Research in Plant Metabolites

Research in Plant Metabolites

valuation of Nitrogen Levels and Planting Density Management on Physiological Traits and Secondary Metabolites in the Medicinal-Industrial Plant Indigo (Indigofera tinctoria L.)

Document Type : Original Article

Authors
Mohammad Reza Zargaran Khouzani 1
Mohammad Hossein Gharineh 2
ahmad koochek zadeh 2
Amin lotfi jalal abadi 2
1 Department of Plant Production Engineering and Genetics, Khuzestan University of Agricultural Sciences and Natural Resources
2 Faculty member, Department of Plant Production Engineering and Genetics, Khuzestan University of Agricultural Sciences and Natural Resources
10.22034/jrpsm.2025.528531.1076
Abstract
Indigofera tinctoria L. is one of the most important dye-yielding plants worldwide, with numerous industrial and medicinal applications. Considering the significant role of agronomic factors in improving both quantitative and qualitative yield of this plant species, the present study was conducted during the 2023 cropping season in Shushhtar County, northern Khuzestan Province, Iran, using a split-plot experimental design based on a randomized complete block design with three replications. The objective was to evaluate the effects of different nitrogen fertilizer rates (25,50,75&100 kg.ha⁻¹) and plant densities (30,40,50 & 60 plants m⁻²) on photosynthetic traits (chlorophyll a ,b ,total chlorophyll & carotenoids) and indigocarmine pigment content.The results indicated that increasing nitrogen application significantly enhanced all photosynthetic traits as well as the secondary metabolite indigocarmine. The highest values of chlorophyll a (1.85 mg g⁻¹), chlorophyll b (0.73 mg.g⁻¹), total chlorophyll (2.58 mg.g⁻¹), and indigocarmine percentage (0.81%) were observed under the treatment of 75 kg N ha⁻¹ and a density of 30 plants m⁻². Moreover, the treatment of 75 kg N ha⁻¹ combined with a planting density of 40 plants m⁻² provided the highest indigocarmine content (5.75 g m⁻²), which was identified as optimal in terms of quantitative performance.Analysis of variance revealed that all main effects were statistically significant at the 1% probability level, while the interaction effect of nitrogen and density was significant at the 5% probability level for all studied traits. The relatively low coefficient of variation (ranging from 3.8% to 7.2%) indicated high accuracy in conducting the experiment. A strong positive and significant correlation (r=0.91) between photosynthetic indices and indigocarmine content suggests a direct role of the photosynthetic system in the synthesis of secondary metabolites.Ultimately, applying 75 kg ha⁻¹ of nitrogen along with a planting density range of 30–40 plants m⁻² was identified as the best treatment in terms of balancing product quality and quantity. These treatments can be proposed as an optimal cultivation model for Indigofera tinctoria L. in similar climatic regions.
Keywords
Agricultural management
Environmental stress
Photosynthesis
Plant pigments and Protective mechanisms
Alizadeh Sahzabi, M., Khoshkhui, M., & Tavili, A. (2007). Effect of nitrogen levels on growth and yield of
Indigofera tinctoria L. under greenhouse conditions. Journal of Agricultural Sciences, 13(3), 25–34.
Arnon, D. I. (1967). Method of extraction of chlorophyll in the plants. Agronomy Journal, 23(1), 112–121.
Bagherpour, E., et al. (2015). Influence of planting date and nitrogen fertilizer on biomass and indigo content
in Isatis tinctoria. Iranian Journal of Medicinal and Aromatic Plants, 31(2), 223–234.
Dadkhah, A., et al. (2009). Optimization of agronomic practices for Indigofera tinctoria. Journal of
Agricultural Science and Technology, 11(3), 215–225.
Ebrahimi, R., Heidari, M., & Paknejad, F. (2015). The effect of different nitrogen levels on physiological
characteristics and yield of safflower (Carthamus tinctorius L.) cultivars. Journal of Crop Production, 8(2), 235–
252.
