Different Amino Acids Inhibit or Promote Rhizome Proliferation and Differentiation in Cymbidium goeringii

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  • 1 Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang 550025, Guizhou, China

The proliferation and differentiation of rhizomes are crucial for the propagation of Cymbidium species. We systematically assessed the effects of different concentrations of 20 amino acids on the proliferation and differentiation of C. goeringii rhizomes. Rhizome proliferation rates were significantly higher in media with 2.0 mmol·L−1 cysteine, 0.5 mmol·L−1 arginine, 0.2 mmol·L−1 asparagine, 1.0 mmol·L−1 proline, and 0.5 mmol·L−1 lysine compared with those in the control. Additionally, 1.0 mmol·L−1 tyrosine, 0.5 mmol·L−1 asparagine, and 0.2 mmol·L−1 aspartate were beneficial for rhizome differentiation. Furthermore, two combinations of amino acids, 0.5 mmol·L−1 arginine + 1.0 mmol·L−1 proline and 0.5 mmol·L−1 arginine + 2.0 mmol·L−1 cysteine, resulted in proliferation rates of 3.05 and 3.01, respectively, after 60 days. The highest differentiation rate (5.39 after 60 days) was observed in media with 0.5 mmol·L−1 asparagine + 0.2 mmol·L−1 aspartate. This study demonstrated that certain combinations of amino acids can effectively promote the proliferation and differentiation of rhizomes during the rapid propagation of C. goeringii.

Abstract

The proliferation and differentiation of rhizomes are crucial for the propagation of Cymbidium species. We systematically assessed the effects of different concentrations of 20 amino acids on the proliferation and differentiation of C. goeringii rhizomes. Rhizome proliferation rates were significantly higher in media with 2.0 mmol·L−1 cysteine, 0.5 mmol·L−1 arginine, 0.2 mmol·L−1 asparagine, 1.0 mmol·L−1 proline, and 0.5 mmol·L−1 lysine compared with those in the control. Additionally, 1.0 mmol·L−1 tyrosine, 0.5 mmol·L−1 asparagine, and 0.2 mmol·L−1 aspartate were beneficial for rhizome differentiation. Furthermore, two combinations of amino acids, 0.5 mmol·L−1 arginine + 1.0 mmol·L−1 proline and 0.5 mmol·L−1 arginine + 2.0 mmol·L−1 cysteine, resulted in proliferation rates of 3.05 and 3.01, respectively, after 60 days. The highest differentiation rate (5.39 after 60 days) was observed in media with 0.5 mmol·L−1 asparagine + 0.2 mmol·L−1 aspartate. This study demonstrated that certain combinations of amino acids can effectively promote the proliferation and differentiation of rhizomes during the rapid propagation of C. goeringii.

Cymbidium goeringii is a famous ornamental flower in China and other Asian countries that has commercially valued characteristics. Recently, studies of C. goeringii have focused mainly on the molecular mechanisms controlling flowering time (Huang et al., 2012; Ren et al., 2020a; Yang et al., 2019; Zhang et al., 2019), floral scent (Ramya et al., 2019), floral organ development (Xiang et al., 2018; Yang et al., 2017), and stress physiology (Ren et al., 2020b). However, this popular species is endangered due to illegal collection and habitat destruction (Chung and Nason, 2007). Currently, the most common propagation method of C. goeringii is through the separation of pseudobulbs; however, this is too inefficient for commercial production. Tissue culture is a rapid and efficient way to propagate C. goeringii. During propagation of C. goeringii, mature seeds are often used as the material to obtain sterile rhizomes for further proliferation and differentiation. The rhizome stage is advantageous for the propagation of C. goeringii (Roy and Banerjee, 2002). However, the proliferation and differentiation rates are slow, and some rhizomes can turn brown, which limits further growth (Paek and Kozai, 1998). Therefore, the optimization of rhizome proliferation and differentiation under culture conditions can promote the rapid propagation of C. goeringii.

Recently, some progress has been made in the study of rhizome proliferation and differentiation of Cymbidium. Activated charcoal (AC) can effectively promote C. sinense rhizome proliferation and prevent browning (Gao et al., 2014; Lee et al., 2011). Additionally, 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid (NAA) can effectively induce rhizome proliferation in Cymbidium (Park et al., 2018; Shimasaki and Uemoto, 1990). Importantly, the combination of kiwifruit juice and sodium citrate can effectively promote rhizome proliferation and prevent browning of the rhizome during the micropropagation of C. goeringii (Huang et al., 2017). In terms of rhizome differentiation, supplementing 6-benzylaminopurine (6-BA) and NAA to the culture medium can promote shoot induction from the rhizome. Park et al. (2018) reported that medium fortified with 20 μM 2,4-D, and 2 μM thidiazuron was most effective for inducing adventitious buds. Cutting the rhizome explants into 1-cm segments resulted in massive shoot formation (Gao et al., 2014). Moreover, a recent study revealed that YUC-mediated auxin biogenesis is involved in shoot regeneration from the rhizome in Cymbidium (Liu et al., 2017).

The rapid propagation technology used for C. goeringii (Park et al., 2018) and other Cymbidium species (Gao et al., 2014; Guha and Rao, 2012; Lee et al., 2011; Liu et al., 2017) has received considerable attention over the years, but little work has focused specifically on the effects of amino acids in tissue culture. As fundamental nutrients, amino acids can be synthesized into many important compounds (such as plant hormones and secondary metabolites) (Krapp, 2015; Tegeder, 2012). Amino acids not only have an important role in the plant nitrogen balance but also control plant growth and development, flowering, and seed yield (Coruzzi and Bush, 2001). Experiments confirmed that amino acid alanine could promote plant growth (Ichihashi et al., 2020). Glutamate can promote rice growth at low concentrations and inhibit rice growth at high concentrations (Kan et al., 2017). Additionally, certain concentrations of amino acids, such as lysine and arginine, can promote the growth of rice plants and tiller bud elongation (Lu et al., 2018; Wang et al., 2019).

