Influence of Phenylacetic Acid and Its Derivatives on Callus Proliferation and Somatic Embryogenesis in Litchi chinensis

Wang, Guo; Liu, Yaoting; Li, Huanling; Zhang, Lei; Li, Zhiying; Wang, Jiabao

doi:10.3390/horticulturae11101200

Open AccessArticle

Influence of Phenylacetic Acid and Its Derivatives on Callus Proliferation and Somatic Embryogenesis in Litchi chinensis

by

Guo Wang

¹,

Yaoting Liu

²,

Huanling Li

¹,

Lei Zhang

³,

Zhiying Li

³ and

Jiabao Wang

^1,*

¹

Environment and Plant Protection Institute, Chinese Academy of Tropical Agriculture Sciences, Haikou 571101, China

²

Hainan Academy of Agriculture and Reclamation Sciences Group Co., Ltd., Haikou 571100, China

³

Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China

^*

Author to whom correspondence should be addressed.

Horticulturae 2025, 11(10), 1200; https://doi.org/10.3390/horticulturae11101200

Submission received: 1 September 2025 / Revised: 18 September 2025 / Accepted: 30 September 2025 / Published: 3 October 2025

(This article belongs to the Special Issue Advancement in Genetics, Biotechnology and Breeding of Tropical and Subtropical Fruit Crops)

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Abstract

The efficiency of in vitro regeneration in litchi (Litchi chinensis Sonn.) is highly influenced by the type and concentration of plant growth regulators (PGRs), particularly auxins. This study evaluated the effects of phenylacetic acid (PAA) and its derivatives—4-chlorophenylacetic acid (CPA) and 4-iodophenylacetic acid (IPA)—on callus proliferation, somatic embryogenesis, and plantlet regeneration in ‘Feizixiao’ litchi, as well as to establish an efficient regeneration protocol. The inclusion of CPA or IPA in callus proliferation medium significantly enhanced the proliferation rate, with 20 mg·L⁻¹ CPA being the most effective. The highest number of somatic embryos per gram of fresh embryonic callus weight (gFW⁻¹) (1131 embryos·gFW⁻¹) was observed with 40 mg·L⁻¹ PAA in proliferation medium. The addition of 10 mg·L⁻¹ IPA to the proliferation medium yielded the highest plantlet regeneration rate (50 plantlets·gFW⁻¹). Supplementing the somatic embryo induction medium with 5 mg·L⁻¹ PAA resulted in 460 somatic embryos·gFW⁻¹ and 86 regenerated plantlets·gFW⁻¹. These findings indicate that PAA and its derivatives are effective PGRs for the in vitro regeneration of litchi, providing a valuable protocol for the propagation of elite cultivars.

Keywords:

plant tissue culture; halogenated auxins; embryo induction; plant regeneration; ‘Feizixiao’ litchi

1. Introduction

Litchi (Litchi chinensis Sonn.) is an economically important fruit tree native to southern China and widely cultivated in tropical and subtropical regions, including China, Vietnam, India, and Thailand [1]. Traditional breeding methods are hindered by the highly heterozygous genetic background and long juvenile period of litchi [2,3], limiting the development of elite cultivars [4]. In vitro regeneration techniques, particularly somatic embryogenesis (SE), offer a promising alternative for rapid propagation and genetic improvement [5,6,7,8]. However, the application of SE in litchi is constrained by challenges such as low embryonic efficiency, genotype dependency, poor germination of somatic embryos, and a high frequency of abnormal embryos [9,10,11]. Therefore, optimizing SE protocols remains a critical research objective.

Plant growth regulators (PGRs), particularly auxins, are pivotal in SE. Synthetic auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthylacetic acid (NAA) are commonly used, but they can cause genetic instability and reduce regeneration efficiency after prolonged exposure [12,13,14,15], highlighting the need for safer alternatives. Phenylacetic acid (PAA), a naturally occurring auxin found in various plants, promotes shoot regeneration, root formation, and somatic embryogenesis in species such as Vanilla planifolia [16] and Decalepis hamiltonii [17]. PAA influences auxin transport, enzyme activity, and defense responses [18,19,20,21], and its activity is thought to be mediated through mechanisms analogous to indole-3-acetic acid (IAA), including potential interactions with auxin receptors and transporters [22,23]. Auxin halogenation can alter their stability, uptake, and activity, potentially leading to their improved efficacy [23,24]. Despite these promising effects, the potential of PAA and its halogenated derivatives in litchi SE, as well as the mechanistic basis for their effects, remain unexplored.

This study specifically aimed to: (1) identify the optimal type and concentration of PAA, CPA, and IPA for enhancing callus proliferation in ‘Feizixiao’ litchi; (2) evaluate their effects on the morphology, yield and subsequent development of somatic embryos; and (3) determine the most effective combinations of these auxins with cytokinins (KT or TDZ) for establishing a high-frequency somatic embryogenesis and plant regeneration system. The findings provide a robust protocol for high-frequency regeneration and contribute to the biotechnological breeding of litchi. Furthermore, the potential implications of using these compounds, including the risk of somaclonal variation in indirectly regenerated plants and the necessity for future genetic fidelity assessments in scaled-up protocols, are discussed.

