Horticulture

In addition, this opens the possibility of customising the buffer species and mixtures added, so that the amounts or proportions of the positive  ion portion of the buffer delivered to the system may be adjusted or controlled by choosing buffer species or mixtures based on the buffering capacity of the negative ion. Conductivity continually increases in fish-only systems because of the build-up of ions which are produced as the by-products of fish metabolism. The inclusion of plants into recirculating aquaponic systems leads to active uptake of waste nutrients and ions by the plants, which counteracts the ionic build-up seen in fish-only systems. The efficiency of plants to take up nutrients and ions in hydroponic systems, and thus maintain zero conductivity accumulation within system waters, is dependent upon whether the correct mix and concentrations of those nutrients are provided. The conductivity curves  from the present study indicate that, in the final third of the experiment, plants within the mixed and potassium treatments maintained slightly lower water conductivities than observed in control or calcium treatments, although overall conductivities did not differ significantly between any treatments across the 21-day course of the research-scale experiment. This apparent lowering of conductivity levels in the last third of the experiment for the mixed and potassium treatments is probably since plants within these treatments had access to increased levels of potassium during their fastest-growing phase.

Potassium is an essential macronutrient to plants  and is known to play a key role in a plant’s ability to synthesise proteins and carbohydrates,and thus grow. Adler, Harper, Takeda, et al.  also argued that when other nutrients limit plant growth, nutrient removal can be increased by adding those nutrients that are most limiting, and therefore, rolling benches other essential nutrients  must be added to the aquaponic system. These nutrients typically include iron, manganese and potassium. The lowered conductivities in the two treatments containing higher additions of potassium  may suggest that the plants in these treatments had a slightly increased ability to achieve elevated carbohydrate and protein synthesis, thus allowing them to remove and assimilate slightly greater amounts of ions from the surrounding water medium and therefore, lowering the conductivity of the system water. This hypothesis also may be supported by the observation that lettuce plants within these two potassium-containing treatments exhibited the highest growth rates and yields  and exhibited the greatest removal rates of nitrate from recirculating waters. In terms of water use, results suggest that those treatments containing potassium were also more efficient,with significantly less water used by the potassium and mixed test treatments, when compared to the control treatment. Again, this result is probably correlated with the plant’s requirement for potassium to achieve carbohydrate and protein synthesis and subsequent growth. The plant’s requirement to transpire more water from those treatments not containing additional potassium may be explained by the fact that the plants may have been transpiring greater amounts of water to try and gain greater access to limiting ions, such as potassium. In conclusion, results from the present study suggest that, when using buffers for maintenance and control of pH in recirculating aquaponic systems, it is advantageous to use buffers with positive ion constituents that are essential to optimal plant growth. Parameters such as plant growth and yield, nitrate removal, conductivity and water use demonstrate that either a potassium-based buffer or a mixture of potassium and calcium-based buffers are the most appropriate buffers to use to achieve optimal plant growth efficiencies in the research-scale aquaponic system, whilst parameters such as fish growth and FCR, dissolved oxygen and pH maintenance are unaffected by the positive ion make-up of the buffer.

In addition, it is apparent from buffer use and pH maintenance parameters that the negative ion constituent of the buffer may affect the amount of buffer required. It is therefore recommended that this work be replicated at a commercial scale and through an entire fish production cycle and that future aquaponic researchers and industry individuals use buffers containing potassium as the positive ion constituent and consider periodically using calciumbased buffers to maintain pH in recirculating aquaponic systems.Plan factories are facilities that enable the year-round production of vegetables and other productions through precise control of the growth condition. The practical implementation of such facilities is needed to decrease its costs. Controlling circadian rhythm is one of optimizing idea to solve the problem of cost. Plant circadian rhythms are composed of a large number of genes. Expressions of these genes build complex feedback loop.The circadian rhythms have important functions in physiological processes of plants including photosynthesis and growth. Therefore precise control of circadian rhythms is able to become a key technology for plant production in artificial environments such as plant factory. When we measure the luciferase  bioluminescence to observe circadian rhythm, the sample what is small seedling or cut leaf inside petri dish is used mainly. However, the cut leaf will change brownish or die after some days. In the leaf of the lettuce, the bioluminescence damps and cannot be measured by its degradation. In addition, a cut leaf is able to grow only to some size into the petri dish. From these reasons, we cannot observe the circadian rhythm in each stage of growth and the responses to the controlled light conditions in the long term. Unfortunately, we have one more problem which LUC bioluminescence only can be measured from the genetically modified lettuce. The lettuce is modified luciferase reporter gene which fused clock gene  was used in this experiment and a firefly luciferase gene into. The genetically modified plants emit bioluminescence in proportion to the expression of CCA1. We cannot treat it for food. Therefore we paid our attention to the delayed fluorescence  of the lettuce. In this study, DF is the light which is emitted immediate after illuminated sample by light emitting diode. When photosynthesis is done, DF is emitted mainly from chlorophyll. DF reflects the physiological state in plant and can be measured in any wild type plant in real time. Therefore it can be one of important indicator of circadian rhythm. However, it is not studied the simultaneous measurement from the same individual because DF has lower bioluminescence intensity than LUC bioluminescence, these spectrums are adjacent each other and the intensity damps exponentially. Because of these reasons, spectrometry is not able to apply to measure of DF and LUC bioluminescence. Therefore, we developed the devise  which measured LUC bioluminescence and DF from lettuce, which cultivated in the state that nearer to vertical farming, at the same time in the long term.Atrazine  is a widely used triazine herbicide over the world. However, studies show that ATZ affects the human endocrine system, lymphatic system, immune system, and reproductive system, and may induce malformations and organism mutations. Although ATZ has been included in the list of endocrine disruptor compounds by the United States, Japan and the European Union and other countries, it is still registered for use in many countries due to its excellent herbicidal efficacy and low price. Therefore, the research on the safety of ATZ’s ecological environment is imminent.

Alfalfa is a kind of forage with high ecological value because of its strong adaptability and regeneration ability. In addition, its root system has nitrogen fixation ability, which can increase the content of soil organic matter and improve the physical and chemical properties of soil. Our previous research has shown that alfalfa has the potential to remediate ATZ contamination. However, excessive pesticide exposure often causes plants to produce some toxic reactions. Chlorophyll content, cell membrane permeability, and accumulation of reactive oxygen species  are commonly used evaluation indicators. Our previous study showed that the accumulation capacity of ATZ in rice leaves at different ages was different,but the mechanism of accumulation on toxic effects is still unclear. Metabolism of pesticides is one of the important ways for plants to slow down the toxic effects. The phase II metabolism  of pesticides in plants requires the consumption of certain endogenous compounds such as glutathione  and glucose, which may disrupt the balance of endogenous metabolites in plants. Cysteine S-conjugates and homoglutathione/glutathione S-conjugates are the major metabolites of ATZ in plants. Therefore, studying the regulation of ATZ on differentially expressed metabolites  and differentially expressed genes  in Cys and GSH metabolism pathways and DEGs related to the generation/elimination of reactive molecular species  in alfalfa is of great significance for the cultivation of ATZ-resistant plant varieties and the construction of transgenic plants with high remediation ability. At present, the research on the fate of organic pollutants in plants is limited to the metabolic process in plants,but the excretion of metabolites by roots is rarely reported. Root exudates refer to various substances released by plant roots into the surrounding environment. A large number of studies have reported that roots can excrete organic acids, phenolic substances, polysaccharides, enzymes and other substances in response to different rhizosphere environmental changes. It is not clear whether the metabolites of ATZ can also be excreted to the external environment and which metabolites can be excreted. The excretion of pesticide metabolites by roots is closely related to the safety of the ecological environment.Per- and polyfluoroalkyl substances  are a group of anthropogenic aliphatic fluorinated chemicals that have been used globally due to their hydrophobic and lipophobic properties. PFASs are persistent in the environment and accumulative in wildlife and humans. Perfluorooctane sulfonate  is one of the well-known PFAS in scientific literature.

It was listed in the Stockholm Convention on Persistent Organic Pollutants in 2009. PFOS was phased out from production and commerce/use in most applications in several countries. Similarly, perfluorooctanoic acid  and long-chain perfluorocarboxylic acids  are believed to be on track to be phased out. Consequently, some substitutes for PFOS/PFOA have emerged, including shorter-chain compounds, ether-PFAS,and fluorotelomers. In particular, 6:2 fluorotelomer sulfonamide alkylbetaine  is an amphoteric PFAS currently used in consumer and industrial products. It was reported in Dupont’s Capstone 1157  surfactant  and AFFFs from Angus Fire,Fire Service Plus, and National Foam. 6:2 FTAB has been detected in various environment media, such as groundwater,soil,earthworms,river water,and sediments. 6:2 FTAB was detected in the surface soil of the long-term fire training site at a high concentration of up to 66,305 ng/g dry weight. Earlier studies demonstrated that PFCA precursors could be taken up by plants from soil/solution and biotransformed to downstream metabolites. 6:2 FTSA, which is also a common PFOS alternative, ebb and flow bench could be bioaccumulated by pumpkin  and then biotransformed to stable products, such as PFHpA, PFHxA, PFPeA, PFBA, PFPrA and TFA. 10:2 fluorotelomer alcohol  could be taken up in wheat and metabolized to PFPeA, PFHxA and perfluorodecanoate  in root while PFDA and perfluoroundecanoic acid  in shoot. 6:2 FTOH  and 8:2 FTOH  could be accumulated by plants, and then transformed to intermediate metabolites and terminal products, such as fluorotelomer saturated acids,fluorotelomer unsaturated carboxylic acids  and PFCAs. Therefore, 6:2 FTAB may be transferred and degraded in plants and further contribute to human exposure through the food chain. However, studies on bioaccumulation and biotransformation of 6:2 FTAB in plants are not reported to date. The biotic and abiotic degradation of 6:2 FTAB has been investigated by several previous studies. The main photochemical degradation intermediate of 6:2 FTAB was 6:2 fluorotelomer sulfonamide,followed by 6:2 FTSA, 6:2 fluorotelomer sulfonamide alkylamine,6:2 FTOH and 6:2 FTUCA, and a large amount of PFCAs have also been observed. In addition to abiotic degradation, the biotic metabolisms of 6:2 FTAB were also investigated, showing different degradation pathways. The main metabolites of 6:2 FTAB biodegraded by strain NB4–1Y isolated from vermicompost were 5:2 fluorotelomer ketone,6:2 FTCA, 6:2 FTOH and 5:2 sFTOH, while 5:3 FTCA, 4:3 FTCA, PFHxA, PFPeA and PFBA were detected at low concentrations. Previous aerobic biodegradation test showed that biodegradation of 6:2 FTAB produced 6:2 FTOH, 6:2 FTCA, 6:2 FTUCA, 5:3 FTCA, 6:2 FTSAm and short-chain PFCAs in the presence of either active or sterilized aerobic sludge, whereas 6:2 FTSA was measured with sterilized sludge only. In addition, 6:2 FTAB could be extensively metabolized in blue mussel and turbot, with 6:2 FTAA being its major metabolite. Formation of 6:2 FTAA, 6:2 FTSAm and 6:2 FTSA indicated that 6:2 FTAB was metabolized in zebrafish. Although it has been confirmed that 6:2 FTAB could be degraded by animals and microorganisms, studies on its transformation mechanism in plants are still limited. It has been demonstrated that 6:2 FTAB had lower bioaccumulation capacity in zebrafish than that of 6:2 FTAA. Previous studies also reported that 6:2 FTAB had toxicity effects on aquatic organisms. The juvenile turbot exposed to 6:2 FTAB was identified that genes involved in the digestion and immune system were suppressed at the transcriptional level. 6:2 FTAB induced developmental toxicity in zebrafish embryos, such as cell apoptosis, oxidative stress and immunotoxicity. 6:2 FTAB and 6:2 FTAA coexposure could disrupt the adult endocrine system and impaired offspring development in adult zebrafish. 

Similar results were found by Koch et al.. Nevertheless, the K content, even in leaves of +K plants, was below the range of 5–40 mg K  indicating an adequate supply of K according to White and Karley. However, since the percentage of K in the DM decreased significantly during growth, this value may not be suitable for predicting K deficiency. Thus, a clear indication of an optimal K concentration should be defined for each growth stage and crop species. Chlorophyll, being an important pigment to absorb light energy,was not affected by K supply. In studies by Armengaud et al.  on Arabidopsis and by Veazie et al.  on Brassica rapa, the chlorophyll content was not affected by K deficit. Presumably, K was directed into the older leaves or side shoots to maintain chlorophyll production by activating specific enzymes. However, several studies revealed a decreased chlorophyll content in leaves of plants under low K supply,which was demonstrated by the decreased photosynthetic efficiency. Similarly, the effect of PEG-induced osmotic stress did not affect the chlorophyll content. However, both chlorophyll a and b content decrease because chlorophyll synthesis is inhibited by water deficit,hydroponic grow system which was obviously not severe in our study.

