NUSANTARA BIOSCIENCE ISSN: 2087-3948 Vol. 6, No. 1, pp. 7-12 E-ISSN: 2087-3956 May 2014 DOI: 10.13057/nusbiosci/n060102

Optimization of in vitro sterilization protocol for obtaining contamination-free cultures of platyphyllos

VAHIDEH PAYAMNOUR1, KAMAL GHASEMI BEZDI2,♥, MOSTAFA MEHRDAD1, AKRAM AHMADI1,♥♥ 1Department of Forest Ecology, Faculty of Forest Sciences, Gorgan University of Agricultural Sciences & Natural Resources, Gorgan, Golestan, Iran. Tel. /Fax. +981714427050, ♥♥email: [email protected] 2Cotton Research Institute of Iran (CRII), Beheshti St, P.O. Box 49175-483, Gorgan, Golestan, Iran. Tel: +98171-2254960. Fax: +98171-2227781, ♥email: [email protected]

Manuscript received: 28 November 2013. Revision accepted: 4 April 2014.

Abstract. Payamnour V, Ghasemi Bezdi K, Mehrdad M, Ahmadi A. 2014. Optimization of in vitro sterilization protocol for obtaining contamination free cultures of Tilia platyphyllos. Nusantara Bioscience 6: 7-12. Tilia platyphyllos is one of threatened of Caspian forests. Tissue culture techniques are applied for culture, regeneration and genetic resources preservation. Utilizing an accurate sterilization procedure is important to reduce the cost, time and energy. The aim of this present study was to provide optimization of in vitro sterilization protocol to obtain contamination-free cultures of T. platyphyllos. Explants were collected randomly from the best individuals of T. platyphyllos, which were located in Tooskestan forest of Gorgan, Iran. Results revealed that the optimum protocol for sterilization was when explants were exposed in pre-sterilizing solution of 600 mg L-1 ascorbic acid, 4 g L-1 captan fungicide and 5% commercial sodium hypochlorite (NaOCl) solution (5% Cl activated) for 20 minutes and then explants were exposed in sterilizing solution containing 600 mg L-1 ascorbic acid and 10% sodium hypochlorite.

Keywords: Explants, pre-sterilization, sterilization, Tilia platyphyllos

INTRODUCTION the landscapes beautiful (Edlin 1976). Tilia platyphyllos can be planted with beech or hornbeam for rehabilitation of The Linden trees, from Genus Tilia belong to the destroyed northern forests of Iran (Sadati et al. 2007). Salicaceae family and order (Sabeti 1965; Sadati Due to the many problems that exist in germination of 2002). Generally, Tilia genus includes woody (about T. platyphyllos seeds, techniques such as tissue culture are 30-40 species) and 10 species of Linden are distributed in important for restoration and conservation of this genetic moderate areas of northern parts of the earth, 4 of which resource (Sadati 2002; Shahrzad 1997; Hartman et al. 4are found in Europe (Magherini and Nim 1993). Tilia 1990; Bewley and Black 1985). The first study of tree platyphyllos is the only species of Tilia genus in Iran, tissue culture was conducted in the mid-1920s (Ghasemi which is one of the important species and has wide Bezdi and Ahmadi 2009). Due to the presence of many distribution (Mozafarian 1998). Natural site of T. microorganisms in the explants, contamination reveals in platyphyllos is located in the northern forests of Iran cultures despite the use of sterilization methods (Skirvin et (Sabeti 1965). al. 1999; Martinez and Wang 2009). Tilia platyphyllos is hydrophobe and hard on the land Tilia platyphyllos is valuable broad-leaved forest tree that prefers cold or moderate climate and also, distributes that much research should be done in tissue culture because its root system (Kunneman and Albers 1991; Haller 1995). the seed germination is negligible due to the hard seed coat. Its longevity is average (Moghadasi 2001) that in some The first reports of the T. platyphyllos tissue culture were species, reaches to 500-1000 years (Kunneman and Albers presented by Barker (1969). He proceeded through callus 1991; Haller 1995). Generally, the are grayish, nut- culture of Tilia americana. Chalupa (1984) was one of shaped and oval-shaped capsule that matures in autumn to those who was very active in T. platyphyllos tissue culture winter, but may also remain on the tree. Each includes a and has done much research in this field. At first, he pod that contains a seed (Brinkman 1974; Pigott and worked on , and used the lateral to grow Huntley 1981). External coating contains a layer with low in vitro culture, in 1984. fiber. Due to many difficulties in tissue culture, Linden can be included as a medical plant because its especially in forest trees, optimizing the sterilization and have medical usage (Moghadasi 2001). procedure of plant material seems necessary (Osterc 2004). The honey produced by bees extracting its nectar is very Analogous investigations were carried out about sterilizing famous in the world (Haller 1995). Its wood has the highest forest trees explants and achieving standard method for efficiency in the sculpture due to its beauty and softness. sterilization (Osterc 2004; Meier-Dinkel 1986; Preil 1997; Usually, these trees are planted in many parks and Langens-Gerrits 1998). In Linden, due to the difficulties in roadsides in Europe, because they grow so fast and make propagating even through laboratory procedures, some