2. Materials and Methods

2.1. Plant Materials and Callus Culture

Embryogenic calli (ECs) were induced from the anthers of ‘Feizixiao’ litchi (Voucher specimen No. CATAS-EPPI-LC-FZX-2019-001, deposited at the Herbarium of the Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, China) collected from trees at the Institute of Tropical Fruits, Hainan Academy of Agricultural Sciences, Hainan Province, China. The anthers were cultured on callus-induction medium [Murashige and Skoog (MS) medium (Phyto Technology Laboratories, Shawnee Mission, KS, USA) [25] supplemented with 2 mg·L⁻¹ benzylaminopurine (BA), 0.5 mg·L⁻¹ NAA, and 3 mg·L⁻¹ 2,4-D]. Cultures were maintained at 25 ± 2 °C in the dark, and calli were subcultured every 40 days [9]. For callus proliferation prior to experiments, ECs were maintained by alternating every 30 days between two media: MS medium with 1 mg·L⁻¹ 2,4-D and MS medium with 1 mg·L⁻¹ 2,4-D, 0.5 mg·L⁻¹ KT, and 5 mg·L⁻¹ AgNO₃. This alternation was carried out for eight cycles (approximately 240 days) before initiating PAA, CPA, and IPA treatments. All media contained 30 g·L⁻¹ sucrose and 7 g·L⁻¹ agar.

2.2. Addition of Regulators to the Proliferation Medium

In the first experiment, 0.2 g of 25-day-old EC (after the pre-culture cycles) was transferred to proliferation medium (MS basal medium supplemented with 1, 5, 10, 20, 40, or 80 mg·L⁻¹ PAA, CPA, or IPA dissolved in distilled water). Each treatment consisted of three replicates (experimental units), with each replicate comprising five Petri dishes. The entire experiment was independently repeated two times (temporal replicates). Callus was weighed on days 0 and 21, and the proliferation rate was calculated over a 3-week incubation period. The control medium contained 1 mg·L⁻¹ 2,4-D. All media were supplemented with 30 g·L⁻¹ sucrose and 7 mg·L⁻¹ agar.

The EC proliferation index was calculated as follows: EC proliferation index = (callus mass on day 21−callus mass on day 0)/callus mass on day 0.

Subsequently, the ECs were subcultured on somatic embryo induction medium [MS medium with 0.1 mg·L⁻¹ NAA, 5 mg·L⁻¹ KT, 0.4 g·L⁻¹ lactalbumin hydrolysate (LH), 0.1 g·L⁻¹ inositol, 60 g·L⁻¹ sucrose, and 10 g·L⁻¹ agar] for 7 weeks. The embryo yield was determined by counting the number of somatic embryos per gram of fresh EC weight (gFW⁻¹).

Somatic embryos were then transferred to maturation medium [MS medium with 0.5 mg·L⁻¹ IAA, 1 mg·L⁻¹ ABA, 100 mL·L⁻¹ coconut water, 60 g·L⁻¹ sucrose, and 10 g·L⁻¹ agar] for 8 weeks. Mature somatic embryos were germinated on regeneration medium (1/2 MS medium with 0.5 mg·L⁻¹ GA₃). The number of regenerated plantlets per gram of fresh EC weight after 6 weeks under a 16 h photoperiod with a light intensity of 50 μmol⁻¹ m²·s⁻¹ was recorded.

2.3. Addition of Regulators to the Somatic Embryo Induction Medium

In a separate experiment, 20-day-old ECs pre-cultured on MS medium with 1 mg·L⁻¹ 2,4-D were transferred to MS medium supplemented with 0.1 mg·L⁻¹ NAA, 0.4 g·L⁻¹ LH, 0.1 g·L⁻¹ inositol, 60 g·L⁻¹ sucrose, 10 g·L⁻¹ agar, 5 g·L⁻¹ KT or TDZ, and 1, 5, 10, or 20 mg·L⁻¹ PAA, CPA, or IPA. The cytokine concentrations were selected based on previous studies [9]. The control medium (CK/T3) contained all components except PAA, CPA, and IPA. For somatic embryo induction, each treatment had six independent replicates, with each replicate consisting of 10 Petri dishes (for a total 60 dishes per treatment). For regeneration, 90 mature somatic embryos (0.5–1.0 mm) per treatment were transferred to regeneration medium and distributed equally across three independent replicate sets (with 30 embryos per replicate). Somatic embryo maturation and regeneration conditions were the same as those described in Section 2.2. All experiments were independently repeated twice.

The PGRs (2,4-D, KT, BA, NAA, LH, IAA, ABA, and GA₃) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). PAA, CPA, and IPA were obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). Sucrose and agar were sourced from Solarbio Life Sciences Co., Ltd. (Beijing, China), and silver nitrate was from Yindian Chemical Co., Ltd. (Shanghai, China).

2.4. Data Analysis

Data were analyzed using one-way analysis of variance (ANOVA). Significant differences among means were determined by Duncan’s new multiple range test at p < 0.05 using DPS Data Processing System software Version 2 (vers. 2, Zhejiang, China) [26]. Data from independent experimental repeats were pooled for analysis after confirming the homogeneity of variances. Results are presented as the mean ± standard deviation (SD).

3. Results

3.1. Effects of Regulators on Callus Proliferation and Morphology

Callus proliferation was significantly enhanced on media supplemented with CPA or IPA compared to control media (Table 1, p < 0.05). In contrast, PAA resulted in lower proliferation rates relative to the control. Calli grown on CPA- or IPA-containing media exhibited darker yellow and coarser particles than those on control or PAA-supplemented media. Proliferation decreased as the PAA concentration increased. At lower PAA concentrations (1–20 mg·L⁻¹), calli appeared light yellow, loose, and fine-grained (Figure 1, red arrow), with abundant oval cells (Figure 2, red arrow) and small cell clusters (Figure 2, pink arrow). Transparent embryos were observed only at 40 mg·L⁻¹ PAA (Figure 1, black arrow).