Fateh, M., Kazemi Arababadi, H., Mohammadi, S., Farhbkhs, F., & Zand, E. (2013). Effect of plant
density and different levels of nitrogen fertilizer on yield components, leaf area, chlorophyll and grain protein of
maize (Zea mays L.) and biomass (Amaranthus retroflexus L.). Iranian Journal of Medicinal and Aromatic Plants
Research, 29(2), 261–268.
Fazeli, R., Amini, M., & Nikooye, B. (2019). Effect of planting date and plant density on growth indices and
essential oil content of basil (Ocimum basilicum L.). Journal of Medicinal Plants, 18(1), 101–112.
Gupta, S. K., Singh, R. P., & Singh, R. (2003). Influence of nitrogen and phosphorus on growth and yield
of Indigofera tinctoria. Indian Journal of Agronomy, 48(3), 178–181.
Hajmirzaei, M. S., Ansari, H., Hashemnia, S. M., & Azizi, M. (2019). Investigation of the interactive effects
of organic fertilizer and irrigation on some morphological and physiological indices of peppermint (Mentha
piperita L.). Iranian Journal of Irrigation and Drainage, 13(6), 1794–1805.
Hernández-Cruz, J. A., Peña-Valdivia, C. B., Sánchez-Reyes, O. O., & Cruz-Ortega, R. (2002).
Photosynthetic pigments, phenols and antioxidant activity in Opuntia spp. under saline stress. Agrociencia, 36(5),
583–591.
Jain, S., Nayak, S., & Joshi, P. (2011). Phytochemical study and physical evaluation of Indigofera tinctoria
leaves. Pharmacie Global, 1, 1–2.
Karimi Farzegi, M., & Manoumi Zadeh, Z. (2014). Effect of planting density and planting date on yield
and yield components of safflower (Carthamus tinctorius L.) in Torbat-e Heydariyeh. Journal of Plant
Environmental Physiology, 9(36), 71–85.
Khoshnam, Z., Amiri-Nejad, M., Ayeen, A., & Parsamotlagh, B. (2021). Evaluation of intercropping
system of indigo (Indigofera tinctoria L.) and roselle (Hibiscus sabdariffa L.) on yield, biodiversity and weed
population dynamics. Journal of Crop and Horticultural Production, 10(2), 109–121.
Koocheki, A., Joorabian, S., & Nassiri Mahallati, M. (2020). Effect of planting date and plant density on
growth and yield of safflower (Carthamus tinctorius L.) under Mashhad conditions. Iranian Journal of Field Crops
Research, 8(1), 1–10. https://doi.org/10.22049/ijfcr.2020.26735.1450
Laitonjam, W. S., & Wangkheirakpam, S. (2011). Comparative study of the major components of the indigo
dye obtained from Strobilanthes flaccidifolius Nees. And Indigofera tinctoria Linn. Journal of Natural Dyes, 3(1),
15–22.
Lichtenthaler, H. K. (1987). Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes.
Methods in Enzymology, 148, 350–382.
Lootfalizadeh, H., et al. (2011). Effects of nitrogen fertilizer on chlorophyll content in legumes. Journal of
Plant Nutrition, 34(10), 1452–1463.
Modafe Behzadi, N., Rashed Mohammadi, P., & Jahan, M. (2018). Influence of planting date and plant
density on morphological characteristics and yield of indigo (Indigofera tinctoria L.). Agricultural Ecology, 10(4),
1067–1079.
Modanlo, M., Amiri, M. B., & Ghanbari, A. (2021). Effects of planting date and nitrogen levels on
morphological traits and essential oil content of lemon balm (Melissa officinalis L.). Journal of Essential Oil
Bearing Plants, 24(2), 321–331.
Nakabayashi, R., & Saito, K. (2015). Integrated metabolomics for abiotic stress responses in plants. Current
Opinion in Plant Biology, 24, 10–16.
