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Article

The Impacts of Plant Growth Regulators on the Rapid Propagation of Gardenia jasminoides Ellis. in Tissue Culture

by
Yang Ai
1,†,
Yunzhu Chen
2,†,
Shuixuan Zhu
1,
Lijuan Jiang
1,
Jingzhen Chen
2,
Changzhu Li
2,
Peiwang Li
2,
Wenbin Zeng
1,
Ding Kuang
3,
Qiang Liu
1,* and
Yan Yang
2,3,*
1
College of Life Science and Technology, Central South University of Forestry and Technology, Changsha 410004, China
2
State Key Laboratory of Utilization of Woody Oil Resource, Hunan Academy of Forestry, Changsha 410004, China
3
Hunan Haitai Bonong Biotechnology Co., Ltd., Yueyang 414000, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Forests 2024, 15(3), 446; https://doi.org/10.3390/f15030446
Submission received: 30 January 2024 / Revised: 21 February 2024 / Accepted: 21 February 2024 / Published: 26 February 2024
(This article belongs to the Section Forest Ecophysiology and Biology)
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Abstract

:
The optimization of explant selection and adjustment of plant growth regulators (PGRs) ratio may enhance the efficiency of micro-propagation of Gardenia jasminoides Ellis. The findings of the study suggest that the shoot tip proved to be the optimal explant for regenerating adventitious buds, with an impressive regeneration rate of 77.78% and the average number of adventitious buds being 2.86. The ideal medium consisted of Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (6-BA) at a 2 mg L−1, indoleacetic acid (IAA) at a 0.2 mg L−1, kinetin (KT) at 0.15 mg L−1, resulting in an outstanding regeneration rate of adventitious buds reaching up to 91.11%. For rooting purposes, the best medium was found be half-strength MS supplemented with indoleacetic acid (IAA) 0.5 mg L−1, achieving an rate for adventitious roots amounting to as high as 97.78%. The culture plantlets ultimately thrived, achieving an impressive transplanting survival rate of 93.33%. The application of PGRs was also found to enhance the regeneration of adventitious buds by increasing the ratios of endogenous hormones ZR/IAA and GA3/IAA. Additionally, it facilitated the differentiation of adventitious roots by elevating the ratios of endogenous hormones IAA/ZR, IAA/GA3, and ABA/GA3. Our study would provide a theoretical reference for the establishment of an efficient gardenia tissue culture system and the industrial production of gardenia.

