An Efficient Method for the Propagation of Bougainvillea glabra ‘New River’ (Nyctaginaceae) from In Vitro Stem Segments

Abstract

Bougainvillea, an evergreen climbing shrub of the Nyctaginaceae family, holds significant ornamental, economic, and medicinal value. Bougainvillea glabra ‘New River’ is widely used in landscapes due to its strong adaptability to the environment, abundance of flowers, and frequent flowering. Traditionally, Bougainvillea glabra ‘New River’ cultivation has relied on methods such as cuttings or grafting, with limited research on in vitro tissue culture propagation. This study aimed to optimize the tissue culture system, exploring a combination of plant growth regulators (PGRs) for Bougainvillea regeneration from in vitro stem segments. The Murashige and Skoog (MS) medium supplemented with indole-3-butyric acid (IBA), 6-benzylaminopurine (6-BA), and 1-naphthlcetic acid (NAA) was employed. The optimal sterilization of Bougainvillea stem segments involved a 30 s treatment with 75% alcohol and 10 min with 1% NaClO. The synergistic effect of 0.1 mg·L⁻¹ of NAA and 2.5 mg·L⁻¹ of 6-BA maximized the shoot sprouting frequency, while 2.5 mg·L⁻¹ of 6-BA and 0.1 mg·L⁻¹ of NAA produced the maximum shoots. Furthermore, 1.5 mg·L⁻¹ of IBA and 0.1 mg·L⁻¹ of NAA induced the highest rooting levels. This work demonstrates the successful adaptation of a greenhouse environment to efficiently regenerate plants in vitro from stem segments. This approach allows for the mass production of Bougainvillea glabra ‘New River’.

1. Introduction

Bougainvillea, a natural climber, is a member of the Nyctaginaceae family and stands out as a multi-flowered plant. It is a very common ornamental plant [1], first explored in Brazil in the 18th century by Louis de Bougainville [2]. This plant is highly favored due to its unique ornamental charm and excellent environmental adaptability. More than 30 regions worldwide have chosen it as their representative flower, such as Zambia, Grenada, Hainan Province in China, and Guam in the United States [3]. At present, important ornamental varieties mainly originate from three basic species, namely Bougainvillea glabra of Choisy, Bougainvillea spectabilis of Willd, and Bougainvillea peruviana of Humboldi and Bonpian [4].

Renowned for its vibrant and ever-present blooms, Bougainvillea glabra ‘New River’ is prominent among ornamental plants in landscaping. Its bracts are purple, and the flowers are lush, blooming almost all year round in tropical regions. It has strong drought resistance and low soil requirements and is easy to plant, so it is widely used in landscape greening. It is easy to make plant landscape ornaments such as potted plants, flower baskets, and flower columns using Bougainvillea glabra ‘New River’ due to its durable and easy-to-shape characteristics. In addition, Bougainvillea glabra ‘New River’ is often used as a rootstock for grafting other Bougainvillea varieties due to its fast growth rate and strong affinity. Beyond its aesthetic appeal, it carries significant economic and medicinal value [5,6]. It has been traditionally employed to address respiratory issues, such as cough, asthma, bronchitis, and whooping cough [7,8]. Despite its South American origin [9], the versatile Bougainvillea glabra ‘New River’ has found its way into landscapes worldwide due to its myriad functions. However, the plant presents challenges concerning reproduction, as its small pollen tube and low pollen and embryo activity hinder successful pollination, fruit setting, and seed production [10,11]. Consequently, asexual propagation methods, including cuttings, grafting, and crimping, have become the primary means of cultivating this plant [12,13].

The conventional methods for propagating Bougainvillea face challenges such as a small proliferation coefficient, a prolonged rooting time, and limitations imposed by seasonal propagation times [14,15]. The traditional cutting propagation method yields a limited number of seedlings within an extended timeframe, particularly for sparse varieties [16]. In contrast, tissue culture techniques can solve these challenges by overcoming environmental constraints, especially difficulties in rooting initiation. This method enables the rapid generation of a substantial quantity of plants with consistent varietal characteristics in a short period. Consequently, it shortens the breeding cycle and reduces the production costs associated with obtaining large quantities of high-quality seedlings [17,18].

