Morphological Analysis, Bud Differentiation, and Regulation of “Bud Jumping” Phenomenon in Oncidium Using Plant Growth Regulators

Abstract

Oncidium has an important market value, with important high-grade cut orchids and potted flowers on the flower market. In the Oncidium cut flowers production industry, there is a common phenomenon that the development of vegetative buds disrupts the normal generation cycle of the inflorescence induction, so-called “bud jumping”. In this study, vegetative bud differentiation and flower bud differentiation were divided into three stages, namely, the initial stage of differentiation, the leaf primordial/flower primordial differentiation stage, and the late stage of leaf bud/flower bud differentiation, as observed by paraffin sectioning. Secondly, we analyzed the differences between the vegetative buds of “bud jumping” plants and the flower buds of normal flowering plants by transcriptome sequencing. The transcriptome analysis results revealed significant differences among plant signaling pathways, particularly in gibberellins, auxins, and cytokinins, which play important roles in this phenomenon’s formation. In conjunction with the transcriptome analysis, the researchers conducted field experiments by applying plant growth regulators on the newborn pseudobulb of young Oncidium plants measuring approximately 49 mm in length. The results showed that the treatment groups of 100 mg/L of gibberellic acid (GA₃) and 100 mg/L GA₃ + 10 mg/L 6-Benziladenine (6-BA) exhibited the highest rate of flower bud differentiation instead of the least “bud jumping” phenomenon, and the “bud jumping” phenomenon was significantly reduced under 25 mg/L, 50 mg/L, and 75 mg/L 3-indoleacetic acid (IAA) treatments. The application of exogenous gibberellins, cytokinins, and auxins can effectively reduce the occurrence of “bud jumping”.

1. Introduction

Oncidium is a genus within the subfamily Epidendroideae of the orchid family (Orchidaceae), which includes around 726 species of orchids family (source of data: World Flora Online), and its flowers’ appearance is thought to resemble a small dancer with a colorful skirt. The flowers are small, dainty, and measure about 1-inch wide and have a large, ruffled labellum or lip. Oncidium flowers are showy, fragrant, usually with yellow to brown lips along with dark brown markings, having high ornamental value. They can be also used as high-grade orchid cut flowers and potted flowers in the flower market, which has important market value. The developing mature Oncidium plant consists mainly of leaves, pseudobulbs, and inflorescences, and the ontogenesis of Oncidium is divided into seven stages (Figure 1a) [1,2]. They are divided into the following stages: (i) The seedling stage of development; (ii) The desheathing stage, i.e., the pseudobulb development stage. (iii) The pseudobulb maturity stage. (iv) The mossing stage, in which flower buds sprout from the base of the pseudobulb. (v) The flowering stage, in which inflorescences progressively develop into the various floral organs. (vi) The adventitious bud sprouting stage, in which adventitious buds (axillary buds) sprout from the base of the pseudobulb in the next cycle of development. (vii) The adventitious bud development stage is where the adventitious bud developed into a vegetative bud, which developed into another new plant. When a pseudobulb develops directly from the mature stage (iii) to the adventitious bud development stage (vii), a phenomenon known as “bud jumping” occurs in the production of cut flowers of Oncidium, which is a common phenomenon of jumping development of vegetative buds interrupting the normal growth cycle of the flower bud differentiation in the production of cut flowers of Oncidium. When the phenomenon of “bud jumping” occurs, the adventitious bud will differentiate into vegetative buds and directly enter the next growth cycle, and if the phenomenon of “bud jumping” occurs repeatedly, the phenomenon of “climbing the stairs” will occur (Figure 1b), which seriously affects the production of cut flowers of Oncidium, leading to a decrease in flower quality and yield.

Leaf bud differentiation is the process by which the leaf primordium of the growth cone at the tip of the plant stem divides into axillary buds and leaves [3]. Flower bud differentiation is a process in which the apical meristematic tissue (SAM) shifts from vegetative to reproductive development during the reproductive growth stage and then develops into the inflorescence meristem (IM) and floral meristem (FM), from which the floral meristem further differentiates into the floral organ primordium [3,4]. Most of the flower buds of the Oncidium are inserted between the stalks of the lateral leaves (the first two leaves at the base of the pseudobulb, except for those on the top of the pseudobulb) and the pseudobulb at the pseudobulb base. To the naked eye, the leaf buds are slightly flattened and thin, unlike the rounded and full flower buds.

Most studies on bud differentiation in Oncidium have concentrated on the relationship between bud differentiation, phytohormones, and the growth environment. Phytohormones play important roles in growth and development, such as vegetative growth, bud differentiation, stage transition, etc. [5]. Wang et al. [6] demonstrated that the content of auxins was very low during the physiological differentiation of flower buds. Although this content subsequently increased, the concentration of auxins in flower buds remained significantly lower than in other bud types. Zeng et al. [7] suggested that relatively high levels of zeatin riboside (ZR) were the main cause of flower bud formation in Phalaenopsis. The application of transcriptomic techniques to investigate bud differentiation and hormone-related studies has been reported extensively [8,9,10]. In a study on flower transition in Phalaenopsis, genes such as PIF4X and TAA2 were found to be highly expressed in flower buds during the initiation and differentiation stages, while GA2ox and PIF4 were expressed at lower levels. It was suggested that PIF4 may be a key gene in promoting flower formation in direct response to gibberellin [11]. Therefore, the intricate interplay of environmental and endogenous physiological factors that initiate the processes of de novo vegetative bud and flower bud differentiation necessitates further comprehensive research.

