Research Article, J Plant Physiol Pathol Vol: 7 Issue: 2
The Effects of Growth Regulators and Illumination Intensity on Anthocyanins Production in Psammosilene tunicoides Callus
Hong-Bian Wei, Yan-Yan Gao, Ming-Sheng Zhang*, Xiao- Hong Wang, Xue Li, Li Tian, Si-Jia Liu and 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 : Ming-Sheng Zhang
School of Life Sciences, Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, 14, Xia-hui Rd., Guiyang City, China
E-mail: [email protected]
Received: February 12, 2019 Accepted: March 28, 2019 Published: April 05, 2019
Citation: Wei H, Gao Y, Zhang M, Wang X, Li X, et al. (2019) The Effects of Growth Regulators and Illumination Intensity on Anthocyanins Production in Psammosilene tunicoides Callus. J Plant Physiol Pathol 7:2. doi: 10.4172/2329-955X.1000199
Psammosilene tunicoides. In this study, the stems, leaves and buds of P. tunicoides were used as explants, adding growth regulators (NAA, 2,4-D and 6-BA) in MS basic medium, and by adjusting the concentrations of growth regulators and the illumination intensity in culture room to induce callus, then the proliferated callus were used to extract anthocyanins. The results showed that the callus induction rate was the highest using buds as explants; it was about 1 to 2 times high of others (stems and leaves). NAA or 6-BA contributed to induce anthocyanins synthesis in the white callus. With the increase of 6-BA concentration, the anthocyanins content increased after slightly decreased, and the anthocyanins synthesis was promoted by increasing NAA concentration from 0.5 mg·L-1 to 1.5 mg·L-1 but decreased thereafter up to 2.5 mg·L-1. The combination screening of growth regulators indicated that 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 anthocyanins formation in callus. The anthocyanins synthesis in callus was repressed when 2,4-D was added into medium. The results will provide reference for the pigments production in callus culture of P. tunicoides.
Keywords: Psammosilene tunicoides; Tissue culture; Callus, Anthocyanins; Growth regulator; Illumination intensity
2,4-D: 2,4-Dichlorophenoxyacetic Acid; 6-BA: 6-Benzylaminopurine; Abs: Absorbance; DW: Dry Weight; FW: Fresh Weight; MS: Murashige and Skoog Medium; NAA: α-Naphthaleneacetic Acid
Psammosilene tunicoides is a single-species genera and Chinese endemic plant; it is also a rare and extinct medicinal plant with high medicinal value. In addition to the root can be used as medicine, its stems, leaves and flowers contain amounts of anthocyanins (secondary metabolites in plants). In recent years, plant anthocyanins have been gradually favored by people because of their prominent effects in anti-oxidation , anti-inflammation , anti-tumor , anti-cancer  and cardioprotection [5,6]. Anthocyanins are abundant in plant of P. tunicoides, but the production of anthocyanins from P. tunicoides has not been reported yet.
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 anthocyanins from plant is often tedious and expensive as the plant is seasonal, and it is also devastating. It is a good method to produce anthocyanins by plant callus culture, and some reports have been made on this aspect [7-10].
In vitro induction of pigments in plant tissue culture is dependent on the culture medium, temperature, pH, rate of aeration, and level of illumination . Earlier studies indicated that induction of anthocyanins was influenced strongly by high sucrose concentration  and low NH4+ nitrogen concentration in the suspension culture media . Most in vitro studies have shown evidence for some effects of light on the pigments accumulation in cultured cells . Others demonstrated that auxin alone significantly inhibited anthocyanins biosynthesis, but co-culture of auxin and cytokinin increased significantly the anthocyanins levels . Therefore, in this test, the authors explored the effects with auxin (NAA, 2,4-D), cytokinin (6-BA) and illumination intensity for anthocyanins biosynthesis in callus culturesof P. tunicoides, so as to provide a theoretical basis for sustainable development and utilization of the anthocyanins resourcesfrom P. tunicoides.
