Acta agriculturae Slovenica, 118/4, 1–7, Ljubljana 2022 doi:10.14720/aas.2022.118.4.1723 Original research article / izvirni znanstveni članek In vitro direct and indirect regeneration of plants from nodal and petiole explants in Pelargonium odoratissimum (L.) Herit. Asghar EBRAHIMZADEH 1, 2, Maliheh FATHOLLAHZADEH 1, Mohammad Ali AAZAMI 1, Mohammad Bagher HASSANPOURAGHDAM 1 Received June 09, 2020; accepted November 03, 2022. Delo je prispelo 9. junija 2020, sprejeto 3. novembra 2022 1 Department of Horticultural Science, University of Maragheh, Maragheh, Iran 2 Corresponding author, e-mail: acebrahimzadeh@gmail.com In vitro direct and indirect regeneration of plants from nodal and petiole explants in Pelargonium odoratissimum (L.) Herit. Abstract: Nodal and petiole explants were employed to study the direct and indirect regeneration from Pelargonium odoratissimum in vitro. Direct shoots regeneration of nodal segments was tried in MS medium containing 1 and 2 mg l-1 BAP and 0.5 mg l-1 IBA. The highest mean shoots number and the greatest shoots per explant number were obtained in the medium containing 2 mg l-1 BAP. Nodal segments were the source of petiole explants and the resulting petioles were cul- tured in ½ MS medium supplemented by 1, 1.5, 2 and 4.5 mg l-1 BAP enriched with 0.1, 1 and 1.5 mg l-1 NAA. With the peti- ole explants, the lowest browning percentage, the highest cal- lus induction and also, the top number of shoots per explant were recorded in 2 mg l-1 BAP + 0.1 mg l-1 NAA medium. The medium supplemented with 0.2 mg l-1 NAA exhibited the de- sired effect on rooting percentage and mean root number and length. The rooted young plants were transferred to the pots containing peat-moss and perlite (1:1) and the acclimatization was successful since, more than 90 % of plants adapted-well in the greenhouse conditions. This in-vitro propagation method- ology would be advisable to the plant production systems and to whom wish to produce the clonal homogenous plants for the commercial ideas and for the detailed molecular studies. Key words: callus induction; MS medium; nodal seg- ments; Pelargonium odoratissimum; shoot regeneration Neposredna in posredna in vitro regeneracija rastlin iz izseč- kov nodijev in listnih pecljev pri muškatki (Pelargonium odo- ratissimum (L.) L Herit) Izvleček: V raziskavi so bili uporabljeni izsečki nodijev in listnih pecljev za preučevanje neposredne in posredne vzgoje rastlin muškatke (Pelargonium odoratissimum (L.) L ̍Hér.) in vitro. Neposredna tvorba poganjkov iz izsečkov nodijev je bile preiskušena na MS gojišču, ki je vsebovalo 1 in 2 mg l-1 BAP in 0,5 mg l-1 IBA. Največje poprečno število poganjkov in največje število poganjkov na izseček je nastalo v gojišču, ki je vsebovalo 2 mg l-1 BAP. Nodiji so bili vir izsečkov listnih pecljev, ki so bili gojeni na polovičnem MS gojišču z dodatkom 1, 1.5, 2 in 4,5 mg l-1 BAP, obogatenega z 0,1, 1 in 1,5 mg l-1 NAA. Z uporabo izsečkov listnih pecljev je bil dosežen najmanjši odstotek por- javitev, najboljša indukcija kalusa in največje število poganjkov pri dodatku 2 mg l-1 BAP + 0,1 mg l-1 NAA v gojišče. Gojišče, kateremu je bil dodano 0,2 mg l-1 NAA je pokazalo zaželjen odstotek ukoreninjenja in največje poprečno število in dolžino korenin. Vkoreninjene mlade rastline so bile posajene v lončke z mešanico šotnega mahu in perlita (1:1). Aklimatizacija je bila uspešna, saj se je več kot 90 % rastlin uspešno prilagodilo raz- meram v rastlinjaku. Takšno metodologijo in vitro razmnože- vanja rastlin bi bilo primerno uvesti v sisteme razmnoževanja, v katerih želimo vzgojiti homogene klonske rastline za komer- cialne namene in podrobnejše molekularne raziskave. Ključne besede: indukcija kalusa; MS gojišče; nodijski iz- sečki; Pelargonium odoratissimum; regeneracija poganjkov Acta agriculturae Slovenica, 118/4 – 20222 A. EBRAHIMZADEH et al. 1 INTRODUCTION Pelargonium odoratissimum is an important aro- matic plant belonging to the Geraniaceae family and is in common growth in the tropics and sub-tropics (Ghanem et al. 2008). This is a perennial herbaceous plant and, is propagated by the semi-hard wood stem cuttings. How- ever, the success rate with this propagation method is quite limited. The oil extracted from the above ground parts of the plant is in great need with the cosmetic, fra- grance and hygienic industries and preparations from the plant have remarkable