A Simple and Highly Efficient Regeneration Protocol for Bougainvillea glabra: Achieving Callus Induction, Shoot Organogenesis, and Elongation on a Single Medium

1. Introduction

Bougainvillea (Nyctaginaceae) comprises roughly 18 wild species that have given rise to over 500 cultivars through interspecific hybridization, bud-sport selection, and open-pollinated seedling screening, widely utilized for landscaping and potted displays across tropical and subtropical regions [1,2,3]. Prized for its remarkable drought tolerance, adaptability to marginal soils, and prolonged blooming, Bougainvillea has become a flagship ornamental with rapidly expanding commercial production. Additionally, its bracts and leaves are rich in betalains, flavonoids, and phenolics with documented antioxidant and anti-inflammatory activities [4,5], highlighting its expanding potential in nutraceutical and pharmacological applications. However, meeting this surging demand is severely constrained by formidable biological bottlenecks in conventional propagation and breeding. The narrow perianth-tube structure characteristic of Nyctaginaceae sequesters the stigma deep within the floral tube [6], resulting in exceptionally low seed-set and frequent embryo abortion under both natural and artificial pollination [1,7]. Consequently, commercial cultivar development relies heavily on serendipitous bud sports, rendering breeding cycles prolonged and unpredictable. Furthermore, traditional cutting and grafting maintain clonal fidelity but cannot generate new genetic variation, while remaining limited by genotype-dependent rooting recalcitrance and seasonal windows. Thus, conventional routes fail to introduce targeted traits—such as novel bract colors, compact architecture, or enhanced stress tolerance—into elite germplasm. Establishing an in vitro plant regeneration system via totipotent callus is therefore not only vital for year-round clonal mass propagation, but also an indispensable bypass around natural reproductive barriers to build a cellular platform for future genetic transformation and molecular breeding.

In recent years, marked progress has been achieved in Bougainvillea in vitro micropropagation. However, regarding explant types and developmental pathways, existing research has predominantly focused on semi-lignified nodal stem segments of B. × buttiana ‘Miss Manila’ [6], B. spectabilis ‘Tongan Red’ [8], and various B. glabra cultivars [3,9]. These systems essentially rely on the outgrowth of pre-existing axillary/latent bud meristems for micropropagation, or halt at non-regenerative callus for secondary metabolite extraction [10], without establishing a genuine totipotent callus-mediated de novo organogenesis system. In the few studies exploring embryonic explants, although Lu et al. [11] achieved callus-mediated shoot regeneration from hypocotyls of B. glabra ‘Bouform 1’, their protocol heavily relied on a multi-stage sequence requiring four distinct culture media (for induction, proliferation, shoot strengthening, and rooting, respectively). Such frequent subculturing between disparate media not only increases labor and contamination risks, but also yielded moderate bud induction (81.3%) and rooting rates (82.5%). More importantly, previous studies neither systematically examined the developmental divergence between mature and juvenile tissues under identical conditions, nor resolved whether callus induction, de novo bud organogenesis, and shoot elongation could be coordinated on a unified single medium. In comparative plant biology, seed-derived hypocotyls represent juvenile tissues characterized by physiological homogeneity, high mitotic competence, and heightened sensitivity to exogenous hormones. In recalcitrant dicotyledonous species such as Astragalus adsurgens [12] and chickpea (Cicer arietinum) [13], hypocotyl-derived callus has been proven to achieve substantially higher regeneration efficiencies (46.6%–66%) than mature leaf callus, recognized as the premier explant to overcome morphogenic recalcitrance. Governed by the classical auxin: cytokinin balance model established by Skoog and Miller [14], exogenous hormone ratios critically dictate cellular dedifferentiation and organogenic fate. Therefore, compared to the lignified stems or nodal buds employed in earlier studies [9,11,15], hypocotyl explants hold immense promise for establishing a streamlined, highly efficient callus-regeneration pipeline.

