Among the Celosia genus, only the flowers of Celosia argentea L. show similarities to a rooster’s crown, giving them the name of cockscomb plants. The variations of C. argentea are categorized into three primary groups: cristata, plumosa, and spicata (Mastuti et al. 2015). Cockscomb is a widely recognized ornamental plant because of its unique crested structure and vibrant colored flowers. Even though cockscomb is commonly known as a decorative plant, it is also a potential candidate for medical research (Sing et al. 2020). Different valuable saponin compounds with beneficial health effects, e.g. celosin A, B, C, D, cristatain, and semenoside A, were found in the seed (Savage 2003; Sun et al. 2011; Vetrichelvan et al. 2002; Wang et al. 2010). Analyzing the ethanol extracts from C. argentea var. cristata (hereby called C. cristata) revealed multiple health benefit compounds (Xiang et al. 2010), among them, antioxidants (4-hydroxyphenethyl alcohol, kaempferol, quercetin) (Anand David et al. 2016; Casadey et al. 2021; Chen and Chen 2013), blood cholesterol-controlling compounds (β-sitosterol) (Lomenick et al. 2015), and cancer development inhibitors (stigmasterol) (Ashraf and Bhatti 2021).

Studies on in vitro tissue culture reported the role of 6-benzyladenine (BAP), naphthaleneacetic acid (NAA), and 2,4-Dicholorophenoxyacetic acid (2,4-D) in the regeneration of C. cristata (Abu Bakar et al. 2014; Mahmad et al. 2015; Taha and Wafa 2012). The response to plant hormones was different between tissue types. Most of these studies used seeds as starting materials. Although this approach is relatively simple compared to other plant tissues as seeds are easy to sterile, for C. cristata, the genetic variation due to the hybridity of commercial seeds could introduce strong variation between samples. To overcome this problem, this study aimed to establish a procedure for callus induction from cockscomb leaves and shoots collected from plants grown on soil, which are more abundant and in principle, contain no genetic variation.

Plant materials

C. argentea var. cristata seeds (Gia Nong Seed Co., Ltd, Vietnam) were sown on soil (Tribat soil - Green Sai Gon Co, Ltd, Ho Chi Minh City, Vietnam) in 8 × 11 cm (diameter × height) pots. Plants were watered twice daily (morning and evening) and kept in greenhouse conditions (30-34°C, the humidity 75-85%, 14 h light/10 h dark). For callus induction from leaves, the fourth and fifth leaves from the shoot were collected from plants with 14-16 fully expanded leaves. To investigate the effect of plant hormones on the callus formation from flowers and inflorescences, newly emerging flowers were used. To evaluate the effect of in vitro callus culture on genetic stability, genomic DNA was extracted from calli maintained for 2 months on 2 mg/L BAP and 2 mg/L NAA.

Surface sterilization

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.

Effect of plant hormones on callus induction

To study the effect of plant hormones on ex vitro leaves, after sterilization, samples were placed on MS medium containing either only 2,4-D (0.5 mg/L, 1 mg/L, 1.5 mg/L, and 2 mg/L); the combination of BAP and NAA (1:1, 2:2, 3:2.5, and 3:3 mg/L); or the combination of BAP with 2,4-D (1:0.5, 1:1, and 2:1 mg/L). Samples were kept in dark condition at 25°C. The results were recorded after 7, 14, and 21 days.

To investigate the callus induction ability of different ex vitro tissues in response to BAP and NAA, all tissues were sterilized prior to being transferred on media containing different combinations of these two hormones (1 mg/L BAP and 1 mg/L NAA; 2 mg/L BAP and 2 mg/L NAA; and 3 mg/L BAP and 3 mg/L NAA). The samples were kept under the same condition as the above experiment. Sample fresh weights were obtained from 14-day-old calli before drying in the incubator at 60°C for three days to determine the respective dry weights.

DNA extraction and PCR amplification

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 analysis

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.

Surface sterilization

Obtaining sterile samples is critical for subsequent in vitro tissue culture. Treating samples with 0.4% NaClO for 25 min gave the highest survival rate of about 96% with only 4% of contamination. The addition of 0.025% of Tween 20 to 0.4% NaClO increased further the survival rate to 98% and significantly enhanced the sterilization effect as no contamination was observed. Longer treatment did not increase the sterilization efficiency. Increasing the NaClO concentration to 0.5% dramatically increased the death rate to 71%.

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.

Callus induction of leaf tissue

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).

Figure 1. Results of callus induction after 7 days on MS medium culture supplemented with various plant hormones. (A) 1 mg/L BAP & 1 mg/L NAA; (B) 2 mg/L BAP & 2 mg/L NAA; (C) 3 mg/L BAP & 2.5 mg/L NAA; (D) 3 mg/L BAP & 3 mg/L NAA; (E) 0.5 mg/L 2,4-D; (F) 1 mg/L 2,4-D; (G) 1.5 mg/L 2,4-D; (H) 2 mg/L 2,4-D; (I) 1 mg/L BAP & 0.5 mg/L 2,4-D; (J) 1 mg/L BAP & 1 mg/L 2,4-D; (K) 2 mg/L BAP & 1 mg/L 2,4-D; (L) MS without plant hormone (Scale bar = 1 cm)

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

Figure 2. Results of callus induction after 14 days on MS medium culture supplemented with various plant hormones. (A) 1 mg/L BAP & 1 mg/L NAA; (B) 2 mg/L BAP & 2 mg/L NAA; (C) 3 mg/L BAP & 2.5 mg/L NAA; (D) 3 mg/L BAP & 3 mg/L NAA; (E) 0.5 mg/L 2,4-D; (F) 1 mg/L 2,4-D; (G) 1.5 mg/L 2,4-D; (H) 2 mg/L 2,4-D; (I) 1 mg/L BAP & 0.5 mg/L 2,4-D; (J) 1 mg/L BAP & 1 mg/L 2,4-D; (K) 2 mg/L BAP & 1 mg/L 2,4-D; (L) MS without plant hormone . (Scale bar = 1 cm). (M) Percentage of callus formation. (N) Callus area. Different letters indicate statistically significant differences (ANOVA, Tukey’s test, P < 0.05)

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.