Generally, the chlorophyll content varies strongly in potato leaves, depending on the cultivar, time of drought stress induction  and leaf development. The decrease in chlorophyll content was caused by reactive oxygen species that are formed as a response to drought stress damaging chloroplasts. This was not demonstrated in our study, perhaps because of the short osmotic stress period. It is also likely that the stress situation occurred at a time when the plants were already fully mature, and therefore less susceptible to abiotic stress. To protect cellular structures and maintain important physiological processes, plants can accumulate low-molecular-weight compounds,which are involved in osmotic adjustment during stress conditions. These include amino acids, measured as free amino acids in leaflets in this study, before, during, and after PEG-induced osmotic stress. In both cultivars at each sampling day, K deficiency increased the free amino acid concentration. This effect might be related to the substitution of K by organic solutes. An increased concentration of organic solutes, such as amino acids, lowers the osmotic potential of plant cells, improving cell hydration and therefore cell turgor to maintain physiological and metabolic processes, which enhance plant growth under stress conditions. Interestingly, the free amino acid content of leaves during PEG induction was not affected by PEG addition in +K plants but reduced in -K+PEG plants compared to -K plants in our study.

A sufficient K supply enhanced amino acid accumulation during PEG-induced osmotic stress compared to that at a low K supply. However, the K contents in the leaves increased during the same period. In accordance with Shabala and Shabala,under K deficiency and PEG-induced osmotic stress, K ions were distributed to developing tissues and accumulated in favour of osmotic compounds since production of these compounds is metabolically more expensive for plants.To characterise the metabolic responses of the plant during stress mitigation under different K supplies, metabolite profiling was used to investigate changes in the concentrations of primary and secondary metabolites. In the metabolomic pathway for Milva, single amino acids showed a higher concentration in -K plants compared to +K plants, which was also shown by higher concentrations of free amino acids in -K leaflets. This increase was even higher when comparing -K+PEG to +K+PEG plants. In some plant species, amino acids are known to increase during drought stress or K deficiency, which was already explained in the previous section. Proline increased under PEG-induced osmotic stress in -K+PEG plants compared to +K+PEG  and -K  plants. Proline concentrations are frequently considered a metabolic stress indicator for several stress factors and its accumulation is often associated with tolerance mechanisms against abiotic stress. Many studies have confirmed that proline accumulates in plant leaves for osmotic adjustment. In this regard, K deficiency alone did not increase the concentration of proline in the sample in our study, which indicates the importance of the proline accumulation under K deficiency when at the same time leaves are exposed to osmotic stress.

Furthermore, the amino acids isoleucine, leucine, and valine accumulated, which has been shown in Arabidopsis thaliana plants exposed to drought stress and was caused by abscisic-acid regulated protein degradation. These amino acids belong to branched chain amino acids formed by pyruvate or aspartate. They are involved in abiotic stress response and act as signaling molecules further regulating gene expression. The increase in concentration of these branched chain amino acids might therefore reflect a response mechanism that is triggered under K deficiency with more pronounced effects when K deficient leaves are exposed to osmotic stress. Another branched chain amino acid is alanine, which is also formed by pyruvate and its accumulation was reported to occur under PEG-stress. Furthermore, Lea et al.  could show that asparagine and lysine accumulate in plant tissues under different stress conditions, revealing stress responses due to K deficiency and PEG, which was also shown in our study. However, the concentrations of amino acids vary strongly due to the stress effect,since individual amino acids react differently to various factors. Therefore, it is difficult to compare studies determining amino acids because of the differences in growth and environmental conditions. Responses of the mentioned free amino acids in potato leaves might provide information related to secondary metabolites of interest.

Accumulations were also observed due to -K for some organic acids involved in the TCA cycle, including α-ketoglutarate and succinate, whereas PEG led to a reduction in both K treatments. In rapeseed, Zhu et al.  showed an increase in α-ketoglutarate and succinate under low K and drought stress conditions. Contrasting results were found by Das et al.  in soybeans, where α-ketoglutarate and succinate decreased. The generally low accumulation of organic compounds and higher contents of TCA intermediates could indicate enhanced respiratory rates and energy metabolism. However, this supports the presumption that K accumulation as an osmotic adaptation against drought stress, is significantly more energy efficient than the formation of organic solutes. K is transported into young leaves via the phloem during stress to fulfill the osmotic function of other solutes. This could explain the lower sugar content in the youngest fully-developed leaves and the increased sucrose content in the -K+PEG plants on the whole plant level, at least for cultivar Milva. However, differences in metabolite profiles vary strongly depending on the type of stress, duration, severity, and cultivar.For potatoes, there is less information on K transporters and how they respond to nutrient deficiencies and abiotic stress. The analysed K transporter genes  belong to the HAK/KUP/KT family, which have different functions in K uptake and transport. In addition, they play a role in stress tolerance and osmotic potential regulation. The expression of related genes is often triggered by a low K supply, enhancing the overall K uptake. The genes of this family are expressed in different plant parts, e.g., in the leaves, roots, or stems, which indicates a connection with the primary uptake of K from the soil, as well as with cellular K homeostasis. 

The transporters studied—StPOT2, StPOT4, and StPOT12—correspond to homologous genes of Arabidopsis thaliana, where they are referred to as AtKUP2, AtKUP4, and AtKUP12, respectively. They belong to different clusters in which the various transporters can be subdivided according to their sequence homology. AtKUP2 and AtKUP4 are assigned to cluster II and described as low-affinity K transporters involved in cell expansion. The transporters of these clusters have many homologous transporters from other plant species and are described in more detail. However, AtKUP12 is assigned to cluster III and has rarely been studied. Our results showed different expressions of the three transporters depending on K supply, PEG-induced osmotic stress, and cultivar. StPOT2 and StPOT4 were more highly expressed in Milva and showed greater expression rates in -K plants at 55 dap. StPOT expression in Agria decreased at 69 dap. The expression rates of all K transporters increased again for all treatments and both cultivars during the recovery phase. Due to the fact, that the plants were already mature and had almost completed their growth cycle, the demand for K in young tissues was no longer as high as at the beginning of plant growth, which could explain the low expression levels of all the three StPOTs in Agria. Thus, Milva was not yet fully matured and was still taking up K. Accordingly, StPOT2 and StPOT4 were still active in Milva since both are related to growth processes. Compared to Agria, higher expression rates of the three StPOTs in Milva could indicate differences in the involved genes, which may vary genotypically. StPOT2 in potato and the homolog gene AtKUP2 in Arabidopsis, as well as AtKUP6 and AtKUP8, play a role in lateral root development by enhancing abscisic acid and responding to osmotic stress. In contrast, AtKUP4 is involved in growth processes by participating in auxin transport, indoor garden which in turn contributes to root hair development. These two transporters are also known to increase their expression more in roots than in leaves, which may explain the lower expression rates in our study.

Increases in the three StPOTs at 82 dap could be related to the increased water uptake of plants in the recovery phase, because more K was available, and therefore, the expression of the three StPOTs increased again. Our results showed that StPOT12 reacted similarly to StPOT2 and StPOT4, but showed higher expression rates in -K Milva plants at 55 dap and up to eight-fold higher expression at 82 dap. Therefore, protein synthesis for StPOT12 may be enhanced, participating more in K uptake compared to proteins of StPOT2 and StPOT4. Zhang et al. also showed a connection between AtKUP12 and increasing K uptake in addition to improving the salt tolerance of Arabidopsis. Therefore, StPOT12 is also involved in abiotic stress. However, the detailed physiological functions remain unclear, and further investigations are needed.Rice, a staple food for more than three billion people worlds over belongs to the genus Oryza of the family Poaceae. Consisting of two cultivated species, O. sativa and O. glaberrima, and 22 wild species, Oryza is a large genus of predominantly tropical aquatic or semi-aquatic grasses. O. sativa is grown worldwide, while O. glaberrima is mostly confined to West Africa. Domesticated since early Holocene,rice cultivation has led to cultivar diversity through phenotypic selection for desirable characters such as grain yield and grain quality. Being widely cultivated, adaptive evolution of rice cultivars over thousands of years has distributed them over wide range of environs, such as deep water to montane ecologies. Being one of the primary centers of origin, India is bestowed with wide diversity of rice landraces, wild congeners and modern cultivars.Estimated between 75000 to 100000, the number of indigenous landraces in India has shrunk over time, although a considerable portion is still conserved for a variety of reasons. In areas of conservation, farmers cherish profound knowledge of landraces such as their uses, properties and peculiarities. In Tamil Nadu, popularly called as the ‘rice granary of South India’, there were about 400 traditional landraces in vogue since olden times, which are still extant and used in households. These landraces brandish several features such as pest and disease resistance  and tolerance to flood,drought  and salt. Some others are suitable for special uses such as fodder and thatching,stamina boosters  and for medicinal uses. Understanding their importance, most of these landraces are now conserved in gene banks across the India, so that they are not destroyed due to climatic vagaries and catastrophes. The practicality of conservation of specialty rice is particularly realized during disasters such as tsunamis, floods, cyclones, etc. when conventional cultivars fail to save the situation. For instance, when the coastal Tamil Nadu was devastated by the Indian Ocean tsunami on 26th December 2004, the salt water ingression from the bay has destroyed most of the standing crops in the affected region.However, soil salinization continues to happen even without disasters along the coastal as well as inlands of India, due to poor quality of irrigation water. Globally, rising salinization limits rice production significantly in those areas, which according to an estimate, accounts for about 33% of irrigated land.

Roots were removed and the fresh weight of the individual shoot vegetation was measured. Basil is commercially important both as fresh and dry and hence both weights were measured and presented. After weighing, cut portions were dried in a Fisher Scientific Isotemp Standard Lab Oven at 60 C for 96 h. Fresh and dry weights of individual basil plants  were used to calculate the fresh and dry crop yield.Nowadays, cyber-physical systems  are drawn the attention within the industry, society and government, due to the enormous impact they have on the economy and the environment,and providing citizens and businesses with a wide range of innovative applications and services. The entry of the CPS in the industry allows high connectivity between the industrial systems and brought great advantages and a wide range of new opportunities to industries but also some questions and problems, such as those related to safety and security. Cyber security is a fundamental discipline that provides confidence in terms that CPS, their information, and supporting communications and information infrastructures are adequately safeguarded.

CPS have many unique characteristics,ebb flow including the need for real-time response and extremely high availability, predictability, and reliability, which impacts cyber security decisions. Besides, an even more critical problem is that the development of these systems has been carried out without taking into account the security aspects, nor the new risks that this automation of processes implies, which put at risk the complete industrial infrastructure,and where any security breaches to these systems could have catastrophic consequences. Taking into account the security from the earliest steps of CPS, i.e., at the design time is crucial to avoid security issues, even though it is a very challenging task. Therefore, security within industrial environments becomes a critical aspect that must be taken into account at all stages of information system development, by obtaining and defining, from early analysis and security requirements  before the system is in place. Security and safety are nonetheless two key properties of CPS  and they share the same goal, protecting CPS from failures. Security and safety refer to different but very important properties,in this paper just focus on those related to security properties for the CPS.Although security requirements are the appropriate solution for many researchers, they are difficult to obtain, analyse and manage by their subjective nature and their description in natural language. For CPS, in addition to software security requirements, we also have physical, control and communication requirements, which make the task of identifying security requirements and translating them into the design of our CPS system even more complicated. 

Therefore, having a common model  is essential, since it facilitates the definition of security requirements for CPS where any particularity of all these elements is taken into account. The high variability of the components involved in a CPS and their possible configurations make it extremely difficult to verify the correctness of the security requirements that reduce the threats and the possible risks. To manage this complexity, we propose the use of Feature Models and a set of reasoning techniques  to verify the correctness of the security requirements and diagnose misconfiguration of the features,according to a catalogue of possible correct configurations. FMs have been previously used for checking security configurations,and the diagnosis of FM configurations is a studied problem by the community. However, how it can be adapted to the specific scenario of CPS is still an open challenge  tackled in this paper. Based on the problems identified, we have developed a CARMEN framework that presents a systematic process to enable from the description of security requirements to the verification and diagnosis for CPS through variability models. CARMEN is focused on the design phase of CPS by presenting a support system for guiding the whole security requirement life-cycle:  creating a meta model which enables the definition of security requirements for CPS based on security recommendations of ENISA  and OWASP  guidelines;  load and update a variability model that encompassed the catalogue of possible correct configurations for CPS;  map both security requirements and variability model resulting in a configuration; verify the correctness of the configuration, and;  if it is not correct, the diagnosis of the configuration to modify for achieving a correct configuration according to requirements.

