Several companies in the United States and the EU have developed recombinant ACE2 and ACE2-Fc fusion proteins for preclinical and clinical testing, although all these products are currently produced in mammalian cell lines . The impact of plant-specific complex glycans on the ability of ACE2-Fc to bind the RBD has been studied using molecular dynamic simulations and illustrates the important role that glycosylation may play in the interaction between the S protein and ACE2 .Griffithsin is a lectin that binds high-mannose glycans, and is currently undergoing clinical development as an antiviral against HIV-1. However, it also binds many other viruses that are pathogenic in humans, including HSV , HCV , Nipah virus , Ebola virus, and coronaviruses including SARS-CoV and MERS , and as recently determined, also SARSCoV-2. A clinical product in development by University of Louisville is currently manufactured in N. benthamiana by Kentucky Bio-processing using a TMV vector. The API is also undergoing preclinical development as a nasal spray for use as a non-vaccine prophylactic against coronaviruses, with clinical evaluation planned for 2020 . This candidate PMP antiviral could be deployed under the EUA pathway if found effective in controlled clinical studies. Griffithsin is an interesting example of a product that is ideally matched to plant-based manufacturing because it is naturally produced by a marine alga. Griffithsin has been expressed with limited success in E. coli and tobacco chloroplasts, ebb flow table but better results have been achieved by transient expression in N. benthamiana using A. tumefaciens infiltration or TMV vectors, with expression levels of up to 1 g kg−1 fresh mass and recoveries of up to 90% .

A TEA model of griffithsin manufactured in plants at initial commercial launch volumes for use in HIV microbicides revealed that process was readily scalable and could provide the needed market volumes of the lectin within an acceptable range of costs, even for cost-constrained markets . The manufacturing process was also assessed for environmental, health, and safety impact and found to have a highly favorable environmental output index with negligible risks to health and safety.In addition to COVID-19 PCR tests, which detect the presence of SARS-CoV-2 RNA, there is a critical need for protein-based diagnostic reagents that test for the presence of viral proteins and thus report a current infection, as well as serological testing for SARS-CoV-2 antibodies that would indicate prior exposure, recovery, and possibly protection from subsequent infection. The most common formats for these tests are the ELISA and lateral flow assay. The design and quality of the binding reagents , along with other test conditions such as sample quality, play a key role in establishing the test specificity and selectivity, which determine the proportion of false positive and false negative results. Although the recombinant protein mass needed for diagnostic testing is relatively small , the number of tests needed for the global population is massive, given that many individuals will need multiple and/or frequent tests. For example, 8 billion tests would require a total of ~2.5 kg purified recombinant protein, which is not an insurmountable target. However, although the production of soluble trimeric full-length S protein by transient transfection in HEK293 cells has been improved by process optimization, current titers are only ~5 mg L−1 after 92 h . Given a theoretical recovery of 50% during purification, a fermentation volume of 1,000 m3 would be required to meet the demand for 2.5 kg of this product.

Furthermore, to our knowledge, the transient transfection of mammalian cells has only been scaled up to ~0.1 m3 . The transient expression of such protein-based diagnostic reagents in plants could increase productivity while offering lower costs and more flexibility to meet fluctuating demands or the need for variant products. Furthermore, diagnostic reagents can include purification tags with no safety restrictions, and quality criteria are less stringent compared to an injectable vaccine or therapeutic. Several companies have risen to the challenge of producing such reagents in plants, including Diamante , Leaf Expression Systems , and a collaborative venture between PlantForm, Cape Bio Pharms, Inno-3B, and Microbix.Resilience is the state of preparedness of a system, defining its ability to withstand unexpected, disastrous events , and to preserve critical functionality while responding quickly so that normal functionality can be restored . The concept was popularized by the 2011 Fukushima nuclear accident but received little attention in the pharmaceutical sector until COVID-19. Of the 277 publications retrieved from the National Library of Medicine22 on July 9th 2020 using the search terms “resilience” and “pandemic,” 82 were evenly distributed between 2002 and 2019 and 195 were published between January and July 2020. Resilience can be analyzed by defining up to five stages of a resilient system under stress, namely prevent, prepare, protect, respond, and recover . Here, prevent includes all measures to avoid the problem all together. In the context of COVID-19, this may have involved the banning of bush meat from markets in densely populated areas . The prepare stage summarizes activities that build capacities to protect a system and pre-empt a disruptive event. In a pandemic scenario, this can include stockpiling personal protective equipment but also ensuring the availability of rapid-response bio-pharmaceutical manufacturing capacity. The protect and respond stages involve measures that limit the loss of system functionality and minimize the time until it starts to recover, respectively. In terms of a disease outbreak, the former can consist of quarantining infected persons, especially in the healthcare sector, to avoid super-spreaders and maintain healthcare system operability . The response measures may include passive strategies such as the adjustment of legislation, including social distancing and public testing regimes, or active steps such as the development of vaccines and therapeutics . Finally, the recover phase is characterized by regained functionality, for example by reducing the protect and response measures that limit system functionality, such as production lockdown. Ultimately, this can result in an increased overall system functionality at the end of a resilience cycle and before the start of the next “iteration” .

For example, a system such as society can be better prepared for a pandemic situation due to increased pharmaceutical production capacity or platforms like plants. From our perspective, the production of recombinant proteins in plants could support the engineering of increased resilience primarily during the prepare and respond stages and, to a lesser extent, during the prevent and recover stages . During the prepare stage, it is important to build sufficient global production capacity for recombinant proteins to mount a rapid and scalable response to a pandemic. These capacities can then be used during the response stage to produce appropriate quantities of recombinant protein for diagnostic , prophylactic , or therapeutic purposes as discussed above. The speed of the plant system will reduce the time taken to launch the response and recovery stages,hydroponic grow table and the higher the production capacity, the more system functionality can be maintained. The same capacities can also be used for the large-scale production of vaccines in transgenic plants if the corresponding pathogen has conserved antigens. This would support the prevent stage by ensuring a large portion of the global population can be supplied with safe and low-cost vaccines, for example, to avoid recurrent outbreaks of the disease. Similarly, existing agricultural capacities may be re-directed to pharmaceutical production as recently discussed . There will be indirect benefits during the recover phase because the speed of plant-based production systems will allow the earlier implementation of measures that bring system functionality back to normal, or at least to a “new or next normal.” Therefore, we conclude that plant-based production systems can contribute substantially to the resilience of public healthcare systems in the context of an emergency pandemic.The cost of pharmaceuticals is increasing in the United States at the global rate of inflation, and a large part of the world’s population cannot afford the cost of medicines produced in developed nations23 . Technical advances that reduce the costs of production and help to ensure that medicines remain accessible, especially to developing nations, are, therefore, welcome. Healthcare in the developing world is tied directly to social and political will, or the extent of government engagement in the execution of healthcare agendas and policies . Specifically, community-based bodies are the primary enforcers of government programs and policies to improve the health of the local population . Planning for the expansion of a bio-pharmaceutical manufacturing program to ensure that sufficient product will be available to satisfy the projected market demand should ideally begin during the early stages of product development. Efficient planning facilitates reductions in the cost and time of the overall development process to shorten the time to market, enabling faster recouping of the R&D investment and subsequent profitability. In addition to the cost of the API, the final product form , the length and complexity of the clinical program for any given indication , and the course of therapy have a major impact on cost. The cost of a pharmaceutical product, therefore, depends on multiple economic factors that ultimately shape how a product’s sales price is determined . Product-dependent costs and pricing are common to all products regardless of platform. Plant-based systems offer several options in terms of equipment and the scheduling of upstream production and DSP, including their integration and synchronization .The efficiency of this translation has a substantial impact on costs, particularly if processes are frozen during early clinical development and must be changed at a subsequent stage. Process-dependent costs begin with production of the API.

The manufacturing costs for PMPs are determined by upstream production and downstream recovery and purification costs. The cost of bio-pharmaceutical manufacturing depends mostly on protein accumulation levels, the overall process yield, and the production scale. Techno-economic assessment models for the manufacture of bio-pharmaceuticals are rarely presented in detail, but analysis of the small number of available PMP studies has shown that the production of bio-pharmaceuticals in plants can be economically more attractive than in other platforms . A simplified TEA model was recently proposed for the manufacture of mAbs using different systems, and this can be applied to any production platform, at least in principle, by focusing on the universal factors that determine the cost and efficiency of bulk drug manufacturing .Minimal processing may be sufficient for oral vaccines and some environmental detection applications and can thus help to limit process development time and production costs . However, most APIs produced in plants are subject to the same stringent regulation as other biologics, even in an emergency pandemic scenario . It is, therefore, important to balance production costs with potential delays in approval that can result from the use of certain process steps or techniques. For example, flocculants can reduce consumables costs during clarification by 50% , but the flocculants that have been tested are not yet approved for use in pharmaceutical manufacturing. Similarly, elastin-like peptides and other fusion tags can reduce the number of unit operations in a purification process, streamlining development and production, but only a few are approved for clinical applications . At an early pandemic response stage, speed is likely to be more important than cost, and production will, therefore, rely on well characterized unit operations that avoid the need for process additives such as flocculants. Single-use equipment is also likely to be favored under these circumstances, because although more expensive than permanent stainless-steel equipment, it is also more flexible and there is no need for cleaning or cleaning validation between batches or campaigns, allowing rapid switching to new product variants if required. As the situation matures , a shift toward cost-saving operations and multi-use equipment would be more beneficial.An important question is whether current countermeasure production capacity is sufficient to meet the needs for COVID-19 therapeutics, vaccines, and diagnostics. For example, a recent report from the Duke Margolis Center for Health Policy24 estimated that ~22 million doses of therapeutic mAbs would be required to meet demand in the United States alone , assuming one dose per patient and using rates of infection estimated in June 2020. The current demand for non-COVID-19 mAbs in the United States is >50 million doses per year27, so COVID-19 has triggered a 44% increase in demand in terms of doses. Although the mAb doses required for pre-exposure and post-exposure COVID-19 treatment will not be known until the completion of clinical trials, it is likely to be 1–10 g per patient based on the dose ranges being tested and experience from other disease outbreaks such as Ebola .

Two clones remained negative after challenges and a third had a low percentage infection. The ultimate evaluation will be in the field where they are exposed to repeated natural infection by the insect vector. Trees of the three clones were planted in November 2007 amongst old trees with greening symptoms. This report is on their current status regarding greening infection as well as growth, production and fruit quality.The objective was to evaluate soil-applied SAR inducers or transgenic enhancement of SAR expression for effect on HLB disease progress in citrus trees challenged by graft-inoculation or psyllid-mediated infection. One yr-old Hamlin sweet orange trees were planted in May 2009 and treated as follows: 1) non-treated check , 2) foliar insecticide to control psyllids, 3) soil-applied imidacloprid/thiamethoxam to induce SAR, 4) soil-applied IMID/THIA plus foliar insecticides, 5) graft-inoculated UTC, 6) graft-inoculated with IMID/THIA. A randomized block design was used with 5 repetitions of 10 tree plots. In 2010, the SAR inducer acibenzolar-S-methyl which does not control psyllids was substituted in treatments 3, 4 and 6. At 24 months after treatments were established in a field site, 105 trees were PCR+ in the trial. Higher numbers of PCR+ trees occurred in the UTC, the UTC with graft inoculation, and the IMID/THIA/ASM with graft-inoculation. A lower number of PCR+ trees occurred in the treatments with SAR inducers, foliar insecticides, and foliar insecticide plus SAR inducers,vertical farming racks but no effect of SAR treatment on rate of HLB disease progress was detected. Transgenics of Duncan grapefruit and Hamlin trees were constructed that expressed the NPR1 gene from Arabidopsis , a key positive regulator of SAR.

Over-expression of AtNPR1 in transgenic lines of these susceptible hosts reduced citrus canker lesions. Resistance to Xanthomonas citri subsp. citri infection was related to expression levels of AtNPR1. Two lines each of ‘Duncan’ grapefruit and Hamlin with the highest expression of AtNPR1 were screened in a greenhouse containing HLB-infected plants and psyllids. Rate of infection progress over 12 months was similar for transgenic lines and non-transgenic controls. Again, an effect of SAR on HLB disease progress was not-detected which confirmed the ineffectiveness of enhanced SAR for HLB disease control. Trioza erytreae is in itself a minor pest of citrus, but its significance is attributed to its ability to vector the citrus African Greening disease pathogen, Candidatus Liberibacter africanus. The population fluctuation of the citrus triozid is correlated to the flushing rhythm of the citrus host, but seasonal fluctuation in infection of populations carrying the Greening pathogen have been difficult to monitor in the past due to limited detection methodologies. This study explored this fluctuation in infectivity in an orchard entirely infected with African greening by using PCR to test individual triozids caught on sticky traps placed weekly in the orchard. Two season’s data are presented from a small sour orange orchard in the Nelspruit district in South Africa. The triozid population fluctuations correlated to previous findings of population peaks following the citrus flush cycles and responses to specific climatic influences. Fluctuations in the percentage infectivity of the T. erytreae populations were observed, with infectivity peaking at or just after the citrus flush seasons, but with peaks in infectivity differing to population peaks.Over the last decade the plant disease Huanglongbing has emerged as a primary threat to citrus production worldwide. HLB is associated with infection by one of a group of phloem-limited bacteria that are readily transmitted by the Asian citrus psyllid, Diaphorina citri.

