Root growth is cyclic, it generally alternates with shoot growth, usually precedes it, and synchronizes with trunk growth. In Israel, Cossman reports growing roots could be found throughout the entire winter season. The most intense root growth periods were in the spring and autumn and of relatively short duration.Monselise also states that root growth alternates in most cases with shoot growth and stresses that soil temperature and soil moisture are important factors. In California, Crider observed that roots do not grow continuously but alternate with shoot growth. Total root elongation and greatest rate of growth was much greater during the first rather than the second and last periods . Total root growth as we1l as the rate of growth during the second or mid-season growth stage was decidedly less than the first and last period. The rate of root elongation was considerably greater during the night rather than during the day. Roots were inactive during the winter and early spring, and Crider suggests that this is the best time to cover crop or interplant. Waynick and Walker observed in California that growth of the roots in the subsoil preceded growth in the upper foot of soil by nearly 28 days in the spring. Root growth preceded shoot growth except in the fall, when the growth cycle seemed to coincide. This condition may vary in other citrus areas. They also noted a difference in root growth with different scions and different aged trees. In October, when the last growth cycle was completed, there was only scattered growth of roots in the upper 45 cm of soil with old trees. Grapefruit Trees showed little development while Valencia trees showed a moderate growth,hydroponic vertical garden but only in the upper 56 cm of soil. In contrast, yearling Valencia trees in the same soil developed a heavy root growth.

Waynick and Walker measured soil temperatures at 15-35 cm, 51 cm, and 66 cm. The soil temperatures at 36 cm and above were consistently lower by 2°C than lower depths until mid-April. The average subsoil temperature in [“Check” appear here in typescript in the margin of the manuscript] late March was 13.8°C, or just above the minimum of 12°C reported by Girton . The soil temperature below 51 cm averaged 1.5°C less than that from 36- 45 cm, and it was in this area that the most active root growth took place. In March, subsoil root growth was first observed. There was no root elongation above 36 cm, but root growth was general between 36-51 cm. The growth rate of the roots varied from .62—1.60 cm daily. Rootlets elongate at the rate of .32-1.27cm per day in an active cycle. Root growth ceased the first week in April, when top growth occurred. Root growth resumed in September and lasted five weeks. In a three week period the rootlets grew as much as 36 cm, and the size of the roots doubled. Their data show growth of the roots is cyclic and precedes top growth. There is a brief rest period when roots cease to elongate and the growth of the top begins. There are 2-3 month periods when the roots fail to elongate. Roots make very poor growth when severed by cultural equipment, even in two year’s time. They emphasize the important relationship of irrigation and supply of nutrient to the growth cycle of the roots, and question the possible undesirable effects of the heavy application of cold irrigation water when the root cycle is just starting. In Florida, Ford states that citrus root concentration fluctuates extensively during the year. It had been assumed that roots grow in alternate cycles to growth flushes, but this did not seem to apply to the situation in Florida. Root concentration was usually lowest in August and highest in September. He stresses the importance of taking root samples at the same time of year. Soil temperatures in Florida citrus orchards are higher than those in California throughout the root zone during the entire year . Tully also noted that in citrus trees shoot and root growth usually alternate. Root systems are generally observed and classified by either complete excavation of the root system, digging trenches, push and pull procedures, removal of soil cores, or use of special technical devices. Factors affecting root distribution are the scion variety, environment, temperature, age of tree, water relations, drainage, salinity, nutrition, diseases, soil types, soil stratification, pruning, cover crop, etc.

Therefore, it is understandable that there is considerable discrepancy in the descriptions of root systems as described by various observers. An old adage is that lateral roots of a tree extend radially from the trunk to a distance equal to that of the height of the tree. A 4.6 meter tall tree would thus have roots extending laterally in all directions to a distance of 4.6 meters. There are, of course, many exceptions to this rule as root distribution is affected by many factors. Under many circumstances, the lateral spread of the roots is such that at any normal orchard spacing there is considerable intermingling of roots from adjacent orchard trees. Perhaps the earliest recorded observations on citrus root systems are those of Mills in California. On excavated trees grown on [“Check” appear here in typescript in the margin of the manuscript] loam soils in Ventura County, he characterizes sweet orange as having a shallow root system. The main mass of the root system of bearing trees on sweet orange stock are concentrated in a horizontal layer about 45 cm thick, the top of which was 20-25 cm from the surface, with crooked tap roots. The tap root of grapefruit was also crooked. The lateral roots develop at the expense of the tap roots, making a prodigious root system. A seven-year-old seedling had a tap root 120 cm long. The largest lateral root started 60 cm below the soil surface and extended for a distance of 8 meters at an average depth of 45 cm below the surface. Over 90 percent of the root system was confined to a layer 25-60 cm below the surface. Grapefruit had a much larger number of fibrous roots than sweet orange, and the roots were deeper beneath the surface, the majority below 38 cm. A nine-year-old navel tree on sour orange had the largest and uppermost lateral, which started 15 cm below the surface and descended immediately at an angle of 45° to 2.5 cm below the surface to a distance 3 M from the tree, when it rose to within 20 cm of the surface. It was then cut by cultivation equipment, grew downward 2.5 cm and horizontally 2.4 meters more. The longest tap root was approximately 3 meters, maybe the deepest recorded in California. Those trees on sour orange with few laterals were less productive than those which had numerous laterals near the surface. Hume states that in Florida’s Hammock soils, sour orange roots are produced abundantly and penetrate well into the soil. He felt that this was an advantage over sweet orange since the trees were not so readily affected by variations in soil moisture. The roots penetrate sufficiently deep to be more or less in contact with a permanent water supply.

Hume also says that under these conditions, sour orange suffers less under prolonged drought under identical conditions than Rough lemon. With Rough lemon the main roots show a wide variation. In all cases the crown-roots extend a considerable distance from the trunk and a strong tap root is produced, but occasionally the large lateral roots lie close to the surface and most of the feeder roots are in the top 38 cm of soil. Hume also states that the root system produced by trifoliate orange is very good,vertical vegetable tower as the roots penetrate well and fibrous roots are produced in abundance. He also cites the California experience of Mills that sweet orange is not deep rooted and that most of the roots are in the top 46 cm of soil. There is lack of evidence in Florida, but in nursery trees, [“Check” appear here in typescript in the margin of the manuscript] root development of trifoliate orange was equal to sour orange. The root system of grapefruit was well developed. In Egypt, El Sawy found the root system of lime heavily branched with abundant fibrous roots. Those trees on citron, sweet lime, and sour orange were sparsely branched with less fibrous roots. The depth of penetration of citron or sweet lime was 41-46 cm, 61-71 cm on lime, and 112-168 cm on sour orange. As far as lateral extension, the citron had the least, followed by sweet lime and those on sour orange. Brown and El Sawy observed in Egyptian orchards that the sour orange adapted to heavy soils and was not readily affected by variations in soil moisture. It provided good root anchorage, roots in loamy soils penetrating to a depth of 110-170 cm with a lateral spread of 4-10 M depending on the scion variety. They made no comment about the root system of sweet orange. The grapefruit and shaddock they felt were well adapted to heavy and moist soils, with their root systems well developed and evenly distributed. The Baladi lime was adapted to sandy soils, but not wet or heavy soils. The root system was richly branched and had more fibrous roots than any of the other stocks. The bulk of the roots in sandy soils penetrated an average depth of 70 cm and occasionally to 250 cm. Lateral roots extended to 7 M. The Rough lemon was adapted to sandy soils where sour orange failed to produce a satisfactory tree. Although it had a strong tap root which penetrated deeply into the soil, there were abundant lateral roots which were well and widely distributed and were nearer to the surface than sour orange. The Rough lemon had many feeder roots. The sweet lemon was also not recommended for heavy soils. Its root system was characterized by downward penetration and lateral expansion similar to that of the Baladi lime, but was sparsely branched and had less fibrous roots in the upper 30 cm of soil. There were no comments on the citron root system other than that it did very poorly on sandy soils. In Israel, Oppenheimer examined the root systems of 19-month-old trees on sweet lime, sour orange, Rough lemon, citron, Baladi , Shamouti, sour orange, pummelo, and grapefruit. He says all Citrus root systems belong to the intensive type. The lemon-lime group are characterized by strong root branches whose horizontal spread does not fall short of their penetration in depth, and with a multitude of fine absorbing rootlets. Single tap roots are rarely found after transplanting. The surface layers of soil are well provided with roots, except the root system of sour lemon, which showed rather fine root branches and a tendency for deep penetration without strong crown root formation in the surface layers. With Rough lemon the lateral roots changed direction from the horizontal and turn downward. He agreed with Hume in praise of Rough lemon, “because of the great foraging power of its roots.” With sweet orange, he observed a tendency to form a strong tap root system which showed good ramification and a considerable development of horizontally growing main roots. He did not agree with observations by Mills in California on this, but did agree with Hume’s Florida observations, perhaps because of soil differences. Hume, in Florida, worked in super drained, very sandy soils, warm enough throughout the year for good root growth and root activity. The sour orange, grapefruit, and pummelo tend to form root systems of a conical shape with well developed penetrating central roots, and a monopodial character. None of the upper roots are strong, and most of the lateral roots descend obliquely. The abundance of fibrous roots on grapefruit produces more fibrous roots than sweet orange or sour orange. In Florida’s deep sandy soils, Savage, Cooper, and Piper examined sixyear-old trees of Parson Brown on 15 root stocks planted at a distance of 30-92 cm. However, some roots were lost in the excavation. Sour orange showed three or more well developed tap roots penetrating 90-125 cm deep. There were numerous small laterals in the upper foot and some of the laterals extended 150 cm from the trunk.

There are many similar top working examples too numerous to mention. Eliminating the lemon trunk, which was usually so susceptible to shellbark, appeared to have the same effect as high budding and eliminated the shellbark. Trees affected by known diseases probably under no circumstances should be top worked. In some instances top working may result in an incompatibility. For example, in 1976 while visiting Japan, the author was shown some satsuma trees on trifoliate orange root stock which had been top worked to Uwa pummelo. An incompatibility occurred at the juncture of the Uwa pummelo and the satsuma. In other instances, the top working may result in the susceptibility to a disease which would not have affected the original combination prior to the top working. To illustrate the point, lemon trees on sweet orange root stock are not susceptible to tristeza. However, if the combination was to be top worked to sweet orange the new combination would be susceptible to tristeza . Trees showing symptoms of tristeza should not be top worked to lemons; but susceptible trees can be top worked successfully if done before tristeza occurs . Growers in Spain, until the occurrence of tristeza, probably did more top working than in any other citrus area of the world. Citrus diseases are easily spread through top working, either by the mechanical means of contaminated tools, or the use of scion varieties in which symptoms of the presence of a pathogen were not expressed. In 1983, Dr. Luis Navaro, Director of INIA at Moncada , Spain,vertical hydroponics told a group of participants at the First International Society of Citrus Nurserymen that of all the Spanish citrus cultivars entered into the new budwood certification program established in Spain, over 50 per cent of the cultivars were carrying three or more viruses.

To make this information more available, Platt prepared a handout on citrus top working compatibility. It is reproduced intact in Table 12. While Platt prepared this information specifically for California growers, citrus growers in other areas will find that many of the recommendations can be appropriately applied to their orchard conditions as well. The largest related incidence of top working in the California citrus industry occurred in the early 1960’s. The incident had nothing to do with tristeza directly, but was associated with the rapid increase in citrus plantings in the 1950’s, particularly in the San Joaquin Valley, following the destruction of orchards in the Los Angeles basin by tristeza. The causal factor was a citrus bud variation referred to as the “acid-navel.” . This was a mutant of the ‘Frost’ navel which was so extensively propagated at that time. The variant was a fruit mutation characterized by smaller fruit, coarser rind, and other undesirable external characters. However, the interior of the fruit had a more watery-appearing flesh with juice of a higher than normal citrus acid content; hence the term “acid-navel.” The fruit characteristics were completely unacceptable for commercial purposes. Since the trees were disease free and no incompatibilities were involved, most of these trees were top worked, generally to another navel orange of the grower’s choice. Hundreds of acres, literally thousands of trees, were top worked. The episode stresses the importance of nurserymen cutting buds for propagation from fruiting trees so that the fruit characteristics of the scion variety can be observed. Inter stocking of citrus trees has not always proved useful. Diseased trees should not be top worked, and when and if top working is necessary, it should be with full knowledge of the consequences involved.

