Carnations (Dianthus caryophyllus L.) possess rigid stems that can withstand long-distance transportation and diverse flower colors. Therefore, they are popular as cut flowers, bedding plants, and pot plants in the global ornamental industry. However, the ornamental industry always demands new carnation cultivars because growers and consumers prefer cultivars with traits that are more novel than the existing ones. Application of Agrobacterium-mediated genetic transformation has made it possible to generate new carnation cultivars to meet the industry’s needs and satisfy the preferences of growers and consumers (Lu et al. 1991; Meng et al. 2009; Ovadis et al. 1999; Shiba and Mii 2005; Van Altvorst, 1995; Zuker et al. 2001). However, achievement of Agrobacterium-mediated genetic transformation always relies on an efficient shoot regeneration method. Despite development of regeneration methods for several carnation cultivars, the methods differed from one cultivar to another cultivar (Arif et al. 2014; Casanova et al. 2008; Gutierrez-Miceli et al. 2009; Karami et al. 2007, Thakur and Kanwar 2018; Thu et al. 2020; Yantcheva et al. 1998; Zhang et al. 2021), suggesting the necessity to develop specific methods for each cultivar.

Generally, regeneration efficacy of carnations is influenced by several factors such as plant growth regulators (PGRs), explant types, and genotypes (Arif et al. 2014; Karami et al. 2007; Thakur and Kanwar 2018; Thu et al. 2020; Zhang et al. 2021). Furthermore, the regeneration of vitrified shoots in carnation has been observed in many studies (Jain et al. 1997; Kevers et al. 1987; Kharrazi et al. 2011; Sato et al. 1993; Thu et al. 2020), which poses a constraint on the production of quality shoots and the success of plant genetic transformation, due to its physiological and morphological malformations. Considering these facts, it is necessary to optimize the above-mentioned factors to establish a shoot regeneration protocol that can efficiently produce quality shoots for each cultivar, enabling genetic improvement through plant genetic transformation.

The carnation cultivars ‘Chabaud and Giant Chabaud’ possess typical floral characteristics such as large flower size, fragrance, and a variety of flower colors, making them popular as cut flowers. Recently, our research group attempted to overexpress acdS gene encoding 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase in the cultivars through Agrobacterium-mediated genetic transformation to promote their flower longevity. However, suitable methods for in vitro shoot regeneration of these cultivars have not been available so far. Therefore, in this study, we aimed to establish an efficient shoot regeneration protocol for these cultivars by optimizing different combinations of PGRs and explant types. Additionally, we assessed the effects of PGRs combination and explant types on the regeneration of normal and vitrified shoots.

Plant materials

The seeds of the carnation cultivars ‘Chabaud and Giant Chabaud’ were surface-sterilized prior to in vitro seed germination following the protocol described by Lee et al. (2023). The sterilized seeds were then cultured on hormone-free Murashige and Skoog (MS) medium containing 3.0% of sucrose and 7.0 g/l of agar. The cultures were placed in a dark condition at 25°C for five days, followed by transfer to a continuous light condition for one day. Afterward, the seeds germinated, and the germinated seedlings with two cotyledons were used as the source materials for the establishment of an in vitro shoot regeneration system.

Effects of explant types and plant growth regulators (PGRs) on shoot regeneration

To determine the efficacy of different explant types on shoot regeneration, cotyledon without shoot apical meristem (SAM) and cotyledon with whole SAM (intact SAM) were excised from the germinated seedlings. Additionally, SAMs were bisected to produce half SAM, and some were injected with microneedles to create destroyed SAMs (Fig. 1).

Figure 1. Illustration of different explant types [cotyledon without shoot apical meristem (cotyledon), cotyledon with half SAM (half SAM), cotyledon with intact SAM (intact SAM), and cotyledon with destroyed SAM (destroyed SAM)]

The four types of explants (cotyledon, intact SAM, half SAM, destroyed SAM) were then cultured on MS medium containing the combinations of plant growth regulators (PGRs): 1.0 mg/l of benzyladenine (BA) and 0.06 mg/l of naphthalene acetic acid (NAA), 1.0 mg/l of BA and 0. 3 mg/l of NAA, and 0.2 mg/l of thidiazuron (TDZ) and 0.5 mg/l of indole-3-butyric acid (IBA), respectively. The culture plates were placed in a culture room with a temperature at 25°C, a 16-h photoperiod, and 70% relative humidity (RH). After four weeks of culture, the percentages of shoot regeneration, average number of shoots per explant, and percentages of vitrified shoots were assessed. Each treatment consisted of 15 explants with three replications.