In orchid production, the use of plant growth regulators (PGRs), water, and fertilizers to regulate plant growth is extensive. Research has widely reported the effects of exogenous gibberellin treatment on various orchid species, including Cymbidium [12] and Phalaenopsis [13]. A certain concentration of gibberellic acid (GA₃) has been shown to promote flowering. Additionally, plant growth regulators such as cytokinin 6-benzylaminopurine (6-BA) and auxin 3-indoleacetic acid (IAA) have been documented to influence flower bud differentiation [14,15]. The balance between plant growth regulators and endogenous hormones can either promote or inhibit flower bud differentiation and leaf bud differentiation in plants. The study of regulating the phenomenon of “bud jumping” in Oncidium through the application of PGRs will serve as a crucial reference for addressing the issue of yield reduction in the production of Oncidium angustifolium cut flowers and enhancing the quality of orchid products.

In this study, we observed a relatively high incidence of the “bud jumping” phenomenon in the Oncidium “Honey Angel” cultivated at the Dongshan Base of Hainan Boda Orchid Technology Co. Almost half of the Oncidium “Honey Angel” plants in the same batch (estimated to total around 50,000 to 60,000 plants) exhibited the “bud jumping” phenomenon, failing to bloom throughout the entire flowering period and resulting in a decline in the overall production of cut flowers. We studied flower buds, leaf buds, and dormant buds in sections to understand the relationship between their differentiation and growth characteristics. Subsequently, the transcriptome was used to analyze the molecular expression differences between flower buds and vegetative buds of Oncidium. Eventually, we conducted a field experiment in which PGRs were applied to Oncidium to study the effects of these PGRs on the production of cut flowers. This study provides a practical guide for the production of the “bud jumping” phenomenon.

2. Materials and Methods

2.1. Experimental Site

The experimental site, located within a shaded greenhouse in Haikou, China, 110°, 19°, experiences summer temperatures ranging from 25 to 42 °C and winter temperatures ranging from 10 to 20 °C. The relative humidity is maintained between 60% and 80%. In summer, the light intensity reaches approximately 35,000 Lux. The experimental seedlings are three-year-old tissue-cultured seedlings that are robust and uniformly grown. The distance between each treatment plot is 0.2 m, and the spacing within each plot is 0.1 m for both rows and plants.

2.2. Plant Materials

The Oncidium “Honey Angel” is a perennial herbaceous plant characterized by its plump, flattened, and oblong–ovoid pseudobulbs. Its leaves are leathery, with 2 positioned atop the pseudobulbs and an additional 2–3 pairs of leaves on both sides, exhibiting an ovate to oblong shape. The flower stalk emerges from the base of the pseudobulb, bearing yellow-hued blooms. These flowers typically feature a prominent labellum (lip) resembling a dancer’s skirt, with smaller petals and sepals. The peak blooming period primarily occurs from October to November. (All of the following experiments were conducted using the Oncidium “Honey Angel”).

2.2.1. Paraffin Section Observation

From October 17 to 29, 2023, new buds of Oncidium “Honey Angel” at various developmental stages were collected. These included undifferentiated buds measuring approximately 6–8 mm in length, buds at the initial stage of primordial differentiation (which were not yet visible to the naked eye but could be identified by the location of the pilot buds), and flower or vegetative buds that were 10–12 mm in length and discernible by sight. Each time, three buds were gathered, rinsed thoroughly with deionized water, and stripped of their bracts before being fixed in a 70% FAA solution. Furthermore, the width, length, thickness, and axillary bud length of the pseudobulbs were meticulously measured and recorded using vernier calipers.

2.2.2. Transcribed Plant Material

Take flower buds and vegetative buds of Oncidium “Honey Angel”, each approximately 10–12 mm in length, with 3 replicates of each type, totaling 6 samples. Carefully strip off the leaf sheath pieces, ensuring that a 10 mm long tip remains attached to each bud. Weigh out approximately 1–2 g of the prepared samples and place them into a freezing tube. Subsequently, freeze the samples in liquid nitrogen and send them on dry ice to Beijing Puri Biotechnology Co. for transcriptome sequencing.

2.2.3. Plant Growth Regulator Spraying Experiment

A total of 288 pots of Oncidium “Honey Angel” plants from the same batch with robust and uniform growth, pseudobulbs starting to form, and in the state of buds waiting to sprout were selected.

2.3. Methodologies

2.3.1. Paraffin Sectioning

The buds were cut longitudinally into sections of approximately 0.5 cm in length and width, and paraffin sections were prepared according to the following procedure: (1) Depending on the buds’ age, they underwent infiltration with varying concentrations of ethanol (50%, 75%, 85%, 95%, and 100%) (Jiangsu Yingke Medical Products Co., Ltd., Taizhou, China). (2) The bud tissue was progressively submerged in melted paraffin wax for a duration of 4 h each time, followed by drying and removal. This immersion, drying, and removal procedure was meticulously repeated three times. (3) Using a microtome, the wax blocks were precisely sliced into sections ranging from 8 to 12 μm in thickness. (4) A small droplet of gelatin adhesive was meticulously placed onto the slide, and the wax tape was carefully flattened onto it without any wrinkles. The slide was then dried in a warm oven and incubated at a temperature of 37 °C for a period of overnight. (5) The slices were systematically immersed in Eco-friendly Dewaxing Transparent Solution I (EDTS I) (Jinkelong (Beijing) Biotechnology Co., Ltd., Beijing, China) for 20 min, followed by an additional 20 min in EDTS II. They were then sequentially submerged in Anhydrous Ethanol I for 5 min, Anhydrous Ethanol II for another 5 min, and finally in 75% ethanol for 5 min, with subsequent rinsing in tap water. (6) The slices were placed in a plant red staining solution for a duration of 2 h, followed by thorough rinsing with tap water to eliminate any excess dye. Subsequently, the slices were briefly immersed in 50%, 70%, and 80% ethanol for 3 to 8 s each. They were then transferred to a plant solid green staining solution for a period of 6 to 20 s, underwent dehydration in three stages using anhydrous ethanol, and were ultimately sealed for preservation.

