Month: December 2022

Several California marketing order and commission commodity promotion and research programs have recently been involved in litigation as a small minority of unhappy producers and handlers have turned to the courts with requests to modify or terminate the programs. Recent court cases involving constitutional challenges include actions against the marketing orders for peaches and nectarines, kiwifruit, plums, apples, grape root stocks, cut flowers, almonds, milk, cling peaches, and table grapes.Marketing order quantity controls can be a powerful economic tool when the commodity group controls most of the production of the commodity and when there are different markets with different price elasticities of demand. Under these conditions, the commodity group can gain a measure of monopoly power and enhance returns through price discrimination. However, since they are unable to control entry, any short-run price enhancement will lead to a longer-run supply response. It is not surprising that quantity controls have been controversial—monopoly pricing practices reduce the welfare of some consumers and may distort resource allocation decisions, while producers face all of the problems of maintaining a cartel. Marketing orders for several California commodities include quantity control provisions , although the use of quantity controls has decreased over time as a result of problems noted above. The federal marketing orders for citrus, with their prorate provisions, were terminated at the end of the 1993-94 crop year after more than 50 years of almost continuous use. The citrus prorates set the amount of lemons and oranges that could be shipped to the domestic fresh market on a weekly basis.The demand facing packers in the fresh market is very inelastic relative to the demands in the processing and export market. Thus,stacking pots price discrimination against the fresh market, by restricting the flow of product to it, is both possible and profitable. Short-run producer price enhancement without any controls on entry led to an acreage response for both lemons and Navel oranges.

As new plantings reached bearing age, the Administrative Committees were forced to divert increasing proportions of the annual crop to exports and processing to maintain fresh market prices. Producer returns from all markets decreased over time, until new plantings were no longer profitable. However, when compared to a competitive solution, prorate resulted in increased acreage and production of citrus, as well as increased exports and processed products . Opponents of the citrus volume regulations, who had been sued in 1983 by the United States for violations of prorate, discovered evidence of over shipments by a large number of competing orange and lemon packing houses.Because of allegations of limitations violations of shipments under the order, a series of lawsuits, investigations, and proposals for penalties under AMAA forfeiture rules threatened to keep the industry in court for years and create economic hardships for many industry participants. To minimize long-term damage to the industry and “to end the divisiveness in the citrus industry caused by over ten years of acrimonious litigation,” the Secretary of Agriculture terminated the California-Arizona citrus marketing orders, effective July 31, 1994, and dismissed all litigation brought pursuant to the AMAA.Raisins provide another good illustration of volume controls in California. California is the largest volume raisin producer in the world, and this industry has operated under a federal marketing order program with volume controls since 1949. Under the raisin marketing order, annual production is divided between free tonnage and a reserve pool, and the Raisin Administrative Committee controls the reserve tonnage. Only free tonnage can be sold on the domestic market, but the RAC can allow packers to buy additional tonnage for free use from the reserve when the RAC determines that such actions are justified by supply and demand conditions. Until 1977, the majority of raisins in the reserve pool were exported at prices that were much lower than for raisins sold on the domestic market. Raisins from the reserve were also used for the school lunch program, government subsidized exports, other government programs, sales to wineries for distilling into alcohol, donations to charity, and cattle feed. Thus, the raisin industry working through the RAC successfully used the reserve pool to practice price discrimination in separate domestic and export markets.

Conditions and markets changed, however, and beginning in 1977, exports were considered free tonnage shipments, and the initial free tonnage was increased to serve favorable export markets. Since 1977, the RAC has often exported reserve pool raisins at prices competitive with world prices but below prices on the domestic market. Finally, the experience of the California almond industry illustrates how changing market conditions can alter the effectiveness of volume controls.The federal marketing order for California almonds includes provisions for market allocation and a reserve pool. At the beginning of each marketing season, the Almond Board of California recommends to the Secretary of Agriculture a maximum annual quantity to be sold in domestic and export markets and the quantity that cannot be sold . The reserve may be designated as either unallocated or allocated reserve. The unallocated reserve is essentially forced storage; nuts can be released from the unallocated reserve as the season progresses or carried over to the following season. The allocated reserve must be utilized in noncompetitive outlets such as almond butter, almond oil, airline samples, or cattle feed. The reserve provision of the almond marketing order was used to encourage export sales through 1972, while maintaining higher prices in the domestic market than in the export market. This price discrimination ended when export markets became an important outlet for California almonds , with price elasticities tending to equalize between domestic and export markets. Recent work indicates that the price elasticity of demand for almonds is now more elastic in the domestic market than in major export markets, leading to the result that short-run revenue maximization through price discrimination could involve restricting sales to export markets . Recent models of acreage response to changing returns indicate that U.S. and Spanish producers each increase production when returns appear favorable . Thus, if the Almond Board were to use the reserve to practice price discrimination and raise world almond prices, increased prices would stimulate production in Spain as well as the United States. As a consequence of these various considerations the almond industry has not implemented volume controls for the past several years. All existing federal marketing orders for California fruits, vegetables, and nuts include provisions for grades and minimum quality standards. However, only ten of the California State marketing programs include quality standards and inspection provisions, and just seven actively use the provisions. Given typical seasonal price relationships for fresh fruit, with high early-season prices, there are strong incentives to ship fruit as early as possible, even though it may not be fully matured. Most consumers are unable to judge the maturity of fruit from appearance and may find that fruit that “looks good” does not “taste good.” The result is an adverse selection problem.

Sellers are aware of the product’s characteristics, but buyers are unaware. In these settings, low-quality products can drive high-quality products from the marketplace. Indeed,fodder growing system representatives of many commodity groups believe that shipments of immature fruit have a negative impact on total sales, because consumers may delay repeat purchases after being dissatisfied with their original purchases. Maturity standards based on sugar content, firmness, and color are used by several marketing orders to determine when fruit is mature enough to be shipped. Minimum quality standards may: increase the retail demand for a product, resulting in higher prices and/or increased sales; reduce marketing margins, with benefits accruing to both producers and consumers; and reduce supply, which with inelastic demand can increase total revenue to producers. Any effective minimum quality standard will restrict the quantity of commodity marketed, but supply control is not the usual focus of such standards. Federal marketing order regulations on grade, size, quality or maturity also applies to imports of the same commodities from other countries during the period the marketing order is in effect. The use of some minimum quality standards has been controversial. Concerns include charges that quality standards are a hidden form of supply control, that quality standards waste edible fruit with the primary impact being on the poorest consumers, and that quality standards are sometimes not equitable because of regional variations in production conditions. While empirical analyses of the economic impact of minimum standards of grade, size, and maturity for California commodities are limited, those available indicate that it is probably relatively small .The purpose of commodity group expenditures on generic advertising and promotion is to increase the demand for the commodity so that more commodity can be sold for the same price, or the same amount can be sold for a higher price. The rationale for mandatory support by all producers is based on the distribution of documented program benefits and the “free-rider problem.” 5 Research completed and underway documents significant increases in product demand as a result of commodity advertising and promotion programs, with net monetary benefits to producers being much greater than costs. For example, Alston et al. estimated that the elasticity of demand with respect to promotion for California table grapes was 0.16.

Using this promotion coefficient, they estimated that the promotional activities of the Table Grape Commission had increased per capita consumption by about 1.5 pounds over that which would have existed in the absence of a promotional program. This increase was about one-third of recent total per capita consumption. The benefits to producers were very high in both the short- and long-run. The short-run marginal benefit-cost ratio was estimated at over 80:1—for every $1 spent on the program, the industry gained net benefits of $80. When producer supply response was factored into the analysis, the benefit-cost ratios decreased. Using a supply elasticity of 5, the average benefit-cost ratio was about 10:1 and the marginal benefit-cost ratio was about 5:1. Studies of the estimated returns from advertising and promotion programs for other California commodities include avocados , prunes , almonds , eggs , raisins , and walnuts . Each of these studies found that advertising and promotion increased the demand for the product and that program returns exceeded costs by a significant margin. The U.S. government has funded agricultural commodity groups, as well as private firms, to conduct promotional programs in export markets. The Market Access Program and its predecessor programs, the Market Promotion Program, and the Targeted Export Assistance Program, have provided matching funds for the promotion of a number of California commodities. Federal allocations of funds to 15 commodity boards, commissions, and other groups promoting only California fruits, nuts, and vegetables totaled $18.67 million in fiscal year 2002. These funds accounted for 18.7 percent of $100 million awarded to all organizations. Organizations that promote products produced in other states as well as California also received large allocations. California organizations receiving 2002 awards greater than $1 million included Blue Diamond Growers/Almond Board of California, California Prune Board, California Table Grape Commission, California Walnut Commission, Raisin Administrative Committee, Sunkist Growers, Inc., and Wine Institute.Research and development provisions are included in most of the California marketing programs. In 1992, there were 28 programs with research expenditures totaling almost$8.5 million . This increased to 38 California programs with a total 2002/03 or 2003/04 budget of $20.2 million. The largest research budgets were for citrus , rice , market milk , almonds , avocados , fresh strawberries , and walnuts . Overall, research expenditures increased from about 7.5 percent of total 1992 commodity group expenditures to just over 10 percent of 2003 expenditures. In terms of the total farm level value of production, research budgets averaged just over 0.1 percent of the 2001 value of covered commodities. Summary statistics on the economic impacts of commodity group research expenditures are limited, but those available indicate attractive rates of return. Most of the research funded by commodity groups operating under state marketing orders and commissions is done at the University of California. A study valuing California agricultural research concluded that the average annual internal rate of return for public investment in California agricultural research and extension for 1949-85 was about 20 percent . Consider, for example, the case of strawberries. California has become the world’s pre-eminent strawberry producer, now accounting for about 80 percent of U.S. fresh and processed production.

California’s agriculture is also a source of demand for both annual and perennial plants and trees, e.g., vegetable transplants, strawberry plants, seeds of all kinds, root stock for trees, and young nursery stock for new plantings and replacements of vines, tree fruits, and nuts. The types of firms producing nursery products vary widely, including extensive field operations, outdoor nurseries, and intensive greenhouse operations.Consumer demand for lettuce is relatively inelastic, and prices vary widely for this perishable commodity depending on acreage and weather-dependent supply conditions. Large grower-shippers operate in the several production areas in California and Arizona, moving with the seasons. The nation’s “salad bowl” is the Salinas Valley in Monterey County, where lettuce is harvested from April through early November. Other coastal areas produce during the same period. The Imperial Valley and other desert areas ship from early December until mid-March. Production on the west side of the San Joaquin Valley fills the market niches between the two major production areas. Field packing, vacuum cooling, and refrigerated transportation are key components requiring coordination for moving lettuce from the field to the consumer with minimal post-harvest loss in quality. Development of value-added pre-package salad greens has reduced shipments of “Iceberg” head lettuce and effectively increased the demand for other greens, including romaine and leaf lettuce.Almost all breeds of beef cattle are raised in California. The dairy sector also contributes a significant quantity of steers, culled cows,hydroponic nft and bulls as animals marketed for beef. Cattle and calves were California’s #1 agricultural commodity until 1980, when the number one position was taken by milk and cream. Later, grapes and nursery products moved ahead of cattle and calves.

Lettuce became the fourth most important crop in 2000. Cattle and calves are now California’s #5 agricultural commodity. More than two-thirds of the state’s land area is essentially non-tillable because of steep slopes or poor soils. These areas are typically used as rangeland for cattle. In addition, cattle are raised an irrigated pasture lands in the foothill areas and on marginal agricultural lands in the Central Valley. The cattle feeding industry is located primarily in the Imperial and San Joaquin Valleys. Cattle are also shipped out of state to feed lots closer to midwest feed supplies. Climate, topography, and overall conditions vary widely within the state, as do the sizes and types of cattle operations. Some are purely cow-calf operations, while others buy and sell animals as stockers, replacements, or feeders to fit the carrying capacity of owned and leased lands. All areas present separate and distinct challenges to cattle production in terms of rainfall, temperature patterns, topography, breeding and calving conditions, transportation, marketing, urban development, and cattle rustling and vandalism.Hay as a commodity category includes alfalfa hay, grain hay, green chop, sudan hay, and wild hay, but alfalfa is by far the most important component, contributing about 85 percent of the value of all hay production. Alfalfa hay acreage in California has averaged about a million acres, but is influenced by profitability of alternative annual crops , trees, and vines. The demand for alfalfa hay is determined to a large part by the size of the state’s dairy herd, which consumes about 70 percent of the supply. Horses consume about 20 percent. Alfalfa hay is grown in every climatic zone of the state. Climate determines the number of cuttings of hay. In the low desert there are as many as eight to ten cuttings per year; in the cool northern intermountain region, farmers harvest only two to four cuttings a year. Most of the crop is not used on the farm where it is produced, but is usually baled and shipped to end users. Pellets and cubes are other forms for equines and export markets. Alfalfa, a perennial crop with a three- to five-year economic life, does best when planted on well drained, deep, medium-textured soils.

