Tag: vertical farming

In good agreement with other studies, Solibacteres, mainly Candidatus Solibacter, previously suggested to be adapted to nutrient-limited environments, was associated with the conventional farming system. Interestingly, taxa capable of degrading xenobiotic compounds were also enriched. The nitrification process was enhanced, as ammonia oxidizing bacteria and archaea wereparticularly induced after applying the conventional fertilization, and that response was consistent over the 3 months of the experiment, similar to longer-term studies. Thaumarchaeota archaea’s enrichment has been previously observed after a long-term application of organic fertilizers and in several long-term fertilization experiments with inorganic N treatment. In addition, several denitrifying bacteria responded to the chemical fertilization, such as Gemmatimonas , Pseudomonas , Achromobacter , Nocardia, and Rhodococcus . In this study, functional profiles were more resistant to intervention than community composition. This agrees with the conclusions of Pan et al., who proposed that the coexistence of organisms with overlapping ecological functions confers functional stability. Fierer et al. found that, under high concentrations of inorganic nitrogen, the relative abundance of the DNA/RNA replication, electron transport, and protein metabolism functions increase. Likewise, Carbonetto et al. evidenced that the relative abundances of intracellular trafficking, secretion and vesicular transport, energy production and conversion, and amino acid transport and metabolism were greater in soils under conventional farming system than in uncultivated soils, consistent with a copiotrophic strategy. Ding et al. reported changes in functional groups associated with nitrogen cycling when conducting a metagenomic analysis, observing the greatest effect for functional groups associated with aerobic ammonia oxidation, nitrite reduction, anaerobic ammonia oxidation, and nitrate reduction. Chen et al., besides, found no significant differences in functional genes, predicted from 16S RNA using PICRUSt, involved in denitrification , nitrification ,blueberries in containers growing and N-fixation when analyzing soils managed over 18 years that included organic and conventional farming.

Similarly, in the present study, when evaluating PICRUSt predicted functions, the Kruskal–Wallis test did not detect any differentially abundant functions between the conventional and organic soil samples, which included those N transformation functions. However, according to LDA, organic soils had greater predicted abundances of glutathione metabolism, which plays an important role in the defense of microorganisms and plants against environmental stresses. In addition, it is also involved in the regulation of sulfur nutrition and plays a key role in the nitrogen-fixing symbiotic interaction. However, the functional results reported here are based on predictions obtained from the 16S rRNA gene, which resulted in NSTI values that were moderately high, as expected for phylogenetically diverse samples such as soil, suggesting that those predictions must be interpreted with caution. In addition, functional differences might have been hidden, as de novo OTUs were eliminated for the analysis to conduct PICRUSt predictions. Nut trees are among of the most important horticultural tree crops. Both production and consumption are increasing dramatically due to strong economic returns and the nutritional value of their products. The world’s tree nut production has increased by 48% over the last 10 years . The world-wide export value of tree nut crops amounted to approximately 34.5 billion dollars in 2019, an increase of ~107% compared to the prior 10-year period. Technical knowledge regarding nut tree production has also rapidly increased as a result of the demand for higher production and quality, multiple destinations of nuts fruit in current consumption and food industry, but also of the growing importance accorded to the nuts in a balanced and healthy diet and in the prevention of various diseases. Among the areas of interest and progress has been the use of root stocks to adapt to climate and edaphic factors including soil borne diseases and abiotic stresses, control scion vigor, increase yield, and improve fruit quality. the selection of the scion cultivar is the grower’s top consideration for long-term productivity and profitability, root stock selection is becoming more important. Now, the root stock scion and interaction per se is considered when planting an orchard. The advantages of selected root stocks have been recognized and utilized in the nut trees’ production, but they do not have a long history of use in many species.

Although nut trees are grown around the world, root stock studies are limited to only a few tree nut species. Initially, most root stocks were open-pollinate seedling, or seed stock. Seed stocks are not as genetically uniform as clonal root stocks, but they have advantages such as deep root system and tolerance to edaphic abiotic stresses. However, seed stocks have high heterozygosity in terms of different traits. Hence, the type of seed and location in which it is grown is important for choosing seed stocks. Seed stocks should be uniform, vigorous, disease resistant, and readily available. Therefore, several studies have been performed to study the growth vigor of seed stocks and improve seed germination in nut trees. In addition to seed stocks, a wide range of clonal root stocks are now being developed. Numerous root stock breeding programs have begun to introduce clonal root stocks to meet important challenges, including excess vigor, low yield, poor nut quality, poor soil, climate change, drought and salt stress, suckering, diseases, and graft incompatibility. Common tree nut root stocks, especially clonal root stocks, and their main characteristics are listed in Table 1. Advances in the development of temperate nut trees root stocks until 2003 were last reviewed by Grauke and Thompson. Given the recent advances in root stock breeding for tree nut crops, this review will focus on the physiological and molecular effects of root stocks on scions under different edaphic and climatic conditions. The main purpose of this review paper is to present studies on various aspects of breeding and physiology of nut trees root stock, as well as, draw a comprehensive vision to accelerate future research in this field using combination of traditional and modern methods. To this end, we first provide overall information on vigor, root stock-scion com-patibility, suckering, and rooting ability which can be useful for tree nut crops researchers and growers. Next, we review water and nutrient uptake on nut trees. In the following, we review phenology and yield related traits which are important in industry and marketing. Then, we comprehensively review abiotic and biotic stresses studies on tree nut crops. Finally, we briefly review root stock-scion transfer of macromolecules and small interfering RNAs in nut trees. Since nut tree crops have a long juvenile period, development of a new variety or root stock may take more than 20 years via classical breeding. Therefore, in the conclusion and perspectives section, we note the future prospects of molecular breeding in nut tree crops using novel technologies for rapid generation advancement.The nut trees growth is strongly controlled by the distribution of organic and inorganic constituents within the tree trunk, canopy, and the root system. The vascular system plays a role in this long-distance signaling.

Hypothetically, root stocks impact scion vigor by controlling water and nutrient transfer and hormones signaling and RNAs which move up through the graft union. Numerous studies have been conducted regarding the effect of root stocks on the growth of nut trees. Pistachio growers and breeders are seeking vigorous root stocks. Kallsen and Parfitt reported ‘Kerman’,planting blueberries in containers the previously primary female pistachio cultivar in California, has a rapid growth habit that produces trunk circumferences larger than that of the root stocks. Matching the scion and root stock growth rates produces stronger graft unions. Highly vigorous root stocks produce more uniform graft unions and reduce bark damage from trunk shaking harvesters by uneven graft unions. They report that UCB1 is a better root stock for ‘Kerman’ as it produces a smoother trunk compared to Pistacia integerrima root stocks. Caruso et al. evaluated one seedling and eight clonal pistachio root stocks and reported that root stock had a significant effect on growth rate of the scion and nut yield. Clones of P. integerrima and P. atlantica are highly to intermediately vigorous root stocks. The pistachio cultivar ‘Bianca’ onto P. integerrima seedling root stock had significantly better growth than on P. terebinthus or P. atlantica clonal root stocks. Scions grown on P. terebinthus root stocks had the least vigor. When ‘Bianca’ scions were budded onto eight in vitro propagated clonal root stocks and observed for 4 years, trunk cross-sectional areas on P. integerrima were three times higher than on P. terebinthus root stocks. Ak and Turker reported the cultivars, ‘Kirmizi’ and ‘Siirt’, grafted onto P. vera, P. khinjuk, and P. atlantica demonstrated different budbreak, flowering time and vegetative growth. P. vera flowered earlier and P. atlantica and P. khinjuk had greater stem diameters. Rahemi and Tavallali studied the effect of ‘Badami’ , ‘Sarakhs’ , and ‘Beneh’ seedling root stocks on growth, yield, and nut quality of the Iranian cultivars, ‘Ohadi’, ‘Kalleh- Ghouchi’, and ‘Ahmad-Aghaei’. ‘Sarakhs’ seedlings had the least vigor, while ‘Badami’ root stocks produced the highest yields and best nut quality. Ghazvini et al. evaluated the ecophysiological characteristics of four seedling root stocks, ‘Badami’, ‘Sarakhs’, P. mutica, and P. atlantica. Photosynthesis, stomatal conductance, and transpiration was highest in trees on the ‘Sarakhs’ root stock and lowest on the P. mutica root stock. P. integerrima is the most vigorous root stock now commonly used in pistachio cultivation but is also the least cold tolerant. It is rapidly being replaced by the more coldand salinity-tolerant hybrids, available as both a seedling and a clone, and P. integerrima × P. atlantica, now available as a clone . In contrast to pistachio, there is no a specific walnut breeding program to select high vigorous root stock. Nevertheless, the major walnut clonal root stocks introduced in the last few years are vigorous. Among the clones of ‘Paradox’ which was introduced by the University of California-Davis, ‘VX2110is highly vigorous and nematodes-tolerant root stock. Furthermore, ‘Grizzly’ clonal walnut root stock has been recently introduced as a highly vigorous root stock. The mother tree of ‘Grizzly’ is a Tulare variety grafted on a seedling Paradox root stock. This root stock showsgood performance in poor soil structure with low nutrition and heavy populations of lesion nematodes. In addition, high vigorous trees are very important for the wood industry. Numerous interspecific hybrids were carried out in Juglans genus between J. regia with J. cinerea, J. nigra, and J. major. Compared to the parent, most of them such as ‘NG230 , ‘NG380 , and ‘MJ2090show high vigor, disease resistance, greater winter-hardiness, and high wood quality. Walnuts are highly vigorous trees with an extended juvenility phase. Dwarf walnut trees could potentially decrease labor costs and increase yields per hectare by allowing increased plant density. Although dwarfing has not generally been the most important objective of walnut root stock breeding programs, identifying sources of this trait is of great interest in countries with high genetic diversity such as Iran, China, Turkey, and Central Asian countries. In these countries, traditional orchards of giant walnut trees are difficult to harvest mechanically. Harvest injuries and death of laborers during manual harvesting have precipitated interest in dwarfing root stocks. Reportedly dwarf walnut trees have a short life span. Therefore, in some countries, breeders are attempting to combine slow-growing scions with vigorous root stocks. Juvenile and mature walnut tree vigor is highly heritable. Wang et al. evaluated Persian walnuts in China and selected six dwarf walnut root stocks; ‘Xinwen 6090 , ‘Xinwen 7240 , ‘Xinwen 9080 , ‘Xinwen 9150 , ‘Xin 9160 , and ‘Xinwen 9170 as potential root stocks for breeding. Analysis of growth traits of ‘semi-cultivated’ local genotypes of Juglans regia on their own roots, in the sands area of south-west Romania, showed that climatic and edaphic factors significantly influenced the annual growth ring width of the trees, but also their adaptability to environmental factors. Precocious and dwarf walnut trees have been evaluated in Iran. These genotypes induce dwarfing and precocity in scions in preliminary experiments, apparently due to a slower growth rate. They have fewer nodes, shorter internodes, and smaller shoot length, smaller root system, and lower sap flow and hydraulic conductivity which are the typic traits of dwarf root stocks in other fruit trees. They also have a better rooting ability and higher grafting success. Dwarfing is a desirable trait for other tree nuts. In China, dwarfing chestnut root stocks are being evaluated. In the USA, Anagnostakis et al. attempted to breed dwarfing chestnut root stocks and suggested that hybrids with Castanea seguinii could be a source of dwarfing. Researchers at the University of Missouri identified various chestnut cultivars as potential sources of dwarfing. Studies of graft compatibility, vegetative growth, and productivity of these trees are continuing to determine if dwarf chestnut root stocks are feasible.

ABA, IAA and ethylene accumulation are altered by polyamine levels, which are positively correlated with fruit susceptibility to B. cinerea during strawberry ripening . Other hormones, such as brassinosteroids and jasmonic acid are present at lower levels during strawberry ripening. BR positively regulates vitamin C levels, sugar and anthocyanin biosynthesis during ripening, while negatively regulating acidity and concentration of other phenolic compounds .Endogenous JA levels are modulated by methyl jasmonate and the JA carboxyl methyltransferase that lead to high levels in white fruit and a decline during ripening, antagonistically to ABA . In strawberry, JA appears to be involved in defence responses against B. cinerea. For example, strawberries treated with MeJA had a delayed and much slower progression of B. cinerea infections .As indicated previously, B. cinerea releases enzymes and metabolites that act as virulence factors but may also induce plant responses that are beneficial for fungal infection . A relevant example of the manipulation of physiological processes in the host by B. cinerea is the interference with specific developmental processes. In tomato plants, B. cinerea infections modified host gene expression to increase susceptibility, such as the induction of senescence in leaves . Moreover, infected unripe tomato fruit show premature expression of genes involved in ethylene synthesis during tomato ripening . These findings suggest that B. cinerea could initiate ethylene production and thereby stimulate early ripening. As strawberries are non-climacteric fruit, ethylene production of B. cinerea may not have substantial effects on strawberry ripening; however,large plastic pots the fungus was also shown to induce genes involved in the biosynthesis of other plant hormones such as ABA. Moreover, B. cinerea can synthesize and secrete ABA that functions as a virulence factor .

