Abstract
Cymbidium faberi, a member of the Cymbidium genus known for its fragrant blooms and graceful foliage, has recently become endangered in the wild due to reproductive challenges. This study aimed to establish systematically a tissue culture system for Cymbidium faberi Rolfe (wild species) by evaluating the effects of various plant growth regulators its propagation stages, including rhizome proliferation, differentiation, shoot strengthening, and rooting. The results showed that 0.5 mg·L−1 thidiazuron significantly promoted rhizome proliferation, achieving a proliferation coefficient of 6.08 after 60 days of culture. For adventitious bud induction, 1.92 mg·L−1 brassinolide was most effective, inducing 6.43 buds per rhizome with an average bud height of 5.25 mm after 90 days of culture. The optimal strategy for shoot growth was using 3.0 mg·L−1 1-naphthaleneacetic acid, resulting in an average shoot height of 6.47 cm after 60 days. The highest rooting rate of 87.5% was achieved with 0.5 mg·L−1 zeatin, producing an average of 3.5 roots per shoot with an average root length of 3.06 cm. This study successfully developed a propagation system for C. faberi and highlighted the significant role of BL in promoting rhizome differentiation. In conclusion, this study provides a robust propagation method to support the conservation and industrial development of C. faberi.
The Orchidaceae is one of the largest and most widespread families of flowering plants, with 704 accepted genera (Royal Botanic Gardens 2024). Cymbidium, an economically important flowering genus in Orchidaceae, includes 145 accepted species worldwide (Royal Botanic Gardens 2024). Cymbidium faberi is known for its pastel floral hues, exquisite color, and fragrant flowers. Global cultural exchange is increasing, and as a result, orchids are steadily spreading over the globe and gaining popularity. C. faberi is listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora due to the need for fungal symbiotic breeding, anthropogenic overharvesting, and habitat destruction (Matters 2020). It is difficult to germinate in natural settings, and its growth cycle is lengthy. The traditional propagation of C. faberi mainly relies on division, but the propagation coefficient is low, making it unable to meet market demands. Plant tissue culture technology is the main technology used for the regeneration and rational development and use of plant resources of endangered orchids.
The mature tissue culture approach of C. faberi uses seeds to produce protocorm by nonsymbiotic germination. According to recent research on nonsymbiotic germination, Fu et al. (2018) discovered that Cymbidium seeds could germinate up to 80% in a medium containing 0.5 mg·L−1 6-benzylaminopurine (BA) + 0.1 mg·L−1 naphthaleneacetic acid (NAA). Therefore, a sterile germination medium can be prepared based on the formulation from the aforementioned literature, allowing seeds to germinate into protocorms, which then form rhizomes, providing abundant experimental materials for subsequent in vitro culture.
Protocorms are first created in C. faberi tissue culture by aseptic germination and quickly develop into rhizomes. After rhizome proliferation, the addition of plant growth regulators (PGRs) induces the differentiation of rhizomes into adventitious buds. These buds then develop into robust shoots, further rooting to obtain complete tissue culture seedlings. These seedlings are subsequently acclimatized and transplanted from the tissue culture bottles to the external environment. To speed up and increase the number of reproductions, this study concentrated on these crucial stages to investigate the effects of various PGRs on the reproduction coefficient of C. faberi explants. The rhizome is a proliferative organ specific to orchids that is a part of the underground transformation stem. Because rhizomes grow slowly, it takes several months to induce buds and develop into seedlings (Huang and Fang 2021; Park et al. 2018). Rhizome growth can be effectively stimulated by adding 0.5 μmol·L−1 melatonin (MT) (Huang et al. 2022) or 0.1 mg·L−1 NAA (Chiang et al. 2010) to the media. In other Cymbidium species, 20 μmol·L−1 2,4-dichlorophenoxyacetic acid (2,4-D) (Park et al. 2018) and 1.0 μmol·L−1 MT (Huang et al. 2022) effectively stimulated the rhizome growth of C. goeringii. NAA also promoted the rhizome growth of C. sinense, C. Jade Hare ‘2011-2’ × C. ensifolium ‘Yinzhen’ (Peng et al. 2023).
