Abstract
Chinese cymbidiums are important flowering ornamental plants. Traditional propagation via seed or division cannot satisfy growers’ demand for commercialization of new cultivars, and in vitro propagation has a low micropropagation efficiency due to the browning of rhizomes. In this study, rhizomes of Cymbidium ‘14-16-13’ and ‘14-16-5’ were cultured on half-strength Murashige and Skoog (MS) medium supplemented with 6-benzyl aminopurine (BAP), NAA (α-napthaleneacetic acid), or BAP with NAA under either the dark or light. The degree of browning was read, and rhizome proliferation or sprouting (sprout numbers) was evaluated. Results showed that there was significant difference in browning grade of rhizomes between ‘14-16-13’ and ‘14-16-5’ regardless of dark and light culture. Dark culture induced rhizome proliferation but failed to induce sprouts. Light culture slightly elevated the degree of browning but induced sprouting. Among the growth regulators evaluated, BAP was more effective for sprout induction. As rhizome browning appeared to be inevitable in micropropagation of the cymbidiums, a compromise between browning and sprout production could be a realistic approach. Our study showed that rhizomes cultured on half-strength MS medium supplemented with 1.5 mg·L−1 BAP were able to produce more than 16 sprouts per vessel even though browning occurred in the rhizomes. Thus, culturing rhizomes in this medium could be a practical solution for in vitro propagation of Chinese cymbidiums.
Cymbidium Sw. is one of the most important orchid genera valued by their attractive flowers, faint fragrance, and elegant straight leaves (Zeng et al. 2020). There are three main types of cymbidiums: epiphytic, terrestrial, and lithophytic (Balilashaki et al. 2022). The terrestrial species, also known as Chinese cymbidiums, including Cym. sinense, Cym. ensifolium, Cym. faberi, Cym. kanran, and Cym. goeringii, have been cultivated since the time of Confucius (551–479 BC) and remain popular as important ornamental flowering plants in eastern Asian (Liu et al. 2017). Traditionally, cymbidiums are propagated through seed germination and the division of pseudobulbs. Due to the poor seed germination rate in the nature and limited availability of pseudobulbs, their propagation has been shifted to micropropagation (Chang and Chang 2000; Huang and Fang 2021; Shimasaki and Uemoto 1991). The micropropagation efficiency of terrestrial Cymbidium species, however, is generally lower compared with epiphytic species and their hybrids (Liu et al. 2017; Shimasaki and Uemoto 1991). Additionally, the routes for in vitro shoot formation are different between temperate and tropical Cymbidium species. Temperate species first form rhizomes from explants before shoot formation, while tropical species first form protocorm-like bodies (PLBs) followed by shoot appearance (Ogura-Tsujita and Okubo 2006).
In vitro culture of axillary buds or tissues of Chinese cymbidiums often leads to the formation of rhizomes (Chang and Chang 1998; Shimasaki and Uemoto 1991). When seeds were in vitro cultured, they initially form PLBs, which then developed into rhizomes (Shimasaki and Uemoto 1991). Rhizomes grow slowly and take several months to produce a limited number of plantlets (Gao et al. 2014; Huang and Fang 2021; Ueda and Torikata 1968), which significantly affects micropropagation efficiency. To improve the efficiency, effects of culture media (Chugh et al. 2009), plant growth regulators (Park et al. 2018), culture methods and conditions (Chang and Chang 2000; Gao et al. 2014; Ueda and Torikata 1968), medium supplementations (Huang and Fang 2021; Lee et al. 2011) on rhizome proliferation and sprouting were investigated. Molecular analysis showed that rhizome sprouting in Cymbidium was correlated with the differential expression of YUC and GH3-like genes, which in turn affected the active IAA content of rhizome in vitro (Liu et al. 2017). These efforts have resulted in some improvement in the micropropagation efficiency, but in general, it still remains difficulty to meet the demand for large-scale propagation.
