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Year 2021, Volume: 42 Issue: 4, 766 - 774, 29.12.2021

Cennet Yaman , Allah Bakhsh , Serkan Uranbey

Abstract

Project Number

project no: 6602b-ZF/17-119

References

  • [1] Boulos J.C., Rahama M., Hegazy M.F., Efferth T., Shikonin derivatives for cancer prevention and therapy, Cancer Letter, 459 (2019) 248–267.
  • [2] Mahmoudi S.Z., Seyedabadi M., Esfahani H.R.M., Amanzadeh Y., Ostad S.N., Anti-inflammatory and analgesic activity of Alkanna bracteosa and Alkanna tricophila, Nat. Prod. Res., 26(6) (2012) 564-569.
  • [3] Güner A., Aslan S., Ekim T., Vural M., Babaç T., Türkiye Bitkileri Listesi, İstanbul:NGBB and Flora Araştırmaları Derneği Yayını, (2012).
  • [4] Davis P.H., Mill R.R., Tan K., Flora of Turkey and the East Aegean Islands, 6. Edinburgh: Edinburgh University press, (1988).
  • [5] Assimopoulou A.N., Karapanagiotis I., Vasiliou A., Kokkini S., Papageorgiou V.P., Analysis of alkannin derivatives from Alkanna species by high-performance liquid chromatography/photodiode array/mass spectrometry, Biomed. Chromatogr., 20 (2006) 1359-1374.
  • [6] Papageorgiou V., Assimopoulou A., Ballis A., Alkannins and shikonins: a new class of wound healing agents, Curr. Med. Chem., 15(30) (2008) 3248-3267.
  • [7] Assimopoulou A.N., Sturm S., Stuppner H., Papageorgiou V.P., Preparative isolation and purification of alkannin/shikonin derivatives from natural products by high speed counter‐current chromatography, Biomed. Chromatogr., 23(2) (2009) 182-198.
  • [8] Tung N.H., Du G., Wang C., Yuan C., Naphthoquinone components from Alkanna tinctoria (L.) Tausch Show significant antiproliferative effects on human colorectal cancer cells, Phytotherapy Research, 27 (2012) 66-70.
  • [9] Jaradat N.A., Zaid A.N., Hussen F.M., Issa L., Altamimi M., Fuqaha B., Nawahda A., Assadi M., Phytoconstituents, antioxidant, sun protection and skin anti-wrinkle effects using four solvents fractions of the root bark of the traditional plant Alkanna tinctoria (L.), Eur. J. Integr. Med., 21 (2018) 88-93.
  • [10] Abdel-Gelil O.E.A., Atwa N.A., Moustafa A.R.A., Mansour S.R., Alkanna species: a promising herbal medicine and its uses, Journal of Food Science and Nutrition Research, 2 (2009) 309-315.
  • [11] Tung N.H., Wang C.Z., Du G.J., Yuan C.S., Uto T., Shoyama Y., Chemopreventive activity of naphthoquinones from Alkanna tinctoria (L.) Tausch in human colorectal cancer cells, J. Gastroenterol Hepatol Res., 5(4) (2016) 2115-2121.
  • [12] Rashan L., Hakkim L., Fiebig H.H., Al-Balushi M., In vitro anti-proliferative activity of the Rubia tinctorum and Alkanna tinctoria root extracts in panel of human tumor cell lines, Jordan J. Biol. Sci., 11(5) (2018) 489-494.
  • [13] Ogurtan Z., Hatipoglu F., Ceylan C., The effect of Alkanna tinctoria Tausch on burn wound healing in rabbits, Deutsche tierärztliche Wochenschrift, 109(11) (2002) 481-485.
  • [14] Alwahibi M.S., Perveen K., Chemical analysis by GC-MS and in vitro antibacterial activity of Alkanna tinctoria extracts against skin infection causing bacteria, Biomedical Research, 28(18) (2017) 7946-7949.
  • [15] Salih M., Mohammed M.S., Basudan O., El Tahir K.E.H., Osman B.I., Ahmed W.J., Evaluation of antipyretic, antinocieptive and sedative effects of Tribulus terrestris, Mimosa pigra and Alkanna tinctoria methanolic extracts, J. Phytopharm., 5 (2016) 1-3.
  • [16] Piekoszewska A., Ekiert H., Zubek S., Arbutin production in Rutagraveolens L. and Hypericum perforatum L. in vitro cultures. Acta Physiologiae Plantarum, 32 (2010) 223-229.
  • [17] Mahjouri S., Movafeghi A., Zare K., Kosari-Nasab M., Nazemiyeh H., Production of naphthoquinone derivatives using two-liquid-phase suspension cultures of Alkanna orientalis, Plant Cell, Tissue and Organ Culture (PCTOC), 124 (2016) 201-207.
  • [18] Bagheri F., Tahvilian R., Karimi N., Chalabi M., Azam M., Shikonin production by callus culture of Onosma bulbotrichom as active pharmaceutical ingredient, Iran J. Pharm. Sci., 17(2) (2018) 495-504.
  • [19] Fu J., Zhao H., Bao J., Wen Z., Fang R., Fazal A., Yang M., Liu B., Yin T., Pang Y., Lu G., Qi J., Yang Y., Establishment of the hairy root culture of Echium plantagineum L. and its shikonin production, 3 Biotech, 10 (2020) 429.
  • [20] Ahmad S., Garg M., Tamboli E.T., Abdin M.Z., Ansari S.H., In vitro production of alkaloids: Factors, approaches, challenges and prospects, Pharmacogn. Rev., 7 (13) (2013).
  • [21] Krol A., Kokotkiewicz A., Szopa A., Ekiert H., Luczkiewicz M., Bioreactor-grown shoot cultures for the secondary metaboliteproduction. In: Ramawat, K.G., Ekiert, H.M., Goyal, S. (Eds.) Plant Cell and Tissue Differentiation and Secondary Metabolites: Fundamentals and Applications, Springer International Publishing, Cham, (2020) 1-62.