Callus proliferation and the number of transparent embryos increased as the CPA concentration increased, peaking at 20 mg·L⁻¹ CPA (Table 1). At this concentration, the callus edges were predominantly differentiated into transparent embryos (Figure 1, black arrow), while the inner callus was compact and coarse (Figure 1, green arrow). Lower CPA concentrations (1–10 mg·L⁻¹) promoted the formation of free single cells (Figure 2, red arrow) and small cell clusters (Figure 2, green arrow), whereas higher concentrations (20–80 mg·L⁻¹) led to larger cell clusters (Figure 2, blue arrow).

Low concentrations of IPA (1 and 5 mg·L⁻¹) also enhanced proliferation, producing creamy yellow, hard, and fine-grained calli (Figure 1, red arrow) with numerous free, small, round single cells (Figure 2, purple arrow). At 10 mg·L⁻¹ IPA, calli became light yellow, compact, and coarse, with an increase in transparent embryos (Figure 1, black arrow) and cell clusters (Figure 2, pink arrow). At 20 mg·L⁻¹ IPA, calli ceased proliferation, with extensive cytoplasmic shrinkage and cell death (Figure 2, yellow and white arrows). Based on these observations and statistical analysis, 5 mg·L⁻¹ IPA was selected for further experiments due to its favorable callus morphology and high embryonic potential.

3.2. Somatic Embryogenesis and Regeneration

The number of somatic embryos increased with the concentration of PAA, CPA, and IPA, with PAA showing the most pronounced effect (Table 1). The highest embryo yields were observed using 40 mg·L⁻¹ PAA, 20 mg·L⁻¹ CPA, and 5 mg·L⁻¹ IPA. Somatic embryos induced by PAA or CPA were primarily spherical (Figure 3, black border), clustered (Figure 3, green border), or trumpet-like (Figure 3, blue border), with few cotyledon embryos (Figure 3, red border). The clustered embryos reached diameters of up to 5 mm, while other embryos were smaller (≈3 mm). In contrast, IPA-induced embryos were predominantly cotyledon, particularly at 5 and 10 mg·L⁻¹, with diameters of approximately 3 mm. However, both spherical and clustered embryos on CPA- and IPA-containing media often turned milky white or brown during maturation (Figure 3, white border).

The regeneration efficiency varied significantly among treatments (Table 1, p < 0.05). IPA-supplemented media yielded the highest number of regenerated plantlets, while PAA and CPA treatments produced lower regeneration rates. Regeneration increased with PAA concentration up to 80 mg·L⁻¹, but decreased at higher CPA and IPA concentrations. Somatic embryos on PGR-supplemented media exhibited delayed greening and germination. Some embryos developed a red coloration (Figure 4C, red border), produced secondary embryos (Figure 4B,D, black arrow), or formed calli (Figure 4D, white arrow). Plantlets often had short, dark green internodes (Figure 4C,D, yellow border).

3.3. Effects of PAA/CPA/IPA Combined with KT or TDZ on Somatic Embryogenesis and Regeneration

3.3.1. PAA Combined with KT or TDZ

The combination of PAA with either KT or TDZ significantly influenced SE and regeneration (Table 2). Low PAA concentrations with KT or high PAA concentrations with TDZ enhanced embryo induction and regeneration, outperforming the CPA and IPA combinations. With PAA and KT, embryo and plantlet numbers peaked at 5 mg·L⁻¹ PAA. With PAA and TDZ, the optimal response was observed at 20 mg·L⁻¹ PAA (PAA-TDZ4). Somatic embryos from PAA treatments were primarily dicotyledonous (Figure 5A) or multicotyledonous (Figure 5B). Regenerated plantlets had shortened internodes (Figure 4E, yellow border), thicker stems (Figure 4F, pink arrow), and occasionally a deep red color (Figure 4E, red border).

3.3.2. CPA Combined with KT or TDZ

CPA combined with KT yielded higher SE and regeneration efficiencies than CPA with TDZ (Table 2). The best results were achieved with 1 mg·L⁻¹ CPA and KT (CPA-KT1), which produced small, uniform dicotyledonous embryos with high regeneration rates. Regenerated plantlets had longer roots (≈7 cm; Figure 4G,H, blue arrow) and stems (≈3 cm; Figure 4G, yellow arrow). Higher CPA concentrations reduced embryo and plantlet numbers, resulting in globular (Figure 5C) or cup-shaped (Figure 5D) embryos with shorter stems and thinner roots. CPA with TDZ produced clustered globular embryos (Figure 5E) with low germination rates and increased callus formation (Figure 4G,F, white arrow).

3.3.3. IPA Combined with KT or TDZ

IPA with KT also outperformed IPA with TDZ (Table 2). The combination of 1 mg·L⁻¹ IPA and KT (IPA-KT1) yielded the highest number of somatic embryos, which were dark green and swollen (Figure 4H, purple border), primarily cotyledon, with stems exceeding 1.5 cm. Higher IPA concentrations reduced the embryo numbers and germination rates, and led to clustered embryos (Figure 5F) with slender leaves (Figure 4G, red arrow). IPA with TDZ produced small, clustered embryos that remained milky white (Figure 4J, black border) and yielded weak plants (Figure 4J, purple arrow) with long roots (Figure 4J, blue arrow). The optimal combination was 1 mg·L⁻¹ IPA and 5 mg·L⁻¹ KT (IPA-KT1), which achieved the highest SE and regeneration efficiency.