The fact that citrus inter stocks, except for preventing certain incompatibilities, have had so little effect on the physiological response of the scion should not imply that they may never be successful. Most citrus inter stocks have been genetically close and oftentimes commercial varieties. Maybe there are non-commercial citrus cultivars or citrus relatives which may produce desirable effects. The success of inter stocks with a tree crop like apples cannot be overlooked. Coincident with any discussion of inter stocks are other horticultural procedures, which may not be so difficult to perform, but perhaps could produce effects similar to inter stocks, and actually be considered as modified inter stocks. The first of these procedures is the ring graft. Considerable interest was generated in the 1930’s through the 1950’s in the technique . Much of the work was done with apples and other tree crops. The procedure is accomplished by using knives such as linoleum knives or grape knives with a curved point at the top of the blade. The knives may have spacers between the blades or handles so that parallel cuts can be made around the trunk or branches of the tree. Considerable pressure is put on the cutting implement so the blades penetrate through the phloem . It is impossible to control the pressure and, as a result, the knife blades cut not only through the phloem but also through the cambium and into the xylem elements. Penetration into the xylem may easily range from 1/32 to 1/8 inches . Thus, not only are the phloem elements severed, but xylem elements also, resulting in a xylem block as well as a phloem block. A single cut through the bark of the tree trunk is referred to as girdling or ringing . The technique is used almost exclusively to increase fruit set in navel oranges, although it is occasionally practiced with some mandarin varieties. The cut is not protected in any manner from desiccation. The resulting severance of both the phloem, cambium, and xylem tissues apparently heals quickly. Furr, Reece, and Hrnciar found there is a return to normal nutrient absorption two weeks after girdling, which suggests that the break in the cut tissues may be at least partially healed over in two weeks.

Curtis and Clark reported that regeneration of new phloem within 10 to 15 days resulted in translocation so nearly normal that no difference could be measured between control and treated shoots in analysis for sugar, starch, nitrogen, and ash. Murneek showed that apple fruit set was increased an equal amount whether girdling affected carbohydrate and nitrogen levels or not. Thus, carbohydrate and nitrogen levels appear to be of little importance in single, knife-cut girdling . Sax reports that phloem blocks induced by killing a small section of the stem with heat prevented the downward movement of vitamins, organic carbohydrates, and grow-regulators . Bonner , in girdling stems of tomato plants, found that in a few days there was an accumulation of thiamin, pyridoxine, pantothenic acid, riboflavin, sucrose, and nitrogen above the girdle. Lewis and McCarty state that it appears possible that the severance of phloem tissues causes a temporary increase or decrease in a growth regulator influencing fruit set. Much of the effects of girdling have thus been done with a single cut. However, other studies of girdling physiology have involved more severe cuts , including the removal of a band of bark from the tree trunk. This is what happens with a ring graft. With a ring graft the strip of bark which is removed from around the tree trunk is immediately replaced with a comparable sized strip of donor bark. The donor strip in this instance is replaced in the proper polar orientation. The ring graft can consist of a bark strip from sweet orange, sour orange, Rough lemon, sweet lime, trifoliate orange, Morton citrange, whatever the researchers’ choice. In unpublished data by the author, plastic electrical tape, or duct tape, was used to hold the donor bark strip in place and prevent desiccation. Since the procedure of a ring graft is somewhat more severe than a simple girdle, the healing process may be a little longer, perhaps weeks, or even months. When the ring of bark is removed from the parent tree, the bark separates at the cambial tissue. Enough cambial initials remain to form new xylem elements to the inside and new phloem tissues to the outside. The strip of donor bark inserted into the removed bark area also has cambial initials on its inner surface. Again,hydroponic vertical farming systems xylem elements are found to the inside, phloem elements to the outside . Roberts was one of the pioneers to explore the use of ring grafts in apples. The author was a graduate student of Roberts in the early 1940’s and recalls in several discussions that Roberts was quick to point out that the effects did not last since the donor bark strip soon became nonfunctional and sloughed off as with a cork cambium. Sax extensively investigated the aspect of bark inversion of the donor bark strip so that the polarity of the conducting cells is at least temporarily reversed and transport of nutrients, metabolites, etc. reduced. Sax considered inverted bark strips as phloem blocks. Reversed polarity is only a temporary problem in bark regeneration . Sax , referring to the work of Roberts , states that the inverted rings checked the growth of the trees, but without the deleterious effect resulting from girdling. He further emphasizes that apparently this work was not continued by Roberts. The author had the pleasure of a long discussion with Sax in 1957, and Sax admitted that the effects of bark inversion were temporary and had very little overall effect on tree growth. In 1956 , the author had conducted some bark inversion experiments with a grower cooperator. The trees were young lemon trees of uncertain destiny since the cooperator indicated the orchard would be removed in one to three years. Replicated treatments were made and the size of the tree trunks above the girdled area measured. The treatments consisted of different widths of inverted bark from 1/4 inch to 2 inches . In some treatments, height of the ring insertion varied. In other treatments, a double ring of bark was inverted, approximately 10-15 cm apart.

Another treatment consisted of an incomplete inverted ring with a 2.5 cm wide strip of normal bark at 180°. See Figures 11 – 21 [Image for Figure 21 could not be located]. Observations were limited because the orchard was pulled before final measurements could be made. In no instance was there any observable difference in tree growth with any treatment as compared to the checks. The width of the inverted ring did not seem to make any difference, the 5 mm was as effective as the 5 cm. Perhaps the use of two inverted rings had more effect than a single inverted ring. The most notable effect was with the incomplete rings where small islands of normal bark were left at 180°. The effectiveness of the blocks was clearly demonstrated since the nutrients and metabolites were translocated to the two normal bark strips resulting in accelerated growth increase in that area. The use of ring grafts using other varieties or species of citrus genetically different from the parent tree produces no permanent effect on the parent tree. Imposing the additional effect of bark inversion to alter polarity is also a temporary effect. Since nearly all commercial citrus trees are budded, it is not surprising that, like other budded or grafted fruit tree combinations, they may be subject to incompatibilities. The terms compatible and congenial refer to healthy long-lived combinations, and incompatible and uncongenial refer to combinations which are not healthy and long-lived. Sometimes the generalization is made that compatibility is a function of the closeness of genetic relationship between the stock and the scion, but there are certainly many exceptions to this hypothesis. If the generally accepted systems of citrus classification are used, there is a wide diversity in the germplasm available within the conservative classification of Swingle , who lists 16 species, and that of Tanaka , who lists 159 species. However, within the Family Rutaceae, subfamily Aurantioideae, there are a total of 33 genera available with considerable interspecific and intraspecific crosses and bigeneric and even trigeneric hybrids available. It is possible to graft any Citrus with Citrus, and apparently many genera with many other genera, but the degree of success may vary greatly. While the author is unaware of Citrus being successfully grafted on any genus outside the subfamily Aurantioideae, he has kept orange buds alive for one year when grafted on Casimiroa edulis, but the buds did not push. In a 1977 conversation with Professor Giovanni Fatta Del Bosco of Palermo, he indicated that mandarin scions will grow, but poorly, on this cultivar.

Seedlings of C. taiwanica, Yuma citrange and some of the Ichang hybrids, for example, also show a low rate of polyembryony and severe cullage is needed in the nursery. Cultivars with a low nucellar rate should probably not be used as root stocks. However, it is possible that if the nucellar seedlings are of considerable root stock interest, clonal propagation by in vitro methods could be considered in the future. In varieties that produce a fairly high percentage of nucellar embryos, most of the seedlings can be recognized as representing the largest proportion of the progeny. They will have the same foliage and branching characters and will in general be of about the same size. They present a uniform and, commonly, easily recognizable type. Mixed with these will be a varying number of seedlings that show different characters, such as different branching and larger or smaller leaves, and that are usually less vigorous and of smaller size. However, in some instances occasional seedlings are more vigorous than the norm. Variations in trifoliate hybrids may be more difficult to detect because the trifoliate leaf character is dominant and all the hybrids exhibit the trifoliate leaf character. Here, in addition, one must look for size of leaves, leaf color, the tendency towards deciduousness, and other factors as well. These differences from the norm, in a large part, are the so-called “variant” seedlings which apparently are mainly produced from gametic embryos. It is these small, off-type seedlings or extremely vigorous seedlings which are variants and should be discarded upon removal from the seedbed,vertical garden indoor or later in the nursery row, in order to obtain uniform seedlings for use as root stocks. In spite of all precautions, some zygotic seedlings do escape culling and are budded and ultimately end up as orchard trees.

Some of these, but not all, may be culled out as budlings in the nursery row because of lack of uniformity as compared to the rest of the progeny. It is unfortunate that most of the people involved in the labor or nursery practices are not trained or qualified to recognize variants. They probably do a better job on the elimination of “goose necks” and “bench roots,” which in reality may not be as important. Seedling progeny of some of the monoembryonic cultivars such as Algerian tangerine and a number of shaddock cultivars, although all genetically different, have been remarkably uniform in the seedbed and nursery. When budded to standard scion varieties and planted in root stock experiments at the Citrus Experiment Station, Riverside, most of the trees at 33 years of age were as uniform in size, appearance, and yield as those budded on nucellar seedlings of other varieties. Certain inherent characteristics persist in the progeny, however. Some seedlings of Algerian tangerine, shaddock, Poorman’s orange and others continue to show a variable percentage of the seedlings with a bark disorder. In testing numerous shaddocks as root stocks for tristeza resistance, the fact that every stock was genetically different did not influence the reaction to tristeza. All were susceptible. Many of the citrus relatives which are monoembryonic also display a high degree of uniformity when grown from seeds. Thus Clausena, Murraya, Triphasia, Citropsis, and other genera will, although possessing one zygotic embryo, produce progeny which appear phenotypically identical to the mother plant. In many of the world’s citrus areas, the importance of strain selection within scion varieties is well recognized and put into practice by the growers and nurserymen. This is perhaps more true in Japan than any other citrus area. Japan’s large Satsuma industry is divided into three fruit categories according to their maturity. The Wase group are early, the Nakates are mid-season, and the Futsu, or common group, are late. Within the Wase group are Okitsu, Miko, Miyagawa and dozens of others.

The other two groups may have a few less strains. Little differences in fruit yield, time of fruit maturity, hardiness, or local preference, etc., are of considerable importance to the grower and the industry without changing the variety. Spain has many strains of Clementines also selected for size, color, seediness, time of maturity, etc. Italy has made similar selections within the Willow Leaf mandarin. Strain selection within a variety has also been extensively practiced in California. Within the Washington navel variety are the Parent, Frost, Newhall, Tulegold, Bonanza, Thomson Imperial , Atwood, Fischer, Dream, Lane Late, etc. Even a wider range of strains may be found within the Eureka and Lisbon lemon varieties. These strain differences are for the growers’ preferential choice; the navels are all sold collectively as navels, and all the lemon strains are sold as lemons. Strain selection, or even varietal selection, while recognized within root stocks, is still generally not practiced, although there could be many advantages in doing so. Thus, for root stock purposes, the citrus industry has just nonchalantly considered a sweet orange a sweet orange, a sour orange a sour orange, or a Rough lemon a Rough lemon. On the other hand, root stocks like Troyer citrange, Sampson tangelo, Cleopatra mandarin, or Rangpur lime, are more specific; they imply one specific cultivar with no recognized variations, or strains, at least at the present time. A grower choosing one of these cultivars for root stocks doesn’t indicate to the nurseryman he wants citrange, tangelo, or mandarin as stock, because it is now a well known fact that there are other citranges, other tangelos, and other mandarins and, unless he specifies, he may get something else. Root stock trials at the Citrus Experiment Station, Riverside, indicate that there are performance differences between different strains of sweet oranges, sour orange, grapefruit, etc., that justify a clonal selection within that species to take advantage of greater yields, variations in tree size, better gummosis resistance, or better nematode resistance , and see root stock yield later in this manuscript. In these trials, and across several scion varieties, the performance of the Rubidoux sour and Brazilian sour was definitely superior to that on African sour and to a lesser extent on Paraguay sour.