It is unknown whether amino acids can promote the growth of C. goeringii rhizomes. This study demonstrated that certain combinations of amino acids can effectively promote the proliferation and differentiation of rhizomes in the rapid propagation of C. goeringii via tissue culture. These findings could provide a theoretical basis and technical support for improving the propagation speed of Cymbidium.

Materials and Methods

In vitro seed germination and rhizome development.

Mature pods (150 d after pollination) of C. goeringii were harvested from a greenhouse at Guizhou University in Guiyang, China. The pods were surface-sterilized by dipping in 70% ethanol for 30 s, immersing in 0.1% (w/v) HgCl2 solution for 10 min, and rinsing five times with sterile distilled water. Then, the pods were cut with a sterile blade, and the seeds were scraped out with the blade for the next experiments. The seed germination capacity was investigated on Murashige and Skoog (MS) media containing 0.5 mg·L−1 NAA, 30 g·L−1 sucrose, 2 g·L−1 AC, and 100 mL·L−1 coconut milk. The coconut milk was obtained from 6- to 7-month-old green coconuts and filtered through filter paper. Seed germination and rhizome development were investigated after 90 d.

In vitro proliferation of rhizomes.

Rhizomes from immature seed were rinsed twice in sterilized water to remove the culture media. Then, 5-mm-long rhizome apical segments were placed on MS media containing 0.2, 0.5, 1.0, or 2.0 mmol·L−1 of nonpolar amino acids (glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, or methionine), polar neutral amino acids (serine, threonine, glutamine, tyrosine, asparagine, or cysteine), polar acidic amino acids (glutamate or aspartate), or polar basic amino acids (histidine, arginine, or lysine). Media were also supplemented with 5 mg·L−1 NAA, 1.5 mg·L−1 6-BA, 2 g·L−1 AC, and 30 g·L−1 sucrose. Medium without amino acids served as a control. After determining the effects of individual amino acids on the proliferation of rhizomes, different combinations of the amino acids that promoted proliferation were examined using the same methods. There were three replicates of each treatment, with 200 rhizomes per replicate. The rhizome proliferation rate was calculated as the rhizome fresh weight after 60 d of culturing compared with the rhizomes before culture.

Shoot differentiation from rhizomes.

The effects of amino acids on shoot differentiation from rhizomes were investigated using the same methods and treatments for amino acids that were used in rhizome proliferation, except 10-mm rhizome apical segments were used. In addition to amino acids, media were supplemented with 5 mg·L−1 6-BA, 0.2 mg·L−1 NAA, and 20 g·L−1 sucrose. Medium without amino acids served as a control. The rhizome differentiation rate was calculated as the average number of shoots per rhizome after 60 d of culturing.

Culture conditions and statistical analysis.

The media were adjusted to pH 5.4 before autoclaving at 121 °C for 20 min at 1.06 kg·cm−2. All cultures were incubated in a controlled environment growth room at 25 ± 2 °C with a 16-h photoperiod under cool white light (40–50 μmol·m−2·s−1). All experiments were set according to a completely random design. The data were analyzed using one-way analyses of variance and Duncan’s multiple range tests at the 5% level.

Results

Effects of nonpolar amino acids on C. goeringii rhizome proliferation and differentiation.

Proline at 1.0 mmol·L−1 and isoleucine at 0.2 mmol·L−1 significantly increased the proliferation of C. goeringii rhizomes compared with the control (Fig. 1A and B). Proliferation rates reached 2.24 and 1.81 under these two treatments, respectively. Although some concentrations of glycine, alanine, valine, and tryptophan resulted in higher proliferation rates compared with the control, the differences were not statistically significant (Table 1). Under these treatments, rhizomes grew well and were light green. Phenylalanine and methionine resulted in proliferation rates similar to those of the control (Table 1). Higher concentrations of leucine inhibited rhizome growth (Table 1). Furthermore, high concentrations of isoleucine and all concentrations of phenylalanine resulted in browning of rhizomes during proliferation (Table 1).

Fig. 1.
Fig. 1.

Effects of different amino acid treatments on Cymbidium goeringii rhizome proliferation. (A) Control. (B) Proline at 1.0 mmol·L−1. (C) Cysteine at 2.0 mmol·L−1. (D) Asparagine at 0.2 mmol·L−1. (E) Aspartate at 2.0 mmol·L−1. (F) Arginine at 0.5 mmol·L−1. (G) Lysine at 0.5 mmol·L−1. (H) Arginine at 0.5 mmol·L−1 + cysteine at 2.0 mmol·L−1. (I) Arginine at 0.5 mmol·L−1 + proline at 1.0 mmol·L−1.

Citation: HortScience horts 56, 1; 10.21273/HORTSCI15441-20

Table 1.

Effects of different nonpolar amino acid treatments on C. goeringii rhizome proliferation and differentiation.

Table 1.

Valine at 0.5 mmol·L−1, leucine at 0.5 mmol·L−1, isoleucine at 0.2 mmol·L−1, tryptophan at 0.2 mmol·L−1, and tryptophan at 1.0 mmol·L−1 had no effect on the differentiation of rhizomes compared with the control (Fig. 1A). All concentrations of glycine, alanine, proline, phenylalanine, and methionine inhibited rhizome differentiation (Table 1). The addition of nonpolar amino acids caused widespread browning during differentiation. The highest level of inhibition occurred under methionine at 2.0 mmol·L−1, but no browning occurred in this treatment (Fig. 2B). In summary, no nonpolar amino acids promoted the differentiation of rhizomes.