  • [22] Praveen N., Murthy H.N., Effects of macroelements and nitrogen source on biomass accumulation and withanolide - A production from cell suspension cultures of Withania somnifera (L.) Dunal, Plant Cell, Tissue and Organ Culture (PCTOC), 104(1) (2011) 119-124.
  • [23] Passinbo H.C., Meira P.R., David J.P., Mesquita P.R., Vale A.E. Rodrigues F.M., Pereira P.A.P., De Santana J.R.F., De Oliveira F.S., De Andrade J.B., David J.M., Volatile organic compounds obtained by in vitro callus cultivation of Plectranthus ornatus Codd. (Lamiaceae), Molecules, (2013) 10320-10333.
  • [24] Fujita Y., Hara Y., Ogino T., Suga C., Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon: I. Effects of nitrogen sources on the production of shikonin derivatives, Plant Cell Rep., 1(2) (1981) 59-60.
  • [25] Boehm R., Sommer S., Li S.M., Heide L., Genetic engineering on shikonin biosynthesis: expression of the bacterial ubiA gene in Lithospermum erythrorhizon, Plant Cell Physiol., 41 (2000) 911-919.
  • [26] Tabata M., Mizukami H., Hiraoka N., Konoshima M., Pigment formation in callus cultures of Lithospermum erythrorhizon, Phytochemistry, 13 (1974) 927-932.
  • [27] Urbanek H., Katarzyna Bergier K., Marian Saniewski M., Patykowski J., Effect of jasmonates and exogenous polysaccharides on production of alkannin pigments in suspension cultures of Alkanna tinctoria, Plant Cell Rep., 15(8) (1996) 637-641.
  • [28] Singh B., Sharma R.A., Antioxidant and antimicrobial activities of Arnebia hispidissima, Am. J. Adv. Drug Deliv., 2(2) (2014) 224-237.
  • [29] Zare K.H., Nazemiyeh H., Movafeghi A., Khosrowshahli M., Motallebi-Azar A., Dadpour M., Omidi Y., Bioprocess engineering of Echium italicum L.: Induction of shikonin and alkannin derivatives by twoliquid- phase suspension cultures. Plant Cell, Tissue and Organ Culture (PCTOC), 100(2) (2010) 157-164.
  • [30] Tatsumi K., Yano M., Kaminade K., Sugiyama A., Sato M., Toyooka K., Aoyama T., Sato F., Yazaki K., Characterization of shikonin derivative secretion in Lithospermum erythrorhizon hairy roots as a model of lipid-soluble metabolite secretion from plants, Front. Plant Sci., 7 (2016) 1066.
  • [31] Akgun I., Ganzera M., Gur C., Senol S., Korkmaz K., Bedir E., Determination of naphthazarin derivatives in 16 Alkanna species by RP-LC using UV and MS for detection, Chromatographia, 70 (2009) 963–967.
  • [32] Yaman C., Uranbey S., Ahmed H.A., Özcan S., Tugay O., Başalma, D., Callus induction and regeneration of Alkanna orientalis var. orientalis and A. sieheana, Bangladesh J. Bot., 48 (2019) 633–640.
  • [33] Murashige T, Skoog F., A revised medium for rapid growth and bio assays with tobacco tissue cultures, Physiol. Plant., 15 (1962) 473–497.
  • [34] Gamborg, O. L., Miller, R. A., Ojima, K., Nutrient requirements of suspension cultures of soybean root cells, Exp. Cell Res., 50 (1968) 151.
  • [35] Gharehmatrossian S., Popov Y., Ghorbanli M., Safaeian S., Iranbakhsh A., Phytochemical and morphological evidences for shikonin production by plant cell cultures of Onosma sericeum Willd, Braz. Arch. Biol. Technol., 59 (2016) 1-7.
  • [36] Bagherieh-Najjar M., Nezamdoost T., Optimization of shikonin production in Onosma dichroantha callus using response surface methodology, Plant Cell, Tissue and Organ Culture (PCTOC), 126 (2016) 399–409.
  • [37] Zhang W.J., Su J., Tan M.Y., Liu G.L., Pang Y.J., Shen H.G., Qi J.L., Yang Y., Expression analysis of shikonin-biosynthetic genes in response to M9 medium and light in Lithospermum erythrorhizon cell cultures, Plant Cell, Tissue and Organ Culture (PCTOC), 101 (2010) 135-142.
  • [38] Shekhawat M.S., Shekhawat N.S., Micropropagation of Arnebia hispidissima (Lehm). DC. and production of alkannin from callus and cell suspension culture, Acta Physiol. Plant., 33 (2011) 1445-1450.
  • [39] Pal M., Chaudhury A., High Frequency Direct Plant Regeneration, Micropropagation and Shikonin Induction in Arnebia hispidissima, J. Crop Sci. Biotechnol., 13(1) (2010) 13-20.
  • [40] Fang R, Wu F, Zou A, Zhu Y, Zhao H, Zhao H., Liao Y., Tang R., Yang T., Pang Y., Wang X., Yang R., Qi J., Lu G., Yang Y., Transgenic analysis reveals LeACS-1 as a positive regulator of ethylene-induced shikonin biosynthesis in Lithospermum erythrorhizon hairy roots, Plant Mol. Biol., 90 (2016) 345-358.
  • [41] Abbasi B., Tian C., Murch S., Saxena P., Liu C., Light-enhanced caffeic acid derivatives biosynthesis in hairy root cultures of Echinacea purpurea, Plant Cell Rep., 26 (2007) 1367-1372.
  • [42] Gupta K., Garg S., Singh J.,, Kumar M., Enhanced production of naphthoquinone metabolite (shikonin) from the cell suspension culture of Arnebia sp. & its up-scaling through bioreactor, 3 Biotech, 4(3) (2014) 263-273.