Under the influence of PGRs, the rhizome of C. faberi can differentiate and produce adventitious buds. More than 90% of the rhizomes initiated shoots of C. faberi by a combination of low concentrations of NAA (0.5 or 1.0 mg·L−1) and high concentrations of 6-BA (2.0 or 5.0 mg·L−1) (Chen et al. 2005). Studies in other plants also showed that bud development of Vanda bicolor was stimulated from the primary bulb by high doses of 6-BA and low NAA levels (Deb et al. 2022). Furthermore, 2.0 mg·L−1 6-BA was useful for rhizome differentiation of Oncidium ‘Sugar Sweet’ (Yang et al. 2010). Recently, the rhizome of C. Jade Hare ‘2011-2’ × C. ensifolium ‘Yinzhen’ elicited more than 16 adventitious buds when exposed to 1.5 mg·L−1 6-BA (Peng et al. 2023). Moreover, C. goeringii can promote young buds with 6-BA and thidiazuron (TDZ) (Park et al. 2018). Many scholars have demonstrated that TDZ can effectively induce adventitious buds in rhizomes (Baskaran et al. 2014; Kher et al. 2014; Lakshmanan and Taji 2000). Previously, Chiang et al. (2010) showed that 0.1 mg·L−1 TDZ was the most beneficial for inducing the number of buds in C. sinense, with the number of buds reaching 5.9, significantly better than that of 6-BA (1.0, 3.0, and 5.0 mg·L−1). However, 3.0 mg·L−1 6-BA had a better effect on the height promotion of small buds. In addition, different species of orchids require various concentrations of PGRs. The treatment of 5.0 μmol·L−1 MT was the most conducive to inducing adventitious buds of C. faberi, whereas only 1.0 μmol·L−1 MT was required for C. goeringii (Huang et al. 2022). In other plants, 4.0 or 5.0 mg·L−1 zeatin (ZT) and TDZ added to the medium increased Trillium govanianum’s adventitious buds induction rate by up to 75% (Chandola et al. 2023). Additionally, Salvia dominica generated 6.3 buds when exposed to 1.2 mg·L−1 TDZ alone (Al-Qudah et al. 2023). Further, 0.4 mg·L−1 TDZ + 0.5 mg·L−1 NAA could effectively induce adventitious buds of ‘RED SUN’ Phalaenopsis aphrodite, and the reduced concentration (0.2 mg·L−1 TDZ + 0.4 mg·L−1 NAA) was more conducive to adventitious bud propagation (Zhang et al. 2022). Bud induction of Citrus suhuiensis was significantly impacted by 3-indole butyric acid (IBA) and kinetin (KT) (Puad et al. 2023). The simultaneous addition of 6-BA, IBA, and TDZ produced more than 94% uncertain bud differentiation in gray poplar (P. tremula × P. alba) (Li et al. 2023a). In a related study of Vanilla odorata, adding 1.0 mg·L−1 6-BA/TDZ and 0.5 to 1.0 mg·L−1 IBA induced 75% adventitious buds (Warner et al. 2023). However, Pang et al. (2007) also discovered that 1,(4-chcorophency)-4,4-dimethy-2,(1,2,3,4-tialol-1-y1)Pentan-3-ol (PP333) could successfully stimulate floral bud differentiation at certain concentrations to support tillering (Guoping 1997). Further research is needed to understand fully the rhizome differentiation and proliferation in the C. faberi tissue culture system. It is clear that during the adventitious bud differentiation and rhizome proliferation stages of various plant species, the kinds and quantities of PGRs differ dramatically.
After the adventitious buds are induced, it is necessary to carry out shoot strengthening and rooting to establish a complete tissue culture system. These two stages of cultivation also need PGRs. A total of 3.0 mg·L−1 6-BA has been demonstrated to be helpful for C. sinense shoot strengthening and root formation (Chiang et al. 2010). Furthermore, the roots and shoots of C. giganteum grew better at 0.909 μmol·L−1 TDZ (Roy et al. 2012). For ‘RED SUN’ Phalaenopsis aphrodite, 0.4 mg·L−1 NAA was found to be efficient in regeneration after boosting the shoot (Zhang et al. 2022), whereas 2.0 μmol·L−1 NAA was used for rooting of C. goeringii (Park et al. 2018). Moreover, 0.5 mg·L−1 IBA promoted the rooting of C. faberi (Tao et al. 2011) and ‘RED SUN’ Phalaenopsis aphrodite (Zhang et al. 2022). The concentration of 0.4 mg·L−1 MT directly induced the rooting of C. geringii (Huang et al. 2022). The rooting of Cleome droserifolia was 85% with 7.42 μmol·L−1 IBA (Ghareb and Mustafa 2023). One hundred percent rooting of Mammillaria vetula was achieved by adding 0.1 mg·L−1 NAA, 0.3 mg·L−1 KT, and 5.0 mg·L−1 ancymidol to the medium at the same time (Lopez-Granero et al. 2023). Recently, 0.1 mg·L−1 NAA has had the best effect on gray poplar (P. tremula × P. alba) (Li et al. 2023a). The preceding results showed that PGRs such as 6-BA, NAA, and IBA are frequently added to the culture medium during the seedling and rooting stages. Because the types and concentrations of PGRs vary greatly and have different effects on cultivation, it is necessary to investigate PGRs systematically.
There have been many applications of new PGRs in tissue culture research, such as BL and 6-phenoxy-1-phenyl-2-(1H-1,2,4-triazol-1-yl) hexan-1-one (Tis108). BL is the active form of brassinosteroids (BRs) that act in the special development process of plants, has a unique physiological effect different from that of traditional hormones, and can exert strong physiological activity at low levels (Zajączkowska and Pacholczak 2024). It is important in promoting plant cell growth and division, seedling growth, and regeneration. It can regulate various processes of plant growth (Rozhon et al. 2019), such as adventitious bud regeneration (Kim et al. 2008), root growth (Bao et al. 2004; Li et al. 2023c; Nolan et al. 2020, 2023; Planas-Riverola et al. 2019). Low concentrations of BL promoted root elongation and lateral root development (Bao et al. 2004), whereas high concentrations inhibited root elongation (Hu et al. 2016; Kurepin et al. 2016). 0.04 μmol·L−1 BL promoted root growth in Arabidopsis thaliana (González-García et al. 2011), and increased the BL concentration to 1.0 μmol·L−1 also promoted rooting in seedlings of Tagetes erecta ‘Marvel’ (Li et al. 2023c). It was further found that 3.0 to 6.0 g·L−1 exogenous BL promoted the growth of the seedlings of Musa acuminata, and the concentration of more than 12 g·L−1 had an inhibitory effect on the seedling growth of the seedlings of Musa acuminata (Zakaria et al. 2018). Additionally, strigolactones (SLs) are a novel class of plant hormones that have been thoroughly investigated in terms of their biological activity and function. Jiu et al. (2021) found that SL influences mesocotyl elongation, seed germination, growth and development, inhibition of branching, and the plant’s response to various stress conditions. This result confirms that SL can control mesocotyl elongation, promote leaf senescence, regulate axillary bud growth in plants, promote leaf senescence, and inhibit branching (Gomez-Roldan et al. 2008). Previous studies and screenings of SL synthesis inhibitors have revealed that 2,2-dimethyl-7-phenoxy-4-(1H-1,2,4-triazol-1-yl) heptan-3-ol (Tis13) and Tis108 have biological activity that can stimulate the formation of lateral roots, induce elongation of root hairs, and promote plant branch growth (Ito et al. 2013; Kawada et al. 2019). According to Jia (2022), the synthetic analog of rac-GR24 and its synthetic inhibitor Tis108 can enhance the growth, development, and nutrient absorption of apples. The effect of Tis108 was less pronounced than that of rac-GR24, suggesting that Tis108 can somewhat lower SL levels in Oryza sativa (Ito et al. 2011). Although PGRs such as BL and Tis108 have some regulatory effect on plant growth, it is unknown how they affect Orchidaceae tissue culture.
