Effect of mannitol, sucrose, and hydrolyzed casein on hypericin content, gland number, and red pigment synthesis in the in vitro culture of Hypericum perforatum cv. Helos

Document Type : Research Paper

Authors

Department of Horticultural Sciences, Faculty of Agriculture, University of Tabriz, Tabriz, Iran.

10.22034/jppb.2025.69043.1381

Abstract

Objective: This study aimed to establish a protocol for enhancing the quantity of hypericin, glands, and red pigments in Hypericum perforatum cv. Helos.
Methods: The effects of hydrolyzed casein (0 and 500 mg/L), mannitol (0, 5, and 10 g/L), and sucrose (20 and 30 g/L) on leaf explants derived from the in vitro plantlets were evaluated. The factors were arranged in a factorial experiment using a completely randomized design with three replications and five samples per experimental unit.
Results: The concentrations of hydrolyzed casein, mannitol, and sucrose markedly influenced the proportions of callus formation and the shoots displaying red pigments. The highest proportion of red pigment-bearing calli was observed in the media containing either the control or 500 mg/L hydrolyzed casein with 20 g/L sucrose, without mannitol. Glands were observed on all shoots that underwent development. The maximum gland number and the highest percentage of shoots with glands were recorded for the explants cultured on media containing 30 g/L sucrose in combination with 5 or 10 g/L mannitol, and similarly for the medium with 20 g/L sucrose and 5 g/L mannitol. These results indicated that the MS composition with sucrose concentration and the addition of hydrolyzed casein significantly enhance hypericin production in the calli and shoots.
Conclusion: Overall, optimization of MS via adjustments to sucrose and the incorporation of hydrolyzed casein increased the production of hypericin, glands, and red pigments in H. perforatum.

