Synergistic soil-less medium for enhanced yield of crops: a step towards incorporating genomic tools for attaining net zero hunger

Globally, industrial farming endangers crucial ecological mechanisms upon which food production relies, while 815 million people are undernourished and a significant number are malnourished. Zero Hunger aims to concurrently solve global ecological sustainability and food security concerns. Recent breakthroughs in molecular tools and approaches have allowed scientists to detect and comprehend the nature and structure of agro-biodiversity at the molecular and genetic levels, providing us an advantage over traditional methods of crop breeding. These bioinformatics techniques let us optimize our target plants for our soil-less medium and vice versa. Most of the soil-borne and seed-borne diseases are the outcomes of non-treated seed and growth media, which are important factors in low productivity. The farmers do not consider these issues, thereby facing problems growing healthy crops and suffering economic losses. This study is going to help the farmers increase their eco-friendly, chemical residue-free, quality yield of crops and their economic returns. The present invention discloses a synergistic soil-less medium that consists of only four ingredients mixed in optimal ratios by weight: vermicompost (70–80%), vermiculite (10–15%), coco peat (10–15%), and Rhizobium (0–1%). The medium exhibits better physical and chemical characteristics than existing conventional media. The vermiculite to coco peat ratio is reduced, while the vermicompost ratio is increased, with the goals of lowering toxicity, increasing plant and water holding capacity, avoiding drying of the media, and conserving water. The medium provides balanced nutrition and proper ventilation for seed germination and the growth of seedlings. Rhizobium is also used to treat the plastic bags and seeds. The results clearly show that the current synergistic soil-less environment is best for complete plant growth. Securing genetic advantages via sexual recombination, induced random mutations, and transgenic techniques have been essential for the development of improved agricultural varieties. The recent availability of targeted genome-editing technology provides a new path for integrating beneficial genetic modifications into the most significant agricultural species on the planet. Clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR/Cas9) has evolved into a potent genome-editing tool for imparting genetic modifications to crop species. In addition, the integration of analytical methods like population genomics, phylogenomics, and metagenomics addresses conservation problems, while whole genome sequencing has opened up a new dimension for explaining the genome architecture and its interactions with other species. The in silico genomic and proteomic investigation was also conducted to forecast future investigations for the growth of French beans on a synergistic soil-less medium with the purpose of studying how a blend of vermicompost, vermiculite, cocopeat, and Rhizobium secrete metal ions, and other chemical compounds into the soil-less medium and affect the development of our target plant as well as several other plants. This interaction was studied using functional and conserved region analysis, phylogenetic analysis, and docking tools.