Because it is a highly water intensive crop, its production cost will be directly affected by higher water prices and pumping costs, reducing the long-term profitability of the crop in the state’s crop mix.Flowers and greens are sold in cut and in potted forms. The major areas of production are the coastal counties where the typical mild climate permits outdoor production and lower-cost greenhouse operations. The major production areas of cut flowers are in the counties surrounding San Francisco Bay, extending southwest to Salinas, and in the coastal regions of San Diego and Santa Barbara Counties. The marketing of cut flowers in California is extremely intricate and complex. Although air shipments are used for transcontinental deliveries, most cut flowers are now precooled and shipped by refrigerated trucks. Increased imports, particularly from Columbia and Mexico, are a concern to California greenhouse growers of the three main cut flowers—roses, chrysanthemums, and carnations. The three have historically accounted for as much as two-thirds of the annual income from cut flowers and cut greens. Potted plants, including the seasonal items—poinsettias, lilies and hydrangeas are favored as consumers bring flowers and greenery into residences and offices. There are now more than 250 species and varieties of foliage plants being offered for sale in the trade.About three-quarters of the California strawberry crop is sold fresh; the remainder is sold for processing. Production of California strawberries runs from mid-February through mid-November and occurs in several growing areas along the southern and central coast. Even though strawberry plants are perennials, growers replant annually to obtain maximum yields and the best quality of fruit. Development of new varieties from an industry-supported fruit breeding program at the University of California has been important to the growth of the California strawberry industry.In 1950, California production of 2 million tons of processing tomatoes accounted for only 36 percent of U.S. production. The combination of favorable climate, good soils, ample water, an excellent highway system, applied technology, and research and development has fostered the growth of the processing industry in California which now produces 9 to 10 million tons annually, and as much as 12.2 million tons in 1999. Prices have fallen, as has acreage, while the industry is undergoing structural changes and reduced profitability. Tomato production is specialized and capital-intensive. Processing has changed from consumer products produced at multi-product plants to now include single product production at specialized “industrial plants” where tomato paste product is packaged in aseptic plastic containers in boxes and drums and shipped throughout the year to end users. Paste is simply a commodity bought and further processed into final consumer products—catsup, sauces, soups, etc.

Processing tomatoes are produced from the Mexican border to the northern Sacramento Valley. Harvest begins in the desert valleys in mid-June and continues northward in the Central Valley through September. A late harvest ends in the southern coastal counties in November. All processing tomatoes are harvested mechanically.California agriculture is large, diverse, complex and dynamic. This chapter documents the industry and its relationship to the rest of the economy. It also provides an overview of unifying forces and trends. Our aim is to supply a convenient compilation of facts and figures from a variety of sources, and to help the reader interpret the wide array of data presented.2 California agriculture is far larger, measured by sales, than that of any other state. California agriculture produces more value than most countries and is larger than, for example, such major agricultural producers as Canada or Australia.California is part of the national and international agricultural markets. Californians consume food that is produced in the state, as well as food that is imported from other states and countries. Agriculture in California is the largest among the states, and produces a variety of animals and animal products, fruit, tree-nuts, vegetables, field crops,hydroponic channel and nursery and floriculture products. The Central Valley accounts for more than half of the State’s gross value of agricultural production.About 93 percent of California’s 101.5 million acres is in rural uses. This rural area is divided evenly between federal and non-federal ownership. The federal land mostly includes national forests, national parks and wildlife areas, and “other land,” such as marshes, open swamps, and bare rock deserts. Roughly 11 percent of the federal rural land is grassland pasture and range used for agriculture. Of California’s 53 million acres of non-federal land, about 80 percent is grassland pasture and range, forest land, and cropland. About 5.5 million acres of California’s non-federal land are defined by the Natural Resources Conservation Service of the U.S. Department of Agriculture as “developed” for residential, industrial, and commercial use. However, the intensity of use varies widely, with much of this land relatively unpopulated. The California Department of Conservation Farmland Mapping and Monitoring Program defines 3.1 million acres of California’s non-federal land as “urban and built-up,” that is, land occupied by structures with a building density of at least one unit to one and one-half acres.

This suggests that roughly 2.4 million acres of “developed” land in the NRCS survey are still relatively rural, or not mapped by FMMP. In total, about 27.7 million acres, including 5 million acres of federal grazing land, are used for agriculture in California. More than half is pasture and range, about 39 percent is cropland, and the remainder is divided between woodland and other land.Conversion of agricultural land to urban uses continues to be a public policy issue in the United States and in California. In California between 1988 and 2000, according to the California Department of Conservation Farmland Mapping and Monitoring Program , about 549,000 acres were converted to urban and built-up uses. At these conversion rates, about 4.2 million acres would be converted in the next 100 years. Of the total acres converted from 1988-2000, 213,000 were formerly cropland and 100,000 were formerly grazing land. Another 235,000 acres were formerly “other land,” as classified by the FMMP. A significant portion of the “other land” was idled farmland previously removed from agricultural production in anticipation of development. This indicates that the figures for cropland and grazing land conversion may be understated. Farmland conversion is a topic of particular interest in the Central Valley, which has over half of the state’s agricultural land and 64 percent of the cropland. The Central Valley has had a lower proportion of its cropland and grazing land converted than the rest of the state. The Valley recorded 43 percent of statewide cropland conversion between 1988 and 2000. Similarly, the Central Valley grazing land, about 44 percent of the state total, contributed only 25 percent of the total grazing land conversions.Farmland conversion to urban uses is associated with population growth. California’s population increased by about 76 percent between 1970 and 2002, while the Central Valley’s population doubled. There is general agreement that state population growth will continue, but little consensus on precise projections of future growth rates. The Bureau of the Census estimates that the state population will be about 50 million by 2025.Nationwide, over the last half-century, the number of farms and the total land in farms have decreased, while the size of an average farm has increased. This trend has been less pronounced in California. While the average U.S. farm doubled in acreage between 1954 and 2002, the average California farm increased by about 13 percent. The official definition of a “farm” was changed in 1954, 1959, and 1974, to remove many of the smallest “farms” from census statistics. Each of these definitional changes decreased the reported number of farms and increased the average farm size. Since1974 a “farm” has been defined in the Census of Agriculture as a place that generates agricultural sales of at least $1,000 annually. Under the current Census of Agriculture definition, the average acreage of California farms decreased by 30 percent between 1974 and 2002. The 2002 Census introduced a new methodology for estimating total number of farms and operators’ demographics. The Census has been conducted via mail returns, and coverage has been always below 100 percent, especially among very small operations. The 2002 methodology accounts for all farms. In 2002, about 80 percent of California farms were less than 180 acres, yet the “average farm” size was 347 acres. These two statistics highlight the fact that a small percent of large farms account for a large percent of total acreage. These large farms include ranches that graze livestock and may generate relatively little total revenue.

The spread of irrigation broadly paralleled the intensification movement. Between 1869 and 1889, the share of California farmland receiving water through artificial means increased from less than one percent to five percent. Growth was relatively slow in the 1890s, but expansion resumed over the 1900s and 1910s. By 1929, irrigated land accounted for nearly 16 percent of the farmland.Data on the value and composition of crop output put California’s agricultural transformation into sharper relief. Between 1859 and 1929, the real value of the state’s crop output increased over 25 times. Growth was especially rapid during the grain boom of the 1860s and 1870s, associated primarily with the expansion of the state’s agricultural land base. Subsequent growth in crop production was mainly due to increasing output per acre and was closely tied to a dramatic shift in the state’s crop mix. After falling in the 1860s and 1870s, the share of intensive crops in the value of total output climbed from less than 4 percent in 1879 to over 20 percent in 1889. By 1909, the intensive share reached nearly one-half, and by 1929, it was almost four fifths of the total.3 Figure 1 provides further documentation of the transformation of California’s crop mix over the late 19th and early 20th centuries. The Figure shows how cropland harvested in California was distributed across selected major crops over the 1879-1997 period. The acreage data reveal that in 1879,nft hydroponic wheat and barley were grown on over 75 percent of the state’s cropland whereas the combined total for the intensive crops was around five percent. By 1929, the picture had changed dramatically. Wheat and barley then accounted for about 26 percent of the cropland harvested and the intensive crop share stood around 35 percent.

In absolute terms, the acreage in the intensive crops expanded over ten times over this half century while that for wheat and barley fell by more than one-third.Data on shipments of California fresh, dried, and canned fruits and nuts reveal the sector’s spectacular expansion over this period. During the 1870s and 1880s, growth rates exceeded 25 percent per year . Shipments continued to grow at robust rates of about eight percent per annum over the 1890s and 1900s. By 1919, California produced 57 percent of the oranges, 70 percent of the prunes and plums, over 80 percent of the grapes and figs, and virtually all of the apricots, almonds, walnuts, olives, and lemons grown in the United States. In addition, California produced significant quantities of apples, pears, cherries, peaches, and other lesser crops. The spectacular growth in California production of specialty crops had important international consequences as traditional Mediterranean exporters of many crops were first driven from the lucrative U.S. market and then faced stiff competition from the upstart Californians in their own backyard of northern Europe. California production significantly affected the markets and incomes of raisin growers in Málaga and Alicante, prune growers in Serbia and Bosnia, and citrus growers in Sicily.Explanations for the causes and timing of California’s structural transformation have long puzzled scholars. The traditional literature yields numerous causal factors, including: increases in demand for income-elastic fruit products in eastern urban markets; improvements in transportation, especially the completion of the transcontinental railroad; reductions in the profitability of wheat due to slumping world grain prices and falling local yields; the spread of irrigation and the accompanying breakup of large land holdings; the increased availability of “cheap” labor; and the accumulation of knowledge about California’s environment and suitable agricultural practices. Yet a careful investigation of the transformation yields a surprising result: much of the credit for the shift to intensive crops must be given to exogenous declines in real interest rates and to “biological” changes as farmers learned more about how to grow new crops in the California environment. Isolated from America’s financial markets, California farmers faced high, even astronomical, interest rates, which discouraged capital investments. Rates fell from well over 100 percent during the Gold Rush to about 30 percent circa 1860. The downward trend continued with real rural mortgage rates approaching 8 to 12 percent by 1890. The implications of falling interest rates for a long-term investment such as an orchard were enormous.

As one Bay Area observer noted in the mid-1880s, the conversion of grain fields to orchards “has naturally been retarded in a community where there is little capital, by the cost of getting land into orchard, and waiting several years for returns.”Calculations indicate that the break-even interest rate for the wheat-to orchard transition was about 10 to 13 percent . These estimates conform fairly closely to the interest rate levels prevailing in California when horticulture began its ascent. A second key supply-side force was the increase in horticultural productivity associated with biological learning. Yields for leading tree crops nearly doubled between 1889 and 1919. When the Gold Rush began, the American occupiers knew little about the region’s soils and climate. As settlement continued, would-be farmers learned to distinguish the better soils from poorer soils, the more amply watered land from the more arid, the areas with moderate climates from those suffering greater extremes. Occasionally overcoming deep-seated prejudices, farmers learned which soils were comparatively more productive for specific crops. California fruit growers engaged in a similar time-consuming process of experimentation to find the most appropriate plant stocks and cultural practices. Existing varieties were introduced from around the world, and new varieties were created. In the early 1870s, USDA plant specialists established the foundation for the state’s citrus industry with navel orange bud wood imported from Bahia, Brazil. Plums and prune trees were brought in from France and Japan; grape vines from France, Italy, Spain, and Germany; and figs from Greece and Turkey. Plant breeders also got in on the act. The legendary Luther Burbank, who settled in California in 1875, developed hundreds of new varieties of plums and other fruits over his long career.In part, the growth of horticultural knowledge occurred through the informal “folk process” highlighted in William Parker’s classic treatment of American agriculture. Over time, the process of research and diffusion became increasingly formalized and institutionalized.As an example, a series of major citrus expositions, held annually in Riverside from the late 1870s on, helped popularize the new Bahia orange variety. An emerging group of specialty farm journals, such as the Southern California Horticulturist, California Citrograph, and California Fruit Grower, supplemented the stalwart Pacific Rural Press to spread information about fruit growing.The California State Board of Horticulture, formed in 1881, provided an active forum for discussion of production and marketing practices, especially through its annual convention of fruit growers. The Agricultural College of the University of California, under the leadership of Eugene Hilgard and Edward Wickson, intensified its research efforts on horticultural and viticultural problems after the mid-1880s.

