Impaginato 475 Adv. Hort. Sci., 2019 33(4): 475­483 DOI: 10.13128/ahsc­8112 Micropropagation of two near threat­ ened orchid. Part 1: Catasetum pileatum cv. Alba S. Zakizadeh, B. Kaviani (*), D. Hashemabadi Department of Horticultural Science, Rasht Branch, Islamic Azad University, Rasht, Iran. Key words: in vitro multiplication, orchid propagation, ornamentals, plant growth regulators. Abstract: Many orchid species are threatened. In this study, a reliable and effi­ cient protocol was outlined for in vitro propagation of Catasetum pileatum cv. Alba, a near threatened orchid species with the proper usage of plant growth regulators (PGRs). Protocorms as explants were cultured on Murashige and Skoog (MS) medium containing different concentrations of kinetin (Kn; 0.00, 0.20, 0.50, 1.00, 2.00, 3.00 and 5.00 mg l­1) and indole­3­butyric acid (IBA; 0.00, 0.10, 0.20, 0.50 and 1.00 mg l­1), either individually or in combination. The fre­ quency of protocorm­like bodies (PLBs) regeneration significantly relied on con­ centrations of PGRs used. A combination of 1.00 mg l­1 Kn and 1.00 mg l­1 IBA was found to be suitable for maximum PLB regeneration (8.63 per explant) and the largest number of leaf (12.70 per explant). The highest rooting frequency with 7.40 roots per explant was achieved on protocorms grown in medium enriched with 1.00 mg l­1 Kn and 0.50 mg l­1 IBA. Plantlets were transplanted to pots filled with a mixture of peat moss, leca and perlite (1:1:1) and transferred to the greenhouse. The plantlets were successfully acclimatized in the green­ house with a survival rate of 80% exhibiting normal developmental patterns. 1. Introduction Many orchid species in all over the world are threatened. These species have been listed in the red data book of the International Union for Conservation of Nature and Natural Resources (IUCN, 2011) because of immethodical collection, illegal trade and biodiversity loss and they have been included in Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), where the international trade is strictly controlled (Chugh et al., 2009; Swarts and Dixon, 2009; Reed et al., 2011). Catasetum pileatum, a rare and near threatened orchid, is a low­ land species, where it occurs as a showy epiphyte. This ornamental species is an impressive plant even out of flower. Orchids are precious as pot and cut flowers not only because of their exotic beauty but also for their long shelf life (Chugh et al., 2009). In vivo propagation of orchids is a slow process and resulted in traits segregation. Also, propagation of orchids by sexual means like seed caus­ (*) Corresponding author: kaviani@iaurasht.ac.ir Citation: ZAKIZADEH S., KAVIANI B., HASHEMABADI D., 2019 ­ Micropropagation of two near threatened orchid. Part 1: Catasetum pileatum cv. Alba. ­ Adv. Hort. Sci., 33(4): 475­483. Copyright: © 2019 Zakizadeh S., Kaviani B., Hashemabadi D. 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 27 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): 475­483 476 es the production of heterozygous plants. Therefore, establishment of protocols for in vitro proliferation of orchids is important and a proper alternative proce­ dure for propagation of orchids. In vitro techniques can be used for conservation of rare and endangered plant species and production of large number of plantlets in short period of time (Engelmann, 2011). Of course, micropropagation of orchids deals with some problems such as high cost of production, low rate of shoot proliferation, poor rooting frequency and phenotypic variations (Bhattacharyya et al., 2016). Different PGRs such as ɑ­naphthaleneacetic acid (NAA), IBA, 1­phenyl­3­(1,2,3­thiadiazol­5­yl)­urea (TDZ), 6­benzyle amino purine (BAP), 6­benzylade­ nine (BA) and Kn have been used for tissue culture of threatened and endangered orchids (Roy et al., 2011; Panwar et al., 2012; Zeng et al., 2012; Baker