© 2018 IJRAR January 2019, Volume 06, Issue 1 www.ijrar.org (E-ISSN 2348-1269, P- ISSN 2349-5138) Effect of BA on high-frequency in vitro flowering in chinensis L. - a tool to early screening of variant types

Sreelekshmi R and E.A. Siril* Department of Botany, University of Kerala, Kariavattom, Thiruvananthapuram 695581, India,

Abstract: Dianthus chinensis L. ( Pink) is a caryophyllaceous, ornamental , grown worldwide on a commercial scale due to perpetual flowering, diversity of colour and wide range of floral forms. In the present study, we forward an in vitro method for induction of flowering of five cultivars of D. chinensis. The different colour varieties viz., Dianthus telstar white, Dianthus telstar pink, Dianthus floral lace light pink, Dianthus floral lace lilac and Dianthus telstar purple picotee cultivars were used in the present study. In vitro culture of nodal explants excised from five selected cultivars on Murashige and Skoog (MS) medium supplemented with 3% sucrose, 0.85% agar and 2.5 µM benzyl adenine (BA) resulted in the axillary bud break and proliferation of shoots. New shoots after 4 weeks of culture were transferred to agar gelled MS medium containing BA produced in vitro flowering. Dianthus telstar white showed maximum flowering response in 2.5µM BA (100%) added medium. Dianthus floral lace light pink showed maximum response on 5µM BA supplemented medium. Other cultivars, Dianthus telstar pink (21.80%), and floral lace lilac (11.07%) and Dianthus telstar purple picotee (21.47%) were flowered in medium containing 2.5µM BA. In vitro flowering in D. chinensis can be used as a tool to screen variants types generated through mutagenic treatments. Also hormonal role in the floral developments can be demonstrated apart from producing without any seasonal effect.

Keywords: BA induced flowering. Dianthus chinensis. In vitro flowering. Micropropagation. Screening of variants

I. INTRODUCTION

The species of the family are well known source of ornamental in gardens. Dianthus chinensis L. (China Pink) is a widely used ornamental crop due to its beautiful flowers and diverse floral colour forms. D. chinensis is a wonderful accent to gardens and home floral arrangements. The dry flower materials are useful for the arrangement of pressed flower crafts, cosmetics and sachets. D. chinensis perform comparatively well to D. caryophyllus in warmer regions, especially in the humid tropical climate conditions. Considering the benefits of this ornamental and floricultural crop and to fulfill the world demand, the workers focused on micropropagation for the large scale production. In vitro multiplication and regeneration protocols were reported from node, shoot tip or explants of D. chinensis (Kantia ad Kothari 2002, Pareek et al. 2004). In vitro techniques essentially help to preserve the genetic diversity of the varieties.

Generally, hybridization technique has been used in this genus to evolve different colour varieties and improving its keeping quality (Nakano and Mii 1993). In Kerala, the southernmost state of India, the seasonal variation seems to influence the cultivation and mass scale production of plantlets, and that may create a difficulty during the identification process of new coloured cultivars from commercial breeding program. In vitro flowering technique provides an opportunity for easy hybrid identification of new throughout the culture period. However, few works are carried out on in vitro flowering nature of Dianthus species (Davis and Upadhyaya 1994). In vitro flower development on D. chinensis were only mentioned in several reports as an observation during the course of micropropagation (Nakano and Mii 1993, Jethwani and Kothari 1996, Kantia and Kothari 2002). Further

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© 2018 IJRAR January 2019, Volume 06, Issue 1 www.ijrar.org (E-ISSN 2348-1269, P- ISSN 2349-5138) experiments to develop a reproducible method of in vitro flowering were not yet attempted in the past. In vitro flowering has been reported to occur either spontaneously or through induction in several plant species in MS basal medium (Kielkowsk 2012). The transition from the vegetative phase to flowering phase depends on the developmental genetic program, which is triggered and modulated by environmental stimuli (Huijser 2011), application of exogenous hormones and also influenced by the effect of internal and chemical factors (Jana and Singh 2011).

