Regeneration of chrysanthemum (Chrysanthemum morifolium) via somatic embryogenesis and screening of clones for agronomic traits

Chrysanthemum morifolium propagation through stem cutting produces weak plants that show delayed anthesis above 20 °C and a reduced flower diameter. Therefore, in this study chrysanthemum plantlets were produced through somatic embryogenic from calli to exploit the somaclonal variation for their improvement. Various explants from variety Dante yellow were cultured on Linsmaier and Skoog (LS) medium with various concentrations of Kinetin (KT) and 2,4-dichlorophenoxyacetic acid (2,4-D) and their combinations for callus induction. Embryogenic calli were proliferated and regenerated by using different plant growth regulators. Regenerated plantlets were acclimatized and evaluated for agronomic characteristics and compared with the mother plant in a replicated field trial. Results revealed that young leaf explants cultured on LS medium containing 9.02 µM 2,4-D and 11.61 µM KT gave calli of greater sizes as compared to other treatments. A combination of 0.44 µM 6-benzylaminopurine (BAP) and 5.37 µM 1-naphthaleneacetic acid (NAA) yielded the highest callus proliferation (0.27 g ± 0.03). A significant number of shootlets (25 ± 0.8) from embryogenic callus was observed on the medium with 0.45 µM 2,4-D. During the field experiment, Clone S84 showed considerable improvement in flower size as compared to the mother plant and was found to be a promising clone for commercialization. Somaclonal variation can be used for crop improvement of chrysanthemum instead of induced mutagenesis. Present study showed the substantial improved flower diameter in regenerated plantlets via somatic embryogenesis. Field screening showed increase in the diameter of flowers and variation in flower colour in somaclonal population. Both features are appealing for the consumers in floriculture industry.


Introduction
Chrysanthemum morifolium is an important floriculture crop from the family Asteraceae (Miler and Jendrzejczyk 2018). Low amounts of ethylene production during its growth phase prolong its shelf life and make it suitable for cut flower production (Zafarullah et al. 2013) and export, providing significant earnings to the producing countries (Chica-Toro and Garzón-González 2018; Roh et al. 2019). Pakistan also has a great export potential by setting its own ornamental plants industry (Anjum et al. 2007). However, more research efforts are needed regarding cultivar improvement and production.
Stem cuttings are commonly used for the propagation of C. morifolium plants in Pakistan. This conventional method produces slow growing plants that are prone to carry those infections which are already present in the mother plant (Mehedi et al. 2020). Rapid initiation of flowering in C. morifolium requires a temperature of 10-20 °C for 3-4 weeks, as above this range anthesis is delayed (Cho and Kim 2020). Many private sector nurseries tried to multiply exotic varieties of C. morifolium in Karachi but these varieties failed to adapt to the hot weather conditions of the city. As a result, some of these nurseries have shifted to the cooler areas of Pakistan that has resulted in an increase in cost of production.
Cross breeding is the most common conventional method for the development of new improved cultivars of chrysanthemum. However, hybrid plants with the preferred traits usually carry other less desirable traits that need several generations to be removed through backcrossing, which significantly increase the labour and cost associated with cross breeding (Su et al. 2019). Besides that, chrysanthemums are complex genetic cultivars with hexaploidy or aneuploidy and are also self-incompatible, which makes the production of pure lines restricted (Pu et al. 2021;Yang et al. 2023). Induced mutagenesis was also found to be effective in chrysanthemum improvement (Kumari et al. 2019). Previously, physical (gamma rays, X-rays, electromagnetic radiations and heavy-ion beams) and chemical (ethyl methanesulfonate and pingyangmycin) mutagens have been successfully used in C. morifolium. However, both are harmful for the environment (Eisa et al. 2022).
Production of in vitro plantlets through somatic embryogenesis offers a rapid and large-scale regeneration system (Natarajan et al. 2020). Moreover, it may induce genetic and/ or epigenetic changes, leading to somaclonal variation that could be exploited for improvement of ornamental plants (Bednarek and Orłowska 2020). Such type of somaclonal variation has been reported in Gentiana decumbens (Tomiczak et al. 2015), Lilium distichum and Lilium cernuum (Fu et al. 2019).
The present study was designed to develop a rapid and reproducible protocol for the regeneration of C. morifolium from somatic embryogenesis and the recovery of somaclonal variants with improved traits in accordance with the demand of floriculture market. To the best of our knowledge, this is the first report of somaclonal variation in C. morifolium without applying any exogenous mutagenic agent.