By the early 1900s, the USDA, the state agricultural research system, and local cooperatives formed an effective working arrangement to acquire and spread knowledge about fruit quality and the effects of packing, shipping, and marketing on spoilage and fruit appearance.These efforts led to the development of pre-cooling and other improved handling techniques, contributing to the emergence of California’s reputation for offering higher-quality horticultural products. This learning process eventually propelled California’s horticultural sector to a position of global leadership.More generally, the example of the state’s horticultural industry highlights the important,nft system if relatively neglected, contribution of biological learning to American agricultural development before the 1930s.A second major transformation took place in the early twentieth century with the increased cultivation of row crops including sugar beets, vegetables, and most notably cotton . These changes represented an intensification of farming with significant capital investments and often led to shifts onto what had been marginal or under-utilized lands. The advent of cotton, which by 1950 had become the state’s most valuable crop, offers another important case study in the continuing evolution of California agriculture.From Spanish times, visionaries attempted to introduce cotton into California on a commercial basis. A variety of factors, including the high cost of labor, the distance from markets and gins, and inadequate knowledge about appropriate varieties, soils, etc. doomed these early efforts. The real breakthrough came during World War I when high prices coupled with government research and promotional campaigns encouraged farmers in the Imperial, Coachella, and San Joaquin Valleys to adopt the crop. Figure2 illustrates acres harvested, bales produced, and yields per acre, from 1910 to 1964. The tremendous absolute increase in California’s cotton acreage since the 1920s contrasts with the absolute decline nationally. California’s acreage in cotton ranked 14th out of 15 cotton-producing states in 1919; by 1959 it ranked second.Several factors distinguished California’s cotton industry from other regions. First, cotton yields were typically more than double the national average. High yields resulted from the favorable climate, rich soils, controlled application of irrigation water, use of the best agricultural practices and fertilizer, adoption of high quality seeds, and relative freedom from pests. Second, the scale and structure of cotton farms was remarkably different in California. From the mid-1920s through the 1950s, the acreage of a California cotton farm were about five times that of farms in the Deep South. As an example of the structural differences between California and other important cotton states, in 1939 farms producing 50 or fewer bales grew to about 17 percent of the output in California, but in other leading cotton states, farms in this class produced at least 80 percent of all cotton output. One-half of the output in California was grown on farms producing more than 200 bales. For the nation as a whole, one-half of the output was raised on farms producing fewer than 13 bales.Thus,it is not surprising that California’s gross income per cotton farm was almost nine times the national average.Other distinctive features of California cotton farms were their more intensive use of power and their earlier mechanization of pre-harvest activities. In 1929, a California farm was almost 20 times more likely to have a tractor than a Mississippi farm.The Pacific Rural Press in 1927 offered a description of the highly mechanized state of many California cotton farms: “[M]en farm in sections…By the most efficient use of tractor power and tools, one outfit with a two-man daylight shift plants 100 acres per day, 6 rows at a time, and cultivates 70 acres 4-rows at a time.”The more rapid adoption of tractors created a setting favorable to further modernization. When picking machines became available, farmers already possessed the mechanical skills and aptitudes needed for machine-based production. The larger size of cotton operations in California and the more intensive use of tractors reflected a fundamentally different form of labor organization than that which dominated the South. By the 1940s, on the eve of cotton harvesting mechanization, most cotton in California was picked on a piece-rate basis by seasonal laborers under a contract system.Although conditions varied, a key ingredient was that a labor contractor recruited and supervised the workers, and dealt directly with the farmer, who might have had little or no personal contact with his laborers. This type of arrangement implied different class and social relationships from those that prevailed in much of the South. The California farm worker was more akin to an agricultural proletarian than to a rural peasant. The proverbial paternalism of southern planters toward their tenants had few parallels in California. As with many crops, California cotton growers also led the way in harvest mechanization. Many of the factors discussed above, including pre-harvest mechanization , relatively high wages, large-scale operations, high yields, a flat landscape, and a relative absence of rain during the harvest season all aided in the adoption of the mechanical harvester. Spindle picking machines first appeared on a commercial basis following World War II. In 1951, over 50 percent of the California crop was mechanically harvested compared to about 10 percent for the rest of the nation. At that time, about 50 percent of all the machines in operation in the United States were at work on California farms.16Similar forces—early adoption of large-scale operations and advanced technologies—characterized California’s livestock economy. The broad trends in livestock production in California since 1850 are reflected in Figure 3, which graphs the number of head of various types of livestock in the state as aggregated into a measure of animal units fed.The region emerged from the Mexican period primarily as a cattle producer.

Samples were collected at 0, 3, and 6 h following illumination with red light. The remainder of the culture was then placed under an IR LED for an additional 12 h, after which a final sample was collected. Upon collection, cells were immediately treated with 475 μL 100% ethanol and fixed for 1 h at room temperature. Cells were then pelleted, washed once with 50 mM sodium citrate , and then resuspended in 500 μL 50 mM sodium citrate to which RNaseA had been added. Following an incubation for 1 h at 37˚C, 50 μL proteinase K was added to a final concentration of 2 mg/mL and cells were incubated at 50˚C for an additional hour. SYTOX green was then added to each sample to a final concentration of 2 μM, and cells were incubated for 1 h at room temperature. Inparallel, control samples were prepared containing exponentially growing cultures of yeast treated with α-factor for for ~3 h prior to fixation and staining. Ploidy was determined by measuring the fluorescence intensity of SYTOX green staining by flow cytometry using a FACSAria III and normalizing to the α-factor-treated samples, which have a ploidy of 1N. For each time point for each independent experiment, 50,000 cells were measured. Analysis of flow cytometry data was performed using FlowJo and Flowing Software .Intact natural landscapes provide ecosystem functions that result in numerous ecosystem goods and services from which humans benefit, including carbon storage, flood protection, and maintenance of species life cycles . However, many of these services are diminished in landscapes that have been converted for agricultural purposes. The provisioning services of these food-production landscapes are clear, and there is increasing recognition that agricultural landscapes can continue to supply or maintain other vital ecosystem services if well managed. These include flood mitigation and carbon storage , pollination ,mobile grow rack and wildlife habitat . Maintaining ecosystem functions to optimize these multiple services in agricultural landscapes is particularly important, given that cropland and pasture currently occupy ~40% of the earth’s land surface, with increases predicted to support a growing global population .

In the past, agricultural production was characterized by growing one or more crops in the same place. Crop rotation would provide inputs of nitrogen, and suppress insects and weeds by breaking their life cycles, yielding modest but stable agricultural inputs . However, this link between ecology and agriculture has become strained over the past few decades a a result of mechanization, new crop varieties, development of agrochemicals, as well as political and economic forces associated with regional agriculture’s supplying international markets . This has led to concerns about the long-term sustainability of food-production systems, and the influence of these practices on the ecosystem services provided to people. An increasing number of studies show how creating diverse agricultural landscapes, through patches of remnant or revegetated native habitat on farmland, even on a small scale, can provide important habitat for native flora and fauna, as well as benefit farm productivity in unexpected ways . For example, fields with uncultivated margins have higher plant and moth diversity as well as more diverse soil macrofauna . Hedgerows have been associated with higher bird and moth diversity, provide movement corridors for fauna and host natural enemies that control agricultural pests . In addition, remnant areas close to agricultural areas improve pollination services with positive consequences for crop yields . Furthermore, agricultural lands store carbon through remnant native vegetation and from the crops cultivated, particularly annual row crops because of their dense planting . Similarly below-ground biomass could be increased by introducing cover crops with deeper roots to increase below-ground biomass while food is still produced . Other ecosystem services from certain types of agriculture include aesthetic landscapes , farm tourism , and the preservation of rural lifestyles . Agriculture can also be the source of ecosystem disservices such as habitat loss and pesticide poisoning of non-targeted species , while soil and nutrient runoff result in losses of soil carbon . The effect of these disservices ranges from the local scale to the regional scale , to the global scale . For example, 20% of the N fertilizer applied in agricultural systems globally moves to aquatic ecosystems . Agricultural production practices in California, which produces roughly half of the fruits, nuts, and vegetables for the U.S, has resulted in widespread nitrate contamination of groundwater aquifers . An emerging body of literature focuses on spatially quantifying ecosystem services and comparing these with patterns of biodiversity across the landscape and agricultural land returns . For example, Nelson et al. 

Assessed these three components under alternative land-use trajectories in the Willamette Basin, Oregon. The study found that a conservation scenario which resulted in high scores for ecosystem services also had high scores for biodiversity, while a development scenario had higher returns to land-owners but lower levels of biodiversity conservation and ecosystem services. Polasky et al. in an assessment of land-use alternatives over a 10-year period in Minnesota found a lack of concordance—the scenarios that created the greatest annual net returns to land-owners also had the lowest social benefits. Agricultural expansion was found to reduce stored carbon, negatively affect water quality, and reduce habitat quality for biodiversity and forest songbirds. The present study has elements similar to Nelson et al. and Polasky et al. . It is conducted at the parcel spatial scale, it is forward looking to 2050, and it addresses ecosystem services and disservices along with biodiversity and financial returns from agriculture. In this study, we examine the assumption that land use change in agriculturally dominated areas provides positive benefits for land-owners and financial agricultural returns at the expense of biodiversity and other ecosystem services, such as carbon storage. We do this by quantifying carbon storage, landscape suitability for birds, ecosystem disservices, and financial returns from agriculture within an area of the Central Valley of California. We ask how these change by 2050 under three alternative scenarios: restoration, urbanization, and enhanced agriculture tailored to the needs of a key species of conservation concern, the Swainson’s Hawk. The study area spans a 72,188-ha area in the Central Valley that includes the Cosumnes River Preserve and surrounding lands up to 50m in elevation, encompassing lands owned privately, by state and federal government, or by non-profit organizations including The Nature Conservancy . Historically, this area was dominated by native grassland, valley oak woodland and savanna, and riparian forest along the once-perennial Cosumnes River. However, conversion to agriculture has resulted in a landscape where only small patches of natural habitat remain. Many of these remnant natural areas are currently experiencing conversion to urban land use from the rapidly growing adjacent cities. Although some of these natural areas are habitat for state- and federally-listed threatened and endangered species and, consequently, development of these lands has resulted in mitigation funding to compensate for habitat losses of imperiled species. Currently, natural vegetation covers 44% of the study area,ebb and flow table based on a vegetation map of the Delta developed by the California Department of Fish and Wildlife in addition to project-based vegetation mapping.

The urban and developed footprint, which currently covers 9% of the study area, has increased by 35% over the last decade . Urban expansion associated with cities at the northern and southern edges of the study area continues to exert development pressure on remaining natural and agricultural lands. Today, the river channel is lined with agricultural levees, and adjacent floodplains are used largely for crops , with agriculture covering 46% of the study area. Agricultural activity in the study area, compared to large-scale agriculture in many other parts of California, has a high diversity of crops across many small parcels. Our agricultural land-use scenario represents an enhanced agriculture which favors compatible crops that are commonly the targets for mitigating habitat loss for the imperiled Swainson’s Hawk through mitigation funding. This scenario is reasonable, given that the region is highly suitable for Swainson’s Hawk nesting and foraging, supporting one of the highest concentrations of birds in the Pacific Flyway. Because B. swainsoni is a species of concern, priority has been placed on managing the landscape for its persistence. Both governmental and nongovernmental land managers currently engage in a number of practices intended to boost populations of avian species of concern, for example, paying farmers to flood fallow fields to provide habitat for migrating waterfowl. Given their conservation priority status, it is possible that management agencies might also pay farmers to enhance habitat for improved outcomes for the Swainson’s Hawk. The widespread use of conservation easements within the study area provides the mechanism and the means to accomplish an enhanced agricultural scenario. The Yolo County Habitat Conservation Plan/Natural Community Conservation Plan is one such policy that points toward management for this species. The Swainson’s Hawk can also be a valuable focal species because of its dependence on tree canopy nesting sites and nearby open-country foraging habitat. Many other raptors, riparian species, and migratory birds also depend on these ecosystem traits. The Swainson’s Hawk is able to forage in specific types of agriculture . In particular, alfalfa is a valuable resource because it is a perennial crop that continually supports high populations of prey whose availability peaks during monthly irrigation and harvesting events. Other crops, such as beet and tomato fields, are also hunted regularly during harvest, though crops such as rice or vineyards are not significantly utilized . The same crop associations are true for a number of other species. Alfalfa, in particular, is considered important habitat for other migratory birds, including shorebirds like Long-Billed Curlew and White-Faced Ibis , both of which are species of conservation concern. The Swainson’s Hawk responds well to protection and restoration of riparian forest habitats , within an agricultural landscape that is used for foraging . Thus, a varying matrix of natural riparian habitat and different forms of agriculture can reasonably be expected to significantly affect the value of the landscape for this species.

The potential for multi-species benefits from single species mitigation or management is rarely evaluated; therefore, we include an assessment of the ramifications of these land-use scenarios for 15 other focal bird species identified by the California Partners in Flight program . These are a suite of species whose requirements define different spatial attributes, habitat characteristics, and management regimes, and represent healthy habitats within our focal landscape . We conducted this study at the parcel scale , which is meaningful because land management decisions for agricultural practices are made at this scale and only a few ecosystem services quantification studies have been conducted at this scale . The average size of parcels in the study area that grow alfalfa, grain, orchard, rice, row crop, or vineyard was 3.88ha . To assess the changes over time among carbon storage, landscape suitability for birds, ecosystem disservices, and returns from agriculture with each of our management scenarios, we first developed a snapshot of the current land cover in the study area. We mapped urban areas using information from the Sacramento Area Council of Governments data from 2005, with a minimum mapping unit of five acres for urban areas and ten acres for rural areas. We defined agricultural land cover data from the CDFW’s Delta vegetation map and from the California Department of Water Resources , with a minimum mapping unit of 10 acres .These were considered sufficiently distinct from each other in terms of foraging value for Swainson’s Hawk. In addition to these agricultural classes, we used four natural vegetation classes along with developed/ urban and water . These land-cover types were intersected with land-owner parcels for the study area . In cases where a parcel contained more than one land use type, the parcel was split into the respective classes to retain this detail. These resultant land- cover data represent the contemporary baseline condition from which we measured changes resulting from the landscape management scenarios. We conducted spatial analysis in ESRI ArcMap version 10.3, in Universal Transverse Mercator projection with North American Datum 1983.We consulted with management stakeholders at the Cosumnes River Preserve to develop three management scenarios to simulate potential changes in the current landscape: extensive restoration, urbanization, and enhanced agriculture designed to benefit the Swainson’s Hawk.

