Abstract
The flowering control of Oncidesa Gower Ramsey ‘Honey Angel’ is important and in-demand by the industry. Therefore, an understanding of the development of inflorescence and vegetative shoot from the leaf axils on the current shoot is required. The internode of a young Oncidesa current shoot between the 0th (at the base of the pseudobulb) and 1st (immediately above the pseudobulb) nodes can enlarge to form a pseudobulb, and the axillary bud on the 0th or -1st (immediately below the 0th node) node can differentiate into an inflorescence bud. The axillary buds on the lower nodes (-2nd to -4th nodes) can remain vegetative. In this study, we investigated the growth and anatomical features of axillary buds at various stages during the growth of the current shoot. We sampled the axillary buds on the 0th to -4th nodes from the current shoots when they were 10, 15, 20, 25, and 30 cm in length for sectioning and anatomical observations. Vegetative buds on the -2nd to -4th nodes grew faster and had more nodes than the inflorescence bud when the current shoot grew from 10 to 25 cm. However, when the current shoot elongated from 25 to 30 cm, the length and node number in the inflorescence bud on the 0th node increased and the inflorescence branch primordia were observable. The length and node number of the inflorescence bud became the same as that of the vegetative buds, which had no further growth, whereas the current shoot grew from 25 to 30 cm. The pseudobulb began to emerge from the leaf sheath (unsheathing) when the current shoot had reached 30 cm in length. Therefore, the time when the pseudobulb started to unsheathe from its subtending leaf was critical for the reproductive growth of Oncidesa Gower Ramsey ‘Honey Angel’ when growth acceleration of the inflorescence bud occurred. Evaluating the current shoot length can be a nondestructive method of estimating the developmental stage of the inflorescence bud.
Some cultivars in the intergeneric genus Oncidesa (Oncidium × Gomesa) are excellent cut flowers (Hu et al. 2018). In Taiwan, Oncidesa Gower Ramsey ‘Honey Angel’ ranks second in cut flower exports, with a value of $16.6 million in 2021 (Council of Agriculture, Taiwan, 2021). However, in Taiwan, Oncidesa growers still face many production challenges, including low prices during the peak flowering seasons. Therefore, effective flowering regulation is necessary (Chen 2019).
Some growers in Taiwan attempt to use plant growth regulators to control the flowering of Oncidesa by altering the development of vegetative or inflorescence buds. The buds at various developmental stages might respond differently to cultural practices or the application of plant growth regulators (Chatield et al. 2000; McArtney and Obermiller 2015). Some Oncidesa growers use 6-benzyladenine (6-BA) to promote vegetative bud emergence, which may increase inflorescence productivity. Some growers use gibberellin acids (GA4 + 7) to control inflorescence growth. However, the results are not consistent and their effectiveness has not been confirmed. Hence, investigations of axillary bud development and morphological features of Oncidesa are required to gain a better understanding of the regulation of flowering.
Oncidesa is a heteroblastic type of sympodial orchid. An enlarged internode called the pseudobulb develops between the third and fourth leaves counted from the top of ‘Honey Angel’. The term “current shoot” is used to describe the developing shoot of Oncidesa (Hew and Yong 2004). Generally, the axillary bud on the node at the base of the pseudobulb, which is hereafter called A0, becomes an inflorescence. After flowering, the axillary buds on the lower nodes of the current shoot develop into new current shoots during the next growth cycle. Furthermore, the former current shoot becomes the “first back shoot” (Hew and Yong 1994; Tanaka et al. 1986).
Inflorescence growth is related to the development of the pseudobulb. Photoassimilates in Oncidium orchids have vital roles in flowering (Yong and Hew 1995). Pseudobulbs store photoassimilates and other nutrients that are used by the developing inflorescence (Wang et al. 2008). To observe the developing pseudobulb inside a young current shoot, the outer leaves must be peeled off, which damages current shoots. Therefore, it is critical to find a nondestructive method of determining the developmental stage of inflorescence.
The development of an inflorescence bud of Oncidesa Sweet Sugar ‘Million Gold’ was studied by Peng et al. (2012). During the initial differentiation phase, the bud first appears as a blunt triangle. Then, during the inflorescence differentiation phase, the growth cone appears with finger-like protrusion bracts at both ends and the growth cone elongates. After the growth cone develops to a certain stage, the inflorescence bud enters the flower primordia differentiation phase, which is the flower primordia form on the main axil of the inflorescence.