2.3.2. Total RNA Extraction and Library Construction, Sequencing, and Data Processing

Initially, RNA was extracted utilizing the standardized protocol provided by the CTAB kit (Genesand Biotech Co., Ltd., Beijing, China). The extracted RNA from all gathered samples underwent rigorous quality assessment to ensure compliance with sequencing standards. Subsequently, a cDNA library was constructed and sequenced on the Illumina NovaSeq 6000 platform. To obtain high-quality transcriptome data, the raw sequences underwent extensive quality control and assembly procedures, resulting in clean reads that were devoid of redundant sequences [16]. The quality of all sample data was meticulously evaluated and optimized using FASTP v0.19.3 [17], with a threshold of Q30 > 85% defining qualifying data. For sequence alignment, the clean reads were mapped to the reference genome using HISAT2 [18]. Only reads exhibiting a perfect match or a single mismatch were subjected to further analysis and annotation based on the reference genome. This process allowed for the retrieval of positional information on the reference genome or genes, as well as sequence-specific features pertaining to the sequenced samples. It is worth noting that the genome sequence of Oncidium “Honeybee” was obtained by our research group, but it is in the process of being published. These analyses were conducted by the authors in collaboration with DynaTech Biosciences, Inc, Beijing, China.

The raw sequence data reported in this paper were deposited in the Genome Sequence Archive [19] in the National Genomics Data Center [20], China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA020078), which are publicly accessible at https://bigd.big.ac.cn/gsa/browse/CRA020078 (accessed on 12 May 2025).

2.3.3. Screening, Functional Annotation and Classification of Differentially Expressed Genes (DEGs)

DEGs (differentially expressed genes) were analyzed using DESeq2 [21], with the criteria of a fold change ≥ 2 and PDR < 0.05 being applied for screening. The screened DEGs were then compared against the COG (http://www.ncbi.nlm.nih.gov/COG/, accessed on 15 February 2025), KOG (https://ftp.ncbi.nlm.nih.gov/pub/COG/KOG/, accessed on 15 February 2025), and KEGG (http://www.genome.jp/kegg/, accessed on 15 February 2025) databases for sequence alignment. Functional classification of the genes was obtained from the COG database, while the KEGG database [22,23,24] was utilized to retrieve pathway information.

2.3.4. qRT-PCR Validation

RNA was reverse transcribed into cDNA using the HiScript II First Strand cDNA Synthesis Kit (+gDNA wiper). The resulting cDNA was then used for subsequent experiments. qRT-PCR-specific primers were designed using Primer 6, and their sequences are presented in Supplementary Table S1. Fluorescence quantitative PCR was performed using the ChamQ SYBR qPCR Master Mix (Q711-02) kit (Shanghai Tulu Harbor Biotechnology Co., Ltd., Shanghai, China), following the manufacturer’s protocol and conducted on ice. The qRT-PCR conditions included an initial denaturation step at 95 °C for 60 s, followed by 40 cycles of amplification (95 °C for 15 s and 60 °C for 30 s). The Actin gene was used as a reference control. The relative mRNA expression level was calculated using the 2^−ΔΔCt method, where Ct represents the cycling threshold of the target gene [25].

2.3.5. Application Test of Plant Growth Regulators

(1)

Test setup of plant growth regulators

Starting from 24 July 2024, the bud-ready Oncidium “Honey Angel” plants were carefully selected from among the vigorously and uniformly growing three-year-old seedlings that were grouped together. The treatments applied included IAA, 6-BA, PP333, GA3, as well as a combination of 6-BA and GA3. The concentration gradients for each treatment were as follows: IAA was administered at concentrations of 25 mg/L, 50 mg/L, and 100 mg/L; 6-BA at 10 mg/L, 25 mg/L, and 50 mg/L; PP333 at 25 mg/L, 50 mg/L, and 100 mg/L; and GA3 at 50 mg/L, 75 mg/L, and 100 mg/L. Each treatment group consisted of 18 pots, which were divided into 3 replicates. The volume sprayed for each concentration treatment group is 300 mL per application. Clear water (mocK) was used as the control treatment.

(2)

Experimental methods and observational indicators

Use a spray bottle for spot spraying and foliar application, spraying the solution from the base of the pseudobulb to the entire plant until the leaves and pseudobulb are completely moistened. A total of four treatments were administered, with each treatment applied every three days; all other management practices and growing conditions remained the same as normal. For seedling vegetative growth, nitrogen fertilizer and balanced fertilizer were used, and potassium fertilizers were gradually added as the seedlings matured, primarily through foliage spraying. Fertilization and pest control measures were implemented once a week.