A morphological marker frequently associated with the low micropropagation efficiency is the browning of rhizomes. Browning has been considered a major constraint in plant tissue culture (Cai et al. 2020; Duan et al. 2019), which is believed to be caused by the accumulation and oxidation of phenolic compounds (Jones and Saxena 2013). The browning results in the inhibition of growth and decrease in the regeneration ability, which restricts the application of tissue culture technology in many species (Arnaldos et al. 2001; Chugh et al. 2009; Mondal et al. 2013). Factors associated with browning include genotype (Hirimburegama and Gamage 1997; Huang et al. 2002), endophyte (Pirttilä et al. 2008), explant (Gow et al. 2009), light (Farrokhzad et al. 2022; Gow et al. 2009), salt concentration (Hildebrandt and Harney 1988), amino acid (Huang and Fang 2021), sugar type and concentration (Dong et al. 2016), temperature (Hildebrandt and Harney 1988), medium pH (Huang et al. 2002; Zhao et al. 2006), and antioxidant agents (Duan et al. 2019; Irshad et al. 2017; Li et al. 2021; Mitsukuri et al. 2009; Teixeira Da Silva 2013). Browning is ubiquitous during micropropagation of Cymbidium, especial in Chinese cymbidiums (Begum et al. 1994; Chang and Chang 1998; Chen et al. 2005; Gao et al. 2014; Huang et al. 2017; Huang and Fang 2021; Tao et al. 2011; Teixeira Da Silva 2013). It is well-known that exogenous plant growth regulators, auxin and cytokinin in particular, play an important role in induction, proliferation, and differentiation of intermediate propagules (Gaspar et al. 1996; Novak et al. 2014). However, there were only a few reports concerning the efforts of auxin and cytokinin on browning of selected plant species (Abohatem et al. 2011; Mohamed and Jayabalan 1996; Roy and Banerjee 2003). Little effort thus far has been made on systematic evaluation of growth regulators, genotypes, and light on rhizome browning of cymbidiums and subsequently shoot initiation.
The objectives of this study were to evaluate the effects of genotype, light, and plant growth regulators on rhizome browning, proliferation, and sprouting and to establish a protocol for in vitro propagation of cymbidiums with limited degree of browning but increased shoot production. We believed that a compromise between browning and sprouting could be a practical approach presently for increasing supply of propagules for commercial production of cymbidiums, particularly those new cultivars.
Materials and Methods
Plant materials.
Cym. ‘14-16-13’ and Cym. ‘14-16-5’ were developed from the same cross of Cym. Jade Hare ‘2011-2’ × Cym. ensifolium ‘Yinzhen’. Morphologically, the two cultivars are similar in growth form and slightly different in flower shapes. Rhizomes were induced from in vitro cultured shoot tips of two cultivars. Shoot tips were derived from seedlings subcultured in a 60-d interval on hormone-free, half-strength MS medium (Murashige and Skoog 1962) supplemented with sucrose 30 g⋅L−1 and carrageenan powder 7 g⋅L−1 at 25 ± 2 °C under dark conditions. The pH of the medium was adjusted to 5.8 with 1 M NaOH or HCl before autoclaving at 121 °C for 20 min. Rhizomes from the two cultivars were cut into 0.8 cm in length and inoculated on a 230 mL glass vessel containing 30 mL of hormone-free half-strength MS medium. In each vessel, 15 rhizomes were inoculated and cultured at 25 ± 2 °C under dark conditions for 60 d. The newly formed rhizomes were used for the following experiments.
Grading of browning.