et al., 2014; Bhattacharyya et al., 2016; Kaviani et al., 2017). Medium composition for in vitro culture of orchids by PLBs is cultivar and species­specific and depends on several factors especially PGRs (Luo et al., 2009). Various explants such as seeds, leaves (foliar explants), nodes, PLBs, protocorms, tubers, shoot tips and floral stalk buds have been used for micropropagation of threatened and endangered orchids (Vij and Aggarwal, 2003; Roy et al., 2011; Panwar et al., 2012; Zeng et al., 2012; Baker et al., 2014; Chen et al., 2015; Bhattacharyya et al., 2016; Kaviani et al., 2017). Among all explants, PLBs and protocorms are more efficient because these can be rapidly multiplied on solid or liquid culture media, and maximum PLBs can be provided in a short period of time (Luo et al., 2003). Asymbiotic seed germination and the use of PLBs induced from vegetative organs are two efficient propagation methods for large­scale propagation of orchids (Zeng et al., 2012). These protocols have been established for many orchid species, and differ­ ent media, primary and secondary metabolites, and PGRs have been used for germination and propaga­ tion (Arditti and Ernst, 1993; Roy et al., 2011). Many protocols for in vitro propagation of orchids, especial­ ly those at risk of extinction, using PLBs as explants and various PGRs, have been reported (Teixeira da Silva et al., 2005, 2006; Firoz Alam et al., 2010; Sinha et al., 2010; Baker et al., 2014). This investigation is the first to report on in vitro multiplication of Catasetum pileatum cv. Alba, a near threatened orchid species, for developing a protocol by proto­ corms as explants and Kn and IBA as PGRs. 2. Materials and Methods Plant material Healthy and sterilized protocorms (0.7 cm long) of Catasetum pileatum cv. Alba grown on Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) were prepared from the Plant Biotechnology Laboratory, Hyrcan Agricultural Sciences and Biotechnology Research Institute, Amol, Iran (Fig. 1A). The protocorms were used as explants and cul­ Fig. 1 ­ Micropropagation of Catasetum pileatum Alba through protocorms. (A) PLBs produced on medium containing 1.00 mg l­1 Kn + 1.00 mg l­1 IBA. (B) Developing PLBs on media enriched with different concentrations of Kn and IBA. (C) Micropropagated shoots from PLBs on medium containing Kn and IBA. (D) Leaves produced on PGRs­free medium (left) and medium containing 1.00 mg l­ 1 Kn + 1.00 mg l­1 IBA (right). (E) Roots produced on PGRs­free medium (left) and medium containing 0.50 mg l­1 Kn + 0.50 mg l­1 IBA. (F) Plantlets produced on media enriched with different concentrations of Kn and IBA. (G) Plantlets transplanted in pots fil­ led with perlite. (H) Greenhouse acclimatized plantlets grown in pots filled with a mixture of leka, peat moss and perlite (in ratio of 1:1:1) (Scale bar = 1 cm). Kaviani et al. ‐ Micropropagation of Catasetum pileatum cv. Alba 477 tured on culture media poured into the culture bot­ tles for in vitro propagation (Fig. 1B). Culture medium and culture conditions The explants were cultured on MS medium con­ taining 3% sucrose and 0.8% Agar­agar. The medium was enriched with various PGRs. The pH of the medi­ um was adjusted to 5.8 with 0.1 N NaOH or HCl prior to autoclaving. All media containing culture tubes were autoclaved at 104 kPa and 121°C for 20 min. To evaluate the effect of PGRs on PLBs regenera­ tion, shoot multiplication and root induction, the explants were cultured on MS medium containing different concentrations of kinetin (Kn; 0.00, 0.20, 0.50, 1.00, 2.00, 3.00 and 5.00 mg l­1) and indole­3­ butyric acid (IBA; 0.00, 0.10, 0.20, 0.50 and 1.00 mg l­ 1), either individually or in combination. For each treatment, three replicates and for each replicate, three explants were taken (totally; 35 treatments, 105 replicates and 345 explants). Following establish­ ment, cultures were