Introduction
How about if human and environmental health were the driving force behind agricultural and food systems? In 2015, the United Nations (UN) General Assembly established the "Zero Hunger" Goal of Sustainable Development, reflecting concerns about the sustainability of global food systems. Accepting that health, environment, and agriculture are interconnected signifies a revolution in academic and popular thinking. Specifically, by incorporating sustainable agriculture objectives into the wider endeavor to eradicate hunger, the Zero Hunger goal reflects a long overdue understanding that industrial agriculture threatens essential ecological processes upon which food supply relies (IPCC 2013;Rockstrom et al. 2009). These well-documented impacts include biodiversity loss, increased pest pressure, soil erosion, soil organic matter losses, greenhouse gas emissions, eutrophication, and water body contamination (Diaz and Rosenberg 2008;Foley et al. 2011). Long regarded as a Faustian deal in the fight against hunger and malnutrition, modern agriculture has failed to fulfill its promise to eradicate hunger. In fact, despite the fact that the globe is awash in "calories," it was not until recently that the Food and Agriculture Organization renamed food security "food and nutrition security," reflecting a rise in micronutrient deficiencies. Today, 815 million people are malnourished, and up to two billion have vitamin deficiencies (FAO et al. 2017;Initiative 2009). In 2003, the World Health Organization classified obesity as an epidemic in both poor and wealthy nations. There is accumulating evidence that both obesity and diet-related chronic disease are major contributors to lost years of healthy life, and instead of contributing to society, unwell individuals are a substantial economic burden (Murray et al. 2013). The consolidation of corporate dominance over global markets and agrifood governance has exacerbated these environmental and health-related problems (Howard2016).
Marker-assisted selection (MAS) has improved the accuracy of identifying genuine conservation units and evolutionary links, hence facilitating the development of various restoration and conservation techniques. The integration of analytical methods like population genomics, phylogenomics, and metagenomics addresses conservation problems, while whole-genome sequencing has opened a new dimension for explaining the genome architecture and its interactions with other species. The genomes of organelles have enabled us to unearth a more comprehensive evolutionary history for each species of interest. Overall, molecular technologies have enabled research pathways concentrating on the synergistic development of crops and assisting in the optimization of various soil-less media for improved and more efficient plant growth (Anuragi et al. 2022).
Using designed nucleases, gene-editing methods based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) produce double strand breaks (DSB) at known DNA regions in the genome. Subsequent repair at the target location adds variation through error-prone non-homologous end joining (NHEJ) to create insertions and deletions (INDELs), with occasional replacement of nucleotides at the repair areas (Sonoda et al. 2006). Alternately, in the presence of a homologous donor DNA template spanning the DSB, the error-free homologous recombination (HR) repair process may be initiated, resulting in targeted gene replacement (Symington and Gautier 2011). The ZFNs and TALENs are based on recognition processes led by proteins. Using these tools, vector synthesis for targeting DNA sequences is a highly difficult and expensive operation. In contrast, the CRISPR/Cas9 system relies on DNA or RNA sequence homology; targeting a specific DNA sequence needs just a 17-23-bp complementary nucleotide sequence known as a guide RNA (gRNA). This adaptability, along with its high efficiency and cheap price, has made CRISPR/Cas9 the most commonly used of the three genome-editing tools (Doudna and Charpentier 2014). CRISPR/Cas9 has been used to delete, activate, and inhibit certain genes in human cells, animal cells, and plant cells (Pennisi 2013). Nicotiana benthamiana (Nekrasov et al. 2013), Arabidopsis, tobacco, sorghum, and rice (Jiang et al. 2013), tomatoes, wheat, soybeans, and maize (Svitashev et al. 2016) (Li et al. 2017) are a few of the examples. In several instances, CRISPR/Cas9-induced mutations have been shown to be transmissible across sexual generations (Odipio et al. 2017).
String beans, common beans, kidney beans, and snap beans are other names for French beans. It is supposed to have originated in America and is a member of the Leguminosae family. The remnants of French beans were discovered during the excavation of ancient towns in Guatemala and Mexico that date back to over 7000 years ago (Chapman and Riddle 2005). It was one of the most common vegetables in Italian homes in 1569, when the Spanish first brought it to Europe's attention. Europeans helped spread it to Asian nations like China and India as well as to Africa. French beans are essentially the unripe fruits and protecting pods of several varieties of common beans that are picked before the seeds within are fully developed and used in our meals (Jabbour and Barbercheck 2008). It is recommended in our diet for a healthy lifestyle since it is thought to be a very rich source of proteins and vitamins. Additionally, it gives our bodies carotenes like neoxanthin, lutein, and violaxanthin, as well as beta-carotenes. It also has antioxidant properties, which help our bodies get rid of dangerous radicals and are good for our cardiovascular system (Michelle et al. 2008). They are also a good source of dietary fiber, which shields colon mucosa from toxins by limiting exposure. Additionally, it contains a lot of zeaxanthin, a dietary carotenoid with beneficial Ultraviolet (UV) filtering properties. It should be a significant dietary supplement because of its enormous health advantages (Dhingra et al. 2012). It is one of the most cultivated crops in India due to its high protein content and various other benefits. In 2010, at a global level, the total production of dry beans was about 19,393 million metric tons in an area of 26.6 million ha (Benton and Grant 2000). In May 2017, the United States Department of Agriculture (USDA) estimates that the global green bean production for 2016-2017 will be 348.04 million Mt, around 2.07 million tons more than the previous projection . Common Indian French beans, which grow within a temperature range of 17.5-27 °C, require cold climate conditions. Temperatures above 30 °C may cause the bean flower buds to fall, and at temperatures above 35 °C, seeds might not form. Apart from this, French beans are very sensitive to night frost. The ideal temperature for French beans is 20-25 °C, but they can be grown within a temperature range of 14-32 °C. High temperatures will cause poor flower development and poor pods (Orphan et al. 2001). However, French beans mature faster in areas with warmer climates. French beans grow well in a variety of soil types, but prefer loamy, silty, and clay loam soils. Soil in the pH range of 5.5-6.0 with cool climatic conditions is suited for bean farming (John et al. 2012 There are various health benefits associated with the French bean. It helps reduce the risk of cardiovascular disease (Roman et al. 2011). Also, it helps in preventing colon cancer, regulating blood sugar levels in diabetics, boosting the immune system, boosting bone health, and improving eye sight. High fiber content and a good source of folic acid help to prevent gastrointestinal issues. Good source of fibers helps in weight management programs. They cut the cancer risk. It also lowers the cholesterol level. It is a good source of calcium, phosphorus, iron, carotene, thiamine, riboflavin, and vitamin (Stefan et al. 2007). Chemical bean cultivation yields a high crop yield and high crop quality. These are made with synthetic ingredients designed to stimulate plant growth. These have the advantages of predictability and reliability. These fertilizers contain a balanced distribution of the three main essential nutrients needed for optimum plant growth: nitrogen, phosphorous, and potassium. Many formulations contain iron, sulfur, and copper (Małgorzata et al. 2016). It has the ability to rely on a small population of agricultural producers to provide food for a much larger non-agricultural population. It also has a low labor cost, resulting in more affordable food. An excessive number of chemical fertilizers, pesticides, herbicides, and insecticides are used on available land to get the maximum yield, which in turn leads to pollution, various diseases, and infections (Watson et al. 2006).
Various disadvantages are also associated with commercial chemical fertilizers. They are more expensive than biofertilizers. They contain ingredients that may be toxic to the skin or respiratory system. These chemicals also need to be mixed and measured accurately; if we use too much chemical fertilizer, it can kill our plants. Chemical fertilizers can build up in the soil, causing a long-term imbalance in the pH and fertility. Fruits and vegetables purchased from farms that promote intensive farming are covered with invisible pesticides; these cannot be washed off easily. That leads to skin allergies, physical deformity, and congenital disease. The consumption of inorganic poisonous vegetables and fruits increases the number of cancer patients (Rehman et al. 2016). Pesticides sprayed on crops not only destroy pests but also contaminate the soil and kill beneficial insects. Heavy use of chemical fertilizers and pesticides affects the workers (who spray the chemicals) and the people residing nearby. Chemical fertilizers destroy the natural flora of microorganisms living in soils that are highly beneficial. Chemical fertilizers encourage plant disease. Chemical fertilizers produce fruits and vegetables with lower nutritional values and less flavor (Table 1, 2, 3, 4, and 5). The microbial inoculants, or in scientific terms, "the biofertilizers," can be generally defined as live and efficient microbial preparations of strains pertaining to nitrogen fixation, phosphate solubilization, or cellulolytic microorganisms that are generally used for the application in terms of inoculation in seed, soil, or composting areas with the objective of increasing the extent of the availability of nutrients in a form that can be easily assimilated by plants (Berry et al. 2006). Biofertilizers are a low-cost source of plant nutrients that are also environmentally friendly and can be used in conjunction with chemical fertilizers. The biofertilizers are bacteria, algae, and fungi, and many can be broadly classified into two categories: nitrogen-fixing biofertilizers (Rhizobium, Azotobacter, Azospirillum, Acetobacter, bluegreen algae, and Azolla) and phosphorus-solubilizing biofertilizers (PSM and Mycorrizae).
Biofertilizers have various advantages over chemical fertilizers (Saeed et al. 2015). They fixed atmospheric nitrogen in the soil and root nodules of legume crops and made it available to the plants. They solubilize the insoluble forms of phosphates like tricalcium, iron, and aluminium phosphate into available forms. They scavenge phosphate from soil layers. They produce hormones and anti-metabolites that promote root growth. They basically result in the decomposition of organic matter and the mineralization of insoluble minerals in soil. Biofertilizers increase the availability of nutrients when applied to seed or soil and improve the yield by 10 to 25% without adversely affecting the soil or environment. They are low-cost and ecofriendly. It is known to induce resistance to nematodes, resulting in improved plant growth (Stolze et al. 2000).