Of course, this was before the incidence of Tristeza.The CRC #343 grapefruit provided better performance results than Duncan, McCarty, Camulos, and several others. The Koethen sweet orange in the Riverside trials appeared preferable to other sweet oranges. In Ventura County, the Olivelands sweet has frequently been preferred. The established performance of root stock cultivars warrants their perpetuation as root stock seed sources and their choice over untested and unproven sources which a nurseryman thinks may be just as good. In California, many citrus nurserymen have, or have had, their own individual seed source trees. Many had been chosen because of past good orchard performance in a specific area, or statewide. Some were the nurseryman’s selection,vertical plant rack others were obtained from the Citrus Research Center at Riverside. In either case, the nurseryman could offer the same clonal selection of root stock year after year with some confidence in its continued good performance. The immediate and rapid acceptance and success of Troyer citrange as a root stock in California almost had disastrous effects. With the increased plantings of young orchards, Troyer seed was in such demand that many nurserymen chose to grow seedlings for future seed sources. Prices were prohibitive for that time , as seed sold in most instances at a rate of ten cents each, or fifty cents per fruit, and three hundred dollars a standard field box . The author observed some of the fruit seedling sources and was able to identify a number of off type trees. The fact that some of the trees were off type didn’t seem to make any difference to the seed supplier. Fruit from the off type trees was harvested along with the fruit from the normal trees, either unknowingly or willingly , and sold as Troyers. While it is one thing to be able to identify any off type trees which can and should be discarded, another situation arises with off types in which there are no discernable visual differences from the mother tree. These may not be hybrid differences, but rather somatic variations which may be just as critical. The author will cite several examples, and keep in mind that this happened before the use of isozymes and other techniques were available for identification purposes. In the 1948 tristeza plantings at Baldwin Park, California , two sources of Bessie sweet orange were used as root stocks and half of the trees per root stock inoculated with tristeza. One of the sources of Bessie was an old source from the Citrus Variety Collection, Riverside. It served as the mother seed source for Bessie seedlings grown in 1924 for 1927 root stock plantings at Riverside . From out of these many seedlings, Dr. Webber had selected seedling #47 as being the largest and healthiest seedling of them all. This should have been the tip off. Seedling #47 was assigned CRC #1693 and also was placed in the Citrus Variety Collection, Riverside. In the Baldwin Park plantings, trees on the Bessie seedling CRC #1693 showed a reaction to tristeza and the trees on the Bessie #245 did not. Obviously the two seed sources were different. Many times my colleagues and I examined the two accessions and could not on leaf and fruit characteristics establish any differences. However, the largest and most vigorous seedling out of a progeny should never have been selected as true to type, and was probably either a gametic or somatic variant. Webber in 1924 also grew a fairly large number of sour orange seedlings for root stock purposes. Out of a population of 389 nursery seedlings, he selected 43 he considered as variants.

He propagated these variants and brought them into fruiting, and also budded the variants to a selected source of Washington navel. The author only wants to make reference to one of these variants. Just prior to my arrival in Riverside in 1946, one of the variants out of the 43 was selected for inclusion in the 1948 citrus root stock-tristeza trials at Baldwin Park . It was merely listed as a “sour orange variant.” The author was always interested in the correct identification of the root stock selections used in his experiments. Examination of this sour orange variant revealed leaf and fruit characters which appeared identical to Rough lemon. Showing foliage and fruit to my colleagues, including Dr. L. D. Batchelor, Dr. H. B. Frost, Dr. E. R. Parker and others, we could not differentiate between the variant and verified sources of Rough lemon. We removed the variant from the sour orange category and placed it in the lemon category, where we thought it more appropriately belonged. Upon being inoculated with tristeza, it proved to be equally, if not more, susceptible than those trees on sour orange. The standard Rough lemons showed no effect of the inoculation. Obviously, the variant was indeed a sour orange variant. How often does this happen? A number of the accessions in the Citrus Variety Collection date back to Dr. Webber’s “largest seedling” in the seedling nursery population. Many other accessions are more recent seedlings. Other seedling accessions may be more true to type than the Bessie, but do we know? Most of the accessions in the variety collection at Lindcove Field Station are seedlings, some of the accessions in the foundation block also. This is adequate cause for concern and alarm. Are they identical to the parent sources? A few years ago, Dr. John Carpenter of the USDA Date and Citrus Station at Indio, California, shocked a group of citrus nurserymen he was speaking to at Riverside, when he said many of you do not have Swingle citrumelo CPB 4475; what you have is a seedling source of Swingle citrumelo, and they are not the same. In recent years there has been considerable interest in C-32 and C-35 citranges . As information was developing and some credence given to the future use of these two root-stocks, some nurserymen obtained seed from Riverside and planted seedlings for future seed sources. The all important question remains, are these seedling sources identical to the original sources at Riverside, and to those tested at South Coast Field Station for tristeza resistance ?

Hanging or lifting irrigation lines where possible, and using covers over the spaghetti tubes, are effective methods to reduce rabbit damage to irrigation. In addition, nurseries may consider encouraging irrigation tubing manufacturers to develop spaghetti tubing more resistant to rabbit gnawing. Nurseries may want to consider an ongoing program to reduce rabbit harborages by using habitat modification where possible. It may be appropriate to target areas that unintentionally provide habitat to reduce suitability. Some measures to reduce influx and suitability may include fencing between natural areas/groves and the nursery; decreasing cover for the rabbits in runways; thinning border vegetation that provides cover, such as the hedges, to reduce hiding places; regularly moving debris piles, recycled wood piles, and storage pallets to reduce breeding and cover within the nursery; and rotating pallets of stock. In the circumstances when trapping is desirable to remove rabbits from an area, the use of traps in runways created by drift fence will be the most effective method. Two species of tree frogs native to the Caribbean have recently become established in the Hawaiian Islands . Since their introduction via the import horticultural trade, the frogs have rapidly expanded their range on the islands of Hawaii, Maui, Oahu, and Kauai. There are two modes of spread for tree frogs. The first is the accidental transport via horticultural products or material from infected nurseries or gardens to uninfected areas. The second mode is the intentional introduction of frogs by citizens into sites that aren’t infested. Theoretically both activities are illegal under Hawaii state law , though enforcement is difficult.

There is a concern on the part of federal, state,vertical farming technology and private agencies/entities managing natural and agricultural resources in Hawaii that introduced Eleutherodactylus frogs pose a serious threat to these resources. E. coqui can reach densities of greater than 24,000/ha and is capable of consuming approximately 114,000 arthropod prey items per hectare in a single night in its native range in Puerto Rico . It is believed that these frogs, once established in native habitats, could prey on endemic arthropods as well as compete indirectly and directly with native birds for limited food resources . Tree frogs may be a vector for plant nematode eggs, and the recent discoveries of frogs in certified nurseries make the frogs a potential quarantine issue that could greatly impact the exportation of disease and pest-free nursery products from the state. Surveys indicate that frog populations have become firmly established in nurseries, parks, residential gardens, resort areas, and lowland forest habitats on the islands of Hawaii and Maui . The number of reported locations has significantly increased on these islands in the last 3 – 4 years . Frog populations have been documented on the islands of Oahu and Kauai , and there is grave concern that these populations will continue to spread. In one horticultural site on the island of Hawaii, one species of tree frog has been documented to obtain densities comparable to the native range . Localized loud vocalization of male frogs throughout the nighttime hours has also been a source of numerous angry complaints from sleepless residents and tourists alike. Restricting the transfer of infected plant materials via the horticultural trade or by the casual public has the potential of stemming further spread of frogs to uninfected areas. However, enforcement of laws dealing with the transportation of alien species within the State of Hawaii has been limited at best. Currently, an enforceable legal mechanism that specifically restricts the movement of plant hosts harboring E. coqui within the state has been proposed by Hawaii Department of Agriculture staff but has not been enacted.

Though the status of an enforceable quarantine on the movement of frogs is currently in question, there is an immediate need to: 1) reduce or eradicate localized frog populations that serve as reservoirs for new infestations and 2) treat infested plant material to insure this situation does not get any worse. Since 1998, research has been conducted with the goal of developing control techniques for these frogs. Current trapping techniques proved in field trials to be inefficient . Cultural practices or hand capture may be effective on a small scale; however, chemicals appear to be the only broad-range and cost-effective immediate method of controlling frog populations. A laboratory study was conducted to screen 35 selected 1) pesticides registered for invertebrate control in ornamental nurseries and floriculture in Hawaii, 2) human pharmaceutical and food products, and 3) surfactants as potential chemicals for E. coqui and E. planirostris frog control . During initial screening, one of two commercially available pesticides containing resmethrin , a synthetic pyrethroid, was found to cause mortality to slightly greater than 50% of tree frogs tested at registered or recommended dosage rates . No surfactants tested were found to cause frog mortality rates greater than 50%. Of the human pharmaceuticals and food products tested, food grade caffeine , applied as a topical spray, proved effective against both E. coqui and E. planirostris. The lowest concentration solution of caffeine and water that resulted in 100% tree frog mortality was a 12.5 mg/ml solution applied to tree frogs topically with a spray bottle. Following the completion of laboratory screening of potential chemicals for Eleutherodactylus control, field trials were conducted on the directed spray application of three different caffeine solutions for controlling introduced Eleutherodactylus frogs in floriculture and nursery crops in Hawaii . The directed spray application of 0.5%, 1.0% and 2.0% caffeine solutions reduced Eleutherodactylus coqui abundance in test situations on or bordering infested ornamental plant nurseries in East Hawaii. Treatment of plots with a single spray application of a 2.0% concentration caffeine solution caused a 100% decline in the relative abundance of Eleutherodactylus frogs and in the relative abundance of frogs adjusted for nightly variation in frog activity before and after treatment on control and treatment plots.

The Hawaii Department of Agriculture’s Pesticide Branch prepared and submitted to the United States Environmental Protection Agency an application for an Emergency Registration for the spray application of caffeine for localized frog control in the State of Hawaii. The U.S. EPA granted the requested registration for a one-year period beginning on 27 September 2001 with the stipulation that data were collected on potential non-target impacts and monitoring of soil and ground water contamination concerns. There has been limited use of caffeine for frog control since the granting of the EPA registration, and the Hawaii Department of Agriculture’s Pesticides Branch has had to be revise and simplify data collection requirements considered cumbersome to potential users. Reporting requirements,vertical tower planter relatively high cost in comparison to other commercially available compounds for the control of pest insects and weeds, and limited data on non-target effects have been concerns raised by potential users of caffeine for frog control. Efforts have been made by other researchers and managers to identify additional compounds for frog control. Anecdotal laboratory observations and field applications suggest that the spray application of a concentrated hydrated lime and water solution may be an additional tool for frog control. As hydrated lime and water solutions are being sprayed on vegetation versus being applied to the ground as a soil supplement, this specific use pattern may need to be registered with the U.S. Environmental Protection Agency. Standardized data collected with some level of scientific rigor showing efficacy is needed to support the legal application of this solution for frog control. Other research is being conducted to determine if natural or synthetic pyrethroid compounds could be used for frog control . In late 2000, several species of potted plants were successfully treated for frogs in test situations using short term drenching with hot water . Since the first documentation of the presence of Eleutherodactylus frogs in Hawaii, populations have spread quickly and local abundance of frogs has grown dramatically, particularly in sites with higher levels of rainfall . Though this issue has garnered significant attention in the local and national media, efforts to control the spread of the frog have been limited. Frogs have, and will continue to, affect the quality of life of citizens who live in infested sites. Citizen frustration with a lack of progress dealing with this situation in infested residential and commercial sites has been building and will continue to build. There is a realistic concern that Eleutherodactylus frogs will be accidentally transported from Hawaii to mainland states within the United States and to other countries in infested cargo. Eleutherodactylus frogs are frequently found in Hawaii at retail nurseries associated with large department and hardware store chains. Eleutherodactylus frogs have been reported in California and Connecticut and these individual frog captures have been attributed to shipments of infested Hawaiian plant products . Though Eleutherodactylus frogs may not become established in many mainland sites within the United States, Eleutherodactylus infesting shipments within Hawaii or infesting shipments going to other Pacific Islands, tropical, and sub-tropical destinations internationally is a realistic quarantine concern. There is a clear need to establish if Eleutherodactylus frogs within certified nurseries are a potential vector for plant nematode eggs , as particular species of plant nematodes are a significant quarantine issue for plant products being shipped from Hawaii to the State of California. It is worth asking the question “why hasn’t there been a significant coordinated effort mounted to deal with introduced Eleutherodactylus frogs in Hawaii?” A primary reason is the lack of techniques to control the spread of frogs; hot water drenching was not tested as a quarantine technique until late 2000, and U.S. EPA emergency registration of the spray application caffeine and water solution for local control was not obtained until late 2001. It would be very hard to enact a quarantine on a pest that impacts a major industry without the ability to control the pest in a simple, cost effective manner. Lack of funding, unclear legal jurisdiction, and bureaucratic inertia are other reasons why the spread of the frogs has continued until it is unrealistic, given current progress dealing with this issue, to believe that frogs will be eradicated from the island of Hawaii . There have been significant efforts by individual staff members of various state, county, federal and non-governmental entities and private citizens to try to stem the spread of invasive frogs and eliminate local populations. USDA APHIS Wildlife Services staff and Hawaii Department of Agriculture staff have written a plan to control and/or eradicate frogs in the State of Hawaii . Funding for this plan is the most realistic means to deal with this situation. It is clear that decisive action to eradicate an introduced pest when it has a limited distribution is far less expensive than eradicating or controlling the pest when it is broadly established. Unfortunately, the window of opportunity for effective action against the Eleutherodactylus frogs in Hawaii is becoming shorter while the problems associated with these pests will become greater as more sites become infested and local frog abundance increases in previously infested sites. Exposure of susceptible plant tissues to non-freezing temperatures below 10–12 ◦C induces a physiological disorder called Chilling Injury . There appears to be two phases in the development of CI. The first phase is initiated in the cold and could involve a change in membrane fluidity or enzyme activity .These secondary symptoms are predicated by primordial events initiated in the cold, and include a host of metabolic and physiological changes that include increased membrane permeability , increased respiration and ethylene production , uneven ripening, disease susceptibility, water soaking and surface pitting . A technique that could detect the earliest physiological changes associated with CI would foster a better understanding of the initial events leading to this disorder, and point to more effective ameliorative action. Magnetic resonance imaging is a nondestructive imaging technique, which is increasingly used to visualize and quantify fruit physiological response to endogenous or exogenous stimuli . MRI uses the magnetic properties of nuclei and their interactions with radio frequency and applied magnetic fields to produce an image . Variations in the chemical composition and integrity of cellular structures can change the movement of water within and among tissues. These changes can be detected as modifications in the relaxation times of the protons in water, which in turn alters the signals used to construct MR images .

