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ISSN : 1225-5009(Print)
ISSN : 2287-772X(Online)
Flower Research Journal Vol.24 No.2 pp.96-102
DOI : https://doi.org/10.11623/frj.2016.24.2.03

Effects of Different Medium Composition and Exogenous Hormones on Browning of Tree Peony (Paeonia suffruticosa Andr.) Callus in Tissue Cultu

Fang Fang Zhou1, Zheng Wang1, Li Yun Shi1, Jia Jia Niu2, Wen Qian Shang1, Dan He1, Song Lin He1
1College of Forestry, Henan Agricultural University, Wenhua Road, Jinshui District, Zhengzhou, Henan, P. R. China
2Institute of Horticulture, Henan Academy of Agricultural Science, Huayuan Road, Jinshui District, Zhengzhou, Henan, P. R. China


Corresponding author: Song-Lin He Tel: +86-0371-63558809 E-mail: hsl213@yeah.net ORCID: https://orcid.org/0000-0002-3462-4730
March 11, 2016 May 13, 2016 June 20, 2016

Abstract

Browning is one of the key factors that influenced the callus subculture of tree peony (Paeonia suffruticosa Andr.). Effects of medium composition and exogenous hormones: macro elements of Murashige and Skoog (MS salts) and iron salt (Fe2+), pH, agar and 6-benzylaminopurine (6-BA), 1-naphthaleneacetic acid (NAA) and kinetin (KT) on the callus browning of P. suffruticosa ‘Shan Hu Tai’ in vitro were studied in this paper. Results showed that the browning of P. suffruticosa callus were more sensitive to KT than 6-BA in different concentrations of 6-BA and KT separately with different concentrations of NAA, and reduced to the lowest (13.3%) under 0.5 mg·L-1 NAA plus 0.3 mg·L-1 KT. 1/4 × MS plus 1/4 × Fe2+ was the best basic medium in which the browning rate was only 18.2%. The browning rate of the callus was the lowest of 4.0% under pH 6.5 and the callus grew better in 7.0 g·L-1 agar than others. This study indicated that the best medium preventing P. suffruticosa callus in vitro from browning was: 1/4 × MS medium supplemented with 6.95 mg·L-1Fe2+, 0.3 mg·L-1 KT, 0.5 mg·L-1 NAA, 6.0 g·L-1 agar and 30 g·L-1 sucrose in pH 6.5.


agar , 6-BA , KT , NAA

초록

    Natural Science Foundation of China
    Project No.31272189
    Project No. 31400596Educational Commission of Henan Province of China
    Project No.2014A220003

    Introduction

    Plant tissue culture relying on the advantage of short-cycle and high propagation coefficient have become one of the most effective ways to solve the problem of rapid propagation in tree peony (Gao et al. 2001; Jaime et al. 2012). Scholars both domestic and abroad had carried out correlative researches and advanced in tree peony in viro (Liu et al. 2012). The browning in organization caused by the phenols became one of the key factors that restricted the development of tissue culture. Paeonia suffruticosa was a species of high phenols, and there also existed serious browning problems in the tissue culture (Lang and Luo 2007; Wang et al. 2009). The browning problem has been a critical factor to the callus differentiation and proliferation of P. suffruticosa in vitro (Chen et al. 2005; Wang et al. 2008; Wang et al. 2009; Wu 2003).

    The callus browning of P. suffruticosa in vitro is influenced by many factors, such as the species, variety, physiological state, selected parts of the material, medium composition, concentration and proportion of exogenous hormones and culture conditions (Fan et al. 2010; He et al. 2005; Wu 2003; Xiao and Yang 2001; Yin et al. 2012; Zhou et al. 2000). In recent years, researchers had focused on exogenous inhibitors and adsorbents, 1-ethenyl-2-pyrrolidinone homopolymer (PVP), AgNO3, vitamin C (Vc) and activate carbon, all that were applied to overcome the browning of P. suffruticosa in vitro but there were few satisfied results (An and Zhao 2005; He et al. 2005; Li et al. 2008; Li et al. 2008; Zhang and Luo 2006). Just a few researches were made on the effects of medium composition on the callus browning of P. suffruticosa in vitro.