Soil-less farming
It is the method of growing crops without soil in mineral solutions or in medium culture, which provides all the necessary nutrients to the plants (Table 6). Soil-less medium is a plantgrowing medium used in soil-less farming that contains a mixture of substrates but does not contain any soil. It generally contains a mixture of organic and inorganic materials that serve individual purposes. Some of the most common soil-less growing mediums include a mixture of peat moss, perlite, vermiculite, sphagnum moss, limestone, and sand. Vermicompost is the product of the composting process using various species of worms, usually red wigglers, white worms, and other earthworms, to create a mixture of decomposing vegetable or food waste, bedding materials, and vermicompost. Vermicompost contains water-soluble nutrients (Cantliffe et al. 2007).

Significance of soil-less farming in the urban context
Land area in urban areas is insufficient to cultivate agriculturally important plants. Ornamental and other plants are grown in pots and other small containers. Use of soil in such containers poses challenges relating to the growth of plants due to lack of aeration, proper nutrients, drainage, water holding capacity, shallow depth, and the limited volume of the container. The challenges associated with poor plant growth when using soil can be overcome by using "soilless" media. Such media provide the appropriate physical and chemical properties necessary for better plant growth and yield. Soil-less medium provides additional benefits, as it protects plants from contaminated soil, soil-borne pests, soil and water salinity, chemical residues in the soil, a lack of fertile soil, and water shortage, which are the main reasons for poor plant growth (Rodriguez et al. 2006). Advantages of soil-less farming 1. Independent of soil fertility: No land fertility or nutrient requirements for crops are needed. 2. Less water usage conserves land and water. 3. Less labor is needed: The labor requirement is reduced due to the lack of tilling, cultivating, fumigating, watering, and other traditional practices. 4. Less affected by environmental change: Plants are not affected by environmental changes such as floods, wind, drought, and climate change. 5. Eradication of soil pathogens is easy: Soil-borne plant diseases are more readily eradicated. 6. Higher yields: The yields from soil-less cultivation are significantly higher as a result of the precise control of the growth elements for the plants, such as nutrition, pH, oxygen, carbon dioxide, light, and temperatures.

Common mixtures used in soil-less medium
1. Peat and perlite in the ratio of 2:1 2. Peat, bark, and sand in the ratio of 2:1:1 3. Peat, bark, and perlite in the ratio of 2:1:1 4. Peat, perlite, and vermiculite in the ratio of 2:1:1 is required for proper care of plants. 5. Drying out: Peat moss, coconut coir, sand, compost, perlite, and vermiculite used mostly in the soil-less medium have low water holding capacities. A potted plant in a soil-less potting mix dries in less than a day in the hot summer sun, making constant watering an essential task. 6. Toxic: The existing medium using vermiculite is toxic because vermiculite contains asbestos, which is toxic to plants and humans. 7. Poor plant support: Coco peat has a higher porosity. It cannot hold the plant for a long time.
The use of soil-less culture is described and explained under "novel carnation seedling culturing substrate," which consists of the following components in percentage by weight: 55-64% earthworm manure, 10% fermented coconut husk, 13-19% vermiculite grains, and 13-19% perlite grains. The medium of the present invention does not involve the use of any fermented materials at all. It also employs a Rhizobium culture, which is not disclosed here; another study reveals a novel organic substrate and a method for preparing it. The medium comprises 58 to 71% of earthworm feces, 25 to 35% of pelhamite, 3.5 to 6.5% of perlite, and 0.05 to 0.5% of trace elements. This medium lacks biofertilizer (chemical or natural) to enhance the growth and yield of the plants. In addition, trace elements are used in the medium, which are partially available to plants, and disclose a soil-less growing medium for strawberries composed of vermicompost, perlite, and cocopeat in the proportions 30:60:10, without the use of chemical fertilizer. No biofertilizer (chemical or natural) is used to enhance the growth and yield of plants. Similarly, a soil-less closed-cycle production of green bean (Phaseolus vulgaris L.) using sub-irrigation with effects on yield, fruit quality, substrate, and nutrient solution parameters is revealed. A soil-less medium for growing green beans consisting of peat + perlite and peat + sand in a 1:3 ration and macro-elements was disclosed at the XXVIII International Horticultural Congress on Science and Horticulture for People (IHC2010). This medium does not contain any water-holding components and is devoid of biofertilizer for enhancing the growth and yield of plants. In addition we have a number of soil-less options in the near future taking into account the biochar advantages (Table 6).
From the above, it is clear that the soil-less medium compositions disclosed in the prior art have one or more limitations, which have been addressed and overcome in the present invention. None of the prior art discloses or anticipates the synergistic soil-less medium composition of the present invention. The present invention though has been carried out for French beans, but can be effectively extended to other crops like tomato, cauliflower, capsicum, pea, and other cash crops.
The genomic and proteomic in silico study was also performed to predict further analysis for the growth of French beans on synergistic soil-less medium. The study was also performed to predict the impact of a mixture of vermicompost, vermiculite, cocopeat, and Rhizobium on the growth of various other plants. To carry out this study, the growth-prompting genes and the respective proteins of French beans were identified. Furthermore, homologous strains were identified, followed by functional and structural analyses of all identified proteins. The evolutionary relationship was studied among French beans and various other plants through phylogenetic analysis. This analysis was done to predict the possibility of growth of other plants on synergistic soil-less medium. A molecular docking study was performed to investigate the ability of chemical components present in synergistic soil-less medium to bind with growth-promoting proteins. This study helps identify the binding site present in growth-promoting proteins. The study will give similar results with the other plants identified with the same growth-prompting genes. This current in silico analysis will help in reducing significant research costs, time, and labor for applying the in vitro findings to various other plant groups.

