METABOLITE PROFILING OF MORINGA USING PY-GCMS AND TOLERANCE EVALUATION TO ALUMINUM ON IN VITRO CULTURE

Article Highlight:

• Four accessions of Moringa oleifera from different regions in Indonesia were evaluated to identify with tolerance to aluminium (Al) stress
• Eight metabolites show a very high correlation with acetic acid (one of the metabolites associated with AlCl₃ stress) including cyclopentene, 2-allyphenol, 4-ethynyl-6-8-dioxane, vinyl ether, ethanone 1-oxiranyl, 2-methylpyridine, 2-butanone, and ethanesulfonic acid.
• This research makes a significant contribution to understanding aluminium tolerance in oleifera by identifying tolerant accessions, clustering relevant traits, and highlighting key metabolites to in vitro culture.

ABSTRACT

Moringa oleifera Linn. has received substantial scientific interest due to its numerous bioactive compounds and its function as a nutritional resource. The absorption of aluminum by plants hinders several metabolic and physiological processes, leading to inhibited plant development and decreased agricultural output. Some accessions from different regions in Indonesia were evaluated to identify those with tolerance to aluminum (Al) stress. Al-tolerant selection was carried out in vitro through the selection method for Al stress by adding 0, 50, 100, 250, and 500 mg/L of AlCl₃ to the media. Furthermore, identifying the metabolite profile of four M. oleifera accessions from four distinct regions in Indonesia: Blora, Bogor, Enrekang, and Bima, has been done using Py-GCMS. Specific metabolites associated with tolerance to Al stress and organic acids need to be identified. The highest survival rate was observed in the Bogor and Blora accessions when exposed to AlCl₃ at concentrations ranging from 0 to 250 mg/L, demonstrating greater tolerance to AlCl₃ than other accessions based on various variable, such as shoot height, number of shoots, number of petioles on a medium containing 100 to 250 mg/L of AlCl₃. The mean value was not statistically different from the control. Acetic acid was identified as one of the metabolites associated with AlCl₃ stress. A total of 21 metabolites were specifically correlated with acetic acid in a positive manner, among which 8 metabolites including cyclopentene, 2-allyphenol, 4-ethynyl-6-8-dioxane, vinyl ether, ethanone 1-oxiranyl, 2-methylpyridine, 2-butanone, and ethanesulfonic acid exhibited a very high correlation.

INTRODUCTION

Aside from its role as a nutritional resource, Moringa oleifera Linn. has received significant scientific interest for having numerous bioactive compound. These substances, contained in seeds, pods, and leaves, exert significant physiological effects on humans. Moringa exemplifies a food source that is rich in essential elements, such as minerals, vitamins, and proteins, making it a reliable and long-lasting source of nourishment(Yuniati et al., 2022).

According to(Srivastava et al., 2023), the environmentally conscious cultivation of Moringa is in line with the ideas of sustainable farming, which makes it a sustainable choice. Humans consume the entire M. oleifera plant, including its leaves, fruits, immature pods, and flowers, as traditional food. Various countries, including India, Hawaii, the Philippines, and other African countries, utilize the flowers, fruits, foliage, and early seed pods of this tree as exceptionally nutritious vegetables(, 2021).

Moringa tree produces between 43 and 115 tonnes of biomass per hectare annually. Its leaves contain high levels of dry matter, crude protein, essential amino acids, and phytoactive substances, such as phenolic acids and flavonoids. According to(Kholif et al., 2016)and(Nouman et al., 2016), they also contain high levels of vitamins A, B, C, and E(Zheng et al., 2022).

Conventional propagation techniques for M. oleifera predominantly involve the cultivation through seedlings or vegetative cuttings.(Debnath & Juran, 2020)have identified several obstacles associated with conventional propagation methods, including a limited quantity of explants, lethargic growth, and heightened vulnerability to pests and diseases. Using in vitro micropropagation, a widely applied technique that has significantly improved the quality of several plant propagations, would be an alternative method(Zheng et al., 2022).

The reduction of arable land from industrialization and conversion has posed a significant obstacle to propagating M. oleifera. To address this challenge, using suboptimal land becomes an alternative option. According to(Fanindi et al., 2020), Indonesia has approximately 108.8 million ha of suboptimal land area as acid dry land, with 62.6 million ha still having the potential for agricultural use. However, inefficient land use is constrained by several abiotic issues, such as low pH soil and high aluminum (Al) saturation. These characteristics inhibit the photosynthetic process and plant growth, resulting in reduced agricultural yields(, 2018);(Fanindi et al., 2020).

Acid-dry land is defined as soil with a pH below 5, indicating acidity. The majority of agricultural land in Indonesia has acidic soil conditions. High rainfall is one contributing factor. Increased rainfall can accelerate the degradation of soil minerals. In moist tropical regions, intensive weathering and nutrient leaching processes cause the soil to be acidic and have high aluminum saturation. Soil acidity can result from prolonged land use and excessive application of chemical fertilizers(Mulyani & Sarwani, 2013).

Acid soils make up approximately 40% of the world’s potentially cultivable areas. Aluminum ranks as the third most plentiful element, behind oxygen and silicon, according to(Riaza et al., 2018). Plants absorption of aluminum hinders several metabolic and physiological processes, resulting in inhibited plant development and decreased agricultural output(Liu et al., 2023).

Soil pH significantly influences plant metabolism, affecting nutrient availability, enzyme activity, and overall biochemical pathways. In acidic soil conditions, essential nutrients, such as phosphorus, calcium, and magnesium become less available, potentially altering the secondary metabolite profile of plants(Sanchez et al., 2006). To comprehensively understand these chemical alterations, a precise and sensitive analytical technique is required.

Pyrolysis gas chromatography and mass spectroscopy (Py-GCMS) is a direct method that analyzes the pyrolytic behavior of biomasses and characterizes their volatile products. Recently, there has been a significant increase in interest in this technique due to its ability to identify all pyrolytic products more efficiently than the GCMS of condensable products (liquid fraction). Identification of these products under various operating conditions necessitates complete condensation(Bensidhom et al., 2021).

According to Inostroza-Blancheteau et al. (2012), organic acids are very important for both tolerance and avoidance pathways because they make stable complexes with Al3+ inside and outside of cells that neutralize it. Various crops excrete distinct varieties of organic acids. For instance, studies have shown that the roots of wheat(Yang et al., 2011)and eucalyptus(Li et al., 2021)secrete large levels of malic acid and acetic acid. Furthermore, researchers have identified citric acid secretion in maize, common bean, and cassia. Additionally, oxalic acid secretion has been discovered in buckwheat(Liu et al., 2023).

The objective of this research was to characterize the metabolite profiles of four M. oleifera accessions obtained from four distinct regions in Indonesia, i.e., Blora, Bogor, Enrekang, and Bima, utilizing Py-GCMS. The research further aimed to assess the responses of these accessions to aluminum chloride (AlCl₃) stress to in vitro culture. Additionally, to identify specific metabolite molecules, excluding organic acids, that are linked to tolerance to Al stress.

MATERIALS AND METHODS

Materials and Experimental Design

The materials used were shoot cultures of 4 accessions of M. oleifera at 6 weeks after culture (WAC). The basal medium used was DKW medium (Driver & Kuniyuki 1984), supplemented with AlCl₃according to the treatment tested. The experimental design was a completely randomized factorial design, with two factors tested: 1) The type of M. oleifera accessions: Blora (Java Island), Bogor (Java Island), Bima (Lombok Island), and Enrekang (Sulawesi Island); 2. Concentrations of AlCl₃(0, 50, 100, 250, and 500 mg/L). This experiment used 12

.....