1. Introduction

The Gardenia jasminoides Ellis, a perennial shrub belonging to the Rubiaceae family, is widely distributed in southeastern and southwestern regions of China [1]. Renowned for its fragrantly aromatic flowers, this plant is extensively cultivated as a houseplant in warm temperate and subtropical gardens. Moreover, it exhibits remarkable adaptability and high survival rates, making it highly valued in pharmacology and horticulture due to its significant economic potential [2]. In fact, it has been officially recognized as one of the first dual-purpose plants used for both food and medicinal purposes in China [3]. Notably, the fruit of G. jasminoides possesses antibacterial and anti-inflammatory properties [4], aids in diabetes treatment [5], regulates blood pressure [6], and improves blood lipid levels [7]. This natural pigment-rich fruit contains crocin which serves as an ideal alternative to artificial pigments by offering superior quality without any associated side effects. Consequently, it can be effectively utilized as a coloring agent for pastries, confections, beverages, and other consumables. Furthermore, G. jasminoides represents a novel woody oil resource with an impressive oil content of approximately 12%, rivaling that of soybean oil. This fruit showcases outstanding antioxidant properties, remarkably surpassing linoleic acid levels found in most edible oils like olive or sunflower oils [8].
The objective of rapid propagation is to achieve the swift and clonal multiplication of superior gene variants in plants that are free from diseases and insects [9]. By placing desired plant buds on suitable growth media under specific conditions, axillary bud production can be increased, making mass reproduction of exceptional plants one of the primary applications of rapid propagation [10]. The MS medium is widely used in tissue culture of G. jasminoides [11]. In addition, by promoting shoot differentiation and rooting, growth regulators play a vital role in tissue-based rapid propagation. The composition and concentration of cytokinins and auxins in the medium significantly influence the formation of adventitious buds. Therefore, optimizing these factors in rapid propagation protocols is crucial for maximizing the production of healthy plants [12]. During long-term propagation, adjustments can be made to 6-BA and indoleacetic acid (IAA) concentrations to optimize shoot yield and quality [13]. Different concentrations of 6-BA have been utilized for regenerating adventitious shoots in Hedera nepalensis [14] and Cymbidium hybrid [15].
The hormones supplied to intact plants and tissue cultures, commonly referred to as exogenous PGRs [16], are crucial for successful in vitro plant regeneration. The optimal levels of exogenous PGRs in the culture medium may vary, and thus, the success of explants’ response to phytohormone supplementation plays a pivotal role [17]. Different exogenous PGRs exhibited varying effects. In the case of Gardeniae fructus, 6-BA demonstrated greater efficacy compared to other cytokinins in the regeneration of adventitious buds [18,19]. Similarly, kinetin was found to be more superior than other growth regulators in promoting the regeneration of adventitious shoots in eggplants [20]. During explant differentiation, different exogenous PGRs exhibit distinct effects. Significant changes in endogenous hormone levels were observed after treating pea plants with the exogenous hormone indole-3-acetic acid (IAA) [21], while the regulation of Robinia pseudoacacia’s height was achieved by altering endogenous hormone levels using exogenous PGRs [22]. In conclusion, it is apparent that the growth modulation of Kinnow mandarin is influenced by its own internal hormonal regulation system [23]. These findings suggest that comprehending the interplay between exogenous PGRs’ concentration on endogenous hormone levels, along with considering tissue sensitivity towards specific hormone groups, may aid in elucidating divergent patterns observed in tissue culture overall [24]. Moreover, the appropriate regulation and equilibrium of endogenous hormone levels by PGRs play a crucial role in plant growth and development. Exogenous PGRs have a significant impact on the overall concentration of both PGRs and other endogenous hormones, thereby enhancing their intricate interplay [25].
In recent years, with the rapid development of traditional Chinese medicine and ornamental flower industries, both wild and cultivated resources of G. jasminoides have been extensively utilized. Plant tissue culture has emerged as a crucial method for large-scale seedling breeding. Therefore, it is imperative to thoroughly investigate the tissue culture and propagation technology of G. jasminoides. The effects of different PGRs on the same plant species during tissue culture may be closely linked to changes in endogenous hormones within the cultured seedlings. Currently, there are no reports on optimizing conditions for rapid propagation of G. jasminoides through tissue culture techniques or examining how exogenous PGRs affect endogenous hormones to enhance adventitious bud and root differentiation. This study aims to establish a rapid tissue culture system for G. jasminoides, while investigating the impact of exogenous PGRs on regenerating adventitious shoots and roots by promoting endogenous hormone activity. By doing so, this research provides technical support and a theoretical foundation for selecting appropriate PGR treatments that can improve the efficiency of rapid propagation in G. jasminoides as well as facilitate factory cultivation of its tissue-cultured seedlings.

2. Results

2.1. Selection of Best Explants for Adventitious Bud Regeneration

Selecting suitable explants can help reduce the costs of micro-propagation in plant industries. In this study, we compared the efficiency of adventitious bud formation among four different explants (shoot tips, stem segments, leaves, and seeds) from G. jasminoides trees that were exposed to the same PGRs. Evidently, there exists a significant disparity in the potential of explants derived from different parts of the same plant to differentiate into adventitious buds (Figure 1A). The shoot tips exhibited the highest regeneration rate of adventitious buds at 77.78% and produced the greatest number of adventitious buds, with an average of 2.86. Their adventitious buds observed in the shoot tip explant appeared around the node below the terminal bud. And the stem segments explants exhibited intermediate levels of regenerating adventitious buds and their quantitative performance, ranking second only to shoot tips. The number of adventitious buds produced by the seed explants was 1 and the regeneration rate was 4.45%, both of which were low, while the leaves fail to undergo differentiation and form adventitious buds (Figure 1B,C). Therefore, the shoot tip is utilized as the optimal explant the best adventitious buds generating efficiency, which are selected for subsequent optimization experiments in PGR treatment.