Preparation of a synergistic soil-less medium for the treatment
The present invention discloses a synergistic soil-less medium for enhanced yield of crops and its preparation method. The following medium was collected from the certified ICCOA farmers association (Salogra Organic Grower Group, Mathiya, Solan, HP).

Mixing of medium substrates
Vermicompost 72 kg, vermiculite 18 kg, and coco peat 18 kg are thoroughly mixed and placed in heap form (108 kg total), out of which 90 kg was further treated with designed treatments, and 18 kg was left untreated (the control). The experiment was performed in triplicate for each designed treatment (T1-T6) ( Table 1).

Addition of Rhizobium to the substrate heap
Preparation of Rhizobium solution: 4 g of Rhizobium culture was dissolved in 1 L (L) of water.
Mixing of Rhizobium solution with substrate heap: The solution was prepared by taking 4 g of Rhizobium culture per 1 L of water and was sprayed on the heap of media. It was covered with a moist gunny bag for 36 h in partial shade and was finally shifted to the various treatments. About 15 L of Rhizobium solution was sufficient to treat the entire heap, which contained 90 kg of media excluding the 18 kg of control media.

Sterilization of reused plastic bags
Plastic bags (30 × 30 × 30 cm) were used, and they were treated with a 1:7 formalin solution for 30 min before drying at room temperature.
Filling of sterilized and Rhizobium treated plastic bags with soil-less medium.
Plastic bags were filled with soil-less medium (the plastic bags contained 6 kg of media per bag).
Testing the effect of the present synergistic soil-less medium on the growth of the plants a. Collection of French bean seeds: French bean seeds of the Falguni variety, which are commercially grown by the farmers, were purchased from the seed shop (100 g). b. Treatment of seeds: Seeds of French beans were treated with 20 g of Rhizobium culture per kg of seeds plus a 20% jaggery solution, followed by drying in partial shade. c. Sowing of seeds: Four treated seeds (not treated in the control) were sown per plastic bag, and after germina-tion, only two vigorous and healthy plants were retained per bag to provide better space for the plant's growth.

Identification of growth promoting genes of Phaseolus vulgaris
The growth promoting genes of Phaseolus vulgaris were identified through extensive literature survey.

Identification of superfamily and conserved region of growth promoting protein
The functional protein sequences of the respective identified genes were extracted from the Phytozome, National Center for Biotechnology Information (NCBI), and Uniprot databases. The BLASTp tool was used to identify homologous protein sequences in the identified proteins. The BLASTp results also helped to retrieve the superfamily and conserved gene regions of various homologous plants. The functional analysis of growth promoting proteins was done with the help of conserved regions of the various homologous plants, as shown in Fig. 1.

Construction of phylogenetic tree
The various proteins of different plants were selected from the BLASTp results to construct the phylogenetic tree. The phylogenetic tree construction was done to study evolutionary relationships among these homologous plants. The   Fig. 1 Flow chart for the in silico analysis of growth promoting proteins of plants identified sequences were subjected to multiple sequence alignment to generate alignment file using Clustal Omega. This file was later uploaded on Interactive Tree of Life (ITOL) server to visualize the tree, as shown in Fig. 1.

Molecular docking analysis
The chemical composition of the synergistic soil-less medium was identified through the literature survey. It contains a combination of various elements responsible for the plant's growth. The molecular docking study was done using the modeled structures of identified proteins and the elements identified in vermicompost, vermiculite, and cocopeat, which compose our synergistic soil-less medium, as shown in Fig. 2. The active ingredients include many macroand micronutrients like calcium (Ca), magnesium (Mg), potassium (K), phosphorus (P), and sodium (Na) (Kurniawan 2019). These retrieved 3D protein structures were then docked with Ca 2+ and Mg 2+ , which are the main components of any plant's structural growth and support. This metal docking was performed using a docking tool specially designed to dock metal ions on receptors, i.e., Metal Ion-Binding Site Prediction and Docking Server (MIB2) (Lin et al. 2016). The docked structures were visualized using Maestro 12.8 (Schrodinger). A few chemical compounds that are secreted by Rhizobium and directly promote plant growth were also identified. The molecular docking of identified protein targets was also done against these identified components, namely, lumichrome, biotin, and thiamine, which are the major components present in the synergistic soilless medium provided by Rhizobium (Streit et al. 1996). The docking was performed using the cavity-detection guided blind (CB) dock tool.