In conclusion, the proliferation and differentiation of rhizomes of Cymbidium is an important link to limiting their rapid reproduction. This study systematically explored the effects of different PGRs and their concentrations on important stages of the tissue culture of C. faberi, including the proliferation and differentiation of rhizomes and the strengthening and rooting of seedlings to optimize the tissue culture system of wild C. faberi. Importantly, we obtained the optimal medium for these different stages and found that 1.92 mg·L−1 BL had an important effect on the differentiation of rhizomes of C. faberi. On the other hand, a low concentration of BL could promote the root growth of C. faberi. This study can provide strong technical support for conserving the germplasm resources of C. faberi and promote the production and industrial development of C. faberi germplasm.
Materials and Methods
Plant materials
Wild C. faberi was manually pollinated on a sunny day in February. Five capsules each with an embryonic age of 150 d were collected from one plant. The capsules were rinsed for 15 min under running water, immersed for 15 min in a cleaning solution made of laundry detergent, and then washed with water. The capsules were then moved to the ultra-cleaning table, where they underwent three rounds of sterile water washing, one soak in 75% alcohol for 5 min, and another three rounds of sterile water washing. The capsules were then soaked in 1% sodium hypochlorite solution for 20 min and washed with five rounds of sterile water. Finally, the capsules were cut in half lengthwise using a sterile scalpel, and all the seeds were scraped onto sterile paper to be mixed. Subsequently, the seeds were dispersed uniformly throughout the medium by shaking them with a small amount of water on their surface. The medium formulation was based on the study by Fu et al. (2018) and consisted of MS + 0.5 mg·L−1 6-BA + 0.1 mg·L−1 NAA + 100 mL·L−1 coconut milk + 1.0 g·L−1 active charcoal (AC) + 20.0 g·L−1 sucrose (Su) + 7.8 g·L−1 agar (Ag). A total of 20 vials were inoculated using tissue culture flasks with a capacity of 500 mL, each containing 100 mL of medium. The number of germinations was counted 4 months after aseptic sowing.
Effects of different PGRs on the rhizome proliferation of C. faberi
Referring to the study of Lee et al. (2011), the control medium for rhizome proliferation was MS + 1.0 mg·L−1 NAA + 0.5 mg·L−1 6-BA + 1.0 g·L−1 AC + 30.0 g·L−1 Su + 7.8 g·L−1 Ag. Different concentrations of PGRs were added separately to the control medium: 0.1, 0.5, 1.0, 1.5 mg·L−1 TDZ; 0.1, 0.5, 1.0, 1.5 mg·L−1 2,4-D; 0.12, 0.24, 0.48, 0.96 mg·L−1 BL; 0.1, 0.5, 1.0, 1.5 mg·L−1 KT; 0.1, 0.5, 1.0, 1.5 mg·L−1 ZT; and 0.1, 0.5, 1.0, 1.5 mg·L−1 IBA (Table 1). Tissue culture bottles with a capacity of 500 mL were used, each containing 100 mL of medium. Each treatment was inoculated with 10 tissue culture bottles of medium, each bottle containing eight rhizomes (1.5 cm).
Culture medium component of Cymbidium faberi for rhizome proliferation, differentiation, shoot strengthening, and shoot rooting.
Effects of different PGRs on the rhizome differentiation of C. faberi
The TDZ concentration was optimized based on the research conducted by Chiang et al. (2010). The control medium for rhizome differentiation was 0.22 mg·L−1 TDZ coupled with MS + 100.0 g·L−1 banana puree + 20.0 g·L−1 Su + 7.8 g·L−1 Ag. PGRs were added to the control-based medium separately at 0.48, 0.96, 1.92, 3.84 mg·L−1 BL; 0.03, 0.15, 0.30, 0.44 mg·L−1 PP333; 0.06, 0.12, 0.24, 0.48 mg·L−1 MT; and 0.17, 0.34, 0.51, 0.68 mg·L−1 Tis108 (Table 1). Tissue culture bottles with a capacity of 500 mL were used, each containing 100 mL of medium. Each treatment was inoculated with 10 tissue culture bottles of medium, each bottle containing eight rhizomes (1.5 cm).
