Tag: greenhouse vertical farming

To date, B. cinerea biocontrol products are mostly Bacillus subtilis-based, but their use in commercial strawberry production is limited because of their insufficient applicability in the field or supply chain . Nevertheless, there is social and scientific interest in using biocontrol against B. cinerea as an alternative to chemical pesticides. Isolates of Colletotrichum gloeosporioides, Epicoccum purpurascens, Gliocladum roseum, Penicillium sp., Trichoderma sp. have displayed high efficiency in controlling B. cinerea and were reported to reduce grey mould incidence on strawberry stamens by 79%–93% and on fruit by 48%–76% . Interestingly, in some experiments, the efficiency of biocontrol by these organisms exceeded the efficacy of control via the fungicide captan. Similar results were obtained for other microbes, such as the yeasts A. pullulans and Candida intermedia , the filamentous ascomycete Ulocladium atrum , or the bacterium Bacillus amyloliquefaciens . Biocontrol via microbes can work via different modes of action, including competition for nutrients, secretion of antibiotic compounds and induction of host defence mechanisms like the up-regulation of chitinase and peroxidase activity . Because biocontrol of B. cinerea relies on a variety of mechanisms, the most significant effects are observed when different organisms are applied in combination . As alternative to applying living microbes, use of extracts or volatiles derived from biocontrol microbes has been suggested . Use of non-synthetic antifungal substances, like phenol-rich olive oil mill wastewater,growing blackberries in containers has also been reported to control B. cinerea growth in vitro and on strawberries . However, these approaches are not implemented on a commercial scale due to high costs compared to the conventional B. cinerea control.

It is common practice to handpick strawberries and place them into clamshells in the field, in order to reduce wounding and bruising of the fruit. Rapid and constant cooling of strawberries at temperatures below 2.5 ºC is another critical strategy to reduce or inhibit reactivation of B. cinerea quiescent infections . Often, strawberries are also stored in modified atmospheres, which are generally low in oxygen and high in carbon dioxide to slow down metabolic processes, senescence and fungal decay . Relative humidity during storage is usually kept around 85%–90% to prevent dehydration of fruit, but limit fungal growth . Novel post harvest treatments of strawberries have been suggested to prevent B. cinerea infections during storage. Examples are edible fruit coatings of chitosan, silk fibroin or methylcellulose that prevent water loss and can include antifungal compounds . MeJA treatment to induce fruit defence mechanisms , ultraviolet and visual light treatment , enrichment of storage atmosphere with chlorine or ozone , and soft mechanical stimulation have also been tested as alternative treatments. Most of these approaches are still in an experimental stage or not yet adaptable to commercial settings.Significant phenotypic variation of incidence or severity of grey mould has been reported; however, F. x ananassa genotypes appear to be universally susceptible and complete resistance has not been observed . Substantial genotypic variation has not been documented and the heritability of resistance to B. cinerea is unknown. Mild phenotypic differences in fruit resistance levels reported in various post harvest studies indicate that genetic variation for resistance may be limited and that its heritability is low. A contributing factor is the intrinsic characteristics of the pathogen, its broad host range, diverse ways of infection and necrotrophic lifestyle, which explain the absence of a gene-for-gene resistance of strawberry to B. cinerea .

Therefore, breeding for escape and tolerance, which includes physiological and biochemical traits, is a more practical option . While limited in scale and scope, earlier studies strongly suggest that the incidence and progression of B. cinerea infections differ between cultivars with soft fruit and those with firm fruit . Hence, previously reported differences amongst cultivars could be the result of the pleiotropic effects of selection for increased fruit firmness and shelf life and the associated developmental and ripening changes, as opposed to direct genetic gains in innate resistance to the pathogen. As discussed, fruit firmness is an important trait associated with resistance to B. cinerea . The strawberry germplasm displays natural variation for fruit firmness and developing cultivars with firmer fruit is an important aim in breeding programmes . Changes in flower morphology could also enhance tolerance to B. cinerea. In strawberry, most B. cinerea infections in fruit appear to originate from primary infections of flowers or secondary infections caused by direct contact with infected flower parts . It was reported that removal of stamen and petals result in lower grey mould incidence . Faster abscission of flower parts, especially petals, has the potential to aid the escape of strawberries from B. cinerea infections . Similarly, plants with pistillate flowers have a lower grey mould incidence in fruit . B. cinerea growth inhibition in stamens is reported to vary within the strawberry germplasm, potentially due to differences in their biochemical composition . Similarly, antifungal compounds in fruit can prevent or limit B. cinerea infections. Several reports indicate that anthocyanin accumulation contributes to tolerance of strawberries to B. cinerea . Anthocyanins do not just improve tolerance to grey mould but also provide health benefits . Breeding for higher anthocyanin content in strawberries is possible and facilitated by existing variation in the germplasm . Inducing anthocyanin accumulation in flowers could also help to limit flower infections. As breeding for higher B. cinerea tolerance will be tedious and likely will not result in complete resistance, complementary approaches should be considered.

Currently, no genetically modified strawberry cultivars are commercially grown; however, several reports show great potential to improve tolerance to grey mould via trans- or cis-genesis. For example, the expression of chitinases or PGIPs from other organisms in strawberries can prevent or slow down fungal infections . Another potential transgenic approach is to increase fruit firmness by altering the expression or activity of pectin degrading enzymes, such as PL or PG . The existing natural variation of PL expression levels and activity in the cultivated strawberry germplasm could be used for cisgenic approaches. Increasing phenolic levels in strawberries by genetic modifications can also be explored as the transcription factor MYB10 was identified as a regulator of anthocyanin levels in strawberries ; Medina-Puche et al., 2014). Transgenic plants with ectopic over expression of MYB10 show elevated anthocyanin levels throughout the entire plant ; however, the resistance of these plants against B. cinerea have not been tested. In summary, these novel breeding approaches should be supported by integrative management strategies including horticultural and agronomic practices, and potentially biocontrol, to achieve maximum control of the disease.Plant gene editing may be the greatest innovation in plant breeding since the Green Revolution. It has already been used to make discoveries in plant biology and has a profound potential to create new crops with desirable characteristics. There are already exciting developments,square pot which show that gene editing may be able to live up to expectations and can be used to produce novel plant phenotypes that would improve agricultural production. Most authorities estimate that food production will have to double in the next 50 years to keep pace with population growth. The focus on global food security, however, is usually on starch-rich cereals and ignores or underestimates the vital importance of horticultural crops. These perishable commodities are often nutrient-dense with bioactive phytochemicals, the consumption of which is needed for a healthy and thriving population. However, an uncomfortable fact is that in addition to losses that may result from disease, drought, extremes of temperature, and other environmental stresses experienced in the field, an additional 25–40%—an average of 33%—of all fruit and vegetables produced globally are never eaten after harvest. This estimate still does not illustrate the extreme losses that can occur in some developing countries, which may be as high as 75%. Current worldwide horticultural crop production is insufficient to meet human nutritional requirements, making post harvest loss and waste all the more unsustainable. Only recently has the need to reduce the loss of horticultural crops after harvest been given the attention it deserves. Although the causes of post harvest loss and waste are complicated, we suggest that technology-assisted breeding for new and improved fruit, vegetables, and ornamentals, compatible with supply chain constraints but delivered at peak quality to the consumer, could be an important part of the solution over the long-term. In this review, we examine the potential for gene editing to make a measurable and robust impact on post harvest waste and loss. Rather than a technical or critical assessment of methodologies or research areas, we focus on connecting the bio-physiology of post harvest produce, the needs of the produce industry, and the wealth of existing molecular research, to suggest a holistic yet straightforward approach to crop improvement. The main focus of the review is the discussion of genes that could influence the quality and shelf-life of produce. First, we examine the steps that are taken to extend shelf-life in the produce supply chain, and the impact of supply chain management on consumer-desired quality traits.

Then we briefly review the CRISPR–Cas9 method to emphasize the flexibility, ease, and power with which traits can be modified. Finally, we take a critical look at remaining barriers which must be overcome to make gene editing for post harvest traits technically and economically viable. This review serves both as an introduction to post harvest and gene editing and as a resource for researchers attempting to utilize the latter for the former.Post harvest waste and post harvest loss are sometimes used interchangeably, but this is incorrect. Post harvest loss is unintentional. It describes the incidental losses that result from events occurring from farm-to-table, such as physical damage, internal bruising, premature spoiling, and insect damage, among others. Produce loss is also described as quantitative because it is measurable. This does not imply that data is easily available, only that it can be assessed. Post harvest waste, in contrast, is intentional. It describes when produce is discarded because it does not meet buyer expectations, even though it is edible. Produce may be rejected by growers, distributors, processing companies, retailers, and consumers for failing to meet desired or established preferences. Produce waste is described as qualitative because it is difficult to measure and assess. Still, in the US, it is estimated that 7% of post harvest losses of fruit and vegetables occur on the farm, while more than twice that, i.e., 17% and 18% are wasted in consumer-facing businesses and in homes, respectively. Produce post harvest loss and waste threatens environmental sustainability, and is especially catastrophic when viewed in the light of the twin challenges of global climate change and increasing population growth. PLW means inefficient use of financial investments in horticulture and more critically, non-renewable natural resources. Technological measures to curb PLW, such as maintaining a cold-chain and use of plastic packaging, additionally have energy and carbon costs. Improving the shelf-life and quality attributes of post harvest crops by genetic modification or smart breeding could be among many solutions to lessen the severity of these problems.Produce must be kept alive from farm to table; however, the biological nature of horticultural produce is often in congruent with modern commercial supply chain operations. Produce and ornamentals are high in water content, and often metabolically active, which makes them highly perishable. This becomes a challenge given the number of food miles fruit, vegetables, and ornamentals can travel in the global supply chain . Modern post harvest supply chains may be separated spatially by thousands of miles, and temporally, by several months. Produce trucked and shipped from the field is often treated: cooled, washed, sorted, dipped, sprayed, or held at desirable temperatures and modified atmospheres to preserve “health”. The majority of produce from mid to large-scale operations may move through a byzantine system of processors, distributors, and trucking and shipping entities. Maintaining an unbroken cold-chain, adequate packing, and shipping are essential to preserving quality and shelf-life. . Produce, even after harvest, respires , transpires water, and, for the “climacteric fruits”, can emit high levels of ethylene, which can be accelerated at high temperatures. Optimizing storage and handling conditions requires managing these biological processes , which may differ for each produce-type or variety, and from how the preharvest environment influences biology at harvest and thereafter.

Nutmeat production. Even though regulated deficit irrigation consistently reduced applied water compared to the control , variation was high enough to prevent the regulated deficit irrigation from having a statistically significant effect on the gallons of irrigation water used to produce 1 pound of nutmeat . Statistical analysis for yield and irrigation water used per pound of nutmeat showed that both block and year effects were highly to very highly significant, presumably as a result of fixed block-to-block variability in the soils as well as the combined effects of year-to-year variation in weather conditions, especially during flowering, and the increasing yields over time. Nut quality. Over 5 years, we found only two statistically significant effects on nut quality under regulated deficit irrigation: a decrease in kernel weight and an increase in the percentage of severe shrivel. Average nut size was 1.18 grams in the regulated deficit irrigation treatment and 1.21 grams in the control . There was severe shrivel in 13.0% of nuts sampled from the regulated deficit irrigation treatment and 9.0% from the control . The non-significant effects measured were nut moisture; percentages of sealed sutures, doubled kernels, twin kernels, blanks, broken kernels, creases, slight shrivels, rupture calluses, gums, molds and stains; and damage by navel orange worm, ants and peach twig borer. For most of the quality factors measured, the effect of year, but not block,plant pot with drainage was also highly to very highly significant . Hull split. Previous research showed that regulated deficit irrigation can increase the rate of hull splitting , but in this study we observed no measurable differences in the duration or extent of hull split between treatments in any year .

Plant water deficit. The SWP values in both treatments were approximately equivalent before and after the regulated deficit irrigation period, but were much lower compared to the control during the hull-split period . This indicates that a well-defined and reproducible plant water deficit was achieved during hull split in the regulated deficit irrigation treatment. For much of the growing season , particularly around harvest time , SWP in the control was also lower than expected for almond trees with non-limited water . This effect may be attributable to a small deficit in water applied by the grower as a result of cutbacks in water availability.The orchard site used in this study presented several difficulties in implementing regulated deficit irrigation as a management technique, in particular the site’s relatively shallow and spatially variable soil with low water-holding capacity, and two comparatively dry years . Both of these factors might lead to an excessive and potentially damaging level of stress when irrigation is reduced, particularly just prior to harvest in almonds, when irrigation must be discontinued to allow for mechanized harvesting. However, using a simple, plant-based approach, consistent water savings of more than 5 inches or about 13% of applied water were achieved with no detectable effects on short- or medium term orchard productivity. When regulated deficit irrigation was compared to the control, there was an annual water savings of 0.4 acre-foot . Although no significant reductions in overall yield or gallons of irrigation water used per pound of nutmeat were observed in our study, significant reductions in yield have been documented in previous deficit experiments with almonds. The negative effects in those studies were not extreme, and the yield reductions were generally associated more strongly with water deficits imposed during post harvest than during hull split. In a 4-year study by Girona et al. , a statistically significant reduction in overall yield was associated with a 40% reduction in water application and a non-significant reduction in kernel dry mass.