Influences of basal media, growth regulators, explant type and photoperiod on callus competency and pigmentation of Alkanna orientalis L.

Year 2021, Volume: 42 Issue: 4, 766 - 774, 29.12.2021

Cennet Yaman , Allah Bakhsh , Serkan Uranbey

Abstract

The goal of this research was in-vitro callus induction and red pigment production of Alkanna orientalis as a medicinal herb which belongs to Boraginaceae family containing valuable naphthoquinone derivates. The two different explants (leaf and leaf base) were subjected to abiotic factors such as different nutrient media (MS, B5 and M9), light and plant growth regulators (IAA and IBA). High frequency reproducible, prolific and compact calli formation was obtained from MS and B5 media supplemented with IAA, whereas high pigmentation was found in leaf base explants on M9 medium. Leaf base explant and dark conditions were found more effective for both callus formation and pigment production. The pigmentation at IBA was more than IAA, and the maximum level of pigmentation was observed on M9 medium with a combination of 1.0 mg/l IBA under dark. The factors that may be the most influential in the production of callus and red color pigment from A. orientalis have been determined. A. orientalis may be considered to be alternative plants for the A/S (alkannin/shikonin) production in vitro.

Keywords

Alkanna orientalis Red pigment Callus Nutrient media Leaf base.

Supporting Institution

Scientific Research Center of Yozgat Bozok University

Project Number

project no: 6602b-ZF/17-119

Thanks

The work was supported by grants from Scientific Research Center of Yozgat Bozok University (project no: 6602b-ZF/17-119).

