In Vitro Callus Culture of Dianthus Chinensis L. for Assessment of Flavonoid Related Gene Expression Prole

Dianthus chinensis L. is an edible, ornamental herb used to prepare the Dianthi Herba, a Chinese traditional rejuvenating medicine. Owing to the rapid proliferation of callus tissues, in vitro production of avonoids has their own specic importance. Callus cultures raised followed by auxin directed biosynthesis of avonoid through related transcript prole were carried out. Murashige and Skoog (MS) medium fortied with 2,4- Dichlorophenoxy acetic acid (2,4- D) or picloram induced formation of friable callus from internode derived cultures of D. chinensis. Culture medium containing 2,4- D (10 µM) produced the highest avonoid content, 4.44 mg quercetin equivalent per gram (QE g − 1 ) under incubation in continuous dark condition, while maximum dry weight yield (0.38 g/ culture) was obtained from 10 µM 2,4- D under 16 h light / 8 h dark condition (50 µmol m − 2 s − 1 irradiance) at 60 days of incubation. The callus raised in light condition in 10 µM 2,4- D selected to analyze avonoid related gene expression prole viz., chalcone synthase (CHS), chalcone isomerase (CHI), avanone-3-hydroxylase (F3H), and avonol synthase (FLS) at specic time intervals. The transcript abundance of CHS, F3H, or FLS gene was higher at 60 days old callus cultures and reaching 11.59, 48.31, and 114.63-fold relative expression than that of initial callus tissues respectively. These understandings are critical for the regulation of targeted phytochemicals as well as their wide exploitation in the eld of biological research.

In recent years, the plant tissue culture technique has become an effective tool to achieve enhanced production of secondary metabolites, especially when resources in the natural stands for the extraction of metabolite is facing shortage or scarcity of speci c bioactive compounds due to increasing the demand (Zhou et al. 2020). Studies on factors involved in vitro production of secondary metabolites and biosynthesis increasingly received attention (Lucho et  Since last three decades, avonoid biosynthetic pathways in plants received due attention in plant science research. The metabolic engineering process aimed to ourish various aspects such as; model plants (to explain the general biosynthetic pathways), oricultural plants (to produce different ornamental plants), plants for increased tolerance to pathogens (engineering of phytoalexin), and crop plants for avonoid enhancement (Tohge et al. 2017;Nabavi et al. 2020). As an important oricultural plant, D. chinensis is known for its extremely diverse color pigmentation and are responsible for production of varied avonoids and anthocyanin derivatives. In view of this the highly proliferated in vitro callus cultures can be treated as tool to dissect and manipulate the genetic route of avonoid production. Many folds enhanced production of metabolite can be targeted by over expression of key genes in the metabolic pathways. The over expression of CHI gene has effectively enhanced avonoid yield in hairy root culture of Glycyrrhiza glabra (Zhang et al. 2009) and Scutellaria baicalensis (Park et al. 2011). Li et al. (2006) states that, CHI gene transferred hairy root cultures of Saussurea involuerata have been expressed an enhanced production of avonoids in contrast to its wild type hairy roots. Moreover, down regulation of avonoids has reported by silencing of CHS gene in transgenic hairy roots of Medicago truncatula (Wasson et al. 2006). These understandings are critical for the regulation of in vitro production avonoids. The relationship between in vitro cell line growth and metabolite production has been partially To assess the stability of avonoid production, the callus (~200mg) raised in 10 µM 2,4-D media were subcultured to MS medium containing 10 µM 2,4-D and were incubated at a photoperiod of 16 h light/ 8 h dark and the subcultures practice continued 4 times at every 60 days interval, and at the end of incubation period avonoid yield was determined.