Effects of different PGRs on shoot strengthening of C. faberi
The control medium for shoot strengthening was MS + 1.0 g·L−1 AC + 30.0 g·L−1 Su + 7.8 g·L−1 Ag. PGRs were added to the control-based medium separately at 0.1, 0.5, 1.0, 1.5 mg·L−1 2,4-D; 0.5, 1.0, 1.5, 2.0 mg·L−1 IBA; 0.12, 0.24, 0.48, and 0.96 mg·L−1 BL; 0.1, 0.5, 1.0, 1.5 mg·L−1 ZT; and 1.0, 2.0, 3.0, and 4.0 mg·L−1 NAA (Table 1). Tissue culture bottles with a capacity of 500 mL were used, each containing 100 mL of medium. Each treatment involved inoculating five tissue culture bottles with six shoots (2.5 cm high) per bottle.
Effects of different PGRs on the shoot rooting of C. faberi
The control medium was 4.0 mg·L−1 NAA coupled with MS, 1.0 g·L−1 AC, 30.0 g·L−1 Su, and 7.8 g·L−1 Ag based on pre-experiments. PGRs were added to the control-based medium separately at 0.1, 0.5, 1.0, 1.5 mg·L−1 2,4-D; 0.5, 1.0, 1.5, 2.0 mg·L−1 IBA; 0.1, 0.5, 1.0, 1.5 mg·L−1 ZT; and 0.12, 0.24, 0.48, 0.96 mg·L−1 BL (Table 1). Tissue culture bottles with a capacity of 500 mL were used, each containing 100 mL of medium. Each treatment involved inoculating five tissue culture bottles with six rootless shoots (2.5 cm high) per bottle.
Effects of different transplanting substrates on seedling growth of C. faberi
The seedlings with consistent growth were selected and transplanted from sterile tissue culture bottles to pots. The height of transplanted tissue culture seedlings was 8 to 10 cm, the number of roots was 3 to 5, and the average length of roots was 3 to 6 cm. The seedlings were placed in a greenhouse from the sterile culture room and subjected to hardening seedlings for 2 d with the lid closed to acclimatize to the light intensity. Then the lid remained half open for 2 d. Finally, a small amount of tap water was poured into the bottle, and the lid was fully opened for 3 d. After hardening, the seedlings were taken out of the tissue culture bottle, and the medium attached to the roots was washed. Then the seedlings were soaked in 0.5 mg·L−1 IBA solution for 3 min and dried. Transplanting substrates used in the experiment were divided into two categories: organic substrates, such as pine bark, coir dregs, moss, and inorganic substrates, such as perlite and vermiculite. Pine bark, moss, and coir dregs were used to separate planting, and there were proportional plantings, including pine bark + moss = 2:3, pine bark + perlite + vermiculite = 3:1:1. The seedlings of C. faberi were planted in plastic pots with drainage holes at the bottom, two seedlings per pot. After planting, the roots were immediately watered. For 10 d following transplanting, water was sprayed on the leaf surface twice daily, once in the morning and once at night. Watering and fertilizing were made every 3 d throughout the later stage. The rate of survival was measured 20 d post-transplant.
Culture conditions
All mediums had a pH of 5.6 ± 0.2. The incubation environment was maintained at 25 ± 2 °C with a 16-h photoperiod under cool white light (40 to 50 μmol·m−2·s−1), and relative humidity was 75% ± 5%.
Data statistics and analysis
Rhizome proliferation.
The number of new growth points and the weight of rhizomes were recorded after 60 d. The quantity proliferation coefficient was calculated using the ratio of the total number of new growth points on all rhizomes to the total number of inoculated rhizomes. The weight proliferation coefficient was calculated using the ratio of the fresh weight of rhizomes after 60 d of culture to the rhizomes before culture.
Rhizome differentiation.
The quantity and height of the new buds on each rhizome were noted 90 d after incubation. Only buds with a height of at least 0.2 cm were counted. The average number of buds induced (the number of newborn buds/rhizomes inoculated) and the average bud height (the sum of the height of buds induced/total number of rhizomes inoculated) were determined.
Shoot strengthening.
The mean fresh weight (the total fresh weight/total number of shoots) and mean height (the sum of shoots height/total number of shoots) of each treatment were measured after a 60-d incubation period.
Shoot rooting.
Following 60 d of incubation, the rooting rate (the ratio of rooted plants to inoculated plants; only roots with a length of at least 0.2 cm were counted), the average number of roots (the total number of roots/the total number of shoots), and the average length of roots (the sum of the root length/the total number of shoots) were computed.
Shoot transplanting.
The survival rate was recorded 20 d after transplantation. The survival rate (%) was determined by dividing the number of surviving shoots by the number of transplanted shoots. Additionally, the leaf color and root growth status were also observed.
The test data were statistically analyzed using SPSS 21.0 and Excel. Duncan’s multiple comparison analysis was used to determine the significance of the differences between the various treatments (P < 0.05). Analysis and plotting were performed using GraphPad Prism 8.
Results
Effects of different PGRs on the rhizome proliferation of C. faberi.
The findings of this study demonstrate that rhizome proliferation and differentiation, seedling growth, and rooting capacity could all be markedly enhanced by adding specific concentrations of PGRs to the culture media. In this study, the seed germinates to form protocorms 4 months after sowing, with the germination rate reaching 82% and then forming rhizomes immediately.
