J Plant Biotechnol (2024) 51:024-032
Published online January 25, 2024
https://doi.org/10.5010/JPB.2024.51.003.024
© The Korean Society of Plant Biotechnology
Correspondence to : e-mail: nthao@hcmus.edu.vn
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Celosia argentea var cristata, commonly known as the cockscomb plant, is a popular ornamental species in Vietnam. Its propagation primarily relies on seeds, enabling widespread cultivation but leading to a notable absence of micropropagation research in the country. This practice poses a potential threat to preserving unique traits susceptible to loss through segregation. To address this gap, this study focused on the impact of plant hormones on callus formation in various aerial tissues - leaves, stems, and newly emerging inflorescences - gathered from plants grown on soil. The calli displayed distinct morphological characteristics under the influence of different combinations of 6-Benzyladenine (BAP), 1-Naphthaleneacetic acid (NAA), and 2,4-Dicholorophenoxyacetic acid (2,4-D). Furthermore, we investigated the genetic stability of C. argentea var cristata calli using random amplified polymorphic DNA (RAPD) markers. The calli persistently cultured on medium containing 2 mg/L BAP and 2 mg/L NAA maintained their genetics stability, as assessed through four RAPD markers: OPA-13, OPA-15, OPA-18 (G), and OPD-2.
Keywords Callus, Cockscomb, ex vitro tissue, Genetic stability, RAPD
Among the
Studies on
The leaves were rinsed with water, and then shaken with 70% ethanol (v/v) for 30 seconds. Leaf samples were subsequently treated with different concentrations of sodium hypochlorite (NaClO) (0.4-0.5%) combined with Tween 20 (0.025-0.05%). The surface sterilization time (10-30 min) was also investigated. The sterilized samples were washed with sterilized water prior to being transferred to plates containing Murashige and Skoog (MS) medium (Murashige and Skoog 1962) supplemented with 3% sucrose and 0.8% agar, pH 5.8.
To study the effect of plant hormones on
To investigate the callus induction ability of different
Calli from leaves, sub-cultured weekly on MS medium supplemented with 2 mg/L BAP and 2 mg/L NAA, were collected after 15, 30, and 60 days for DNA extraction. Leaves of the same plant used for callus induction were used as control. The DNA samples were extracted following the CTAB method as previously described (Doyle 1991; Ly et al. 2020). Ten RAPD primers, i.e. OPA-7, OPA-10, OPA-13, OPA-14, OPA-15, OPA-16, OPA18, OPA20, OPD2, and OPA18 (G) (with the sequence AGGTGACCGG) (Hariyati et al. 2013; Joshi et al. 2011) were used to evaluate the genetic stability. The negative control used DEPC-treated water instead of genomic DNA. Polymerase chain reactions (PCRs) were performed according to the manufacturer’s manual (GoTaq® Green Master Mix kit, Promega, USA). The thermal cycle started at 92°C for 2 min, followed by 35 cycles of 92°C-30 s, 41°C-30 s, and 72°C-2 min. The final extension step was held at 72°C for 5 min. Amplified products were migrated on 1% agarose gel at 80 V for 35 min, and DNA size was determined according to the HyperLadder™ 1kb (BIO-33053, Meridian Bioscience Inc, UK).
Statistical difference was determined by ANOVA (P < 0.05) followed by Tukey’s HSD post hoc test using SPSS v20 (IBM, USA). In addition, the callus area was measured by ImageJ software v1.8.0 (Schindelin et al. 2012). In all experiments, each treatment was repeated three times.
Obtaining sterile samples is critical for subsequent
This procedure was applied to sterilize other tissue types including stem and inflorescence. Consistent results were observed with stem tissue as sterilized samples showed high vitality, and no contamination was observed. For inflorescence tissues, this protocol caused a higher death ratio with 16% of samples contaminated.