The research on the tissue culture of Bougainvillea dates back to the early 20th century, with significant progress achieved by various researchers [19,20]. Past studies on Bougainvillea tissue culture have predominantly focused on explant selection, basic medium formulation, and hormone application [21]. Recent investigations have shifted toward understanding the tissue culture variations among different Bougainvillea varieties, aiming to develop a more efficient system that lowers costs and enhances benefits. For instance, Kumari et al. [22] conducted in vitro root culture and domestication studies on challenging-to-root Bougainvillea varieties, Mahatma Gandhi and Refulgens. Jain et al. [23] provided a protocol for the in vitro multiplication of the Bougainvillea cultivar Mahara. Tao et al. [24] explored the tissue culture conditions for Bougainvillea buttiana ‘Miss Manila’, effectively improving the proliferation and rooting rates. Establishing a rapid, simple, and reproducible tissue culture propagation system for Bougainvillea glabra ‘New River’ is crucial for its widespread application.

2. Materials and Methods

2.1. Plant Material

The plant materials were acquired from a tree nursery of the Institute of Tropical Agriculture and Forestry, Hainan University, Danzhou, Hainan Province, China (109°29′02″ E, 19°30′17″ N). Fresh stem segments (2–3 cm long) with 1–2 axillary buds were excised from young green branches of Bougainvillea glabra ‘New River’ that were healthy and free of pests and diseases and were used as explants. All plant plant growth regulators including 6-benzyladenine (6-BA, available ingredient 99%), 1-naphthaleneacetic acid (NAA, 99%) and indole-3-butyric acid (IBA, 99%) were provided by the Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Beijing Lanjieke Technology Co., Ltd. (Beijing, China) supplied the agar (strength 1200 g/cm²) and sucrose (99%).

2.2. Sterilization of Explants

The stem segments underwent pretreatment with liquid soap for 5 min, followed by thorough rinsing with tap water lasting 30 min. The dust-free stem segments were then surface-sterilized with 75% ethanol for 30 s and then rinsed three times with sterilized distilled water. Next, the stem segments were further sterilized with sodium hypochlorite for 4, 6, 8, 10, or 12 min (

Table 1. Treatment combinations for explant sterilization of Bougainvillea glabra ‘New River’.

Explant Sterilization
Treatment	Treatment Time with 75% Ethanol (s)	Concentration of NaClO 1 (%)	Treatment Time with NaClO (min)
S1	30	1	4
S2	30	1	6
S3	30	1	8
S4	30	1	10
S5	30	1	12
S6	30	2	4
S7	30	2	6
S8	30	2	8
S9	30	2	10
S10	30	2	12

¹ Sodium hypochlorite.

) and washed 5–6 times with sterile water. The surfaces of the explants were drained of moisture with sterilized filter paper, and the cut surfaces of the explants that were exposed to the sterilizing solution were clipped off. Finally, the disinfected stem segments were introduced into Murashige and Skoog (MS) medium [25] supplemented with 2.5 mg·L⁻¹ of 6-benzylaminopurine (6-BA) and 0.1 mg·L⁻¹ of 1-naphthlcetic acid (NAA). The contamination rate, survival rate, and death rate of the explants were documented after a 15-day culture period [26].

The pH of the medium was adjusted to 5.8 ± 0.2 using a 1 mol·L⁻¹ NaOH solution before solidifying it with 8 g·L⁻¹ agar. The medium was then autoclaved at 121 °C for 20 min. The cultures were kept in a room with a 12 h light and 12 h dark cycle, and the light intensity was maintained at 1500–2000 lux using a cool white fluorescent lamp. The relative humidity and temperature were maintained at 55%–60% and 25 ± 2 °C, respectively.

This experiment was undertaken in February, April, June, August, October, and December to investigate the impact of the season of collection on the sterilization of explants.

2.3. Culture Conditions for Adventitious Shoot Induction

The sterile explants were cultured on 30 mL of MS medium supplemented with 30 g·L⁻¹ of sucrose and with various concentrations of PGRs (NAA: 0, 0.05, 0.1, or 0.15 mg·L⁻¹; 6-BA: 0, 1.5, 2.0, or 2.5 mg·L⁻¹) (

Table 2. Treatment combinations for axillary bud induction of Bougainvillea glabra ‘New River’.