antimicrobial, antifungal and insecticidal activities (Ghanem et al. 2008; Gupta et al. 2002). Plants regeneration and propagation by the in vitro culture methods have been defined as reliable ways to the breeding and the economical production of medicinal plants. Shoot regeneration with both the direct and indi- rect ways were experienced to have enough plant materi- als with the traits of interest. The need for the disease-free plants besides mass-production have been the main ideas to resort to in vitro propagation of Pelargonium odorat- issimum. However, there is no universal protocol suit- able for Pelargonium spp in vitro propagation (Wojtania et al. 2004). In the in vitro conditions, regeneration rate is dependent upon genotype, explants type, media com- ponents, plant growth regulators (PGRs), light intensity and quality and, the interaction of these factors (Arshad et al. 2012). In vitro direct and indirect regeneration of Pelargonium graveolens L’Hér. has been reported by leaf and nodal explants (Satyakala et al. 1995; Saxena et al. 2000) as well as shoots formation from calli samples de- rived from the leaf sections of ‘Bipuli’ cultivar (Gupta et al., 2002). With Pelargoniun radula L Herit micropropaga- tion (Zuraida et al. 2013), the utmost shoots regenera- tion rate was acquired for nodal explants in a medium containing 0.5  mg l-1 BAP+1  mg l-1 IBA. Saxena et al. (2000) were successful to obtain the highest shoots num- ber per leaf explants in a medium enriched with 5  mg l-1 Kinetin+1  mg/L NAA. For the nodal segments, the best results were acquired from the combination of 8 mg l-1 Kinetin +1 mg l-1 NAA. Krishna Raj et al. (1997) re- ported that somatic embryogenesis of P. odoratissimum ‘Frensham’ cultivar was successful with the young leaf petioles. Hammerchlag and Bottino (1981) noted that callogenesis from the young seedlings of nodal segments was dependent upon the phenological stage of growth and the younger seedlings produced the highest num- ber of shoots in vitro. Adventitious shoots regeneration from the petiole explants of Pelargonium × hederaefolium ‘Bonette’ cultivar was stimulated by the high amounts (9-18 µM) of thidiazoron and the related embryogenesis and organogenesis crosstalk was studied in detail. Rooting behavior in vitro is another criterion in plants micropropagation which is mainly dependent on auxin and cytokinin equilibrium with the dominancy of auxin type PGRs (Zuraida et al., 2013). Rooting in P. gra- veolens and P. radiattum (Andrews) Pers was the best in half strength MS medium with 0.1 mg l-1 NAA (Zuraida et al., 2013). In spite of great attempts on Pelargonium species in vitro cultures; there are scattered information on P. odoratissimum propagation methods in vitro. The improvements in tissue culture method with the Pelar- gonium species would be the basal steps in the enhanced production of high valued secondary metabolites from these species. Moreover, the in vitro cultural methods will assist molecular biologists and biotechnologists to manipulate the species for the optimized production of the desired constituents. The present experiment was planned to study the potential direct and indirect regen- eration capability of nodal and petiole explants of P. odo- ratissimum in vitro. This methodology would be possibly a reliable procedure for the commercial in vitro produc- tion of this high-valued crop for the coming molecular and breeding programs. 2 MATERIAL AND METHODS 2.1 PLANT MATERIAL AND STERILIZATION Nodal explants were taken from mother plants grown under a reference university research greenhouse in Maragheh, Iran. The homogenous mother pot plants (2 years old) available in our greenhouse were employed for sampling of the uniform nodal section of about 5 to 8 mm in diameter from the middle part of shoots. 30 nodal segments (3 treatments and 10 replications per treatment) were rinsed in running water for 45 minutes. Superficial sterilization of nodal cuttings was completed in a laminar-airflow cabinet as followed: immersion in 70 % ethanol for one minute, followed by sodium hypochlo- rite, 15 % containing 2 drops of Triton X-100 for 13 min- utes. Subsequently, the explants were rinsed three-times in distilled water and treated with 70 % alcohol for 20 seconds. Finally, the samples were placed on sterile paper to get dried. The dried sterile plant material was cut into 0.5-1 cm segments and transferred to MS medium. Peti- ole explants were acquired from the shoots grown from nodal segments in vitro. The sterile petiole samples were divided into 0.5 cm explants. Acta agriculturae Slovenica, 118/4 – 2022 3 In vitro direct and indirect regeneration of plants from nodal and petiole explants in Pelargonium odoratissimum ... 