    To explore the effects of medium composition on the browning of P. suffruticosa callus in vitro, the axillary buds of ‘Shan Hu Tai’ was used as plant materials in this study. The medium composition included the macro elements of Murashige and Skoog (MS salts), iron salt (Fe2+ ), agar, pH (measured with a pHS-3B meter, Hongyi Instrument Co., Shanghai, China), and different concentrations and proportions of exogenous hormone including 6-benzylaminopurine (6-BA), 1-naphthaleneacetic acid （NAA） and kinetin (KT) (all above reagents were from the Shanghai Just Scientific Co. Ltd., Shanghai, P. R. China). The study are expected to select the most appropriate medium that could control the browning effectively and offer a strong foundation for building the complete regeneration system of P. suffruticosa in vitro.

    Materials and Methods

    Plant material and culture conditions

    ‘Shan Hu Tai’, a cultivar of P. suffruticosa, with characteristics of dwarf and compact type, strong growth potential, high flowering rate, long florescence, crown-shaped flower and moderate blossom, was selected as the source of material. The initial explants were the axillary buds of ‘Shan Hu Tai’, then the petioles of the initial explants were cut into 1.0 cm in length after cultured 4 weeks in vitro and cultured on callus induction medium containing 1/2 × MS salts, 1.0 mg·L-1 6-BA, 0.5 mg·L-1 NAA, 30 g·L-1 sucrose, 1.0 g·L-1 PVP, 0.5 g·L-1 Lactoalbumin hydrolysate (LH), and 7.0 g·L-1 agar in the range of pH 5.8 - 6.0 with 0.1 M NaOH before autoclaving at 121℃ for 15 min at 0.2 MPa. After 40 d, under the same conditions, the callus was cut into 1.0 cm × 1.0 cm in size and obtained for the following experiments (Fig. 1A).

    All the treatments were kept at 24 ± 1℃ under white fluorescent lamps at a light intensity of 36 μmol·m-2·s-1 with a 12 h photoperiod.

    Exogenous hormones treatment

    The callus was cultured under the following 24 different treatments: 6-BA at the four concentrations of 0.0 mg·L-1, 0.5 mg·L-1, 1.0 mg·L-1 and 2.0 mg· L-1 combining separately with NAA at the three concentrations of 0.5 mg·L-1, 1.0 mg·L-1 and 2.0 mg·L-1; and KT at the four concentrations of 0.3 mg·L-1, 0.5 mg·L-1, 1.0 mg·L-1 and 1.5 mg·L-1 separately combining with NAA at three the concentrations of 0.5 mg·L-1, 1.0 mg·L-1 and 2.0 mg·L-1. All treatments were on the medium of 1/2 × MS + PVP (1.0 g·L-1) + LH (0.5 g·L-1) + sucrose (30.0 g·L-1) + agar (7.0 g·L-1), pH 5.8 - 6.0. The treatments adding NAA separately were served as the controls.

    MS salts and Fe2+ treatment

    The callus was trained under the 10 treatments: macro elements at the medium of 1/4 × MS and 1/2 × MS separately adding the Fe2+ salt at the five concentrations: 6.95 mg·L-1, 13.9 mg·L-1, 20.85 mg·L-1, 27.8 mg·L-1 and 34.75 mg·L-1. Additional ingredients of the medium contained 1.0 mg·L-1 6-BA, 0.5 mg·L-1 NAA, 1 g·L-1 PVP, 0.5 g·L-1 LH, 30.0 g·L-1 sucrose and 7.0 g·L-1 agar, and all were adjusted to pH 5.8 - 6.0.

    pH treatment

    The callus was cultured under the eight pH conditions: 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 on the medium of 1/2 × MS salts, 1.0 mg·L-1 6-BA, 0.5 mg·L-1 NAA, 1 g·L-1 PVP, 0.5 g·L-1 LH, 30.0 g·L-1 sucrose and 7.0 g·L-1 agar.