Callus growth index and water accumulation
The growth capacity of callus in terms of growth index (GI) was determined as the ratio of the accumulated and the initial biomass. The callus Growth index of callus raised in medium containing 2,4-D (10 µM) at every 10 days interval was calculated by the following formula; GI = Wf -Wi/ Wi. Where, GI represents growth index, Wf and Wi represents the nal and initial fresh weight of callus (Godoy-Hernandez and Vazquez-Flota, 2006).
In vitro cultures of D. chinensis are highly vulnerable to varying degrees of hyperhydricity (Sreelekshmi and Siril 2020; Sreelekshmi et al. 2021a and b), thus callus mass accumulates high proportion of water leading to increment in fresh weight. The water accumulation of cultures raised in medium containing 2,4-D (10 µM) was determined at every 10 days interval using the formula; Callus moisture content (%)= Fresh weight -Dry weight×100/ Dry weight.

RNA isolation and cDNA synthesis
The e ciency of metabolite production speci c to expression of avonoid synthesis gene was measured by isolating the callus biomass raised in medium containing 10µM 2, 4-D at 1, 20, 40, 60, or 80 days intervals. Three biological replicates for each culture were randomly selected, ground in liquid nitrogen, and sample (100 mg) from each, pooled for RNA isolation using trizol reagent (Ambion by Life Technologies, Invitrogen, Carlsbad, USA) method (Simms et al. 1993). The concentration and integrity of RNA sample was quanti ed by NANO DROP ONE (Thermo Scienti c, USA) and agarose gel (1.6 %; SRL, Mumbai, India) electrophoresis. Five hundred nanogram of RNA were converted in to cDNA using Primer Script TM 1 st strand cDNA Synthesis Kit AB-1453/B (Thermo Scienti c, USA) according to the manufacturer's instructions. The 20 µl nal reaction mixture was incubated at 42 °C for 30 min followed by inactivation at 95 °C for 2 min.

Analysis of expression of genes associated with avonoid biosynthesis
The expression of key genes associated with avonoid biosynthesis pathway was determined via quantitative real-time PCR (qRT-PCR (qTOWER 2.2, Analytic Jena, Germany) and data analysis has performed by aj, qPCRsoft 3.0. The gene expression analysis of selected genes (CHS, CHI, F3H, and FLS) in different culture systems were analyzed by using, the gene speci c primers which were designed on the basis of sequence data procured from D. caryophyllus GenBank (National Center for Biotechnology Information; NCBI). The RT speci c primers were designed using PrimerQuest tool (Integrated DNA Technologies, Inc., Iowa 52241, USA) under default parameters. The designed primer sequences were again sorted by checking the speci cation of PCR primer status in Sequence Manipulation Suite; Version 2 software (Stothard 2000), and purchased (Geno Biosciences Pvt. Ltd, Noida, India). The designed primer sequences were duly tabulated to relative expression study ( Table 1). The speci city of RT speci c primers was found by melting curve analysis and single band product visualization by agarose gel electrophoresis. The expression of gene of interest was con rmed by using actin (ACT) as the internal

Statistical analysis
All the experiments on callus cultures were conducted using a randomized complete block design (RCBD) method. Every experiment consists of four replication blocks of 10 culture tubes or 6 culture asks for each treatment. To record callus induction as well as avonoid production, parameters viz., percentage of response, fresh weight (g), dry weight (g), and total avonoid (44 mg QE g -1 DW) were recorded every 20 days of culture. The data subjected to analysis of variance (ANOVA) and the differences among the mean values were compared with Duncan's Multiple Range Test (DMRT;, p<0.05) using IBM SPSS Statistics V22.0 and all results were expressed as mean ± SD. The RNA isolation was conducted with pooled replications, and data represented the mean relative expression of three repeats with standard error bars.

