Impaginato 81 Adv. Hort. Sci., 2022 36(1): 81­86 DOI: 10.36253/ahsc­10158 Influence of 6­benzylaminopurine spray time after pinching on growth and flow­ ering of Veronica dahurica Steven J.­H. Kim 1, H.J. Oh, S.Y. Kim, G.U. Suh (*) Division of Plant Resources, Korea National Arboretum, 12519 Yangpyeong, Korea. Key words: Cytokinin, foliar application, foliar spray, multiple shoots. Abstract: Veronica dahurica Steven (family Scrophulariaceae) is an ornamental plant from Korea. The aim of the present study was to produce multiple­ branched plants by using 6­benzylaminopurine (BA) spray application at several time intervals after pinching. All 10 cm long plants were sprayed with 0, 500, 1000, and 2000 mg·L­1 BA at 0, 7, and 14 days after pinching. Growth character­ istics were examined 10 weeks after pinching and flowering time was recorded. The number of branches was highest in the group sprayed with 1000 mg·L­1 BA at 0 d after pinching. The greatest plant height was observed in the group treat­ ed with 1000 mg·L­1 BA 14 d after pinching and the minimum plant height was observed in the group sprayed with 500 mg·L­1 BA at 0 d after pinching. The groups sprayed with higher BA concentrations and with longer intervals between pinching and spraying showed greater delay in the time to first flower. The flower length was decreased in the pinched and BA­treated group com­ pared with the control. Thus, BA application and pinching could promote multi­ ple branch induction and control flowering time in V. dahurica Steven. 1. Introduction The genus Veronica L. (family Scrophulariaceae) comprises about 500 species. It is distributed across most of the Northern Hemisphere and in many parts of the Southern Hemisphere and has a wide ecological range, from alpine to coastal vegetation and dry to aquatic vegetation (Albach et al., 2004; Albach et al., 2005). Veronica spp. have the characteristics of graceful and bountiful flowers, long­blooming, easy­care perennials, and they are commercially used in gardens and as cut­flowers (Areal et al., 2008; Hawke, 2010). Veronica dahurica Steven is distributed across East Asia, in Siberia, Far East Russia, China, North and South Korea, and Mongolia (Choi, 2016). The stems of V. dahurica are erect and upright, with simple hairs, and occur individually or in small groups. The plant grows to 30­50 cm in height. The opposing leaflets are 18­25 mm in length and 15­22 mm in width and have a deltoid shape with deeply cut tips on the edge of the leaf. The flowers are racemule, white or pink colored, with short hairs on (*) Corresponding author: arboseed@korea.kr (1) Present address Honam National Institute of Biological Resources, 99, Gohadoan­gil, 58762 Mokpo­si, Korea. Citation: KIM J.­H., OH H.J., KIM S.Y., SUH G.U., 2022 ­ Influence of 6‐benzylaminopurine spray time after pinching on growth and flowering of Veronica dahurica Steven. ­ Adv. Hort. Sci., 36(1): 81­86 Copyright: © 2022 Kim J.­H., Oh H.J., Kim S.Y., Suh G.U. This is an open access, peer reviewed article publi­ shed by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 21 December 2020 Accepted for publication 12 August 2021 AHS Advances in Horticultural Science Short note https://doi.org/10.36253/ahsc-10158 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2022 36(1): 81­86 82 the apical part. The corolla is 5­7.5 mm in length (KPNI, 2019). Multiple branching has commercial advantages for flower production, because it improves yield and quality, controls plant size, and inhibits loathing (Zieslin et al., 1975; Barbier et al., 2017; Kim et al., 2020). Pinching and BA foliar spray are the most use­ ful methods for production of multiple­branched plants (Lee et al., 2006). Pinching is the removal of the apical bud to release the lower axillary buds from apical domi­ nance. Pinching increases branching and stimulates axillary bud development by decreasing auxin pro­ duction in the apical bud