2.2. Types of Adventitious Buds’ Regeneration of Shoot Tip Explants

Further investigation into the regenerative effect of various types of PGRs on adventitious buds in explants of shoot tips, which have been identified as the optimal explants in G. jasminoides. It was observed that IAA exhibited superior efficacy compared to NAA in promoting the differentiation of shoot tips’ adventitious buds. Compared to the S1 (NAA) treatment group, the S3 (IAA) treatment group exhibited a significant 22% increase in adventitious regeneration rate and a 0.51 cm increment in budding plant height. Analogously, in comparison to the S2 (NAA + KT) treatment group, the S4 (IAA + KI) treatment group demonstrated a remarkable 37% enhancement in adventitious regeneration rate and a 0.72 cm elevation in budding plant height. Remarkably, within the IAA experimental groups, the inclusion of KT kinetin (S4 group) resulted in a significant increase in conductivity by 9% and budding plant height by 0.48 cm compared to the absence of KT kinetin (S3 group) (Table 1). Consequently, this investigation concludes that a combination of 6-BA + IAA + KT should be selected as the optimal PGR for regeneration adventitious buds G. jasminoides shoot tip explants (Figure 2).
As the best combination of PGR types, 6-BA + IAA + KT, has been identified as an effective blend for regenerating adventitious buds of shoot tips. An orthogonal experiment was conducted to investigate the optimal concentration. The experimental findings reveal that a concentration of 2 mg L−1 6-BA yields a significantly higher rate of adventitious bud regeneration compared to concentrations at 3 or 4 mg L−1, which may indicate that a high concentration of cytokinin (6-BA) could effectively impede the differentiation of adventitious buds. The P3 experimental group consisted of a combination of 2.0 mg L−1 6-BA, 0.2 mg L−1 IAA, and 0.15 mg L−1 KT, which resulted in the highest rate of adventitious bud regeneration at an impressive 91.11%. Additionally, this treatment yielded the greatest number of buds with an average count of 4.2 (Table 2 and Figure 3).

2.3. Effects of PGRs on Endogenous Hormones in Regenerated Adventitious Buds

Furthermore, the endogenous hormone content and their ratios in adventitious buds derived from shoot tip explants were compared under different combinations of PGRs in the P1 to P9 experimental groups. The aim was to elucidate the underlying mechanism by which PGRs enhance the regeneration efficiency of shoot tip explants in G. jasminoides. The results demonstrated that PGR treatment significantly influenced the endogenous hormone content in adventitious buds (Figure S1), particularly altering the ratios of ZR/IAA, GA3/IAA, and ABA/GA3 among these hormones (Figure 4A–C). The ZR/IAA to GA3/IAA ratio was highest in group P3, which also exhibited the highest rate of adventitious bud regeneration when considering (Figure 3). By examining the correlation between endogenous hormone content and the rate of adventitious bud regeneration, a significant negative correlation was observed between endogenous hormone IAA and the rate of adventitious bud regeneration, while ZR/IAA and GA3/IAA showed significant positive correlations with this rate (Figure 4D). In conclusion, the promotion of adventitious bud regeneration by PGRs primarily occurs through the modulation of endogenous hormone ratios such as ZR/IAA and GA3/IAA.

2.4. Effects of PGRs on Regeneration of Adventitious Roots

The shoot tip explants of G. jasminoides were subjected to optimized PGR treatment and transferred to a rooting medium for a duration of 35 days following the regeneration of adventitious shoots. Subsequently, the effects of various PGR treatments on the rooting efficacy of G. jasminoides were compared (Figure 5A). The results demonstrated that the inclusion of IAA (0.5 mg L−1) in the culture medium significantly enhanced both the regeneration rate (98%) and length (5.90 cm) of adventitious roots in G. jasminoides compared to other experimental groups (Figure 5B,C). Moreover, although the average number of roots was slightly lower than that observed in the IBA + IAA group, it remained noteworthy. The regeneration rate of adventitious roots was significantly lower when IBA (0.5 mg L−1) was used as an auxin, compared to the CK treatment (53.33%). This observation suggests that the addition of inappropriate exogenous hormones inhibits adventitious root production. Interestingly, the IBA+IAA treatment group exhibited a higher number of roots (Figure 5D), albeit with shorter root length and absence of primary root formation. In conclusion, IAA (0.5 mg L−1) demonstrated superior efficacy in inducing adventitious roots in G. jasminoides shoot tip explants.
Further analysis of the effects of different exogenous PGRs on endogenous hormone levels in the adventitious roots of G. jasminoides shoot tip explants revealed that the IAA/GA3, IAA/ZR, ABA/GA3 ratios and IAA were significantly higher in the IAA experimental group, which exhibited the highest regeneration rate of adventitious roots, compared to other experimental groups (Figure S2). Conversely, the IBA experimental group displayed significantly lower IAA/GA3, IAA/ZR, and ABA/GA3 ratios, indicating a poorer effect on adventitious root formation (Figure 6A−C). The findings of this study suggest that an elevated endogenous hormone ratio of IAA/GA3, IAA/ZR, and ABA/GA3 can enhance the differentiation process of adventitious roots in G. jasminoides. Additionally, the correlation analysis between endogenous hormone content and adventitious root regeneration rate revealed a significant positive association with IAA, IAA/GA3, IAA/ZR, and ABA/GA3, particularly with IAA/GA3 and IAA/ZR (Figure 6D). Consequently, the supplementation of exogenous growth regulator IAA (0.5 mg L−1) enhanced the ratio of endogenous hormones IAA/ZR, IAA/GA3 and ABA/GA3 to facilitate adventitious root differentiation.