Materials and Methods
Induction of white callus
The young stems, leaves and buds from asepsis seedlings of P. tunicoides were inoculated on MS basic medium  containing 2,4-D (0.5 to 1.5 mg·L-1), 6-BA (0.5 to 2.0 mg·L-1), sucrose (30 g·L-1) and agar (7 g·L-1) by orthogonal experiment L9(33) (Table 1). The pH of the medium was adjusted to 5.8 before autoclaving at 121°C for 20 min. The culture conditions were (25 ± 1)°C and illumination intensity of 1000 lx with 12 h·d-1 photoperiod. The explants were placed respectively on Petri dishes containing MS medium, 5 explants per Petri dish with five repetitions for callus induction. After 25 days, significant differences among the treatments were estimated by analysis of variance (ANOVA).
|Serial number||Explants（g）||2,4-D (mg·L-1)||6-BA (mg·L-1)||FW (g)|
|Means with different letters indicate significant level at p=0.05.|
Table 1: The callus induction of P. tunicoides by L9(33).
Induction of anthocyanins by growth regulators
This study determined the optimal conditions of pigment production using auxin (NAA) and cytokinin (6-BA). The experiments concerning anthocyanins induction in the white callus were performed on MS medium supplemented 30 g·L-1 sucrose and combining different concentrations growth regulators (6-BA and NAA), addition of 6-BA and NAA ranging from 0 to 2.5 mg·L-1 in increments of 0.5 mg·L-1 (Table 2).
|6-BA (mg·L-1)||FW (g)||Abs||NAA (mg·L-1)||FW (g)||Abs|
|0.5||1.97 ± 0.03a||0.396 ± 0.01a||0.5||2.13 ± 0.1a||0.326 ± 0.03a|
|1.0||1.51 ± 0.06b||0.402 ± 0.02a||1.0||2.08 ± 0.1b||0.216 ± 0.03b|
|1.5||1.26 ± 0.06c||0.384 ± 0.02ab||1.5||1.99 ± 0.08bc||0.226 ± 0.03b|
|2.0||1.01 ± 0.05d||0.276 ± 0.04bc||2.0||1.80 ± 0.09c||0.174 ± 0.03b|
|2.5||0.96 ± 0.06d||0.191 ± 0.03c||2.5||1.65 ± 0.07c||0.098 ± 0.02c|
|Data represents mean value ± standard deviation (SD), means with different letters indicate significant level at p=0.05. The same as below.|
Table 2: Effects of different concentrations 6-BA or NAA on fresh weight of callus and anthocyanin induction of P. tunicoides.
Induction of anthocyanins by illumination intensity
This experiment was based on the callus which cultivated on MS medium with 1.5 mg·L-1 NAA and 0.5 mg·L-1 6-BA, and then set different illumination intensity (0, 500, 1000, 1500, 2000, 2500 lx) to induce anthocyanins. Each illumination intensity treatment with 10 Petri dishes which were inoculated fresh callus (each Petri dish contained 0.5 gram callus) and light-dark cycle of 12 h.
Determination of callus growth
The growth of callus was monitored by determination of fresh and dry biomass. Using filter paper to absorb the surface water on the callus, just until the point when free liquid was no longer expressed in paper and then to record fresh weight (FW). After that, the callus was dried in high temperature drying oven for 24 h, then the dry weight (DW) was calculated.
Extraction of anthocyanins
Weighing 2 gram of fresh callus used to extract anthocyanins, and extraction solvent was 1.0% HCl-MeOH (1:50 w/v). Treating callus was extracted overnight in test tube (25 mL) at 4°C for anthocyanins measurement . The extract was filtered through filter paper to 10 mL brown glass flask volumetric, and anthocyanins content was expressed from absorbance measurement.
During the white callus induction phase, the total number and fresh/dry weight of callus in each treatment were counted. During the anthocyanins production phase, the weight of callus which had induced and accumulated anthocyanins were counted to calculate the average anthocyanins content of each treatment. But in this paper, we adopted the direct method (A=A530) to determinate the content of anthocyanins in the same solvent . All extracts were prepared in triplicate. In order to find the characters conditions of anthocyanins 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
Effects of explants and growth regulators for callus induction
The callus initiated from different explants was slight red color and friable on media containing different factors, which combination leading optimal callus formation of P. tunicoides. It was indicated that the explants with the treatment of cut were inducted to produce callus after one week, and the callus of buds grew obviously better than others (A, B, C in Figure 1). Because the suitable conditions for promoting anthocyanins production in callus were not provided at this stage, the anthocyanins contents in callus were less and the colors of callus were lighter (almost white after 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 formation was obtained in 0.5 mg·L-1 2,4-D and 1.0 mg·L-1 6-BA, which the callus fresh weight was 3.11 g (Serial No. 8 in Table 1). The result not much differed from those of , 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, the biomass for callogenesis were well and quickly. The callus was soft in texture, friable in structure and white in color after 25 days. Callus induction from various tissues was different . 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 .