In order to compare between experiments, central and public data storage is fundamental. The use of standardized metadata combined with open controlled vocabularies or ontologies is crucial to being able to interoperate between different data types. The “findable, accessible, interoperable, reusable” data principles are aimed at improving the data ecosystem to allow researchers to better locate and integrate data. In the phytobiome sphere, the National Microbiome Data Collaborative is a new initiative to make microbiome data FAIR and aims to use standards such as the Environment Ontology to describe environmental characteristics of samples and the microbial ecosystems embedded within them. One of those limitations surrounding reference databases is the paucity of experimentally validated data that links microbial and plant metabolism, protein function, and DNA sequence. As an example, whereas microbial genes are assigned putative functions based on sequence homology, their actual activity may deviate from these annotations leading, to incorrect interpretations and predictions of ecosystem function . Furthermore, standardized analysis is critical and can be achieved by using centrally updated, state-of-the-art software tools. KBase, the U.S. Department of Energy Systems Biology Knowledge base, has offered a public data storage and analysis dashboard that allows the generation of so-called narratives in which a dataset undergoes a string of analyses . In addition,vertical farming tower for sale large-scale field datasets are increasingly taking advantage of supercomputer resources and ML algorithms that are required to filter noise and generate sensible interpretations from billions of data points.The development of the above mentioned technologies and experimental platforms will improve our understanding of the plant–microbe–atmosphere–soil ecosystem at high spatial and temporal resolution. The combination and integrated use of the discussed tools will further provide opportunities for novel approaches to plant root microbiome research.

An example of an integrated approach is the combination of UAVs equipped with advanced imaging capabilities to study QTL or GWAS populations growing in the field. This would streamline and scale current experimental procedures, so that new genetic markers for various above- and below ground phenotypic characteristics could be identified. These, in turn, could be correlated to microbiome community profiles in roots and leaves. Another example is the combined usage of SynComs and single plants in EcoFabs for advanced root and microbe imaging resolved over space and time complemented with metabolite analysis,enabling systematic examination of the role of specific microbes and metabolites in modifying root architecture. This approach can help identify novel, specific microbial products that can be used to influence important plant traits known to affect field performance . Microbial model systems can then be engineered to produce promising compounds for tests on plants in soil. Finally, we foresee EcoPODs and EcoTrons being used for time-series experiments that span several weeks and months, possibly years, in which high-throughput omics together with continuous environmental sensor measurements can provide in depth yet broad-scale datasets that can be used for training artificial intelligence algorithms related to biogeochemical cycling in relationship to climate. Due to the many direct and indirect ties between local plant– microbe–soil–ecosystem well-being and systems-wide ecological health, technological improvements in phytobiome research are directly translatable to improvements in climate change research. The above mentioned advances in instrumentation and methodology push precision agriculture and precision phytobiome research forward and allow for improved and more sustainable crop productivity under rapidly changing and increasingly extreme climatic conditions. These advances will have impacts in food and energy security and bio-safety as well as environmental conservation and bio-remediation.Seasonally dry tropical forests are dominated by deciduous species coexisting with a small number of evergreen species . Trees withstand the dry season through two mechanisms of drought resistance: desiccation delay and desiccation tolerance .

Two important traits related to desiccation delay are leaf shedding which reduces water loss, and depth of rooting , which determines the sources of water and nutrients used by vegetation . Although previous reports suggested that evergreen species access relatively deeper water sources than deciduous species ,more recent reports suggest that access to water is more related to tree size than phenology . However, there is relatively little information regarding differences among deciduous species having different timing or leaf shedding behavior, even though it is well known that leaf senescence behavior varies greatly among tropical dry forest tree species. Flushing and leaf abscission result from complex interactions between plants and their environment; in many species, the main abiotic factors driving these processes are solar radiation, air relative humidity, vapor pressure deficit, precipitation and soil water content . Four main categories of leaf shedding phenology have been proposed by Williams et al. : evergreen species, which retain a full canopy throughout the year; partially deciduous species, which lose up to 50 % of their canopy during the dry season; semi-deciduous species, which lose more than 50 % of their canopy during the dry season; and deciduous species, in which all leaves are lost during the dry season as they remain leafless for at least 1 month. Most tropical dry forest species are thought to deploy the majority of their root systems relatively deep in the soil profile where moisture tends to be greater and of longer duration . However, in northern Yucatan the hard upper limestone layer, beginning immediately below the shallow soil, impedes root growth, limiting downward growth to crevices and rhizoliths, and the occasional cavities filled with soil material . Rock crevices allow roots to grow far deeper than they would in unfractured bedrock . Thus, in the seasonally dry tropical forests of northern Yucatan, the ability of tree species to grow deep roots and access additional sources of water beyond topsoil could be a crucial characteristic related to variation in phenology and the relative abundance of contrasting tree species. Sources of water used by trees can often be identified by comparing the isotopic composition of water from stems with potential water sources, because there is usually no isotopic fractionation of either hydrogen or oxygen isotopes during water uptake . When trees take water from more than one source, the proportion of water absorbed from each source can be calculated using isotope mixing models .

Such models were developed to cope with multiple sources and allow the input of ancillary data that are known about the system to constrain model outputs, thereby providing results that are restricted to real possibilities. Sources of water used by native trees in northern Yucatan have been studied using these approaches, and large variation in the depth of water uptake among deciduous and evergreen species has been observed . Furthermore,hydroponic vertical farm using these same isotopic approaches along a forest age chronosequence in northeastern Yucatan, evergreen trees were found to access deeper water sources than deciduous species in early succession . Thus, integrating rooting depth as a component of tropical dry forest tree strategies appears especially promising in complex karstic Yucatecan soils. Water-use efficiency , the ratio of carbon gained in photosynthesis relative to water loss during transpiration , is another key factor when considering the costs and benefits of a deep rooting system. Leaf carbon isotopic composition can be used to assess WUE in certain circumstances, and is often positively related to WUE because a high photosynthetic rate per unit stomatal conductance is usually associated with relatively low internal CO2 concentration and reduces discrimination against 13CO2 by rubisco . Although d13C has been used alone to infer WUE, its combination with analysis of isotopic composition leaf organic oxygen improves interpretation of leaf d13C values by allowing analysis of whether variation in d13C is due to changes on the photosynthetic activity or stomatal activity . When humidity increases, the isotopic enrichment of leaf water decreases, causing a reduction in d18O . Theory and empirical data also demonstrate that d18O correlated negatively with stomatal conductance . In shallow soils of northern Yucatan, Querejeta et al. showed that individuals of the same tree species differing in age had different WUE, with younger trees having greater WUE than older ones, indicating that these techniques hold promise for integrating potential differences in water sources with leaf physiological activity. This study focuses on phenological variation between two dominant tropical dry forest species in relation to the depth of water uptake. We hypothesize that the late deciduous habit in P. piscipula and the early deciduous habit in G. floribundum may be determined by their ability to take water from different sources. P. piscipula may have access to deeper sources than G. floribundum. However, due to the restrictions for root growth imposed by the hard bedrock, both species will likely extract most of their water from shallow sources. We also hypothesize that differential use of water sources is linked to key ecophysiological measures of plant performance, including the timing of leaf fall, leaf size, leaf water potential and the balance of carbon gain and water loss as interpreted by leaf stable isotopic composition.Topsoil, calcium carbonate rock layers, soil pockets and plant tissue samples were collected in three sampling campaigns: October 2007 , January 2008 and May 2008 . Topsoil, bedrock and soil pocket samples were obtained from recently exposed walls. Sampling of the lower portion of the walls was restricted by the rock materials produced during blasting events, moreover, roots were not often observed growing in this layer; thus, soil pockets and rock samples were taken only from 0 to 5 m depth.

Because topsoil was removed before rock blasting, soil samples were taken from areas up to 40 m away from the wall being sampled. In addition, gravimetric water content was evaluated by taking 20 samples from topsoil and each rock layer, and a variable number of samples from soil pockets depending on presence of these features in the wall being sampled and drying at 105 C. The eight water sources considered go down from the upper soil layer down to the ground water . Non-transpiring woody shoots were collected from five individuals of each species from vegetation within the quarry. Four stem samples of 5–10 mm width and 50–80 mm long were obtained from each tree. The same trees were sampled at each sampling campaign. No leaves or green tissue were included in the sample to avoid contamination of xylem water by isotopically enriched water that had undergone evaporation from the plant . Samples were preserved in hermetic capped vials wrapped with parafilm and stored in the freezer until processed. Groundwater samples were taken from an open well 2 km from the quarry. Water was extracted from topsoil, soil from pockets, rock and plant stem samples using a cryogenic vacuum distillation line for at least 60 min for stems and 40 min for soil and rocks . Water content of soil, bedrock and tree stems was calculated from sub-samples taken right before the water extraction and oven dried for 24 h at 80 C and 105 C . Stable isotopic composition of oxygen and hydrogen analyses were conducted at the Centre for Stable Isotope Bio-geochemistry at the University of California, Berkeley using a chromiumreactor interfaced with a continuous flow isotope ratio mass spectrometer . d18O and d2 H values were reported in delta notation relative to the Vienna Standard Mean Ocean Water . Values of substrate and stem water d18O and d2 H were plotted in a bi-variate relationship with the Meteoric Water Line determined for this region by Socki et al. , to evaluate the role of water sources across seasons. The contribution to tree water uptake from the different sources during the three sampling campaigns was calculated using Iso-Source software . This software calculates ranges of source-proportional contributions to a mixture based on the isotopic signatures of the mixture and each of the sources. Both d18O and d2 H data were used for model calculations. The data set for modeling was grouped as follows: Laja 0–200 cm was disregarded because is not a significant source of water at any time , February data were not analyzed to avoid misinterpretations due to unsampled water sources , ground water was taken out of the analyses because root systems of P. piscipula and G. floribundum were not observed growing beyond 5-m depths and, bedrock from 200–400 cm was grouped as a single source because their isotopic values were very similar. Because sampled trees were growing \1 km from where rock samples were taken, we assumed that the isotopic compositions of rock were similar in both places.

Please refer to other reviews that discussed aspects of this process . Rhizobacteria was shown to induce the accumulation of sesquiterperne synthase transcripts . Sesquiterperne, used in cosmetics and perfumery, is one component of vetiver root essential oil synthesis. The authors showed that the root associated bacteria metabolized the vetiver oil and produced additional compounds, which suggested that each distinct rhizobacteria community contributed to a signature composition of commercial vetiver oil. For two decades, it was thought that the plant pathogenic fungus Rhizopus microsporus produced the antimitotic polyketide macrolide rhizoxin. Partida-Martinez and Hertweck uncovered that it was actually an endosymbiont, Burkholderia sp., of the fungus that produced rhizoxin. The authors demonstrated that in the absence of Burkholderia, no rhizoxin was produced in the fungal culture. Transmission electron microscopy demonstrated that Burkholderia sp. was localized in the fungal cytosol . Rhizoxin inhibits mitosis and leads to cell cycle arrest, and has potential as an antitumor drug. Further investigation elucidated rhizoxin derivative structures and obtained stable analogues by inhibition of a putative P-450 monooxygenase . Ryan et al. summarized applications for bacterial endophytes, including production of bio-materials such as poly-3-hydroxybutyrate and poly-3-hydroxyalkanoate . Catalán et al. showed that diazotrophic endophyte Herbaspirillum seropedicae could accumulate 36% of its biomass as PHB and could constitute a cost-effective mean for producing bio-material.