    Agar treatment

    The callus were cultured under the ten agar concentrations : 3.0 g·L-1, 3.5 g·L-1, 4.0 g·L-1, 4.5 g·L-1, 5.0 g·L-1, 6.0 g·L-1, 7.0 g·L-1, 8.0 g·L-1, 9.0 g·L-1 and 10.0 g·L-1 on the medium of 1 /2 × MS salts, 1 .0 mg·L-1 6-BA, 0.5 mg·L-1 NAA, 1.0 g·L-1 PVP, 0.5 g·L-1 LH, and 30.0 g·L-1 sucrose and all media were adjusted to pH 5.8 - 6.0.

    Growth measurements

    30 pieces callus were inoculated in each treatment (5 replicates of 6 pieces of callus per culture vessel that was 100 mL Erlenmeyer flask) and all treatments were repeated 3 times. The callus from each treatment was removed after 30 d, and the browning level was recorded every 2 d in the first 10 d, then every 5 d during the following 20 d. The browning rate of callus was calculated as: (browning callus number/total inoculated number) × 100.

    The browning degree of the callus referred to the following four levels : level 0 (no browning and the surrounding medium of the callus presented white)， level 1 (some began appearing drab), level 2 (most were browning and the surrounding medium turned up brown), level 3 (all were browning to death).

    Data analysis

    The analysis of variance was performed with Data Processing System (DPS) software version 3.01 (Refine Information Tech. Co., Ltd., China) and Excel 2010, and significant differences between the treatments was assessed by D uncan’s multiple range test ( DMT) a t P ≤ 0.05.

    Results and Discussion

    Effects of different combinations of exogenous hormones on the browning of P. suffruticosa callus

    The browning rates of the callus cultured under 0.3 mg·L-1 KT plus NAA were lower than those of others (Table 1), under which the callus grew even better than others and the browning level were mainly at 0 - 1. The browning rates and levels of the callus increased gradually as the concentrations of 6-BA increasing at the same concentration of NAA. The browning rates and levels of the callus were much lower under 0.3 mg·L-1 KT than others under the same concentration of NAA. The browning levels of the callus under different KT concentrations were higher than those of callus under different 6-BA concentrations in the same concentration of NAA.

    The browning rate and level of the callus reached the lowest (13.3%, level 0 - 1) under the treatment of 0.5 mg·L-1 NAA plus 0.3 mg·L-1 KT, followed by the treatment of 2.0 mg·L-1 NAA plus 0.3 mg·L-1 KT (20.0%, level 0 - 1). However, the browning rate and level of the callus went up to the highest (65.0%, level 3) under the treatment of 2.0 mg·L-1 NAA plus 0.5 mg·L-1 KT.

    The activities of the cell division and the callus growth could be induced only when the basic medium matched with appropriate plant hormones (Tan and Dai 2001). Zhang et al. (1999) inferred that selecting the best growth regulator to accelerate the growth and differentiation of explants was an effective approach to overcome browning. The Exogenous hormone had great regulation effect on the browning of ginkgobiloba callus (Xu et al. 1999). Wang and Yue (2009) showed that 6-BA at the concentrations of 2.0 mg·L-1 was easy to lead to browning, but 2.0 mg·L-1 KT plus 2.0 mg·L-1 NAA brought less effect to tissue browning of Iris germanica. Zhang et al. (2002) found that adding 6-BA had no effect on the browning in black pine calli. In this study, we found that the callus browning of P. suffruticosa ‘Shan Hu Tai’ was also affected greatly and more sensitive to the change of KT concentration than that of 6-BA. Our result also clearly showed that the browning rates and level of callus was significantly lower (13.3%, level 0 - 1) under 0.3 mg·L-1 KT plus 0.5 mg·L-1 NAA than others (Fig. 1B). It may be related to the varieties of the plant.

