Impaginato 485 Adv. Hort. Sci., 2019 33(4): 485­493 DOI: 10.13128/ahsc­8115 Micropropagation of two near threat­ ened orchid. Part 2: Phalaenopsis amabilis Blume var. Grandiflora M. Mohammadi, B. Kaviani (*), Sh. Sedaghathoor Department of Horticultural Science, Rasht Branch, Islamic Azad University, Rasht, Iran. Key words: in vitro multiplication, orchid propagation, ornamentals, plant growth regulators. Abstract: Phalaenopsis is one of the most popular orchids in the world, through the development of many artificial hybrids. In this research, a reliable and effi­ cient protocol is presented for in vitro proliferation of Phalaenopsis amabilis Blume cv. Grandiflora. Protocorm­like bodies (PLBs) were cultured on Murashige and Skoog (MS) medium containing different concentrations of kinetin (Kn; 0.00, 0.50, 1.00, 2.00 and 3.00 mg l­1) and indole­3­butyric acid (IBA; 0.00, 0.10, 0.20, 0.50 and 1.00 mg l­1), either individually or in combination and activated charcoal (AC; 0.00, 0.50 and 1.00 g l­1). A combination of 0.20 mg l−1 IBA and 2.00 mg l­1 Kn on medium containing 1.00 g l­1 AC was found to be suit­ able for maximum leaf number (6.16±0.503 per explant). The highest rooting frequency with 7.13±0.153 roots per explant was achieved on medium enriched with 0.50 mg l­1 IBA and 0.50 mg l­1 Kn on medium containing 1.00 g l­1 AC. The largest number of callus (9.10±0.611) was induced on explants cultured in medium containing 0.20 mg l−1 IBA and 0.50 mg l­1 Kn on medium without AC. The plantlets were successfully acclimatized in the greenhouse with a survival rate of 95% exhibiting normal developmental patterns. 1. Introduction Phalaenopsis Blume, known as moth orchid, is a genus of approxi­ mately 60 species native to tropical rainforests of South and South­East Asia, Australia and New Guinea (Winkelmann et al., 2006). Phalaenopsis as a cut and pot flowering plant is one of the most popular orchids in the trade and hobbyists through the development of many artificial hybrids. They are epiphytic plants, and consist of only a few leathery leaves (Sinha et al., 2010). Large scale natural clonal propagation is not possible in Phalaenopsis. Therefore, the establishment of protocols for in vitro proliferation of orchids is the only method for high frequency regeneration of these plants. In vitro techniques can be used for storage of rare and endangered plant species and production of large number of plantlets in short period of time (Engelmann, 2011). In vitro multiplication of orchids deals with some problems such as high cost of production, low rate of shoot prolifer­ (*) Corresponding author: kaviani@iaurasht.ac.ir Citation: MOHAMMADI M., KAVIANI B., SEDAGHATHOOR SH., 2019 ­ Micropropagation of two near threa‐ tened orchid. Part 2: Phalaenopsis amabilis Blume var. Grandiflor. ­ Adv. Hort. Sci., 33(4): 485­493. Copyright: © 2019 Mohammadi M., Kaviani B., Sedaghathoor Sh. 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 9 December 2018 Accepted for publication 8 July 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(4): 485­493 486 ation, poor rooting frequency and genetic variations (Bhattacharyya et al., 2016). Several methods for in vitro propagation of Phalaenopsis through callus induction and cell suspension culture were developed (Tanaka, 1992; Arditti and Ernst, 1993; Tokuhara and Mii, 2001; Sinha et al., 2010). Park et al. (2002) devel­ oped an efficient in vitro propagation method for Phalaenopsis by using protocorm­like bodies (PLBs) derived from leaf explants. Medium composition for in vitro culture of orchids by PLBs is species­specific and depends on several factors (Luo et al., 2009). Kuo et al. (2005) reported a protocol for regenerating a Phalaenopsis cultivar by direct somatic embryogene­ sis. This method was not so efficient and feasible for commercial propagation because of low frequency regeneration of different cultivars of Phalaenopsis hybrid. On the other hand, many protocols for in vitro propagation of orchids, especially those in danger of extinction, using PLBs as explants and various PGRs, have been reported (Sinha et al., 2010; Teixeira da Silva, 2006; Baker et al., 