and inhibiting lateral bud growth (Barbier et al., 2017). Pinching is commonly used to improve yield by inducing branches, decreas­ ing plant height to produce dwarf plants, and control­ ling flowering time. This method also increases the cytokinin content. Exogenously applied cytokinin, like foliar application, has the same effect as pinching. Plant growth regulators (PGRs), such as cytokinins, play important roles in the control of herbage type and flowering. Cytokinin regulates gynoecium formation and female gametophyte development, embryo development and seed size, pavement cell morphogenesis, axillary bud release, and nutrient uptake. This promotes vascular cambial development, nodulation, chloroplast development, cell division, and phloem development. It also inhibits lateral root formation, senescence, and cell proliferation in the root apical meristem (Kieber and Schaller, 2018). In the present study, the use of foliar sprays of dif­ ferent concentrations of BA and pinching to improve the branching and flowering of V. dahurica are evalu­ ated for commercial use on ornamental plants. In addition, the interaction and use of different growing environments is examined to further manipulate the production of V. dahurica, particularly for use as flowering pot plants. 2. Materials and Methods Plant materials Seeds of V. dahurica were collected from the Useful Plant Resources Center, Korea National Arboretum on 12th September 2018 and stored in a 4°C chamber. V. dahurica seedlings were grown on 128­cell trays filled with a commercial soilless sub­ strate (Baroker; Seoul Bio, Eumseong, Korea) on 7th May 2019, and transplanted into 11.5 cm diameter containers filled with the same substrate on 26th June 2019. The Baroker consisted of 68% coir dust, 15% peat moss, 7% perlite, 6% vermiculite, and 4% zeo­ lite, at pH 5.5­6.0. Two weeks after transplanting, plants were fertilized with 1000 mg·L­1 Peters Professional 20­20­20 (Everris, Geldermalsem, The Netherlands). All plants were grown in a greenhouse. Pinching and BA foliar spray application V. dahurica plants of 10 cm length, with 2­3 nodes and 2 leaves, were selected. All plant materials were pinched at every second internode from the basal stem, and then foliar­sprayed with 10 mL of 0, 500, or 2000 mg·L­1 BA (Duchefa, Haarlem, The Netherlands), using a hand­pump sprayer, at 0, 7, or 14 spraying days after pinching (SDP). The BA solu­ tions were prepared in 10 mL of 99 % ethanol by diluting with distilled water. Growth conditions The experiment was conducted between 5th July 2019 and 13th September 2019. Pinching and BA treatments were applied once at 10 weeks of culture under greenhouse conditions. The mean values of relative humidity and temperature in the greenhouse are shown in figure 1. Irrigation was carried out by drip watering at 500 mL daily in September and twice daily from July to August. Plant growth and flowering The variables considered consisted of height and width of plant, number of nodes, length and width of leaf, number of leaves, lateral branch length, number Fig. 1 ­ Principal component loading pattern of six traits of Amaranthus accessions. Kim et al. ‐ BA foliar spray application effects for V. dahurica Steven 83 of lateral branches and flower characteristics. These parameter were evaluated 10 weeks after treatment. The time to first flowering was recorded. Statistical analysis The experiment was conducted with twelve plantlets per three replicates and three replications per treatment, in a completely randomized block design, unless specified otherwise. Significant differ­ ences were determined using Duncan’s multiple range test (DMRT) with SAS software (Version 9.4; SAS Institute, Cary, NC, USA). 