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��ࡱ�>��	�����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������[�	�R���bjbj����<�ΐΐ�Y%�������:	:	������������8��k,�?���"������edE>G>G>G>G>G>G>$�@�lCk>i�\$��\$\$k>������>�7�7�7\$�����E>�7\$E>�7�7w:�:�����@czb���<38�:1>�>0?�:zDt5�zD�:�:&zD��:l�V��7������k>k>,6����?\$\$\$\$��������������������������������������������������������������������zD���������:	I:	The effects of growth regulators and illumination intensity on anthocyanin production in Psammosilene tunicoides callus

Hong-Bian Wei, Ming-Sheng Zhang*, Yan-Yan Gao, Xiao-Hong Wang, Li Tian, Jian-Dong Liu
School of Life Sciences, Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang, 550025 Guizhou, People�s Republic of China
* Corresponding Author: School of Life Sciences, Guizhou University, 14, Xia-hui Rd., Guiyang City, China. Fax: 86-851-83856374, E-mail: mshzhang@163.com.
Hong-Bian Wei: School of Life Sciences, Guizhou University, Guiyang, 550025 Guizhou, People�s Republic of China. E-mail: 13984322059@163.com.
Yan-Yan Gao: School of Life Sciences, Guizhou University, Guiyzng, 550025 Guizhou, People�s Republic of China. E-mail: yygaoluck@163.com.
Xiao-Hong Wang: School of Life Sciences, Guizhou University, Guiyzng, 550025 Guizhou, People�s Republic of China. E-mail: swuwxhong@163.com.
Li Tian: School of Life Sciences, Guizhou University, Guiyzng, 550025 Guizhou, People�s Republic of China. E-mail: 1254886015@qq.com.
Jian-Dong Liu: School of Life Sciences, Guizhou University, Guiyzng, 550025 Guizhou, People�s Republic of China. E-mail: 534875761@qq.com.
Abstract
Psammosilene tunicoides is a specific monotypic genus medicinal plant with high medicinal value in China. In addition to the root can be used as medicine, its stems, leaves and flowers contain amounts of anthocyanin . In resent years, because of the prominent effect of plant anthocyanin in antioxidation, anti-inflammatory, cardioprotective, anticancer and antitumor, so they have been favored gradually. In order to make full development and utilization of anthocyanin resources, we have designed in this study. The plant tissue culture technology was used to induce callus from the stems, leaves and buds of P. tunicoides in this experiment. By adjusting the concentration of growth regulators (NAA, 2,4-D and 6-BA) in MS basic medium, and the illumination intensity, making the callus of P. tunicoides to produce high levels of anthocyanins. The results show that the bud induction rate was about 1 to 2 times high of others. NAA or 6-BA contributed to induce red anthocyanin synthesis in the white callus. With the increase of 6-BA concentration, the anthocyanin content increased after slightly decreased, and the anthocyanin synthesis was promoted by increasing NAA concentration from 0.5 mg l-1 to 1.5 mg l-1 and decreased thereafter up to 2.5 mg l-1 NAA. The combination screening of growth regulators indicated the combination of 1.5 mg l-1 NAA+0.5 mg l-1 6-BA was the appropriate condition in the range of 0.5 mg l-1 to 2.5 mg l-1. The illumination intensity of 2000 lx was fit for anthocyanin formation in callus. The synthesis of anthocyanin in callus was repressed while adding 2,4-D into medium. The enhance of anthocyanin induction in callus by elicitors is largely due to 0.5 mg l-1 6-BA, 1.5 mg l-1 NAA and 2000 lx illumination intensity. And 2,4-D had significantly inhibition to the expression of anthocyanin genes.
Key words: Psammosilene tunicoides, tissue culture, callus, anthocyanin, growth regulator, illumination intensity
INTRODUCTION
In recent years, the natural plant pigment has been favored gradually. And it as additives in foods and drinks is a significant factor influencing food manufacture, cosmetics industry and health care. Anthocyanin as a natural colorant has widely used in people's daily life, not only because it is less harmfulness than artificial color, but it also exhibit significant antioxidant activity (Claudia et al., 2011). The potent chemoprevention attribute of anthocyanins and related flavonoids have recently been demonstrated in anti-inflammatory (Wang and Mazza, 2002), cardioprotective (Morazzoni and Magistretti, 1990; Brindle and Timberlake, 1996) and anticancer (Castonguay et al., 1997), antitumor (Bagchi et al., 2004).
Anthocyanins belong to the plant secondary metabolites exist widely in plant roots, stems, leaves, flowers, fruits and storage organs with colors from scarlet to blue. But extraction of the anthocyanin from plant is often tedious and expensive as the plant is seasonal. Therefore, anthocyanin production by tissue cultures (Zhang and Shintaro, 1999) has been widely reported in many medicinal plant species, such as Alkanna tinctoria (Giovanni et al., 1994) and Carthamus tinctorius (Gao et al., 2000). Psammosilene tunicoides is a characteristic monotypic genus medicinal plant with high medicinal value in China. In addition to the root can be used as medicine, its purple flowers and stems also can provide as experimental materials.
Callus culture is an effective and safe approach to produce pigments (Smetanska, 2008). In vitro induction of pigments in plant tissue culture is dependent on the culture medium, temperature, pH, rate of aeration, and level of illumination (Aksu and Eren, 2005). Earlier studies indicated that induction of anthocyanin was strongly influenced by high sucrose concentration (Hennayake et al., 2006) and low NH4+ nitrogen concentration in the suspension culture media (Piovan and Filippini, 2007). Most in vitro studies have shown evidence for some effects of light on the accumulation of pigments in cultured cells (Boehm et al., 1991). Others demonstrated that auxin alone significantly inhibited anthicyanin biosynthesis. Co-culture of auxin and cytokinin significantly promoted the cytokinin induced increase in anthocyanin levels (Ji et al., 2014). Therefore, in this research we investigated the different auxin (NAA, 2,4-D), cytokinin (6-BA) and illumination intensity on the effects of anthocyanin biosynthesis for producing high anthocyanins using P. tunicoides callus cultures.