Organic contaminants naturally biodegrade,plastic pots 30 liters except for chlorinated compounds such as polychorinated biphenyl and 4-chloronitrobenzene , which tend to be persistent and recalcitrant in the environment. Recent reports on chlorinated compound degrading rhizosphere bacteria are presented. Natural PCB-degrader bacterial populations were cultured from several plant species growing in a contaminated site. High numbers of culturable PCB-degrader colonies were isolated from roots of Austrian pine and goat willow . Most of the PCB metabolizing bacteria are Rhodococcus sp. However, the isolates were first selected based on cultivability and morphology thus possibly leading to a biased representation of PCB-degrading bacterial communities. In addition, since the PCB degradation capability of these strains was tested in liquid medium, the benefits of plant association was not addressed. Narasimhan et al. investigated the role of plant secondary compounds in stimulating rhizobacteria growth and PCB removal efficiency. The authors identified that the Arabidopsis root exudates consisted mostly of phenylpropanoids, such as flavonoids, lignins and indole compounds. Wild type and mutant Arabidopsis lines over producing flavonoids sustained higher counts of flavonoid-utilizing P. putida strain than bacteria grown on Arabidopsis mutant not producing flavonoids. More interestingly, close to 90% of PCB was degraded in soil adhering to the roots indicating that direct contact with roots and the exudates resulted in bacteria growth and bio-degradation enhancement. Genetically engineered microbes were also applied towards remediation. The PCB degradation efficiency of a recombinant strain of P. fluorescens expressing a bph operon under the control of nodulation genes from Sinorhizobium meliloti, was examined . Resting cell PCB-degradation experiments indicated that the recombinant strain metabolized different cogeners of PCB more efficiently than the Burkholderia sp. strain LB400. However, no plant-bacteria potted experiments were performed to measure PCB degradation. Böltner et al. enriched rhizosphere microbes and demonstrated that four Sphingomonas strains were capable of rhizoremediating hexachlorocyclohexane .

Potted experiments showed that 30% of lindane was removed with corn seedlings inoculated with the Sphingomonas strains. Whereas, in unplanted soil, sterile planted soil, and uninoculated-unplanted soil, less than 3% of lindane was removed. Liu et al. successfully inoculated a gfp-tagged strain of Comamonas sp. onto the roots of alfalfa as demonstrated by quantitative competitive -PCR and confocal laser scanning microscopy . The rhizosphere community shifted due to addition of 4CNB and the Comamonas strain was characterized using denaturing gradient gel electrophoresis . In outdoor potted experiments, the symbiosis enhanced phytotoxicity resistance, and removed 4CNB faster than the plant control without the strain in a 24-hour period. However, the control without the strain removed 60% of the 4CNB compared to 99% in experiment with strain inoculation. It seemed likely that if the experiment was extended beyond the 24-hour measurements, the 4CNB removal might be similar between the control and the treatment.However, certain volatile organic compounds such as trichloroethylene and BTEX are released into the atmosphere through the plant’s vascular system. The endophyte-plant interaction was used to degrade these volatile organic pollutants and minimize evapotranspiration . Barac et al. demonstrated that the genetically modified endophytic strain of Burkholderia cepacia together with yellow lupine reduced evapotranspiration of toluene by 50-70%, and reduced phytotoxicity. The same group inoculated poplar cuttings with two strains of B. cepacia, an endophyte and a soil isolate, expressing the toluene monooxygenase , and showed significantly less toluene being transpired in the poplar inoculated with the endophytic B. cepacia . In addition, the authors indicated evidence for horizontal gene transfer of the recombinant plasmid encoding the toluene degradation pathway from the inoculant B. cepacia to the endogenous microbial community, and suggested that it would be possible to eliminate selection of an appropriate endophyte since the bio-degradation gene will be transferred to the endogenous microorganisms.

However, the authors touched on the issue of low horizontal transfer efficiency but did not elaborate on solutions for the lack of control over the time scale of the transfer and recipients of the genes. The authors also did not comment on the risks associated with the spread of exogenous genes into a new environment. However, the risk caused by a gene originally isolated from the environment, such as the tom gene should be low compared to risks from synthetic genes. Moore et al. isolated endophytic bacteria from two poplar varieties that were grown at a field trial site phytoremediating BTEX in the groundwater. There were differences in spatial compartmentalization of strain localization in the root, shoot and leaves suggesting there were species-specific and non-specific associations between bacteria and plants. However, the study did not examine the reasons for the strain localization and association with the plants and whether remediation efficiency was affected by bacterial community. Heavy metal contamination is a persistent problem since the metals, unlike organic compounds, do not biodegrade. Several recent reviews address role of plant growth promoting rhizobacteria in metals remediation , and various plant bacteria systems that have been applied towards remediation of metals contamination . Removal of metals from soil or groundwater using rhizosphere bacteria and plants has been demonstrated recently. Willowseedlings inoculated with different strains of rhizosphere Streptomyces and Agromyces and the strains ability to produce indole acetic acid and siderophore was measured. No correlation was observed between IAA and siderophore production and metal accumulation. Only plants inoculated with Agromyces terreus exhibited reduced phytotoxicity due to cadmium and zinc. Metal accumulation in leaf biomass was measured . However, the data did not demonstrate a marked difference between the inoculated plants and the non-inoculated control. Perhaps,round plastic pots most of the phyto accumulated metals were immobilized in the roots where most of the rhizosphere strains would be located. No root metal concentrations were measured. Wu et al. utilized an engineered symbiosis between recombinant P. putida and sunflower plants for adsorption of cadmium. The recombinant P. putida expressed a synthetic phytochelatin, EC20, and exhibited inherent cadmium resistance. The inoculation of the P. putida strain enhanced plant growth and resulted in 40 % more cadmium accumulation from hydroponic solutions than the non-inoculated control. No field study was conducted. Ryan et al. demonstrated that a recombinant strain of P. fluorescens F113rifPCB with arsenic resistance genes and PCB degradation capabilities protected M. sativa when grown in soil supplemented with sodium arsenate. No arsenic removal nor PCB degradation efficiency were assessed with this plant-bacteria system. Since most of the metals tend to accumulate in the top 20 cm layer of the soil , grass species with high fine-root biomass providing large surface area for bacteria colonization and metal accumulation, in the top soil layer, would be ideal for remediation of metal contaminated sites. The plants serve as concentrators of metals, and further treatment of the plant biomass accumulated with metals would be required. Overall, the use of the plant-bacteria system would be more cost-effective than excavation of the soil contaminated with low levels of heavy metals. The successful demonstrations of laboratory-scale phyto- or rhizo- remediation do not always translate into adequate removal of contaminants in field-scale. The reasons include heterogeneity of the field site, unpredictability of environmental conditions, inability to sustain bacterial population and the remediating microbes being out competed by endogenous organisms. There are several reasons that plant-bacteria interactions are advantageous when applied to remediation. The plant-associated bacteria population would be more competitive than the native soil microorganisms since plant exudates provide nutrients.

Plants would act as natural pumps that draw contaminated soil pore water towards the plant-associated remediating bacteria. Rhizoremediation is an ideal strategy for cleanup of mixed-contaminants. Recombinant rhizosphere bacteria with specific genes targeting pollutants present at the site can be inoculated into plant roots. For example, Lee et al. engineered two strains of rhizobacteria with TCE degradation capability, and surface expression of synthetic phytochelatins for improved heavy metal resistance, thus enhancing the rate of TCE degradation. An excellent review by Gerhardt et al. addressed strategies to overcoming challenges of field application of phyto- or rhizo-remediation such as stressors to the microbes, complexity of the field conditions, regulatory acceptability and the use of genetically modified organisms . Research that look at the impact of GMOs on native bacteria and bacterial community changes post introduction of willow trees for rhizoremediation of PCB contamination are starting to emerge and will elucidate questions regarding the impact on the native microfauna. Studies that compare remediation efficiencies of natural attenuation, bio-augmentation, phytoremediation and rhizoremedation, under field conditions , assess bacterial colonization and activity on plant roots, and measure allometric relationships of tree trunk size and various root parameters provide invaluable information for determining field application options and evaluating contaminant removal. More extensive, long-term field comparisons of control plots with treatment plots for remediation efficiency are necessary to validate laboratory observations and to gain public and regulatory agency confidence for this promising application. Another application of plant-microbe interaction drawing increasing attention is carbon sequestration, where atmospheric C is deposited as plant root material, incorporated into the soil microorganisms and soil organic matter. It is hypothesized that increase in CO2 leads to an increase in rhizodeposition, and wider C/N ratio thus retarding decomposition . Several reviews are dedicated to presenting methods for measuring CO2 fluxes in different soil compartments , mechanism of root carbon stabilization , effects of elevated CO2 on below ground carbon storage , and carbon sequestration by roots . This nascent field is still at the exploratory stage where most of the research is focusing on understanding the effects of elevated CO2 levels on microbial community, below-ground plant material production, and biomass decomposition, as well as land management practices and plant species on the long term potential of C rhizodeposition. Several researchers explored the possibility of different land management practices to enhance C sequestration. Bailey et al. compared ex situ incubations of soil samples from five different ecosystems . The restored prairie samples had the highest total soil carbon and also the highest fungal-to-bacterial activities . The authors asserted that increased F:B ratios correlated with higher amount of carbon stored in the soil. Also, that invasive land management decreased fungal biomass and thus the carbon stored in the soil. Soil samples were collected and CO2 respiration experiments were conducted in a laboratory setting over a 6-hour period, thus the measurements might not be representative of field conditions. Heinemeyer et al. described contradicting results in their in situ study of the ability of Lodgepole pine associated mycorrhiza to store soil C over a period of 1- year, where the fungus was thought to return plant surplus C directly back to the atmosphere. Verburg et al. compared the net ecosystem carbon exchange between the atmosphere and two experimental non-native cheat grass varieties in a 2-year study in the Desert Research Institute’s Ecologically Controlled Enclosed Lysimeter Laboratories. They showed that fertilization increased C uptake initially, however, C loss through soil respiration was also enhanced. Bazot et al. found similar results, at a grassland ecosystem of Free Air CO2 Enrichment is Eschikon, Switzerland, where increased N supply to the plants enhanced allocation of fixed C to the shoots and reduced below ground carbon allocation and rhizodeposition.

All of the farms in our survey follow agroecological production practices which include a focus on building soil health through, most commonly, cover cropping, compost application, and no-till practices. These practices produce synergistic effects of adding fertility to the soil through organic matter amendments and boosting water holding capacity. Soil building practices are a response to the impetus to remediate toxins present in urban soils , a prerequisite to intensive cultivation and unique consideration of the urban farm environment. Overall, production practices on our urban farms seek to conserve, protect and enhance natural resources. Our survey respondents described numerous strategies for enabling diversified, intensive production of fruits, vegetables, and other agricultural products. These strategies span both short and long-term, from planting in raised beds with imported soil, to building soil health in situ via heavy applications of compost, manure, and cover crops for several years leading up to vegetable crop production. There is a growing interest in using no-till practices, which are among the suite of practices associated with “carbon farming” for enhancing soil carbon sequestration . This illustrates a synergistic opportunity for urban food policy and urban climate policy, showing where urban food production and city Climate Action Plans 4 can converge and generate mutual support . Farmers are also engaged in innovative resource recycling and resource use efficiency and other strategies to enhance resilience such as installing rainwater catchment systems in concert with swales and soil health practices to optimize use of this scarce resource.

Farms are planting native flowers and shrubs to attract beneficial insects,fodder sprouting system rather than purchasing chemical inputs for pest management. From a city planning perspective, the impetus to remediate storm water overflows and maintain corridors for essential pollinators are two priorities that can be met through incentivizing and planning spaces for UAE.East Bay urban farms reflect multiple scales and forms of diversity including agrobiodiversity, organizational and participant diversity, diversified sources of capital, labor and land arrangements, as well as diversified modes of exchange. Diversity among operations technically doing the same thing- growing food in cities- signals the fluid, flexible, peripheral, and at times revolutionary nature of urban food production spaces, which may conflict with or resist the institutional, political-economic status quo . Urban farms rely on diverse revenue streams from their diversity of activities beyond sale of produce. These activities, including educational services and community events, are important to elevate in policy conversations. Valuing and therefore protecting urban food production spaces requires thinking differently about them in a context like the San Francisco Bay Area. One stakeholder suggested considering urban farms as museums, providing essential cultural and educational offerings to city residents . The quality of the food and the value of the education, health, and community building, are strong arguments for including urban farms in an urban-agroecological framework for city planning and efforts to improve CFS. The diversity of land access agreements and labor sources used by urban farmers in the East Bay underscores equity considerations in urban agroecological transitions. Farms rely heavily on donated land and volunteer and citizen labor. Even 50% of the for-profit enterprises reported relying on volunteer labor, speaking to both the precarious economics of running an economically viable for-profit food production business in the city, and the interest among young people and aspiring farmers in gaining agroecological cultivation skills through arrangements where they donate their labor free of charge. Volunteer labor substitutes for revenue to a certain degree, allowing farms to exist and distribute food informally without needing to generate much revenue or provide many jobs.