Bud and shoot germination
The germination process was accelerated after sowing the treated French bean seeds in the growth medium in plastic bags, as bud germination was observed in the T4 Rhizobium treatment on the third day and showed the best result (Fig. 3), followed by Trichoderma (T1), PSB (T2), and VAM (T3) treatments on the 5th and 6th days, respectively, when compared to the control, where bud germination was lacking even on the 8th day of seed sowing.

The shoot elongation
The shoot elongation was observed longest in the T4 treatment (4 cm) (

Days to flowering
The days to flowering were reduced, and it was observed that the flowering process was initiated on the 14th day of the germination process in treatment T4 (Fig. 5), followed by other treatments, as compared to control, where a prolonged process of flowering was observed on the 20th day of seed germination (Fig. 6).

Pod emergence
The data shows that pod emergence was observed in treatment T4, followed by T1 and T3, as compared to the control, where no visible pod was observed on the 27th day of the germination period (Table 3; Fig. 7 a and b).

Root length and number of roots
The observations pertaining to root length (cm) clearly display that treatment T4 was recorded with the longest root and number of roots (2.7 cm and 5) on the 30th day, followed by T5 (1.9 cm) and T2 (1.8 cm) when compared to the control (1.6 cm and 3) (Table 4). Thus, from the above results, it is clear that the present synergistic soil-less medium (vermicompost + coco peat + vermiculite + Rhizobium) is best with regard to bud germination, shoot length, plant height, flowering, pod emergence, number of pods, root length, and number of roots (Table 5). It also increased the longevity, productivity, and flower initiation of all French bean plants, and, therefore, the result is most satisfactory. The production of food for direct or indirect human consumption is the most notable outcome of food systems. Thus, the amount to which the food generated by food systems contributes to human health, and well-being through good diets is an essential measure of the food system's own health (Barilla 2017). It is also critical to the investigation of potential paths to zero hunger. Similar to the many solutions proposed by agricultural experts based on insufficient definitions of sustainability and productivity, there is no uniform definition of a "healthy diet." A global analysis of dietary patterns indicates that "a diet of minimally processed, close to nature, mostly plant-based foods is definitively connected with health promotion and illness prevention" (Katz & Meller 2014). Other definitions of healthy diets include macro-and micronutrient sufficiency, limitations on additives, trans fats, added sugars, salt, and general variety (HLPE 2017;Blesh et al. 2019).
In addition, it is crucial for genomics-assisted breeding to improve our capacity to comprehensively query nucleic acid-dependent information in the cell. In this regard, molecular markers have proven to be incredibly valuable genetic tools for producing genetic, physical, and phenotypic maps in a wide range of food crop species. With the progress of technology, it is now feasible to identify mutations by scanning enormous numbers of plants for uncommon, abnormal, or normal genetic variation in specific target genes that have been shown to be associated with critical features (Yadav et al. 2022). In a floating system, composts made from different raw materials, such as vineyard waste, tomato waste, leek waste, and olive mill cake, can replace peat in the production of infant green vegetables. In addition to enhancing crop output and product quality, using 25% compost as part of the growing medium while growing young green vegetables in a floating system also inhibits Pythium irregulare (Gimenez et al. 2020). The scientists came to the conclusion that without having any negative growth impacts, bark-based substrates for tomato and basil plants may be replaced with hardwood and sugarcane bagasse biochar in amounts of 50% and 70%, respectively. The analysis of nutritional solution and hydraulic characteristics of substrates and mixes, the interactions between microbes and plant growing media, the impact of soil-less and environmental variables on plant development and photosynthesis, and the build-up of secondary metabolites (Tuzel et al. 2020; Huang et al 2020). Additionally, soil-less media component has been addressed for enhancing plant development and yield (Prasad et al. 2019). A similar study supports our result which ensures the yield, growth index, and leaf greenness of tomato and basil plants cultivated in biochar-infused/ soil-less mixes were comparable to or higher than those grown on commercial substrates based on bark Yu et al. 2020;Thakur et al. 2023).

Limitations/obstacles in tagging zero hunger with genomic tools: off-target effects
Although genomic technology is a priority among researchers, there are several issues with market regulation that need to be resolved. The off-target impacts are one of the main problems. Off-target effects of these genomic tool systems Fig. 7 a, b Rhizobium treatment (T4) shows better result in pod emergence on the 27th day when compared to the treatment T6 (control) (a) (b) have been reported in several papers , which may have hazardous consequences on the products and present security issues. Precision genome editing is urgently needed to control the expression of a gene if its disruption results in fitness costs and impacts plant growth and development. Furthermore, advancements in agricultural production and the eradication of food poverty depend heavily on the discovery and characterization of novel genes with desirable agronomic or nutritionally significant features. The majority of discussions have centered on how deploying genome editing technology in human germline cells will affect society, bioethics, and the law. Most scientists concur that CRISPR-Cas9 should be permitted for use in developing human disease models and studying the molecular processes of diseases, but that it should be outlawed for eugenic or enhancing goals (Butt et al. 2020). It is anticipated that therapeutic genome editing in human embryos will not be feasible in the near future when ethical considerations, safety issues, and application challenges are taken into account collectively. Once genome editing technologies have attained an acceptable level of safety for clinical applications in the prevention of hereditary illnesses, social, legal, and bioethical issues should be thoroughly examined. The deliberate damage principle is affected because the likelihood of inheriting a non-target genetic change outweighs any potential therapeutic benefits . Regulating regulations that could prevent germline genome editing violations must be re-evaluated (Macintosh 2019). It is anticipated that CRISPR-Cas9 (Kaur et al. 2018;Kim et al. 2021;Li et al. 2021) will continue to be used therapeutically and grow rapidly in the medical industry. Therefore, it is critical to create more precise and organized approaches, such as unbiased and biased off-target detection techniques, gRNA engineering, and modification, enhanced Cas variants, effective CRISPR system delivery techniques, anti-CRISPR protein development, and efficient base-editing and prime editing systems (Naeem et al. 2020). Recently developed base-editing and prime editing tools have proved to be less hazardous with almost negligible off-target effects in plants and animals, thus facilitating the development of risk-free genetically engineered (GE) food crops to achieve the global zero hunger goal. To attain the worldwide aim of ending hunger, newly created base-editing and prime editing techniques have been shown to be less dangerous with almost little off-target impacts on plants and animals. But if the problems raised here can be resolved, it is obvious that CRISPR-Cas9 will be used in germline cells in the future (Hirsch et al. 2019). For therapeutic use in germline cells, genomic tools must be totally dependable. The public's conscience should not be overlooked, and pertinent legal, social, and ethical legislation should be put into place as quickly as possible, even while research on the use of CRISPR-Cas9 for clinical purposes is still being conducted (Table 6).

