Nature Agriculture

The purpose of classification of farming systems is to develop strategies and interventions relevant to the various systems which may vary in the types and degrees of severity of constraints, resource bases and enterprise patterns. Blanket recommendations of technologies and improved management practices could be one of the reasons for low adoption of interventions by agricultural systems which are highly diverse in agro-ecological and socio-economic conditions.Dairy farming systems in Ethiopia have been extensively characterized. Since dairy cattle genetic improvement strategies in Ethiopia target the peri-urban/urban system and the major milk sheds for introduction of exotic crossbred dairy cattle, the major distinguishing characteristics of the three highland systems is the herd genetic structure, the urban and peri-urban systems are characterized as cross bred-based systems keeping high grade exotic crossbred cows whereas the rural or traditional system is a local cattle-based system.

The studies cited above have however focused on the systems in and around the big cities and the major milk sheds. On the other hand, studies on regional towns have indicated that the crossbred herd composition in the urban/peri-urban system is less than reported in the above cited studies. Despite the extensive characterization of dairy farming systems, a comprehensive characterization of dairy systems in the highlands across the value chain supported with valid statistical analyses is not available in the literature.Secondly, the topology of farms within systems and the underlying determinants for within-system variation/diversity in herd genetic composition are not known to confirm if farms within systems could be considered as a uniform unit to target development interventions. In this study, we analyzed farms sampled form of two states in the wet highlands of Ethiopia to validate the classification of the smallholder dairy farming systems, establish the genetic structure of the three systems and identify within-system determinants of variations in herdgenetic structure based on a series of statistical analyses taking the small holder system in Ethiopia as a case study.

Questionnaire survey on herd genetic composition, herd size, production resources,and household characteristics based on farmers’ recall were collected from February to September 2015 using structured questionnaires. The data from West Shoa and West Gojam zones were analyzed separately to serve as are plication of the study. The genotypes of animals were classified as local, low grade, medium grade and high grade crossbreds based on the exotic blood level of the animals. Low grades were defined as those having about 25% exotic blood resulting from first cross cows backcrossed to local breed sire, medium grade crosses were first crosses with 50% exotic blood resulting from crossing of local cows and exotic bulls , and high grades were animals with about 75% or above exotic blood resulting from first cross cows crossed with pure exotic sire commonly through AI. The level of exotic blood level for cows supplied by government ranches and research institutes was established from the cow certificate provided by the institute. For animals that were born on the farm, exotic blood levels were estimated from the dam and sire breed type as recalled by the owner and from records of AI service providers.Discriminant function analysis was used to reclassify the 180 farms in each zone into their original rural, peri-urban and urban categories.

The model was composed of discriminant functions based on linear combinations of four predictor variables which represented the genetic compositions of herds. The predictor variables were local, low grade, medium grade and high grade crossbred animals in the herds. The variables that provided the best discrimination between the groups were selected based on their statistical significance. The classifications were cross-validated applying leave-one-out analysis where each farm was classified by the functions derived from all farms other than the farm to be reclassified. Multinomial logistic regressions were conducted to estimate the probability of keeping the various genotypes across the three farming systems. Factors that would determine adoption of the various genotypes by farmers in the three farming systems were identified through generalized linear regression analysis fitting Poisson distribution with log transformation of the number of animals as a dependent variable. All analyses were conducted using SPPS version 20 .

Using discriminant function analyses based on herd genetic structures as predictor variables, 180 farms in each of West Shoa and West Gojam zones were reclassified into rural, peri-urban and urban systems . The predictor variables that contributed significantly to the reclassification were number of local, low grade,medium grade and high grade crossbred animals in the herds in West Shoa,and number of local and high grade crossbred animals in West Gojam. In West Shoa, the classification function coefficients for number of local animals were marginally higher for the rural and peri-urban system than for the urban system, the coefficients for the number of low grade crossbreds was higher for the rural system than for the peri-urban and urban systems, whereas the coefficients for the high grades for the rural system were lower than for the peri-urban and urban systems.

Many therapeutic proteins or N glycoproteins synthesised in plants differ in their glycosylation patterns from those derived from the mammalian systems.This may also induce increased allergenicity or reduced immunogenicity. The glycosylation pathways in plants can be altered for humanising the plant-derived vaccines or therapeutic proteins. Environmental issues of plant vaccines include gene transfer and exposure to antigens or selectable marker proteins, while risks to human health include oraltolerance, allergenicity, inconsistent dosage, worker exposure and unintended exposure to antigens or selectable marker proteins in the food chain. These risks are controllable through appropriate regulatory measures at all stages of production and distribution of a potential plant-made vaccine . The potential and prospects of plant made pharmaceuticals is restricted by the potential of transgene spread from crops through outcross, challenges in transgene bio containment, unpredictable impact of epigenetic events on transgene expression etc.

