min. The treated seeds were then rinsed four times with distilled water. The sterilization procedure was conducted within a laminar airflow hood (Hoten LaminAir HVR 2448, USA).

2.3.2 Seeds germination

For germinating Cleome droserifolia seeds, a half-strength MS medium (Duchefa, Haarlem, the Netherlands) foritified with 3% sucrose (wv−1) was employed. The medium’s pH was tuned to 5.7 ± 0.1, and it was solidified using 0.3% phytagel (Duchefa, Haarlem, the Netherlands) before undergoing autoclaving at 121 °C and 1.06 kg cm−2 for 15 min. Then, 7–8 seeds were placed in each aseptic glass jar (375 ml) containing the medium. After that, the jars were incubated in a dark and humid environment for 7 days to stimulate germination, followed by a transfer to a light environment. Seedlings emerged within two weeks and were used as explants for subsequent in vitro propagation.

2.3.3 Shoot initiation and multiplication

Cotyledonary node explants, obtained from 15-day-old seedlings, were placed in 375 ml jars containing 50 ml of MS medium. The medium was supplemented with 0.27 µM α-naphthaleneacetic acid (NAA) along with various concentrations of cytokinins (Sigma Cell Culture, min 90%, St. Louis, USA). Specifically, Kinetin (Kin) was used at concentrations of 1.16, 2.32, 4.60, and 9.30 µM, N6-2- isopentenyl adinine (2iP) at concentrations of 1.23, 2.46, 4.90, and 9.80 µM, and thidiazuron (TDZ) at concentrations of 1.14, 2.27, 4.55, and 9.10 µM, individually. As a control, plant growth regulators (PGRs) free MS medium was utilized. The cultured jars were maintained under controlled conditions of 25 ± 1 °C and a 16-hour light/8-hour dark photoperiod using cool white fluorescent lamps providing 2500–3000 lx. After a culture period of 30 days, assessments were conducted for survival and growth percentages, as well as the average number and length (cm) of shoots per explant.

2.3.4 Roots induction and hardening

Shoots measuring 5–6 cm in length were introduced to diverse root induction media. Different strengths of MS media (full, ½, and ¼ MS medium) were enriched with 2.46 to 9.80 µM of indole-3-butyric acid (IBA; Sigma Cell Culture, min. 90%, St. Louis, USA) or 2.69 to 10.74 µM of NAA to initiate rooting. A control group utilizing free MS medium was included. Following the adjustment and autoclaving of the medium pH, the cultures were incubated. After a four-week cultivation period on the rooting media, parameters such as shoot length, average number of roots, and root length were documented. Once the plantlets had successfully rooted, they were gently cleansed with distilled water to eliminate any nutrient medium residues. Subsequently, the plantlets were transplanted into plastic pots containing a mixture of autoclaved sand, soil, and peat moss (1:1:1 ratio, Peat moss, PROMIX®). To ensure plant stability, the pots were irrigated and enveloped with polyethylene bags. These bags were gradually removed to reduce humidity after a six-week span. The plants were then allowed to grow under open conditions.

2.3.5 Experimental design and statistical analysis

The experiments were meticulously organized using a completely randomized desigened, encompassing a minimum of ten replicates for each treatment. These experiments were repeated thrice. Statistical evaluation involved Analysis of Variance (ANOVA) alongside Duncan’s multiple range test [40], as modified by Snedecor and Cochran [41], were used to evaluate the obtained data. Significance was attributed to means sharing a common letter when P ≤ 0.05, indicating no significant differences.

3 Results

3.1 Molecular identification of Cleome droserifolia by DNA barcoding

The classification and identification of the endangered Cleome droserifolia (Rorida droserifolia) plant was carried out using three DNA barcodes; [ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit (rbcL), maturase K (matK) and trnH-psbA] to conserve this endangered plant. The forward and the reverse primer pairs of the three used barcodes produced expected amplicon sizes of approximately 700, 950 and 500 bp for rbcL, matK and trnH-psbA, respectively. Sequences were submitted to the GenBank database after removing the peripheral noisy parts and were given the following accession numbers OM863545, OM863546 and OM863547 for the nucleotide sequences. BLASTn tool of the National Centre of Biotechnology Information (NCBI) was used to identify the obtained sequences by comparing the newly generated sequences of the three barcodes; rbcL, matK and trnH-psbA of Cleome droserifolia (Rorida droserifolia) with the library of the previously submitted GenBank accessions. The species of the highest identity percentages of the BLAST matching results and their phylogenetic trees analysis are presented in Tables 2, 3 and 4; Figs. 3, 4 and 5. The alignments of the three barcode sequences against GenBank accessions produced a query coverage between 74 and 100% for rbcL (Table 2) and 100% for each of matK (Table 3) and trnH-psbA (Table 4).