Dianthus spp. are commonly used as model plants for various in vitro morphogenesis experiments. In vitro flowering studies on D. chinensis will serve as an experimental model to elucidate physiological and molecular aspects of flower development. In vitro production of flowering shoot in German Red carnation (D. caryophyllus) by the effect of uniconazole, a triazole based plant growth retardant and gibberellic acid (Davis and Upadhyaya 1994) were reported. However, the effect of BA on in vitro flower induction in D. chinensis was not explored. Previous reports in other herbaceous plants suggests widely used cytokinin, BA can be a potent inducer of in vitro flowering (He and Loh 2002, Corbesier et al. 2003). Application of BA promotes flower bud induction in Arabidopsis mutants and conformed that the genes, TWIN SISTER OF FT (TSF) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) are necessary for the flowering response, thus clearly conformed cytokinin regulated molecular itinerary to flowering (Aloia et al. 2011). BA treatment activate the transcription of TSF gene but not FT in , finally the overall gene expression leads to the flowering response. The aim of this work was to study the effect of BA on in vitro flowering nature of some selected D. chinensis cultivars. In vitro flowering of D. chinensis provide, a suitable experimental system for studying phase transition from vegetative to floral development.

II. MATERIALS AND METHODS

Plant material and Culture conditions

The five selected colour varieties viz., Dianthus telstar white, Dianthus telstar pink, Dianthus floral lace light pink, Dianthus floral lace lilac and Dianthus telstar purple picotee of D. chinensis were procured from District Horticultural Nursery, Thiruvananthapuram, Kerala, India and maintained in the glass house of Department of Botany, University of Kerala served as the source for explants. Fresh shoots were collected from different cultivars during October-November and nodal segments (1-2 cm) were excised. They were thoroughly washed in running tap water for 30 min, followed by washing with 2% polysorbitol detergent solution (Labolene Mfg. Fischer Fine Chemicals, Mumbai, India) for 10 min, and then treated with 0.1% carbendazim fungicide (Bavistin Mfg.BASF, Mumbai, India) for 15 min followed by washing 3-4 rinses with distilled water. These explants were surface sterilized with 0.1% HgCl2 (Sisco Research Laboratories, Mumbai, India) solution for 3 min and washed with sterile distilled water for 4-5 times to remove trace of HgCl2. The surface sterilized explants were trimmed at the cut ends and placed in 25×150mm test tubes containing 15 ml MS medium supplemented with 3% sucrose and 0.85% agar (Sisco Research Laboratories, Mumbai, India) and varying type and conc. (µM) of cytokinins.

Effect of cytokinins on culture establishment

The first experiment was conducted to standardize the most suitable type and conc. of cytokinin for the in vitro culture establishment and proliferation of shoots for the five selected cultivars of D. chinensis. MS medium containing 3 % sucrose and cytokinins viz., BA (MW=225.25; Sigma–Aldrich, Bangalore, India), kinetin (MW=215.21; Sigma– Aldrich, Bangalore, India) or 2-iP (MW=203.2; Sigma–Aldrich, Bangalore, India) were used at different concentrations (0.5, 2.5, 5.0 or 7.5µM) for the in vitro multiplication. After 30 days of culture, % response of explant, IJRAR19J1471 International Journal of Research and Analytical Reviews (IJRAR) www.ijrar.org 11

© 2018 IJRAR January 2019, Volume 06, Issue 1 www.ijrar.org (E-ISSN 2348-1269, P- ISSN 2349-5138) mean number of shoots developed and mean shoot length (cm) were recorded. Thus nascent shoots developed after 30 days of culture were excised and segmented having a single node (1-2cm) were subcultured on to 2.5µM BA for further multiplication. The experiments were repeated and observations were recorded every 30 days of culture.

