Efficient Plant Regeneration via Indirect Organogenesis in Carnation (Dianthus caryophyllus semperflorens flore pleno) Cultivars

Nine standard carnation cultivars – ‘Cameron’, ‘Eskimo’, ‘Grand Slam’, ‘Liberty’, ‘Mariposa’, ‘Noblesse’, ‘Tabasco’, ‘Tabor’, and ‘White Liberty’ – were studied. The in vitro leaf explants obtained from axillary buds were excised into 0.5–0.7 cm pieces, followed by incubation with the axial side down on the Murashige and Skoog (1962) (MS) basal medium. In all experiments, culture conditions included a temperature of 25 ± 2 °C under a PPFD of 80 µmol·m⁻²·s⁻¹ using fluorescent light with a 16-h photoperiod.

The leaf segments were incubated on the MS media containing 30 g·dm⁻³ of sucrose, solidified with 0.7% (w/v) Plant agar (Duchefa Biochemie) at pH 5.8, before autoclaving and supplemented with different concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) (0, 0.2, 0.5, and 1 mg·dm⁻³), 1-naphthaleneacetic acid (NAA) (0 and 0.5 mg·dm⁻³), 6-benzylaminopurine (BA) (0, 0.2, and 0.5 mg·dm⁻³), and CH (0 and 200 mg·dm⁻³) in 16 different combinations (Table 1). For callus induction leaf explants were cultured in 80-mm Petri dishes containing 25 ml of fresh MS medium.

For shoot regeneration, three-week-old calluses were transferred to the MS media supplemented with BA (0, 2, 3, and 5 mg·dm⁻³), NAA (0, 0.2, and 0.6 mg·dm⁻³), As (0, 20, 40, and 80 mg·dm⁻³), and AgNO₃ (0 and 5 mg·dm⁻³) in 10 different combinations, in culture jars (10 × 6 cm). AgNO₃ was filter-sterilized through a 0.24-μm Millipore membrane and added to the autoclaved culture media. The other compounds were added prior to the autoclaving at 121 °C for 15 min.

To eliminate hyperhydricity of regenerated shoots caused by high concentrations of cytokines, hyper-hydric shoots were transferred to a modified MS recovery medium containing 0.275 g·L⁻¹ of NH₄NO₃ with 0.5 mg·dm⁻³ gibberellic acid (GA₃), 0.3 mg·dm⁻³ BA, and 0.1 mg·dm⁻³ NAA based on a pilot experiment (data not shown) for about two weeks. GA₃ was filter-sterilized through a 0.24-μm filter and added to the media after autoclaving.

The regenerated shoots were cut off from the base and transferred for elongation to an agar-solidified MS elongation medium supplemented with 1 mg·dm⁻³ of kinetin (KIN) and 0.1 mg·dm⁻³ of NAA for three weeks. For root induction, the shoots with 4–7 leaves of 2–3 cm long were transferred to the ½ MS medium (rooting medium) supplemented with 0.26 mg·dm⁻³ of indole-3-butyric acid (IBA). After 14 days, root formation was observed (Figure 1G–I). The rooted plantlets with 5–8 leaves and a height of 3–4 cm were removed from the jars, and the agar was washed away gently from the roots with tap water. The plantlets were transplanted to plastic trays (50 × 40 × 10 cm) containing perlite, coco peat, and peat moss (1:1:1, v/v). They were then kept in a greenhouse under controlled conditions (a temperature of 25 ± 2 °C under a PPFD of 100–110 µmol·m⁻²·s⁻¹ and a relative humidity of 90%) for acclimatization.

Figure 1

The leaves of in vitro-regenerated plants and donor plants were finely chopped in 500 µl of ice-cold nuclei extraction buffer (PVP: 10 mg, DNA-extraction buffer: 1 cc, RNase: 15 µl, and PI: 30 µl). Then, 2 ml of nuclei staining solution (CyStain^® PI Absolate P) was added to the crude mixture. To eliminate cell debris, the suspension was filtered with a 50-µm filter, and the nuclei were collected into pre-chilled tubes. The ploidy level of the samples was analyzed by a flow cytometer (BD FACS Calibur, Biosciences, USA) using a 488-nm argon laser to excite the PI fluorochrome and an FL-2 detector with a 585/42 bandpass filter. Samples were run on low pressure, and 104 nuclei were counted within the double gate (Nalousi et al. 2019).

Each treatment contained 12 explants cultured in 80-mm Petri dishes with three replications. Different callus induction and plant regeneration parameters were collected, and the significant differences in means were statistically analyzed by one-way analysis of variance (one-way ANOVA). For non-normal distribution data, logarithmic transformation was performed prior to further analysis. Values represent means ± SD and different letters show a significant difference (p < 0.05) based on Duncan’s multiple range test (DMRT).

Significant differences were observed in callus formation depending on different combinations or concentrations of PGRs and genotype (Tables 1 & 2). The highest callus induction frequency was observed in the ‘Noblesse’ cultivar with 91.67% and the ‘White Liberty’ cultivar with 90.33%. The results indicated that in treatments with a high callus growth rate, callus induction frequency significantly decreased. In this regard, ‘Noblesse’ and ‘White Liberty’ cultivars with the highest callus frequency percentage showed a slow growth rate (Table 2). And vice versa, in cultivars with a high callus growth rate (earlier callus induction), such as ‘Cameron’ and ‘Eskimo’, a lower callus induction frequency was observed. The MS medium supplemented with 0.2 mg·dm⁻³ of 2,4-D and 200 mg·dm⁻³ of CH showed the highest quality of organogenic calli (the calli with the highest rate of plant regeneration with a green-yellow color and a semi-compact characteristic) among the cultivars (Figure 1A, B).

After four weeks, some green spots were observed on the surface of the calli (Figure 1C). After eight weeks, shoot regeneration was observed. Addition of As to the media significantly increased the percentage of shoot induction and the shoot number in all tested cultivars. While, in the MS medium supplemented with 3 mg·dm⁻³ of BA, 0.6 mg·dm⁻³ of NAA, and 5 mg·dm⁻³ of AgNO₃ without As, the mean shoot regeneration rate was 53.96% among the cultivars. Adding 40 mg·dm⁻³ of As significantly increased the regeneration rate to 78.83% (Table 3). The highest rate of regeneration was observed in ‘Noblesse’, ‘Cameron’, and ‘Tabasco’ cultivars with an average regeneration rate of 95.24%. The ‘Cameron’ cultivar with 28.3 shoots per explant showed the highest number of shoots in each ex-plant. In high concentrations of BA (> 3 mg·dm⁻³) and As (> 40 mg·dm⁻³), the rate of regeneration and the number of shoots decreased. In the MS medium supplemented with 3 mg·dm⁻³ of BA, 0.6 mg·dm⁻³ of NAA, 5 mg·dm⁻³ of AgNO₃, and 40 mg·dm⁻³ of As, the highest rate of regeneration was observed in all the cultivars (Table 4).

In the majority of regenerated shoots, hyperhydricity was observed (Figure 1E). Such shoots were recovered from hyperhydricity on the recovery medium after two to three weeks (Figure 1F). Subsequently, the recovered plantlets were transferred to the MS elongation medium on which the shoots of all cultivars increased by approximately 2–3 cm after three weeks (Figure 1G). All shoots were rooted after two weeks when transferred to the rooting medium (Figure 1H). More than 80% of the plantlets survived transfer to greenhouse condition and acclimatized successfully (Figure 1I–K).

The genetic stability assessment using the flow cytometry analysis did not detect significant differences between in vitro-regenerated and control plants in terms of DNA ratios (Figure 2).

Figure 2