8 NUSANTARA BIOSCIENCE 6 (1): 7-12, May 2014 researches have been done (Shahrzad 1997; Mehrdad et al. the medium was adjusted to 5.7 before autoclaving, and 2007, 2011). Shahrzad (1997) harvested the twigs of T. ascorbic acid added to the medium after autoclaving. platyphyllos from three provinces of Tehran (botanic The buds were used as explants for accessing best garden), Mazandaran (Waz forests) and Gilan (Shafaroud protocol to obtain contamination free-cultures of T. forest) in different seasons. She found that the best method platyphyllos. Different treatments of time and material for sterilization of this species was washing explants with were used for pre-sterilizing and sterilizing of explants. 70% ethanol, with 1 and 2% sodium hypochlorite (NaOCl) Then, viability (in percent) of explants was investigated and 1% HgCl2 and the best time to harvest buds was from after sterilizing and transferring to a MS basal medium the fall to early winter. Also, she stated that suitable buds using a completely randomized design with three for culturing were apical buds or near the terminal end replications. Data obtained from this research were buds. Also, Mehrdad et al. (2011) studied the analyzed statistically using one-way analysis of variance micropropagation of Tilia begonifolia, in vitro. (ANOVA) and the significant differences among means Therefore, this experiment was undertaken with an were assessed using Duncan’s multiple range test with objective of finding out the optimized in vitro sterilization MSTATC software. protocol to obtain contamination-free cultures of T. platyphyllos. RESULTS AND DISCUSSION

MATERIALS AND METHODS Variance analysis results of different factors in order to pre-sterilizing of explants, ascorbic acid amounts at In the present research, twigs of Tilia platyphyllos were sterilizing solutions, necessary time for sterilizing used as a source plant. Twigs were harvested from treatments, sodium hypochlorite concentrations of Tooskestan forest of Gorgan, Iran, transferred to the sterilizing solutions and interaction effects of these factors laboratory and were cleaned. At first, water and soap water on viability (in percent) of explants are shown in Table 2. were used to eliminate surface pollution. In order to reduce According to the results, viability (percent) of explants pollution, pre-sterilizing was carried out, in which explants under pre-sterilizing factors, ascorbic acid amount, were set at a definite volume of sterilized water (dependent sterilizing time, sodium hypochlorite concentration and on explants volume) with 300 and 600 mg L-1 ascorbic their interaction effects were significant (p≤0. 01). acid, 2 and 4 g L-1 captan (1, 2, 3, 6-tetrahydro-N- trichloromethyl thiophthalmide) fungicide and 5% and 10% Table 2. Variance analysis of pre-sterilizing effect, ascorbic acid, commercial sodium hypochlorite (NaOCl) and then were time of pre-sterilizing, sodium hypochlorite concentration and shacked in 10, 20 and 30 minutes (Table 1). Then, explants their interaction effects on viability percent of T. platyphyllos were exposed to running water for washing. explants

Source of variation Degree of Mean of Table 1. Type and amount of used compound at pre-sterilizing (S.O.V) freedom (df) Squares (MS) solutions Pre-sterilizing (P) 7 0.220**