To explain in detail each of these steps, the paper has been organised as follows: Section 2 reviews the most relevant papers in the area. Section 3 details a case study based on Agriculture 4.0 and introduces the possible cyber-risks to which this type of systems are subject. Section 4 presents the proposed framework for the diagnosis of this type of system. Section 5 applies the proposed framework to the case study to show its applicability; and, finally, conclusions are drawn, and future work is outlined in Section 6.Feature-Oriented Domain Analysis  have become mature fields in the Software Product Line  arena in the last decades. Several are the scenarios where SPLs based on feature model analysis have been applied,and different researchers highlight the advantages of these systems since the use of Model-Driven Engineering  methodology and the SPL paradigm is becoming increasingly important. The complexity and the high variability of a CPS, and how SPL can help were analysed in Arrieta et al.  and Beek et al.,detecting the points of variability using feature model analysis. The analysis of the variability of CPS can also support the testing. Security is an understudied field in SPL area. Different approaches have been presented to manage the variability and specify security requirements from the early stages of the product line development. Similarly, other approaches addressed the idea of including the security variability into an SPL. In Fægri and Hallsteinsen,the authors established a software architecture as a reference to develop SPL, dealing with information security aspects. SPLs are currently being targeted for application in CPS, as for some researchers, no standard provides a structured co-engineering process to facilitate the communication between security engineers. 

For other researchers, information security must be a top priority when engineering C-CPS as the engineering artefacts represent assets of high value, and the research is focused on the generation of new security requirements stemming from risks introduced by CPS. On the other hand, there are approaches focused on security as a use case, such as in Arciniegas et al.  and the methodology SecPL,where is highlighted the importance of specifying the security requirements and product-line variability. These are annotated in the design model of any system. Other researchers developed a security requirements engineering framework for CPS, which is an extension of SREP. The capacity to support the high variability in the security context though Feature Models appeared in previous papers,where the authors analysed which vulnerabilities could be used to simulate attack-defence scenarios, but these simulations were not oriented towards more complex scenarios, such as cyberphysical systems.As seen in the introduction, nowadays CPS require an adequate security configuration. Therefore, some researchers are focused on the development of ontologies and security requirements. Some researchers have developed security tools based on ontologies capable of being integrated with the initial stages of the development process of critical systems. On the other hand, requirements have been analysed not only from the software side but also from the hardware perspective, including sensors and network security. Therefore they propose the development of a security requirements framework for CPS, analysing the existing ones, and concluding that currently there is no suitable requirement framework for this type of systems. Other researchers consider that CPS have unique characteristics that limit the applicability and suitability of traditional cyber security techniques and strategies, and therefore propose the development of a methodology of cyber security requirements oriented towards weapons systems. This methodology allows us to discover solutions that improve dimensions,being possible to use automated coding tools. 

Therefore, we can conclude that at present different researchers have found the need to develop requirement grammars to control the security risks associated with CPS. Moreover, derived from the complexity of the CPS, feature models have been previously used in the context of security.The earth is facing the risks of increasing soil pollution caused by heavy metals, which is particularly critical for humans as pollution concentrates on farm land where food is produced. Among the heavy metals, cadmium  is generally considered more detrimental than others in terms of toxicity and mutagenic effects on plants and humans. Cadmium has a similar structure to several essential elements, such as iron, manganese, and zinc, and it can enter the plant rely on the transporters of these elements; therefore, it can be transferred to all living organisms through the food chain, which raises concerns over risks on public health. Although some plant species, called hyper accumulators, greenhouse benches can accumulate exceptionally high amounts of Cd without obvious damage symptoms,most crops, including horticultural crops, are sensitive to Cd. The mechanisms of Cd toxicity in horticultural crops, as well as prevention of Cd accumulation in shoot tissues, have been frequently explored. Cd is absorbed from the environment into plants mainly by the roots and transported through the xylem to the plant shoots, where it can be distributed to various plant tissues. Oxidative damage is a typical symptom caused by Cd toxicity in plants. The Cd indirectly induced reactive oxygen species  can react with lipids and proteins, leading to lipid peroxidation and membrane damage. Cd accumulation also alters chloroplast ultra structure and inhibits the electron transport chain, resulting in decreased chlorophyll content and reduced photosynthetic rate. An efficient way to improve plant Cd tolerance is to minimize root-to-shoot Cd2+ transportation or lower the Cd2+ concentration in the cytoplasm.

Several metal transporters on the root epidermal cell membrane relating to Cd transportation into root cells have been reported. For example, the gene encoding a natural resistance-associated macrophage protein 5  is mainly expressed in the rice root epidermal cells, which is an important plasma membrane-localized transporter for Cd2+. When Cd enters the root cells, it can be chelated or precipitated by specific small molecules, such as metallothionein,in the plant cytoplasm and transported to the vacuole, thereby reducing Cd2+ toxicity. Furthermore, the plant uses its antioxidant defense system to protect itself from Cd-induced oxidative damage. For example, the antioxidant enzymes in plants, such as superoxide dismutase,catalase,ascorbate peroxidase,and glutathione reductase,scavenge ROS through increased activity to maintain proper cellular ROS levels under Cd stress. Due to the high mobility nature in plant tissues, Cd can replace Fe from proteins and disturb nitrogen and sulfur metabolism. Nitrogen metabolism is central to the plant response to heavy metals and plays a vital role in Cd tolerance. Cd stress in plants can disrupt amino acids metabolism, as manifested by the accumulation of several specific free amino acids that are beneficial for Cd tolerance. For example, previous study found that proline accumulated in plants under Cd stress, which was conducive to the of ROS elimination and functioned as a protein compatible solute. Furthermore, the amino acids have been confirmed to serve as signaling molecules and affect synthesis and activity of some enzymes, which play an important role in maintaining plant cell metabolism and stability. Several studies indicated that amino acids rich in carboxyl, amino, thiol, and phenolic groups are crucial for metal detoxification due to their ability to form complex metal cations to decrease their toxicity. Autophagy, a highly conserved mechanism in plant cells, targets the cellular components for degradation in the vacuole by the resident hydrolytic enzymes. This recycling system involves the degradation of cytoplasmic proteins and organelles, promoting the maintenance of nutrient remobilization and other metabolic processes. Studies have demonstrated that autophagy can be induced during abiotic stress; meanwhile, it plays an essential role in plant tolerance to various abiotic stresses, such as heat,salt,and drought. Studies on the function of autophagy-related genes  against Cd toxicity in animals are relatively clear, but lacking in plants. Previous studies provided evidence for autophagy induction in plant cells under Cd stress. However, the specific mechanisms of autophagy in the Cd tolerance of plants have yet to be explored. Here, three MdATG10-overexpressing apple lines were used to understand the involvement of autophagy in plant Cd tolerance. Results showed that MdATG10 over-expression enhanced the autophagic activity and improved the tolerance of apple plants in response to Cd stress.

However, it is important to bear in mind that even if minimising the fertiliser input had a less relevant impact on costs, it is key to preventing leaching nutrients and preserving the over-polluted aquifers in the region.Industrialization during the last centuries has led to extended areas of contamination with increased metal concentration levels. During the last three decades, eco-friendly, gentle soil remediation techniques using different plant species have been developed to address this problem. Plant characteristics such as metal tolerance, accumulation behaviour, biomass production, and root morphology determine metal extraction capacities. A number of plant species known as “hyperaccumulators” share the ability to grow on metalliferous soils and to accumulate large amounts of heavy metals in their aerial organs, to concentration levels exceeding that of normal plants >100 times without suffering phytotoxic effects. One species, Noccaea rotundifolia ssp. cepaeifolia, was found to accumulate up to 21,000 mg kg
1 Zn. Reeves and Brooks  found Zn concentrations up to 17,300 mg kg
1 in leaves of N. rotundifolia in the vicinity of a PbeZn mine in the Cave del Predile  area, Northern Italy. Reeves and Brooks  reported that this species was also able to accumulate Pb. The highest Pb concentrations were found in shoots of N. rotundifolia in Cave del Predile. The same authors reported N. rotundifolia being a Cd hyperaccumulator. 

In contrast, Koubova et al. using pot and hydroponic experiments showed that the potential of N. rotundifolia to accumulate Pb in the plant shoots biomass was negligible and did not confirm the potential Pb hyperaccumulation ability of this species.For this experiment a soil  from Arnoldstein with moderate Zn, Pb and Cd contamination and loamy texture was used as control. As this soil was moderately contaminated with Cd, we also studied Cd accumulation in shoots as well as the labile fractions and mobilisation of Cd in the rhizosphere. Part of the soil material was spiked to obtain a highly Zn, Pb and Cd contaminated soil. The desired amount of metals was dissolved in deionized water,25 liter pot then sprayed on the soil in polyethylene bags and subsequently thoroughly mixed by shaking the plastic bag. For equilibration, soils were incubated at 40 C for 3 weeks with several wetting and drying cycles using deionized water. Selected characteristics of the experimental soils are shown in Table 1. To study changes of labile metal fractions, pH and DOC concentration in the N. rotundifolia rhizosphere we conducted a rhizobox experiment using the design of Wenzel et al.  in 4 replicates. Eight seedlings were planted on top of a soileroot compartment with an opening slit at the bottom. After 6 weeks the number of plants was reduced to four, and the plant compartment was transferred on top of a rhizosphere soil compartment attached to a vertical root-only compartment that is separated from rhizosphere soil compartment by a nylon membrane  and a transparent acrylic window. After another 7 weeks, sufficient root coverage of the nylon membrane separating roots from rhizosphere soil was achieved.

Subsequently,the rhizosphere compartment was separated from the root layer. Roots adjacent to the membrane were washed in an ultrasonic bath and rinsed using bi-distilled water.The experimental soil used in this experiment was obtained from an arable field near Wiesen, Austria ; selected characteristics of this loamy soil are given in Table 2. Airdried soil was passed through a 2-mm sieve. Cation exchange capacity,organic matter content,plant available P based on the Olsen method  and plant available K using 1 M ammonium acetate  were determined. Part of the sieved soil was maintained to provide non-polluted control soil,other batches were enriched with Zn or Pb to yield nominal concentrations of 100, 500, 1000, 2000 and 3000 mg kg
1  or 250, 500, 1000 and 2000 mg kg
1  by mixing the soil with ZnO or PbO, respectively. Metal oxides were used for spiking to avoid mass flow of metals between compartments through irrigation. The near-total  Zn and Pb concentrations in Wiesen soil  were 31.9 and 4.89 mg kg-1 respectively. To meet the presumable high zinc requirement of N. rotundifolia, we also spiked the control soil to obtain 100 mg kg-1. The metal spiking procedure was carried out as described in chapter 2.2. The near total,ammonium nitrate-extractable,and calcium nitrate extractable  concentrations of Zn and Pb in the soils, soluble P,soil pH and dissolved organic carbon were determined after three weeks equilibration and are presented in Table S1. A split-root system technique was used to study the effect of localized Zn and Pb patches on the root foraging behaviour of N. rotundifolia according to a modified method of Langer et al.. Half-liter pots, which were separated into 2 equal compartments, were filled with homogeneous or heterogeneous pairs of control  or Pb- and Zn-enriched soil treatments as shown in Table 3 and Fig. S1.

The metalenriched patches in heterogeneous treatments were placed on the right-hand side of the pots. Experimental pots were equipped with a mesh permeable  for root growth but stabilizing the paired substrate patches supporting proper separation during the harvesting procedure. The free-draining pots were placed on a grid to avoid mixing of percolating water from different patches. Twelve day-old seedlings were placed individually at the contact zone of the paired soil patches. The experiments were carried out at a soil moisture of 80e85% of maximum water holding capacity in a greenhouse with a completely randomized design and 4 replications. At harvest,fresh root biomass that developed in each split substrate patch as well as shoots were harvested. Root morphological characteristics were measured on fresh root material. Fresh roots were weighed and cut into pieces. An aliquot of fresh roots from each part of the pot  was randomly selected and kept in ethanol  for root scanning; in addition, a  sub-sample was dried at 65 C for 2 days to determine the dry weight factor. Detection of root morphological characteristics in different diameter classes was done by scanning the roots using a modified Optical Scanner ; images were processed by using WinRHIZO®. To increase the contrast of the roots against the background on the scanned images, they were stained with 5% Giemza solution for 10 min at about 40 C on a heating plate. Data analyses were performed with ANOVA  in SAS software, followed by LSD and Duncan-test. The differences in root characteristics between patches were evaluated using a paired t-test.N. rotundifolia tended to produce less shoot and root dry matter in the homogeneous as compared to the heterogeneous Zn and Pb treatments. 

