Nitrogen (N) is an essential nutrient required for growth and development of crops that will eventually effect on the yield potential. It is a major element in the structural, genetic and metabolic composites in plant cells, such as proteins, hormones, enzymes, chlorophylls and vitamins (Uchida 2000). Developing crops require an adequate N supplement for attaining a better vegetative growth and a desirable yield (Lemaire et al. 2008). Further, it induces the flower development of vegetable crops, by contributing to the metabolism and effects on the quality, size, colour and taste of the fruits (Wahocho et al. 2016).
Nitrogen is a limiting factor in the production of crops under many agro-eocological systems, unless a symbiotic association with N-fixing microorganisms is available. Thus, inorganic nitrogenous fertilizers have become an essential input in the commercial agricultural operations that is supplied in the form of nitrate (NO3−) and ammonium (NH4+) ions, those are the amplest inorganic forms of N in the agricultural lands (Barunawati et al. 2013). Those fertilizers are applied in the liquid- or solid-forms as the foliar- or soil-applications. Being the high solubility, applied N is lost from the soil, thus, a frequent application is necessary in order to obtain a highest growth, development and yield from the crops.
The excessive use of N fertilizers has negative impact on the environment. Only a limited proportion of the N applied to the soil is essentially taken up by the plants, while the rest is lost, by gaseous emission due to soil denitrification and ammonia volatilization, washing in runoff or leaching (Raun and Johnson 1999; Good et al. 2004; Vos 2009; Schum and Jansen 2014). Ammonium nitrogen (NH4+ ions) being positively charged has good sorption behavior with the negatively charged clay or silt particles (Eturki et al. 2012). Despite their water solubility, they bind with those particles, preventing the leaching (Nommik and Vahtras 1982) however, subjected to leaching in the sandy soils with poor contents of organic matter, clay and silt fractions (Hallberg 1989). However, due to the soils lacking positively charged sites for adsorbing negatively charged nitrate nitrogen (NO3− irons), they are extensively leached-down being the main mode of loss specially from the sandy soils (Xiong-Hui et al. 2011). The problem gets worsen in the areas with hot/arid climate with higher temperature levels where the higher decomposition rate limits the available organic matter contents to the very low level as reported in the Kalpitiya area in Sri Lanka, where the sandy regosols is the predominant soil type (Jayasingha et al. 2013). Thus, farmers often tend to apply excessive amounts of nitrogen fertilizers, while further increments are applied during rainy season to compensate the losses due to leaching (Jayasingha et al. 2013). Furthermore, if the area prevails higher temperature levels, the problem is severe since heavy irrigation is demanded to cool-down the environment. Thus, a frequent use of N fertilizer in high doses is required to maximize the crop production. Such continuous and rigorous application of N fertilizer and over-irrigation have led to significant levels of contamination of groundwater all over the world and the issue is serious in commercial cultivations in many parts of the globe (Gusman and Mari 1999; Zebarth and Milburn 2003; Jalali 2005; Singh and Craswell 2021). The contaminated water creates the social problems that eventually impacts on the human health. High levels of NO3− in the drinking water and food products may lead to severe health issues such as methaemoglobinemia, birth defects or the risk of cancer development (Lawrence and Kuruppuarachchi 1986; Bawatharani et al. 2004; Gao et al. 2012; Yu et al. 2020). Furthermore, these interconnected factors cause to enhance the cost of production. Therefore, precautions have to be taken in such agricultural lands to prevent leaching of N.
The negative environmental impact of nitrogen fertilizers can be reduced exploring two strategies. Development of high-efficiency fertilizers and optimized nutrient management is one approach (Hopkins et al. 2008; Singh and Craswell 2021) where the N demand depends on the size of the crop and nitrogen use efficiency (NUE) (Ladha et al. 2005; Prieto et al. 2017). The second focuses on varietal selection for tolerance to lower N availability in the soil (Schum and Jansen 2014; Mastrodomenico et al. 2019). In order to practice the environmental friendly sustainable agriculture, the fertilizer requirement needs to be optimized by using cultivars which can tolerate low N levels and high utilization efficiency (Cormier et al. 2016). In this scenario, effective germplasm screening procedures are required. The genotypes with higher NUE will remain their growth and reproduction stable under reduced N supplement.
There is a wide range of methodological approaches available for selection (Miflin 2000). The most basic approach is screening under field conditions. However, owing to the interaction with the environment, a particular variety will perform differently in the field, resulting in a high level of inconsistency (Zhang et al. 2013). It requires more time, labor and space compared to the other methods (Miflin 2000; Panis et al. 2020). In recent years, advanced genotyping and phenotyping technologies have been introduced. Marker-assisted selection (MAS) is one of most efficient approach that can be used to screen thousands of genomic regions of a crop (Collard and Mackill 2008; Lidder and Sonnino 2012). In parallel, high throughput phenotyping techniques, including hyperspectral, multispectral, thermal sensors have been introduced in the recent past (Araus et al. 2018). However, these advanced techniques are more complex and require high capital cost.
One of the most high-throughput, efficient, and cost-effective strategies for selecting the stress-tolerant plants is tissue culture-based in vitro assay of the genetic resources (Jathunarachchi et al. 2021). In vitro screening facilitates a simpler, more convenient, and more detailed analysis of specific stress responses in a highly controlled environment in an effective mode (Rai et al. 2011). The testing factor is changed in the absence of external environmental threats while all other factors remain the same. The approach has been successfully utilized to screen genetic resources in different aspects including NUE (Hajari et al. 2015; Schum et al. 2017; Langholtz et al. 2021).
Chili (Capsicum annum), belongs to family Solanaceae, is one of the most extensively grown horticultural crop in South Asian region. Chili contains high carotenoids, ascorbic acid (vitamins A and C) and phenolic compounds giving antioxidant, antimicrobial and anti-cancer properties (Dini 2018; Hernandez-Alvarez et al. 2020) that helps preventing the parasitic and heart diseases, improving the circulation, regulating the cholesterol level and controlling the obesity by increasing the energy outflow (Dini 2018). The burning sensation of capsaicin (Ringkamp 2008; Yenigun and Thanassi 2019) is the specific characteristic feature of chili pods that are used as vegetables and spice in fresh, cooked, or processed forms as powder, curry paste, sauce and pickle (Padmanabhan et al. 2016). The FAO estimates that 45.0 million tons of chilies are produced globally each year (FAOSTAT, 2021). Thus, expansion of cultivation is important to meet the demand causing a minimal impact to the environment.
Kalpitiya agricultural zone in Sri Lanka located between 79° 40’– 79° 50’ Easting longitude and 8° 20’ – 8° 90’ Northern latitude (Arachchi et al. 2014) that contains sandy Regosols, is experiencing the aforementioned obstacles in cultivating chili which is one of the most cultivated crops in the area. The issue has marked serious where the ground water is unsuitable for the human consumption (Arachchi et al. 2014, Jayasingha et al. 2013). The main contaminant is N. However, the vegetables cultivated in the Kalpitiya area produce and supply an estimated 20% of the country’s vegetables. Continuation of the production by applying sustainable strategies is a timely requirement. Cultivation of the highly responsive chili varieties with high NUE is a potential solution to overcome the aforementioned problems. Therefore, the present study was undertaken to screen the commercially cultivated chili varieties tolerant to reduced N supplements under in vitro conditions as a preliminary approach to select the varieties capable of minimizing the usage of N fertilizer.