** Code of pre- Ascorbic Captan Commercial Ascorbic acid (S) 2 0.192 ** sterilizing acid fungicide sodium P×S 14 0.030 solution (mg L-1) (g L-1) hypochlorite (%) Time of sterilizing (T) 2 0.788** ** 1 300 2 5 P×T 14 0.038 ** 2 300 2 10 S×T 4 0.059 P×S×T 28 0.008** 3 300 4 5 Sodium hypochlorite 5 2.111** 4 300 4 10 concentration (N) 5 600 2 5 P×N 35 0.035** 6 600 2 10 S×N 10 0.024** 7 600 4 5 P×S×N 70 0.005** 8 600 4 10 T×N 10 0.057** P×T×N 70 0.006** S×T×N 20 0.008** Then, to achieve suitable sterilizing of explants and P×S×T×N 140 0.003** successful culture, the effects of different concentrations of Error 864 0.003 commercial sodium hypochlorite (0, 10, 20, 30, 40 and Total 1295 50%) and time of sterilizing (5, 10 and 20 minutes) were Note: **): significantly different at the 1% level. investigated. Likewise, different concentrations (0, 300 and 600 mg L-1) of ascorbic acid in the media were studied to eliminate the harmful effects of phenol compound that exist The presented data in Table 3 showed the results in explants at micro-propagation culture. The MS medium obtained from means comparisons of pre-sterilizing factor (Murashige and Skoog 1962) containing 30 g L-1 sucrose effect as ascorbic acid amount and their interaction with T. and 6.7 g L-1 agar was used for all experiments. The pH of platyphyllos explants viability by Duncan’s multiple range

PAYAMNOUR et al. – In vitro sterilization protocol for Tilia platyphyllos 9 test at the 5% level. As presented, factors were investigated The results obtained from a mean comparison of at five different groups. No. 7 pre-sterilizing treatment was sterilizing time effect on T. platyphyllos explants viability the best (first group) and showed 17.6% viability, which are presented in Table 4. Time of 5 minutes for sterilizing consisted of 600 mg L-1 Ascorbic acid, 4 g L-1 captan with 15.4% viability placed in the first group, 10 minutes fungicide and 5% commercial sodium hypochlorite. No. 8 with 11.9% in the second group and 20 minutes with 6.9% treatment showed 16.1% viability and placed in second viability in the third group. Therefore, it could be group Duncan’s multiple range test which consisted of 600 concluded that the viability percentage of explants mg L-1 ascorbic acid, 4 g L-1 captan fungicide and 10% decreased with the increasing time of sterilizing, and commercial sodium hypochlorite. Table 3 clearly indicated existing material at a sterilizing solution had a negative that these two groups revealed higher viability than other effect on explants viability. Thereupon, it is better to groups. In contrast, No. 1 pre-sterilizing treatment showed reduce the treating time with sterilizing solutions, if it is the minimum percentage of explants viability (6.7%) which possible. contained at least concentration of ascorbic acid, captan The data of the interaction between pre-sterilizing fungicide and sodium hypochlorite. factors and time of exposing at sterilizing solution on T. The results of mean comparison of ascorbic acid platyphyllos explants viability are shown in Table 4, that amount effect on T. platyphyllos explants (Table 3) showed indicated the highest value of free-contamination explants that under study factors set at two different groups with the (24.8% and 24.3%, respectively) were obtained when highest percentage of explants viability (13.8%) were explants were at a sterilizing solution for 5 minutes (No. 7 observed when higher amount of ascorbic acid (600 mg L- and No. 8 pre-sterilizing codes). It was evident that the pre- 1) were used. The results clearly indicated that, 0 and 300 sterilizing solution had less effect than sterilizing time. On mg L-1 ascorbic acid were non-significant and placed in the the other hand, with the increase of captan fungicide and second group with 10.1 and 10.2% viability, respectively. ascorbic acid in pre-sterilizing solution, viability increased, From the mentioned results, it can be concluded that with but increasing the time more than optimum time, negative the increase of ascorbic acid until 300 mg L-1, viability effects of solution increased and viability decreased increased a little, but the viability showed significant extremely. As, with increasing the time from 5 to 10 increase when ascorbic acid changed from 300 to 600 mg L-1. minutes, viability decreased and with increasing the time to The interaction between pre-sterilizing factor and 20 minutes, viability decreased even more. ascorbic acid in sterilizing solution on T. platyphyllos explants viability (Table 3) revealed that data were Table 4. The means comparison of explants viability (%) of T. classified into 18 groups according to viability. The highest platyphyllos in relation to pre-sterilizing factor effect, time of viability percentage (24.1) at No. 7 pre-sterilizing treatment sterilizing and their interactions was observed that contained 600 mg L-1 ascorbic acid, 4 g L-1 captan fungicide and 5% commercial sodium Pre- Time of sterilizing (minute) Mean of pre- -1 sterilizing sterilizing hypochlorite and sterilizing solution with 600 mg L code 5 10 20 factor ascorbic acid showed significant increase viability than 1 7.3ij 6.9ij 6.1jk 6.7 e other treatments. On the other hand, No. 1 pre-sterilizing fgh efg j d -1 -1 2 10.6 11.4 6.5 9.5 treatment containing 300 mg L ascorbic acid, 2 g L def fgh j d captan fungicide and 5% commercial sodium hypochlorite 3 12.8 12.5 6.7 10.1 c def j c and sterilizing solution without ascorbic acid showed the 4 15.5 12.6 6.6 11.5 cd gh k d minimum amount of viability. 5 14.6 9.4 4.2 9.4 6 13.5cde 10.2gh 6.5j 10.1 d Table 3. The means comparison of explants viability (%) of T. 7 24.8a 18.5b 9.6gh 17.6 a platyphyllos in relation to pre-sterilizing factor effect, ascorbic 8 24.3a 15.2c 9.0hi 16.1 b acid amount and their interactions Mean 15.9a 11.89b 6.88c -