Plant cell culture bioreactors or greenhouses and use of plant virus expression systems to produce vaccine proteins in large quantities can be thought of as safer alternatives. The infamous escape of transgene in case of Prodigene and Starlink corn are examples. ProdiGene produced a transgeniccorn that expressed a vaccine for preventing bacteria-induced diarrhoea in pigs,but in 2002, ProdiGene failed to eradicate plants that had seeded from their previous season’s transgenic corn crop which contaminated non-transgenic soybeans.In 2003,the Animal and Plant Health Inspection Service of theUS Department of Agriculture made it mandatory for engineered plants producing pharmaceuticals to be grown under permit. Inefficient transgenebio containment is a serious hurdle to commercialisation of molecular pharmingusing plants. Regulatory hurdles remain a barrier to molecular farming, further increasing the cost and time, which otherwise are major advantages of plant-made vaccines.

Purification, quality controls for vaccine approval are major cost factors in vaccine production . Containment of the recombinant material is a concern which needs to be carefully monitored, to prevent these from entering the food chain and environment. The recombinant plant-based vaccines produced in transgenic plants must undergo a tight regulatory process before commercialisation.The paradigm of plant-made vaccines has evolved from vaccines consumed by world’s poorest populations through fresh producederived from their local farm, to eating engineered fruit or vegetable sprescribed by a health care worker, to a plant product derived from batch processed,freeze-dried plant tissues prescribed by a health care worker to current aradigm that PMVs are not food materials that need to meet still-evolving regulations of national regulatory authorities for drug administration and Department of Agriculture . Plants producing pharmaceuticals are regulated by USDA and the regulatory framework is developed by the FDA and Centre for Veterinary Medicine. The antigen present in edible vaccines is considered as a chemical, that does not comply with FDA rules concerning nutritional additives, but is recognized as non-GRAS.

These vaccines, under the category of food, would be included as a genetically modified food and thus are not considered a high health risk. Due to this ambiguity, a legal void currently exists with respect to regulations for standardizing edible vaccine commercialization. It is not yet clear what part of the vaccine discharges the antigen itself, the transgenic, modified fruits or the transgenic seeds . In the presence of this legal uncertainty, every country is expected to evaluate whether the entrance of edible vaccines is permitted. In 2005, the World Health Organization delivered a report on the implementation of good agricultural practices for the development of biopharmaceuticals. This report includes detailed information about methods of quality control for medicinal plants, testing to assess identity and purity, and recommended materials for plants in biopharmaceuticals. Bio-farming or molecular farming is attractive because of its flexibility, scalability,low manufacturing cost, no toxicity or pathogenic contamination, but many projects are at various developmental stages and not many are yet available to the pharmaceutical industry.

Optimization of lab protocols for up-scaling the production of therapeutics at commercial level is important for clinical use.Plant metabolic engineering is a highly significant technology for production of high-value pharmaceutical compounds. Fusion proteins for multicomponent vaccines against multiple diseases are a potential tool to incorporate into immunization programmes. Unlike genetically engineered microbial systems such as viruses, which pose more risks to the environment and humans, and have more chances of escape, difficulty in controlling and monitoring such escapes or unintended presence, plants are immobile. Control, containment and monitoring of genetically engineered plants are easier and containment can be achieved by regulating pollen transmission.

The challenges to develop and produce vaccines and therapeutics are immense, due to the ever-increasing or rapidly evolving pathogens, resulting in greater demand than supply. Rapid development and large-scale production of vaccines is the need-of-the-hour in case of an unexpected global calamity of a pandemic of massive scale. Plants offer a safe alternative for low-cost as well as large-scale production system for the vaccines, especially in developing countries. The review describes the strategies, advantages, challenges and prospects in the production of plant-based vaccines. A vaccine provides immunity against a disease. Vaccines used for immunization against diseases can be categorised into live attenuated vaccines, inactivated vaccines,subunit, recombinant or conjugated vaccines, toxoid-based vaccines, viral vector-based vaccines and nucleic acids-based vaccines.Live attenuated vaccine uses weakened or attenuated form of a pathogen, that causes a disease. They provide strong long-term immune response as they are similar to natural infection.

Attenuated vaccine may have small amount of the live virus which may be risky for people with weak immune system. Also, they require cold storage facilities. Live attenuated vaccines are used against smallpox,chickenpox, Measles, Mumps and Rubella etc. .Inactivated vaccines use killed or inactive pathogen and require booster doses of vaccine for immunity . Flu, pertussis, polio, rabies vaccines etc. are inactivated vaccines.Subunit, recombinant, polysaccharide and conjugate vaccines use part of the pathogen such as protein, capsid , sugar moiety etc. Subunitvaccines comprise of purified antigen derived from the pathogen, while conjugate vaccines, consist of a polysaccharide component of the pathogen that is poorly immunogenic, so that it is chemically linked to a protein. Recombinant subunit vaccines are safer since they do not have a pathogen and can also be scaled up. Since subunit vaccines consist of small fractions of the pathogen, immunogenicityis greatly decreased with respect to those derived from whole cells,generating the need for co-administering adjuvants to attain immune-protection.

They can be used in patients with weak immune systems and require booster doses to maintain immunity. These vaccines are used against Hepatitis B,Whooping cough, Human Papilloma Virus etc.Diphtheria and tetanus vaccines are toxoid vaccines .Viral vector-based vaccines such as Adenovirus or measles virus vectors are highly versatile platforms for vaccine development. Viral vector-based vaccines can be used for different viruses, delivered without additional adjuvants and can be administered as intramuscular, intranasal, intradermal and oral vaccination.High yield production processes and means of up scaling have been established for these vaccines so that they can be used immediately in case of apandemic outbreak. But viral vectors are genetically modified organisms considered as potential risks to human health and environment and unsafe due to persistent replication of attenuated vaccines. Viral vectors can integrate into the host genome, or undergo recombination during production, leading to emergence of uncharacterised or novel pathogens.

