Impaginato 375 Adv. Hort. Sci., 2019 33(3): 375­379 DOI: 10.13128/ahs­24026 Direct shoot regeneration of three Petunia cultivars A.N. Vakili 1, H. Bagheri 1 (*), P. Azadi 2 1 Department of Biotechnology, Bu‐Ali Sina University, 6517838695, Hamedan, Iran. 2 Department of Genetic Engineering, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran. Key words: BAP, leaf disk, organogenesis, Petunia hybrida, TDZ. Abstract: A tissue culture system for acquiring high­efficiency regeneration of Petunia was optimized. Leaf explants of Alvan, Large Flower Alvan (LF Alvan) and Mahalat cultivars of Petunia hybrida were cultured separately on MS medi­ um including various concentrations of TDZ and BA without auxin in order to assess direct shoot regeneration. Alvan showed the highest frequency of shoot regeneration (100%) and the highest mean number of shoots per explant (25.33) on MS containing 2 mg/l TDZ. For LF Alvan cultivar the highest percent­ age of shoot organogenesis (100%) and the highest mean number of shoots per explant (18.20) were observed when MS medium containing 1 mg/l BA was used. With the Mahalat cultivar the maximum rate of direct regeneration was obtained on MS supplemented with 0.5 and 1 mg/l BA (80%). The mean num­ ber of shoots per explant (9.63) was obtained when 2 mg/l TDZ was used. Regenerated shoots were successfully elongated (2 to 3 cm in length) and transferred into half­strength MS as the rooting medium supplemented with 0.1 mg/l NAA. The shoots were successfully rooted, acclimatized and transferred to the greenhouse. 1. Introduction Petunia (Petunia hybrida) is well known as an economically important ornamental plant and is grown worldwide for its beautiful and fragrant flowers. Propagation techniques with modern approaches intend to give a hand to scientists to provide demands of ornamental industry (Rout et al., 2006). An efficient plant regeneration system is necessary for the success­ ful genetic transformation (Ntui et al., 2010). There are several reports for in vitro shoot regeneration of Petunia hybrida species from several explants including leaf (Preece, 2000; Ntui et al., 2010; Abu­Qaoud et al., 2010; Khan et al., 2011; Abu­Qaoud, 2012; Burbulis et al., 2015), somatic cells (Rao et al., 1973) cotyledon (Dulien, 1991), embryo (Dimasi­Theriou et al., 1993), protoplast (Auer et al., 1992; Auer et al., 1999; Abu­Qaoud et al., 2010), petal (Razdan, 2003), and microspore (Li et al., 2013). Various factors could affect organogenesis in P. hybrida such as light (Reuveni and Evenor, 2007), sugar and CO2 (Qu et al., 2007), ethylene (*) Corresponding author: bagheri.hedayat@gmail.com Citation: VAKILI A.N., BAGHERI H., AZADI P., 2019 ­ Direct shoot regeneration of three Petunia cultivars. ­ Adv. Hort. Sci., 33(3): 375­379 Copyright: © 2019 Vakili A.N., Bagheri H., Azadi P. This is an open access, peer reviewed article published 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 1 October 2018 Accepted for publication 23 April 2019 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2019 33(3): 375­379 376 (Dimasi­Theriou et al., 1993), nitrogen and calcium (Frett and Dirr, 1996) and also hormonal combina­ tions (Ying et al., 2005; Xiao­Feng et al., 2009; Xian­ Chun, 2010) Petunia regeneration happens directly and indirectly by combinations of auxins and cytokinins in medium culture (Michalczuk and Michalczuk, 2000; Ziv et al., 2005). Adventitious bud formation from somatic cells of P. hybrida was induced by exogenous cytokinins such as BA (6­benzyladenine), Zeatin, Kinetin and TDZ (Thidiazuron) (Rao et al., 1973; Thirukkumaran et al., 2009). It is reported that TDZ acted different from traditional cytokinins and was able to accomplish both the cytokinin and auxin requirements of differ­ ent plant species for regeneration (Murthy