Nematiyan, A., & Ghoushchi, F. (2011). Effect of planting density and nitrogen fertilizer on yield and yield
components of medicinal plant Aloe vera (Aloe barbadensis Mill). Journal of Crop Ecophysiology (Islamic Azad
University, Ahvaz Branch), 3(10), 85–93.
Nohong, M., et al. (2019). Growth response of Indigofera zollingeriana to nitrogen fertilization. Tropical
Agriculture Research, 31(2), 123–132.
Noroozi, Y., Ghobadi, M., Saedi, M., Rezadoust, H., Kahrizi, D., & Dougan, H. (2024). Effect of nitrogen
application and harvest cutting on yield, quality, and essential oil composition of thyme (Thymus vulgaris L.).
Medicinal Plant Eco-Phytochemistry, 2(12), 27–44.
Omid Beigi, A. (2021). Comprehensive guide to the production and processing of medicinal plants (Vol. 1).
Behnashr Publications (Astān-e Quds Razavi).
Sajed Gollojeh, K., Khomari, S., Shekhzadeh, P., Sabaghnia, N., & Mohebodini, M. (2020). The effect of
foliar spray of Nano silicone and salicylic acid on physiological traits and seed yield of spring rapeseed at water
limitation conditions. Journal of Crop Production, 12(4),137–156. https://doi.org/10.22069/ejcp.2020.16396.2221
Sales, P. J., Da Silva, J. B., & De Souza Filho, A. P. S. (2016). Light competition and plant spacing affect
biomass production and essential oil composition in Lippia alba (Mill.) N.E. Brown. Industrial Crops and
Products, 87, 209–216.
Setyaningrum, D., Budiastuti, M. T. S., Pujiasmanto, B., Purnomo, D., & Supriyono. (2020). Light
intensity and biofertilizers effect on natural indigo production and nutrient uptake of Indigofera tinctoria L. Indian
Journal of Agricultural Research, 54(5), 578–584.
Shabahang, J., Radmehr, M., & Ahmadvand, R. (2014). The effect of planting density on growth indices
and grain yield of wheat (Triticum aestivum L.) cultivars. Journal of Crop Production and Processing, 4(12), 133–
143.
Soleimani Sardo, M., Afsharmanesh, Gh. R., & Roodbari, Z. (2018). Evaluation of planting date and plant
density on yield and yield components of mung bean (Vigna radiata L.) in Jiroft region. Journal of Agronomy
and Plant Breeding in Dry Regions, 1(1), 27–34.
Tahmasebi, A., Kamali, H. R., & Zakari Darbaghi, Z. (2025). Evaluation of plant density effect on the
nutritional quality of seeds in four quinoa (Chenopodium quinoa Willd.) cultivars under hot and humid conditions
Journal of Research in Plant Metabolites, 3(1), 5–19. https://doi.org/10.22034/jrpsm.2025.517031.1064
Torrecilla, J. M., et al. (2011). Secondary metabolite accumulation under nitrogen stress in medicinal plants.
Plant Physiology and Biochemistry, 49(10), 1123–1130.
Zahedifar, M., et al. (2022). Soil chemical analysis in agricultural research. Iranian Journal of Soil Research,
36(4), 450–460.
Zarei, Sh., Hosseibi, P., Kahrizi, D., & Safi Aldin Ardabili, S. M. (2021). Effect of nitrogen on yield and
yield components of camelina (Camelina sativa) under different planting dates. Iranian Journal of Crop Research,
19(4), 311–325.
Zargaran Khouzan, M. R., & Daei Naseri, S. M. A. (2025). Nile: A deep dive into the history of natural
and synthetic indigo. Arshak Publishing.
Zhang, Y., Wang, X., Liu, Y., & Zhao, W. (2014). Leaf photosynthetic characteristics and chlorophyll
fluorescence parameters of Paeonia lactiflora under different light intensities. Photosynthetica, 52(3), 447–455.