2.2 REGENERATION The basal medium was MS supplemented with 3 % sucrose and 0.7 % agar along with 500 mg l-1 myoinositol. For some instances, we used also ½ MS medium. Nodal segments were cultured in MS medium supplemented by BAP (1 and 2 mg l-1) and IBA (0.5 mg l-1). Petiole explants were cultured in ½ strength MS medium containing BAP (1, 1.5, 2 and 4.5 mg l-1) and NAA (0.1, 1, 1.5 mg l-1). To prevent tissue browning, 50 mg l-1 citric acid was added to the medium. Three explants were placed in every 9 cm petri-dish. The cultures were placed under dark con- ditions in a growth chamber for two weeks under 16:8 hours photoperiod regime at 23 ± 1 ºC and 20 ± 1 ºC. 2.3 PROLIFERATION STAGE Two weeks after the cultures establishment; the nodal segments were subcultured in the MS basal me- dium with the initial treatment combinations. Three suc- cessive subcultures were done at two week intervals. The number and length of shoots were recorded twice per week. In the second phase; and 4 weeks after establish- ment of the petiole explants, the tender creamy colored calli were developed. Callus formation percentage was measured for each sample. Calli were subcultured on the ½ MS medium with the initial treatment combina- tions as well. Two weeks after calli were sub-cultured; the shoots were produced. Later, the number and length of the shoots were measured. Petiole explants were taken from the shoots produced from the nodal segments in vitro. For the indirect regeneration stage, the derived calli were cultured on the initial ½ MS medium. 2.4 ROOTING The shoots derived from the proliferation stage were cut from the distal ends and were cultured on ½ MS me- dium containing 0.1 and 0.2 mg l-1 NAA combined with 2  g l-1 activated charcoal. Rooting percentage and the number and length of the roots were recorded about ten days after transferring to the rooting medium. 2.5 ACCLIMATIZATION OF THE NEWLY- FORMED SEEDLINGS Warm (50-60 ºC) water was used to remove the agar debris from the young roots and, the plantlets were trans- ferred to the pots containing peat moss: perlite (1:1) and were placed in a growth chamber under 16:8 photoperi- od regime and 23±1 ºC and 20±1 ºC temperature regime during the day and night, respectively. 2.6 STATISTICAL ANALYSIS Experimental design for the nodal segments was arranged with 3 treatments and 10 replications in a completely randomized design (CRD) and for petiole explants with 6 treatments and 5 replications with 3 ex- perimental units as factorial experiment based on CRD. Each experiment was repeated at least twice and the re- ported data are the means of two experiments. Data were analyzed by SAS (version 9) and mean comparisons were done by LSD (Least Significant Difference) test at the 0.05 probability level. 3 RESULTS 3.1 DIRECT SHOOT REGENERATION Two weeks after the explant cultures; the shoots were visible on nodal explants. For the multiplication/ proliferation, shoots were cultured with the initial treat- ments. Treatments and growth regulators concentration and combinations had inevitable effects on the growth and developmental response of plant samples. Mean comparisons revealed that 2  mg l-1 BAP+0.5 mg l-1 IBA and 2 mg l-1 BAP (alone) had sig- nificant effects on reducing the browning percentage in samples (Figure 2). With the nodal section explants, the proximal ends were responded to the media and, we ob- served the browning disorder. BAP proved to be the desirable compound for ini- tiating the shoot growth in terms of total shoot number per explant and mean shoot number in nodal segments of Pelargonium odoratissimum (Figure 1) Calli were produced on ½ MS medium containing different combinations of BAP and NAA. Calli were in- duced from the explants during 25 to 30 days under dark conditions. All treatment combinations (NAA+BAP) had positive effects on the calli production from petiole explants. Mean comparisons revealed that 1.5 and 2 mg l-1 BAP+0.1 and 1 mg l-1 NAA had the highest callus in- duction potential (Table 1). In the present experiment, the explant browning was overcome with using 50 mg