These safety concerns might also delay clinical studies in case of a pandemic. Viral vector-based vaccines are highly complex and comparatively cost-intensive .Nucleic acid-based vaccines employ antigen-encoding plasmid DNA or RNA or messenger RNA or viral replicons. Due to the ease of antigen manipulation they are also versatile. Vaccine can be developed against various pathogens such as virus, bacteria or parasite and administered as intramuscular or intradermal injections. A eukaryotic expression cassette carrying the antigen is inserted into a bacterial plasmid for propagation in E. coli . Minimal DNA constructs devoid of a bacterial backbone, such as the semi-synthetic minicircle DNA and the fully synthetic Doggy boneTM, have been developed to avoid safety issues related with selectable marker. DNA vector vaccine provides relatively low immune-genicity, since DNA vaccines must cross both plasma and nuclear membranes for protein expression, unlike the RNA vaccines which upon crossing plasmamembrane are translated. Encapsulation of DNA vaccines in lipid nanoparticles,adsorption to polymers and use of molecular adjuvants like cytokines can enhance the uptake of DNA vaccines and enhance the immune response. DNA vaccines have long-term persistence, however, potential risk of genomic integration of exogenous DNA into the host genome or chromosomes may result inmutagenes is and oncogenesis or new diseases. Molecular adjuvants like cytokines may also have undesirable, side-effects such as inflammation or autoimmunity.DNA vector-based antigen expression is the first effective vaccine against Ebola virus, Zika virus etc. and used against human pathogens such as HIV, influenza virus, malaria, hepatitis B virus, respiratory syncytial and herpes simplex virus .

The arrival of most of the pests, such as army worms, was detected in the minor season when there were prolonged dry spells.The pests had the capacity to devour large tracks of maize, increasing the production cost of the poor-resourced farmer. Apart from army worm infestation,diseases like maize streak and stem borer were also causing distress to the farmers.The presence of these pests and diseases were facilitated by changing climate in the Municipality.The respondents were asked what they did when their crop failed; 23%of them reported that they sold other farm produce, 15% of the respondents engaged in trading activities, whereas 12.3% traded in farm animals with about20.5% of farmers doing virtually nothing. Six percent of the respondents lived on borrowing money from friends and relatives. Precipitation was low in the early 1960s but rose steadily in the mid-1960s,falling suddenly in the 1970s. Rainfall, however, progressively rose and somewhat stabilised in the mid-1970s and declined again in the early 1980s.

There was no clear pattern from 1988 to 2015. It was also obvious that from 1988 to2015, the rainfall amount hardly crossed 1000 mm unlike the period between1960-1982. This confirms the farmers’ perception that the current rainfall pattern is rather irregular and unpredictable.The averages of 1960-1982 were higher than that of the present, 1988-2014. From the months of January to March, the mean of 1960-82 was greater than that of 1988-2014. The farmers were right that lately, the amount of rainfall in March had drastically fallen as the amount from 1960-1982 was well over 100 mm , whereas the average amount for theperiod between 1988 and 2014 had reduced to 71.0 mm, which most probably accounted for the reason why majority of the farmers sowed in April .Between 1988 and 2014 rainfall averages had appreciated from April to June,with a deficit in July, rising again in August. Currently, the amount of rainfall in November has decreased to the detriment of farmers. This means that presently,crops do not receive much rainfall as compared to 30 years ago. It can also be inferred from Table 2 & Table 2 that presently, August is getting wetter and the major and minor seasons are progressively merging. It is also clear that the first and the last quarters of the year are getting drier over the last couple of decades with the last five years being the worst.The future seems to be rather bleak looking at the trend in 1960-1982 with almost no rains in November and December.

The average total amount of the period 2010-2014 showed a reduction of 22% over the past five years with a decline between 1960-82 and 1988-2014 of6%. The month of March which was meant for planting of crops now obtained64.5 mm of rainfall against 114.7 mm in the past buttering farmers assertion that climate had changed. On rainfall regularity, the pattern of precipitation in the Municipality is chararcterised by uncertainty. Based on the above tables the amount of rainfall in the month of August increased but figures from 2015 deviated from the trend. The precipitation received in the August,2015 farming season was 0.00 mm. Table 2 also shows a reduction in rainfall amounts between June and August. This phenomenon might have had a negative effect on maize production in 2015, resulting in the lowest yields of maize recorded for the Municipality in the last six years. A report by indicated that even though other contributing factors exist, rising temperature and irregularity in precipitation are the major causes of the continuous reduction in maize yields. Annual rainfall for Nkoranza South Municipality since the 2008/9 farming season for the month of March had never risen above100 mm but rose to 11 mm in 2015 which confirms that the nature of rains in the transitional zone is irregular. According to , declines in total precipitation and increasing irregular rainfall patterns make farming more hazardous,increasing the likelihood of crop failures and reducing agricultural production.