maintained at 24±2°C, 70­80% RH, and 16­h photoperiod of 50­60 μmol m­2 s­1 irra­ diance provided by cool­white fluorescent tubes. Assessment of characteristics After 60 days, the effect of PGRs on advanced PLBs development was assessed by using PLBs num­ ber, plantlet height, leaf number, root length, root number and viability percentage. PLBs germination percentage was calculated by the following formula: % sprouted PLBs = Number of sprouted PLBs x 100 Total PLBS Hardening and acclimatization In vitro rooted plantlets were taken out from cul­ ture vessels and washed thoroughly under running tap water to remove substrate residual and trans­ planted to plastic dishes containing perlite for 15­20 days (Fig. 1G). Then, plantlets were transferred to plastic pots (18 cm height × 12 cm diameter) filled with a potting mixture of Leca (Light Expanded Clay Aggregate), peat moss and perlite (in ratio of 1:1:1) (Fig. 1H). All the pots were then transferred to the greenhouse with temperature 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 polyethylene bags to retain mois­ ture inside and were opened gradually during 2 weeks. Survival rate (%) was recorded after 60 days from transfer to greenhouse conditions. Plantlets were initially covered with a polythene sheet to maintain relative humidity (90%). The number of sur­ viving plants was recorded after 4 weeks from trans­ fer. Experimental design and data analysis The experiments were established in a completely randomized design with three replicates per treat­ ment (totally; 345 explants). PGRs­free MS medium was used as control in the experiments. Data were subjected to analysis of variance (ANOVA) and means were compared by the LSD test at P < 0.05 using the SPSS ver. 17 (SPSS Inc., USA). 3. Results Assessment of suitable conditions for PLBs regenera‐ tion The main goal of this study was the maximum regeneration of PLBs during clonal proliferation. LSD test showed significant differences among different concentrations of Kn, also reciprocal effect of Kn and IBA for PLBs number (p˂0.01). LSD also showed that the effect of IBA was no significant on PLBs number when applied individually (Table 1). The effects of Kn and IBA in the MS medium on the PLBs multiplication and growth are shown in figure 1 and Tables 2, 3 and 4. In the current study the formation of PLBs and shoot buds from the explants was observed within 45 Table 1 ­ Analysis of variance of the effect of different concentrations of Kn and IBA on measured characters of Catasetum pileatum Alba grown in vitro condition Source of variations df Plantlets height Number of PLBs per protocorm Leaf number Root number Root length Viability percentage Kn 6 50.8** 10.61** 30.88** 0.870** 23.52** 457** IBA 4 7.16 NS 1.66 NS 3.916** 15.57** 24.04** 258* Kn × IBA 24 6.29** 2.60** 4.32** 0.647** 7.73** 167* Error 70 3.42 1.126 1.72 0.241 0.973 83.8 cv (%) ­ 21.5 19.97 20.2 9.714 10.91 11.01 *, **: Significant at the 0.05 and 0.01 probability level, respectively, NS: Not significant at p=0.05. Adv. Hort. Sci., 2019 33(4): 475­483 478 days of culture establishment. The regeneration of PLBs are closely related with the concentration of both Kn and IBA when used in combination, howev­ er, Kn had more important role than IBA when these are applied individually. When the explants were cul­ tured in medium supplemented with Kn individually, formation of more PLBs was observed than when explants were cultured in medium containing IBA individually (Tables 2 and 3). Among the tested Kn concentrations, 1.00 mg l­1 proved beneficial in induc­ ing the highest frequency of 6.76 per explant gener­ ating PLBs (Table 2). When the explants were grown in medium enriched with Kn and IBA (1.00 mg l­1 from both of them) the largest number of PLBs (8.63 per explant) was observed (Table 4). Minimum PLBs regeneration (with average of 4.00 PLBs per explant) was