2.5. Transplanting of Culture Plantlets after Bud and Root Regeneration

The cultured plants derived from shoot tip explants of G. jasminoides were transplanted into culture pots with various transplanting substrates after acclimating to the greenhouse environment, aiming to compare the effects of different substrate types on the survival rate of tissue-cultured plantlets (Figure 7). The experimental results demonstrate that the optimal transplanting substrate is a blend of loess soil, peat soil, perlite, and vermiculite in a ratio of 2:2:1:1. This exquisite mixture boasts an impress live transplanting survival rate of 93.33%, while earthworm castings exhibit the lowest survival rate at a mere 31.11% (Table 3). Taken together, the successful and healthy growth of cultivated G. jasminoides plants can be achieved through PGR treatment of adventitious shoots and roots regeneration from shoot tip explants.

3. Discussion

3.1. Optimization of Rapid Propagation System of Gardenia jasminoides

Furthermore, the selection of appropriate PGRs is a pivotal factor in plant tissue culture. The cytokinins (6-BA and KT) and auxins (IAA and NAA) are recognized as the primary PGRs involved in bud development during tissue culture [26,27,28]. However, the specific type and concentration of PGR required for regenerating adventitious buds vary across different plant tissues. For instance, 6-BA alone exhibited the highest efficacy in regenerating adventitious buds in Piper nigrum and Mentha piperita, with a success rate of 85% and 100%, respectively [29,30]. In contrast, the combined application of 6-BA and NAA played a pivotal role in successfully regenerating adventitia formation in chrysanthemum [31,32,33]. In this study, we identified that the optimal hormone ratio for regenerating adventitious buds was 6-BA (2 mg L−1) + IAA (0.2 mg L−1) + KT (0.15 mg L−1), resulting in a remarkable regeneration rate of up to 91%. Moreover, our findings demonstrated that the combination of PGRs (6-BA, KT, and NAA) achieved the highest regeneration rate during adventitious bud formation.
The application of different types of PGRs during the root formation stage of tissue culture seedlings can have varying effects on root regeneration. Auxin PGRs, including IAA, IBA, and NAA, have been observed to promote lateral root development [30,31]; however, their specific impact varies across different plant species due to variations in the affinities of auxin receptors involved in root formation [34]. The optimal PGRs for adventitious root regeneration in G. jasminoides was determined to be IAA at a concentration of 0.5mg L−1. However, there generation of adventitious roots in G. jasminoides was inhibited by IBA, resulting in a regeneration rate that was 41% lower than that observed in the control treatment (53%). The use of IAA alone was also found to be more favorable for adventitious root regeneration in Pogostemon erectus [35]. Conversely, IBA alone exhibited the highest efficacy in regenerating adventitious roots in Prunus dulcis Mill. [36]. Furthermore, the combined treatment of NAA + IBA demonstrated superior hormone-induced adventitious root formation in bamboo [35,36,37].