Besides hormones, increased oxidative reactions caused by the pathogen may influence ripening progression .Defence mechanisms can be divided into preformed and induced defences. In strawberries, preformed defence compounds are especially abundant in the unripe stage, as reviewed in the section on quiescence of B. cinerea. Even though plants accumulate defence compounds, B. cinerea has mechanisms to cope with these metabolites by efflux and detoxification of inhibitory substances. ATP-binding cassette transporters are used by B. cinerea to facilitate the efflux of antifungal compounds, such as stilbenes . B. cinerea is capable of detoxifying inhibitory substances, like epicatechin by secretion of laccases . Active B. cinerea infections can result in a reduction of specific secondary metabolites. It has been reported that levels of flavan-3-ol, benzoic acid and phenylpropanoids drop in B. cinerea-infected strawberries . Strawberries respond to B. cinerea infection by triggering defences. In some cases, preformed and induced defences can overlap such as in the case of PGIPs. An endogenous PGIP appears to be constitutively expressed in fruit from various strawberry cultivars . However, this PGIP and six additional ones show higher expression levels upon infection with B. cinerea . Overexpression of FaPGIP1a and FaPGIP2a in cisgenic plants conferred enhanced resistance to grey mould . Other enzymes induced by B. cinerea infections are chitinases. Expression of the chitinases FaChi2-1 and FaChi2-2 peaked 16 hpi in B. cinerea-infected strawberries . Furthermore, heterologous expression of Phaseolus vulgaris chitinase cH5B in strawberry resulted in higher resistance to infection . Another study demonstrated that application of heat-inactivated cells of the yeast Aureobasidium pullulans promoted tolerance to B. cinerea in strawberries . This primed resistance is probably due to the fruit’s perception of chitin from the yeast leading to induction of chitinases or other plant immune responses. Moreover, fruit defence responses may be primed using mechanical stimulation as it was reported for strawberry leaves . Induced defences include accumulation of secondary metabolites and ROS. For instance, strawberries accumulate proanthocyanins around infection zones possibly to restrict fungal growth . The surroundings of infection sites generally display higher ROS production . ROS can serve as an effective defence against pathogens but also can lead to cell death, which is considered beneficial for necrotrophic fungi . B. cinerea itself produces ROS to induce host cell death, deplete plant antioxidants and increase lipid peroxidation . It is therefore interesting that, in unripe tomato fruit ROS production leads to resistance against B. cinerea, whereas in ripe fruit it seems to promote susceptibility . Future research will likely shed more light on the role of ROS in induced defences of strawberry fruit.

Basal immunity is activated upon fungal infection. Degradation of fruit cell wall pectins can produce demethylated oligogalacturonides that trigger basal immune responses . Expression of the F. x ananassa pectin methylesterase 1 FaPE1 in Fragaria vesca resulted in reduced methyl-esterification of oligogalacturonides in fruit. This reduced esterification activated basal defences via the salicylic acid signalling pathway that led to a higher resistance to B. cinerea . Involvement of SA signalling in responses against B. cinerea was previously suggested when strawberry plants and fruit treated with SA showed decreased post harvest decay . B. cinerea can suppress the expression of plant defence responses by hijacking the host sRNA regulatory pathways . In strawberry fruits, B. cinerea infections can alter the expression of microRNAs involved in the regulation of defence genes, including the plant intracellular Ras group-related LRR protein 9-like gene . Interestingly, B. cinerea can also take up plant sRNAs during its interaction with the host. For instance, transgenic plants expressing sRNA that targets B. cinerea DCL1 and DCL2 show significantly reduced fungal growth in strawberries . The suppression of fungal growth via host sRNA is not well understood, and it is yet to be demonstrated that this mechanism of defence naturally occurs in plants.The diverse arsenal of infection mechanisms employed by B. cinerea explains its extremely wide-host range. It is therefore not surprising that entirely resistant strawberry genotypes do not exist . Several authors have analysed field resistance of strawberries to B. cinerea by quantifying disease development without artificial inoculation. A multi-year study of three strawberry cultivars found a significant effect of year, cultivar and cultivar by year interaction on the incidence of B. cinerea infections . Moreover, there was a positive correlation between row density and disease. Other studies investigated field resistance in annual winter production systems and found that variation of B. cinerea incidence between years was larger than genotype differences within years . Even though field resistance assessments investigate conditions similar to commercial production, considerable variability between environmental conditions and years can interfere with the detection of genotype differences.Due to the confounding effects of different non-genetic variables in field studies,squre planter pots assessment of post harvest resistance to B. cinerea infections has been pursued to determine genotype differences between strawberry cultivars or species. A large study of grey mould development during post harvest storage of non-inoculated fruit reported variation in disease incidence and speed of progression amongst cultivars, but no complete resistance was observed .

Another approach to reducing environmental effects in disease tests is to inoculate fruit with B. cinerea conidia suspensions. Bestfleisch et al. tested quantitative resistance in 107 accessions of wild and cultivated strawberry. In this study, two wild ecotypes of F. virginiana showed high resistance to B. cinerea infections and slow disease progression. Such high tolerance in wild species was also reported in B. cinerea-inoculated leaves and fruit of F. chiloensis accessions from Chile . In these wild accessions, B. cinerea grew much slower. Comparative studies of disease progression indicated that fruit from the cultivar Chandler developed lesions at 24 hpi, while fruit from an F. chiloensis ecotype developed symptoms at 72 hpi . Fruit were entirely covered with mould at 6 days post-infection for the cultivar Chandler and at 9 dpi for the F. chiloensis ecotype. Considering that some accessions, particularly wild ecotypes, show reduced grey mould incidence and progression, there might be genetic sources of resistance against B. cinerea that could be used to increase resistance in strawberry. However, information about resistance mechanisms is mostly based on assumptions or empirical data. Differences in ripening patterns have been suggested as a potential explanation for resistance. For instance, some strawberries ripen from inside to outside, leaving the skin, which is the entry point of infections, unripe and thus resistant for a longer time . Some more tolerant cultivars remain white or unripe around the calyx , which is where many B. cinerea infections tend to initiate. Another mechanism of resistance could be the presence of fungal inhibitors or the induction of PR proteins. FcPR5 and FcPR10 are highly induced in resistant F. chiloensis accessions when compared to commercial F. x ananassa cultivars . Based on sequence homology, FcPR5 probably possesses anti-fungal activity, and FcPR10 is likely a ribonuclease. These findings reflect that even though efforts have been made to explore resistance mechanisms of strawberry to B. cinerea, very little is known. Therefore, more research is necessary to better understand the biology of strawberry interactions with B. cinerea infections using diverse germplasm accessions.Many disease management strategies have been implemented for the control of B. cinerea in strawberry as further described below. However, even combined approaches are only capable of reducing disease incidence and severity but cannot completely prevent or eliminate grey mould in strawberries .Historically, B. cinerea infections in strawberry production have been managed by agronomic and horticultural practices, such as removal of senescent plant material to avoid inoculum buildup . Preventing contact of fruit with soil is another common practice to avoid B. cinerea infections, as most of the inoculum is present on the ground and soil moisture promotes conidia germination . Selecting the right irrigation system could help reduce grey mould incidence; mainly, the use of drip irrigation and micro-sprinklers results in limited inoculum spread and reduction of water films on the fruit . As canopy characteristics influence microclimates , nitrogen fertilization can lead to dense canopies and favour grey mould .

Similarly, shorter plant spacings promote higher incidence of B. cinerea in the field . Additionally, plastic tunnels can avoid airborne inoculum and B. cinerea incidence is lower in non-fungicide treated tunnels than in fungicide treated fields , but tunnels favour powdery mildew and complicate harvest. In summary, cultural practices are essential to limit preharvest B. cinerea infections of strawberries, especially in organic agriculture.In modern production, pesticide applications are the most common management practice for B. cinerea control . In the previous two decades, the main pesticides used in strawberry production against B. cinerea belonged to the Fungicide Resistance Action Committee Groups 1 and 2, as well as captan . However, due to increasing fungicide resistance and new legal restrictions, producers have been forced to diversify their fungicide regimen . The frequency and timing of fungicide applications are crucial for B. cinerea control. One application of fenhexamid at anthesis can be as efficient as multiple weekly applications . Additionally, alternation and combination of different fungicides with different modes of action are recommended . Resistance of B. cinerea to fungicides is a real challenge in horticulture and fungicide resistance profiles can shift considerably even within a single season . A screen of 13 B. cinerea isolates in Louisiana showed that all were partial to full resistance to FRAC 1 fungicides, and several of the isolates also had different levels of resistance to FRAC 2 fungicides . A larger survey of 1890 B. cinerea isolates revealed that 7 isolates from different locations were resistant to all single-action site FRAC fungicides groups that are registered for B. cinerea control . B. cinerea resistance to fungicides is usually associated with over expression of efflux transporters or with modification of fungicide targets. These resistance mechanisms are acquired via mutations and recombination that occur frequently in B. cinerea due to heterokaryosis, sexual reproduction and the presence of abundant transposable elements in its genome . Efflux of fungicides or accumulation of altered fungicide targets has also been shown to lead to multi-resistances . The presence of resistant isolates against the most common multi-action site fungicides reinforces the need for innovative management practices. A new generation of RNA-based fungicides has been proposed, which relies on the application of sRNA or dsRNAs that target B. cinerea virulence genes to reduce fungal infections in strawberries . However, these RNA-based fungicides remain far from commercialization, which is why fungicide resistance management such as mixture and rotation of different fungicides or testing local isolates for resistance is necessary .

Usage would best be confined to lemons and in areas where salinity and gummosis may be critical factors. Hybrids of pummelos with other promising root stock cultivars should be considered.Almonds are California’s top agricultural export — 80% of those consumed worldwide are grown here. As water resources become increasingly scarce due to population growth, environmental needs and periodic drought, it will become more difficult both monetarily and politically to obtain sufficient water for crop irrigation. Drought tolerance in almonds has been documented in previous studies, but substantial irrigation is still required to maintain current production levels. Over the last 14 years there has been a steady increase in both bearing acres and yields — about 70 pounds per acre in almond yield improvement annnually , indicating a steady improvement in cultural practices, among them, irrigation. There is a pressing need to reliably maintain current almond production with less water. Surface-water allotments for irrigation during drought are often significantly reduced because precedence is given to other uses . Water reserves in California were low following the droughts of 2007, 2008 and 2009. In fact, spring 2008 was the driest on record . The current basis for estimating the irrigation need of a crop is to combine the water lost from the soil with the water lost through leaves , into an overall loss, the crop evapotranspiration . ETc is calculated by multiplying a weather based reference crop ET , by a crop coefficient , to give the final estimate . Research in the late 1980s and 1990s estimated the average seasonal ETc for almonds at 40 to 42 inches ,square pots with estimated seasonal irrigation requirements of 36 to 38 inches under typical soil and rainfall conditions of the southern San Joaquin Valley . But later field research suggested that almond ETc may average from 48 to 54 inches .

Reasons for the higher recent estimates probably reflect the many changes that have occurred in almond culture over the past two decades. Almond orchards are now intensively managed with pressurized rather than surface irrigation systems, and crop water status can also be monitored directly using midday stem water potential . SWP is measured directly on leaves sampled in the orchard using a pressure chamber, and it indicates the level of physiological water stress that is being experienced by the trees at the time of sampling, much as blood pressure or temperature can be a measure of any physiological stress in humans . Furthermore, nitrogen fertility management is more intensive than it was when the earlier research was conducted, and pruning practices have changed to manage canopy light differently, both producing more foliage and potentially higher ETc. In fact, a higher ETc rate and higher yields may both be responses to more intensive almond management. The ETc method of irrigation scheduling aims to maintain the crop in a non-stressed condition by supplying enough water to satisfy ETc. Alternative methods have been proposed that attempt to reduce unnecessary vegetative growth in orchard and vine crops in order to make water use more efficient; they include deficit irrigation, partial root-zone drying and regulated deficit irrigation . The objective of regulated deficit irrigation is typically to irrigate so that trees experience mild-to-moderate levels of water stress, in order to achieve an optimal horticultural balance between vegetative growth, which is very sensitive to stress, and fruit production, which is less sensitive . Previous studies in almonds and other crops have shown the beneficial effects of regulated deficit irrigation, including control of excessive vegetative growth, reduced hull rot and improved hull split in almonds , increased fruit density in prunes and pears and reduced vegetative growth in peaches . Previous studies of regulated deficit irrigation have created stress by applying a fraction of ETc, but for this 5-year study we used a plant-based indicator of stress and set a target level of mild-to-moderate stress during the hull-split period. We undertook this study to determine whether meaningful reductions in consumptive water use could be achieved with minimal impacts on orchard productivity.