While previous tissue-culture studies on Bougainvillea have almost exclusively utilized stem segments, the organogenic potential of leaf explants—which are abundant, non-destructive, and accessible year-round—remains largely unaddressed. To evaluate this convenient vegetative tissue and elucidate the developmental determinants governing totipotency, the present study systematically investigated young leaves (representing terminally differentiated tissues) alongside seed-derived juvenile hypocotyls. Our primary objective was to establish a simple, highly efficient, and complete callus-mediated whole-plant regeneration system for B. glabra Choisy ‘Light Violet’ (hereafter referred to as B. glabra). To achieve this goal, three specific tasks were addressed: (1) to systematically evaluate and contrast the callus induction and de novo organogenic competence of young leaf versus juvenile hypocotyl explants; (2) to optimize a streamlined hormone regime capable of coordinating callus induction, de novo bud organogenesis, and shoot elongation on a unified single medium, followed by root induction on a separate medium; and (3) to establish an effective ex vitro acclimatization and transplanting procedure to ensure whole-plant survival. Ultimately, this system not only provides a robust, high-efficiency micropropagation protocol for B. glabra, but also establishes an indispensable cellular recipient platform for genetic transformation. This technological advance facilitates the reverse-genetic dissection of bract pigmentation, flowering periodicity, and stress tolerance mechanisms, while opening a promising new avenue for precision breeding and genetic improvement in Bougainvillea, thereby circumventing the longstanding bottlenecks of conventional cross-breeding and vegetative cutting propagation.

2. Materials and Methods

2.1. Plant Material

Both the seeds and donor plants of B. glabra Choisy ‘Light Violet’ (hereafter referred to as B. glabra) used in this study were obtained from the Bougainvillea Science and Technology Expo Park in Sanya Yazhou Bay Science and Technology City (Sanya, China). All tissue culture experiments were conducted at the Tissue Culture Laboratory, Sanya Research Institute, Hainan Academy of Agricultural Sciences.

2.2. Culture Medium Preparation

Culture media were prepared following the standard method described by [12]. Full-strength MS basal medium containing 4.4 g/L MS basal salts and 30 g/L sucrose was used for callus induction and shoot differentiation, whereas half-strength MS (1/2 MS) medium with 30 g/L sucrose was utilized for rooting. Appropriate plant growth regulators (PGRs) were supplemented into the media according to the experimental treatments. The medium pH was adjusted to 5.8 using 1 M NaOH or HCI prior to the addition of 3.0 g/L Phytagel as the gelling agent. The media were then autoclaved at 121 °C for 20 min. After autoclaving, approximately 15–20 mL of the medium was poured into each sterile Petri dish, and 70–80 mL was dispensed into each culture bottle. All chemicals and culture reagents were purchased from Sanya Lvhengyuan Biotechnology Co., Ltd. (Sanya, China).

2.3. Leaf Explant Preparation and Callus Induction

Young, fully expanded leaves were collected and pooled from 5–10 healthy donor shrubs of B. glabra cultivated in the Bougainvillea Science and Technology Expo Park and washed thoroughly to remove surface debris. Leaves were first rinsed twice with sterile water, surface-sterilized with 1% sodium hypochlorite for 2 min, and finally rinsed five times with sterile water. Preliminary experiments showed that 75% ethanol pretreatment caused serious browning injury to young Bougainvillea leaves, so ethanol treatment was abandoned. For fragile leaf explants, mild disinfection is a feasible strategy for reducing chemical phytotoxicity while accepting a certain level of contamination [16,17]. Through this sterilization protocol, the contamination rate of leaf explants was successfully maintained below 20%. After removal of the midrib, the lamina was cut into pieces of approximately 1 cm², which were inoculated onto callus induction medium (MS + 30 g/L sucrose + 3 g/L Phytagel, pH 5.8) supplemented with one of the 27 hormone combinations of 6-BA with 2,4-D or NAA, and cultured in the dark at 25–28 °C and 50–60% relative humidity for 7-8 weeks. Approximately 100 young leaves were processed per experimental batch. For each hormone combination, three replicate Petri dishes were set up with 6–10 explants per dish, and three independent biological replicates were performed. Callus obtained from the best-performing induction treatments was subcultured onto six proliferation media differing in 6-BA, 2,4-D, NAA, or KT (kinetin) content (Table 1) to identify the combination supporting the most vigorous, non-browning callus growth. Proliferated leaf callus was subsequently transferred to the same four differentiation media later used for hypocotyl-derived callus (Table 3) to test its capacity for adventitious bud formation. For each treatment in both the proliferation and differentiation stages, three replicate Petri dishes containing 6–10 explants each were evaluated across three independent biological replicates.