MATERIALS AND METHODS
Induction of white callus
This study was carried out using young stems, leaves and buds by aseptic seeding of P. tunicoides cultivated on MS basic medium (Murashige and Skoog, 1962) containing 6-BA, 2,4-D, 3% (w/v) sucrose and 0.7% (w/v) agar. The pH of the medium was adjusted to 5.8 before autoclaving at 121 ! for 20 min. The culture conditions were (25 � 1) ! and illumination intensity of 1000 lx during 12 h/d photoperiod. Choice of explants from the seeds sprouted about 30 days later. Explants were placed respectively on Petri dishes containing MS medium, 5 plants per Petri dish with five repetitions for callus induction. After 25 days, significant differences among the variants were estimated by analysis of variance (ANOVA).
Induction and accumulation of anthocyanin
This experiment determined the optimal conditions of pigment production using auxin (NAA) and cytokinin (6-BA). Experiments on anthocyanin induction in the white callus were performed on MS medium supplemented 3% (w/v) sucrose and different concentrations and combination of the growth regulators 6-BA and NAA, addition of 6-BA ranging from 0 to 2.5 mg l-1 in increments of 0.5 mg l-1, with NAA ranging from 0 to 2.5 mg l-1. In addition, we studied the effects of 2,4-D on the accumulation of anthocyanin from callus that have been inducted anthocyanin.
The effects of illumination intensity for anthocyanin
As early as 1995, an article was reported that betalain pigmentation in callus was induced by blue and blue/UV lights (Kishima et al., 1995). The different illumination intensity was detected by review of the literature. In this experiment, the effect of different illumination intensity (0, 500, 1000, 1500, 2000, 2500 lx) for anthocyanin induction was examined with callus cultivated on MS medium with 6-BA and NAA. The callus was weighed about 0.5 g fresh weight (FW) to every Petri dish with five repetitions.
Determination of callus growth
The growth of callus was monitored by measurement of fresh and dry biomass. Water on the surface of the callus was absorbed with filter paper, just until the point when free liquid was no longer expressed in paper and then fresh weight (FW) was recorded. After that, biomass was stoved in high temperature drying oven after 24 h, then dry weight (DW) was calculated.
Extraction of anthocyanin
The callus quantity (1 g, FW) was weighted as described previously for extracting anthocyanin. Extraction was performed using 1.0% HCl-MeOH (1:50 w/v). Treating callus was extracted overnight in test tube (25 ml) at 4 ! for anthocyanin measurement (Fang, 1998). The extract was filtered through filter paper to 10 ml brown glass flask volumetric, and anthocyanin content was expressed from absorbance measurement.
Statistical analysis
During the white callus induction phase, the total number and fresh/dry weight of callus in each treatment were counted. During the anthocyanin production phase, the weight of callus which had induced and accumulated anthocyanin were counted to calculate the average anthocyanin content of each treatment. But in this paper, we adopted the direct method (A=A530) to determinate the content of anthocyanin in the same solvent (Huo et al., 2005). All extracts were prepared in triplicate. In order to find the characters conditions of anthocyanin production, a P-value < 0.05 was considered statistically significant by Tukey�s post hoc text. Correlation was evaluated using the Pearson correlation analysis. All experimental data were expressed as means � standard deviation (SD) of five independent replications.