Our study took place in a micro-sprinkler-irrigated, 270-acre almond orchard near Orland in the northern Sacramento Valley, which was planted with ‘Nonpareil’ and ‘Carmel’ trees spaced at 12 feet by 24 feet . The orchard was divided into five approximately equal blocks; two were planted in 1993 and three in 1999. From the first year of the experiment , the canopy shaded area in midsummer at noon was greater than 50% in all blocks, so all blocks were considered to exhibit fully developed crop water requirements . The five blocks were each subdivided into two sections to match the existing irrigation system design, with control and regulated deficit irrigation treatments assigned to the sections on alternating sides. Two rows of ‘Nonpareil’ almond trees in the center of each section were designated as the experimental plots, with two trees from each block used as the monitoring trees for SWP measurements. The rows averaged approximately 69 trees per block, and monitoring trees were positioned approximately one-third and two-thirds of the way into each row . SWP values were initially taken on weekly field visits using a pressure chamber, and were collected biweekly during the hull-split period. Leaves, still on the tree, were covered with an aluminized Mylar bag for a minimum of 10 minutes prior to measurements . Meters were installed on a single lateral line in each irrigation section to measure water applications. In 2004 and 2005, block-specific recommendations for regulated deficit irrigation were communicated to the grower, who was responsible for dayto-day irrigation management. In 2005, the orchard exhibited defoliation due to Alternaria leaf spot, and the grower was reluctant to withhold water from the large regulated deficit irrigation plots. In 2006, a separate irrigation system that could be monitored and controlled via a satellite-linked Internet service was installed for the experimental ‘Nonpareil’ row and the two adjacent Midday SWP and water meter data were collected weekly from early April until the hull-split period. Visual surveys were made weekly starting in mid-June to anticipate the beginning of hull split. Irrigation was reduced once the onset of hull split was observed in blank nuts, generally about a week before the onset of hull split in normal nuts. Before and following the hull-split period,drainage plant pot the water amounts applied to the regulated deficit irrigation and grower control treatments were equivalent. During the hull-split period, SWP was measured twice weekly and irrigation was adjusted to achieve a target mild-tomoderate stress level of −14 to −18 bars in each block.

By the last year of the study , block-specific irrigation was not necessary because the target SWP could be achieved using about the same level of deficit irrigation in all the treatment blocks. The target levels of midday SWP employed in this field trial were set to achieve mild-to-moderate water stress during the regulated deficit irrigation period. For almonds, Shackel reported about a 50% reduction in midsummer stomatal conductance with SWP values of −14 to −18 bars compared with a non-stressed SWP above −10 bars . Irrigation was returned to normal once visual surveys indicated 90% hull split in each block. The grower commercially harvested entire rows, and a weighing trailer was used to determine gross harvest weight in the field. We collected a 4-pound sub-sample from each of the blocks and used them to convert harvest weights into nutmeat yields. In this field trial, regulated deficit irrigation was limited to the hull-split phase of almond growth and development. ETc is typically highest during midsummer, so the opportunity is greatest at this time to impose crop stress in order to achieve significant irrigation reductions. In addition, Teviotdale et al. reported that both hull split and nut harvestability are improved and hull rot is reduced when regulated deficit irrigation is imposed during the hull-split period. Other stages of almond growth and development have shown greater susceptibility to negative impacts on tree growth and nut production . Crop stress is also difficult to impose from leaf-out through midMay due to rainfall, lower ETc rates and generally sufficient soil moisture.Soil moisture. We installed neutronprobe access tubes to measure the change in stored soil moisture from early spring to late summer, in order to quantify the contribution of soil water to the crop’s water needs . We installed two grids of 16 tubes in a single block, each in the southwest quadrant of a single monitoring tree for both the regulated deficit irrigation and control treatments. The tubes were arranged in 4-by-4 grids with overall dimensions of 6 feet by 12 feet . The grid spacing was measured from the center of the tube, with 2-foot spacing in the north-south direction and 4-foot spacing in the east-west direction. We tried to install the tubes to an overall depth of 60 inches and measure volumetric soil water content at 1-foot intervals, at depths of 8, 18, 30, 42 and 54 inches . However, due to the widespread variability in soils — including areas with significant gravel content, soil stratification and a shallow, temporarily perched water table — we achieved a depth of 54 inches for only 22 of the 32 tubes.

The remaining tubes were installed to a depth of 42 inches . Soil moisture readings were taken two or three times per season, typically around full bloom, in late summer and post harvest. The shallow water table receded during the course of each growing season, especially during the drought years of 2007 and 2008; it did not appear to influence orchard water status significantly during our study. If capillary flow of water from the shallow water table had contributed significantly to crop consumptive use, midday SWP would not have responded to the withholding of irrigation water during hull split. In addition, the gravel content and hard pan appeared to be barriers to deeper root development, so the roots may not have reached the soil water. Soil type. Soil types were variable throughout the orchard, but the majority of acreage consisted of three types: Cortina very gravelly sandy loam, Hillgate loam and Redding gravelly loam . These soils are described by a USDA land capability rating of 3 or 4, which generally groups soil types based on restrictions for field crops. The Redding soil typically has a restrictive layer at 20 to 40 inches , and the other soils extend to below 80 inches . Based on a nominal 60-inch soil profile, all have low available water — approximately 3.5 inches for the Cortina and Redding soils and 8 inches for the Hillgate soil . The two grids of neutron-probe access tubes were positioned in either a Cortina or Redding soil type. Ground cover. Ground cover varied between mowed resident vegetation in spring and winter, and bare ground in summer. Vegetation around the neutron-probe access tubes, where a mower could not be used, was controlled with herbicides each spring to match the surrounding vegetation. Reductions in water use Water savings. An average water balance summary for 5 years of this study showed overall savings of 4.8 inches of applied water in the regulated deficit irrigation regime . The neutron-probe readings showed an average seasonal contribution of approximately 5.0 inches of stored water in the control and 4.5 inches in the regulated deficit irrigation treatment, amounting to about 11% of overall consumptive water use. All in season precipitation was assumed to be an effective contribution. When the savings in applied water were combined with the contribution from soil storage, the regulated deficit irrigation regime resulted in a total average annual consumptive-water-use savings of 5.3 inches over the 5-year period, and yearly savings ranged from 10% to 15%, or 5.2 to 6.1 inches . Yield increases. Yields increased in both treatments during the 5-year study, with no clear trend of any reduction due to regulated deficit irrigation . The orchard’s increasing yields can be attributed to its relatively young age and continuing canopy growth. Canopy growth is typically very sensitive to deficit irrigation, so it is noteworthy that plant-based regulated deficit irrigation did not have a negative impact on yields over time, presumably because the deficit period was after the main period of vegetative growth.

Wolf also studied characteristic differences observable in cross-sections of one year-old roots of the same three species . The trifoliate orange is easily distinguished from the sour orange and the Yuzu by the large vessels in the wood, these being much larger and more numerous than similar vessels in the other two species, and by the more numerous groups of bast fibers in the bark, which form three or four broken concentric rings. In the Yuzu, only a few scattered groups of bast fibers are present in the bark, whereas in the sour orange the groups of bast fibers are numerous and close together in the inner row, with only a few scattered groups farther out, a condition intermediate between that of Yuzu and the trifoliate orange, but clearly differentiating the sour. In the differentiation of trifoliate stock from the Yuzu, Wolf also found that the Yuzu roots, when bruised, emitted a strong penetrating odor, disagreeable to many, and that the odor of the trifoliate is fainter and milder. The author also noticed some differences in the color of roots and their morphological appearance. In Israel, Cossman also studied the anatomy of citrus roots, including the root structure of sweet lime, Rough lemon, sour lemon, citron, ‘Baladi’ sweet orange, ‘Shamouti’ sweet orange, sour orange, grapefruit and shaddock. Cossman felt that characters which might be of taxonomic importance were the mode of lignification of the pith, the configuration of the protoxylem strands, suberization in the endodermis, and the thickening of the walls of the epiblema . Later, Hayward and Long described in detail the anatomy of the seedling and roots of the Valencia orange. In Israel, Green, Vardi, and Galun studied the plastomes of various citrus species and several citrus relatives. They found a resemblance between the plastomes of cultivars of lemon, orange, sour orange, grapefruit and pummelo. The plastomes of other citrus species, such as mandarin and citron,drainage gutter differed from each other as well as the plastomes of the above citrus species.

Furthermore, within the citrus relatives examined, the plastomes of the trifoliate orange and Microcitrus spp. were distinct from each other as well as from the citrus cultivars tested. They felt the result of their study constituted a useful tool for the identification of plastomes in hybrid plants of Citrus developed from protoplast fusion, i.e., somatic hybridization. Some excellent work on the anatomy of Citrus has been published by Schneider . However, most of this is developmental anatomy. He has also published crucially important papers on the seasonal production of xylem and phloem in the sweet orange tree trunk and the ontogeny of lemon tree bark , the relationship of the phloem to certain destructive diseases such as the Buckskin disease of peach and cherry to tristeza , and the incompatibilities and decline of lemons . Schneider et al. were extremely helpful in the early detection and diagnosis of these pathological and physiological problems. However, for some reason, Schneider ignored the structure of the xylem as it might differ between citrus species and how it might aid in identification. Some excellent work has been done on the structure of wood as an aid to identification with forest trees, both conifers and deciduous. One might consult Jane and [text incomplete] . The importance of knowledge of wood structure cannot be emphasized more than the convincing and convicting evidence provided by a wood expert in the trial of the kidnapper of the Charles Lindbergh baby in the early 1930’s. The expert from the regional U.S. Forest Products Laboratory at Madison, Wisconsin, successfully established that the wood in the ladder used by the kidnapper came from the attic of the kidnapper’s home, from which several pieces of wood were missing.Wagnon, Dobbins, and Breece used foliar gland characters in the identification of peach and nectarine varieties. It is possible that the nature, size, number, and arrangement of oil glands in Citrus leaves may also be useful in identification. Hirano , and Gianotti reported on the numbers and variation in stomata in Citrus and some related genera. This technique also might be of some benefit.

Nothing has been done recently with anatomical structure as an aid to root stock identification or taxonomic relationships. Clearly this method will become more complicated as more hybrids involving bigeneric and even trigeneric crosses are made. Furthermore, a microscope, good laboratory, technique, and a thorough knowledge of plant anatomy are required. The first attempt to identify root stocks by colorimetric chemical reactions was apparently that made by Henricksen , who based his method on the presence of varying quantities and kinds of glucosides containing phenol in all citrus roots. He used extracts from root pieces and, with ferric chloride as an indicator, found that the different color reactions or precipitations obtained were more or less characteristic for the four species he worked with, namely sour orange, grapefruit, sweet orange, and Rough lemon. Color density was greatest on sour orange and lightest with Rough lemon. Some confusion existed between sweet orange and Rough lemon. One, of course, needs known standard samples for comparisons. Halma and Haas developed a similar but more extensive method of identifying citrus species by employing colorimetric chemical tests with samples of dried bark since most of the reactive agent seemed to be concentrated there. A number of tests were used in these experiments, but the one that gave most consistent results was the Almen test developed by Cohn for carbolic or salicylic acid, which is practically the same as Millon’s reagent for albumens and phenols. Their experiments also indicated that three other reagents in various forms, molybdic acid, titanium chloride, and ferric chloride, were of value when identification was doubtful. The results obtained by these investigators was sufficiently uniform within commonly accepted limits of the species to lead Halma and Haas to “suggest the possibility that these colorimetric differences may be useful in citrus classification.” In a later paper, Halma described the preparation and use of the Almen test as it had been modified since its first use by Halma and Haas . The tests were only carried out with lemon, Rough lemon, grapefruit, sweet orange, and sour orange. Marloth , in South Africa, made extensive studies and experiments on the use of the four colorimetric reagents in identifying Citrus species, working mainly by the methods suggested by Halma and Haas . Both groups were able to distinguish between the commercial lemon and Rough lemon, and Marloth was able to separate grapefruit and pummelo. As the inroads of tristeza in Brazil became more prevalent and the relationship of root stock-scion combinations became more evident, Bacchi also used these colorimetric tests to distinguish root stocks.

He attempted to identify 15 species, hybrids, and cultivars and found that the reactions obtained were somewhat different from those described by Halma and Haas and Marloth . The differences between sweet orange and sour orange were quite apparent, but the situation becomes more complex with other species and varieties. Bacchi therefore proposed the separation of root stock species and varieties into four groups: sour orange, sweet orange, lemon, and “all the others.” When tristeza began to threaten California orchards and a variety of root stocks appeared to be involved, Masters made a review of laboratory tests for the determination of Citrus root stock varieties. He refined the technique somewhat and was more specific in his color chart,macetas para fresas which is perhaps the best available; it is reproduced here for the benefit of those who wish to conduct such colorimetric tests . Masters was the first person to point out that there is a difference in color reaction between above-ground and underground samples, and for these reasons an addendum is attached. Masters also proposed the use of ultraviolet light and fluorescence as an additional aid. Some of the differences between above-ground and below-ground samples may be pointed out, such as: above-ground sour orange extracts are clear with the ferric chloride test, belowground samples may become cloudy; sweet orange extracts show poor fluorescence above-ground and good fluorescence below-ground. Some of the differences in bark sample location may account for the discrepancies between previous investigators. Certainly it makes a difference as to how much the bark sample is scraped or washed to remove soil particles. The presence of contaminants such as fertilizer, pest control residues, fungicides, and other chemical agents may also make a difference in the color reactions. Of course, the importance of having knowns to compare unknowns with is critical to the tests. Furr and Reece also used a modification of the root stock color tests for the identification of hybrid and nucellar citrus seedlings with a reasonable degree of success. Similar tests were also used by Nishiura, Matsushima and Okudai to identify species and also distinguish hybrids from nucellar seedlings. Nakamura and Nakayama and Krishnamurthy, Singh and Deo also used the tests for studying phylogenetic relationships of the citrus species. Although these chemical tests were somewhat primitive by today’s standards, remarkable results were obtained by someone with care and experience. As chemical techniques and procedures improved, so have the diagnostic aids. Selle proposed a method of clearly identifying sour orange root stock from other stocks by paper chromatography. Essentially the method consisted of taking a piece of root stock bark, placing it upon a sheet of filter paper and hitting it with a hammer. Or, he used a bark extract made with a solution of ethyl alcohol-normal butyl alcohol-acetic acid and water, and placed drops of the extract on filter paper. The spots were allowed to dry, sprayed with a dilute solution of ninhydrin and again allowed to dry. The spots were examined with a long wave ultraviolet lamp and a characteristic flame pattern was observed for fluorescence. The hammer technique gave the most striking results. Selle also developed a spot chromatographic method. Root sections were taken and the bark removed, cut into very small pieces, placed in a bottle, and treated with 2,2-dimethoxypropane.