Effect of BA on in vitro flowering

To test the effect of BA on in vitro flowering of D. chinensis, 3 cm long microshoots of different cultivars were aseptically excised from cultures developed in MS medium containing 2.5µM BA. These shoots were planted on agar gelled (0.85%) MS medium supplemented BA (2.5 or 5µM). All the cultures were incubated at 25±2°C in a culture room with 50 μmol m−2s−1 irradiance provided by cool white fluorescent tubes (40 W; Philips, India) and were exposed to a photoperiod of 16-h and 55±5% of relative humidity. The frequency of in vitro flowering along the subculture I, II and III was recorded at the end of four week incubation in each subculture.

Effect of auxins on in vitro flowering

The effect of auxin on in vitro flowering was studied using excised shoots (>3cm) of D. chinensis floral lace light pink cultivar. Agar gelled MS medium containing 3% sucrose and different conc. of IBA (0.5, 1 or 2.5µM) was tested. Each treatment was analyzed as triplicate and was repeated tree times. Observations on in vitro flowering were recorded after 4 weeks of culture.

Effect of sucrose on in vitro flowering

To evaluate the effect of sucrose (SISCO Research Laboratories, Mumbai, India) concentrations on in vitro flowering of D. chinensis floral lace light pink cultivar the excised shoot (>3cm) were used. The individual shoots were transferred to agar gelled (0.85%) MS medium containing different concentrations (1, 2, 3, 4 or 5% w/v) of sucrose along with 2.5µM BA was tested.llf shoot, percentage of flowering and number of flowers per

Experimental layout and statistical analysis: Experiments on in vitro response and in vitro flowering were conducted by three replication blocks of 8 culture tubes each. After four weeks of culture, percentage of response, shoot number and shoot length were recorded. In case of in vitro flowering experiments, data on percentage flowering, number of flowers per culture was recorded after four weeks of culture. Data scored in percentages were subjected to arcsine transformation before analysis, and then converted back to percentages for presentation in the tables (Snedecor and Cochran 1962). The observed data was subjected to analysis of variance (ANOVA) and the differences among the mean value were compared with Duncan`s Multiple Range Test (DMRT) (P<0.005) using SPSS ver. 22.

III. RESULT AND DISCUSSION

Culture establishment of selected cultivars

The effect of different cytokinin concentrations on in vitro response on D. chinensis was examined. All the selected cultivars showed almost similar response to different cytokinin concentrations. The addition of different cytokinins in MS basal medium significantly enhanced the shooting response. The shoot bud development at the nodal region was observed in 5 d-old cultures. Emergence of adventitious shoot bud from intermodal region was observed in 14 days after culture from the lower cut end of explants. Cultures on BA supplemented medium showed development of axillary shoots and adventitious shoot buds. Adventitious bud development could not find in hormone free medium.

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© 2018 IJRAR January 2019, Volume 06, Issue 1 www.ijrar.org (E-ISSN 2348-1269, P- ISSN 2349-5138) ANOVA revealed significant (˂0.001) interaction between cytokinin type and concentrations on both explant response and shoot induction (Table 1).

Table 1. Effect of MS medium supplemented with different concentration of cytokinins on nodal explants of D. chinensis L.