In vitro plant growth and rooting, acclimatization, and transplantation

The normal shoots derived from intact SAM of both cultivars, especially those cultured on the media containing the combinations of BA and NAA, were individually transferred to glass bottles containing hormone-free MS medium, 3.0% sucrose, and 7.0 g/l agar for plant growth and rooting. Similarly, the vitrified shoots obtained from the other explants cultured on all PGR combinations were also transferred to glass bottles containing the same media composition. The bottles were placed in the same culture room described above for six weeks. After six weeks, the plants that did not normalize were discarded, and those that grew well and normalized were carefully removed from the bottles, and any adhering agar was gently washed off with running tap-water. Then, the plants were transplanted into small plastic pots filled with peat-based soil, followed by placing these pots in a small plastic house for one week to maintain RH at around 80%. Once acclimatized, they were transferred to a greenhouse set up with a temperature at 25°C, a 16-h photoperiod, and 70% RH.

Statistical analysis

The data were analyzed using SPSS V. 11.09 (IBM Corporation, Armonk, NY, USA) and are presented as the mean of three replicates. To assess the difference among the mean values, Duncan Multiple Range Test (DMRT, p < 0.05) were performed.

Effects of explant types and PGR combinations on shoot regeneration of carnation cv. Giant Chabaud

The four types of explants (cotyledon, intact SAM, half SAM, and destroyed SAM) were cultured on MS medium containing the combinations of 1.0 mg/l of BA and 0.06 mg/l of NAA, 1.0 mg/l of BA and 0. 3 mg/l of NAA, and 0.2 mg/l of TDZ and 0.5 mg/l of IBA, respectively. All explant types, except cotyledon, responded to the PGR combinations and started regenerating shoots after two weeks of culture, and the shoots continued to grow well over time (Fig. 2a-f).

Figure 2. Shoot regeneration from different explant types of carnation cv. Giant Chabaud [half SAM (a: 2 weeks and b: 3 weeks), intact SAM (c: 2 weeks and d: 3 weeks), and destroyed SAM (e: 2 weeks and f: 3 weeks) cultured on the combination of BA (1.0 mg/l) and NAA (0.06 mg/l)

After four weeks of culture, the shoot regeneration percentages, average number of shoots, and percentages of normal and vitrified shoots were assessed, revealing variations in response to different explant types and PGR combinations (Fig. 3, Table 1). The highest shoot regeneration percentages were observed from intact SAM, while the percentages observed from half SAM were significantly higher than those from destroyed SAM for all PGR combinations. However, the highest average number of shoots per explant was induced from half SAM, and the number of shoots induced from destroyed SAM was also higher than those induced from intact SAM across all PGR combinations. Specifically, half SAM induced its highest number of shoots (9.37) when cultured on the combination of TDZ (0.2 mg/l) and BA (0.5 mg/l), The highest number of shoots induced by destroyed SAM was 8.23, which occurred on the media containing the combination of BA (1.0 mg/l) and NAA (0.06 mg/l). Intact SAM induced its highest number of shoots (5.65) on the media containing the combination of BA (1.0 mg/l) and NAA (0.3 mg/l). However, when cultured on the combination of BA (1.0 mg/l) and NAA (0.06 mg/l), 29.17% and 63.64% of the shoots regenerated from half-SAM and destroyed SAM were observed to be vitrified, while only 6.9% of the shoots from intact SAM exhibited vitrification. Furthermore, higher percentages of vitrified shoots (44.44% and 90.91%) were observed from half SAM and destroyed SAM respectivery when cultured on the combination of BA (1.0 mg/l) and NAA (0.3 mg/l), whereas vitrified shoots were not induced at all from intact SAM. When the explants were cultured on the media containing the combination of TDZ (0.2 mg/l) and BA (0.5 mg/l), the percentage of vitrified shoots increased to 59.26% and 92% from half-SAM and destroyed SAM, respectively, and the percentage of vitrified shoots from intact SAM also increased to 26.67%. Therefore, culturing intact SAM on the media containing the combination of BA (1.0 mg/l) and NAA (0.3 mg/l) was observed to be suitable for production of quality shoots.