The quantity and weight proliferation coefficients were noted and tallied to comprehend the impact of distinct PGRs on the growth of C. faberi rhizomes (Supplemental Table 1). This investigation showed that the C. faberi rhizomes treated with six PGRs alone had a variable influence on proliferation (Figs. 1 and 2; Supplemental Table 1). The 0.5 mg·L−1 TDZ treatment was the most beneficial for increasing the proliferation coefficient of rhizomes, with the number of new growth points reaching 6.08 and the weight gain multiple reaching 2.03 (Fig. 1A). The rhizomes were also tender green without browning (Fig. 2B). Additionally, the rhizome weight proliferation coefficient and the number of new growth points of the control treatment were 1.35 and 3.88, respectively. The most effective treatments among each PGRs for promoting new growth points were 0.5 mg·L−1 TDZ (Figs. 1A and 2B), 0.24 mg·L−1 BL (Figs. 1C and 2D), 0.5 mg·L−1 ZT (Figs. 1E and 2F), and 0.1 mg·L−1 IBA (Figs. 1F and 2G). The number of new growth points was substantially higher than that of the control. Moreover, the weight of C. faberi rhizomes under 0.1 and 0.5 mg·L−1 TDZ (Fig. 1A), 1.0 and 1.5 mg·L−1 KT (Fig. 1D), 0.1 mg·L−1 ZT (Fig. 1E), and 0.1 and 1.5 mg·L−1 IBA (Fig. 1F) was considerably higher than that of the control. Regarding both the quantity and weight proliferation coefficient, there was no discernible difference between the control and different 2,4-D concentrations (Fig. 1B and Supplemental Table 1). On the other hand, the number of neonatal points of rhizomes at various KT concentrations (Figs. 1D and 2E) and the control did not differ statistically. A low BL concentration promoted the growth of C. faberi rhizomes more than a high BL concentration. A high BL concentration resulted in the browning of rhizomes. Browning of the rhizomes was more pronounced under high concentrations of BL than under other PGRs (Fig. 2I). Additionally, rhizomes also appeared when the concentration of TDZ increased to 1.5 mg·L−1. The 0.5 mg·L−1 TDZ treatment resulted in the highest quantity and weight proliferation coefficients (Supplemental Table 1). The optimal concentration of TDZ to promote the weight and new growth point of the C. faberi rhizomes was 0.5 mg·L−1.
Effect of different plant growth regulators on the quantity and weight proliferation coefficients of Cymbidium faberi rhizomes after 60 d of incubation. (A–F) The left Y axis (green) is the quantity proliferation coefficient, and the right Y axis (blue) is the weight proliferation coefficient. Different lowercase letters indicate significant differences at the P < 0.05 level.
Citation: HortScience 59, 9; 10.21273/HORTSCI17803-24
Effect of different plant growth regulators on the proliferation of Cymbidium faberi rhizomes after 60 d of incubation. Bars = 0.5 cm.
Citation: HortScience 59, 9; 10.21273/HORTSCI17803-24
Effects of different PGRs on the rhizome differentiation of C. faberi.
The average number and height of buds emerging from these rhizomes under various treatments for PGRs were noted and analyzed to investigate the effect of PGRs on the differentiation of C. faberi rhizomes (Supplemental Table 2). These two indicators can fully evaluate the ability of different treatments to induce adventitious buds (Figs. 3 and 4; Supplemental Table 2). The results showed that different treatments of BL (Figs. 3A, 3E, and 4B), PP333 (Figs. 3B, 3F, and 4C), MT (Figs. 3C, 3G, and 4D), and Tis108 (Figs. 3D, 3H, and 4E) promoted adventitious buds induced by rhizomes of C. faberi. The number of adventitious buds induced by the rhizomes of C. faberi under the control treatment was only 2.16, and the bud height was 3.63 mm (Figs. 3 and 4A). 1.92 mg·L−1 BL was best suited for rhizome differentiation; bud height was 5.25 mm (Fig. 3E), and the number of buds induced from rhizomes was 6.43 (Fig. 3A). Some rhizomes were able to grow up to 10 buds. The second-best differentiation effect was achieved by0.03 mg·L−1 PP333 (Fig. 3F and Supplemental Table 2). Additionally, the concentration of PP333 at 0.15 mg·L−1 significantly increased the number of adventitious buds (Fig. 3B), and 0.03 mg·L−1 PP333 was beneficial to the growth of adventitious buds height of 5.55 mm (Fig. 3F), which was higher than that of other PGRs. Under various treatments of PGRs, the number of adventitious buds caused by 0.96, 1.92, and 3.84 mg·L−1 BL (Fig. 3A) and 0.15, 0.30, and 0.44 mg·L−1 PP333 (Fig. 3B) was considerably higher than that of the control. Furthermore, although the number of adventitious buds induced under MT (Fig. 3C) and Tis108 (Fig. 3D) treatments increased, no significant difference existed between them and the control (Supplemental Table 2). Similarly, there was no significant difference in adventitious bud height between MT treatment and control (Fig. 3G). The height of adventitious buds under 0.17 and 0.34 mg·L−1 Tis108 treatment was significantly higher than that of the control (Figs. 3H and 4E). Browning was positively associated with the differentiating effect of PGR on rhizomes. The control showed the least amount of browning (Fig. 4A). Rhizomes treated with BL showed the most severe browning (Fig. 4B), with PP333 showing the second-most severe browning (Fig. 4C). The rhizomes treated with MT (Fig. 4D) and Tis108 (Fig. 4E) showed browning, but not obviously. The findings demonstrated that all four PGRs had greater differentiating effects on rhizomes than the control, with 1.92 mg·L−1 BL being the most helpful in increasing adventitious buds, with a significantly higher number than other treatments (Supplemental Table 2). The most effective treatment for adventitious bud height was 0.03 mg·L−1 PP333; however, there was no statistically significant difference between it and the second-ranked treatment, 1.92 mg·L−1 BL. To induce adventitious buds in rhizomes, 1.92 mg·L−1 BL was shown to have the most efficacy.