Explant used and callus induction
Greenhouse-grown plants of Dante yellow, an exotic variety of ornamental C. morifolium, was used as the source of explants. The explants were washed with liquid detergent under running tap water for 5-7 min, and were then surface sterilized in a laminar flow cabinet with 70% (v/v) ethanol for 1 min followed by 20% (v/v) commercial bleach mixed with 2 drops of tween20 detergent for 5-7 min with gentle stirring, and finally rinsed four times with sterile distilled water.

Morphological and developmental features of callus
Morphology of different types of calli and their developmental features were studied using 4X magnification under a dissecting microscope. The detailed structure of embryogenic callus was observed using compound microscope with 100× magnification.

Regeneration of embryogenic callus
Embryogenic calli (0.1 g) selected according to their morphology were cultured for regeneration on the LS media detailed in Table 2, at 24 ± 2 °C under a 16/8 h light/dark photoperiod with a 108 µmol m −2 s −1 light intensity, in 5 replications. Plantlets were regenerated from the embryogenic calli after 4 weeks and were then transferred onto LS medium augmented with IBA (0.49 µM) for elongation and rooting under same growth conditions as recommended by Waseem et al. (2009).

Acclimatization of plants
Regenerated plants with well-developed shoots and roots were transferred to three different potting mixtures and conditions [T-1: Cocopeat + weekly spray of ½ strength Hoagland's solution, T-2: Garden soil + weekly spray of ½ strength Hoagland's solution, T-3: Cow manure + garden soil (1:3)] to optimize high survival rate of plants. Plantlets were taken out of the vessels one hour before planting after removing dead leaves, and they were washed with running tap water to remove agar from their root zone.
For T-1, polythene bags with drainage holes were used to give roots more space to expand, whereas for T-2 and T-3 germinating trays were used. Garden soil, cocopeat and cow manure were autoclaved to make media safe and contamination-free for the tissue cultured plantlets. The experiment was performed under plastic covers to maintain a high amount of moisture. Plantlets were acclimatized under growth room conditions used for the regeneration of embryogenic calli and transferred to the nursery after 4 weeks.

Preparation of replicates and screening of somaclones
A total of 120 acclimatized (R 0 ) plantlets were transferred to the nursery. Plantlets were pinched after 4 weeks and again after 7 weeks for making more branches for replications as described by Bala (2015). By 11 weeks three replicates were taken from each plantlet and dipped in powdered IBA from the lower end and transferred onto germinating trays sprayed with Hoagland's solution. After 2-3 weeks, three replicated plantlets (R1) with well-developed roots were ready for the replicated yield trial using a Randomized Complete Block Design (RCBD). Planting was done in late December and data was collected in late March. Initially, water was provided for one week, then 1 g/L of NPK (1:1:1) solution was sprayed every week until 6 weeks. The nursery was then covered with a black plastic sheet for flower induction.
In the greenhouse three replicated blocks each of 1 m 2 were made, distanced from each other by 0.5 m. Each block consisted of 120 regenerated somaclonal plants and 10 mother plants of variety Dante yellow, used as control, to evaluate and compare the performance of the somaclonal plants. Plant to plant distance was 2.5 inches and row to row distance was 5 inches. Different important parameters [Stem thickness (mm), Plant height (cm), Flower color, Flower diameter (inches) and Suckers produced per clone] of 3 replicated clones were recorded after approximately 3.5 months of growth.