2014; Kaviani et al., 2017). Various PGRs like α­naphthaleneacetic acid (NAA), indole­3­butyric acid (IBA), 1­phenyl­3­(1,2,3­thiadia­ zol­5­yl)­urea (TDZ), 6­benzyle amino purine (BAP), 6­ benzyladenine (BA) and kinetin (Kn) have been applied for micropropagation of rare and endangered orchids (Roy et al., 2011; Panwar et al., 2012; Zeng et al., 2012; Bhattacharyya et al., 2016; Kaviani et al., 2017). Many explants such as seed, leaf, node section, protocorm, PLB, tuber, shoot tip and inflorescence have been used for in vitro proliferation of endan­ gered orchids (Sinha et al., 2010; Roy et al., 2011; Panwar et al., 2012; Zeng et al., 2012; Baker et al., 2014; Chen et al., 2015; Bhattacharyya et al., 2016). PLB is more efficient because of maximum multiplica­ tion in a short period of time (Luo et al. 2003). This study describes an efficient and reliable protocol for high frequency regeneration and callus induction of Phalaenopsis amabilis Blume var. Grandiflora, a rare and near endangered orchid species by PLBs. 2. Materials and Methods Source of explant Leaves (0.5­1 cm long) were excised from young Phalaenopsis amabilis Blume var. Grandiflora plants growing in the greenhouse of Hyrcan Agricultural Sciences and Biotechnology Research Institute, Amol, Iran. The leaves were washed under running tap water for 15­20 min and rinsed thoroughly with dis­ tilled water. These were surface sterilized with HgCl2 (0.1% w/v) for 10 min followed by NaOCl (20%) for 15 min with 1 drop of Tween 20, then rinsed with sterile distilled water. Finally, leaves were sterilized in ethanol 75% for 1 min and washed 3­4 times with sterilized distilled water and finally excised to seg­ ments of 5­7 mm as primary explants for culture in Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing 3% sucrose and 0.8% agar. The medium was supplemented with 0.20 mg l­1 NAA along with 3.00 mg l­1 BAP (appropriate types and concentrations of PGRs obtained before for maxi­ mum production of PLBs; data not shown). Healthy and sterilized PLBs (Fig. 1A) produced in the Plant Biotechnology Laboratory, Hyrcan Agricultural Sciences and Biotechnology Research Institute, Amol, Iran, were excised and used as secondary explants for in vitro propagation. Culture medium and growth conditions The explants (PLBs) were cultured in MS medium containing 3% sucrose and 0.8% agar. The medium was enriched with various PGRs and activated char­ coal (AC). The pH of the medium was adjusted to 5.8 with 0.1 N NaOH or HCl prior to autoclaving. All media contained in culture bottles were autoclaved at 104 kPa and 121°C for 20 min. To evaluate the effect of PGRs and AC on shoot multiplication (i.e. increasing the number of leaves and their development) and root induction, the explants were cultured on MS medium containing different concentrations of kn (0.00, 0.50, 1.00, 2.00 and 3.00 mg l­1) and IBA (0.00, 0.10, 0.20, 0.50 and 1.00 mg l­1), either individually or in combination. Medium was supplemented with or without activat­ ed charcoal (AC; 0.00, 0.50 and 1.00 g l­1). For each treatment, three replicates and for each replicate, three specimens (explants) were taken (totally; 75 treatments, 225 replicates and 675 specimens or explants). Following establishment, cultures were maintained at 24±2°C, 70­80% RH, and 16­h photope­ riod of 50­60 μmol m­2 s−1 irradiance provided by cool­white fluorescent tubes. After 60 days, the effect of PGRs and AC on advanced PLBs development was assessed by measur­ ing number of leaves per explant, leaf length, leaf width, number of roots per explant, root length, num­ ber of explants with callus and viability percentage. Plant development and acclimatization In vitro rooted plantlets were taken out from cul­ ture vessels and washed thoroughly under running tap water to remove adherent nutrient and transplanted to plastic pots (18 cm height × 12 cm diameter) filled Kaviani et al. ‐ Micropropagation of Phalaenopsis amabilis var. Grandiflora 487 with a potting mixture of leca (Light Expanded Clay Aggregate), peat moss and perlite (1:1:1). All the pots were then transferred to the greenhouse with tem­ perature of 24±2°C to 20±2°C