Comparison of root allocation patterns revealed no significant difference in the partitioning of root mass in any of the homogeneous Zn and Pb soil treatments, with approximately 50% allocation into the left and right sides of all pots. We found preferred partitioning of root mass into the low Zn and Pb halves  of heterogeneous soil treatments with a large difference of metal concentrations between low and high metal compartments. In such Zn treatments,approximately 35% of the total root mass was found in the Znenriched half patch with higher Zn concentration;similarly, in the highly heterogeneous Pb treatments  38 and 32% of total root mass respectively, allocated to the Pb-enriched half. In heterogeneous treatments with less pronounced Zn concentration differences between the high and low metal compartments,we found no difference in root dry matter allocation, however, increased root length and related larger surface area in the compartment with lower Zn concentration. The average root diameter decreased with increasing Zn concentration in soil in heterogeneous treatments except in 100/ 3000. We found no significant differences in root length and surface area between the compartments of heterogeneous Pb treatments. However, specific root area  and specific root length  were significantly higher in the Pbenriched patch in two treatments with the largest Pb concentration difference between compartments. Lead and Zn concentrations in N. rotundifolia shoots increased with increasing soil metal concentration, following a linear function for Pb but approaching a plateau in the case of Zn, with only small differences between homogeneous and heterogeneous treatments. The only considerable deviation from this pattern was found for the heterogeneous Zn treatments 100/3000 and 500/3000 where the shoot Zn concentrations in the heterogeneous treatments were clearly below the fit expected from all other data, and lower than the corresponding homogenous treatments. 

In the homogenous Zn treatments, the plant Zn contents  increased with increasing Zn supply, approaching a plateau at high soil Zn concentrations. In the heterogeneous treatments, the Zn contents follow a more irregular pattern, with lower than expected values in the treatments 100/1000 and 100/3000.The shoot Pb contents showed a linear positive response to increasing Pb concentrations in soil, with generally larger contents in the heterogeneous treatments.In heterogeneous treatments with smaller differences in Zn concentration between compartments, toxicity stress was indicated by changes in root morphology, including decreased root length and smaller root surface area in the Zn-enriched patches with higher Zn concentrations even though allocation of total root dry matter was not different. Toxicity was also indicated by decreasing average root diameter as the overall  Zn concentration in the split-pot study was increased. In contrast, spiking the moderately polluted ARN-B soil with Zn,Cd,and Pb  resulted in a ~32% increase of shoot dry matter. Overall, our findings provide evidence for high tolerance of N. rotundifolia to Zn in hydroponic solution and even on soil from its natural habitat spiked with a metal cocktail, whereas this species appeared to be more sensitive to Zn-only spikes in an initially low-metal agricultural soil. Taking all evidence together we classify this species as highly tolerant Zn accumulator plant. N. rotundifolia exhibits similar tolerance to Zn exposure as N. caerulescens, raspberry cultivation pot albeit at lower levels of Zn accumulation in shoots. In the hydroponic Pb dose-response experiment, N. rotundifolia accumulated enormous Pb concentrations in its roots, with almost 81.8 g kg
1 at 1000 mM Pb in solution. However, only a small proportion of Pb was transferred to shoots, with ~1250 mg kg
1 in the 1000 mM Pb treatment.Only at this largest concentration the commonly accepted hyperaccumulator threshold of Pb was exceeded. Apart from the high Pb concentrations  in the soils of the split-pot experiment, Pb concentrations in shoots did not exceed 100 mg kg-1 thus remaining one order of magnitude below the hyperaccumulation threshold.Even in the spiked soil the Pb shoot concentrations in the rhizobox experiment did not exceed 86 mg kg-1. This is in contrast to previous reports of Pb hyperaccumulation by N. rotundifolia, with Pb shoot concentrations up to 8200 mg kg-1 in field-collected specimen from the Southern Alps in adjacent regions of Italy and Austria. 

In all experiments of our study, the shoot: root concentration quotient of Pb was clearly <1, decreasing to values near 0.01 in the highest Pb treatments of the hydroponic study. Similarly, the bioconcentration factor for Pb remained clearly below 1 in all experimental setups. N. rotundifolia responded to Pb with decreased root and shoot dry matter production, starting from the lowest level of exposure. On the other hand, we found increased dry matter production upon spiking a moderately metal-polluted soil from the plant’s native environment with a cocktail containing Cd, Zn and Pb, resulting in a soil concentration of 1650 mg Pb kg-1. As for Zn, the root biomass of N. rotundifolia was evenly distributed in all homogenous and heterogeneous treatments unless the concentration difference between the compartments was very high. We found no significant differences in root surface area and root length between the compartments of heterogeneous Pb treatments. However, specific root surface area  and specific root length  were significantly higher in the Pb-enriched patch of the two treatments with the largest soil Pb concentration difference between compartments. Taking together all evidence obtained in our study,we challenge earlier reports on Pb hyperaccumulation properties of field-collected N. rotundifolia specimen from the same site. Our findings support the doubts regarding the identification of hyperaccumulation solely based on field-collected specimen related to potentially ineffective washing procedures prior to analysis. In that case aerial contamination from emissions or soil dust derived in the vicinity of the collected plant specimen cannot be excluded and may bias the results towards meeting hyperaccumulation thresholds.

The article analyzed the potential of increasing fodder productivity, seed quality, and control and the potential of fodder production in different aspects of open fields like using barren lands from salinity or water scarcity. Significant industrial development has been noticed in recent years for high-tech controlled environment fodder production, especially in the United States, Europe, and Australia. Many start-up industries evolved in the market with advanced technologies for large-scale hydroponic fodder production with artificial lighting. However, minimal research activities have been reported in terms of technological development like environmental control, optimization in energy and water use, automation, and control. There is an information gap in the potential of CEFP and their challenges from engineering aspects, especially the energy use in HVAC and lighting systems. The critical analysis of thermal environmental control techniques and challenges for CEFP is essential for researchers, industry, and producers. A comprehensive review would be beneficial for understanding the prospects and limitations of CEFP for various stakeholders.

Livestock units are expected to increase up to 50%, and arable land per capita will decrease by approximately 23% in 2050 compared with the number in 2012.CEFP would increase as the arable land demand for human food production with the increased world population. A critical analysis of thermal environment control for CEFP would benefit various stakeholders from academia to industry. Therefore, this article presents a comprehensive analysis of multiple aspects of CEFP for livestock production. Section 2 presents hydroponic growing methods and their application for CEFP, and different facility types are analyzed in Section 3. Section 4 includes the analysis of previous studies on nutritional aspects, including the pros and cons for livestock productivity. Indoor environmental requirements and techniques for sustainable barley fodder system production are presented in Section 5, and potential option and control techniques are presented in Section 6. Finally, Section 7 includes a comprehensive analysis of the prospect, challenges, and research needs to achieve sustainability goals for the CEFP. Hydroponics is a method of growing plants using nutrient solutions or water without soil. Different types of leafy vegetables and fodder could be produced using hydroponic systems. The growing system could be open systems, where the nutrient solution or water is not recycled, and closed systems recycle excess water from growing systems. The open hydroponic systems are not practically feasible due to wastage of water and nutrients.

An experiment study showed that using macro and micro-nutrients with water does not significantly affect the yield of wheat fodder grown hydroponically for seven days. So, hydroponic fodder grown in CE comes without fertilizers and chemicals, making it a sustainable solution for livestock production. Based on the solution/water feeding methods, six different hydroponic techniques are available for growing plants, such as nutrient film system , deep water culture system , aeroponics system , ebb and flow system , wick system , and drip hydroponic system. Fig. 3 describes the basic working principles for various watering methods for hydroponic production and their associated advantages and disadvantages. NFS, EFS, and DWCS are commonly used for leafy green production in CEA applications. The aeroponic system is the advanced form of the hydroponic system, which could be challenging for CEFP. Table 1 shows critical influencing indicators to develop a decision matrix for watering technologies in hydroponic production. The information presented in Table 1 is generated from Verner et al.. WS is relatively simple, with no energy demand components as no motor is required for water movement, but not suitable for large-scale production. DWCS system is also simple and less energy-intensive, but a large volume of water is needed for operation and is prone to water-borne diseases. In general, the hydroponic fodder production technique is slightly different from the techniques used for fruit and vegetable production. The standard technique for fodder production is spraying solution or water at certain intervals and draining excess water to the tank for recycling . However, the performance of these techniques for fodder production has not been extensively studied.

In general, the concept of the NFS is most promising for draining the water in a hydroponic fodder production system. The slope of the growing bed is a critical parameter in the NFS that needs to be optimized for better performance. Growing trays in NFS are positioned at some angle or slope to facilitate a nutrient solution to drain into the nutrient solution reservoir and recirculate. NFS with a 1.5% slope was found ideal for lettuce production, and a relatively steeper slope was found beneficial for tomato productivity. The impact of growing trays slope on the productivity of green wheat fodder in the NFS system was evaluated with two different light sources . The study reported that the highest yield of wheat fodder after seven days of cultivation could be achieved with growing trays sloped at 6.5%. Matos et al. designed a hydroponic fodder production system based on the concept of the DWCS, but the study did not evaluate the performance compared with other standard techniques. Fig. 5 shows a complete CEFP unit with NFT system with 8% slope and other cirtical components for automated production system. To our best knowledge, no research has been conducted to assess the performances of the various techniques for supplying nutrient/water for fodder production. Also, the water-saving potential of an innovative approach for CEFP needs to be investigated to minimize the possibility of molds and waterborne disease contamination with fodder. Greenhouses could be used for fodder production to minimize energy-intensive artificial lighting. FAO recently supported a project for hydrophobic fodder production and established 79 simple greenhouses structures across seven regions in Namibia. The project began in 2020, benefited around 3,350 households, and saved their livestock from dying. Different low-tech greenhouse structures are mainly used for fodder production based on local conditions and resources. A study reported that a high-tech greenhouse requires about 8-15 kWh of electricity to produce 600 kg of hydroponic maize fodder daily, which could be minimized using a low-tech net house. S´ anchez del Castillo et al. evaluated the effect of different densities for growing wheat and barley in a low-tech poly-covered greenhouse in Mexico.

The low-cost hydroponic systems made of locally available materials are common in developing countries. In Malawi, hydroponic fodder has been produced in a simple poly-covered greenhouse with wooden frame shelving for trays. The major challenge in low-cost fodder production systems is controlling or adjusting the temperature, humidity, and air circulation, especially during the dry, hot summer months. The yield and type of fodder grown in these low-tech facilities are highly dependent upon the season and climatic condition of the locality/region. Modular farms are flexible and self-contained systems that allow growing fodders or vegetables without soil or sunlight. Hydroponic fodder could be grown in stackable vertical trays in warehouse-like structures or shipping containers with the help of artificial lighting. Grov Technologies in Utah, USA, has designed different vertical growing units for warehouse-type facilities to grow with advanced technologies like the Internet of Things , cloud, robotics, and machine learning for lighting and spectrum management to maximize potential yield. They claimed that their olympus tower could produce 2,300 to 2,700 kg of sprouted wheat or barley grass per day using less than five percent of water and replacing 35 to 50 acres of land compared to traditional farming. Similarly, two young farmers set up a warehouse hydroponic facility to produce high-quality green fodder for livestock in the Canary Islands in Spain. An 80 m2 facility could produce 1,000 kg of barley per day, providing a continuous supply of green fodder for 300 goats. The report indicates that the system could save 90% more water than traditional methods. Other industries have also been marketing the stackable vertical farming system to grow fodder in indoor spaces like warehouse-type structures or retrofitted abandoned buildings to grow livestock fodder. Agritom based in Australia, claimed that their custom type machine could produce from 100 kg to 1,200 kg of fodder per day, with a footprint ranging between 4.5-29.0 m2. HydroGreen is a local chain ag-tech company founded by farmers in South Dakota in the USA, manufacturing a fully automated vertical growing system that produces high-quality nutritional green fodder. They reported that about 2,700 square foot buildings could replace about 600 acres of land and produce 5.0 tons of fodder daily using a fraction of the water used in traditional crop production.

Fodder Tech, a company based in Utah, manufactures and markets different sizes and modular fodder production systems. The stackable vertical farming system could produce 9,000 kg of fresh fodder per day. A fully automated shipping container type system is becoming popular for growing fodder under extreme climates. Many companies are marketing the shipping containers type systems with a production capacity range of 50-2,500 kg per day, depending on the size of units. Agritom is currently marketing nine different sizes of the container system with LED lighting systems having rated production capacity per day between 50 kg to 2,250 kg. They reported that a 30 m2 system could produce 1,000 kg of green fodder per day compared with 1.0 hectares of land required in the traditional farming system. Fodder Tech is marketing various sizes of shipping containers with a capacity range from 50 kg to 900 kg per day. Table 2 summarizes some current industry leaderships for manufacturing and marking the CEFP systems. It is often said that the need for green fodder for livestock is as important as the need for good nutritious food for humans. Green fodder is always considered an inevitable,hydroponic barley fodder continuous, and economical source of nutrients for livestock from normal production perspectives. Compared with other available dry roughages, fodder is a natural, highly palatable, and digestible feed enriched with micro-nutrients resulting in improved nutrient digestibility, health, and performance of animals. Therefore, ensuring continuous and secured fodder supply is considered a major driver of sustainable and profitable livestock production. Hydroponics is a successful growing technique that offers a constant supply of green fodder all year round, even in all sorts of worse climatic conditions for sustainable livestock production. These plant materials and fodders are abundant in protein and energy, easily digestible, and utilized by most animals. It is reported that sprouted barley produced in hydroponic systems contained higher fiber, protein, and minerals than the barley grain. The nutritive value of animal feed could be analyzed in terms of dry matter , crude protein , crude fiber , ether extract , ash content , neutral detergent fiber , and acid detergent fiber . Thadchanamoorthy et al. showed that at 10th day after planting, the nutritive value of sprouted maize were greater compared to maize grain.Usually, when compared with respective grain/seed, hydroponic fodder has fewer contents of organic matter and non-fibrous carbohydrates,whereas increased contents of CP, NDF, ADF, and Ca.