Fig. 2.
Fig. 2.

Effects of different amino acid treatments on Cymbidium goeringii rhizome differentiation. (A) Control. (B) Methionine at 2.0 mmol·L−1. (C) Asparagine at 0.5 mmol·L−1. (D) Asparagine at 0.5 mmol·L−1 + tyrosine at 1.0 mmol·L−1. (E) Asparagine at 0.5 mmol·L−1 + aspartate at 0.2 mmol·L−1. (F) Shoots developed from the tips of the rhizomes in Huabao medium supplemented with activated charcoal (AC) and naphthalene acetic acid (NAA) after 2 months.

Citation: HortScience horts 56, 1; 10.21273/HORTSCI15441-20

Effects of polar neutral amino acids on C. goeringii rhizome proliferation and differentiation.

As shown in Table 2 and Fig. 1, cysteine at 2.0 mmol·L−1 (Fig. 1C) and asparagine at 0.2 mmol·L−1 (Fig. 1D) significantly increased rhizome proliferation rates of C. goeringii compared with the control, with proliferation rates of 2.35 and 2.33, respectively. Higher concentrations of asparagine inhibited rhizome growth (Table 2). Cysteine at 0.5 and 1.0 mmol·L−1 also resulted in significantly higher rhizome proliferation compared with the control (Table 2). Different concentrations of serine had no effect on rhizome proliferation (Table 2). However, threonine 0.2 mmol·L−1 and tyrosine 1.0 mmol·L−1 significantly promoted the proliferation of rhizomes compared with the control (Table 2). Glutamine was not beneficial to rhizome proliferation (Table 2). Rhizome browning was not observed under any polar neutral amino acid treatments.

Table 2.

Effects of different polar neutral amino acid treatments on C. goeringii rhizome proliferation and differentiation.

Table 2.

The differentiation of rhizomes was significantly promoted under asparagine at 0.5 mmol·L−1 (Fig. 2C), which resulted in four buds per rhizome (Table 2). Tyrosine (at 0.2, 0.5, 1.0, or 2.0 mmol·L−1), asparagine (at 1.0 or 2.0 mmol·L−1), and cysteine (at 0.5 mmol·L−1) also significantly promoted rhizome differentiation compared with the control, resulting in more than three buds per rhizome (Table 2). Different concentrations of serine and threonine significantly inhibited the differentiation of rhizome compared with the control (Table 2). Glutamine had no effect on rhizome differentiation compared with the control (Table 2). All concentrations of serine and threonine significantly inhibited rhizome differentiation compared with the control, resulting in fewer than two buds per rhizome (Table 2). The addition of nonpolar amino acids caused browning during differentiation, but not during proliferation.

Effects of polar acidic amino acids on C. goeringii rhizome proliferation and differentiation.

Low concentrations of glutamate (0.2 mmol·L−1 and 0.5 mmol·L−1) significantly promoted rhizome proliferation of C. goeringii compared with the control (Table 3). The proliferation rate was 1.82 when glutamate was added at 0.2 mmol·L−1. Under this treatment, rhizome growth was normal and the rhizome was green. The proliferation rate decreased under increasing glutamate concentrations, with inhibition under 1.0 mmol·L−1 and increasing inhibition under higher concentrations (Table 3). Aspartate did not increase rhizome proliferation, and higher concentrations of aspartate inhibited rhizome growth and resulted in rhizome browning (Fig. 1E).

Table 3.

Effects of different polar acidic amino acid treatments on C. goeringii rhizome proliferation and differentiation.

Table 3.

Low concentrations of aspartate (0.2 mmol·L−1) significantly increased rhizome differentiation compared with the control, resulting in more than four buds per rhizome (Table 3). Glutamate resulted in lower rhizome differentiation rates compared with the control (Table 3). Both polar acidic amino acids caused severe browning during rhizome differentiation.

Effects of polar basic amino acids on C. goeringii rhizome proliferation and differentiation.

Rhizome proliferation under histidine treatments did not differ significantly from that under the control, except for at 2.0 mmol·L−1, when proliferation was significantly lower compared with that under the control of C. goeringii (Table 4). The addition of arginine at 0.5 mmol·L−1 significantly increased proliferation rates compared with the control, resulting in a proliferation rate of 2.32 (Fig. 1F). Proliferation rates decreased with increasing arginine concentrations, and rhizome browning occurred. Lysine at 0.5 mmol·L−1 also resulted in higher rhizome proliferation compared with the control, with a proliferation rate of 2.21 (Fig. 1G). No rhizome browning occurred under lysine treatments.

Table 4.

Effects of different polar basic amino acid treatments on C. goeringii rhizome proliferation and differentiation.

Table 4.

All concentrations of histidine and lysine significantly inhibited rhizome differentiation (Table 4). Low concentrations (0.2 mmol·L−1) of arginine had no significant effect on rhizome differentiation compared with the control; however, with increasing arginine concentrations, the inhibition of rhizome differentiation became obvious (Table 4). All polar basic amino acids except arginine at 0.5 mmol·L−1 caused widespread browning during differentiation.

Effects of amino acid combinations on C. goeringii rhizome proliferation and differentiation.