day/night (light intensity of 3500 Lux, RH of 80­90% and 14­h photoperiod) for acclimatization. The pots were covered with polyeth­ ylene bags to retain moisture inside and were opened gradually during 2 weeks. Plantlets were initially cov­ ered with a polythene sheet to maintain relative humidity (90%). The number of surviving plants was recorded after 12 weeks of transfer. Experimental design and data analysis The experiments were established in a completely randomized design with three replicates per treat­ ment (totally 675 explants). PGRs­free MS medium was used as control in the experiments. The results were expressed as mean±SD. Data were subjected to analysis of variance (ANOVA) (except for acclimatiza­ tion records) and means were compared by the LSD test at P<0.05 using the SPSS ver. 17 (SPSS Inc., USA). 3. Results The effect of PGRs and AC on the leaf growth (number, length and diameter) and root growth (number and length) of Phalaenopsis amabilis Blume var. Grandiflora is shown in Tables 1­4 and figure 1. PLBs produced on MS medium containing 0.20 mg l­1 NAA + 3.00 mg l­1 BAP (appropriate types and con­ centrations of PGRs obtained before for maximum production of PLBs; data not shown), were used as primary explants (Fig. 2A). These PLBs were produced after 60 days of culture of leaf explant on this medi­ um (Fig. 2B). These PLBs were used as secondary explants and cultured on media supplemented with Fig. 1 ­ Micropropagation of Phalaenopsis amabilis Blume cv. Grandiflora through protocorm­like bodies (PLBs). (A) PLBs produced on MS medium containing 0.20 mg l­1 NAA + 3.00 mg l­1 BAP. (B) Micropropagated shoots from PLBs on medium containing 0.50 mg l­1 IBA + 2.00 mg l­1 Kn. (C) Plantlets produced on medium supplemented with 0.20 mg l­1 IBA + 2.00 mg l­1 Kn + 1.00 g l­1 AC. (D) Length of leaf obtained on medium containing 0.50 mg l­1 IBA together with 2.00 mg l­1 Kn and 1.00 g l­1 AC. (E) Width of leaf obtained on medium containing 0.50 mg l­1 IBA together with 1.00 mg l­1 Kn 1.00 g l­1 AC. (F) Plantlets produced on media contai­ ning 1.00 g l−1 AC together with different concentrations of IBA and Kn. From left to right: 0.10 mg l−1 IBA + 0.50 mg l−1 Kn, 0.50 mg l−1 IBA + 0.50 mg l−1 Kn, 0.20 mg l−1 IBA + 1.00 mg l−1 Kn, 0.10 mg l−1 IBA + 1.00 mg l−1 Kn and 0.50 mg l−1 Kn without IBA. (G) Number and length of roots obtained on medium enriched with 0.50 mg l−1 IBA plus 0.50 mg l−1 Kn and 1.00 g l−1. (H) Greenhouse acclimatized plantlets in pots filled with leca, peat moss and perlite (in ratio of 1:1:1). Fig. 2 ­ (A) PLBs and callus formation. (B) PLBs development and multiplication in MS medium containing 0.20 mg l­1 NAA + 3.00 mg l­1 BAP. (C) Callus formation from the explants cultured in media enriched with 0.50 mg l­1 IBA + 3.00 mg l­1 Kn (left) and 0.20 mg l­1 IBA and 0.50 mg l­1 Kn (right). (D) Callus formation from the explants cultured in media enriched with different concentrations of IBA and Kn (from left to right: 0.50 mg l­1 IBA + 3.00 mg l­1 Kn, con­ trol, 1.00 mg l­1 IBA + 1.00 mg l­1 Kn and 0.20 mg l­1 IBA and 0.50 mg l­1 Kn). Adv. Hort. Sci., 2019 33(4): 485­493 488 different concentrations of PGRs and AC. ANOVA showed significant differences between various con­ centrations of PGRs and AC on most measured para­ meters (Table 1). Effect of PGRs and AC on multiplication parameters Advanced shoot development was significantly affected by the composition of the medium. The media containing 0.20 mg l­1 IBA and 2.00 mg l­1 Kn along with 0.50 g l­1 AC, and without AC were suitable for leaf number (Tables 2, 3, Figs. 1B, C). The highest leaf length (4.66±0.702 cm per explant) and leaf width (3.13±0.603 cm per explant) were obtained in media enriched with 0.50 mg l−1 IBA along with 2.00 mg l­1 Kn and 0.50 mg l­1 IBA along with 1.00 mg l­1 Kn, respectively (Table 4, Figs. 1D, E). MS medium enriched with 0.20 mg l­1 IBA and 2.00 mg l­1 Kn along with 1.00 g l­1 AC was the most appropriate medium for leaf number (6.16±0.503 per explant) (Table 4). Among all concentrations of IBA, Kn and AC used Table 2 ­ Effect of different concentrations of Kn and IBA without AC on the studied