In the UA literature, reliance on volunteer labor comes under criticism for being a product of the “neoliberal city,” where responsibility for action falls to the individual rather than the state, and the equity concerns around who is able to volunteer their time are problematized . By reporting the common use of volunteers on East Bay urban farms, we do not seek to promote or valorize this practice, but rather recognize it as a necessary interim step occurring in our study context in the absence of dramatic local government intervention or radical reforms to address community food insecurity: those who are willing and able are participating through civic engagement in urban farms to produce, harvest and distribute healthy food to those in need. Many volunteers are retired or recent graduates, seeking opportunities to contribute meaningfully to their communities. The volunteers we have communicated with generally report positive experiences and enjoyment from their time digging in the soil. Despite this, it is vital to acknowledge that the goals of food sovereignty underlying agroecology, especially the Nyéléni declaration, imply that food producers need to be able to earn a living to secure other basic needs, farm revenue is needed to sustain operations, and community members need to be able to pay. However, in cities where wages are stagnating relative to the cost of living and the right to remain is under threat to rising property values and rents , affordability of food impacts growers and consumers alike. The critique in the literature against charity in the food system is that the dependence on charitable donations in the food space are a patch for the destructive neoliberal state, which has shifted the burden of social well-being onto the nonprofit sector. Heynen, critiquing the depoliticization of hunger and poverty through charity, asserts that “[c]harity, however well intentioned, has become the means by which the welfare state was successfully rolled back” . At the same time, in exploring the radical democratic politics of groups like Food-Not-Bombs, Heynen describes the kind of anarchist philosophy of mutual aid and cooperativism through food sharing that we see in the East Bay agroecosystem.

Farms are not just distributing food to the hungry in hidden basements or exploiting free labor, but engaging in highly visible work, inviting those who visit or consume farm outputs to work, cook, learn, teach, share and get political. The reality is that growing food in cities has particular challenges, increasing the costs of farming on top of issues already outlined regarding the cost of land and labor . We find that the importance of donated money and time to further the anti-hunger and advocacy efforts of farms is not counter to the transformational goals of AE broadly or UAE in particular. In this way, we seek to nuance the premise that volunteer labor is universally problematic and counter-productive to radical food system reform efforts, aligning instead with some urban agroecological scholarship that argues for improved work-life balance through living wage jobs that afford more people opportunities to pursue hobbies and interests and volunteer their time supporting community efforts that align with their values . Pimbert outlines three dimensions of urban agroecological transformation that are needed, including economic, with new forms of organization and relocalized wealth production as well as “creation of free time for citizens to shape and re-govern urban spaces” . Volunteerism has a place in a transformed, equitable, environmentally sustainable local food system,microgreen fodder system although reliance on it as the primary source of labor is undesirable. Our findings around labor in particular stand in contrast to the often-referenced benefit of urban agriculture as a job creation tool . At least in the current political economic landscape of the East Bay, urban farms do not generate enough economic revenue or city investment in order to hire many full time positions; this remains a goal of many operations and opportunity for policy intervention, especially with respect to enhancing the resilience of urban agroecosystems to economic disturbance.Farms in our case study display a strong focus on reducing hunger and promoting food equity, namely through culturally appropriate diets, and the emphasis on human and social values. Due to the plethora of produce going home with volunteers, circulating at neighborhood crop swaps, and gleaned or harvested by community members that is not weighed and tracked before it is consumed, it is understandably difficult to quantify the “food security” impacts of urban agriculture . While food security may be difficult to quantify, it is nevertheless being addressed by urban farms in unique ways . In school gardens, for example, produce that is not used for classroom cooking demonstrations sometimes goes home with students or families excited to find culturally relevant crops growing in their neighborhood. Supporting healthy, diversified and culturally appropriate diets are an important element of agroecology. The diversity and quality of produce grown, especially when it is an item that might not otherwise be available to a family in a “food desert,” contribute greatly to the value produced on urban farms. One farmer interviewed described how one school garden site serves students from Hispanic, African American, Middle Eastern, Asian, and Eastern European families. The garden teacher spoke about the diversity of crops relevant to various cultural food traditions; for example, the chayote plants were of particular interest to Latinx students excited to bring them home to their mothers, while African American students eagerly collected bunches of collards, and Middle Eastern mothers came to the garden in person to collect fava beans and figs. In this way, urban agroecology contributes to food security and nutrition as well as biodiversity. It also serves to reaffirm cultural identity and a sense of place for immigrant and refugee families.

Agroecology places a strong emphasis on human and social values, such as dignity, equity, inclusion and justice contributing to improved livelihoods of [urban] communities . Our study demonstrated that the majority of farm respondents placed food security, education, and environmental sustainability above profit, sales and yield. Forty percent of respondents self-identified as “Educational” farms, and most others offer educational workshops and demonstrations as part of their focus on horizontal knowledge-sharing.The majority of our study respondents were also women. As a grassroots movement, urban agroecology can empower women to become their own agents of change.Our results suggest the opportunity to reconceptualize and refocus the urban food policy discussion in U.S. cities around urban agriculture in a way that includes and values their social, educational, and cultural services. Urban farms are recreational and cultural heritage sites bearing comparison to public parks and museums, while also producing invaluable healthy food in areas that most need it. They provide important respite, social connection, and stress reduction to urban residents, often particularly in need of peaceful spaces. In the words of one farmer, “Urban farms can be havens of peace, health, and community, but it requires heavy involvement and advocacy from those communities for the long term in order to be successful” . Agroecology calls for responsible and effective governance to support the transition to just, equitable and sustainable food and farming systems. In an urban environment, this requires the creation of enabling policies that ensure equitable land access and producer control over access to land, especially among the more vulnerable and historically marginalized populations. Land access is expressed most frequently as an obstacle to scaling urban food production by survey respondents, and it is certainly more of a challenge for lower-income and minority groups interested in cultivating their own “commons” . There are examples among our East Bay survey respondents of collective governance at the farm and community level, such as one farm site which is owned cooperatively by three non-profit organizations that collectively serve minority and formerly incarcerated populations, aspiring beginning farmers, and the local community through a cooperative goat dairy, fruit tree nursery, and annual vegetable production plots. City and county governance bodies have an opportunity to strengthen the resilience of urban agriculture operations and opportunities for farmer collaboration by providing subsidies and incentives for social and ecosystem services. City-level efforts to compensate or recognize farmers for ecosystem services such as soil remediation and carbon sequestration, for example, are not yet realized. Further examples of responsible governance from our data include recommendations for public procurement programs to source food from aggregated urban produce . Our respondents are engaged in circular and solidarity economies, key features of agroecology, including bartering, sharing, and exchanging resources and produce with those in their social networks. They are also interested in collaborating in a localized effort to strengthen the link between producers and consumers by aggregating produce and sharing distribution .

In the dark, the STM cell densities were similar to that of WT M. loti . However, high intensity blue light prevented the growth of WT M. loti, but after 72 h, the growth of the STM strains was partially restored. The A610 values of the WT and STM strains were significantly different at 144 h . We also measured growth parameters in plants inoculated with the STM strains and exposed to blue light. For the shaded roots, no significant difference between the WT M. loti and STM strains was observed in shoot length , root length , or nodule number . For the unshaded plants, the nodule number of plants inoculated with WT M. loti was 40% of that of the shaded roots. For plants inoculated with the STM strains, the nodule count in unshaded roots was >65% of the number for the shaded roots . Taken together, these results suggest that both sets of rhizobial blue light receptors are required to inhibit nodulation.Because blue light perception by both the root and rhizobia leads to an inhibition of nodulation, we inoculated RNAi plants with the STM mutants under the same growth conditions used in Table 1 and assessed nodulation status 21 dai. As shown in Table 2, root nodules were formed in cry-RNAi plants with M. loti or in EV plants inoculated with STM strains, supporting the results shown in Table 1 and Figure 5H. The nodulation of RNAi-targeted Ljcry1A and Ljcry2B plants when inoculated by mllpp1 or mlphl was increased compared with EV plants inoculated with mllpp1 or mlphl. In RNAi-targeted Ljcry1A and Ljcry2B plants inoculated with the mllpp1 strain or the mlphl strain,dutch buckets nodulation was enhanced compared with EV plants inoculated with a WT M. loti strain .

The increase in nodule number was additive, indicating that the inhibition of nodulation by light is caused by blue light perception by both the host plant roots and rhizobia.Previous studies showed that nodulation in Trifolium subterraneum L. Woogenellup is either inhibited or not inhibited in the light, depending on the rhizobial strain . On the other hand, in Pisum sativum and P. vulgaris , root nodule numbers were decreased when roots were exposed to light. Our data support both of these seemingly disparate conclusions because, in L. japonicus, inhibition of nodulation by light is caused by blue light perception by both the host plant roots and rhizobia. Bonomi et al. reported that a short time exposure of rhizobia to white light prior to inoculation enhanced nodulation in Pisum sativum. In our study, a light treatment was given after inoculating with rhizobia, which may explain the differences in results. Using a split-root system, we found that the number of nodules formed on unshaded root systems was significantly lower compared with shaded root systems and even on a single root, which showed that nodule inhibition was not systemic . Recently, Chen et al. reported that the shoot lengths of Oryza sativa seedlings were inhibited by blue light and, in Lactuca sativa, shoot biomass was also decreased under blue light . Furthermore, shoot and hypocotyl lengths of lettuce, radish, and pepper decreased in response to increasing the quantity of both blue light and red light . Although the number of nodules did not differ between the red and blue treatments, the shoot lengths of blue light–treated plants in shaded L. japonicus roots were significantly decreased compared with red light–treated plants . In lettuce, root growth was decreased by blue light irradiation . In contrast, in rice, root growth was not different between roots either exposed to blue light or not . Our observation of root growth was that no large difference occurred whether the roots were shaded or unshaded in L. japonicus .

Taken together, these results show that overall shoot growth is inhibited by blue light irradiation, whereas the effect of blue light irradiation on root growth depends on the plant species. Under both white and blue light, a significant difference was seen in the shoot lengths of inoculated plants that had their roots shaded compared with uninoculated plants grown under the same conditions. These data lead us to conclude that the shoot growth of shaded roots was positively affected by the presence of root nodules. We also found that the inhibition of nodulation in white light was caused by its blue component and that root nodule number reduction under blue light was not related to the lack of a carbon source . Similarly, for isolated P. vulgaris roots, blue light inhibited nodulation more than red light or white light did . Our data are thus consistent with the absence of a significant effect of red light on root nodule number in Pisum sativum and, also, show that number of root nodules following blue light treatment was significantly reduced . Further implicating the key role of blue light, we found that nodulation was significantly increased in roots depleted of cry1A and cry2B . Because the expression of all the Ljcry1 and Ljcry2 genes was down-regulated in both cry1A and cry2B RNAi plants, we conclude that blue light inhibits nodulation via one or both cryptochromes 1 and 2. On the other hand, reduced expression of the three phototropin genes in L. japonicus MG20 had no effect on nodulation, indicating that blue light perception affects nodule development in the roots of this legume through cryptochromes and not phototropins. We also demonstrated that M. loti growth is inhibited specifically by blue light and that the rhizobial blue light receptors LPP1 and photolyase are involved in its suppression . Moreover, the growth of the STM strains under blue light did not lead to a full recovery of the levels of the dark-grown strains , which means that both rhizobial photoreceptors are involved. Finally, when the Ljcry1A and Ljcry2B-targeted RNAi plants were inoculated with the STM strains, an additive increase in nodule number was observed .

