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Article

Mutagenesis and Flowering Promotion through Sodium Azide In Vitro Culture of Cymbidium faberi Rolfe

by
Zhengjing Wu
*,
Sujuan Liu
,
Bingjie An
,
Hao Zhang
,
Jingjing Wu
,
Chenfang Li
and
Yuan Long
College of Horticulture and Plant Protection, Henan University of Science and Technology, Luoyang 471000, China
*
Author to whom correspondence should be addressed.
Horticulturae 2024, 10(8), 889; https://doi.org/10.3390/horticulturae10080889
Submission received: 24 June 2024 / Revised: 29 July 2024 / Accepted: 21 August 2024 / Published: 22 August 2024
(This article belongs to the Section Floriculture, Nursery and Landscape, and Turf)
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Abstract

:
Cymbidium faberi Rolfe is one of the traditional Chinese orchids with important ornamental value, and the cultivation of Cymbidium faberi Rolfe mutant strains with different appearances is essential to increase its economic value. However, at present, their acquisition largely relies on natural mutation. The objectives of this research were to mutagenize Cymbidium faberi Rolfe protocorm-like bodies (PLBs) and shoots in vitro using sodium azide (NaN3) and to screen and evaluate mutants in the mutagenized seedlings using morphological characteristics. Cymbidium faberi Rolfe PLBs and shoots were used as mutagenic materials. Mutations were induced by the addition of 0.0 (control), 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg·L−1 and 0.0 (control), 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 mg·L−1 NaN3 to a shoot-growth-inducing medium. The mortality rates of the PLBs and shoots increased with an increase in the NaN3 concentration. At 14 d of co-cultivation, the PLBs and shoots were most efficiently mutagenized with 1.5 mg·L−1 and 4.0 mg·L−1 NaN3, respectively. After the explants were cultured for 3 months, changes in leaf and flower morphology were observed in some mutants: shorter and thicker leaves, shorter node length, reduced height, and mid-translucent leaves compared with controls. Some Cymbidium faberi Rolfe bloomed prematurely, with single flowers with large, thick petal sepals and small inflorescences. Colors included light green throughout, with some exhibiting purple stamens. This suggests that NaN3 can effectively mutagenize Cymbidium faberi Rolfe PLBs and shoots to satisfy people’s demand for this plant’s ornamental properties while increasing its economic value.

1. Introduction

Cymbidium is a terrestrial plant of the genus Cymbidium Sw. in the family Orchidaceae, native to China. It is also known as Summer Cymbidium and Nine-section Orchid [1], and is a second-class-protected wild plant with a long history of cultivation. It is a tall plant, with fragrant and attractive flowers and elegantly erect leaves [2,3], which makes it of significant ornamental and economic value [4] in East Asia [5].
The natural fruiting rate of wild Cymbidium faberi Rolfe is only 7.5% [6]. Cymbidium faberi Rolfe capsules, like most Orchidaceae, contain millions of seeds; however, the seeds are extremely tiny and are devoid of any metabolic mechanisms, have no endosperm or cotyledons, and contain an underdeveloped embryo within the testa. The testa contains only lipid droplets and a small amount of protein and lacks the support nutrients needed for germination processes [7,8,9]. This plant requires specific fungi for natural germination [10], and although large numbers of seeds are produced, few germinate in nature and wild Cymbidium faberi Rolfe resources are becoming increasingly scarce. Therefore, the study of Cymbidium faberi Rolfe in vitro tissue culture and the establishment of a regeneration system are extremely helpful for the conservation, innovation, and utilization of Cymbidium faberi Rolfe germplasm resources. It has been demonstrated that media with low inorganic salt concentrations are suitable for Cymbidium faberi Rolfe seed germination, and coconut juice promotes the induction and proliferation of PLBs [11]. The addition of banana juice to the medium facilitates the differentiation of PLBs [12]. There are fewer systematic studies on Cymbidium faberi Rolfe in terms of fast propagation via histoculture. In this study, a complete system of Cymbidium faberi Rolfe regeneration was established from seed germination to the proliferation of PLBs, seedling emergence, rooting, and final transplantation.
In cultivated plants, NaN3 is probably one of the most effective chemical mutagens [13] that can cause point mutations, especially AT to CG, which can lead to amino acid changes, alter protein function, and change plant phenotypes [14]. Moreover, it is a potent point mutagen with the advantages of high efficiency, no residual toxicity, and a low price and has been widely used in flower breeding [15] and in orchids such as Phalaenopsis aphrodite Rchb. f. [16], Dendrobium nobile Lindl. [14,17], and Oncidium hybridum [18]. NaN3 can cause base insertions and/or deletions in replicating DNA molecules and base substitutions, and the mutagenic efficiency of NaN3 is increased when the number of replicating DNA molecules increases during the rapid cell division of the mutagenized material, causing an increased likelihood of point mutations [19,20,21]. The induction of shoots from Cymbidium faberi Rolfe PLBs involves a large number of DNA replications and was selected for mutation induction in this study. The concentration of the mutagen and the time of application also play an important role in the mutagenesis process [22], and the likelihood of generating mutations increases with each given mutagen concentration level and co-culture time. However, if the concentration is increased to very high levels and the application period is prolonged, negative effects may occur [23], such as an increase in Cymbidium faberi Rolfe seedling mortality and browning/damage rates; therefore, suitable mutagenic conditions need to be determined for PLB tissues.
Cymbidium faberi Rolfe has undergone a long period of natural selection and adaptation during its evolutionary process. Its genome is relatively stable and it mostly reproduces asexually, with a small chance of genetic mutation and a relatively low rate of natural mutation. Artificial mutagenesis can increase the mutation rate in plants. In this study, we explored the conditions of Cymbidium faberi Rolfe in histoculture, established a Cymbidium faberi Rolfe regeneration system, and utilized NaN3 mutagenesis to increase the rate of genetic variation in Cymbidium faberi Rolfe. This will enrich the genetic diversity of Cymbidium faberi Rolfe and provide important materials and means to breed Cymbidium faberi Rolfe varieties that are more economical, ornamental, or pest-resistant and to provide support for the enrichment of Cymbidium faberi Rolfe germplasm resources.