    Effects of different combinations of MS salts and Fe2+ s alt medium on the browning of P. suffruticosa callus

    The browning rates and levels of ‘Shan Hu Tai’ callus cultured under different Fe2+ concentrations except 13.9 mg·L-1 in 1/4 × MS salts were significantly lower than those of callus in 1 /2 × MS salts (Table 2 ). As the concentration of MS salts was definite and the Fe2+ concentration ranged from 6.95 mg·L-1 to 34.75 mg·L-1, the browning rates and levels of callus increased. The browning rates and levels of callus under 6.95 mg·L-1Fe2+ were obviously lower than that of the controls. The browning rates and levels of callus under 1/4 × MS salts with 6.95 mg·L-1Fe2+ were the lowest (18.2%, level 0 - 1) (Fig. 1C) and the highest (85.7%, level 3) under 1 /2 × MS salts with 3 4.75 mg·L-1Fe2+.

    The basic medium ensured the minimum physical activity of the culture survival (Pan et al. 2004). Zhang et al. (1999) and Lin et al. (2007) pointed out that high concentration of inorganic salts in medium would cause plenty of phenol generated and lead to browning in explants, and the decrease of concentration inorganic salts could reduce the phenol and relieve the browning of explants. Similar conclusions of the relationship between the browning and Fe2+ were made in the test of the tea tree callus induced from the stem (Zhao 2008). It was confirmed that the browning rate decreased greatly as the concentration of the inorganic salts halved in the DKW medium of the walnut in vitro (Liu 2002). Xu and Li (2006) also found that the browning ratio of Phalaenopsis sp. explant in vitro on 1/2 × MS was less than that cultured on MS, and doubling the content of Fe2+ could increase the browning rate of the explants. We also found that the browning rates of callus increased with the Fe2+ concentration in the range of 6.95 mg·L-1 to 34.75 mg·L-1, and 1/4 × MS salts was more suitable than 1/2 × MS salts for P. suffruticosa callus.

    Effects of different pH values on the browning of P. suffruticosa callus

    The differences of the callus browning rates and level under different pH were distinct in different cultural time and pH conditions (Table 3). As the culture time extended, the browning rate of callus decreased except pH 4.0. As the pH increased, the browning rate of callus decreased first and then increased after 30 d. As the increasing of the pH value, the state of the callus became more stable. The callus browning rate and level was the lowest (4.0%, level 0) under pH 6.5 and pH 6.0 after 30 d. However, the callus browning rates and levels in pH 6.5 were lower than that in pH 6.0 during the whole culture time. The browning rate and level of the callus was the highest under pH 4.0 (43.8%, level 2 - 3).

    Low pH value could decrease the activity of polyphenol oxidase and utilization rate of substrate, and relieve the browning of explants (Lin et al. 2007). In this study, we found that the callus browning rate and level of P. suffruticosa decreased first and then increased after 30 d as the pH decreased, the callus browning rate and level of P. suffruticosa was the highest in the pH (4.0), and the suitable pH value (6.0) was benefit to the growth of P. Suffruticosa callus (Fig. 1D). Researches have shown that pH condition affected the state of medium greatly, and the medium of P. suffruticosa were in liquid in the study. It could be inferred that the high humidity of the medium state was harmful to the growth of the callus, and it led the browning of P. suffruticosa callus worsen in the later growth state at pH 4.0.

    Effects of different concentrations of agar on the browning of P. suffruticosa callus

    There were significant differences in the callus browning rates and level under different concentrations of agar (Table 4). The callus browning rate and level in P. suffruticosa showed different tends as time went by. They decreased continually in the range of 6.0 - 8.0 g·L-1 and 4.0 g·L-1, and increased in the range of 9.0 - 10.0 g·L-1 of the agar. The contrary trend obviously existed between the ranges of 3.0 - 3.5 g·L-1 and 4.5 - 5.0 g·L-1 in the agar. As the concentrations of agar increased, the browning rates and level of callus decreased first and increased then after 30 d. The browning rate and level was the lowest (5.0%, level 0) under 6.0 g·L-1 agar after 30 d, and we found new callus generated from the original browning parts under 6.0 g·L-1 agar (Fig. 1E). The browning rate and level of the callus was the highest (56.2%, and level 3) under 10.0 g·L-1 agar after 30 d (Fig. 1F).