However, the temporal progression of infection, time to symptom expression , and how they relate to transmissibility remains unresolved. We graft inoculated sweet orange trees with C. Liberibacter asiaticus, then at different times after inoculation, we inspected plants for HLB symptoms, measured bacterial infection levels in plants, and measured acquisition by psyllid adults that were confined on the trees. Plant infection levels increased rapidly over time, saturating at uniformly high levels near 200 days after inoculation – the same time at which all infected trees first showed disease symptoms. Pathogen acquisition by vectors was positively associated with plant infection level and time since inoculation, with acquisition occurring as early as the first introduction 60 days after inoculation. These results suggest that there is ample potential for psyllids to acquire the pathogen from trees during the asymptomatic phase of infection. If so, this could limit the effectiveness of tree rouging as a disease management tool and would likely explain the rapid spread observed for this disease in the field. The diseases Huanglongbing and citrus tristeza are both phloem limited and have significant economic impact on citrus production wherever they are found. Studies of host resistance have indicated that Poncirus trifoliata has tolerance/resistance to both diseases, suggesting there may be some common factors in the two kinds of resistance. We have conducted studies of host gene expression changes that occur in response to infection to gain further insight. Controlled inoculation by grafting infected budwood was used to infect potted greenhouse plants of Cleopatra mandarin , US-897 , and US-942 with CTV and with Candidatus Liberibacter asiaticus, the pathogen associated with HLB. Stem and leaf tissues was collected at 10, 20, and 30 weeks after inoculation. DNA-free RNA was subjected to real-time one-step SYBR Green RT-PCR analysis and relative gene expression was determined using the 2–ΔΔCt -method. Differences in gene expression between mockinoculated and Citrus tristeza virus -inoculated plants were generally not prominent. Differences in gene expression between mock-inoculated and Lasinoculated plants were most pronounced in susceptible Cleopatra plants and at the later stages of infection. Transcripts for a constitutive disease resistance protein were induced in response to Las in susceptible plants, but showed higher expression levels independent of infection in the tolerant genotypes.

A gene for a Myb-like transcriptional regulator family protein which is associated with resistance to some phytoplasma, responded strongly to Las, but also responded to CTV in tolerant plants. Transcript levels for other genes, such as a 2-oxoglutarate and Fe- dependant oxygenase and a plastidic glucose transporter , were considerably higher in US-897 and US-942 plants compared with Cleopatra plants independent of infection with either pathogen. It is hypothesized that these genes play a role in the resistance or tolerance of trifoliate orange and its hybrids to HLB and CTV.The Asian citrus psyllid is fairly well established in all counties in southern California and is spreading up the coast into Ventura and Santa Barbara Counties, but is not established in the major citrus production area of the Central Valley. The current status of Huanglongbing in California remains as only one backyard tree in Los Angeles County. This tree was identified as being infected with Candidatus Liberibacter asiaticus in March 2012 and removed. Despite continued surveys in that area,vertical rack system no additional trees have been found positive for HLBassociated pathogens.As a means of early diagnosis, adult and nymph ACP are collected and tested to determine whether HLB-associated bacteria are present in a localized area. The current approved diagnostic method for HLB in the United States is q-PCR; however, as demonstrated in Florida, this method may detect HLB-associated bacteria too late for the implementation of an effective eradication strategy. Hence, the Citrus Research Board has made the development of early HLB diagnostic methods their highest research priority. Methods being pursued involve the detection of molecules or compounds unique to Liberibacter infection within the host plant including volatile organic compounds , proteins secreted by the bacteria, small RNAs, and metabolomic compounds. These methods are very promising and are being tested against the approved q-PCR assay in time-course experiments conducted within a containment facility on the University of California, Davis campus. In addition, the Jet Propulsion Laboratories/NASA will monitor the reflectance properties of leaves for changes during the course of an infection. If this method is successful, JPL will further evaluate whether a large scale survey can be conducted by air or satellite. These new methods are also being field tested in Los Angeles County in the neighborhood where the CLas-infected tree was found in 2012 and in Texas in the vicinity of previous detections of HLB-positive trees. Candidatus Liberibacter is a genus of plant entophyte gram-negative bacteria in the Rhizobiaceae family vectored by psyllids. Most of the members of the genus are associated with damaging plant diseases and are subject to phytosanitary regulation. Ca. L. asiaticus, Ca. L. africanus and Ca. L. americanus are associated with Huanglongbing, one of the most destructive diseases of citrus. The disease is present in different countries of Asia, America and Africa but the three pathogens and their vectors Diaphorina citri and Trioza erytreae have a more complex distribution. The disease continues to spread rapidly and is a threat to the Mediterranean Basin countries, where the disease has not been reported. Early detection of the citrus Liberibacter in host plants and vector insects is essential to prevent its introduction and spread and for HLB control and management in countries where the disease is present. Diagnostic methods have to cope with the uneven distribution of the bacterium in plant tissues associated with irregular vector transmission, low bacterial titer during early stages of infection in host plants and vector insects and require differentiation of the individual species. By using the InCheck Platform , which combines multiplex PCR and microarray hybridisation on a single chip, we are developing a diagnostic assay to identify and discriminate among the species of Liberibacter in plant DNA extracts. The design of the probes on the microarray module for the identification and discrimination was based on the sequences of the following species: Ca. L. asiaticus, Ca. L. africanus, Ca. L. americanus, Ca. L. solanacearum, Ca. L. europeus, Ca. L. crescens and outgroup species within the Rhizobiaceae family.

The theory of natural spread of psorosis by insects in Argentina was stated by Rodríguez Pujol and Beñatena, researchers from the Concordia Experiment Station of the National Institute of Agricultural Technology , in 1965. In 1966, Rodríguez Pujol mentioned natural spread of psorosis in seedlings in the citrus region along the Uruguay River in Argentina. Timmer and Beñatena reported 0.1 – 1% of the root stock seedlings in the nursery were rogued because of psorosis. Timmer and Garnsey observed in Texas natural spread of Citrus ringspot virus in grapefruit at a rate less than 2%. Beñatena and Portillo showed psorosis transmission from diseased plants to healthy citrus seedlings by Toxoptera citricidus and T. aurantii winged individuals, by wingless adults of Toxoptera spp., by colonies of Toxoptera spp. and Aphis citricola, with a long incubation period and erratic symptom appearance. Since then, there have been advances in the study of the causal agent, diagnostic methods, and possible transgenic lines tolerant and /or resistant to CPsV but little has been mentioned about the natural spread of this virus. This work confirms the aerial spread by insects of CPsV in the field, both in a plot of certified citrus plants free of CPsV and in trials for testing aerial and/or soil spread of the virus in the Uruguay River citrus region in Argentina.Citrus psorosis virus is the causal agent of Psorosis, an important disease causing significant economic losses in Argentina and Uruguay. CPsV is the type member of the genus Ophiovirus, family Ophioviridae. Its genome has three singlestranded RNAs of negative polarity, encapsidated with a coat protein . As there is no natural resistance to Psorosis, transgenic Pineapple sweet orange plants were obtained expressing an intron-RNA hairpin from the cp gene . This RNA transcript induces the post-transcriptional gene silencing mechanism, degrading all RNA with cp gene sequences, thus preventing the progress of the infection. When ihpCP citrus trees were CPsV-challenged by graft-transmission, at least two SwO lines did not show any symptoms and CPsV was not detected by molecular and serological analysis. The hallmark of the PTGS is the presence of small RNAs , which were detected in the transgenic tissues indicating that resistance is PTGS-mediated. Our interest is to evaluate whether ihpCP is able to transfer this feature of resistance to susceptible grafted-tissues. For that, non-transgenic buds were grafted on the two resistant ihpCP lines used as rootstocks, which had previously been inoculated with CPsV. In another assay, a susceptible CP-line, expressing the complete CP-mRNA, was grafted on the same two ihpCP resistant rootstocks. Both, the non-transgenic and transgenic CP were susceptible. New flushes showed characteristic symptoms and TAS-ELISA and qRT-PCR analysis were positive.

My research tests for the possibility that foreign investors are drawn to so-called “pollution havens”—regions in which environmental regulations are either less severe or not well enforced. This research focuses on the manufacturing sectors in four countries: Mexico, Venezuela, Ivory Coast and Morocco. In the late 1980s, the share of foreign assets in manufacturing varied from 38 percent in Ivory Coast to seven percent in Venezuela. Foreign investment accounted for about 15 percent of total assets in Moroccan manufacturing, and ten percent in Mexico. The analysis of pollution havens and foreign investors is divided into three parts. First, we examine whether foreign investors in these four developing countries are attracted toward “dirtier” sectors, defined as product groups where either pollution emissions are high or pollution abatement costs are high . Second, we then compare the energy efficiency of domestic enterprises and multinationals in these same countries. This allows us to see whether foreign investors played an important role in improving the environment by using more energy-efficient technology as well as cleaner sources of energy. Finally, we examine whether the pattern of outward U.S. foreign investment is skewed toward sectors with high pollution abatement costs. We first analyzed whether the pattern of foreign investment in the four developing countries provides any evidence of pollution havens. Is there more foreign investment in sectors with high pollution abatement costs or high emissions, after controlling for other factors that affect the pattern of foreign investment? This means that we need to control for such factors as the degree of competition,dutch bucket hydroponic the size of the domestic market, domestic wages and other factors that make the market attractive.

Once we control for these factors, we then analyze whether there is more foreign investment in sectors where pollution abatement costs in the United States are high, using annual, sector-level data drawn from U.S. surveys on pollution abatement costs among manufacturing plants. The results suggest that pollution abatement costs in the U.S. have no impact on the pattern of foreign investment in these four countries. In other words, there is no more investment in Mexico in petroleum refining or cement, sectors with high abatement costs, than there is in other sectors. As a follow-up, we used emissions data from the U.S. toxic release inventory to analyze whether or not there is more foreign investment in these four developing countries in areas of manufacturing where U.S. pollution emissions are high. As measures of emissions, we use total particulates , a measure of air pollution; biological oxygen demand , which is a broad measure of water pollution; and total toxic releases . Total particulates, which capture small and large dust particles, are closely related to phenomena such as the London smog and to air pollution in cities with emissions from fuel and diesel oil combustion, energy-intensive processes such as steel and cement, two-stroke engines, coal use, and burning of wood and residues. Analysis in the World Bank and elsewhere indicates that the release of particulates is the main air pollution problem in many third world cities . Biological Oxygen Demand indicates how discharges to water bodies deplete their oxygen levels, and is widely accepted as a broad measure of water pollution. Total toxic releases is an unweighted sum of releases of the 320 compounds in the U.S. EPA’s toxic chemical release inventory. All of these measures are by weight. Emissions are divided by the total output of the firm, measured in monetary terms, to arrive at sector-specific emission intensities for the three pollutants. Table 1 reports the correlations between these three measures of emission intensity and U.S. pollution abatement costs . Correlation coefficients are a measure of the statistical relationship between two variables. They range from –1.0 to 1.0 .

A correlation coefficient near zero implies little relationship between the two variables. Table 1 shows that, in a comparison among different manufacturing sub-sectors within the United States, there is no significant correlation between air pollution, water pollution and toxicity. Thus, although these three measures of pollution are very broadly defined, there is no general tendency that a sector which pollutes in one medium also pollutes in another medium. However, Table 1 does report a statistically significant correlation between abatement costs and toxic releases. Industries which on average have high abatement costs typically also emit toxic substances. If we use these alternative measures of pollution intensity in our analysis, we again find no relationship between high emissions in the U.S. and the pattern of foreign investment abroad. The second part of the study examines the behavior of foreign and domestic enterprises in these four host countries. We find that multinational firms are significantly more efficient in their use of energy than domestic plants. In addition, multinationals tend to use cleaner types of energy, such as electricity and natural gas. Even if we take into account the fact that multinational enterprises are typically younger than domestic firms, we still find that multinational firms of the same vintage are more energy efficient. To the extent that energy use is a good proxy for pollution emissions, this suggests that multinationals in developing countries tend to use cleaner technologies than domestic firms. To test whether energy use is a good proxy for emissions, we explore the relationship between energy use and toxicity across sectors, using U.S. data. We show that even in the U.S., where respectable air pollution control programs have been in place for more than 20 years, and the choice of fuels and electricity is highly varied, there is a strong statistical relationship between air pollution coefficients and energy use. As in Table 1, we use three measures of emissions: particulates, which measure air pollution; BOD, which measures water pollution; and toxics.