Arginine at 0.5 mmol·L−1 + proline at 1.0 mmol·L−1 (Fig. 1I) and arginine at 0.5 mmol·L−1 + cysteine at 2.0 mmol·L−1 (Fig. 1H) obviously promoted the growth of rhizomes more than other treatments, resulting in proliferation rates of 3.05 and 3.01, respectively. Other combinations also promoted the proliferation of rhizomes, and browning was only observed under the amino acid combinations of cysteine + threonine and cysteine + glutamate (Table 5).

Table 5.

Effects of amino acid combination treatments on C. goeringii rhizome proliferation.

Table 5.

Asparagine at 0.5 mmol·L−1 + tyrosine at 1.0 mmol·L−1 (Fig. 2D) and asparagine at 0.5 mmol·L−1 + aspartate at 0.2 mmol·L−1 (Fig. 2E) resulted in significantly higher rhizome differentiation rates compared with the other combinations tested, with differentiation rates of 5.34 and 5.39, respectively (Table 6). All the combinations of amino acids caused a certain degree of browning. After treatment with amino acids, the differentiated buds can grow into seedlings suitable for transplanting out of bottles (Fig. 2F).

Table 6.

Effects of amino acid combination treatments on C. goeringii rhizome differentiation.

Table 6.

Discussion

Amino acids are important physiological active substances in plants that have an important role in regulating growth. We systematically investigated the effects of 20 amino acids on the proliferation and differentiation of C. goeringii rhizomes formed by aseptic seeding. Our results indicate that different types and concentrations of amino acids vary in their effects on the growth of C. goeringii rhizomes. The addition of cysteine, arginine, asparagine, proline, and lysine significantly promoted the proliferation of C. goeringii rhizomes without browning. Furthermore, arginine combined with cysteine or proline resulted in the highest proliferation rates of C. goeringii rhizomes. Conversely, the addition of amino acids during differentiation often caused browning. However, tyrosine, asparagine, and aspartate increased the differentiation rate of rhizomes, and asparagine combined with aspartate resulted in the highest differentiation rate.

Amino acids can promote the growth and development of plants, but excess concentrations inhibit growth (Chen et al., 2017). We found that the effects of many amino acids on the growth of rhizomes were in accordance with this rule. Increasing the content of cysteine in vivo can help improve the ability of plants to resist oxidative and environmental stress (Domínguez-Solís et al., 2004). This experiment showed that cysteine, alone or together with other amino acids, could promote rhizome growth. The reason for this may be that cysteine, as an antioxidant, can effectively inhibit the activities of catalase and polyphenol oxidase, thereby inhibiting browning and promoting rhizome growth. Lu et al. (2018) showed that arginine and lysine could promote rice growth and tillering bud elongation. During this experiment, arginine and lysine, alone or in combination, could promote rhizome proliferation. The reason may be that lysine can increase the activities of glutamic-oxaloacetic transaminase and alanine transaminase in plants and change the nitrogen metabolism of plants.

Plants mitigate stress by controlling the absorption, synthesis, and degradation of amino acids (Bottari and Festa, 1996). We found that asparagine can also alleviate rhizome browning during the proliferation stage and promote rhizome proliferation and differentiation. Proline can increase the adaptability of plants to external stresses, enhance or stabilize the activity of detoxifying enzymes, and stimulate the accumulation of stress response proteins (Matysik et al., 2002). During this experiment, proline alleviated rhizome browning and promoted rhizome proliferation.

Many amino acids, including phenylalanine, resulted in C. goeringii rhizome browning and inhibited rhizome growth. It has been reported that phenylalanine can inhibit Arabidopsis seed germination and seedling growth (Voll et al., 2004), and high concentrations of phenylalanine inhibit rice growth (Lu et al., 2018). Therefore, our results are consistent with those of other studies. Tyrosine is an essential aromatic amino acid required for synthesis of proteins and diverse natural products (Schenck and Maeda, 2018). We found that tyrosine promotes rhizome differentiation, which has not been observed before.

In conclusion, arginine, lysine, proline, and cysteine, which mainly belong to polar basic amino acids (arginine and lysine) and neutral amino acids (proline and cysteine), are suitable for rhizome proliferation of Cymbidium goeringii. However, only polar neutral amino acids (asparagine or tyrosine) and polar acidic amino acid (aspartate) are suitable for rhizome differentiation of Cymbidium goeringii. In our study, one or two amino acids were investigated in each treatment. Therefore, it is unclear whether the addition of three or more amino acids together could further promote rhizome differentiation and proliferation. Future studies should investigate the effects of more amino acid combinations and other additives. Whether different amino acids can promote the seedling rooting of C. goeringii also needs to be investigated.

Literature Cited

  • Bottari, E. & Festa, M.R. 1996 Asparagine as a ligand for cadmium (II), lead (II) and zinc (II) Chem. Spec. Bioavail. 8 75 83 doi: 10.1080/09542299.1996.11083272

    • Search Google Scholar
    • Export Citation
  • Chen, Z.Y., Chen, T.P., Li, H.J. & Chui, S.Y. 2017 Research progress in molecular mechanism of amino acids absorption and transport by plants Mol. Plant Breed. 15 5166 5171 doi: 10.13271/j.mpb.015.005166

    • Search Google Scholar
    • Export Citation
  • Chung, M.Y. & Nason, J.D. 2007 Spatial demographic and genetic consequences of harvesting within populations of terrestrial orchid Cymbidium goeringii Biol. Conserv. 137 125 137 doi: 10.1016/j.biocon.2007.01.021

    • Search Google Scholar
    • Export Citation
  • Coruzzi, G. & Bush, D.R. 2001 Nitrogen and carbon nutrient and metabolite signaling in plant Plant Physiol. 125 61 64 doi: 10.1104/pp.125.1.61