These results thus demonstrate that the inhibition of nodulation by light is caused by blue light perception by both host plant roots and rhizobia. With regard to nodulation, the number of infection threads per plant in L. japonicus is severely reduced in plants grown under blue light . Nodule size classes also differed between the shaded and unshaded plants. In the latter, a large population of smaller-sized nodules and only a few large ones developed on the illuminated roots compared with the shaded controls , suggesting that the inhibition of L. japonicus MG20 root nodulation under blue light results from one or both reduced or abnormal infection. Nodule fresh weights are also lower in the illuminated plants. At least two hypotheses can be invoked to explain these results. The first is based on the fact that light perception reduces rhizobial growth, resulting in insufficient size of the rhizobial population needed for inducing the earliest stages of nodule formation. This scenario is consistent with the studies on Rhizobium leguminosarum bv. viciae 3841 in which white light was reported to interfere with the synthesis of bacterial surface determinants that are needed for competent infection and nodulation of pea . Inhibition of attachment to root surfaces might also result in fewer infection events with a concomitant reduction in nodule size and weight,grow bucket which we observed. An alternative or additional mechanism is based on the observations of Grobbelaar et al. that light perception affects nodule development independently of the early stages of attachment, root hair deformation, and infection thread formation, which would mean that darkness is not required for nodule initiation but for nodule organogenesis. Our data are also consistent with this mechanism because we observed a larger size class of nodules in the 0.5- to 1-mm range and a reduction in the group of 1-mm-sized nodules in the shaded vs. unshaded roots. In addition, the fresh weight per nodule under shaded conditions was greater than when the roots were illuminated. However, the fact that fewer mature nodules were observed on unshaded roots could solely be due to the abortion or lack of the initial nodulation attempts under blue light. We are currently examining which of these two mechanisms is more likely to explain the downstream effects of blue light in the M. loti–L. japonicus symbiosis. Higher plants developed avoidance mechanisms to survive under conditions of biotic or abiotic stress. It is well-recognized that plants utilize light as the trigger for these responses. For example, in the case of anthogenesis and light-induced germination, the plant determines the timing by light perception through phytochromes and cryptochromes to increase the possibility of survival of descendants . In root negative phototropism, plant roots bend opposite to the light direction via phototropins . In the shade avoidance syndrome, plants grow taller or bend toward the light to avoid shade through the action of phytochromes . In shade avoidance in the case of root nodulation and mycorrhization, host plants suppress these interactions under suboptimal light conditions through the phytochrome system to avoid expending energy . Therefore, we can consider inhibition of nodulation by light as one of several avoidance responses plants use to conserve energy in response to environmental stress. Sun et al. reported that the light from the surrounding environment of herbaceous plants enters the interior of the stem. Then, via an internal light-conducting system, light of wavelengths between 710 and 940 nm, which includes far-red light, is conducted axially and efficiently from leaves and stems toward underground roots, whereas lower wavelengths of light , such as blue, are not.

These authors also stated that this internal light environment might be of crucial importance for the phytochrome-regulated metabolic activities of plant stems and roots. Because the phytochrome system is involved in the response to FR light conducted to the root, another system, such as blue light perception, may be utilized for avoidance mechanisms in response to stress. Clearly, more studies are needed.As nanotechnology rapidly evolves,engineered nano materials are entering air, waters, soils,and sediments where they could adversely affect organisms to ecosystems.Actual environmental impacts of ENMs have not been documented, and there are uncertainties about the potential for, and how to evaluate, impacts.ENM ecotoxicology elucidates hazards and their mechanisms.The scope overlaps with conventional ecotoxicology, although ENMs are particulate and diverse,with varying cores, native or acquired surface chemistries,conditional agglomeration or dissolution,and size- plus composition-dependent electronic properties,affecting their reactivity and biological interactions.Focusing ENM ecotoxicology invokes exposure scenarios relevant to ENM production,use,disposal,and product release .Scenarios consider environmental fate and transport,bio-availability,and ENM uptake into ecological receptors.In conventional chemical toxicology, observed and perceived exposures often diverge. In ENM ecotoxicology, water is emphasized, while soil and sediment impacts have received less attention.Where do exposures occur? What ENM forms and quantities are involved? Which ecological receptors are affected?Which local exposure conditions prevail? As with conventional chemical risk assessment, such questions unite hazard and exposure assessments.Standardized test regimens do not derive from scenarios, since ENM test conditions are predefined for standardized end points.Ideally, ENM hazards are studied at realistic exposures for ecologically relevant receptors. An example would be studying real soils under controllable yet realistic conditions, that is, in greenhouses or lysimeters.However, requiring absolute realism in all ENM ecotoxicology would pose scientific challenges associated with measuring ENMs analytically in environmental media; measuring toxicity across a representative range of environmental conditions; characterizing environmental ENM forms and their transformations so that toxicity is measured for representative materials; ENMs altering physical or chemical exposure media conditions; and few efficient approaches for estimating hazards and exposures necessary to evaluate risks before ENM products develop. Another challenge is internal to the scientific community: multiple dissimilar working definitions of environmental relevance intruding on scholarship, including peer review.Environmental relevance remains undefined, leading to categorization of research around a few selected concepts.Previously, over 600 published studies were examined to compare modeled or measured environmental concentrations of ENMs versus concentrations administered in ENM ecotoxicity assessments.The study found nominal concentration disparities, but also infrequent testing at low ENM concentrations. The study noted uncertainties in ENM exposure modeling, and that other toxicity testing conditions beyond ENM concentration including aqueous chemistry, biological receptor, system complexity, and ENM form relate to real-world conditions. However, the study did not establish what constitutes environmental relevance in the ecotoxicology of ENMs.

NA has been shown to be an important divalent metal chelator and is involved in metal transport and homeostasis in plants.Previous evidence also showed over expression of genes involved in NA synthesis increased nickel tolerance in tobacco and Arabidopsis thaliana.Liao et al. revealed that NA and histidine have the highest binding constants for Cu2+ in chicory and tomato.Thus, the observed up-regulation of NA and histidine in leaves is a possible Cu2+ detoxification mechanism for lettuce plants. Phytochelatins and metallothioneins have been shown to play an important role detoxifying excess Cu2+.PCs are synthesized from reduced glutathione in a transpeptidation reaction.In addition, GSH is involved in a plethora of cellular processes, including defense against ROS,and sequestration of heavy metals.Glycine and glutamate are the main constituents of GSH. The observed elevated levels of glycine may indicate GSH and PCs are upregulated to detoxify excess Cu2+. Pidatala et al. also observed that glycine and glutamate increased in response to Pb. Increasing Tolerance. In addition to chelating copper, lettuce plants must employ other strategies to increase their tolerance to higher copper levels. The level of spermidine and putrescine, which are important polyamines, were elevated in all treated plants . Previous studies showed that putrescine and spermidine play an important role in plant stress response to diverse environmental stresses by acting as antioxidants to scavenging free radicals.Therefore, elevated polyamines may contribute to enhanced tolerance of lettuce to copper. In this study,hydroponic nft channel ethanolamine levels were increased in all NP treated plants. Rajaeian et al. suggested that EA increased salt tolerance of tobacco plants by stimulation of antioxidative responses.

Kogan et al. showed pretreatment with ethanolamine enhanced the tolerance of Helianthus annuus L. to salt stress.Elevated K+ is another possible important tolerance mechanism. Increased K+ in plants can lower ROS production by reducing activity of NAD oxidases and maintaining photosynthetic electron transport.K+ has been implicated in regulating plant stress responses. Guard cells take up mainly K+ .It has been demonstrated that K+ triggers the stomata to open. In guard cells of open stomata, K+ was 2−4 times higher, malic acid 6 times higher, and citric acid 3 times higher, compared to closed stomata.It has been repeatedly hypothesized that organic acid synthesis would accompany stomatal opening.The accumulated K+ in guard cells may promote stomata opening. Increased stomatal opening/ transpiration is expected to promote photosynthesis and thereby increase plant growth.Borowski and Michalek showed that foliar application of potassium salts to spinach leaves resulted in more intensive gas exchange in leaves and, as a consequence of that, increased leaf yield. Antioxidant Defense. As reported before, Cu generates ROS in cells through the Fenton reaction. Our study also showed ROS was triggered by Cu2 NPs . ROS scavenging enzymes and antioxidant molecules are a common plant response to ROS stress.Phenolic acids and ascorbic acid are important low molecular antioxidants.Previous studies indicate ROS stress increases accumulation of antioxidant molecules.Up-regulated low molecular weight antioxidants can serve as scavengers of free radicals to protect plants from oxidative damage.Interestingly, our results showed the levels of three phenolic compounds and dehydroascorbic acid, which are important antioxidant molecules, were significantly decreased in all nanopesticidetreated lettuce leaves . GABA levels also decreased. It is possibly that biosynthesis of these metabolites was activated in response to ROS stress induced by Cu at an early stage of defense. However, since the stress was sustained for one month, this induced the imbalance between ROS and the antioxidant defense system.

Therefore, the antioxidant system was impaired due to the continuously generated ROS and limited ability to regulate them.Exposure to copper-based nanopesticides is likely to increase. For lettuce, exposure via foliar application, as intended, did not result in visible leaf damage. In fact, in several cases leaf biomass increased significantly. Cu2 nanopesticides can clearly enter stomata, even when aggregated. We demonstrated that Cu was translocated to the roots, although almost all the Cu mass was accumulated in leaves. Despite no visible damage, metabolomics revealed some significant changes in levels of amino acids, organic acids, carbohydrates and other important metabolites, particularly in leaves. The effect in roots was much smaller. The plants may be up-regulating some of these metabolites to increase the tolerance of plant to Cu2 nanopesticide. Metabolomics can be used as a sensitive and powerful tool to understand the response of plants to nanoparticles at a molecular level. However, it is not clear if the observed metabolic changes were entirely induced by Cu ions or if NPs also contributed. Future work should address how best to use Cu ions as a control at the same level of bio-availability, to better distinguish the contribution of nano-Cu from that of the Cu ions to the observed metabolomics changes.Micro-irrigation has become the optimal standard for irrigation and fertigation of horticultural crops in Australia, due to increased water scarcity and higher costs of fertilizers over the last decade. Intensive fertigation schedules have been developed to increase yield and quality of many permanent horticultural crops, including mandarin. This combines drip irrigation and fertigation to deliver water and nutrients directly to the roots of the crop, with the aim of synchronizing the applications with crop demand and maintaining the desired concentration and distribution of ions and water in the soil . The overall aim of these interventions is to develop an irrigation and nutrient management program that increases yield and fruit quality, while reducing leaching. The fundamental principle of drip fertigation is to apply water and nutrients regularly to a small volume of soil at a low application rate and at a high frequency to closely meet crop demand .

However, the potential for movement of water and mineral nutrients, especially nitrogen , below the root zone and into the ground- and then surface-waters using these approaches is still high. This is due to a number of factors: amount and intensity of precipitation, the large amounts of water and nutrients being applied, the limited capacity of roots to take up these nutrients, and to the ability of irrigators to manage drainage and hence leaching. Citrus is one of the important horticultural crops being grown under intensive fertigation systems in Australia. The vast majority of citrus plantings are oranges , with the rest split between mandarins , lemons and limes , and grapefruit . About 75% of the Australian citrus industry is located in the Murray-Darling Basin, utilising the lighter-textured free-draining soils adjacent to the Murray, Darling and Murrumbidgee rivers,nft growing system and thus potential off-site effects of poorly managed fertigation may have wider implications. Irrigated horticulture has, in general, been identified as the major source of nitrogen in drainage waters in the Murray Darling Basin . A significantly high nitrate level has been reported in drainage water and soil solution under grapevines in the Murray Darling Basin. These values are significantly higher than the Australian environmental trigger value for nitrate . Leaching of nitrates from soils under perennial horticulture may pose a potential threat to groundwater. The main sources of nitrate in mandarin production are mineral fertilizers. Nitrate is removed from the soil by plant uptake or through decomposition by micro-organisms in the process of denitrification. In well-aerated soils typical of this region, denitrification is often negligible because of a lack of favourable conditions . Nitrate, being an anion, moves freely in these mineral soils, and hence has the potential to leach into groundwater and waterways if fertigation is not well scheduled . Several researchers have reported substantial leaching of applied N in citrus cultivation under field conditions . Syvertsen and Jifon found that N leaching was higher under weekly fertigated orange trees than under daily or monthly fertigated trees. Syvertsen and Sax reported that increasing the number of fertigation events could significantly reduce N leaching. However, they observed 38–52% leaching of N from fertilizer, and the nitrogen use efficiency ranging between 25% and 44% in Hamlin orange trees. Other researchers have reported that nitrate accumulates toward the boundary of the wetted volume for most combinations of drip emitter discharge, input concentrations, and volumes applied. These studies suggest that there is a need for efficient tools, capable of describing and quantifying nitrate leaching, as well as nitrate uptake by crops, which in turn would help in designing and managing drip irrigation systems and achieving a high N fertilizer use efficiency, thereby limiting the export of this nutrient as a pollutant to downstream water systems. In addition to nitrate leaching, salinity is also an important factor influencing the sustainability of the citrus production worldwide, as citrus species are relatively salt sensitive. The reported value of the average threshold electrical conductivity of saturation extract and slope for oranges are 1.7 dS m 1 and 16%, respectively . Salt damage is usually manifested as leaf burn and defoliation, and is associated with accumulation of toxic levels of Na+ and/or Cl in leaf cells. Under drip irrigation there are many factors influencing the distribution of soil water and salts, and hence the water use efficiency , such as water quality, dripper discharge rate , irrigation water depth , and irrigation frequency . Simulation models have been valuable research tools in studies involving complex and interactive processes of water flow and solute transport through the soil profile, as well as the effects of management practices on crop yields and the environment .