et al., 1998; Sanikhani et al., 2006). The highest frequency of direct shoot organogenesis of Daady Blue and White Dreams cultivars of P. hybrida was obtained on MS medium supplemented with different concentra­ tion of TDZ (Abu­Qaoud, 2012). Also, TDZ alone pro­ vided the highest percentage of shoot organogenesis and mean number of shoot per explant of P. hybrida cv. Mitchell (Thirukkumaran et al., 2009). It is also reported that exogenous cytokinin especially BA could control the commitment of Petunia leaf explants to induce shoots in tissue culture (Auer et al., 1992; Abu­Qaoud et al., 2010). Therefore in this study, we investigated the effect of TDZ and BA as well as genotype on direct shoot regeneration of three Petunia cultivars. This efficient regeneration system is very useful in genetic transformation pro­ jects of P. hybrida. 2. Materials and Methods Seed germination Seeds of three local cultivars of Petunia, Alvan, Large Flower Alvan (LF Alvan) and Mahalat, were sterilized with 70% ethanol for 30s, and sodium hypochlorite solution 1% for 10 minutes. They rinsed 3 times with sterilized water and cultured on MS medium. Seeds were grown under 25 ± 2 °C with 16/8 hour photoperiod, under fluorescent illumina­ tions (40 μmol m­2s­1). Organogenesis The newly formed leaves were cut 6­8 mm in length, and then cultivated on 5 modified MSmedia: MS medium without hormones (MS1), MS + 0.5 mg/l BA (MS2) [Sigma­Aldrich, Steinheim, Germany], MS + 1 mg/l BA (MS3), MS + 1 mg/l TDZ (MS4) [Sigma­ Aldrich, Steinheim, Germany] and MS + 2 mg/l TDZ (MS5). Abu­Qaoud et al., (2010) got more regenera­ tion when they used 0.8 mg/l BA. Therefore we selected 0, 0.5 and 1 mg/l BA to better estimate BA effect. Also as Thirukkumaran et al., (2009) reported more regeneration with 2 mg/l TDZ, we selected 0, 1 and 2 mg/l TDZ to investigate its effect. Moreover, the MS was supplemented with 30 g/l sucrose and solidified with 7 g/l agar [Duchefa, Haarlem and The Netherlands]. The optimum pH of all culture media was considered 5.8 which adjusted with 1N NaOH before sterilization. Then all media were sterilized using autoclave at 121°C for 20 min. Explants were placed on regeneration medium with the adaxial side upward. The cultures were incubated at 25±2°C, with a light to dark period of 16/8 hours under cool­white fluorescent light at 40 μmol m­2 s­1. Explants were sub­cultured every two weeks. They were investigat­ ed using binocular Stereo Microscope, regarding to the mean number of explants inducing shoots and the mean number of induced shoots and buds per explants after 4­5 weeks on regeneration medium. Rooting and acclimatizing Regenerated shoots were transferred into half­ strength MS supplemented with 30 g/l sucrose, 0.1 mg/l NAA [Duchefa, Haarlem, and The Netherlands] and solidified with 7 g/l agar. The rooted plantlets rinsed under tap water and planted on the plastic pots with combination of sterile peat moss and per­ lite mixture (2:1). They kept in greenhouse condi­ tions. Statistical analysis The experiment was done based on completely randomized design with three replications and 10 leaf explants in each replication. Data were normal­ ized through arcsin (√x) and (√x+0.5) transformation in SPSS. The normalized data were analyzed using SAS statistical analysis package and were compared via Duncan’s multiple range test at P ≤ 0.01 and P ≤ 0.05. 