3.2. Effects of PGRs on Endogenous Hormones

Previous studies have demonstrated that PGRs influence the regeneration of adventitious buds by modulating endogenous hormone levels within these buds. Furthermore, alterations in the ratio of endogenous hormones can provide a more comprehensive reflection of plant growth compared to individual hormones [38]. Endogenous plant hormones play a pivotal role in facilitating adaptation to dynamic environments through their regulation of plant growth, development, and nutrient allocation [39]. For example, the Cytokinins play a pivotal role in promoting cellular division and growth, thereby facilitating bud differentiation [40]. Conversely, the auxin facilitates callus formation and root development in plants [41]. Numerous studies have demonstrated that exogenous application of auxin and cytokinin can modulate the endogenous plant hormone levels, thereby influencing the efficacy of plant tissue culture [42,43]. This study investigated the impact of varying ratios of PGRs on endogenous hormone levels in adventitious shoots and roots. The application of PGRs resulted in an increase in the ZR/IAA and GA3/IAA ratios, specifically during the regeneration phase of adventitious buds. These findings align with previous research demonstrating a significant positive correlation between the rate of adventitious bud regeneration and ZR/IAA levels in Fraxinus mandshurica [44]. And the IAA/ZR, IAA/GA3, and ABA/GA3 ratios of endogenous hormones were enhanced by exogenous IAA treatment to augment the regeneration rate of adventitious roots. In the rooting experiment of tissue culture seedlings of Eucommia ulmoides, a higher IAA/ZR ratio was found to stimulate greater formation of adventitious roots [45].

4. Materials and Methods

4.1. Materials

The G. jasminoides’ explants (young shoot tips, stems and leaves, and mature seeds) were obtained from the G. jasminoides mother tree in 2022, all the G. jasminoides cuttings plant of the Gardenia fructus large variety named Linhai No. 1 were obtained from Hunan Haitai Bonong Biotechnology Co., Ltd. (Yueyang City, Hunan, China) in 2020 and cultivated in Gardenia Germplasm Resource plantation (113°3′30.24′′, 28°7′32.70′′) of Hunan Academy of Forestry in Changsha City, Hunan Province, China. a plantation with an annual average temperature of 17.2 °C (highest of 39 °C and lowest of −2 °C) and an annual average rainfall of 1361.6 mm. The plantation received regular fertilization and watering.

4.2. Selection of Best Explants Type for Adventitious Bud Regeneration

The different gardenia explants (shoot tips, stem segments, leaves, and seeds) were subjected to and transferred onto a medium composed of MS medium + 6-BA (3 mg L−1) + IAA (0.2 mg L−1) + KT (0.1 mg L−1) + sucrose (30 g/L) + agar (6 g/L). Thirty culture flasks were transferred for each type of explant, and after a 4-week culturing period, their efficiency in adventitious bud regeneration was compared. The regeneration frequency was calculated using the formula “regeneration rate = (number of induced adventitious bud explants/total number of inoculated explants) × 100%”. The height of the budding plantlets and the number of adventitious buds were recorded in each experimental group, while maintaining an incubation temperature at 23 ± 2 °C and a photosynthetic photon flux density (PPFD) ranging from 30 μmol m−2s−1 to 40 μmol m−2s−1 provided by light-emitting diodes (LEDs). The PGRs used in this study, including 6-BA, IAA, NAA, IBA and KT, were purchased from Yuanye Bio-Technology Co., Ltd. in Shanghai, China.

4.3. Screening Best PGR for Adventitious Bud Regeneration

The shoot tip explants, which were selected from test 4.2. due to their superior efficiency in adventitious bud regeneration compared to other explants, were cultured in a medium containing various types and ratios of PGRs for a duration of 5 weeks to regenerate the formation of adventitious buds. Thirty culture flasks were transferred for each treatment involving different types and ratios of PGRs, with three shoot tip explants being transferred into each flask. The height of budding plantlets and the number of adventitious buds in each experimental group were recorded.

4.4. Regeneration of Adventitious Roots

The plantlets cultured from G. jasminoides’ shoot tip explants after optimal PRGs treatment for bud regeneration were transferred to 1/2MS medium supplemented with IBA at a concentration of 0.5 mg L−1, IAA at a concentration of 0.5 mg L−1, or a combination of IBA (0.5 mg L−1) and IAA (0.25 mg L−1) for one week under dark conditions, followed by four weeks under light conditions for adventitious roots regeneration. Thirty culture flasks were used for each PGR treatment. The regeneration rate, number of roots, root length, and growth status of shoot tip explants culturing plantlets adventitious shoots were quantified and compared among different treatment groups with various PGRs.