HYDRUS-2D has been used extensively in evaluating the effects of soil hydraulic properties, soil layering, dripper discharge rates, irrigation frequencies, water quality, and timing of nutrient applications on wetting patterns and solute distribution . Although these studies demonstrate well the importance of numerical modelling in the design and management of irrigation and fertigation systems for various crops, most studies involving salinity and nitrate leaching are based on either an analysis of hypothetical scenarios, or are carried out for annual crops. Hence, there is a need to carry out modelling studies for perennial horticultural crops such as mandarin, using experimental results from field studies involving modern irrigation systems such as drip. The objectives of the present investigation were to evaluate water, salt , and nitrate movement in soil below young mandarin tree using HYDRUS-2D, and to evaluate various irrigation and fertigation strategies for controlling deep drainage and nitrate leaching, whilst maintaining soil salinity below the threshold for mandarin. This approach will help us understand the best irrigation and fertigation management practices to be adopted in future practical applications, with the goal to increase root water and nutrient uptake.The field experiment was conducted at the Dareton Agricultural and Advisory Station , located in the Coomealla Irrigation Area, 3 km from Dareton and 10 km from Went worth in New South Wales . The research station forms part of the Sunraysia fruit growing district of NSW and Victoria located in the Murray Darling Basin. An experimental site with an intensive fertigation system, consisting of various mandarin varieties budded onto a number of root stock varieties , was established in October 2005. The trees were planted at a spacing of 5 m-2 m. The actual monitoring and measurements were initiated in August 2006. The trees were managed and fertilized following current commercial practices, although the amounts of applied fertilizer varied. The soils of the site are alkaline , with red sandy loam from the surface to 90-cm depth, and loam below . The total organic carbon content is very low in the first 30 cm, and below 0.25% in the remainder of the root zone. The climate is characterized as dry, with warm to hot summers and mild winters. The total rainfall during the experimental period from 21 August 2006 to 20 August 2007 was 187 mm , which was slightly below average for the area.Mild frost conditions occur during the winter months. Weather data were collected from an automated weather station located within the research station.The HYDRUS-2D software package was used to simulate the transient two-dimensional movement of water and solutes in the soil. This program numerically solves the Richards’ equation for variably-saturated water flow, and advection–dispersion equations for both heat and solute transport. The model additionally allows specification of root water uptake, which affects the spatial distribution of water, salts and nitrate between irrigation cycles. The solute transport equation considers the advective–dispersive transport in the liquid phase, as well as diffusion in the gaseous phase.

Of special interest have been further investigations of alterations in this kinetic pattern that occur as the membrane complex is gradually disassembled. The disappearance of the period 4 behavior is a sensitive indicator of the loss of ability to. store oxidizing equivalents; however, under many circumstances rapid rereduction of the primary electron donor continues to occur by components that have yet to be identified. Photo system I reaction centers behave qualitatively differently from those of PS II. Instead . of the large increase in fluorescence yield seen upon closing PS II reaction centers through illumination or chemical reduction of the electron acceptors, it has been difficult to demonstrate any contribution to this variable fluorescence from PS I. Now, using isolated and enriched PS I reaction center preparations we have seen a small decrease in fluorescence yield associated with closing the reaction centers. By contrast with the behavior of PS II, this variable fluorescence’ occurs’ upon oxidation of the primary electron’ donor chlorophyll, P700. These results for both· PS I and PS II are’ consistent with, but· do not prove, a model in which the increased fluorescence yield comes from the reversal of the primary process leading to the repopulation of the chlorophyll excited singlet state following charge recombination. We are carrying out time-resolved fluorescence decay measurements to try to define the mechanism more precisely. !.r Model studies were carried out on electron donor/acceptor complexes synthesized by covalent linkage of a photo-excitable ruthenium complex to a benzoquinone. The linker consisted of oligo peptides containing zero to four prolines.

By monitoring the effect of increasing Si the physical separation of the electron donor from the electron acceptor using fluorescence yield and time-resolved fluorescence relaxation,ebb and flow tray we could follow the competing electron transfer in the range from 10-9 to 10-6 sec. It is clear from these studies, carried out by Miller Research Institute Fellow Dr. Kirk Schanze, that electron transfer can occur rapidly and efficiently across longer non-aromatic bridging groups than had been suspected. In ongoing studies using kinetic EPR spectroscopy we will investigate the lifetime of the charge-separated species that simulates the initial stages of photosynthetic charge separation. Much evidence has now accumulated that the storage of the four oxidizing equivalents needed to produce 02 from water photosynthetically occurs in a complex containing four manganese atoms. The nature of this complex, which is presumed to involve interaction with a protein environment, is still unknown. Even the kinetics is in dispute. Optical spectroscopic changes have been interpreted to result from three successive one-electron oxidations from Mn+3 to Mn+4 prior to O2 release, whereas EPR, X-ray spectroscopy and other approaches favor a mechanism with different oxidation state changes for the three preliminary steps. We have used X-ray absorption and EXAFS, in conjunction with low temperature EPR measurements to investigate these questions. Absorption edge energy studies on Mn are suggestive that, So, the most reduced state of the complex~ has the .same oxidation state for Mn as Sl, where one electron has been removed by the PS II reaction center. Presumably this electron has come from an associated ligand that does not directly involve Mn. The second step, from Sl to S2, definitely involves Mn oxidation, perhaps by two equivalents transferredone to the reaction center and one to rereduce the ligand. The third step, from S2 to S3, again . involves no apparent change in the Mn oxidation state, but the final step S3 to S4 and back to So returns the complex back to the fully reduced state. The state S4, which should be the most oxidized but is unstable, has not yet been trapped for investigation.

EXAFS measurements give information about the local coordination environment of the Mn in the water splitting complex. Two of the three shells previously identified for Sl are attributed to 0 or N atoms, and the third shell to Mn atoms. Further enhancement of signal quality has revealed a fourth shell, apparently also resulting from Mn. A possible model involving a distorted cubic arrangement with Mn and either 0 or N atoms at alternate comers has been proposed by Brudvig and Crabtree [Proc. Natl. Acad. Sci. USA 83, 4586-4588 ] to account for these and other spectroscopic data. We find that the coordination in Sl does not differ significantly from that in S2, by the EXAFS criteria; however, analogous investigations of S3 are still under way. Previous proposals that sulfur or chlorine atoms are involved as Mn ligands are not consistent with the EXAFS data. We have also shown that resolved super hyperfine structure of a low temperature EPR signal associated with S2 does not result from interactions with CI nuclei in the immediate coordination environment. Until the successful isolation of the water-splitting complex has been accomplished, further spectroscopic studies such as these will be our principal source of information about the enzyme that accomplishes this important aspect of photosynthesis. The information gained is expected to be valuable for designing bio-mimetic systems for using light to split water into hydrogen and oxygen as alternative energy sources. It is of interest that the genes for Crt biosyntheis are regulated differently from the other photosynthetic’ genes in response to light intensity and O2 • We have found that under steady-state high O2 the level of mRNA from the Bam Ill-H fragment was relatively high and it increased when the cells were shifted from anaerobic to aerobic conditions. We have evidence suggesting that crt A in the Bam HI-H is activated by O2 • This gene is responsible for the oxidation of spheroidene to spheroidenone. Such regulation in response to O2 may be related to the function of Crt. It has been long known that Crt has two functions: harvesting light and protecting cells including the photosynthetic apparatus from photooxidative damage which only occurs in presence of both high light and O2 • Activation of crt A and other Crt genes by O2 may be part of the protective mechanism by which Crt scavenges O2 radicals in the cell.

Here we have also found another type of protective mechanism in response to high light. That is, in contrast to the decrease in the levels of mRNA for LH, RC and Bchl biosynthesis, an increase in light intensity raised the levels of mRNA from a number of crt genes located in the Bam HI-M, Bam Ill-G and Bam Ill-H fragments. Although the increase in transcription of crt genes in response to high light seems to be a plausible protective mechanism, we do not rule out the possibility that regulation may also occur post-transcriptionally, including the activation of Crt biosynthetic enzymes by light and O2 • The 0.4 kb small transcript from the Bam Ill-I also showed a response to high light in the opposite fasion to the mRNAs for LH and RC. It did not hybridize to either regions genetically mapped as crt F or crt E . The response of this transcript to O2 is very similar to that of mRNAs for LH and RC, but is very different from that of mRNAs for Crt biosynthesis. On the other hand, transcript level increases in response to high light as do the mRNAs for Crt biosynthesis except to a greater extent, but it is very different from that ‘of mRNAs for LH and RC in this respect.Other interesting results in this study are the finding of multiple transcripts for the RC-H as shown by Northern hybridization. The 1.2 and 1.4 kb transcripts of the RC-H gene, 764 bp in size, are probably initiated from the middle of the ORF F1696. We do not know whether they have different initiation sites,4×8 flood tray or if the 1.2 kb transcript is the product of processing of the 1.4 kb transcript. The relatively long 5′ non-coding region may contain important regulatory sequences. Assay for transcripts from all of the ORF putative genes near the LH and RC gene clusters in the Bam Ill-C -EcoRI-B, and Bam Ill-F fragments resulted in the detection of mRNA from only C2397. We suggest that the putative genes in both fragments are either not expressed under the define growth conditions, or their mRNAs, if present, are below the limits of detection by our methods. Our research has been directed towards the understanding of the mechanism controlling the coordinate expression of genes encoding photosynthetic components. While the photosynthetic processes are primarily carried out in the chloroplasts of eucaryotic photosynthetic cells, the genes encoding the components are distributed into both the nuclear and the chloroplast genome. The coordination of expression of these physically separated genomes is the subject of our investigations. We are determining if the chloroplast or its encoded components play a role in the light response of nuclear genes whose produc.ts· are involved in photosynthesis. The photosynthetic apparatus is a highly organized and specialized structure made up of many components. The activity of the system is dependent on the activity and arrangement of the individual components. The cell must balance the production of the individual components to optimize the photosynthetic activity of the system.

This balancing act is not simply a maintainence of equal numbers of all components since some components in the apparatus are needed in great excess over others. Also, control must be exerted on the production of these components as a function of time. For example, the production of Light Harvesting Chlorophyll Binding Protein must not begin before chlorophyll biosynthesis since the protein is rapidly destroyed in the absence of chlorophyll. Even though the chloroplasts of photosynthetic organisms contain their own genome, many of the functions carried out in the organelle are encoded in the nucleus. Some of the proteins which function in chloroplast are encoded partially in the chloroplast and partially in the nucleus. The expression of both these genomes is regulated by light. Light stimulates mRNA synthesis as well as DNA synthesis in Euglena gracilis. Presumably” this light response is mediated by photo pigments in the cells which receive the light and convert it into a signal which ‘control nucleic acid activity in both genome compartments. We are studying a series of bleached mutants of Euglena which lack various photo pigments as well as have different levels of chloroplast DNA. Using these variants, we hope to determine the involvement of the photo pigments and chloroplast DNA in the stimulation of nuclear DNA and RNA synthesis. First, we are characterizing the cell cycle response to light of these various mutants by flow cytometry. Wild type Euglena conditioned in the resting medium in the dark can be stimulated to reenter the cell cycle by light. We are comparing the kinetics of reentry into the cell cycle of the bleached mutants with that of the wild type to discern the role of the photo pigments and chloroplast supplied functions in regulating the DNA replication response in the nucleus. We are also comparing the light stimulated expression of nuclear encoded chloroplast functions in wild type vs bleached mutants to determine if the chloroplast and/or the missing photo pigments play a role in the regulation of the nuclear genes. Can nuclear genes coding for photosynthetic components be regulated by light in the absence of functional chloroplasts? At present we are using heterologous probing to clone the nuclear genes for the light harvesting chlorophyll alb proteins in Euglena. We have been testing conditions that will allow plant protein-DNA complex formation. Tobacco root and leaf protein extracts have been tested for their DNA-binding capacities after • _ separation of the proteins by electrophoresis and transfer to a nitrocellulose membrane. We have seen a number of proteins which show DNA-binding capacities, and have noted large differences in the DNA-binding pattern of extracts from the two tissues. We are presently characterizing the nature of these proteins and assessing the contribution of chloroplast DNA-binding proteins to the binding pattern observed in the case of the leaf extracts. Even if a major contribution in this difference comes from the leaf chloroplastic proteins, the two smaller MW proteins present only in the root extract represent a true difference in the DNA binding patterns of the proteins from root and leaf tissue.