parameters of in vitro grown Phalaenopsis amabilis Blume cv. Grandiflora Means with different letters on the same column are significantly different (p<0.05) based on LSD test. Table 1 ­ Effect of different concentrations of Kn, IBA and AC on the studied parameters of in vitro grown Phalaenopsis amabilis Blume cv. Grandiflora *, **: Significant at the 0.05 and 0.01 probability level, respectively, NS: Not significant at p=0.05. Source of variations df Mean of squares Leaf number Leaf length Leaf width Root number Root length Callus number Viability percentage AC 2 6.526 ** 5.563 ** 6.881 ** 6.38 ** 5.87 ** 3.402 ns 76.00 ns IBA 4 14.71 ** 8.818 ** 4.708 ** 23.50 ** 48.60 ** 5.224 ** 1000 ** Kn 4 48.81 ** 9.746 ** 9.030 ** 5.54 ** 11.13 ** 49.2 ** 1026 ** AC × IBA 8 0.206 ns 1.139 ** 0.417 * 3.29 ** 2.35 ** 9.66 ** 291 ** AC × Kn 8 1.238 ** 1.560 ** 0.3782 ns 1.63 ** 1.434 * 12.87 ** 297 ** IBA × Kn 16 7.043 ** 4.208 ** 2.805 ** 6.35 ** 4.43 ** 20.60 ** 651 ** AC × IBA × Kn 32 1.326 ** 1.587 ** 0.697 ** 2.89 ** 2.066 ** 10.90 ** 662 ** Error 150 0.453 0.304 0.197 0.597 0.602 1.517 99.66 CV − 19.09 20.83 25.01 15.79 17.6 25.47 12.47 PGRs (mg l­1) Leaf number Leaf length (cm) Leaf width (cm) Root number Root length (cm) Callus number Viability percentageIBA Kn 0.00 0.00 2.66±0.681 d­g 1.73±0.153 h 1.16±0.306 fg 4.03±0.513 bc 3.33±0.603 hi 4.76±2.454 cd 70.00±10.000 bc 0.00 0.50 3.76±0.624 bcd 2.03±0.208 e­h 0.93±0.153 g 4.33±0.929 a­c 3.10±0.624 i 5.03±0.854 cd 63.30±10.000 c 0.00 1.00 2.93±0.265 c­g 2.13±0.520 d­h 1.20±0.265 e­g 4.00±0.529 bc 3.53±1.206 f­i 5.10±1.539 cd 73.30±10.000 a­c 0.00 2.00 2.20±0.493 fg 2.86±0.503 b­e 1.56±0.153 d­g 4.86±1.358 a­c 4.73±0.400 bcdef 4.53±0.737 cd 90.00±15.275 a 0.00 3.00 2.83±0.208 d­g 2.40±0.208 c­h 2.00±0.100 c­e 3.76±0.416 c 3.30±0.231 hi 7.96±2.166 ab 80.00±10.000 ab 0.10 0.00 2.06±0.200 g 1.83±0.321 f­h 1.66±0.100 c­g 4.80±0.889 a­c 4.20±0.794 b­i 3.63±1.332 cd 96.60±15.275 a 0.10 0.50 3.20±0.929 c­g 2.30±0.802 c­h 1.56±0.361 d­g 4.56±0.100 a­c 3.93±0.493 c­i 3.16±0.954 d 80.00±15.275 ab 0.10 1.00 3.10± 0.643 c­g 2.43±0.781 c­h 2.43±0.265 a­c 5.16±0.800 a­c 5.13±0.702 bcd 5.73±2.452 bc 70.00±10.000 bc 0.10 2.00 4.10±0.624 a­c 2.26±0.153 c­h 2.86±0.794 ab 4.53±1.415 a­c 4.43±0.451 b­h 3.76±3.694 cd 73.30±20.000 a­c 0.10 3.00 3.00±0.300 c­g 1.80±0.153 gh 1.26±0.351 d­g 4.06±0.493 bc 4.20±1.012 b­i 4.60±1.193 cd 83.30±10.000 ab 0.20 0.00 2.56±0.300 efg 2.40±0.854 c­h 1.16±0.361 fg 4.96±1.450 a­c 5.30±0.351 b 3.56±0.781 cd 73.30±10.000 a­c 0.20 0.50 3.76±0.306 bcd 2.43±0.321 c­h 1.80±0.529 c­f 5.40±0.700 ab 4.20±0.462 b­i 9.10±0.611 a 73.30±10.000 a­c 0.20 1.00 3.73±0.723 b­e 3.10±0.700 a­d 2.03±0.500 cd 5.40±0.493 ab 6.60±0.458 a 4.76±1.858 cd 83.30±10.000 ab 0.20 2.00 5.10±0.889 a 2.76±0.493 b­g 3.06±0.153 a 5.63±1.015 a 5.10±0.586 bcd 3.40±0.473 d 80.00±10.000 ab 0.20 3.00 3.73±0.153 b­e 3.66±0.794 ab 1.96±0.529 c­f 4.90±0.651 a­c 4.96±1.365 bcde 4.60±0.651 cd 80.00±15.275 ab 0.50 0.00 2.83±0.153 d­g 2.16±0.306 d­h 1.33±0.814 d­g 4.90±0.954 a­c 5.23±0.361 b 4.73±0.896 cd 90.00±5.774 a 0.50 0.50 3.46±0.300 b­e 2.33±0.153 c­h 1.70±0.058 c­g 5.20±0.721 a­c 4.40±1.250 b­h 3.93±0.404 cd 80.00±10.000 ab 0.50 1.00 3.66±0.361 b­e 2.90±0.854 a­e 2.06±0.200 b­d 4.50±0.971 a­c 5.20±1.552 bc 4.86±1.973 cd 80.00±10.000 ab 0.50 2.00 4.60±0.361 ab 3.63±0.721 ab 1.33±0.351 d­g 4.56±0.635 a­c 4.66±0.961 b­g 3.40±0.833 d 80.00±10.000 ab 0.50 3.00 3.20±0.503 c­g 2.20±0.458 c­h 1.30±0.265 d­g 5.06±1.250 a­c 3.40±0.513 g­i 4.73±1.249 cd 80.00±10.000 ab 1.00 0.00 2.86±0.503 d­g 2.26±0.306 c­h 1.56±0.100 d­g 4.56±1.457 a­c 3.73±1.350 e­i 4.43±1.058 cd 80.00±10.000 ab 1.00 0.50 3.13±0.458 c­g 2.13±0.764 d­h 1.66±0.208 c­g 4.63±1.358 a­c 4.20±1.286 b­i 5.73±3.134 bc 90.00±10.000 a 1.00 1.00 3.03±0.833 c­g 2.83±0.306 b­f 1.96±0.751 c­f 4.33±0.681 a­c 3.86±0.987 d­i 4.93±1.102 cd 73.30±10.000 a­c 1.00 2.00 4.56±0.723 ab 3.20±0.351 a­c 1.63±0.800 c­g 4.70±0.651 a­c 3.86±1.845 d­i 4.03±2.364 cd 80.00±5.774 ab 1.00 3.00 3.26±0.854 c­e 3.90±0.208 a 1.40±0.850 d­g 4.56±0.404 a­c 4.06±0.416 b­i 4.20±0.513 cd 90.00±15.275 a Kaviani et al. ‐ Micropropagation of Phalaenopsis amabilis