Axillary Bud Induction
Treatment	Plant Growth Regulators in MS 1 Medium (mg·L−1)
	6-BA 2	NAA 3
CK	0	0
A1	1.5	0.05
A2	1.5	0.10
A3	1.5	0.15
A4	2.0	0.05
A5	2.0	0.10
A6	2.0	0.15
A7	2.5	0.05
A8	2.5	0.10
A9	2.5	0.15

¹ Murashige and Skoog. ² 6-benzylaminopurine. ³ α-naphthaleneacetic acid.

). A daily observation of the explants’ development was conducted. After a 30-day culture period, the shoot sprouting frequency (%), the average number of shoots per explant, and average shoot length (mm) were measured and counted [24].

2.4. Culturing of Shoot Multiplication

When the axillary buds of the induced culture of Bougainvillea grew to a length of 3–5 cm, the stem segments with axillary buds were cut off for proliferative cultures. The axillary buds were then cultured on a shoot multiplication medium [MS medium plus 30 g·L⁻¹ sucrose and 8 g·L⁻¹ agar or supplemented with two types of PGRs: 6-BA (1.5, 2.0, or 2.5 mg·L⁻¹); NAA (0.05, 0.1, or 1.5 mg·L⁻¹)] (

Table 3. Treatment combinations for shoot multiplication of Bougainvillea glabra ‘New River’.

Shoot Multiplication
Treatment	Plant Growth Regulators in MS 1 Medium (mg·L−1)
	6-BA 2	NAA 3
M1	1.5	0.05
M2	1.5	0.10
M3	1.5	0.15
M4	2.0	0.05
M5	2.0	0.10
M6	2.0	0.15
M7	2.5	0.05
M8	2.5	0.10
M9	2.5	0.15

¹ Murashige and Skoog. ² 6-benzylaminopurine. ³ α-naphthaleneacetic acid.

). The proliferation coefficients and growth status of adventitious shoots were observed and counted after culturing for 30 d [27].

2.5. Induction and Acclimatization of Roots

Shoots that were propagated from stem segments had grown into plantlets within 30 d. The plantlets were subsequently isolated and incubated in an MS medium containing 30 g·L⁻¹ of sucrose and different amounts of plant growth regulators (PGRs), namely, IBA (0, 0.5, 1.0, or 1.5 mg·L⁻¹) and NAA (0, 0.05, 0.1, or 0.15 mg·L⁻¹) (

Table 4. Treatment combinations for root induction of Bougainvillea glabra ‘New River’.

Root Induction
Treatment	Plant Growth Regulators in MS 1 Medium (mg·L−1)
	IBA 2	NAA 3
CK	0	0
R1	0.5	0.05
R2	0.5	0.10
R3	0.5	0.15
R4	1.5	0.05
R5	1.5	0.10
R6	1.5	0.15
R7	2.0	0.05
R8	2.0	0.10
R9	2.0	0.15

¹ Murashige and Skoog. ² indole-3-butyric acid. ³ α-naphthaleneacetic acid.

) [28]. The medium was supplemented with 8 g·L⁻¹ of agar to solidify it. The average number of roots per plantlet and the average length of roots (measured in millimeters) were recorded after 60 days. Complete plantlets with numerous healthy roots were selected for acclimatization. Subsequently, the plantlets were transferred to a greenhouse for 15 days. Following this, they were cautiously removed from the rooting medium, delicately rinsed with tap water, and dried on tissue paper to prevent any contamination or infection. Finally, the plantlets were transplanted into plastic pots filled with a mixture of peat and river sand at a ratio of 2:1 (v/v) after high-pressure sterilization. Water was sprayed on the leaf surfaces every morning and evening at regular intervals to maintain humidity and promote plantlet growth. The rates of plant survival were calculated after 30 d [29].

2.6. Data Analysis

The experiments were conducted using a randomized approach. Thirty stem segments were chosen for each sterilization procedure. For additional tests, ten stem segments were chosen for each treatment. The experiments were reproduced thrice. The data were tabulated, arranged, and condensed utilizing Microsoft Excel 2019, while graphs were generated employing Origin 2021. The data underwent one-way analysis of variance (ANOVA) and multiple-range tests (LSD) using IBM SPSS Statistics 25.