2.4. Seed Germination and Hypocotyl Explant Preparation

Mature, undamaged fruits of B. glabra were harvested from approximately 5–10 healthy, mature donor shrubs and their seeds extracted. Plump, visually healthy, uncontaminated seeds were selected, washed free of surface debris with detergent, and rinsed under running tap water for 20–30 min. In a laminar-flow hood, seeds were transferred to sterile bottles, washed three times with sterile water (1 min each), surface-sterilized in 1% (w/v) sodium hypochlorite solution with a few drops of Tween-20 for 25–30 min, and then rinsed five times with sterile water (1 min each). Seeds were dried on sterile filter paper before inoculation onto the germination medium. Sterilized seeds were inoculated onto seed germination medium (MS + 30 g/L sucrose + 3 g/L Phytagel, pH 5.8) at a density of 3–5 seeds per Petri dish and cultured in the dark at 25–28 °C and 50–60% relative humidity for 10–14 days, yielding sterile seedlings approximately 4–5 cm tall. Roots and cotyledons were excised, and the remaining hypocotyls were cut into segments 0.5–1 cm long for use as explants. Dark germination was used to promote hypocotyl elongation and to minimize premature chlorophyll accumulation and lignification, both of which are associated with reduced callus competence in dicotyledonous hypocotyls. In total, approximately 100 B. glabra seeds were processed to obtain hypocotyl explants.

2.5. Hypocotyl-Derived Callus Induction

Hypocotyl segments were inoculated (6–10 explants per dish; plastic dishes 90 mm diameter × 20 mm height) onto MS basal medium supplemented with 30 g/L sucrose, 3 g/L Phytagel (pH 5.8), and one of six combinations of 6-BA, 2,4-D, and KT (Table 2). Cultures were maintained in the dark at 25–28 °C and 50–60% relative humidity for 20–30 days, with the medium refreshed every 15 days. Callus induction rate and callus morphology were recorded. The hormone combinations tested spanned a gradient from cytokinin-dominant (0.5 mg/L 6-BA + 1 mg/L KT) through balanced 6-BA/2,4-D ratios to auxin-only (2.5 mg/L 2,4-D alone), following the classical auxin: cytokinin framework of Skoog & Miller [18], in order to identify the hormonal window that favored callus proliferation without premature organ commitment. For each hormone combination, three replicate Petri dishes were employed (6–10 explants per dish), and the entire experiment was conducted with three independent biological replicates.

2.6. Adventitious Bud Differentiation

Callus obtained from the best-performing induction treatments was subcultured onto differentiation medium (MS + 30 g/L sucrose + 3 g/L Phytagel, pH 5.8) supplemented with one of four hormone combinations (Table 3), and cultured at 25–28 °C, 50–60% relative humidity under a 16-h photoperiod, with the medium refreshed every 15 days. For each hormone combination, three replicate Petri dishes were evaluated with 6–10 callus pieces per dish, and three independent biological replicates were performed. Adventitious bud induction rate was recorded after 30 days.

2.7. Shoot Strengthening and Rooting

Vigorous adventitious buds were excised at the base and cultured on shoot-strengthening medium (MS + 2 mg/L 6-BA + 0.2 mg/L 2,4-D + 30 g/L sucrose + 3 g/L Phytagel) at 25–28 °C, 50–60% relative humidity, and a 16-h photoperiod for approximately 30 days, yielding shoots with 8–10 true leaves. Strengthened shoots were then transferred to one of five rooting media (Table 4) under the same culture conditions, and rooting rate was recorded after approximately 30 days. For each hormone treatment, six culture bottles were used with 3–4 strengthened shoots per bottle, and three independent biological replicates were performed.

2.8. Acclimatization and Transplanting

For acclimatization, rooted plantlets were first hardened in culture vessels with the lids gradually opened for approximately 3–7days under culture room conditions. The plantlets were then gently removed, and residual Phytagel was washed from the roots with running water. Subsequently, the plantlets were transplanted into plastic pots containing sterilized soil. Survival and growth were assessed after approximately 30 days.

2.9. Data Recording and Statistical Analysis

Callus induction, adventitious bud induction, and rooting rates were calculated for each biological replicate as the percentage of responding explants relative to the inoculated explants. Data from three independent biological replicates are expressed as the mean ± standard deviation (SD), along with cumulative response ratios (pooled n/N). Differences in response frequencies among treatments across the three replicates were analyzed statistically using Fisher’s exact test, with the significance level defined as P < 0.001. Different lowercase letters in the figures and tables denote statistically significant differences. Callus morphology (volume and texture) and shoot/root vigor were assessed visually at each subculture interval and are reported descriptively alongside the quantitative induction rates.