var. Grandiflora 489 half­concentration of IBA and Kn (Fig. 1F). All other media differed significantly and gave lower root length growth rates. Among all treatments, 0.50 mg l­ 1 IBA plus 0.50 mg l­1 Kn and 1.00 g l­1 AC was found to be the most effective for root formation (7.13±0.153 per explant) (Table 4, Fig. 1G). However, the root number (7.06±0.777 per explant) produced in medi­ um containing 0.20 mg l­1 IBA plus 1.00 mg l­1 Kn and 0.50 g l­1 AC was noticeable (Table 3). In most cases, minimum root number was recorded in media with­ out IBA. Among all concentrations of IBA, Kn and AC used individually, maximum root number (5.28±0.564 per explant) was produced in medium containing 0.20 mg l­1 IBA (data not shown). The in vitro rooted plantlets were successfully acclimatized in the greenhouse (Fig. 1H). Pots were filled with leca, peat moss and perlite (in ratio of 1:1:1). Acclimatization was achieved in 4­6 weeks, and at this stage plants attain the height of about 12­ 16 cm. Acclimatization of micropropagated plantlets to the natural conditions requires several anatomical, morphological and physiological changes especially in xylem, leaves and photosynthesis. The hardened plantlets were maintained in the Hyrcan Agricultural individually, maximum leaf number (4.52±0.33 per explant) was produced in medium containing 2.00 mg l­1 Kn (data not shown). Production of leaf was relatively high by all PLBs grown on medium contain­ ing 2.00 mg l­1 Kn in combination with all concentra­ tions of IBA with or without AC (Tables 2, 3, 4). Thus, the optimal concentration of Kn was 2.00 mg l­1. Also, the optimal concentrations of IBA were 0.20 and 0.50 mg l­1. These concentrations in combination with each other recorded maximum shoot and root pro­ duction. Media supplemented with 1.00 g l­1 AC was most suitable for in vitro leaf growth since it resulted in the largest leaf number and development (Table 4, Figs. 1D, E, F). Advanced root development was significantly affected by the composition of the medium, when measured through root length and root number. All treatments of PGRs and AC, individually and in com­ bination had significant effects (P<0.01) on root growth (Table 1). Root length was highest (6.66±0.709 cm per explant) in presence of 0.20 mg l­ 1 IBA plus 2.00 mg l­1 Kn and 1.00 g l­1 AC medium (Table 4). However, no statistically significant differ­ ence in root length was detected between this and Table 3 ­ Effect of different concentrations of Kn and IBA along with 0.50 mg l­1 AC on the studied parameters of in vitro grown Phalaenopsis amabilis Blume cv. Grandiflora Means with different letters on the same column are significantly different (p<0.05) based on LSD test. PGRs (mg l­1) Leaf number Leaf length (cm) Leaf width (cm) Root number Root length (cm) Callus number Viability percentageIBA Kn 0.00 0.00 3.40±0.950 cd 1.66±0.872 f 1.73±1.210 b­e 3.86±0.907 gh 3.20±0.493 g 4.00±0.624d­g 90.00±10.000 a 0.00 0.50 2.66±0.907 d 1.76±0.252 ef 0.80±1.250 f 3.23±1.286 h 3.33±0.351 efg 5.60±1.054 bcd 80.00± 0.000 ab 0.00 1.00 3.63±0.819 bcd 2.60±0.265 a­f 1.30±0.208 c­f 4.10±0.751 f­h 3.16±0.346 g 6.70±1.136 abc 80.00±20.817 ab 0.00 2.00 2.90±1.106 d 3.10±0.306 abc 1.60±0.513 b­e 5.13±0.889 b­g 3.23±0.458 fg 3.00±0.666 g 70.00±15.275 b 0.00 3.00 2.90±0.917 d 2.33±0.961 b­f 1.33±0.513 c­f 4.80±0.907 b­g 3.33±0.954 efg 3.73±1.159 d­g 70.00±10.000 b 0.10 0.00 3.20±0.351 d 2.33±0.265 b­f 1.13±0.115 ef 5.80±1.002 a­e 5.06±1.212 a­d 3.26±1.332 fg 90.00±5.774 a 0.10 0.50 2.66±0.777 d 2.10±0.265 def 1.60±0.100 b­e 6.16±0.473 abc 3.76±0.751 d­g 5.50±0.721 bcd 90.00±10.000 a 0.10 1.00 2.86±0.702 d 2.43±0.721 b­f 2.06±0.208 abc 5.83±0.200 a­e 4.23±0.557 c­g 6.56±0.656 abc 90.00±10.000 a 0.10 2.00 5.13±0.666 a 2.10±0.611 def 2.30±0.265 a 5.96±1.682 a­d 3.86±0.100 c­g 3.40±1.353 efg 90.00±15.275 a 0.10 3.00 3.33±1.193 d 2.53±0.666 b­f 1.56±0.306 b­f 4.90±0.917 b­g 4.70±1.266 a­e 3.53±1.159 d­g 73.30±10.000 ab 0.20 0.00 2.96±1.277 d 2.90±0.458 a­d 1.20±0.265 def 3.73±0.520 gh 5.96±1.106 ab 3.90±0.656 d­g 70.00±10.000 b 0.20 0.50 3.26±0.473 d 2.63±0.436 a­f 1.93±0.451 a­d 6.10±1.411 abc 4.63±1.229 b­f 5.36±0.666 b­e 