This shift of nutrients is good for dairy animals from an animal nutrition perspective. Moreover, another study also revealed that hydroponic wheat fodder increased the DM, CP, EE, CF, K, and Na contents by 5.0%, 44.0%, 132.5%, 176.9%, 150%, and 320%, respectively, compared with conventional wheat fodder. Another study evaluated the yield and nutritional value of three barley cultivars that were harvested on the 6th, 8th, 10th, and 12th days. The study reported that the DM content of barley grain was 93.6% on the 6th day and decreased to 91.1% on the 12th day, whereas CP remained the highest on the 12th day of harvesting. Also, hydroponically produced green fodder exhibits various enzyme activities that break down complex proteins into albumin, globulin, and amino acids, resulting in higher protein quality . Studies also revealed that germination accelerated amylase and lipase activity in hydroponic fodder, increasing sugar and fatty acid content. The fatty acid concentrations in hydroponic fodder, particularly ω-3, ω-6, and stearic acid, increased linearly with the growing period. The sprouting process can significantly increase vitamins A, E, and β-carotene content compared to their grain. One study showed a 700% increase in vitamin E in fodder from grain; however, it is suggested to exercise caution about these numbers—while these numbers indicate a significant increase, based on dairy cow intake requirements, they may not be high from a biological perspective.

From the aquaponic facility at Caldwell, two water samples were collected per week: one from the fish tank and the other from the plant bed which was used to grow Romaine Lettuce, watercress, lettuce and green peppers. Similarly, from the facility at Bryan, three water samples were collected: one from the Tilapia tank, one from the Gold- fish tank, and the other from the main plant bed where lettuce and kale were cultivated. The aquaponic facility at Grimes was one of the largest in the state and four water samples were collected each week: one each from the main growth tank which was used for breeding Tilapia and certain shrimps, one from the tank which bred bluegill fish, one from the tank which was used to grow plant seedlings and the other from the main greenhouse where tomatoes, lettuce and collard greens were grown. All of these tanks were connected together with continuous water flow between them, and water purifiers were installed in each of these tanks to ensure that the recycled water meets the optimal water quality requirements needed for growth of fish and plants. A set up where the experiments were recorded have been shown in Fig. 1. The wastewater from the fish tank is connected to the main set-up, where extra nutrients are added to the aquaponic solution to optimize plant growth. After collecting the water samples, they were sent to the Soil, Water, and Forage Testing Laboratory, Texas A&M University to determine the nutrient concentration for each of the samples. The method used to carry out each of these nutrient concentrations in the laboratory is described as follows in Table 1. This process was carried out each week for 9 months and then the data were analysed. Before explaining the analysis part, a pipeline on how the data was processed to design the Decision Support System has been elaborately stated in Fig. 2 below.

As mentioned above, due to the limited size of the data-set, it is not possible to make accurate inferences taking all the predictors into account. Thus, several dimensionality reduction techniques have been used on the data-set to select the top predictors that define the nutrient concentration of the aquaponic solution. The predictors which had zero variance were removed from the data-set. Then, a correlation matrix was constructed between the predictors, and one of the two predictors, which had higher than 90% pair-wise correlation among them, stacking pots was removed. Next, all the predictors with less than 5% importance in the data-set were eliminated as they would likely incorrectly skew any inferencing made from the data-set. As the primary goal was to bring down the size of the data-set to 5 primary chemical predictors, Recursive Feature Elimination technique with XGBoost classifier was used, which ranked the predictors in the order of their importance. This resulted in bringing down the size of the data-set to 5 chemical predictors. In addition to this, 2 categorical predictors were also appended to the data-set, one storing the month and the other storing the place in which the observations were recorded. Therefore, a total of 143 observations with 7 predictors were used to design classification rules and carry out inferences.To perform any classification rules on a given data-set, Data Visualization is an important tool as it aids in understanding the structure of the data. It equally helps in choosing the classification techniques that can be used on the data-set depending on the separability between the classes. In this case, Principal Component Analysis was used. As PCA treats the entire analysis as an unsupervised learning approach and performs an orthogonal transformation of the data, Principal Components were calculated to visualize the variance in the data-set. The loading matrix was analysed to determine the predictor that contributes the most in each Principal Component and therefore, an inference can be drawn about the relative importance of each predictor from their holding values depending on the value of correlation, which was observed between the predictors and the respective PCs.

To further explore the interpretation from the PCs, the pairwise PC plots have been studied to infer which classifiers would suit the best depending on the pattern of separability of the data. All the data points belonging to class 0 have been color-coded in red and the data points belonging to class 1 have been color-coded in green to have a clear visual understanding of the binary distribution of the data.As stated above, the size of the data-set poses a serious issue while designing the classifier in this context, due to which, the classifiers have been trained and tested on the same data. This method of error estimation is referred to as Bolstered Error Estimation. One of the main reasons for choosing this type of estimator is its low bias as well as low variance. This also results in a faster estimator compared to other resampling methods like the bootstrap . The error estimates have been calculated for each of these estimators for the four popular classification rules by varying the size from small to moderate-sized data-sets. The basic idea is to bolster the original empirical distribution of the available data utilizing suitable Bolstering kernels placed at each data point location. For this case, a uniform zero-mean, spherical Bolstering kernel fi⋄ was chosen for analysis, with covariance matrices of the form σi2Ip. In each case, there would be a family of Bolstered estimators corresponding to each value of where i varies from 1 to n. Larger values of σi would result in wider Bolstering kernels resulting in lower variance estimators, but after a point, bias starts to increase. Therefore, choosing the values of standard deviations for the Bolstering kernels is a challenge and several approaches have been attempted to find the best error estimator in this case which minimizes the bias-variance trade-off.

As stated before, the entire approach was treated as an unsupervised approach and the K-means algorithm was used to classify the observations into two classes. Out of the 143 observations used for analysis, 84 were classified into class 0 and 59 into class 1. As it was difficult to derive inferences considering all the predictors, dimensionality reduction techniques were used. ‘Carbonate ’ was dropped as the variance was zero throughout. Then, a correlation matrix was constructed between the rest of the predictors. This led to the removal of 6 predictors from the data-set namely magnesium, both the measures of hardness, alkalinity, Total Dissolved Salts, and conductivity. Then, Extra Trees classifier was used to find the percentages of importance for each of the 14 predictors. Now, the predictors that had less than 5% importance were removed from the data-set. From the list of chemical predictors, Nitrates and Phosphorus, with the importance of 4.51% and 4.44% respectively, and from the list of chemical properties, pH, SAR, and charge balance with the importance of 1.2%, 0.93%, and 0.93% respectively were removed. Thus, the final list of chemical predictors used in the analysis was as follows: Potassium, Boron, Bicarbonate, Sulfate, and Chloride concentrations in the solution . In addition to this, 2 categorical predictors were also appended to the data-set, one storing the month and the other storing the place in which the observations were recorded. Therefore, a total of 143 observations with 7 predictors were used to design classification rules and to carry out inferences. Next, visualizations were carried out on the data-set to gauge the separability of the data and the classifiers which can be used for inferencing.

As inferred from Fig. 5, the first three PCs go on to explain 46.34%, 34.40%, and 9.36% of the total variance in the data-set respectively. The loading matrix was analysed to get an idea about which predictor contributes the most in each PC and therefore, an inference can be drawn about the relative importance of each predictor from their holding values. From Table 2, it can be inferred that the PLACE_CLASS, MONTH_CLASS, and the bicarbonate variables had the highest holding values in the 1st, 2nd, and 3rd PC respectively, indicating their strong correlations with the respective PCs. From this, one of the strongest inferences that can be drawn is that both the categorical predictors, one storing the place and the other storing the month in which the observations were recorded, are the most important predictors as they show high holding values in the first two PCs which explain about 80% of the variance in the data-set. To further explore the interpretation from the PCs, the pairwise PC plots have been studied to infer which classifiers would suit the best depending on the pattern of separability of the data. All the data points belonging to class 0 have been color-coded in red and those belonging to class 1 in green, giving a clear visual understanding that the data follows a binary distribution. From the PC plots, it has been observed that most of the discriminatory information is contained in the first PC, as expected, as it explains most of the variance in the data-set. Next, due to the low sample size,grow lights there is a high probability of over-fitting the data because of which, the notion of different Bolstered error estimators has been introduced in this domain. The performance of linear classifiers like LDA and Linear SVM along with the performance of non-linear classifiers like CART and 3-NN have been used along with these Bolstered error estimators, and results have been discussed below. The value of the depth of the decision tree was chosen as 2 due to the low volume of the data-set as a higher value of the depth would result in over-fitting the data and lead to unreasonably high accuracy on the training data-set.

The value of the K for the KNearest Neighbor was chosen to be 3 using Elbow method as the mean cluster distance on the training data-set was optimal.Eq. holds in the case of linear classifiers like LDA and linear SVM where the Bolstering kernels are given by uniform circular distributions. As the decision boundary in the above case is a hyperplane, it is possible to find analytical solutions as in . However, when the error estimate is made for non-linear classifiers like CART and KNN, an approximate solution is needed which is obtained by applying Monte-Carlo integration. The error estimates for all the four classifiers i.e. LDA, KNN, CART and Linear SVM have been tabulated and the results are shown below. The value of N i.e. the number of randomly sampled data points has been varied from 20% to 100% in increments of 10 for each of the classifiers. As shown in Fig. 7, the Bolstered Error Estimates reduced from 8% to 2.5% for LDA when the size of the sample set increased. For the nonlinear classifiers, it can be observed that they over-fit the training set due to their minuscule size thereby increasing the chance of the classifier performing badly on the testing set. Therefore, a decision not to proceed with either of these classifiers was made using Bolstered Error re-substitution as it is very likely to over-fit when the size of the data-set is small. Here, the opposite is observed when a linear SVC is used with different values of penalty parameters . Irrespective of the amount of data, the error estimates for each of the classifiers are around 50% which makes it unsuitable to be used as an ideal classifier for the separation of data between the classes as it showcases an ideal case of under-fitting.As the name suggests, in this type of error estimation, the classifiers are trained on each subset separately. Every subset contains all the data points belonging to one particular class except one which is used as the test data point. Therefore, in a binary classification problem, if m data points belong to class 0 and n data points belong to class 1, there will be a total of m-1 subsets for class 0 and n-1 subsets for class 1; and the classifiers will be trained separately on all of them. In the end, an aggregate of the error on each of these subsets is calculated to find out the total Bolstered Leave One Out Error estimate for each of these classifiers.

Bacopa monnieri Wettst., an extensively documented medicinal plant, for its nootropic properties had witnessed a burgeoned interest towards analyses of plant growth, role of culture conditions and elicitations on metabolites yield and their bio-activities.The growing disease burden of neurological disorders, especially in developing countries like India , further augment the need to explore the phyto-pharmacological importance of this plant. Last decade had witnessed extensive studies on the phyto-constituents in the extract of Bacopa monnieri, beyond saponin . Such studies are significant, owing to the vast therapeutic application, beyond nootropic ability, exhibited by the phytocompounds of Bacopa monnieri . However, some of the trivial bottlenecks, encountered while cultivation of Bacopa monnieri could be the variation in yield of phytocompounds. Such variation often evident in plant growth are primarily due to agro-climatic and genotypic variations along with seasonal variation in the yield of phytocompounds and the bio-activities of extract and herbal formulations.Tissue culture techniques have been optimized for the production of therapeutic phytocompounds rich biomass and studies are underway to explore a sustainable scalable solution to mass production of biomass rich in phytocompounds. These concerns obtrude exigencies for exploring alternative robust cultivation strategies. In order to circumvent the impact of cultivation conditions vis-a-vis improving the growth rate and yield of pytocompounds, there is a need to explore some innovative cultivation techniques.