3. Results

3.1. Callus Induction and Proliferation from Bougainvillea Leaf Explants, but Failure to Differentiate Adventitious Buds

Leaves are among the most commonly used explants in plant tissue culture because they are abundant, easy to collect, and do not depend on the poor and unpredictable seed-set that limits access to Bougainvillea seed. Following surface sterilization (1% NaClO for 2 min), the contamination rate of leaf explants was maintained below 20%, and over 80% of explants remained viable and aseptic, providing sufficient material for subsequent culture (Table S1). We therefore first attempted callus induction using young B. glabra leaves, cut into approximately 1 cm² pieces after removal of the midrib, and cultured in the dark on media containing 18 6-BA/2,4-D (Table S2 a-r) combinations and 9 6-BA/NAA (Table S2 A-I) combinations for 2 months (Figure 1A; Figure S1). Among the 6-BA/2,4-D combinations tested, 0.5 mg/L 6-BA + 1 mg/L 2,4-D and 0.5 mg/L 6-BA + 3 mg/L 2,4-D gave the best callus induction, while among the 6-BA/NAA combinations, 2 mg/L 6-BA + 0.5 mg/L NAA was most effective (Table S2). All treatments produced visible callus by day 20 and continued to enlarge through day 60, showing that Bougainvillea leaf tissue, like hypocotyl tissue, is broadly responsive to exogenous auxin/cytokinin stimulation.

The induced leaf callus obtained from the treatment 0.5 mg/L 6-BA + 1 mg/L 2,4-D (the yellow box in Figure 1A) was then subcultured onto six proliferation media differing in 6-BA, 2,4-D, NAA, or KT content (Figure 1B, Table 1). Medium c (2 mg/L 6-BA + 0.2 mg/L 2,4-D, the yellow box in Figure 1B) produced the best proliferation, with callus that was large in volume, uniformly green, and free of browning after 30 days. Medium d (1 mg/L 6-BA + 0.1 mg/L 2,4-D) also produced large-volume, green callus, but was visibly less vigorous than medium c. Conversely, the callus induced by culture media a (1 mg/L 6-BA + 0.5 mg/L NAA) and b (0.5 mg/L 6-BA + 0.25 mg/L NAA) was large in volume but slightly browned, while the callus induced by culture media e (3 mg/L 2,4-D + 0.05 mg/L KT) and f (1.5 mg/L 2,4-D + 0.025 mg/L KT) was browned, with no noticeable change in callus volume induced by culture medium e.

However, when leaf-derived callus proliferated on medium c was transferred to the same four differentiation media that were later found effective for hypocotyl callus (Table 3), none of the treatments produced adventitious buds, even after extended subculture; the callus either persisted indefinitely as an undifferentiated, proliferating mass or gradually browned and lost viability. This outcome, obtained despite testing the identical hormonal window (2 mg/L 6-BA + 0.2 mg/L 2,4-D) that later proved sufficient to induce shoot differentiation from hypocotyl callus, indicated that the failure of leaf callus to regenerate shoots was not simply a matter of an unexplored hormone combination but reflected an intrinsic developmental constraint of the leaf-derived tissue itself. We therefore turned to seed-derived hypocotyls, a less differentiated and more juvenile tissue, as the explant source for building a complete callus-mediated regeneration system, as described in the remaining Results sections below.

3.2. Seed Germination and Hypocotyl Explant Collection

Aseptic germination on seed germination medium produced sterile seedlings approximately 4–5 cm tall within 10–14 days of dark culture (Figure 2A). Removal of the roots and cotyledons yielded uniform, physiologically juvenile hypocotyl segments suitable for callus induction. Low contamination was observed among the sterilized seed batches used in this study, consistent with the low microbial burden of internal seed tissue relative to field-collected stem material.

3.3. Callus Induction from Hypocotyl Explants

All six hormone-ratio media tested induced callus formation from hypocotyl explants; however, callus morphology and growth vigor differed markedly among treatments (Table 2; Figure 2B). The combinations 2 mg/L 6-BA + 0.5 mg/L 2,4-D and 2 mg/L 6-BA + 0.2 mg/L 2,4-D produced notably larger, more compact callus than the other treatments, and the latter combination uniquely induced spontaneous bud-point differentiation by day 25 of culture (the red box in Figure 2B). In contrast, 0.5 mg/L 6-BA + 1 mg/L KT, 0.5 mg/L 6-BA + 2 mg/L 2,4-D, and 0.5 mg/L 6-BA + 4 mg/L 2,4-D produced only small-volume callus after 25 days, and 2.5 mg/L 2,4-D alone resulted in callus browning accompanied by unwanted rooting. This pattern indicates that callus induction per se was largely hormone-insensitive across the range tested, whereas callus quality and developmental competence were highly sensitive to the specific 6-BA:2,4-D ratio.