Of these, 78% indicated that the nature of the change was erratic.The majority of the respondents established that the severity of this episode was devastating over the past 5 years. This had therefore made their farming operations riskier as there was no irrigation system in the Municipality, bringing farming activities to a near halt whenever the rain failed .About 65.5% of the farmers admitted that unguided activities of unscrupulouschainsaw operators had led to the cutting down of trees which might have resulted in reduction in rains. On the other hand, 25.5% attributed the changes in precipitation trend to nature itself and 0.5% of the respondents believed that the changes had come due to excessive emission of GHGs into the atmosphere.Another 5% of the farmers also placed the blame on bushfires whereas 3.2% indicated that they did not know what had caused this change.When they were further probed about what could be done to reverse the situation of the erratic rainfall, 64.1% responded that there should be afforestation to replace the felled trees. Others had the view that only God could intervene to restore the situation whilst some farmers called for bush fire control.

Of the several crop models, the FAO CROPWAT and SMACS models can easily be adopted with very little field data demand. The latter has also been tested for the same study area for observed climatology of 42 years covering the period 1961-2002, under no-climate change conditions. FAO’s CROPWAT is a monthly crop model which considers daily rainfalls applied on selected days of each decade. SMACS is a daily-moisture accounting model adaptable for different crops, and externally coupled with daily rainfall and temperature generation model. Another advantage of the SMACS model is that it easily enables/allows external coupling with GCM outputs with possible disaggregation to daily values using weather generators.Beyond impact assessment, the SMACS model can be used as a decision support system as it can also be usedto calculate water balances such as actual crop ET, excess surface runoff and actual soil moisture besides yield and crop stress indicators. It is therefore suitable to evaluate and investigate the potential of and promote rainwater harvesting and conservation agriculture practices as potential adaptation measures.

The water requirement of the crop at a given time of the growing season is calculated by multiplying the reference evapotranspiration with a crop coefficient, whose values are published by FAO . SMACS model is considered due to easy adaptability to simulate soil moisture balances, crop water demand and stress for current climatology as well as post climate change conditions.In the SMACS model, all precipitation in excess of surface runoff to potential transpiration is used as the environmental indicator of water stress and yield potential -. In the crop models considered in the study, water stress is assumed in the crops to affect growth by limiting photosynthesis in direct proportion to the ratio of actual to potential transpiration. The common approach for estimating crop yield reduction is based on FAO experience, which addressed the relationship between crop yield and water use by proposing a simple equation where relative yield reduction is linearly related to the corresponding relative reduction in evapotranspiration .Future climate changes, as well as differences in climates from one location to another, may involve changes in climatic variability as well as changes in the means. In this study, a synthetic weather generator is used to systematically change the within-year variability of temperature and precipitation, without altering long-term mean values.

For precipitation, both the magnitude and the qualitative nature of the variability can get manipulated. The synthetic daily weather series serve as input to the three crop simulation models. Due to its transboundary linkage and since it exhibits similar agro-ecological characteristics extending and covering Southern Zambia, eastern Namibia, western Zimbabwe and north-eastern Botswana, the site was considered as an ideal site for assessing the climate change impacts of rain fed farming systems. Furthermore, the study site is a vast agricultural area where rain fed agriculture is currently practiced.The area is characterized by the presence of heavy clay soils. It is understood that during the rainy season they are liable to become sticky, waterlogged and poorly aerated, and they become hard during dry periods. They are reasonably fertile and are capable of retaining both water and nutrients . Successful cultivation and good management in some areas have shown that these soils are able to make a significant contribution to food production.Because of low drainage, harvesting rainwater excesses can be practiced in such soil conditions.The climate of the project area, like most of the north and south-eastern Botswana, is sub-tropical and semi-arid. The climate records at Kasane and Pandamatenga compiled by the Department of Metrological Services have been available for the study. The monthly air temperature variation in the project area computed from 1971-2000 recorded data indicates a mean daily minimum and maximum temperature of 15˚C to29˚C, respectively.

The highest temperatures are prevalent during November to March of the year while June and July experience the lowest temperatures.Figure 2 shows the annual temperature variations and general trend in the daily maximum and minimum temperature data of Francistown, a nearby climatic station for the period 1971 to 2000. Wind direction is normally easterly, north-easterly and south-easterly. The incidence of higher winds is greater around September,October and November with average speeds of above 180km/day.Rainfall is characterized by short periods of heavy rain, which cause flush flooding. The men annual rainfall in the study area is generally about 550 mm. The study attempted to establish and understand the degree of susceptibility of crop to failures in terms of soil moisture availability to meet the evapo-transpirative demands of crops studied. Using the SMACS model, the following indices, namely risk, resilience and reliability were used to investigate the available moisture failure rates in rain fed conditions, where rainfed agriculture is practiced in the study site. In this study, daily simulations from climatic data of 1971-2000 were made for the study area, considering commonly grown crops: maize,sunflower,and sorghum.