Although morphological observations of vegetative growth and inflorescence bud formation in Gomesa Boissiense have been studied by Tanaka et al. (1986), it has been considered that the development of inflorescence of Gomesa Boissiense (an autumn-flowering hybrid) was promoted by long days and that it flowered once every year. In contrast, Oncidesa Gower Ramsey ‘Honey Angel’ produces inflorescences twice every year. Although the flowering habits of Gomesa Boissiense and Oncidesa Gower Ramsey are different, Oncidesa should have a similar floral developmental pattern as Gomesa because it is an intergeneric genus between Oncidium and Gomesa. In Gomesa Boissiense, the node number increased in the main axis, followed by floret primordia differentiation on the branches (Tanaka et al. 1986).
During this study, we focused on the correlation between current shoot growth and the developmental status of the axils. This nondestructive method of determining the axils growth is the first step to establishing the flowering regulation technique of Oncidesa Gower Ramsey. We report our findings of the growth of vegetative and flower buds of ‘Honey Angel’ to clarify the relationship between current shoot growth and axillary bud development.
Materials and Methods
Plant materials.
Mature plants of vegetatively propagated Oncidesa Gower Ramsey ‘Honey Angel’ used during this study were cultivated in a commercial shade house in Taichung, Taiwan (24°11′ N, 120°45′ E). Plants were grown in 23.5-cm pots filled with gravel under 50% to 70% shade. Current shoots that were 10, 15, 20, 25, and 30 cm in length were collected for anatomical observation on 5 Jan 2021 (Fig. 1). The pseudobulb emerged from the leaf sheath when shoots reached 30 cm (Fig. 1E). There were 15 single shoot replications for shoot length and axillary bud length measurements at each growth stage. However, there were only five representative samples available for measuring the length of A0 on the 10-cm current shoot.
Appearance of Oncidesa Gower Ramsey ‘Honey Angel’ when the current shoot was 10 (A), 15 (B), 20 (C), 25 (D), and 30 cm (E). The 30-cm shoot was at the unsheathing stage.
Citation: HortScience 58, 3; 10.21273/HORTSCI16960-22
Measurements.
Length of the axillary bud at the base of the pseudobulb (A0) and buds on the descending nodes, A-1, A-2, A-3, and A-4, were measured by the vernier scale after removing the outer leaves. A0 on the 10-cm current shoot was young and very tender; therefore, it was easily damaged when removing the outer leaves. Therefore, A0 on the 10-cm current shoot was cut off, along with some tissues of the outer leaves; then, the bud length was measured using Image J (National Institutes of Health, Bethesda, MD) after paraffin sectioning.
Except for A0 on the 10-cm current shoot, axillary buds were measured before being severed from the current shoot. After paraffin sectioning, the node number of buds was determined by the number of bracts attached to the main axis. Additionally, we observed the morphological changes of the axillary buds.
Bud anatomical study.
The structures of the current shoot are illustrated in Fig. 2. Axillary buds on the 0th to -4th nodes (A0 to A-4) were collected from current shoots at various lengths for the next sectioning, with five single-bud replications each. The buds were fixed in an FPGA solution (5% volume/volume formalin, 5% volume/volume propionic acid, 15% volume/volume glycerol, 35% volume/volume alcohol) for 1 d and transferred to 50% alcohol three times for 10 min each time. Then, samples were dehydrated by a tertiary-butanol (TBA) series (10%, 20%, 35%, 55%, and 75% each for 1.5 h and 100% for 8 h; safranin O powder was added to 100% TBA) and placed into a 75 °C oven, infiltrated, and embedded with paraffin wax (Leica Biosystems, Nussloch, Germany) for 1 d. Samples were longitudinally sectioned into slides with a thickness of 10 to 15 μm using a rotary microtome (CE-25; Caesar Instrument Co. Ltd., Taiwan). The sections were stained with 6 g⋅L−1 hematoxylin (hematoxylin solution gill no. 3; Sigma Chemical Co., St. Louis, MO) and observed and photographed using an optical microscope (Eclipse E600; Nikon Instrument Inc., Tokyo, Japan).