Figure 3. Shoot regeneration from the different explant types of carnation cv. Giant Chabaud cultured on combinations of different plant growth regulators. These photos were taken 30 days after culture. The scale bar denotes a length of 1 cm

Table 1 . Effects of combinations of plant growth regulators (PGRs) and explant types on the regeneration of carnation cv. Giant Chabaud.

PGR combination (mg/l)	Explant type	Shoot regeneration (%)	Average no. of shoot/explant	Vitrified shoot (%)
1.0 BA + 0.06 NAA	(1) Cotyledon	0g	0f	0h
	(2) Half SAM	83e	9.17a	29.17e
	(3) Intact SAM	96.67b	4.59e	6.9g
	(4) Destroyed SAM	75.43f	8.23b	63.64b
1.0 BA + 0.3 NAA	(1) Cotyledon	0g	0f	0h
	(2) Half SAM	93.1c	7.37c	44.44d
	(3) Intact SAM	100a	5.65d	0h
	(4) Destroyed SAM	73.33f	6.05d	90.91a
0.2 TDZ + 0.5 IBA	(1) Cotyledon	0g	0f	0h
	(2) Half SAM	90d	9.37a	59.26c
	(3) Intact SAM	100a	4.86e	26.67f
	(4) Destroyed SAM	83.33e	7.2c	92a

Data represent the means of three replicates. Means with the same letters are not significantly different by the Duncan Multiple Range Test (DMRT, p < 0.05)..

Effects of explant types and PGR combinations on shoot regeneration of carnation cv. Chabaud

As observed for cv. Giant Chabaud, cotyledons from cv. Chabaud did not exhibit any response when cultured on MS medium containing the same PGR combinations until four weeks of culture, while the other explant types (half SAM, intact SAM, and destroyed SAM) successfully regenerated shoots (Fig. 4). The shoot regeneration percentages induced from half SAM and intact SAM were 95-100% for all PGR combinations, but those induced from destroyed SAM were 83-86%, revealing a variation of regeneration efficiency depending on the explant types. When considering the average number of shoots per explant, half SAM induced more shoots than intact SAM, and destroyed SAM induced the lowest average number of shoots, except for the combination of BA (1.0 mg/l) and NAA (0.3 mg/l). This finding indicates that half SAM has a higher regenerative capacity than intact SAM, which, in turn, has a higher regenerative capacity than destroyed SAM (Table 2).

Figure 4. Shoot regeneration from the different explant types of carnation cv. Giant Chabaud cultured on combinations of different plant growth regulators. The photos were taken 30 days after culture. The scale bar denotes a length of 1 cm

Table 2 . Effects of combinations of plant growth regulators (PGRs) and explant types on the regeneration of carnation cv. Chabaud.

PGR combination (mg/l)	Explant type	Shoot regeneration (%)	Average no. of shoot/explant	Vitrified shoot (%)
1.0 BA + 0.06 NAA	(1) Cotyledon	0d	0f	0h
	(2) Half SAM	96.67b	9.66a	93.1b
	(3) Intact SAM	100a	6.25cd	10f
	(4) Destroyed SAM	85c	5.76cd	100a
1.0 BA + 0.3 NAA	(1) Cotyledon	0d	0f	0h
	(2) Half SAM	100a	9.73a	90c
	(3) Intact SAM	95b	6.05cd	5g
	(4) Destroyed SAM	86.67c	6.92c	84d
0.2 TDZ + 0.5 IBA	(1) Cotyledon	0d	0f	0h
	(2) Half SAM	100a	8.27b	93.33b
	(3) Intact SAM	100a	5.6d	100a
	(4) Destroyed SAM	83.33c	4.2e	80e

Data represent the means of three replicates. Means with the same letters are not significantly different by the Duncan Multiple Range Test (DMRT, p < 0.05).