After 7 days, the emergence of small calli was observed at the edges of leaf samples except those treated with 1 mg/L BAP & 1 mg/L NAA, 1 mg/L 2,4-D, and 1 mg/L BAP & 0.5 mg/L 2,4-D (Fig. 1A, F, and I). In all other treatments, the calli were small, and white (Fig. 1). On the control medium, leaf samples were alive but did not generate callus (Fig. 1L).
After 14 days on callus induction media, the formation of callus was observed in all treatments (Table 1) while most samples on the control medium showed necrosis (Fig. 2L). The combination of BAP and NAA showed the highest callus formation efficiency with only the exception of the 1 mg/L BAP & 1 mg/L NAA treatment (Fig. 2M). Root development was also observed in samples treated with either 2 mg/L BAP & 2 mg/L NAA and 3 mg/L BAP & 3 mg/L NAA (Fig. 2B, D). Similar 100% of callus formation was also observed in the treatment with 1 mg/L BAP & 0.5 mg/L 2,4-D. All other treatments using either 2,4-D alone or higher 2,4-D concentrations in combination with BAP showed significantly lower callus formation efficiency (Fig. 2M). It must be noticed that calli formed on media containing 2,4-D had yellow color while the combination of BAP and NAA induced the formation of white and or light yellow calli. Although multiple treatments showed the maximum callus formation efficiency, the highest callus size was only observed in the treatment with 2 mg/L BAP & 2 mg/L NAA. In all other treatments, the calli were only half in size (Fig. 2N).
Table 1 . Effects of various plant hormones on cultured tissue of C. cristata derived from leaves after 14 days. Different letters indicate statistically significant differences (ANOVA, Tukey’s test, P < 0.05)
Concentration of plant hormones (mg/L) | Percentage of callus induction (%) | Observation | ||
---|---|---|---|---|
BAP | NAA | 2,4-D | ||
1 | 1 | - | 23.33 ± 5.77e | White and small callus |
2 | 2 | - | 98.33 ± 2.89a | Some of the white callus had roots |
3 | 2.5 | - | 95.33 ± 4.51a | Yellow-brown callus |
3 | 3 | - | 100a | White callus |
- | - | 0.5 | 63.33 ± 7.64bc | Yellow-brown callus |
- | - | 1 | 41.87 ± 10.96d | Yellow-brown callus |
- | - | 1.5 | 19.37 ± 12.21e | Yellow-brown callus |
- | - | 2 | 67.23 ± 2.54b | Yellow-brown callus |
1 | - | 0.5 | 100a | Greyish-white calluses and leaf tissues were dark green |
1 | - | 1 | 64.4 ± 5.11bc | Yellow-brown callus |
2 | - | 1 | 49.03 ± 1.67cd | Yellow-brown callus |
The formed calli continued to increase in size after 21 days while most of the samples on the control medium were dead (Fig. 3). However, the development speed of calli was different between treatments with the fastest speed observed in samples treated with 2 mg/L BAP & 2 mg/L NAA. The yellow color of samples on media containing 2,4-D became darker and some samples turned brown. Stronger effects were observed on 2,4-D-only treatments compared to treatments where BAP was added. In all treatments, calli induced from leaf samples were compact.
The calli induced from leaf tissue on MS media supplemented with 2 mg/L BAP and 2 mg/L NAA had the highest fresh weight and dry weight after 14 days, 102 ± 23 mg and 12 ± 2 mg, respectively (Fig. 4B, C). When the calli were induced at concentrations of 1 mg/L BAP and 1 mg/L NAA, they turned brown after two weeks. Fresh weight and dry weight of calli induced on MS medium containing 1 mg/L BAP and 1 mg/L NAA was 43 ± 14 mg and 5 ± 2 mg. The exposure of thin layers of leaf to 3 mg/L BAP and 3 mg/L NAA resulted in severe browning in all samples similar to samples on the control medium without any plant hormones.