After standing for 30 minutes, single drops were placed on filter paper and the spots observed as they dried. Complete identification of all the root stocks was not obtained by using dimethoxypropane alone, and he got better differentiation by adding anhydrous aluminum chloride to the solution. He was thus able to identify sour orange, sweet orange, grapefruit, tangelo, mandarin, Rough lemon, trifoliate orange and Troyer citrange. The memory of the past is not always too reliable, but the author is quite certain that in conversation with Selle, he indicated he could also tell with the root bark what the scion variety was budded on it. Unfortunately, with Selle’s sudden death, perhaps this information was lost. Pieringer, Edwards, and Wolford and Kesterson et al. studied the application of gas-liquid chromatography to the citrus leaf oils for the identification of kinds of Citrus. Kesterson and his coworkers included eleven kinds of citrus and their data demonstrated that the oil composition for the different species is quite variable. They list the most prevalent and distinguishing features for each type oil in order of importance for sour orange, grapefruit, tangelo, mandarin, sweet orange and Rough lemon. They state that, “The percent of composition within species is shown to be sufficiently different to distinguish one variety from another.” Limits of normal deviation, tree variability, and seasonal variations are all factors which may affect leaf oil properties and they feel additional work will establish these limits. Pieringer, Edwards and Wolford studied the leaf oils of eight different citrus varieties and two sources of sour orange as subjected to four methods of instrumental analyses. These were: infrared and ultraviolet spectrophotometry, gas chromatography, and measurement of refractive indices. Some methods were more effective than others in separating closely related cultivars. They felt gas chromatography more successfully differentiated the varieties, whereas infrared and ultraviolet spectrophotometry appeared to be limited to the identification of Citrus species. The value of the refractive index was not fully determined. Burger , on page 109 of this thesis, obtained chromatograms with high pressure liquid chromatography of the phenolic present in the root stocks he worked with. Using this method, he could distinguish between Troyer and Carrizo citranges, which most other researchers could not do.

The unique character of Rough lemon under these conditions is the relatively high concentration of feeder roots at the 76-152 cm depths. On the poorly drained soils of the east coast of Florida, the results would be different. Results would also be different on the cooler and heavier soils of California. Frequent concentration in this zone may be greater than the total amount of roots in the upper 25 cm of soil. With sour orange the feeder roots have been found 3-4 M deep at nine years of age, which compares favorably with Rough lemon. Mature trees on sour orange usually have fewer feeder roots below 76 M than Rough lemon and differ from Rough lemon in having more feeder roots concentrated in the 0-25 cm zone and less in the 76-152 cm zone. Sweet orange differs from Rough lemon and sour orange in that the sweet orange has the greatest concentration of feeder roots of any stock used commercially.The high concentration of roots in rather limited zones may account for why growers in sandy soils claim sweet orange must be irrigated more frequently than Rough lemon. Ford also reports that 30-year-old trees on grapefruit roots had a shallow root development.He also says that Cleopatra is deep rooted. Temple oranges on Cleopatra at nine years of age had roots to 5.2 M. In regard to Webber’s comments regarding citrus root systems, the author is taking the liberty of interspersing his own comments since Webber did not live to the termination and removal of many of his root stock trials in 1960. At the time of the conclusion of these experiments, the root system of each tree was carefully analyzed by Kirkpatrick and Bitters , in which the roots of every tree and combination were counted, sized, and measured. Also, during the tristeza era in Southern California, when thousands of trees were pushed and pulled, it provided an opportunity to observe numerous root systems under diverse soil and environmental conditions. At the time of Webber’s demise ,best vertical garden system sour orange was still the world’s most popular root stock and the most widely used root stock in California. Webber points out that the sour orange has commonly been considered to be best adapted to growth on low, moist, and fairly heavy soils.

It first became established in Florida on such soils. On the very light sandy soils of the so-called “ridge” section of central Florida, it has been a failure. In California, especially with lemons , it was equally successful on light sandy soils such as in the Upland area. In root stock experiments it gave better results on a fairly light sandy soil at Riverside than a much heavier loam soil at Fillmore. His explanation to these different reactions was the difference in climatic and soil conditions as they reacted on the deep rooted sour orange. In California, where alkali in sub-soils and high water table is sometimes a problem, such as in the Imperial Valley, a deep rooted tree like sour orange may be seriously injured. Aeration may be a problem in high water tables and root asphyxiation may occur, or damage from high salt content. As the lower roots die back, they may be attacked by soil organisms like Phytophthora and cause further damage. A shallower-rooted tree would sustain little or no damage. The sour orange develops one or more tap roots which can grow to considerable depths, but lateral root development is more limited than in Rough lemon, sweet orange, or grapefruit. The tap root of sour orange cut back at transplanting from the nursery commonly branches and forms a small group of tap roots. Halma found one mature Eureka lemon grove in which the average number of main roots per tree was fifteen for 64 trees, of which 65 per cent were typical tap roots, whereas sweet orange was devoid of tap roots. Deep tap root penetration on deep soils renders the sour orange, to some degree, resistant to the effects of drought. Hume states that both nursery and grove trees on sour orange suffer much less in periods of protracted drought under identical conditions than Rough lemon, but Evans states in Dade County, Florida, orchard trees on grapefruit and sour orange may be actually dying of drought when adjacent trees on Rough lemon are satisfactory. Hume’s statement would not apply, perhaps, to results on the deep, sandy “ridge” soils of Florida which were planted mostly after 1926.

Webber further states the sweet orange does not develop a well differential tap root and is usually moderately shallow rooted. It does, however, have an abundant system of lateral roots. The author’s observations in California would agree. Mills states the sweet orange is a surface growing stock which has few or no penetrating roots, which does not agree with the observations of Ford . Webber makes no comment on mandarin root systems. However, when the old root stock plantings at Riverside were pulled, the author found the most extensive and massive root systems of all stocks observed with Washington navel, Valencia, grapefruit, and Eureka and Lisbon lemons to be on Cleopatra mandarin. It penetrated nearly as deeply as sour orange, had extensive laterals, but moderate fibrous roots on the sandy loam soil at Riverside which is underlain by a caliche hard pan at 1 M or more in depth. Dancy, Clementine, and Oneco were similar in structure to Cleopatra, but to a slightly lesser extent. Even the tractor driver removing the trees commented that the trees on Cleo were the most difficult to remove. Webber also made no statement regarding the pummelos. With all scion varieties at Riverside the root system of the pummelos was similar to that of sour orange, with deep penetrating multiple tap roots, a moderate lateral root system extending somewhat obliquely as in sour orange, and a scarcity of fibrous roots. On Rough lemon root stock, he states it develops a very wide spreading abundant system of lateral roots and commonly exhibits no marked tap root. The trees in these experiments did have abundant spreading laterals, profuse fibrous roots, but no tap roots. The root systems were not nearly as extensive as sweet orange, Cleopatra mandarin and several other stocks, and may account for the smaller tree size on the Rough lemon stocks. Webber also made no comment regarding lime root stocks. In the root-stock experiments at Riverside, the root system of sweet lime was similar to, but inferior to, Rough lemon. There were no tap roots, the laterals did not extend as far, but there were abundant fibrous roots. In other California experiments, the West Indian Lime had a root system similar to that described by El Azzouni and Wali , no tap root, extensive surface laterals with limited extension, and profuse fibrous roots.

The Rangpur lime had a somewhat similar root system but did not duplicate the mandarin root stocks in character. The citron also had no tap root, no extensive surface laterals and only fair amounts of fibrous roots. Many of these trees were “leaners”, further indicating that they had a weak root system. Relative to trifoliate orange, Webber remarks that it develops a well-branched root system with very abundant fibrous roots, but that the roots did not extend laterally as far as Rough lemon or sweet orange. The author’s observations on many trees indicate the complete absence of deep laterals,growing strawberries very shallow penetration, a very sharp angle of diversion from the trunk area and a moderate amount of fibrous roots. Most of the laterals were flattened in proximity to the trunk area with no surface laterals and many fibrous roots. No references were made to any of the citranges, although Morton, Savage, Rush, Coleman and Cunningham were in the plantings and Troyer and Carrizo were in later plantings. Most of the citranges have a poorly developed tap root system which only penetrates to a depth of about 1 M. They have, however, many laterals which emerge at an oblique angle and descend downward and may penetrate deeper than the tap roots. Fibrous roots are less extensive than the sweet orange parent, and in the surface layers less than the trifoliate orange parent. The root systems of Troyer, Carrizo, Savage and Morton were more extensive than the others observed and agrees well with Savage, Cooper, and Piper . No reference was made to Sampson tangelo. As expected from its parentage, its root system was extensive, but not as extensive as either mandarin or grapefruit. The tap root system was not well developed but the surface and subsurface laterals were prolific, and fibrous roots were moderate. Yuzu was very deep rooted with good surface laterals, but lacking in fibrous roots. The author would agree with Webber’s description of Calamondin, which has very large penetrating tap roots, at least several extending straight down to 150-180 cm or more. There were few laterals, only near the surface, and a scarcity of fibrous roots. Of 25 root stocks Webber observed, he felt Calamondin exhibited the most marked tap root system, followed by C. webberii, and sour orange next. The author’s observations would agree with this except, the shaddocks would also have to be included.

The root systems of C. macrophylla and C. pennivesiculata are very similar to the lemon-lime group, that is no pronounced tap roots. Extensive trees on Severinia buxifolia were the smallest of any observed and it was surprising that the roots could support andanchor the tree. There was no tap root per se, but extensive surface laterals, most of which were less that 5 cm in diameter and did not extend more than 150-180 cm from the tree trunk, and very few fibrous roots. The root systems of cuttings varied tremendously with the scion cultivar. Those of navel were the poorest, consisting of only 3-5 very large surface laterals with little penetration and few fibrous roots. Those of grapefruit cuttings were intermediate. The Valencia cuttings, however, were very similar to sweet orange root stocks except they didn’t penetrate as deeply. There were lots of surface laterals and lower tier laterals and extensive fibrous root development. Huberty states that the dominant factors in determining root distribution of plants appeared to be irrigation and soil types and sub-soils. However, in an irrigation experiment at the Citrus Experiment Station which provided for irrigation on a two, four, and six-weeks’ schedule, no noticeable difference was apparent in the amount, or the pattern, of the roots as affected by the various irrigation treatments. A marked difference was shown by the type of root stock. Smith, Kinnison, and Carns in Arizona report the effect of variable frequency of irrigation treatments on the root development of young Marsh grapefruit trees on sour orange root stock in the light sandy soils of the Yuma Mesa. In these experiments, irrigation intervals of one, two, three, four, five, and six to nine weeks were followed during the summer irrigation season for three years. The weekly irrigation schedule followed on Plot I kept the surface soil moist and at a lower range of temperature than in the other plots, and permitted an extensive root development the first year in the top 15 cm of soil. The root development in the top 15 cm of Plot II, irrigated every two weeks, was quite pronounced, but in the remaining plots it was appreciably less. This related condition prevailed for several years and then indicated a tendency toward relatively shallow root development irrespective of soil-moisture conditions. The effect of increasing the interval between irrigations seemed to limit the total root structure rather than to force development into the lower soil depths where favorable moisture conditions existed. Huberty also points out that part of the root system of a 25-year-old Washington Navel orange tree on sweet orange root was exposed by careful digging. This tree was planted on a contour row in a sandy loam soil exceeding 150 cm in depth. The longest root found was growing along the tree row in soil which was not cultivated and to which irrigation water was not directly applied.