However, most of the shoots regenerated from half SAM and destroyed SAM, which were cultured on all PGR combinations, were observed to be verified. Similarly, all shoots regenerated from intact SAM that were cultured on the combination of TDZ (0.2 mg/l) and IBA (0.5 mg/l) exhibited vitrification, but those regenerated from the explants cultured on BA and NAA combinations showed only 5-10% vitrification. Therefore, for cv. Chabaud, culturing intact SAM on the combination of BA (1.0 mg/l) and NAA (0.3 mg/l) would be the most suitable for the production of quality shoots.

In vitro plant growth and rooting, acclimatization, and transplanting

For both cultivars, the normal shoots derived from intact SAM cultured on BA and NAA combinations rapidly grew well with many roots and exhibited healthier when cultured on hormone-free MS medium (Fig. 5a). However, the vitrified shoots showed a delayed growth and did not induce roots after four weeks of culture, resulting in no conversion to normal plants. Therefore, only the normal shoots were acclimatized and transferred to the greenhouse. The regenerated plants thrived in the greenhouse and eventually produced flowers (Fig. 5b).

Figure 5. In vitro growth and rooting of the shoots derived from intact SAM of the carnations (a) and flowering of the in vitro regenerated plants under the greenhouse (b)

In vitro shoot regeneration methods have been developed in several carnation cultivars; however, the method developed for one cultivar was not suitable for another cultivar, as factors such as plant growth regulators, explant types, and genotypes influence shoot regeneration efficiency (Arif et al. 2014; Karami et al. 2007; Thakur and Kanwar 2018; Thu et al. 2020; Zhang et al. 2021). In this study, we attempted to develop an in vitro shoot regeneration method for the carnation cultivars ‘Chabaud and Giant Chabaud’ by optimizing different combinations of PGRs and explant types. We observed that cotyledon (without SAM) could not induce shoots with any PRG combinations used, while the other cotyledons with half SAM or intact SAM or destroyed SAM highly induced shoots for both cultivars. This result suggested that cotyledons were unable to induce shoots if they do not contain SAM, indicating the important role of SAM in shoot regeneration. Previous studies have reported the success of shoot regeneration from cotyledons in carnations (Jain et al. 1997; Meng et al. 2009; Zhang et al. 2021). However, it should be noted that the cotyledons used in those studies contained half SAM or intact SAM, and therefore, the shoot regenerations were solely derived from SAM. In cv. Giant Chabaud, we observed variation in shoot regeneration percentages depending on explant types and PGR combinations. The highest percentage (93.1%) from half SAM was observed in the combination of BA (1.0 mg/l) and NAA (0.3 mg/l), and that (100%) from intact SAM was noticed in the combinations of BA (1.0 mg/l) and NAA (0.3 mg/l), as well as TDZ (0.2 mg/l) and IBA (0.5 mg/l). In the case of destroyed SAM, the highest percentage (83.33%) was observed with TDZ (0.2 mg/l) and IBA (0.5 mg/l). However, the PGR combinations that gave the highest regeneration percentages for cv. Giant Chabaud did not give the same results for cv. Chabuad, suggesting the influence of genotypes on shoot regeneration percentages even using the same PGR combinations and explant types.

In cv. Giant Chabaud, the combination of BA (1.0 mg/l) and NAA (0.06 mg/l) induced a higher number of shoots from half SAM or destroyed SAM compared to the combination of BA (1.0 mg/l) and NAA (0.3 mg/l). However, BA (1.0 mg/l) and NAA (0.3 mg/l) combination resulted in higher number of shoots in cotyledon with intact SAM than the BA (1.0 mg/l) and NAA (0.06 mg/l) combination. In cv. Chabaud, the number of shoots obtained from half or intact SAM were not significantly different between the two PGR combinations, but BA (1.0 mg/l) and NAA (0.3 mg/l) combination yielded more shoots in destroyed SAM. Moreover, the explants responded differently to the combination of TDZ (0.2 mg/l) and IBA (0.5 mg/l) for both cultivars. This indicated significant influences of explant types, PGRs, and genotypes in shoot regeneration. Previous studies also reported the influences of these factors on shoot regeneration in carnations (Kallak et al. 1997; Kanwar and Kumar 2009; Sharma et al. 2016; Thu et al. 2020). Interestingly, we noticed that the status of the SAM plays a crucial role in shoot regeneration, as cotyledons with half SAM and destroyed SAM were able to regenerate more shoots than those with intact SAM. It was likely that cutting or destroying of SAM stimulated cell division faster than in intact SAM, resulting in the induction of a higher number of shoots per explant.