For stem tissue, the combination of 2 mg/L BAP and 2 mg/L NAA also showed the highest callus formation with fresh and dry weight of 34 ± 3 mg and 3.7 ± 0.3 mg. Contrary to leaf tissues, callus from stem tissues in 3 mg/L BAP and 3 mg/L NAA treatment did not undergo cell death and had the fresh weight was similar to that of 1 mg/L BAP and 1 mg/L NAA (Fig. 4B). The stem dry weight also followed a similar tendency as observed in fresh weight.
For inflorescence tissues, in all concentrations, there was calli formation but there is no difference in weight, with fresh weight ranging from 4-6 mg (Fig. 4B), and dry weight ranging from 0.7-1.2 mg (Fig. 4C). Although the medium containing 1 mg/L BAP and 1 mg/L NAA showed the highest callus induction efficiency, the difference was insignificant.
RAPD primers OPA and OPD are commonly used to assess the genetic stability of different plant species including rice (Azim et al. 2022; Devi and Reddy 2015), black gram (Arulbalachandran et al. 2010), and
Of the 10 RAPD primers used, OPA14, OPA16, and OPA20 did not appear any bands (Fig. 5). The primer, OPA10, only gave 1 band, and OPA7, OPA18 gave 2 bands. Meanwhile, primers OPA13, OPA15, OPA18 (G), and OPD2 gave 6-8 bands. With all primers used, no genetical variation was observed between calli 15, 30, and 60 days on medium containing 2 mg/L BAP and 2 mg/L NAA (Fig. 5).
The combination of 1 mg/L BAP and 1 mg/L NAA was reported to allow shoot growth after 7 days (Abu Bakar et al. 2014). In this study, leaf samples treated with 1 mg/L BAP and 1 mg/L NAA showed very low callus induction efficiency after 7 days. In addition, the results of callus formation in response to 2,4-D in this study were also different from that reported for
The plant hormones auxin and cytokinin play an essential role in plant growth and development (Jones and Ljung 2011). Auxin promotes root growth, while cytokinin stimulates the growth of shoots (Mohamad et al. 2022). A balance in effect between internal and external auxin and cytokinin is necessary to induce callus. Shifting the balance toward auxin or cytokinin during
Differences in callus color in response to different plant hormone treatments suggested different metabolite compounds synthesized in these calli. Mastuti et al. (2021) determined the compounds present in yellow calli using high-performance liquid chromatography (HPLC) method. The authors discovered that in the yellow
In our study, all treatments, either with or without BAP, stimulated the formation of compact calli from
For further application of the callus culture of
This research is funded by University of Science, VNU-HCM under grant number SH-CNSH 2023-08.
T.-H.N., P.N.D.Q., and V.A.L., conceived the research project; T.-H.N., and V.A.L., designed the experiments; N.-A.T.-N., and M.Y.H., performed the experiments; N.-A. T.-N., and H.H.D., analyzed data; N.-A.T.-N., M.Y.H., V.A.L., and T.-H.N., wrote the article. All authors reviewed and approved the final manuscript.
J Plant Biotechnol 2024; 51(1): 24-32
Published online January 25, 2024 https://doi.org/10.5010/JPB.2024.51.003.024
Copyright © The Korean Society of Plant Biotechnology.