obtained in media without Kn. Assessment of suitable conditions for plantlets height and leaf number The effect of PGRs in the MS medium on the plantlets growth (plantlets height and leaf number) is shown in figure 1C and Tables 1­4. After 60 days of culture of protocorms on media fortified with differ­ ent concentrations of PGRs, plantlets height and leaf number were measured. Plantlets growth was signifi­ cantly affected by the composition of the medium. Growth of plantlets from the explants without the addition of Kn to the culture medium was relatively poor. The plantlets growth rate ranged from 5.00 to 17.00 cm on the regeneration media (Table 4). MS medium fortified with 1.00 mg l­1 Kn and 0.50 mg l­1 IBA was the most appropriate medium for plantlet height (16.76 cm per explant) (Table 4). Treatment with 1.00 mg l­1 Kn in combination with 0.50 mg l­1 IBA produced plantlets with 13.36 cm long. Among all levels of Kn and IBA used individually, Kn at 1.00 mg l­ 1 was noted better induction of plantlet on explants than the other levels (Tables 2 and 3). The plantlets produced on medium without Kn was least (Tables 3 and 4). MS medium fortified with 1.00 mg l­1 Kn and 1.00 mg l­1 IBA was the most appropriate medium for leaf number (12.70 per explant) (Fig. 1D, Table 4). The media containing 1.00 mg l­1 Kn and 0.50 mg l­1 IBA was suitable for leaf number, too (Table 4). Among all concentrations of IBA and Kn used individually (Tables 2 and 3), maximum leaf number (9.36 per explant) was produced in medium containing 1.00 mg l­1 Kn. Thus, the optimal concentration of Kn was Table 2 ­ Mean comparison of the effect of different concentrations of Kn on measured characters of Catasetum pileatum Alba grown in vitro condition Table 3 ­ Mean comparison of the effect of different concentrations of IBA on measured characters of Catasetum pileatum Alba grown in vitro condition Means with different letters on the same column are significantly different (p<0.05) based on LSD test. Means with different letters on the same column are significantly different (p<0.05) based on LSD test. Kn (mg l­1) Plantlets height (cm) Number of PLBs per protocorm Leaf number Root number Root length (cm) Viability percent­ age 0.00 6.22 d 4.01 d 4.75 d 5.32 a 8.68 bc 75.30 c 0.20 7.27 cd 4.80 c 5.58 cd 5.12 ab 10.28 a 85.30 b 0.50 9.72 b 5.41 bc 6.73 b 5.08 abc 10.68 a 84.00 b 1.00 11.84 a 6.76 a 9.36 a 5.38 a 9.14 b 93.30 a 2.00 9.02 b 5.18 bc 6.32 bc 4.95 bc 9.26 b 80.00 bc 3.00 8.38 bc 5.73 b 6.57 b 4.75 c 6.93 d 82.00 bc 5.00 7.62 c 5.27 bc 6.11 bc 4.81 bc 8.30 c 82.00 bc IBA (mg l­1) Plantlets height (cm) Number of PLBs per protocorm Leaf number Root number Root length (cm) Viability percent­ age 0.00 7.96 c 5.00 b 6.15 b 4.33 d 8.22 c 83.80 ab 0.10 8.66 abc 5.43 ab 6.20 ab 4.62 cd 9.32 b 88.09 a 0.20 9.15 ab 5.37 ab 6.17 b 4.80 bc 10.34 a 83.80 ab 0.50 9.14 ab 5.68 a 6.92 ab 6.53 a 9.62 b 81.40 b 1.00 8.00 bc 5.05 ab 7.00 a 5.00 b 7.70 c 78.50 b Kaviani et al. ‐ Micropropagation of Catasetum pileatum cv. Alba 479 treatments of PGRs, individually and in combination had significant effects (P<0.01) on root growth (Table 1). Root length was highest (14.06 cm per explant) in 0.50 mg l­1 Kn along with 0.50 mg l­1 IBA (Fig. 1E), however, this medium was not significantly different compared to 0.50 mg l­1 Kn along with 0.20 mg l­1 IBA medium with induction of 12.90 cm long for root (Table 4). All other media were significantly different and gave lower root length growth rates. Among all treatments, 1.00 mg l­1 Kn plus 0.50 mg l­1 IBA was found to be the most effective for root for­ 1.00 mg l­1. Also, the optimal concentrations of IBA were 1.00 and 0.50 mg l­1. These concentrations in combination with each other recorded maximum leaf production. This means that the PGRs acted synergis­ tically. Assessment of suitable conditions for root character‐ istics and