    Bad state of cell growth, deteriorated culture conditions and accumulation of harmful substances would cause the polyphenol compounds oxidized and tissue browning under the effect of polyphenol oxidase (Xu et al. 1999). High concentration of agar could poison the materials in vitro and caused the callus browning for reason that the hard medium restricted the spread of nutrients and phenol, reduce moisture content of the medium and made the harmful material piled up (Lin et al. 2007; Tan and Dai 2001). It just explained the phenomenon that the browning rate and level increased as the concentration of agar ranged from 6.0 g·L-1 to 10.0 g·L-1. However, Xu and Li (2006) also found that reducing the content of agar increased the level of browning. The browning in vitro might be a kind of stress response to the environment stress for plant cell (Feng et al. 2015). This study showed that moderate hardness of medium was beneficial to inhibit browning of P. suffruticosa callus, either underweight or overweight of the medium hardness was not conducive to P. suffruticosa callus growth and even increased the browning rate and level. We also concluded that the P. suffruticosa callus in vitro might have a stress adaptation to different agar concentrations based on the phenomenon that the callus browning rates and level declined with the agar concentration from 4.0 g·L-1 to 8.0 g·L-1 during the later culture time.

    The browning rate and level of P. suffruticosa callus decreased greatly under low concentration of inorganic salts(macro elements and Fe2+ ) combining with appropriate concentration of KT and NAA, and the proper pH value and agar dosage could prevent effectively the browning of P. suffruticosa ‘Shan Hu Tai’ callus. We also found an interesting phenomenon that the results about agar (6.0 g·L-1) and pH (6.0) were just similar to the best wild environment of faintly acid and sandy soil for P. suffruticosa, it just prove that the medium we concluded is close to the natural growing conditions of P. suffruticosa. And the results build a strong foundation for overcoming the callus browning and further study in P. suffruticosa.

    Acknowledgements

    We thank Meng XY, Lv BY, L i XJ, Xie MJ and Yang DJ for their contributions to this research. This work was funded by the “Natural Science Foundation of China (Project No. 31272189 and Project No. 31400596)” and “Educational Commission of Henan Province of China (Project No. 2014A220003)”.

    Figure

    669_F1.jpg

    Callus initial (A); Callus under 0.5 mg·L-1 NAA plus 0.3 mg·L-1 KT after 30 d (B); Callus under 1/4 × MS salts plus 1/4 × Fe2+ after 30 d (C); Callus under pH 6.0 after 30 d (D); Callus under 6.0 g·L-1 agar after 30 d (E); Callus under 10.0 g·L-1 agar after 30 d (F).

    Table

    Changes of callus browning rates in different exogenous hormone combinations of NAA, 6-BA and KT medium in Paeonnia suffruticosa ‘Shan Hu Tai’.

    zRange (browning rate of the callus) = Xmax (browning rate of the callus) – Xmin (browning rate of the callus)
    yValues are the means of 90 explants (three replicates of thirty explants) and different letters show significant differences between treatments at P ≤ 0.05 by DMRT. The follows were the same.

    Changes of callus browning rates in different combinations of MS salts and Fe2+ salt medium in P. suffruticosa ‘Shan Hu Tai’.

    zValues are the means of 90 explants (three replicates of thirty explants) and different letters show significant differences between treatments at P ≤ 0.05 by DMRT. The follows were the same.

    Changes of callus browning rates in different pH mediums in P.suffruticosa ‘Shan Hu Tai’.

    zValues are the means of 90 explants (three replicates of thirty explants) and different letters show significant differences between treatments at P ≤ 0.05 by DMRT. The follows were the same.

    Changes of callus browning rates in different agar concentrations medium in P. suffruticosa ‘Shan Hu Tai’.

    zValues are the means of 90 explants (three replicates of thirty explants) and different letters show significant differences between treatments at P ≤ 0.05 by DMRT. The follows were the same.

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    2. Journal Abbreviation : 'Flower Res. J.'
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      Doi Prefix : 10.11623/frj.
      ISSN : 1225-5009 (Print) / 2287-772X (Online)
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