Nhat-Anh Tran-Nguyen・My Y Huynh・Hong Hanh Doan・Phuong Ngo Diem Quach・Thanh-Hao Nguyen・ Vi An Ly
Faculty of Biology and Biotechnology, University of Science, Ho Chi Minh City, Vietnam
Vietnam National University, Ho Chi Minh City, Vietnam
Laboratory of Molecular Biotechnology, University of Science, Vietnam National University, Ho Chi Minh City, Vietnam
Correspondence to:e-mail: nthao@hcmus.edu.vn
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Celosia argentea var cristata, commonly known as the cockscomb plant, is a popular ornamental species in Vietnam. Its propagation primarily relies on seeds, enabling widespread cultivation but leading to a notable absence of micropropagation research in the country. This practice poses a potential threat to preserving unique traits susceptible to loss through segregation. To address this gap, this study focused on the impact of plant hormones on callus formation in various aerial tissues - leaves, stems, and newly emerging inflorescences - gathered from plants grown on soil. The calli displayed distinct morphological characteristics under the influence of different combinations of 6-Benzyladenine (BAP), 1-Naphthaleneacetic acid (NAA), and 2,4-Dicholorophenoxyacetic acid (2,4-D). Furthermore, we investigated the genetic stability of C. argentea var cristata calli using random amplified polymorphic DNA (RAPD) markers. The calli persistently cultured on medium containing 2 mg/L BAP and 2 mg/L NAA maintained their genetics stability, as assessed through four RAPD markers: OPA-13, OPA-15, OPA-18 (G), and OPD-2.
Keywords: Callus, Cockscomb, ex vitro tissue, Genetic stability, RAPD
Among the
Studies on
The leaves were rinsed with water, and then shaken with 70% ethanol (v/v) for 30 seconds. Leaf samples were subsequently treated with different concentrations of sodium hypochlorite (NaClO) (0.4-0.5%) combined with Tween 20 (0.025-0.05%). The surface sterilization time (10-30 min) was also investigated. The sterilized samples were washed with sterilized water prior to being transferred to plates containing Murashige and Skoog (MS) medium (Murashige and Skoog 1962) supplemented with 3% sucrose and 0.8% agar, pH 5.8.
To study the effect of plant hormones on
To investigate the callus induction ability of different
Calli from leaves, sub-cultured weekly on MS medium supplemented with 2 mg/L BAP and 2 mg/L NAA, were collected after 15, 30, and 60 days for DNA extraction. Leaves of the same plant used for callus induction were used as control. The DNA samples were extracted following the CTAB method as previously described (Doyle 1991; Ly et al. 2020). Ten RAPD primers, i.e. OPA-7, OPA-10, OPA-13, OPA-14, OPA-15, OPA-16, OPA18, OPA20, OPD2, and OPA18 (G) (with the sequence AGGTGACCGG) (Hariyati et al. 2013; Joshi et al. 2011) were used to evaluate the genetic stability. The negative control used DEPC-treated water instead of genomic DNA. Polymerase chain reactions (PCRs) were performed according to the manufacturer’s manual (GoTaq® Green Master Mix kit, Promega, USA). The thermal cycle started at 92°C for 2 min, followed by 35 cycles of 92°C-30 s, 41°C-30 s, and 72°C-2 min. The final extension step was held at 72°C for 5 min. Amplified products were migrated on 1% agarose gel at 80 V for 35 min, and DNA size was determined according to the HyperLadder™ 1kb (BIO-33053, Meridian Bioscience Inc, UK).
Statistical difference was determined by ANOVA (P < 0.05) followed by Tukey’s HSD post hoc test using SPSS v20 (IBM, USA). In addition, the callus area was measured by ImageJ software v1.8.0 (Schindelin et al. 2012). In all experiments, each treatment was repeated three times.
Obtaining sterile samples is critical for subsequent
This procedure was applied to sterilize other tissue types including stem and inflorescence. Consistent results were observed with stem tissue as sterilized samples showed high vitality, and no contamination was observed. For inflorescence tissues, this protocol caused a higher death ratio with 16% of samples contaminated.
After 7 days, the emergence of small calli was observed at the edges of leaf samples except those treated with 1 mg/L BAP & 1 mg/L NAA, 1 mg/L 2,4-D, and 1 mg/L BAP & 0.5 mg/L 2,4-D (Fig. 1A, F, and I). In all other treatments, the calli were small, and white (Fig. 1). On the control medium, leaf samples were alive but did not generate callus (Fig. 1L).