The Bittersweet differed from sour orange in fewer laterals and very few fine fibrous roots. However, the tap roots penetrated slightly deeper than sour orange. With the sweet oranges, Pineapple and Parson Brown, the root systems were very similar. Both showed well developed central roots, usually two, penetrating about the same depth as sour orange, about 90-125 cm deep, with numerous small laterals the full length of the tap roots. The laterals in the upper 30 cm of soil were not as long as sour orange. There were abundant fibrous roots in the first 30 cm. Their data did not agree with what Mills observed in California’s cooler and loamier soils, but rather with Hume’s observations in Florida, that roots of sweet orange and sour orange are about equal in development, which Oppenheimer also reports in the sandy soils of Israel. Rough lemon had the most vigorous root system of all 15 stocks. It had exceptionally large lateral roots with a spread of 150 cm or more from the trunk. The central tap extended as deeply as sour orange, but the fibrous roots were not as abundant around the crown as sweet orange or sour orange. For Duncan and Bowen grapefruit they found an abundance of fibrous roots and many large vigorous laterals and two or more large penetrating tap roots extending 92 cm with a mass of fibrous roots the full extension. The abundance of fibrous roots agrees with observations by Mills and Oppenheimer . The trifoliate orange had deeply penetrating central roots and numerous laterals with abundant fibrous roots in the upper 31-46 cm of soil. The root system was similar to sour orange except for the smaller spread of the laterals. Cleopatra mandarin had a cone-shaped root system with well developed central roots penetrating 125 cm or more. There were long fine-textured lateral roots in the upper 61 cm of soil well supplied with fibrous roots. Its root system didn’t differ much from sour orange except the tap root was straight, slightly longer, and less divided. The root system of Suen Kat was similar to Cleopatra. The root system of Morton Citrange had the tap root dividing into several which penetrated vertically and then fanned out,vertical farming equipments descending obliquely to about 91 cm. The laterals were similar to sour orange, with a good fibrous system.

The Rusk citrange was similar in root structure to Morton except there were fewer fibrous roots.There were no tap roots. The root system resembled grapefruit in fibrous roots, but grapefruit had a pronounced tap root. The Cuban Shaddock had a root system almost identical to Rough lemon except it had more fibrous roots. The calamondin had a group of vigorous central roots which penetrated deeply, in fact, more deeply than any of the other stocks observed. Yuzu displayed the smallest root system of all 15 stocks observed. It penetrated deeply, and there was a scarcity of laterals. Sweet orange cuttings had a shallow root system with many vigorous laterals which did not penetrate more than 61-91 cm. Savage, Cooper, and Piper thus concluded that Rough lemon and Cuban Shaddock had the most extensive root system, the calamondin the deepest, and sweet lime and grapefruit had the greatest amount of fibrous roots. In [date lacking] [“Check” appear here in typescript in the margin of the manuscript], Ross reported that trifoliate orange produces a well-branched root system with an abundance of fibrous roots which are very sensitive to drying. Robinson grew ten scion varieties on Cleopatra mandarin and sour orange in Norfolk sand in Florida, and at nine years of age reported that Cleopatra produced a deep tap root with numerous fibrous roots superior to sour orange. In India, Burns and Kulkarni exposed roots of Santara and Mosambi on Jamburi from fiveyear-old trees growing poorly. With the Santara trees 320 cm high, the lateral roots extended 42 M and average 2.5 M. The root spread was greatest parallel to the tree rows. With the Mosambi trees having a height of 2.1 M the diameter spread of the roots was 1.8 M. A 14-year-old tree of Santara had roots with a radius of 3 M with secondary roots of 4.9 M. The laterals were about 23 cm deep. He concluded Jamburi was surface rooting with a fair spread of laterals, but few deep penetrating roots under the tropical conditions of India. In Ceylon , Gandhi examined the root systems of Mosambi trees planted 5.5 x 5.5 M. The spread of the crown roots was 3.7 M, or less than 1.8 M between trees. Roots had spread 2.7-4.9 M from the crown and at four years the roots were intermingled. Grapefruit trees on Rough lemon at five years had a lateral spread of 4.9 M. Fifteen year-old Santara trees planted 4.6 x 4.6 M had roots which completely intermingled and extended trunk to trunk. The Santara trees on Rough lemon at 14 years had roots extending a radius of 3 M, some 4.9 M, and a similar situation occurred with Mosambi. He only studied the lateral spread of surface roots. He did not feel it necessary to ascertain the distribution of fibrous roots since he felt only the exposure of laterals to a depth of 10-20 cm was enough to give a general idea of the nature of the fibrous roots.

Gandhi also stated that the depth of rooting, lateral spread of main and subsidiary roots and their branching, and quantity of fibrous roots are specific characters. These specific characters may vary with different species, but the location of the fibrous roots on the root system did not seem to be a specific character. He felt it appeared to be more a result of environment and cultural treatment. Montenegro in Brazil found that sour orange seedlings had more superficial and less horizontal extensive root systems than Caipira sweet orange, and poorer feeder root systems as compared to the well-developed system of the Cravo tangerine. The root system of Caipira sweet orange was deep and had a vigorous feeder root system. The most vigorous root systems were Florida Rough lemon, sweet orange and Cravo tangerine. The least vigorous root systems were Rangpur lime, sweet lime, and trifoliate orange. Hamlin sweet orange scions strongly stimulated feeder root development in root stocks. This effect was moderate with Pera and slight in Baianinha sweet orange. Root development was more vigorous in well-drained and aerated soil than in imperfectly drained,vertical farm tower shallow layered soil. Exocortis and tristeza checked root development. Extensive root development was not necessarily associated with large tree crowns or high fruit yields. As root stocks for the sweet orange varieties Hamlin, Pera, and Baianinha, Rangpur lime stock showed poorer feeder root development than Caipira, Pera, Cravo tangerine, or Florida Rough lemon. Vigor was medium on Sampson tangelo which is deep rooted like Florida Rough lemon. Trifoliate orange on clay soils produced fewer roots than Florida Rough lemon, Cravo tangerine, or Caipira orange. With Baianinha scions Caipira was more abundantly rooted than Rangpur lime, Brazilian Rough lemon, or sweet lime. Nucellar clones of Baianinha on Caipira had excellent root development superior to that of an old clone on Pera orange or Cleopatra mandarin. Seedlings of Caipira orange rooted more deeply than those of sour orange and Cravo tangerine, and in these instances the roots tended to be massed around the trunk base. Ten-year-old Hamlin oranges on either trifoliate orange or sweet lime had 90 per cent of the root system within 90 cm of the surface, but for all of the remaining ten-year-old combinations of scion and stocks the top 60 cm of soil contained 90 per cent of the roots. On light soils the roots of grafted and seedling trees of Baianinha extended down to 90 cm.

In Japan, Okuchi et al. excavated a single tree of Satsuma mandarin on trifoliate orange which was 22 years of age. Roots extended down to 120 cm so the distribution was similar to that observed by others. Most roots were within 1 M of the trunk. The horizontal and vertical distribution of the roots showed 93 per cent within 2 M of the trunk and 94 per cent within 60 cm. Most feeder roots were within 20 cm of the surface with only a few at deeper depths. In Egypt, El Azzouni and Wali excavated the whole root system of 15- year-old Washington navels on Rough lemon, lime and sour orange. Roots were graded according to diameter and dry weights taken. There was some variability among trees on the same species. Roots of all stocks extended laterally outside the 5 M zone allocated. Sour orange extended laterally the most, with lime the lowest, and Rough lemon intermediate. The roots outside the 5 M radius were considered minor compared to the total root distribution. There was a high percentage of fibrous roots on all stocks. Woody roots .75 cm in diameter and greater extended 2.4 M from the trunk and beyond the 5 M radius, so greater extension was needed. Sour orange roots extended vertically 1.5 M, although Rough lemon and lime extended to 1.2 M with Rough lemon deeper than lime. Much of the root system of all three stocks was located in the 15-30 cm depth. There were considerable roots at .6 M, with lime having more roots in this zone than either Rough lemon or sour orange. Lime had the shallowest roots, Rough lemon intermediate, and sour orange the deepest. There were no direct orientation effects. Sour orange had the highest weight of total fibrous roots, with Rough lemon intermediate and lime the lowest. In contrast, the net weights of lime showed the highest percent of fibrous roots followed by Rough lemon and sour orange. All these stocks had fibrous roots located in the first .3 M and second .6 M, and with lime most were in the first .3 M, especially at 15 cm, even though the orchard was cultivated. Sour orange had the greatest root weight, followed by Rough lemon and lime, in that order. There were no tap roots on any of the stocks. Ford found Rough lemon roots penetrated to a depth of 4.3-5.2 M at 15 years of age in the warm deep well-drained Lakeland fine sand of Florida’s central ridge. At 25 years, the total amount of feeder roots was greater at the 76-152 cm zone than the 0-25 cm zone. Fifty per cent of the feeder roots were below 76 cm, and 15 per cent below 2.7 M. At 18 years sour orange feeder roots only penetrated occasionally to a depth of 5.2 M in sandy soils. Trees on Rough lemon had more feeder roots below 76 cm than trees on sour orange. The feeder roots of grapefruit were confined to 2.1-2.4 M zones. Temple oranges on Cleopatra had feeder roots to 5.2 M at nine years of age. Sweet orange at 15 years had root penetration to 3.4 M and had the greatest total weight of feeder roots per unit volume of soil than any other root stock observed. He reports Rough lemon feeder roots 7.6 M from the trunk. However, Ford reported Rough lemon roots had extended laterally a distance of 16.8 M. At Riverside, the author also observed the greatest lateral extension of roots on Rough lemon. One root extended parallel to the irrigation furrow a distance of over 9.1 M and was over 2.5 cm in diameter at the point at which it was severed during its removal. Ford reports that citrus trees in central Florida are very deep rooted. The root system of Rough lemon may penetrate to depths of 3 M at 6-8 years of age. Although 40 per cent of the feeder root system of a young five-year-old tree may be in the upper 25 cm of soil, by the time the trees are 20-30 years of age more than 50 per cent of the feeder root system may be below 76 cm in the soil, and of this amount 19 percent may be found below 2.7 M with roots extending down to 4.3-5 M.

Within the Rutaceae, subfamily Aurantioideae, Citrus grows poorly on the Tribe Clauseneae, subtribes Micromelinae, Clauseninae, and Merrilliinae. It does somewhat better with the Tribe Citreae, subtribe Triphasiinae, but there are no promising combinations as yet. As might be expected, Citrus grafts best within the subtribe Citrinae, but again, there are many incompatibilities. In this subtribe, Citrus can grow successfully on such genera as Poncirus, Severinia, Microcitrus, Citropsis, Eremocitrus, and Clymenia, either directly, or through or with the use of certain inter stocks. Within the Balsamocitrinae, Citrus has been grafted successfully on Swinglea, Aegle, and Feronia. Conversely, Hesperethusa grows very well on sweet orange, and Pleiospermium grows well on C. taiwanica. A more detailed discussion of the role of citrus relatives as citrus root stocks may be found in this text. This section will deal primarily with incompatibility problems with Citrus on Citrus, and the closely allied genera Poncirus and Fortunella and their hybrids with Citrus. A great deal of reference material is available on graft incompatibilities of tree crops in general. Among the best of these are Hartmann and Kester , Mosse , Herrero , and Scaramuzzi . There are no comprehensive publications dealing exclusively with incompatibilities with Citrus and therefore an attempt will be made to emphasize this area of discussion. Herrero suggested four categories of incompatibility which included the following: 1) graft combinations where the buds failed to grow out; 2) graft combinations where incompatibility was due to virus infection; 3) graft combinations with mechanically weak unions, in which the cause of death was usually breakage, and ill health,vertical grow rack if shown it was due to mechanical obstruction at the union; and, 4) graft combinations where ill health was not directly due to abnormal union structure but was usually associated with abnormal starch distribution. Whether this is the cause of ill health or the effect is not known.

Regardless of whose system of incompatibility categories are followed, it is a recognized fact that Citrus has incompatibilities which may fall into any of these designated groupings. It is essential, therefore, that the types of incompatibility be defined for clarification purposes. According to Hartmann and Kester , translocated incompatibility includes those cases in which the incompatible condition is not overcome by the insertion of a mutually compatible inter stock because some labile influence can apparently move through the inter stock. This type of incompatibility involves phloem degeneration, and can be recognized by the development of a brown line or necrotic area in the bark, and often designated as bud union crease. Consequently, restriction of movement of carbohydrates occurs at the graft union, with an accumulation of starch above the bud union and a reduction below. Usually a swelling of the scion immediately above the bud union results in a bulge, but not always. Reciprocal combinations may be compatible. In the various combinations in this category, the range of bark tissue breakdown can extend from virtually no union at all, to a mechanically weak union with distorted tissues, to a strong union with normally connected tissues. One component of the combination may carry the virus and be symptomless, but the other component may be susceptible to it. Such is the case with tristeza. The sweet orange component alone is not affected by it, and the sour orange component alone is also not affected by the disease. When sweet orange is grafted on sour orange and the virus is present, the adverse reaction occurs. Some lethal metabolic substance is produced in the sweet orange scion which, when translocated to and below the bud union, causes, according to Schneider , hyperplasia and hypertrophy, callusing of the sieve plates, necrotic sieve tubes, and other anatomical abnormalities of the vascular system in the sour orange stock, or in any other tristeza susceptible stock. The reciprocal is not affected, so the toxic substance is not produced in the sour orange leaves, only in certain scions, and only affecting certain root stocks. The use of a resistant intermediate does not prevent the toxic material from being translocated through it . A translocated incompatibility can be introduced into a compatible combination by the introduction into the system of a third component. Any time a tree is top worked, the compatibility of the two previously existing compatible components may change. Eureka lemon trees on Rough lemon root may be considered as compatible and tolerant to tristeza. In a 1976 visit to Japan, the author observed similar difficulties. The most popular tree combination is Satsuma mandarin on trifoliate orange root stock. Due to over planting and overproduction and resulting low prices, growers are indiscriminately top working to miscellaneous new varieties, and numerous incompatibilities are now appearing. Whenever the insertion of a third component is made, the performance of the resultant combination is much in doubt unless previous experience indicates otherwise.