acclimatization Advanced root development was significantly affected by the composition of the medium, when measured through root length and root number. All 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 Plant growth regulators (mg l­1) Plantlets height (cm) Number of PLBs per protocorm Leaf number Root number Root length (cm) Viability percentage Kn IBA 0.00 0.00 5.63 jk 3.56 jk 5.20 f­h 5.26 d­g 5.73 o 73.33 e­g 0.00 0.10 6.60 h­k 3.83 ijk 4.46 h 4.13 k­n 6.83 mno 80.00 c­g 0.00 0.20 5.10 k 4.46 e­k 4.73 gh 4.93 e­j 11.23 cd 86.60 a­e 0.00 0.50 5.20 k 4.63 e­k 4.66 gh 7.00 a 11.80 bc 70.00 fg 0.00 1.00 6.60 h­k 3.56 jk 4.70 gh 5.30 c­g 7.83 k­m 66.60 g 0.20 0.00 6.50 i­k 5.36 c­h 5.40 e­h 4.56 g­n 10.90 c­g 90.00 a­d 0.20 0.10 8.73 c­i 5.93 b­f 5.56 d­h 4.43 h­n 12.93 ab 100.00 a 0.20 0.20 6.83 f­k 4.30 e­k 5.53 d­h 4.80 f­l 10.13 d­h 73.33 e­g 0.20 0.50 6.70 g­k 5.36 c­h 5.80 c­h 7.26 a 9.43 f­j 83.30 b­f 0.20 1.00 7.60 d­k 4.06 h­k 5.60 c­h 4.53 g­n 8.00 j­m 80.00 c­g 0.50 0.00 9.43 c­i 4.66 e­k 6.13 b­h 4.40 h­n 7.63 l­n 86.60 a­e 0.50 0.10 9.50 c­i 4.16 g­k 7.73 bc 5.10 d­i 9.76 d­i 83.30 b­f 0.50 0.20 10.30 cd 7.36 ab 6.46 b­h 4.30 j­n 12.90 ab 86.60 a 0.50 0.50 10.60 bc 5.73 b­h 6.13 b­h 6.63 ab 14.06 a 86.60 a­e 0.50 1.00 8.80 c­i 5.13 d­h 7.20 b­f 5.00 d­j 9.06 h­l 76.66 d­g 1.00 0.00 9.53 c­i 6.00 b­f 8.00 b 3.86 mn 9.33 g­k 93.30 abc 1.00 0.10 9.86c­e 6.63bcd 6.56b­h 4.86e­k 9.13h­l 96.60ab 1.00 0.20 13.36 b 5.53 c­h 7.40 b­e 5.63 cde 11.00 c­f 100.00 a 1.00 0.50 16.76 a 7.10 abc 12.16 a 7.40 a 8.23 i­m 96.60 ab 1.00 1.00 9.66 c­g 8.63 a 12.70 a 5.16 d­h 8.00 j­m 80.00 c­g 2.00 0.00 8.56 c­j 4.66 e­k 6.40 b­h 4.36 i­n 8.96 h­l 76.66 d­g 2.00 0.10 10.40 b­d 6.20 b­e 6.80 b­g 4.76 f­l 9.50 e­j 86.60 a­e 2.00 0.20 9.90 c­e 5.10 e­j 5.86 b­h 4.4 6 h­n 11.06 c­e 70.00 fg 2.00 0.50 7.50 d­k 5.33 c­h 5.90 b­h 5.76 cd 9.70 d­i 76.66 d­g 2.00 1.00 8.73 c­i 4.60 e­k 6.66 b­g 5.40 c­f 7.10 mno 90.00 a­d 3.00 0.00 7.83 c­k 5.50 c­h 5.86 b­h 3.83 n 7.66 l­n 76.66 d­g 3.00 0.10 7.23 e­k 5.93 b­e 6.80 b­g 4.46 h­n 7.60 l­n 90.00 a­d 3.00 0.20 9.40 c­i 6.93 a­c 7.66 bcd 4.56 g­n 6.20 no 80.00 c­g 3.00 0.50 9.60 c­h 5.90 b­g 6.56 b­ h 6.06 bc 6.96 mno 86.60 a­e 3.00 1.00 7.86 c­k 4.40 f­k 5.96 b­h 4.83 f­k 6.23 no 76.660 d­g 5.00 0.00 8.23 c­k 5.26 c­j 6.10 b­h 4.03 l­n 7.33 mn 90.00 a­d 5.00 0.10 8.33 c­j 5.33 c­h 5.50 e­h 4.63 f­m 9.50 e­j 80.00 c­g 5.00 0.20 7.16 e­k 4.93 d­k 5.53 d­h 4.93 e­j 9.86 d­h 90.00 a­d 5.00 0.50 7.63 c­k 5.83 b­g 7.26 b­f 5.63 cde 7.16 mno 70.00 fg 5.00 1.00 6.76 g­k 5.00 e­k 6.16 b­h 4.83 f­k 7.66 l­n 80.00 c­g Means with different letters on the same column are significantly different (p<0.05) based on LSD test. 480 Adv. Hort. Sci., 2019 33(4): 475­483 mation (7.133 per explant) (Table 4). Root production in this medium was not significantly higher than fol­ lowing media. The root number (7.26 and 7.00 per explant) produced in media containing 0.20 mg l­1 Kn plus 0.50 mg l­1 IBA and 0.50 mg l­1 IBA without Kn, respectively was noticeable (Table 4). In most cases, minimum root number was recorded in media with­ out IBA. Among all concentrations of IBA and Kn used singly (Tables 2 and 3), maximum root number (6.53 per explant) was produced in medium fortified with 0.50 mg l­1 IBA. The in vitro rooted plantlets (Fig. 1F) were suc­ cessfully acclimatized in the greenhouse. Pots were filled with leca, peat moss and perlite (in ratio of 1:1:1) (Figs. 1G and H). Acclimatization was achieved in 4­6 weeks, and at this stage plants attain the height of about 10­15 cm. Acclimatization of micro­ propagated plantlets to the natural conditions requires several anatomical, morphological and phys­ iological changes especially in xylem, leaves and pho­ tosynthesis. The hardened plantlets (Fig. 1H) are maintained in the Hyrcan Agricultural Sciences and Biotechnology Research Institute, Amol, Iran with 80% field