Impaginato 479 Adv. Hort. Sci., 2018 32(4): 479-485 DOI: 10.13128/ahs-21833 Factors affecting in vitro propagation of some genotypes of Himalayan cedar [Cedrus deodara (Roxb. ex Lamb) G. Don.] Z. Nazemi Rafi, H. Salehi (*) Department of Horticultural Science, School of Agriculture, Shiraz University, Shiraz, Iran. Key words: auxin, conifers, cytokinins, micropropagation, pinaceae. Abstract: Four genotypes of Himalayan cedar were grown in vitro for assessing shoot proliferation. This experiment consist three parts. Initially, the explants (leafy and defoliated shoot-tips) of mature plants were disinfected and cul- tured on different basal media (LP, MS and WPM) that supplemented with ben- zyladenine (BA) at different concentrations for 6 weeks. Leafless explants pro- duced the highest number of shoots and the longest shoots for four genotypes. There was no significant difference between the culture mediums and benzy- ladenine concentrations. In second phase, the influence of benzyladenine (2.5, 5, 10, 20 µM) and thidiazuron (TDZ) (0.4, 0.8, 1.6 µM) with combination of dif- ferent auxin (NAA) concentrations (0, 1, 2, 3 µM) was determined on axillary shoot proliferation of the leafless explants of four genotypes grown on WPM. For all thidiazuron concentrations, significant differences between genotypes were detected. In general, with all genotypes, the use of 0.8 μM thidiazuron in the absence or presence of auxin (2 μM) led to the highest length and number of axillary shoots per explant, respectively. Finally, in another experiment, the following cytokinin treatments were investigated for axillary shoot multiplica- tion of the CD1 genotype: thidiazuron (0, 0.1, 0.2, 0.4, 0.8 μM) and N6-[2- Isopentenyl] adenine (2iP) (0, 0.1, 0.2, 0.3, 0.4, 0.5 μM) in combination with benzyladenine (2.5 µM). The best results were obtained in thidiazuron (0.8 µM) with combination of benzyladenine (2.5 µM). This protocol is considered as the first successful report on culture establishment of some genotypes of mature C. deodara trees. 1. Introduction The Himalayan cedar [Cedrus deodara (roxb. ex lamb) G. Don.], which belongs to the Pinaceae family, is a beautiful, evergreen, and ornamental tree growing widely on the gradient of the Western Himalayas (Champion et al., 1965). Commercial seed bearing of C. deodara begins about 30 to 45 years of age, and good seed crops are borne every 3 years (Tewari, 1994). regeneration through seeds in Cedrus deodara is quite slow and undependable. Generally, there is a preference for propagation of mature trees, which helps improve afforestation management, breeding projects and production of elite tree genotypes. However, maturation of most tree species is a major limiting factor for the use of micropropagation in (*) Corresponding author: hsalehi@shirazu.ac.ir Citation: NAZEMI rAfI Z., SAlEHI H., 2018 - Factors affect- ing in vitro propagation of some genotypes of Himalayan cedar [Cedrus deodara (Roxb. ex Lamb) G. Don.]. - Adv. Hort. Sci., 32(4): 479-485 Copyright: © 2018 Nazemi rafi Z., Salehi H. 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 26 October 2017 Accepted for publication 18 May 2018 AHS Advances in Horticultural Science Adv. Hort. Sci., 2018 32(4): 479-485 480 afforestation projects (lin et al., 1991). There are a few reports on in vitro propagation of mature conifers in the last 20 years (Gupta and Durzan, 1985; Dumas and Monteuuis, 1995; Parasharami et al., 2003; Andersone and Ievinsh, 2005; Malabadi and van Staden, 2005; Cortizo et al., 2009; De Diego et al., 2010). few reports on in vitro propagation of c e d a r s ( C e d r u s l i b a n i A . r i c h . a n d C . a t l a n t i c a Manetti) are available (Piola and rohr, 1996; Piola et al., 1998, 1999; renau-Morata et al., 2005). There are only two reports on the in vitro culture of deodar