Observations and data recording for the emergence of buds were conducted on the 7th, 14th, and 30th days after the fourth treatment spraying. The morphology and locations of the buds were examined to differentiate between flower buds and vegetative buds through visual inspection. The number of buds was documented, with each pseudobulb counted as one plant. Pseudobulbs from plants that had already bloomed were excluded from the count. The emergence of buds on each plant was appropriately labeled. Using the results from the 30th day, the percentage of flower buds and vegetative buds was calculated relative to the total number of buds.

2.4. Data Analysis

All measurements were performed in triplicate. Univariate values were analyzed by ANOVA using R software (https://www.r-project.org/) (IBM Corporation, Armonk, NY, USA) and means were compared using Duncan’s new multiple range test (p < 0.05).

3. Results

3.1. Morphological Characteristics of Adventitious Bud and Leaf/Flower Bud Differentiation in Oncidium “Honey Angel”

The term “lead” is used by Tanaka et al. [15] to refer to the basal part of the pseudobulb that does not produce roots, and all subsequent bud formations occur on it. In this study, we investigated the phenotypic characteristics of bud differentiation and development in the phenomenon of “bud jumping” in Oncidium. We employed microscopic observation of paraffin sections and morphologically categorized leaf/flower bud differentiation into three distinct periods (Table 1).

Table 1. Cellular and phenotypic characteristics of developing bud differentiation.

Periods	Cellular Level	External Circumstances
Initial stage of leaf bud differentiation	The growth cone located at the stem tip has a mountain cone-like shape, where its width is broader than its height. The cells within its apical meristematic tissue are tightly packed, whereas the cells situated at its lower base are more loosely arranged and exhibit a tendency to converge and develop towards the stem tip (Figure 2a).	A new round of bud differentiation in Oncidium was observed in mid-July (Northern Hemisphere summer) at the experimental site buds emanating from the bases of the four axils of the pseudobulb “lead”, which are very small and are adventitious buds for which it is uncertain which one is the pilot bud at this time (Figure 3a)
Leaf primordial differentiation stage	Significant elongation at the tip of the growth cone in the meristematic tissue at the stem tip, with rounded, expanded, and loosely arranged cells and a tendency to develop a convex protrusion (Figure 2b).	Adventitious buds on the “lead” continued to grow and develop, and the pioneer buds have not yet exposed their leaf sheaths and need to be plucked off the pseudobulb cotyledons to be visible (Figure 3b)
Late leaf bud differentiation (visually distinguishable)	The meristematic tissue at the stem end had differentiated into the first and second cotyledon morphology (Figure 2c).	The first and second axillary buds from the “lead” stop growing, while the third or fourth axillary buds develop into vegetative buds. At this stage, the cotyledons of the pseudobulb can only be observed by gently separating them, revealing flattened leaf buds that are beginning to emerge (Figure 3d)
Initial stage of bud differentiation	The cellular morphology and external phenotype observed in the initial stages of flower bud differentiation were similar to those seen in leaf bud differentiation, characterized by closely positioned axillary leaves (Figure 2d).	Therefore, these features could not be used to distinguish between flower and vegetative buds (Figure 3a)
Differentiation stage of inflorescence primordium	The meristematic tissue at the tip of the stem began to differentiate into reproductive tissue, exhibiting a notable protrusion at the apex of the growth cone. The basal cells became rounded, and there was a tendency for finger-like projections to develop at both ends of the growth cone later (Figure 2e).
Late stage of flower bud differentiation	The middle rounded stem end continues to grow, and its ends develop into angular protuberances that differentiate into bud primordia (Figure 2f). Subsequently, the flower organs, including calyx and petal primordia, gradually differentiate, and each floret begins to develop on both sides, alongside the differentiation of the inflorescence primordium initiated (Figure 3e).	At this point, when the leaves are plucked, the round-tipped buds can be seen to appear (Figure 3c)

Table 1. Cellular and phenotypic characteristics of developing bud differentiation.

Periods	Cellular Level	External Circumstances
Initial stage of leaf bud differentiation	The growth cone located at the stem tip has a mountain cone-like shape, where its width is broader than its height. The cells within its apical meristematic tissue are tightly packed, whereas the cells situated at its lower base are more loosely arranged and exhibit a tendency to converge and develop towards the stem tip (Figure 2a).	A new round of bud differentiation in Oncidium was observed in mid-July (Northern Hemisphere summer) at the experimental site buds emanating from the bases of the four axils of the pseudobulb “lead”, which are very small and are adventitious buds for which it is uncertain which one is the pilot bud at this time (Figure 3a)
Leaf primordial differentiation stage	Significant elongation at the tip of the growth cone in the meristematic tissue at the stem tip, with rounded, expanded, and loosely arranged cells and a tendency to develop a convex protrusion (Figure 2b).	Adventitious buds on the “lead” continued to grow and develop, and the pioneer buds have not yet exposed their leaf sheaths and need to be plucked off the pseudobulb cotyledons to be visible (Figure 3b)
Late leaf bud differentiation (visually distinguishable)	The meristematic tissue at the stem end had differentiated into the first and second cotyledon morphology (Figure 2c).	The first and second axillary buds from the “lead” stop growing, while the third or fourth axillary buds develop into vegetative buds. At this stage, the cotyledons of the pseudobulb can only be observed by gently separating them, revealing flattened leaf buds that are beginning to emerge (Figure 3d)
Initial stage of bud differentiation	The cellular morphology and external phenotype observed in the initial stages of flower bud differentiation were similar to those seen in leaf bud differentiation, characterized by closely positioned axillary leaves (Figure 2d).	Therefore, these features could not be used to distinguish between flower and vegetative buds (Figure 3a)
Differentiation stage of inflorescence primordium	The meristematic tissue at the tip of the stem began to differentiate into reproductive tissue, exhibiting a notable protrusion at the apex of the growth cone. The basal cells became rounded, and there was a tendency for finger-like projections to develop at both ends of the growth cone later (Figure 2e).
Late stage of flower bud differentiation	The middle rounded stem end continues to grow, and its ends develop into angular protuberances that differentiate into bud primordia (Figure 2f). Subsequently, the flower organs, including calyx and petal primordia, gradually differentiate, and each floret begins to develop on both sides, alongside the differentiation of the inflorescence primordium initiated (Figure 3e).	At this point, when the leaves are plucked, the round-tipped buds can be seen to appear (Figure 3c)