3. Results and Discussion Effects of BA and SDP on growth characteristics V. dahurica was cultivated with 0, 500, 1000, or 2000 mg·L­1 BA foliar spray application at 0, 7, or 14 SDP, and plant growth was evaluated 10 weeks after treatment. BA foliar application groups, which were co­treated with pinching, successfully formed lateral branches, while the effect of pinching treatment alone was similar to that of the control. Maximum lateral branch formation was observed in the group treated with 1000 mg·L­1 BA spray on 0 day after pinching, with a mean of 13.92 secondary branches per plant and a branch production efficiency approxi­ mately 2.93­fold that of the control (Table 1). Both pinching and BA foliar spray application can promote branch production by controlling apical dominance through cytokinin signaling (Barbier et al., 2017; Kieber and Schaller, 2018). BA foliar spray application is recommended to promote branch production in many ornamental plants such as Ardisia pusilla (Lee et al., 2006), Echinacea cultivars (Latimer and Freeborn, 2009), and Kalanchoe species (Currey and Erwin, 2012). Pinching is also recommended for branch production in A. pusilla (Lee et al., 2006), Targets exeta L. (Meena et al., 2015), and Elsholtizia (Sohn and Kim, 2003). However, even when the con­ centration of BA was increased with increasing SDP, the multiple­branch production effect was not greater than that of the treatment immediately after pinching. Lee et al. (2006) reported that 0 to 7 SDP increased branch production in Ardisia pusilla, but that branch production decreased at SDP>7. In our treatments, plant height was similar to or less than that in the control; however, lateral branch length was decreased in all treatment groups compared with that in the control (Table 1, Fig. 2). Leaf charac­ teristics were also investigated in our experiments. Compared with the control, leaf number and leaf width were increased in almost all or all treatment groups, respectively, and leaf length was decreased in almost all treatment groups (Table 1). All treat­ ments showed a decrease in the number of nodes compared with that in the control. Pinching and cytokinin application can be economically used to enhance production of plant stems, flowers, and Table 1 ­ Growth effects of pinching and spray application on V. dahurica 10 weeks after treatment SDP= spraying days after pinching; BA= 6­benzylaminiopurine; z Mean separation within columns by Duncan´s multiple range test at p＜0.05. SDP (days) BA (mg·L­1) Plant No. of nodes (per/plantlet) Leaf Secondary branch Height (cm) Width (cm) Length (cm) Width (cm) Number (per/plantlet) Length (cm) Number (per/plantlet) Control 29.08 abz 38.42 a 14.00 a 6.27 a 3.57 c 177.94 d 29.43 a 4.75 f Pinching 28.69 ab 37.39 a 11.67 bc 6.33 a 3.76 bc 197.17 cb 28.52 a 6.78 ef 0 500 20.65 c 23.87 c 10.53 bc 6.99 a 4.24 ab 298.97 b 12.44 e 11.64 abc 1000 24.86 bc 30.54 b 11.61 bc 7.08 a 4.48 a 407.36 a 15.60 cd 13.920 a 2000 27.25 ab 23.44 c 7.00 d 4.71 b 3.86 bc 175.67 d 18.86 bcd 11.06 abc 7 500 26.59 ab 29.56 b 6.69 d 4.61 b 3.76 bc 215.61 cd 16.47 b 10.59 bcd 1000 24.60 bc 27.68 bc 7.8 d 5.01 b 3.79 bc 191.08 cd 16.11 bc 11.48 bcd 2000 28.22 ab 31.97 b 13.00 ab 4.23 b 3.75 bc 239.28 bcd 12.56 bcd 8.00 de 14 500 24.76 bc 30.57 b 12.28 ab 4.70 b 4.34 ab 256.72 bc 13.37 e 8.92 cde 1000 30.56 a 32.53 b 12.77 ab 4.82 b 4.28 ab 432.14 a 12.86 de 12.86 ab 2000 24.55 bc 30.98 b 11.36 bc 7.04 a 4.13 ab 370.58 a 18.10 bc 12.67 ab Adv. Hort. Sci., 2022 36(1): 81­86 84 seeds by decreasing the planting density and increas­ ing the number of branches. Small dwarf plants were grown as pot plants by reducing the plant height (Table 1, Fig. 2). Effects of BA and SDP on flowering Pinching treatment alone resulted in the highest flowering percentage per plantlet after 10 weeks of culture (86%), followed by BA 2000 mg·L­1 spray application at 0 SDP, BA 1000 mg·L­1 at 0 SDP, BA 1000 mg·L­1 at 7 SDP, BA 1000 mg·L­1 at 14 SDP, BA 500 mg·L­1 at 7 SDP, and the control. There were significant differences in days to first flowering among treatment groups. Pinching delayed the first flowering (24.69 days after pinching) com­ pared with the control (14.14 days after pinching). BA 500 mg·L­1 (0 SDP: 30.94 days/ 14 SDP: 29.17 days) and 1000 mg·L­1 (0 SDP:30.72 days/ 14 SDP: 26.06 days) at 0 SDP and 14 SDP increased the number of days to first flowering compared with the control and BA 0 mg·L­1 with pinching groups. However, other treatments decreased the number of days to first flowering compared with the BA 0 mg·L­1 with pinch­ ing and control groups. In the flower development stage, the first increase in cytokinin occurred in the bud induction stage, followed by a dramatic increase in the transition stage. Exogenous cytokinin treat­ ment can promote floral transition through transcrip­ tional induction of the floral activators TWINSISTER OF FT (TSF), FLOWERING LOCUS D (FD), and SOCI (D’Aloia et al., 2011; Winterhagen et al., 2020). The number of flowers and buds also differed among treatments. The BA 2000 mg·L­1 at 0 SDP treatment produced the highest flower number, fol­ lowed by BA 1000 mg·L­1 at 14 SDP. However, the highest number of buds was observed in the BA 2000 mg·L­1 at 14 SDP treatment group, followed by BA 1000 mg·L­1 at 7 SDP, pinching, and BA 1000 mg·L­1 at 14 SDP (Fig. 2, 3). The observed increases in flower number and buds are due to the increase in branch number and the promotion of flowering by cytokinin (Kumar et al., 2002; D’Aloia et al., 2011). Both the flower length and the peduncle length decreased with BA foliar spray application and pinch­ ing treatments. The effect of the interaction between pinching and BA on the flower length was found to be significant. The smallest flower length (10.49 cm) was recorded with application of BA 1000 mg·L­1 at 7 SDP compared with a flower length of 21.62 cm in the control group. With BA 2000 mg·L­1 treatment at 0 SDP, the peduncle length of V. dahurica Steven was significantly lower than that of the control (Fig. 3). Ferrante et al. (2006) reported that a 500 µM BA spray application reduced inflorescence length in Salvia splendens Kerr Gawl ‘Flamex 2000’. However, the effect of BA on flower size depends on BA concen­ tration, method of application, and plant species (Pobudkiewicz, 2008) and can also affect other vari­ ables such as tepal length, flower diameter, and inflo­ rescence length (Pobudkiewicz and Nowak, 1994; Ferrante et al., 2006; Pobudkiewicz and Treder, 2006). 4. Conclusions The present study revealed the commercial utility of pinching and BA foliar spray application for induc­ tion of multiple branching and control of flowering in V. dahurica. Treatment with 1000 mg·L­1 BA at 0 days after pinching produced the highest branch number after 10 weeks. Pinching delayed the first flowering time compared with that in the control, but some BA foliar applications accelerated the first flowering time. Pinching and BA foliar application also had the effect of reducing flower length and peduncle length. We anticipate that this knowledge will be beneficial for enhancing branching and controlling flowering Fig. 2 ­ Flowering characteristics of V. dahurica with pinching and BA spray application. Kim et al. ‐ BA foliar spray application effects for V. dahurica Steven 85 time and characteristics of V. dahurica in commercial applications. Acknowledgements This study was supported by the ‘Plant Material Selection and New Cultivar Development for Wildflower Industrialization (KNA1­2­32,17­7)’ pro­ ject funded by the Korea National Arboretum. References ALBACH D.C., CHASE M.W., 2004 ­ Incongruence in Veroniceae (Plantaginaceae): evidence from two plas‐ tid and a nuclear region. ­ Mol. Phylogenetics Evol., 32: 183­197. ALBACH D.C., JENSEN S.R., ÖZGÖKCE F., GRAYER R.J., 2005 ­ Veronica: chemical characters for the support of phy‐ logenetic relationships based on nuclear ribosomal and plastid DNA sequence data. ­ Bioch. System. Ecol., 33(11): 1087­1106. AREAL F.J., TOUZA J., MACLEOD A., DEHNEN­SCHMUTZ K., PERRINGS