cedar with the sole use of the seeds (Bhatnagar et al., 1983; Tamta and Palni, 2004). Among regeneration methods, axillary bud induc- tion is preferable for most maintenance of genetic stability and less mutation risk (Vasil and Vasil, 1980; Pierik, 1987). The enhancement of reliability of tissue culture system is being achieved through improving of medium component or correction of the conditions of environment, or both. However, the genotype has a significant impact on the accuracy and repeatability of tissue culture and its effect must be evaluated (Sul and Korban, 1994). In this domain, cytokinins and aux- ins play important roles for maintenance and accept- able growth of cultures. During the recent decades, several synthetic compounds have been introduced for induction of regeneration potential of plants (Guo et al., 2011). Among the compounds, TDZ (Tang and Newton, 2005) and BA (Datta et al., 2006) are highly effective on Pinaceae family such as Eastern white pine and lodgepole pine, Virginia pine, red spruce, and Taxus wallichiana Zucc., respectively. To our best knowledge, no report document the in vitro vegetative regeneration of this species from mature tissues. Our goal was to build methods for the in vitro propagation of C. deodara from adult trees, and to show the effect of genotype on its micropropagation. 2. Materials and Methods Experiment 1. Plant materials Actively growing shoots (4-6 cm long) were col- lected from mature (20-25 years old) C. deodara trees (genotype not specified) in a seed orchard near the School of Agriculture, Shiraz University, Iran. This was done from September 2015 to November 2016. The shoots were wrapped with wet paper toweling, enclosed in plastic bags and then kept at 4°C until 1 day before use. Surface sterilization of explants In this experiment, two kinds of explants with the similar length were used. The first type of explants retained their needles (leaves) to full length (fig. 1 a). In the second type of explants, the leaves were trimmed to a quarter of their initial length (fig. 1 a). Both types of explants were soaked in tap-water for at least 2 h. Then, they were submerged for 30 min- utes in an aqueous solution of 2% benomyl to reduce fungal contamination. Afterwards, they were treated with 70% ethanol for 2 minutes followed by 15% Clorox (containing 5.25% sodium hypochlorite) with 0.2% ‘Tween-20’ for 15 minutes for surface steriliza- tion. finally, they were rinsed three times with sterile distilled water. Both kinds of explants were cut into 1-2 cm pieces under sterile conditions and subse- quently cultured on different nutrient media. Culture establishment The explants were cultured on three different cul- ture media: lP (Quoirin and lepoivre, 1977), woody plant medium [WPM] (lloyd and McCown, 1980) and MS (Murashige and Skoog, 1962), that were supple- mented with benzyladenine (BA) at different concen- trations (0, 0.63, 1.25, 2.5, 5 µM). Then, the cultures were placed in a growth chamber at 25±2°C under a 16 h photoperiod provided by cool white fluorescent lamps (30 μm m-2 s-1) for 6 weeks. At the end, the num- ber and length of proliferated shoots were measured. Experiment 2. Plant materials and aseptic culture This experiment consisted of two phases. In the first phase, the effect of different growth regulators on axillary shoot proliferation of four genotypes was fig. 1 - In vitro proliferation of Cedrus deodara (roxb. ex lamb) G. Don. (A) Different types of explants: explants retained their needles (leaves) and, defoliated explants; (B) Axillary shoot proliferation on WPM medium containing 0 . 