Newly formed rhizomes of ‘14-16-13’ after removing the apical meristem, were cut into about 0.8 cm in length and evenly inoculated on a glass vessel containing 30 mL of the medium which were prepared in the same way as mentioned above. Twenty rhizomes were inoculated in each vessel, and a total of 12 vessels were prepared. They were cultured under 12-hour photoperiod with a light intensity of 18 μmol·m−2·s−1 provided by cool-white, fluorescent lamps at 25 ± 2 °C. Rhizome browning was monitored daily, and browning was graded according to the criteria as follows: Grade 0, no browning in rhizomes and in the culture medium (Fig. 1A); Grade 1, browning occurring in the incision surface of rhizomes (arrow), no browning in the medium (Fig. 1B); Grade 2, obviously browning occurring in the rhizomes, but their growth or differentiation (arrow) was normal, medium became slight browning (Fig. 1C); Grade 3, rhizomes became serious browning and their growth or differentiation (arrow) was inhibited, medium became serious browning (Fig. 1D).
Criterion of rhizome browning grading in Cymbidium. (A) Grade 0, rhizome not browning, medium not browning; (B) Grade 1, incision surface of rhizome browning (arrow), medium not browning; (C) Grade 2, rhizome obvious browning but its growth or differentiation (arrow) was normal, medium slight browning; (D) Grade 3, rhizome serious browning and its growth or differentiation (arrow) was inhibited, medium serious browning.
Citation: HortScience 58, 6; 10.21273/HORTSCI17119-23
Rhizome browning of two cultivars cultured with different growth regulators in the dark.
Newly formed rhizomes of ‘14-16-13’ and ‘14-16-5’, after removing the apical meristem, were cut into ∼0.8 cm in length and evenly inoculated on glass vessels containing 30 mL of half-strength MS medium supplemented with 0.5 mg⋅L−1 NAA, 0.5 mg⋅L−1 BAP, 0.5 mg⋅L−1 BAP with 0.1 mg⋅L−1 NAA, or no hormones at all. The choice of the growth regulators and their concentrations was based on our preliminary studies with cymbidiums. Twenty rhizomes were inoculated in each vessel, with a total of 12 vessels per treatment, which were cultured in the dark. The initial fresh weight of the rhizomes per vessel was weighed with an analytical balance in a laminar flow. Each vessel was considered as an experimental unit. The experiment was arranged as a completely randomized design with 12 replications. After 45 d of culture, rhizome browning was scored based on the aforementioned criteria. The final fresh weight of the rhizomes per vessel was weighed. A proliferation coefficient was calculated as the final fresh weight divided by the initial fresh weight.
Rhizome browning of two cultivars cultured with different growth regulators under light.
This experiment was conducted in the same way as that performed in the dark, but rhizome explants were cultured under 12-hour photoperiod at a light intensity of 18 μmol·m−2·s−1 provided by cool-white, fluorescent lamps. After 45 d, rhizome browning was scored as mentioned previously. The number of sprouts induced from rhizomes in each vessel was recorded.
Effects of different BAP concentrations on rhizome browning and sprouting under light.
To identify an appropriate concentration of BAP for limited browning, but increased shoot regeneration, rhizomes of ‘14-16-13’ with 0.8 cm in length without the apical meristem were inoculated on half-strength MS medium supplemented with 1.0, 1.5, and 2.0 mg·L−1 BAP as well as 2.0 mg·L−1 BAP + 500 mg·L−1 vitamin C (VC), respectively. The medium was prepared in the same way as mentioned previously except for half-strength MS + 2.0 mg⋅L−1 BAP + 500 mg⋅L−1 VC, in which 1 mL filter-sterilized solution of VC (500 mg⋅mL−1) was added to 1 L half-strength MS medium containing 2.0 mg⋅L−1 BAP when its temperature reached 45–50 °C. In each culture vessel, 20 rhizomes were inoculated. A total of 12 vessels were prepared for each treatment. Again, the experiment was arranged as a completely randomized design with 12 replications. The inoculated material was cultured at 25 ± 2 °C under 12-hour photoperiod provided by cool-white, fluorescent lamps at a light intensity of 18 μmol·m−2·s−1. After 45 d of culture, the browning grade and the number of sprouts formed from rhizome per vessel were recorded.
Statistical analysis.