l-1 of citric acid in ½ MS medium. Our results shows that the lowest browning rate in petiole explants was achieved in the medium containing 2 mg l-1 BAP+0.1 mg l-1 NAA (Table 1). The results revealed that the survival rate and re- generation potential in petiole explants were related to Acta agriculturae Slovenica, 118/4 – 20224 A. EBRAHIMZADEH et al. the ratios and amount of plant growth regulators applied. Two weeks after subculture, the shoots were developed. The result showed that the highest number and length of the shoots was obtained in the medium containing 2 mg l-1 BAP+0.1 mg l-1 NAA (Table 1). The greatest proliferation rate with nodal explants was recorded in the medium containing 2 mg/L BAP and with petiole explant in the medium supplemented by 2 mg l-1 BAP+0.1 mg l-1 NAA (Figure 2 & Table 1) 3.2 ROOTING AND ACCLIMATIZATION The shoots derived from the nodal and petiole ex- plants were cultured in ½ MS medium enriched with 0.1 and 0.2 mg l-1 NAA plus 2 g l-1 active charcoal. Figure 3 shows that the highest rooting percentage was observed in 0.2 mg l-1 NAA. 0.2 mg l-1 NAA was the concentration of choice for both root number and rooting percentage as well. Furthermore, mean comparisons revealed that, for the root number, there was a difference between NAA concentrations, and explant type. So that, with 0.2  mg l-1 NAA, the top root number was belonged to the nodal explants (Figure 4). The results in figure 5 shows that with both nodal and petiole explants; 0.2 mg l-1 NAA had more root length. Eventually, the intact rooted plantlets were selected for the acclimatization stage. The plantlets were trans- Callogenesis (%) Browning percentage (%) Mean shoots number Mean shoots length (cm)Treatment combination 45.66de79.36ab1.78bc0.45c1.5 mg l-1 BAP +1.5 mg l-1 NAA 25.32e92.66a0.66c0.21c2 mg l-1 BAP + 1 mg l-1 NAA 100a15.35d19.14a3.33a2 mg l-1 BAP + 0.1 mg l-1 NAA 73.03bc58.96bc4.36b1.22b1mg l-1 BAP + 1 mg l-1 NAA 93.24ab40.66c2.78bc1.35b1.5 mg l-1BAP + 1 mg l-1 NAA 59.63cd56.38c2.02bc1.12b4.5 mg l-1 BAP + 1 mg l-1 NAA Table 1: Mean comparison for the effects of treatment combination on callogenesis, and shoots induction in petiole explants of Pelargonium odoratissimum in vitro Different letters in columns are significant (p ≥ 0.01) based on LSD test Figure 1: Treatment combination effects on total shoot num- ber per explant and mean shoot number in nodal segments of Pelargonium odoratissimum in vitro (based on LSD test) Figure 2: Treatment combination effects on browning rate of nodal segments of Pelargonium odoratissimum in vitro (based on LSD test) Figure 3: The effect of NAA concentrations on rooting per- centage of Pelargonium odoratissimum in vitro (p ≥ 0.01) Acta agriculturae Slovenica, 118/4 – 2022 5 In vitro direct and indirect regeneration of plants from nodal and petiole explants in Pelargonium odoratissimum ... ferred to the pots containing peat moss-perlite (1:1), and were placed in a growth chamber under 16:8 hrs. light: darkness period, and 23±1 ºC and 20±1  ºC day: night temperature regime. After one month, the survived plantlets percentage was recorded and later, the acclima- tion of the potted plants was quite successful (more than 90 %). 4 DISCUSSION The results showed that the sole cytokinin com- pared to the co-application of cytokinin:auxin had more promising effect on mean shoots number. It seems that cytokinins at higher concentrations, weaken the apical dominance and hence, stimulate auxiliary shoot induc- tion. These findings are concomitant with the reports of Zuraida et al. (2013) with diverse Pelargonium species. From the production viewpoint, in vitro micropropaga- tion is a crucial step for the optimized proliferation rates, and furthermore, the fast and mass multiplication of the shoots provide the plant material needed for the coming stages of propagation route. The internal hormonal ratios and, especially auxin (IAA) amount were the major fac- tors affecting the multiplication in direct organogenesis from Jasmine nodal segments and the MS medium hav- ing 3 mg l-1 BAP and 1 mg l-1 NAA was the best treatment (Farzinebrahimi et al., 2014). In Pelargonium capitatum plants incubated under dark conditions for 4 weeks and cultured in the medium containing 0.5 mg l-1 NAA+1 mg l-1 BAP+1 mg l-1 Zeatin; the full (100 %) direct shoot re- generation was achieved (Hassanein and Dorion, 2005). Epinosa et al. (2006) produced the