Soil-less media in insect pest management
It is not well understood how certain antagonists, the diversity of microbial communities, and the activity of the whole microflora affect disease suppression (Postma 2004). However, maintaining disease suppression in hydroponics offers a viable method of pathogen management. Three primary methods are available: (i) enhancing the suppressiveness utilizing a mixed culture of microorganisms with complementary ecological features and antagonistic abilities along with disinfection approaches; (ii) using antagonistic microorganisms; and (iii) modifying substrates to encourage the establishment of the suppressive microflora. According to Paulitz and Bélanger (2001) and Postma (2004), the introduction, establishment, and interaction of the biocontrol agent with the root environment are assumed to be made easier by the biological vacuum and the constrained volume of the matrix of soil-less substrates. As a result, many bacteria (Bacillus, Serratia, and Actinomycetes), fungi (Trichoderma, nonpathogenic Fusarium), and oomycetes (Pythium) have been investigated as bio-control agents in soil-less cropping systems. Associating numerous antagonistic drugs with complementary and/or synergistic modes of action against one or more diseases could increase suppressiveness, enhancing the efficacy of biocontrol (Spadaro and Gullino 2005). This is true in cases that are naturally suppressive, where the suppression is brought on by intricate interactions between many microbes working together. Examples include soils that inhibit Fusarium wilts and in which fluorescent Pseudomonas and non-pathogenic Fusarium were found to be the primary antagonists (Alabouvette et al. 2006). For soil-less cultures, it is essential to establish long-term control strategies, such as traditional biological control but also novel types of experiments, such as the reuse of substrates (with their suppressive microflora) or the use of suppressive ready-to-use substrates. Because many environmental factors can be regulated, maintaining the microflora in soil-less culture is much simpler than on soil ( Table 7).

Identification of superfamily and conserved region of growth promoting protein
The 5 growth-promoting genes retrieved from the extensive literature survey were cysteine (C)-rich receptor-like kinases (CRK)8, CRK11, CRK28, CRK31, and TFL1y for French beans. The functional proteins for all 5 genes were acquired from the NCBI, Phytozome, and Uniprot servers. Furthermore, the conserved region of protein sequences, the superfamily, and the functional analysis of the five identified proteins were done using the BLASTp tool. The results indicated that among 5 proteins, the 3 functional superfamilies SPS1 for genes CRK8, CRK28, and CRK31, DnaJ for CRK11, and PEBP for TFL1y were identified, as mentioned Table 7 Futuristic biochar based synergistic media that can be used as good soil supplements and nutrient enrichment source Soilless media options for future The integrated approach for soil-less module Sustainable synergistic biochar options for significant effect on nutrient efficiency, uptake, and plant growth Reference Utilization of yellow pine sawdust Compost made from tree leaves and branches is added before mixing When the soil needs to be remedied, utilizing a mixture of biochar and compost is preferable to using biochar alone since it has a higher N and P supply for plants and greater enzymatic activity Azeem et al. (2021) Choosing hardwood as an excellent option combining with compost made from galip nut pulp Using biochar and compost alone enhanced the contents of N and P in the soil, while using biochar alone produced the maximum P content in the soil Hannet et al. (2021) Combining yellow pine sawdust Compost made from tree leaves and branches is added before mixing When the soil needs to be remedied, utilizing a mixture of biochar and compost is preferable to using biochar alone since it has a higher N and P supply for plants and greater enzymatic activity Azeem et al. (2021) Integrating hardwood media Combining with compost made from galip nut pulp Using biochar and compost alone enhanced the contents of N and P in the soil, while using Biochar alone produced the maximum P content in the soil Hannet et al. (2021) Yellow pine sawdust Compost made from tree leaves and branches is added before mixing When the soil needs to be remedied, utilizing a mixture of biochar and compost is preferable to using biochar alone since it has a higher N and P supply for plants and greater enzymatic activity Azeem et al. (2021) Hardwood Compost integration with galip nut Using biochar and compost alone enhanced the contents of N and P in the soil, while using biochar alone produced the maximum P content in the soil Hannet et al. (2021) Yellow pine sawdust Compost prepared from tree leaves and branches Utilizing a mixture of biochar and compost is preferable to using alone since it has a higher N and P supply for plants and greater enzymatic activity Azeem et al. (2021) Utilizing oak residue Combining plant biomass, sheep manure, and co-composting Higher yield and productivity and ecological safety Oldfield et al. (2019) Page 13 of 23 86 338-600 (Haas et al. 2003) LGVPDMKKPFVRGN-HYFIVNVLIPKNIS-GTERVLVEQLASLR TIGR02349 DnaJ 67-422 (Haas et al. 2003) 359-624 (Haas et al. 2003) 4 Cysteine (C)-rich receptor-like kinases 31 Phvul.007G049100 Phaseolus vulgaris 347-612 (Haas et al. 2003) in Table 8. These identified superfamilies were having different functions on the growth of plants. The three different corresponding proteins of the identified superfamilies SPS1, DnaJ, and PEBP were serine/threonine kinase, chaperone protein DnaJ, and phosphatidylethanolamine-binding protein (PEBP), respectively. This functional analysis of the proteins was done to study the growth of French beans and various homologous plants. The phosphatidylethanolaminebinding protein (PEBP) has grown as a result of neo-and sub-functionalization in plant evolution. The members of this gene have distinct activities due to differential expression and differential protein complex building, which allows them to mediate the crosstalk between the two reproductive events in geophytes in response to environmental and endogenous signals. We suggest that the PEBPs be thought of as conductors of geophyte reproductive development in light of this research (Khosa et al. 2021). The network of protein-serine/threonine kinases in plant cells appears to function as a "central processing unit" (CPU), accepting input data from receptors that detect environmental factors, phytohormones, and other external factors and transforming it into useful outputs like adjustments in metabolism, gene expression, and cell growth and division (Mills et al. 1992).
DnaJ belongs to the molecular chaperone hsp40 family, also known as the J-protein family, whose members control the hsp70s' activity. DnaK (hsp70) becomes ADP-bound and interacts steadily with the polypeptide substrate after DnaJ (hsp40) binds to it and activates its ATPase activity. The hsp70 chaperone mechanism works in the cell to fold developing proteins, move polypeptides across organelle membranes, coordinate stress responses, and designate particular proteins for destruction (Frydman 2001;Walsh et al. 2004).