After 14 days on callus induction media, the formation of callus was observed in all treatments (Table 1) while most samples on the control medium showed necrosis (Fig. 2L). The combination of BAP and NAA showed the highest callus formation efficiency with only the exception of the 1 mg/L BAP & 1 mg/L NAA treatment (Fig. 2M). Root development was also observed in samples treated with either 2 mg/L BAP & 2 mg/L NAA and 3 mg/L BAP & 3 mg/L NAA (Fig. 2B, D). Similar 100% of callus formation was also observed in the treatment with 1 mg/L BAP & 0.5 mg/L 2,4-D. All other treatments using either 2,4-D alone or higher 2,4-D concentrations in combination with BAP showed significantly lower callus formation efficiency (Fig. 2M). It must be noticed that calli formed on media containing 2,4-D had yellow color while the combination of BAP and NAA induced the formation of white and or light yellow calli. Although multiple treatments showed the maximum callus formation efficiency, the highest callus size was only observed in the treatment with 2 mg/L BAP & 2 mg/L NAA. In all other treatments, the calli were only half in size (Fig. 2N).
Table 1 . Effects of various plant hormones on cultured tissue of C. cristata derived from leaves after 14 days. Different letters indicate statistically significant differences (ANOVA, Tukey’s test, P < 0.05).
Concentration of plant hormones (mg/L) | Percentage of callus induction (%) | Observation | ||
---|---|---|---|---|
BAP | NAA | 2,4-D | ||
1 | 1 | - | 23.33 ± 5.77e | White and small callus |
2 | 2 | - | 98.33 ± 2.89a | Some of the white callus had roots |
3 | 2.5 | - | 95.33 ± 4.51a | Yellow-brown callus |
3 | 3 | - | 100a | White callus |
- | - | 0.5 | 63.33 ± 7.64bc | Yellow-brown callus |
- | - | 1 | 41.87 ± 10.96d | Yellow-brown callus |
- | - | 1.5 | 19.37 ± 12.21e | Yellow-brown callus |
- | - | 2 | 67.23 ± 2.54b | Yellow-brown callus |
1 | - | 0.5 | 100a | Greyish-white calluses and leaf tissues were dark green |
1 | - | 1 | 64.4 ± 5.11bc | Yellow-brown callus |
2 | - | 1 | 49.03 ± 1.67cd | Yellow-brown callus |
The formed calli continued to increase in size after 21 days while most of the samples on the control medium were dead (Fig. 3). However, the development speed of calli was different between treatments with the fastest speed observed in samples treated with 2 mg/L BAP & 2 mg/L NAA. The yellow color of samples on media containing 2,4-D became darker and some samples turned brown. Stronger effects were observed on 2,4-D-only treatments compared to treatments where BAP was added. In all treatments, calli induced from leaf samples were compact.
The calli induced from leaf tissue on MS media supplemented with 2 mg/L BAP and 2 mg/L NAA had the highest fresh weight and dry weight after 14 days, 102 ± 23 mg and 12 ± 2 mg, respectively (Fig. 4B, C). When the calli were induced at concentrations of 1 mg/L BAP and 1 mg/L NAA, they turned brown after two weeks. Fresh weight and dry weight of calli induced on MS medium containing 1 mg/L BAP and 1 mg/L NAA was 43 ± 14 mg and 5 ± 2 mg. The exposure of thin layers of leaf to 3 mg/L BAP and 3 mg/L NAA resulted in severe browning in all samples similar to samples on the control medium without any plant hormones.
For stem tissue, the combination of 2 mg/L BAP and 2 mg/L NAA also showed the highest callus formation with fresh and dry weight of 34 ± 3 mg and 3.7 ± 0.3 mg. Contrary to leaf tissues, callus from stem tissues in 3 mg/L BAP and 3 mg/L NAA treatment did not undergo cell death and had the fresh weight was similar to that of 1 mg/L BAP and 1 mg/L NAA (Fig. 4B). The stem dry weight also followed a similar tendency as observed in fresh weight.