According to Hartmann and Kester , localized incompatibility includes combinations in which the incompatibility reactions apparently depend upon actual contact between stock and scions, and separation of the components by insertion of a mutually compatible inter stock overcomes the incompatibility symptoms. In the incompatible combination, the union structure is generally mechanically weak, with continuity of cambium and vascular tissues broken, although in some instances the union is strong and the tissues join normally. Oftentimes external symptoms develop slowly, appearing in proportion to the degree of anatomical disturbance at the bud union. Root starvation eventually results due to translocation difficulties across the defective graft union. In some cases actual breakage may occur due to the masses of parenchymatous tissue rather than normally differentiated tissues which are commonly found at the bud union. In Citrus, an example of this would be the decline of Eureka lemon on Troyer citrange root stock, first pointed out by Bitters, Brusca, and Dukeshire , and later confirmed by Weathers et al. . Weathers states that this incompatibility cannot be prevented by an intermediate stem piece such as sweet orange. However, this was successfully done by Nauriyal, Shannon and Frolich . The author in 1951 budded Cook Eureka on a Frost Valencia intermediate on Troyer citrange root stock involving numerous trees, and those trees, as of 1987, are still vigorous, healthy, and productive. Mechanically weak unions and breakage occurs readily; for example, Figures 22 [Image could not be located] and, Limoneria 8A Lisbon on Citrus taiwanica and also Frost Lisbon on Ponkan mandarin. There are also delayed symptoms of incompatibility. In these instances, the stock-scion combination grows in a normal fashion for perhaps 15 to 20 years or longer, and then the trees decline. One of the best examples of delayed incompatibility is the socalled “black-line” of walnuts in California, Oregon, and France. Serr reports that when varieties of Juglans regia are grafted onto seedling root stocks of J. hindsii, the northern California black walnut, or onto Paradox roots , affected trees grow normally, bearing good crops, until they reach an age of 15 to 20 or more years of age, then the difficulty begins. This disorder has recently been identified by Mircetich, Refsguard, and Matheron as being caused by a graft-transmitted virus, namely, the cherry ring spot virus. Comparable types of delayed incompatibility occur commonly in citrus. Examples are the decline of Satsuma mandarin on Troyer citrange at 16 to 18 years, Washington navel on trifoliate orange and Morton citrange at 15 to 20 years,vertical planting tower and a lemon bud union disorder of both Eureka and Lisbon cultivars on Sampson tangelo, Cleopatra mandarin, and many other stocks at 5 to 10 years of age.

The role that viruses may play in these disorders is unknown. Hoy, et al. discovered that prune brown line disease was caused by the tomato ring spot virus, and that the organism was spread by the dagger nematode. The disease affected French prune, Empress prune, and President plum cultivars on Myrobalan plum and peach root stocks, but Marianna #2624 was not affected. The above mentioned citrus maladies are accompanied by a swelling or bulge at the bud union and compression girdling results. The swelling and girdling occurs above the union with the lemons on Sampson tangelo, and below the union with the Satsumas on Troyer or the navels on trifoliate orange. While swellings at the bud union are often believed to be a positive indication of incompatibility , in Citrus there are many combinations in which the scion severely overgrows the stock, and others in which the stock severely overgrows the scion, and yet the combinations are healthy, vigorous, and productive at 30, 40, or even 50 years of age. Obviously, other factors may be involved, as with other tree crops. A more extensive discussion of individual incompatibilities will be detailed later in this section. While the actual time required for bud union continuity to take place may vary with the environment, time of budding, and other factors, it is clear from Mendel’s investigations that the process can be essentially completed in 45 days. His observations are in complete agreement with those of Randhawa and Bajwa , who found that the normal union of Malta Blood and Jaffa sweet oranges on Karna khatta root stock was complete in about six weeks. This time factor is in sharp contrast to the work of Malik , who states that with the Valencia sweet orange and Kinnow mandarin budded onto Rough lemon root stock, that practically no union takes place six months after budding and even after a year has lapsed, the bud union is not 100 per cent complete. Mendel also points out that normal wood and bark elements are laid down after the formation of a common cambium; a connection of the vessels and sieve tubes is thus established. Thereafter the exact border between the graft mates cannot be determined. Thus, nothing can be detected anatomically at the border zone between the scion and the root stock which would indicate a disturbance of the sap streams, be it in the wood or in the bark, proving the progress of the union was a normal one. The initial union of Malta Blood red sweet orange on Karna khatta described by Randhawa and Bajwa was normal, but they point out that Bajwa and Singh and Kirpal Singh and Singh indicate that this combination exhibits incompatibility in the orchard, which suggests a delayed type of incompatibility, or perhaps the entry of a pathogen. Whereas the work of Mendel was concerned only with the anatomy of the actual union process, the work of Goldschmidt-Blumenthal was conducted on trees 20 years old. She investigated the bud-union anatomy of Shamouti orange on Rough lemon, sour lemon, sweet lime, citron, Goliath shaddock, Duncan grapefruit, sour orange, Baladi sweet orange, and Shamouti sweet orange. The grafted root stocks did differ in their anatomical structures, especially in the number and width of vessel clusters, the amount of parenchymatous tissue, the structure of rays, and the number of idioblasts. The Shamouti scions also displayed differences in the number and cross-sectional area of the vessels, amount of parenchymatous tissue, size of rays, and the number of idioblasts. The relative water conductive area of the Shamouti scions was markedly affected by the root stock. Shamouti scions on Shamouti, Baladi, sour orange, Rough lemon, and grapefruit stocks showed a good correlation between bud union morphology and the anatomical structure. In the remaining combinations, there appeared to be no correlation. The predicting of compatible or incompatible combinations and ultimate root stock performance by either anatomical, chemical, or other rapid methods would be of great value to the root stock research worker in developing improved root stocks. However, none of these methods have proved satisfactory so far. Various laboratory methods have been developed for evaluating stock-scion incompatibility in young apple nursery trees without growing trees to maturity by Evans and Hilton . These include water conductivity measurements through the graft union, macroscopic evaluation of the external appearance of the graft union, microscopic evaluation of the graft union, and breaking-strength tests. A biochemical method used by Samish was based upon the presence of a glucoside associated with pear-quince incompatibility. There is a low prunasin content in one quince stock and a high prunasin content in another. Lapins was able to detect some symptoms of stock-scion incompatibility of apricot varieties on peach seedling root-stocks by macroscopic examination of discontinuity of inner bark tissue at the graft union of the young trees, but found no other correlation of anatomical structure to compatibility.

The author was somewhat surprised during several visits to Florida in the 1960’s, that in an area where Rough lemon was then the predominant root stock, several large nurseries did not have their own seed source trees. Some relied upon in-trained personnel going into the hammocks and collecting fruit from feral trees. Such practices undoubtedly resulted in some hybrids and off types being gathered and contaminating the total seed source. Fortunately, the inroads of the burrowing nematode led to the selection of resistant or tolerant lemon types like Estes and Milam. The future status of Rough lemon as a root stock in Florida is uncertain since Rough lemon is very sensitive to “young tree decline.” Whether or not there is any clonal tolerance within a root stock to this disease is currently unknown, but certainly a better history of known seed sources might have been helpful. Citrus industries around the world have recently had the foresight to recognize the importance of clonal variation and the necessity of selecting superior performing cultivars of both scions and root stocks as perpetuated, controlled, and supervised foundation plantings of disease-free, true to type sources of scion bud wood, root stock budwood, and seed sources. Perhaps California was the first to see the need for and initiate such a program. Readers should consult Calavan, Mather and McEachern on the registration, certification, and indexing of citrus trees. Citrus areas such as Florida, Spain, South Africa, Morocco, and others have also either successfully established certification programs as standard practices,vertical garden growing or the programs are in the process of being developed and adopted. In agricultural crops it is generally considered important to exercise some selection of the plants or seeds used in propagation in order to eliminate inferior individuals.Some selection has been commonly practiced in the propagation of citrus nursery stock seedlings .

Proof of the desirability of such selection has clearly been pointed out by Webber , and Webber and Barrett . Such experiments were continued in part up until 1960, but the principal conclusions were published by Webber . As the sour orange, during Webber’s period, was perhaps more generally used than any other root stock the world over and was the principal root-stock in use in California, he chose to present the data from his selection tests in considerable detail. In considering these results, Webber pointed out that it should be remembered that the percentage of nucellar embryony of the sour orange apparently varies from 70 to 80 per cent as he determined from the examination of different lots of seedlings. These percentage figures for sour orange have been confirmed not only by Webber , but also by Frost , Toxopeus , Torres , Ueno, Iwamasa and Nishiura , and Frost and Soost . Webber has shown that, in a lot of 389 unselected sour orange seedlings worked with buds from one highly selected tree of the Washington navel, the seedlings that were the largest at the time of budding produced, in general, the largest trees in the orchard. In this lot of trees, the area of the cross section of seedling trunk , when compared and correlated with the area of cross section of the one-year old budding trunk two inches above the bud union, gave a correlation coefficient of +0.736 ± 0.016, showing that among the young budded trees the large ones had been mainly produced on large stock seedlings. When the same trees were eight-year-old orchard trees , a similar comparison gave a correlation of +0.437 ± 0.028. These correlations are high enough to show clearly that in an ordinary lot of unselected stock seedlings the size of the seedling at the time of budding has a significant influence on the size of the budlings and of the orchard trees. In the same lot of trees, when the size of the one-year-old budlings was compared with that of the eight-year-old orchard trees, a correlation of +0.622 ± 0.021 is found to exist, showing that, in general, the large budlings produced large orchard trees. If vigorous, large-sized orchard trees are desired, these figures indicate the importance of a rigid selection to eliminate the small seedlings and small budlings in the nursery. Irrespective of the species or variety of root stock used, variations in size such as Webber found will exist. A careful examination of such a lot of sour orange seedlings also reveals that there are other differences than size: e.g., in type of branching, length of internodes, form and size of leaf, etc.

Size variations of seedlings are probably due in appreciable measure to environmental and accidental causes, such as crowding of seedlings in the seedbed, but the other types of variation in seedlings grown in proximity in the nursery are not likely to be caused by environment. Such variations, in most plants, are more likely to be due to different heritage from the two parental lines commingled in the offspring. In Citrus, however, where a high percentage of the seedlings of most good root stock types are developed from nucellar embryos, variations due to the crossing of different types are not so frequent. Here a large percentage of seedlings come from nucellar embryos and are likely to be genetically of the same type and thus to exhibit similar characters. If one studies carefully a field of nursery seedlings of a sour orange variety it will become evident that the great majority of them, some 70 to 80 per cent of the total, are very much alike, and the other 20 to 30 per cent will differ from this prevailing type in various characters, such as reflexed branches, short bunchy growth, smaller leaves, rounded leaves, more pointed leaves, narrower leaves, and the like. These off type seedlings, which are mainly smaller and apparently weaker than the general run of seedling of the prevailing or normal type, are designated “variants” because they do vary. While the seedlings of the prevailing type are evidently in greater part from nucellar embryos, it is probable that the seedlings of the variant types are mainly from embryos which are of zygotic origin, and that these seedlings are therefore genetically different from the nucellar-embryo seedlings of the prevailing type. All of these seedlings are from the sour orange, but Webber asks the question “are all equally satisfactory to use as root stocks?” An examination of the lot of 389 nursery seedlings of sour orange that Webber referred to revealed that 43 of them should be classed as variants because of characters, visible in the nursery, differing from the prevailing type. Webber numbered all of these seedlings and budded them at the same time with buds from one selected tree of Washington navel, and the resultant trees were then grown in the orchard at the Citrus Experiment Station, Riverside. With one or two exceptions, all of the trees classed as variants produced orchard trees showing some degree of dwarfing, and most of them produced marked dwarfing. These variants apparently occur normally among citrus seedlings,growing strawberries vertical system regardless of the source from which they are derived, but the number of variants produced can vary with the root stock cultivar. Since the majority of them are constitutionally weak and below normal size, a rather large proportion of them die before reaching the age and size for budding. Many are doubtless discarded from the seed bed because they are too small to transplant successfully, but a sizeable number of them, if not recognized and discarded, are finally budded and transplanted into orchards, causing irregularities in size and lowered production.