In Arabidopsis, AtTic20-I and AtTic110 are expressed to a lower extent in roots than in leaves, similar to pea . These results seemingly contradict those of Hirabayashi et al., who concluded a comparable expression level of Tic20-I in shoots and roots. However, they used a non-quantifiable approach in contrast to our quantitative analysis. Furthermore, in our experiments the overall expression of AtTic20-I and AtTic110 differs notably from that in pea, AtTic110 RNA being about 3.5 and 6 times more abundant than AtTic20-I in leaves and roots, respectively. We also designed specific primers for the second Tic20 homolog in Arabidopsis, AtTic20-IV, and our quantitative method was sufficiently sensitive to precisely define its RNA levels in Arabidopsis leaves and roots, allowing direct comparison with the expression of AtTic20-I and AtTic110 . Transcription of AtTic20-IV had also been investigated in parallel to AtTic110 by Teng et al., who observed a differential ratio of expression using two different methods, of which one was not even sensitive enough to detect AtTic20-IV. A very recent study also investigated the expression of AtTic20-IV, however, without any quantification of their data. Our data show that AtTic20-IV is present in leaves and roots with transcript levels similar to AtTic20-I, but less abundant than AtTic110. Interestingly, and in accordance with the data presented by Hirabayashi et al., transcript levels of AtTic20-IV in roots are higher than those of AtTic20-I, while the opposite is true in leaf tissue. It can be speculated that the observed expression pattern reflects tissue-specific differentiation of both genes. AtTic20-IV may still partially complement for the function of AtTic20-I, as becomes evident from the viability of attic20-I knockout plants and the yellowish phenotype of attic20-I mutants over expressing AtTic20- IV. However, the severe phenotype of attic20-I plants, in conjunction with the observed differential expression pattern, clearly indicates specific functions of the two homologs. Furthermore, hydroponic nft a higher AtTic110 expression rate as observed in antisense attic20-I lines might indicate another possible compensatory effect .

The expression pattern of the three investigated genes was found to be similar in Arabidopsis growing hydroponically with or without sucrose or on soil . However, gene expression was generally higher in plants growing without sucrose.Semi-quantitative analysis of Tic20 and Tic110 on protein level was performed using immunoblots of envelope membranes isolated from two-week-old pea and four week-old Arabidopsis plants. In parallel, calibration curves were generated using a series of known concentrations of over expressed and purified proteins . After quantification of immunoblots from envelopes, amounts of PsTic20, PsTic110, AtTic20 and AtTic110 were determined using the corresponding calibration curve. The amount of PsTic110 in IE was found to be almost eight times higher than that of PsTic20 , which differs strikingly from the similar transcript levels of the two genes detected in leaves , indicating profound differences in post translational processes such as translation rate and protein turnover. In Arabidopsis, the absolute amount of AtTic110 is nearly the same as in pea , however, Arabidopsis envelopes represent a mixture, containing both outer and IE vesicles. Thus, the relative amount of AtTic110 is possibly higher than in pea. Surprisingly, the amount of AtTic20 is more than 100 times lower than that of AtTic110, showing an even greater difference in comparison to the observed RNA expression levels . Taking the different molecular size of Tic110 and Tic20 into account , we still observe 20 times more AtTic110 than AtTic20 protein. In pea, we found 1.4 times more Tic110 RNA than Tic20, whereas in Arabidopsis the ratio of Tic110 to Tic20 is 20.3. The number of channel forming units must even be more different, since Tic110 was shown to form dimers, whereas Tic20 builds very large complexes between 700 kDa and 1 MDa. Thus, two Tic110 molecules would be necessary to form a channel in contrast to Tic20, which would require many more molecules to form the pore. Though we cannot exclude that Tic20 might be subject to degradation by an unknown protease in vivo, protease treatments with thermolysin of right-side out IE vesicles in vitro clearly shows that Tic20 is very protease resistant,even in the presence of detergent. In contrast, Tic110 is easily degraded already without addition of detergent .

This argues against more rapid degradation of Tic20 compared to Tic110 during preparation of IE. The difference in Tic110 to Tic20 ratios both on the RNA and protein level between pea and Arabidopsis may be due to the different age of the plants or the different needs under the given growth conditions, and suggests that there is no strict stoichiometry between the two proteins. Moreover, the low abundance of Tic20 in comparison to Tic110 in envelopes clearly demonstrates that Tic20 cannot be the main channel of the Tic translocon as previously suggested, since it cannot possibly support the required import rates of some highly abundant preproteins that are needed in the chloroplast.Experimental data suggested a common complex between Tic110 and Tic20 in chloroplast envelope membranes using a cross-linking approach. However, the interaction was not visible in the absence of Toc components, making a stable association unlikely. Furthermore, no evidence for a common complex was found by Kikuchi et al. using solubilized chloroplasts of pea and Arabidopsis for two-dimensional blue native/SDS-PAGE analysis. Likewise, the difference in Tic110 to Tic20 ratios both on the RNA and protein level between pea and Arabidopsis indicates that a common complex, in which both proteins cooperate in translocation channel formation in a reasonable stoichiometry, is improbable. To clarify this issue, we addressed these partly conflicting results by using IE vesicles, which should minimize the possible influence of the interaction with Toc components on complex formation. Pea IE vesicles were solubilized in 5% digitonin and subjected to 2D BN/ SDS-PAGE. Immunoblots revealed that both Tic20 and Tic110 are present in distinct high molecular weight complexes : Tic110-containing complexes migrate at a size of ~ 200-300 kDa, whereas Tic20 displays a much slower mobility in BN-PAGE and is present in complexes exceeding 700 kDa, in line with the results from Kikuchi et al.. However, at a similar molecular weight of 250 kDa on BN-PAGE not only Tic110 but also Hsp93, Tic62 and Tic55 were described. The molecular weight of a complex containing all of these components would be much higher. Therefore, components of the Tic complex might associate with Tic110 very dynamically resulting in different compositions under different conditions, or alternatively, there are different complexes present at the same molecular weight.Tic22, the only Tic component located in the intermembrane space, is a potential candidate, since both proteins were identified together in cross-linking experiments.

However, only minor amounts of Tic20 and Tic22 were shown to co-localize after gel filtration of solubilized envelope membranes. A second candidate for common complex formation is PIC1/Tic21: Kikuchi et al.demonstrated that a one-megadalton complex of Tic20 contains PIC1/Tic21 as a minor subunit. PIC1/ Tic21 was proposed to form a protein translocation channel in the Tic complex, mainly based on protein import defects of knockout mutants and on structural similarities to amino acid transporters and sugar permeases. An independent study by Duy et al. favours the hypothesis that PIC1/Tic21 forms a metal permease in the IE of chloroplasts,hydroponic channel rendering the import-related role questionable. This discrepancy will have to be addressed in the future. To test the complex formation of Tic20 in vitro without the involvement of other proteins, we used Tic20- proteoliposomes for 2D BN/SDS-PAGE analysis, similarly to IE vesicles . The migration behaviour of the protein resembles that observed in IE: the majority of the protein localizes in high molecular weight range, however, the signal appears more widespread and a portion is also detected at lower molecular weights, possibly as monomers. This observation reveals that Tic20 has the inherent ability to homo-oligomerize in the presence of a lipid bilayer. The less distinct signal could be due to different solubilization of Tic20 by digitonin in IE vesicles vs. liposomes, or could be an indication that additional subunits stabilize the endogenous Tic20 complexes, which are not present after the reconstitution. However, we interpret these observations as support for the hypothesis that the major component of the one megadalton complex in IE are homo-oligomers composed of Tic20.In silico analysis of Tic20 predicts the presence of four hydrophobic transmembrane helices positioning both N- and C-termini to one side of the membrane . According to these predictions, three cysteins in PsTic20 face the same side, while the fourth would be located in the plane of the membrane. We used pea IE vesicles prepared in a right-side-out orientation to determine the topology of Tic20 employing a Cys-labelling technique. To this end, the IE vesicles were incubated with a membrane-impermeable, Cys-reactive agent that adds a molecular weight of 5,000 Da to the target protein for each reactive Cys residue. In our experiments PEG-Mal did not strongly label any Cys residues of Tic20 under the conditions applied , indicating the absence of accessible Cys residues on the outside of the membrane. Only one faint additional band of higher molecular weight was detectable , possibly due to a partially accessible Cys located within the membrane. In the presence of 1% SDS, however, all four Cys residues present in PsTic20 are rapidly PEGylated, as demonstrated by the appearance of four intense additional bands after only five minutes of incubation. The observed gain in molecular weight per modification is bigger than the expected 5 kDa for each Cys, but this can be attributed to an aberrant mobility of the modified protein in the Bis-Tris/ SDS-PAGE used in the assay. Our results support a four transmembrane helix topology in which both the C- and N-termini are facing the stromal side of the membrane , with no Cys residues oriented towards the intermembrane space. Cys108 is most likely located in helix one, Cys227 and Cys230 are oriented to the stromal side of helix four and Cys243 is located in the stroma.

This topology is also in line with green fluorescent protein-labelling studies by van Dooren et al.indicating that the N- and C-termini also of the Toxoplasma gondii homolog of Tic20 face the stromal side of the inner apicoplast membrane.Tic20 was identified more than a decade ago but since then no heterologous expression and purification procedure has been reported, which could successfully synthesize folded full-length Tic20. Here, we report two efficient Escherichia coli based systems for Tic20 expression and purification from both pea and Arabidopsis: codon optimized PsTic20 was over expressed in a S12 cell lysate in presence of detergents, and AtTic20 over expression was successfully accomplished by adaptation of a special induction system. Following these steps, both pea and Arabidopsis proteins could be purified to homogeneity by metal affinity purification . Using the purified protein, we performed structural characterization studies of Tic20 by subjecting it to circular dichroism spectroscopy . The recorded spectra of PsTic20, displaying two minima at 210 and 222 nm and a large peak of positive ellipticity centered at 193 nm, are highly characteristic of a-helical proteins, and thus demonstrate that the protein exists in a folded state after purification in the presence of detergent. The secondary structure of Tic20 was estimated by fitting spectra to reference data sets resulting in an a-helical content of approximately 78%, confirming in silico predictions.To better characterize Tic20 in a membrane-mimicking environment, heterologously expressed and purified AtTic20 was reconstituted into liposomes in vitro. Initially, flotation experiments were performed to verify a stable insertion. In the presence or absence of liposomes, Tic20 was placed at the bottom of a gradient ranging from 1.6 M to 0.1 M sucrose. In the presence of liposomes, Tic20 migrated to the middle of the gradient, indicating a change in its density caused by interaction with liposomes. In contrast, the protein alone remained at the bottom of the gradient . Proteoliposomes were also treated with various buffers before flotation , to test whether the protein is firmly inserted into the liposomalmembrane or just loosely bound to the vesicle surface. None of the applied conditions changed the migration behaviour of Tic20 in the gradient , indicating that Tic20 was deeply inserted into the liposomal membrane. Thus, proteoliposomes represent a suitable in vitro system for the analysis of Tic20 channel activity.Even though Tic20 has long been suggested to form a channel in the IE membrane, this notion was solely based on structural analogy to other four-transmembrane helix proteins, and no experimental evidence has been provided so far.

Despite successfully expressing thaumatin in yeast , bacteria, fungi, and transgenic and transfected plants, biotechnological large-scale production facilities have yet to be established. Molecular farming, the production of recombinant proteins in plants, offers several advantages over bioreactor-based systems. In this application, plants are thought of as nature’s single use bioreactors, offering many benefits such as reduced upstream production complexity and costs , linear scalability, and their inability to replicate human viruses. specifically, open-field growth of plants has the potential to meet the market’s need for a large-scale, continuous demand of a commodity product at a competitive upstream cost. It has been marked suitable for this operation as plants can be easily adapted on an agricultural scale to yield several metric tons of the purified protein per year. Here, we present a feasibility study for a protein production level of tens of metric tons per year. The success of a new product in the biotechnology process industry depends on well-integrated planning that involves market analysis, product development, process development, and addressing regulatory issues simultaneously, which requires some decisions to be made with limited information. This generates demand for a platform to help fill in those gaps and facilitate making more informed process and technology decisions. Process simulation models can be used in several stages of the product life cycle including idea generation, process development, facility design, and manufacturing. For instance, based on preliminary economic evaluations of new projects, they are used to eliminate unfeasible ideas early on. During the development phase of the product, as the process undergoes frequent changes, such models can easily evaluate the impact of these changes and identify cost-sensitive areas.Additionally, PSMs are widely used in designing new manufacturing facilities mainly as a tool for sizing process equipment and supporting utilities,ebb and flow tray as well as for estimating the required capital investment and cost of goods.