Fig. 3
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Phylogenetic tree of Cleome droserifolia using the cpDNA marker: ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL)

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Phylogenetic trees and BLAST result for the three partial gene sequences clear that the plant under study is closely related to previously published Cleome species. BLAST results with the rbcL gene revealed that the highest identity percentage between Cleome droserifolia and the other published sequences of the species in the same genus showed 99.56% identity with Cleome amblyocarpa, 99.42 with Cleome violacea, 98.83 with Cleome arabica and the identity percentage with the same species of Cleome droserifolia (Rorida droserifolia) of the previously published accessions were 98.68 (accession # KU739619.1), 98.93 (accession # KR998500.1) and 99.04 (accession # OM112196.1) with query coverage of 100, 82 and 75%, respectively (Table 2).

Table 2 DNA barcode of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) downloaded from GenBank database including plant species with similarity percentage of more than 98.49% with Cleome droserifolia
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For matK gene; the BLAST results revealed 99.71, 97.83, 96.15, 96.68, 96.01 and 96.24% identity percentages with Cleome droserifolia, Cleome breyeri, Cleome ornithopodioides, Cleome monochroma, Cleome turkmena and Cleome chilensis, respectively (Table 3).

Table 3 DNA barcode of maturase K (matK) downloaded from GenBank database including plant species with similarity percentage of more than 95.65 with Cleome droserifolia
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It is clear from Table 4 that, Cleome anomala and Cleome moritziana showed the highest similarity percentages with Cleome droserifolia for the trnH-psbA gene which was 96.59 and 95.28%, respectively. Also, the phylogenetic trees for the three used genes show that, the closely related species were grouped in one cluster and the relatively distantly related species were scattered in different groups (Figs. 3, 4 and 5).

Fig. 4
figure 4

Phylogenetic tree of Cleome droserifolia using the cpDNA marker: maturase K (matK)

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Table 4 DNA barcode of trnH-psbA downloaded from GenBank database including plant species with similarity percentage of more than 85.30% with Cleome droserifolia
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Fig. 5
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Phylogenetic tree of Cleome droserifolia using the cpDNA marker: trnH-psbA

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3.2 In vitro propagation of Cleome droserifolia

3.2.1 In vitro shoot induction and multiplication

In this study, Cleome droserifolia seeds were successfully germinated on ½ MS medium after 7 days of dark incubation, achieving a germination rate of approximately 60% (Fig. 6a). Cotyledonary node explants from 15-day-old seedlings were cultured on MS medium fortified with 0.27 µM NAA and different concentrations of cytokinins, namely Kin, 2iP, and TDZ, to induce shoot formation (Table 5).

Table 5 Effect of Murashige and Skoog (MS) medium fortified with 0.27 µM α-naphthaleneacetic acid (NAA) in addition to Kinetin (Kin), N6-(2-isopentenyl) adenine (2iP), or thidiazuron (TDZ) on the in vitro multiplication of Cleome droserifolia. All treatments gave 100% of survived explants
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All tested treatments exhibited 100% survival of explants. Among the three cytokinins investigated, the highest shoot induction response, mean number of shoots, and shoot length were observed on MS medium fortified with Kin compared to 2iP or TDZ (Table 5) (Fig. 6).

Fig. 6
figure 6

In vitro propagation of Cleome droserifolia; a in vitro germination of the seeds, b in vitro multiplication of the shoots, c vitrification of the multiplied shoots with high concentrations of cytokinins, d rooted plantlet, and e acclimatization of transplants in the greenhouse

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In media containing 2iP or TDZ, the initiation of shoot bud formation was delayed compared to the medium with Kin. Notably, the medium fortified with 2.32 µM Kin + 0.27 µM NAA demonstrated the most favorable outcomes for shoot induction, achieving an 81% success rate (Table 5) (Fig. 6b). Additionally, this concentration of Kin resulted in the highest mean number of shoots (8.16) and the longest shoot length (4.5 cm).

Higher concentrations of Kin showed a decreasing trend in shoot induction percentage, as well as the mean number and length of shoots per explant. Similar trends were observed with 2iP, where increased concentrations led to reduced shoot induction and mean number of shoots. Compared to Kin or 2iP, the use of TDZ resulted in the lowest percentage of shoot induction, following the pattern observed in the control medium.

Employing elevated concentrations of the cytokinins tested in this experiment resulted in the vitrification of plants, as shown in Fig. 6c. Among the various combinations, MS medium fortified with 0.27 µM NAA + 2.32 µM Kin yielded the most healthy and normal shoot growth.