Effect of different plant growth regulators on the differentiation of Cymbidium faberi rhizomes after 90 d of incubation. (A–D) Average number of buds induced per rhizome. (E–H) Average bud height. Different lowercase letters indicate significant differences at the P < 0.05 level.
Citation: HortScience 59, 9; 10.21273/HORTSCI17803-24
Effect of different plant growth regulators (PGRs) on the differentiation of Cymbidium faberi rhizomes after 90 d of incubation. (A–E) Optimal concentration of each PGR for differentiation. (F–J) Enlarged image of a single rhizome. Bars = 0.5 cm.
Citation: HortScience 59, 9; 10.21273/HORTSCI17803-24
Effects of different PGRs on the shoot strengthening of C. faberi.
The effects of several PGRs on C. faberi shoots were investigated by statistically analyzing the differences in each shoot’s height and weight after 60 d of incubation (Supplemental Table 3). The results showed that the shoots under control treatment weighed 0.12 g and were 1.65 cm tall (Figs. 4 and 5A). The sturdiness of C. faberi shoots was affected by 2,4-D MT (Figs. 3C, 3G, and 4D),(Figs. 5A, 5F, and 6B), IBA (Figs. 5B, 5G, and 6C), BL (Figs. 5C, 5H, and 6D), ZT (Figs. 5D, 5I, and 6E) and NAA (Figs. 5E, 5J, and 6F). Among them, shoots treated with 0.1, 0.5, 1.0, and 1.5 mg·L−1 2,4-D showed larger heights than the control (Figs. 5A and 6B). On the other hand, only 0.1, 0.5, and 1.0 mg·L−1 2,4-D could significantly increase shoot weight, and the shoot weight was inhibited under the 1.5 mg·L−1 treatment (Fig. 5F). When the concentration of IBA was 0.5, 1.0, 1.5, and 2.0 mg·L−1, the height of the shoots was higher than that of the control (Figs. 5B and 6C). However, the weight was lower than the control when the concentration was raised to 2.0 mg·L−1 (Fig. 5G). At lower BL concentrations (0.12 and 0.24 mg·L−1), shoot height increased more slowly. However, a notable rise in shoot height was observed when the concentration was raised to 0.48 mg·L−1 (Figs. 5C and 6D). The shoot weights were greater than the control at all four BL concentrations. However, the impacts on shoot weights were not statistically significant at 0.12 and 0.96 mg·L−1 concentrations (Fig. 5H). Compared with the control, each concentration of ZT had a significant effect on shoots height (Fig. 5D), and 0.5 mg·L−1 ZT had the best effect (Fig. 6E). In terms of seedling weight, only 0.5 mg·L−1 ZT had a significant promoting effect (Fig. 5I). Further research revealed that 3.0 mg·L−1 NAA was the most conducive to the strong growth of C. faberi shoots among the four PGRs mentioned earlier (Supplemental Table 3). The height and weight of the shoots were 6.47 cm (Fig. 5E) and 0.28 g (Fig. 5J), respectively. In summary, the medium containing 3.0 mg·L−1 NAA exhibited the greatest impact on shoot strengthening.
Effect of different plant growth regulators on shoot strengthening of Cymbidium faber after 60 d of incubation. (A–E) Shoot height. (F–J) Shoot fresh weight. Different lowercase letters indicate significant differences at the P < 0.05 level.
Citation: HortScience 59, 9; 10.21273/HORTSCI17803-24
Effect of different plant growth regulators (PGRs) on shoot strengthening of Cymbidium faberi after 60 d of incubation. (A–F) Optimal concentration of each PGR for shoot strengthening. Bars = 0.5 cm.
Citation: HortScience 59, 9; 10.21273/HORTSCI17803-24
Effects of different PGRs on the shoot rooting of C. faberi.
When shoots were cultivated for 60 d with various PGRs, the number and length of their roots were counted (Supplemental Table 4). The effects of these PGRs on seedling rooting were investigated by evaluating the rooting rate, average number of roots, and root length (Figs. 7 and 8; Supplemental Table 4). Under control conditions, the number of roots was 1.17, the root length was 1.33 cm, and the rooting rate was 19.45% (Supplemental Table 4). Treatment with 2,4-D, IBA, ZT, and BL generally enhanced root number and length (Figs. 7 and 8). Among these, 2,4-D treatments at various concentrations significantly increased root number (Fig. 7A and E) and length (Figs. 7I and 8B), with a rooting rate of 78.13% observed at 0.5 mg·L−1 2,4-D, higher than other concentrations. IBA treatments did not significantly improve shoot rooting compared with the control (Fig. 7G, F, and J). Furthermore, more roots were formed by the 0.1, 0.5, and 1.0 mg·L−1 ZT treatments than by the control (Fig. 7G; Supplemental Table 4). The results also indicate that the highest rooting rate, 87.5%, was achieved with 0.5 mg·L−1 ZT (Fig. 7C), which also produced the highest number of roots at 3.5 (Fig. 7G). However, no rooting was observed at high ZT concentrations of 1.5 mg·L−1. Additionally, increasing BL concentration gradually decreased rooting ability, with a rooting rate of 68.75% at 0.12 mg·L−1, and no rooting at 0.96 mg·L−1 (Fig. 7D, H, and L). Significantly longer root lengths compared with the control were noted with treatments of 1.0 mg·L−1 2,4-D, 0.5 mg·L−1 ZT, and 0.12 mg·L−1 BL (Supplemental Table 4). No significant difference was observed in the rooting effects of any IBA treatments. The longest roots, measuring 3.25 cm, were achieved with 1.0 mg·L−1 2,4-D treatment (Fig. 7I; Supplemental Table 4). In summary, 0.5 mg·L−1 ZT was the most effective PGR for increasing the number and rate of roots, followed by 0.5 mg·L−1 2,4-D. The longest root lengths were seen with 1.0 mg·L−1 2,4-D, although differences were not significant compared with 1.5 mg·L−1 IBA, 0.5 mg·L−1 ZT, and 0.12 mg·L−1 BL treatments (Supplemental Table 4). Thus, 0.5 mg·L−1 ZT is recommended for rooting C. faberi (Figs. 7and 8).