2. Materials and Methods

2.1. Plant Material and Explant Preparation

The Cymbidium faberi Rolfe seeds used in this experimental study were obtained from Tongbai County, Nanyang City, Henan Province. They were aseptically inoculated onto an induced seed germination medium and cultured for 3 months to obtain sterile PLBs as explants for mutagenesis. NaN3 solution was prepared in the laboratory and the dosage was self-adjusted according to the desired concentration during the test. The whole process was carried out in a sterile environment. The drugs and reagents used in the test were purchased from Beijing Chembase Technology Co., Ltd., Beijing, China.

2.2. Building a Cymbidium faberi Rolfe Regeneration System

Seed germination (Figure 1a): Seed capsules were sterilized with 2% sodium hypochlorite (NaOCl) solution for 8 min with shaking vibration and then rinsed with sterile water 3 times. The capsules were cut open, and the seeds were removed with a small sterile spoon and rinsed with sterile water 3 times. Then, the seeds were transferred to conical flasks and placed on a magnetic stirrer, before being rotary cut with a sterilized blade and inoculated with Murashige and Skoog (MS) (0.7% agar) medium + 10 g·L−1 sucrose + 10 g·L−1 glucose + 3 g·L−1 peptone + 0.2 mg·L−1 6-BA (6-Benzylaminopurine) + 0.2 mg·L−1 NAA (naphthaleneacetic acid). The seeds developed into PLBs after 120 d.
PLB proliferation: Different combinations of plant growth regulators (PGRs) were screened for their effects on the proliferation of PLBs (Table 1). Here, 1/2 MS (0.7% agar) was used as the basic medium with 10 g·L−1 sucrose; 10 g·L−1 glucose; 3 g·L−1 peptone; 1 g·L−1 activated charcoal; 100 mL·L−1 coconut milk; and different concentrations of combinations of PGRs, NAA, 6-BA, 2,4-D (2,4-Dichlorophenoxyacetic acid), and IBA (indole butyric acid). Then, 24 vials were connected to each formulation, and each vial was inoculated with six 5 mm unbranched PLBs and placed in a sterile culture room. After 60 days, the mean number of new PLBs, the average new length, and the average new diameter were recorded.
PLB shoot induction: Sterile PLBs were inoculated with a scalpel by cutting into 1 cm stem segments in the 1/2 MS (0.7% agar) medium + 10 g·L−1 sucrose + 10 g·L−1 glucose + 3 g·L−1 peptone + 1 g·L−1 activated charcoal + 100 mL·L−1 coconut water + 1 mg·L−1 6-BA + 1 mg·L−1 KT (Kinetin) + 1 mg·L−1 TDZ (Thidiazuron) + 1 mg·L−1 NAA + 0.1 mg·L−1 2,4-D to induce shoots.
Rooting induction and seedling transplantation: The seedlings were inoculated in 1/2 MS (0.7% agar) medium + 3 g·L−1 peptone + 15 g·L−1 sucrose + 1 g·L−1 activated charcoal + 100 mL·L−1 coconut water with different combinations of concentrations of NAA, IAA, and 6-BA to induce the rooting of PLBs that had shoots. Each vial was inoculated with 6 explants, 12 vials per treatment, and 4 vials per replication, and the experiment was repeated three times. The rooting rate was observed and recorded after 50 days, and the root growth was recorded. After 80 d, the seedlings were transplanted. After 2 d of seedling refining, the roots were soaked in 0.1% potassium permanganate for 30 s and then rinsed. They were then transplanted into a special substrate for orchids. The survival rate of Cymbidium faberi Rolfe seedlings was recorded after 30 d.