Numerous overhead costs like the construction of thegreenhouse, a viable model or tray systems or buckets to name a few. These maybe a disadvantage; however, the literature shows that the larger-scale hydroponicsystems tend to be more cost-effective than small-scale land-based farms. Thecost analysis of using the hydroponic grower as compared to traditional farmingis shown in Table 1. As it is presented, the payback period starts in the thirdyear. On the other hand, the traditional method allows the payback period withinone year. The harvest under a traditional method may be affected by weathercondition and other causes extraneous to the traditional farmer. Table 1 showsthe cost analysis of vertical farming using hydroponics and its comparison totraditional land-based farms.

As presented in Table 1, on the second year, the total remaining balance inVertical Hydroponic Farming method will be $59954.94. The remainingloan balance is $48,000 which could be paid in full. And the remaining balancewill be $11954.94 less the total operational expenditure of $8010.47. On the thirdyear, the initial investment will be $3944.47. Therefore, the Return On Investment will be in the second year. Moreover, the remaining area of land could be utilized for planting other crops. For the traditional farmingmethod, although the loan was paid immediately, it is shown that the remainingbalance on the second cropping is smaller than that of the VHF method. However,in the traditional method, third cropping is not possible because the landmust recoup from the exhaustion of nutrients due to the two cropping seasonsand the weather condition during the third cropping is volatile and inappropriatefor onion production. More so, using the traditional method, the entire10,000 m2 farmland area was used while in VHF system, the remaining lot space could be used for other agricultural and live stocks production. The VHF is alsofree from unfavorable weather condition as the onion grows under an artificialenvironment and controlled physical surroundings.

In many studies, the plant’s growth rate is higher in Vertical farming using hydroponicstechnology. Using the system will provide the farmer control of thefarm allowing more significant results. With hydroponics, the farmer will have full control over the farm together with more optimal results. The growth ofonion is shown in Tables 2-4 below. Hydroponic onion did as well if not evenbetter than the plants grown in soil. Notwithstanding the higher cost of initialcapital, increase crop yields would compensate the investment . The systemcan also serve as an alternative to land-based farming in areas where the landsoil is poor in quality. When the technology is applied correctly, the growth ofthe plants is observable hence could provide earlier harvest resulting in threecropping seasons per year. Table 2 shows the data yielded by the experiments.The Table 2 presents the growth of onion planted in a vertical grower applyingthe hydroponic system of providing nutrients to the onion. It is observedthat there is a 2.5 cm average bulb diameter yielded within a 30 day period. Fromthe 2 cm initial bulb development, it grew to 3.1 on the 30th day of the experiment.On the next scheduled experiment in May 2017, the initial bulb diameter onthe first day developed to 4.1 cm on the 30th experimental day.

This goes toshow that compared to the first experimental phase the bulb development ofonion in the second experiment is relatively bigger. The result is attributed toextraneous variables which the researchers were not able to control in the firstphase of the experiment. On the average, the onion bulb development in thesecond experiment yielded an average growth of 3 cm in a 30-day period.The results of the final and last phase of the experiment are shown in Table 4.As it is depicted in the table. There was a 0.02 cm increased in the developmentof bulb diameter compared to Table 3 Phase 2 experiment yielding only a 4.1 cmgrowth of onion bulb smaller than the result of July 2017 3rd and last phase ofthe experiment.The three phases of laboratory experiments scheduled in three different crop seasons clearly showed that the technology is adaptable to onion growing. Theresults of the experiments are similar to that found out by the researchers of theNational Aeronautics and Space Administration.The present study affirmsthe result of the NASA researches where onion, like any other vegetables,can be grown using artificial simulated environment.The Figure 5 shows theactual observation of the study.The Figure 5 showed the hydroponic technology as applied by the scientistshaving the vision to bring the technology in outer space where there is no landor soil where plant or vegetables could be transplanted to sustain their food.The NASA researchers are checking hydroponic onions with Bibb lettuceto his left and radishes to the right.

Free radicals may mediate the conversion of Amadori compounds to AGEs . It has been reported that catechins display potent scavenging activity towards free radicals when compared to the positive control aminoguanidine, removing free radicals mainly through the phenolic hydroxyl group of the B-ring . These findings suggest that catechins are involved in the inhibition of AGE formationat each stage of the route, ranging from the Amadori compounds to the formation of AGEs. Furthermore, the antioxidant activity of procyanidin varies depending on the degree of polymerization, the binding site, and the tertiary structure of the polymer, and the antioxidant capacity increases as the degree of polymerization rises . This blocks chain oxidative reactions by acting as a free radical scavenger that quenches free radicals .

Procyanidin is thought to be involved in the inhibition of AGE formation at each step because the AGE formation route involves stepwise oxidative reactions. Here, the structure-activity relationship of the isolated compounds reveals that compound 7, a dimer of compound 6 , shows higher activity than compound 6. There is a difference between compounds 8 and 9 in that the former has protocatechuic acid attached to procyanidin B, and the latter has gallic acid attached to procyanidin B. However, compound 9, which has more hydroxyl groups, showed higher activity. In addition, compound 10 was a trimer with the highest degree of polymerization and the highest activity among these compounds. In the comparison of extracts, when the extracts of immature persimmons were compared with those of ripe persimmons, the activity in Tone Wase persimmons that had not undergone astringency removal was higher than that in immature persimmons. However, this activity was greatly reduced by astringency removal. Regarding Fuyu persimmons, activity in ripe persimmons was lower than that in immature persimmons.