We report the rank correlations between those emissions and six different factor inputs in Table 2: the share of unskilled labor in total value of shipments, the skilled labor share, capital share, manufactured input shares, raw material input shares, and the share of energy inputs in total output. Energy use is highly correlated with different measures of emissions. The correlation between energy use and particulates is .58; between toxics and energy use the correlation varies between 0.52 and 0.55. The correlation with BOD is lower at 0.22,dutch buckets system though also significantly different from zero. Table 2 also shows that the correlation between pollution and energy use is much higher than for other factor inputs. Yet even if energy intensity could provide a good proxy for emissions across industries, energy intensity may not be a good proxy for differences in emissions between plants within the same industry. To investigate this issue, we used a cross section of U.S. manufacturing firms to examine the relationship between different types of factor inputs and plant-specific emissions, one industry at a time. The strength of the relationship between energy use and emissions varies with the type of industry. Specifically, particulates are highly correlated with energy use at the plant level for only four industries: chemicals, petroleum refining, lumber and wood products, and non-electrical machinery. Two of the most polluting activities in manufacturing—chemicals and petroleum refining—are included in these four sectors. Consequently, we repeated the analysis, restricting ourselves to only those four sectors where energy use serves as a reasonable proxy for emissions. We find that in those sectors, foreign firms are indeed more efficient in their use of energy.The third part of the study shifts the focus from developing countries to the United States. We examined the pattern of outbound U.S. foreign investment, asking whether, after controlling for other factors, there is any indication that most foreign investment originating in the United States and relocating abroad is located in pollution-intensive sectors. We find some evidence in support of this hypothesis, but the results are too weak to make strong claims.The Citrus Variety Improvement Program of INTA, in Argentina was established 29 years ago as an industry insurance policy. Since 1984, the Concordia Experiment Station has maintained citrus mother trees tested for freedom from disease and trueness to type. The objective is to secure propagating material of commercial varieties for industry needs. Varieties are selected from research projects and/or introductions and are subject to quarantine controls and a sanitation process. The selected trees are shoot-tip grafted and indexed for tristeza, psorosis, exocorThis, cachexia, citrus variegated chlorosis, canker and huanglongbing. Two hundred and eighteen scion and rootstock varieties are maintained and evaluated annually for their agronomic characteristics and sanitary status by visual observation and biological, immunochemical and/or molecular diagnostics. Data obtained on origin, botanical characteristics, agronomic performance, sanitary status and availability of propagation material is recorded. The information is available at INTA’s website . Basic material is exported to citrus producing countries and sent to germplasm banks on request. The isolation, annual evaluations, pest monitoring and disease diagnosis guarantee the quality of the material offered by the program to the Argentine citrus industry.Lemon cultivars recovered by shoot-tip grafting were compared with the original 30-40 year old nucellar clones located in a germplasm block in Tucuman, Argentina. Cultivars evaluated were Frost Eureka, Limoneira 8A Lisbon, Feminello Santa Theresa, and Genoa EEAT. These were grafted on Poncirus trifoliata Flying Dragon, except for Frost Eureka which was grafted on 79 AC Cleopatra mandarin x Swingle citrumelo [Citrus reshni xdue to its incompatibility with trifoliate rootstock. The selected clones were naturally infected with CTV, an endemic disease, and Frost Eureka was also infected with one viroid .

Trees propagated with STG budwood were indexed and confirmed to be free of CTV, psorosis, and citrus viroids before planting. A field trial was planted in November 2007, with four randomized blocks per treatment and four replicate trees per block. Tree height, canopy volume, trunk circumference, fruit yield and quality were evaluated. CTV infection was monitored annually in spring of 2009, 2010 and 2011, and thereafter in spring and fall of 2012 and 2013. Direct immuno printing ELISA with 3DF1+3CA5 monoclonal antibodies was used to detect CTV in field samples. In 2009, Limoneira 8A had the highest incidence of CTV infected plants followed by Genoa EEAT while Feminello Santa Theresa remained CTV free. In 2013, 100% of STG lemon lines were infected with CTV. Cumulative yield after four harvests of Limoneira 8A, Feminello Santa Theresa, and Genoa EEAT was no different from trees budded from their respective mother trees. In contrast, the STG line of Frost Eureka yielded 80% more fruit than the source tree with the viroid. In conclusion, CTV did not substantially affect the growth and yield of the lemon cultivars tested, while the viroid in Frost Eureka considerably reduced tree productivity.Shoot-Tip Grafting is an important part of a comprehensive certification program. Reduction in the time for certification to an average of two years is possible. Components of a highly successful STGing program include good scheduling, dedicated STGing personnel, a comprehensive testing program, a reliable source of seed, and a clean area for working including specialized tools and equipment. Proficiency in both the bud stick technique and the leaf-stripping technique will give flexibility in scheduling and allow faster completion times.Laboratory tests on young STGs should be used for a early indication of failure; follow up testing will determine if elimination of pathogens was successful. Precision trimming starts with the use of Gelrite for a solid base of STG set up, allowing the STGs to be evaluated in a horizontal position with a dissection microscope. Detailed notes are taken weekly and micro-trimming can prevent the selection of root sprouts by accident. Most STGs begin sprouting at two to three weeks. Small STGs grafted into root stocks will grow faster in the greenhouse and be ahead of STGs grown for a longer period in culture tubes in the laboratory. Increased sucrose can speed the development of some selections, but the increased number of root sprouts and associated trimming can be disadvantageous. The transfer to the greenhouse may be a problem area; it is best if personnel involved with STGing do the grafting and follow-up. Kinkoji is used for grafting of STGs as it is easy to work with year around and has increased our success rate.

Together they establish water use objectives and reporting standards for indoor and outdoor residential and commercial use; require SWRCB and CDWR to adopt long-term standards for efficient water use; update urban water management plans to include the reliability of the water supplies and strategies for meeting current and future water needs; require urban water suppliers to conduct a water supply and demand assessment and make water shortage contingency plans available to customers; and require water suppliers to declare emergency measures to ensure sufficient water for human consumption, sanitation and fire protection. AB 2371 was enacted on Sept. 28, 2018. It continues to enforce many current landscape water conservation practices in and out of drought, including hydrozoning, water budgeting, storm water collection, use of recycled water and irrigation equipment maintenance. In addition, it requires the Contractors State License Board to update the C-27 landscape contractors’ exam as needed to include questions on new and emerging landscape irrigation efficiency practices; allows potential purchasers of housing units containing in-ground landscape irrigation systems to require irrigation system inspections; and requires the formation of a working group to examine and consider updating current consumer information on landscape water use. It also requires CDWR, following a public hearing every 3 years,hydroponic nft channel to update MWELO or determine that an update is not needed and consider revising and updating the WUCOLS database.

UC continues to play a major role in providing objective information to policymakers as they formulate and update legislation on water conservation in commercial and residential landscapes. UC also continues to advance the science to conserve water and help ensure that legislative targets are met. Due to continued improvements in the efficiency of sprinkler and drip irrigation systems , ETAF was further reduced in 2015 from 0.7 to 0.55 for residential landscapes and from 0.7 to 0.45 for commercial landscapes . Conservation on this scale will rely heavily on implementing best practices that decrease water loss, identifying new species of drought-resistant landscape plants and improving irrigation system performance. In practice, many irrigation systems fall far short of the irrigation efficiencies used in the current MWELO. Bijoor et al. found that smart irrigation systems were more effective at reducing water loss than irrigation systems operated by conventional timers and that the difference exceeded water savings realized from selecting a warm-season turf species over a cool-season species. Reid and Oki continue to screen a wide variety of landscape plants for their drought resistance to expand the palette of California-friendly landscape plants. Work by their team has already led to the identification of hundreds of drought-resistant plants included in MWELO. UC ANR Specialist Amir Haghverdi is leading a project to further define water requirements of turf under deficit irrigation and reclaimed water regimes; evaluate the performance of soil moisture–sensing and ET-based smart landscape irrigation technologies on water use effectiveness under deficit irrigation; and monitor turf responses to multiple levels of water stress using multi-spectral and thermal remote-sensing techniques.

David Fujino and the Western Center for Agricultural Equipment established the Smart Landscape project at UC Davis in collaboration with more than 20 organizations and companies. Smart Landscape provides workshops and on-site training for landscapers and students on new water-saving technologies. Several UC faculty, specialists and advisors are involved in various projects throughout the state alone and in cooperation with the U.S. Forest Service and other groups to determine the long-term durability of a wide variety of underused landscape trees under warmer, drier conditions due to climate change and urban heat islands.Horticulture likely originated 20,000years ago. There are over 100 species of horticultural crops, consisting of diverse fruits, vegetables, and tubers, many of which are of high economic value with enormous production volume worldwide. The amounts of fruits, vegetables, and tubers produced in 2018 were 868, 1089, and 832 million tons respectively , and the increased demand from a growing, and afuent global population, is predicted to drive further expansion of horticultural output. Horticultural crops not only provide basic calories , but also, are among the most crucial sources of fiber, organic acids, micro- and macro minerals, vitamins, and antioxidants in human diets. Healthy attributes, and a wide range of tastes, textures, and favors make horticultural crops attractive. Starch is critical to human society given its versatile uses. Starch is the dominant energy source in the human diet, providing over 50% of our daily caloric needs. In the food industry, starch is widely used as a thickener, stabilizer, lipid replacer, defoaming agent, gelling agent, emulsifer, and dietary fiber, and in the pharmaceutical industry, starch is used as an excipient for drug delivery . In addition to these diverse uses, starch is an excellent renewable material for making ethanol biofuels and degradable ‘bioplastic’ products. Starch is almost ubiquitous in higher plants, including horticultural crops, in ways that may or may not be noticed.

For instance, potato, sweet potato, yam, and cassava are starchy, but spinach, lettuce, and ripe tomatoes, berries, and citrus are not, yet starch is likely to be important to the growth, development and fitness of all of these crops, as they are in better studied models. The widely accepted view is that starch accumulates either in a transitory state, or for long-term storage starch. Transitory starch follows a diurnal pattern: it is synthesized and accumulated directly from the products of photosynthesis in the leaf and in the stem during the daytime, and is then degraded into sugars as an energy source for the following night. In comparison, storage starch is defined as that located in perennating organs such as seeds, grain, embryos and tubers , where it provides sustenance for the next generation during germination and sprouting in sexual and asexual propagated crops, respectively. A third class of starch: ‘transitory-storage starch’ has been proposed. It describes starch that is accumulated and degraded during development in the storage organ. Transitory-storage starch is a feature of many species including horticultural crops of economic value such as tomato, banana, kiwi, strawberry, nectarine, and apple fruit . Starch accumulates as semi-crystalline, water insoluble granules that vary in diameter from 1 to 100μm depending on species. Starch is organized into two glucan polymers: amylose and amylopectin. Amylose and amylopectin consist primarily of linear chains of glucoses joined by α-1,4-glycosidic bonds. In amylopectin,vertical farming racks the α-1,4-glucan chains are branched more frequently through α-1,6-glycosidic bonds, compared to amylose. The branching of the amylopectin chains is such that chains of diferent lengths are produced: short, medium and long chains, and the frequency with which each fraction occurs influences starch functionality. Side chains of amylopectin form clusters around branching points, and two adjacent chains make up a double helix. These physical features of amylopectin polymers leads to a semi-crystalline granule; amylose with a randomly coiled conformation, fills the matrices within the granule. Amylopectin and amylose account for around 25 and 75% of the starch in major heterotrophic storage organs, respectively, while the starch in leaf tissues is approximately 5 – 10% amylose.Amylose and amylopectin are synthesized by the coordinate action of a group of four key enzymes. The core starch biosynthetic enzymes include ADP-glucose pyrophosphorylases , starch synthases , starch branching enzymes , and de-branching enzymes , of which there are many isoforms. In brief, AGPases initiate the frst step of starch biosynthesis by catalyzing the formation of ADP-glucose. SSs elongate the glucan chains in amylose and amylopectin; SBEs branch the glucan chains, while the DBEs shorten and modify the starch chains which enable a higher-order semicrystalline structure to form. SBEs, the focus of this review, hydrolyze α-1,4-linked glucan chains, and attach the newly-created ‘free’ chain to another glucan chain within the starch granule, via an α-1,6-linkage. Through this action, SBEs largely determine the proportion of the relatively unbranched amylose to the highly-branched amylopectin. Two major classes of SBEs are bio-functionally known: SBE1 and SBE2 , and they vary in terms of their substrate selectivity, whereas the function of SBE3 awaits verifcation across a broader set of species. SBE1 preferentially branches ‘amylose-like’ long glucan chains as the substrate, while SBE2 prefers a more branched substrate.SBEs are the key players in the regulation of the amylose-to-amylopectin proportion in plants. However, their functions in many harvested horticultural crops have been under-investigated, although evidence points to the importance of starch in determining the post harvest quality of these crops.