2.3. NaN3 Mutagenesis

The mutagenized material was inoculated in induced shoot medium supplemented with NaN3, 1/2 MS (0.7% agar) medium + 10 g·L−1 sucrose + 10 g·L−1 glucose + 3 g·L−1 peptone + 1 g·L−1 activated charcoal + 100 mL·L−1 coconut milk + 1 mg·L−1 6-BA + 1 mg·L−1 KT (Kinetin) + 1 mg·L−1 TDZ (Thidiazuron) + 1 mg·L−1 NAA + 0.1 mg·L−1 2,4-D. The treatment groups E0–E6 were each inoculated with approximately 1 cm of PLB material and treated with 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg·L−1 NaN3, respectively. The following concentrations of NaN3 were added to the medium of treatment groups F0–F7: 0.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 mg·L−1. Subsequently, approximately 1 cm of the material was inoculated for mutagenesis. After 60 d of induction culture, the differentiation rate, mortality rate, and leaf growth of all treatments were observed and recorded.

2.4. Data Analysis

All experiments were repeated three times. Excel 2021 was used to preliminarily organize the experimental data. SPSS 26.0 software was used for one-way ANOVA. Duncan’s multiple range test was used to test the significance of the differences between the means. The significance level was set at 0.05. The results were expressed as mean ± standard deviation.
Index determination method: New branches = number of new growth branches/number of surviving explants; new growth length = new growth length/number of surviving explants; new growth diameter = new growth diameter/number of surviving explants; PLB differentiation rate = number of PLBs differentiated and sprouted/number of inoculated viable PLBs × 100%; mortality = dead explants/inoculated explants × 100%; leaf growth length = total leaf growth length/number of surviving leaves × 100%.

3. Results

3.1. Effect of PGRs on Proliferation of PLBs

The proliferation culture of Cymbidium faberi Rolfe PLBs was carried out using different combinations of PGRs at different concentrations, and the proliferation status of each treatment is shown in Table 2. Newly expanded bumps began to appear on the surface of the PLBs by 20 days of incubation, followed by gradual elongation and the growth of new PLBs (Figure 1b). In the comparison of treatments B-1 and B-3, it was observed that the diameters of the PLBs became smaller after increasing the concentration of 2,4-D. Elevated concentrations of 2,4-D inhibited the thickening of PLBs. In the comparison of treatments B-1 and B-4, it was observed that all of the indicators of PLBs decreased after replacing NAA with IBA. The effect of NAA in inducing the proliferation of PLBs was better. In treatments B-1 and B-2, it was observed that the newly formed branches of PLBs increased and the diameter of PLBs increased after adding 0.5 mg·L−1 6-BA, and the newly formed length of PLBs increased after adding 1.0 mg·L−1 6-BA. Combining all indexes, the B-1 formulation was finally selected as the concentration combination of PGRs for the proliferation of PLBs.