This ultimately helps companies decide on building a new facility versus outsourcing to contact manufacturers. There are currently few published data-driven simulations of techno-economic models for plant-based manufacturing of proteins for pharmaceutical, biofuel, commercial enzyme, and food safety applications. However, to the best of our knowledge, no studies have proposed or assessed the feasibility of plant-based protein bio-production platforms on the commodity scale in tens of metric tons per year. The feasibility of production at this scale is critical for the emergence of thaumatin as a sugar substitute. Here, we present a preliminary process design, process simulation, and economic analysis for the large-scale manufacturing of thaumatin II variant by several different molecular farming production platforms.The base case scenario assumes an annual production capacity of 50 MT thaumatin. To achieve this level of production in a consistent manner, manufacturing is divided into 157 annual batches. Upstream production is attainable through open-field, staggered plantation of Nicotiana tabacum plants. Each batch has a duration of 45 days and a recipe cycle time of 2 days. A full list of process assumptions can be found in Table S1. The proposed design achieves the expression of thaumatin in N. tabacum leaves using magnICON® v.3. This technology developed by Icon Genetics GmbH allows for the separation of the “growth” and the “expression” phases in a manufacturing process. Moreover, this process obviates the need to use agroinfiltration, which requires more capital and operational costs for inoculum preparation and implementation of expensive units for the infiltration process, containment of the genetically engineered agrobacteria, and elimination of bacteria-derived endotoxins. In this design, transgenic N. tabacum or N. benthamiana plants carry a double-inducible viral vector that has been deconstructed into its two components, the replicon and the cell-to-cell movement protein. Background expression of recombinant proteins prior to induction remains minimal; however, inducible release of viral RNA replicons—from stably integrated DNA proreplicons—is triggered upon spraying the leaves and/or drenching the roots with a 4% ethanol solution resulting in expression levels as high as 4.3 g/kg fresh weight in Nicotiana benthamiana.

Nonetheless, Nicotiana tabacumhas several advantages that make it more suitable for large-scale open field production such as field hardiness, high biomass yields, well-established infrastructure for large-scale processing, plentiful seed production, while attaining expression levels up to 2 g/kg FW. Furthermore, it is unlikely that transgenic tobacco material would mix with material destined for the human food or animal feed chain, unless it is grown in rotation with a food crop, but further development of strict Good Agricultural Practice for transgenic plants should overcome these issues.An alternative upstream facility design scenario was developed to evaluate the process economics of a more controlled supply of thaumatin by growing the plant host in a 10-layer vertical farming indoor environment. Nicotiana benthamiana is chosen as a host because it is known to be a model for protein expression for both Agrobacterium and virus-based systems, but its low biomass yield and difficulties regarding adaptation in the field hinder its application for open outdoor growth. However, this species grows very well in indoor, controlled environments and has high recombinant protein production. This upstream production facility uses the same method of expression and follows the same schedule as the base case upstream facility.Transient expression in plants is a method of recombinantly producing proteins without stable integration of genes in the nuclear or chloroplast genome. The main advantages of using this method are reducing the extensive amount of time needed to develop a stable transgenic line and overcoming bio-safety concerns with growing transgenic food crops in the field expressing heterologous proteins. Transient expression is attainable through several systems including biolistic delivery of naked DNA, agrobacteria, and infection with viral vectors. Notably, the use of viral vectors has been marked suitable for application on a field-scale due to the flexibility of production, and the quick accumulation of target proteins while achieving high yields. A new report has shown efficacy in delivering RNA viral particles using a 1–3 bar pressure, 1–4 mm atomizer nozzles spray devices in the presence of an abrasive to cause mechanical wounding of plant cell wall. GRAS notices GRN 738 and GRN 910 describe production of thaumatin in edible plant species and N. benthamiana, respectively. The expression of thaumatin in leaf tissue of the food crops Beta vulgaris , Spinacia oleracea , or Lactuca sativa is generally lower than in N. benthamiana.

However, despite having lower expression levels, the absence of pyridine alkaloids that are present in Nicotiana species is a major advantage for production in food crops because of the significant downstream resources needed to remove alkaloids in Nicotiana-based products. The ultimate solution may be a high-expressing engineered Nicotiana host devoid of alkaloid biosynthesis, but that option was not modeled in this study. The transient production facility is designed to produce 50 MT of purified thaumatin in spinach, annually, over 153 batches due to longer turnaround time required for S. oleracea compared to N. tabacum crops. Each batch has a duration of 67.8 days and a recipe cycle time of 1.94 days. The proposed base case upstream field production facility,4×8 flood tray displayed in Figure 1, consists of a 540 acre block of land divided into 22 plots, each of which is suitable for growing 318,000 kg FW of N. tabacum, carrying 477 kg of thaumatin, accounting for downstream recovery of 66.8%. It is assumed that the facility is located in a suitable climate where the growth of N. tabacum is attainable throughout the year, ignoring variations in production between batches . Each batch starts with direct seeding of transgenic N. tabacum plants in the field . The seeds are left to germinate for two weeks followed by vegetative growth for 3 more weeks post germination . A fertigation stream is applied to deliver the necessary nutrients for optimal plant growth. After a total of 35 days post seeding, a tractor sprayer applies 4900 L of a 4% ethanol solution to the plot’s crop, triggering the synthesis and accumulation of thaumatin in plant biomass. The plants are incubated for 7 more days, during which time they continue to uptake nutrients and express thaumatin. After 42 days from seeding, the batch is harvested through two mechanical harvesters and four hopper trucks at a rate of 17,000 kg/h and transported to downstream processing facility using a conveyer belt . The plot undergoes a turnaround period of three days for which the labor and equipment cost is included. No pesticides, fungicides, or herbicides costs are added due to the assumption that not enough growing degree days are accumulated during the batch cycle duration , for disease-causing organisms to be a concern. Transgenic Nicotiana benthamiana seeds are germinated in soilless plant substrate at a density of 94 plants per tray . Seedlings are then grown hydroponically, under LEDs, until reaching manufacturing maturity after 35 days. Induction occurs in a separate hydroponic reservoir, where plants are root drenched and sprayed with 0.01 L of 4% ethanol per kg FW plant tissue. The plants are left to grow for 7 additional days during the incubation period. N. benthamiana biomass is mechanically harvested at a rate of 54,000 kg/h and transported to downstream processing facility using a conveyer belt . The indoor upstream facility flowsheet can be found in Figure S1. The total facility footprint was calculated to be 83,000 m2 . The base case downstream processing facility is designed to purify and formulate 318.5 kg/batch of thaumatin with 98% purity.

A DSP batch starts with shredding plant biomass using two industrial shredders , each processing 40,000 kg of plant biomass/h. This step is designed to homogenize the leaves and stems to facilitate the extraction process. Shredded plant material is then mixed with an acetate buffer in a 0.8 L of buffer to 1 kg of biomass ratio. This step leverages stability of thaumatin at low pH to precipitate host plant proteins that aren’t stable under acidic conditions. The extraction buffer consists of 50 mM acetic acid and 150 mM sodium chloride mixture at a pH of 4.0. The resulting plant slurry is then fed into a screw press to separate most of the dry plant material. A screw press is recommended for this step because it minimizes the amount of extraction buffer needed by forcing out more plant sap with the increasing pressure inside the chamber. The crude extract stream obtained from the screw press unit is sent to three parallel P&F filtration units for initial clarification, each having a membrane area of 190 m2 . Furthermore, the model assumes the use of food-grade filtermembranes designed to include 10 filter sheets with decreasing particle retention size from 25 to 0.1 µm. The acetate buffer is applied once again as cake wash with a 0.2 L buffer to 1 L extract ratio. Diatomaceous earth is added to this step as a filter aid in a 6:100. The stability of thaumatin at low pH and high temperatures facilitates the precipitation of more host cell proteins as well as other undesired plant-derived compounds. Using seven heating tanks , the plant extract is then heated to 60 ◦C for 60 min. Following heat incubation, the stream is sent to a P&F filtration unit to capture the heat-precipitated proteins. It is assumed that a 90% reduction of N. tabacum total soluble proteins is attainable following the heat incubation and precipitation steps. Concentrating the thaumatin stream prior to the ultrafiltration/diafiltration step is necessary to avoid processing large liquid volumes ~573,000 L further downstream. It has been reported that thaumatin experiences a loss in sweetness when heated above 70 ◦C at a pH of 7.0; therefore, the product stream undergoes concentration by evaporation prior to neutralizing the solution since the protein can sustain higher temperatures at a low pH.The triple effect evaporation unit is designed to evaporate 90% of the water content in the stream at 109 ◦C, 77 ◦C, and 40 ◦C in the first, second, and third effect, respectively, over 4 h. The exiting stream is then neutralized with 1:1 molar ratio and mixed in V-101 for 30 min and sent to the P&F filtration unit to remove any precipitated materials. An additional 1.5% loss of thaumatin during this step is assumed. Because soluble impurities such as nicotine and other pyridine alkaloids are abundant in N. tabacum plants, a UF/DF step is necessary to eliminate small molecules. The UF/DF unit consists of 4 stacked cassette holders, each containing twenty 3.5 m2 cassettes.

The potential for a Mars mission in the early 2030s underscores the urgency of developing a road map for advantageous space bio-technologies.The replicative capacity of biology reduces mission launch cost by producing goods on-demand using in situ resources , recycling waste products , and interacting with other biological processes for stable ecosystem function . This trait not only lowers initial launch costs, but also minimizes the quantity and frequency of resupply missions that would otherwise be required due to limited food and pharmaceutical shelf-life on deep space missions. Biological systems also provide robust utility via genetic engineering, which can provide solutions to unforeseen problems and lower inherent risk . For example, organisms can be engineered on-site to produce a pharmaceutical to treat an unexpected medical condition when rapid supply from Earth would be infeasible . A so-called “bio-manufactory” for deep space missions based on in situ resource utilization and composed of integrated biologically-driven subunits capable of producing food, pharmaceuticals, and biomaterials will greatly reduce launch and resupply cost, and is therefore critical to the future of human based space exploration .The standard specifications for Mars exploration from 2009 to 2019 are not biomanufacturing-driven due to the novelty of space bioengineering. Here, we outline biotechnological support to produce food, medicine, and specialized construction materials on a long-term mission with six crew-members and surface operations for ∼ 500 sols flanked by two interplanetary transits of ∼ 210 days . We further assume predeployment cargo that includes in situ resource utilization hardware for Mars-ascent propellant production , which is to be launched from Earth to a mission site. Additional supplies such as habitat assemblies , photovoltaics , experimental equipment, and other non-living consumables will be included. The proposed bio-manufactory would augment processes for air generation and water and waste recycling and purification—typically associated with Environmental Control and Life Support Systems —since its needs overlap but are broader,grow hydroponic and drive a wider development of an array of ISRU, in situ manufacturing , food and pharmaceutical synthesis , and loop closure technologies .

Food, medicine, and gas exchange to sustain humans imposes important ECLSS feasibility constraints . These arise from a crewmember physiological profile, with an upper-bound metabolic rate of ∼ 11–13 MJ/CM-sol that can be satisfied through prepackaged meals and potable water intake of 2.5 kg/CM-sol . Sustaining a CM also entails providing oxygen at 0.8 kg/CM-sol and recycling the 1.04 kg/CM-sol of CO2, 0.11 kg of fecal and urine solid, and 3.6 kg of water waste within a habitat kept at ∼ 294 K and ∼ 70 kPa. Proposed short duration missions lean heavily on chemical processes for life support with consumables sent from Earth . As the length of a mission increases, demands on the quantity and quality of consumables increase dramatically. As missions become more complex with longer surface operations, biotechnology offers methods for consumable production in the form of edible crops and waste recycling through microbial digestion . Advancements in bio-manufacturing for deep space exploration will ensure a transition from short term missions such as those on the ISS that are reliant on single use-single-supply resources to long-term missions that are sustainable.Efficiency gains in a bio-manufactory come in part from the interconnection and modularity of various unit operations . However, different mission stage requirements for assembly, operation, timing, and productivity can lead to different optimal biomanufactory system configurations. A challenge therefore exists for technology choice and process optimization to address the high flexibility, scalability, and infrastructure minimization needs of an integrated biomanufactory. Current frameworks for biomanufacturing optimization do not dwell on these aspects. A series of new innovations in modeling processes and developing performance metrics specific to ECLSS biotechnology is called for, innovations that can suitably capture risk, modularity, autonomy, and recyclability. Concomitant invention in engineering infrastructure will also be required.An estimated ∼ 10,000 kg of food mass is required for a crew of six on a ∼ 900 days mission to Mars . Food production for longer missions reduces this mission overhead and increases food store flexibility, bolsters astronaut mental health, revitalizes air, and recycles wastewater through transpiration and condensation capture . Pharmaceutical life support must address challenges of accelerated instability [ ∼ 75% of solid formulation pharmaceuticals are projected to expire mid-mission at 880 days ], the need for a wide range of pharmaceuticals to mitigate a myriad of low probability medical risks, and the mismatch between the long re-supply times to Mars and often short therapeutic time windows for pharmaceutical treatment.