Morphology of Oncidesa Gower Ramsey ‘Honey Angel’ when the current shoot was 10 cm (A), 15 cm (B), 20 cm (C), 25 cm (D), 25 cm with pseudobulb swelling (E), and 30 cm with pseudobulb unsheathing (F) (Ax: axillary buds).
Citation: HortScience 58, 3; 10.21273/HORTSCI16960-22
Statistical analysis.
CoStat software (version 6.4; CoHort Software, Monterey, CA) was used to statistically analyze data. We used SigmaPlot software (version 10.0; Systat Software, San Jose, CA) to plot figures. The least significant difference test at 5% probability was used to compare treatments when the analysis of variance showed significant differences between means.
Results
Development of pseudobulbs.
The current shoots at 10 to 30 cm were selected for observation (Fig. 1). After removing the outer leaves, we found that some shoots at 20 and 25 cm had already developed small column-shaped pseudobulbs (Fig. 2C and D). Pseudobulbs swelled and had a smooth surface when the current shoot length was 25 cm (Fig. 2E). The pseudobulb continued to develop and unsheathed when current shoots were approximately 30 cm in length (Fig. 1E). The unsheathing pseudobulb was spindle-shaped (Fig. 2F). The pseudobulb started to elongate from 3 to 44 mm as the current shoot grew from 20 to 30 cm in length (Fig. 3). Regarding the 25-cm shoots, among a total of 15 buds observed, 13 (87%) were similar to those in Fig. 2D, with the pseudobulbs being approximately 5.8 mm in height; however, two (13%) were like those in Fig. 2E, which apparently swelled (data not shown). However, compared with the size of the pseudobulb at the unsheathing stage, the sizes of the pseudobulb at the 25-cm stage were still much smaller (Fig. 2D–F).
The growth curve of the Oncidesa Gower Ramsey ‘Honey Angel’ pseudobulb. Each point represents the mean ± SE of 15 replications.
Citation: HortScience 58, 3; 10.21273/HORTSCI16960-22
When current shoots reached the 30-cm unsheathing stage, there were evident increases in the length, width, and thickness of the pseudobulbs. Among them, the pseudobulb length increased most obviously (Fig. 3).
Development of axillary buds.
Axillary buds develop on the nodes beneath the pseudobulb. When the current shoot was 10 to 15 cm, most of them had not developed a pseudobulb. There are usually three leaves above the pseudobulbs of current shoots at the 20- to 30-cm stages. The fourth leaf counted basipetally from the top of a current shoot was the one that was at the base of the pseudobulb and subtended A0 and the fifth leaf covered A-1. Hence, by counting the leaf number, we could estimate the A0 bud position before pseudobulb development.
When the current shoots grew from 10 to 25 cm, the lengths of the axillary buds that might develop into an inflorescence, A0 and A-1, especially A0, were smaller than the vegetative buds, A-2 to A-4, below them (Fig. 4). The A0 length was 2 mm shorter than that of A-1 during an early stage of the current shoot growth (Fig. 4). However, when the current shoot elongated from 25 to 30 cm, and while the pseudobulb was swelling, a rapid increase in length was observed for A0 and A-1, with an average of 7.1 to 13.7 mm, and their lengths were ultimately close to those of A-2 to A-4 (average of 13.3 mm) (Fig. 4). A-1 on the 30-cm current shoot was 4.8 mm longer than A-1 on the 25-cm shoot. Furthermore, A0 had a greater increase in length (8.3 mm), when the current shoot elongated from 25 to 30 cm (Fig. 4). When the current shoot was 10 to 15 cm, lower-position axillary buds, A-3 and A-4, were approximately 1 to 2 mm longer than A-2. The length of A-3 and A-4, in contrast, did not change as the current shoot grew from 25 to 30 cm, whereas the length of A-2 significantly increased by 3 mm (Fig. 4). At the unsheathing stage, the lengths of A0 to A-4 were the same (Fig. 4).
The growth curve of axillary buds originated from the 0th to -4th node of Oncidesa Gower Ramsey ‘Honey Angel’ on the current shoot. Each point represents the mean ± SE of 5 to 15 replications. NS, not significantly different. ***Significantly different at P ≤ 0.001 (A0-A-4: axillary buds). Means in the same column followed by different lowercase letter indicate significant differences among the axillary bud (Ax) lengths according to the least significant difference at P ≤ 0.05. Means within a row in each parameter followed by different uppercase letters are significantly different at P ≤ 0.05 according to the least significant difference test.