In our study, the overall treatment difference in kernel dry mass of 2.5% was statistically significant but relatively minor. At this site, even though the grower annually applied what many consider full ETc, the SWP values indicated that the orchard trees were experiencing mild-to-moderate stress during much of the season, particularly around harvest. According to a previous study, mild to-moderate stress may not be unusual in commercial almond production . Therefore, it is difficult to determine how much water might be saved statewide if our recommendations for regulated deficit irrigation were widely adopted. Our plant-based strategy for regulated deficit irrigation is based on targeted stress levels at specific stages of crop development. If current grower practice already achieves this stress level, then the water savings shown in this study may not be realized. It will be important to further document current practices in terms of both ETc and SWP in order to have a more reliable estimate of the potential water savings from using regulated deficit irrigation in almond orchards. The water savings in our study might also be improved upon. Depending on winter rainfall and soil type, a plant based strategy might allow irrigation to be reduced for longer periods of the season in many almond-producing areas of the state. The contribution from rainfall is another important consideration; during this study there were 2 years of below-average rainfall, and the average annual contribution to crop consumptive water use from soil storage was only 11%. In less droughty years, or on soils with a higher water-holding capacity, water savings from plant-based regulated deficit irrigation might have been greater.Coyote tobacco.Each seed capsule contains more than one hundred seeds and a thrivmg plant may ripen hundreds of capsules. Diminutive seed size confers certain advantages. Tiny seeds incorporate into soil and are insulated from the scorching effects of fire. Tobacco plant foliage may serve as adispersal agent. With a swish of the summer wind, the small seeds spill from the capsules and many adhere to the plant’s sticky stems and leaves.

Dry, wind-blown or hand-tossed tobacco foliage inevitably travels with a cargo of seeds. During high water the small seeds may also raft downstream attached to tobacco fohage and stems associated with flotsam. Although coyote tobacco seeds may germinate the spring after they ripen, the seeds can also wait for optimum conditions, mamtaining their viability for many years—perhaps more than one hundred . Careful investigations reveal that the coyote tobacco seed bank is finely tuned to disturbance, especially by fire. Maximal seed germination is triggered both by an organic compound found in smoke and by the effects of fire, which remove potential competitors and their germination-inhibiting chemicals. Most large stands of coyote tobacco result from range and woodland fires. In the absence of fire, other triggers influence seed germination. Soil disturbance, the removal of plant debris, and competitive vegetation result in a hedge-betting strategy: i.e., the germination of a much smaller portion of the seed bank. Thus, without fire, coyote tobacco sometimes appears along graded roadsides, in washes, sand dunes, stock corrals, and in other disturbance-prone environments where the seeds persist . Coyote tobacco has a vast, mostly inland range that extends from Mexico north to Canada, and from the east slope of the Rocky Mountains to the rain shadowed lower east slopes of the Cascade Mountains of Washington and Oregon. Coyote tobacco is the only native species of tobacco found throughout much of the Great Basin. In CaUfomia its range extends west across the Cascade and the Sierra Nevada mountains to include much of the state TTie scattered populations of coyote tobacco along the Oregon-CaUfomia borderland nearly surroimd the vicinity of the Upper Klamath River and illustrate the wide diversity of environments to which the species is adapted. East of the Upper Klamath River in southeastern Oregon and northeastern CaUfomia,large plastic pots coyote tobacco behaves as it does throughout much of the basin and range country, populating burned rangeland and then disappearing a few years later. In the absence of fire, it appears primarily along graded roadsides, occasionally in dense stands that dwindle within a year or two . Not far to the southeast of the Upper Klamath River, the campground and roadsides of Lava Beds National Monument host a scattered crop of tobacco nearly every year . When a fire swept through the area in 1999, the number of plants increased dramatically and then declined to the occasional scattered individual . A short distance to the northwest of the Upper Klamath River, but well above it, coyote tobacco may be found on Siskiyou Summit, the highest point on Interstate 5. On this pass the vegetation is wind-scoured in winter, but the summer season is generally mild with occasional showers that tend to skirt the drier valleys on both sides. In the summer of 2003, coyote tobacco plants, like a line-up of hitchhikers, occupied both sides of the highway. With access to open habitat, and fertilized with trucker-supplied nitrogenous waste, the plants set tens of thousands of seeds among the roadside foam cups, wads of paper, and plastic bags . To the south and southwest of the study area, coyote tobacco is also found in the Shasta Valley, and in the vicinity of Mt. Shasta . The Klamath River is one of only three rivers that cut entirely through the Cascade-Sierra uplift.

Along the southern Oregon and northern Califomia borderland, the Cascade Range serves as a semi-permeable biogeograpic boundary between two biogeographic regions: the Great Basin Floristic Province and the California Floristic Province .The trough created by the Klamath River as it slices through the Cascade Mountains functions as a corridor connecting portions of the two provinces. The study area includes the landscapes adjacent to and above the Upper Klamath River where the river transitions from the Great Basin Floristic Province to the California Floristic Province. Throughout the Cascade Mountains portion of the Upper Klamath River, the ranges of species characteristic of each region interweave, influencing the kinds and abundance of plants available for cultural use. Since the seed banks of coyote tobacco are seldom manifest as actual plants in the landscape, it is difficult to assess the geographic range of the species on the basis of a single season of observations. For instance, an intensive one-season plant survey of the Irongate Reservoir vicinity in 1996 did not note coyote tobacco, although under the right circumstances the plants are quite common. The Upper Klamath River Project^ allowed participants many opportunities to access vast portions of the landscape for many consecutive growing seasons, enabling us to spot widely dispersed and infrequently occurring stands of wild tobacco. Within the study area, coyote tobacco is a regular summertime resident to the east, on the edge of the dry, Great Basin-like Butte Valley along the OregonCalifornia border. Most August mornings, a sharp eye spots a few small, dusty plants flowering just off the local dirt access roads. The plants respond well to the soil disturbance associated with machine-piled logging slash. One year, robust plants were confined to the vicmity of these piles; the next year, except for a hidden seed bank, they were as absent as the loggers. A dusty ranch with aged outbuildings, adjacent to the Upper Klamath River, is the locale for another small, semi-permanent population. Each year a few plants germinate in the cow- and ATV-churned soil. Single plants and small patches of plants appear and disappear along the rough roads that parallel the river along the Oregon-California border . On one occasion, when a culvert pipe was replaced upslope from Irongate Reservoir, hundreds of plants densely carpeted the area disturbed by the backboe; the next year there were only two plants. The following year there were none, and none have been observed at this location since. While recently burned areas and roadsides are recognized as common habitats for tobacco, riversides are seldom considered to be accommodating to the plants. However, where the Upper Klamath River cuts through the Cascades, coyote tobacco occasionally appears just below the winter-spring high-water line along the banks of the river. After the flood of 1997, a patch of just a few large plants grew up in a silted side channel not far above the 1-5 crossing. Large leaves from these flood-awakened plants were cheerfully harvested by local Shasta tribal member Mary Carpelan . One small gravel bar along the river seems to grow a crop of coyote tobacco year after year. The plants grow best between the river’s high-water mark and its summer low-water edge, holding to the places where occasional floods scour shade-producing riparian shrubs and trees from the bank.

This chromatograph is reproduced in Figure 74 [Image could not be located] The use of, or combination of, other diagnostic aids may also prove valuable. Thus, Albach and Redman used the composition of flavones in citrus fruits as an aid in citrus classification, but these compounds might be a factor in bark identification as well. Dreyer used citrus fruit bitter principles for chemotaxonomy in the Rutaceae. Esen and Soost found that based on the occurrence or absence of browning in young shoot extracts, Citrus taxa can be classified into two phenotypes: browning and nonbrowning. The ability to cause browning in shoot extracts has been shown to be due to a single gene , which controls the production of a substrate of polyphenol oxidase. Esen and Soost suggest the technique might not only be useful as a genetic marker, but also as a taxonomic criterion, when used with other procedures. Another very helpful chemical technique to aid in citrus identification, particularly in distinguishing between nucellar and zygotic seedlings, is the use of isozymes. This procedure is based on the horizontal starch gel electrophoresis of heterozygous loci found in leaf extracts of the cultivars to be analyzed,cultivo frambuesa whether known cultivars or new hybrid progenies. Perhaps the first researchers to use the method as a practical approach in Citrus were Ueno , Ueno and Nishiura , and Ueno and Nishiura . Ueno and Nishiura were very successful in identifying hybrid and nucellar seedlings in progeny obtained from a breeding program extending over a 10 year period at the Fruit Tree Research Station at Okitsu, Japan. The progeny of the crosses were categorized by analysis of the leaf enzymes using peroxidase isozyme electrophoresis.

Ueno extended this technique to confirm the identity of Citrus species and varieties in the collection at the Okitsu Station. Ueno and Nishiura used the same procedure to study the graft hybrids, Kobayashi mikan , Kinkoji-unshiu , and Takagi-mikan . They were able to establish that the Kobayashi mikan and the Kinkoji unshiu were tree graft hybrids, but that the Takagi-mikan was not. Torres, Soost, and Diedenhofen were the first to report clearly the allozyme systems in Citrus. They reported 19 codominant identifiable alleles distributed among four loci controlling three enzymes. These three gene enzyme systems were glutamate oxaloacetate transaminase , phosphoglucose isomerase , and phosphoglucose mutase . Using this technique, Soost, Williams, and Torres were clearly able to distinguish between zygotic and nucellar five-month-old citrus seedlings. The seedlings were from a cross using King mandarin as the female parent and Parson’s Special mandarin as the pollen parent. Of the 128 seedlings obtained from the cross, and using the two genetically defined markers , they found 18 to be nucellar and 110 to be zygotic. Fortunately, all of these seedlings were planted in the orchard for further observations. The work of Soost and Torres extends and amplifies the work of Torres, Soost and Diedenhofen . Three additional gene-enzyme systems, malate dehydrogenase , hexose kinase and isocitrate dehydrogenase , were determined and used for possible identification of cultivars in the subgenus Eucitrus. Additional taxa were analyzed for glutamate oxaloacetate transaminase , phosphoglucose isomerase and phosphoglucose mutase . Examples of the use of the three latter enzyme systems are presented in the paper as well as considerable references to the numerous cultivars in the Citrus Variety Collection at the Citrus Research Center, Riverside.

Torres, Soost, and Mau-Lastovicka emphasize that isozymes of Citrus provide molecular tags to determine the genetic origin of citrus seedlings. A very large proportion of all possible seedlings, either from selfing or crossing, can now be distinguished with a great deal of certainty as to their genetic origin. Citrus leaf extracts were analyzed by starch gel electrophoresis for the previously mentioned enzymes of hexokinase , isocitrate dehydrogenase , leucine aminopeptidase , malate dehydrogenase and malic enzyme . The isozyme technique is wonderful for distinguishing between nucellar and zygotic seedlings, but for other uses it does have limitations. Sometimes it may accurately identify the genetic makeup of many cultivars, in others, not. However, in some cases it is extremely helpful in that although it cannot tell one of the parentage of a cultivar, it can tell one what it is not. Sibs out of the same cross cannot be identified from each other. Orange cultivars cannot be accurately identified, or with little confidence. Carrizo and Troyer citrange have not been definitely identified. Eureka, Lisbon, and Villafranca lemon strains cannot be distinguished, etc. Many of the various chemical tests are rather complicated and not only need special supplies and equipment, but also trained technicians. If such tests are necessary, the grower should obtain the services of a professional laboratory with experience in these techniques. Furthermore, these tests are not practical for use by the nurserymen who grow thousands of trees or hundreds of thousands of trees. One should use root stocks which are highly nucellar, such as Troyer and Carrizo citrange, Rough lemon, sweet orange, etc. Using root stocks like Sacaton citrumelo and Citrus taiwanica, which only have about a 50% nucellar rate, can provide nothing but trouble. The nurseryman cannot possibly effectively discard all the zygotes and hence, tree variation and poor performance in the orchard is to be affected. The sweet orange, Citrus sinensis, achieved prominence only in California .