4.5. Transplanting of Culturing Plantlets

The healthy plantlets, which were cultured and rooted successfully, were carefully selected and transplanted into various transplanting substrate matrixes (loess soil, peat soil, perlite, and vermiculite). In the greenhouse, each matrix accommodated 30 plantlets under an illumination time of 12 h per day (The PPFD is 30 μmol m−2s−1 to 40 μmol m−2s−1 and the temperature maintained at 25 ± 2 °C). After a period of 30 days for adaptation and growth, the survival rates were calculated.

4.6. Determination of Endogenous Hormones

The endogenous hormones of adventitious buds and roots of G. jasminoides’ cultured plantlets were quantified using High-Performance Liquid Chromatography (HPLC) [46]. The endogenous hormone content of different PGR treatments for adventitious buds and roots regeneration was determined using the internal standard method, with indoleacetic acid (IAA), abscisic acid (ABA), gibberellic acid 3 (GA3), zeatin (ZR), and 6-Benzylaminopurine (6-BA) standards obtained from Yuanye Bio-Technology Co., Ltd., Shanghai, China.

4.7. Data statistics and Analysis

The effect of different treatments (Such as explants selection test, PGR groups for bud and root regeneration) was analyzed using one-way analysis of variance (ANOVA), and the means were compared for significant differences using Duncan’s Multiple Range Test (DMRT), a post hoc test, at a significance level of p < 0.05. All statistical analyses were conducted utilizing SPSS 26.0 (IBM SPSS, New York, NY, USA).

5. Conclusions

In this study, we established and optimized a rapid propagation system for micropropagation of G. jasminoides. Our findings demonstrate that the shoot tips as the most suitable site for regeneration adventitious buds. Further the optimal medium for adventitious shoot regeneration consists of MS supplemented with 6-BA (2 mg L−1), IAA (0.2 mg L−1), KT (0.15 mg L−1), 6 g/L agar, and 30 g/L sucrose, whereas the optimal rooting medium is 1/2MS supplemented with IAA (0.5 mg L−1), 6 g/L agar, and 30 g/L sucrose. And the blend mixture of loess soil/peat soil/perlite: vermiculite (2:2:1:1) was the best transplanting substrate. In the process of tissue culture optimization, it was also found that exogenous PGRs promoted adventitious bud regeneration by increasing the ratios of endogenous hormones ZR/IAA and GA3/IAA. It also promoted adventitious root differentiation by increasing the ratio of endogenous hormones IAA/ZR, IAA/GA3, and ABA/GA3. In future research, we should focus on the relationship between PGRs and endogenous hormones in the regeneration of G. jasminoides, so that the types and levels of PGRs can be more accurately mastered in rapid reproduction. The results of this study will provide technical support for the construction of an efficient tissue culture and rapid propagation system and an improved variety in the industrial production of G. jasminoides.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f15030446/s1, Figure S1: Effect of different PGRs on the contents of four endogenous hormones in adventitious buds; Figure S2: Effect of different PGRs on the contents of four endogenous hormones in adventitious roots.

Author Contributions

Conceptualization, Q.L. and Y.Y.; methodology, Y.A., Y.C. and S.Z.; writing—original draft preparation, Y.A.; writing—review and editing, Q.L.; visualization, W.Z. and D.K.; supervision, J.C., P.L. and C.L.; project administration, Q.L.; funding acquisition, L.J. and Y.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Science and Technology Innovation Program of Hunan Province (2023NK4298), the Innovation Demonstration Project of Chenzhou City (2022sfq53), the Natural Science Foundation of Hunan Province (2021JJ31143), the Key Scientific and Technological Innovation Platform of Hunan Province (2023PT1002) and the State Key Laboratory of Utilization of Woody Oil Resource (GZKF202202).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments

We thank the State Key Laboratory of Utilization of Woody Oil Resource for providing the testing platform.