3.2. Effect of PGRs on Induced Rooting of Cymbidium faberi Rolfe Seedlings

Rooting and transplanting is a key step in the rapid propagation of Cymbidium faberi Rolfe tissue culture. PLB-induced Cymbidium faberi Rolfe seedlings (Figure 1c) were inoculated on rooting medium and rooted one month later (Figure 1d). As shown in Table 3, with the increase in the NAA concentration and the decrease in the 6-BA concentration, the rooting rate gradually increased. After adding IAA, the rooting rate was further improved, indicating that NAA and IAA coordinated with each other and promoted the rooting of Cymbidium faberi Rolfe. The rooting rates for the six media were different, and the Cymbidium faberi Rolfe seedlings that had been rooted by the different formulations were transplanted into orchid-specific substrates, in which the roots induced by the high concentration of NAA were thicker and survived better. The combination of 2 mg·L−1 NAA, 1 mg·L−1 IAA, and 1 mg·L−1 6-BA resulted in the highest rooting rate, and the survival rate of yellow-green thick roots was also significantly higher than that of other treatments. One month later, the Cymbidium faberi Rolfe seedlings (Figure 1e) had new shoot growth (Figure 1f), and these were subsequently identified as induced rooting PGRs.

3.3. Mutagenesis of PLBs through Low Concentration of NaN3

In orchid mutagenesis breeding, most researchers use the 50% lethal dose (LD50) as the optimal mutagenic dose. However, there are also cases where the mutagenic effect cannot be evaluated with the half-lethal dose, and after orchid PLB mutagenic treatment, sometimes, materials treated with different doses survive but growth is stagnated, and the use of the half reduction dose (the 50% reduction dose, RD50) as the optimal mutagenic dose has been reported.
When PLBs were used as explants for mutagenesis (Table 4), PLB lethality was the highest under the 3 mg·L−1 NaN3 treatment, and although it did not reach half lethality, the differentiation rate was significantly reduced and the mutagenesis efficiency was low, making it unsuitable for increasing the concentration of NaN3 any further. With an increase of 0.5–2.5 mg·L−1 in NaN3 concentration, the mortality rate of the PLBs increased and the differentiation rate decreased significantly. An increase in NaN3 concentration may inhibit the differentiation of some PLBs into buds, which is not conducive to the formation of mutant buds. The differentiation rate of PLBs at 1.5 mg·L−1 NaN3 was 49.81%, and the amount of PLB differentiation was close to half of the reduction dose; hence, it was determined that 1.5 mg·L−1 NaN3 was the most suitable mutagenic concentration for PLBs as explants.
The mutant buds in this experiment are celandine buds that are significantly different in appearance from normal celandine buds (Figure 2). There are gold-line striped leaves, yellow-green leaves, albino leaves, spiral leaves, and dwarf seedlings.

3.4. Mutagenesis of Shoots by High Concentration of NaN3

PLB-induced buds were used as explants for mutagenesis. The degree of differentiation of the buds is relatively high, and the mutagenesis efficiency of low concentrations on already differentiated buds is low, so the selected NaN3 concentrations are relatively high. As shown in Table 5, the mortality rate of Cymbidium faberi Rolfe buds was very high when co-cultured with high concentrations of NaN3. All of the shoots treated with 10 mg·L−1 NaN3 died, and although some shoots treated with 9 mg·L−1 NaN3 survived, their growth was almost stagnant, and the leaf growth was zero at 60 days. Under the treatment of 4~5 mg·L−1 NaN3, the Cymbidium faberi Rolfe buds were close to the half-lethal dose, among which the leaf growth of 4 mg·L−1 NaN3 treatment was close to half of the control group. The damage to Cymbidium faberi Rolfe buds was relatively small, which was the critical point that Cymbidium faberi Rolfe buds could withstand for in vitro mutagenesis. Therefore, when using buds as mutagenic materials, 4 mg·L−1 NaN3 is the optimal mutagenic concentration.

3.5. NaN3 Promotes Flowering in Cymbidium faberi Rolfe

We found that some of the NaN3-treated PLBs rapidly entered the blossom season. Some of the material showed early blossom during the co-culture of Cymbidium faberi Rolfe shoots with NaN3 (Figure 2e–h). The flower shape varied, with some flowers having a normal shape and others with small or curled petals. The flower color was mostly light green, with some showing purple. However, the flower blooming time was earlier than normal.