Note: Means separated by Duncan’s multiple range test at alpha = Pre- -1 Mean of pre- Ascorbic acid (mg L ) 0.05. Means with the common letters were not significantly sterilizing sterilizing different (at 5% level) and they were in the same group. code 0 300 600 factor

n mn i-l e 1 4.4 6.4 9.3 6.7 lm k-h g-k d 2 7.4 10.1 11.0 9.5 The data in Table 5 showed the mean comparison of 3 9.9h-k 7.5lm 12.9d-g 10.1 d the sodium hypochlorite effect on viability of T. 4 8.3klm 11.5f-i 14.8cd 11.5 c platyphyllos explants. The viability of explants at 10% and 5 7.3lm 8.4klm 12.5efg 9.4 d 20% sodium hypochlorite was 25.6% and 19.5% at 6 9.4h-l 9.0jkl 11.8fgh 10.1 d sterilizing solution, respectively. Whereas, no explants 7 15.4c 13.5c-f 24.1a 17.6 a were alive in sterilizing solution without sodium 8 18.9b 15.3c 14.3cde 16.1 b hypochlorite and 100% of them were contaminated. It is Mean of 10.1 b 10.2 b 13.8 a - quite clear that increasing commercial sodium ascorbic acid hypochlorite, explants viability decreased and too much of Note: Means separated by Duncan’s multiple range test at alpha = commercial sodium hypochlorite might cause the viability 0.05. Means with the common letters were not significantly till zero. Thus, it can be concluded that sodium different (at the 5 % level) and they were in the same group.

10 NUSANTARA BIOSCIENCE 6 (1): 7-12, May 2014 hypochlorite is necessary, but should be used in minimum sterilizing solution without using sodium hypochlorite did amount. not show any viability. Concerning the interaction between the pre-sterilizing factors and the sodium hypochlorite amount on explants Table 6. The means comparison of explant viability (%) of T. viability (Table 5), the data indicated that the highest platyphyllos in relation to ascorbic acid, time of sterilizing and viability (40.9%) was related to pre-sterilizing No. 7 and their interactions 10% sodium hypochlorite. Viability difference between the Mean of pre- first and second groups showed the effect of commercial Time of sterilizing (minute) sterilizing sodium hypochlorite in pre-sterilizing solution that 5 10 20 factor demonstrated the minimum necessity of using commercial Ascorbic 0 11.8c 11.2cd 7.4e 10.1 b sodium hypochlorite at pre-sterilizing solution. To study acid (mg 300 14.7b 10.1d 5.7f 10.2 b the negative effects of commercial sodium hypochlorite we L-1) 600 19.6a 14.3b 7.5e 13.8 a increased up to 50% at sterilizing solution and it caused a Mean 19.8a 11.9b 6.9c remarkable reduction of explants viability. In all pre- Note: Means separated by Duncan’s multiple range test at alpha = sterilizing method, without using sodium hypochlorite in 0.05. Means with the common letters were not significantly sterilizing solution, there was not any viability observed. different (at 5% level) and they were in the same group.