As a result, activity in immature persimmons can be considered to be higher than that of the persimmons that we usually eat. This is thought to be due to the effect of tannins. First, when the Fuyu ripens, acetaldehyde generated inside the fruit forms polymers by cross-linking the soluble tannins, making the tannins insoluble. The Tone Wase has a high soluble tannin content of 1–2% when ripe. The anaerobic treatment of persimmon with carbon dioxide or ethanol increases the acetaldehyde content, rendering the tannins insoluble. Based on the above and the other results of this study, high activity was observed in immature persimmons containing soluble tannins and in ripe Tone Wase persimmons that had not undergone astringency removal, but activity was not observed in ripe Tone Wase persimmons that had undergone astringency removal and contained insoluble tannins or in ripe Fuyu persimmons. Therefore, it is presumed that soluble tannins have an inhibitory effect on AGE formation, while insoluble tannins may not have such activity. In the beverage and flavoring experiments, the extracts of immature persimmons and gallic acid had no inhibitory effect on deterioration. For TPA, deterioration was inhibited at lower sample concentrations except for dodecane, and for GA, the concentration that showed the greatest inhibition was 5 mg/L.

It is believed that low concentrations did not provide sufficient antioxidative potency to protect the citral and failed to inhibit its deterioration. The following two points can be considered as possible reasons for the failure to sufficiently inhibit deterioration when concentrations became too high. First, it is thought to be due to the acidic nature of the sample, which led to its deterioration. Other causes include the fact that gallic acid is also an acid, and the extracts of immature persimmons that contain it are also thought to be acidic. The deterioration may have been accelerated due to the more acidic nature of the sample added as its concentration increased. Second, pro-oxidants may be produced due to the generation of other radicals associated with the elimination of oxidizing radicals. In a previous study, the inhibition of the deterioration of citral was investigated using ubiquinol as an antioxidant.

The results showed that neither too low nor too high a concentration sufficiently inhibited deterioration, indicating the existence of an optimal concentration . Although it is difficult to elucidate the detailed mechanism, similar phenomena have been observed for other antioxidants, such as carotenoids . In addition, there were no deteriorated substances such as p-methyl acetophenone , which exhibited a petroleum scent, and the deteriorated substances formedwere myrcenol and linalool oxide . As neither of the two deteriorated substances was cyclized, it is thought that the deterioration of p-menthadien-8-ol was inhibited at the preliminary stage of its formation. In the presence of the emulsifier SY-Glyster ML-750, citral was converted into alcohols by the Cannizzaro reaction and proceeded to become linalool, which was ultimately converted into linalooloxide and myrcenol.

Its structure has a cis-trans structural isomer, which refers to a compound that has two isomers: geranial and neral . Citral is an essential oil component abundant in lemongrass and lemon balm and is used as a component of citrus fragrances because of its strong lemon-like scent. It is used as a flavoring agent in cosmetics and foods and as a raw material in the manufacture of other flavors and fragrances. The bioactivity of citral is reported to have antimicrobial, anti-inflammatory, analgesic, and sedative effects . Citral is easily oxidized under acidic and oxidative stress conditions, resulting in not only the loss of its lemon scent but also the production of undesirable off-flavors. In addition, citral gradually changes from a pale yellow to stronger yellowish color as it deteriorates. The mechanism of the formation of off-flavors in citral, as shown in Figure 2, is described as follows: First, p-menthadien-8-ol is formed by cyclization, which is then oxidized to p-cymen-8-ol, followed by the formation of p-methyl acetophenone via α- p-dimethyl styrene.

This p-methyl acetophenone has a lower threshold than the other products , a very strong deterioration odor, and is known to cause a signifificant quality loss in citrus products . The compound, p-menthadien-8-ol, has a lemon aroma, but compounds of the latter structures cause the deterioration of odors similar to p-methyl acetophenone.Isolation of Functional Compounds Used persimmons were obtained by separating the peel and flesh of each. Their immature persimmon was chopped by a commercial blender and homogenized after, finishing with a total volume of 3 L with water. Cold methanol was added to the water solution, and the mixture was allowed to stand 1 week in the dark to obtain crude extracts. The crude extracts were obtained by filtration and vacuum concentration. The crude extracts were extracted with hexane, then with 1-butanol to obtain hexane, 1-butanol, and water extracts. The isolation of 1-butanol extracts was performed by repeatEmulsions, which are thermodynamically metastable dispersion systems, are one of the forms of application of flavors in foods. Emulsions can change the rate of chemical reactions due to the presence of interfacial films that inhibit the reaction between the lipid and water phases .

The deterioration of citral may occur under both acidic and oxidative conditions, suggesting that the stability of citral may be improved by the formation of an emulsion. In this study, three types of alkanes , with citral mixed into them, were used for the oil phase, while SY-Glyster ML-750, an emulsifier, was used for the water phase. SY-Glyster ML-750 is a polyglyceryl fatty acid ester made by esterifying polyglycerin-10 and lauric acid. SY has been used as an excellent emulsifier in whipped cream, coffee whiteners, margarine, shortening, chocolate, milk drinks, and functional lipids. In this study, we investigated the optimal alkane and the inhibitory effect of citral deterioration caused by thinned persimmons in an emulsified form. Citral is a combination of the isomers geranial and neral. It is a lemon-scented aroma component that is widely used as a fragrance and flavoring in cosmetics and food. However, citral is susceptible to oxidation and deterioration, which causes the loss of its lemon scent, leading to the deterioration of food quality. Therefore, the addition of antioxidants is necessary. In this study, myrcenol and linalooloxide were detected as deteriorated substances using GC-MS, but other deteriorated substances were not tested. This formation route is thought to be caused by the effect of the SY-Glyster emulsifier .