4. Discussion

The in vitro regeneration of many plant species, including litchi, represents a promising means of improving the quality of planting materials [6,7]. While direct organogenesis might be preferable for minimizing somaclonal variation in commercial micropropagation [27], somatic embryogenesis remains a powerful tool for mass propagation and genetic studies due to its high multiplication potential and suitability for synthetic seed production [5,10]. However, the potential for somaclonal variation in plants derived from somatic embryogenesis necessitates future genetic fidelity assessments using molecular markers before its large-scale deployment [28].

In established litchi regeneration systems, calli exhibit genotypic variations in growth and morphology [13,29,30]. This study demonstrates that PAA and its derivatives significantly influence these parameters in ‘Feizixiao’. The enhanced callus proliferation after CPA and IPA treatments (Table 1) suggests that halogenation alters the properties of auxin, potentially increasing its stability or its affinity with auxin receptors or transporters relative to PAA [23,24]. The superior somatic embryo yield with 40 mg·L⁻¹ PAA (1131 embryos·gFW⁻¹) aligns with findings in geranium, where PAA accelerated somatic embryo induction [31]. This promoting effect could be attributed to PAA’s auxin-like activity, which potentially regulates endogenous auxin transport [22] and enhances the morphogenesis of early somatic embryos. The high regeneration rates associated with lower IPA concentrations (5–10 mg·L⁻¹, 46–50 plantlets·gFW⁻¹) might be due to IPA’s weaker auxin activity [23,24], allowing for better progression from embryo maturation to germination. This is in contrast to stronger auxins such as 2,4-D, which can inhibit subsequent plant development at higher concentrations [14,32,33]. The morphological differences observed (Figure 1, Figure 2 and Figure 3) likely reflect underlying changes in cellular patterning and hormone gradients triggered by the different auxins.

The combination of PAA derivatives with cytokines further modulated somatic embryogenesis outcomes. The consistently better performance of PAA compared to CPA and IPA when combined with KT or TDZ (Table 2), as well as the higher efficiency of KT combinations overall, underscores the importance of the cytokinin–auxin balance for initiating growth and differentiation [28]. The optimal result for simultaneous embryo induction and plant regeneration was achieved with 5 mg·L⁻¹ PAA and 5 mg·L⁻¹ KT (PAA-KT2: 460 embryos·gFW⁻¹ and 86 plantlets·gFW⁻¹). This synergistic effect might involve the cytokine-mediated activation of cell division genes and auxin-driven embryonic pathway genes.

PAA biosynthesis from phenylalanine shares similarities with IAA pathways, and there is evidence of analogous metabolism and signaling mechanisms [22,23]. The high frequency of bud regeneration from PAA-treated somatic embryos indicates its direct participation in somatic embryogenesis events, possibly through modulating the expression of key transcription factors such as LEC, WUS, or BBM [6]. The red coloration and secondary embryo that were observed (Figure 4) might be linked to stress responses or altered phenylpropanoid pathway activity induced by the PGR treatments [23].

While the current findings are promising, further physiological, biochemical, and molecular studies are needed to fully characterize the regulatory mechanisms involved. Transcriptomic or proteomic analyses comparing the effects of PAA, CPA, and IPA could identify key genes and proteins involved in their action, providing deeper insights into SE regulation in litchi.

5. Conclusions

In this study, we established an efficient somatic embryogenesis system for ‘Feizixiao’ litchi using PAA and its derivatives. CPA (20 mg·L⁻¹) significantly enhanced callus proliferation. PAA (40 mg·L⁻¹) in proliferation medium yielded the highest number of somatic embryos, while IPA (10 mg·L⁻¹) in proliferation medium promoted the highest regeneration rate. For the somatic embryo induction medium, the combination of 5 mg·L⁻¹ PAA and 5 mg·L⁻¹ KT proved most effective for simultaneous embryo induction and plant regeneration. These findings demonstrated that PAA and its derivatives can serve as potent alternatives to synthetic auxins such as 2,4-D in litchi tissue culture, providing a robust protocol for the micropropagation of elite cultivars. Future studies should focus on elucidating the molecular mechanisms underlying PAA-induced somatic embryogenesis and assessing the genetic fidelity of regenerates to facilitate commercial application.

Author Contributions

Conceptualization, J.W.; methodology, H.L.; validation, G.W. and Y.L.; formal analysis, G.W.; investigation, L.Z.; resources, Z.L.; data curation and writing—original draft preparation, G.W.; writing—review and editing, J.W.; supervision, H.L.; project administration and funding acquisition, J.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (Grant No. 32402522), the Science and Technology Special Fund of Hainan Province (Grant No. ZDYF2023XDNY052), the China Agricultural Industry Technology system (Grant No. CARS-32) and the Hainan Province Agricultural Industry Technology System (Grant No. HNARS-08). The APC was funded by the China Agriculture Research System (Grant No. CARS-32).

Institutional Review Board Statement

This study did not include any experiments with human participants or animals performed by any of the authors.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

Yaoting Liu was employed by the company Hainan Academy of Agriculture and Reclamation Sciences Group 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.