establishment rate. Assessment of suitable conditions for viability per‐ centage Significant difference was observed between the Kn (P<0.01), IBA and combination of Kn and IBA (P<0.05) levels and viability percentage of proto­ corms (Table 1). It has been observed that among the combinations and concentrations of PGRs, 0.20 mg l­1 Kn with 0.10 mg l­1 IBA and 1.00 mg l­1 with 0.20 mg l­1 IBA were the most effective for viability percentage (100.00) (Table 4). Viability percentage in these two media was significantly higher than that of other media. Least viability percentage (66.60) was observed in PLBs cultured on medium containing 1.00 mg l­1 IBA without Kn (Table 4). 4. Discussion and Conclusions Current study revealed that the addition of exter­ nal PGRs in appropriate concentrations induced PLBs formation from the protocorm explants cultured in the MS medium. In orchids, PLB regeneration from explants such as shoot and root tips and leaf and stem segments is a proper method of in vitro prolifer­ ation (Seeni and Latha, 2000; Dohling et al., 2012). Consonant with our findings, Roy et al. (2011) demonstrated that the frequency of PLBs regenera­ tion of orchid Vanda coerulea significantly relied on kinds and concentrations of PGRs used. These researchers showed that a combination of 1.00 mg l­1 NAA and 0.85 mg l­1 BAP was found to be suitable for maximum PLB regeneration. Luo et al. (2008) showed that 0.50 mg l­1 Kn was proper for PLB formation of orchid Dendrobium densiflorum. The regeneration and proliferation of multiple PLBs are closely related with the type and concentration of cytokinins used. Among all cytokinins used for in vitro PLBs regenera­ tion of orchids, BA, BAP, TDZ and Kn have the most application (Luo et al., 2008; Chugh et al., 2009; Firoz Alam et al., 2010; Roy et al., 2011; Panwar et al., 2012; Baker et al., 2014; Bhattacharyya et al., 2016; Kaviani et al., 2017). When the explants were grown in medium enriched with both of Kn and IBA the maximum PLBs was obtained. Contrary to our find­ ing, BAP individually was better than in combination with NAA for the maximum PLBs formation of orchid Oncidium (Kalimuthu et al., 2007). Study on Vanda coerulea revealed that when the protocorms were cultured on BAP or NAA alone, at any concentration recorded low proliferation rate. This means that the PGRs acted synergistically (Roy et al., 2011). Baker et al. (2014) reported the highest PLBs regeneration of orchid Catasetum on MS medium containing a com­ bination of 0.50 mg l­1 BA plus 0.50 mg l­1 NAA. Present study demonstrated that the production of more than seven PLBs was observed in the media supplemented with 0.50 mg l­1 kn along with 0.20 mg l­1 IBA and 1.00 mg l­1 Kn along with 0.50 mg l­1 IBA. Similar findings were found by Panwar et al. (2012) through study on orchid Eulophia nuda. The combination, concentrations and the ratio between the PGRs are critically important for the for­ mation of shoots and PLBs in orchids (Dohling et al., 2007; Baker et al., 2014; Bhattacharyya et al., 2016; Kaviani et al., 2017). Investigation on orchid Eulophia nuda showed that the maximum shoot multiplication was achieved on MS medium containing 2.00 mg l­1 BA and 1.00 mg l­1 Kn after 4 weeks of cultures (Panwar et al., 2012). The type, concentrations and different combinations of PGRs plays an important role during micropropagation of many orchid species (Arditti and Ernst, 1993; Panwar et al., 2012). In orchids, the use of protocorm and PLB as the explants is the most appropriate and simplest method for in vitro propagation. Protocorm contains meristematic cells and can differentiate to a new shoot. Therefore, protocorm can be used to enhance proliferation and simultaneous production of orchid plantlets (Teixeira da Silva et al., 2005). Protocorms Kaviani et al. ‐ Micropropagation of Catasetum pileatum cv. Alba 481 are being