Subsequently, we quantified citral, myrcenol, and linalooloxide. Then, in the water phase, the SY-Glyster ML-750 solution was used as an emulsifier, and TPA, a crude extract of the peel of Tone Wase persimmons, was added as an antioxidant, and gallic acid was added as a comparator substance at the respective concentrations. The control was prepared without the antioxidant TPA or GA. All of the abovementioned components were emulsified to produce emulsions, which were stored at 37 ◦C for 4 weeks, and the amounts of citral and deteriorated substances were measured and compared by gas chromatography every week. In this experiment, we investigated the oxidative stability of citral induced by the addition of antioxidants and the optimal combinations between the different types of alkanes and antioxidant concentrations.All the isolated compounds 1 to 12 showed higher activity than the positive control aminoguanidine in inhibiting the formation of AGEs. In particular, compounds 6 to 10 showed very high activity. These were catechins and procyanidins, which are polymers of catechin. Dicarbonyl compounds, such as glyoxal and methylglyoxal, are intermediates in the AGE formation route. It has been reported that catechins mainly trap dicarbonyl compounds through either the C6 or C8 position of the A-ring . Moreover, free radicals are also closely related to the formation of AGEs because their formation process generates a large number of free radicals .

Our results are consistent with the previous studies indicating high level of landrace accession polymorphism. Landraces from Ethiopia were highly polymorphic and formed a distinct and separate grouping from Australian cultivars, breeding lines, and European cultivars, which cluster together, probably revealing their pedigree and breeding history . Further development of molecular markers and application in genetic diversity studies could enhance our understanding of white lupin genetic resources and promote their use in modern breeding. The Greek landscape is characterized by a mountainous terrain, with numerous peninsulas and islands, and is considered a hotspot of L. albus diversity. Such isolated regions, justify divergence of local landraces, explaining the finding that landraces from Andros and Lemnos islands and from Mani peninsula are clustered separately from the cultivars examined. Additionally, landraces from Andros island are distinctly diversified from the rest accessions, implying that Andros constitutes a genetic pool of unique genes, potentially useful for white lupin breeding. Eventual exchange of landraces between Greek farmers and dispersal of their cultivation farther than their local point of origin may explain landrace sub-clusters on the dendrogram.

It is also a common practice for smallholder farmers to use a portion of the seed yield for the next growing season, exclusively, or in mixtures with high yielding cultivars. Long periods of recycling the seed of a commercial variety, along with potential adaptation pressure and selection by the farmers, promote the generation of new variability, resulting in new landraces that are well adapted to the specific geoclimatic conditions and to their traditional management and uses . Moreover, potential gene flow from neighboring white lupin cultivar crops could justify that landraces appear genetically in close proximity with commercial germplasm. Quinolizidine alkaloids are predominantly found in high levels in natural populations and landraces, making lupin a repulsive choice for lambs and goats, that feed on pasture . That justifies the presence of white lupin natural populations throughout uncultivated lands, that shelter underutilized genetic diversity. QA biosynthesis occurs in the vegetative upper part of the plant, and they are subsequently transported to the seeds. Modern breeding strategies focus on creating elite cultivars, with low alkaloid content , as well as cultivars that retain QAs in vegetative tissues, thus producing hardy plants with “sweet” seeds . Their biosynthesis is regulated by five different loci, with pauper being thoroughly investigated thus far, on account of its contribution to QA synthesis regulation. Regarding the accessions under examination, the “sweet” pauper marker was detected in all cultivars, in breeding lines GRKML and GRKAL, and in GR26 landrace.

Absence of the “sweet” pauper marker from the vast majority of the landraces is in accordance with their high QA content. However, presence of the marker in high QA genotypes, like the cultivar “Multitalia”, makes this marker a weak predictor of low QA content with broad applicability. In Mediterranean farming systems, white lupin is considered a winter crop and sowing takes place in autumn, in order for the crop to take advantage of the late winter rainfalls. Thus, sufficient seed filling is succeeded before the early dry spells that occur in May, which have been more frequent and severe in Mediterranean ecosystems, due to the climate change . Early flowering is considered to be an effective stress escape mechanism, in that it promotes fulfillment of the plants biological cycle, prior to terminal drought stress . Additionally, genotypes non-responsive to vernalization, suggest crops suitable for spring sown cultivation, in northern regions, with long lasting winter. While white lupin germplasm native to the Balkan peninsula has been previously characterized as vernalization responsive , among the local landraces under investigation, GR3, GR7, GR12, GR16, GR19, GR21, and GR26 hold both SEPALLATA 3 and FLOWERING LOCUS T early flowering alleles, implying a vernalization independence-promoting regional microclimate at the collection sites . Moreover, the early flowering allele of SEPALLATA 3 was detected in all commercial varieties, except for Ulysse.