3. Results Effect of BA on organogenesis Direct shoot formation was obtained in all three cultivars after 4­5 weeks. No regeneration occurred on MS1 medium which means hormones are neces­ sary to induce shooting (Tables 1, 2). When 0.5 mg/l BA (MS2) was used no differences in frequency of regeneration was observed among cultivars. The low Vakili et al. ‐ Regeneration of three Petunia cultivars 377 mean numbers of shoot per explant (5.05 and 6.21) were observed in MS2 for Mahalat and Alvan culti­ vars, respectively. When 1 mg/l BA (MS3) was used differences were observed in all three cultivars and LF Alvan cultivar showed 100% shoot regeneration (Table 2), with a mean number of 18.20 shoots per explants (Fig. 1 a). Effect of TDZ on organogenesis Significant differences were observed among three cultivars when TDZ concentration was increased (Tables 3, 4). The low shoot regeneration frequency was obtained on MS4 and MS5 media for Mahalat cultivar (Table 4). Alvan cultivar showed the highest percentage of shoot regeneration (100%) and mean number of shoots per explant (25.33) on MS with 2 mg/l TDZ (Fig. 1 b) and the lowest one (6.61) was belong to Mahalat cultivar on MS with 1 mg/l TDZ. 4. Discussion and Conclusions We could show that auxin is not necessary for direct shoot regeneration of three cultivars of P. hybrida. It is already reported that the number of shoot per explants dramatically increased when explants exposed to the medium containing BA (Auer et al., 1992). The highest shoot regeneration rate (45%) and the maximum average number of shoots per explant (7.5) from Petunia leaf explants on MS with 2 mg/l BA + 0.5 mg/l NAA has also been report­ ed (Abu­Qaoud et al., 2010). In the current study the highest shoot regeneration frequency in Alvan culti­ var and the mean number of shoots per explant in both Alvan and Mahalat cultivars were observed when 2 mg/l TDZ was used which is in conformity with Thirukkumaran et al. (2009). The importance of TDZ on regeneration and shoot induction frequency Table 1 ­ Effect of modified MS medium supplemented with different concentration of BA on shoot regeneration from leaf explants of P. hybrid The values represent the mean ± standard error of three replicates. Different letters are showing considerable differences at P≤0.05. Fig. 1 Plant regeneration from leaf explants of different culti­ vars of Petunia hybrida. (a) Direct shoot regeneration of LF Alvan on MS + 1 mg/l BA (bar: 2 mm); (b) Direct shoot regeneration of Alvan on MS + 2 mg/l TDZ (bar: 4 mm); (c) Root formation after 2 weeks on rooting media (bar: 5 mm); (d) A 4 weeks old plantlet after transfer to the pot (bar: 1 cm). MS Media Frequency of regeneration The mean number of shoots per explant Cultivars Cultivars MS1 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00 ± 0.00 e 0.00 ± 0.00 e 0.00 ± 0.00 e MS2 83.33 ± 2.8 b 83.33 ± 1.8 b 80.00 ± 3.1 b 6.21 ± 0.12 d 13.31 ± 0.85 b 5.05 ± 1.00 d MS3 80.00 ± 3.7 b 100.00 ±0.00 a 80.00 ± 1.1 b 10.12 ± 0.41 bc 18.20 ± 0.85 a 7.76 ± 0.56 cd Table 2 ­ Effect of modified MS medium supplemented with different concentration of TDZ on shoot regeneration from leaf explants of P. hybrid The values represent the mean ± standard error of three replicates. Different letters are showing considerable differents at P≤0.05. MS Media Frequency of regeneration The mean number of shoots and buds per explant Cultivars Cultivars Alvan LF Alvan Mahalat Alvan LF Alvan Mahalat MS1 0.00 ± 0.00 d 0.00 ± 0.00 d 0.00 ± 0.00 d 0.00 ± 0.00 f 0.00 ± 0.00 f 0.00 ± 0.00 f MS2 83.33 ± 3.4 b 80.00 ± 1.0 b 66.66 ± 2.1 c 16.25 ± 1.00 b 12.00 ± 1.21 c 6.61 ± 0.08 e MS3 100.00 ± 0.00 a 83.33 ± 0.8 b 70.00 ± 1.7 c 25.33 ± 1.02 a 14.31 ± 0.96 bc 9.63.00 ± 0.11 d Adv. Hort. Sci., 2019 33(3): 375­379 378 and the mean number of shoots per explant was also investigated in Daddy blue and Dreams white geno­ types (Abu­Qaoud, 2012). This study showed that a cytokinin source of TDZ or BA may be enough for direct shoot regeneration of three mentioned culti­ vars of P. hybrida. Application of TDZ instead of both auxin and cytokinin requirements for organogenesis in the wide range of plant species has been support­ ed (Murthy et al., 1998). Probably TDZ tends to make