Effects of growth regulators for anthocyanin induction in callus
The effects of hormones combinations for anthocyanins induction in plants culture has previously been reported , but it has no reported that induce anthocyanins from P. tunicoides. In this study, it has been recognized that anthocyanins induction and formation in P. tunicoides callus can be regulated by 6-BA, NAA or 6-BA and NAA combination. NAA or 6-BA alone inhibited significantly the multiplication of callus and the induction of anthocyanins with the increase of its concentration (Table 2), but the co-treatment both NAA and 6-BA promoted significantly callus growth and anthocyanins formation (Table 3), and 0.5 mg·L-1 6-BA and 1.5 mg·L-1 NAA was the best combination (Serial No. 11 in Table 3), this result is consistent with which co-treatment of auxin and cytokinin would significantly enhance the anthocyanins  And the same time, according to the spectrogram of pigment extracting liquid of P. tunicoides callus, we found the anthocyanins characteristic absorption peak (n=529.0355, Figure 2).
|1||0.5||0.5||2.09 ± 0.02ab||0.43 ± 0.05ab|
|2||0.5||1.0||2.39 ± 0.12a||0.40 ± 0.05abc|
|3||0.5||1.5||2.07 ± 0.03ab||0.43 ± 0.02ab|
|4||0.5||2.0||1.37 ±0.05ef||0.41 ± 0.02ab|
|5||0.5||2.5||0.84 ± 0.07j||0.40 ± 0.04abc|
|6||1.0||0.5||1.86 ± 0.11abc||0.38 ± 0.04abc|
|7||1.0||1.0||2.13 ± 0.12ab||0.25 ± 0.04bc|
|8||1.0||1.5||2.08 ± 0.03ab||0.32 ± 0.02bc|
|9||1.0||2.0||1.58 ± 0.09cd||0.13 ± 0.01d|
|10||1.0||2.5||0.97 ± 0.07ij||0.15 ± 0.03d|
|11||1.5||0.5||2.43 ± 0.08a||0.57 ± 0.05a|
|12||1.5||1.0||1.68 ± 0.04bc||0.29 ± 0.03bc|
|13||1.5||1.5||1.55 ± 0.03de||0.44 ± 0.04ab|
|14||1.5||2.0||1.32 ± 0.07fj||0.43 ± 0.02ab|
|15||1.5||2.5||1.21 ± 0.10jh||0.35 ± 0.04bc|
|16||2.0||0.5||1.82 ± 0.06bc||0.43 ± 0.03ab|
|17||2.0||1.0||2.03 ± 0.03ab||0.15 ± 0.03d|
|18||2.0||1.5||1.14 ± 0.12jh||0.36 ± 0.04bc|
|19||2.0||2.0||1.03 ± 0.07ij||0.36 ± 0.03bc|
|20||2.0||2.5||0.95 ± 0.10ij||0.20 ± 0.03cd|
|21||2.5||0.5||1.79 ± 0.13bc||0.33 ± 0.07bcd|
|22||2.5||1.0||2.09 ± 0.08ab||0.24 ± 0.03bcd|
|23||2.5||1.5||1.73 ± 0.12bc||0.31 ± 0.03bcd|
|24||2.5||2.0||1.10 ± 0.12hi||0.21 ± 0.04cd|
|25||2.5||2.5||1.02 ± 0.03ij||0.13 ± 0.03d|
Table 3: Effects of different concentrations and combinations of growth regulators on fresh weight of callus and anthocyanin induction of P. tunicoides.
Effects of illumination intensity for anthocyanins formation in callus
The callus of P. tunicoides was cultured on MS medium with optimum growth regulators combination (0.5 mg·L-1 6-BA and 1.5 mg·L-1 NAA) to induce anthocyanins, then combined with different illumination intensity treatment (light-dark cycle of 12 h). The results showed that Illumination is necessary for anthocyanins formation in callus (Figure 3). With the increase of illumination intensity, the anthocyanins content (Expressed in Abs. The same below) increased, the highest content at 2000 lx (Abs was 0.53), then decreased when up to an illumination intensity 2500 lx (Table 4). Some reports indicated that pigments production were greater in the light than in the dark , the reason is that the genes associated with anthocyanins biosynthesis could be activated by light irradiation , the expression of anthocyanins genes were regulated by UV-B receptors and were also stimulated by blue and red light [24,25] and signal transduction mechanisms as a role of complexity contacted with light-mediated control of anthocyanins gene expression . In this way, a large of proteins that was an indication of increased gene expression would be produced by the red callus.