The inability of FTa1 primer set to hybridize in three landraces from Andros island and in cultivars Frieda and Celina,suggest contingent mutations located at the primer binding site, in the genomes of those accessions . The GIGANTEA early flowering promoting allele was detected only in GR5, GR10, and Frieda cultivar. However, further experimentation on the aforementioned landraces is needed, in order to examine the predictive power of those molecular markers, and their potential implementation in white lupin breeding programs. Alleles that confer resistance to anthracnose, were previously found only in Ethiopian landraces, which are distinctly related to European improved germplasm. Resistanceconferring alleles, located in antr04_1/antr05_1 and antr04_2/antr05_2 loci, were also detected in the Greek landraces examined, with GR23 and GR25 from Andros, having both of them in their genome . Summarizing these results, molecular markers linked to important agronomic traits have been identified in white lupin germplasm from the Greek rural areas.

Studies revealed that the application of Mo in beans, via seeds, increases the mass of the nodule ; moreover, its foliar spray increases nodule formation, nitrogen contents, the grain number, the grain mass, and overall productivity . The enhanced benefits may be attributed to the acceleration of N absorption and assimilation processes via BNF . Mo is a crucial element for more than 40 enzymes, four of which have been found in plants, including nitrate reductase and nitrogenase synthesis . Thus, plants receiving increased Mo will have increased N productivity levels based on the higher activities of the abovementioned enzymes. Mo bound to a unique pterin compound, named Mo cofactor, binds to diverse apoproteins and aids in anchoring the Mo center at the correct position within the holo-enzyme for its correct interaction with other components of the electron transport chain . Thus, Mo supply can strengthen plant metabolism at different growth stages through an improved enzymatic and non-enzymatic antioxidant defense system and also enhance other pharmacological properties.

Moreover, Mo is also an essential mineral for the human body as it is part of important metabolic enzymes such as xanthine oxidase sulfite oxidase, aldehyde oxidase, and mitochondrial amidoxime reducing component.With this background, we hypothesized that Mo application can enhance the nutritional and pharmacological value of Canavalia species by improving their N content and resulting in primary and secondary metabolite production. Thus, the present study aimed to evaluate the impact of Mo seed priming on three Canavalia species/cultivar sprouts. To the best of our knowledge, no study has been conducted to assess the influence of Mo seed priming on Canavalia ensiformis var. gladiata, Canavalia ensiformis var. truncata Ricker, and Canavalia gladiata var. alba Hisauc. Herein, we evaluated the impacts of Mo treatment on growth, N content, and phenolic metabolism as well as on the concentrations of several phytochemical compounds. We further examined the role of Mo in the enhancement of pharmacological properties of Canavalia species. Overall, our study contributed to an understanding of the biochemical basis of Mo that induces high growth and tissue quality of different Canavalia species/cultivars.

The variations in the concentration of nutrients and phytochemicals were quantified in both Mo-exposed and unexposed seeds of Canavalia. The quantity of total lipids was significantly increased in CA1, a non-significant change was observed in CA2, while a decrease was recorded in CA3. Furthermore, a significant increase in fiber content was recorded in CA1, a decrease was found in CA3, while there was no change in the fiber content of CA2. Upward trends in total proteins, flavonoids, saponins, and glycosides were noticed .The results showed a slight increase in N production in the sproutings of three species, as shown in Figure 2A. Moreover, the quantification of nitrogen reductase activity showed its increase in CA3 and decrease in CA1 and CA2 in Mo-treated plants, as shown in Figure 2B. Next, we studied the effect of Mo on two pathways of glutamate synthesis, i.e., glutamate-dehydrogenase and glutamine synthetase /glutamate synthase pathways. The activities of GDH, GOGAT, and GS were measured . The results showed an increase in GDH and GOGAT activity in the three species. The rise in GDH activity was significant in CA3, while it was non-significant in other species. Inversely, GS activity was decreased after Mo treatment and the differences were highly significant in the three species/cultivars. Further, the impact of Mo treatmenton dihydrodipicolinate synthase and cystathionine γ- synthase activity was explored.

The experiment showed that the activity of DHDPS significantly increased in CA3 and non-significantly in CA1 and CA2 . Moreover, a notable increase in the activity of CGS was measured in sprouts of CA3 . Furthermore, amino acids, i.e., asparagine, glutamine, glycine, glutamic acid, isoleucine, arginine, tyrosine, lysine, serine, alanine, proline, histidine, leucine, isoleucine, valine, cystine, threonine, tryptophan, and methionine, were quantified in Mo-treated Canavalia species and data were compared to untreated controls. The changes in each amino acid content due to Mo exposure are shown in Table 2. Overall, an increase in amino acid production was detected in the three species. The quantity of cystine, isoleucine, leucine, glycine, alanine, and proline was increased significantly in CA1; asparagine, glutamine, glutamic acid, alanine, proline, cystine, and lysine were enhanced in CA2; and asparagine, glutamine, glycine, histidine, methionine, cystine, isoleucine, tyrosine, lysine, threonine, and tryptophan were enhanced in CA3. Variable trends were noticed among different sprouts.