3. Results

3.1. Treatment S4 Improved Sterilization

After 15 d of cultivation in the MS media, the explants showed different degrees of contamination, survival, and mortality rates after each sterilization treatment. When the disinfection concentration of NaClO was kept constant, the contamination rate of the explants significantly decreased, the survival rate first increased and then decreased, and the mortality rate significantly increased with the increase in the disinfection time (Figure 1;

Table 5. Effects of different sterilization treatments on explants.

Treatment	Contamination Rate (%)	Survival Rate (%)	Mortality Rate (%)
S1	66.67 ± 3.50 a	33.33 ± 5.80 d	0.00 ± 0.00 e
S2	44.44 ± 5.10 b	54.44 ± 2.30 c	1.11 ± 1.93 d
S3	22.22 ± 1.70 cde	77.78 ± 11.50 ab	0.00 ± 0.00 e
S4	16.67 ± 0.00 def	83.33 ± 5.80 a	0.00 ± 0.00 e
S5	15.56 ± 4.00 ef	80.00 ± 3.00 ab	4.44 ± 5.09 c
S6	24.44 ± 2.30 c	75.56 ± 12.10 ab	0.00 ± 0.00 e
S7	23.33 ± 3.50 cd	76.67 ± 6.50 ab	0.00 ± 0.00 e
S8	14.44 ± 5.10 f	81.11 ± 3.50 ab	4.44 ± 1.93 c
S9	15.56 ± 2.30 ef	74.44 ± 4.00 ab	10.00 ± 0.00 b
S10	11.11 ± 1.70 f	70.00 ± 3.00 b	18.89 ± 3.85 a

The different letters indicate significant differences between treatment combinations (p < 0.05).

). The lowest contamination rate (11.11%) was observed in the S10 treatment, while the highest survival rate (83.33%) was observed in the S4 treatment. Furthermore, the lowest mortality rate (0) was observed in the S1, S3, S4, S6, and S7 treatments. By combining the analyses of contamination, survival, and mortality, it is inferred that the best way to sterilize the stem segments of Bougainvillea glabra ‘New River’ was to apply a 75% alcohol treatment for 30 s and a 1% NaClO treatment for 10 min.

3.2. Spring Is the Best Time for Collecting Explants

A notable disparity in the contamination levels of the explants was observed across the samples/material obtained from various seasons (Figure 2). In February and April, the contamination rate was low, the survival rate of the explants was high, the axillary bud sprouting rate was high, the sprouting was fast, the bud color was positive, and the growth was ‘good’. It was easier to disinfect and sterilize these materials. With the arrival of summer, the temperature rose, more rain was recorded, and germs were easy to breed. In June and August, the contamination rate rose linearly, the survival rate dropped, and it was difficult to sterilize and clean the explants. The contamination rate eased in October–December as the temperature dropped after the fall. Therefore, spring is the best time to retrieve explants.

3.3. A8 Treatment Produced a High Shoot Sprouting Frequency

The axillary buds of Bougainvillea were stimulated in an MS medium with varying doses of PGRs. After one week of incubation, a yellowish-green callus began to grow at its base, which was accompanied by the sprouting of lateral buds. After 30 days (Figure 3;

Table 6. Effect of combinations of different concentrations of plant hormones on the induction of axillary buds.

Treatment	Shoot Sprouting Frequency (%)	Mean Shoot Length (mm)	Mean Number of Shoots
CK	63.33 ± 11.55 d	4.00 ± 0.27 ab	1.27 ± 0.25 e
A1	76.67 ± 5.77 cd	3.20 ± 0.17 cd	2.00 ± 0.17 bc
A2	80.00 ± 0.00 abc	3.00 ± 0.30 cde	2.30 ± 0.27 ab
A3	80.00 ± 0.00 abc	4.30 ± 0.27 a	1.67 ± 0.15 cd
A4	83.33 ± 11.55 ab	3.20 ± 0.53 cd	2.30 ± 0.44 ab
A5	90.00 ± 10.00 ab	2.60 ± 0.18 de	2.00 ± 0.10 bc
A6	86.67 ± 15.28 ab	3.15 ± 0.10 cde	2.00 ± 0.30 bc
A7	90.00 ± 0.00 ab	2.55 ± 0.09 e	1.67 ± 0.21 cd
A8	96.67 ± 5.77 a	3.57 ± 0.59 bc	2.67 ± 0.31 a
A9	93.33 ± 11.55 ab	2.55 ± 0.20 e	2.00 ± 0.20 bc

The different letters indicate significant differences between treatment combinations (p < 0.05).