Zhang et al. (2021) reported that BA (1.0 mg/l) and NAA (0.06 mg/l) combination were more effective in regenerating shoots from cotyledon with half SAM than BA (1.0 mg/l) and NAA (0.3 mg/l) combination. However, the maximum number of shoots obtained from their study was higher than that obtained from this study. It could be due to the difference of cultivars used in their study and our study. Surprisingly, Zhang et al. (2021) did not obtain any shoot when intact SAM were cultured on the same PGR combination, which was totally different from our work, as intact SAM from both cultivars induced the reasonable number of shoots in our study.

The occurrence of vitrified shoots in carnations has been extensively studied (Jain et al. 1997; Kevers et al. 1987; Kharrazi et al. 2011; Sato et al. 1993; Thu et al. 2020), posing challenges for producing quality shoots and successful plant genetic transformation due to their physiological and morphological abnormalities. In our study, we also observed vitrified shoots, specifically in those derived from cotyledons with half SAM or destroyed SAM, which exhibited symptoms of vitrification. Surprisingly, shoots from intact SAM did not show vitrification. The status of SAM significantly influenced the regeneration of quality shoots. Additionally, the use of certain PGR combination showed a notable influence on the induction of vitrified shoots. The combination of TDZ and IBA significantly induced a higher number of vitrified shoots compared to BA and NAA combinations. Notably, while intact SAM hardly induced vitrified shoots with BA and NAA combinations, they did induce vitrified shoots when cultured with the TDZ and IBA combination. Furthermore, we observed that genetic factors also play a significant role in the induction of vitrified shoots. For example, cv. Giant Chabaud induced 26.67% of vitrified shoots, while cv. Chabaud induced 100% of vitrified shoots from the same explant type and PGR combination. Previous studies have reported that vitrified shoot induction in carnations is influenced by imbalanced PGRs, genetic factors, as well as the age and physiological status of the explants (Jain et al. 1997; Kevers et al. 1987; Kharrazi et al. 2011; Sato et al. 1993; Thu et al. 2020). Taken together, the cotyledons with half SAM or destroyed SAM are not suitable for use as explants in regenerating quality shoots, as most of the shoots derived from these explants were vitrified. Additionally, the combination of TDZ and IBA has proven unsuitable for the regeneration of quality shoots. Therefore, the study suggests that culturing the cotyledons with intact SAM in BA and NAA combinations, particularly in BA (1.0 mg/l) and NAA (0.3 mg/l) combination, will be helpful for production of quality shoots in these carnations.

We aimed to establish an effective in vitro regeneration method for the carnation cultivars ‘Giant Chabaud and Chabaud’ by evaluating the impacts of explant types and PGR combinations. We found that both explant types and PGRs significantly influenced the regeneration of normal shoots, and there was noticeable variation in shoot regeneration depending on the genotypes. For both cultivars, culturing intact SAM in the combination of BA (1.0 mg/l) and NAA (0.3 mg/l) gave a satisfactory number of normal shoots without exhibiting vitrification. As a result, we recommend the use of intact SAM with the combination of BA (1.0 mg/l) and NAA (0.3 mg/l) for regeneration of normal shoots for these carnation cultivars. Moreover, the developed method holds great promise for in vitro propagation and genetic transformation of these carnations, providing valuable insights for future research and applications.

This work was supported by the National Research Foundation (NRF) Grant funded by the Korean government (MSIT) (No. 2021R1A2C2008951).This research was supported by Kyungpook National University Research Fund, 2023