We aimed to develop a better understanding of the role of SBEs in fruits, tubers, and leafy greens in physiological processes by exploring SBE sequence relationships, expression, and starch phenotypes in diverse crops.SBEs have three classes of isozymes including two functional SBE classes and one putative class 3 SBE . SBE1 isoforms appeared earlier than SBE2 and SBE3 in the viridiplantae, but plant SBE1 and SBE2 are more homologous to each other, than to SBE3. SBEs have been identified and relatively well-characterized in cereal crops, tubers, and Arabidopsis thaliana over the last two decades ,but, as mentioned, little attention has been paid to the diverse group of species that are classified as horticultural crops. Within each class of SBE, the cereals grouped together, while most non-cereals formed another cluster . This pattern is due to the divergence of monocots from dicots around 200 million years ago. In contrast to the presence of ‘a’ and ‘b’ sub-isoforms of SBE2 in cereal crops, horticultural plant species generally have one SBE2 isoform. It was also observed that not all species have a known or predicted class 3 isoform. The SBE sequences contained within diverse organs, i.e., fruits, tubers, roots, and leafy vegetables , clusThered together based on their respective plant families. Te class 1 SBE is absent in Arabidopsis thaliana, and so it was not surprising that this SBE class is not present in the Brassicaceae. However, the class 1 SBE is also absent in apple , and European olive , but these species all have two class 2 SBE isoforms . In addition, banana contains at least four types of SBE2, and transcripts corresponding to these SBE2s have been identifed, indicating that they are expressed.Starch Branching Enzymes belong to the α-amylase family of enzymes, specifically the glycoside hydrolase family 13 super family, with multiple isoforms encoded by diferent genes . The overall structure of the SBE polypeptide is highly conserved: all SBEs possess a central α-amylase catalytic domain , and an NH2- terminus, and a carboxyl- terminus. The SBE NH2-terminus contains two conserved domains: a chloroplast transit peptide for plastid-targeting, and a CBM48 domain for binding to starch. The C-terminus contains the residues that determine substrate preference and catalytic activity. The central region of the enzyme contains the “A” catalytic domain, that is made up of 8–barrels. Notably, the class 3 SBE may not directly participate in starch biosynthesis in Arabidopsis, but it has a demonstrated function in mediating cesium toxicity of photosynthesis. However, the role of SBE3 is unlikely to be conserved. In potato, StSBE3 has a unique coiled-coil motif which is absent in the AtSBE3 polypeptide . Notably, the CBM48 domain is also deficient in AtSBE3 . It is possible that the StSBE3 may interact and complex with other starch biosynthetic enzymes through its coiled-coil domain, in a similar way to the SS4-PTST2 interaction in Arabidopsis, the GBSS-PTST1 interaction in rice or the SBE containing protein complexes in cereal endosperm , rendering an assistant function in starch biosynthesis. This species-specific mode of action of SBE3 may reveal a novel function of SBEs generally. Indeed, although all SBEs are predicted to form complexes with starch phosphorylases , the starch synthases and isoamylase , interactions with other proteins show differences depending on the species and SBE isoform.Four conserved regions critical for catalysis, named Regions 1-4 , are found within the catalytic A-domain . Regions 1-3 are directly involved in catalysis, while Region 4 is involved in direct substrate binding. SBE1 and 2 have largely invariant residues, but the residues in the SBE3 isoform of many species have substitutions at these sites. Post-transcriptional phosphorylation of the SBE-protein complexes formed with other starch biosynthetic enzymes has been found in cereal crops and in cassava, while experimental evidence of this regulation in the majority of horticultural crops is absent. SBE1 and SBE3 have fewer possible phosphorylation amino acid sites than SBE2 .

Human pathogen–plant models should be developed for the purpose of breeding efforts to enhance food safety based on enteric pathogen strain– plant commodity variety pairs identified from prominent or recurring foodborne illness outbreaks. At the same time, plant genetic resources that may facilitate genome-wide association studies should not be excluded. Furthermore, the use of human pathogens in routine assays requires highly trained personnel and laboratory/greenhouse bio-safety conditions according to NIH guidelines, in addition to considerable costs associated with the handling of microbial hazards in contained facilities. These approaches will require collaborative efforts among food safety experts, plant–microbe interaction biologists, microbiologists, and crop breeders for successful advancements in the field. Roots are not only vital for anchorage and for acquisition of water and nutrients from the soil, but are also engaged in complex physical and chemical interactions with the soil. Plant roots release approximately 11–40% of their photosynthetically fixed carbon, commonly known as root exudates, into the soil . Root exudates and mucilage act as nutrient sources and as signaling molecules for soil microorganisms, thus shaping the microbial community in the immediate vicinity of the root system . In turn, microbial processes promote plant growth by aiding in nutrient acquisition, plant growth hormone production and bio-control of plant pathogens . The physicochemical characteristics of the surrounding soil are also affected by interactions between roots and the microbial community. This interplay between the different rhizosphere components is affected by spatio-temporal processes,hydroponic grow system which culminates in dynamic feedback loops that maintain the complex rhizosphere environment with physical, chemical and biological gradients that are distinct from the bulk soil .

Understanding these intricate rhizosphere relationships is vital in devising strategies to increase plant productivity and comprehend localized bio-geochemical processes. In many rhizosphere studies, the use of pots and containers is predominant as it allows the plants to be cultivated under controlled conditions and at low cost. Compared to field studies, growth of plants in defined spaces also offers advantages in ease of handling, monitoring and sampling . Much of what we know of the rhizosphere microbiome has resulted from such pot-grown plants. However, since the rhizosphere and roots are still out of view in the soil, destructive sampling of the root is required prior to analysis. Destructive sampling may result in the loss of three-dimensional spatial information on rhizosphere processes over time, which is increasingly being recognized as a critical parameter. On the other hand, soil free techniques such as hydroponics and aeroponics can provide visual access to the rhizosphere circumventing the need for destructive sampling. Other alternatives are gel-based substrates which can maintain rhizosphere transparency as well as the 3D architecture of roots and have been applied successfully in high throughput imaging, phenotyping and trait mapping platforms . Nonetheless, the root phenotype and traits of plants grown under soil-free conditions are known to differ from those of soil-grown plants . These soil substitutes do not also accurately simulate the heterogeneous nature of soil aggregates, thus complicating extrapolations for field relevance. Sophisticated imaging approaches such as magnetic resonance imaging and X-ray computer tomography can be used to analyze root systems in the soil with minimal disturbance but they are low throughput, expensive and may not be easily accessible . It is apparent that structural changes in design caThered to solving specific challenges in the rhizosphere are indeed necessary.

To overcome these challenges relating to the rhizosphere in soil, specialized plant growth chamber systems have been designed, and successful implementation has led to multiple variations of similar designs. These specialized systems often have a visible rhizosphere which enables coupling with other technologies thereby increasing the breadth of experimental techniques applicable to the rhizosphere system. This review discusses representative growth chamber systems designed to study major rhizosphere processes and interactions in soil.Specifically, the reviewed growth systems are selected based on the following criteria: the growth chamber is amenable for use with soil/soil-like substrates and therefore, hydroponics, aeroponics and agar/gel-based systems are not discussed except in microfluidic-based platforms, it is built with the intention to maintain growth of the plant and has architectural features distinct from conventional pots, and lastly it is able to be set up in a laboratory; i.e., field measurement systems and observation platforms are not included. For instance, a minirhizotron, consisting of a camera mounted in a glass tube submerged in the soil which provides non-destructive root imaging over time will not be discussed as it is out of the scope of this review. Through our assessment of lab-based chamber systems, we identify unique advantages and challenges associated with each system . We hope that future fabrication designs can benefit and improve on designs that work well. Lastly, we offer our perspectives on areas in which technological advances are needed to fill current knowledge gaps. In studying rhizosphere processes, the myriad of complex interactions among members of the rhizosphere are often dissected to two interacting variables such as root-and-soil or root-and-microbes, etc. Each of these interactions inherently operates under distinct parameters and requires specifically designed platforms to effectively answer different research questions. This review is structured in a way that first describes each rhizosphere process briefly and then reports on the specific growth chamber systems designed to facilitate experiments for answering related research questions.

The major rhizosphere processes discussed below include root system architecture, physicochemical gradients in the soil, exudation patterns by the roots and interactions between roots and nematodes, fungi or bacteria.Root system architecture encompasses structural features that provide spatial configuration such as root length, width, spread and number and is an important rhizosphere parameter in regulating soil porosity, and nutrient and water uptake efficiency by plants . Plants have been observed to “sense” and direct root growth toward nutrient sources in soil, and the RSA of a plant exhibits great malleability in response to environmental stimuli which in turn, influences microbial communities . For instance, bean plants grew deeper roots under drought conditions to enhance water foraging capabilities while low phosphate conditions stimulated the formation of dense lateral roots involved in P uptake from upper soil layers . Given that most soils are heterogenous, understanding the RSA of plants becomes critical in improving resource use efficiency and agricultural yields . Often, RSA in pot-grown plants is investigated by excising the roots via mechanical means such as root washing or blowing with compressed air . These methods are, however,ebb and flow trays time-consuming, cause inevitable damage of fine root hairs and result in loss of spatial and temporal information . An appealing alternative for studying RSA is the use of rhizotrons. Rhizotrons were initially constructed as underground facilities designed for viewing and measuring roots in the field . In the lab, the rhizotron implies a chamber constructed using two vertical sheets with at least one or both of the sheets being transparent and/or removable . This allows repeated visual inspections of individual roots; a feature unachievable with destructive sampling. In some cases, the word “rhizobox” is used for a similar set up although this was first introduced in as compartmentalized systems to separate the root and soil compartments . Rhizotrons/rhizoboxes are often constructed with PVC or acrylic materials and come in many sizes to accommodate different plants with soil or soil-less substrates . Root growth and morphology in the rhizotron can be tracked by a variety of methods ranging from manual tracing onto a plastic sheet, using handheld or flat bed scanners to fully automated time-lapse imaging camera systems .Data can be subsequently analyzed with a wide range of software packages . Affordable and robust RSA imaging platforms using rhizotrons have also been developed for increased accessibility in low-income countries . The versatile construction of a rhizotron design for RSA studies has inspired many variations. For instance, ara-rhizotrons were designed to enable the study of 3D canopy competition with simultaneous root growth observation in an Arabidopsis plant population . The horizontal and radial design of HorhizotronTM and mini-Horhizotron consisting of transparent quadrants attached to a central chamber were developed to study lateral growth of roots in a semi-3D space and to perform post-transplant assessment . The separated quadrants can also be used with different soil substrates simultaneously to study substrate effects on root growth . A rhizotron fitted with water-tight gasket seals has also been used successfully to investigate the RSA of plants under water-logged conditions . Despite the continuous real-time visual read-out, most rhizotron designs suffer from inevitable loss of information from roots occluded by soil particles. The GLO-Roots system overcomes this by imaging from both sides of the rhizotron while using bio-luminescent roots to create higher contrast against the soil, enabling quantitative studies on RSA . Following advances in engineering and device fabrication, more rhizotron variants adapted to specific plant growth conditions can be envisioned.In a typical topsoil, approximately half is composed of solid minerals and organic matter while the rest is a fluctuating composition of water and gas filled spaces influenced by environmental conditions and uptake/release of solutes from plants .

Changes in gaseous and hydrologic parameters, such as ions, O2 and moisture among others, create a spatially complex environment that influences microbial communities and overall plant health. These physicochemical fluxes are heterogeneously distributed along roots and vary with root types and zones . Often, they exist as gradients in the rhizosphere , thus emphasizing the need for non-destructive sampling in order to accurately capture processes occurring at biologically relevant times and scales. Rhizotron chambers with a visually accessible rhizosphere allows in situ and continuous mapping of these gradients in the soil through the use of different types of imaging methods. For instance, photo luminescence-based optical sensors enable in situ, repeated detection of small molecule analytes in addition to pH , O2 and NH4 . Methods like zymography to detect enzyme activity and diffusive gradients in thin film can be used to map solute concentrations in the soil down to sub-mm scales with high spatial resolution more realistically than traditional destructive approaches. For example, transport and distribution of water in the rhizosphere soil has been imaged on both 2D and 3D planes by coupling a rhizotron with neutron radiography and tomography, respectively and showed varying moisture gradients along the root system with higher water uptake at the rhizosphere compared to bulk soil. On the other hand, if the rhizotron slabs are thin enough , even simple imaging solutions based on light transmission can be set up to capture water uptake by roots in sand . Despite trade-offs in method sensitivity between these two studies, a rhizotron set up is critical in both designs and illustrates its adaptability to multiple equipment.Roots exude a substantial amount of photo synthetically fixed organic carbon into the soil consisting of a wide variety of compounds such as sugars, organic acids, and primary and secondary metabolites . Together with mucilage and border cells , root exudates provide a major source of nutrients for the rhizosphere microbiome . Root exudation is regulated under genetic control as well as in response to environmental conditions in the soil such as nutrient limitations or increase in toxicity . Exudate patterns are also recognized as one of the strongest drivers shaping the rhizosphere microbiome . As a central player in the rhizosphere ecosystem, it is imperative to understand root exudation patterns to unravel subsequent impacts to the surrounding soil and microbial community. Improvements in analytical instrumentation have made it possible to move from targeted to untargeted explorations with mass spectrometry to create root exudate fingerprints in its entire complexity . Regardless, the impact of such techniques relies partly on our exudate sampling techniques. Detection of exudates in real-time is difficult due to rapid biotransformation and sorption to the soil matrix. As such, common collection methods rely on root washing in hydroponic systems to overcome complications in the soil matrix and preserve native exudation profiles. However, a comparison between a soil-based collection method and hydroponic methods showed varied responses particularly in amino acid exudation although the underlying cause was not elucidated . It is possible that the differing growth conditions between hydroponics and soil, which include differences in gas concentrations, mechanical impedance and microbial spatial composition, can elicit differing root exudation responses to the same environmental stimuli.