In Southern California during the period from 1900-1950 it was probably the principal root stock in use, except on heavy soils and the sandy soils of the Coachella Valley. Since 1950, its use as a root stock has steadily declined so that by about 1970 it was seldom planted, but is in certain situations. As a root stock, its performance in California was very satisfactory, with all major commercial scions performing comparable to those same scion trees on sour orange or better. Such trees were superior in performance to trees on Rough lemon, grapefruit, Cleopatra mandarin, trifoliate orange, etc., see Webber , Wutscher , and Castle . Sweet orange was used to a very limited extent in Florida and Australia. It was well tested in South Africa by Marloth , but never achieved acceptance. The sweet orange consists of a homogeneous group of cultivars, one of the most uniform of the citrus species. Maximum variability may exist between the Valencia and the navel, the red- pigmented “blood” oranges , the pink pigmented varieties , the blond oranges, the acidless oranges, and the seedy varieties that have been used for root stocks. They are commonly and collectively referred to in California as ‘sweets,’ ‘seedy sweets,’ or ‘Mediterranean sweets.’ According to origin, they may be ‘Blackman’, ‘Koethen’, ‘Hinckley’, ‘East Highland’, ‘Olivelands’, etc. In Florida, seedy varieties like ‘Pineapple,’ ‘Parson Brown,’ ‘Homosassa,’ and ‘Florida common’ were some of the sources used. The sweet orange varieties grow readily from seed,plastic gutter but the seeds are easily injured by drying out and must be handled carefully . All sweet orange varieties are highly nucellar , and require a minimum amount of rogueing in the seed beds and nursery seedlings to remove the variants. The seedlings grow more slowly, when young, than those of the sour orange, and produce low-branching bushy trunks which may require more shaping in the nursery prior to budding than seedlings of Rough lemon, Troyer citrange, Cleopatra mandarin and others. The scion buds, after insertion, grow rapidly and produce large budlings with most scions —but not as large as on Rough lemon, Rangpur lime, Alemow, etc. The sweet orange may be grown from cuttings more easily than the sour orange, but not as easily as cuttings of the Rough lemon. As a root stock, the sweet orange in California is only medium in cold resistance, more hardy than Rough lemon, but maybe not as good as sour orange. When mature trees are frozen to the ground, as in a Florida freeze, it sprouts readily from the base of the trunk.The sweet orange usually gives normal bud unions with all varieties of sweet orange, mandarin, grapefruit, lemon and lime. That is to say the stock and scion are usually nearly equal in size, particularly with young trees. With older trees, there may be a tendency for a slight bulge at the union and the stock may be slightly smaller than the scion, but not to the degree attained on sour orange stock. This is generally true with grapefruit and lemon scions. In some instances, Eureka lemon trees on sweet orange may show a slight bud union overgrowth. For illustrations of these bud unions, see Figure 76 [Image could not be located]. The sweet orange grows fairly well on some heavy soils, but is best adapted to growth on rich sandy loams. Trees on sweet orange do not do well on very sandy soils such as in the Coachella Valley of California, nor extremely heavy or calcareous soils such as Porterville or Ducor adobes. On heavier soils that are poorly drained, the trees may develop severe iron chlorosis symptoms, and this is also true on the calcareous soils. Embleton, et al. and Wutscher found that trees on sweet orange root may have higher leaf levels of nitrogen, phosphorous and copper than trees on other root stocks.

The trees of sweet orange root stock do not commonly develop a well differentiated tap root like the sour orange root stock and is usually moderately shallow rooted, rarely penetrating to the depth that sour orange root stocks do, although an occasional lateral may do so. The sweet orange does, however, develop an abundant system of lateral roots which generally penetrate deeper than those of Rough lemon in California soils. Illustrations of these root systems may be found in the section on roots. Navel orange cuttings produced only a few very large surface laterals which have little penetration. On the other hand, Valencia orange cuttings produce a more abundant root system similar to those obtained on sweet orange seedlings. There would appear to be no disadvantage in using Valencia orange cuttings for orchard trees in areas where sweet orange could satisfactorily be used as a root stock. Trees on sweet orange root stock are large and vigorous, producing standard sizes in combination with all commercial citrus varieties in California. In most areas of California, the sweet orange trees are larger than those on sour orange root stock, but are smaller than trees on Sampson tangelo or Troyer citrange. Also, in California, they have, on the better soils, produced larger trees than those on Rough lemon, but in the sandy soils of Florida they are generally smaller. Yields on sweet orange root stock are good, generally among the higher echelon with all scion varieties except with navel oranges, where trees on sour orange, Troyer citrange, or the non-commercial Morton citrange out-yielded them. Rios Castaño, Torres, and Camacho report that orange trees on sweet orange root stock in Columbia were low in production, but offer no explanation. The sweet orange combinations are not as precocious in bearing as trees on trifoliate orange or Alemow, but the trees under favorable situations are long-lived and bear well into the advanced age of 50-60 years or longer. One orchard at the Citrus Experiment Station, Riverside must be 80 years and is still productive, although tree size is getting out of hand. There have been few loses from gummosis in the old orchard, but some trees have declined from psorosis. In California, fruits on the sweet orange stocks mature at the normal season for the variety; they are thin skinned, juicy, and of high quality, and hold up well in all physical and chemical characters to the extreme end of the long harvesting season. Percent juice, soluble solids and citric acid content of the fruits are essentially identical to those obtained on sour orange—with all varieties and in all areas of California . Wutscher , however, reports that the acid content of fruit on sweet orange root stock in Texas was higher than that of fruit on sour orange. This is not true in California. Fruits on sweet orange root stock are thus intermediate in quality, being superior to those grown on Rough lemon, sweet lime, or Alemow. They are, however, of poorer quality than those grown on trifoliate orange or Troyer and Savage citranges. Granulation of the fruit is generally not a serious problem as compared to other stocks.

The root terminated 9.1 M from the tree trunk and was 11.3 M long. At no place was the root more than 46 cm below the ground surface, and at the free end was only 15 cm below the surface. In the imperfectly drained east coastal soils of Florida, Ford reported that stabilizing the water table at a lower level increased the total rooting area and the newly developed roots survived without periodic destruction. Lowering the water table from 76-178 cm doubled the quantity of feeder roots in four years and increased the size of the tree. Cahoon, Harding, and Miller found that the higher the tree yields, the more feeder roots found in the irrigated row middles. In several high-producing orchards the amount of roots found between trees actually exceeded those found under the trees.Cahoon, Huberty, and Garber report on a differential furrow irrigation treatment applied to a Washington Navel orange orchard from 1934-1957 on sweet orange stock. The treatments were frequent versus infrequent. In 1957 root samples were taken to a depth of 122 cm. Trees irrigated on a frequent schedule produced fewer deep roots than the trees irrigated on an infrequent interval. The difference was more evident at the 61-92 cm levels. Samish in Israel reported essentially the same thing. Cahoon and Stolzy in California used a neutron moderation method to estimate root distribution as affected by irrigation and root stocks. They encountered troublesome problems with soil moisture variability,square plant pots soil profiles, etc. Ford found poor root growth in the leached zone of certain acid soils of the imperfectly drained Florida flat woods.

In laboratory tests poor root growth was not corrected by the application of adequate water and nutrients. In laboratory tests the Rough lemon produced the best feeder and lateral roots, even better than sour orange. Damage to the roots was more severe at low pH 5.0. Roots of Cleopatra were severely damaged at high and low pH 5.0-6.5. Ford also states that the relatively poor feeder root growth of trifoliate orange together with the root damage that occurred at pH 5.0 when flooded suggests this stock should be carefully evaluated. On the other hand, the satisfactory tolerance of Rangpur lime to flooding warrants further study. Ford says the citrus root system is capable of rapid and deep growth in sandy soils but will not grow into or exist long in a soil saturated with water. When the water table is within 60 cm of the surface, roots are confined to a shallow zone. Fluctuating water tables have a pronounced affect on the root system. He compared roots from trees in orchards with 1.8 M deep drain lines to an adjacent undrained orchard. In the undrained orchard the highest per cent of roots were at 0-50 cm, less at 25-50 cm, and almost none below 50 cm. In the drained orchard there was good rooting to 50 in. and some rooting even to 180 cm, but less as the distance from the chain line increased. Stabilizing the water table at a lower level increased the total rooting area and newly developed roots survived. Lowering the water table from 75 cm to 180 cm doubled the quantity of feeder roots in four years and increased the size of the trees. The feeder root concentration in the deep rooting zone 75-180 cm was greater than in the 0-25 cm level. In Israel, Cossman established a close correlation between the vigour of stocks on sandy soil and the osmotic pressure of their root cells. The slow-growing group represented by pummelo, grapefruit, and sour orange have remarkably low figures for their osmotic pressure.

The roots of this group are easily outclassed by the retentive forces of the soil particles whenever the wilting range is approached. In Texas, Adriance stresses the importance of the tap root system of sour orange, but points out that the major portion of the root system was between 46-61 cm. He emphasizes the importance of environment, natural habitat of species, aeration, water table, salt content and stratified soils. Adriance and Hampton examined the root systems of trees on sour orange grown on different soil types and subjected to different cultural practices. A poor-stunted tree grown on a very dense and compact soil had a spread of lateral roots 1.8-2.1 M. There were few roots 1.2-2.5 cm in diameter in the upper 21 cm of soil, a minimum of fibrous roots down to 61 cm, and no roots below that zone. A medium sized tree grown on a compacted soil and underlain with caliche at 125 cm showed roots were small but up to 2.5 cm in size and were well distributed although they did not penetrate deeply. A large tree grown on a good textured soil to a depth of 152 cm had roots down to 152 cm and below. Another tree in a tilled orchard which was disked to a depth of 10 in. had good roots around the tree, but there was little lateral spread beyond that distance. A tree under nontillage had roots with a lateral spread of 5.5-6.1 M. In California, Crider found that citrus roots were found to be distributed largely according to the character and the previous cultivation treatment of the soil. In the case of a 25-year-old tree there were practically no roots below 1.2 M due to a tenacious subsoil. A 30-year-old tree on a dry sandy soil showed good root development to a depth of 2.7 M. In a well-cultivated and fertilized orchard, with young trees 3-6 years old, 50 per cent of the roots were in the first 46 cm of soil. On the other hand older indifferently handled trees showed greater root accumulation in the 30-60 cm and 60-90 cm layers.

Young stresses the importance of soil texture, drainage, aeration, and moisture relationship to citrus root development. In Florida, Ford observed Hamlin and Valencia oranges on Cleopatra and Rough lemon at 15-21 years of age growing on a red sandy clay some 46 cm-4.8 M below the soil surface. Trees on the Cleopatra were 46-92 cm taller than the trees on Rough lemon where the roots penetrated into the clay. The height of the trees on Rough lemon decreased as the clay was closer to the surface. A restriction in root growth imposed by the clay did not consistently increase feeder root concentration above the clay. Root growth ceased when the clay percentage was above 28 per cent. Feeder root concentration of 15-year-old Hamlins and Valencia on Cleopatra growing in deep sandy soil was greater than Rough lemon, even though the trees were smaller than the same scions on Rough lemon. At Riverside, much of the area occupied by the citrus root stock trials initiated by Webber was underlain with impenetrable hardpan. At one location where the hardpan was approximately 1 M from the surface, some of the deep-tap-rooted trees such as sour orange had their tap roots growing down to the hardpan and then fusing together in a solid plate like a pedestal and then the roots diverged at a lateral angle. In the Azusa-Covina area of California where many of the soils are alluvial sandy loams, especially adjacent to washes which were subject to flooding and were underlain with sand and gravel substrata. In such soils where the alluvium was deep, the roots of sour orange penetrated to a depth of 2 M or more with few laterals. However,vertical farming equipment as the trees in the orchard approached the stream bed the sour orange roots penetrated only to a depth of 60 cm or less with no tap roots, but a well developed system of surface laterals . Fertilizers and nutrition also play a big part in citrus root development. In Florida’s deep sandy soils, Ford, Reuther, and Smith found nitrogen was the primary element influencing root development in two fertilizer experiments after six years of differential treatment. The high nitrogen plots had 37 per cent less feeder roots than the low nitrogen plots to a depth of 1.5 M. Neither potassium or magnesium had any appreciable effect on root development. In the second plot there were 38 per cent less feeder roots at 13-89 cm in the high nitrogen regime as compared to low nitrogen levels. They felt a direct salt concentration [“Check” appear here in typescript in the margin of the manuscript] was responsible for the effects. In California, Cahoon et al. examined the effects of various types of nitrogen fertilizer on root density and distribution as related to water infiltration in a long-term fertilizer experiment on a sandy loam soil. They found that various nitrogen treatments, particularly the long-term application of sodium nitrate, and ammonium sulfate reduced root concentrations in the first 10 cm of soil. In tropical Trinidad Gregory found the root systems of Marsh grapefruit on sour orange were more vigorous and extensive on manured trees than unmanured trees. The manured trees had several lateral roots which exceeded the average spread of the branches and extended 106 cm from the trunk on 3-year-old trees. Most were shorter, and feeder roots occurred 8-46 cm from the trunk. The unmanured trees had shorter roots and the main feeding roots were only 8-31 cm from the trunk.