After checking normal distribution of the collected data, analysis of variance was performed using SPSS 24 (IBM Corporation, Somers, NY). If significant differences occurred among treatments, means were separated by Duncan’s multiple comparison method at P < 0.05 level. Student’s t test was used to determine cultivar differences in browning, differentiation, or sprout numbers at P < 0.05 level.
Results
Browning grade and rhizome proliferation coefficient affected by genotypes and growth regulators in the dark culture.
Cultivars exhibited different levels of browning depending on growth regulator treatments. For example, the degrees of browning in ‘14-16-13’ cultured with 0.5 mg⋅L−1 NAA or 0.5 mg⋅L−1 BAP + 0.1 mg⋅L−1 NAA was significantly higher than those of ‘14-16-5’ (Table 1). However, there were no significant differences in proliferation coefficient between cultivars cultured at growth regulator treatment except for the treatment of 0.5 mg⋅L−1 BAP + 0.1 mg⋅L−1 NAA. Culture medium supplemented with growth regulators increased the level of browning except for the treatment supplemented with 0.5 mg⋅L−1 NAA. On the other hand, different growth regulators appeared to have a significant effect on rhizome proliferation coefficients. The proliferation coefficient of rhizomes cultured on medium supplemented with 0.5 mg⋅L−1 NAA was significantly higher than that of the other treatments, indicating that NAA plays a role in rhizome proliferation. A distinct characteristic of dark culture was that new rhizomes were produced from existing rhizomes, but no shoots or sprouts were induced from the rhizomes regardless of cultivars or growth regulator treatments (Fig. 2).
The browning grade and proliferation coefficient of Cymbidium rhizomes affected by cultivars and growth regulators during in vitro culture under dark conditions.
The browning and proliferation of rhizomes after 45 d of culture on half-strength Murashige and Skoog (MS) medium supplemented different hormones. Rhizomes of ‘14-16-5’ cultured with (A) 0 mg⋅L−1 BAP + 0 mg⋅L−1 NAA, (B) 0.5 mg⋅L−1 NAA, (C) 0.5 mg⋅L−1 BAP, and (D) 0.5 mg⋅L−1 BAP + 0.1 mg⋅L−1 NAA as well as rhizome of ‘14-16-13’ cultured with (E) 0 mg⋅L−1 BAP + 0 mg⋅L−1 NAA, (F) 0.5 mg⋅L−1 NAA, (G) 0.5 mg⋅L−1 BAP, and (H) 0.5 mg⋅L−1 BAP + 0.1 mg⋅L−1 NAA. BAP = 6-benzyl aminopurine; NAA = α-napthaleneacetic acid.
Citation: HortScience 58, 6; 10.21273/HORTSCI17119-23
Browning grade and sprouting were affected by genotypes and growth regulators when cultured under light.
The browning grade of rhizomes cultured with each growth regulator treatment under 12-h photoperiod at the light intensity of 18 μmol·m−2·s−1 appeared to be elevated to some extent compared with corresponding rhizomes cultured under the dark (Fig. 3, Table 2). Significant differences in browning grade occurred between cultivars. Rhizomes of ‘14-16-13’ had significantly higher levels of browning than those of ‘14-16-5’. The results also showed that the addition of NAA alone in medium had little effect on browning grade, whereas supplement of BAP alone significantly increased the browning grade. In contrast to the dark culture, rhizomes cultured with 0.5 mg⋅L−1 BAP or 0.5 mg⋅L−1 BAP + 0.1 mg⋅L−1 NAA produced sprouts, of which BAP alone induced significantly higher numbers of shoots (Fig. 3, Table 2). However, there were no differences in mean sprout numbers between cultivars.