  • Domínguez-Solís, J.R., López-Martín, M.C., Ager, F.J., Ynsa, M.D., Romero, L.C. & Gotor, C. 2004 Increased cysteine availability is essential for cadmium tolerance and accumulation in Arabidopsis thaliana Plant Biotechnol. J. 2 469 476 doi: 10.1111/j.1467-7652.2004.00092.x

    • Search Google Scholar
    • Export Citation
  • Gao, R., Wu, S.Q., Piao, X.C., Park, S.Y. & Lian, M.L. 2014 Micropropagation of Cymbidium sinense using continuous and temporary airlift bioreactor systems Acta Physiol. Plant. 36 117 124 doi: 10.1007/s11738-013-1392-9

    • Search Google Scholar
    • Export Citation
  • Guha, S. & Rao, I.U. 2012 Nitric oxide promoted rhizome induction in Cymbidium shoot buds under magnesium deficiency Biol. Plant. 56 227 236 doi: 10.1007/s10535-012-0081-7

    • Search Google Scholar
    • Export Citation
  • Huang, W.T., Fang, Z.M., Zeng, S.J., Zhang, J.X., Wu, K.L., Chen, Z.L., Jaime, A.T. & Duan, J. 2012 Molecular cloning and functional analysis of three FLOWERING LOCUS T (FT) homologous genes from Chinese Cymbidium Intl. J. Mol. Sci. 13 11385 11398 doi: 10.3390/ijms130911385

    • Search Google Scholar
    • Export Citation
  • Huang, W.T., Zeng, L.T., Wu, B.W. & Fang, Z.M. 2017 Effects of different organic additives and sodium citrate combination on rhizomes proliferation of Cymbidium goeringii J. Anhui Agr. Univ. 44 1112 1118 doi: 10.13610/j.cnki.1672-352x.20171214.010

    • Search Google Scholar
    • Export Citation
  • Ichihashi, Y., Date, Y., Shino, A., Shimizu, T., Shibata, A., Kumaishi, K., Funahashi, F., Wakayama, K., Yamazaki, K., Umezawa, A., Sato, T., Kobayashi, M., Kamimura, M., Kusano, M., Che, F.S., Brien, M.O., Tanoi, K., Hayashi, M., Nakamura, R., Shirasu, K., Kikuchi, J. & Nihei, N. 2020 Multi-omics analysis on an agroecosystem reveals the significant role of organic nitrogen to increase agricultural crop yield Proc. Natl. Acad. Sci. USA 117 17259, doi: 10.1073/pnas.1917259117

    • Search Google Scholar
    • Export Citation
  • Kan, C.C., Chung, T.Y., Wu, H.Y., Juo, Y.A. & Hsieh, M.H. 2017 Exogenous glutamate rapidly induces the expression of genes involved in metabolism and defense responses in rice roots BMC Genomics 18 186, doi: 10.1186/s12864-017-3588-7

    • Search Google Scholar
    • Export Citation
  • Krapp, A. 2015 Plant nitrogen assimilation and its regulation: A complex puzzle with missing pieces Curr. Opin. Plant Biol. 25 115 122 doi: 10.1016/j.pbi.2015.05.010

    • Search Google Scholar
    • Export Citation
  • Lee, O.R., Yang, D.C., Chung, H.J. & Min, B.H. 2011 Efficient in vitro plant regeneration from hybrid rhizomes of Cymbidium sinense seeds Hort. Environ. Biotechnol. 52 303 308 doi: 10.1007/s13580-011-0187-4

    • Search Google Scholar
    • Export Citation
  • Liu, Y., Zhang, H.L., Guo, H.R., Xie, L., Zeng, R.Z., Zhang, X.Q. & Zhang, Z.S. 2017 Transcriptomic and hormonal analyses reveal that YUC-mediated auxin biogenesis is involved in shoot regeneration from rhizome in Cymbidium Front. Plant Sci. 8 1866, doi: 10.3389/fpls.2017.01866

    • Search Google Scholar
    • Export Citation
  • Lu, K., Wu, B.W., Wang, J., Zhu, W., Nie, H.P., Qian, J.J., Huang, W.T. & Fang, Z.M. 2018 Blocking amino acid transporter OsAAP3 improves grain yield by promoting outgrowth buds and increasing tiller number in rice Plant Biotechnol. J. 16 1710 1722 doi: 10.1111/pbi.12907

    • Search Google Scholar
    • Export Citation
  • Matysik, J., Alia, A., Bhalu, B. & Mohanty, P. 2002 Molecular mechanisms of quenching of reactive oxygen species by proline under stress in plants Curr. Sci. 82 525 532 doi: 10.1038/416238a

    • Search Google Scholar
    • Export Citation
  • Paek, K.Y. & Kozai, T. 1998 Micropropagation of temperate Cymbidium via rhizome culture HortTechnology 8 283 288 doi: 10.21273/HORTTECH.8.3.283

  • Park, H.Y., Kang, K.W., Kim, D.H. & Sivanesan, I. 2018 In vitro propagation of Cymbidium goeringii Reichenbach fil. through direct adventitious shoot regeneration Physiol. Mol. Biol. Plants 24 307 313 doi: 10.1007/s12298-017-0503-2

    • Search Google Scholar
    • Export Citation
  • Ramya, M., Park, P.M., Chuang, Y.C., Kwon, O.K., An, H.R., Park, P.M., Baek, Y.S., Kang, B.C., Tsai, W.C. & Chen, H.H. 2019 RNA sequencing analysis of Cymbidium goeringii identifies floral scent biosynthesis related genes BMC Plant Biol. 19 337, doi: 10.1186/s12870-019-1940-6