Citation: HortScience 58, 3; 10.21273/HORTSCI16960-22
The regression analysis showed a positive relationship between the current shoot length and A0 length (R2 = 0.71) (Fig. 5A). There was a similar trend for the pseudobulb length and A0 length (r = 0.95) (Fig. 5B).
Relationship between the A0 bud length and current shoot length (A). Relationship between the A0 bud length and pseudobulb length (B).
Citation: HortScience 58, 3; 10.21273/HORTSCI16960-22
The number of nodes on A0 remained unchanged when the current shoot grew from 10 to 20 cm, whereas the node number increased from 6.3 to 10.9 as the shoot grew from 20 to 30 cm. The node number on A-1 was 7.0, which was significantly lower than those of axillary buds on lower nodes when the current shoot was 10 cm. However, the number of A-1 nodes increased to 9.8 when the current shoot elongated to 15 cm; thereafter, it remained unchanged. The node number of A-2 to A-4 remained approximately 10.3 when the current shoot grew from 10 to 30 cm (Fig. 6).
The node number of the axillary buds on the 0th to -4th node of Oncidesa Gower Ramsey ‘Honey Angel’ current shoot. Each point represents the mean ± SE of four to five replications. NS, not significantly different. Significantly different at *P ≤ 0.05 and **P ≤ 0.01, respectively (A0-A-4: axillary buds). Means in the same row followed by a different lowercase letter are different at P ≤ 0.05 according to the least significant difference test.
Citation: HortScience 58, 3; 10.21273/HORTSCI16960-22
Inflorescence differentiation.
It was difficult to distinguish the inflorescence bud, A0, from the vegetative buds, A-2 to A-4, when the length of the current shoot was 10 to 20 cm (Figs. 7A–C and 8). At a shoot length of 25 to 30 cm, the cytoplasm in the axils on some A0 buds became denser, and branch primordia formed (Fig. 7D and E). We examined five A0 buds when the current shoot was 25 cm; two (40%) had developed branch primordia (Fig. 7D) and three (60%) looked similar to other lower axillary buds (Fig. 8), remaining vegetative. Nine replications of A0 buds were obtained for sectioning when the current shoot was 30 cm. Six of them (67%) had developed branch primordia (Fig. 7E), whereas three of them (33%) remained vegetative (Fig. 8). As stated, A0 formed branch primordia and could be distinguished from vegetative buds as the current shoot grew from 25 to 30 cm in length, when the pseudobulb started swelling. However, during this study, no branch primordium of A-1 was evident. Morphological features of A-1 appeared the same as those of the vegetative buds below it (A-2 to A-4) (Fig. 8).
Longitudinal section of the bud on the 0th node of Oncidesa Gower Ramsey ‘Honey Angel’ when the current shoot was 10 (A), 15 (B), 20 (C), 25 (D), and 30 cm (E). SP = shoot apex; B = bract; BP = branch primordium. The 30-cm shoot was at the unsheathing stage.
Citation: HortScience 58, 3; 10.21273/HORTSCI16960-22
Longitudinal section of a vegetative bud of Oncidesa Gower Ramsey ‘Honey Angel’ when the current shoot was 30 cm at the unsheathing stage. The A-2 bud is shown. SP = shoot apex; B = bract.
Citation: HortScience 58, 3; 10.21273/HORTSCI16960-22
Discussion
During this experiment, inflorescence initiation was only observed in A0, similar to that reported for Oncidesa Gower Ramsey (Shyu and Lin 1997), Oncidesa Goldiana (Hew and Yong 1994), and Gomesa Boissiense (Tanaka et al. 1986). Bracts formed on the main axis of the reproductive bud, covering branch primordia (Fig. 7D and E). Similarly, at the early inflorescence differentiation stage of Rhyncholaeliocattleya Shinfong Princess, bract primordia developed and became a closure area called the “sheath”; the growth rate of the sheath was faster than that of the shoot apex, which played a role in protecting the differentiating inflorescence (Wu et al. 2009). The bract number increased during the inflorescence development of Gomesa Boissiense, which indicated the increased node number (Tanaka et al. 1986).