RESULTS AND DISCUSSION
Induction of callus from P. tunicoides
The callus initiated from different explants were slight red colors and friable on medium containing different factors, which combination that yielding optimal callus formation. It was indicated that the explants with the treatment of cut were inducted callus firstly after one week, and the callus of buds grew obviously better than others (Fig.1 A, B, C). Since there are no optimal condition to culture anthocyanin, the anthocyanin of callus could not be accumulated. So the white callus was gradually increased in the culture during 25 days.
The result showed that the main influence factor of callus induction was explants (Serial No. 7 to 9 in Table 1). The great number of callus forming was obtained in 0.5 mg/L 2,4-D and 1.0 mg/L 6-BA (Serial No. 1 in Table), which the callus fresh weight was 3.11 g inoculated onto treatment medium. The result not much differed from those of (Li et al., 2011), who obtained callus from young stems of P. tunicoides on MS medium supplemented with various hormones in dark condition. In the inoculation of buds media, biomass for callogenesis were well and quickly. The callus was soft in texture, friable in structure and white after 25 days. Callus induction from various tissues were different (Abdul et al., 2014). The article was reported that soft and friable callus were developed from shoot primordia, hard and compact callus developed from 2 weeks old buds of Zingiber officinale cultured on MS medium (Vincent et al., 1992).
Induction of anthocyanin from callus
The effects of hormones combinations on anthocyanins induction in plants culture has previously been reported (Meyer and van Staden, 1995). But has no reported about inducing anthocyanin from P. tunicoides. We have demonstrated in this test that anthocyanin can be induced by 6-BA and NAA. It has been recognized that anthocyanin formation in callus was under three different systems involving 6-BA, NAA and 6-BA + NAA combination. According to the spectrogram of pigment extracting liquid of P. tunicoides callus, we found the anthocyanins characteristic absorption peak (n=529.0355, Figure 2).
Effect of 6-BA or NAA to the multiplication of P. tunicoides callus and anthocyanin were shown in Table 2. NAA or 6-BA alone inhibited significantly callus growth and multiplication with the increase of its concentration. The callus cultured in 1.0 mg/L 6-BA (0 mg/L NAA) or 0.5 mg/L NAA (0 mg/L 6-BA) exhibited the highest of callus multiplication. But 2.0 mg/L 6-BA and 1.5 mg/L NAA resulted in more anthocyanins production (Abs=0.399 and 0.326, respectively), high concentrations of NAA such as 2.0 and 2.5 mg/L resulted in low anthocyanin production. (Table 2).
Co-treatment of auxin and cytokinin would significantly enhance the anthocyanin (Ji et al., 2014). On this basis, adding different concentrations NAA (0.5 to 2.0 mg/L) to MS medium supplemented with 6-BA (0.5 to 2.0 mg/L) were tested to see if they would increase anthocyanin content. The results indicate that the combination effect of 0.5 mg/L 6-BA and 1.5 mg/L NAA was the appropriate condition in the range 0.5 to 2.5 mg/L (Table 3). So auxin and cytokinin displayed the interaction of controlling anthocyanin synthesis. But in the medium with 1.5 mg/L NAA, we just added low concentration 6-BA to induct maximum anthocyanin. Too high 6-BA concentration inhibited anthocyanin synthesis. And during in the subculture (0.5 mg/L 6-BA and 1.5 mg/L NAA), callus were white and anthocyanin formation was stable gradually.
Accumulation of anthocyanin from callus
The anthocyanin accumulation of callus culture was not suppressed by used growth regulators at the present combination and concentration. In other cases, 2,4-D was observed to inhibit the accumulation of anthocyanin in vitro (Makunga et al., 1997). Then we cultured callus formed anthocyanin upon the addition of different concentrations 2,4-D (0, 0.5, 1.0, 1.5, 2.0 mg/L) to find optimal condition. Our results show that P. tunicoides anthocyanin can be maximum accumulated without the addition of 2,4-D (picture N in Figure 3 and Table 4), but the callus weight in anthocyanin accumulation medium supplement with 2,4-D was higher than the medium without 2,4-D (Table 4). In other words, 2,4-D could reduce anthocyanin yield in P. tunicoides callus culture but increased callus biomass. This may be due to the influence of 2,4-D as suppressor to increase the incorporation of phenylalanine into amino acids. The phenylalanine was reduced by this conjugation reaction, and it was acted on by the enzymes of the phenylpropanoid pathway. The products of phenylpropanoid metabolism were the precursors for anthocyanin biosynthesis (Makunga et al., 1997). As we known that anthocyanin formation were controlled by anthocyanin genes that included structure genes (CHS, CHI, F3H, DFR) and regulatory genes (MYB10, bHLH3). There are other reported that 2,4-D inhibited the transcription of regulatory genes, the structure genes that were regulated by the regulatory genes also decreased. Therefore, the anthocyanin genes expression were inhibited by 2,4-D (Ji et al., 2014).
The requirement of P. tunicoides callus for light in order to induce anthocyanin is shown in Figure 3 (G, H, I, J, K, L). We found that callus exposed to illumination intensity (2000 lx, light-dark cycle of 12 h) accumulated anthocyanin to the highest Abs (0.528, Table 4). With the increase of illumination intensity, the Abs (pigment content) increased, and decreased there after up to a illumination intensity 2500 lx. Previous experiment with C. tinctorius anthocyanins were performed under light and dark conditions. It was shown that pigment production was greater in the light than in the dark (Gao et al., 2000). The reason is that the genes associated with anthocyanin biosynthesis could be activated by light irradiation. In this way, a large of proteins that was an indication of increased gene expression would be produced by the red callus. Signal transduction mechanisms as a role of complexion contacted with light-mediated control of anthocyanin gene expression. Some reports showed that the expression of anthocyanin gene was regulated by UV-B receptors and was also stimulated by blue and red light (Kishima et al., 1995 and Molchan et al., 1996).
According to our observation, P. tunicoides callus hardly grew in anthocyanin accumulation medium, and the callus became dead after produced anthocyanin. The direct cause behind this could be that anthocyanin belong the result of the secondary metabolism. During the process of secondary metabolites accumulation, the cell growth and production forming would be inhibited until the secondary metabolites to achieve the maximum, except the release of secondary metabolites or other regulate of artificial methods. End products caused the feedback inhibition of the first or the previous enzyme to result in the decrease of anthocyanin induction. Two reasons lead to plant cell could not growth normally, even dead between the slight toxicity and abound of end products.