Statistical analysis
In the present study, callus proliferation, shoot regeneration on different media and replicated field trial were evaluated through One-Way Analysis of variance (One-Way ANOVA) and Duncan multiple range test (DMRT) by using SPSS version 16 at p < 0.05. For frequency distribution of somaclones histograms were made by using SPSS version 16.
A heat-map was constructed through TBtools-master using euclidean distance matrix and average linking.

Callus induction
All explants cultured on LS medium with 11.61 µM KT and 9.02 µM 2,4-D produced callus. Profuse callusing was produced from young leaf explants and the entire explant turned into callus within 2 weeks, while other explants showed callus only on the margins. A heat-map was made by using simultaneous clustering of all varying concentrations and combinations of callus inducing PGRs (Fig. 1). The heat-map shows two broad groups of PGRs (concentrations/combinations) where the group with 4.52 µM 2,4-D alone and the combination of 9.02 µM of 2,4-D with 11.61 µM of KT represent the best media compositions for callus induction. The cluster of the second group is further divided into two sub-groups: a first sub-group that represents PGR concentrations producing a low amount of callus and a second one containing PGR concentrations that responded poorly for callus induction. Further microscopic investigations revealed that the callus produced on young leaf explants was only embryogenic in nature (Fig. 2a). These observations contrast with previous results by Shinoyamo et al. (2004) and Tymoszuk et al. (2014), where a low concentration of 2,4-D and a high concentration of KT were most efficient for inducing embryogenic callus on young leaf explants. In another study, Naing et al. (2013) reported the use of equal quantities of KT plus 2,4-D as the best combination for inducing embryogenic callus on leaf explants in 37 days.
Noticeably, in the present study a substantial amount of embryogenic calli were produced using a comparatively lower amount of PGRs than reported by Tymoszuk et al. (2014), who used 4 mg L −1 2,4-D along with KT for ligulate florets of Chrysanthemum grandiflorum. The present study revealed that a significant amount of callus was induced on LS medium with lower concentrations of 2,4-D alone (2.26, 4.52, 6.78 and 9.04 µM), among which 4.52 µM 2,4-D gave a relatively higher amount of callus. Conversely, LS medium with high concentrations of 2,4-D (11.31, 13.57, 15.83 and 18.09 µM) induced roots with a significantly lesser amount of callus on the margins of explants. The callus induced by 2,4-D was compact and pale in appearance and non-granulated. Moreover, experiments on the regeneration of such type of callus revealed that it was non-regenerable in nature.
It was observed that LS medium with eight varying concentrations of KT (2.32,4.64,6.96,9.29,11.61,13.93,16.26 and 18.58 µM) did not produce any type of undifferentiated mass of callus with all explants used. Similar results were also observed by Naing et al. (2013) who reported that somatic embryos (SE) were induced by the addition of cytokinins such as Thidiazuron (TDZ), KT, BA plus 2,4-D, while cytokinin alone was not useful for embryogenic callus induction.

Callus morphology
Morphologically, three diverse kinds of calli were produced by the combination of 9.02 µM of 2,4-D and 11.61 µM of KT. Young leaf explants showed the formation of granulated, yellowish-pale coloured callus (Fig. 2b). Further experiments showed that the callus produced only from young leaf explants was regenerable and showed an increased somatic embryogenesis potential, whereas fully expanded leaves gave whitish-pale coloured callus (Fig. 2c), present only on the margins and insufficient in quantity. The third type of callus was mucilaginous, translucent, shiny and soggy in appearance and produced randomly among the above defined calli (Fig. 2d). This is the first report of mucilaginous callus on C. morifolium explants.