    • Search Google Scholar
    • Export Citation
  • Ren, R., Gao, J., Lu, C.Q., Wei, Y.L., Jin, J.P., Wong, S.M., Zhu, G.F. & Yang, F.X. 2020a Highly efficient protoplast isolation and transient expression system for functional characterization of flowering related genes in Cymbidium orchids Intl. J. Mol. Med. 21 2264, doi: 10.3390/ijms21072264

    • Search Google Scholar
    • Export Citation
  • Ren, R., Wei, Y.L., Ahmad, S., Jin, J.P., Gao, J., Lu, C.Q., Zhu, G.F. & Yang, F.X. 2020b Identification and characterization of NPR1 and PR1 homologs in Cymbidium orchids in response to multiple hormones, salinity and viral stresses Intl. J. Mol. Sci. 21 1977, doi: 10.3390/ijms21061977

    • Search Google Scholar
    • Export Citation
  • Roy, J. & Banerjee, N. 2002 Rhizome and shoot development during in vitro propagation of Geodorum densiflorum (Lam.) Schltr Sci. Hort. 94 181 192 doi: 10.1016/S0304-4238(01)00373-9

    • Search Google Scholar
    • Export Citation
  • Schenck, C.A. & Maeda, H.A. 2018 Tyrosine biosynthesis, metabolism, and catabolism in plants Phytochemistry 149 82 102 doi: 10.1016/j.phytochem.2018.02.003

    • Search Google Scholar
    • Export Citation
  • Shimasaki, K. & Uemoto, S. 1990 Micropropagation of a terrestrial Cymbidium species using rhizomes developed from seeds and pseudobulbs Plant Cell Tiss. Org. 22 237 244 doi: 10.1007/bf00033642

    • Search Google Scholar
    • Export Citation
  • Tegeder, M. 2012 Transporters for amino acids in plant cells: Some functions and many unknowns Curr. Opin. Plant Biol. 15 315 321 doi: 10.1016/j.pbi.2012.02.001

    • Search Google Scholar
    • Export Citation
  • Voll, L.M., Allaire, E.E., Fiene, G. & Weber, A.P. 2004 The Arabidopsis phenylalanine insensitive growth mutant exhibits a deregulated amino acid metabolism Plant Physiol. 136 3058 3069 doi: 10.1104/pp.104.047506

    • Search Google Scholar
    • Export Citation
  • Wang, J., Wu, B.W., Lu, K., Wei, Q., Qian, J.J., Chen, Y.P. & Fang, Z.M. 2019 The amino acid permease 5 (OsAAP5) regulates tiller number and grain yield in rice Plant Physiol. 180 1031 1045 doi: 10.1104/pp.19.00034

    • Search Google Scholar
    • Export Citation
  • Xiang, L., Chen, Y., Chen, L.P., Fu, X.P., Zhao, K.G., Zhang, J. & Sun, C.B. 2018 B and E MADS-box genes determine the perianth formation in Cymbidium goeringii Rchb.f Physiol. Plant. 162 353 369 doi: 10.1111/ppl.12647

    • Search Google Scholar
    • Export Citation
  • Yang, F.X., Zhu, G.F., Wang, Z., Liu, H.L., Xu, Q.Q., Huang, D. & Zhao, C.Y. 2017 Integrated mRNA and microRNA transcriptome variations in the multi-tepal mutant provide insights into the floral patterning of the orchid Cymbidium goeringii BMC Genomics 18 367, doi: 10.1186/s12864-017-3756-9

    • Search Google Scholar
    • Export Citation
  • Yang, F.X., Zhu, G.F., Wei, Y.L., Gao, J., Liang, G., Peng, L.Y., Lu, C.Q. & Jin, J.P. 2019 Low-temperature-induced changes in the transcriptome reveal a major role of CgSVP genes in regulating flowering of Cymbidium goeringii BMC Genomics 20 53, doi: 10.1186/s12864-019-5425-7

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  • Zhang, J.X., Zhao, X.L., Tian, R.X., Zeng, S.J., Wu, K.L., Jaime, A.T. & Duan, J. 2019 Molecular cloning and functional analysis of three CONSTANS-Like genes from Chinese Cymbidium J. Plant Growth Regul. 39 3 79 84 doi: 10.1007/s00344-019-10044-9

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Contributor Notes

This work was supported by the grants from the National Natural Science Foundation of China (32060064/31301250) and High-level Talent Funding of Guizhou University (702646193301).

Z.F. is the corresponding author. E-mail: zmfang@gzu.edu.cn.

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    Effects of different amino acid treatments on Cymbidium goeringii rhizome proliferation. (A) Control. (B) Proline at 1.0 mmol·L−1. (C) Cysteine at 2.0 mmol·L−1. (D) Asparagine at 0.2 mmol·L−1. (E) Aspartate at 2.0 mmol·L−1. (F) Arginine at 0.5 mmol·L−1. (G) Lysine at 0.5 mmol·L−1. (H) Arginine at 0.5 mmol·L−1 + cysteine at 2.0 mmol·L−1. (I) Arginine at 0.5 mmol·L−1 + proline at 1.0 mmol·L−1.

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    Effects of different amino acid treatments on Cymbidium goeringii rhizome differentiation. (A) Control. (B) Methionine at 2.0 mmol·L−1. (C) Asparagine at 0.5 mmol·L−1. (D) Asparagine at 0.5 mmol·L−1 + tyrosine at 1.0 mmol·L−1. (E) Asparagine at 0.5 mmol·L−1 + aspartate at 0.2 mmol·L−1. (F) Shoots developed from the tips of the rhizomes in Huabao medium supplemented with activated charcoal (AC) and naphthalene acetic acid (NAA) after 2 months.