Results And Discussion
In vitro callus induction Callus induction from intermodal or leaf segments cultivated on different auxin supplemented media showed distinct growth characters (Table 1). When explants were cultured on full strength MS medium containing 2.5 or 5 µM 2, 4-D or picloram, induction of friable callus (100%) was noticed ( Fig. 1a and b), while the explants cultured on the media containing NAA, IAA, or IBA produced roots with low callus proliferation (Fig. 1c-e). In contrast to picloram 2,4-D facilitated higher callus biomass accumulation both in internode (2.13 g/ culture) and leaf explants (0.78 g/ culture). The response of 5 µM 2, 4-D also showed distinct callus morphology after 40 days cultures (Fig. 1f). The internodal segment showed superior response with creamy friable callus, while leaf explant showed inferior callogenic response and the developed callus was white, nodular type ( Fig. 1g and h). Friable greenish-yellow callus was induced (100%) after 1 week from the cut ends of the internode cultured on MS medium containing 2, 4-D (2.5 µM) (Fig. 1i), and highly proliferative creamy callus formation occurred after one month of incubation. In contrast, the auxin-free cultures failed to produce callus irrespective of explants used. The loosely aggregated, friable callus mass induced by the intervention of 2,4-D is capable to grow rapidly with high cell proliferation (Souza et al. 2011). Due to superior response and high fresh weight, the internode derived calli were selected for further avonoid production experiments. Similar callus proliferation response was reported in D. chinensis (Nontaswatsri et al. 2008 (Table 2 and 3). Among the different incubation conditions, 2, 4-D at light condition was found to be the most effective for callus induction, producing rapidly growing friable calli (Fig. 2a). The friable greenish callus was formed from all the treatments within 1 week of culture. After 30 days, the callus tissues turned creamy to yellow colored, and later (80 d The higher FW (11.0 g/ culture; Fig. 2b) was achieved on 10 µM followed by 15 µM 2, 4-D (9.0 g/ culture) containing medium at 16 h light/ 8 h dark condition (Fig. 2c) and least (3.2 mg g -1 ) fresh biomass of callus formed at 1 µM 2,4-D during 80 days incubation under complete dark condition (Fig. 2d). As the duration of incubation increased, the biomass accumulation also increased with respect to auxin concentration in light or dark condition. At dark condition, friable white callus (Fig. 2d) was proliferated on 10 µM 2,4-D and upon extending the days of incubation to 80 days, colour of callus turned to yellowish brown ( Fig. 2e and f). A gradual increase of dry weight was observed with increasing concentration of 2,4-D, regardless of incubation condition, whether it is light or dark, whereas the maximum dry weight of callus (0. compared with other auxins. Dry weight accumulation of callus was found to be maximum at 60 th day of incubation in all the auxin concentrations. Thereafter, it declined possibly due to unavailability of nutrients or deterioration of callus due to accumulation of auto-toxic metabolic products. Alwash et al. (2018) reported the quick callogenic response characterized by the formation of friable, granular, or greenish yellow to creamy callus in D. caryophyllus using 2,4-D (1 mg L -1 ) and BA (0.5 mg L -1 ) added medium.

Callus growth index and moisture content
Growth index (GI) of callus raised in 10µM 2, 4-D were analyzed from 10 th day to 80 th day of culture at 10 days intervals. The growth curve of callus biomass showed sigmoid pattern at 10 µM 2, 4-D. Time course monitoring of callus growth suggests that an initial slow growth (lag phase) for rst 30 days and there after sudden increase in GI (log phase) ending at day 60 (Fig. 3a). Subsequently the linear phase was shifted to stationary phase after 60 th day of incubation. Callus maintained in medium containing 2, 4-D (10 µM) for 80 days recorded 11 g FW/ culture and indicated 55-fold increase over the initial callus mass. Similarly, the callus moisture content increased signi cantly (p<0.05) and proportionally to the fresh weight accumulation in 10µM 2,4-D medium (Fig. 3b). Moderate level of moisture was accumulated up to 50 th day of incubation, there after upraised exponentially with increasing the period of incubation.