8 μ M T D Z a n d 2 μ M N A A ( a f t e r 4 w e e k s ) ; ( C ) Elongation of axillary bud on EM medium after 90 days. Nazemi Rafi and Salehi - In vitro propagation of Himalayan cedar 481 studied. In the second phase, axillary shoot multipli- cation of genotype CD1 was studied. To examine the effect of genotype on proliferation rate, 110 to 130 actively growing shoots with 4-6 cm length were col- lected from four mature 20-25 year-old C. deodara trees (genotypes CD1 to CD4) in a seed orchard near the School of Agriculture, Shiraz University, Iran. The shoots were defoliated, cut into 1-2 cm in length pieces and used as initial explants (n=114 per geno- type). Surface sterilization was carried out in the same manner as in experiment 1. for culture estab- lishment, the explants were cultured on WPM medi- um supplemented with growth regulators [BA, NAA and thidiazuron (TDZ)] for shoot induction and prolif- eration. The medium was supplemented with BA (0, 2.5, 5, 10, 20 µM) and TDZ (0, 0.4, 0.8, 1.6 µM) alone or in combination with NAA (1, 2, 3 µM). The effect of TDZ and BA on shoot proliferation of four geno- types (CD1 to CD4) was investigated. The conditions, under which the cultures were incubated, were same to those of experiment 1. Elongation of induced shoots Axillary shoots formed on explants (from geno- type CD1) were isolated and transferred to elonga- tion media (EM). The EM was growth regulator-free half strength WPM, and it was supplemented with 3 g l-1 activated charcoal (AC), 15 g l-1 sucrose and 8 g l-1 agar. The explants were maintained in the culture medium, and then subcultured into glass jars (150 ml) containing 40 ml of the fresh EM. The environ- mental conditions were same as that in culture establishment. The subculturing procedure was repeated every 4 weeks for 3 months. The culture conditions (temperature and light) were same as those used for culture establishment experiment. Shoot multiplication After 3 month, the shoots grown on EM were cut to the same length and then transferred to shoot multiplication medium containing TDZ (0, 0.1, 0.2, 0.4, 0.8 µM) and 2iP (0, 0.1, 0.2, 0.3, 0.4, 0.5 µM) in combination with 2.5 µM BA for 6 weeks. Statistical analysis All experiments were conducted as factorial based on a completely randomized design with four replica- tions, each replicate comprised of four jam glasses, four explants per glass. All experiments were repeat- ed twice. Shoot proliferation rate and shoot length were recorded at the end of the fourth week. SPSS statistical software was used for analyzing data, and the one-way ANOVA with Tukey’s test (P<0.05) was used for comparing means. Three-way ANOVA was applied to examine the interactions of TDZ, BA and genotype. 3. Results Experiment 1 The effects of different culture media (lP, MS and WPM) on two types of explants were investigated. The defoliated explants showed the best results, and there was a significant difference for shoot prolifera- tion between defoliated and leafy explants, in all three culture media. The lowest frequency in shoot proliferation belonged to the leafy explants in MS medium (0.06). According to the results, WPM medi- um showed the highest number and length of prolif- erated shoots, but without significant difference as compared with lP. Moreover, leafless explants pro- duced higher and longer proliferated shoots than leafy explants. There was no significant difference between different amounts of BA on length and number of proliferated shoots (data not shown). Experiment 2 When all four genotypes (CD1, CD2, CD3, CD4) were grown at different levels of cytokinin treat- ments, shoot proliferation from leafless explants was observed. The highest mean number of shoots per explant was observed at 0.8 μM thidiazuron for genotypes CD1 (4.6), CD2 (4.4), CD3 (2.4), and at 0.4 μM thidiazuron for CD4 (2.4) (fig. 2). Genotypes CD1 and CD2 were the most responsive genotypes over all cytokinin treatments. furthermore, the highest fig. 2 - Effect of TDZ concentrations on shoot proliferation of four genotypes