References

  • [1] Boulos J.C., Rahama M., Hegazy M.F., Efferth T., Shikonin derivatives for cancer prevention and therapy, Cancer Letter, 459 (2019) 248–267.
  • [2] Mahmoudi S.Z., Seyedabadi M., Esfahani H.R.M., Amanzadeh Y., Ostad S.N., Anti-inflammatory and analgesic activity of Alkanna bracteosa and Alkanna tricophila, Nat. Prod. Res., 26(6) (2012) 564-569.
  • [3] Güner A., Aslan S., Ekim T., Vural M., Babaç T., Türkiye Bitkileri Listesi, İstanbul:NGBB and Flora Araştırmaları Derneği Yayını, (2012).
  • [4] Davis P.H., Mill R.R., Tan K., Flora of Turkey and the East Aegean Islands, 6. Edinburgh: Edinburgh University press, (1988).
  • [5] Assimopoulou A.N., Karapanagiotis I., Vasiliou A., Kokkini S., Papageorgiou V.P., Analysis of alkannin derivatives from Alkanna species by high-performance liquid chromatography/photodiode array/mass spectrometry, Biomed. Chromatogr., 20 (2006) 1359-1374.
  • [6] Papageorgiou V., Assimopoulou A., Ballis A., Alkannins and shikonins: a new class of wound healing agents, Curr. Med. Chem., 15(30) (2008) 3248-3267.
  • [7] Assimopoulou A.N., Sturm S., Stuppner H., Papageorgiou V.P., Preparative isolation and purification of alkannin/shikonin derivatives from natural products by high speed counter‐current chromatography, Biomed. Chromatogr., 23(2) (2009) 182-198.
  • [8] Tung N.H., Du G., Wang C., Yuan C., Naphthoquinone components from Alkanna tinctoria (L.) Tausch Show significant antiproliferative effects on human colorectal cancer cells, Phytotherapy Research, 27 (2012) 66-70.
  • [9] Jaradat N.A., Zaid A.N., Hussen F.M., Issa L., Altamimi M., Fuqaha B., Nawahda A., Assadi M., Phytoconstituents, antioxidant, sun protection and skin anti-wrinkle effects using four solvents fractions of the root bark of the traditional plant Alkanna tinctoria (L.), Eur. J. Integr. Med., 21 (2018) 88-93.
  • [10] Abdel-Gelil O.E.A., Atwa N.A., Moustafa A.R.A., Mansour S.R., Alkanna species: a promising herbal medicine and its uses, Journal of Food Science and Nutrition Research, 2 (2009) 309-315.
  • [11] Tung N.H., Wang C.Z., Du G.J., Yuan C.S., Uto T., Shoyama Y., Chemopreventive activity of naphthoquinones from Alkanna tinctoria (L.) Tausch in human colorectal cancer cells, J. Gastroenterol Hepatol Res., 5(4) (2016) 2115-2121.
  • [12] Rashan L., Hakkim L., Fiebig H.H., Al-Balushi M., In vitro anti-proliferative activity of the Rubia tinctorum and Alkanna tinctoria root extracts in panel of human tumor cell lines, Jordan J. Biol. Sci., 11(5) (2018) 489-494.
  • [13] Ogurtan Z., Hatipoglu F., Ceylan C., The effect of Alkanna tinctoria Tausch on burn wound healing in rabbits, Deutsche tierärztliche Wochenschrift, 109(11) (2002) 481-485.
  • [14] Alwahibi M.S., Perveen K., Chemical analysis by GC-MS and in vitro antibacterial activity of Alkanna tinctoria extracts against skin infection causing bacteria, Biomedical Research, 28(18) (2017) 7946-7949.
  • [15] Salih M., Mohammed M.S., Basudan O., El Tahir K.E.H., Osman B.I., Ahmed W.J., Evaluation of antipyretic, antinocieptive and sedative effects of Tribulus terrestris, Mimosa pigra and Alkanna tinctoria methanolic extracts, J. Phytopharm., 5 (2016) 1-3.
  • [16] Piekoszewska A., Ekiert H., Zubek S., Arbutin production in Rutagraveolens L. and Hypericum perforatum L. in vitro cultures. Acta Physiologiae Plantarum, 32 (2010) 223-229.