3.2.2 In vitro rooting and acclimatization of plantlets

The in vitro propagated shoots were transferred to various media for root induction. Full, half, and quarter-strength MS media were tested along with different concentrations of IBA (2.46, 4.96, 7.42, and 9.80 µM) or NAA (2.69, 5.37, 8.10, and 10.74 µM). Full and half MS media didn’t stimulate rooting after 4 weeks of culture. Also, IBA was more effective than NAA in stimulating root formation.

Among the tested conditions, quarter-strength MS medium supplemented with varying IBA concentrations successfully induced root development after 4 weeks. The summarized impact of IBA and NAA on root induction is presented in Table 6. Shoots cultured on the control medium (¼ MS without IBA) showed no response to root stimulation. The highest root induction percentage (85%) was observed with shoots cultured on ¼ MS medium fortified with 7.42 µM IBA. This same medium yielded an average of 11.6 roots per shoot, with roots measuring around 9.17 cm and shoots reaching 5.6 cm in length (Fig. 6d). The medium containing 9.80 µM IBA produced the second-highest rooting percentage (75%), along with 13.3 mean roots per shoot and 8.75 cm root length.

Table 6 Effect of quarter-strength Murashige and Skoog (¼ MS) medium fortified with different concentrations of indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA) on the rooting of Cleome droserifolia multiple shoot
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Rooting percentage gradually increased with rising IBA and NAA levels, reaching a peak at 7.42 and 8.10 µM NAA, after which it decreased upon using 9.80 µM IBA or 10.74 µM NAA. The differences in the mean number and length of roots and shoot length between the highest tested IBA concentrations (4.96, 7.42, and 9.80 µM) were not statistically significant. However, the concentration of 7.42 µM IBA resulted in the highest rooting percentage at 85%. Following root induction, plantlets were gradually transferred to greenhouse conditions, exhibiting a survival rate of about 65% (Fig. 6e).

4 Discussion

4.1 Molecular identification of Cleome droserifolia by DNA barcoding

DNA barcoding analysis of plant species is playing a pivotal role in designing biodiversity conservation strategies and enhancing species identification and classification [27]. Compared to RNA and protein macromolecules, DNA markers are more reliable for species identification due to DNA’s stability [42]. DNA barcoding utilizes specific regions of nuclei, plastids, or mitochondria to create an accurate tool for species taxonomy and precise authentication of unknown species [43]. It relies on short DNA sequences, which are then matched to existing barcode sequences in comprehensive databases of known species [44]. Unlike morphological identification methods, DNA barcoding is not restricted by morphological traits and doesn’t necessitate specialized training. It aligns seamlessly with standard databases [27, 45].

In this study, utilizing phylogenetic tree analysis and BLAST matching of the three barcode markers (rbcL, matK, and trnH-psbA), Cleome droserifolia was conclusively identified within the Cleome genus, achieving a 100% certainty level. Prior research indicates that a DNA barcoding attempt is successful when BLAST results show an identity percentage of 95% or higher and correspond to a single genus on the generic level. At the species level, success is achieved when the identity percentage is also above 95%, and a single species is matched [46].

In our study, the highest identity percentages between Cleome droserifolia and other published Cleome species ranged between 96.59% and 99.71%. Specifically, the matK sequence displayed 99.71% identity with Cleome droserifolia, and the rbcL sequence showed identity percentages of 99.04% (accession # OM112196.1), 98.93% (accession # KR998500.1), and 98.68% (accession # KU739619.1) with the same species, Rorida droserifolia. Regarding the trnH-psbA marker, successful identification was achieved only on the genus level. Notably, this study introduced a new barcode sequence for the trnH-psbA marker into the GenBank database.

Therefore, based on the obtained data, the authentication of Cleome droserifolia proved successful at both the species and genus levels using the rbcL and matK markers, while the trnH-psbA marker achieved genus-level identification. The phylogenetic trees constructed using the three markers (rbcL, matK, and trnH-psbA) consistently positioned Cleome droserifolia within the Cleome genus.

Hence, this study effectively authenticated Cleome droserifolia on both species and genus levels. Here, DNA barcoding was employed as a swift and cost-effective molecular method, augmenting morphological identification for precise Cleome droserifolia authentication. It underscores DNA barcoding’s potential to contribute to building comprehensive barcoding libraries for endangered species, aiding species classification [47].