of Cedrus deodara. Bars with the same letters indicate no significant difference at Tukey test (P=0.05). Adv. Hort. Sci., 2018 32(4): 479-485 482 mean number of axillary shoots per explant of all genotypes (4.31) was obtained when 0.8 μM TDZ was used in combination with 2 μM NAA and the highest mean length of axillary shoots of all genotypes (11.2 mm) was obtained when 0.8 μM TDZ was applied alone (Tables 1, 2, fig. 1 b). NAA had a positive effect on quality attributes (like color, vitality, and size). However, increasing the concentration of NAA to 3 μM reduced the number and length of proliferated shoots (Tables 1, 2). Then, axillary shoot multiplica- tion of genotype CD1 was studied (fig. 3). The high- est mean number of axillary shoots per explant (4.13) and mean length of axillary shoots (10.38 mm) were obtained when 0.8 μM TDZ was applied (Table 3). The best result of shoot proliferation obtained for CD1 genotype on WPM medium supplemented with 0.8 μM TDZ (fig. 2). The same result was obtained for CD1 genotype on WPM medium supplemented with 2.5 μM BA (fig. 4). 4. Discussion and Conclusions Propagation of conifers from mature explants has always been difficult. In general, most studies on the induction of embryogenesis and/or organogenesis in conifers includes the culture of seed or zygotic tis- Means with the same letters (small letters for interactions and capital letters for main effects) indicate no significant difference at Tukey’s test (P=0.05). Table 1 - Effects of different concentrations of TDZ, BA and NAA on the number of proliferated shoots in Cedrus deodara Treatments NAA Mean values 0 1 2 3 Control 2.41± 0.02 lm† 2.16±0.02 pq 2.31±0.05 mno 1.97±0.01 r 2.21 f TDZ (µM) 0.4 3.75±0.00 b 3.31±0.03 e 3.44±0.03 de 3.06±0.03 f 3.39 B 0.8 3.38±0.03 de 3.50±0.00 cd 4.31±0.03 a 3.63±0.03 bc 3.70 A 1.6 2.25±0.00 nop 2.50±0.00 jkl 2.63±0.03 ij 2.00±0.00 r 2.34 Ef BA (µM) 2.5 2.69±0.03 hi 3.06±0.03 f 3.40±0.03 de 2.50±0.00 jkl 2.91 C 5 2.38±0.03 lmn 2.44±0.03 klm 2.44±0.03 klm 2.56±0.03 ijk 2.45 E 10 2.44±0.03 klm 2.88±0.03 g 2.81±0.03 gh 2.31±0.03 mno 2.61 D 20 2.25±0.00 nop 2.38±0.03 lmn 2.19±0.03 opq 2.06±0.03 qr 2.22 f Mean values 2.66 B 2.71 AB 2.87 A 2.45 C 2.67 Means with the same letters (small letters for interactions and capital letters for main effects) indicate no significant difference at Tukey’s test (P=0.05). Table 2 - Effects of different concentrations of TDZ, BA and NAA on the length of proliferated shoots in Cedrus deodara Treatments NAA Mean values 0 1 2 3 Control 0.0 5.80±0.06 h 5.55±0.06 hij 4.88±0.04 klm 4.50±0.09 mn 5.18 D TDZ (µM) 0.4 8.75±0.05 c 6.92±0.01 fg 8.12±0.09 d 7.12±0.12 fg 7.73 B 0.8 11.20±0.14 a 7.75±0.07 de 9.56±0.05 b 6.81±0.22 g 8.83 A 1.6 4.96±0.07 klm 5.24±0.08 ijk 5.20±0.08 i-l 4.67±0.02 lm 5.02 D BA (µM) 2.5 6.82±0.16 g 7.32±0.10 efg 5.57±0.10 hij 5.08±0.08 jkl 6.20 C 5 7.15±0.19 fg 7.43±0.10 ef 5.93±0.11 h 5.18±0.15 i-l 6.42 C 10 5.65±0.16 hi 5.93±0.08 h 4.07±0.09 no 3.12±0.08 q 4.69 D 20 3.44±0.12 pq 3.84±0.17 op 3.90±0.13 op 2.16±0.07 r 3.34 E Mean values 6.62 A 6.17 AB 5.79 B 4.79 C 5.84 fig. 3 - Multiple shoot formation on subculture of Cedrus deodara on WPM with 0.8 μM TDZ in combination with 2.5 μM BA. Nazemi Rafi and Salehi - In vitro propagation of Himalayan cedar 483 sues (Tang et al., 2006; Humánez et al., 2011). To our best knowledge, this is the first report of successful induction of axillary shoots on explants taken from adult C. deodara. There have been few reports on the effect of different kinds of basal media on the axillary shoot proliferation in cultures of explants from mature conifer trees (Andersone and Ievinsh, 2002; De Diego et al., 2010; renau-Morata et al., 2005). In the present investigation, shoot proliferation rates were generally greater on basal WPM than on basal lP and MS media. Shoot buds