balance among endogenous growth regulators that is essential for inducing specific modes of regeneration. It was found that many factors such as genotype and exogenous growth regulators have the capability to influence on biochemical pathways controlling the endogenous cytokinin content (Krikorian, 1995). In the present study a significant difference in regenera­ tion frequency was observed among studied cultivars probably due to the different level of endogenous hormones. For LF Alvan cultivar, the maximum regen­ eration frequency (100%) and the highest number of shoots per explants (18.20) were obtained when BA concentration was increased from 0.5 to 1 mg/l while the other two cultivars showed less reaction. These findings confirm the report of Jamshidnia and Sayed Tabatabaei (2013), and Burbulis et al., (2015) on dif­ ferences in shoot regeneration frequency among three different genotypes of Petunia. Here we report an efficient direct shoot regeneration system in Petunia hybrida using leaf explants of Alvan cultivar. This cultivar can be considered as a suitable cultivar for transformation experiments. To conclude, the present study provided an effi­ cient direct shoot regeneration system without auxin in Petunia using leaf explants that could be improve transformation studies. Acknowledgements This work was supported by Bu­Ali Sina University, Hamedan, Iran, and Novin Giti Gene Biotech. Co. Biotechnology Incubator Center of National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran. References ABU­QAOUD H., 2012 ­ Improving adventitious shoot regeneration from cultured leaf explants of Petunia hybrida using thidiazuron. ­ Afr. J. Biotechnol., 11(51): 11230­11235. Table 3 ­ Analysis of variance of different concentrations of TDZ on shoot regeneration of P. Hybrida *, **, significant at 5% and 1% levels, respectively. Table 4 ­ Effect of TDZ on shoot regeneration of P. Hybrida using Duncan’s multiple range test MS1= MS medium without hormones, MS4= MS + 1 mg/l TDZ; MS5= MS + 2 mg/l TDZ. Means compared using Duncan’s multiple range test. The Values represent the mean ± standard error of three replicates. Different letters are showing considerable differents at P≤ 0.05. Source of Variation DF Mean squares P­value Frequency of regeneration Mean number of shoots per explant Frequency of regeneration Mean number of shoots per explant TDZ 2 3.1897 ** 11.4806 ** 0.000 0.000 Cultivar 2 1.3250** 6.5896 ** 0.009 0.004 TDZ × Cultivar 4 0. 8015* 4.2010 ** 0.022 0.009 Error 18 0. 215 0.9080 Total 26 MS Media Frequency of regeneration The mean number of shoots and buds per explant Cultivars Cultivars Alvan LF Alvan Mahalat Alvan LF Alvan Mahalat MS1 0.00 ± 0.00 d 0.00 ± 0.00 d 0.00 ± 0.00 d 0.00 ± 0.00 f 0.00 ± 0.00 f 0.00 ± 0.00 f MS4 83.33 ± 3.4 b 80.00 ± 1.0 b 66.66 ± 2.1 c 16.25 ± 1.00 b 12.00 ± 1.21 c 6.61 ± 0.08 e MS5 100.00 ± 0.00 a 83.33 ± 0.8 b 70.00 ± 1.7 c 25.33 ± 1.02 a 14.31 ± 0.96 bc 9.63 ± 0.11 d Vakili et al. ‐ Regeneration of three Petunia cultivars 379 NTUI V.O., AZADI P., SUPAPORN H., MII M., 2010 ‐ Plant regeneration from stem segment‐derived friable callus of “Fonio” (Digitaria exilis (L.) Stapf.). ­ Sci. Hortic., 125: 494­499. PREECE J.E., 2000 ­ Shoot organogenesis from petunia leaves, pp. 167­175. ­ In: TRIGIANO R.N. (ed.) Plant tis‐ sue culture concepts and laboratory exercises. CRC Press, Boca Raton, FL, USA, pp. 472. QU Y.H., LIN C., ZHOU W., LI Y., CHEN B., CHEN G.Q., 2007 ­ Effect of CO2 concentration and moisture content of sugar‐free media on the tissue cultured plantlets in a large growth chamber. ­ Commun. Nonlinear Sci., 14: 322­330. RAO P.S., HANDRO W., HARADA H., 1973 ­ Hormonal con‐ trol of differentiation of shoots, roots and embryos in leaf and stem cultures of Petunia inflata and Petunia hybrida. ­ Physiol Plant., 28: 