Conflicts of Interest

Authors Ding Kuang and Yan Yang are employed by the company Hunan Haitai Bonong Biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The company Hunan Haitai Bonong Biotechnology Co., Ltd. had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Figure 1. Effects of explants from different parts on adventitious bud regeneration. Note: (A) Regeneration of Plant growth regulators adventitious shoots in different explants. (B) Adventitious bud regeneration rate. (C) The average number of adventitious buds. Different letters above bars within statistically significant differences among explants by Duncan’s multiple range test at p < 0.05.
Figure 1. Effects of explants from different parts on adventitious bud regeneration. Note: (A) Regeneration of Plant growth regulators adventitious shoots in different explants. (B) Adventitious bud regeneration rate. (C) The average number of adventitious buds. Different letters above bars within statistically significant differences among explants by Duncan’s multiple range test at p < 0.05.
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Figure 2. Effects of different types of plant growth regulators on the regeneration of adventitious buds in G. jasminoides.
Figure 2. Effects of different types of plant growth regulators on the regeneration of adventitious buds in G. jasminoides.
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Figure 3. Effects of different ratios of plant growth regulators on the regeneration of shoot tips’ adventitious buds in G. jasminoides. BU: adventitious bud.
Figure 3. Effects of different ratios of plant growth regulators on the regeneration of shoot tips’ adventitious buds in G. jasminoides. BU: adventitious bud.
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Figure 4. Effects of plant growth regulators on endogenous hormones during the regeneration of adventitious buds in G. jasminoides. Note: (A–C) The relative amount of endogenous hormones. (D) correlation between endogenous hormone content and the rate of adventitious bud regeneration. Ad%: Percentage of adventitious bud regeneration. Different letters above bars within statistically significant differences between different times of the treatments at the p < 0.05 level according to Duncan’s multiple range test. **and * means the character indexes be significantly different at 0.01 or 0.05 level.
Figure 4. Effects of plant growth regulators on endogenous hormones during the regeneration of adventitious buds in G. jasminoides. Note: (A–C) The relative amount of endogenous hormones. (D) correlation between endogenous hormone content and the rate of adventitious bud regeneration. Ad%: Percentage of adventitious bud regeneration. Different letters above bars within statistically significant differences between different times of the treatments at the p < 0.05 level according to Duncan’s multiple range test. **and * means the character indexes be significantly different at 0.01 or 0.05 level.
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Figure 5. Effects of plant growth regulators on regeneration of adventitious roots. Note: (A) Growth status of adventitious roots. (B) Adventitious root regeneration rate. (C) Average root length. (D) Average number of roots. Different letters above bars within statistically significant differences between treatments by Duncan’s multiple range test at p < 0.05.
Figure 5. Effects of plant growth regulators on regeneration of adventitious roots. Note: (A) Growth status of adventitious roots. (B) Adventitious root regeneration rate. (C) Average root length. (D) Average number of roots. Different letters above bars within statistically significant differences between treatments by Duncan’s multiple range test at p < 0.05.
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Figure 6. Effects of plant growth regulators on endogenous hormones during adventitious root regeneration in G. jasminoides. Note: (A–C) The relative amount of endogenous hormones. (D) Correlation between endogenous hormone content and the rate of adventitious root regeneration. Ro%: Percentage of adventitious root regeneration. Different letters above bars within statistically significant differences between different times of the treatments at the p < 0.05 level according to Duncan’s multiple range test. **and * means the character indexes be significantly different at 0.001, 0.01 or 0.05 level.