Hydroponic culture, also called soil-less culture is obtruding as a well-accepted technique for improving growth and biomass yield in vegetables and herbs.Due to the slow, but steady integration and application of hydroponic technique for medicinal plants, scant information highlighting the role of hydroponic nft cultivation on growth and saponin yield is available. Maneeply et al. proposed the positive effect of NFT and DFT with higher growth and saponin accumulation than field cultivation, in hydroponic culture of Bacopa monnieri, in Hoagland’s solution. The need to explore alternative media composition and effect of abiotic factors that may synergistically affecting the growth of Bacopa monnieri have not been explored. In current study, the effect of Murashige and Skoog media, ‘Leafy 200’ hydroponic media and light intensity were explored, to design a robust bacosides production strategy using hydroponics.Bacopa monnieri  was chosen among the available geographical variants, for the study, due to comparatively higher saponin yield, reported previously.These plants were maintained in a pot, for a year, within campus premises. The chemicals, growth hormone and basal media were purchased from HiMedia, and methanol for phytochemical extraction was purchased from Qualigens Fine Chemicals Pvt. Ltd, . The experiments were conducted in phytajars containing 20 mL of Murashige and Skoog media. Hydroponic media, used in the study with brand name ‘Leafy 200’, was purchased from GreenLoop .

The amount of powdered media used to constitute 1x media solutions, were as per manufacturer’s instruction. The saponin standard ‘Bacopaside I’ used in the study was purchased from Natural Remedies, Bangalore .The milliQ water, used in the study, had resistivity of 18.3 Mohms cm 1 medium with 0.8% agar. Surface sterilization involved the treatment of explant with 0.1 % mercuric chloride for 30 s, followed by washing in sterile distilled water. The plant material was further treated with 0.1 % sodium hypochlorite for 40 s and again washed in sterile distilled water. Finally, the explant was washed with 70% sterile ethanol followed by washing with water. The sterile plants were inoculated on MS Agar media. The media also contained antibiotics viz., ampicillin and amphotericin B ; and 1mg.L 1 phytohormone BAP , as reported previously . The phytajars with sterile plants were propagated in the growth chamber maintained at 25 2 C, illuminated with white light with photoperiod of 12 h.The statistical design of experiment with 4-run experiment was performed and the phytajars with explants were incubated under specified conditions. The harvested plants, after 9-day acclimatization and growth under four different experimental conditions showed significant variations in morphology of the plants . It was observed that propagation of plant in MS liquid media under white and blue/red light showed significant variations in morphology and. Previous studies have reported the photomorphogenesis, due to three main families of photoreceptors, viz. phytochromes, blue-light receptor and less explored UV-B photoreceptors . Studies by Najafabadi et al. , highlighted the significance of red light on the growth and hypericins from in vitro culture of Hypericum perforatum. In another study, Macedo et al. , reported the contrasting effect of red and blue light on the leaf mass density in the tissue culture of Alternanthera brasiliana. Studies by Chrysargyris et al. , indicated the effect of nitrogen supplementation during hydroponic culture on plant growth, chlorophyll content and yield of essential oil, highlighting the need of plant specific optimization of the media.

Ronga et al. , reported the benefits of digestate on dry matter, aromatic compound and sesquiterpenes. The result in our studies showed more number of leaves and adventitious shoots in hydroponic media illuminated with white light. The leaf area and thickness of the leaves of plant cultured in MS media with white light were more other culture conditions. The explant in MS media under blue/red light showed stunted growth with small leaves. The effect of ‘Leafy 200’ hydroponic media was also evident in the morphologies of the harvest and showed morphological alternation and. The plant propagated in ‘Leafy 200’ hydroponic media under white light showed adventitious rooting from the nodes. Also in comparison to plant propagated under white light in MS media, the number of nodes and leaves in plant in ‘Leafy 200’ and white light are more.The results for the first time highlight the effect of light intensity and commercial hydroponic media on the morphogenesis of Bacopa monnieri. Further the effect of culture conditions on total saponin yield as also compared .The result of ANOVA showed insignificant variation in the yield of saponin cultured in both, MS media and hydroponic media . The results suggest that the yield of saponin is not significantly affected on changing the light intensity with either media. The study is important as it suggest hydroponic media ‘Leafy 200’ to be a cost effect substitute of costly tissue culture technique. Moreover, previous studies have shown that hydroponics or soil-less cultivation have provided a robust solution to the growing concern of variable plant growth or yield of phytocompounds thereof. Recently, Maneeply et al reported the high yield of active compounds and higher production of Bacopa sp. through hydroponic technique.

The strategy may be extended to further optimize hydroponic cultivation condition of highly demanded nootropic plant Bacopa monnieri. Moreover, the study to explore the molecular regulation of media components and light intensity for this high value plant will assist cultivars and farmers to mass cultivate the plant under optimized conditions. The effect of different abiotic components on bacosides yield was observed .Wastewater hydroponic system is the integration of wastewater treatment into hydroponic plant production . In a wastewater hydroponic system, nutrients generated from wastewater treatment through physical and microbial degradation are absorbed by plants. Using such synergies offer several advantages over other bioremediation/phytoremediation techniques such as constructed wetlands by producing value-added crops . As a wastewater-based hydroponic technology, it requires less area, it is inexpensive and can be implemented onsite . The hydroponic component serves as a secondary or tertiary treatment step for wastewater treatment thus minimizing the requirements for further purification of wastewater to levels acceptable for disposal standards. Hence, linking the two systems may offer sustainable options in which renewable resources such as water and nutrients recovered from domestic wastewater can be used for crop production. This will reduce energy and input costs which are commonly involved in conventional wastewater treatment plants and commercial hydroponic crop production systems, respectively . The use of wastewater effluents as nutrient sources for hydroponic channel crop production has been widely used as a form of domestic wastewater disposal . This practice has been recognised as one of the sustainable methods for wastewater management. Several studies have shown the potential of different types of wastewater as a source of fertilizer and irrigation water for the cultivation of green plants in the hydroponic system . In these studies, a number of crop species, including leafy vegetables , fruit crops and ornamental plants grown in hydroponic system have been reported to be suitable for wastewater hydroponic system. Results from these studies showed variation in nutrients removal, yield/biomass production and crop quality when compared to either conventional wastewater treatment or commercial hydroponic crop production system .

This variation is caused by a number of factors including, crop selection in terms of adaptability to grow in hydroponic systems and their tolerance to grow under wastewater of different qualities, the source and composition of wastewater used for agricultural irrigation and fertilization . The method of wastewater treatment before reuse is also reported to have an effect on crop performance irrigated with wastewater. Decentralised wastewater treatment systems which involve the combination of treatment and disposal of wastewater has gained attention in recent years . Such an approach allows for the reuse of treated wastewater within the source of generation and is designed to operate at a small-scale level, thereby offering sustainable opportunities for wastewater treatment especially for developing countries . This system involves a number of treatment stages/processes for the breakdown of waste fractions in a wastewater treatment plant. Among those processes, anaerobic digestion is regarded as the most important component of the DEWATS system due to its low requirement on energy inputs, low sludge production and the opportunity to recover nutrients from the effluents produced . This high nutrient load of wastewater generated through anaerobic digestion has prompted its use for agriculture, including the hydroponic cultivation of crops. Generally, higher plants require both macro and micro-nutrients to grow and reproduce to their full potential. The most essential elements required in sufficient amounts for tomato cultivation include, N, P, K, Ca, Mg, Fe, Zn. The demand for these nutrients is known to be higher in hydroponically grown crops compared to their field-grown counterparts. This is because field produced crops source a portion of their nutrients supply from the soil, whereas, in hydroponic production system, the nutrient requirements of the crop are fulfilled by the nutrient solution . Integrated wastewater treatment and hydroponic plant production systems that solely depend on wastewater to supply nutrients for tomato plants have been reported to be deficient in nutrients such as nitrogen, phosphorus, potassium and calcium .

This is attributed to the low content of nutrients such as N, P, K, Ca and Mg in wastewater effluents. In addition, the complex nature of tomato plants in terms of nutrient management which is highly variable than leafy vegetables are also known to be the limiting factor for growth and yield performance in wastewater hydroponic systems. The nutrient demand of tomatoes changes as the plants grow from germination, vegetative to the reproductive stage . From germination to first flower development, N, P, K are the most critical nutrients required by the plants while, K, Ca, Mg, Zn and Mn are required in sufficient amounts during fruit set. Most investigations have shown that the plant nitrogen content decreased significantly when digestates from organic manure were used as an alternative to mineral fertilizers under hydroponic systems , as well as in pot experiments . The reduction in N content has been related to differences in the composition of the digestate. However, NH4þ is known to be the dominant source of N for domestic wastewater hence its conversion to nitrate is required for hydroponically grown tomatoes . This is necessary because of the high sensitivity of tomatoes to high NH4þ –N levels. The low content and unavailability of Mg and P in wastewater effluents as a result of struvite formation and precipitation during anaerobic digestion were also reported to be the growth-limiting factor for hydroponically grown tomatoes . However, other studies have reported that supplementation of wastewater with deficient nutrients improved plant growth in hydroponically grown plants. The addition of P and micro-nutrients particularly, iron increased the shoot biomass of lettuce .

The tension meter was suggested to be placed vertically because the results from the tensile experiment were affected by the inclined angle of the tension meter. The tension meter was pull up slowly at a constant speed until the root of hydroponic lettuce was separated from the planting board. Then the maximum pulling force in this experiment was recorded. Thirty samples were randomly selected for the experiment. The sketch map and experimental field of the measurement method were shown in Fig. 3.Shear experiment was carried out to determine the necessary cutting force of a product with different cutting speeds and cutting positions when shearing the hydroponic lettuce root. The cutting force plays an essential role in designing cutting devices of harvesting machinery. The experiments were performed by using an electronic universal testing machine controlled by a computer, as shown in Fig. 4. The cutting angle was selected as 20 according to the actual cutting operation requirements. The root tip of hydroponic lettuce was connected with a tension meter. Then the root of hydroponic lettuce was slowly straightened. The tension value of tension meter is recorded as the specified tension when the root was just in a flat state, the sketch map and experimental field were shown in Fig. 5a and b, respectively. A single bevel blade with knife edge angle of 25 and made of carbon steel 0.5 mm thick was used in this study because of its strength and durability.

The hydroponic lettuce root could not be cut off when the cutting speed was less than 500 mm/min, but the cutting force tends to be stable with little change when the cutting speed was more than 900 mm/min. Therefore, the loading speeds of the cutting blade were selected as 500 mm/min, 600 mm/ min, 700 mm/min, 800 mm/min, 900 mm/min, respectively. The average MRL of hydroponic lettuce is 95 mm, the cutting positions were selected as 0 mm, 30 mm, 60 mm, and 90 mm away from the planting board, respectively. These position corresponds to MED0, MED3, MED6, and MED9, as shown in Fig. 1. The hydroponic lettuce root was fixed on a shearing fixture and it was cut with different cutting speeds. The root was moved to the next position after completing shear experiment of one position. Every experiment was repeated 3 times in different cutting speeds and different cutting positions combination, then average results were compared statistically.One-Way analysis of variance with Microsoft Office Excel 2016 software and Multi-Way analysis of variance with Design Expert 8.0 software were used for the analysis of variance of experimental data. In addition, a root cutting force model was built by a multivariate regressive method. The model was checked by an F-test in Design Expert 8.0 software.The moisture content was determined by the direct drying method.

The main distribution range of CD of hydroponic lettuce was from 300 to 359 mm, which accounts for 87% of the total samples; the main distribution range of OL was from 50 to 89 mm, 4×8 flood tray which accounts for 90% of the total samples; the main distribution range of pH was from 170 to 210 mm, which accounts for 83% of the total samples; the main distribution range of TH was from 450 to 569 mm, which accounts for 77% of the total samples; the main distribution range of TRL was from 240 to 359 mm, which accounts for 83% of the total samples; the main distribution range of MRL was from 80 to 109 mm, which accounts for 90% of the total samples; the main distribution range of SDi was from 11.0 to 14.9 mm, which accounts for 90% of the total samples; the main distribution range of MED3 was from 25.00 to 34.99 mm, which accounts for 94% of the total samples; the main distribution range of MED6 was from 45.00 to 59.99 mm, which accounts for 80% of the total samples; the main distribution range of MED9 was from 40.00 to 54.99 mm, which accounts for 83% of the total samples; the main distribution range of the total weight was from 130 to 179 g, which accounts for 80% of the total samples; the main distribution range of the net weight was from 120 to 169 g, which accounts for 77% of the total samples. The histograms of these data obtained from physical properties experiments on hydroponic lettuce were shown in Fig. 6, and the geometric parameters of hydroponic lettuce were shown in Table 1. The longest MRL was 114 mm. Therefore, the depth of the nutrient tank should be greater than 114 mm to avoid the influence on the growth of lettuce. The varying of PH and CD were significant. The maximum values of PH and CD were suggested to be referenced when designing harvesting machinery to avoid the failure of harvesting. The MED can provide support for the design of the cutting devices. The MED with 63.21 mm is required to be acceptable when the cutting device is fully open. In addition, planting density and root extension of hydroponic lettuce will also affect the growth of the crop. Therefore, OL,TRL, and TH can be referenced when determining the plant spacing and cultivation mode. To select a planting board with suitable strength, the weight should be used as a reference standard.