Cytokinin % of Cytokinin type concentration Shoot number Shoot length response (µM) - 0 16.3±2.7f 1.0±0.0f 1.2±0.1e BA 0.5 51.7±2.5e 2.5±0.3e 1.7±0.2e a a a BA 2.5 100.0±0.0 6.3±0.3 7.6±0.2 BA 5 89.1±4.0b 7.2±0.6a 4.5±0.3b BA 7.5 87.0±2.7b 7.5±0.8a 2.7±0.4cd KIN 0.5 43.3±1.9e 2.8±0.2de 4.5±0.3b KIN 2.5 76.7±1.1cd 4.0±0.3bcd 7.3±0.2a cd cde b KIN 5 75.1±1.9 3.7±0.3 5.0±0.3 KIN 7.5 66.7±2.0d 2.7±0.2e 3.2±0.2cd 2-iP 0.5 46.6±1.9e 1.3±0.3f 2.8±0.4cd 2-iP 2.5 90.0±0.0b 2.7±0.2e 7.3±0.3a 2-iP 5 83.7±2.7bc 4.5±0.3bc 3.5±0.3c cd b d 2-iP 7.5 75.1±1.9 5.2±0.6 2.5±0.3 F value Main effect Df (n-1) 12 57.3*** 28.9*** 58.9*** Cytokinin Type Df (n-1) 2 45.4*** 51.6*** 13.7*** Cytokinin conc. Df (n-1) 3 106.7*** 34.0*** 163.4*** Variety X Conc. Df (n-1) 11 5.8*** 9.7*** 6.4***

*** Significant at P<0.001 level, Means within column followed by same letters are not significantly (P<0.05) different as determined by Duncan’s Multiple Range Test

BA (2.5µM) supplemented MS medium found to be best medium for shoot induction in all the cultivars (6.3shoots per plants) with 100% response and minimal hyperhydric syndrome. Shoots developed in this medium showed active growth and thus attained 7.6 cm length (Fig. 1a) after 30 d of culture. Cultures on higher BA concentration showed higher rate of multiplication but morphologically abnormal shoots with glassy nature (hyperhydricity). BA showed superior response over other cytokinins like 2-iP or KIN on all the cultivars. It may be due to its easy permeability or metabolism and induction of zeatin like hormones with in the tissue (Lodha et al. 2015). A single shoot with an average length of 7.3cm was developed on medium supplemented with 2-iP (2.5 µM) (Fig. 1d). Cultures raised were further subcultured on MS medium supplemented with 2.5µM BA showed stabilized production of nascent microshoots.

In vitro flowering of different cultivars

The individual shoots (>3cm) of five cultivars of D. chinensis from proliferated shoot cultures were excised and cultured on agar gelled (0.8%) MS medium supplemented with 2.5 or 5 µM BA and 3% sucrose. Effect of BA was significant (P<0.001) to in vitro flowering of five cultivars of D. chinensis. However, flowering response of five cultivars varied (Table 2) with respect to BA concentrations (Fig. 1b) and maximum response (100%) was recorded in cultivar telstar white at I stage culture and during II stage culture, this cultivar showed 88.7% flowering response. Initiation of flower buds at terminal position of in vitro established shoot was noticed within two week of cultures. The flower buds at the early stage were small, dark green protrusions, later elongated, with membranous, light green sheath. Three weeks after development, these terminal buds were fully opened with expanded corolla characterized by irregular, zig-zag margins. Flowering response of different cultivars were varied significantly, where Dianthus floral lace light pink cultivar flowered 100% in 2.5µM BA added medium at 1st subculture. Cultures raised in BA added

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© 2018 IJRAR January 2019, Volume 06, Issue 1 www.ijrar.org (E-ISSN 2348-1269, P- ISSN 2349-5138) medium also showed development of nascent shoots from the nodal region of main shoot. In vitro produced flowers in all cultivars were persisted more than one weeks and later corolla was withered followed by detachment of pedicel. Cultivar telstar white showed 10-12 days longevity and 10-15 days for floral lace light pink flowers. The colour and size of in vitro formed flowers were almost similar to that of source plant used. Interestingly, seed settings were also occurred in in vitro formed flowers. In vitro flowering response of five D. chinensis cultivars were recorded and showed following features;

Dianthus Telstar White: The white flowered D. chinensis responded excellently to in vitro propagation system as well as in vitro flowering. This cultivar showed 100% flowering response with higher number of shoot and shoot length (7.22cm) in 2.5µM BA (Fig. 1c) containing medium. The multiple shoots developed on BA added medium also led production of floral buds. In 5µM BA concentration this cultivar showed 94.43% flowering and reduction in flower induction with respect to increasing subculture was noticed.

