Introduction

Ranunculus asiaticus L. originates from European countries, near the Mediterranean, and has been adapted to agricultural environments, including changes in climate and soil (Beruto and Debergh 2004;Dhooghe et al. 2012). R. asiaticus cultivars have been used as cut flowers or garden and potted flowers due to their ornamental values (Bernstein et al. 2005;Cerveny et al. 2012). R. asiaticus cultivars include adorable and well-formed flowers with various colors and long stems that are applicable for the ornamental plants (Bernstein et al. 2005;Shahri and Tahir 2011;Valdes-Aguilar et al. 2009). As these attributes are associated with the ornamental values of bulbous flowers, improved agronomical techniques are constantly required to promote floriculture (Sajjad et al. 2017).

The application of plant hormones, such as GA and BA, have contributed to plant growth and development processes, via various complex signaling patterns (Gupta and Chakrabarty 2013;Yruela 2015). Plant growth regulators have been used to induce pre- or post-production of flower bulbs (Gupta and Chakrabarty 2013;Manimaran et al. 2017;Miller 2012;Sajjad et al. 2017;Yruela 2015). Various well known GA roles in plants include cell elongation, breaking seed-coat dormancy, seed germination, and flowering. These functions are related to quantitative and qualitative improvements in bulbous flowers (Gupta and Chakrabarty 2013;Manimaran et al. 2017;Yruela 2015). Several flower bulb species have been subjected to a wide range of GA₃ concentrations in horticultural studies. For example, appropriate amounts of GA₃ improved plant growth, flower number, and days to flowering (Chakraborty and Choudhuri 2016;Esfahani et al. 2016;Guney et al. 2016;Ramzan et al. 2014;Sarkar et al. 2014). Meanwhile, experiments with BA have been used to evaluate the quantity and quality of flower in bulb species (Asgari et al. 2014;Janowska 2014;Khan et al. 2011;Pogroszewska et al. 2007;Sajjad et al. 2015;Sharma et al. 2009). These two hormones have shown positive growth and developmental effects on bulbous flowers (Emami et al. 2011;Han 1995;Janowska 2013;Kim and Miller 2009). However, the effect of these hormones on Ranunculus remains unknown so far.

Here, we applied GA₃ and BA in combination or individually to identify the effects on the growth of eight Ranunculus cultivars; Giallo Millepetali (GM), Bianco Millepetali (BM), Arancio Millepetali AM, Rosa SC (RS), Arancio Pratolino(AP), Giallo Pratolino (GP), Bianco Pratolino (BP), and Rosa Ch Pratolino (RC). Our results showed an overall acceleration in flowering initiation in these cultivars by use of the plant hormones. Moreover, the combination of GA₃ + BA affected other growth factors, including plant height, flowering and flower size and number.

Materials and Methods

The study was conducted in December 2016 at the experimental greenhouse (35°83´ N, 127°03´ E) of the floriculture research division, National Institute of Horticultural and Herbal Science, Republic of Korea. Bulbs of R. asiaticus were obtained from Bioancheri Creations (Camparosso Mare, Italy), and we selected eight cultivars: Giallo Millepetali (GM), Bianco Millepetali (BM), Arancio Millepetali AM, Rosa SC (RS), Arancio Pratolino(AP), Giallo Pratolino (GP), Bianco Pratolino (BP), and Rosa Ch Pratolino (RC) for the experiment. Gibberellic acid (GA₃) and benzyl adenine (BA) were purchased from Sigma-Aldrich Co. (St Louis, MO, US). To evaluate the effects of GA₃ combined with BA on plant growth characteristics, we used different combinations of these hormones: GA₃ 50 mg·L^-1 + BA 100 mg·L^-1 and GA₃ 100 mg·L^-1 + BA 50 mg·L^-1. Also, the two hormones were applied individually at 50 and 100 mg·L^-1 to bulbs by soil drench (50 mL/pot) and the water was used for controls. Eight replicates per treatment were established. After treatment bulbs were transplanted into pots and stored in moist conditions at 7 °C for 2 weeks to induce sprouting and then planted in a greenhouse. Plant height, flower and leaf yield, flower size, days to flowering, and flowering duration were used to measure the growth of R. asiaticus cultivars. Data are expressed as means and standard errors (SE) of each treatment group. Data were statistically analyzed using ANOVA in SAS (version 9.2; SAS Institute, Cary, NC). Differences between treatment groups were considered significant at p ≤ 0.05.