The fact that those trees budded on the variant stocks were dwarfed should not have been overlooked. Dwarfing is not objectionable if the trees are uniform, healthy, and productive. Small tree size can be compensated for by planting more trees per acre. While Webber was aware that many citrus hybrids produce true to type offspring, there is no evidence that he attempted to find this out with these variants. Even if a variant with desirable dwarfing characters didn’t come true to type from seed, it could be propagated by asexual means, as with the East Malling stocks for apples. This variant planting survived Webber’s death in 1946 and was still in existence when the author arrived in Riverside in the fall of 1946. Unfortunately, the plot was terminated a few years later before the author became fully acquainted with it. One of these variants has already been discussed and was used in the 1948 tristeza trials . Variants may be a practical approach to dwarfing, but of course not with sour orange variants because of the tristeza problem with this cultivar and most of its hybrids. There may be some other problems in that perhaps some of the variants are not fruitful. The author selected dwarf variant seedlings out of seedbeds of Rough lemon, Rangpur lime and a few other cultivars with the idea of later using them for dwarfing root stocks. However, after 10 to 12 years in the orchard, they had not flowered or fruited and were discarded. One enterprising nurseryman in Southern California who was interested in growing dwarf citrus trees for home use was growing Rough lemon seedlings and throwing away the nucellars and only budding on the variants. Of course, this practice does not assure any degree of uniformity as far as size of tree is concerned, but it does demonstrate that the use of variants may be a practical approach to dwarf citrus trees. The fairly high correlation that Webber found between sizes of nursery seedlings and sizes of eight-year-old orchard trees was caused, in large part, by the presence of these variants. When the 43 variants were excluded and the size of the nursery seedlings, as shown by area of cross section of trunk of the remaining 346 trees, was compared at different intervals with the trunk and size of the budlings, the following gradually decreasing correlations were obtained: with one-year-old budlings, +0.459 ± 0.026; three-year-old trees, +0.125 ± 0.036; six-year-old trees, +0.010 ± 0.036; and with eight-year-old trees, -0.021 ± 0.037. In the nucellar population, consisting of the seedlings after the variants were removed, the small degree of correlation at first existing had disappeared by the end of the sixth year in the orchard. This is corroborated by the fact that with the same population the correlation of trunk size of seedlings with the trunk size of stocks and with volume of tops, of the eight-year-old trees, had fallen -0.054 ± 0.032 and -0.012 ± 0.036, respectively, indicating that there was no correlation. The yield of the trees during the first eight-year period does, however, show a low correlation. Here, with the population of 346 trees , when area of trunk cross section of stock seedlings was compared with the total five-year yields of the eight-year-old orchard trees grown on them, there was obtained a correlation of +0.135 ± 0.035. Although this is a positive correlation, it is so low as to be of doubtful significance. In view of the fact that, after the variants are removed, the variation in size of the remaining seedlings shows no significant correlation with the size of the orchard trees at the end of eight years, it seems clear that the important factor in nursery selection is removal of the variants. This can be achieved with fair success by discarding the small seedlings when they are dug from the seedbed, and by a further elimination of the small seedlings and variant types in the nursery just before the budding, and of weak trees after the budding. Unfortunately, many of the citrus nurseries have become extremely large and this physical work is done by untrained persons who are unaware of this problem. Some variants do get by and end up as orchard trees. With the population of the 389 sour orange trees that Webber referred to above, the correlation of area of cross section of scion trunks of the one-year-old budlings with that of the eight-year-old orchard trees gave a coefficient of +0.622 ± 0.021; a similar correlation for the 346 trees remaining after the variants had been eliminated was reduced to +0.182 ± 0.034. When, with the entire population of 389 trees, budling size was correlated with total five-year yield of fruit per tree in the eight-year-old orchard, the coefficient was +0.233 ± 0.025; and with variants removed, this was reduced to +0.233 ± 0.034. It seems, thus, that even with the variants removed, there is an appreciable correlation between tree size and yield of orchard trees.

This result demonstrated that SlARF6A targets the promoters of CAB, RbcS, and SlGLK1 genes and positively regulates chlorophyll accumulation, chloroplast development and photosynthesis.Motif analysis showed that the SAMS1 promoter contains the conserved ARF binding site, the TGTCTC box. The transient expression assays showed that the LUC/REN ratios were significantly decreased compared with that of the control, suggesting that SlARF6A negatively regulates the expression of SAMS1 genes . ChIP-qPCR was carried out to confirm the binding of SlARF6A with the SAMS1 promoter in vivo, and the results showed that the promoter sequences containing the TGTCTC of SAMS1 were significantly enriched compared with those with the negative control anti-IgG . The direct binding of SlARF6A protein to the SAMS1 promoter was further verified by EMSA. The results indicated that the SlARF6A protein directly bound to the TGTCTC motif in the SAMS1 promoter . Taken together, SlARF6A can target the SAMS1 promoter and negatively regulate the expression of SAMS1 genes. The data demonstrate that SlARF6A plays an important role in ethylene production and fruit ripening.In this study, we functionally characterized the transcription factor SlARF6A in tomato. However, there are two very similar SlARF6 genes in the tomato genome, namely, SlARF6A and SlARF6B. We also examined the function of SlARF6B using genetic approaches and found no obvious phenotypes in the transgenic RNAi and over expression tomato plants . This may be related to the fact that SlARF6B lacks the AUX/IAA domain in the C-terminus of the protein .Previous studies reported that chlorophyll accumulation increased in Arabidopsis roots when they were detached from shoots,lettuce vertical farming which was repressed by auxin treatment. Mutant analyses showed that auxin inhibits the accumulation of chlorophyll through the function of IAA14, ARF7, and ARF19 in Arabidopsis.

In tomato, SlARF4 plays an important role as an inhibitor in chlorophyll biosynthesis and sugar accumulation via transcriptional inhibition of SlGLK1 expression in tomato. In this study, over expression of SlARF6A resulted in enhanced chlorophyll accumulation and chloroplast development, whereas down regulation of SlARF6A decreased chlorophyll accumulation and chloroplast number in tomato . These results demonstrate that SlARF6A positively regulates chlorophyll accumulation and chloroplast number in tomato. Our study also showed that SlARF6A directly targeted the SlGLK1 promoter and positively regulated SlGLK1 expression . Nguyen et al. reported that over expression of SlGLK1 and SlGLK2 produced dark-green fruits and increased chlorophyll accumulation and chloroplast development. The fact that the phenotypes of SlGLK1 over expression plants resembled those described in the OE-SlARF6A plants further suggests that SlARF6A positively regulates SlGLK1 to improve chlorophyll accumulation and chloroplast development in tomato leaves and fruits. Although SlGLK1 and SlGLK2 have similar functions, SlGLK1 functions largely in leaves, while SlGLK2 functions in fruits. However, SlGLK2 does not account for the chlorophyll phenotypes in OE and RNAi-SlARF6A plants because the ‘Micro-Tom’ variety possesses two null alleles of SlGLK2. In our study, down regulation of SlARF6A reduced SlGLK1 expression and chlorophyll accumulation, whereas over expression of SlARF6A increased SlGLK1 expression and chlorophyll accumulation in leaves and fruits of tomato plants . The data demonstrate that SlGLK1 may be involved in chlorophyll accumulation not only in tomato leaves but also in fruits. Further study is needed to elucidate the important role of SlGLK1 in tomato fruit using CRISPR/Cas9 technologies. The chlorophyll a/b-binding proteins are the apoproteins of the light-harvesting complex of photosystem II . CABs are normally complexed with xanthophylls and chlorophyll, functioning as the antennacomplex, and are involved in photosynthetic electron transport. Meng et al. reported that SlBEL11 directly acted on the promoter of CABs to suppress their transcription. Silencing of SlBEL11 increased the expression of CAB genes, resulting in enhanced chlorophyll accumulation and stability in thylakoid membranes of chloroplasts in green tomato fruit.

In our study, SlARF6A targeted the promoter of CABs, which positively regulated chlorophyll accumulation, chloroplast development and photosynthesis in tomato . Our data further demonstrate important roles of CABs in chloroplast activity and photosynthesis in tomato. Rubisco, a key enzyme in the fixation of CO2, is the ratelimiting factor in the photosynthesis pathway under conditions of saturating light and atmospheric CO2. The RbcL and RbcS genes encode two subunits that form the Rubisco enzyme. The RbcL and RbcS genes are localized to the chloroplasts and to the nucleus, respectively. Our study showed that over expression of SlARF6A increased the expression of the RbcS gene. Moreover, SlARF6A directly targeted the RbcS promoter and positively regulated RbcS expression . In addition, SlARF6A positively affected photosynthesis in the fruits and leaves of tomato plants . Our study demonstrates that SlARF6A has important roles in photosynthesis via the direct regulation of the RbcS gene in tomato. Interestingly, RNA-Seq data showed that the expression levels of SlARF4 and SlARF10 genes were not altered in RNAi-SlARF6A and OE-SlARF6A plants, suggesting that SlARF6A may act on chlorophyll accumulation independently of SlARF4 and SlARF10. However, studies indicate that ARFs must form dimers on palindromic TGTCTC AuxREs to form a stable complex, leading to the possibility that SlARF6A, SlARF4 and SlARF10 could form dimers with each other to regulate chlorophyll metabolism. Further study could focus on the interactions among SlARF6A, SlARF4, and SlARF10 to comprehensively elucidate the effects of the transcriptional regulation of ARFs on chlorophyll accumulation in tomato.Down regulation of SlARF4 increased the photosynthesis rate and enhanced the accumulation of starch, glucose and fructose in tomato fruits. In this study, the increased chlorophyll accumulation and photosynthesis rate in OESlARF6A plants resulted in the increased contents of starch and soluble sugars in fruits . Starch is a dominant factor in the nutrients and flavor of fruits. AGPase catalyzes the first regulatory step in starch synthesis, converting glucose-1-phosphate and ATP into ADP-glucose. This critical catalytic reaction is also a limiting step during starch biosynthesis in potato tubers. Knockdown of SlARF4 increases the expression of AGPase genes and starch content. In this study, SlARF6A was positively correlated with the expression of AGPase genes , suggesting the important role of AGPase genes in starch biosynthesis in tomato. However, the EMSA failed to detect any binding between SlARF6A and the promoters of AGPase genes, even though auxin-responsive motifs were detected in the promoters of AGPase S1 and AGPase S2 genes.

Evidence suggests that sucrose induces the expression of AGPase genes in leaves and fruits in tomato. In this study, over expression of SlARF6A led to increased sucrose content in tomato fruits, while the RNAi-SlARF6A fruits displayed decreased sucrose accumulation . The altered accumulation of starch in OE-SlARF6A and RNAi-SlARF6A lines may be explained by the altered expression of AGPase genes influenced by sucrose in tomato. Over expression of SlARF6A also resulted in increased glucose and fructose content, vertical grow shelf which was likely due to the increased starch content degraded into increased contents of soluble sugars in tomato fruits. Our results are consistent with the notion that incipient starch content determines soluble sugars in the process of fruit development. Our study also provides a valuable method to improve the nutritional value of tomato fruits via regulation of SlARF6A expression.The tomato ARF2A gene was reported to positively regulate fruit ripening. Over expression of ARF2A in tomato resulted in blotchy ripening, and silencing of ARF2A led to retarded fruit ripening. Overexpression of ARF2A in tomato promoted early production of ethylene and expression of ethylene biosynthesis and receptor genes. In this study, SlARF6A negatively regulated fruit ripening and ethylene biosynthesis in tomato fruit . S-adenosyl-L-methionine , synthesized by SAM synthetase from ATP and methionine, is a substrate for ethylene biosynthesis . SAM is converted to ACC by the ACS enzyme, and ACC is then converted to ethylene by ACO. The level of SAM is tightly controlled to integrate developmental signals into the hormonal control of plant development. In Arabidopsis, over expression of SAMS1 increases the SAM and ethylene levels, whereas sam1/2 mutants show the opposite phenotype in seedlings. Similarly, in tomato plants, over expression of SAMS1 results in higher concentrations of ACC and ethylene compared with those in WT plants. These data indicate the important role of the SAMS1 gene in ethylene biosynthesis in plants. In this study, SlARF6A directly targeted the SAMS1 promoter and negatively regulated SAMS1 expression . The regulatory mechanism by which SlARF6A affects fruit ripening and ethylene production in tomato fruits can be explained by the interaction between SlARF6A and the SAMS1 promoter. It is interesting that ethylene and auxin interact with each other to control some plant developmental processes. For example, ethylene controls root growth through regulation of auxin biosynthesis, transport and signaling, while the formation of hypocotyl apical hooks is also regulated in a similar fashion in Arabidopsis. In tomato, knockdown of IAA3 results in both auxin and ethylene phenotypes, suggesting that IAA3 might be the molecular connection between ethylene and auxin. Liu et al. reported that the ethylene response factor SlERFB3 integrated ethylene and auxin signaling through direct regulation of the Aux/IAA27 gene in tomato. Our results indicate that SlARF6A negatively regulates ethylene biosynthesis and that the interaction of SlARF6A and SAMS1 represents an important integrative hub mediating ethylene-auxin cross-talk in tomato. In summary, our results demonstrate that SlARF6A regulates chlorophyll level and chloroplast development by directly binding to the promoters of the SlGLK1, CAB1, and CAB2 genes. SlARF6A also directly targets the RbcS gene promoter, activating RbcS expression and increasing the photosynthetic rate. The increased chlorophyll accumulation and chloroplast activity improve photosynthesis, resulting in the increased accumulation of starch and soluble sugars in tomato. In addition, SlARF6A can act directly on the promoter of SAMS1 and negatively regulate its expression, thereby influencing ethylene production and fruit ripening.