Pharmaceutical production for longer missions can mitigate the impact of this anticipated instability and accelerate response time to unanticipated medical threats. In early missions, FPS may boost crew morale and supplement labile nutrients . As mission scale increases, FPS may meet important food and pharmaceutical needs . A biomanufactory that focuses on oxygenic photoautotrophs, namely plants, algae and cyanobacteria, enhances simplicity, versatility, and synergy with intersecting life support systems and a Martian atmosphere has been shown to support such biological systems . While plant-based food has been the main staple considered for extended missions , the advent of cultured and 3D printed meat-like products from animal, plant and fungal cells may ultimately provide a scalable and efficient alternative to cropping systems . FPS organisms for Mars use must be optimized for growth and yields of biomass, nutrient, and pharmaceutical accumulation. Providing adequate and appropriate lighting will be a challenge of photo autotrophic-centric FPS on Mars . Developing plants and algae with reduced chloroplast light-harvesting antenna size has the potential to improve whole-organism quantum yield by increasing light penetration deeper into the canopy, which will reduce the fraction of light that is wastefully dissipated as heat and allow higher planting density . Developing FPS organisms for pharmaceutical production is especially complicated, given the breadth of production modalities and pharmaceutical need . Limited resource pharmaceutical purification is also a critically important consideration that has not been rigorously addressed. Promising biologically-derived purification technologies should be considered for processing drugs that require very high purity . Developing FPS growth systems for Mars requires synergistic biotic and abiotic optimization, as indicated by lighting systems and plant microbiomes. For lighting, consider that recent advancements in LED efficiency now make LEDs optimal for crop growth in extraterrestrial systems.The ideal spectra from tunable LEDs will likely be one with a high fraction of red photons for maximum production efficiency, but increasing the fraction of shorter wavelength blue photons could increase crop quality . Similarly, higher photon intensities increase production rates but decrease production efficiency. Understanding the associated volume and power/cooling requirement trade offs will be paramount to increasing overall system efficiency. For microbiomes, consider that ISS open-air plant cultivation results in rapid and widespread colonization by atypicaly low diversity bacterial and fungal microbiomes that often lead to plant disease and decreased plant productivity .

Synthetic microbial communities may provide stability and resilience to the plant microbiome and simultaneously improve the phenotype of host plants via the genes carried by community members. A subset of naturally occurring microbes are well known to promote growth of their plant hosts , accelerate wastewater remediation and nutrient recycling , and shield plant hosts from both abiotic and biotic stresses , including opportunistic pathogens . While SynCom design is challenging, the inclusion of SynComs in life support systems represents a critical risk-mitigation strategy to protect vital food and pharma resources. The application of SynComs to Mars-based agriculture motivates additional discussions in tradeoffs between customized hydroponics versus regolith-based farming,growing lettuce hydroponically both of which will require distinct technology platforms and applied SynComs.Our biomanufactory FPS module has three submodules: crops, pharmaceuticals, and functional foods . The inputs to all three submodules are nearly identical in needing H2O as an electron donor, CO2 as a carbon source, and light as an energy source, with the required nitrogen source being organism-dependent . H2O, CO2, and light are directly available from the Martian environment. Fixed nitrogen comes from the biomanufactory ISRU module. The submodules output O2, biomass, and waste products. However, the crop submodule chiefly outputs edible biomass for bulk food consumption, the pharmaceutical submodule synthesizes medicines, and the functional foods submodule augments the nutritional requirements of the crop submodule with microbially-produced vitamins . These outputs will be consumed directly by crew-members, with waste products entering the LC module for recycling. All submodules will have increased risk, modularity, and recyclability relative to traditional technological approaches. Increased risk is associated with biomass loss due to lowerthan-expected yields, contamination, and possible growth system failure. Increased modularity over shipping known pharmaceuticals to Mars derives from the programmability of biology, and the rapid response time of molecular pharming in crops for as-needed production of biologics. Increased recyclability stems from the lack of packaging required for shipping food and pharmaceuticals from Earth, as well as the ability to recycle plant waste using anaerobic digestion. At a systems integration level, FPS organism care will increase the crew time requirements for setup, maintenance, and harvesting compared to advance food and pharmaceutical shipments. However, overall cost impacts require careful scrutiny: crop growth likely saves on shipping costs, whereas pharmaceutical or functional food production on Mars may increase costs relative to shipping drugs and vitamins from Earth.Maintaining FPS systems requires cultivation vessels/chambers, support structures, plumbing, and tools. Such physical objects represent elements of an inventory that, for short missions, will likely be a combination of predeployment cargo and supplies from the crewed transit vehicle . As mission duration increases, so does the quantity, composition diversity, and construction complexity of these objects. The extent of ISM for initial exploration missions is not currently specified . Nevertheless, recent developments imply that ISM will be critical for the generation of commodities and consumables made of plastics , metals , composite-ceramics , and electronics as mission objects, with uses ranging from functional tools to physical components of the life-supporting habitat . Plastics will make up the majority of high-turnover items with sizes on the order of small parts to bench-top equipment, and will also account for contingencies . Biotechnology—specifically synthetic biology—in combination with additive manufacturing has been proposed an a critical element towards the establishment of off world manufacturing and can produce such polymeric constructs from basic feed stocks in a more compact and integrated way than chemical synthesis, because microbial bioreactors operate much closer to ambient conditions than chemical processes .

The versatility of microbial metabolisms allows direct use of CO2 from Mars’ atmosphere, methane from abiotic Sabatier processes , and/or biologically synthesized C2 compounds such as acetate, as well as waste biomass. A class of bio-plastics that can be directly obtained from microorganisms are polyhydroxyalkanoates . While the dominant natural PHA is poly , microbes can produce various copolymers with an expansive range of physical properties . This is commonly accomplished through co-feeding with fatty acids or hydroxyalkanoates, which get incorporated in the polyester. These co-substrates can be sourced from additional process inputs or generated in situ. For example the PHA poly-lactic acid can be produced by engineered Escherichia coli , albeit to much lower weight percent than is observed in organisms producing PHAs naturally. PHA composition can be modulated in other organisms . The rapid development of synthetic biology tools for non-model organisms opens an opportunity to tune PHA production in high PHB producers and derive a range of high-performance materials. Before downstream processing , the intracellularly accumulating bio-plastics need to be purified. The required degree of purity determines the approach and required secondary resources. Fused filament fabrication 3Dprinting, which works well in microgravity , has been applied for PLA processing and may be extendable to other bio-polyesters. Ideally, additive manufacturing will be integrated in-line with bio-plastics production and filament extrusion.Figure 4 depicts the use of three organism candidates from genera Cupriavidus, Methylocystis, and Halomonas that can meet bio-plastic production. This requires a different set of parameters to optimize their deployment, which strongly affects reactor design and operation. These microbes are capable of using a variety of carbon sources for bio-plastic production, each with a trade-off. For example, leveraging C2 feed stocks as the primary source will allow versatility in the microbe selection, but may be less efficient and autonomous than engineering a single organism like Cupriavidus necator to use CO2 directly from the atmosphere. Alternatively, in the event that CH4 is produced abiotically for ascent propellant , a marginal fraction of total CH4 will be sufficient for producing enough plastic without additional hardware costs associated with ISRU C2 production.

The product of NO2 ! was measured using the UV-Vis spectrophotometer at 540 nm. The content of H2O2 was determined after extraction by homogenizing plant tissues with 2 mL cold acetone . After centrifugation at 5000g at 4 ” C for 10 min, a 1.0 mL aliquot of the supernatant was mixed with 0.1 mL of 5% TiSO4 and 0.1 mL ammonia. After centrifugation, the titanium-peroxide complex pellet was resuspended in 3.0 mL of 2 M H2SO4, and absorbance was determined at 415 nm with a standard curve generated with known concentrations of H2O2.Plasma membrane integrity was evaluated by staining roots with Evans blue solution as in Yamamoto et al. with minor modifications. After the PPCP treatment, roots were stained with Evans blue solution for 15 min, and the stained roots were washed thoroughly with deionized water. The trapped Evans blue was released by extracting the roots in 5.0 mL of N,N-dimethylformamide. Absorbance of the supernatant was determined spectrophotometrically at 600 nm. The level of lipid peroxidation in roots and shoots of cucumber was measured in terms of malondialdehyde, which was determined according to the reaction with thiobarbituric acid as described in Yamamoto et al. . Historical staining for lipid peroxidation was conducted with Schiff’s reagent .For the measurement of reduced glutathione and oxidized glutathione , plant tissues were homogenized in 5 mL of cold 5% meta-phosphoric acid on ice. The homogenate was centrifuged at 12,000 g for 15 min at 4 ” C, and the supernatant was used for analysis of GSH and GSSG . A 0.5-mL aliquot of the supernatant was added to a reaction mixture containing 100 mM PBS , 0.2 mM NADPH and 1 mM 50 50 – dithiobis-2-nitrobenzonic acid . The reaction was started by the addition of 3 U of glutathione reductase, and absorbance at 412 nm was measured after 5 min. For GSSG, 2- vinylpyridine was added to the neutralized supernatant to mask GSH. Simultaneously,grow hydroponic the same volume of water was added for the total glutathione assay. The GSH concentration was obtained by subtracting the GSSG from the total GSH.Fresh plant tissue samples were frozen in liquid nitrogen, and homogenized in 50 mM PBS containing 1 mM EDTA and 1% PVP, with the addition of 1 mM ascorbate for ascorbate peroxidase.

The homogenate was centrifuged at 12,000 g for 20 min at 4 ” C, and the supernatant was used for the following enzyme assays . Protein content in enzyme extracts was determined by Coomassie brilliant blue G-250 with a standard curve using bovine serum albumin as the standard. Measurement of superoxide dismutase activity was carried out by inhibiting photochemical reduction of nitro blue tetrazolium . The assay mixture contained 50 mM PBS , 13 mM methionine, 75 mM NBT and 2 mM riboflavin. After addition of 100 mL of enzyme extract, the glass tubes were placed under light for 15 min, and then read at 560 nm. One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the reduction of NBT. To determine ascorbate peroxidase activity, a 200 mL aliquot of enzyme extract was added to the reaction mixture of 1 mM ascorbate and 0.3 mM H2O2 in 50 mM PBS. The absorbance changes were monitored at 290 nm for 3 min as ascorbate was oxidized, the enzyme activity was calculated using the extinction coefficient of 2.8 mM! 1 cm! 1 for ascorbate. Peroxidase activity was monitored by oxidation of 0.2% guaiacol using 0.3% H2O2 after addition of 50 mL enzyme extract. The enzyme activity was calculated using an extinction coefficient of 26.6 mM! 1 cm! 1 . Glutathione S-transferase activity was determined in 2 mL of a reaction mixture containing 50 mM PBS , 1 mM 1-chloro-2,4-dinitrobenzene , 5 mM GSH and 100 mL enzyme extract. The GST activity was measured spectrophotometrically at 340 nm based on GSH-CDNB adduct synthesis using extinction coefficient 9.6 mM! 1 cm! 1 for GSH-CDNB.To evaluate the sensitivity of cucumber plants to PPCPs, an initial dose-response experiment was carried out. Statistical analysis showed no significant difference in the biomass of plants grown in 0.5, 5 ng L! 1 PPCP-spiked and control solutions . However, treatment with PPCPs at 50 mg L! 1 progressively caused an increase of leaf necrosis . The level of chlorophyll a and chlorophyll b decreased with increasing PPCP treatment rates . Meanwhile, root activity decreased by 15.4% and 28.2% after exposure to 5 and 50 mg L! 1 PPCPs, respectively, as compared with the control .Previous studies showed that reactive oxygen species were produced when plants were exposed to xenobiotics .