Table 5. The means comparison of explant viability (%) of T. platyphyllos in relation to pre-sterilizing factor effect, sodium Table 7. The means comparison of explant viability (%) of T. hypochlorite and their interactions platyphyllos in relation to ascorbic acid, sodium hypochlorite and their interactions Mean of Pre- Sodium hypochlorite (%) pre- Mean of sterilizing Sodium hypochlorite (%) sterilizing pre- code 0 10 20 30 40 50 factor sterilizing 0 10 20 30 40 50 1 0t 15.9ni 12.41jkl 5.5o-r 4.2p-s 2st 6.7 e factor 2 0t 21.8ef 14.4hij 9.63lmn 7.96mno 4.14rst 9.5 d Ascorbic 0 Oh 22.9b 17.3c 11.5e 6.9f 2.2g 10.1 b 3 0t 2fg 17.2gh 11.1klm 8.3mno 3.7grs 10.1 d acid (mg 300 0h 22.8b 17.6c 11.1e 7.2f 2.4g 10.2 b 4 0t 26.7d 20.2fg 3.3ijk 6.8n-q 2.2rst 11.5 c L-1) 600 0h 31.1a 23.5b 15.4d 7.8e 3g 13.8 a 5 0t 2fg 15.7hi 10.9klm 7nop 2.8rst 9.4 d Mean 0f 25.6a 19.5b 12.7c 8d 2.6e 6 0t 23.5e 17.6gh 11klm 6.7n-q 1.7st 10.1 d Note: Means separated by Duncan’s multiple range test at alpha = 7 0t 41a 30.4c 2fg 12jkl 2.6rst 17.6 a 0.05. Means with the common letters were not significantly 8 0t 36b 28cd 2fg 11klm 2.4rst 16.1 b different (at the 5% level) and they were in the same group. Mean of 0f 25.6a 19.5b 12.7c 8d 2.6e - sodium hypochlorite Table 8. The means comparison of explant viability (%) of T. Note: Means separated by Duncan’s multiple range test at alpha = platyphyllos in relation to time of sterilizing (minute), sodium 0.05. Means with the common letters were not significantly hypochlorite and their interactions different (at the 5% level), and they were in the same group. Mean of Sodium hypochlorite (%) pre- As for the interaction effect between ascorbic acid sterilizing treatment and time of exposing explants to sterilizing 0 10 20 30 40 50 factor solution on explants viability (Table 6), the data indicated Time of 5 0h 31.6a 24.5b 17.8d 14e 4.4g 15.8 a that an increase of ascorbic acid in sterilizing solution had a sterilizing 10 0h 25.8b 21.8c 13.96e 7.2f 2.6g 11.9 b direct effect (increasing viability) and time of exposing (minute) 20 0h 19.4d 12.1e 6.25f 2.7g 0.8h 6.9 c explants to sterilizing solution had the reverse effect Mean 0f 25.6a 19.5b 12.7c 8d 2.6e (reducing viability) on explants viability. The highest Note: Means separated by Duncan’s multiple range test at alpha = viability (19.6%) was observed in 5 minutes-exposure to 0.05. Means with the common letters were not significantly the sterilizing solution containing 600 mg L-1 ascorbic acid. different (at the 5% level) and they were in the same group. Also, the minimum viability (5.7%) was observed in 20 minutes-exposure to the sterilizing solution containing 300 mg L-1 ascorbic acid. The results showed in Table 8 revealed that the highest The data obtained from the interaction between ascorbic percentage of viability (31.6%) was related to treatment acid amount and sodium hypochlorite at sterilizing solution with 10% sodium hypochlorite for 5 minutes. Other on explant viability (Table 7) pointed out that the highest treatments pointed out that the time of sterilizing, increased viability (31.1%) was related to 600 mg L-1 ascorbic acid the negative effects of sodium hypochlorite in sterilizing and 10% sodium hypochlorite. It can be concluded that the solution and with the increasing time and sodium minimum usage of commercial sodium hypochlorite and hypochlorite, viability decreased (severely). the maximum amount of ascorbic acid (600 mg L-1) was Also, the results of the mean comparison of interaction necessary to increase explant viability. The explants in effects between pre-sterilizing factors, ascorbic acid amounts, time of sterilizing and sodium hypochlorite amounts in sterilizing solution (the data were not shown)