For inflorescence tissues, in all concentrations, there was calli formation but there is no difference in weight, with fresh weight ranging from 4-6 mg (Fig. 4B), and dry weight ranging from 0.7-1.2 mg (Fig. 4C). Although the medium containing 1 mg/L BAP and 1 mg/L NAA showed the highest callus induction efficiency, the difference was insignificant.
RAPD primers OPA and OPD are commonly used to assess the genetic stability of different plant species including rice (Azim et al. 2022; Devi and Reddy 2015), black gram (Arulbalachandran et al. 2010), and
Of the 10 RAPD primers used, OPA14, OPA16, and OPA20 did not appear any bands (Fig. 5). The primer, OPA10, only gave 1 band, and OPA7, OPA18 gave 2 bands. Meanwhile, primers OPA13, OPA15, OPA18 (G), and OPD2 gave 6-8 bands. With all primers used, no genetical variation was observed between calli 15, 30, and 60 days on medium containing 2 mg/L BAP and 2 mg/L NAA (Fig. 5).
The combination of 1 mg/L BAP and 1 mg/L NAA was reported to allow shoot growth after 7 days (Abu Bakar et al. 2014). In this study, leaf samples treated with 1 mg/L BAP and 1 mg/L NAA showed very low callus induction efficiency after 7 days. In addition, the results of callus formation in response to 2,4-D in this study were also different from that reported for
The plant hormones auxin and cytokinin play an essential role in plant growth and development (Jones and Ljung 2011). Auxin promotes root growth, while cytokinin stimulates the growth of shoots (Mohamad et al. 2022). A balance in effect between internal and external auxin and cytokinin is necessary to induce callus. Shifting the balance toward auxin or cytokinin during
Differences in callus color in response to different plant hormone treatments suggested different metabolite compounds synthesized in these calli. Mastuti et al. (2021) determined the compounds present in yellow calli using high-performance liquid chromatography (HPLC) method. The authors discovered that in the yellow
In our study, all treatments, either with or without BAP, stimulated the formation of compact calli from
For further application of the callus culture of
This research is funded by University of Science, VNU-HCM under grant number SH-CNSH 2023-08.
T.-H.N., P.N.D.Q., and V.A.L., conceived the research project; T.-H.N., and V.A.L., designed the experiments; N.-A.T.-N., and M.Y.H., performed the experiments; N.-A. T.-N., and H.H.D., analyzed data; N.-A.T.-N., M.Y.H., V.A.L., and T.-H.N., wrote the article. All authors reviewed and approved the final manuscript.
Table 1 . Effects of various plant hormones on cultured tissue of C. cristata derived from leaves after 14 days. Different letters indicate statistically significant differences (ANOVA, Tukey’s test, P < 0.05).
Concentration of plant hormones (mg/L) | Percentage of callus induction (%) | Observation | ||
---|---|---|---|---|
BAP | NAA | 2,4-D | ||
1 | 1 | - | 23.33 ± 5.77e | White and small callus |
2 | 2 | - | 98.33 ± 2.89a | Some of the white callus had roots |
3 | 2.5 | - | 95.33 ± 4.51a | Yellow-brown callus |
3 | 3 | - | 100a | White callus |
- | - | 0.5 | 63.33 ± 7.64bc | Yellow-brown callus |
- | - | 1 | 41.87 ± 10.96d | Yellow-brown callus |
- | - | 1.5 | 19.37 ± 12.21e | Yellow-brown callus |
- | - | 2 | 67.23 ± 2.54b | Yellow-brown callus |
1 | - | 0.5 | 100a | Greyish-white calluses and leaf tissues were dark green |
1 | - | 1 | 64.4 ± 5.11bc | Yellow-brown callus |
2 | - | 1 | 49.03 ± 1.67cd | Yellow-brown callus |
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