Keywords

Main Subjects


Akyüz B. 2025. Effect of different carbon sources and concentrations on in vitro propagation of chestnut. Plant Cell Tissue Organ Cult. 160(2): 25. https://doi.org/10.1007/s11240-024-02960-w
Al-Asadi Ahmed ZR, Al-Mayahi Ahmed MW, Awad Khairullah M. 2024. Effects of dicamba and casein hydrolysate on in vitro growth and shoot regeneration of date palm (Phoenix dactylifera L.) cv. Barhee. Folia Oecol. 51(1): 56-65. https://doi.org/10.2478/foecol-2024-0006
Al-Khateeb AA, Solliman MED, El-Beltagi HS, Mohasseb HAA, Al-Khateeb SA. 2019. Evaluation of plant growth regulators and casein hydrolysate addition on secondary products production in cultures of date palm. Fresenius Environ Bull. 28: 2957-2967.
Amiri S, Kazemitabar SK. 2011. Enhancement of callus induction and regeneration efficiency from embryo cultures of Datura stramonium by adjusting carbon sources and concentrations. Afr J Biotechnol. 10(50): 10101-10107. http://dx.doi.org/10.5897/AJB10.816
Bělonožníková K, Černý M, Hýsková V, Synková H, Valcke R, Hodek O, Ryšlavá H. 2023. Casein as protein and hydrolysate: Biostimulant or nitrogen source for Nicotiana tabacum plants grown in vitro? Physiol Plant. 175(4): e13973. https://doi.org/10.1111/ppl.13973
Coste A, Pop C, Halmagyi A, Butiuc-Keul A. 2019. Secondary metabolites in shoot cultures of Hypericum. In: Ramawat KG, Ekiert HM, Goyal S. (eds) Plant cell and tissue differentiation and secondary metabolites. Reference Series in Phytochemistry. Cham: Springer, pp. 1-36. https://doi.org/10.1007/978-3-030-30185-9_9
Dorani E, Sadeghi Z. 2024. In vitro propagation of Physalis alkekengi L. using axillary buds. J Plant Physiol Breed. 14(1): 67-76.‏ https://doi.org/10.22034/jppb.2024.58985.1322
El-Azraq BM, Hassan HMS, El-Mekawey MA, Ali MA. 2018. Effect of light, explant type, growth regulators and mannitol on (Capparis spinosa L.) callus induction and active ingredients production. Sinai J Appl Sci. 7(2): 89-98. http://dx.doi.org/10.21608/sinjas.2018.78907
Gao F, Peng C, Wang H, Shen H, Yang L. 2021. Selection of culture conditions for callus induction and proliferation by somatic embryogenesis of Pinus koraiensis. J For Res. 32: 483-491. https://doi.org/10.1007/s11676-020-01147-1  
Ghassemi B, Nayeri FD, Hosseini R. 2015. The impact of carbon source, explants and growth regulators on callogenesis and organogenesis of Artemisia annua. Not Sci Biol. 7(4): 475-485. http://dx.doi.org/10.15835/nsb.7.4.9596
Grzegorczyk-Karolak I, Hnatuszko-Konka K, Krzemińska M, Olszewska MA, Owczarek A. 2021. Cytokinin-based tissue cultures for stable medicinal plant production: Regeneration and phytochemical profiling of Salvia bulleyana shoots. Biomolecules. 11(10): 1513. https://doi.org/10.3390/biom11101513
Jan R, Khan MA, Asaf S, Lee IJ, Kim KM. 2020. Modulation of sugar and nitrogen in callus induction media alter PAL pathway, SA and biomass accumulation in rice callus. Plant Cell Tissue Organ Cult. 143: 517-530. https://link.springer.com/article/10.1007/s11240-020-01938-8
Kalashnikova EA, Kirakosyan RN, Abubakarov HG, Zaitseva SM. 2022. The effect of the hormonal composition of the nutrient medium and endogenous polyphenols on the formation of callus tissue Ipomoea batatas (L.). Problems of Biological, Medical and Pharmaceutical Chemistry 25(11): 46-58. https://doi.org/10.29296/25877313-2022-11-08
Khakpour S, Motallebi-Azar A, Hosseini B, Alizadeh-Salte S, Hasani A. 2015. Optimization of micropropagation by different concentration of vitamins and sucrose in St. John’s Wort (Hypericum perforatum). Plant Breed Seed Sci. 71(1): 67.
Khaliluev MR, Bogoutdinova LR, Baranova GB, Baranova EN, Kharchenko PN, Dolgov SV. 2014. Influence of genotype, explant type, and component of culture medium on in vitro callus induction and shoot organogenesis of tomato (Solanum lycopersicum L.). Biol Bull Russ Acad Sci. 41: 512-521. https://doi.org/10.1007/s11240-018-1395-8
Koperdáková J, Košuth J, Čellárová E. 2007. Variation in the content of hypericins in four generations of seed progeny of Hypericum perforatum somaclones. J Plant Res. 120(1): 123-128. https://doi.org/10.1007/s10265-006-0031-6
Kwiecień I, Szydłowska A, Kawka B, Beerhues L, Ekiert H. 2015. Accumulation of biologically active phenolic acids in agitated shoot cultures of three Hypericum perforatum cultivars: ‘Elixir’, ‘Helos’ and ‘Topas’. Plant Cell Tissue Organ Cult. 123: 273-281. https://doi.org/10.1007/s11240-015-0830-3
Linjikao J, Inthima P, Limmongkon A, Kongbangkerd A. 2024. Mannitol and sorbitol concentration optimization for effective Epipactis flava Seidenf. in vitro slow growth storage. In Vitro Cell Dev Biol- Plant. 60(4): 496-507. http://dx.doi.org/10.1007/s11627-024-10437-w
Malik K, Birla D, Yadav H, Sainger M, Chaudhary D, Jaiwal PK. 2017. Evaluation of carbon sources, gelling agents, growth hormones and additives for efficient callus induction and plant regeneration in Indian wheat (Triticum aestivum L.) genotypes using mature embryos. J Crop Sci Biotechnol. 20: 185-192. https://doi.org/10.1007/s12892-017-0046-0
Martinez ME, Jorquera L, Poirrier P, Díaz K, Chamy R. 2021. Effect of the carbon source and plant growth regulators (PGRs) in the induction and maintenance of an in vitro callus culture of Taraxacum officinale (L) Weber Ex FH Wigg. Agronomy. 11(6): 1181. https://doi.org/10.3390/agronomy11061181
Matter MA, Hanafy MS, Aly UI. 2017. Effect of methyl jasmonate and mannitol application on growth and eugenol content in callus cultures of carnation. Plant Tissue Cult Biotechnol. 27(2): 227-240. http://dx.doi.org/10.3329/ptcb.v27i2.35028
Mohammadrezakhani S, Rezanejad F, Ganjalikhani F. 2022. Quercetin and gamma-aminobutyric acid content and in vitro germination in pollen grains of Citrus species at different temperatures. J Plant Physiol Breed. 12(2): 11-19.‏ https://doi.org/10.22034/jppb.2022.16294
Rajesh K, Reddy SN, Reddy APK, Singh BG. 2014. A comparative study of plant growth regulators on morphological, seed yield and quality parameters of greengram. Int J Appl Biol Pharm Technol. 5(3): 103-109.
Rashidi M, Saremi-Rad A, Froozesh P, Ghasemo Omran VO. 2018. Effect of plant growth regulators on Fittonia verschaffeltii regeneration at in vitro conditions. J Plant Physiol Breed. 8(2): 59-68.‏ https://doi.org/10.22034/jppb.2018.9742
Sadat-Hosseini M, Soleimani A. 2024. Callus induction, shoot and root regeneration in Hyssopus officinalis using sodium nitroprusside and plant growth regulators. J Med Plants Byprod. 13(4): 932-939. https://doi.org/10.22034/jmpb.2023.363600.1611
Singh PR, Singh LJ. 2021. In vitro propagation for improvement of medicinal plants: a review. J Pharmacogn Phytochem. 10(1): 1484-1489.
Ullah M, Khan MS. 2022. Effects of carbon sources and growth regulators on the tissue culture of sugarcane. Sarhad J Agric. 38(1): 312-321. http://dx.doi.org/10.17582/journal.sja/2022/38.1.312.321