The cellular growth of the leaf bud shoot axis tissue was closer to the growth cone at the stem tip, shorter and denser the closer it was (Figure 2g). The cells of the bud axis tissue of the flower buds grew toward the stem tip and on both sides and were elongated and relatively sparse (Figure 2h). The length of the growth cone of adventitious buds during dormancy is longer than the height (Figure 2i), and there is no clear trend in cell growth of the bud axis similar to that of Figure 2g,h.

3.2. Relationship Between Bud Differentiation and Pseudobulb Development in Oncidium “Honey Angel”

In the initial stage of bud differentiation, the base of the pseudobulb typically contains four adventitious buds, which are quite small at this stage. As the pseudobulb matures and expands, one of the adventitious buds becomes the pioneer bud, which will differentiate into either a flower bud or a vegetative bud. The data presented in

Table 2. Growth indexes of different tissues in several stages.

Period of Bud Differentiation	Length of Buds (mm)	Length of Pseudobulbs (mm)	Thickness of Pseudobulbs (mm)	Width of Pseudobulbs (mm)
Initial stage of bud differentiation	6.37 ± 0.2 c	49.40 ± 0.5 b	7.04 ± 0.1 b	10.83 ± 0.9 c
Leaf primordium differentiation stage	8.73 ± 0.9 b	54.07 ± 1.3 b	13.45 ± 3.8 a	15.21 ± 3.9 b
Late stage of bud differentiation	11.76 ± 1.7 a	60.54 ± 1.8 a	12.84 ± 0.7 a	19.22 ± 1.8 a
Initial stage of flower bud differentiation	6.40 ± 0.0 c	49.83 ± 0.5 b	7.05 ± 0.1 b	11.53 ± 0.6 c
Differentiation stage of inflorescence primordium	9.54 ± 0.4 b	50.20 ± 1.1 b	10.81 ± 1.2 a	16.14 ± 1.0 ab
Late stage of flower bud differentiation	11.46 ± 1.0 a	61.47 ± 2.1 a	13.95 ± 0.1 a	19.07 ± 0.3 a

Note: Three buds were tested, with different letters in the same column indicating significant difference (p < 0.05).

indicate that the length, thickness, and width of the pseudobulbs increase with the progression of bud differentiation; furthermore, as the pseudobulbs mature, a greater number of buds becomes differentiated. When observing the state of buds with the naked eye, the buds need to be exposed to the leaf sheaths and have obvious morphological features, so they can be clearly distinguished at the late stage of bud differentiation when the buds are about 10–12 mm. At the young stage of pseudobulb (length of 49 mm or so), when most of the axillary buds are in the state of adventitious buds waiting to sprout, it is easiest to produce the effect of exogenous phytohormone spraying on the plants at this period.

3.3. RNA Sequencing, Quality Control, and Functional Annotation and Classification

To further investigate the reasons behind the formation of the “bud jumping” phenomenon, RNA-Seq analysis was conducted on vegetative buds from plants exhibiting this phenomenon and flower buds from normal flowering plants. The results (

Table 3. Growth indexes of different bud differentiation stages.

Samples	Raw Reads	Clean Reads	GC Content (%)	% ≥ Q30	Mapped Reads	Gene Transcripts
OFB1	42,848,732	20,441,097	45.21	95.16%	33,594,880 (82.17%)	12,954
OFB2	41,237,108	19,801,580	45.29	95.05%	32,618,072 (82.36%)	12,979
OFB3	46,854,894	22,485,145	45.22	94.76%	36,864,547 (81.98%)	13,168
OVB1	42,178,172	20,269,341	44.88	94.98%	33,099,228 (81.65%)	13,253
OVB2	44,292,884	21,251,957	44.81	94.86%	34,730,115 (81.71%)	13,114
OVB3	40,630,874	19,481,320	45.58	95.47%	31,975,871 (82.07%)	13,000

O denotes Oncidium “Honey Angel”, OVB is the vegetative bud, OFB is the flower bud. Numbers 1–3 indicate three biological replicates.

) indicated that the GC content ranged from 44.84% to 45.58%, the Q30 value exceeded 94.86%, and the alignment with the reference gene sequence was between 81.65% and 82.36%. In OFB1-3, there are 12,954 to 13,168 gene transcripts, while in OVB1-3, there are 13,000 to 13,253 gene transcripts. Among them, 14 gene transcripts have zero expression in OVB1-3 but are expressed in OFB1-3, and 9 gene transcripts are expressed in OFB1-3 but have zero expression in OVB1-3. A total of 2442 deferentially expressed genes (DEGs) were identified, comprising 1161 up-regulated genes and 1281 down-regulated genes, using a fold change rate of ≥2 and a p-value of <0.05 as the screening criteria (Supplementary Figure S1). The DEGs were subsequently annotated in various databases, with a total of 2363 DEGs annotated. Of these, 867 were mapped to the Clusters of Orthologous Genes (COG) database, 873 to the KEGG database, and the majority, totaling 2307, were annotated in the eggNOG database (Supplementary Figure S2).