Phylogenetic analysis through tree construction
The PEBP protein of the TFL1y gene was further analyzed to identify homologous proteins in various other plants using the BLASTp tool. The various proteins were selected for the construction and analysis of the phylogenetic tree, as shown in Fig. 8 a and b. The phylogenetic tree shows that the closest related protein of growthpromoting genes of Phaseolus vulgaris is Lablab purpureus, as it is under the same internal node, while Vigna unguiculata is the outgroup of the clade these three are making. Going further up the tree, we see Glycine soja and Spatholobus suberectus together in another clade that is monophyletic to the previously mentioned clade. Cajanus cajan is the outgroup of this bigger clade, and Mucuna pruriens is the next closest species to Phaseolus vulgaris. Hyacinth bean, black-eyed pea, wild soya bean, Caulis spatholobi, and velvet bean are mostly leguminous  (Kwak et al. 2008) plants, and they need and require Rhizobium to grow. The results of this analysis show that these homologous plants will show almost similar results for growth on synergistic soil-less medium as that of Phaseolus vulgaris (French beans). The phylogenetic tree construction of the other two proteins, serine/threonine kinase and chaperone protein DnaJ, of the superfamilies SPS1 and DnaJ, respectively, has already been reported (Quezada et al. 2019).

Molecular docking interaction between growth promoting proteins and chemical components present in synergistic soil-less medium
It is reported that the receptor-like kinases are great candidates for in silico analysis. These RLKs are conserved upstream signaling molecules that control a variety of biological functions, such as plant development and stress tolerance. RLKs of the key class of cysteine (C)rich receptor-like kinases (CRKs) are essential for plant disease resistance and cell death. In the study by Quezada et al. (2019), the genes of cysteine-rich receptor-like kinase are identified and studied. The genes were then used to draw a phylogenetic tree, which exhibited 5 subgroups, of which group 5 sequences were not present in Phytozome (v13). So, one gene from each group was selected for 3D homology protein modeling. Cysteine (C)-rich receptor-like kinases 08, 11, 28, and 31 of groups I, II, IV, and III respectively, were selected. The 3D structures of these proteins were created using Swiss Model Expasy (Chen et al. 2021).

Molecular docking interaction between growth promoting proteins and metals present in synergistic soil-less medium
The docking score given by the MIB2 shows high affinity and a stable bond forming between the cysteine (C)-rich receptor-like kinases and the metal ions, as mentioned in Table 9. These could mean that metal ions are maybe acting as catalysts, promoters, or even just activators for these proteins, increasing the activity of these proteins, which in turn increases several biological processes that lead to plant development and stress adaptation in French beans. The vermicompost, vermiculite, and cocopeat help in the faster uptake and accumulation of Ca, Mg, K, P, and N; among these, Ca and Mg are the fastest metals to accumulate (Chaulagain et al. 2017). According to the docking results, the CRK8 gene product shows a significant binding affinity towards Mg ions and not with Ca ions, and so do CRK28 and 31's products too. Contrary to this, the CRK11 gene product shows more affinity towards Ca ions. TFL1y's protein shows a very small difference, meaning it could bind with both ions. All the CRK proteins get metal ions attached to the adjacently positioned amino acids, while the Mg ion binds with TFL1y's protein in closely positioned amino acids that are not adjacent in the primary sequence of the protein structures. For the CRK11 gene product, the binding site of metals is really close to each other, so it could be assumed that it will prefer only binding with Ca ions if both are available. Figure 9 a-h show how the metal ions are interacting with the 3D-modeled proteins. This is visualized in the MIB2 results Complete and circular phylogenetic tree and b. zoomed in, highlighted and rectangular phylogenetic tree are visualization of PEPB protein phylogenetic analysis by ITOL tool themselves, and the interaction shown here is the one chosen with the highest scores, indicating the best score. These metal ions, after binding to these 2-3 amino acids, might change the function of the proteins by slightly changing the surface charge, hydrophobicity, electrostatic potential, and surface topography. Mg and Ca ions are well known enzyme activators, structure promoters, and Lewis's acids. These metal bindings might also change proteins' properties like adsorption, aggregation, stability, protein-protein recognition, protein folding, solubility, and membrane binding. This means that because of the presence of these metal ions, these proteins responsible for upregulation of stress responses and growth-resulting biological processes are increasing or getting affected positively.
Molecular docking interaction between growth promoting proteins and chemicals secreted by Rhizobium present in the synergistic soil-less medium.
CB dock results show that lumichrome and CRK 28 and CRK 31 gene products are making a significantly stable binding, therefore giving a very strong and positive indication that their interaction is responsible for the faster growth of the French beans here. After understanding the function of the superfamily of these proteins, it is safe to deduce that cellular and tissue-level functions are getting faster, as these proteins act as transporters of information and help in communicating responses, leading to lesser lag periods in the growth of the plant tissues and smaller delays and dormancy periods. The biological processes get faster, and metabolism, gene expression, and cell growth and division are regulated more efficiently. All the other docking interactions are also quite stable with high scores, therefore indicating that lumichrome, biotin, and thiamine are helping to increase the functionality of the protein structures. Figure 10 a-l show that even though lumichrome, biotin, and thiamine are different ligands, they get attached in the same domains for every protein (except PEBP-thiamine). This is also shown in Table 10 with the same cavity size. It means that all three must be attacking the same functional domain of the proteins and availing similar effects too; the only difference among the ligands could be the fact that they have different binding affinity, compared to each other with the proteins. So, in the presence of all 3 ligands, lumichrome would replace the other two as they have the same binding site, and lumichrome shows the highest affinity towards all 4 proteins and therefore enacts better results than biotin and thiamine.