70.00±15.275 b 0.20 1.00 3.80±1.044 bcd 2.23±0.351 c­f 2.26±0.289 ab 7.06±0.777 a 6.06±1.710 a 5.43±1.514 b­e 76.60±20.817 ab 0.20 2.00 5.80±1.002 a 2.16±0.557 c­f 1.83±0.814 b­e 5.56±0.557 a­f 5.26±0.902 abc 8.56±3.029 a 70.00±15.275 b 0.20 3.00 3.83±0.800 bcd 3.23±0.611 ab 1.73±1.150 b­e 4.50±1.436 d­h 4.00±0.800 c­g 3.16±0.624 fg 90.00±10.000 a 0.50 0.00 3.10±1.539 d 2.10± 0.950 def 1.36±0.681 c­f 5.40±1.234 b­f 4.40±1.290 c­g 7.30±0.702 ab 70.00±10.000 b 0.50 0.50 4.63±1.629 abc 2.70±0.153 a­e 1.63±0.586 b­e 6.23±1.320 ab 3.80±2.022 d­g 4.33±0.643 d­g 73.30±10.000 ab 0.50 1.00 2.86±0.709 d 2.73±1.210 a­e 1.90±0.611 a­f 4.93±0.700 b­g 4.53±0.709 c­g 5.16±1.229 c­f 80.00±10.000 ab 0.50 2.00 4.76±0.473 ab 3.56±0.709 a 1.46±0.709 c­f 4.66±0.624 c­h 4.00±0.954 c­g 4.76±0.833 c­g 86.60±10.000 ab 0.50 3.00 3.53±0.361 bcd 2.93±1.595 a­d 1.70±0.458 b­e 4.13±1.007 f­h 4.56±0.900 b­g 4.90±0.436 c­g 86.6±10.000 ab 1.00 0.00 2.86±0.681 d 3.03±0.306 a­d 1.56±0.252 b­f 4.73±0.794 b­h 3.66±1.234 d­g 3.20±2.219 fg 70.00±10.000 b 1.00 0.50 3.36±0.900 cd 2.36±0.755 b­f 1.66±0.208 b­e 4.53±0.361 d­h 4.56±0.436 b­g 5.60±3.005 bcd 90.00±10.000 a 1.00 1.00 3.40±0.794 cd 2.93±0.300 a­d 1.93±0.755 a­d 4.43±0.987 e­h 4.53±1.405 c­g 4.63±3.233 c­g 70.00±15.275 b 1.00 2.00 4.73±0.379 ab 2.90±0.153 a­d 1.46±0.200 c­f 4.13±1.493 f­h 4.26±0.624 c­g 3.93±0.666 d­g 73.30±10.000 ab 1.00 3.00 3.76±0.907 bcd 2.73±0.100 a­e 1.63±0.451 b­e 4.20±1.795 f­h 4.20±0.755 c­g 3.93±0.153 d­g 90.00±11.547 a 490 Adv. Hort. Sci., 2019 33(4): 485­493 Sciences and Biotechnology Research Institute, Amol, Iran with 95% field establishment rate. Effect of PGRs and AC on viability percentage Significant differences were found in viability per­ centage among the different concentrations of PGRs alone and in combination with each other, also with AC concentrations. The rate of produced plantlets was highest when IBA at 0.10 mg l­1 alone was added to the media (Table 2). Least viability percentage (63.30±10.00) was observed in PLBs cultured on media containing 0.50 mg l­1 Kn without AC and 1.00 mg l­1 IBA along with 1.00 mg l­1 Kn with 1.00 g l­1 AC (Tables 2, 4). Effect of PGRs and AC on callus production LSD test did not show significant differences among different concentrations of AC for callus pro­ duction. A combination of 0.20 mg l­1 IBA and 0.50 mg l ­1 Kn induced highest callus production (9.10±0.611) (Figs. 2C, D), which differed significantly from the other tested combinations, being this rate two or three­fold higher than in the other treatments (Tables 2, 3, 4). There was no any direct correlation between increasing PGRs and AC concentrations and increase in callus production. In most cases, mini­ mum callus formation was observed in the explants cultured on media without IBA or Kn with or without AC (Tables 2, 3, 4, Figs. 2C, D). 4. Discussion and Conclusions The present investigation demonstrated that the addition of external PGRs in proper concentrations induced leaf formation from the PLBs explants cul­ tured in the MS medium. The regeneration of multi­ ple shoots (leaves in some orchids like Phalaenopsis amabilis) has been reported to be closely related with the type and concentration of cytokinins used (Amoo et al., 2014). Development of multiple shoots from PLBs has been successfully achieved in some orchids such as Cymbidium, Dendrobium, Catasetum, Phalanoepsis, Habeneria and Satyrium (Talukdar, 2001; Sheelavanthmath and Murthy, 2001; Mahendran and Bai, 2009; Baker et al., 2014; Kaviani et al., 2017). In Dendrobium huoshanense, Kn was reported to be more effective for plantlet regenera­ tion from PLBs than other cytokinins (Luo et al., 2009). Kn was also used for shoot multiplication of Means with different letters on the same column are significantly different (p<0.05) based on LSD test. Table 4 ­ Mean comparison of the effect of different concentrations of Kn and IBA on measured characters of Catasetum pileatum Alba grown in vitro condition PGRs (mg l­1) Leaf number Leaf length (cm) Leaf width (cm) Root number Root length (cm) Callus number Viability percentageIBA Kn 0.00 0.00 3.30±0.896 e­h 2.30±0.529 ef 1.40±0.462 d 4.50±0.802 