Dianthus Telstar Pink: is an annual pink coloured cultivar showed low levels of flowering response in 2.5µM concentration (21.08%). The microshoots grew actively and elongated to 5.12cm size with terminal as well as axillary bud development.

Dianthus Floral Lace Light Pink: The perennial white with pink double shaded cultivar showed 100% flowering response in first transfer of in vitro shoot clump (~6.5cm ) to 2.5µM BA added medium. However, low levels of flowering response were observed on 5µM BA added medium.

Dianthus Floral Lace Lilac: Medium containing 2.5µM BA resulted in development of healthy shoots with needle like leaves and flowers (11%). Whereas, addition of 5µM BA was deleterious to flower development. The cultivar showed basal callusing and hyperhydricity on 5µM BA concentration.

Dianthus telstar purple picotee: The typical D. chinensis bicolor cultivar (annual) showed low response (21%) to flowering on 2.5µM BA.

Table 2. A comparison on in vitro flowering of five D. chinensis cultivars on MS medium supplemented with two different concentrations of BA

Variety BA Conc. % Flowering (µM) I Subculture II Sub culture III subculture

Dianthus Telstar White 2.5 100.00a 88.87a 83.30 5 94.43a 88.87a 83.30

Dianthus Diamond Pink 2.5 5 21.80b 5.53b 0.00 11.07bc 0.00b 0.00 Dianthus floral lace light 2.5 100.00a 94.43a 83.30 5 11.07bc 0.00b 0.00 Dianthus Telstar Pink 2.5 11.07bc 0.00± 0.00 5 0.00c 0.00± 0.00 Dianthus Telstar Purple Picotee 2.5 21.47b 5.53b 0.00 5 0.00c 0.00b 0.00 F value Main effect Df (n-1)= 9 69.01*** 120.70*** - Cultivars (n-1)= 4 114.54*** 198.80*** - BA Conc. Df (n-1)= 1 6.07* 45.07*** - Cultivars X BA Conc. Df (n-1)= 4 39.22** 61.52*** -

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© 2018 IJRAR January 2019, Volume 06, Issue 1 www.ijrar.org (E-ISSN 2348-1269, P- ISSN 2349-5138) *** Significant at P<0.001 level, **Significant at P<0.01 level. *Significant at P<0.05 level, Means within column followed by same letters are not significantly (P<0.05) different as determined by Duncan’s Multiple Range Test

Growth regulators have many important roles in diverse developmental process ranging from germination to formation of shoots, roots or flowers (Wang et al. 2001). Previous reports point out that in vitro flowering was stimulated by the addition of cytokinin and also by the activation of endogenous cytokinin in ascending xylem sap (Patil and Jeyanthi 1995). The requirement of cytokinin for the growth and development of flower buds has been reported in both monocots and dicots (Wang et al. 2001). Cytokinin has been recognized as an important component of a multi-factored flowering stimulus and recognized as an important signal to induction of flowering (Bonhomme et al. 2000, Lindsay et al. 2006). Experiments on cytokinin contents in leaf extract and leaf exudate proposed that this particular hormone might be involved in flowering as systemic signals (Bernier and Perilleux 2005). Studies on cytokinin on ornamental plants revealed that besides the role in axillary shoot proliferation, cytokinin can increase number of flower, flower diameter, flower longevity, and number of days to flowering (Pobudkiewicz 2007). Mallikarjuna and Rajedradev (2009) reported that medium containing BA produced higher number of multiple shoots coupled with in vitro flowering in Holarrhena antidysenterica than Kin added medium. Similar results were also available in Bacopa chamaedryiedes (Haque and Ghosh 2013) and Ipomea quamoelit (Ghosh and Haque 2013). BA has been reported to be effective in floral induction of a number of plant species, such as orchids (Naor et al. 2004), bitter melon (Wang et al. 2001), Perilla frutesceus (Zhang 2007), Dioscorea zingiberensis (Huang et al. 2009) and dwarf tomato (Nanna 2009). According to Vadawale et al. (2006), the flower buds of Vitex negundo were produced by the combined effect of both BA and Kin. In case of Scoparia dulcis, addition of Kin in the medium induced flower buds and were failed to produce functional flowers. Kim et al. (2000) reported that an intergeneric orchid hybrid Doritaenopsis sprayed with BA at a lower conc. had much flowers than high BA treatments. The German carnation (D. caryophyllus) produced adventitious flowering from in vitro shoots containing 8.9µM BA and 2-7µM NAA. High efficiency microshoot induction and plant regeneration were reported from leaf explants of D. chinensis (Kantia and Kothari 2002). Fragmented flower buds, individual petals and receptacles were used for the induction of adventitious shoots in different cultivars of D. caryophyllus.