4.2 In vitro propagation of Cleome droserifolia

Conserving natural plant resources often involves utilizing seedling materials for in vitro cultures due to their rich genetic diversity [48]. This approach can significantly boost the propagation rate of native species [49]. Seedling explants from the Cleomaceae family have garnered significant attention for micropropagation due to their ability for prolific shoot multiplication, as seen in species like Cleome viscosa [35]; Cleome spinosa [50] and Cleome dendroides [51]. In this study, cotyledonary node explants from 15-day-old Cleome droserifolia seedlings were employed for shoot induction and multiplication. These explants were effectively cultured on a medium containing NAA at 0.27 µM along with varying concentrations of cytokinins such as Kin, 2iP, and TDZ. The significance of auxins and cytokinins in promoting cell division and organ induction is well established [52]. Auxins, a crucial class of plant hormones, play a pivotal role in the formation of in vivo organs. The movement of auxins from apical shoots to basal plant tissues is essential for growth and various developmental processes [53]. Atta et al. [54] reported that, the addition of auxin in the tissue culture medium triggers the regulation of cell division, from which founder cells appears. Notably, In this work, the addition of lower concentrations of NAA (0.27 µM) promoted the regeneration of shoot buds by participating in meristem formation.

Similarly, cytokinins consider essential group of plant growth regulators (PGRs), influence growth by stimulating direct shoot initiation, cell division, and multiplication [55]. The type and amount of cytokinin significantly affect shoot multiplication. In this experiment, the highest mean number of shoots was noticed with kin followed by 2iP and TDZ. Elevated concentrations of cytokinins adversely impacted shoot multiplication and growth. Several researchers, including Ashraf et al. [56] and Karolak et al. [57], have observed that lower cytokinin concentrations are more suitable for stimulating shoot bud development, while higher concentrations can inhibit the number and frequency of shoot induction. This observation is consistent with the findings of Koroch et al. [58], who noted that higher concentrations of BA hindered shoot growth and multiplication in Echinacea purpurea. Similar trends have been identified in other medicinal plants such as Aspilia africana [59], Prunella vulgaris [60], and Elephantopus scaber [61]. Additionally, in this study, higher concentrations of cytokinins were linked to plant vitrification. Thus, the combination of low auxin and cytokinin concentrations appears to be most effective for stimulating meristem growth and facilitating the emergence of new shoots in Cleome droserifolia. For many medicinal plants, optimal cytokinin concentrations enhance shoot numbers, but the introduction of low levels of both auxin and cytokinin promotes a higher proportion of shoot proliferation [62,63,64,65]. This synergistic effect likely supports meristematic growth as apical meristems naturally produce endogenous auxins, and adding exogenous auxin in low quantities aids this meristematic growth [57].

In conclusion, the best medium for shoot induction and multiplication of Cleome droserifolia through in vitro propagation seems to be MS medium fortified with 2.32 µM Kin and 0.27 µM NAA.

In the realm of plant propagation, in vitro root induction stands as a critical bottleneck. It’s widely acknowledged that auxins are essential for root development and growth [66]. In this study, roots successfully emerged after four weeks of cultivation. Several factors influence root development, encompassing plant species and the type and concentration of auxins. In the case of Cleome droserifolia, IBA and NAA were employed to encourage root formation on a quarter strength MS medium. Interestingly, IBA proved to be more effective than NAA in this regard. IBA, being a natural auxin with heightened stability and low oxidative propensity, showcases notable root-inducing properties [67]. Among the tested media, the most effective one for prompting root formation in in vitro multiplied shoots of Cleome droserifolia was found to be ¼ MS medium fortified with 7.42 µM IBA. This particular medium exhibited an impressive 85% rooting rate along with the highest average number and length of roots per explant, as well as the mean length of axillary shoots. This outcome aligns with the findings of Hassan [33], who achieved a 93.3% rooting rate for Cleome droserifolia shoots with 7.42 µM IBA, albeit using full-strength MS medium. Following successful rooting, the established plantlets were transitioned to a greenhouse setting for further cultivation.

5 Conclusions

The Cleome droserifolia plant faces a multitude of challenges, including ecological disasters, over-collection, over-grazing, and difficulties in obtaining its seeds. These issues have collectively placed this plant species in a vulnerable position, leading to its decline and potential disappearance from its natural habitat. In response, we have undertaken two distinct ex-situ plant conservation strategies. Firstly, we employed DNA barcode analysis using three specific DNA barcodes, including a novel barcode that has been introduced for the first time in the GenBank database. This approach provides a robust method for accurately identifying the Cleome droserifolia plant. Secondly, we successfully established an efficient in vitro propagation protocol for Cleome droserifolia through this study. These endeavors mark crucial initial steps in the broader context of plant conservation, facilitating both research initiatives and public awareness efforts. It’s important to note that further investigations involving additional nuclear markers will be necessary to comprehensively authenticate the plant’s identity. This study is aligned with one of the central goals of the 2030 Agenda and the Sustainable Development Goals (SDGs) — namely, the preservation and safeguarding of threatened species. By contributing to the conservation of Cleome droserifolia, we are actively participating in the broader mission to protect and prevent the extinction of endangered plant species.