c­f 3.46±0.436 de 5.80±0.208 a­c 90.00±15.275 a 0.00 0.50 3.10±0.493 e­h 2.56cdef ± 0.656 1.60±0.451 d 3.90±0.529 f 3.20±1.153 e 4.56±1.422 bc 73.30±10.000 a­c 0.00 1.00 3.40±0.666 d­g 2.73±0.802 cdef 2.20±0.608 a­d 4.83±1.464 b­f 4.16±1.210 c­e 5.66±0.451 a­c 80.00±10.000 a­c 0.00 2.00 3.43±0.473 d­g 2.33±0.361 def 1.90±0.737 bcd 4.90±0.917 b­f 3.73±1.069 c­e 4.43±1.779 bc 70.00±11.547 bc 0.00 3.00 3.06±1.453 e­h 2.20±1.405 ef 2.30±0.153 a­d 4.33±0.321 def 4.53±0.208 b­e 3.86±0.265 c 73.30±10.000 a­c 0.10 0.00 3.00±0.611 e­h 2.30±0.416 ef 1.50±0.321 d 5.36±0.493 b­e 5.10±0.458 a­d 3.83±0.666 c 80.00±15.275 abc 0.10 0.50 2.83±0.945 gh 3.16±1.401 cde 2.00±0.208 a­d 4.73±0.764 b­f 4.53±0.643 b­e 6.90±1.007 ab 90.00±10.000 a 0.10 1.00 3.26±1.277 e­h 2.23±0.794 ef 2.10±0.200 a­d 5.86±0.451 abc 6.13±0.436 ab 5.80±1.097 a­c 73.30±10.000 a­c 0.10 2.00 5.03±1.290 ab 3.46±0.551 bc 2.63±0.400 ab 5.76±0.208 abcd 5.13±1.168 a­d 5.20±0.889 a­c 90.00±5.774 a 0.10 3.00 2.90±1.650 fgh 3.06±0.200 cdef 2.06±0.513 a­d 4.73±0.200 b­f 4.50±0.624 b­e 4.26±1.217 bc 90.00±10.000 a 0.20 0.00 2.26±0.850 h 2.90±0.208 cdef 1.43±0.351 d 6.10±0.950 ab 4.70±0.473 b­e 3.80±1.332 c 90.00±10.000 a 0.20 0.50 3.80±0.929 b­g 3.20±0.416 cde 1.90±0.416 bcd 4.40±0.473 def 4.06±0.666 c­e 6.90±0.300 ab 80.00±10.000 a­c 0.20 1.00 3.23±0.513 e­h 2.60±0.794 cdef 2.56±0.896 abc 6.10±0.700 ab 6.66±0.709 a 5.03±1.350 bc 76.60±10.000 a­c 0.20 2.00 6.16±0.503 a 2.73±0.907 cdef 2.86±0.058 b 4.46±0.643 c­f 5.40±0.361 a­c 4.36±1.790 bc 70.00±10.000 bc 0.20 3.00 4.20±0.208 b­f 3.43±0.666 bcd 2.00±0.208 a­d 4.93±0.850 b­f 5.20±0.850 a­c 3.80±1.836 c 90.00±10.000 a 0.50 0.00 3.00±0.153 e­h 2.20±0.651 ef 1.80±0.058 bcd 5.36±0.252 bcde 4.66±0.850 b­e 6.16±1.159 a­c 76.60±10.000 ac 0.50 0.50 4.63±0.702 bcd 2.70±0.306 cdef 1.56±0.987 d 7.13±0.153 a 4.40±1.124 c­e 5.26±0.586 a­c 86.60±11.547 ab 0.50 1.00 3.73±0.436 b­g 2.80±0.737 cdef 3.13±0.603 a 4.10±0.416 ef 3.86±0.493 c­e 4.46±1.909 bc 73.30±10.000 a­c 0.50 2.00 4.93±0.814 abc 4.66±0.702 a 1.43±0.551 d 4.76±0.681 b­f 4.36±0.751 c­e 4.03±0.889 c 80.00±5.774 a­c 0.50 3.00 3.66±0.404 c­g 2.90±0.300 cdef 1.76±0.306 c 4.50±0.854 c­f 4.40±0.252 cde 5.10±0.751 a­c 90.00±10.000 a 1.00 0.00 3.06±0.569 e­h 3.00±0.300 cdef 2.06±0.231 a­d 5.00±0.971 b­f 3.96±0.862 c­e 4.13±0.751 c 80.00±15.275 a­c 1.00 0.50 3.33±1.114 d­g 2.53±1.124 cdef 1.66±0.709 cd 4.86±0.757 b­f 5.06±0.529 a­d 7.73±1.210 a 70.00±10.000 bc 1.00 1.00 3.86±0.404 b­g 1.96±0.700 f 2.06±0.265 a­d 4.96±0.451 b­f 4.86±0.850 b­e 4.90± 1.253 bc 63.30±10.000 c 1.00 2.00 4.30±0.416 bcde 2.26±0.777 ef 1.86±0.115 bcd 4.76±0.557 b­f 4.30±0.361 c­e 4.56±0.929 bc 90.00±10.000 a 1.00 3.00 4.10±0.458 b­g 4.46±0.321 ab 1.70±0.458 bcd 4.83±0.651 b­f 3.80±0.624 c­e 3.80±0.458 c 90.00±10.000 a Kaviani et al. ‐ Micropropagation of Phalaenopsis amabilis var. Grandiflora 491 some other orchids (Saiprasad et al., 2004; Malabadi et al., 2005; Panwar et al., 2012). In Satyrium nepalense, protocorm developed multiple shoots directly on the medium supplemented with cytokinins. In most of the orchids the presence of cytokinins alone promoted optimal shoot prolifera­ tion (Mahendran and Bai, 2009). BA is known to pro­ mote seedling leaf formation in Paphiopedilum (Huang et al., 2001; Chen et al., 2015). Bhattacharyya et al. (2016) reported that when the explants were grown in medium containing cytokinin and auxin, a higher rate of response frequency of shoot buds and PLBs was observed in all PGRs combinations. Also according to Seeni and Latha (2000) and Roy et al. (2011), PGRs in orchids act more efficiently when used in combination. Therefore, cytokinin and auxin are supposed to act synergistically. Effectiveness of AC on shoot multiplication and leaf growth has been demonstrated in some orchids (George and Ravishankar, 1997; Thomas and Michael, 2007; Hossain et al., 2010; Roy et al., 2011; Zeng et al., 2012; Panwar et al., 2012). Study on Paphiopedilum wardii evidenced that the plantlet growth in vitro was significantly affected by AC along with PGRs (Zeng et al., 2012). Roy et al. (2011) showed that healthy plantlets of Vanda coerulea were induced from PLBs when