), the A8 treatment induced the highest rate of bud germination (96.67%) and the largest coefficient of differentiated adventitious shoots (2.67). It was followed by the A9 treatment combination, which produced a high bud germination rate of 93.33%. The A3 treatment produced the longest average bud length of 43 mm, followed by the control, with 40 mm. However, the bud germination rate (63.33%) and the coefficient of differentiated adventitious shoots (1.27) in the controls were significantly less than those induced by the treatments containing PGRs. The optimal medium for inducing adventitious shoots of Bougainvillea glabra ‘New River’ was MS medium supplemented with 2.5 mg·L⁻¹ of 6-BA and 0.1 mg·L⁻¹ of NAA (treatment A8).

3.4. High Shoot Multiplication in M8 Treatment

Following 30 days of cultivation, a substantial quantity of shoots and calluses were generated in the MS medium enriched with plant growth regulators (PGRs) at different doses. The M8 treatment induced a significantly higher number of shoots (mean = 4.67) with robust growth and larger leaves compared with the other treatment combinations. It was followed by the M7 treatment combination, which had a high mean number of shoots of 4.43 (Figure 4,

Table 7. Effects of combinations of different concentrations of plant hormones on the proliferation of axillary buds.

Treatment	Multiplication Coefficient	General Plantlet Growth
M1	2.30 ± 0.50 c	Robust growth; large leaves
M2	3.00 ± 0.30 bc	Average growth; larger leaves
M3	3.67 ± 1.15 abc	Robust growth; large leaves
M4	3.33 ± 0.25 abc	Average growth; small leaves
M5	3.67 ± 0.58 abc	Poor growth; small leaves
M6	4.00 ± 1.04 ab	Average growth; larger leaves
M7	4.43 ± 0.76 a	Poor growth; large leaves
M8	4.67 ± 0.47 a	Robust growth; large leaves
M9	4.00 ± 0.53 ab	Poor growth; large leaves

The different letters indicate significant differences between treatment combinations (p < 0.05).

). The proliferating culture caused nearly twice as many shoots as the inducing culture. The MS medium with a concentration of 2.5 mg·L⁻¹ of 6-BA and 0.1 mg·L⁻¹ of NAA (treatment M8) was determined to be the most effective medium for the proliferation of Bougainvillea glabra ‘New River’ shoots.

3.5. High Root Induction Efficiency in R8 Treatment

The various combinations of hormone treatments differed in their effectiveness in inducing the rooting of adventitious shoots. The rooting rate (36.67%) of the R8 treatment combination (MS medium plus 1.5 mg·L⁻¹ of IBA and 0.1 mg·L⁻¹ of NAA) was the highest (Figure 5;

Table 8. Effects of combinations of different concentrations of plant hormones on rooting of plants.

Treatment	Rooting Percentage (%)	Mean Number of Roots
CK	16.67 ± 5.77 cd	2.23 ± 0.21 d
R1	10.00 ± 10.00 e	3.23 ± 0.32 abc
R2	13.33 ± 5.77 de	3.07 ± 0.12 bc
R3	10.00 ± 10.00 e	3.73 ± 0.35 a
R4	20.00 ± 0.00 bcd	3.57 ± 0.47 ab
R5	23.33 ± 5.77 abcd	2.97 ± 0.06 c
R6	30.00 ± 10.00 abc	3.27 ± 0.31 abc
R7	30.00 ± 10.00 abc	3.33 ± 0.15 abc
R8	36.67 ± 11.55 a	3.43 ± 0.49 abc
R9	33.33 ± 5.77 ab	3.03 ± 0.06 bc

The different letters indicate significant differences between treatment combinations (p < 0.05).