4. Discussion

PGRs play an important role in in vitro culture, and different types and ratios can regulate cell differentiation, proliferation, and elongation [24]. In this study, we found that NAA favored the proliferation of PLBs. The induction of PLBs is necessary for the exogenous application of growth factors [25]. In this experiment, two growth-hormone-based PGRs, NAA and IBA, were selected for comparison. When 1 mg-L−1 was added, the proliferation efficiency of PLBs in NAA was higher than that of IBA, which was more suitable for PLB propagation and was consistent with the results of previous studies [2,26,27]. The rooting of isolated adventitious buds and the domestication of plants are critical steps in micropropagation. Some Orchidaceae studies have reported that NAA is favorable for rooting [28,29]. The rooting rate of Cymbidium faberi Rolfe shoots increased with increasing NAA concentration and decreasing 6-BA concentration in the 1/2MS medium in this study, which is consistent with the previously reported results.
Our experiments yielded partially strain-mutated Cymbidium faberi Rolfe plants, suggesting that NaN3 is effective as a chemical mutagen for the in vitro mutagenesis of Cymbidium faberi Rolfe. The viability of Cymbidium faberi Rolfe tissues and the mutagenesis rate are affected by NaN3 concentration due to cytogenetic aberrations and physiological instability [30], as well as destabilization between growth regulators and promoter inhibitors [31].
Cymbidium faberi Rolfe is difficult to flower, with new seedlings typically growing for more than 3 years to complete nutrient accumulation, produce flower shoots, and undergo 5 months of vernalized dormancy before flowering. Orchidaceae are highly valued in the global horticultural market for their ethereal appearance and ecological diversity [32]. In this study, Cymbidium faberi Rolfe showed early flowering after NaN3 mutagenesis, which greatly shortened the time required for Cymbidium faberi Rolfe to flower and significantly enhanced its ornamental and economic value.
Some studies have reported that NaN3 induces changes in flower color, flower shape, and leaf shape in terrestrial orchids (Spathoglotis plicata) [33] and bluebells (Browalia speciosa) [34]. In the present study, NaN3 also changed the flower shape, flowering period, and color of Cymbidium faberi Rolfe flowers. Plant flowering, the transition from nutrient growth to reproductive growth [35], is a key event in the plant life cycle, and NaN3 caused some Cymbidium faberi Rolfe to flower early. It is speculated that sodium azide, as a respiratory inhibitor, leads to a shortage of ATP [36,37,38,39], which inhibits nutrient growth and coerces the plant’s transition to reproductive growth, causing Cymbidium faberi Rolfe to appear to flower early. In addition, the flowering period is the most critical transition period in the entire life cycle of the plant and is a highly sensitive period to external stresses [40]. It has been proposed that due to the ability of plants to regulate their own growth and developmental processes in response to changes in the external environment, many plants flower early when they are subjected to unfavorable conditions, such as abiotic stresses, drought [41], high salinity [42], and low temperature [43]. This phenomenon is known as “adversity-induced flowering” [44]. Environmental stresses can stimulate and regulate gene networks and promote the formation of flower shoots [45,46], which have an impact on the flowering process of plants. NaN3 can also be regarded as an external stress to Cymbidium faberi Rolfe, and it may be that this stress stimulates Cymbidium faberi Rolfe to enter the flowering stage earlier.
Cymbidium faberi Rolfe cultured using NaN3 mutagenesis not only exhibited changes in the flowering period but also demonstrated new variations in flower shape. In the future, it is also possible to change the flower color by adjusting the pH of the medium and the endogenous hormone of the plant, which is of great significance for the cultivation of ornamental plants and the development of the market.