Conclusion
The soil-less medium composition contains a unique synergistic mixture of four natural growth substrates (vermicompost -66.2%; vermiculite -16.5%; coco peat -16.5%; and Rhizobium-0.8%).The said combination has not been disclosed in any prior art and is novel. The technical advancement of the present invention lies in disclosing a synergistic soil-less medium for enhanced yield of crops that exhibits the best physical and chemical characteristics for plant growth. It provides nutritional benefits to plants due to the presence of vermicompost, vermiculite, and cocopeat in the ratio of 2:0.5:0.5:0.02. Vermicompost with an efficient water-holding capacity is used to provide a sufficient amount of water to plants. Plastic bags and seeds are also treated with Rhizobium to promote plant growth. Plants are grown in an eco-friendly and economical manner by using reused and sterilized plastic bags instead of costly containers. As per the prevailing practice, the growth media are used in various ratios, but under the present study, the ratios of vermiculite and coco peat were reduced and the ratio of vermicompost was increased with the objective of having an appropriate water holding capacity and therefore avoiding drying of the media. The technical advancement of the present invention lies in disclosing a synergistic soil-less medium for enhanced crop yields. The ratio of vermiculite, coco peat, and vermicompost to Rhizobium is optimized to provide nutrients, reduce toxicity, increase water holding capacity, avoid drying of the media, and conserve water.
The in silico analysis helps to identify various other growth-promoting genes and their corresponding proteins in French beans and also in other plants. Most of the plants identified through phylogenetic analysis, such as the hyacinth bean, black-eyed pea, wild soya bean, Caulis spatholobi, and velvet bean, are leguminous plants, the same as French beans. It is already well known that the leguminous plants need Rhizobium to grow well, so they can also grow well in manure. The screened CRK8 gene product shows a significant binding affinity towards Mg ions; almost similar results were observed for the products of CRK genes 28 and 31. It was also observed that these 3 genes (CRK8, CRK28, and CRK31) belonged to the same superfamily, SPS1, during the identification of the conserved region. The CRK11 gene product shows more affinity for Ca ions. In the case of the CRK11 gene product, the binding sites for both the metals Ca 2+ and Mg 2+ are comparatively very close to each other, so there is a strong competition between the two ions for these binding sites. The Ca 2+ was showing the highest docking score with the CRK11 gene product, as compared to the Mg 2+ . This result shows that Ca 2+ is preferred first for binding on the CRK11 gene product site, even if Mg 2+ is also available. The Ca 2+ strongly binds to binding sites and changes the property of the target protein, so the Mg 2+ does not get available sites to bind. These results indicate that due to the presence of these metal ions, the proteins become responsible for upregulating the stress response and growth-resulting biological processes also increase and get affected positively. Among the 3 chemicals selected for the docking study, lumichrome for CRK28 and CRK31 gene products gave significantly stable binding. All the other docking interactions were also effective and scored highly. These significant results indicate that lumichrome, biotin, and thiamine chemicals increase the functional properties of the protein responsible for the growth. The lumichrome, biotin, and thiamine get attached to the same domains in the case of the CRK gene protein, except in the case of PEBPthiamine. Among the selected 3 ligands, lumichrome can replace the other two ligands as they have the same binding site, and lumichrome shows the highest affinity towards all 4 proteins. The Rhizobium supports the fixing of nitrogen; the Mg ions are best utilized by CRK 08; and the Ca ions are best utilized by CRK 11. The overall results of the current study conclude that the designed media can be a suitable source for the growth of various plants as the growth-promoting proteins are supporting these metals and elements as a result of the in silico study.
The synergistic medium of the present invention exhibits better physical and chemical characteristics than existing conventional media. The medium provides balanced nutrition and proper ventilation for seed germination and the growth of seedlings. It has the advantage of being rich in nutrients, economical, and eco-friendly, as it reduces environmental pollution owing to the use of vermicompost, which is obtained by converting bio-waste into useful products. Furthermore, various genomic sequencing techniques like CRISPR exist. Researchers are interested in the CRISPR/Cas9 system because it makes changing the DNA of many living creatures safer and simpler. Gene editing has transformed agriculture by reducing biotic and abiotic stressors and increasing productivity.