Figure 6. Effects of plant growth regulators on endogenous hormones during adventitious root regeneration in G. jasminoides. Note: (A–C) The relative amount of endogenous hormones. (D) Correlation between endogenous hormone content and the rate of adventitious root regeneration. Ro%: Percentage of adventitious root regeneration. Different letters above bars within statistically significant differences between different times of the treatments at the p < 0.05 level according to Duncan’s multiple range test. **and * means the character indexes be significantly different at 0.001, 0.01 or 0.05 level.
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Figure 7. Transplanting of Gardenia jasminoides Ellis. culture plantlets.
Figure 7. Transplanting of Gardenia jasminoides Ellis. culture plantlets.
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Table 1. Effects of different PGR types on the regeneration of adventitious buds in Gardenia jasminoides.
Table 1. Effects of different PGR types on the regeneration of adventitious buds in Gardenia jasminoides.
ExperimentsPGR (mg L−1)Adventitious Bud
Regeneration Rate (%)		Average Budding Plant Height (cm)Growth Status
6-BAIAANAAKT
S12 0.2 55.33 ± 4.04 b0.65 ± 0.05 dSmall amount of browning, forming callus
S22 0.20.149.00 ± 3.46 b0.92 ± 0.06 cFormation of callus
S320.2 77.67 ± 4.04 a1.16 ± 0.07 bHealthy
S420.2 0.186.66 ± 6.65 a1.64 ± 0.06 aHealthy, forming a small amount of callus
Note: values represent means ± SD. Means followed by the same letter in the column are non-significantly different by DMRT (p = 0.05). PGR: plant growth regulator.
Table 2. Effects of different PGRs ratios on the regeneration of adventitious buds in Gardenia jasminoides Ellis.
Table 2. Effects of different PGRs ratios on the regeneration of adventitious buds in Gardenia jasminoides Ellis.
ExperimentsPGR (mg L−1)Adventitious Bud
Regeneration Rate (%)		Average Number
of Buds		Budding Plant Height (cm)
6-BAIAAKT
P120.10.0572.22 ± 8.39 bc3.80 ± 0.30 ab0.89 ± 0.05 c
P220.150.177.78 ± 5.09 b3.15 ± 0.29 bcd0.69 ± 0.04 d
P320.20.1591.11 ± 3.85 a4.20 ± 0.17 a1.13 ± 0.02 ab
P430.10.167.78 ± 5.09 bcd3.12 ± 0.38 cd1.05 ± 0.12 b
P530.150.1560.00 ± 3.33 cde3.61 ± 0.48 abc1.20 ± 0.05 a
P630.20.0558.89 ± 5.09 de3.34 ± 0.37 bcd0.89 ± 0.07 c
P740.10.1552.22 ± 5.09 e1.44 ± 0.18 e0.74 ± 0.0 d
P840.150.0551.11 ± 8.39 e1.42 ± 0.46 e0.65 ± 0.05 d
P940.20.153.33 ± 8.82 e2.75 ± 0.18 d0.65 ± 0.10 d
Note: values represent means ± SD. Means followed by the same letter in the column are non-significantly different by DMRT (p = 0.05); PGR: plant growth regulator; R(Ad): range analysis of adventitious bud regeneration rate.
Table 3. Effects of different transplanting substrate on the transplantation of Gardenia jasminoides culture plantlets.
Table 3. Effects of different transplanting substrate on the transplantation of Gardenia jasminoides culture plantlets.
ExperimentsTransplanting SubstrateSurvival Rate (%)
Peat SoilEarthworm CastingsPerliteVermiculiteLoess Soil
W11 56.67 ± 3.34 e
W21 1 65.56 ± 1.93 cd
W31 1 67.78 ± 1.92 c
W42 11 65.55 ± 3.85 cd
W5 1 54.44 ± 1.93 e
W6 11 60.00 ± 3.33 de
W7 1 1 57.78 ± 1.92 e
W8 211 57.78 ± 5.09 e
W9 174.45 ± 3.85 b
W102 11293.33 ± 3.34 a
Note: values represent means ± SD. Means followed by the same letter in the column are non-significantly different by DMRT (p = 0.05); PGR: plant growth regulator.
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Ai, Y.; Chen, Y.; Zhu, S.; Jiang, L.; Chen, J.; Li, C.; Li, P.; Zeng, W.; Kuang, D.; Liu, Q.; et al. The Impacts of Plant Growth Regulators on the Rapid Propagation of Gardenia jasminoides Ellis. in Tissue Culture. Forests 2024, 15, 446. https://doi.org/10.3390/f15030446

AMA Style

Ai Y, Chen Y, Zhu S, Jiang L, Chen J, Li C, Li P, Zeng W, Kuang D, Liu Q, et al. The Impacts of Plant Growth Regulators on the Rapid Propagation of Gardenia jasminoides Ellis. in Tissue Culture. Forests. 2024; 15(3):446. https://doi.org/10.3390/f15030446

Chicago/Turabian Style

Ai, Yang, Yunzhu Chen, Shuixuan Zhu, Lijuan Jiang, Jingzhen Chen, Changzhu Li, Peiwang Li, Wenbin Zeng, Ding Kuang, Qiang Liu, and et al. 2024. "The Impacts of Plant Growth Regulators on the Rapid Propagation of Gardenia jasminoides Ellis. in Tissue Culture" Forests 15, no. 3: 446. https://doi.org/10.3390/f15030446

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