In Florida’s deep sandy soils, Spencer found that phosphate applications markedly reduced the concentration of feeder roots, especially in the surface 30 cm of soil. Reductions in root growth were not noted in the deeper soil zones even at the highest phosphate rates. Similar observations were made by Smith and Ford . Smith and Specht suggested an increase of iron chlorosis in Florida was mainly caused by an accumulation of copper in the soil with consequent root damage. Since copper accumulates primarily in the top soil they suggested trees became chlorotic because of root damage in that area. Chelated iron applied to seedlings in soil solution did not overcome the stunted root system associated with high copper levels. Ford had shown that in Florida 70 per cent of the feeder root system of healthy trees growing in deep sandy soils are located below 25 cm. Ford found that feeder root damage in orange trees affected with severe iron deficiency was not confined to the topsoil. Feeder root damage like copper toxicity was found to a depth of 1.5 M in groves located near lakes and swamps. Soil pH in the 0-25 cm zone was below 1.5 M with the subsoil at pH 3.9-4.4. All the groves had a high concentration of copper in the topsoil. The application of FeEDTA chelate to chlorotic trees which showed extreme root damage to a depth of 1.5 M resulted in pronounced new growth of roots in the subsoil. The increase of root growth was proportionately greater with an increase in depth so that often there were more new roots in the 75-150 cm zone than the 25-75 cm zone. Where iron chelate resulted in new leaf and shoot growth there was a corresponding increase in feeder root growth which occurred mostly below the 25 cm depth. If feeder roots were found in the 0-25 cm zone under chlorotic trees, treatment resulted in an increase of the number of feeder roots on the laterals. If there were no lateral roots in the surface 25 cm, then no new feeder roots were present after treatment. Changes in soil pH greatly influenced the distribution of feeder roots throughout the entire root profile. Ford says that in general, root concentration is highest when nutrition elements are low but not deficient. At high levels of applications of fertilizers the concentration of roots is reduced for all major elements. The correlation did not apply to the micro-elements. A deficiency of iron severely reduced the root system and an excess of copper and manganese prevented growth of the feeder roots due to toxicity in certain soil horizons. He suggested that from the standpoint of the root system the lowest level of nutrients consistent with high yield and healthy trees was the best. The effect of excessive accumulations of micro nutrients like Cu, Zn, and Mn on mycorrhiza and in turn, on root development, has yet to be fully evaluated.

Demographic items included self-reported sex, race and ethnicity, age, marital status, employment status, residential zip code, and sheltering-in-place status given the COVID-19 pandemic. The Alcohol Use Disorders Identification Test-Concise , a widely used 3-item self-report measure based on the 10-item original AUDIT, assessed hazardous or harmful alcohol consumption in the past 3 months. A score of 4+ for men and 3+ for women indicated significant problems with alcohol consumption. The AUDIT-C has been found to be a valid screening test for heavy drinking and/or active alcohol abuse or dependence. The Drug Abuse Screening Test-10 , a 10-item self-report measure adapted from the 28-item DAST, assessed consequences related to drug abuse, excluding alcohol and tobacco in the past 3 months. The last item of the DAST-10 regarding medical problems resulting from drug use was not reassessed because it was an exclusion criterion in the study screener; hence, the total possible range for the sample was 0-9, not 0-10. Total scores of 3+ indicated significant problems related to drug abuse. The DAST-10 has moderate test-retest reliability, sensitivity, and specificity. For the AUDIT-C and DAST-10 measures at post treatment, the reference period was the past 2 months, to reflect the period of intervention. Craving was assessed with a single item asking, “In the past 7 days, how much were you bothered by cravings or urges to drink alcohol or use drugs?”, with response options of not at all , a little bit , moderately , quite a bit , and extremely . The Brief Situational Confidence Questionnaire, a state-dependent measure,vertical farms assessed self-confidence to resist the urge “right now” to drink heavily or use drugs in different situations reported on visual analog scales anchored from 0% “not at all confident” to 100% “totally confident.”

The Patient Health Questionnaire-8 item , an 8-item scale, assessed depressive symptoms, and the Generalized Anxiety Disorder-7 item , a 7-item scale, assessed symptoms of generalized anxiety disorder. Both the PHQ-8 and GAD-7 have good internal consistency and demonstrated convergent validity with measures of depression, stress, and anxiety. A total of 2 items assessed the history of therapy for mental health or substance use concerns. Lifetime psychiatric diagnoses were assessed using 10 items plus a write-in option for others. A single item assessed currently taking prescribed medications for a psychiatric diagnosis. The treatment feasibility and acceptability of W-SUDs were assessed post treatment using the Usage Rating Profile-Intervention Feasibility and Acceptability scales, the 8-item Client Satisfaction Questionnaire-8 questions, and the 12-item Working Alliance Inventory-Short Revised. The URP-I item response options ranged from strongly disagree to strongly agree; the items were summed for a total score within each scale, with one feasibility item reverse coded. The CSQ-8 items have 4-point rating scales with response descriptors that vary. Internal consistency exceeds 0.90, and the total sum score ranges from 8 to 32, with higher total scores indicating higher satisfaction. The WAI-SR has three 4-item sub-scales, with 5-point rating scales, that reflect development of an affective bond in treatment and level of agreement with treatment goals and treatment tasks. Serious adverse events occurring in the 8 weeks after the start of the study were assessed for hospitalization related to substance use, suicide attempt, alcohol or drug overdose, and severe withdrawal . Positive endorsements were followed up with questions about the timing, diagnosis, and resolution. If additional details were needed to determine whether the event was study related, a team member reached out to the participant. Serious adverse events were reported to the study’s Data Safety Monitoring Board within 72 hours of the team learning of the event. Participants’ W-SUDs app use, including days of app use, number of check-ins, and number of messages sent, was collected via the Woebot app, as were module completion rates, lesson acceptability ratings indicated on a binary scale , and mood impact after tools utilization . In addition, on a daily basis, the W-SUDs app assessed mood, cravings or urges to use, and pain. In-the-moment emotional state was reported through emoji selection with a default menu of 19 total moods, including options for negative , positive , and average mood , with an additional ability to type in free text emotion words and/or self-selected emoji expressions. Cravings were assessed as not at all , a little bit , moderately , quite a bit , or extremely .

Physical pain was rated on a scale of 0 to 10. Descriptive statistics were used to describe the sample and examine the ratings of program feasibility and acceptability. Paired samples ttests and McNemar nonparametric tests examined within-subject changes from preto post treatment on measures of substance use, confidence, cravings, mood, and pain. Change scores were calculated , and bivariate correlations were used to examine associations between changes in AUDIT-C and DAST-10 scores and changes in use occasions, confidence, and depression and anxiety scores. ttests were conducted to examine changes from pre- to post treatment in substance use, confidence, mood, and pain by whether participants were currently in therapy or taking psychiatric medications. Post treatment survey completion was 50.5% , with better retention among those with a higher CAGE-AID score at screening . Retention was lowest among those with a CAGE-AID score of 2 and higher for those scoring 3 or 4 . Retention was unrelated to participant demographic characteristics, previous use of Woebot, psychiatric diagnoses, primary problematic substance, depressive symptoms, pain, cravings, confidence, substance use occasions, AUDIT-C scores, or DAST-10 scores . Missing data on individual survey items was minimal. In a single instance, a participant’s average score values were imputed when missing 1 item on the PHQ-8. Participants were prompted to report craving and pain ratings within the W-SUDs app on a daily basis. The data were aggregated so that if participants provided multiple ratings within a day, the scores were averaged. To examine changes over time, generalized estimating equationlinear models were run with week entered as a factor, setting week 1 as the reference category. A total of 1571 mood ratings were entered into the W-SUDs app by 90 of the 101 participants, with each participant entering on average 17.5 mood ratings or 2.2 per week. A total of 1399 craving and 1403 pain ratings were entered into the W-SUDs app by 87 of the 101 participants , with each participant providing an average of 16.1 ratings for cravings and 16.1 ratings for pain. Table 2 shows the number of participants providing craving ratings for each week and summarizes the generalized estimating equation model analyzing craving ratings over time. Compared with week 1, craving ratings were significantly lower at weeks 4 through 9. By weeks 8 and 9, craving ratings were reduced by approximately half of the sample’s mean rating at week 1.

In contrast, pain ratings did not differ significantly by week and over the 9 weeks averaged, on a scale of 0 to 10.W-SUDs, an automated conversational agent,vertical plant tower was feasible to deliver, engaging, and acceptable and was associated with significant improvements pre- to post treatment in self-reported measures of substance use, confidence, craving, depression, and anxiety and in-app measures of craving. The W-SUDs app registration rate among those who completed the baseline survey was 78.9% , comparable with other successful mobile health interventions. As expected, the use of the W-SUDs app was highest early in treatment and declined over the 8 weeks. Study of engagement with digital health apps has been growing, with no consensus yet on ideal construct definitions. Simply reporting the number of messages or minutes spent on an app over time may undermine clarity and genuine understanding of the type and manifestation of app utilization related to clinical outcomes of interest. Further research in this area is warranted. The observed reductions from pre- to post treatment measures of depression and anxiety symptoms were consistent with a previous evaluation of Woebot conducted with college students self-identified as having symptoms of anxiety and depression. Furthermore, in this study, treatment-related reductions in depression and anxiety symptoms were associated with declines in problematic substance use. Declines in depressive symptoms observed from pre- to post treatment were greater among the participants in therapy. This study also examined working alliance, proposed to mediate clinical outcomes in traditional therapeutic settings. Traditionally, working alliance has been characterized as the cooperation and collaboration in the therapeutic relationship between the patient and the therapist. The role of working alliance in relationally based systems and digital therapeutics has been previously considered; the potential of alliance to mediate outcomes in Woebot should be further validated in future studies adequately powered to examine mediators of change. Measures of physical pain did not change with the use of W-SUDs as reported in pre- and post treatment measures or within the app; however, the sample’s baseline ratings of pain intensity and pain interference were low. Although not a direct intervention target, pain was measured due to the potential for use of substances to self-treat physical pain and the possibility that pain may worsen if substance use was reduced, which was not observed here. Within-app lesson completion and content acceptability were high for the overall sample, although there was a wide range of use patterns. Most participants used all facets of the W-SUDs app: tracked their mood, cravings, and pain; completed on average over 7 psycho educational lessons; and used tools in the W-SUDs app. Only about half of the sample completed the post treatment assessment, with better retention among those screening higher on the CAGE-AID. That is, those with more severe substance use problems at the start of the study, and hence in greater need of the intervention, were more likely to complete the post treatment evaluation. None of the other measured variables distinguished those who did and did not complete the post treatment evaluation. This level of attrition is commensurate with other digital mental health solution trial attrition rates. By addressing problematic substance use, including but not limited to alcohol, the W-SUDs intervention supports and extends a growing body of literature on the use of automated conversational agents and other mobile apps to support behavioral health.

A systematic review of mobile and web-based interventions targeting the reduction of problematic substance use found that most web-based interventions produced significant short-term improvements in at least one measure of problematic substance use. Mobile apps were less common than web-based interventions, with weaker evidence of efficacy and some indication of causing harm . However, mobile interventions can be efficacious. Electronic screening and brief intervention programs, which use mobile tools to screen for excessive alcohol use and deliver personalized feedback, have been found to effectively reduce alcohol consumption and alcohol-related problems. However, rigorous evaluation trials of digital interventions targeting non-alcohol substance use are limited. Furthermore, although a systematic review concluded that conversational agents showed preliminary efficacy in reducing psychological distress among adults with mental health concerns compared with inactive control conditions, this is the first published study of a conversational agent adapted for substance use. Study strengths include study enrollment being double the initial recruitment goal, reflecting interest in W-SUDs. Most participants reported lifetime psychiatric diagnoses, and approximately half of the participants endorsed current moderate-to-severe levels of depression or anxiety. From pre- to post treatment with W-SUDs, participants reported significant improvements in multiple measures of substance use and mood. The delivery modality of W-SUDs offered easy, immediate, and stigma-free access to emotional support and substance use recovery information, particularly relevant during a time of global physical distancing and sheltering in place. More time spent at home, coupled with reduced access to in-person mental health care, may have increased enrollment and engagement with the app. Although further data on recruitment and enrollment are warranted, these early findings suggest that individuals with SUDs are indeed interested in obtaining support for this condition from a fully digitalized conversational agent. This study had a single-group design, and the outcomes were short term and limited to post treatment, thus limiting the strength of inferences that can be drawn. The sample was predominately female and identified as non-Hispanic White, and the majority were employed full-time. Non-Hispanic White participants reported higher program acceptability on 2 of the 4 measures compared with participants from other racial or ethnic groups. Future research on W-SUDs will use a randomized design, with longer follow-up, and focus on recruitment of a more diverse population to better inform racial or ethnic cultural programmatic tailoring, using quotas to ensure racial or ethnic diversity in sampling.

Herrero made extensive studies of the size and areas occupied by fibers, vessels, parenchyma and rays in the xylem of hardy fruit trees. The width, distance between, and relative numbers of uniseriate and multiseriate rays in the cambium were also determined. He could not correlate any of these anatomical characteristics with incompatibility. Robitaille and Carlson , working with five apple species on six clonal root stocks, also report no significant correlation between anatomical characteristics and graft take. However, when histograms of the poorer groups were compared to those of the better groups, their results did contradict those of Herrero , since they found that when the xylem structure of two species is sufficiently different, those two species will not intergraft with much success. With regard to citrus, Mendel and Cohen in Israel studied the starch level in the trunk as a measure of compatibility between stock and scion. They found no clear indication of impaired carbohydrate movement through the bud union of Shamouti orange on seven root stocks inducing low growth vigor. The starch level in the bark and wood of the root stock portion of the budded trees was negatively correlated with tree size. They concluded that the starch level results from the growth vigor of the tree, and is not a factor which affects it. In California, Jensen, Wilcox, and Foote found greater amounts of starch above the bud union of lemons on sour orange stock as compared to those on grapefruit. This again is probably the effect of impaired translocation. Mendel and Cohen said that an early evaluation of orchard tree performance is possible if clear correlations can be established between the behavior of, or rate of,vertical hydroponic farming physical processes in a young root stock or budling and the later performance of the budded trees. This certainly will not work in the case of delayed incompatibility or virus-induced incompatibility.