CONCLUSION
Final conclusion, the present work demonstrated that growth regulators as 6-BA and NAA have its greatest influence on stimulating anthocyanin synthesis pathway to primary metabolism in P. tunicoides callus culture. The enhance of anthocyanin induced by elicitors is largely due to 0.5 mg/L 6-BA, 1.5 mg/L NAA and 2000 lx illumination intensity. Besides, 2,4-D had significantly inhibition to the expression of anthocyanin genes.

ABBREVIATIONS
Abs          Absorbance
DW          Dry weight
FW          Fresh weight
MS          Murashige and Skoog
NAA        �-Naphthaleneacetic acid
6-BA        6-Benzylaminopurine
2,4-D        2,4 Dichlorophenoxyacetic

AUTHORS  CONTRIBUTION
HBW and MSZ conceived and designed the study. HBW carried out the induction of anthocyanin from callus and drafted the manuscript. XL performed the statistical analysis and coordination and helped to draft the manuscript. XLL and LT participated in the induction and proliferation the callus. All authors read and approved the final manuscript.

ACKNOWLEDGEMENTS
This research was funded to MSZ by a grant from the National Key Research and Development Project in China (No. 2016YFC050260403), the Major Special Project of Science and Technology Plan in Guizhou of China (No. 2017-5411-06 and 2017-5788), the Science and Technology Support Plan Project in Guizhou of China. (No. 2018-2797), the Special Fund of Science and Technology Innovation Talent Team Construction in Guizhou of China (No. 2016-5624), the Project of High-level Innovative Talents in Guizhou of China (No. 2015-4031), the Major Projects of Innovation Group in Guizhou of China (No. 2016-023) and the Modern Industrial Technical System Construction Project of Chinese Medicinal Materials in Guizhou of China (Grant No. GZCYTX-02). 
Declarations
This research belongs to the research project of the tutor, which is accomplished by the unified guidance of the tutor in the same laboratory. There is no conflict of interest. All the data and materials in this paper are reliable and available from our lab.

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