Historically, from 1950-2002, alfalfa and cotton have been among California’s top commodities in terms of total value . In 1950 cotton was ranked third in terms of value of production in California with a value of $202 million. By 2001, cotton had slipped to the eighth most valuable commodity in California in value of production. The trend has been downward during the period 1950-2002. Hay was ranked fifth in 1950 in California with a value of production of $121 million. In 2001, hay was ranked seventh in value of production just ahead of cotton. Models are developed for California alfalfa and cotton acreage, production, and consumption. Both single equation and systems of equations are estimated. The data consist of 33 annual observations from 1970 to 2002. In some models, there were slightly fewer observations due to lags in the specifications. A brief description of the alfalfa market is given prior to reporting the estimations of the models. In addition, some issues related to the nature of the data are discussed. Alfalfa hay acreage in California has averaged about a million acres per year during the past 30 years . Alfalfa contributes about 85 percent of the value of all hay production in California. Alfalfa is influenced by profitability of alternative annual crops such as cotton, tomatoes, trees, and vines. The demand for alfalfa hay is determined to a large degree by the size of the state’s dairy herd, which consumes about 70 percent of the supply. Horses consume about 20 percent. Alfalfa is a perennial crop with a three to five-year economic life. Since it is a water intensive crop,grow table its profitability is strongly influenced by water and water costs. In addition, alfalfa is important in crop rotations because of its beneficial effects on the soil .

Alfalfa production in California has been increasing annually since the mid nineties . It reached a peak in 2002 at 8.1 million tons. The increase in production has been primarily due to the upward trend in yields and not to increases in acreage. Alfalfa real grower price in California, using a 1983/84 base, has exhibited a downward trend since the early eighties . In 2002 the real grower price was about $60 per ton. A three-equation system for alfalfa was developed and estimated. Iterative three stage least squares are used to estimate a model consisting of acreage, production, and demand relationships for alfalfa. We assume that the market for alfalfa is in equilibrium, that is, that quantity demanded is equal to production. We further assume that stocks are included in the demand for alfalfa. Thus, the three endogenous variables are: acreage, production, and alfalfa price. The estimators will be asymptotically efficient given that the model is specified correctly. The gain in efficiency is due to taking into account the correlation across equations. And three-stage least squares will purge the correlation that exist between endogenous variables on the right hand side of the equations in the model with the error terms. Cotton is the most important field crop gown in California. Growers in California grow two types of cotton: Upland, or Acala and Pima. Upland cotton makes up about 70 to 75 percent of the California cotton market and is the higher-quality cotton. Upland has a worldwide reputation as the premium medium staple cotton, with consistently high fiber strength useful in many apparel fabric applications. Export markets are important, attracting as much as 80 percent of California’s annual cotton production in some years making it California’s second highest export crop . Historically, California cotton, in terms of value of production, was the third highest ranking crop in California in 1950 below cattle and calves and dairy products.

In 2001 cotton was ranked the eighth highest valued crop below milk and cream, grapes, nursery products, cattle and calves, lettuce, oranges, and hay . There has been a downward trend in cotton acreage and production in California since 1979. California growers produced 3.4 million bales of cotton on l.6 million acres in 1979. In 2002 they produced about 2 million bales of cotton on 700,000 acres . Cotton yields have experienced an upward trend since 1979 . Nominal producers’ prices in California for cotton exhibit an upward trend since the 1970s, but real producers’ prices in California has exhibit a downward trend since the mid seventies . Recently the World Trade Organization ruled against U.S. cotton subsidies. U.S. cotton subsidies totaled about $10 billion in 2002 and the WTO ruled that the subsidies created an unfair competition for Brazil, which filed the complaint. California producers received about $1.2 billion in subsidies in 2002. California cotton is not as subsidized as cotton in other states, such as Texas, because subsidies are based on price and California’s higher-quality cotton is more expensive . Acreage, production, and demand equations are estimated for California cotton. Single equation and system of equations models are developed and estimated. In this report we aggregated the different cotton varieties. Disaggregated models of cotton were also estimated because of changes in the cotton industry and to allow for different impacts for subsidized and unsubsidized varieties. The number of observations in the disaggregated models present in the next section are limited due to the relatively recent introduction of Pima in California. The estimated models indicate that the short-run own-price elasticity of alfalfa acreage is inelastic but more elastic when ample water is available. By applying water marginally through out the growing period, a producer can obtain more cuttings of alfalfa. Alfalfa yields are also responsive to increases in prices. The own price elasticity of yields is 0.08 and highly significant.

Alfalfa yields are negatively related to the previous year’s cotton price. Production is positively related to own price with an estimated elasticity of 0.44 and significant. Production was negatively related to risk with an elasticity of risk equal to -0.75. Demand for alfalfa is a derived demand and is positively related to the number of cows and milk price support and negatively related to its own price. The estimated own-price elasticity of cotton acreage is 0.53 and highly significant. Cotton acreage decreases with an increase in risk in growing cotton and as price of alfalfa increases. The short-run own-price elasticity of cotton production is 0.497 and the long-run estimate is 0.503. The own-price elasticity of cotton demand is – 0.684. Rayon is a substitute for cotton. The empirical results support the fact that alfalfa and cotton are rotating crops in California. In recent years there has been an increase in Pima acreage relative to the traditional Upland variety in California. Upland cotton prices have a positive impact on acres planted to Upland. When Pima prices increase, the acres planted to Upland decrease. A similar situation applies to Pima acreage. That is, an increase in Upland prices causes a decrease in Pima acreage. Thus, the empirical results support that hypothesis that relative prices of Upland and Pima have a significant impact on the adoption of the two varieties. Future research needs to focus on the collection of more data related to the consumption of California cotton and alfalfa, stocks and inventories, and interstate trade of alfalfa between California and Oregon and Nevada. California is one of the major producers of rice in the US. The other most important states are Arkansas, Louisiana, Mississippi, Missouri and Texas. The market in California appears to be fully integrated with the southern states, as suggested by an empirical check of the law of one price. This conclusion is hardly surprising,growing lettuce hydroponically given that rice is a storable and easily transportable commodity. Figure 1 illustrates the law of one price between California and Arkansas. A simple ordinary least squares regression of California rice price on Arkansas price gives an R2 of 0.939 and an estimated slope coefficient between 0.80 and 0.94 . Moreover, a simple cointegration test suggests the absence of unit roots in the disturbances. Thus, California price and Arkansas rice prices move together over the long run. Market integration suggests that a US level model can be useful to describe California rice production. In this study, however, we present both national and state models. Prior to reporting the estimated production function for rice, a brief discussion of some aberrations of the rice market will be explained. Around 1976-77 there was a price collapse that caused producers to rotate to other crops or not plant rice at all. This lead to decreases in rice production. In the early eighties rice prices collapsed again and this caused many growers to forfeit their crop to the government because the price was below the value of the government loan. This was not only the case with rice, but other program crops such as wheat and corn. In an attempt to reduce acreage and sell off the rice that the government had claimed, the government implemented the 50/92 plan. Subsides were directly linked to production. Thus, if a grower did not produce he was not paid. The 50/92 program allowed the grower to produce on 50% of his acreage and receive 92% of the subsidies that he would receive if he had produced on 100% of his land. This reduced production allowed the government to reduce the stocks of commodities that they had to claim in 1981-82. The 50/92 program ran until about 1988. Since then subsidies have been decoupled from production to prevent problems like this from happening again. The 50/92 program was popular in the south, especially in Texas where their production was lower and they had low fixed costs of land, but in California it was only widely used for a few years.

Policy variables are incorporated into some of the models below. The estimated US price expectation elasticity of supply is 0.45 which is also inelastic but is not significant. The estimated coefficient of Thailand price of rice is 0.27 with a t-ratio of about two. The index for price support is positive but not significant. There is also a positive and significant time trend in the supply of rice. According to the estimated price coefficient , the elasticity of demand of US rice implies that an increase of 1% in price results in a decrease of 0.36% change in the quantity demanded. As the price of Thailand rice increases, the demand for US rice increases, but again the estimated coefficient is not significant. The income elasticity is 0.33 and the estimated coefficient of Japanese income is 0.34 as expected. Both coefficients are not significant, however. Rice producers in California and throughout the United States respond positively to increases in rice prices. The short-run price elasticity of production, based on a partial adjustment model, for the US was estimated to be 0.23. When policy variables were included in the production equation the price elasticity dropped to 0.18 .The production equation was an aggregated one. For a disaggregated approach that estimates how rice producers respond to different support programs, see McDonald and Sumner. The estimated own-price elasticity of demand for rice was found to be inelastic for a SUR system. The income elasticity for rice was estimated to be 0.74 in a single-equation demand function . US rice producers export less when the US price increases . They export more when the Thailand rice price increases since Thailand is a major competitor in the world market. California is the second leading producer of fresh tomatoes in the US, after Florida. Figures 1-3 compares fresh tomatoes planted acreage, production and nominal price for US, Florida and California. California accounts for about 95 percent of the area harvested for processing tomatoes in the United States—up from 79 percent in 1980 and 87 percent in 1990. The other major producers are Texas, Utah, Illinois, Virginia, and Delaware and Florida. In Figure 1, total U.S. fresh tomato acreage has declined over the period 1960 to 2002, but acreage in California and Florida has remained steady. The declined in acreage has come from the states of Texas, Utah, Illinois, Virginia, and Delaware . Figures 4-6 illustrates the trends for California and US planted acreage, production and nominal prices for processed tomatoes.Tomato growing is based on grower-processor contract agreements. The majority of production is traded this way with the spot market playing a marginal role. Most initial processing is by firms that manufacture tomato paste, a raw ingredient.

The seasonal NO3–N concentrations in the domain varied from 0.01–7.03 mmol L 1 . Hutton et al. reported higher mobilization of nitrate at a shallower depth under drip irrigation of grapevine, and seasonal root zone nitrate concentrations ranging between 0 and 11.07 mmol L 1 in the Murrumbidgee Irrigation Areas in Australia. As the season continued and plant uptake was reduced, excess water further mobilised nitrate–nitrogen out of the root zone, as is evident from 27/04/07 and beyond . At the end of the crop season, little nitrogen remained in the soil system, and what did remain was well beyond the reach of the plants. This nitrogen is expected to continue leaching downwards over time and become a potential source of nitrate–nitrogen loading to the ground water. Additionally, peak NO3  –N concentrations in the soil profile L 1 and in drained water L 1 were significantly higher than the Australian environmental standard for protection of 80% NO3 –N L 1 and 95% of species NO3–N L 1. The NO3–N concentrations in the soil solution also occasionally exceeded the level of Australian drinking water quality standard for nitrate NO3–N L 1. High levels of nitrate–nitrogen below the crop root zone are undesirable, as some recharge to groundwater aquifers can occur, in addition to flow into downstream rivers, which are used for drinking water and irrigation. These findings are consistent with other studies , in which high nitrate concentrations in drainage water under drip and furrow fertigated irrigation systems have been reported.Estimated water balance components above and below the soil surface under a mandarin tree are presented in Table 4. It can be seen that in a highly precise drip irrigation system, a large amount of applied water drained out of the root zone, even though the amount of irrigation applied was based on estimated ETC. This drainage corresponded to 33.5% of applied water,hydroponics growing system and occurred because highly permeable light textured soils, such as those found in this study, are prone to deep drainage whenever the water application exceeds ETC.

The drainage amount in our study falls within the range of recharge fluxes to groundwater reported by Kurtzman et al. under citrus orchards in a semiarid Mediterranean climate. Mandarin root water uptake amounted to 307.3 mm, which constitutes about 49% of applied water. Root water uptake slightly increased when the model was run without considering solute stress , which is not a significant difference. It further substantiates the results obtained for seasonal ECsw in Fig. 6, where salinity remained below threshold over the season. Evaporation accounted for 17.7% of the total water applied through irrigation and rainfall. The modelling study overestimated the sink components of the water balance by 4.79 mm . There were major differences between water input and output from January 2007 onwards . During this period, irrigation and precipitation significantly exceeded tree water uptake , which eventually resulted in deep drainage from March 2007 onwards. Therefore, current irrigation scheduling requires adjustment during this period. This illustrates how simulations were helpful in evaluating the overall water dynamics in soil under the mandarin tree. The nitrogen balance is presented in Table 5. The nitrogen fertilizer was applied either in the form of NH4 + or NO3, but NH4 + transforms quickly to NO3  through the process of nitrification. Model simulations showed that nitrification of NH4 + was very rapid and most of the NH4 + –N converted to NO3  before it moved to a depth of 20 cm, and no traces of NH4 + were observed below this depth. It is apparent that the nitrification of NH4 + took place in the upper soil layer, which contains organic matter and moisture that supports microorganisms , facilitating the nitrification of NH4 + . Though NH4 + was initially nitrified to NO2 and consequently to NO3, NO2 was short-lived in the soil and decayed to NO3 quickly. Therefore, the simulated plant NH4 + –N uptake was only 0.71 kg ha 1 . Hence, the NO3–N form was responsible for most of the plant uptake, corresponding to about 85% of the applied nitrogen.