Effect of different plant growth regulators on rooting of Cymbidium faberi shoots after 60 d of incubation. (A–D) Rooting rate (%). (E–H) Average root number. (I–L) Average root length. Different lowercase letters indicate significant differences at the P < 0.05 level.
Citation: HortScience 59, 9; 10.21273/HORTSCI17803-24
Effect of different plant growth regulators (PGRs) on the rooting of Cymbidium faberi shoots after 60 d of incubation. (A–E) Optimal concentration of each PGR for rooting. Bars = 0.5 cm.
Citation: HortScience 59, 9; 10.21273/HORTSCI17803-24
Effects of different transplanting substrates on seedling growth of C. faberi.
The materials for this experiment, tissue culture seedlings of 0.5 mg·L−1 ZT, were the seedlings with the highest rooting rate in the rooting studies described earlier. Statistical analysis was performed to examine how different transplanting substrates affected the growth of C. faberi seedlings. The results (Table 2; Fig. 9) indicated that distinct transplanting substrates had varying impacts when cultured under the same circumstances. Using pine bark alone has poor water retention but good air permeability and will not cause root rot, and only some roots will break. As a green organic compound fertilizer, moss has a special water-absorbing structure, which can play a role in water and fertilizer retention (Lueth and Reski 2023). Under the treatment of moss, the leaves of the C. faberi are verdant, and some of the roots appear to rot. Moss + pine bark could make the survival rate of C. faberi seedlings the highest, and the survival rate of C. faberi seedlings was as high as 100% 20 d after transplanting, and the root growth was healthy. Coir dregs also retain water, although the root can grow rapidly. Still, because the coconut shell has adsorption of nitrogen, the seedlings absorb less nitrogen (Wang and Yang 2015), the roots grow slender and black under long-term cultivation, and the final survival rate is low. In addition, the treatment of pine bark + perlite + vermiculite could adjust the pH of the substrate, make up for the shortcomings of poor water retention of pine bark, and improve the survival rate of seedlings, reaching 99.50% 20 d after transplanting. The fleshy roots were thick and strong, and the survival rate was higher than that of pine bark alone. In summary, the optimal substrate ratio was found to be pine bark + moss at a 2:3 ratio.
Effects of matrix components on the growth of Cymbidium faberi (20 d after transplanting).
Cymbidium faberi of different periods after transplanting: (A) 20 d after transplanting, (B) 1 year after transplanting, (C) flowering stage, and (D) fruiting stage.
Citation: HortScience 59, 9; 10.21273/HORTSCI17803-24
The high seedling survival rate in this study, achieved by treating each group of seedlings with five substrates in varying ratios, demonstrates the adaptability of tissue culture-grown seedlings. This finding is supported by Chen et al. (2005), who showed that domesticated tissue-cultured plants have no significant issues.
Discussion
In this study, the effects of various plant growth regulators (PGRs) on the rhizome proliferation, differentiation, shoot strengthening, and rooting of C. faberi were thoroughly investigated. In rhizome proliferation experiments, the control was MS medium incorporated with 1.0 mg·L−1 NAA + 0.5 mg·L−1 6-BA. We designed the concentration ratio because Lee et al. (2011) concluded that 1.0 mg·L−1 NAA + 0.5 mg·L−1 6-BA added to the Hyponex medium was suitable for shoots induced from hybrid rhizomes of C. sinense. Our previous pre-experiment used this PGR combination to induce adventitious buds from rhizomes. Still, we found that no buds were obtained, but it was more suitable for the proliferation of rhizomes, with a quantity proliferation coefficient of 3.88 (Supplemental Table 1). Therefore, we added PGRs such as TDZ on 1.0 mg·L−1 NAA + 0.5 mg·L−1 6-BA to find the best medium for rhizome proliferation to further improve the efficiency of rhizome proliferation.