  • Bottari, E. & Festa, M.R. 1996 Asparagine as a ligand for cadmium (II), lead (II) and zinc (II) Chem. Spec. Bioavail. 8 75 83 doi: 10.1080/09542299.1996.11083272

    • Search Google Scholar
    • Export Citation
  • Chen, Z.Y., Chen, T.P., Li, H.J. & Chui, S.Y. 2017 Research progress in molecular mechanism of amino acids absorption and transport by plants Mol. Plant Breed. 15 5166 5171 doi: 10.13271/j.mpb.015.005166

    • Search Google Scholar
    • Export Citation
  • Chung, M.Y. & Nason, J.D. 2007 Spatial demographic and genetic consequences of harvesting within populations of terrestrial orchid Cymbidium goeringii Biol. Conserv. 137 125 137 doi: 10.1016/j.biocon.2007.01.021

    • Search Google Scholar
    • Export Citation
  • Coruzzi, G. & Bush, D.R. 2001 Nitrogen and carbon nutrient and metabolite signaling in plant Plant Physiol. 125 61 64 doi: 10.1104/pp.125.1.61

  • Domínguez-Solís, J.R., López-Martín, M.C., Ager, F.J., Ynsa, M.D., Romero, L.C. & Gotor, C. 2004 Increased cysteine availability is essential for cadmium tolerance and accumulation in Arabidopsis thaliana Plant Biotechnol. J. 2 469 476 doi: 10.1111/j.1467-7652.2004.00092.x

    • Search Google Scholar
    • Export Citation
  • Gao, R., Wu, S.Q., Piao, X.C., Park, S.Y. & Lian, M.L. 2014 Micropropagation of Cymbidium sinense using continuous and temporary airlift bioreactor systems Acta Physiol. Plant. 36 117 124 doi: 10.1007/s11738-013-1392-9

    • Search Google Scholar
    • Export Citation
  • Guha, S. & Rao, I.U. 2012 Nitric oxide promoted rhizome induction in Cymbidium shoot buds under magnesium deficiency Biol. Plant. 56 227 236 doi: 10.1007/s10535-012-0081-7

    • Search Google Scholar
    • Export Citation
  • Huang, W.T., Fang, Z.M., Zeng, S.J., Zhang, J.X., Wu, K.L., Chen, Z.L., Jaime, A.T. & Duan, J. 2012 Molecular cloning and functional analysis of three FLOWERING LOCUS T (FT) homologous genes from Chinese Cymbidium Intl. J. Mol. Sci. 13 11385 11398 doi: 10.3390/ijms130911385

    • Search Google Scholar
    • Export Citation
  • Huang, W.T., Zeng, L.T., Wu, B.W. & Fang, Z.M. 2017 Effects of different organic additives and sodium citrate combination on rhizomes proliferation of Cymbidium goeringii J. Anhui Agr. Univ. 44 1112 1118 doi: 10.13610/j.cnki.1672-352x.20171214.010

    • Search Google Scholar
    • Export Citation
  • Ichihashi, Y., Date, Y., Shino, A., Shimizu, T., Shibata, A., Kumaishi, K., Funahashi, F., Wakayama, K., Yamazaki, K., Umezawa, A., Sato, T., Kobayashi, M., Kamimura, M., Kusano, M., Che, F.S., Brien, M.O., Tanoi, K., Hayashi, M., Nakamura, R., Shirasu, K., Kikuchi, J. & Nihei, N. 2020 Multi-omics analysis on an agroecosystem reveals the significant role of organic nitrogen to increase agricultural crop yield Proc. Natl. Acad. Sci. USA 117 17259, doi: 10.1073/pnas.1917259117

    • Search Google Scholar
    • Export Citation
  • Kan, C.C., Chung, T.Y., Wu, H.Y., Juo, Y.A. & Hsieh, M.H. 2017 Exogenous glutamate rapidly induces the expression of genes involved in metabolism and defense responses in rice roots BMC Genomics 18 186, doi: 10.1186/s12864-017-3588-7

    • Search Google Scholar
    • Export Citation
  • Krapp, A. 2015 Plant nitrogen assimilation and its regulation: A complex puzzle with missing pieces Curr. Opin. Plant Biol. 25 115 122 doi: 10.1016/j.pbi.2015.05.010

    • Search Google Scholar
    • Export Citation
  • Lee, O.R., Yang, D.C., Chung, H.J. & Min, B.H. 2011 Efficient in vitro plant regeneration from hybrid rhizomes of Cymbidium sinense seeds Hort. Environ. Biotechnol. 52 303 308 doi: 10.1007/s13580-011-0187-4

    • Search Google Scholar
    • Export Citation
  • Liu, Y., Zhang, H.L., Guo, H.R., Xie, L., Zeng, R.Z., Zhang, X.Q. & Zhang, Z.S. 2017 Transcriptomic and hormonal analyses reveal that YUC-mediated auxin biogenesis is involved in shoot regeneration from rhizome in Cymbidium Front. Plant Sci. 8 1866, doi: 10.3389/fpls.2017.01866

    • Search Google Scholar
    • Export Citation
  • Lu, K., Wu, B.W., Wang, J., Zhu, W., Nie, H.P., Qian, J.J., Huang, W.T. & Fang, Z.M. 2018 Blocking amino acid transporter OsAAP3 improves grain yield by promoting outgrowth buds and increasing tiller number in rice Plant Biotechnol. J. 16 1710 1722 doi: 10.1111/pbi.12907