The development of inflorescence of Gomesa Boissiense could be classified into three stages. First, bracts on the main axis formed and increased in number. Then, branch primordia formed on the axils of the main axis, covered by the bracts. During the third stage, bract primordia were differentiated on branches with increased node numbers (Tanaka et al. 1986).
Regarding Oncidesa Sweet Sugar ‘Million Gold’, during the initial differentiation phase of the reproductive bud, the apex of the bud had a dome shape and no protruding bracts. However, during the inflorescence primordium differential phase, bracts emerged and covered the apex, and the shoot apex started to elongate (Peng et al. 2012). During our previous investigation of Oncidesa Gower Ramsey ‘Honey Angel’, we found that bracts had formed and covered the apex of A0 when the current shoot was 5 cm (data not shown). During this experiment, we observed a similar bud shape when the current shoot was 10 to 20 cm (Fig. 7A–C).
The length and node number of A0 did not increase until the current shoot length reached 20 to 25 cm (Figs. 4 and 6). This suggested that the activation of inflorescence of Oncidesa Gower Ramsey ‘Honey Angel’ occurred in the current shoot at 20 to 25 cm in length.
During this study, the branch primordia became observable on the inflorescence bud after the pseudobulb had started swelling (the unsheathing stage), but the floret primordia had not appeared (Fig. 7D and E). Hence, the reproductive bud of ‘Honey Angel’ still did not enter the flower primordia differentiation phase when the current shoot was 30 cm in length. Chang and Lee (1999) noted that, for Oncidesa Gower Ramsey, floret primordia did not appear until the pseudobulb had unsheathed for 9 weeks.
The development and nutritional status of a pseudobulb have great correlations with the development of the inflorescence bud (Chang and Lee 1999; Hew and Yong 1994). When the current shoot length of ‘Honey Angel’ was shorter than 15 cm, the main change in morphology was the growth of leaves. Pseudobulbs had not developed during this phase, and the inflorescence bud had not activated either. Then, the pseudobulb started to elongate after the current shoot reached 20 cm in length (Fig. 3); furthermore, the inflorescence bud length (Fig. 4) and node number (Fig. 6) significantly increased.
The R2 value of the correlation between the current shoot lengths and the A0 length was 0.71 (Fig. 5A), whereas the r value of the correlation between the pseudobulb lengths and A0 length was 0.95 (Fig. 5B). This suggests that the pseudobulb length is a more accurate indicator for assessing the developmental stage of the inflorescence bud. However, removal of the leaf sheath is necessary and would destroy the shoot when measuring the pseudobulb length before plants enter the unsheathing stage. Although the R2 of the relationship is lower, the measurement of the current shoot length does not need to destroy the plants, which is a better means of assessing the development stage of the inflorescence bud.
Floral meristem and vegetative meristem are homologous structures; therefore, it is difficult to distinguish between them before buds undergo the floral transition stage. For many species, the floral meristems develop with leaf-like bracts (Bennett and Leyser 2006). Similar results were obtained during this study; before the branch primordium emerged, inflorescence buds (Fig. 7A–C) looked the same as vegetative buds (Fig. 8).
The change in the axillary bud length during this study (Fig. 4) agreed with that observed by Tanaka et al. (1986) when studying Gomesa Boissiense. During the early stage of axillary bud growth, the growth rates of A-2 and A-3 were higher than those of the upper nodes A0 and A-1.
A new Oncidesa shoot usually emerges from the -2nd to -3rd nodes (Hew and Yong 1994; Shyu and Lin 1997); however, the bud on the -2nd node has a better chance of developing into a shoot during the next growth phase (Shyu and Lin 1997). During our observations, the vegetative bud on the -2nd (A-2) node was largest when the current shoot was 30 cm in length. The bud on the -3rd node was the second largest, followed by the buds on the -4th and -1st nodes (Fig. 4). These results support the conclusion that the bud on the -2nd node was more mature and had the best opportunity to emerge at a later growing stage.
As the results of this study show, most inflorescence buds were differentiated from A0. Inflorescence branch primordia formed when the current shoot length was 25 or 30 cm. Pseudobulbs started to enlarge during this stage. The growth rate of the vegetative buds on lower nodes slowed. These results provide insight regarding the development of leaf axils of Oncidesa Gower Ramsey ‘Honey Angel’. The correlation of the developmental stage of inflorescence with the appearance of the current shoot provided the base information for regulating largely seasonal flowering and improving the profits of the cut flower industry.