The study showed that TDZ, 2,4-D, BL, KT, ZT, and IBA had varying effects on the rhizome proliferation of C. faberi. In previous studies, TDZ was used more for rhizome budding (Baskaran et al. 2014; Kher et al. 2014; Lakshmanan and Taji 2000). In this study, low concentrations of TDZ (0.5 mg·L−1) significantly increased the number of new growth points in rhizomes (up to 6.47) (Figs. 1A and 2B), supporting Lu’s (1993) hypothesis that low doses of TDZ can promote tissue culture. However, higher concentrations of TDZ led to rhizome browning and inhibited proliferation. Rhizome browning seriously when TDZ concentration reached 1.5 mg·L−1 (Fig. 2H) demonstrated that browning will inhibit further growth. Similarly, elevated concentrations of BL caused severe browning, inhibiting further growth (Fig. 2I). Although BL has been shown to enhance proliferation in other species (Kuno and Ji 1996), in this study, only low concentrations were effective, and the weight proliferation effect was not significant compared with the control. Overall, TDZ was the most effective for C. faberi rhizome proliferation, whereas BL was less effective.
The differentiation of rhizomes and the concentration of PGRs have a complicated relationship (Lu et al. 1994). The results of adventitious bud induction by PGRs in this work demonstrated that BL, PP333, and Tis108 promote rhizome differentiation in C. faberi (Figs. 3 and 4). The best differentiation impact on rhizomes was caused by BL at high concentration (1.92 mg·L−1), some rhizomes produced up to 10 adventitious buds (Fig. 4B), which was substantially different from that of the control (Fig. 3A). Similar studies in other plants such as Nicotiana tabacum (Kim et al. 2008) and Brassica oleracea var. boturytis (Kawada et al. 2019) have demonstrated BL’s ability to promote adventitious bud regeneration. It was also found that PP333 was second only to BL in differentiation (Figs. 3B, 3F, and 4C). Similar reports have shown that PP333 can promote tillering and change leaf structure (Guoping 1997; Li et al. 2023b). Consistent with the results in this paper, PP333 had the best effect on the height of adventitious buds, which was significantly higher than that of other treatments (Supplemental Table 2). Furthermore, related studies have shown that specific MT concentrations promote the induction of adventitious buds in C. goeringii and C. faberi rhizomes (Huang et al. 2022), and Tis108 regulates mesocotyl elongation (Hu et al. 2010). The findings of this study demonstrated that Tis108 (Figs. 3D, 3H, and 4E) and MT (Figs. 3C, 3G, and 4D) promoted the differentiation of C. faberi rhizomes. Both PP333 and BL had stronger impacts on rhizome differentiation compared with MT and Tis108. Additionally, adventitious bud induction was positively associated with browning intensity, with high BL concentrations causing more severe browning.
According to the findings of the C. faberi, 2,4-D, IBA, BL, ZT, and NAA promoted shoot strengthening to varying degrees (Figs. 5 and 6). NAA treatments significantly enhanced shoots, with 3.0 mg·L−1 NAA having the strongest effect, producing the highest shoot height of 6.47 cm (Figs. 5E and 6F). As the most commonly used auxin, NAA was also used in the shoot strengthening stage of ‘RED SUN’ Phalaenopsis aphrodite, but the concentration was only 0.4 mg·L−1 (Zhang et al. 2022). In contrast, this study found that C. faberi needed a higher concentration of NAA. It has been demonstrated that NAA plus 6-BA works well and effectively strengthens seedlings in space Phalaenopsis (Guo et al. 2016) and Paphiopedilum henryanum (Li et al. 2020). The results indicate that different Cymbidium species have different requirements for the types and concentrations of PGRs.
Various doses of 2,4-D, BL, and ZT were found to influence shoot rooting (Figs. 7 and 8). The highest rooting rate was observed with 0.5 mg·L−1 ZT (Figs. 7C, 7G, 7K, and 8D), significantly greater than other PGRs, although ZT’s effects on shoot roots in C. faberi tissue culture are not well documented. Similar studies on the rooting effect of ZT have been conducted in other plants, such as ZT promoting root development of Fragaria ananassa (Debnath 2006). Prior research on orchid shoot rooting relied on TDZ (Roy et al. 2012), 6-BA (Chiang et al. 2010), MT (Huang et al. 2022), NAA (Park et al. 2018). The rooting of C. faberi shoots was likewise observed to be positively impacted by 2,4-D (Fig. 7A, E, and I) and IBA (Fig. 7B, F, and J). In the study of ‘RED SUN’ Phalaenopsis aphrodite (Zhang et al. 2022) and C. faberi (Tao et al. 2011), IBA was also reported to have similar functions. However, this study showed that IBA did not significantly promote rooting. Furthermore, BL is thought to be advantageous for root growth because it can stimulate cell division and elongation during root growth (Nolan et al. 2020, 2022; Planas-Riverola et al. 2019). According to previous research (Bao et al. 2004; Hu et al. 2016; Kurepin et al. 2016), a high concentration of BL inhibited root growth, whereas the rooting of BL-treated shoots in this study was consistent with previous studies. A low concentration of BL had a greater induction effect on roots than a high concentration. This study can be used as a preliminary attempt to study the application of BL in orchid tissue culture, and the mechanism of BL in vitro remains to be further explored.
Conclusion
The results of this study provide a comprehensive basis for improving the propagation efficiency of C. faberi. Specifically, 0.5 mg·L−1 TDZ is optimal for rhizome proliferation, 1.92 mg·L−1 BL for adventitious bud induction, 3.0 mg·L−1 NAA for shoot strengthening, and 0.5 mg·L−1 ZT for rooting. Additionally, the study highlights the nuanced effects of BL, in which high concentrations promote bud induction but low concentrations hinder rooting. These findings contribute to the broader understanding of PGR applications in orchid tissue culture and offer practical guidelines for the effective propagation of C. faberi. Further research is recommended to explore the underlying mechanisms of BL in vitro and to refine the application of these PGRs for other orchid species.
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