Developmental stages of somatic embryos
The 4X magnification of a dissecting microscope and the 100X magnification of a compound microscope unveiled five developmental sequences of somatic embryos (SE). The first structure formed was longitudinal in appearance and termed pro-embryonic mass (Fig. 3a). It was subsequently converted into a globular structure (Fig. 3b) and then to a heart-shaped structure (Fig. 3c) that was bilaterally symmetrical in appearance. Heart-shaped structures were also observed under the compound microscope (Fig. 3d). Torpedo embryos were formed as the fourth stage (Fig. 3e), while the fifth and final stage was a cotyledonary structure (Fig. 3f) that later gave rise to the primary shoot.
On the other hand, embryogenic calli transferred to the same medium that had been best for their induction (i.e., 9.02 µM 2,4-D with 11.61 µM KT) turned brown, whereas callus induced in LS medium containing 0.45 µM 2, 4-D started rooting. Thus, this medium was not suitable for callus proliferation. An important finding of this study is the 170% increase in embryogenic callus production within 2 weeks only. It was observed that the embryogenic callus of C. morifolium was only multiplied when cultured in the form of (0.1 g) clusters onto LS medium, while isolated embryos turned brown within a few days on the same culture conditions. This appears to be the first report of the effect of cluster size on the multiplication of embryogenic callus of C. morifolium.

Conversion of embryogenic callus into plantlets
There are several studies regarding the conversion of embryogenic callus of C. morifolium into plantlets on a medium without PGRs (May and Trigiano 1991;Tanaka et al. 2000;Shinoyama et al. 2004;Ilahi et al. 2007;Naing et al. 2013). However, in the present study media without PGRs showed a low frequency of embryogenic callus conversion into plantlets. Thus, the effect of different PGRs was investigated for callus regeneration, and a low concentration of 2,4-D (0.45 µM) was found to yield a significant conversion of embryogenic callus into plantlets (25 ± 0.8) within 4 weeks ( Fig. 4b; Table 2). However, this is contradictory to the results of Lema-Rumińska and Niedojadło (2014), who reported that cytokinin are crucial PGR for the conversion of somatic embryos of chrysanthemum into plantlets.
Interestingly, callus was converted into plantlets only when cultured in the form of 0.1 g clusters. When small plantlets were cultured on different concentrations and combinations of PGRs, only a low concentration of IBA (0.49 µM) showed a 3.5-fold increase in shoot length along with the induction of roots after 6 weeks (Fig. 4c). In other formulations, plantlets started to turn into callus again from the basal region within 4 weeks. Roots also developed on the same elongation medium with 0.49 µM IBA.

Acclimatization of plantlets and survival percentage
The present study showed that out of three different potting mixtures, sterilized cocopeat sprayed with ½ strength of Hoagland's solution weekly (T-1) was the best for acclimatization as it gave 100% of survival rate of plantlets. Moreover, plantlets were strong and new leaves also emerged within 4 weeks (Fig. 4d). Contrary to that, medium based on sterilized garden soil sprayed weekly with ½ strength of Hoagland's solution gave only 60% survival of plantlets with weak stem development that needed extra support of wooden sticks. Additionally, there was no sign of new leaf emergence even after 4 weeks (Fig. 4e). Garden soil proved to be a compact medium and showed dryness as compared to the other two treatments. Earlier, soil and sand were reported to be the least desirable media for the acclimatization of in vitro developed plantlets of ornamental plants such as Gloxinia, Saintpaulia and Gerbera jamesonii (Kashyap and Dhiman 2011;Singh et al. 2017). The mixture of cow manure with garden soil (1:3) (T-3) showed only 40% of plant survival and proved to be the least suitable for the acclimatization of plantlets. A schematic illustration of the whole protocol developed for in vitro multiplication and acclimatization of chrysanthemum variety Dante yellow is presented in Fig. 5.