Figure 3. Results of callus induction after 21 days on MS medium culture supplemented with various plant hormones. (A) 1 mg/L BAP & 1 mg/L NAA; (B) 2 mg/L BAP & 2 mg/L NAA; (C) 3 mg/L BAP & 2.5 mg/L NAA; (D) 3 mg/L BAP & 3 mg/L NAA; (E) 0.5 mg/L 2,4-D; (F) 1 mg/L 2,4-D; (G) 1.5 mg/L 2,4-D; (H) 2 mg/L 2,4-D; (I) 1 mg/L BAP & 0.5 mg/L 2,4-D; (J) 1 mg/L BAP & 1 mg/L 2,4-D; (K) 2 mg/L BAP & 1 mg/L 2,4-D; (L) MS without plant hormone. (Scale bar = 1 cm)

The effect of BAP and NAA on callus induction from different types of thin layer tissues

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.

Figure 4. Thin cell layer of leaf, shoot, and inflorescence tissues after 14 days on MS medium supplemented with various combinations of BAP and NAA. (A) Callus formation after 14 days of treatment on different media (Scale bar = 1 cm). (B and C) Fresh weights and dry weights of samples observed in (A). Different letters indicate statistically significant differences (ANOVA, Tukey’s test, P < 0.05)

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.

PCR amplification using RAPD primers

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 Rhynchostylis retusa (L.) (Ray et al. 2006). While several RAPD markers, such as OPH and OPT, were successfully used to detect the genetic variation of C. argentea, the use of OPA and OPD haven’t been reported in this species. To evaluate the effectiveness of these primers in the evaluation of the genetic stability of C. cristata, ten OPA and OPD primers were chosen to investigate the genetic stability of C. cristata calli.

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).

Figure 5. Electrophoresis results of RAPD primers. (-): negative control without DNA template; (L): genomic DNA of leaf sample; (15): genomic DNA of callus after 15 days; (30): genomic DNA of callus after 30 days; (60): genomic DNA of callus after 60 days; (M): HyperLadder™ 1kb

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 in vitro explants. Guadarrama-Flores et al. (2015) noted that single use of 2,4-D at 1.5 mg/L and 2 mg/L did not form callus (Guadarrama-Flores et al. 2015). Whereas, in this study, the use of 2 mg/L 2,4-D led to the highest rate of calli formation out of the four 2,4-D treatments (Fig. 2M). Many factors could affect the result of plant tissue culture, among them, the source of explant (Yildiz 2012). In contrast to Mastuti et al. (2021) study, this study used ex vitro explants rather than in vitro explants (Mastuti et al. 2021). Such differences in the source of the explant explained the different responses to plant hormones. According to Yildiz (2002), ex vitro explants presented more challenges for plant tissue culture research than in vitro explants (Yildiz 2002). For the first time, this study reported the effect of plant hormones on the callus induction of different C. cristata ex vitro tissues.

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 in vitro tissue culture could induce roots or shoots from the callus (Jones and Ljung 2011). In this study, the root formation from calli in the treatment of 2 mg/L BAP & 2 mg/L NAA and 3 mg/L BAP & 3 mg/L NAA (Fig. 2-B, D) suggested a shift toward auxin effect in these samples.

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 Celosia calli, miraxanthin V was the main betaxanthin compound, followed by 3-methoxytyramine betaxanthin. But in the red calli, amaranthin and isoamaranthin were the main betacyanin (Mastuti et al. 2021). Apart from betacyanin, red calli also contains betaxanthin with a lower content (Mastuti et al. 2021). The study of Guadarrama-Flores et al. (2015) on cell suspension culture of C. argentea var. plumosa identified two main betaxanthins, vulgaxanthin I and miraxanthin (Guadarrama-Flores et al. 2015). The yellow callus also had betacyanins like amaranthin and betanin, as well as dihydroxylated betanidin and decarboxy-betanidin (Guadarrama-Flores et al. 2015). In our study, the yellow color observed in calli treated with 2,4-D suggested similar compounds could be found in these samples, although further confirmation by experiment is required.

In our study, all treatments, either with or without BAP, stimulated the formation of compact calli from ex vitro tissues. A part of this result was supported by different works on tissue culture using in vitro C. argentea as the compact callus was observed in samples grown on media containing BAP (Abu Bakar et al. 2014). However, in the study by Mastuti et al. (2021), when using MS medium supplemented with 1 mg/L BAP and 0.1 mg/L 2,4-D, tissue samples formed friable callus (Mastuti et al. 2021). Such difference, again, emphasized the importance of tissue origin on the properties of callus.

For further application of the callus culture of C. cristata, genetic stability is one of the most important factors affecting the quality of subsequent work. Very few studies focused on the genetic variation of this plant species. Recently, Abdulhakeem et al. (2022), used several RAPD markers, including OPH05, OPB17, OPB04, and OPT17, to identify the diversity of C. argentea mutants (Abdulhakeem et al. 2022). In the same year, Kurucz et al. used 10 RAPD primers to evaluate the stability of mutant-factor-treated C. argentea (Kurucz et al. 2022). In our work, we used RAPD to evaluate the genetic stability of calli continuously grown on callus induction medium over a period of 60 days. Among the 10 tested primers, the four which showed polymorphic bands showed the potential for evaluating the genetic variation of C. cristata.

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.