cultured on MS medi- um presented the lowest organogenic response, and this was perhaps as a result of the comparatively high nitrate concentration as compared to the lP medium or WPM. Tuskan et al. (1990) showed that the extra nitrate had a negative effect on the organogenic response during micropropagation. Therefore, low nitrogen content of the medium is a major factor for promoting organogenesis in conifer species (Tang et al., 2001; Schestibratov et al., 2003). Explant type was an important factor affecting axillary bud prolif- eration in in vitro culture. Piola et al. (1998) demon- strated that the accumulation of ABA in needles seems to be the major cause of bud dormancy in micropropagation of C. libani. In this experiment, defoliated explants of C. deodara also showed better response on proliferation medium. In in vitro bicentennial cedar micropropagation, it was found that the accomplishment of the protocol depends on genotype (renau-Morata et al., 2005). Among two cytokinin treatments examined, TDZ induced the highest number of proliferated shoots for all genotypes. After investigating the effect of three TDZ concentrations on shoot proliferation of four genotypes, the significant difference for their interaction was found. However, in our investigation, BA showed no significant genotypic differences for shoot proliferation. TDZ has gotten more attention in recent years due to its ability to assist in vitro regen- eration of conifers (Mathur and Nadgauda, 1999; Sul and Korban, 2004; renau-Morata et al., 2005; Tang and Newton, 2005; Cortizo et al., 2009; De Diego et al., 2010; Humánez et al., 2011). TDZ can decrease the enzyme activity related to oxidative stress during formation of adventitious shoots (Tang and Newton 2005). recent reports on mature stone pine dis- played the superiority of TDZ over other cytokinins in advancing axillary shoot proliferation (Cortizo et al., 2009). It was shown that high concentrations of cytokinin in the medium, particularly BA led to the low regeneration response, which may be attributed t o t h e t o x i c e f f e c t s o f h i g h c o n c e n t r a t i o n s o f cytokinins (Sarmast et al., 2012). In this report, the presence of NAA with either TDZ or BA improved the incidence of shoot organogenesis in adult tissues of C. deodara. This has been also observed in other conifer species (Sul and Korban, 2004; Zhu et al., 2010). As this study shows, the type of explants and cul- ture media has a very important role in success of culture establishment of Cedrus deodara. It was proven that defoliation of the explants has positive effect on shoot proliferation. The effects of genotype and growth regulators on proliferation of axillary In each column, means with the same letters indicate no signifi- cant difference at Tukey’s test (P=0.05). Table 3 - Effects of different concentrations of TDZ and 2iP in combination with 2.5 µM BA on shoot multiplication of Cedrus deodara Treatments Number of proliferated shoots length Control 0.0 2.44± 0.07 e 6.69± 0.12 e TDZ (µM) 0.1 3.38± 0.16 bc 7.26± 0.16 de 0.2 3.44± 0.21 bc 7.80± 0.27 cd 0.4 3.88± 0.16 ab 9.93± 0.24 a 0.8 4.13± 0.13 a 10.38± 0.11 a 2iP (µM) 0.1 2.50± 0.20 e 6.58± 0.11 e 0.2 2.69± 0.12 de 7.38± 0.18 de 0.3 3.00± 0.10 cde 7.93± 0.21 cd 0.4 3.25± 0.18 bcd 8.38± 0.15 bc 0.5 3.56± 0.12 abc 8.88± 0.14 b fig. 4 - Effect of BA concentrations on shoot proliferation of four genotypes of Cedrus deodara. Bars with the same letters indicate no significant difference at Tukey test (P=0.05). 484 Adv. Hort. Sci., 2018 32(4): 479-485 shoots have been studied and low concentration of TDZ combined with NAA has positive effects on the number of proliferated axillary shoots. furthermore, two of the four genotypes showed better response to cytokinins. 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