References Cited
Bennett, T & Leyser, O. 2006 Something on the side: Axillary meristems and plant development Plant Mol Biol. 60 843 854 https://doi.org/10.1007/s11103-005-2763-4
Chang, YC & Lee, N. 1999 Growth, morphology and anatomy of pseudobulb and inflorescence in Oncidium Gower Ramsey (in Chinese with English abstract) J Chin Soc Hortic Sci. 45 87 99 https://doi.org/10.6964/JCSHS.199903.0087
Chatield, SP, Stirnberg, P, Forde, BG & Leyser, O. 2000 The hormonal regulation of axillary bud growth in Arabidopsis Plant J. 24 159 169 https://doi.org/10.1046/j.1365-313x.2000.00862.x
Chen, PB. 2019 Effects of shoot excision on the growth and flowering of Oncidesa & carbohydrate mobilization within detached inflorescence of Oncidesa (MS. Thesis; in Chinese with English abstract) National Taiwan University Taipei, Taiwan https://doi.org/10.4324/9781351119146-4
Council of Agriculture, Taiwan 2021 Trade statistics https://agrstat.coa.gov.tw/sdweb/public/trade/TradeCoa.aspx. [accessed 5 Oct 2022]
Hew, CS & Yong, JWH. 1994 Growth and photosynthesis of Oncidium ‘Goldiana’ J Hortic Sci. 69 809 819 https://doi.org/10.1080/14620316.1994.11516517
Hew, CS & Yong, JWH. 2004 A brief introduction to orchid morphology and nomenclature 11 23 Hew, CS & Yong, JWH The physiology of tropical orchids in relation to the industry. World Scientific Singapore https://doi.org/10.1142/9789812567215_0002
Hu, CJ, Lee, N & Lee, YI. 2018 Meiotic defects and premature tapetal degeneration are involved in the low fertility of Oncidesa Gower Ramsey, an important cut-flower orchid HortScience. 53 1283 1287 https://doi.org/10.21273/HORTSCI13011-18
McArtney, S & Obermiller, JD. 2015 Effect of notching, 6-benzyladenine, and 6-benzyladenine plus gibberellin A4+A7 on budbreak and shoot development from paradormant buds on the leader of young apple trees Technol Prod Rep. 25 233 237 https://doi.org/10.21273/HORTTECH.25.2.233
Peng, F, Tian, M, Wang, CX & Wei, PX. 2012 Morphological and anatomical characteristic along with nutrient content changes during flower bud differentiation in Oncidium (in Chinese with English abstract) J Zhejiang A&F Univ. 29 7 11 https://doi.org/10.1007/s40415-022-00792-6
Shyu, HE & Lin, RS. 1997 Studies on growth and flowering of Oncidium (in Chinese with English abstract) Hortic Natl Chun Hsin Univ. 22 123 134 https://doi.org/10.17660/ActaHortic.1988.218.16
Tanaka, M, Yamada, S & Goi, M. 1986 Morphological observation on vegetative growth and flower bud formation in Oncidium Boissiense Sci Hortic. 28 133 146 https://doi.org/10.1016/0304-4238(86)90133-0
Wang, CY, Chiou, CY, Wang, HL, Krishnamurthy, R, Venkatagiri, S, Tan, J & Yeh, KW. 2008 Carbohydrate mobilization and gene regulatory profile in the pseudobulb of Oncidium orchid during the flowering process Planta. 227 1063 1077 https://doi.org/10.1007/s00425-007-0681-1
Wu, GL, Sun, Y, Shen, GZ & Xi, DJ. 2009 Studies on anatomy of flower bud differentiation and development in Cattleya (in Chinese with English abstract) J Zhejiang Univ. 35 173 179 https://doi.org/10.1002/j.1537-2197.1951.tb14806.x
Yong, JWH & Hew, CS. 1995 The importance of photoassimilate contribution from the current shoot and connected back shoots to inflorescence size in the thin-leaved sympodial orchid Oncidium Goldiana Plant Sci. 156 450 459 https://doi.org/10.1086/297267