cultured on medium fortified with 3.00 g l­1 AC, 5.36 µM NAA and 3.80 µM BAP. In our study, there was no difference between 0.50 and 1.00 g l­1 AC for leaf growth (Tables 3, 4). Supplementation of AC in the media significantly influenced plantlet growth (shoot multiplication and root growth) over the control (Roy et al., 2011). This finding confirmed our results on the effect of AC on leaf growth parameters. In fact, a combination of 0.20 mg l­1 IBA and 2.00 mg l­1 Kn on medium contain­ ing 0.50 and 1.00 g l­1 AC was found to be suitable for maximum leaf number. This positive effect of AC on shoot multiplication has been attributed to the ability of AC to absorb phenolic compounds released by the plantlets into the media and regulate the pH level (Pan and van Staden, 1998; Eymar et al., 2000). A positive linear relationship was found between AC concentration and plantlet growth of Vanda coerulea Griff ex. Lindl. (Blue Vanda) (Roy et al., 2011). In Paphiopedilum spicerianum , 1.0 mg l ­1 NAA, 10% banana homogenate and 0.50 g l­1 AC was the most effective to promote seedling formation (Chen et al., 2015). AC might also act as a growth promoter that inhibits harmful effects of some compounds produced during seedling formation (Roy et al., 2011). Our study showed the positive effect of IBA on root formation. In Satyrium nepalense and Dendrobium nobile, IBA resulted in a better rooting efficiency over NAA in terms of rooting frequency and root number (Mahendran and Bai, 2009; Bhattacharyya et al., 2016). According to these authors, maximum rooting efficiency (86% or 5.4 roots/shoot) was obtained in medium fortified with 2.00 mg l­1 IBA in Dendrobium nobile (Bhattacharyya et al., 2016), while the highest number of roots per shoot (6.40) was achieved at 9.84 mM IBA in Satyrium nepalense D. Don. (Mahendran and Bai, 2009). The effectiveness of IBA in rooting has been shown for some other orchids like Vanilla planifolia (Giridhar et al., 2001), Cymbidium aloifolium (L.) SW. and Dendrobium nobile Lindl. (Nayak et al., 2002), Cymbidium pendulum (Nongdam et al., 2006), Satyrium nepalense (Mahendran and Bai, 2009), Vanda teres (Firoz Alam et al., 2010) and Eulophia nuda Lindl. (Panwar et al., 2012). A maximum 90% response for root formation and highest number of roots (5.50) with length of 5.30 cm per shoot was obtained on IBA (2.46 mM) treated shoots of Eulophia nuda Lindl. (Panwar et al., 2012). Study of Baker et al. (2014) on micropropagation of Catasetum demonstrated that the largest number of root (7.16) and root length (193.40 mm) were obtained on MS medium supplemented with 0.50 mg l−1 BA along with 0.50 mg l­1 NAA. Our results are in line with previous findings, as maximum root length and root number were obtained in medium contain­ ing both IBA and Kn. Our study showed the positive effect of AC on root growth. Similarly, Roy et al. (2011) evidenced a positive influence of AC on root growth of Vanda coerulea. Some other researches demonstrated that the presence of AC in the media stimulated rooting in Vanilla planifolia (George and Ravishankar, 1997), Cymbidium sinense (Chang and Chang, 2000) and Paphiopedilum spicerianum (Chen et al., 2015). Addition of AC in rooting medium maintained the pH level, increased the nitrogen uptake and stimulated the rooting of in vitro shoots (Eymar et al., 2000; Panwar et al., 2012). In the present work, minimum callus formation was obtained frequently on media without IBA or Kn with or without AC. In Eulophia nuda and on medium containing higher concentration of BA, the explants produced callus at the base of shoots while lesser number of shoots were differentiated on medium with lower BA concentration (Panwar et al., 2012). Also, in medium with higher concentration of BA Adv. Hort. Sci., 2019 33(4): 485­493 492 combined with Kn lower shoot production with more callus induction was observed. In conclusion, Phalaenopsis amabilis Blume var. Grandiflora is a scarce and near threatened orchid. Many of orchid’s species and cultivars are threat­ ened, rare, vulnerable, endangered, indeterminate or in danger of extinction. 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