The browning and sprouts differentiation of rhizome after 45 d of culture on half-strength Murashige and Skoog (MS) medium supplemented different hormones. Rhizomes of ‘14-16-5’ cultured with (A) 0 mg⋅L−1 BAP + 0 mg⋅L−1 NAA, (B) 0.5 mg⋅L−1 NAA, (C) 0.5 mg⋅L−1 BAP, and (D) 0.5 mg⋅L−1 BAP + 0.1 mg⋅L−1 NAA as well as rhizomes of ‘14-16-13’ cultured with (E) 0 mg⋅L−1 BAP + 0 mg⋅L−1 NAA, (F) 0.5 mg⋅L−1 NAA, (G) 0.5 mg⋅L−1 BAP, and (H) 0.5 mg⋅L−1 BAP + 0.1 mg⋅L−1 NAA. BAP = 6-benzyl aminopurine; NAA = α-napthaleneacetic acid.
Citation: HortScience 58, 6; 10.21273/HORTSCI17119-23
The browning grade and average number of differentiated sprouts of Cymbidium rhizomes affected by cultivars and growth regulators during in vitro culture under a 12-h photoperiod with light intensity of 18 μmol·m−2·s−1.
Browning grade and sprout numbers were induced by different BAP concentrations when cultured under light.
Because 0.5 mg⋅L−1 BAP induced both cultivars to produce a comparable number of sprouts, rhizomes of ‘14-16-13’ were induced by BAP at 1.0, 1.5, and 2.0 mg⋅L−1 as well as BAP at 2.0 mg⋅L−1 with 500 VC to optimizing sprout induction (Table 3). The degree of browning was comparable among the treatments except 1.0 mg⋅L−1 BAP, which had significantly lower browning (Fig. 4). The mean numbers of sprouts induced by the growth regulator treatments were also similar except for 2.0 mg⋅L−1 BAP without addition of VC, which had significantly lower numbers of sprouts.
The browning grade and average number of differentiated sprouts of rhizomes of Cym. ‘14-16-13’ affected by different concentrations of BAP during in vitro culture under a 12-h photoperiod with light intensity of 18 μmol·m−2·s−1.
The browning of ‘14-16-13’ rhizomes and their sprouting after 45 d of culture on half-strength Murashige and Skoog (MS) medium supplemented different concentrations of BAP with or without vitamin C. (A) 1.0 mg⋅L−1 BAP, (B) 1.5 mg⋅L−1 BAP, (C) 2.0 mg⋅L−1 BAP, and (D) 2.0 mg⋅L−1 BAP + 500 mg⋅L−1 vitamin C. BAP = 6-benzyl aminopurine; NAA = α-napthaleneacetic acid.
Citation: HortScience 58, 6; 10.21273/HORTSCI17119-23
Discussion
Browning has been considered as a limiting factor influencing plant micropropagation (Abohatem et al. 2011; Duan et al. 2019; Gow et al. 2009; Kaewubon et al. 2015; Li et al. 2021; Mahendran and Bai 2015). There are two types of tissue browning, enzymatic and nonenzymatic (Martinez and Whitaker 1995). Enzymatic browning is mediated by a group of enzymes including peroxidase, polyphenol oxidase, and L-phenylalanine ammonialyase that are largely inactive in cell membranes (Li et al. 2015; Ru et al. 2013). The substrates of these enzymes are phenolic compounds that stay in vacuoles. When tissues are wounded, the enzymes meet with substrates, resulting in the oxidation of phenolic compounds to unstable o-quinones and then polymerize to the appearance of browning pigments, such as melanins (Holderbaum et al. 2010; Martinez and Whitaker 1995). The abundance of the substrates and activity of the enzymes in wounded tissues determine the quickness and degree of browning (Martinez and Whitaker 1995). Nonenzymatic browning includes the Maillard reaction and auto-oxidation reaction with phenolic compounds resulting in the formation of iron-phenol complexes (Martinez and Whitaker 1995). Cymbidiums and orchids in general are rich in phenolic compounds (Lv et al. 2022; Shubha and Chowdappa 2016). The excision of rhizomes likely activates those enzymes, resulting the browning. Gao et al. (2014) also reported the incised parts of the rhizomes of cymbidium became brown after 20 d of culture in bioreactors. Thus, enzymatic browning has been an obstacle for in vitro propagation of cymbidiums.