References

Hussain, S.Z.; Naseer, B.; Qadri, T.; Fatima, T.; Bhat, T.A. Litchi (Litchi chinensis): Morphology, taxonomy, composition and health beneffts. In Fruits Grown in Highland Regions of the Himalayas: Nutritional and Health Beneffts; Hussain, S.Z., Naseer, B., Qadri, T., Fatima, T., Bhat, T.A., Eds.; Springer International Publishing: Cham, Switzerland, 2021; pp. 181–191. [Google Scholar]
Litz, R.E. Somatic embryogenesis from cultured leaf explants of the tropical tree Euphoria longan Stend. J. Plant Physiol. 1988, 132, 190–193. [Google Scholar] [CrossRef]
Litz, R.E.; Raharjo, S. Dimocarpus longan longan and Litchi chinensis litchi. In Biotechnology of Fruit and Nut Crops; Litz, R.E., Ed.; CABI: Wallingford, UK, 2005; pp. 628–636. [Google Scholar] [CrossRef]
Chapman, K.R. Sapindaceae. Lychae. In Tropical Tree Fruits for Australia, Queensland Department of Primary Industries, Brisbane; Page, P.E., Ed.; Queensland Department of Primary Industries: Brisbane, Australia, 1984; pp. 179–191. [Google Scholar]
Qin, Y.Q.; Wang, D.; Fu, J.X.; Zhang, Z.K.; Qin, Y.H.; Hu, G.B.; Zhao, J.T. Agrobacterium rhizogenes-mediated hairy root transformation as an efficient system for gene function analysis in Litchi chinensis. Plant Methods 2021, 17, 103. [Google Scholar] [CrossRef] [PubMed]
Qin, Y.Q.; Zhang, B.; Wang, S.Q.; Guo, W.Y.; Zhang, Z.K.; Qin, Y.H.; Zhao, J.T.; Hu, G.B. Establishment of somatic embryogenesis regeneration system and transcriptome analysis of early somatic embryogenesis in Litchi chinensis. Hortic. Plant J. 2025, 11, 535–547. [Google Scholar] [CrossRef]
Qin, Y.Q.; Zhang, B.; Luo, X.L.; Wang, S.Q.; Fu, J.X.; Zhang, Z.K.; Qin, Y.H.; Zhao, J.T.; Hu, G.B. Development of an Agrobacterium tumefaciens-mediate transformation system for somatic embryos and transcriptome analysis of LcMYB1’s inhibitory effect on somatic embryogenesis in Litchi chinensis. J. Integr. Agric. 2025, 24, 594–609. [Google Scholar] [CrossRef]
Wang, S.J.; Wang, G.; Li, H.L.; Li, F.; Wang, J.B. Agrobacterium tumefaciens-mediated transformation of embryogenic callus and CRISPR/Cas9-mediated genome editing in ‘Feizixiao’ litchi. Hortic. Plant J. 2023, 9, 947–957. [Google Scholar] [CrossRef]
Wang, G.; Li, H.L.; Wang, S.J.; Sun, J.H.; Zhang, X.C.; Wang, J.B. In vitro regeneration of litchi (Litchi chinensis Sonn.). Afr. J. Biotechnol. 2016, 15, 1026–1034. [Google Scholar] [CrossRef]
Das, D.K.; Rahman, A.; Kumari, D.; Kumari, N. Synthetic seed preparation, germination and plantlet regeneration of Litchi (Litchi chinensis Sonn.). Am. J. Plant Sci. 2016, 7, 1395–1406. [Google Scholar] [CrossRef]
Huang, M.Q.; Guo, W.Y.; Wu, X.Y.; Qin, Y.Q.; Sabir, I.A.; Zhang, Z.K.; Qin, Y.H.; Hu, G.B.; Zhao, J.T. Somatic embryogenesis and plant regeneration of Litchi chinensis Sonn. cv. ‘Zili’ from immature zygotic embryos. Plant Cell Tissue Organ. Cult. 2024, 156, 39. [Google Scholar] [CrossRef]
Yu, C.H.; Chen, Z.G.; Lu, L.X.; Lin, J.W. Somatic embryogenesis and plant regeneration from litchi protoplasts isolated from embryogenic suspensions. Plant Cell Tissue Organ. Cult. 2000, 61, 51–58. [Google Scholar] [CrossRef]
Raharjo, S.H.T.; Litz, R.E. Somatic embryogenesis and plant regeneration of litchi (Litchi chinensis Sonn.) from leaves of mature phase trees. Plant Cell Tissue Organ. Cult. 2007, 89, 113–119. [Google Scholar] [CrossRef]
Ma, X.Y.; Yi, G.J.; Huang, X.L. Leaf callus induction and suspension culture establishment in litchi (Litchi chinensis Sonn.) cv. Huaizhi. Acta Physiol. Plant. 2009, 31, 401–405. [Google Scholar] [CrossRef]
Li, S.; Feng, J.; Xiang, X. Induction of embryogenic callus and subculture proliferation of Litchi chinensis leaves. Guangdong Agric. Sci. 2021, 48, 41–49. [Google Scholar] [CrossRef]