The monthly N applications were slightly higher than plant uptake during the flowering and fruit growth periods . However, the monthly uptake was slightly higher than the N application between these periods. High frequency of N applications in small doses resulted in similar nitrogen uptake efficiency in citrus as in other studies . Similarly, Scholberg et al. reported doubling of nitrogen use efficiency as a result of frequent application of N in a dilute solution. Slightly higher uptake was recorded when fertigation was applied in second last hour of an irrigation event , as compared to when it was applied early in the irrigation event . Hence, it can be concluded that timing of fertigation does not have a major impact in a normal fertigation schedule with small and frequent N doses within an irrigation event in light textured soils. Similar results were also obtained in our earlier study in a lysimeter planted with an orange tree , which revealed that timing of fertilizer N applications in small doses in an irrigation event with a low emitter rate had little impact on the nitrogen uptake efficiency. Nitrate–nitrogen leaching accounted for only 15% of the applied nitrogen . Monthly N balance revealed that most of the N leaching happened between March 2007 and August 2007, which was correlated with the extent of deep drainage occurring during this period. NO3 –N losses ranging from 2% to 15% were illustrated by Paramasivam et al. and Alva et al. , attributable in part to an improved management of N, which could be a contributor in the current estimation.In our study, it is evident that there were significant deep drainage and nitrate–nitrogen leaching losses , which could be reduced by appropriate management. Hence, different simulations involving the reduction of irrigation and fertigation applications during the whole or part of the crop season were conducted, to optimize water and nitrogen uptake and to reduce their losses from the soil .

Increasing the irrigation frequency with short irrigation events while maintaining the same irrigation volume, had no impact on deep drainage and N leaching . However, the seasonal salinity increased by 11% compared to the standard practice. This confirms that the current irrigation schedule followed with respect to the irrigation frequency seems to be optimal under the experimental conditions. In S2, Dr_W and Dr_N were reduced by 14.4% and 19%, respectively, but salinity increased by 11%. However, a sustained reduction in irrigation by 20% eventually reduced the Dr_W and Dr_N by 28.1 and 38.3%, respectively, at the expense of a 4.9% decline in plant water uptake, but with a 4% increase in N uptake. However, salinity increased by 25.8% compared to the normal practice, which would likely have a significant impact on plant growth. Scenarios S4 and S5 were based on decreasing the nitrogen application by 10% and 20%, resulting in a decrease in N leaching by 7.4% and 14.8%, respectively, along with a much higher reduction in plant N uptake , suggesting that the reduction in the fertilizer application alone is not a viable option to control N leaching under standard conditions. A combined reduction in irrigation and fertigation by 10% further reduced N leaching by 5.5%,flood table compared to reducing irrigation alone , but at the same time plant N uptake was reduced by 5% more than in S2. Similarly, reducing irrigation and N application by 20% produced a pronounced reduction in N leaching and water drainage , but it also resulted in a decrease in plant N uptake by 15.8% and water uptake by 4.8%, compared to normal practice. At the same time, salinity increased by 25.8%, which is similar to S3. The reduction in plant water and N uptake would have a major impact on plant growth and yield, and would adversely impact the sustainability of this expensive irrigation system. Hence, reducing fertilizer applications does not seem to be a good proposition under the current experimental conditions, as it results in an appreciable decline in plant N uptake. However, Kurtzman et al. reported that a 25% reduction in the application of N fertilizer is a suitable agro-hydrological strategy to lower the nitrate flux to groundwater by 50% under different environmental conditions. Rather, reducing irrigation alone seems to be a better option to control the deep drainage and N leaching losses under the conditions encounThered at the experimental site. Additionally, it is worth noting that in S3 and S7 the salinity during a period between October and December at a depth of 25 cm, and during December at a depth of 50 cm, increased considerably, and was higher than the threshold level , confirming that a sustained reduction in irrigation and fertigation is not a viable agro-hydrological option for controlling water and N leaching under the mandarin orchard. However, it seems unnecessary to reduce irrigation applications uniformly across the season as suggested by Lido9n et al. . Rather, irrigation could more profitably be reduced only during a particular time period when excess water was applied. The water and N balance data in our study revealed that an imbalance between water applications and uptake happened during the second half of the crop season, i.e., from January till August 2007, resulting in maximum drainage and N leaching , coinciding with the fruit maturation and harvesting stage. Hence, there is a need to reschedule irrigation within this period, rather than reducing water applications throughout the entire season.

Keeping this in mind, the following 5 scenarios were executed, in which irrigation was reduced during the second half of the crop season, i.e., between January and August, by 10%, 20%, 30%, 40%, and 50%, respectively. Scenarios S10, S11, and S12 showed an enormous potential for reducing water and N losses. In S10, Dr_W and Dr_N were reduced by 8% and 4% more than in S7, N uptake was increased by 6.9% , and salinity was also 4% less than in S7, which seems quite promising. On the other hand, in S11 and S12, the Dr_W and Dr_N were reduced to a greater extent than in S10, and soil salinity increased substantially , due to a considerable reduction in the leaching fraction. This is also shown in Fig. 12, which shows that monthly soil solution salinity in S11 and S12 at the 25 and 50 cm soil depths increased dramatically between January and August. Although ECsw remained below the threshold level, except at a 50 cm depth in S12 during March 2007, there is a significant likelihood of it increasing further in subsequent seasons, which would ultimately impact the growth and yield of mandarin trees. Hence, under current conditions, Scenario S10 represents the best option to control excessive water and N losses, and high salinity, and to increase the water and N efficiency for mandarin trees. Other permutations and combinations, involving fertilizer reductions along with S10, did not provide further improvements in controlling water and N leaching. It is concluded that simulations of irrigation and fertilizer applications, using HYDRUS, can be helpful in identifying strategies to improve the water and N efficiency for drip irrigation systems of perennial horticultural crops.Ex situ seed banks play an important role in accessing and propagating seeds for restoration of degraded and fragmented native habitats and for the conservation of threatened species. Accordingly, many botanic gardens and conservation agencies worldwide have established seed banks to address ongoing concerns about habitat destruction and in particular the loss of biodiversity. As well as providing seeds for use in conservation and restoration programs, seed banks provide unique opportunities for studies of the biology, ecology, and evolution of native species as well as opportunities for the commercialization of species with horticultural and agricultural potential. Given the value of these collections and in many cases the irreplaceable nature of the accessions held, it is imperative that seed banks are managed in ways that minimize age-related seed losses. Consequently, the maintenance of seed viability during long-term storage is a major focus of seed banks. However, the longevity of seed viability varies greatly among plant species and is significantly impacted by seed storage conditions. Therefore, the success of long-term ex situ seed conservation is dependent on regular monitoring using standardized and reproducible approaches.

DNA sequencing was performed using ABI BigDye Terminator according to the manufacturer’s protocol, except that 5-μl reactions were performed with 0.25 μl of BigDye on an ABI 3730xl DNA sequencing machine with 50 cm arrays. DNA sequences were analyzed with CodonCode Aligner v. 2.0.6 . We detected three types of polymorphism in our sequences – single feature polymorphism , insertions and deletions and variable number tandem repeats . Most of the SFPs that had been detected using the Affymetrix GeneChip were due to a single nucleotide polymorphism , but in five cases due to a single base indel. Since Haploview cannot handle missing values, missing bases were substituted prior to data analysis with an appropriate single nucleotide. Because all single-base indels could be tagged with SNPs from the same marker locus , we use the term SNP throughout the text. Both indels and VNTRs were excluded from data analysis, unless otherwise noted in the text.One hundred and ninety two RILs derived from a cross between an F1 of cv. Valmaine × cv. Salinas 88 and cv. Salinas were genotyped with EST-derived markers. Selection of markers for this first round of genotyping was based on the molecular linkage map developed from an interspecific cross between L. sativa cv. Salinas and Lactuca serriola accession UC96US23. Twenty markers were selected to evenly cover linkage group 2 in intervals of approximately 10 to 20 cM. After preliminary mapping of the resistance gene, the region containing Tvr1 was saturated with markers originating from a microarray-based study also carried out on the Salinas × UC96US23 population.

Marker polymorphism was tested with HRM analysis,25 liter pot plastic unless the difference between segregating alleles could be visually observed using gel electrophoresis. If polymorphism could not be observed with HRM analysis, PCR products from the two parental genotypes were sequenced and new primers were designed for HRM. Statistical analysis of the linkage between molecular markers and die back resistance was performed by MapManager QTX software. Dieback resistance for each RIL was considered as a bi-allelic qualitative trait and used for linkage analysis.Association mapping was performed on a set of 68 accessions from seven horticultural types of lettuce . In the first step, markers closely linked to the Tvr1 gene were amplified from each accession and sequenced. In the second step, the sequenced amplicons were analyzed for polymorphism with the CodonCode software and inputted into Haploview v. 4.2. Intra-locus SNPs were tagged in Haploview with the Tagger function at r2 = 1. Untagged SNPs from all markers and a representative SNP for each tag were then entered into TASSEL v. 2.0.1. TASSEL was subsequently used to test for association between individual SNPs and resistance to die back while accounting for the population structure. Both p-values for each SNP and percent of phenotypic variation explained by the model were calculated with TASSEL after 100,000 permutations. Prior to association analysis, the population structure in the set of 68 accessions was assessed with thirty EST-SSR markers distributed throughout the genome using the computer program STRUCTURE 2.2. Ten runs of STRUCTURE were done by setting the number of populations from 1 to 15. For each run, the number of iterations and burn-in period iterations were both set to 200,000. The ad hoc statistic was used to estimate the number of sub-populations. The optimum number of sub-populations was subsequently used to calculate the fraction of each individual’s genome that originates from each of the five sub-populations.

The qk values obtained from STRUCTURE were used as covariates in the statistical model given by TASSEL.The level of genetic variation at the nucleotide level was estimated as nucleotide polymorphism and nucleotide diversity . To test the neutrality of mutations, we employed Tajima’s D test, which is based on differences between π and θ. Analyses of genetic variation and estimates of haplotype diversity were carried out using DnaSP v. 5.00.04 software. Linkage disequilibrium between pairs of SNP loci in the genome was calculated with Haploview and the values were pooled over the entire data set. Decay of LD with distance was estimated using a logarithmic trend line that was fitted to the data. Distances between markers were calculated from their respective positions on the consensus molecular linkage map. The consensus map was created with JoinMap v. 2.0 from the Salinas × UC96US23 map and the × Salinas map . SNPs with frequency < 5% were excluded from the analysis.The resistance-SNP association observed in the set of 68 accessions was detected through sequencing of PCRamplicons from individual accessions. In order to accelerate and simplify the test of association, we developed a primer pair that allowed detection of polymorphism in the marker Cntg10192 through high-resolution melting analysis. These primers amplify a 185 bp product that contains all three SNPs detected in the marker Cntg10192 at the positions 54, 72, and 100. The first two SNPs match perfectly with the resistance allele, while the third SNP explains 40.9% of the trait variation. As with the first two SNPs, the third SNP has a C ⇔ T substitution. All susceptible genotypes carry the T allele, while resistant genotypes have either the T or C alleles at the third SNP. It appears that the T allele in the resistant material is associated with the resistance present in cv. Salinas and most of the other iceberg cultivars, whereas the C allele is associated with the resistance present in the three lines that originate from the romaine-like primitive accession PI491224.