    • Search Google Scholar
    • Export Citation
  • Matysik, J., Alia, A., Bhalu, B. & Mohanty, P. 2002 Molecular mechanisms of quenching of reactive oxygen species by proline under stress in plants Curr. Sci. 82 525 532 doi: 10.1038/416238a

    • Search Google Scholar
    • Export Citation
  • Paek, K.Y. & Kozai, T. 1998 Micropropagation of temperate Cymbidium via rhizome culture HortTechnology 8 283 288 doi: 10.21273/HORTTECH.8.3.283

  • Park, H.Y., Kang, K.W., Kim, D.H. & Sivanesan, I. 2018 In vitro propagation of Cymbidium goeringii Reichenbach fil. through direct adventitious shoot regeneration Physiol. Mol. Biol. Plants 24 307 313 doi: 10.1007/s12298-017-0503-2

    • Search Google Scholar
    • Export Citation
  • Ramya, M., Park, P.M., Chuang, Y.C., Kwon, O.K., An, H.R., Park, P.M., Baek, Y.S., Kang, B.C., Tsai, W.C. & Chen, H.H. 2019 RNA sequencing analysis of Cymbidium goeringii identifies floral scent biosynthesis related genes BMC Plant Biol. 19 337, doi: 10.1186/s12870-019-1940-6

    • Search Google Scholar
    • Export Citation
  • Ren, R., Gao, J., Lu, C.Q., Wei, Y.L., Jin, J.P., Wong, S.M., Zhu, G.F. & Yang, F.X. 2020a Highly efficient protoplast isolation and transient expression system for functional characterization of flowering related genes in Cymbidium orchids Intl. J. Mol. Med. 21 2264, doi: 10.3390/ijms21072264

    • Search Google Scholar
    • Export Citation
  • Ren, R., Wei, Y.L., Ahmad, S., Jin, J.P., Gao, J., Lu, C.Q., Zhu, G.F. & Yang, F.X. 2020b Identification and characterization of NPR1 and PR1 homologs in Cymbidium orchids in response to multiple hormones, salinity and viral stresses Intl. J. Mol. Sci. 21 1977, doi: 10.3390/ijms21061977

    • Search Google Scholar
    • Export Citation
  • Roy, J. & Banerjee, N. 2002 Rhizome and shoot development during in vitro propagation of Geodorum densiflorum (Lam.) Schltr Sci. Hort. 94 181 192 doi: 10.1016/S0304-4238(01)00373-9

    • Search Google Scholar
    • Export Citation
  • Schenck, C.A. & Maeda, H.A. 2018 Tyrosine biosynthesis, metabolism, and catabolism in plants Phytochemistry 149 82 102 doi: 10.1016/j.phytochem.2018.02.003

    • Search Google Scholar
    • Export Citation
  • Shimasaki, K. & Uemoto, S. 1990 Micropropagation of a terrestrial Cymbidium species using rhizomes developed from seeds and pseudobulbs Plant Cell Tiss. Org. 22 237 244 doi: 10.1007/bf00033642

    • Search Google Scholar
    • Export Citation
  • Tegeder, M. 2012 Transporters for amino acids in plant cells: Some functions and many unknowns Curr. Opin. Plant Biol. 15 315 321 doi: 10.1016/j.pbi.2012.02.001

    • Search Google Scholar
    • Export Citation
  • Voll, L.M., Allaire, E.E., Fiene, G. & Weber, A.P. 2004 The Arabidopsis phenylalanine insensitive growth mutant exhibits a deregulated amino acid metabolism Plant Physiol. 136 3058 3069 doi: 10.1104/pp.104.047506

    • Search Google Scholar
    • Export Citation
  • Wang, J., Wu, B.W., Lu, K., Wei, Q., Qian, J.J., Chen, Y.P. & Fang, Z.M. 2019 The amino acid permease 5 (OsAAP5) regulates tiller number and grain yield in rice Plant Physiol. 180 1031 1045 doi: 10.1104/pp.19.00034

    • Search Google Scholar
    • Export Citation
  • Xiang, L., Chen, Y., Chen, L.P., Fu, X.P., Zhao, K.G., Zhang, J. & Sun, C.B. 2018 B and E MADS-box genes determine the perianth formation in Cymbidium goeringii Rchb.f Physiol. Plant. 162 353 369 doi: 10.1111/ppl.12647

    • Search Google Scholar
    • Export Citation
  • Yang, F.X., Zhu, G.F., Wang, Z., Liu, H.L., Xu, Q.Q., Huang, D. & Zhao, C.Y. 2017 Integrated mRNA and microRNA transcriptome variations in the multi-tepal mutant provide insights into the floral patterning of the orchid Cymbidium goeringii BMC Genomics 18 367, doi: 10.1186/s12864-017-3756-9

    • Search Google Scholar
    • Export Citation
  • Yang, F.X., Zhu, G.F., Wei, Y.L., Gao, J., Liang, G., Peng, L.Y., Lu, C.Q. & Jin, J.P. 2019 Low-temperature-induced changes in the transcriptome reveal a major role of CgSVP genes in regulating flowering of Cymbidium goeringii BMC Genomics 20 53, doi: 10.1186/s12864-019-5425-7

    • Search Google Scholar
    • Export Citation
  • Zhang, J.X., Zhao, X.L., Tian, R.X., Zeng, S.J., Wu, K.L., Jaime, A.T. & Duan, J. 2019 Molecular cloning and functional analysis of three CONSTANS-Like genes from Chinese Cymbidium J. Plant Growth Regul. 39 3 79 84 doi: 10.1007/s00344-019-10044-9

    • Search Google Scholar
    • Export Citation
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