Giridhar, P.; Ramu, G.D.V.; Ravishankar, G.A. Phenyl acetic acidinduced in vitro shoot multiplication of Vanilla planifolia. Trop. Sci. 2003, 43, 92–95. [Google Scholar] [CrossRef]
Giridhar, P.; Vijayaramu, D.; Obulreddy, B.; Rajasekaran, T.; Ravashankar, G.A. Influence phenylacetic acid on clonal propagation of Decalepis hamiltonii Wight and Arn: An endangered shrub. Vitr. Cell. Dev. Biol.-Plant 2003, 39, 463. [Google Scholar] [CrossRef]
Ghanti, S.K.; Sujata, K.G.; Rao, M.S. The effect of phenylacetic acid on bud induction, elongation and rooting of chickpea. Biol. Plant. 2009, 53, 779–783. [Google Scholar] [CrossRef]
Qiao, G.R.; Li, H.Y.; Liu, M.Y.; Jiang, J.; Yin, Y.F.; Zhang, L.; Zhuo, R.Y. Callus induction and plant regeneration from anthers of Dendrocalamus latiflorus Munro. Vitr. Cell. Dev. Biol.-Plant 2013, 49, 375–382. [Google Scholar] [CrossRef]
Sugawara, S.; Mashiguchi, K.; Tanaka, K.; Hishiyam, H. Distinct characteristics of indole-3-acetic acid and phenylacetic acid, two common auxins in plants. Plant Cell Physiol. 2015, 56, 1641–1654. [Google Scholar] [CrossRef]
Sumayo, M.S.; Son, J.S.; Ghim, S.Y. Exogenous application of phenylacetic acid promotes root hair growth and induces the systemic resistance of tobacco against bacterial soft-rot pathogen Pectobacterium carotovorum subsp. Carotovorum. Funct. Plant Biol. 2018, 45, 1119–1127. [Google Scholar] [CrossRef]
Johnson, C.F.; Morris, D.A. Regulation of auxin transport in the pea (Pisum sativum L.) by phenylacetic acid: Effects on the components of transmembrane transport of indol-3yl-acetic acid. Planta 1987, 172, 400–407. [Google Scholar] [CrossRef]
Cook, S.D. An historical review of phenylacetic acid. Plant Cell Physiol. 2019, 60, 243–254. [Google Scholar] [CrossRef]
Iwase, A.; Takebayashi, A.; Aoi, Y.; Favero, D.S.; Watanabe, S.; Seo, M.; Kasahara, H.; Sugimoto, K. 4-Phenylbutyric acid promotes plant regeneration as an auxin by being converted to phenylacetic acid via an IBR3-independent pathway. J. Plant Biotechnol. 2022, 39, 51–58. [Google Scholar] [CrossRef] [PubMed]
Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 1962, 15, 473–497. [Google Scholar] [CrossRef]
Tang, Q.Y. Data Processing System-Experimental Design, Statistical Analysis and Data Mining, 2nd ed.; China Science Publishing & Media Ltd.: Beijing, China, 2007; Available online: https://www.sciencep.com (accessed on 1 January 2020).
Bregitzer, P.; Campbell, R.D.; Wu, Y. Plant regeneration from barley callus: Effects of 2,4-dichlorophenoxyacetic acid and phenylacetic acid. Plant Cell Tissue Organ. Cult. 1995, 3, 229–235. [Google Scholar] [CrossRef]
Al-Mayahi, A.M.W. The Efect of Phenyl Acetic Acid (PAA) on Micropropagation of Date Palm Followed by Genetic Stability Assessment. J. Plant Growth Regul. 2022, 41, 3127–3137. [Google Scholar] [CrossRef]
Wang, G.; Liu, Y.T.; Gao, Z.Y.; Li, H.L.; Wang, J.B. Effects of amino acids on callus proliferation and somatic embryogenesis in Litchi chinensis cv. ‘Feizixiao’. Horticulturae 2023, 9, 1311. [Google Scholar] [CrossRef]
Fu, L.F.; Tang, D.Y. Induction pollen plants of litchi tree (Litchi chinensis Sonn.). Acta Genet. Sin. 1983, 10, 369–374. [Google Scholar] [CrossRef]
Slimmon, T.; Qureshi, J.A.; Saxena, P.K. Phenylacetic acid induced somatic embryogenesis in cultured hypocotyl explants of Geranium (Pelargonium × hortorum Bailey). Plant Cell Rep. 1991, 10, 587–589. [Google Scholar] [CrossRef]
Puchooa, D. In vitro regeneration of litchi (Litchi chinensis Sonn.). Afr. J. Biotechnol. 2004, 3, 576–584. [Google Scholar]
Tanida, K.; Shiota, H. Anise-cultured cells abolish 2,4-dichlorophenoxyacetic acid in culture medium. Plant Biotechnol. 2019, 36, 209–212. [Google Scholar] [CrossRef]