Total avonoid production
Flavonoid production through callus culture on 2, 4-D added MS medium showed signi cant result both in light and dark condition (Table 5). Among different concentrations of 2, 4-D, 10 µM found to be the most effective to produce callus as well as avonoid. The highest avonoid accumulation in 2,4-D forti ed medium were noticed in 10 µM concentration at dark condition (4.44 mg QE g -1 DW) on 60 th day of incubation. Flavonoid content of callus sampled showed steady increase up to 60 th day of sampling, thereafter production slightly declined. In comparison to the initial avonoid content (0.40 mg QE g -1 DW), cultures raised in 2, 4-D (10 µM) containing medium after 60 days incubation in dark, 11.1-fold increase achieved. 10 µM 2,4-D at 16 h light/ 8 h dark incubation resulted 3.85 mg QE g -1 DW leading 11.3-fold avonoid production compared to initial 10 days culture and 19.25-fold to control set. The incubation condition did show any signi cant role on avonoid production and callus maintained in light condition produced slightly decreased concentration of avonoid (Table 5) However, cultures raised in 10 µM 2, 4-D and incubated in light condition (16 h light/ 8 h dark) has promoted enhanced biomass accumulation, thus contributed signi cantly to highest avonoid yield.
The stability assay of avonoid production on 10 µM 2,4-D added medium at light condition revealed the stable metabolite production during four consecutive subcultures (Fig. 4). By considering the stability and avonoid yield, 10 µM 2, 4-D under 16 h light condition was selected as the best hormone treatment for callus culture in D. chinensis. Our study indicated that calli at 60 days of incubation had high accumulation of total avonoids. It is noticed that callus adapted to grow well in light condition, consequently obtain higher biomass, and seems to conducive avonoid production and is in accordance qRT-PCR analysis on avonoid related genes expression in callus culture The qRT-PCR ampli cation curve of CHS, CHI, F3H or FLS genes were generated using callus sampled in different time intervals and cycle threshold (CT) was determined at speci c threshold uorescent values. The speci city of 40 cycle ampli cation for each primer set along with ACTIN primers in callus sample was con rmed by the single peak melting curve of qRT-PCR products (Fig. 6). The presence of single bands with expected size in agarose (1.6 %; SRL, Mumbai, India) gel electrophoresis also showed the quality and speci city of PCR products.
The high concentration of avonoid accumulated as well as proliferated callus tissues raised in MS medium containing 10µM 2,4-D, and incubated at normal photoperiod (16h light/8 h dark) was harvested at different time intervals (1, 20, 40, 60, (Li et al. 2018). However, the UV exposure treatment on hairy root cultures of Fagopyrum tataricum compared to untreated control, 30-40 fold FLS transcript abundance in avonoid pathway was reported (Huang et al. 2016). In the present study, nal gene expression of FLS under 2,4-D treatment showed the highest transcript abundance at 60 th day of growth phase, which was 114.63-fold higher than control. These much fold change of avonoid gene (FLS) due to the gene ux, which targeted towards many pathways and might be signi cantly contributed net result of enhanced production of avonoids. As such, based on expression pro le it is entailed that 2,4-D steered callus growth, couple avonoid accumulation by up-regulating a series of avonoid biosynthesis-related genes. The work forwards, an excellent idea on over production of diverse avonoid compound through the highly proliferated callus lines of D. chinensis.

Conclusion
In this study, we have developed an e cient callus culture system for in vitro study on avonoid biosynthesis and found that 2,4-D had a signi cant effect on CHS, CHI, F3H, and FLS transcript level on speci c time intervals. MS medium containing 10µM 2,4-D enhanced 11.59, 48.31, and 114.63 folds CHS, F3H, and FLS transcript abundance respectively at 60th days of culturing. The study forwards an idea that serves to enhance the avonoid production by focusing on transcript abundance in different callus phases. The developed culture system should provide a future research to apply new bio-technique tools on constructing novel avonoid regulating sequence of the e cient, proliferated cell lines of D. chinensis Declarations Tables