highest indirect or- ganogenesis of Prunus serotina Ehrh. plants from the leaf explants incubated under dark conditions for three weeks. Possibly, darkness affects the internal hormonal balance and positively interacts with the auxin:cytokinin exogenous applications in favor of higher organogen- esis potential. Furthermore, darkness ameliorates the deteriorative effects of phenolics and hence declines the browning rates in the plant samples in vitro (Sukhumpin- ij et al., 2010). Our results showed that for the scented geranium, BAP alone was enough for the shoot multiplication. The idea is that for the optimized shoots proliferation in ge- ranium, the high amounts of cytokinins per auxins are functional and essential. Calli production is dependent upon the morpholog- ical characteristics of the plant tissues and the type and concentration of growth regulators employed. Thiruven- gadam et al. (2006) reported that MS medium supple- mented with 1 mg l-1 2, 4-D attained the maximum callus induction. Khodadadi et al. (2015) reported the improved calli production and shoot regeneration of Momordica char- antia L. leaf explants by increasing NAA and kinetin concentrations up to 1 mg l-1. However, high concentra- tions of the growth regulators mentioned had quite nega- tive effects on callogenesis. Benazir et al. (2013) realized that the greatest callogenesis percentage in Pelargonium graveolens was obtained by 20 µM IBA+10 µM KIN and 20 µM IBA+10 µM BAP as well as by 20 µM IAA+10 µM KIN. Overall, the balanced auxin to cytokinin ratio is the essential factor impacts calli production and the further proliferation of leaf explants of scented geranium (Bena- zir et al., 2013). Furthermore, Charlwood and Charl- wood (1991) noted that the callogenesis was improved in scented geranium whenever they used more than 1 mg l-1 of auxins and cytokinins. Callogenesis response in different organs and tissues is dependent on the endog- enous and exogenous hormonal balance, so, the logical cytokinin:auxin ratios with the dominancy of cytokinins are vital for the petiole explants callogenesis. The diverse responses of the leaf and petiole explants to the varying concentrations of BA and NAA in the me- Figure 4: The effect of NAA concentrations on root number of Pelargonium odoratissimum in vitro (p ≥ 0.0) Figure 5: The effect of NAA concentrations on root length of Pelargonium odoratissimum in vitro (p ≥ 0.) Acta agriculturae Slovenica, 118/4 – 20226 A. EBRAHIMZADEH et al. dium possibly are due to the differences in the internal hormonal content or because of the different sensitivity of the organs to the growth regulators employed (Koroch et al., 2002). Arshad et al. (2012) demonstrated that the highest survival rate in P. capitatum was obtained in a medium enriched with 2 mg l-1 BA+1 or 2 mg l-1 NAA. In another study, Saxena et al. (2000) said that using 0.5 mg l-1 BAP instead of kinetin along with 0.1 mg l-1 NAA dras- tically increased the survival rate in P. graveolens. Shoots proliferation and growth need suitable rates and amounts of auxin and in this case, NAA. The need for the low concentrations of auxins (0.1  mg l-1 NAA) for the formation and elongation of the shoots is maybe due to the near sufficiency of internal auxin. Saxena et al. (2000) reported that cytokinin/auxin rate plays a pivotal role in both direct and indirect organogenesis process of the plants in vitro. The same idea has been reported by Brown and Charlwood (1986). The later scientists noted that organo- genesis responses and shoot growth in scented geranium tissue culture was dependent on the exogenous applica- tions of growth regulators and specially was responsive to BAP and NAA. Generally, shoot induction in vitro is highly responsive to the cytokinins type and concentra- tion. This bio-molecules stimulate the organogenesis in the potentiate calli cells and hence, encourage the shoots induction and initiation. Cytokinins also increase cell di- vision rates and more specially enhance the adventitious shoots formation via combating the apical dominancy. Koroch et al. (2002) reported that the medium con- taining 4.4 µM BAP+0.05 µM NAA achieved the highest regeneration rate and the greatest number of shoots. Moreover, Saxena et al. (2000) demonstrated that the maximum number of shoots in rose-scented geranium was obtained by a medium enriched with 0.5  mg l-1 BAP+0.1 mg l-1 NAA. Ghanem et