Figure 1. Phenotypic responses of litchi callus to different plant growth regulator treatments. CK, control (1 mg·L⁻¹ 2,4-D). P1–P6, PAA treatments: 1 (P1), 5 (P2), 10 (P3), 20 (P4), 40 (P5), and 80 (P6) mg·L⁻¹. C1–C6, CPA treatments: 1 (C1), 5 (C2), 10 (C3), 20 (C4), 40 (C5), and 80 (C6) mg·L⁻¹. I1–I6, IPA treatments: 1 (I1), 5 (I2), 10 (I3), 20 (I4), 40 (I5), and 80 (I6) mg·L⁻¹. Key morphological features: red arrow, embryonic callus; black arrow, proembryo; green arrow, granular and hard calli. Scale bar = 1000 μm.

Figure 2. Cellular morphology of litchi calli under various plant growth regulator treatments. CK, control (1 mg·L⁻¹ 2,4-D). P1–P6, PAA at 1 (P1), 5 (P2), 10 (P3), 20 (P4), 40 (P5), and 80 (P6) mg·L⁻¹. C1–C6, CPA at 1 (C1), 5 (C2), 10 (C3), 20 (C4), 40 (C5), and 80 (C6) mg·L⁻¹. I1–I6, IPA at 1 (I1), 5 (I2), 10 (I3), 20 (I4), 40 (I5), and 80 (I6) mg·L⁻¹. Arrows: red, embryonic cells; pink, embryonic cell clusters; green, small cell clusters; blue, large cell clusters; purple, small round single cells; yellow, cytoplasmic clumping; white, apoptotic cells. Scale bar = 100 μm.

Figure 3. Somatic embryogenesis of litchi under different plant growth regulator treatments. CK, control (1 mg·L⁻¹ 2,4-D). P1–P6, PAA at 1 (P1), 5 (P2), 10 (P3), 20 (P4), 40 (P5), and 80 (P6) mg·L⁻¹. C1–C6, CPA at 1 (C1), 5 (C2), 10 (C3), 20 (C4), 40 (C5), and 80 (C6) mg·L⁻¹. I1–I2, IPA at 1 (I1), 5 (I2), 10 (I3), 20 (I4), 40 (I5), and 80 (I6) mg·L⁻¹. Borders: black, spherical embryos; blue, trumpet-like embryos; red, cotyledonary embryos; green, clustered embryos; white, abnormal/ milky embryos. Scale bar = 90 mm.

Figure 4. Regeneration of litchi plantlets influenced by plant growth regulators. (A) Control (CK; 1 mg·L⁻¹ 2,4-D). (B–D) Plantlets regenerated from callus proliferation media supplemented with PAA (B), CPA (C), and IPA (D). (E–J) Plantlets from somatic embryogenesis media supplemented with PAA (E,F), CPA (G,H), and IPA (I,J). Borders: red, red embryos; yellow, short dark-green stems; blue, light-green somatic embryos; white, new calli; purple, dark-green swollen embryos; black, secondary milky embryos. Arrows indicate the following: white, new calli; pink, thick stems; yellow, elongated stems; blue, long roots; purple, thin weak plants.

Figure 5. Morphological diversity of mature somatic litchi embryos. (A) Dicotyledonous. (B) Multicotyledonous. (C) Globular. (D) Cup-shaped. (E) Globular mass. (F) Agglomerate. Scale bar = 2500 μm.

Table 1. Effects of phenylacetic acid (PAA), 4-chlorophenylacetic acid (CPA), and 4-iodophenylacetic acid (IPA) on litchi callus proliferation, somatic embryogenesis, and plant regeneration.

Treatment	PGR Concentration (mg·L⁻¹)				Callus Proliferation (Fold)	No. Somatic Embryos (gFW⁻¹)	No. Regenerated Plantlets (gFW⁻¹)
Treatment	2,4-D	PAA	CPA	IPA	Callus Proliferation (Fold)	No. Somatic Embryos (gFW⁻¹)	No. Regenerated Plantlets (gFW⁻¹)
CK	1				8.12 ± 0.19 f	232 ± 9.61 f	13 ± 4.50 b
P1		1			8.21 ± 0.18 f	144 ± 11.93 i	4 ± 0.39 e, f
P2		5			8.09 ± 0.25 f	225 ± 8.083 f	2 ± 0.07 e, f
P3		10			6.99 ± 0.18 f	285 ± 5.86 d,e	3 ± 0.73 e, f
P4		20			6.18 ± 0.17 h	366 ± 11.06 c	3 ± 0.10 e, f
P5		40			4.61 ± 0.18 j	1131 ± 8.89 a	11 ± 0.53 b, c
P6		80			3.43 ± 0.22 k	1028 ± 20.53 b	13 ± 1.03 b, c
C1			1		11.23 ± 0.00 c	164 ± 1.00 h	0 f
C2			5		10.94 ± 1.27 c, d	273 ± 6.08 e	9 ± 0.20 c, d
C3			10		12.24 ± 0.93 b	131 ± 6.08 i	6 ± 2.64 d, e
C4			20		13.25 ± 0.41 a	289 ± 6.56 d	0 f
C5			40		10.31 ± 0.12 d	193 ± 1.73 g	0 f
C6			80		3.94 ± 0.31 j, k	73 ± 4.58 k	0 f
I1				1	9.38 ± 0.49 e	238 ± 3.61 f	6 ± 1.89 d, e
I2				5	9.46 ± 0.92 e	293 ± 6.08 d	46 ± 7.05 a
I3				10	6.45 ± 0.44 g, h	271 ± 6.25 e	50 ± 5.11 a
I4				20	5.39 ± 0.67 i	194 ± 8.72 g	14 ± 0.88 b
I5				40	3.93 ± 0.59 j, k	106 ± 3.61 j	5 ± 1.29 d, e
I6				80	1.90 ± 0.45 l	70 ± 7.37 k	0 f

Data are presented as mean ± SD (n = 3). Different lowercase letters indicate significant differences among treatments based on Duncan’s multiple range test (p < 0.05). The control (CK) contained 1 mg·L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D).

Table 2. Effects of PAA, CPA, and IPA combined with KT or TDZ on somatic embryogenesis and plant regeneration in litchi.