  • [17] Mahjouri S., Movafeghi A., Zare K., Kosari-Nasab M., Nazemiyeh H., Production of naphthoquinone derivatives using two-liquid-phase suspension cultures of Alkanna orientalis, Plant Cell, Tissue and Organ Culture (PCTOC), 124 (2016) 201-207.
  • [18] Bagheri F., Tahvilian R., Karimi N., Chalabi M., Azam M., Shikonin production by callus culture of Onosma bulbotrichom as active pharmaceutical ingredient, Iran J. Pharm. Sci., 17(2) (2018) 495-504.
  • [19] Fu J., Zhao H., Bao J., Wen Z., Fang R., Fazal A., Yang M., Liu B., Yin T., Pang Y., Lu G., Qi J., Yang Y., Establishment of the hairy root culture of Echium plantagineum L. and its shikonin production, 3 Biotech, 10 (2020) 429.
  • [20] Ahmad S., Garg M., Tamboli E.T., Abdin M.Z., Ansari S.H., In vitro production of alkaloids: Factors, approaches, challenges and prospects, Pharmacogn. Rev., 7 (13) (2013).
  • [21] Krol A., Kokotkiewicz A., Szopa A., Ekiert H., Luczkiewicz M., Bioreactor-grown shoot cultures for the secondary metaboliteproduction. In: Ramawat, K.G., Ekiert, H.M., Goyal, S. (Eds.) Plant Cell and Tissue Differentiation and Secondary Metabolites: Fundamentals and Applications, Springer International Publishing, Cham, (2020) 1-62.
  • [22] Praveen N., Murthy H.N., Effects of macroelements and nitrogen source on biomass accumulation and withanolide - A production from cell suspension cultures of Withania somnifera (L.) Dunal, Plant Cell, Tissue and Organ Culture (PCTOC), 104(1) (2011) 119-124.
  • [23] Passinbo H.C., Meira P.R., David J.P., Mesquita P.R., Vale A.E. Rodrigues F.M., Pereira P.A.P., De Santana J.R.F., De Oliveira F.S., De Andrade J.B., David J.M., Volatile organic compounds obtained by in vitro callus cultivation of Plectranthus ornatus Codd. (Lamiaceae), Molecules, (2013) 10320-10333.
  • [24] Fujita Y., Hara Y., Ogino T., Suga C., Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon: I. Effects of nitrogen sources on the production of shikonin derivatives, Plant Cell Rep., 1(2) (1981) 59-60.
  • [25] Boehm R., Sommer S., Li S.M., Heide L., Genetic engineering on shikonin biosynthesis: expression of the bacterial ubiA gene in Lithospermum erythrorhizon, Plant Cell Physiol., 41 (2000) 911-919.
  • [26] Tabata M., Mizukami H., Hiraoka N., Konoshima M., Pigment formation in callus cultures of Lithospermum erythrorhizon, Phytochemistry, 13 (1974) 927-932.
  • [27] Urbanek H., Katarzyna Bergier K., Marian Saniewski M., Patykowski J., Effect of jasmonates and exogenous polysaccharides on production of alkannin pigments in suspension cultures of Alkanna tinctoria, Plant Cell Rep., 15(8) (1996) 637-641.
  • [28] Singh B., Sharma R.A., Antioxidant and antimicrobial activities of Arnebia hispidissima, Am. J. Adv. Drug Deliv., 2(2) (2014) 224-237.
  • [29] Zare K.H., Nazemiyeh H., Movafeghi A., Khosrowshahli M., Motallebi-Azar A., Dadpour M., Omidi Y., Bioprocess engineering of Echium italicum L.: Induction of shikonin and alkannin derivatives by twoliquid- phase suspension cultures. Plant Cell, Tissue and Organ Culture (PCTOC), 100(2) (2010) 157-164.
  • [30] Tatsumi K., Yano M., Kaminade K., Sugiyama A., Sato M., Toyooka K., Aoyama T., Sato F., Yazaki K., Characterization of shikonin derivative secretion in Lithospermum erythrorhizon hairy roots as a model of lipid-soluble metabolite secretion from plants, Front. Plant Sci., 7 (2016) 1066.