Marker Cntg10192, therefore, not only allows for the detection of alleles associated with die back resistance, but also separates alleles of different origins. To further investigate polymorphism in this genomic region we sequenced two accessions from L. serriola, a wild species closely related to cultivated lettuce. One of the accessions is resistant to the disease, while the other one is susceptible.The resistant accession has a haplotype similar to cv. Salinas but instead of the T allele at position 54, it carries the C allele. The three SNPs at the marker Cntg10192 can thus distinguish four different haplotypes; three resistant and one associated with susceptibility . Haplotype R1 has the T-T-T allele combination at positions 54, 72, and 100. Haplotype R2 carries the T-T-C combination, while haplotype R3 carries the C-T-T alleles. Disease susceptibility was always associated with the S1 haplotype that carries the C-C-T combination. All four haplotypes can easily be separated through high-resolution melting analysis .Nucleotide polymorphism was observed in all nine markers that were sequenced from the region flanking the Tvr1gene. The rate of nucleotide substitutions in a set of 68 accessions translates into ~1 SNP per 149 bp between pairs of randomly selected sequences. This SNP frequency was somewhat lower when only coding regions were considered . These values are well within the range observed for other plant species. For example,25 litre plant pot the average SNP frequency is 60 bp in aspen, 87 bp in potato, 104 bp in maize, 130 bp in sugar beet, 232 bp in rice, 435 bp in sorghum, 585 bp in tomato, and 1030 bp in soybean. Both nucleotide polymorphism and nucleotide diversity of lettuce are similar to that observed in maize , potato , and sugar beet , but larger than in tomato , and soybean. If results from the analyzed region correspond to those for the whole genome, sequence variation in lettuce is relatively high for a selfing species. It was previously observed that selfing species have generally lower levels of sequence variation than outcrossing species because of smaller effective population sizes. Although polymorphism in lettuce appears to be considerably larger than in selfing soybean and tomato, it is similar to that observed in rice, which is also a self-pollinating species. The ratio of nucleotide diversity in coding and non-coding sequences was not analyzed in detail, since data from only four markers are available. However, the ratio haplotype frequencies, and calculated using the across these markers appears to be smaller than in DNAsp software.

This measure of diversity is analogous to Arabidopsis , soybean the heterozygosity at a single locus, and is at its maximum , maize , and potato . when haplotypes observed in the sample occur at equal This difference is likely due to a higher level of functional frequencies. Diversity based on haplotypes ranged constraint on the perigenic sequence of lettuce. Meas- from 0.593 in QGG19E03 to 0.809 in marker Cntg11275, ures of haplotype diversity were based on estimated with an average value of 0.732 ± 0.024. These values are higher than in rice , soybean , and human . It is possible that the high level of diversity is related to the way that selection of the 68 accessions was performed. We included die back resistant and susceptible material from all predominant horticultural types, thereby selecting haplotypes at similar frequencies. It would be interesting to observe how haplotype diversity changes in different genomic regions and/or for a different set of accessions. To test the neutrality of mutations, Tajima’s D was calculated for all surveyed markers. The average Dwas larger than in soybean , potato , and sorghum . A positive D value indicates a deficit of low-frequency alleles relative to what is expected. Since large D values can be caused by a population subdivision, it is possible that the presence of sub-populations in the analyzed set of lettuce accessions affects both haplotype diversity and the D values. When neutrality of mutations was tested in individual markers, three markers closely linked to the Tvr1 gene had Tajima’s D values significantly higher than expected . Again, the population structure or selection at the Tvr1 locus or the marker itself could have caused departures from neutrality.The decay of LD for the Trv1 region was relatively slow when measured both within individual markers and between markers flanking Tvr1. Estimated values of r2 were ~0.322 at 900 bp, and ~0.124 at 8 cM. A fitted logarithmic curve shows that the r2 value of 0.2 is reached somewhere between 0.5 cM to 1 cM. LD of SNP markers observed in some other selfing species was similar; LD in Arabidopsis was 250 kb or 1 cM and in soybean was ~50 kb. Intra-locus LD decayed very little in tomato, with the log trend showing r2 > 0.6 at 900 bp. However, it is problematic to compare decay of LD across species due to the large variability in LD quantification. LD depends on a combination of many factors, such as the origin of the population, selected set of accessions, analyzed genomic region, molecular marker system, and presence of unidentified sub-populations. Hyten compared four different soybean populations for levels of LD decline. While in the domesticated Asian G. max population LD did not decline along the 500 kb sequenced region, the wild Glycine soja population had a large LD decline within the LD block size averaging 12 kb. Comparable observations were not only made in the selfing Arabidopsis, but also in the out crossing maize and aspen. Our results show a large difference between estimates of LD when analyses were performed across all horticultural types or within each individual type. While the estimate of r2 at a distance of 8 cM was 0.124 for the whole set, it was 0.247 for romaine type and 0.345 for crisp head lettuce. Because only a relatively small part of the genome was analyzed in the present work, it is not possible to calculate LD at distances over 8 cM. However, the trend for the logarithmic curve suggests that LD could reach more than 15 cM in romaine and probably more than 25 cM in crisp head types before declining to the value of r2 < 0.2. When only iceberg types were included in the analysis, LD was still at its maximum at a distance of 8 cM . Although these observations come from a limited number of individuals, they are supported by the fact that the modern iceberg-type lettuce has an extremely limited genetic diversity that is frequently associated with extensive LD.We identified the genomic region carrying resistance against die back and nine markers closely linked with the Tvr1 gene through linkage analysis. We subsequently used this information to test the linked markers for association with the disease resistance on a set of 68 diverse accessions.

Both of these examples used sense transgenes, therefore this type of silencing effect commonly is referred to as co-suppression. Several other groups working with similar systems have reported analogous results . In contrast, antisense silencing was shown in tobacco to be not graft-transmissible regardless of whether the signal originated in the scion or root stock . In tomato grafting experiments with the ACC oxidase gene, antisense silencing of scion ACC oxidase was not seen early after graft establishment, however after several weeks a graft-transmissable silencing was observed . This time lag may account for why the earlier experiments concluded that there was no silencing in grafted antisense lines. A high level of expression of the target gene in the scion was necessary for the detection of silencing by Northern hybridization, as a result of expression of the antisense construct in the root stock, a situation similar to the nitrate reductase experiments discussed earlier . Thus, experimental time lines, the levels of target gene expression, and the model organisms used may be important determinants of the efficacy of antisense silencing in grafted systems. It has also been shown that even when target gene are not present in the recipient graft, transgenic siRNAs can accumulate from donor grafts . Arabidopsis containing a GFP inverted-repeat silencing construct as the donor was grafted withWT or GFP-expressing scions as recipients. The sRNAs identified in scion tissues included siRNAs generated as a result of the GFP construct and a substantial population of endogenous sRNAs from the root stock donor as well. Size classes ranging from 21 to 25 nt were most abundant, and the 24-nt class directed epigenetic modification of the GFP signal in the scion. The massively parallel deep sequencing methods used by this group showed that if a silencing target was not present in the recipient ,plastic planters bulk then siRNAs generated from hairpin-GFP in the root stock were still present in the scion, albeit at levels several of orders of magnitude lower.

This could be why previous experiments using less sensitive detection techniques, such as Northern blots, did not detect mobility of the signal. A recent report has shown that beyond the 24-nt siRNAs mentioned above, all size classes of siRNAs can trigger homologous sequence-specific methylation of targets at long-distances, at least in Arabidopsis . What facilitates the movement of sRNAs? sRNAs and associated RNPs are small enough to be translocated based on their size, since experiments have shown that a 27-kDa GFP is able to diffuse into the vascular system . Results of experiments where movement proteins are included indicate that spreading of the silencing signal is at least partially dependent on the size of the plasmodesmatal apertures . Alternatively, movement of the silencing signal might be selective, perhaps requiring protein– protein, or protein–nucleic acid interactions in order to obviate the apparent plasmodesmatal aperture size exclusion limit. This view is supported by experiments involving mutants deffective or deficient in the ability to move signals . Regardless of uncertainties related to the mechanism of sRNA movement, the evidence demonstrates that movement does indeed occur through the phloem component of the vascular system and is mediated by plasmodesmata, at least to some degree. Many experiments have been performed regarding the mobility of RNAs, both large and small, but whether the same pathways that are used for the movement of mRNA are used for miRNA or siRNA movement has not been determined. The emerging idea that sRNAs are involved in physiology, defense, and development, both cell autonomously and for long-distance signaling, is becoming more widely accepted . Given the variability in mobility detected across several studies, it seems that plasmodesmata-based transport of sRNAs is a regulated process. However, the molecular mechanisms that mediate sRNA mobility and whether they are cis or trans-acting are unknown.

Researchers have successfully employed strategies that utilize the expression of siRNAs in order to protect the plant root zone from pests and pathogens . For example, in soybean, resistance strategies that target soybean cyst nematode genes, including those associated with stimulating root growth in infected plants, sperm production, and female development have been tested . By grafting these plants to WT scions, systemic protection may be achieved in a manner similar to the virus resistance reported in tobacco and more recently in cassava in experiments demonstrating control of the devastating Cassava brown streak Uganda virus . Aside from pathogen resistance, down–regulation, and/or epigenetic modification of transcripts and genetic networks in the scion or the root stock also appear to be possible through the use of siRNAs and could influence scion-specific characteristics, such as flowering time, fruit production or quality, or root characteristics, such as tuberization in potatoes .In addition to RNAs, proteins may be transported over long distances in a regulated fashion. Certain motifs, reminiscent of nuclear localization signals, allow protein entry into CC and subsequently into the phloem for long-distance movement. Despite the evidence for selective and regulated processes for protein long-distance translocation, there is also evidence that shows nonspecific “leakage” of supposedly cell-autonomous proteins into sieve tubes and subsequently into sink tissues. Xylem vessels,which mainly transport water and low molecular weight inorganic and organic solutes, have been shown to contain proteins, although at lower concentrations than in phloem sap . Proteins targeted to the apoplast may inadvertently enter xylem or phloem vasculature and subsequently be transported to and unloaded in sink tissues. Examples of movement of proteins include exogenous viral movement proteins, endogenous transcription factors and xylem/phloem proteins . Some of the first studies of xylem protein transport involved viral movement proteins , but as knowledge has progressed, more researchers have been able to demonstrate mobility of endogenous plant proteins. For many years, proteins had been observed in the phloem, but the idea of a coordinated, selective, and regulated process of trafficking, influencing not only development, but plant responses to environmental cues is a more recent idea that has gained support .

Mobile proteins or non-cell-autonomous proteins may be encoded by as many as 20% of the genes in Arabidopsis . A comprehensive analysis of phloem sap proteins in pumpkin and cucumber using high resolution 2-D gel electrophoresis and partial sequencing by mass spectrometry identified several hundred proteins in the phloem, and the majority of these proteins may have roles in stress and defense reactions . Models of the mechanics underlying protein mobility in the vasculature include the structures associated with the vascular tissue. Within the phloem, SE, which lack a nucleus, ribosomes, and a vacuole, depend on neighboring CC for maintenance of their metabolic tasks . Because mature SE cells cannot synthesize proteins, the likely origins of proteins in the phloem are immature SE or CC. Structurally different from the plasmodesmata that connect mesophyll cells, specialized plasmodesmata between CC and SE are branched with all of the branches on the CC side funneling to a single opening on the SE membrane side. The requirements for specificity of transport between CC and SE are not completely known but accumulating evidence points to the importance of these branched plasmodesmata. Reviews from two research groups establish plasmodesmata as the “gatekeepers” of macromolecular transport into the SE . The specific mechanisms governing the regulation of plasmodesmatal apertures are still a mystery, but fluorescently labeled dextrans and GFP expression have been used to study plasmodesmatal size exclusion limits and their function under differing conditions. Through grafting,collection pot the vascular networks of both root stock and scion become connected and what is mobile in the root stock vascular networks is likely to become mobile in the vascular networks of the scion. In a thorough heterografting experiment involving 11 interspecific and intergeneric Cucurbit graft combinations, several structural P-proteins appeared in the recipient phloem exudate, as shown by SDS-PAGE and Coomassie staining. The results effectively demonstrated the direction of transmission was dependent on the combination of heterograft used, with some graft partners taking the role of donor or acceptor, and some able to perform both roles . This has clear implications for choosing of graft partners for GE-modifified root stocks. Fluorescence microscopy of graft junctions has shown sieve tube bridges connecting scion external bundle phloem to internal bundle root stock phloem when mobility was demonstrated. This observation identified physical continuity within the phloem as a prerequisite for mobility of proteins, but did not resolve the selective directionality observed .When two Cucurbit structural P-proteins, PP1 and PP2 were examined in intergeneric grafts, RT-PCR and Northern blots demonstrated that protein products rather than mRNA transcripts were translocated across the graft junctions. In addition to structural proteins, RNA-binding proteins appear to be abundant in the phloem translocation stream. Phloem sap collected and analyzed from four different sources all contained sRNAs of 18–25 nt sizes with various abundance profiles for each species. Fractionation of the phloem sap from pumpkin, cucumber, and lupine also identified a small∼27 kDa protein that bound strongly to 18–24 nt ssRNA. After cloning the pumpkin PSPR1 gene, microinjection studies demonstrated that PSPR1 specifically shuttled a high percentage of the ssRNAs across cell boundaries. In these studies, co-injection and subsequent movement of a 20-kDa fluorescent dextran showed that plasmodesmatal aperture was at least 20 kDa. Apparently, dilated plasmodesmata alone were not sufficient to allow the movement of ssRNAs between cells, since use of another protein shown to increase plasmodesmatal apertures was not sufficient to allow the movement of the ssRNAs .