Results and Discussion

Growth characteristics of R. asiaticus cultivars

Flower size and plant height are the important factors to evaluate ornamental quality of bulbous plants. R. asiaticus L., known as the “Turban Buttercup”, has an attractive color and flower size (Bernstein et al. 2005;Beruto and Debergh 2004;Cerveny et al. 2012;Dhooghe et al. 2012), which is an important part of its ornamental value. Especially, the long stem of R. asiaticus has been reported to be useful as the cut-flowers (Bernstein et al. 2005;Shahri and Tahir 2011;Valdes-Aguilar et al. 2009).

We compared eight R. asiaticus cultivars each other to investigate the growth characteristics. GM, BM, AM, and RC cultivars generally showed higher plant height and first flower size than AP, GP, BP, and RC cultivars (Fig 1 and 2). The comparison between R. asiaticus cultivars has not been fully studied. The comparison on growth characteristics between cultivars is required to apply and cultivate various R. asiaticus cultivars. Our results showed growth patterns of R. asiaticus cultivars on the basis of plant height and first flower size for the further research.

Effects of GA₃ and BA

Agronomical techniques for enhancing and accelerating flower growth and development are requisite factors in floriculture. Applications of hormones, such as gibberellic acid (GA₃) and benzyl adenine (BA), produce quantitative and qualitative improvements that are related to cultivating and harvesting in the horticultural industry (Gupta and Chakrabarty 2013;Manimaran et al. 2017;Miller 2012;Yruela 2015). The combination of two hormones has shown positive effects on plant growth, including flowering, in bulb flowers (Emami et al. 2011;Han 1995;Janowska 2013;Kim and Miller 2009). In this study, we used GA₃, BA, and combinations of the two hormones to assess the effects on growth characteristics of R. asiaticus cultivars.

Table 1 showed the correlation analysis of growth and flowering traits of R. asiaticus cultivars by GA₃ and BA. The plant height was significantly affected between cultivars and hormone effects in the correlation analysis (p ≤ 0.001). In detail, effects on growth and flowering traits of R. asiaticus cultivars by GA₃ and BA treatments were indicated in Fig. 1 and 2. Despite some negative effects of plant height of R. asiaticus cultivars, the treatments of GA₃ generally increased the plant heights, especially AP, GP and PM with significant difference (p ≤ 0.05). On the other hand, BA treatments showed positive and negative variations of plant height. Decreases of plant heights were observed in concentrations at BA 100 and 200 mg·L^-1 treatments R. asiaticus cultivars, except for GM and AM, with significant difference (p ≤ 0.05). Ranunculus has been utilized in several forms, including cut, pot, and garden flowers (Bernstein et al. 2005;Cerveny et al. 2012). Plant height, including stalk length of flowers, is one of the major factors of ornamental applications. In general, GA₃ increased plant height, including stem length, in allium, gladiolus, iris, lily, tulip, as well as Ranunculus sp. (Chakraborty and Choudhuri 2016;Dogra et al. 2012;Guney et al. 2016;Khan et al. 2011;Mayoli et al. 2009;Ramzan et al. 2014;Sajid et al. 2009;Sajjad et al. 2015;Sarkar et al. 2014). GA₃ has been applied to Ranunculus sp. in shade treatments to increase flower stem length (Mayoli et al. 2009). The effects of GA₃ on plant length were also observed in R. asiaticus cultivars in this study. In contrast, no significant effects of BA applications on length of bulbous flowers were reported in previous studies (Asgari et al. 2014;Khan et al. 2011;Sajjad et al. 2015). However, the plant height of bulb flowers was enhanced by BA treatments in another study (Sharma et al. 2009). Applications of BA did not affect the stem length of R. asiaticus cultivars in this study.