3.4. Differentiation of Adventitious Buds from Callus

Hypocotyl explants cultured on 2 mg/L 6-BA + 0.2 mg/L 2,4-D (the optimal callus induction medium; red box in Figure 2B) were used for subsequent differentiation screening experiments. When callus was subcultured onto four differentiation media, only 2 mg/L 6-BA + 0.2 mg/L 2,4-D (the red box in Figure 2C) induced adventitious buds, with a short induction time and an average differentiation rate of 90.4 ± 2.5% (Figure 2D and Figure 2E; Table 3). The remaining combinations (0.5 mg/L 6-BA + 1 mg/L KT, 2 mg/L KT + 0.2 mg/L NAA + 0.2 mg/L IAA, and 0.5 mg/L KT + 0.05 mg/L NAA + 0.05 mg/L IAA) allowed the callus to continue enlarging but failed to induce shoot differentiation, indicating that a moderate cytokinin/auxin ratio dominated by 6-BA and a low concentration of 2,4-D, rather than KT- or NAA/IAA-based combinations, was required to redirect callus development toward organogenesis in this species.

3.5. Shoot Strengthening and Rooting

Adventitious buds strengthened on MS + 2 mg/L 6-BA + 0.2 mg/L 2,4-D produced robust shoots bearing 8–10 true leaves within about 30 days (Figure 3A). Among five rooting media tested, only 1/2 MS + 1.25 mg/L NAA + 0.75 mg/L IBA (Figure 3B and Figure 3C; Table 4) induced rooting, giving an average rooting rate of 95.2 ± 0.7% and vigorous, well-branched root systems; auxin-only or single-auxin combinations at the concentrations tested (NAA alone, IBA + IAA, 2,4-D alone, or IAA alone) failed to induce roots.

3.6. Acclimatization and Transplanting

After pre-acclimatization with culture-vessel lids gradually loosened and subsequent transfer to sterilized soil, plantlets showed vigorous growth after 30 days (Figure 3D), indicating that the regenerated plants transitioned successfully from in-vitro to ex-vitro conditions.

Overall, the five-stage pipeline described above (callus induction, adventitious bud differentiation, shoot strengthening, rooting, and acclimatization) converted seed-derived hypocotyl segments into acclimatized, soil-grown plants within approximately 115–125 days from seed sterilization, with the callus induction and differentiation steps together accounting for roughly half of this timeline. On average, each explant yielded 3–5 regenerated plantlets.

4. Discussion

Addressing the longstanding recalcitrance of Bougainvillea callus to shoot organogenesis, the present study successfully established an efficient, complete, and streamlined whole-plant regeneration protocol using seed-derived juvenile hypocotyls as explants. Among the tested plant growth regulator combinations, the regime of 2.0 mg/L 6-BA and 0.2 mg/L 2,4-D effectively promoted compact, actively dividing callus that spontaneously initiated bud primordia, ultimately achieving a high adventitious bud differentiation rate of 90.4 ± 2.5% on the same medium. Following shoot elongation, robust rooting was achieved on 1/2 MS medium supplemented with 1.25 mg/L NAA and 0.75 mg/L IBA (95.2 ± 0.7% rooting rate), followed by successful ex vitro acclimatization and soil establishment. Taken together, these results demonstrate a complete, highly reproducible plant regeneration pipeline that progresses through a totipotent callus intermediate, delineating a specific 6-BA-dominant, low-2,4-D hormonal window that facilitates the callus-to-shoot transition in B. glabra. Furthermore, by coordinating callus induction, de novo bud differentiation, and shoot elongation on a unified single medium, this streamlined protocol substantially overcomes the operational complexity of conventional multi-stage subcultures, providing a reliable platform for the mass micropropagation and future biotechnology of this species.