5. Conclusions

In this study, a regeneration system for Cymbidium faberi Rolfe was established using the plant tissue culture technique to observe the proliferation and rooting response of PLBs after the addition of different PGRs. Based on the measured effects of PGRs on the proliferation of PLBs, we found that the proliferation rate was significantly elevated with the addition of PGRs compared to that of untreated PLBs. The addition of 0.5 mg·L−1 6-BA induced more branches and larger diameters in the PLBs, the replacement of IBA with NAA increased the proliferation efficiency of PLBs, and the thickening of PLBs in Cymbidium faberi Rolfe was inhibited by elevated 2,4-D concentrations. From this result, we determined that the optimal formulation for the proliferation of PLBs is 1/2 MS + 1 mg·L−1 NAA + 1 mg·L−1 6-BA + 0.1 mg·L−1 2,4-D. We also evaluated the effect of PGRs on the rooting of Cymbidium faberi Rolfe seedlings: decreasing the 6-BA concentration and increasing the NAA concentration resulted in an increased rooting rate, which was even further improved with the addition of IAA. Thus, the optimal rooting formulation was determined to be 1/2 MS + 2 mg·L−1 NAA + 1 mg·L−1 IAA + 1 mg·L−1 6-BA. The induction of mutations was the focus of this study. The addition of NaN3 to the medium for the mutagenic culture of PLBs and shoots revealed that rhizomes were more suitable as the base material for Cymbidium faberi Rolfe mutagenesis. The addition of 1.5 mg·L−1 NaN3 was more effective in mutagenesis during the induction of shoots from PLBs to produce leaf and flower shape variants. Leaf shape variants include the popular mid-translucent, tiger-spotted, and dwarfing variants. Flowering variants include shortened pavilions, two flowers on one stalk, an increased number of flowers, and missing petals. We also found that some of the Cymbidium faberi Rolfe plants mutagenized with NaN3 exhibited an early entry into flowering. This suggests that sodium azide is effective as a chemical mutagen in the in vitro mutagenesis of Cymbidium faberi Rolfe. Since rarity is precious and Cymbidium faberi Rolfe is an ornamental flower, changes in its leaf shape and flower shape will significantly increase its ornamental and economic value. It also means that a new variety may be born. This study provides ideas for regulating early flowering, which will help the development of future research on regulating flowering time in plants.

Author Contributions

Conceptualization, Z.W.; Software, Y.L.; Validation, C.L.; Investigation, H.Z.; Resources, J.W.; Data curation, B.A.; Writing—original draft, S.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Henan Provincial Science and Technology Research Project in the Agricultural Field of Henan Province: 182102110066.

Data Availability Statement

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

Conflicts of Interest

The authors declare no conflict of interest.