458­463. RAZDAN M.K., 2003 ­ Introduction to plant tissue culture. Science 170. Regeneration in vitro by ethylene. ­ Plant Cell Tissue Organ Cult., 32: 219­225. REUVENI M., EVENOR D., 2007 ­ On the effect of light on shoot regeneration in petunia. ­ Plant Cell Tissue Organ Cult., 89: 49­54. ROUT G.R., MOHAPATRA A., JAIN S.M., 2006 ­ Tissue cul‐ ture of ornamental pot plant. A critical review on pre‐ sent scenario and future prospects. ­ Biotechnol. Adv., 24: 531­560. SANIKHANI M., FRELLO S., SEREK M., 2006 ­ TDZ induces shoot regeneration in various Kalanchoe blossfeldiana Poelln. cultivars in the absence of auxin. ­ Plant Cell Tissue Organ Cult., 85: 75­82. THIRUKKUMARAN G., NTUNI V.O., KHAN R.S., MII M., 2009 ­ Thidiazuron: an efficient plant growth regulator for enhancing Agrobacterium‐mediated transformation in Petunia hybrida. ­ Plant Cell Tissue Organ Cult., 99: 109­115. XIAN­CHUN Z., 2010 ­ Effect of the plant hormone ratio on tissue culture of Petunia hybrida. ­ J. Anhui Agric. Sci., 17: 17­20. XIAO­FENG F., GUO­DONG Z., JUN­QUAN X., 2009 ­ Study on callus induction and plant regeneration of Petunia hybrida. ­ Northern Hort., n. 06. YING Z., FENG­XIA L., HUIMING Z., LI Z., 2005 ­ Tissue cul‐ ture regeneration system of three cultivars of fragrant Petunia. ­ J. Shenyang Agric. Univ., n. 04. ZIV M., GANDELMAN M., GERA A., 2005 ­ Expression of viral resistance in transformed petunia plants regener‐ ated in vitro. ­ Acta Horticulturae, 683: 243­247. ABU­QAOUD H., ABU­RAYYA A., SAMI Y., 2010 ­ In vitro regeneration and somaclonal variation of Petunia hybrida. ­ J. Fruit Ornam. Plant Res., 18(1): 71­81. AUER C.A., LALOUE M., COHEN J.D., COOKE T.J., 1992 ­ Uptake and metabolism of benzyladenine during shoot organogenesis in Petunia leaf explants. ­ J. Plant Growth Regul., 11: 105­114. AUER C.A., MOTYKA V., BREZINOVA A., KAMINEK M., 1999 ­ Endogenous cytokinins accumulation and cytokinins oxidase activity during shoot organogenesis of Petunia hybrida. ­ Physiol. Plant., 105: 141­147. BURBULIS N., BLINSTRUBIENE A., JONYTIENE V., 2015 ­ In vitro regeneration from leaf explants of Petunia hybri­ da L. ­ Propag. Ornam. Plants, 15(2): 47­52. DIMASI­THERIOU K., EONOMOU A.S., SFAKIOTAKIS E.M., 1993 ­ Promotion of petunia (Petunia hybrida L.) regen‐ eration in vitro by ethylene. ­ Plant Cell Tiss. Organ Cult., 32: 219­225. DULIEU H., 1991 ­ Inheritance of the regeneration capacity in the genus Petunia. ­ Euphytica, 53: 173­181. FRETT J.J., DIRR M.A., 1986 ­ Effect of nitrogen and calci‐ um stock plant nutrition on Petunia x hybrida leaf and anther explant growth in vitro. ­ Sci. Hort., 28: 289­ 298. JAMSHIDNIA M., SAYED TABATABAEI B.E., 2013 ­ Callus induction and regeneration from shoot apex and leaf disc cultures of three commercial petunias. ­ Adv. Crop Sci., 3: 444­453. KHAN R.S., ALAM S., IQBAL M., AZADI P., NAKAMURA I., MII M., 2011 ­ Botrytis cinerea‐resistant marker‐free Petunia hybrida produced using the MAT vector sys‐ tem. ‐ Plant Cell Tissue Organ Cult., 106: 11­20. KRIKORIAN A.D., 1995 ­ Hormones in tissue culture and micropropagation, pp. 774­796. ­ In: DAVIES P.J. (ed.) Plant hormones: physiology, biochemistry and molecu‐ lar biology. Kluwer Academic Publishers, pp. 833. LI F., LI C., LI M., YU M., FANG C., WANG S., 2013 ­ In vitro culture of Petunia hybrida microspores and agrobac‐ terium‐mediated transient expression of β‐glu‐ curonidase (GUS) reporter gene. ­ Int. J. Agric. Biol., 15: 1098­1104. MICHALCZUK B., MICHALCZUK L., 2000 ­ The effect of light quality on regeneration rate and plantlet development in transgenic petunia ‘Revolution’ (Surfinia type). ­ Acta Horticulturae, 530: 397­401. MURTHY B.N.S., MURCH S.J., SAXENA P.K., 1998 ­ Thidiazuron: a potent regulator of plant morphogene‐ sis. ­ In Vitro Cell Dev. Biol. Plant., 34: 267­275.