However, little is known if exposure to PPCPs would induce intracellular ROS production in higher plants. It was found that even at 0.5 mg L! 1 , PPCPs induced H2O2 accumulation in both shoots and roots, and the maximum elevation occurred at the highest concentration , where H2O2 levels were about 3.0- and 3.2-fold higher in shoots and roots, respectively, than those from the control treatment . Additionally, O2 .- did not increase significantly after exposure to PPCPs at 0.5 mg L! 1 , which could be due to the fast conversion of O2 .- to H2O2.Analysis of Evans blue uptake and malondialdehyde content showed that PPCPs caused oxidative damage to the plasma membrane and lipid fraction in plant seedlings; however the damage was less pronounced in the leaves. A significant increase in Evans blue uptake was found at 50 mg L! 1 PPCPs in the root , and histochemical staining clearly indicated that cell death occurred in the root tip that is the most active and sensitive region of the root. Lipid peroxidation measured as MDA increased in all of the stressed plants, and its content was higher in roots than in shoots in all of the PPCP treatments . These results were further confirmed by histochemical analysis using Schiff’s reagent to detect lipid peroxidation in plants .After 7 d of cultivation, SOD showed the maximal activity in roots after exposure to PPCPs at 5 mg L! 1 and decreased thereafter . Ascorbate peroxidase showed a dose-dependent response, increasing about 2.0- and 1.1-fold in roots and shoots, respectively, after exposure to PPCPs at 50 mg L! 1 . Peroxidase is among the enzymes with a potential role in the detoxification of a variety of xenobiotics, and GSTs often detoxify exogenous compounds by conjugation with GSH. The total POD and GST activities both increased appreciably after exposure to PPCPs. The GSH content increased in leaves after exposure to PPCPs, while the root showed a maximal GSH content at 0.5 mg L! 1 PPCPs, followed by decreases thereafter . The decreases of GSH in roots may be due to GSH serving as an antioxidant for preventing oxidative damage, and also acting to detoxify PPCPs by conjugation. The GSSG content displayed little change when PPCP concentrations were low . However, when the PPCP concentration was increased to 50 mg L! 1 , there was a significant increase in GSSG content .Results from the present study illustrated the physiological, biochemical and molecular mechanisms involved in the detoxification of PPCPs in plants by considering especially homeostasis of ROS and anti-oxidant metabolism.

The results clearly showed that PPCP-induced morphological indicators changed at elevated PPCP concentrations , and the impact was more pronounced in roots than shoots. The enhanced sensitivity of roots to PPCP toxicity may be due to the greater accumulation of PPCPs in the root . Similar observations were previously reported in alfalfa, lettuce,and pepper after PPCP exposure . In addition, Christou et al. suggested that PPCPs in a mixture displayed a different uptake pattern compared with that when exposed individually. The present study also showed that roots consistently accumulated PPCPs to a higher level than shoots when exposed to mixed PPCPs . In shoots, growing lettuce hydroponically the relative accumulation of individual PPCPs differed somewhat from that in Wu et al. , where carbamazepine and diazepam were found at the highest levels, followed by meprobamate and trimethoprim. The discrepancy between the studies may be attributed to the different growth conditions, plant cultivars, and sampling intervals. Acetaminophen, however, was not detected in the plant tissues , likely owing to its rapid metabolism after uptake . Recent studies showed that contact with PPCPs was capable of inducing a complex set of physiological responses in higher plants . Generally, contents of leaf pigments, including chlorophyll and carotenoids, provide valuable information about the physiological status of a plant. Here, a clear leaf necrosis and reduction in contents of chlorophyll and carotenoids were observed at higher PPCP concentrations . Findings from this and previous studies together suggest that PPCPs may significantly affect plant growth. Prior to the induction of whole plant morphological effects, stress may also lead to physiological, biochemical and molecular changes within the plant. However, little information is available about the toxic effects in plants from a mechanistic perspective. A direct result of stress-induced cellular changes is the enhanced ROS accumulation, consequently imposing oxidative stress to bio-molecules . Although additional research is needed to establish oxidative stress as the primary mechanism of PPCP toxicity to higher plants, it is clear that oxidative stress is involved in the development of PPCP-induced toxic symptoms. In this study, changes in ROS levels were observed in comparison to the control after exposure to PPCPs , and the response occurred at much lower concentrations than that for morphological effects. Overproduction of ROS can cause cell damage and is the final consequence of oxidative stress. In the current study, the increase in ROS production coincided with the increase in membrane damage and lipid peroxidation in the cucumber plants, indicating the presence of oxidative stress. In a previous study, exposure to 10 mg L! 1 of diclofenac, sulfamethoxazole, trimethoprim or 17a-ethinylestradiol did not induce significant lipid peroxidation in alfalfa leaves . Exposure to mixed PPCPs was found to exacerbate cytotoxicity to a rainbow trout gonadal cell line as compared to exposure to individual compounds . These results indicated that studies using individual PPCPs might underestimate the actual environmental impacts of trace organic contaminants that usually occur as mixtures. During the period of time in which cucumber plants were exposed to the PPCP mixture, an overall induction of enzymatic and non-enzymatic antioxidant systems was observed. Superoxide dismutase constitutes the first line of defense against ROS, which can dismutate O2 .- into the more stable H2O2 . In this study, the root experienced more significant oxidative damage as a result of greater ROS accumulation than shoots, and elevated activities of SOD were observed in treatments below 5 mg L! 1 of PPCPs .

Above the 5 mg L! 1 treatment rate, SOD activity in roots was significantly inhibited. In a previous study,increased SOD activity was detected after exposure to paracetamol , while decreased SOD activity was found under triclosan and galaxolide stress in wheat seedlings. A possible explanation for the decreased SOD activity may be that the oxidative stress exceeded the capability of the enzymatic machinery. It must be noted that there exist three forms of SOD isoenzymes: copper/zinc containing SOD, manganese containing SOD and iron containing SOD, which are all localized in different cellular compartments. Further research should take into account the specific roles of different SOD isoenzymes from different cellular compartments after exposure to trace xenobiotics. Using ascorbate as a reductant, H2O2 was reduced to water by ascorbate peroxidase , the major component of the ascorbate-glutathione cycle . Thus the increase in APX activity in the PPCP treated cucumber plants may be related to the functioning of the ascorbate-glutathione cycle that detoxifies H2O2 thereby preventing further damage. This was consistent with previous observations in Brassica juncea with acetaminophen treatment . It has been shown that APX may be responsible for the fine modulation of ROS for signaling, whereas CAT might be responsible for the removal of excess ROS during stress. The significantly increased catalase activity with increasing PPCP concentrations reaffirmed that the cucumber plants experienced serious oxidative stress when in contact with the PPCP mixture. Beside anti-oxidation, another key role of POD and GSTs is their ability to inactivate toxic compounds . For example, Agostini et al. demonstrated the capacity of peroxidases to degrade the pesticide 2,4-dichlorophenol in a cell culture of Brassica napus. Xia et al. suggested that the induced activity of GST by chlorpyrifos indicated formation of glutathione S-conjugates to detoxify the insecticide in plants.

High-Frequency Nitrate required separate injections of monoammonium phosphate and Ca2 in order to avoid precipitation of Ca32. All fertigations lasted 24 h, starting and ending at 7:00 AM, and injection of the non-MAP fertilizers took place between 11:00 PM and 3:00 AM. The soil at the site is classified as a Milham sandy loam derived from granitic and sedimentary alluvium, deep and well-drained. In an existing experiment , five blocks are laid out as a randomized complete blocks design, with eight strip-plot treatments of 15 trees. Treatments assessed had K applications of 224.17 kg ha 1 and P applications of 74.46 kg ha 1. Four blocks were studied for N2O surface emissions, and three of these included soil gas and solute measurements.Static gas chambers were used, of 20.3 cm diameter PVC piping 11 cm high placed over PVC collars installed 5 cm deep directly after fertigation, with aluminum insulation, rubber bottom seals, and manual internal fans. Two drippers were sampled in each plot per event, and different drippers were studied at each fertigation event; those studied were situated about 2 m from the nearest tree trunk, to represent average temperature, moisture and root presence. Chambers were positioned at 4 distances from drippers, along a transect into the center of the alley, at 0, 20, 50 and 90 cm. The maximum extent of the wetting zone at surface rarely exceeded 60 cm from drippers. Emissions from each collar were used to describe emissions from rings around the dripper,hydroponic growing systems with borders midway between distances. Three gas samples were taken over 10-min intervals. To calculate fluxes the ideal gas law was used as by Parkin and Venterea including ambient temperatures from a CIMIS weather station 2.5 km from the orchard, and an elevation of 120 m, corresponding to 0.985 atm of pressure.

Nonlinearity was not apparent with these short flux times.Surface emissions of N2O were expected to derive almost entirely from N fertigations and the first subsequent irrigations in the Standard treatment , with strong seasonal dependence that informed the sampling schedule . Output rates of drippers were checked. Gas samples were taken around peak emissions on the first day after fertigation, and thenceforth from 8:00–14:00 to represent daily averages. Emissions for statistical comparisons were the sums of observed measurements per plot, considered as describing a whole day. However, in order to be comparable to Standard, emissions from the HF applications required interpolations of unmeasured events, which were averages of each measured event’s emissions with the previous or the next. Previous experience of the patterns of emission demanded a different approach in order to estimate total growing season emissions per treatment. First day emissions were calculated assuming that N2O emissions increased linearly from baseline at 7 A.M. until peak ambient temperature, which was close to the time of measurement, then a Q10 adjustment was made using hourly ambient temperatures for the remainder of the day and night . Both of these manipulations sought to avoid overestimation. Q10 values were derived from seasonal diurnal measurements. To estimate the emissions over two additional days after sampling, an exponential curve of decline was fit to measured points averaged by treatment, as emissions = y0 + a, which had the best fit to data. Baseline rates were calculated by treatment to describe time periods when the upper soil was dry, starting 5–7 days after each fertigation, where the curve of decline ended. These were extended until the first day of fertigation for emissions calculations.Soil gas, moisture, temperature and N-distribution profiles were assessed in 3 experimental blocks. 1/800 OD brass tubing was used to take gas samples from depths of 5,10, 15, 20, 30, 45, 60 and 80 cm in the soil, at 20 cm from drippers.

The tubing was reinforced with steel wire, for insertion, after which the wire was withdrawn and replaced with a second, close-fitting internal brass tube of 3/ 3200 OD to minimize internal air volume. Tubes were topped with septa consisting of Nalgene tubing of 1/800 inner diameter injected with a dual plug of silicone and butyl rubber; butyl rubber blocks N2O diffusion while silicone maintains a good seal through syringe use. A volume of air corresponding to the tube volume was extracted before sampling. 5-mL gas samples were taken from 5 and 10 cm depth, and 10-mL samples from 15 cm and greater depths. For samples at 5 and 10 cm, tubes with closed ends and lateral cuts near their tip were inserted at 45 to the soil surface, allowing the surface soil to be tamped down at the entrance point without compacting the soil to be sampled. On this sandy loam, samples could be taken 5 h after irrigation without apparent resistance due to soil water. Gas samples were stored in glass vials with silicone sealant applied over the butyl septum before evacuation to 45 mTorr. All samples were analyzed for N2O using a 63Ni electron capture detector , and larger vials were tested for CH4 and CO2 using a flame ionizing detector , both on the same gas chromatograph . Simultaneously, a custom-built thermo couple probe of diameter 3/1600 took soil temperature at 5, 10, 15, 20, 30, 45 and 60 cm . A neutron probe provided measurements of volumetric water content at 20, 30, 45, 60 and 90 cm, using aluminum access tubes, and a soil moisture count curve fitted to measurements from this soil . A Theta probe was used to measure uvol in the upper 10 cm. Texture and bulk density were tested at each site at 0–20, 20– 30, 30–45, 45–60 and 60–90 cm. To compare the vertical distribution of dissolved NH4 + and NO3
in the soil after fertigations, high-flow ceramic soil solution samplers were installed at 15, 30 and 60 cm depth, 20 cm from the dripper laterally. Evacuation down to 600 mm Hg of vacuum was accomplished with an automotive brake pump, and samples were collected 6 h later, but on the third days after fertigation, samples adequate for analysis were only extracted from 38% of samplers, with fewer in summer, and very few in general from 15 cm depth.

Beginning in June, exchangeable NH4 + and NO3
were also assessed using soil samples from 0 to 5, 12.5–17.5 and 27.5–32.5 cm on each day. Approx. 20 g fresh weight soil was extracted in 80 mL of 2 M KCl, with one hour of shaking. These and the samples of soil solution were run for colorimetric analysis of NH4 + and of NO3.N2O concentrations by depth were combined with moisture readings, bulk density and texture to estimate the soil diffusion coefficient for nitrous oxide using the Unified Diffusivity Model— Buckingham Burdine Campbell . The Campbell parameter “b” was estimated using the clay fraction . Net production or consumption of N2O was estimated for each measured point in the soil except the deepest, using the one-dimensional mathematical model of Yoh et al. and field measurements described above.The soils were collected in late August after a month of irrigations without fertilizer. One replicate per plot was used, and results analyzed as an RCB consistent with the field experiment. pH was tested with 1 g:2 mL slurry in deionized distilled water, using samples from 0 to 20 cm depth at 20 cm from drippers. Treatment effects on nitrification rate were tested with the same samples using an ammonium oxidation assay citing Berg and Rosswall. Treatment effects on denitrification were tested with the same samples using a modified denitrification enzyme activity procedure with N2 as a flushing gas, with glucose, and without chloramphenicol. 5% head space acetylene, generated from calcium carbide,hydroponic farming was added to a round of duplicate samples; in the first test on the soils, 10% head space acetylene had appeared to impede the entire denitrification process. Head space gas samples were nonlinear by 60 min, so data from 20 and 40 min were used. The microcosms were also tested at 24 h to describe Denitrification Potential ; by 48 h, N2O declined in some microcosms. Soils from UAN treatments were tested for N2O production over a 36-h incubation at 3% O2 and 50% WFPS according to the method of Zhu et al. with some modifications. Samples of 15 g dry weight underwent a 24-h pre-wetting at 25% WHC, then were put into 180 mL Erlenmeyer flasks with butyl rubber stoppers and twice evacuated to 1000 mTorr and flushed for a minute with N2. Flasks were then injected with O2 for a 3% O2 head space, then fertigated to 50% WHC with NH4 + or NO3, with and without acetylene, and pure water . 5 mL head space samples assessed N2O production rates through measurements at 5 and 15 h; by 25 h they had declined in NH4 + microcosms, although N2O production continued.Estimated growing season N2O emissions were greatest from HF UAN fertigation, followed by Standard UAN, and lowest from the HF NO3 treatment, but comparisons between the two UAN and the two HF treatments only showed a significant difference between the HF systems . The failure to find differences in the Standard UAN—HF UAN comparison may have been due to high variability within and between blocks for the Standard UAN plots; in the UAN comparison the significance of blocking was p < 0.69 vs. p < 0.12 in the HF comparison. CVs over the measured period were 52% in Standard, 24% in HF UAN and 27% in HF NO3. Emissions from the irrigations following Standard UAN fertigations totaled 26% of the treatment’s growing-season emissions. In previous work, little effect had been seen in subsequent irrigations, even in loam soils . In the HF treatments such residual effects were indistinguishable from effects of the next fertigation.