Based on COG enrichment analysis (Supplementary Figure S3), it is evident that the DEGs were most significantly enriched in carbohydrate transport and metabolic functions, followed by signaling mechanisms and general function predictions. KEGG enrichment (Figure 4) results indicated that these DEGs were significantly enriched in biosynthetic pathways, particularly those related co-factors, phenylpropanes, and steroids, as well as metabolic pathways such as starch and sucrose, fructose, and mannose. Additionally, DEGs were enriched in phytohormone signaling pathways, phytopathogen interactions, carbon sequestration pathways in photosynthetic organisms, and pentose and glucuronide interconversion pathways. Taken together, these findings demonstrated that the primary differences in differentiation between the two buds at this stage primarily involve the metabolic and synthetic transport of sugars and the transduction of phytohormone signals.

3.4. Phytohormone Signaling Regulation

Endogenous phytohormones play crucial roles in the regulation of vegetative growth, bud differentiation, stage transition, and other aspects of plant growth and development [5]. These hormones include auxins, gibberellins (GAs), cytokinins (CTKs), abscisic acid (ABA), and others. According to the hormone regulation hypothesis, auxins, CTKs, and GAs are pivotal regulators of the transition from vegetative growth to reproductive growth, and the regulation and microexpression of these hormones play a non-negligible role in influencing the occurrence of the phenomenon of “bud jumping”. In the significant difference pathway analysis, it was found that genes related to auxins, GA, brassinosteroid (BR), salicylic acid (SA), and CTK signaling and synthesis were involved in the regulation of “bud jumping” in Oncidium. In the actual study (Figure 5), AUX1 homologs, the auxin receptor TIR1, and TAA homologs were highly expressed in vegetative buds. The AUX/IAA family of auxin signaling regulators was predominantly expressed in vegetative buds as well. The downstream GH3 and SAUR genes and the transporter protein PIN were all up-regulated in expression. CYP735A, an important gene in the major pathway for synthesizing trans-zeatin, was also prominent [26]. The high expression of LONELY GUY (LOG1), another gene involved in CTK biosynthesis, in flower buds, along with the variable expression of LOG1 in both types of buds, suggests that the amount of synthesized CTK in buds was subject to fine-tuned regulation. Furthermore, the ratio of CTK content plays a crucial regulatory role in bud differentiation. In the GA hormone pathway, the gibberellin oxidase-related gene GA20ox, which was involved in GA synthesis, was expressed in both types of buds. Notably, GA20ox2 in Arabidopsis accelerates flower opening [27]. Interestingly, GA2ox, which inactivates GAs, was highly expressed in flower buds. The regulation of these genes maintains a delicate balance of GA content between flower buds and vegetative buds. In summary, by combining gene function annotation and enrichment information, after in-depth excavation and analysis of auxin, GA, and CTK biosynthesis and metabolic pathways and phytohormone signaling pathways, we found that the genes related to these three hormones have an important role in regulating plant vegetative bud and flower bud differentiation.

In addition, the study also found that the genes BZR1 and CYCD3 related to the BR signaling pathway were down-regulated, while the downstream CTH4-based genes were up- and down-regulated, and the genes NPR5 and PAL related to the SA hormone were down-regulated, while NPR1 was up- and down-regulated. These hormone signaling and synthesis-related genes are involved in the regulation of “bud jumping” formation.

3.5. qRT-PCR Quantitative Analysis

For qRT-PCR quantification, OhGene009120 (IAA), OhGene015963 (GA20ox), OhGene001497 (PIF4), and OhGene011730 (SAUR) were selected (Figure 6). The results revealed that the expression trends of these three genes were consistent with the transcriptional data, suggesting a certain degree of reliability in the transcriptional data.

3.6. Foliar Application Test of Plant Growth Regulators

By the 30th day, no flower opening was observed in any of the PGR treatment groups or the control group. However, it was notable that the highest number of flowering branches was observed in the 100 mg/L GA₃ treatment group for the Oncidium “Honey Angel” variety (Supplementary Figure S4a). In the 6-BA treatment group, a symbiosis of flower bud and vegetative bud was observed on a single pseudobulb (Supplementary Figure S4b), while the 50 mg/L treatment group exhibited the emergence of new buds from aged pseudobulbs (Supplementary Figure S4c). Based on the observation of the pioneer buds’ location, no bud transformation due to the application of exogenous phytohormones was detected during this period.

In terms of flower and vegetative bud rates, all five treatments had some effect on bud differentiation. As shown in

Table 4. Effects of different concentrations of plant growth regulators on bud differentiation of Oncidium “Honey Angel”.