Effects of 2,4-D for callus proliferation and anthocyanins accumulation
Under the above optimum conditions to induce anthocyanins of P. tunicoides (Including optimum combination of growth regulators and light intensity), adding 2,4-D (0, 0.5, 1.0, 1.5, 2.0 mg·L-1) to the media. The results reflected that the callus proliferated in varying degrees with the increase of 2,4-D concentration, and the anthocyanins content in callus decreased significantly (Table 4). In other words, 2,4-D strongly inhibited the accumulation of anthocyanins in callus (M and N in Figure 3). This may be due to the influence of 2,4-D as a suppressor to increase the incorporation of phenylalanine into amino acids, the products of phenylpropanoid metabolism were the precursors for anthocyanins biosynthesis, the phenylalanine was reduced by this conjugation reaction, and it was acted on by the enzymes of the phenylpropanoid pathway  is generally known that anthocyanins formation were controlled by anthocyanins genes that included structure genes (CHS, CHI, F3H, DFR) and regulatory genes (MYB10, bHLH3), 2,4-D inhibited the transcription of regulatory genes, the structure genes that were regulated by the regulatory genes also decreased. Therefore, the anthocyanins genes expression were inhibited by 2,4-D .
|FW (g)||Abs||Illumination intensity
|0.0||5.24 ± 0.09c||1.30 ± 0.02a||0||2.38 ± 0.07b||0.13 ± 0.05e|
|0.5||6.07 ± 0.06ab||0.08 ± 0.016b||500||3.17 ± 0.06a||0.23 ± 0.02de|
|1.0||6.60 ± 0.07a||0.04 ± 0.004b||1000||3.00 ± 0.10a||0.32 ± 0.02cd|
|1.5||5.96 ± 0.15b||0.07 ± 0.004b||1500||3.12 ± 0.08a||0.38 ± 0.03bc|
|2.0||5.90 ± 0.18b||0.04 ± 0.004b||2000||2.97 ± 0.10a||0.53 ± 0.03a|
|2500||3.09 ± 0.09a||0.44 ± 0.02ab|
Table 4: Effects of different concentrations of 2,4-D and illumination intensity on fresh weight of callus and anthocyanin accumulation of P. tunicoides.
According to our observation, the callus of P. tunicoides hardly grew in anthocyanins accumulation medium, and the callus became dead after produced anthocyanins. The direct cause behind this could be that anthocyanin belongs to 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 anthocyanins induction. Two reasons lead to plant cell could not growth normally, even dead between the slight toxicity and abound of end products.
Final conclusion, the present work demonstrated that the buds of P. tunicoides are suitable explants for inducing callus, the optimum medium is MS+0.5 mg·L-1 2,4-D+1.0 mg·L-1 6-BA+30 g·L-1 sucrose+7 g·L-1 agar. 0.5 mg·L-1 6-BA and 1.5 mg·L-1 NAA is the best combination for callus growth and anthocyanins formation. 2,4-D has a certain promoting effect for callus proliferation, but it strongly inhibits anthocyanin formation in callus. 2000 lx illumination intensity is the most favorable for anthocyanin synthesis in callus.
Conflict of Interest
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.
This research was funded to Ming-Sheng Zhang 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).
- Claudia SG, Livia SC, Tatiana CC, Catia HC, Norma A, et al. (2011) Establishment of anthocyanin-producing cell suspension cultures of Cleome rosea Vahl ex DC. Plant Cell Tissue Organ Cult 106: 537-545.
- Wang J, Mazza G (2002) Inhibitory effects of anthocyanins and other phenolic compounds on nitric oxide production in LPS/IFN-c-active RAW 2647 macrophages. J Agric Food Chem 50: 850-857.
- Bagchi D, Sen CK, Bagchi M, Atalay M (2004) Anti-angiogenic, antioxidant and anti-carcinogenic properties of a novel anthocy-anin-rich berry extract formula. Biochemistry 69: 75-78.
- Castonguay A, Gali HU, Perchellet EM, Gao XM, Jalbert M, et al. (1997) Antitumorigenic and antipromoting activities of ellagic acid, ellagitannins, and oligomeric anthocyanin and procyanidin. Int J Oncol 10: 367-373.
- Morazzoni R, Magistretti MJ (1990) Activity of Myrtocyan, anthocyanoside complex from Vaccinium myrtillus (VMA), on platelet aggregation and adhesiveness. Fitoterapia 61: 13-21.