Growth traits related with the flowering are important to evaluate ornamental values of flowers because these factors affect the floricultural industry. Days to flowering, flower size and number of flowers were significantly affected between cultivars and hormone effects in the correlation analysis (p ≤ 0.05, p ≤ 0.001) (Table 1). We investigated days to flowering, first flower height, and number of flowers from R. asiaticus cultivars by GA₃ and BA treatments (Fig. 1). R. asiaticus cultivars treated with GA₃ generally flowered earlier with significant results (p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001). Conversely, the days to flowering were significantly longer in R. asiaticus cultivars treated with BA at the higher concentration (p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001). GA₃ and BA have been shown to affect days to flowering in flowers of iris, gladiolus, tulip, and Ranunculus sp. (Dogra et al. 2012;Mayoli et al. 2009;Ramzan et al. 2014;Sajjad et al. 2015;Sarkar et al. 2014;Taha 2012). Most studies reported that GA₃ treatments reduced the days to flowering (Dogra et al. 2012;Mayoli et al. 2009;Ramzan et al. 2014;Sajjad et al. 2015;Sarkar et al. 2014;Taha 2012). Our results for GA₃ treatments were similar to those in previous research. Sajjad et al. (2015) showed that BA-treated Gladiolus initiated flowering later, compared with controls. Delayed periods of first flowers of R. asiaticus cultivars were also observed in this study. Overall, GA₃ treatments increased first flower height of R. asiaticus cultivars. In particular, the first flower size was significantly increased by GA₃ treatment at the concentration of 100 and 200 mg·L^-1 compared with the controls (p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001). By contract, the decrease of first flower height was observed by BA treatment in R. asiaticus cultivars. Mayoli et al. (2009) demonstrated that GA₃ increased flower head diameters of Ranunculus sp. Studies of the effects of BA on flower growth demonstrated that bulb flowers showed no significant improvement in terms of floret features (Asgari et al. 2014;Janowska 2014;Pogroszewska 2007;Sajjad et al. 2015). A similar tendency was observed in the R. asiaticus cultivars in this study. The number of flowers was significantly affected by the application concentrations in the correlation analysis (p ≤ 0.001) (Table 1); however it is difficult to find proportional changes in numbers of flowers of R. asiaticus cultivars by treatments of GA₃ and BA at each concentration. Some significant increases of number of flowers were observed in the hormone treatments. In particular, BA 10 mg·L^-1 treatments showed the significant increases of number of flowers in some R. asiaticus cultivars (p ≤ 0.05). The positive effects of GA₃ and BA applications have been reported in bulbous plants, including allium, calla, gradiolus, narcissus, and Ranunculus (Asgari et al. 2014;Mayoli et al. 2009;Pogroszewska 2007;Sarkar et al. 2014; Zanowska et al. 2014). Especially, only GA₃ 100 mg·L^-1 treatment showed the significant increase of number of Ranunculus flowers, except for higher concentrations (Mayoli et al. 2009). The increases of number of flowers by GA₃ and BA treatments were also observed in our research.

Effects of GA₃ + BA

We applied the mixture of GA₃ and BA to evaluate the combinational growth effects on R. asiaticus cultivars (Fig. 3). Some treatments showed positive effects the plant height. The significant increase were detected in combinations of GA₃ + BA at AP, GP, and RC cultivars compared with controls (p ≤ 0.05). Days to flowering was generally reduced by treatments of the mixture of BA and GA₃, except for AM cultivar. The cultivars, BP and RC, showed the decreases of days for flowering. In particular, the combinations of GA₃ 100 mg·L^-1 + BA 50 mg·L^-1 significantly reduced days to flowering of BP cultivar (p ≤ 0.05). The improvements of first flower heights of R. asiaticus cultivars were partly observed in some cultivars such as GM, AM, AP and RC with the significant results (p ≤ 0.05). Overall, combined treatments not significantly showed positive effects of numbers of flowers of R. asiaticus cultivars; however some treatment were affected in RS and RC.

A combination of GA4+7 and BA was shown to elongate stem lengths in tulips (Kim and Miller 2008). This effect was also observed in our study in several cultivars. The combined treatment of GA₃ and BA decreased the days to flowering of R. asiaticus cultivars. Even though there are a limited number of studies of the combined effects of GA₃ and BA, our data may provide sufficient information to control flowering in these plants. The hormone treatments GA₃ + BA partly induced greater flower heights and diameters in this study. A combination of hormones was used to evaluate the growth characteristics of Calla flowers, but showed no positive effects on flower size (Janowska 2013). Even though our results were partly similar to those of Janowska (2013), we observed the positive effects on flower heights and diameters in one R. asiaticus cultivars. The application of using GA₃ plus BA have revealed the higher yield of Calla flowering (Janowska 2013). Even though these are no significant differences in data, the increase in the flower number was observed in the GA₃ plus BA treatments of R. asiaticus cultivars.

In conclusion, we measured growth characteristics of R. asiaticus cultivars treated with individual or combined GA₃ and BA. Each hormone and the mixture of them positively affected the growth and flowering characteristics, such as plant height, flower size, flower yield, and days to flowering in R. asiaticus cultivars. In particular, days to flowering was shortened by treating GA₃. On the other hand, BA treatments delayed days to flowering of R. asiaticus cultivars. Moreover, our results showed the growth characteristics on the cultivars of R. asiaticus. More specific and diverse research is needed to understand the positive role of hormones in plant cultivation. This study contributes towards further research of related bulbous flowers.