). Although the rooting rate of R3 (10%) was lower than that of R8, the mean number of roots for R3 (3.73) was higher than that of R8 (3.43) and was the highest among all combinations. The control group had a higher rooting rate (16.67%) than the R1, R2, and R3 treatment combinations. In addition, the controls exhibited no callus formation and had a much lower number of roots than the treatment groups supplemented with plant growth regulators (PGRs). The findings demonstrate that the most effective medium for Bougainvillea glabra ‘New River’ root formation was the MS medium with a concentration of 1.5 mg·L⁻¹ of IBA and 0.1 mg·L⁻¹ of NAA (treatment R8).

The plantlets with well-developed roots were selected for acclimatization. After 30 d, approximately 82% of the plantlets had survived, indicating that Bougainvillea glabra ‘New River’ plantlets regenerated in vitro could grow into healthy plants in a conventional environment (Figure 6).

4. Discussion

4.1. Selection of Suitable Plant Material

Selecting an appropriate explant from a healthy and vigorous plant is the first step in plant tissue culture [30]. In this study, shoot stem segments from young green branches of Bougainvillea glabra ‘New River’ that were healthy and free of pests and diseases were used as explants, from which adventitious shoots were induced, and plants were regenerated after shoot development and rooting in the culture media. It has been shown that shoot-tip explants are often easily sterilized to death due to their delicate nature and low survival rate [21]. However, fully lignified stem segments carry more bacteria and cannot be easily sterilized, which results in a higher rate of contamination. Although there are cases of obtaining sterile seedlings via the seed route [31], it is less efficient for obtaining Bougainvillea explants as it is difficult to obtain seeds due to the small pollen tubes and low embryonic activity of Bougainvillea. Bougainvillea leaf explants have also been induced to form calluses, but they did not show embryoid differentiation or the development of adventitious shoots [32].

Therefore, using shoot stem segments as explants is highly efficient compared with terminal buds, fully lignified stem segments, seeds, and leaves. Shoot stem segments require a short period of time for the culture cycle and are easy to obtain, stable, and help in the rapid propagation and popularization of Bougainvillea glabra ‘New River’. However, the optimal explant may vary among different varieties, as may the explants selected for different culture purposes.

4.2. Effects of NaClO Concentration and Sterilization Time on Explants

Establishing an aseptic system of propagation is key to plant tissue culture; therefore, effective sterilization of the explants is needed [15]. In this study, explants of Bougainvillea glabra ‘New River’ were disinfected with 75% ethanol combined with 1% NaClO. The results show that the duration and concentration of NaClO treatment had a greater impact on the contamination rate of explants, the viability rate, and the mortality rate. An increase in the concentration of NaClO had a greater detrimental effect on the explants. With an increase in the concentration and the prolongation of the soaking time, the degree of injury to the stem segments increased, the recovery of growth was slow, and, in some cases, it was difficult to resume growth. Overall, healing was inhibited as browning expanded from the base to the entire stem segment, which was finally killed. In this case, a short-span time gradient experiment was set up to obtain precise information on an effective treatment time that could efficiently reduce the contamination rates while minimizing tissue damage during the application of disinfectants to the explants [33]. Ascorbic acid, PVP, and antioxidants could be added optionally to reduce the browning phenomenon of explants during tissue culture [34]. As it is difficult to disinfect the surface occupied by the explants of ectomycorrhizal bacteria, adding appropriate amounts of Tween to the disinfectant solution is usually recommended. In addition, bacteriostatic agents or antibiotics can be added to the culture medium to prevent the growth of endophytic bacteria in the explants [32].