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© 2018 IJRAR January 2019, Volume 06, Issue 1 www.ijrar.org (E-ISSN 2348-1269, P- ISSN 2349-5138)

Fig. 1 Effect of BA on in vitro propagation and flowering on five different cultivars of Dianthus chinensis L. a Culture of original nodal explant on MS medium having BA (2.5µM) after 2 weeks (Scale bar 0.5cm). b. In vitro flowering of different Dianthus cultivars on BA (2.5µM) supplemented medium (Scale bar 0.8cm). c. 100% in vitro flowering of Dianthus white cultivar on 2.5µM BA supplemented medium. (Scale bar 0.5cm). d. Axillary shoot development from 2.5µM 2-iP treated medium (Scale bar 0.5cm). Effect of auxin on in vitro flowering

The treatment with auxin accompanied with elongation of shoots after one week. It is noticed that auxin did not show any particular effect on in vitro flowering. Flowering response of microshoots cultured on different IBA supplemented medium varied significantly (Table 3). Only 53.33% of flowering was observed on 0.5µM concentration of IBA (Fig. 2b) and decreased with increasing concentrations. According to Franklin et al. (2000) auxin is an effective class of hormones to induce flowering and maturation of flowers. In contrast to this auxin concentration is either ineffective or inhibitory to flowering in D. chinensis. The callus development and enhanced root production was noticed in auxin (NAA, IAA or IBA) added medium and was inefficient to in vitro flowering process. Some cultures of Dianthus floral lace light pink showed flower development along with basal callusing on IBA treated medium (Fig. 2c). The reports on Bambusa arundinacea by Joshi and Nadganda (1997) explained flowers could only produce in the absence of roots, where shoot were cultured on optimum BA concentration.

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© 2018 IJRAR January 2019, Volume 06, Issue 1 www.ijrar.org (E-ISSN 2348-1269, P- ISSN 2349-5138) Table 3. Effect of MS medium supplemented with different concentrations of IBA on in vitro flowering of D. chinensis L.

IBA conc.(µM) Shoot length (cm) % of flowering No of flowers per culture 0 7.3±0.17ab 100.0±0.00a 4.3±0.33a 0.5 7.5±0.29ab 53.3±3.33b 1.7±0.33b 1 8.5±0.29a 43.3±3.33c 1.3±0.33b 2.5 7.0±0.58b 20.0±0.00d 1.0±0.00b F value Df (n-1)=3 3.2* 203.2*** 27.9*** *** Significant at P<0.001 level, *Significant at P<0.05 level, Means within column followed by same letters are not significantly (P<0.05) different as determined by Duncan’s Multiple Range Test