The present study provides new insight into the link between auxin signaling, chloroplast activity, and ethylene biosynthesis during tomato fruit development. Our data also provide an effective way to improve fruit nutrition of horticulture crops via regulation of chlorophyll accumulation and photosynthetic activity.For chlorophyll content determination, the fruits at different developmental stages and leaf tissues were collected and examined based on the methods described by Powell et al.. To determine chlorophyll auto- fluorescence, pericarp was peeled off tomato fruits and observed with a TCS SP2 laser confocal microscope . For transmission electron microscopy, pericarp tissues were examined with an FEI Tecnai T12 twin transmission electron microscope according to the method described by Nguyen et al.. For measurements of photosynthesis rates, the green mature fruits and leaves were measured via a PAM-2500 pulse-amplitude modulation fluorometer . The chlorophyll fluorescence parameter was measured based on the method described by Maury et al..For sugar extraction, 1 g of fruit tissue was collected and ground under liquid nitrogen. Subsequently, 10 mL of 80% ethanol was used for extraction three times at 80℃ for 30 min. After centrifugation, samples were completely evaporated under vacuum and then dissolved in 4 mL of distilled water. Using the dissolved samples, HPLC was carried out to determine the content of sucrose, fructose and glucose. Starch content determination was performed using fruit pellets. Four milliliters of 0.2 M KOH was used to dissolve the pellet by incubating the sample in a boiling water bath for 30 min. Then, 1.48 mL of 1 M acetic acid with 7 units of amyloglucosidase was employed to hydrolyze each sample for 45 min. Finally, 10 mL of distilled water was adopted to dissolve the sample, and then the dissolved sample was used for starch content measurement. For metabolite measurement, HPLC analysis was conducted using an Agilent 1260 Series liquid chromatograph system with a vacuum degasser, an autosampler, a binary pump, and a diode array detector controlled by Agilent ChemStation software. A precolumn and a Waters XBridge Amide column were used for analysis. The separation was performed via an isocratic solvent system with solvent A and solvent B , while the mobile phase was maintained at 75% B for 15 min for elution. The column temperature was maintained at 38 °C, and the solvent flow rate was 0.6 mL/ min. Meanwhile, the injection volume was 10 μL for each sample. With a drift tube temperature at 80 °C, the detection system for HPLC was an ELSD 2000, and air was used as the carrier gas with a flow rate of 2.2 L/min. Finally, the contents of glucose, fructose, sucrose and starch in tomato fruits were determined based on the methods described by Geigenberger et al..

Our results show that the greatest variation in stem water d18O and plant water sources occurred during the frontal season and initiation of the dry season in February, whenG. floribundum was shedding old leaves and growing new leaves, but P. piscipula maintained its leaves from the previous wet season . Contrary to what was expected, P. piscipula took water primarily from shallow sources regardless of the month, although some contribution from deeper sources has the potential to occur. Rain also appeared to be an important source for this species. This implies that P. piscipula could have a very well developed shallow root system that allows rapid water uptake after a precipitation event. On the other hand, G. floribundum took water from topsoil and bedrock, the latter being a more important source in the dry season. This suggests a deeper root system than G. floribundum. Overall, our results indicate that the contrasting early and late dry season leaf loss phenology of these two species is not simply determined by rooting depth, but rather a more complicated suite of species-based characteristics based on opportunistic use of dynamic water sources, the balance between carbon gain and water loss, and maintenance of water potential at the end of the dry season. These results are consistent with other studies demonstrating a broad array of coordinated strategies for dealing with seasonal drought in tropical forests . A primary factor determining differences in leaf loss phenology between the two studied species appears to be the maintenance of water potential. G. floribundum consistently exhibited more positive water potential values than P. piscipula, suggesting that G. floribundum has a limited capacity to tolerate negative water potential and moderates water use in a manner that maintains bulk leaf water potential at relatively more positive values compared to P. piscipula . This could provide an advantage of maximizing carbon gain during the dry season when light availability is high.

Leaf organic d13C and d18O values support this observation, because P. piscipula showed consistently higher d13C values than G. floribundum ,vertical farm coupled with lower d18O indicating that the decrease in photosynthetic carbon isotope discrimination was associated with greater stomatal conductance and greater photosynthesis . Greater photosynthesis in P. piscipula is consistent with maintaining a canopy of leaves later into the dry season. Thus, our results are most consistent with maintenance plant water potential to maximize carbon gain during the onset of the dry season. The observation that P. piscipula appeared to use shallower water sources and maintained its canopy of leaveslater into the dry season was not expected. Part of this pattern is driven by the capability of P. piscipula to utilize dynamic sources of water, such as the cold front precipitation during the frontal season . This makes sense, because the Laja bedrock layer was a poor source of water at all times, and soil pockets, which are available, but heterogeneous in distribution, were always better sources of water than rock layers . Water content of soil/ bedrock sources changed along the year suggesting a different seasonal contribution to plant water uptake. The hg of topsoil in the wet and frontal seasons were very similar and three times greater than values measured in the dry season. The Sascab bedrock layer could be a significant source of water in the wet and frontal seasons, but not in the dry season. Soil pockets had two times more water than topsoil in the dry season suggesting that they could be an important source of water for trees during the dry season. In the dry season, the rock profile had hg between 1 and 5 % but nearly all were less than 1 %. These values were slightly lower than those reported by Querejeta et al. and Hasselquist et al. in nearby areas, which suggest that the bedrock was subjected to a greater evaporation during this study. The d18O values of water in this study integrated processes ranging from evaporation of soil and bedrock water sources, transpiration of tree species, and precipitation events. In the wet season, enriched values of d18O of water in topsoil 10–15 cm and trees revealed the occurrence of a depleted precipitation event that occurred on October 21, 1 day before sampling, bringing 19.7 mm of water . Furthermore, a frontal system including cold front #3, the tropical wave #37, as well as the remnants of tropical storm Kiko that formed in the pacific, converged on the study area days before the wet season sampling in October 2007 .

Hurricanes, tropical storms and cold fronts generally have lower stable isotope ratios than convective precipitation events . For example, Perry et al. recorded d18O values of -9.91 % for precipitation during tropical storm Mitch in 1998, and precipitation events ranging from -6 to -10 % for d18O have been recorded in the vicinities of the study area . Consequently, depleted oxygen values in soil 15–30 cm and P. piscipula and G. floribundum trees could be accredited to precipitation originated from these events. Soil pockets also showed more negative values than rock, suggesting that depleted rain water reached this layer. During dry season measurements in February 2008, the d18O of topsoil 0–15 cm was more positive than groundwater suggesting another depleted source of water. Cold front #29, which occurred 4 days before sampling and brought 33.9 mm , could be the main source of water. The strength of the dry season promoted the enrichment of all water sources respect to earlier samplings. However, the more negative value of topsoil from 0 to 5 cm could be affected by dew water since this soil sampling was done early in the morning. More negative d18O values in topsoil than ground water have also been observed by Saha et al. in similar environmental conditions in Miami, associated with water condensation occurring at night in the upper soil layers. Condensation has been shown to deplete d18O soil water 10–15 cm depth by 5 % . Condensation can also account for up to 47 % of total transpiration . Surface dew is easily generated when temperatures go below the dew point at night or in early morning . Under tropical conditions in Tahiti, Clus et al. reported average dew yields of 0.102 mm of dew during the dry season. Therefore, condensed water could be an important source for P. piscipula. Overall, our results indicate that variation in phenology between these two deciduous tropical dry-forest tree species, which vary in the timing of their deciduousness, is not akin to the relatively large variation in rooting depth that can occur between tropical evergreen and deciduous species , but rather reflects the diversity of plant physiological strategies that occur in tropical forest .Insertional mutagenesis is a powerful strategy for gene identification and functional genomics in plants . While the T-DNA approach is applicable to the model plants Arabidopsis and rice, where effective transformation methods are available,nft vertical farming it may not be feasible in many other plant species whose transformation is inefficient.

Transposon can be alternatively used for insertional mutagenesis in those plants, since the generation of new insertions occurs through crossing or propagation rather than through transformation. Supported by the United States National Science Foundation-Plant Genome Program . S.Q. was supported by the Research Start-up Grants of Zhejiang Academy of Agricultural Sciences , China. P.B.F.O. was supported by EU FP5 and FP6 projects CerealGene Tags and CEDROME . J.-S. J. was supported by the World Class University and the Crop Functional Genomics Center projects, Korean Ministry of Education, Science and Technology, Korea.The maize Ac-Ds transposable element has been shown to be active in the plant kingdom widely .Ds insertion libraries have been generated in Arabidopsisand rice . However, the current strategies of transposon tagging are usually slow and labor intensive and have several drawbacks. For example, in the presence of Ac transposase , transposed Ds elements may continue secondary transpositions. Unstable Ds insertions and serial transposition events may cause untagged mutations because imprecise excision or a transposition footprint can result in a mutation that is no longer associated with the transposon . Another problem is that the Ac-Ds transposable elements are highly active in rice and can transpose early in newly transformed callus cells , which results in many sibling plants carrying the same Ds insertions and consequently decreasing gene tagging efficiency. In the present study, we constructed 12 Ac-Ds transposon tagging vectors based on three approaches: AcTPase controlled by glucocorticoid binding domain/VP16 acidic activation domain/Gal4 DNA-binding domain chemical inducible expression system; deletion of AcTPase via Crelox site-specific recombination that was initially triggered by Ds excision; and suppression of early transposition events in transformed rice callus through a dual-functional hygromycin resistance gene in a novel Ds element. We have tested these vectors in transgenic rice and characterized the transposition events. Our results showed that these vectors are useful in functional genomics of rice and they will be useful for other crop plants as well.We constructed Ac-Ds transposon tagging vectors using a GVG-inducible expression system . The vectors pJJ86 and pDs-Ac-GVG carry an in cis two-element system that consists of Ds, 35S:GVG that expresses the chimeric GVG transcription activator, and AcTPase controlled by a GVG-inducible promoter. The inducible promoter is transactivated through interaction between GVG and the 4xGAL4-upstream activating sequence .

The transactivating activity of GVG is regulated by treatment with the steroid chemical dexamethasone . The Ds element in pJJ86 contains the 4x CaMV 35S enhancers for activation tagging , while the Ds in pDsAc-GVG does not. Excision of Ds from pJJ86 can be detected because in the resulting T-DNA fragment, the β-glucuronidase gene is driven by a CaMV 35S promoter. We also constructed a two-vector tagging system in which GVG-inducible AcTPase and Ds are in separate vectors . The strategy of the two-vector system is that transgenic plants carrying the GVG-inducible AcTPase and Ds are generated, respectively, and the AcTPase and Ds are combined in F1 by genetic crosses. In this case, Ds is mobilized in the presence of AcTPase in F1 plants, but stabilized after it is uncoupled from AcTPase in the subsequent generation. To test whether the inducible Ac-Ds system is functional in rice, we transformed rice cultivar Nipponbare with pJJ86. Independently transformed rice calli were cultured for 5 d on media with DEX to induce expression of AcTPase. Because Ds transposition can be detected by GUS activity, the DEX-treated calli and untreated controls were stained for GUS activity. DEX treatment of pJJ86-transformed calli exhibited stronger GUS staining than controls , indicating that the DEXinducible system in this vector is functional in rice. At the same time, there was low background of GUS activity in the untreated rice calli , suggesting that some background transposition occurred in the pJJ86 transformants.Because the Ac-Ds transposable elements are active in newly transformed callus cells and early transposition events lead to the same Ds insertions in sibling plants, we constructed a novel Ds element, designated HPT-Ds, and used the hygromycin resistance gene to suppress transposition. The pHPT-Ds1 vector carrying HPT-Ds and GVGinducible AcTPase in cis is shown in Figure 1E. The HPT gene in HPT-Ds has the same intron and triple splice acceptors as in the gene-trap Ds . Because HPT-Ds is immediately downstream of maize ubiquitin 1 promoter in T-DNA, the Ubi:HPT-Ds fusion confers hygromycin resistance, and transformed rice cells are thereby selected on hygromycin media. In case of transposition, HPT-Ds in the rice genome may not have a promoter nearby for transcription and the rice cells lose hygromycin resistance and can be counter-selected by hygromycin. To examine the efficacy of the HPT-Ds element, we made a test construct containing Ubi:HPT-Ds and confirmed the function of the Ubi-driving HPT gene in a rice transformation experiment. A total of 250 rice calli were transformed using a particle bombardment method and hygromycin-resistant cells were selected from 30 callus explants after 50 d of selection on hgromycin media. In constructing the pHPT-Ds1 vector, HPT-Ds was cloned between Ubi and GUS so that transposant cells canbe detected by GUS assay. The pHPT-Ds2 vector is similar to pHPT-Ds1 except that pHPT-Ds2 carries a Bar gene and transposition can be selected by herbicide resistance . pHPT-Ds1 was introduced into rice cultivar Nipponbare.