There were important seasonal effects; higher temperature caused earlier and higher peaks of N2O emission, as well as higher event totals .The average precipitation at the site is 18 cm. The winter of 2013–2014 which followed our work was the driest on record in California; the experimental site received only 2 cm of precipitation and so was not monitored for emissions. However, previous work in the orchard , monitoring UAN-derived N2O emissions from static sprinklers at similar application rates, included a winter with 12 cm precipitation. Of the 730 g N2O-N yearly average emission, those data allowed an estimate that 18.5 g N2O-N ha
1 were emitted in winter. The latter quantity, if it had been seen in all the treatments studied here, would increase estimated N2O emissions by 3.7–8.5%. At the same time, Mediterranean perennial crop systems usually see more winter rainfall, and are capable of supporting cover crops in the winter. In such cases, post-season emissions can be expected to constitute a larger portion of the total and cover crop management, can be an important control, as studied in vineyards .To analyze the transport and fate of applied fertilizer, NH4 + and NO3 in soil solution were sampled by suction lysimeters and in soil by KCl extraction . Urea from Standard UAN was hydrolyzed to NH4 + and further oxidized to mobile NO3 in the soil profile, which by the third day led to 14x higher total NO3
concentrations at 60 cm depth under Standard UAN than under HF NO3 . Low dissolved NO3 in the HF NO3 treatment may be ascribed to higher diffusion to outer parts of the wetting zone , greater root uptake of nitrate through mass flow soon after fertigation, and the observed high retention of nitrate in the upper part of the soil . NH4 + in solution near surface agreed with differences in application strength , but such differences were not apparent in KCl-extractable NH4 + , which described much greater quantities.In general, a better understanding of the spatial origin and fate of N2O under different conditions should lead to improved fertilization and fertigation practices. In this investigation, net N2O production by depth was calculated from soil gas concentrations, soil diffusion parameters, temperatures and water contents  along a one-dimensional vertical profile at 20 cm from the dripper. A 1-D model cannot give a mass balance for N applied with water from a point source; unaccounted lateral diffusion will probably amount to a net loss and lead to underestimates of production. However this problem was judged to be ameliorated by conditions observed before the experiment.

Plants were harvested 45 days after germination when the differences in root characters could be efficiently measured among different recombinant lines. The data for different shoot characters were recorded and the tubes containing roots were stored at 4°C until processing. The roots were washed and recovered without damage using a Xotation technique . The shoot characters measured were longest leaf length , maximum width of the longest leaf , leaf area , plant height , number of tillers , and dry shoot biomass . The root characters measured were number of roots greater than 30 cm , longest root length , total length of roots greater than 30 cm , shallow root weight , deep root weight , dry root biomass , and root biomass to shoot biomass ratio .The shoot and root data were subjected to the analysis of variance for each year .The overall rooting ability of each genotype was calculated by ranking each genotype for individual root traits in each replication in each of the 3 years. Genotypes with the highest values were ranked 5 and those with the lowest values were ranked 1. Subsequently, all the ranks of root characters for each genotype were summed providing a measure of the rooting ability index for each genotype at different replications . The genotypic rank sums averaged across the years were subjected to the non-parametric Quade analysis developed for randomized complete block designs to differentiate genotypes for overall rooting ability.Phenotyping of recombinants was necessary to test the general applicability of the consensus 1RS-1BS map in locating a 1RS region showing better rooting ability. Various shoot and root traits were studied using two parents and three recombinants covering the whole 1RS-1BS map. There were significant differences among years for all shoot characters measured, except for the maximum width of the longest leaf .

Significant differences were found among the genotypes for shoot characters, except for maximum width of the longest leaf and leaf area. Genotype £ year interaction was significant only for the number of tillers per plant . This interaction was due to changes in the magnitude of the genotypic means across different years rather than changes in ranking of the means. Therefore, shoot characters in Table 2 are represented by means averaged across years. Pavon 1RS.1BL was taller,growing tomatoes hydroponically had longer leaf length and a greater root-to-shoot ratio than Pavon 76. Since Pavon 1RS.1BL and Pavon 76 had similar shoot biomass , greater root-to-shoot biomass ratio in the former genotype indicated greater root biomass in 1RS.1BL than Pavon 76 . Leaf area in 1B + 2 was the highest followed by Pavon 1RS.1BL . Despite significant differences observed among the genotypes for shoot characters, the differences were relatively small, except for the shoot biomass in 1B + 2 in the third year which was due to greater number of tillers per plant and plant height . Otherwise, the rest of the genotypes did not show large differences for combined as well as for individual years for most of the shoot traits. There were significant differences among years for all root characters measured . Significant differences were found among the genotypes for all the root characters measured, except for longest root length. The genotype £ year interaction was significant only for the number of roots greater than 30 cm. Therefore, means for root characters were averaged across 3 years . The number of roots greater than 30 cm and root biomass in Pavon 1RS.1BL was greater than those in Pavon 76, which confirmed the results reported earlier . The number of roots greater than 30 cm in Pavon 1RS.1BL, 1B + 2 and 1B + 38 was similar, but greater than those in Pavon 76 and T-14 . A similar trend was observed for the total length of roots greater than 30 cm. Shallow root weight was highest in Pavon 1RS.1BL followed by 1B + 2 , and 1B + 38 . The lowest shallow root weight belonged to T-14 . Deep root weight in Pavon 1RS.1BL, 1B + 2, and 1B + 38 were similar, but greater than those in Pavon 76 and T-14.

The greater dry root biomass observed in Pavon 1RS.1BL as compared to Pavon 76 was because of a combination of greater shallow and deep root weight in the former than in the latter genotype .Genetic mapping in wide hybrids has been performed for many plant species, particularly in diploids including barley , chickpea , lentils , onion , Nicotiana species , and tomato . In the early days of genetic mapping with molecular markers, wide hybrids were the approach of choice, for it guaranteed much higher levels of polymorphism than in intra-specific hybrids. Despite notable instances of non-Mendelian segregation and skewed distribution of recombination, wide hybrids produced useful genetic maps with higher marker saturation at considerably less cost and eVort . The use of wide hybrids in allopolyploids is more complicated than in diploids as allopolyploids tend to have some kind of chromosome pairing control system in place that limits crossing over to homologues. Hence, homoeologous pairing may be low or even non-existent. In this study, recombinants produced by crossing over were used. These recombinants were selected from a population of 103 such recombinants developed by Lukaszewski . The entire recombinant population was selected from a population of ca. 17,000 progeny with the Ph1 system disabled. If the assumption is made that crossing over in the Ph1+ and Ph1¡ wheats is the same, and they appear to be, except for the absence of multiple crossovers per arm , then the sample analyzed here would be equivalent to a population of 136 back cross progeny, a sensible number giving the maximum resolution level of 0.7 cM. The physical distribution of 68 recombinant breakpoints used in the present study is shown in Fig. 3. With a total of 20 physical and molecular markers, we constructed the combined genetic map of 1RS-1BS recombinant breakpoints in Pavon 76 background. The genetic map produced here has an average density of 2.5 cM. The maps shown in Figs. 2 and 4 represent only the physical 40% of the distal ends of the 1S arms as no recombination took place in the proximal 60% of the arm. Any loci in this region would show complete linkage with the centromere.

The advantage of using these recombinant breakpoints in developing a genetic map is in their physical differences from one another. Here, we used two methods to map. Firstly, we used the recombinant breakpoints to develop a genetic map of the arm, and secondly, we used the map to classify the breakpoints. Each interval or genetic distance between two markers is represented by one or more recombinant breakpoints that most often include both reciprocal configurations of the chromosomes. This physical separation of the breakpoints further refines the genetic map to 0.7 cM level. The reciprocal nature of chromosomes with breakpoints in any given interval will permit allocation of identified genetic loci to very narrow physical segments of rye chromatin. This may be a useful tool in dissecting the genetic components of a particular gene of interest or an important agronomic trait present on 1RS. Somers et al. detected an average of one single nucleotide polymorphism per 540 bp ESTs in wheat and demonstrated the reliability of designing PCR primers for locus-specific amplicon production. In this study, we targeted a single chromosome arm, 1BS,hydroponic growing supplies and expected that most of the 91 EST loci allocated to this arm could be converted to 1BS locus-specific amplicon markers. Given that the recombination was intergeneric, we expected that most of these markers would be polymorphic between wheat and rye, and so would produce a highly saturated map. However, only four PCR-based markers showed reliable polymorphism, though we cannot rule out sequence-based polymorphisms that we could not detect. The sequences of the 91 EST loci on wheat 1S arms were used to search rice orthologs using TIGR rice version 4.0 gene models, and 54 of these sequences had rice blastx hits of e-20 or better, comprising 52 rice gene models. Of the 52 models, were clustered on chromosome 5 of rice, which is known to besyntenic to chromosomes 1S of Triticeae . In the present study, two gene models with genebank accessions viz; BE497177 and BF483588 were found to be represented as markers, Xucr_4 and Xucr_8, respectively . The rice annotation of Xucr_8 is described as a drought induced 19 protein which can be helpful in studying drought. However, a high frequency of insertions and deletions in rice and maize make these genomes more Xuid at the DNA sequence level than indicated by Southern analyses . Testing a total of 16 SSR and eSSR markers yielded four polymorphic amplified products, which were comparable to the previous studies . Röder et al. found different band sizes for cultivar and synthetic wheat, though the differences were comparable. Only 1 out of 9 eSSR markers could be mapped on 1BS-1RS. This low success rate may be due to low polymorphism between wheat and rye 1S arms. Whether this indicates high conservation of genic regions across species we cannot tell at this point. Peng and Lapitan showed amplification of 15 wheat eSSR markers in rye as well as barley. They detected polymorphism between wheat and barley but did not mention polymorphism between wheat and rye. A comparison of the current 1RS- 1BS map with the SSR and eSSR maps of the 1B chromosome shows a good agreement in marker order, location, and relative positions in its distal part, regardless of the projection mode used. A similar approach was used to generate a genetic map of ph1b-induced 2R-2B intergeneric recombinants, with a similar success , validating the wide-hybrid approach to mapping. The integration of physical and molecular markers in the present 1RS-1BS map also provided the alignment of recombinant breakpoints over each map interval.

This information offers great potential to study agronomic traits affected by the introduction of alien 1RS chromatin into wheat. Our working hypothesis was if a 1B + line shows some specific trait then this trait should be absent in its complementary T-line, and vice versa. To check the applicability of this concept, we looked at different root characters in wheat. Studies during the past few years showed an increased root biomass in wheat with the alien 1RS chromosome arm over standard spring wheat Pavon 76 and found a positive correlation of increased root biomass with grain yield . Recent studies in rice and maize have also correlated the QTLs for root traits with QTLs for yield under Weld conditions . A major limitation to study roots is the difficulty in making observations as the process is very laborious and time consuming. In the present study, we tested only three recombinant chromosomes with breakpoints selected to divide the recombining portion of the arm into three segments of roughly similar lengths. The experiments were conducted in 3 years at the same months of the year, in replicated trials, to determine the magnitude of the genotype £ year interaction and the repeatability of results from the sand-tube technique. Significant variation was observed from year to year , with considerably higher means in the third year. This might have been due to relatively lower temperatures in the third year providing better conditions for vegetative growth. The significant genotype £ year interaction observed for number of roots greater than 30 cm was due to only one genotype, T-14, producing more roots only in year 2. Otherwise, other genotypes showed similar trends for this character across the 3 years. The lack of signiWcant genotype £ year interaction for most of the root characters examined indicated high repeatability for these root traits. Quade analysis used on rank sums separated the five genotypes in two groups viz., Pavon 1RS.1BL, 1B + 2 and 1B + 38 containing distal rye chromatin with higher rooting ability and Pavon 76 and T-14 lacking the distal rye segment with lower rooting ability . Overall, the wave genotypes examined showed relatively small variation for shoot characters but they differed in root characteristics including root biomass.