4.3. MS Medium with PGRs Successfully Induced Shoot Regeneration and Multiplication from the Stem Segments

PGRs are crucial to the growth and development of plant tissues or organs in laboratory settings [35]. Appropriate cytokinins combined with other growth hormones can significantly enhance the rate of triggered and proliferated axillary buds of explants [27,28]. 6-BA and NAA are the predominant hormones utilized for beginning culture and promoting proliferation. In the present study, the induction and proliferation of axillary buds were enhanced as the overall concentration of 6-BA and NAA increased. This indicates that higher concentrations of 6-BA and NAA can effectively promote the induction and proliferation of axillary buds, which agrees with Tao’s results [24]. When the 6-BA concentrations were 1.5 mg·L⁻¹ and 2.0 mg·L⁻¹, the proliferation coefficient increased with the increase in the NAA concentration. However, when the 6-BA concentration was 2.5 mg·L⁻¹, the proliferation coefficient increased and then decreased with the increase in the NAA concentration. This indicates that a higher hormone concentration was not better, and too high a concentration inhibited the proliferation of sprouts. When the concentration of NAA was constant, the rate of explant germination increased with the increase in the 6-BA concentration. This suggests that axillary bud induction is facilitated by suitably increasing the content of 6-BA in the induction medium. Numerous other plants, including Pentas lanceolata and Lepturus repens, have also been shown to exhibit similar effects [36,37].

4.4. IBA Played a Crucial Role in Root Induction of Bougainvillea glabra ‘New River’

The present investigation assessed the impact of PGRs on the in vitro rooting of Bougainvillea glabra ‘New River’ plantlets that were propagated from stem segments. IBA and NAA are commonly employed in root induction studies in histoculture [38]. Different concentrations of IBA and NAA were used to promote the rooting of plants, which induced a significant increase in the number of rooted plants as well as the root coefficient (compared with the control). IBA is essential for the initiation of root formation in Bougainvillea glabra ‘New River’. The rate at which rootless histocultured seedlings developed roots progressively rose when the IBA dose was raised from 0.05 mg·L⁻¹ to 1.5 mg·L⁻¹. The optimal concentration of IBA for root induction in plants was found to be 1.5 mg·L⁻¹, but variations in the NAA concentration did not have a significant impact. Some studies demonstrated that the rooting impact of combining numerous auxins was superior to that of using them alone [39]. Furthermore, NAA not only stimulates rooting but also enhances germination. Meanwhile, IBA is primarily employed to induce rooting due to its more potent actions, resulting in a greater number of roots being formed [33]. Importantly, the successful acclimatization of plantlets is a significant step for in vitro plant propagation. In this study, the plantlets with roots were acclimatized in a greenhouse and cultivated in plastic pots filled with peat and river sand at a ratio of 2:1 (v/v) after high-pressure sterilization. Approximately 82% of the plantlets had survived after 30 d.

5. Conclusions

This work successfully developed a rapid in vitro propagation method for Bougainvillea glabra ‘New River’ via the investigation of essential processes such as explant sterilization, axillary bud induction, proliferation, rooting, and domesticated transfer (Figure 6). Bougainvillea glabra ‘New River’ shoot stem segments, with robust growth and free of pests and diseases, were collected in the spring season of the year and were used as explants. In summary, the explants were first sterilized with 75% alcohol for 30 s + 1% NaClO for 10 min and then inoculated onto MS + 2.5 mg·L⁻¹ of 6-BA + 0.1 mg·L⁻¹ of NAA to induce axillary bud sprouting. When the axillary buds had sprouted, the new shoots were cut and inoculated onto a culture medium containing MS + 2.5 mg·L⁻¹ of 6-BA + 0.1 mg·L⁻¹ of NAA for proliferation to obtain the regeneration buds. Then, the regenerated shoots were cut and cultured on MS + 1.5 mg·L⁻¹ of IAA + 0.1 mg·L⁻¹ of NAA for rooting. After rooting, the seedlings were acclimatized in a greenhouse for 15 d and transplanted into a sterilized substrate of peat and river sand in a 2:1 (v/v) ratio. The survival rate of transplanting reached 82%. From a commercial perspective, the cost of producing each plant via tissue culture technology is estimated at approximately CNY 0.2, and a stem segment explant can produce approximately 300 plants within 3 months. Although the cost of single-plant-seedling cultivation is slightly higher compared with traditional hard branch cuttings, it has a large reproductive capacity, high seedling rate, good variety traits, and short time consumption. Moreover, it brings higher benefits per unit of time and is more suitable for factory production. The results of this experiment provide technical support for the application of the rapid propagation of Bougainvillea glabra ‘New River’. However, there is still no breakthrough in inducing plant regeneration via the healing pathway, which can be further explored by adjusting the composition of the basic medium, using different hormones, and adjusting the hormone concentration ratios and other conditions.