4.1. Hypocotyls, Not Leaves, Are the Productive Explant for Callus-Mediated Regeneration

The physiological state and differentiation potential of the explant are core determinants of success in plant tissue-culture regeneration, and the present study provides a direct, within-species comparison of the two. Both leaf and hypocotyl explants formed callus readily under suitable hormone conditions; leaf explants exhibited consistently high callus initiation across the tested 6-BA/auxin combinations, and leaf callus could be brought to a large-volume, non-browning proliferative state on 2 mg/L 6-BA + 0.2 mg/L 2,4-D (Table 1), yet only hypocotyl-derived callus proved capable of redifferentiating into adventitious buds. Critically, this divergence cannot be attributed to an unexplored hormonal window: leaf callus was challenged with the same 2 mg/L 6-BA + 0.2 mg/L 2,4-D regime, and the same set of differentiation media that later proved sufficient to drive a 90.4 ± 2.5% bud-differentiation rate from hypocotyl callus, yet it produced no adventitious buds under any of them. This points to an intrinsic difference in developmental competence between the two tissues rather than a simple deficiency of the culture medium. Leaf mesophyll cells are terminally differentiated and photosynthetically specialized, and dedifferentiating them fully enough to re-enter an organogenic program is intrinsically more difficult than reprogramming the physiologically juvenile, mitotically active cells of a hypocotyl, a pattern broadly consistent with tissue-culture studies of other woody and semi-woody species in which juvenile tissues outperform mature leaves as regeneration substrates [19,20,21]. Practically, this result also has value on its own terms: it rules out leaf explants as a shortcut around bougainvillea’s poor seed-set for building a transformation-competent callus system, and it indicates that any future attempt to regenerate Bougainvillea from leaf tissue will likely require a qualitatively different strategy for example, additional dedifferentiation cues such as gibberellin or an extended pre-culture period rather than simple re-optimization of the 6-BA:2,4-D ratio used here.

4.2. Auxin 2,4-D Plays a Crucial Role in Bougainvillea Tissue Culture

The combined results of leaf and hypocotyl tissue culture experiments indicate that auxin 2,4-D plays a vital role in the entire process of callus induction, proliferation, and differentiation in Bougainvillea. In the leaf callus induction stage, the combination of 6-BA and 2,4-D showed markedly better induction effects than the combination of 6-BA and NAA, with 2,4-D-mediated callus proliferation exhibiting higher efficiency and better growth. In the hypocotyl regeneration system, a unified medium containing 2.0 mg/L 6-BA and 0.2 mg/L 2,4-D coordinated the entire sequence of callus induction, de novo bud differentiation, and shoot elongation in a single culture step; other auxin ratios could not complete the regeneration process. This demonstrates the important role of auxin 2,4-D in Bougainvillea tissue culture. This indicates that Bougainvillea tissue cells exhibit a highly specific response to 2,4-D. Appropriate concentrations of 2,4-D can precisely regulate cell dedifferentiation and redifferentiation processes, maintaining high organogenic competence of callus tissue. It is a key hormone in the Bougainvillea tissue culture regeneration system and can provide an important target for subsequent research on the hormone regulation mechanism of Bougainvillea.

4.3. Comparison with Previous Research

The central conceptual difference between this system and earlier Bougainvillea protocols lies in the establishment of a streamlined, high-efficiency callus-mediated de novo organogenesis pathway. Most previous micropropagation protocols using nodal segments [6,9] or shoot tips regenerated shoots directly from pre-existing axillary meristems. While Gong et al. [3] reported that semi-lignified stem segments could form basal callus capable of shoot differentiation, their adventitious bud differentiation rate was remarkably low (only 25.6%) and required a complex series of multi-stage transfers across five disparate media formulations. Likewise, our callus-derived organogenesis pathway resembles that of Lu et al. [11] wherein compact callus forms at explant cut ends to yield adventitious buds; this shared pattern further substantiates the biological reliability and feasibility of directly differentiating adventitious buds from hypocotyl-derived callus. Nevertheless, while their protocol relied on a cumbersome four-medium sequence, our system achieves a superior differentiation efficiency while coordinating callus induction, shoot differentiation, and subsequent growth on a single medium formulation, markedly streamlining the entire procedure. Additionally, using adventitious buds from three calla lily (Zantedeschia hybrida) cultivars, previous work revealed significant inter-cultivar divergence during the plant regeneration stage [22] suggesting that adventitious bud differentiation in Bougainvillea may likewise be cultivar-dependent. Furthermore, while Nasrat et al. (2022) [10] successfully induced callus from nodal segments on WPM, their cultures were maintained strictly for secondary metabolite and antioxidant extraction, halting at the callus proliferation stage without any shoot regeneration. In contrast, our protocol demonstrates a callus-mediated de novo organogenesis pathway that circumvents multi-step subcultures by coordinating callus induction, bud differentiation, and shoot elongation on a unified single medium.