PAYAMNOUR et al. – In vitro sterilization protocol for Tilia platyphyllos 11 implied that No. 7 pre-sterilizing with 600 mg L-1 ascorbic losses (George 1993; Altan et al. 2010). Fungi are from acid in sterilizing solution containing 10% sodium contaminations in plant tissue culture that may arrive with hypochlorite for 10 minutes and 5 minutes showed 68.3% explants, airborne or enter a culture (Babao et al. 2001; and 65% viability, respectively and they were the optimum Altan et al. 2010). In the present study, a fungicide was treatments for sterilization of T. platyphyllos explants. used to reduce fungal contaminations observed during the Meanwhile, Viability was not observed in treatments research. Observed fungi were identified and were reported without sodium hypochlorite and also with high in the other research that consisted of Penicillium sp., concentration of sodium hypochlorite (over 50%). Fusarium sp. and Alternaria sp. (Mehrdad et al. 2010). Shahrzad (1997) reported that using fungicide reduced Discussion remarkably fungal contaminations, which agrees with these Tilia platyphyllos is one of the valuable and high results. longevity (sometimes until 1000 years) plant species that The effectiveness of sterilization may depend on the exist individually under ecological and economic value of type, concentration and time of treatment with the sterilant beech and oak trees (Sabeti 1965). These typical trees are (Roxas et al. 1996; Tomaszeska-Sowa and Figas 2014). planted in parks and streets in Europe and America. The The best sterilization method was found for a solution leaves of this tree which are processed in tea and the honey containing 600 mg L-1 ascorbic acid and subsequently, 10% produced by bees which extract nectar from their v/v commercial sodium hypochlorite while the optimal are popular in the world, as well (Sadati et al. 2007). This time was 5 minutes. The explant viability decreased with species has been distributed from 100 to 1600 meters and the increment of sterilization time and materials in sterilizer even, sometimes, to 2400 meters above sea level (Sheikh solution that has a negative effect on explant viability. Al-Eslami and Namiranian 2002). Because of the wide Researchers working on sodium hypochlorite concentration range of altitude distribution and genetic variability and percentage of the surviving explants (Sharifkhani et al. preservation of this species and efforts in preserving the 2011) agree with mentioned results. Also, Researchers natural structure of Iran forests, the importance of this working with NaOCl reported that using sodium species is clear for everyone. Many studies demonstrated hypochlorite for 5-10 minutes was considered a suitable that producing T. platyphyllos seedlings have encountered time (Miller and Lipschutz 1984; Naik and Chandra 1993; serious problems such as weak germination in nurseries Gopal et al. 1998; Villafranca 1998; Badoni and Chauhan until now. It was an interesting fact that although mature 2010). seeds are healthy, their germination is low (Tabari et al. Due to poisonous effects resulted from high 2007). Although there are problems, using biotechnology concentration of sodium hypochlorite, all explants except tools such as tissue culture might be a proper solution. complete seeds that contained embryo preservative Tissue culture and regeneration of T. platyphyllos are membrane turned yellow and dried after several days. difficult and the success of regeneration is very low. Preservative membrane causes material transferring, Hence, regeneration may also be an effective technique for slowly. Sodium hypochlorite is necessary, but it should be multiple stock plants of this species. used at least amount. Increment of sodium hypochlorite One of important problems in case of T. platyphyllos or concentration will decrease the viability of T. platyphyllos every plant, which their explants collected from in vivo explants. Similar results were recorded by Shahrzad (1997) special forest areas, is the control of contamination at in using sodium hypochlorite and sterilizing time, but in that vitro condition. It is necessary to note, that pre-sterilizing is project HgCl2 was used. However, Shahrzad (1997) preliminary stage that can reduce contamination of explants reported that the optimum concentration of sodium in vitro. Although, this stage was conducted in laminar hypochlorite 1% and 2%, but in this study, the best airflow, which is not sterile, but this contamination could concentration of this sterilizer solution was 0.5%. not be included in the data in the independent stage for calculations. According to this point and achieved experiments in this study, it could not eliminate pre- CONCLUSION sterilizing effect from calculations and considered it as one of dependent on sterilizing stage. Therefore, with used In sterilization of T. platyphyllos, it was concluded that material at pre-sterilizing, eight treatments of pre- superabundant of phenolic compound was reduced by sterilizing were adopted and included in this investigation. using ascorbic acid. The optimum protocol was using pre- The present study demonstrated that the best sterilization sterilizing solution of 600 mg L-1 ascorbic acid, 4 g L-1 method was using 4 g L-1 captan and 600 mg L-1 ascorbic captan fungicide and 5% commercial sodium hypochlorite acid and 5% v/v commercial sodium hypochlorite. In (NaOCl) solution for 20 minutes. The results of present general, increasing chlorox concentration increased research will be applicable for propagation in large scale, percentage of contamination-free explants that presented conservation, breeding, and further genetic improvement results were coordinated with those of Abou Dahab et al. programs. (2005). Sharifkhani et al. (2011) demonstrated that the most suitable concentration of sodium hypochlorite for sterilization is 5%, which agrees with these results. A wide range of contamination has been identified in plant tissue culture that causes considerable economic

12 NUSANTARA BIOSCIENCE 6 (1): 7-12, May 2014

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