Screening of somaclones
Among the agronomic characteristics assessed in the somaclonal population, substantial variation was observed in flower diameter, flower color and suckers produced per clone, while stem thickness (mm) and plant height (cm) were less variable. Screening of all somaclones showed that 15 clones had a significantly increased flower diameter as compared to the mother plant (at p < 0.05) while six somaclones showed a substantial decrease in the flower diameter ( Table 3).
Colour of a flower is the primary feature for appeal to consumers, even more than flower scent (Nasri et al. 2021). In this study, four clones showed phenotypic variation in flower colour as compared to the mother plant (Fig. 6a). In somaclone S4 the petals were devoid of red pigmentation and showed only yellow colour inflorescence (Fig. 6b). In contrast, somaclone S68 showed red inflorescences only (Fig. 6c). However, both showed a significant decrease in the mean diameter of the flower as compared to the mean diameter of the field-grown plants (0.83 ± 0.06 inches). Similarly, somaclones S4 and S68 respectively showed 37.5% and 40% decrease (p < 0.05) in flower diameter. It was also observed that somaclone S42 showed less red portion in the centre as compared to the field-grown plant (Fig. 6d). Somaclone S55 exhibited interestingly abnormal petals with a flat semi-full inflorescence (Fig. 6e), and a 19.27% decrease in flower diameter. Similar to the present result, flat semi-full inflorescences were also observed by Miler and Kulus (2018). However, in their study chrysanthemum 'Alchimist' was first treated with microwaves.
Flower diameter is a very important parameter for determining the quality of flowers (Singh et al. 2019). In the present study, regenerated Clone S84 showed the highest (45%) increase in the diameter of flowers, with 1.5 ± 0.5 inches as showing less red portion in the centre. e Clone S55 showing some interestingly abnormal petals in flat semi-full inflorescence compared to the mother plant which was 0.83 ± 0.24 inches. Somaclones S18, S19, S21, S23, S26, S29, S30, S35, S41, S44, S59, S66, S67 and S69 also showed a slight increase in flower diameter. However, clones S4, S11, S14, S17 and S20 exhibited a significant decrease in flower diameter. Frequency distribution of somaclones for flower diameter was positively skewed with mesokurtosis (Fig. 7a). The values of mean, median and mode of regenerated flower diameter were 0.91, 1 and 1 inches, respectively, which were significantly higher than the mean value of flower diameter of field-grown plants (0.83 ± 0.06 inches). This result showed an increase in the flower diameter of somaclonal plants as compared to the field-grown plant.
In the present study, frequency distribution for suckers produced per clone was positively skewed with leptokurtosis (Fig. 7b). The values of mean, median and mode for the number of suckers produced per somaclone were 4.57, 4, and 4, respectively, which were all greater than the mean value of suckers produced per field-grown plant (3.57 ± 0.2), indicating that somaclones showed an increase in the production of suckers per plant as compared to field-grown plant.
Frequency distribution for the diameter of the stem was also positively skewed with platykurtosis (Fig. 7c), and the mean value of the stem diameter of field-grown plants was (3.69 ± 0.1 mm) identical to the mean value of the stem diameter of cloned plants (3.7 mm). The value of mode for cloned plants (4 mm) indicated that a large number of clones had an improved stem diameter. However, statistical analysis of variance and DMRT showed the variation among somaclones and compared with that mother plant to be nonsignificant at p < 0.05.
Frequency distribution for the height of cloned plants was the only parameter which showed no change (Fig. 7d) as the mean value of field-grown plants was (21.2 ± 1.7 cm) identical to the mean value of cloned plants (21.44 cm). Most of the parameters represented an increase as compared to their field-grown plant, which indicates that plantlets cloned via induction of somatic embryogenesis resulted in crop improvement.
A noteworthy result of this study was the easy protocol for plant regeneration via embryogenic callus induction. Further, the experiment for field trial concluded that regenerated plantlets showed positive somaclonal variations. Clone S84 showed an increase in flower diameter as compared to the field-grown plants. Hence, plant tissue culture technique has a great potential to improve chrysanthemum through somaclonal variation. This study will be helpful for the improvement and establishment of floriculture in Pakistan.
Author contributions SA: conducted the experiments, data collection, data analysis and wrote the results and discussion. SR and SS: conceived and designed the research. TSB: collected references and literature. AA: conducted the experiment and data collection. NH: reviewed