They also found that the total phenolic compounds in Citrus buds and branches accumulate with scion growth, reach a peak with growth cessation, and decrease during the rest period between growth flushes. TPC, rather than phenols, exhibit highest correlation with growth. There was a significant inverse relationship found between the TPC level in the bark of young seedlings and with the size of five-year-old root stock seedlings, and with budded trees three years after budding on these root stocks. They concluded the level of TPC in the bark of young seedlings eight to ten months old is a fairly reliable indication of the performance of young trees until entering bearing age. In Australia, Bevington, Greenhalgh, and McWhirter used reciprocal ring grafts of trifoliate orange and sour orange with Eureka lemon to study the incompatibility problem. They found an initial disturbance of tissue in the outer phloem of affected trees as early as two months after grafting. Whether this technique is suitable to study incompatibility in other combinations, or in a delayed incompatibility, is unknown. However, better indices of ultimate root stock performance are needed. Compared to the amount of anatomical work done on incompatibilities of deciduous fruit trees, the number of anatomical studies on similar problems with citrus has been relatively little when one considers the size of the industry and the problems that confront it. Most of the anatomical work on mature trees pertinent to bud union disorders has been done at the Citrus Research Center in Riverside, California. Without going into detail, the most informative of these are: Schneider on the anatomy of tristezaaffected trees, Schneider on phloem studies of sour orange, Schneider on effect of trunk girdling on phloem, Schneider on phloem studies of sweet orange, Schneider on ontogeny of lemon bark, Schneider on sour orange necrosis, Schneider and Wallace relevant to anatomical studies of top worked trees, and Schneider on dealing with a bud union problem on C. macrophylla root stock.In addition, a better understanding of the anatomical problems involved with the incompatibilities associated with lemon tree decline may be found in Schneider et al. . Also, investigations of graft incompatibility among Persea species by Aaouine has an excellent discussion of tree crop incompatibilities and hundreds of references.

False bud unions occasionally occur in citrus, but their cause is not always definable. For example, in some areas of California subject to heavy surface flooding, bud unions may have been buried under a foot or more of soil and for obvious reasons never excavated. In looking for such bud unions in association with tristeza studies and proper root stock identification, sometimes constrictions in the scion variety under the soil line closely resembled a bud union. In another case, an orchard was sprayed with 2-4-D for weed control purposes and apparently over dosed. At the soil line, the root stock was severely constricted and appeared to be a second union. The author has grown most of his research trees and exercised close surveillance over their propagation, yet occasionally a tree has appeared to have an intermediate stem piece. In Spain during 1968, the author also observed several trees in an orchard which appeared to have an inter stock , but the grower assured him the trees had never been top worked. While such incidences are rare, they may occasionally occur. Usually the external location of the bud union agrees exactly with the internal location. The author has observed, however, some bud unions in which the external appearance of the bark of the stock is so distinct from that of the scion variety that the external union of the two tissues is 4.5 cm away from the internal union, and in some instances more . Also, it is not uncommon to find scion suckers arising on the root stock and as much as 2.5 to 5 cm below the union, particularly with trifoliate orange stock and its hybrids . One might attribute some of these occurrences to double budding and eventual sprouting of the latent buds, but the frequency and diversity of such observations makes one wonder if all such observations can be reconciled with such a procedure. Occasionally, graft hybrids arise at the bud union and result from an adventitious bud which initiates from the union of stock and scion tissues. Examples of this phenomenon are the Kinkoje-unshiu and Kobayashi-mikan of Japan. The famous “Arancio bizzarria” of Nati is another illustration and so is that of sweet lime plus sour orange described by Casella . Probably graft hybrids occur more frequently than suspected, but the common cultural practice of removing suckers from the bud union area most likely eliminates most of them before they are recognized as such. The possibility of using certain graft hybrids as root stocks should be considered.

They would of course have to be vegetatively propagated. Lee first pointed out that Valencia oranges in the Philippines had poor bud unions on Calamondin. He also pointed out that Oneco mandarin had good bud unions on this stock. DeLeon indicated that Tahiti lime, pummelo, grapefruit, and lemon did poorly on this stock in the Philippines,vertical gardening systems but recommended it for limes other than Tahiti, as well as for mandarins. Temple orange and Marsh grapefruit were designated as incompatible on calamondin by Traub and Friend and Friend, Mortensen, and Stansel . Later, Friend and Mortensen classified the calamondin as an unreliable root stock in the Lower Rio Grande Valley and in the Winter Garden area of Texas. In California, Webber pointed out that Eureka and Lisbon lemons, Washington navels, Valencia orange and Marsh grapefruit did very poorly on calamondin and that many of the trees died at an early age. He makes no statement regarding the bud union, which usually shows some external symptom of the disorder, and failed to point out that Satsumas on this root stock performed satisfactorily. Marloth mentions that in South Africa, Marsh grapefruit trees on calamondin made such poor growth that they were removed after two years. Nagami kumquat trees on calamondin root stock were compatible according to Traub and Friend . Tanaka states that the calamondin was used as a root stock in the Changchou area of China, although it was not specified as with what scions, but Webber implied that it was probably with mandarin scions. Baker reported that Meyer lemon did well on calamondin. Olson reports that kumquat hybrids have not done well on sour orange root stock, and Robinson and Savage indicated that in Florida limequats succeeded on any common stock except sour orange. Traub and Friend stated that calamondin and Eustis limequat were uncongenial on sour orange root stocks. A bud union disorder of calamondin was called to the attention of Weathers and Calavan when it was noticed in a citrus variety collection at Santa Barbara, California. Affected trees were found on root stocks of grapefruit, sweet orange, and Troyer citrange. Subsequently, other affected trees were discovered on root stocks of trifoliate orange, sour orange, and lemons in other citrus areas of California. Almost all calamondin trees over four years of age, and on roots other than their own, show the abnormality. Calamondin trees growing as seedlings or cuttings show little or no evidence of the abnormality. They concluded it was a bud-perpetuated, nontransmissible disorder. The author observed bud union abnormalities with Valencia orange trees on calamondin in the tristeza plantings at Baldwin Park , and in root stock plantings made in the Coachella Valley of California , but the trees were not in distress after 13 years of age. However, Marsh grapefruit and Eureka lemons on calamondin in the desert area were in severe decline within three to four years, but Dancy tangerines on calamondin stock performed well. A similar bud union disorder of both old-line and nucellar-line Red Blush grapefruit on calamondin and other kumquat hybrid root stocks was described in Texas by Cooper and Olson , and Olson . Many other instances of failure of calamondin as a root stock were reviewed by Olson and Webber and will be discussed in more detail later in the text. A bud union disorder with Nippon orangequat on sweet orange was also noted by Weathers and Calavan in California. Similar bud union abnormalities were observed with Eustis limequat on Sunshine tangelo, grapefruit, and sour orange by Olson .

Weathers and Calavan in California obtained no evidence of virus transmission to any inoculated seedlings of sweet orange, sour orange, calamondin, West Indian lime, Sunshine tangelo, Rangpur lime, or Palestine sweet lime when they used buds from severely affected calamondin trees as a source of inoculum. They also grew nucellar seedlings of calamondin and propagated them on root stocks of sour orange, sweet orange, grapefruit, Troyer citrange, Morton citrange, trifoliate orange, Willow leaf mandarin, Wekiwa tangelo, and Rangpur lime. Some of these seedlings were inoculated with buds from affected calamondin trees, others left as checks. Scions from affected old-line calamondin were propagated on seedlings of sour orange, sweet orange, grapefruit, Troyer and Morton citranges, trifoliate orange, Willow leaf mandarin, Rangpur lime, Wekiwa and Sampson tangelos, Ponkan mandarin and Rough lemon. Wekiwa and Sunshine tangelos were also grafted with seedlings of calamondin, and others were inoculated from affected trees. With the nucellar lines on sweet orange, grapefruit, Troyer and Morton citrange, trifoliate orange, and Rangpur lime there were no symptoms of decline after three years. With the nucellar line, calamondin on Wekiwa tangelo, Willow leaf mandarin, and sour orange, both inoculated and check trees declined. In the buds from old-line calamondin, all those except on Rough lemon wilted and many died. With the Wekiwa and Sunshine tangelo on calamondin inoculated with old-line calamondin buds, there were no effects after three years. The time period for observations may have been too short for some symptoms to develop. All this is very confusing. Weathers and Calavan suggest that if a virus is involved, it must be seed transmitted , and either carried in the root stock seedlings of the calamondin seedlings. The causal agent could also be transmissible by a vector or by mechanical means. In Texas, Olson noted Shary Red grapefruit, Webb Red Blush grapefruit, and Valencia trees on calamondin had a bud union disorder. Shary Red grapefruit or Valencia orange on lemonquat showed symptoms similar to those on calamondin.

Webber obtained almost the same correlation for a population of 1,506 ten-year old orchard trees of Washington navel on sweet orange stocks, from which all small budlings had been vigorously eliminated before planting . Here the area of cross section of scion trunk of the orchard trees, taken one year after planting, correlated with the area of cross section of trunk when nine years of age and with average total seven year yield per tree, gave coefficients of +0.158 ± 0.017 and +0.229 ± 0.016, respectively . These results, together with other data, led Webber to conclude: “It thus seems that it is fairly safe to assume that after all variants are excluded from a population a small correlation still exists between budling size and tree size and between budling size and orchard yield, amounting to a coefficient of approximately +.l5 to +.18 in the former, and +.22 to +.23 in the latter. These are positive correlations, but are small and thus of rather doubtful significance and leave one somewhat in doubt whether any selection other than that intended to eliminate the variants is important.” In the experiments with sour orange trees referred to previously, detailed records of each tree were retained and it was thus possible to arrange and test the results of different methods of selection . Records of the entire population of 346 trees were used and two methods of selection were tested, one based on the diameter of the stock seedling just prior to budding, the other on the diameter of the scion trunk of the one-year-old budlings just before they were transplanted to the orchard. In the selection based on seedling size,vertical farming systems seedlings 2.1 cm or more in diameter were graded as firsts , and those 2.0 cm or less in diameter were graded as seconds .

In the selection based on budling size, a similar classification was employed: budlings having scion trunks 1.8cm or more in diameter were graded as firsts , and those having scion trunks 1.7cm or less in diameter were graded as seconds . The average total five-year yields of all the trees in each grade were then brought together for comparison. An examination of Table 1 shows that, when the selection was based on diameter of seedling trunk, there were 237 seedlings in grade one and 109 in grade two and the average five-year yield of trees in grade one was 56.2 pounds per tree greater than that of trees of grade two. When the selection was based on the diameter of the budling trunk, there were 242 trees in grade one and 104 trees in grade two. The average five-year yield of the trees grown from grade-one budlings was 47.2 pounds per tree greater than that of trees from grade-two budlings. The elimination of variants is of primary importance in the cultivation of citrus, as trees propagated on variant seedlings remain, with few exceptions, permanently dwarfed and unproductive. In the studies reported above, differences in size of trees and in yields, as the result of selection after elimination of variants, were especially marked during the first few years. These differences gradually minimized, but in most cases were still apparent after 30 years in the orchard. The benefit resulting from the selection of variant-free progenies and larger-sized progenies was interpreted by Webber as due to the holdover influence of large size and vigor in the young seedlings, which maintain that advantage apparently throughout the life of the orchard. It was not supposed that the heritage of the plants was influenced by the selection, but rather that the effect was similar to that obtained by the selection of large seeds and enabling the tree to get off to a good start. Careful studies on root stock size as it affects the size and yield of orchard trees were also carried out by Mendel in Israel. Mendel used stocks of the Palestine sweet lime budded to Shamouti sweet orange.