Treatment Group	Flower Bud Rate%	Vegetative Bud Rate%	Adventitious Bud Rate%
mock	50.00 ± 0 d	38.89 ± 9.6 abc	11.11 ± 9.6 ab
IAA (25 mg/L)	77.78 ± 9.6 abc	5.56 ± 9.6 d	16.67 ± 0 ab
IAA (50 mg/L)	66.67 ± 16.7 abcd	11.11 ± 9.6 d	22.22 ± 9.6 a
IAA (100 mg/L)	66.67 ± 16.7 abcd	16.67 ± 16.7 d	16.67 ± 0 ab
6-BA (10 mg/L)	83.33 ± 16.7 ab	5.56 ± 9.6 d	11.11 ± 9.6 ab
6-BA (25 mg/L)	78.89 ± 7.7 abc	21.11 ± 7.7 cd	0 b
6-BA (50 mg/L)	57.94 ± 8.4 cd	42.06 ± 8.4 ab	0 b
GA3 (50 mg/L)	64.44 ± 17.1 abcd	12.22 ± 10.7 d	23.33 ± 25.2 a
GA3 (75 mg/L)	61.11 ± 9.6 bcd	22.22 ± 19.3 bcd	16.67 ± 16.7 ab
GA3 (100 mg/L)	88.89 ± 9.6 a	0 d	11.11 ± 9.6 ab
GA3 (100 mg/L) + 6-BA (10 mg/L)	88.89 ± 19.3 a	5.56 ± 9.6 d	5.56 ± 9.6 ab
GA3 (100 mg/L) + 6-BA (25 mg/L)	77.78 ± 9.6 abc	16.67 ± 16.7 d	5.56 ± 9.6 ab
GA3 (100 mg/L) + 6-BA (50 mg/L)	55.56 ± 9.6 cd	44.44 ± 9.6 a	0 b

Adventitious buds are buds that are not visually distinguishable. Different letters in the same column indicate significant difference (p < 0.05).

, among the Oncidium “Honey Angel”, the highest flowering bud percentage was recorded in the 100 mg/L GA₃ and 100 mg/L GA₃ + 10 mg/L 6-BA, both achieving 88.89%. The 10 mg/L 6-BA and 25 mg/L 6-BA treatment groups exhibited the second highest flower bud percentages of 83.33% and 78.89%, respectively; both were significantly different from the control (mock) group. The vegetative bud percentage increased with higher concentrations in the IAA-treated group. Flower bud percentage was 77.78% and vegetative bud percentage was 5.56% in the 25 mg/L IAA-treated group, both of which were significantly different from the mock group. In the 6-BA treatment group, vegetative bud percentage increased in all treatment groups with increasing concentration, but only the 10 mg/L 6-BA treatment group was significantly different from the mock group, and the vegetative bud percentage was higher in the 50 mg/L 6-BA treatment group than the mock group, while flower bud percentage decreased with increasing concentration. Significant differences in vegetative bud percentage were observed in the treatment groups of 100 mg/L GA₃ + 10 mg/L 6-BA, 100 mg/L GA₃ + 25 mg/L 6-BA, among others, with the most notable result being the 100 mg/L GA₃ treatment, which resulted in 0% of vegetative bud. The results from the combined treatment groups indicated that a higher GA/6-BA ratio correlated with an increased flower bud rate and a decreased vegetative bud rate.

In conclusion, the treatment groups of 25 mg/L IAA, 10 mg/L 6-BA, and 100 mg/L GA₃ combined with 10 mg/L 6-BA demonstrated significant effects in suppressing the phenomenon of “bud jumping” in the Oncidium “Honey Angel” variety. Among these, the latter two groups exhibited superior efficacy in addressing this phenomenon. The PGR spraying experiments indicated that these PGRs play a crucial regulatory role in the differentiation of axillary buds into either flower buds or vegetative buds during the development of newly formed pseudobulbs.

4. Discussion

Oncidium is a perennial herb that taxonomically belongs to the Orchidaceae family of compound-stemmed sympodial orchid and epiphytic orchid. Based on Hew and Yong’s methodology [28], the growth cycle of O. Goidiana can be divided into four main stages of vegetative growth, bud stage, seedling stage, sheath stage, and pseudobulb stage, and two stages of reproductive development, completion of flower bud differentiation and flowering stage. According to Chin et al. [1], the mossy stage is subdivided before the reproductive growth stage, when the pioneer buds are flower buds. Leaf bud differentiation is completed at the stage of vegetative growth, while flower bud differentiation is completed at the stage of reproductive development; when the bud differentiation is in the jump development, the phenomenon of “bud jumping” occurred. General flower bud differentiation has been classified into seven stages, and Peng et al. [29] classified the flower bud differentiation of O. Milliongolds into six stages, of which the undifferentiated stage of flower buds and the stage of inflorescence primordial differentiation were similar to the results of the present study. Instead, the author categorized the subsequent differentiation and development of flower organs, including buds, calyxes, and petals, entirely within the late stage of flower bud differentiation.