PGR	PGR Concentration (mg·L⁻¹)						No. Somatic Embryos (gFW⁻¹)	No. Regeneration Plantlets (gFW⁻¹)
PGR	NAA	KT	TDZ	PAA	CPA	IPA	No. Somatic Embryos (gFW⁻¹)	No. Regeneration Plantlets (gFW⁻¹)
CK (T3)	0.1	5	-	-	-	-	227 ± 6.08 g, h	13 ± 2.37 e
PAA-KT1	-	5	-	1	-	-	320 ± 5.00 c	53 ± 3.61 b
PAA-KT2	-	5	-	5	-	-	460 ± 4.00 a	86 ± 3.61 a
PAA-KT3	-	5	-	10	-	-	215 ± 2.00 i	29 ± 4.62 c
PAA-KT4	-	5	-	20	-	-	0 n	0 g
PAA-TDZ1	-	-	0.5	1	-	-	0 n	0 g
PAA-TDZ2	-	-	0.5	5	-	-	83 ± 1.00 m	9 ± 1.53 e, f
PAA-TDZ3	-	-	0.5	10	-	-	140 ± 2.00 l	28 ± 3.46 c
PAA-TDZ4	-	-	0.5	20	-	-	340 ± 2.00 b	85 ± 5.03 a
CPA-KT1	-	5	-	-	1	-	346 ± 15.62 b	49 ± 7.12 b
CPA-KT2	-	5	-	-	5	-	307 ± 8.19 d	19 ± 1.28 d
CPA-KT3	-	5	-	-	10	-	203 ± 11.27 j	3 ± 4.99 f, g
CPA-KT4	-	5	-	-	20	-	136 ± 9.54 l	2 ± 2.71 g
CPA-TDZ1	-	-	0.5	-	1	-	227 ± 7.00 g, h	2 ± 3.34 g
CPA-TDZ2	-	-	0.5	-	5	-	215 ± 6.24 i	2 ± 3.06 g
CPA-TDZ3	-	-	0.5	-	10	-	166 ± 5.29 k	0 g
CPA-TDZ4	-	-	0.5	-	20	-	134 ±5.57 l	0 g
IPA-KT1	-	5	-	-	-	1	291 ± 4.58 e	31 ± 4.06 c
IPA-KT2	-	5	-	-	-	5	264 ± 7.00 f	11 ± 3.80 e
IPA-KT3	-	5	-	-	-	10	217 ± 2.65 h, i	5 ± 4.17 f, g
IPA-KT4	-	5	-	-	-	20	194 ± 7.00 j	2 ± 3.33 g
IPA-TDZ1	-	-	0.5	-	-	1	231 ± 5.29 g	3 ± 5.24 f, g
IPA-TDZ2	-	-	0.5	-	-	5	195 ± 5.57 j	2 ± 3.40 g
IPA-TDZ3	-	-	0.5	-	-	10	162 ± 5.29 k	1 ± 2.43 g
IPA-TDZ4	-	-	0.5	-	-	20	138 ± 3.61 l	1 ± 2.06 g

Data are presented as mean ± SD (n = 3). Different lowercase letters indicate significant differences among treatments (Duncan’s test, p < 0.05). Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; NAA, 1-naphthylacetic acid; KT, kinetin; TDZ, thidiazuron.

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Wang, G.; Liu, Y.; Li, H.; Zhang, L.; Li, Z.; Wang, J. Influence of Phenylacetic Acid and Its Derivatives on Callus Proliferation and Somatic Embryogenesis in Litchi chinensis. Horticulturae 2025, 11, 1200. https://doi.org/10.3390/horticulturae11101200

AMA Style

Wang G, Liu Y, Li H, Zhang L, Li Z, Wang J. Influence of Phenylacetic Acid and Its Derivatives on Callus Proliferation and Somatic Embryogenesis in Litchi chinensis. Horticulturae. 2025; 11(10):1200. https://doi.org/10.3390/horticulturae11101200

Chicago/Turabian Style

Wang, Guo, Yaoting Liu, Huanling Li, Lei Zhang, Zhiying Li, and Jiabao Wang. 2025. "Influence of Phenylacetic Acid and Its Derivatives on Callus Proliferation and Somatic Embryogenesis in Litchi chinensis" Horticulturae 11, no. 10: 1200. https://doi.org/10.3390/horticulturae11101200

APA Style

Wang, G., Liu, Y., Li, H., Zhang, L., Li, Z., & Wang, J. (2025). Influence of Phenylacetic Acid and Its Derivatives on Callus Proliferation and Somatic Embryogenesis in Litchi chinensis. Horticulturae, 11(10), 1200. https://doi.org/10.3390/horticulturae11101200

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Influence of Phenylacetic Acid and Its Derivatives on Callus Proliferation and Somatic Embryogenesis in Litchi chinensis

Abstract

1. Introduction

2. Materials and Methods

2.1. Plant Materials and Callus Culture

2.2. Addition of Regulators to the Proliferation Medium

2.3. Addition of Regulators to the Somatic Embryo Induction Medium

2.4. Data Analysis

3. Results

3.1. Effects of Regulators on Callus Proliferation and Morphology

3.2. Somatic Embryogenesis and Regeneration

3.3. Effects of PAA/CPA/IPA Combined with KT or TDZ on Somatic Embryogenesis and Regeneration

3.3.1. PAA Combined with KT or TDZ

3.3.2. CPA Combined with KT or TDZ

3.3.3. IPA Combined with KT or TDZ

4. Discussion

5. Conclusions

Author Contributions

Funding

Institutional Review Board Statement

Informed Consent Statement

Data Availability Statement

Conflicts of Interest

References

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