Our results showed that cymbidium genotypes differed significantly in rhizome browning. The grade of browning in ‘14-16-13’ was significantly higher than ‘14-16-5’ even when rhizomes were cultured on medium without growth regulators (Tables 1 and 2). Genotypic differences in browning have been documented in other cymbidium cultivars and other crops. Cym. ‘Xiaofeng’, which was developed from the cross between Cym. Maureen Carter ‘Dafeng’ and Cym. sinense ‘Qijianbaimo’, had the same morphological appearance as Cym. sinense ‘Qijianbaimo’ and enabled it to be efficiently micropropagated. However, Cym. sinense ‘Qijianbaimo’, one of its parents, was not easily propagated due to its browning resulting in low regeneration ability (Liu et al. 2017). Ficus carica ‘Seungjung Dauphine’ showed less incidence of tissue browning and had excellent budbreak after 4 weeks of culture initiation. But other F. carica cultivars (Banane, Brunswick, Violet Dauphine, and King) had poor budbreak and even the death of cultured explants after the first 5 weeks of culture; the difficulty in budbreak was mainly attributed to the release of phenolic compounds from dissected buds (Kim et al. 2007). Genotypic differences in browning may be due to the differences in the abundance of phenolic compounds and/or polyphenol oxidase activities between the cultivars. Phenolic compounds and polyphenol oxidase activities in cymbidium cultivars will be determined in our future research.
Rhizomes cultured in the dark showed slightly lower grades of browning compared with those cultured under light (Tables 1 and 2). This is likely attributed to light-enhanced activities of polyphenol oxidase and/or the production of phenolic compounds. However, dark culture only induced the proliferation of rhizomes, that is, increased fresh weights but was unable to induce sprouting. On the other hand, rhizome culture under light resulted in sprouting. Light has been well documented for regulation of plant regeneration due to the role of phytochrome (Ikeuchi et al. 2016; Reuveni and Evenor 2007). Light and cytokinins have been documented to connect with the developmental process (Chory et al. 1994), including regeneration. This is true in the present study, as rhizomes cultured under light with BAP significantly increased sprout numbers (Table 2).
The higher number of sprouts induced by BAP under light might indicate that Chinese cymbidiums could be practically propagated in vitro from browned rhizomes, meaning a compromise between browning and sprout numbers is needed for micropropagation of the cymbidiums. Although other cytokinins need to be tested, our study showed that BAP is effective in inducing sprouting. BAP has been shown to be the optimal hormone to delay fresh-cut lettuce from browning, and RNA-sequencing analysis suggested that the delay was attributed to BAP-mediated transcriptional regulation of phenolic-related metabolite biosynthesis (Liu et al. 2022). It is possible that BAP at an appropriate concentration delays biosynthesis of the phenolic compound, and the delay provides a window for BAP to induce tissue differentiation and sprouting.
To optimize the sprout induction by BAP, BAP at different concentrations was evaluated. BAP at 1.5 mg⋅L−1 induced more than 16 sprouts per vessel, although sprout numbers induced by the other BAP treatments were not significantly different from it except 2.0 mg⋅L−1 BAP, which resulted in a decrease in sprout numbers. However, addition of vitamin C to this medium increased sprout numbers, suggesting the role of vitamin C in minimizing detrimental effects of phenolic compounds, which has been documented in apple by El-Shimi (1993) and in pistachio by Marín et al. (2016). Nevertheless, under the current circumstance, we believe that culture of rhizomes on half-strength MS medium supplemented with 1.5 mg⋅L−1 BAP under light is a protocol for in vitro culture of Chinese cymbidiums. It is certain that further investigation is warranted to understand the browning process and methods for suppressing polyphenol oxidase activity and improving the micropropagation efficiency of this group of orchids.
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