The hormonal requirements identified here are broadly consistent with the classical auxin: cytokinin ratio model first articulated by Skoog & Miller [18]: elevated cytokinin relative to auxin favors shoot formation, elevated auxin relative to cytokinin favors rooting, and comparable concentrations of both favor continued callus proliferation. In the present system, a cytokinin-dominant medium (2 mg/L 6-BA) combined with a low concentration of 2,4-D (0.2 mg/L) gave the best combination of callus quality and subsequent shoot differentiation, whereas low cytokinin (0.5 mg/L 6-BA) combined with a higher 2,4-D concentration (2 mg/L) supported vigorous callus growth but not differentiation, and 2,4-D alone shifted development toward unwanted rooting and browning precisely the auxin-dominant, root-promoting outcome the classical model predicts. This pattern also mirrors the auxin-dominance effect reported for Bougainvillea axillary-bud induction in recent response-surface optimization work [8], where 2,4-D exerted the strongest influence among the three hormones tested.

In quantitative terms, the 95 ± 0.7% rooting rate achieved on 1/2 MS + 1.25 mg/L NAA + 0.75 mg/L IBA compares favorably with the 82% rooting rate reported for nodal-segment-derived B. × buttiana ‘Miss Manila’ plantlets on 1/2 MS + 2.0 mg/L IBA alone [6], and exceeds the 66.7% rooting rate reported by Li et al. (2025) [8]. Likewise, the 90.4 ± 2.5% adventitious bud differentiation rate obtained from hypocotyl callus substantially outperforms the 25.6% differentiation rate reported by Gong et al. [3] and the 81.3% rate reported by Lu et al. [11], demonstrating that seed-derived juvenile hypocotyls possess exceptional morphogenic totipotency when cultured under an optimized 6-BA:2,4-D balance.

4.4. Future Perspectives

The establishment of this simplified, single-medium regeneration protocol provides a practical and reliable platform for both mass clonal propagation and future biotechnology in Bougainvillea. However, several specific objectives remain to be explored. First, while the present system proved highly effective for B. glabra, plant tissue-culture response is frequently genotype-dependent; evaluating this streamlined protocol across a broader range of elite commercial cultivars—particularly economically valuable bicolored and interspecific hybrids—will be an important next step. Second, future studies should incorporate histological and cytological analyses to characterize the precise cellular origins of bud primordia at the cut surfaces, alongside flow-cytometric and molecular marker assessments to confirm genetic and ploidy fidelity during in vitro culture. Finally, this protocol establishes the indispensable cellular recipient tissue required for downstream genetic enhancement; testing the competence of this actively dividing hypocotyl-derived callus for Agrobacterium-mediated transformation or CRISPR/Cas-mediated gene editing will pave the way for targeted trait improvements in this globally important ornamental genus.

5. Conclusions

In this study, initial attempts using young Bougainvillea glabra leaves readily induced proliferating callus but completely failed to undergo adventitious shoot organogenesis (Figure 4A). In contrast, seed-derived juvenile hypocotyls successfully broke this developmental arrest, establishing a complete and highly efficient callus-mediated whole-plant regeneration system (Figure 4B). This optimized protocol achieves compact callus induction, high-frequency de novo shoot differentiation (90.4 ± 2.5%), robust root induction (95.2 ± 0.7% on 1/2 MS + 1.25 mg/L NAA + 0.75 mg/L IBA), and successful ex vitro acclimatization. Crucially, the entire sequence from hypocotyl callus induction and shoot differentiation to shoot elongation was coordinated on a unified, single culture medium (MS + 2.0 mg/L 6-BA + 0.2 mg/L 2,4-D), substantially streamlining the operational process and enhancing efficiency. Beyond establishing a standardized, high-efficiency micropropagation pipeline for B. glabra, this robust protocol provides an indispensable cellular platform and recipient system for future Agrobacterium-mediated genetic transformation, CRISPR/Cas9 genome editing, and precision molecular breeding in this economically and ornamentally important genus.