Four plots of 20 trees each on small stocks and four plots of 20 trees each on large stocks were set out in alternate arrangement in a test orchard and their comparative development recorded. Mendel found that the difference between the groups of small and large stocks which, at the time of selection in May 1931, amounted to 43.6 per cent , decreased gradually until the time of transplanting into the test orchard in March 1934, when the difference was only 7.3 per cent. At this time, a second selection was made, which raised the difference to 20.5 per cent. Following this, Mendel stated that the difference between the small and large stocks was reduced to 5.1 per cent in June 1939. The growth of the scions, on the whole, showed the same tendencies as that of the stocks, and differences between the groups with regards to yield were not significant from the first year of fruiting onward. He concluded that the elimination of deviating phenotypes was therefore all that mattered in the selection of sweet lime stocks. This does not agree with the root stock data presented by the author where different scions and different root stocks were involved, variants were supposedly eliminated, and yet the difference in response of size of budlings was still evident after 33 years with most of the combinations. Mendel’s conclusion somewhat confirms Webber’s findings in his earlier experiments , but not the latter. Mendel, however, worked with Palestine sweet lime, which develops a very high percentage of nucellar embryos and thus reproduces nearly true to type through the seed, exhibiting only about 2 per cent of variant seedlings. It is difficult to believe that Webber didn’t eliminate most of the variants in all the combinations he had. As Webber pointed out, the variant seedlings are usually small and weak, and are mostly eliminated in transplanting from the seedbed to the nursery, or are usually easily detected and eliminated in the nursery at the time of budding. This same condition holds with the Rough lemon, Sampson tangelo, and such citranges as the Morton, Troyer, Savage, Rusk, and others, the embryos of which are from 98 to 100 per cent nucellar.

With such stock varieties, selection to eliminate the small number of variants is less troublesome; but with seedling stocks of some sweet oranges, sour orange, grapefruit, some mandarin varieties, Citrus macrophylla, etc., where the proportion of variants may range from 20 to 30 per cent, removal of the variant seedlings becomes highly important. Furthermore, experience indicates that in transplanting seedlings from the seedbed to the nursery, and to some degree in transplanting budlings to the orchard, the small plants are more likely to die than the larger ones,growing vegetables in vertical pvc pipe and for this reason also, the small plants should commonly be discarded. Gardner and Horanic also report on the relation of citrus nursery tree size 1/2 to 7/8 inches in diameter to ultimate size of tree and production. They found in a 17- year-old root stock test block of two varieties on seven root stocks, that although the tree size and yield were profoundly influenced by the root stock variety, there was not pronounced correlation between the initial sizes studied and ultimate size, or between initial size and yield, within the various root stocks. There was, of course, a positive relationship between final tree size and yield. When the seedbed stock is being dug and transplanted, the policy of discarding all small individual plants should be rigorously followed. The proportion that should be destroyed will depend upon the degree of variation exhibited by the particular lot of seedlings. If they are grown from seeds taken at random from trees of varying types as with the “bulk seed” so commonly used by many nurserymen, it is probable that 20 per cent or more of the total number should be eliminated.If the seedlings are grown from seed from selected trees known to produce fairly uniform seedlings, the percentage of discards should be smaller. If the seed is from special varieties in which nucellar embryony is high, such as in Palestine sweet lime, Rough lemon, C. macrophylla, or Troyer citrange, then the percentage of discards at the seedbed may be limited merely to the size that can be properly handled at transplanting. In instances where C. taiwanica, Sacaton citrumelo , or other low polyembryonic types are used, cullage may run as high as 50 per cent. At this time also, all malformed seedlings, such as extreme cases of “goose-neck” or “bench root,” should be discarded. However, if care is not exercised in transplanting the seedlings, small seedlings in particular can have the tap root markedly bent on insertion into the planting hole. Maybe this isn’t too serious. We know of no record where growers have willfully planted budlings with bench roots. However, it was intentionally done at Riverside with Valencia oranges on Morton citrange stock in 1944. There were three five-tree plots with normal root systems and three five-tree plots having bench roots. There were never any observed differences between these two lots of trees. When the trees were pulled in 1960, 26 years later, the trees were the same size, there was no difference in yield, and the ultimate root systems looked alike at time of pulling. While elimination of bench roots is advisable, it is not critical if some occur in the nursery. It is of course difficult to eliminate bench roots in balled nursery trees or container grown trees, but not in bare root trees. When the seedlings growing in the nursery have reached the size and age for budding, preferably just before the budding is started, the nursery should be examined, row by row, and all the inferior or off type plants removed. At this time also, a considerable number of the small plants should be eliminated. The budder or his helpers can do this at time of budding. This is the last time that the characters of the seedlings can be examined, and it is the most important time to do a thorough job. If there was a fair elimination at the seedbed, the rogueing in the nursery should not require the destruction of another 5 to 10 per cent of the total number of plants.

When the budded trees have reached the age and size for transplanting into the orchard, they may vary considerably in size, owing to differences in manipulation, in the time the buds started growth, etc. If, however, the seedling stocks have been carefully selected, all should produce good trees, except rarely where the buds did not heal well, or were defective. At this time all particularly undersized, weak, or sickly budlings should be eliminated. Nurserymen in California should be aware that in some areas where the nursery is located adjacent to natural vegetation, natural spread of stubborn disease is particularly high, and obviously infected trees should also be discarded. With properly selected stocks and good buds, the elimination of budlings should be slight, probably not more than 5 to 10 per cent. Even at this time, the trees have a relatively low value in comparison with the value they will have after a few years in the orchard, and the importance of having every tree a good tree, in a long-term crop like citrus, can scarcely be over estimated. The nursery practices described by Platt and Opetz deal primarily with field grown nurseries, which for many citrus growing areas are now obsolete. The trend since 1973 has been toward container grown citrus. South Africa, since about 1985, grows only container grown plants. Spain and Australia are principally into container growing, with field grown nurseries rapidly disappearing. Container growing is more efficient, cheaper, requires half the growing period of field grown trees, plus other advantages. Perhaps the greatest advantage of container citrus is that each plant is treated as an individual, not a mass of thousands. Each plant can receive more careful care, treatment, and scrutiny. The individual handling of each plant should result in better observation and the likelihood that off types and variants will be more readily noticed and discarded. The fact that certified trees must be identified and logged adds additional confidence.

The nucleotide sequence of the putative DNA-binding domain of SlARF6A was amplified and fused to that of the glutathione S-transferase tag in a pGEX-4T-1 bacterial expression vector and expressed using Escherichia coli strain BM Rosetta . Isopropyl-β-D-thiogalactopyranoside was used to induce recombinant protein expression, and a GST-Tagged Protein Purification Kit was used to purify the protein. Purified recombinant proteins and biotin-labeled fragments of the target promoters were used to conduct EMSA with a Light Shift Chemiluminescent EMSA kit based on the method described in detail by Han et al.. The Pierce Biotin 3’ End DNA Labeling Kit was employed to label the probe containing the TGTCTC sequence with biotin. The unlabeled same sequence was used in the assay as a competitor. To generate the mutant probe, the TGTCTC DNA fragment was changed to AAAAAA. Biotin-labeled DNA was assayed via a ChemiDoc™ MP Imaging System based on the manufacturer’s procedures. All primers for the EMSA are listed in Supplementary Table S3.Many varieties of Citrus grown commercially will reproduce reasonably true to type from seed through a phenomenon known as nucellar embryony as described by Frost and Soost . These include sweet orange, Rough lemon, Cleopatra mandarin, Troyer citrange and others. Other varieties such as the pummelos, citrons, Algerian tangerine, Temple orange, etc. are monoembryonic and will not reproduce true to type from seed since they only produce hybrid progeny. Still others, such as certain lemons, Mexican lime, nansho-daidai, yuzu, etc. have relatively low polyembryony. On the other hand, some varieties such as the Washington navel orange, Bearss lime, Satsuma mandarin,vertical hydroponic and Pixie mandarin are seedless, or nearly so, and it would be impossible to obtain seed in adequate quantities.

Seedling trees of most varieties are vigorous upright growers, extremely thorny, and are late in coming into bearing. Many citrus species and varieties also do not inherently possess adequate hardiness to cold, resistance to soil-borne diseases, tolerance to salinity or high water tables, and other desirable qualities that would enable them to survive long in their planted environment as seedlings. Consequently, some are therefore occasionally propagated by some vegetative means such as cuttings, layers, or marcots, but generally by grafting or budding onto a root stock of some closely related variety, species, or even genera, or hybrids thereof, to take advantage of the root stock’s influence. The latter method is the one generally used in the propagation of citrus varieties, and the general technique is described by Platt and Opetz . The root stock and the scion interact with each other to produce stionic effects which may have a three-way influence. First, there is the influence of the scion upon the stock. For example, the scion may increase the sodium uptake of the root stock. The depth of root penetration or the extent and configuration of the root system may vary on a given root stock, depending upon the scion variety budded upon it. The root stock may greatly alter the scion. It may dwarf or invigorate it. Yields may be increased or decreased; fruit size may be altered; fruit quality can be affected; hardiness of the scion may also be influenced; and maturity and precociousness of the scion are other considerations. The union of a stock and a scion may give rise to a combination which may be affected by an external factor which by itself affects neither the stock nor the scion individually. Such a situation exists when the virus disease tristeza is present. The sweet orange by itself is not measurably affected; the sour orange by itself is not viscerally affected. However, when sweet orange is budded upon sour orange and tristeza virus is present, the combination will decline or die from the disease. If sour orange is budded upon sweet orange, there is no expression of the disease, even though the virus is present.

While it has long been recognized that the stock and scion have a reciprocal influence on each other, there must be a certain affinity or congenial relationship between them for healthy development of the composite plant. Different root stocks vary in their adaptability to grow on different soils and under different climatic conditions, as well as with different scion varieties. All of these factors will be thoroughly discussed in depth individually at a more appropriate place in this monograph. It is ridiculous to assume that any one root stock will have the general qualities to meet each grower’s needs, yet each individual grower’s specific root stock need is a critical choice for the success of his orchard. The successful choice of a root stock is important because it is to be a permanent part of that orchard and cannot be changed at will like a cultural practice, a fertilizer program, and irrigation schedule or pest control procedures. A considerable fund of information has accumulated in recent years throughout the citrus producing regions of the world concerning root stock reactions under different conditions. A recent comprehensive, but concise, review of much of the body of knowledge has been made by Wutscher . The last previous detailed treatment of the subject was by Webber and Batchelor and Rounds . The purpose of the present monograph, therefore, is that it will emphasize the importance of careful root stock selection for different commercial citrus varieties. It is an attempt to evaluate the response of the root stock to the influence of its total environment; describe the difficulties commonly encountered in choosing desirable root stocks; stress the need not only of considering the results of scientifically planned field experiments, but also the findings and observations of discriminating growers; and review critically the results of root stocks and related experiments. In addition, much original data and observations on trials conducted by the writer during a 40 year period at the Citrus Research Center of the University of California, Riverside are presented.

These are based on nearly 500 root stocks and 100,000 trees grown by the author and supplemented by numerous observations in commercial orchards throughout the world. It is just as important to use carefully selected root stocks of superior performance as it is to use selected superior fruit varieties. The selection of a root stock should be for the purpose of enhancing the merits of a scion variety, or adapting it to its total environment, rather than merely to follow local custom. The selection of improved fruit varieties has been in progress for centuries, but the choice of the best root stocks to use has not received much attention prior to a hundred years ago, and most of it has been in the last fifty years. Although propagation by budding and grafting was understood many centuries ago in China and elsewhere, it was mostly considered a curiosity rather than a practical measure,vertical lettuce tower and most commercial citrus trees throughout the world were grown as seedlings. Schenk states that citrus was budded in China before the time of Christ. The author finds this acceptable but extremely difficult to document. In Han Yen-chih’s “Chü Lu” written in 1178 A.D., and translated into English in 1923, he does describe the grafting process. However, he does indicate the method for grafting trees will be found in the work called “Ssu Shih Tsuan Yao,” which I have not been able to find a record of. Greek and Roman references are numerous. According to Condit the grafting and budding of fruit trees were common practices in the time of Theophrastus, who said, “The ingrafted part uses the other as an ordinary plant uses the ground. Whenever they have split the trunk, they insert the scion which they have fashioned to a wedge-shape; then with a mallet they drive it in to fit as snugly as possible.” Virgil in his Georgics and Ecologues, according to Condit , provided an explicit as well as a poetic account of grafting. Briefly it is as follows: “Nor is there one sole way to graft and bud, for where young eyes from the trees bark swell forth, bursting their slender sheaths, a slit is made just as the knot; and here they fasten in the shoot from stranger tree and bid it thrive in the moist sapwood. Or, smooth trunks are gashed and wedges through the solid timber driven. Then fruit scions set; in no long time the tall trees skyward lifts its laden boughs and sees with wonder what strange leaves it bears and fruitage not its own.” According to Gallesio , Palladius, who is thought to have written at some time in the 5th century states, “They graft the citron in April in warm districts and in May in colder latitudes, placing the graft not upon the bark, but opening the stem or trunk near the ground.” Also, wood cuts from Ferrari’s “Hesperides” clearly show the grafting technique being practiced in an ancient herbarium . In the early history of citrus culture, especially in the western world, consumption of the fruit was principally restricted to the area in which it was produced. As faster and more convenient transportation developed, new consumer markets developed, which in turn stimulated new plantings. New plantings created new problems.