- Brindle P, Timberlake CF (1996) Anthocyanins as natural foodcolors: Selected aspects. Food Chem 58:103-109.
- Giovanni M, Carmela G, Antonio M, Roberto B, Pietro DL (1994) Pigment production from in vitro cultures of Alkanna tinctoria Tausch. Plant Cell Rep 13: 406-410.
- Zhang W, Shintaro F (1999) Production of anthocyanins by plant cell cultures. Biotechol Bioproc Engg 4: 231-252.
- Gao W Y, Fan L, Paek KY (2000) Yellow and red pigment production by cell cultures of Carthamus tinctorius in a bioreactor. Plant Cell Tissue Organ Cult 60: 95-100.
- Smetanska I (2008) Production of secondary metabolites using plantcell cultures. Adv Biochem Eng Biotechnol 111: 187-228
- Aksu Z, Eren AT (2005) Carotenoids production by the yeast Rhodotorulamucilaginosa: Use of agricultural wastes as a carbon source. Proc Biochem 40: 2985-2991.
- Hennayake CK, Takagi S, Nishimura K, Kanechi M, Uno Y, et al. (2006) Differential expression of anthocyanin biosynthesisgenes in suspension culture cells of Rosa hybrida cv. Charleston. Plant Biotechnol 23: 379-385.
- Piovan A, Filippini R (2007) Anthocyanins in Catharanthus roseus in vivo and in vitro: a review. Phytochem Rev 6: 235-242.
- Boehm H, Boehm E, Rink E (1991) Establishment and characterization of a betaxanthin-producing cell culture from Portulaca grandiflora. Plant Cell Tissue Organ Cult 26: 75-82.
- Ji XH, Wang YT, Zhang R, Wu SJ, An MM, et al. (2014) Effect of auxin, cytokinin and nitrogen on anthocuanin biosynthesis in callus cultures of red-fleshed apple (Malus sieversii f. niedzwetzkyana). Plant Cell Tissue Organ Cult 120: 325-337.
- Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tabacco tissue cultures. Plant Physiol 15: 473-497.
- Fang YM, Smith MAL, Pepin MF (1998) Benzyl adenine restores anthocyanin pigmentation in suspension cultures of wild Vaccinium pahalae. Plant Cell Tissue Organ Cult 54: 113-122.
- Huo LL, Su P, Lv YH (2005) Measurement of total anthocyanins in Mulberry by UV-visible spectroscopy. Liquor Making 32: 88-89.
- Li Y, Wei W, Tian SD, Liu TX, Zhang ZS (2011) The optimization of plant hormones in the cultivation of Psammosilene tunicoides callus. Lishizhen Medicine and Materia Medica Research 22: 109-111.
- Abdul RZ, Othman AN, Kamarulzaman FLI, Ahmad A (2014) Establishment and optimization growth of shoot buds-derived callus and suspension cell cultures of Kaempferia parviflora. Am J Plant Sci 5: 2693-2699.
- Vincent KA, Hariharan M, Mathew KM (1992) Embryogenesis and plantlet formation in tissue culture of Kaempferia galangal L, a medicinal plant. Phytomorphology 42: 253-256.
- Meyer HJ,van Staden JV (1995) The in vitro production ofanthocyanin from callus cultures of Oxalis linearis. Plant Cell Tissue Organ Cult 40: 55-58.
- Xu ZR, Li CL, Sun Y, Li YH (2008) Screening the genes associated with anthocyanin biosynthesis in 'Tsuda' and 'Yurugi Akamaru' Turnip. Lett Biotechnol19: 693-696.
- Kishima Y, Shimaya A, Adachi T (1995) Ebidence that blue light induces betalain pigmentation in Portulaca callus. Plant Cell Tissue Organ Cult 43: 67-70.
- Molchan J, Jenkins G, Schafer E, Weiss D (1996) Signal percep-tion, transduction and gene expression involved in anthocyanin biosynthesis. Crit Rev Plant Sci 15: 525-557.
- Lin Y, Wang YH, Li B, Tan H, Li DN, et al. (2018) Comparative transcriptome analysis of genes involved in anthocyanin synthesis in blueberry. Plant Physiol Biochem 127: 561-572.
- Makunga NP, Staden JV, Cress WA (1997) The effect of light and 2,4-D on anthocyanin production in Oxalis reclinatacallus. Plant Growth Regul 23: 153-158.