And the size of the planting hole in the planting board could be determined according to the maximum value of SDi of hydroponic lettuce. Meanwhile, it can be seen that the dispersion of physical parameters of hydroponic lettuce was small from the Coefficient of Variation less than 0.15. Therefore, harvesting machinery can be designed according to the main distribution range of physical parameters of hydroponic lettuce.The moisture content of hydroponic lettuce was high, but the value of different parts were quite different. The moisture content of leaf was considerably higher than root and stem, the average moisture contents were 95.73%, 91.71%, 91.85% for leaf, stem, and root, respectively, as shown in Table 2. The CV of moisture content of stem, root, and leaf were less than the reference value of 0.15, which means that the data were reliable in this study. Xiang et al. concluded that the moisture content of fresh-cut lettuce leaves was 96.8% during storage, which was similar to the result of this study. In addition, leafy vegetables with high moisture content were more likely to wither and rot. Therefore, the automatic equipment should finish harvesting and packaging as soon as possible to avoid the impact on the quality of the product. Besides, although the moisture content of the stem was the lowest, it was easier to break because of its small toughness. Therefore, more protection should be given to this part in harvesting and packaging.The maximum force and the minimum force of pulling hydroponic lettuce out of the planting board were 16.5 N and 9.6 N, respectively. The value of the average pulling force was 13.03 N with standard deviations of 1.85 N. The experimental data were within the normal range with CV was less than 0.15 specified in the evaluation data dispersion. The main distribution range of the pulling force of hydroponic lettuce was from 11.0 to 15.9 N, which accounts for 83% of the total samples, as shown in Fig. 7.

Shandong Agricultural University Hu reported the pulling force of spinach was 14 N with the soil moisture content was 12.3% during harvest. The harvesting style of lettuce was comparable with spinach. Therefore, the harvesting machinery of spinach can be referred to when designing the harvesting machinery of lettuce. In addition, the planting board may be pulled up together with the hydroponic lettuce due to the light weight of the planting board. Therefore, the planting board was suggested to be fixed during the experiment and the actual harvest to avoid harvest failure.Thirty results were obtained through the shear experiment, as shown in Table 3. The scatter distribution was shown in Fig. 8. The maximum and minimum root cutting force were 27.43 N and 1.41 N, respectively. The maximum root cutting force appeared under the combination of cutting position of 0 mm and cutting speed of 500 mm/min. The minimum root cutting force appeared under the combination of cutting position of 90 mm and cutting speed of 900 mm/min. Generally, the root cutting force of hydroponic lettuce increased with the increase of cutting speed, and also increased with the distance from the planting board decreased.The variance analysis of the regression equation for the root cutting force was shown in Table 4. The F-value of the model of the regression equation was 86.95, and the F-value as a reference can be obtained from the standard F distribution table, which was 18.51. This result means that the model was significant . Meanwhile, the effect of cutting position and cutting speed on root cutting force were significant with P-value less than 0.05. In addition, the F-value of cutting position and cutting speed were 148.85 and 25.04, respectively. The influence of cutting position on the root cutting force was more significant than cutting speed . The response surface method in Design Expert 8.0 was used to summarize the regulation of root cutting force. The response surface figure between the cutting speed, cutting position, and root cutting force was shown in Fig. 9. It can be concluded: for the same cutting speed, the farther the cutting position from the planting board, the smaller the root cutting force, ebb flow tray and for the same cutting position, the higher the cutting speed, the smaller the root cutting force. The minimum root cutting force was 1.41 N with the 900 mm/min of cutting speed and 90 mm of the distance between the cutting position and the planting board. Du et al. and Li et al.have reported the trend of root cutting force of cabbage with cutting position: root cutting force decreases with the cutting position close to the outer most leaf of cabbage, which consistent with the results of this study.

Meanwhile, the trend of response surface was unanimous with the result of variance analysis.The value of lettuce characteristic research can be used in the harvesting stage and packaging stage of hydroponic lettuce processing. The ideal result used minimum force to finish harvesting without damaging the hydroponic lettuce. The average value of the moisture content of hydroponic lettuce was more than 90%. To ensure the quality of lettuce, the harvesting and packaging processing should be shortened as much as possible. The minimum value of the pulling force of 16.5 N can be applied effectively to ensure the hydroponic lettuce can be pulled out successfully. The planting board should be fixed to avoid pulling out together with hydroponic lettuce during harvest. The values of the root cutting force indicated the necessary forces to cut off the root with different cutting positions and different cutting speeds. The F-value of cutting position was 148.85, but the F-value of cutting speed was 25.04. The influence of cutting position on the root cutting force was more significant . To save energy, a further away from the planting board position should be selected to cut.Hydroponic growth system is a versatile platform to study plant behavior under controlled conditions. It is a convenient system for not only controlling the plant nutrition but also provides an excellent model for researchers to study root system and its responses under different environmental conditions. Certain hydroponic systems have been developed for Arabidopsis and other crop species to study their behavior under various environmental stress conditions. To our best knowledge, only few studies were available on trees species which were grown hydroponically under controlled environment. Based on existing knowledge, we have modified/ customized hydroponic system for Pongamia seedlings. Pongamia is known to possess elongated tap root and lateral root system. To accommodate Pongamia tap root system and without facing the root inhibiting effect, we have designed new experimental system. Our method was uniquely designed to perform tree species stress studies with minimal resources. Pods of P. pinnata accession TOIL 12 were obtained from Tree Oil India Limited , Zaheerabad, Hyderabad, Telangana.

Leaf area, photosynthetic potential and crop growth rate are important indexes for representing the photosynthetic capacity of a population. Wu et al.  suggested that the leaf area, photosynthetic potential and population growth rate of superhybridized japonica rice were significantly greater than those of Shanyou 63 control rice at the middle and late stages. Huo et al.  reported that, with a delayed sowing date, the LAI significantly decreased at the JS, HS and dough stage and slightly decreased at the MS. The photosynthetic potential significantly or extremely significantly declined from sowing until the JS and from the JS to the HS. The cropgrowth rate markedly increased from sowing until the JS, and no significant difference was observed from the JS to the HS, whereas the rate decreased significantly from the HS to the MS. The net assimilation rate noticeably increased from sowing until the JS and from the JS to the HS but noticeably decreased from the HS to the MS. However, few reports exist on the effects of seedling age at transplanting on the photosynthetic production characteristics of mechanically transplanted HLMS.

In this research, with increasing seedling age, the LAI decreased for both cultivars at the HS and MS, the photosynthetic potential also decreased from the TS to the JS and from the JS to the HS, and the crop growth rate decreased from the TS to the JS, flood table except for 6 Liangyou 9368 in 2015. No obvious pattern of differences in crop growth rate was observed from the JS to the HS, but the rate was greater for the young seedlings than for the old seedlings from the HS to the MS. With increasing seedling age, the Pn markedly decreased at the HS, but there was no significant difference in Pn between the 13- and 20-day-old 6 Liangyou 9368 seedlings.Selenium  is an essential micro-nutrient for maintaining human health . When enters in metabolism, Se can enhance the anti-carcinogenic capacity of the human body . Vegetables and cereals are important sourcesof Se for humans. However, the low bioavailability of Se in the soil in some areas restricts Se accumulation in vegetables and cereals , which leads to inadequate Se intake levels to possibly prevent cancer . Se deficiency in the diet is a worldwide problem, especially in China, the UK, Eastern Europe and Australia . Therefore, there is an increasing demand for Se-enriched food . Vegetables play important roles in the human diet.

The consumption of Se-rich vegetables could be a safe and effective way to solve the problem of Se deficiency. It is known that plants can uptake Se in the form of selenate, selenite and organic species . The exogenous application of Se has been particularly effective in increasing the Se concentration in plants, but this effect varies among species . An excessive intake of Se can also lead to chronic toxicity for humans, with the recommended daily maximum intake of dietary Se not exceeding 400 mg per day . The target to regulate the Se concentration in vegetables is still therefore unknown. Consequently, there is an urgent need to develop adaptive agricultural strategies to regulate Se uptake and distribution in vegetables. Nitrate is one of the main forms of nitrogen used for plant growth and development and is widely used in vegetable production, especially in leafy vegetables grown in hydroponic systems . Lettuce  is the main crop grown in greenhouses and is consumed worldwide due to its flavour and high levels of phytochemicals. However, lettuce is a hyperaccumulator of nitrate and easily accumulates a large amount of nitrate in its leaves . The daily consumption of vegetables with high amounts of nitrate is associated with a higher risk for cancers and methemoglobinemia . In our previous study, we reported that exogenous Se application had a positive effect in restricting nitrate accumulation in hydroponic lettuce . However, little is known about the relationship between Se accumulation and nitrate reduction in plants under different forms of exogenous Se.

Light is not only the driving force for photosynthesis but also serves as the transduction signal to regulate metabolism in plants . Compared with light intensity and light duration, light spectra have more complex roles in regulating plant growth and development . To date, red and blue light emitting diodes  have been proven to be the most efficient artificial light source for driving photosynthesis and are widely used in vegetable production . Previous studies reported that the light spectral composition plays an important role in regulating the accumulation of mineral elements, such as N, P and K in plants . However, few studies have focused on the effect of light spectra on Se uptake, translocation and accumulation under different forms of exogenous Se treatment. In this study, we conducted a selenate concentration screening experiment and then comparatively investigated the effects of the light spectral composition and Se forms on the uptake, translocation and accumulation of Se, nitrate metabolism and photosynthetic performance of lettuce grown hydroponically. The main objectives of this study were to:  investigate the effect of the combination of light spectra and Se forms on the accumulation and distribution of Se and nitrate reduction and  investigate the relationship between the light spectral composition and the nitrate/ Se content in lettuce under different forms of exogenous Se treatment.

The results of this study are crucial to understanding and revealing the mechanisms that are responsible for Se uptake, distribution and toxicity in plants. Furthermore, the information from this study can provide guidance on producing high nutritional quality vegetables with safe Se concentrations.In the main light experiment, the plants and previous growth conditions were the same as above. At the end of the dark period of day 21, similar size seedlings were transplanted into 25-L containers with 0.5 μmol L–1 selenite  or 10 μmol L–1 selenate  in Hoagland solution. These plants were grown under different light spectra for 25 days. There were five different LED light treatments: monochromatic red LED light , monochromatic blue LED light , combined red and blue LED light with a red to blue ratio at 4 , 8  and 12 , respectively. The plants exposed to FL were used as controls. The details of the light spectra from the light sources used in this study are summarized in Appendix A. The light intensity at the plant canopy was monitored every other day using a light intensity metre . The light intensity was maintained at 200 mmol m–2 s−1 by changing the distances between the light sources and plants. Other environmental factors were the same as those at the seedling stage. There were three replicates with a total of 48 plants per treatment. The nutrient solution with the same Se treatment was replaced every 5 days.The RCF and TF could be used to reflect the capacities for Se uptake, accumulation and translocation in response to the light spectra and exogenous Se. The RCF and TF of Se were significantly affected by the light spectral composition and applied forms of Se.

Regardless of the concentration difference, the RCF for exogenous selenite was approximately 5 times higher and the TF was 6.8–11.9 times lower than that for exogenous selenate.With regard to the selenate treatment, the values of RCF and TF in the plants exposed to red and blue LED were lower than those under FL. The RCF of Se under R/B=8 was markedly higher but the TF of Se was lower than that under the other LED light spectral treatments. Interestingly, these parameters for the plants under R/B=4 changed in an opposite direction compared to those for R/B=8, as shown by the lowest RCF and highest TF under R/B=4. These results indicate that R/B=8 was more efficient in promoting Se absorption, while R/B=4 was more effective in promoting Se transportation in lettuce plants. In contrast, the highest and lowest RCF of Se under exogenous selenite was observed in the plants exposed to R and R/B=4, respectively. However, the RCF under the other light treatments was comparable to that under the FL treatment. Compared with FL,rolling benches red and blue LED light  led to significant decreases in the TF of Se in the plants treated with exogenous selenite, and the lowest TF of Se was observed under R.No significant difference was observed for the total nitrogen  content under different light spectra and exogenous Se treatments . However, the nitrate contentsand N assimilation enzyme activities in the lettuce leaves were markedly affected by the Se forms and light spectral composition . The nitrate contents in the lettuce treated with selenate were lower than those under selenite, indicating that compared with selenite, exogenous selenate was more efficient in retarding nitrate accumulation in the lettuce plants. Relative to FL, the nitrate contents were significantly lower under red and blue LED light. Interestingly, regardless of the Se form, the lowest nitrate content was observed in the plants exposed to R/B=8. NR activity was significantly affected by the light spectra, applied Se forms and their interaction.