Probably the general use of grafted or budded trees first became an accepted practice as a result of the outbreaks of “foot-rot,” in the middle of the 19th century, as pointed out by Fawcett as it occurred in the Azores in 1834. It was observed in the Mediterranean area that the sour orange was resistant to the disease and could be successfully used as a root stock in place of the susceptible sweet orange varieties. The introduction of seedless commercial varieties such as the Valencia Orange, Pera, or Marsh grapefruit to the industry also gave impetus to propagation by budding. Since the middle of the 20th century, considerable attention has been given to the choice of the best root stocks for the different varieties and soil conditions. However, for the most part, relevance has been placed on the results obtained in a given area with one stock, or at best with only two or three others for comparison. For nearly half a century in California, root stock choice was based on the adage, “Use sweet orange on the light soils and sour orange on the heavy soils.” If a root stock gave commercially successful results, it was generally considered satisfactory, with little incentive to search for a better one. This attitude still prevails in many citrus producing countries today. The greater part of the information available in any locality, with reference to successful stocks, was based on the experience of the growers, and often this is the most reliable available. It must be recognized, however, that such local experiences are inadequate, as they do not include replicated trials with a sufficient number of stocks or scion sources over a long enough period of time to supply valid comparisons. This is a situation which has prevailed in nearly every citrus growing area, but since the 1940’s growing emphasis has been given to systematic trials, especially in the United States and Brazil. Probably the one single factor which has given more impetus than any other to the recent emphasis on citrus root stock trials around the world is the occurrence of tristeza, and the intricate response of various stock-scion combinations to its presence. While the disease has primarily affected the sweet orange and certain other scions on sour orange root stock, many other root stocks and combinations can be affected. See the section on tristeza elsewhere in this monograph. Studies on the etiology of tristeza focused greater attention on other diseases caused by transmissible agents which are often influenced in their reaction by certain root stock-scion combinations or the root stock itself . The inroads of disease usually necessitate growing citrus where other citrus has previously grown, and the complex replant problem becomes a serious consideration. Rising production costs economically demand greater production per acre either in tons of fruit or pounds of soluble solids. Greater production necessitates new markets and the competition increases demands for better quality fruit, both for fresh fruit and for concentrate or other purposes. In order to solve these problems, it becomes increasingly necessary to obtain the greatest production at the least possible cost.

A total of 26 stably transformed callus lines were obtained. In the condition without DEX treatment, five calli were randomly selected from each callus line and GUS-assayed for detection of transposant cells. Transposant cells were detected in 84.6% of callus lines but mosaic GUS patterns occurred at low frequency as compared with the GUS patterns of untreated pJJ86 calli . GUS assays were also carried out on 14 of pHPT-Ds1 transformed plantlets; 57.1% plantlets contained transposant cells that were rarely distributed in the tissue . The results of pJJ86 transformants and pHPT-Ds1 transformants indicated that there was background transposition activity in the rice calli and plantlets selected from hygromycinmedia, and that the growth of rice cells containing HPT-Ds transposition events were partially suppressed by hygromycin counter selection. To characterize the HPT-Ds excision events, rice genomic DNA of eight GUS-positive pHPT-Ds1 transformants was extracted and examined in nested polymerase chain reaction reactions using Ubi- and GUS-specific primers . Reconstructed Ubi:GUS sequence containing the HPT-Ds empty donor site was confirmed by sequencing the 657-bp PCR product . These results suggested that HPT-Ds elements in the pHPT-Ds1 transformants excised from the T-DNA. To get more information about the background transposition in the GVG-inducible AcPTase system, we constructed pHPT-Ds3 and pHPT-Ds4 by removing the 35S:GVG from pHPT-Ds1 and pHPT-Ds2, respectively. According to the GUS assay results of pHPT-Ds3 transformed callus lines, 57.1% of the callus lines showed somatic transposition. The mosaic GUS patterns of pHPT-Ds3 transformants were similar to those of pHPT-Ds1 transformants and the transposition frequency was a little lower than 84.6% of the pHPT-Ds1 calli. Our explanation for the results of pHPT-Ds1 and pHPT-Ds3 is that the background transposition in the GVG-inducible Ac-Ds system was primarily due to a low-level leaky expression of 4xUAS:AcTPase.

The system of bacterial Cre-lox site-specific recombination was shown to be a useful tool for the generation of chromosomal rearrangements in plants . To stabilize transposed HPT-Ds,indoor vertical farming we used Cre-lox system to delete AcTPase after HPT-Ds transposition. pHPT-Ds5 and pHPT-Ds6 carry AcTPase flanked by two lox sites and the Cre gene that is separated from the upstream Ubi by HPT-Ds. To control AcTPase, the two vectors were designed in such a way that HPT-Ds can transpose in the rice genome and excision of HPT-Ds reconstructs Ubi:Cre and Cre recombinase mediates lox-lox recombination and thereby deletes AcTPase . For examination of rice cells containing deletion events, GUS and Bar were used in pHPTDs5 and pHPT-Ds6, respectively. We transformed pHPT-Ds5 into rice cultivars Taipei 309 and Nipponbare. Three of four Taipei 309 transformants and 23 of 30 Nipponbare transformants showed transposition as shown by mosaic GUS patterns . Genomic DNA of the Taipei 309 transformants was examined in nested PCR reactions using Ubi- and Cre-specific primers. Reconstructed Ubi:Cre sequence containing the HPT-Ds EDS was confirmed by sequencing the 0.6-kb PCR product . A 4.3-kb fragment containing the HPT-Ds full donor site in T-DNA was also amplifified in PCR of the transformants. Additionally, the pHPT-Ds6 vector was transformed into Nipponbare and the Ubi:Cre sequence was detected in genomic DNA of five of the eight pHPT-Ds6 transformants . Using adaptor-ligation PCR , we successfully cloned the rice genomic sequences flanking the HPT-Ds terminus from one pHPT-Ds5-transformant and one pHPT-Ds6- transformant . BLAST analysis showed that the flanking sequences were from the rice chromosomes 6 and 4, respectively, thereby confirming the reinsertion of excised HPT-Ds in the rice genome.

In analysis of T1 populations of pHPT-Ds5 plants, four of six T1 families showed transposition based on spotted GUS staining of the leaf tissues. These results indicated that the HPT-Ds element in pHPT-Ds5 and pHPTDs6 transformants transposed in rice and that the transposition restored Cre expression and induced deletion of AcTPase.During plant transformation and selection, HPT expression relies on the upstream Ubi promoter to confer resistance to hygromycin in selection media. In case of transposition, the HPT gene may be inactive because the 5 flanking sequence of HPT-Ds at a new genomic site may not be able to provide promoter activity. It is conceivable that most of the transposant cells become sensitive to hygromycin. Therefore, the counter-selection nature of the HPT gene in HPT-Ds can be used to diminish transposant cells in newly transformed rice calli on hygromycin media. In testing pHPT-Ds1 and pHPT-Ds3, it was observed that early transposition events in transformed calli and plantlets were suppressed by hygromycin. Few transposant cells in the calli and plantlets were able to grow under the hygromycin selection pressure, which might be due to escaping transposant cells or because of promoter activity of the 5 transposon flanking sequence. Because transposition requires transposase, an important theme in transposon tagging research is how to efficiently control transposase activity. It was reported that AcTPase driven by strong promoters mediated high-frequency Ds excision in several dicot plants . Strong double enhancers of CaMV 35S promoter adjacent to wildtype Ac element induced high-frequency Ac excision in rice transformation . In the present study, we have used the GVG-inducible promoter to control AcTPase expression and transposition was induced to high levels by DEX treatment of pJJ86 transformed callus.

However, we also observed a leaky expression of AcTPase in the GVG-inducible Ac system in the transformants of pJJ86, pHPT-Ds1 and pHPT-Ds7 based on GUS assay results. Our explanation is that the transposition background was primarily from a low level of leaky expression of 4xUAS:AcTPase. Consistently, in the pHPT-Ds3 and pHPT-Ds5 vectors that do not have 35S:GVG, 57.1% of the pHPT-Ds3 transformants and 76.6% of the pHPT-Ds5 transformants still showed transposition in somatic cells. In spite of the wild type Ac element having a weak promoter that supports only 0.2% expression of the CaMV 35S promoter , the wildtype Acitself can transpose in rice with a relatively low activity for three successive generations . This indicates that a weak expression of AcTPase can cause transposition events. In Southern blot analysis of genomic DNA of pHPT-Ds7 and pHPT-Ds8 transformants, the 5.4 kb hybridizing band represented the HPT-Ds at FDS in T-DNA. For the hybridizing bands larger or smaller than 5.4 kb, we explain that some of the bands might be from transposed HPT-Ds. The pHPTDs7 transformants showed transposition in somatic cells as suggested by GUS assay results. Because the rice genomic DNA for Southern hybridization was extracted from few leavesof a transformant, transposition in other leaves might not have been detected in the results. Also, since a rice transformant may have more than one T-DNA copy and may contain rearranged T-DNA,hydroponic vertical farming the hybridizing bands larger or smaller than 5.4 kb might possibly be from transgene rearrangement. Nevertheless, the efficacy of the HPT-Ds element when it was brought together with the GVG-inducible-AcTPase and the Cre-lox recombination system in pHPT-Ds7 and pHPT-Ds8 was confirmed by GUS assay and Southern blot analysis. For inducible Ac-Ds system, it was reported that in Arabidopsis AcTPase controlled by a heat shock promoter transactivated Ds upon heat shock treatment of flowering plants and the transposition was subsequently stabilized by release of the heat shock treatment . The heat shock method used in Arabidopsis seems impractical for rice because of the difficulty of heat shock treatment of a large number of rice plants. But for the GVG-inducible Ac-Ds system, the transgenic rice plants can be treated with DEX by hydroponics or by spray to induce transposition to higher frequency given that the treatment condition is optimized. Because the Cre-lox-based strategy will help delete AcTPase and thereby stabilize transposed HPTDs elements, we will be able to use GVG-inducible AcTPase to induce higher levels of transposition while using the Cre-lox system to stabilize transposition. The pHPT-Ds7 and pHPTDs8 vectors contain both GVG-inducible AcTPase and Crelox systems and therefore provide a good solution to major drawbacks in the Ac-Ds system. Further work needs to be done with the pHPT-Ds8 vector to determine how to enhance transposition by DEX induction and how to use the Bar gene to select Basta-resistant transposant progeny. In summary, we have constructed a series of Ac-Ds transposon tagging vectors and tested individual approaches to control AcTPase expression and transposition in transgenic rice. The pJJ86 and pDs-Ac-GVG vectors were made for testing GVGinducible AcTPase; the pHPT-Ds1 vector was for testing both GVG-inducible AcTPase and HPT-Ds that contains a dualfunctional HPT gene; the pHPT-Ds5 and pHPT-Ds6 vectors were for testing the deletion of AcTPase via Cre-lox recombination.

The pHPT-Ds7 and pHPT-Ds8 vectors contain all the features of GVG-inducible AcTPase, HPT-Ds and Cre-lox recombination and were tested for comprehensive control of AcTPase and HPT-Ds. The Ac-Ds transposon tagging vectors described in the present paper are publicly available, and provide useful resources for the functional genomics of a wide range of plants and especially for that of monocot plants.Introgressions from wild species are important resources for broadening the genetic base of cultivated species, particularly for traits where little variability currently exists. This is certainly the case for cultivated tomato , an economically important vegetable crop species with limited genetic variability . The genetic diversity of tomato has been augmented through introgression of alleles from several closely related wild species . One of these species, Solanum habrochaites, has been an important source of favorable alleles for horticultural traits such as yield, fruit size, and fruit quality . This wild species also contains genes for resistance to major tomato diseases such as late blight, bacterial canker, gray mold, and early blight . In cultivated tomato, genetic diversity is particularly lacking for resistance to late blight disease caused by Phytophthora infestans . Late blight is an economically important and devastating disease of both tomato and potato because it results in approximately $5 billion in annual crop losses and chemical control costs . S. habrochaites has genetic resistance to P. infestans. QTL for quantitative resistance to P. infestans from S. habrochaites have been mapped on each of tomato’s 12 chromosomes . Three of these QTL were then fifine-mapped by Brouwer and St.Clair using near-isogenic lines . QTL affecting horticultural traits including plant height, plant shape, maturity, yield, and fruit size were co-located and/or linked with each of these resistance QTL, suggesting the potential for linkage drag in crosses between S. lycopersicum and S. habrochaites. Subsequently, we mapped the QTL on S. habrochaites chromosome 11 at higher resolution using sub-NILs and detected multiple closely linked QTL controlling both foliar and stem resistance to P. infestans within a 9.4-cM region . To gain a better understanding of the genetic basis of QTL controlling horticultural traits and their linkage relationships with QTL for resistance to P. infestans, we used this same set of sub-NILs in the present study to map loci controlling horticultural traits and determine linkage relationships among them and with P. infestans resistance QTL. We also sought to identify useful breeding material with improved late blight resistance in this set of sub-NILs. In the present study, we further investigated the P. infestans resistance QTL lb11 region identified by Brouwer and St. Clair , conferred by a S. habrochaites introgression on tomato chromosome 11 as a potential source of useful quantitative resistance to late blight disease of tomato. Specifically, our goals in this study were to: assess the effects and extent of linkage drag of QTL controlling horticultural traits with P. infestans resistance QTL on S. habrochaites chromosome 11; identify markers closely linked to P. infestans resistance QTL and to positive alleles at horticultural QTL to facilitate MAS breeding; and identify potentially useful breeding lines for future breeding of tomato cultivars with improved quantitative resistance to late blight disease.We developed a set of sub-near-isogenic lines in S. lycopersicum for a chromosome 11 introgression containing resistance QTL from P. infestans-resistant S. habrochaites accession LA2099 via marker assisted selection during back crossing and selfing generations, as described by Johnson et al. . Methods used for genomic DNA extractions, genotyping with chromosome 11 PCR-based markers , primer sequences, enzymatic reaction conditions, and restriction enzymes used for each marker were described by Johnson et al. . We genotyped 1902 BC6S1 progeny to identify recombinant subNIL progeny for the chromosome 11 introgression from S. habrochaites; of these progeny, a subset of 852 progeny was used to construct a linkage map for the introgressed region .