Auxins, CTKs, and GAs are antagonistic and mutually reinforcing interactions, making the balance of their ratios crucial for the process of bud differentiation. The transduction of auxin signaling plays an important role in influencing differentiation within bud meristematic tissues. The AUX/IAA gene family is integral to the regulation of plant morphogenesis, and the concentration of auxin is vital for the differentiation of tissues and organs, such as SAMs [30]. AUX/IAA interacts with the auxin receptor TIR1 in auxin signaling, and ubiquitination degradation of AUX/IAA proteins represses ARF transcription factors and regulates downstream gene expression through ARF family transcription factors [31]. The transporter protein PIN is also involved in the regulation of axillary bud development [32,33]. In this study, ARF1 was up-regulated and down-regulated for both upstream genes, but its downstream genes, GH3 and SAUR, were up-regulated, and with the involvement of PIN promoting the transduction and transport of growth hormone and its responsive role in buds. The expression level of these genes may be more conducive to the regulation of flower bud differentiation within the SAM. Gibberellin synthesis in flower buds is regulated by GA20ox, while GA2ox inactivates gibberellin synthesis, resulting in compromised GA content. The PIF family of proteins, which are photosensitive and interact with pigments to sense gibberellin signals and mediate photo-regulatory responses, are closely associated with flowering signals [33]. Notably, PIF4 is highly expressed in flower buds. CTK is a central regulator of plant axillary bud germination and growth. In the ctk synthesis pathway, cytokinin hydroxylase CYP735A catalyzes the hydroxylation of isopentenyl adenosine phosphate (ATP/ADP/AMP hydroxylation) to form trans-zeatin nucleoside [26,34]. This compound is then converted to the free base CTK by the cytokinin-activating enzyme LONELY GUYs (LOGs) [35]. CYP735A is highly expressed in flower buds, contributing to an increase in CTK synthesis precursors. LOGs are present in varying degrees in both vegetative and flower buds, significantly influencing CTK promotion of bud meristem differentiation [36,37]. Auxin promotes the accumulation of gibberellins [38], and both gibberellins and CTKs facilitate auxin transport. Accelerated auxin transport, in turn, enhances the degradation or glycosylation of CTKs [39]. Shi [40] utilized transcriptome analysis to investigate the mechanisms of flower formation in Passiflora, concluding that auxins, CTKs, and GAs play crucial regulatory roles in the transition to flower formation in this genus. When BR is present, transcription factors such as BZR1 are activated to regulate the expression of downstream CTH4 and CYCD3 genes in order to participate in processes such as cell elongation, photosynthesis, and phytohormone synthesis [41]. NPRs are required to interact with transcription factors to induce the expression of pathogenesis-related genes and thereby promote defense responses, but the regulatory role of NPR1 and NPR5 expression has not been extensively studied. Of course, these five hormones also interact with each other to regulate bud differentiation and development. In this study, these hormone-related DEGs were analyzed, but the molecular mechanisms underlying the formation of “bud jumping” in Oncidium still require further research to be answered. At present, auxins, CTKs, and GAs are the most likely to affect the formation of “bud jumping”.

The application of PGRs influences the levels of endogenous hormones, thereby affecting bud differentiation. The use of exogenous CTK impacts not only the synthesis, transport, and signaling of endogenous CTK but also the accumulation and response of auxin [42]. Additionally, there have been reports indicating that spraying exogenous GA can increase the concentration of endogenous GA [12,42]. Similarly, two exogenous phytohormones, GA₃ and 6-BA, exhibit both promotional and inhibitory effects on flower and vegetative bud differentiation. Although these hormones are typically regarded as promoters, they can sometimes demonstrate antagonistic effects on various developmental processes [43,44]. In studies of apple flower induction, higher CTK/GA ratios tend to favor flowering [45]. In this experiment, the flower bud formation rate was highest, while the vegetative bud formation rate was lowest in the treatment group of GA₃ (100 mg/L) combined with 6-BA (10 mg/L) for the Oncidium “Honey Angel”. Conversely, a higher GA/CTK ratio promoted flower bud differentiation. These results indicate that specific concentrations of exogenous GA/CTK hormone treatments can interact with endogenous hormones to establish a new equilibrium, which is more favorable for promoting flower bud differentiation in Oncidium. CTKs were associated with the breakage of axillary trophic and inflorescence meristematic tissues, and the emergence of lateral branches was found when Phalaenopsis was sprayed with 6-BA during the trophic growth stage [46]. The results of the present experiment indicate that as the concentration of the 6-BA treatment group increases, the rate of vegetative bud also increased. During bud differentiation, the maintenance of the apical meristem was usually regulated by the interaction of several hormones, especially the two endogenous hormones IAA and CTK [47]; CTK was directly involved in the growth of axillary buds [48]. In the experiment, the Oncidium “Honey Angel” exhibited fewer vegetative buds in the IAA-treated group compared to the control group. This suggests that exogenous auxin sprays and endogenous auxin can reach an alternative equilibrium, inhibiting the differentiation of vegetative buds. This was related to Tanaka’s [49] suggestion that auxins can inhibit the development of axillary buds by inhibiting the biosynthesis of CTK. In Oncidium, bud differentiation responded to different concentrations of PGRs, which acted antagonistically to the endogenous hormones of the plant and promoted the differentiation of flower buds to a certain extent, while exceeding the maximum value also promoted the differentiation of trophic buds.

In this study, the researchers discovered a significant standard deviation in the vegetative bud rate and adventitious bud rate among certain treatment groups. This variability may be attributed to environmental factors, such as unpredictable weather conditions characterized by windy and rainy spells from July to September, as well as operational variables during the exogenous hormone application experiment. For example, when the PGRs are sprayed in the morning, varying amounts of rainfall may occur in the afternoon. These conditions have had a substantial impact on the efficacy of the exogenous hormone.

5. Conclusions

In this study, CTKs, IAA, GAs, BRs, and SA were identified from the phytohormone signaling pathways in the transcriptome data as potential contributors to the “bud jumping” phenomenon in Oncidium. This finding was further validated through an exogenous phytohormone application experiment. Due to the technical requirements of production, 6-BA, IAA, and GA₃ were selected for spraying experiments. Additionally, it was also found that GA₃ at a concentration of 100 mg/L, either alone or in combination with 10 mg/L of 6-BA, minimized the occurrence of “bud jumping” and increased cut flower yield.