Given that the ssRNAs were approximately 8 kDa, their lack of movement when KN1 was provided suggested a sequestration mechanism or a more complex ssRNA-binding protein interaction than is currently presumed. In an informative experiment, rice thioredoxin a major phloem sieve tube protein with basic antioxidant functions, was expressed in E. coli and fluorescently labeled with FITC . In tobacco, the labeled, heterologously expressed RPP13-1 protein was observed to migrate beyond the site of injection. However, the similarly purified and labeled E. coli homolog of RPP13-1 was not phloem-mobile under duplicate conditions, suggesting significant sequence or structure requirements for movement. Co-injection of rice RPP13-1 and FITC-labeled dextrans established that RPP13-1 increased the plasmodesmatal size exclusion limit to 9–20 kDa, from ∼1 kDa. Furthermore, two mutants of RPP13-1 that were deficient for mobility were identified and crystal structure prediction studies suggested that charged clusters of residues on the outer surface were responsible for binding and/or transport of RPP13-1 through the companion cell-plasmodesmata complex . Aoki et al. demonstrated the importance of protein structure for mobility using two heat shock proteins , CmHsc70-1 and CmHsc70-2, that had been isolated from pumpkin phloem sap. In microinjection experiments, CmHsc70-1 and CmHsc70-2, interacted with plasmodesmata, increasing the size exclusion limit and thereby, enhanced their own cell-to-cell transport. The C-terminal region of these HSPs potentiated their noncell-autonomous mobility through the plasmodesmata. A gainof-function experiment in which the C-terminal cucumber HSP motif was fused to a human Hsp70 protein established that the fusion protein, but not WT human Hsp70, could move from cellto-cell following micro-injection into pumpkin cotyledons, much like the movement of injected intact CmHsc70-1 and CmHsc70- 2. Interestingly, fusing the HSP C-terminal motif to GFP did notresult in cell-to-cell migration, suggesting that at least in this case, the targeting motif was only active in the context of highly conserved HSPs . Unlike nuclear localization signals or ER-targeting peptides, vascular system targeting peptides may have several different motifs, perhaps suggesting specialized interactions with different families of proteins, and/or selective import/export mechanisms. While targeting motifs appear to be important in regulating mobility, a non-regulated diffusion-based mechanism in the symplast from one cell to another is supported by the observation that protein size influences non-targeted movement of GFP but differences appear to be species- and developmental stage-dependent. Earlier studies indicated that non-regulated diffusion is limited to ∼50 kDa proteins in mature leaves and 60 kDa proteins in developing leaves . Unregulated diffusion-based movement across the sieve tube element–companion cell complex has been observed when CC specific promoters regulate 27 kDa GFP expression.While it was perhaps not surprising to detect the GFP in the vascular system due to the porous end plates of the SE, unloading of the GFP into the mesophyll sink cells was unexpected. Using the same promoter, GFP-fusions as large as 67 kDa subsequently were shown to traffic from CC to SE in root tips, although larger variants were restricted to a zone of cells adjacent to the mature protophloem.

A promising approach that can be used to guide “breeding for manipulation” is the use of plant and robot geometric models to co-design tree structures and machines to optimize manipulation reachability and throughput . Also, the use of large numbers of simpler, cheaper actuators that approach plants from different positions has shown promise in terms of reachability , and could be adopted to increase overall throughput.Agricultural robotics enable sensing and interacting with crops at fine spatial scales, even at the level of individual plants or plant parts. Thus, they enable high-throughput phenotyping for breeding improved crop cultivars, and ultra-precise farming, which is a key technology for increasing crop production in a sustainable manner. They can also generate crop-related data that can be used to increase food safety and traceability, and to optimize crop management. Furthermore, agricultural robots can reduce our dependence on unskilled farm labor, which is diminishing in many countries. Also, the emerging paradigm of replacing large conventional agricultural machines with teams of smaller autonomous vehicles could open up possibilities for dramatically changing the way we cultivate crops. Small machines reduce drastically soil compaction and are not necessarily restricted to crop rows; hence, they could be used to establish alternative, productive crop patterns that incorporate mixed cropping, which is known to reduce pest pressures and increase biodiversity. To accomplish their tasks, agricultural robotics face significant challenges. Their mechanical embodiments, electronics, and their sensing, perception and control software must operate with accuracy, repeatability, reliability and robustness under wide variations in environmental conditions; diversity in cropping systems; variation in crop physical and chemical characteristics and responses to environment and management,blueberry containers due to intraspecies biological variation; diversity and complexity of plant canopy structures. Essentially, agricultural robotics must combine the advanced perception and manipulation capabilities of robotic systems, with the throughput, efficiency and reliability of hard automation systems, in a cost-effective manner.

Sleep timing and duration in humans are determined in part by a master circadian clock entrained to local time by retinal inputs encoding environmental light-dark cycles. The clock is phase delayed by light in the evening and early night, and advanced by light in the late night and early morning. With industrialization and on-demand access to electric lighting, exposure to evening light has increased, while exposure to natural light during the day has decreased. The expected net effect is a delay in the phase at which the circadian clock aligns with local time, and thus a delay in the timing of the circadian sleep-wake cycle. Depending on an individual’s social schedule , this may result in a significant misalignment between biological time and social time, a state known as social jetlag. If sleep onset is delayed, but wake onset is fixed by the social schedule, then nocturnal sleep will be restricted. Epidemiological studies have uncovered associations between short sleep and population health, while experimental studies support a causal role for sleep restriction in metabolic and other health disorders currently described as epidemic. This leads to conjecture that a significant portion of the population in industrialized societies may sleep less than is physiologically optimal and that this may contribute to negative trends in population health. Although the logic supporting this conjecture is compelling, empirical support for the argument that industrialization has caused chronic sleep restriction is weak because information about sleep duration prior to widespread availability of electric lighting is anecdotal and based on self-report. Self-reports typically overestimate sleep, compared to objective measures such as actigraphy and polysomnography. Consequently, the degree to which sleep duration has declined with industrialization may be overestimated and is controversial. Trends over the past several decades within already industrialized societies are equivocal, with some studies showing increases, others showing decreases, and some showing no systematic change in sleep length.Another way to estimate the impacts of industrialization on sleep is to study sleep in indigenous communities living traditional lifestyles without electric lighting. Recently, several studies have used wrist-worn accelerometers and light sensors to examine sleep patterns in communities with little reliable access to electric lighting.

The findings generated from these studies have been variable. Daily sleep duration was observed to be unexpectedly short in traditional hunting and gathering societies in Africa and South America, an agrarian society in Madagasgar, and a pastoralist society in Namibia. By contrast, sleep duration was comparatively long in a traditional horticultural society of Papa New Guinea, and in an Argentinian society who were traditionally hunter-gatherers and showed a marked seasonal variation. In cases where societies are transitioning to electricity, groups with access to on-demand artificial lighting showed a delay in nocturnal sleep timing compared to groups without access . In some, but not all cases, delayed sleep was associated with reduced sleep duration. These results indicate that lifestyle may be an important determinant of habitual sleep duration and provide evidence for an effect of on-demand electric lighting on sleep timing and duration. To further examine the impact of lifestyle and electric lighting on sleep, we used actigraphy to measure sleep timing and duration in indigenous Ni-Vanuatu living traditional, small-scale subsistence lifestyles on Tanna Island, Vanuatu, in south pacific Melanesia. This study population provides some unique advantages, including homogeneity of ethnicity and lifestyle on the island, little seasonal variation in climate and daylength, and the availability of an electric grid in coastal but not inland villages, permitting a within-society comparison of sleep with and without access to on-demand electric lighting. Also, the latitude of Tanna Island, and thus the annual variation in photoperiod, is very close to the latitude of several hunter-gatherer societies previously shown to exhibit short sleep, thus permitting cross-cultural comparisons with a natural control for daylength.We recruited 91 adults living on Tanna Island to participate in our study. Forty-five of the participants lived in coastal villages with on-demand access to electricity, and 46 participants lived in villages that were up to 10 km inland and beyond the electric grid. Some data were lost due to equipment failure or were excluded due to non-compliance , leaving final sample sizes of 39 coastal participants and 43 inland participants .

Participants in both communities live similar lifestyles and rely primarily on small-scale farming for a livelihood.Data were collected from males, females, and females who were currently breastfeeding. It was expected that breastfeeding would lead to higher levels of sleep disruption due to mother-infant co-arousal, therefore breastfeeding females were maintained as a separate sample. Other females were not breastfeeding at the time of data collection and the ages of their children were over 2 years. Because fathers on Tanna Island typically take a less active role than the mother in infant rearing, it was deemed acceptable to collect males as one homogenous group irrespective of “father” status. Age and birthdays are not commonly tracked, so when documentation of age was unavailable, participant ages were estimated visually or relative to the birth of peers. Of those included in the final analysis, demographic variables and other sample characteristics are provided in Table 1. We obtained research permits from the Vanuatu Cultural Centre as well as permission from the elders and chiefs in the host communities. Participants were recruited by word of mouth. We explained the details of the study and obtained informed consent verbally from each participant, as outlined by the Office of Research Ethics at Simon Fraser University, Burnaby BC, Canada. Gifts equivalent to $5 CAD were given for participation. All procedures were performed in accordance with relevant guidelines and regulations and approved by Simon Fraser University.Sources of light at night were most often a singular incandescent light bulb inside dwellings powered by electrical grid ,best indoor plant pots and/or small solar powered LED lights . Solar torches were placed on the floor to facilitate household duties or were carried by hand when walking through the village. Light intensity provided by the torches did not exceed 2 lux, measured at 1-meter distance using the Actiwatch-2 light sensor . During the dates of this study, all participants from communities with access to electricity reported using artificial lights. Although seven participants living in electric communities did not have working electricity at the time of data collection, they each used solar torches, and reported exposure to electric light. Most of the participants from villages without electricity either owned or shared solar torches for use at night, and daily evening use was reported by 84% of participants in non-electric communities. Electronic devices were almost non-existent with the exception of some basic mobile phones, which were not a common source of light at night, especially in villages of the electric grid as there was no ready access to charging stations.Sleeping arrangements were variable, but most sleeping spaces consisted of blankets or foam mattresses on grass woven mats on the floor of the dwelling . Floors were commonly wood plank, or hard packed ground. Co-sleeping is typical on Tanna Island; all participants in this study shared sleeping quarters with multiple children or adults. Sleeping spaces were shared by immediate family , and sometimes with extended family as well . Sleeping arrangements are often flexible and can change readily. Traditional homes are made of local natural materials, carefully constructed to withstand cyclones Separate dwellings are used for sleeping and cooking.

A few dwellings incorporated cement or tin components . Little time is spent inside during the day. Temperature and humidity in typical sleeping spaces were measured with iButtons sampling at 20-minute intervals throughout the study interval. Te iButtons were placed in representative huts in one electric village and one non-electric village. Temperature in the non-electric village during this period averaged ~24.6 °C in the day and ~22.9 °C at night, with an average daily range of 3.9±1.8°C. Electric villages were slightly warmer and drier, averaging ~25.9 °C in the day and ~23.9 °C at night, with a daily range of 3.6±1.8 °C. Te daily temperature minimum occurred at ~06:20h, and the maximum between 13:20–14:00h, in both communities.Although coffee beans are exported from Tanna Island, cafeinated beverages are not commonly used or readily available in the villages studied. All men drink a beverage of kava root, which, although only a few of its constituents have been studied, has been found to have mild sedative, anxiolytic, and antinociceptive/analgesic properties. Sixty-five percent of male participants reported drinking kava on a daily basis. Women are not traditionally permitted to drink kava for leisure nor are they permitted to participate in Kava ceremonies. Food consists primarily of locally cultivated foods, such as root vegetables , seasonal fruit , and on occasion purchased rice, and chicken, beef, fish or pork for ceremonies/celebrations. Cooking takes place over a fire, or with hot stones in an earth oven cooking pit. Breakfast and dinner times were regimented but flexible, and men who attended the nakamals would often eat late. Lunch times were not regimented . Although both community types practice primarily subsistence horticultural lifestyles, 23% of the population of each community type report spending some time participating in wage labour. Farming is the primary daily activity for 93% of the non-electric community members compared to 41% of the electric community members, who report spending more time on other daily activities within the village . Most women report their primary evening activity to be caring for children and attendance at church groups, whereas men report spending time in the nakamal for kava drinking . Bislama is the national language in Vanuatu, but many distinct indigenous oral languages exist on Tanna, and can vary even between nearby villages. Although some terms exist to refer to times of the solar day, residents of Tanna Island do not quantitatively track time within a day , and there are no designated work and free days. Alarm clocks were not used by the participants of this study. Chickens and small pigs wander freely through the villages and often alert residents to sunrise. Individuals do report taking days of from agrarian responsibilities, but working in the garden on these days is replaced with obligations for religious worship . Formal education is not common, but is increasing in prevalence, especially for villages in close proximity to a school, which is the case for electric villages. Morning social obligations included early awakening by some women to prepare children for school , or to travel to Wednesday market to sell produce to make money for school fees. We did not collect information on the prevalence of school attendance but as noted, electric villages where closer to schools or to roads where vehicles travel.