CPK10, CPK30, and CPK32 are subgroup III Ca2+-sensor protein kinases . The activity of CPKs can be enhanced in response to nitrate within 10 min. They have all been identified as master regulators that orchestrate primary nitrate responses. Analysis of the single cpk10, cpk30, and cpk32 mutants has shown that they only trivially affect nitrate-responsive genes. However, in the double mutants cpk10 cpk30, cpk30 cpk32, and cpk10 cpk32 and the triple mutant cpk10 cpk30 cpk32, nitrate-responsive marker genes were reduced. Transcriptomic analysis showed that CPK10, CPK30, and CPK32 modulated various key cellular and metabolic functions immediately activated by nitrate. Furthermore, CPK10, CPK30, and CPK32 can phosphorylate NLP7 at Ser205 in vivo in the presence of nitrate, and trigger the nitrate-CPK-NLP signaling network.Recently, three other nitrate regulatory genes NRG2, CPSF30-L, and FIP1 were identified using a forward genetics method. Two independent NRG2 T-DNA insertion lines showed reduced induction for nitrate-responsive sentinel genes , indicating that NRG2 plays an essential role in nitrate signaling. At the physiological level, NRG2 affects accumulation of nitrate in plants. Further investigation revealed that it regulates nitrate uptake by roots and the translocation of nitrate within plants. These effects might be achieved through modulating NRT1.1 and NRT1.8 as the expression of both genes was altered in the mutants. Genetic and molecular data suggest that NRG2 can regulate the expression and work upstream of NRT1.1, but function independently, with NLP7 in regulating nitrate signaling. In addition,indoor growers transcriptomic analysis showed that four clusters in the differentially expressed genes in nrg2 mutant were involved in the regulation of nitrate transport and response, confirming that NRG2 plays essential roles in nitrate regulation.

Interestingly, NRG2 can directly interact with NLP7 in vitro and in vivo, as revealed by yeast two hybrid and BiFC experiments.In addition, the Arabidopsis genome harbors 15 members that are homologous with the NRG2 protein. All members of the NRG2 family contain two unknown conserved domains: DUF630 and DUF632. Whether and which other members of the NRG2 family are involved in nitrate signaling and what functions the two domains play are interesting and pertinent directions for future research. The CPSF30 gene encodes 28-kD and 65-kD proteins. The 28-kD protein was identified as a cleavage and polyadenylation specificity factor; the protein contains three characteristic CCCH zinc finger motifs and functions as both an endonuclease and an RNA-binding protein. An additional YTH domain, along with the three zinc finger motifs, are contained in the 65-kD protein. A mutant allele of CPSF30, cpsf30-2 with a G-to-A mutation in the first exon of gene CPSF30, was identified by genetic screening and used to explore the functions of CPSF30. The expression of nitrate-responsive genes can be down regulated in response to nitrate in cpsf30-2 compared to wild-type and restored to wild-type levels in a complemented CPSF30-L/cpsf30-2 line, indicating that CPSF30-L is involved in nitrate signaling. CPSF30-L can regulate nitrate accumulation and assimilation at the physiological level. Transcriptomic analysis showed that genes involved in six nitrogen-related clusters, including nitrate transport and assimilation, were differentially expressed in the cpsf30-2 mutant. Further study revealed that CPSF30 could work upstream of NRT1.1 and independently of NLP7. CPSF30 can also affect NRT1.1 mRNA 30 UTR alternative polyadenylation. All these results demonstrate that CPSF30 plays an important role in the primary nitrate response. FIP1, a factor interacting with poly polymerase 1, was identified as a positive nitrate regulatory gene using the fifip1 mutant and a FIP1/fifip1 line.

Nitrate-induced expression of NIA1, NiR, and NRT2.1 is repressed in the fifip1 mutant and can be restored to the wild type in the FIP1/fifip1 line. Furthermore, FIP1 can affect nitrate accumulation through regulating the expression of NRT1.8 and nitrate assimilation genes. Further research found that FIP1 could interact with CPSF30 and both genes can regulate the expression of CIPK8 and CIPK23. In addition, FIP1 can affect the 3 0 UTR polyadenylation of NRT1.1, a similar function to CPSF30. CPSF30, FIP1, and some other components such as CPSF100 can form a complex involved in poly processing. Together, these findings suggest that the complex composed by CPSF30 and FIP1 may play important roles in nitrate signaling. In the extant literature, key molecular components involved in primary nitrate responses, covering nitrate sensors, transcription factors, protein kinases, and polyadenylation specificity factors, have been identified. Methodologically, this has been achieved by using forward and reverse genetics as well as systems biology approaches . In summary, in the presence of both ammonium and nitrate , NRT1.1 functions as a sensor. NLP7, NRG2, and CPSF30 have been revealed to work upstream of NRT1.1. NRG2 can interact with NLP7 whilst NLP7 can interact with, and be phosphorylated by, CPK10. In addition, NLP7 binds to the promoter of NRT1.1 as revealed by ChIP and EMSA assays. NRT1.1 works upstream of, and regulates, TGA1/TGA4. Furthermore, CIPK23 interacts with and phosphorylates NRT1.1. CPSF30 can interact with FIP1 and regulate the expression of both CIPK8 and CIPK23. NIGT1.1 can suppress NLP7-activated NRT2.1. In the presence of nitrate but absence of ammonium , NRT1.1 works only as a nitrate transporter, but not as a nitrate regulator. The other nitrate regulatory genes, including NRG2, NLP7, CPSF30, FIP1, LBD37/38/39, SPL9, NIGT1s, CIPK8, and CIPK23, still play an important role in the nitrate signaling.

Serving as an important molecular signal, nitrate also regulates plant growth and development and has been particularly well studied in the context of root system architecture. Root system architecture controls the absorption and utilization of nutrients and affects the growth and biomass of plants. Lateral root growth is dually regulated by nitrate availability, including local induction by NO3− and systemic repression by high NO3. Several key genes and miRNAs functioning in nitrate-regulated root architecture have been characterized. The ANR1 gene, encoding a member of the MADS-box family of transcription factors,danish trolley was the first gene to be identified as an essential component in nitrate-regulated root growth. Nitrate can inhibit the growth of lateral roots when seedlings are grown on media with higher nitrate concentrations compared to lower nitrate concentrations . However, ANR1 down regulated lines obtained by antisense or co-suppression exhibited reduced lateral root length when grown on media with various nitrate concentrations, indicating the enhanced sensitivity of lateral root growth to nitrate inhibition in those lines. Over expression of ANR1 in roots resulted in increased lateral root growth and this phenotype was strongly dependent on the presence of nitrate, suggesting post translational control of ANR1 activity by nitrate. Interestingly, the expression of ANR1 in nrt1.1 mutants was dramatically diminished and these mutants exhibited reduced root elongation in nitrate-rich patches, similar to what was observed with the ANR1-repressed lines. This suggests that NRT1.1 works upstream of ANR1 in terms of local nitrate-induced lateral root growth. Recently, the auxin transport role of NRT1.1 was characterized in lateral root primordia when seedlings were grown on media without nitrate or with low nitrate concentrations; under these conditions, NRT1.1 represses the growth of pre-emerged LR primordia and young LRs by inhibiting the accumulation of auxin. Subsequently, Gojon’s lab revealed that the NRT1.1-mediated regulation of LR growth was dependent on the phosphorylation of NRT1.1 and the non-phosphorylated form of NRT1.1 could transport auxin in the absence of nitrate or in low nitrate concentrations. Further investigation indicated that in the presence of nitrate, the promoter activity of NRT1.1 was stimulated and mRNA stability was increased, while protein accumulation and auxin transport activity were repressed in LRPs, resulting in accelerated lateral root growth. Altogether, NRT1.1 offers a link between nitrate and auxin signaling during lateral root development. However, the mechanisms by which nitrate induces the expression of NRT1.1 while repressing NRT1.1 protein accumulation and auxin transport activity in LRPs remain unclear. Previous reports have also documented that several genes involved in hormone biosynthesis or response regulate the root system architecture response to changes in nitrate availability. NRT2.1, a high-affinity nitrate transport gene, is induced by nitrate and sugar. Wild-type seedlings grown on media with high carbon/nitrogen ratios exhibited significantly repressed lateral root initiation compared to a standard growth medium.

However, the repression of lateral root initiation was diminished in nrt2.1 mutants under high C/N ratios where this phenotype was not dependent on nitrate uptake. These results demonstrate that NRT2.1 plays an important role in lateral root initiation under high C/N ratios. In addition, nrt2.1 mutants exhibited significantly reduced shoot-to-root ratios compared to wild-type and nrt2.2 mutant seedlings when grown in common hydroponic conditions . The reductions in shoot-to-root ratios were even greater for nrt2.1 nrt2.2, suggesting that both genes are involved in regulating plant growth with NRT2.1 playing a more important role. Moreover, nrt2.1 mutants exhibit reduced LR growth on media with limited nitrogen and this reduction was more severe in nrt2.1 nrt2.2 double mutant plants, indicating that both genes are important regulators involved in lateral root growth. Recently, Gutierrez’s lab determined that induction of NRT2.1 and NRT2.2 was directly regulated by TGA1/TGA4 in response to nitrate treatment. Further investigation showed that tga1 tga4 plants and nrt2.1 nrt2.2 plants exhibited similarly decreased LR initiation compared with wild-type plants, indicating that NRT2.1 and NRT2.2 work downstream of TGA1/TGA4 to modulate LR initiation in response to nitrate. Lateral root emergence was also affected in tga1 tga4 and nrt2.1 nrt2.2 mutants, and tga1 tga4 mutants displayed larger reductions in LR emergence than nrt2.1 nrt2.2 mutants, revealing that additional pathways are required for LR emergence controlled by TGA1/TGA4 besides NRT2.1 and NRT2.2. Moreover, primary roots in tga1 tga4 mutants were shorter than in wild-type and nrt2.1 nrt2.2 plants, suggesting that the modulation of primary root growth by TGA1/TGA4 is independent of NRT2.1 and NRT2.2. The protein kinase CIPK8 is not only involved in primary nitrate response, but also in long-term nitrate regulation on root growth. In the presence of nitrate, cipk8 mutants exhibited longer primary root length compared to the wild type, indicating that CIPK8 modulates primary root growth in a nitrate-dependent pathway.

Furthermore, the key nitrate regulator NLP7 has also been found to control root growth under both N-limited and N-rich conditions besides its essential roles in the primary nitrate response . nlp7 mutants developed longer primary roots and higher LR density on N-rich media. Interestingly, transgenic lines with over expression of NLP7 also exhibited increased primary root length and lateral root density under 1, 3, and 10 mM nitrate conditions.The underlying inter-phenotype mechanisms regulating root growth in the mutant and over expression lines are still unknown. These findings indicate that NLP7 plays an important role in nitrate-regulated root development. Recently, it has been shown that the Ca2+-sensor protein kinases CPK10, CPK30, and CPK32 are also involved in nitrate-specific control of root development. In response to nitrate, icpk mutants had reduced lateral root primordia density and reduced lateral root elongation compared to the wild type. In the last few years, microRNAs have emerged as important regulators involved in nitrate-regulated root growth. It has been reported that miR167 targets and controls expression of the auxin response factor ARF8, and both miR167 and ARF8 are expressed in the pericycle and lateral root cap. Levels of miR167 were repressed under nitrogen treatment, leading to accumulation of ARF8 in the pericycle. In contrast to wild-type plants, which displayed increased ratios of initiating vs. emerging lateral roots in response to nitrogen treatment, the miR167a over expression lines and arf8 mutants were insensitive to nitrogen in terms of lateral root emergence. These results indicate that the auxin response factor-miRNA regulatory module miR167/ARF8 plays an important role in controlling lateral root growth in response to nitrogen . In addition, miR393 was induced by nitrate treatment, specifically cleaved the auxin receptor AFB3 transcript, and modulated the accumulation of AFB3 mRNA in roots under nitrate treatment . The primary root of the wild type was shorter when treated with KNO3 compared to KCL, however the primary root of the miR393-overexpression line andafb3 mutant were insensitive to nitrate treatments. miR393/AFB3 also controlled lateral root growth as well as primary root growth. The miR393 over expression line and afb3 mutant showed diminished densities of initiating and emerging lateral roots compared to the wild type, which exhibited increased growth of lateral roots in response to nitrate treatments.