© 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 Dianthus chinensis L. cultivars- 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. (China Pink) is a caryophyllaceous, ornamental plant, 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 flowers without any seasonal effect. Keywords: BA induced flowering. Dianthus chinensis. In vitro flowering. Micropropagation. Screening of variants I. INTRODUCTION The species of the family caryophyllaceae are well known source of ornamental plants 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 flower 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 leaf 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 cultivar 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 IJRAR19J1471 International Journal of Research and Analytical Reviews (IJRAR) www.ijrar.org 10 © 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 leaves, 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.