C., PALMIERI M.G., SPENCE N.J., 2008 ­ Integrating drivers influencing the detection of plant pests carried in the international cut flower trade. ­ J. Environ. Manag., 89(4): 300­307. BARBIER F.F., DUN E.A., BEVERIDGE C.A., 2017 ­ Apical dominance. ­ Current Biology, 27(17): 864­865. CHOI K.S., 2016 ­ Phylogeny and biogeography of the Veronica L. subgenus Pseudolysimachium (W.D.J. Koch) Buchenau. ­ Ph.D. Thesis, Yeungnam University, Gyeongsangbuk­do, Korea. CURREY C.J., ERWIN J.E., 2012 ­ Foliar applications of plant growth regulators affect stem elongation and branch‐ Fig. 3 ­ Growth and flowering characteristics in pinching and BA application groups 10 weeks after treatment. A) Control. B) Pinching. C) BA 500 ppm 0 week after pinching. D) BA 1000 ppm 0 week after pinching. ing of 11 kalanchoe species. ­ HortTechnol., 22(3): 338­ 344. D’ALOIA M., BONHOMME D., BOUCHE F., TANSEDDAJ K., ORMENESE S., TORTI S., COUPLAND G., PERILLEUX C., 2011 ­ Cytokinin promotes flowering of Arabidopsis via transcriptional activation of the FT paralogue TSF. ­ Plant J., 65: 972­979. FERRANTE A., MENSUALI­SODI A., SERRA G., TOGNONI F., 2006 ­ Evaluation of postproduction performance of Salvia splendens potted plants for interiors use. ­ Acta Horticulturae, 723: 415­420. HAWKE R., 2010 ­ Comparative studies of Veronica and Veronicastrum. ­ Plant Evaluation Notes, 33. KIEBER J.J., SCHALLER G.E., 2018 ­ Cytokinin signaling in plant development. ­ Develop., 145: dev149344. KIM J.­S., YON G.H., KIM B.G., CHOI J.­S., KIM E.A., KO Y.K., LEE I.Y., 2020 ­ Application of Mevalocidin as a new plant growth regulator for thinning and lateral shoot induction. ­ Weed and Turfgrass Sci., 9(1): 29­42. KPNI, 2019 ­ Korea National Arboretum. Pocheon, Korea. ­ Korean Plant Names Index, http://www.nature.go. kr/kbi/idx/searchIndex.do. KUMAR R., SINGH K., REDDY B.S., 2002 ­ Effect of planting time, photoperiod, GA3 and pinching on carnation. ­ J. Ornam. Hortic. (India), 5(2): 20­23. LATIMER J., FREEBORN J., 2009 ­ New uses of PGRs in orna‐ mentals: configure (6‐BA) increases branching of herba‐ ceous perennials. ­ Proceedings of the 36th Annual Meeting of the Plant Growth Regulation Society of America, pp. 88­93. LEE C.H., KWON O.K., LEE K.S., 2006 ­ Pot plants production of multiple‐branched Ardisia pusilla as influenced by 6‐ benzylaminopurine related to spray time after pinching. ­ Hortic., Environ. Biotech., 47(4): 203­210. MEENA Y., SIROHI H.S., TOMAR B.S., KUMAR S., 2015 ­ Effect of planting time, spacing and pinching on growth and seed yield traits in African marigold (Tagetes erec­ ta) cv. Pusa Narangi Gainda. ­ Indian J. Agric. Sci., content of potted Elsholtzia ciliata and E. splendens. ­ J. Korean Soc. Hortic. Sci., 44(6): 939­946. WINTERHAGEN P., HEGELE M., TIYAYON P., WUNSCHE J.N., 2020 ­ Cytokinin accumulation and flowering gene expression are orchestrated for floral meristem devel‐ opment in longan (Dimocarpus longan Lour.) after chemical flower induction. ­ Scientia Horticulturae, 270: 109467. ZIESLIN N., HURWITZ A., HALEVY A.H., 1975 ­ Flower pro‐ duction and the accumulation and distribution of car‐ bohydrates in different parts of Baccara rose plants as influenced by various pruning and pinching treatments. ­ J. Hortic. Sci., 50(4): 339­348. 86 Adv. Hort. Sci., 2022 36(1): 81­86 85(6): 797­801. POBUDKIEWICZ A., 2008 ­ The influence of growth retar‐ dants and cytokinins on flowering of ornamental plants. ­ Acta Agrobotanica, 61(1): 137­141. POBUDKIEWICZ A., NOWAK J., 1994 ­ The influence of flur‐ primidol on the growth of the CMM dwarf Dianthus caryophyllus L. cv. Snowmass. ‐ J. Fruit Ornam. Plant Res., 2(4): 136­142. POBUDKIEWICZ A., TREDER J., 2006 ­ Effects of flurprimidol and daminozide on growth and flowering of oriental lily ‘Mona Lisa’. ­ Scientia Horticulturae, 110(4): 328­333. SOHN K.H., KIM K.S., 2003 ­ Effect of pinching and short‐ day treatment for height, flowering, and essential oil