applied by many researchers as explants for in vitro propagation of many rare and endangered orchid species (Deb and Temjensangba, 2006, Teixeira da Silva et al., 2006; Roy et al., 2011; Dohling et al., 2012; Baker et al., 2014; Bhattacharyya et al., 2016; Kaviani et al., 2017). PLB production was induced from many explants such as protocorm, shoot tip, node, root tip, leaf and stem segments dur­ ing in vitro propagation of orchids (Dohling et al., 2012; Baker et al., 2014; Bhattacharyya et al., 2016; Kaviani et al., 2017). The regeneration and propagation of multiple shoots are closely related with the type and concen­ tration of cytokinins used (Amoo et al., 2014). The differentiation of multiple shoots from PLBs has been reported in some orchids such as Cymbidium, Dendrobium, Catasetum, Phalanoepsis, Habeneria and Satyrium (Talukdar, 2001; Sheelavanthmath and Murthy, 2001; Mahendran and Bai, 2009; Hossain et al., 2010; Baker et al., 2014; Kaviani et al., 2017). Cytokinins have a wide range of functions including regulatory role on various physiological and develop­ mental processes (Werner et al. , 2001). In Dendrobium huoshanense C.Z. Tang et S.J. Cheng, Kn was reported to be more effective for plantlet regen­ eration from PLBs than BAP, N­benzyl­tetrahydropy­ ranyladenine (BPA), isopentenyl adenine (2­iP), TDZ and Zeatin (Zt) (Luo et al., 2009). The best response appeared on the medium enriched with 4.50 mg l−1 Kn. Kn was also used for shoot proliferation of some other orchids (Saiprasad et al., 2004; Malabadi et al., 2005; Martin and Madassery, 2006; Panwar et al., 2012). Contrary to our findings, study on Dendrobium nobile revealed that when explants were cultured in MS medium supplemented with BAP alone, forma­ tion of PLBs was done but direct shoot formation was not observed (Bhattacharyya et al., 2016). Study of Mahendran and Bai (2009) demonstrated that among the cytokinins used for multiple shoot induction of Satyrium nepalense D. Don. TDZ was found to be superior. In this study, protocorm developed multiple shoots directly on the medium supplemented with cytokinins. In most of the orchids the presence of cytokinins singly promoted optimal shoot prolifera­ tion (Mahendran and Bai, 2009). In the present study, addition of external PGRs (both of Kn and IBA) in suitable concentrations induced plantlets growth and leaf formation from PLBs cultured in the MS medium without callus formation. The main advan­ tage of direct organogenesis without an intervening callus phase is that somaclonal variation is reduced (Roy et al., 2011). When the media was supplement­ ed with IBA singly the response was poor. Similar finding was reported by Roy et al. (2011) worked on Vanda coerulea. Bhattacharyya et al. (2016) showed that when the explants were grown in medium con­ taining cytokinin and auxin, a higher rate of response frequency (92.6%) of shoot buds and PLBs was observed in all PGRs combinations. In Eulophia nuda Lindl., maximum shoot multiplication and elongation were obtained on MS medium containing 2.00 mg l−1 BA and 1.00 mg l­1 Kn (Panwar et al., 2012). PGRs in orchids act more efficiently when used in combina­ tion (Seeni and Latha, 2000; Roy et al., 2011). Similar with our findings, IBA resulted in a better rooting efficiency over NAA in terms of rooting frequen­ cy and number of roots induced per shoot in Satyrium nepalense D.Don. and Dendrobium nobile (Mahendran and Bai, 2009; Bhattacharyya et al., 2016). As, maxi­ mum rooting efficiency (86% or 5.4 roots/shoot) was obtained in medium supplemented with 2.00 mg l−1 of IBA after 8 weeks of culture (Bhattacharyya et al., 2016). The highest number of roots per shoot (6.40) was achieved at 2.00 mg l−1 IBA (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 alofolium (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. 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