  • [31] Akgun I., Ganzera M., Gur C., Senol S., Korkmaz K., Bedir E., Determination of naphthazarin derivatives in 16 Alkanna species by RP-LC using UV and MS for detection, Chromatographia, 70 (2009) 963–967.
  • [32] Yaman C., Uranbey S., Ahmed H.A., Özcan S., Tugay O., Başalma, D., Callus induction and regeneration of Alkanna orientalis var. orientalis and A. sieheana, Bangladesh J. Bot., 48 (2019) 633–640.
  • [33] Murashige T, Skoog F., A revised medium for rapid growth and bio assays with tobacco tissue cultures, Physiol. Plant., 15 (1962) 473–497.
  • [34] Gamborg, O. L., Miller, R. A., Ojima, K., Nutrient requirements of suspension cultures of soybean root cells, Exp. Cell Res., 50 (1968) 151.
  • [35] Gharehmatrossian S., Popov Y., Ghorbanli M., Safaeian S., Iranbakhsh A., Phytochemical and morphological evidences for shikonin production by plant cell cultures of Onosma sericeum Willd, Braz. Arch. Biol. Technol., 59 (2016) 1-7.
  • [36] Bagherieh-Najjar M., Nezamdoost T., Optimization of shikonin production in Onosma dichroantha callus using response surface methodology, Plant Cell, Tissue and Organ Culture (PCTOC), 126 (2016) 399–409.
  • [37] Zhang W.J., Su J., Tan M.Y., Liu G.L., Pang Y.J., Shen H.G., Qi J.L., Yang Y., Expression analysis of shikonin-biosynthetic genes in response to M9 medium and light in Lithospermum erythrorhizon cell cultures, Plant Cell, Tissue and Organ Culture (PCTOC), 101 (2010) 135-142.
  • [38] Shekhawat M.S., Shekhawat N.S., Micropropagation of Arnebia hispidissima (Lehm). DC. and production of alkannin from callus and cell suspension culture, Acta Physiol. Plant., 33 (2011) 1445-1450.
  • [39] Pal M., Chaudhury A., High Frequency Direct Plant Regeneration, Micropropagation and Shikonin Induction in Arnebia hispidissima, J. Crop Sci. Biotechnol., 13(1) (2010) 13-20.
  • [40] Fang R, Wu F, Zou A, Zhu Y, Zhao H, Zhao H., Liao Y., Tang R., Yang T., Pang Y., Wang X., Yang R., Qi J., Lu G., Yang Y., Transgenic analysis reveals LeACS-1 as a positive regulator of ethylene-induced shikonin biosynthesis in Lithospermum erythrorhizon hairy roots, Plant Mol. Biol., 90 (2016) 345-358.
  • [41] Abbasi B., Tian C., Murch S., Saxena P., Liu C., Light-enhanced caffeic acid derivatives biosynthesis in hairy root cultures of Echinacea purpurea, Plant Cell Rep., 26 (2007) 1367-1372.
  • [42] Gupta K., Garg S., Singh J.,, Kumar M., Enhanced production of naphthoquinone metabolite (shikonin) from the cell suspension culture of Arnebia sp. & its up-scaling through bioreactor, 3 Biotech, 4(3) (2014) 263-273.
There are 42 citations in total.

Details

Primary Language English
Subjects Structural Biology
Journal Section Natural Sciences
Authors

Cennet Yaman 0000-0002-2364-8171

Allah Bakhsh 0000-0003-3561-7863

Serkan Uranbey 0000-0002-0312-8099

Project Number project no: 6602b-ZF/17-119
Publication Date December 29, 2021
Submission Date May 5, 2021
Acceptance Date November 6, 2021
Published in Issue Year 2021Volume: 42 Issue: 4

Cite

APA Yaman, C., Bakhsh, A., & Uranbey, S. (2021). Influences of basal media, growth regulators, explant type and photoperiod on callus competency and pigmentation of Alkanna orientalis L. Cumhuriyet Science Journal, 42(4), 766-774.