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Horticulturae 10 00889 g001
Figure 1. Cymbidium faberi Rolfe fast propagation system construction. (a) Sprouting PLBs from Cymbidium faberi Rolfe seeds; (b) PLBs expand and begin to multiply; (c) PLBs differentiate into shoots and begin to form seedlings; (d) rooting is induced; (e) one month after transplantation; (f) new shoots grow.
Figure 1. Cymbidium faberi Rolfe fast propagation system construction. (a) Sprouting PLBs from Cymbidium faberi Rolfe seeds; (b) PLBs expand and begin to multiply; (c) PLBs differentiate into shoots and begin to form seedlings; (d) rooting is induced; (e) one month after transplantation; (f) new shoots grow.
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Figure 2. Partially mutated Cymbidium faberi Rolfe plants. (a) Leaf mesophyll penetration; (b) interspersed yellow-green leaf veins; (c) leaf blades curled up; (d) dwarfed seedlings; (e–h) mutagenized early flowering.
Figure 2. Partially mutated Cymbidium faberi Rolfe plants. (a) Leaf mesophyll penetration; (b) interspersed yellow-green leaf veins; (c) leaf blades curled up; (d) dwarfed seedlings; (e–h) mutagenized early flowering.
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Table 1. PGR formulation for inducing proliferation of Cymbidium faberi Rolfe PLBs.
Table 1. PGR formulation for inducing proliferation of Cymbidium faberi Rolfe PLBs.
TreatmentsNAA (mg·L−1)6-BA (mg·L−1)2,4-D (mg·L−1)IBA (mg·L−1)
B-00000
B-110.50.10
B-211.00.10
B-310.50.20
B-400.50.11
Table 2. Effect of PGRs NAA, 6-BA, 2,4-D, and IBA on proliferation of Cymbidium faberi Rolfe PLBs.
Table 2. Effect of PGRs NAA, 6-BA, 2,4-D, and IBA on proliferation of Cymbidium faberi Rolfe PLBs.
TreatmentsNew BranchesNew Growth Length (mm)New Growth Diameter (mm)
B-000.26 ± 0.04 d0.04 ± 0.05 e
B-13.10 ± 0.23 a4.87 ± 0.01 b0.96 ± 0.04 a
B-22.80 ± 0.17 a5.31 ± 0.06 a0.82 ± 0.03 b
B-32.47 ± 0.14 b4.53 ± 0.04 c0.59 ± 0.04 d
B-42.31 ± 0.17 b4.76 ± 0.05 b0.68 ± 0.02 c
Note: The values represent the means (±SD). The mean values within a column followed by the same letter are not significantly different at p < 0.05 according to the analysis of variance (ANOVA).
Table 3. Effect of plant growth regulators on rooting and transplanting survival of Cymbidium faberi Rolfe seedlings.
Table 3. Effect of plant growth regulators on rooting and transplanting survival of Cymbidium faberi Rolfe seedlings.
TreatmentsNAA
(mg·L−1)		IAA
(mg·L−1)		6-BA
(mg·L−1)		Rooting
Rate (%)		Transplant
Survival Rate (%)		Root Growth
Status
D-10.2028.80 ± 0.80 e87.41 ± 0.77 dWhite slender
D-20.50210.18 ± 0.80 de90.29 ± 0.44 cWhite slender
D-310218.98 ± 3.21 c96.64 ± 0.37 bGreen thick
D-40.21212.96 ± 0.80 d90.47 ± 0.24 cWhite slender
D-520149.54 ± 3.21 b96.21 ± 0.77 bWhite thick
D-621173.15 ± 2.12 a98.31 ± 0.37 aYellow-green thick
Note: The letters in the table indicate significant differences at the 0.05 level.
Table 4. Effect of low concentration of NaN3 on the mutagenesis of Cymbidium faberi Rolfe PLB regeneration buds.
Table 4. Effect of low concentration of NaN3 on the mutagenesis of Cymbidium faberi Rolfe PLB regeneration buds.
TreatmentsNaN3 (mg·L−1)PLB Mortality (%)Differentiation Rate (%)
E-00.08.33 ± 0.41 d80.56 ± 0.24 a
E-10.516.67 ± 0.41 c71.17 ± 0.28 b
E-21.019.44 ± 0.48 c58.27 ± 0.34 c
E-31.536.11 ± 0.63 b49.81 ± 0.52 d
E-42.041.67 ± 0.72 ab41.34 ± 0.14 e
E-52.548.61 ± 0.24 a26.28 ± 0.40 f
E-63.048.33 ± 0.41 d10.68 ± 0.40 g
Note: The letters in the table indicate significant differences at the 0.05 level.
Table 5. Effect of high concentrations of NaN3 on mutagenesis of Cymbidium faberi Rolfe buds.
Table 5. Effect of high concentrations of NaN3 on mutagenesis of Cymbidium faberi Rolfe buds.
TreatmentNaN3 (mg·L−1)Leaf Growth Length (mm)Bud Mortality Rate (%)
F-00.016.33 ± 0.88 a0
F-14.06.04 ± 0.85 b48.61 ± 0.24 d
F-25.04.38 ± 0.25 c52.78 ± 0.24 d
F-36.03.38 ± 0.33 d66.67 ± 0.42 c
F-47.01.42 ± 0.19 e80.56 ± 1.04 b
F-58.00.96 ± 0.38 e84.72 ± 0.24 b
F-69.00 ± 0 f93.06 ± 0.24 a
F-710.00 ± 0 f1 ± 0 a
Note: The letters in the table indicate significant differences at the 0.05 level.
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Wu, Z.; Liu, S.; An, B.; Zhang, H.; Wu, J.; Li, C.; Long, Y. Mutagenesis and Flowering Promotion through Sodium Azide In Vitro Culture of Cymbidium faberi Rolfe. Horticulturae 2024, 10, 889. https://doi.org/10.3390/horticulturae10080889

AMA Style

Wu Z, Liu S, An B, Zhang H, Wu J, Li C, Long Y. Mutagenesis and Flowering Promotion through Sodium Azide In Vitro Culture of Cymbidium faberi Rolfe. Horticulturae. 2024; 10(8):889. https://doi.org/10.3390/horticulturae10080889

Chicago/Turabian Style

Wu, Zhengjing, Sujuan Liu, Bingjie An, Hao Zhang, Jingjing Wu, Chenfang Li, and Yuan Long. 2024. "Mutagenesis and Flowering Promotion through Sodium Azide In Vitro Culture of Cymbidium faberi Rolfe" Horticulturae 10, no. 8: 889. https://doi.org/10.3390/horticulturae10080889

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