Effect of Sucrose on flower induction The different concentrations of sucrose had significant effect on multiple shoot formation as well as flower induction in vitro (Table 4). Multiple shoot development was occurred in medium containing different concentrations of sucrose. Cent percent flowering and maximum number of flowers (4.33) per shoot was observed when the medium was supplemented with 3% (w/v) sucrose (Fig. 2a) than 1, 2, 4 or 5% concentration. These results were in line with previous findings on Perella fluritence (Zhang 2005), Anethum graveolens (Jana and Singh 2011), Pentanema indicum (Sivanesan and Jeong 2007), Pisum sativum (Franklin et al. 2000) and Gentiana trifolia (Zhang and Leung 2002). The concentration and the nature of carbon source in the MS medium have an important influence on reproductive bud stimulation. Sucrose is one of the best carbon sources for in vitro flowering. Possibly, addition of sugar stimulates the development floral buds (Singh et al. 2006). Optimum concentration sucrose for flowering was closely followed by D-glucose and fructose. The lower concentration of carbon source was deleterious to shoot regeneration and also promoted hyperhydricity. Interaction of both sucrose and cytokinin influence the flower bud induction as reported in Sinapis alba by moving between shoot and root (Havelange et al. 2000). Light period or day length is most predictive factor for plant reproduction (Bernier and Perilleux 2005). It was observed that 16/8 h light/dark cycle with 40µm-2s-1 light intensity (fluorescent tube) is better than other light period on in vitro flowering. It was cleared that under 14/10 h light/ dark cycle buds failed to blooming. Cultures maintained under dark condition did not produce any flower buds. In the present study we observed that, the change in the BA concentration and genotype difference in cultivars influenced the flowering of D. chinensis in vitro when all the other parameters were kept at the optimal level.

Table 4. Effect of MS medium supplemented with different concentration of sucrose on in vitro flowering of D. chinensis L.

No of flowers per Shoot length (cm) % of flowering Sucrose conc. (g/l) culture 0 1.3±0.17d 0.0±0.00d 0.0±0.00c 10 2.0±0.29d 0.0±0.00d 0.0±0.00c 20 4.7±0.17c 10.0±5.77c 0.7±0.33c 30 7.3±0.17a 10.00±0.00a 4.3±0.33a 40 8.0±0.29a 56.7±3.33b 2.0±0.58b 50 6.5±0.29b 16.7±3.33c 1.0±0.00c

F value df(n-1)=5 141.3*** 172.5*** 29.3*** *** Significant at P<0.001 level, Means within column followed by same letters are not significantly (P<0.05) different as determined by Duncan’s Multiple Range Test

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© 2018 IJRAR January 2019, Volume 06, Issue 1 www.ijrar.org (E-ISSN 2348-1269, P- ISSN 2349-5138)

Fig. 2 Effect of sucrose and auxins on in vitro flowering of Dianthus chinensis L. a 100% in vitro flowering of Dianthus floral lace light pink cultivar on 3% sucrose supplemented medium (Scale bar 0.8cm). b Dianthus flower development on 0.5µM IBA treated medium showing rooted shoot (Scale bar 0.5cm). c In vitro flower development on IBA treated medium showing basal white callusing (Scale bar 1.6cm).

IV. CONCLUSION Through the present study we report for the first time an efficient in vitro flowering system for D. chinensis- a plant of high ornamental and medicinal importance. The present study revealed that the highly effective role of BA to multiplication and in vitro flowering of the D. chinensis. MS medium with 2.5µM BA along with 3% sucrose was found to be more effective for in vitro flowering. The Dianthus white cultivar showed comparatively good explant response and 100% flowering in MS medium supplemented with BA. Additions of IBA were ineffective or inhibitory to in vitro flowering in D. chinensis. Through the present work, in vitro flowering in D. chinensis was demonstrated. The developed method significantly contributes to identification of variant phenotypes and in vitro screening of desired floral morphs. Further, the in vitro flowering model system developed through the study can be used to elucidate regulation mechanism of flowering at genic level.

V. ACKNOWLEDGMENTS

The authors are grateful to Dr. Suhara Beevy S, Associate Professor and Head, Department of Botany, University of Kerala, Kariavattom, Thiruvananthapuram, for providing the facilities. The authors also thank University of Kerala for financial support in the form of Junior Research Fellowship (No.AcVI(1)/715/BOT/13289/2015).

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