al. (2008) wrote that leaf and petiole explants of P. graveolens had the suitable regeneration rate at the MS medium filled with 1 mg l-1 BAP+0.1  mg l-1 NAA. The essentiality of using at least 0.5 to 1 mg l-1 BAP for the shoots elongation in the nodal explants of scented geranium has been emphasized by Zuraida et al. (2013). Satyakala et al. (1995) noted that the maximum number and length of the nodal cutting for the same plant need 1 mg l-1 BAP and IAA. The highest regenera- tion percentage with P. hortorrum was obtained in the medium supplemented with 1 mg l-1 BAP and/or Zeatin plus 0.2 mg l-1 NAA (Hassanein and Dorion, 2005). Gen- erally, the direct and indirect regeneration in rose–scent- ed geranium is responsive to the endogenous and exog- enous PGRS, explant type and the chemical properties of the growing medium. The most probable idea is that root formation in Pelargonium species in vitro is most dependent upon the shoots related attributes. Zuraida et al. (2013) noted that 0.2 mg l-1 auxin (IAA and IBA) was the best for the high- est rooting percentage in Pelargonium citrosum and P. radula which is in line with our results. Auxins alone or in combination with the low concentration of cytokinin are able to initiate the root primordia (Zuraida et al., 2013). It seems that, deciding on the type and concentra- tion of exogenous auxin application for in vitro prolifera- tion and production is quite dependent on the internal hormonal content of the plant tissue. So, owing to the results obtained from the present experiment, 0.2 mg l-1 NAA produced the highest root number as well as the longest roots. 5 CONCLUSION The current experiment was conducted to reach a reliable fast and easy propagation method for the direct and indirect regeneration of Pelargonium odoratissimum nodal and petiole explants under in vitro conditions. Overall, an established in vitro culture regeneration pro- tocol is dependent on several factors such as; plant spe- cies, explant type and media composition. In the present study, the best treatment for direct regeneration from the nodal segments was obtained by 2  mg l-1 BAP and for indirect regeneration from the petiole explants. The opti- mized treatment combination was 2 mg l-1 BAP+1 mg l-1 NAA. Moreover, the results showed that nodal explants compared to petioles had greater regeneration rate and have been considered more suitable for Pelargonium regeneration purposes. All in all, difficulty in petioles regeneration rate, along with the highest probability of somatic mutations and the low rate of shoots prolifera- tions, makes this explant not suitable candidate for the in vitro proliferation of Pelargonium and even makes the indirect regeneration method more time-consuming and expensive. However, since the calli are the initial and easy starting materials for the production of secondary me- tabolites with suspension cultures, so, the study of cal- logenesis and calli production and optimization for the accelerated secondary metabolites production will be a motivation for using the petiole explants with in vitro commercial insights. Moreover, the results showed that explant type and PGRS concentration were the main fac- tors influencing regeneration potential and rooting be- havior in Pelargonium in vitro culture. In such a way, the highest regeneration rate was traced and recorded with nodal segments. The easy protocol experienced with the present experiment could be employed for the clonal propagation and suspension cultures of this high-valued ornamental medicinal species. Acta agriculturae Slovenica, 118/4 – 2022 7 In vitro direct and indirect regeneration of plants from nodal and petiole explants in Pelargonium odoratissimum ... 6 ACKNOWLEDGMENTS We thank Dr Libia Acened Chaparro Torres (Poly- technic University of Cartagena, Spain) for her invalu- able help. 7 REFERENCES Arshad, M., Silvestre, J., Merlina, G., Dumat, C., Pinelli, E., and Kellerhoff, J. (2012). Thidiazuron- induced shoot organogenesis from mature leaf explants of scented Pelargonium capitatum cultivars. Plant Cell Tissue and Organ Culture, 108, 315-322. https://doi. org/10.1007/s11240-011-0045-1 Benazir, J.F., Suganthi, R., Chandrika, P., and Mathithu- milan, B. (2013). In vitro regeneration and transfor- mation studies on Pelargonium graveolens (Gera- nium) an important medicinal and aromatic plant. 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