The NR activity of the plants treated with selenate was higher than that under the selenite treatments. The highest and lowest NR activity were observed under R/B=8 and B, respectively, after exogenous selenate and selenite application. Unlike the changes in NR activity, NiR activity was mainly affected by the light spectral composition. The highest NiR activity was observed under R/B=8, while this parameter under the other light treatments was comparable to that under FL. The activities of GS and GOGAT under LED light  were higher than those under FL. The highest GS activities were observed under R/B=8, while the GOGAT activities were the highest in the plants exposed to B for both the exogenous selenite and selenate treatments.Under the exogenous selenate and selenite treatments, the Se and nitrate contents in the lettuce leaves  were both negatively correlated with the percentage of red light from the light sources . However, there was a significant linear relationship between the Se and nitrate content under exogenous selenate and selenite combined with different light spectral compositions . These results indicate that the accumulation of Se and nitrate were regulated by the light spectral composition and that a higher ratio of red light was not conducive to Se and nitrate accumulation in lettuce.Se is an essential mineral element for both humans and animals and is mainly acquired from plants . Exogenous Se application can increase the concentration of Se in the edible parts of plants. However, Se has not been proven to be an essential mineral element for plants, and excessive Se can be toxic. In this study, the effect of selenate on lettuce growth was concentrate-dependent: low concentration  promoting growth and high concentration  inhibiting growth.

These results are consistent with those of previous studies showing that a small amount of Se is beneficial for plant growth, while excessive doses could induce a decrease in the photosynthetic capacity, ultimately leading to reduced biomass or even plant death . In the present study, the significant effect of the light spectra and the concomitantly marked interaction between light spectra and Se forms on most of the plant biomass parameters indicate that the light spectrum plays an important role in the process of Se regulation of plant growth. Under mixed red and blue LED light, the higher biomass of selenatetreated plants observed in our study is partly attributed to their high photosynthetic capacity, as shown by higher Pn under selenate than under selenite . Similar results were also reported in wheat by Kaur and Sharma . Compared with other trace elements, Se is arguably one of the most interesting elements because of the very narrow window between deficiency and toxicity . An excessive intake of Se in the diet can also be harmful to human beings and animals . The ability to control Se concentration at safe levels in the edible parts of plants is important, alongside promoting Se accumulation in plants. In plants, mineral element uptake and translocation are regulated by many factors. Light can strongly affect plant cell potentialsor fluxes of ions other than those associated only with energy . Previous studies have found that the uptake capacity of mineral elements of plant tissue was affected by the light spectral composition .

Higher antioxidant activity has been measured in ginseng leaves and fruits than in roots, possibly in association with the 4e9-fold higher phenolic contents of fruits and leaves than roots. However, the beneficial effects of these phenolic compound-rich fruits and leaves remain unclear. Short-term hydroponic-cultured ginseng is a widely available, eco-friendly plant that cultivated in pesticide-free indoor farms. Unlike typically grown ginseng, which is usually sold in the form of dried roots, sHCG is harvested as a whole plant, consisting of roots, stems, and leaves. Ginseng roots contain high levels of ginsenosides, whereas the stems and leaves contain high levels of phenolic compounds; therefore, sHCG has a nutritional advantage over ginseng because all parts of the plant are used. Our previous study reported that sHCG, in the form of 1-year-old ginseng seedlings grown hydroponically for 21 days, exhibited higher antioxidant activity than 5-year-old ginseng plants. Contents of total ginsenosides and four ginsenosides  were significantly higher in sHCG than in 5-year-old ginseng. Additionally, sHCG has an almost fourfold higher total phenolic content compared to 5-year-old ginseng.

However, the beneficial physiological effects of sHCG are not clear. Several variable factors determine the extractability of saponin, such as temperature, solvents, extraction time, and the liquid to solid ratio. However, it is difficult to determine which factor is most important due to their complex interactions. Therefore, led grow lights response surface methodology  has been used to determine optimal extraction conditions in studies attempting to maximize the yield of saponins from medicinal plants and Rg1 and phenolics from RG. However, to date, no study has evaluated the extraction conditions for sHCG and the bio-activity of the extract. In this study, the optimal extraction conditions for sHCG were evaluated using RSM modeling, with the aim of maximizing the yields of bioactive compounds abundant in sHCG but present at lower levels in white ginseng  and RG. Additionally, pilot-scale sHCG extracts were produced using the optimal extraction conditions for future commercial production and the effects of the extracts on TNF-a/IFN-ginduced TARC levels in human keratinocytes were evaluated as indicators of AD disease severity.Fifty-one phenolic compounds were extracted from sHCG, WG, and RG, and analyzed using ultra-high-performance liquid chromatography-tandem mass spectrometry by reference to a database of mass-fragmentation patterns. The CGA content was markedly higher in sHCG than in WG and RG.

HPLC-UV quantification revealed a CGA content of 12.7 mg/100 g dry sHCG, which was 2.5- and 13.3-fold higher than the contents in WG and RG, respectively . Similarly, Chung et al. reported that of 23 phenolic compounds, CGA and p- and m-coumaric acids were the major phenolic compounds detected, particularly in leaves of ginseng aged 3e6 years. Because only roots of WG and RG were analyzed, the higher content of CGA in sHCG may be attributed to the leaves. CGA is biosynthesized and stored during the early stages of leaf development in other plants, such as coffee trees and perforate St John’s wort. Therefore, it is reasonable to assume that the ginsenoside and CGA contents are higher in sHCG containing leaves and stems cultured in the early growth period than in conventionally grown ginseng and RG. In addition to CGA, 22 ginsenosides of sHCG were profiled in our previous study, and 4 ginsenosides  were selected as representative bioactive compounds. These four ginsenosides and CGA are known to be anti-inflammatory, antioxidant and immune enhancing agents. AD is a chronic inflammatory skin disease caused by impaired immune regulation, genetic abnormalities, and hypersensitivity to environmental factors such as allergens. Keratinocytes are epidermal cells that play a critical role in maintaining epidermal barrier function, thus protecting the body from allergens and pathogens. When stimulated by immune triggers , keratinocytes release inflammatory mediators  and macrophage-derived chemokines.

TARC/CCL17 and thechemokines promote penetration of inflamed tissue by T cells. In particular, TARC/CCL17, which has high affinity for CCR4þ T cells, is found only in AD lesions; higher levels are associated with more severe AD. To examine the AD-preventing effects of CGA and the four ginsenosides, it was evaluated whether secretion of TNF-a/IFN-ginduced TARC/CCL17 was decreased by the treatment of HaCaT cells with CGA and the four ginsenosides. Treatment of HaCaTs with 2 and 4 mM CGA signifificantly reduced TNF-a/IFN-g-induced production of TARC/CCL17 by 32% and 45%, respectively . Also, Re, Rg1, Rb1, and Rd at 10 mM significantly reduced TNF-a/IFN-ginduced TARC/CCL17 production in HaCaTs by 28%, 38%, 31%, and 22%, respectively .The milk thistle Silybum marianum Gaertn., synonymous Carduus marianus L., is an annual or biennial plant. The plant is native to the Mediterranean region; however, it was naturalized elsewhere in the world . The fruits of this plant accumulate isomeric mixture of flavonolignans in their outer shells. Silibinins, isosilibinins, silydianin and silychristin are the major flavonolignans produced by S. marianum fruits. The standardized extract of the fruits is known as silymarin, which is an important pharmaceutical raw material used for oral treatment of liver disorders. This plant was placed in 2016 at the sixth position among the top-selling herbal dietary supplements in the natural and health food market and the sixteenth position in the mainstream multi-outlet channel market in U.S.A., at about $9.968 million and $17.077 million, respectively .

In consideration of the market requirements for a standardized product with a high content of bioactive principles, several efforts are directed to the setup of suitable growing conditions for stimulation of plant secondary metabolite production.Therefore, the development of an alternative growing system could be an effective tool to overcome the drawbacks linked to open-field cultivation . Hydroponics is a growing system, in which the nutrient elements that are normally found in the soil are dissolved in a proper quantity of the irrigation water supplied to the plants. Hydroponic is also known as ’soilless culture’, because the plants are cultivated in pure nutrient solutions or in artificial growing media  instead of the common agricultural soil . With more isolated condition and well-defined composition in this technique, the management of important growing parameters such as surrounding climate or nutrition represents a significant tool for the regulation of secondary metabolism. In particular, a proper change in the composition of the nutrient solution could stimulate the secondary metabolism and favor the accumulation of bioactive compounds inside the tissues.A further major advantage of hydroponics is the possibility to expose the plants to stress factors that can elicit an increase in the concentrations of secondary metabolites .

Among the most remarkable representatives of elicitors is salicylic acid which is a natural plant stress mediator that can highly improve the production of pharmaceutically active compounds in plants . It successfully enhanced silymarin production in S. marianum in vitro cultured cells in MS liquid medium but not previously used in hydroponic culture. Nutrient film technique is one of the hydroponic types in which the plants are housed in net pots that are placed on channels having the nutrient solution. The nutrient solution is pumped though the channels and constantly running along the bottom of the channel and the roots are not completely submerged . Hydroponic culture of medicinal plants aiming at production of important secondary metabolites is rarely reported.Nuclear magnetic resonance spectroscopy can provide useful qualitative and quantitative information in analysis of complex mixtures such as plant extracts. Quantitative proton nuclear magnetic resonance spectroscopy  can offer an overview of the sample composition through quantification of multiple metabolites without the need for chromatographic separation. This method, orthogonal to the high-performance liquid chromatography  analysis, allows quantification of targeted compounds without the need of reference materials . So, the objectives of the present study were to establish a hydroponic culture protocol for S. marianum as one of the medicinally valuable plant species using nutrient film technique; enhance plant capacity to accumulate flavonolignans using salicylic acid as elicitor and determine the influence of the hydroponic system, vertical grow system as a different cultivation technique and elicitation on seed productivity and silymarin yield which was achieved by comparing the flavonolignan content in different cultures using qHNMR and HPLC techniques.

In addition to, provide a demonstration of the effect of salicylic acid on biosynthetic pathway leading to flavonolignan production.Different salicylic acid treatments were supplied to each single plant and the plants were randomized on one nutrient film apparatus so that each single plant was a biological repetition. Salicylic acid was added at concentrations of 100, 200 and 400 lM, into the nutrient medium when the plant is at BBCH growth stage 75. Nonelicitor treatment was considered as the control culture. The mature fruits of the cultured S. marianum plants were collected at BBCH growth stage 88, 5 d after the elicitor addition. Sampling was performed individually from each single plant and further fruit analysis was performed at single plant level.In the past 40 years, numerous strategies have been developed to improve plant productivity using in vitro culture techniques such as elicitation . However, there are no reports for the use of these strategies in hydroponic system. In the present study, hydroponic culture was successfully established from the seedlings at BBCH growth stage 14 using nutrient film technique system . The results of measuring different factors for flower-head and seed proved that milk thistle productivity significantly affected by hydroponic system and elicitation treatments . Considerable increase in these factors was achieved by elicitation of the hydroponically cultured plant using 200 mM salicylic acid than field-grown plant . The most interesting observations for the hydroponic culture was its ability to produce fast plant growth with high biomass. In addition to, the feasibility to control the growing environment and to change the composition of the nutrient solution, when it was necessary to treat the nutrient solution with elicitors, because the nutrient elements are readily available at the root zone and can be easily taken up by the plants. The flavonolignans contents in the fruits of different cultures of S. marianum were analyzed using both qHNMR and HPLC. Both techniques were reported to be used interchangeably to quantitate flavonolignans in the silymarin complex .Quantitative analysis in NMR depends on the fact that the integrated intensity of a signal due to the analyte nuclei is directly proportional to its molar concentration and to the number of nuclei that give rise to this signal. The direct proportionality of the analytical response and molar concentration is one of the main advantages of qNMR as a method for quantification over other spectroscopic methods. In UV spectroscopy, for example, the concentration is related to the molar absorptivity which is different for each molecule. Therefore, we have to obtain pure reference standard for each compound of interest. In qNMR, a single standard can be used to quantify many components in a mixture, which can be even structurally unrelated to the analytes under investigation, contains the nucleus of interest and has a resonance that does not overlap those of our target analytes. Silibinin was used as a reference standard.

The 1 H NMR data of the compounds under investigation in this study has been previously published . The chemical structures of flavonolignans under investigation in the present study are shown in Fig. 3. The flavonolignan contents in the fruits of hydroponically cultured plant are shown in Table 1. Silychristin, silibinin and isosilibinin were the major flavonolignans detected in the fruits of the cultured plants while silydianin was not detected.  estimated the silymarin content in the field-grown fruits collected from Cairo – Alexandria Road between 11.02 and 15.54 mg/g DW in which silychristin and silibinin/ isosilibinin were 2.77–3.17 and 3.03–6.38 mg/g DW respectively. Taking in consideration the variation due to re-cultivation in different habitat in Beni-Suef governorate. Elicitation of the hydroponic culture with salicylic acid at different concentrations  enchanced the production of these flavonolignans in the fruits. A dose of 100 mM salicylic acid added to the hydroponic cultures of S. marianum for 5 d increased total silymarin content 1.4-fold higher than the control. The highest content of total silymarin was observed after addition of 200 mM salicylic acid for 5 d. This is considered as 1.7-fold higher than in the control.