Chimeras Composition, Behavior, and Terminology.—A plant chimera is a combination of tissues of two or more genetic constitutions in the same individual plant or part of a plant (fig. 5-10). The term chimera is not applied to ordinary budded and grafted plants, but is restricted to forms in which the combined genetic types (components) grow together, side by side, in the same part of the plant. These components, like the stock and scion of a budded tree, maintain separate genetic constitutions in their respective cell lineages, but their close association results in mutual physiological influence and often compels compromise between conflicting growth tendencies. As a rule, the components of a chimera no doubt differ in chromosome or gene constitution; occasionally, they may differ in plastid constitution only, as in some green-and-white variegated forms. The term graft hybrid correctly refers to chimeras resulting from graftage. There is little evidence in higher plants for the existence of hybrids produced by actual fusion of somatic cells of different genetic types. Chimeras are classified according to the relative positions of the components. If one component forms an outer covering surrounding an inner core of another component, the combination is a periclinal chimera (Jones, 1934). If one component does not surround the other, but both extend to the surface or the center of an organ, the chimera is sectorial. If the arrangement is externally sectorial, but one component does not extend to the center of the shoot, the chimera is often called mericlinal (Jorgensen and Crane, 1927). If the two genetic types are irregularly mixed throughout the plant, as in some forms of variegation, they form a mosaic or mixed chimera. Chimeras may be classified also on the basis of their origin (Frost, 1926a). If a chimera is produced by growth from tissues of two varieties or species, at a bud or graft union, it is synthetic; if it is produced by genetic change in a cell or cells originally like the rest of the plant, it is autogenous. All these kinds of chimeras seem to occur in citrus trees; in view of their importance in relation to bud variation and variety improvement, their characteristics are discussed in detail below. Developmental Relationships in Chimeras.—Chimeras can be analyzed on the basis of the arrangement of the component genetic types in leaf, stem, and flower tissues, together with the ways in which the various parts are developed from the meristems, or growing points, of the plant. One growing-point cell layer, or primary histogen (Dermen, 1945) may produce one or more layers of the mature tissue; in the fruit, tissue of superficial origin may be deeply buried. In older studies, evidence for chimeras was usually based on the morphological and histological appearance of the tissues. Some of the best-known cases in citrus can be recognized in this way. Differences in chromosome number, when they occur, provide a much more accurate means of identifying the cell lineages. If the epidermis, for example, carries a tetraploid complement of chromosomes, while the hypodermal tissue carries the diploid number, tissue derivation can be determined in the developing parts by chromosome counts, and often by cell size. During the past twenty-five years, this method has been used in several plant studies. In Datura, extensive investigation has shown that three primary histogenic layers are regularly present in the apical meristems, and that these layers give rise to certain tissues of the stem, leaves, and fruits (Satina, Blakeslee, and Avery, 1940; Satina, 1945). Dermen (1947, 1953a, 1953b) employed the same method to trace the origin of tissues in cranberry, apple, and peach, where three histogenic layers are also the usual basis for development. In many of these fruits, and in Datura, the polyploid condition was induced by colchicine. The number of histogenic layers and the regularity of their behavior are incompletely known in most families of plants. A review by Gifford (1954) showed that in a majority of the dicotyledons studied there were two primary outer layers and presumably one core layer, while in monocotyledons only one outer layer was present in many cases. Citrus appears to have three histogenic layers. In dicotyledonous plants, the epidermis is regularly developed from the outermost growing-point layer (the dermatogen, or Layer I): Layer II produces a marginal zone of leaf tissue, or even all the leaf parenchyma inside the epidermis, as well as the gametes and at least part of the associated tapetal and nucellar cells. In certain green-and-white chimeras a green-producing Layer II usually produces a larger part of the leaf tissue than does a white-producing Layer II (Rischkow, 1936). In some exceptional cases Layer I, instead of Layer II, gives rise to the gametes (Jones, 1934, p. 42). Differences in growth-rate tendencies between the components of a chimera may affect the regularity of tissue differentiation and the stability of the chimeral pattern. Sectorial chimeras are generally unstable; in the formation of lateral buds, one component is often eliminated or a periclinal arrangement may be formed. The conditions producing mosaic chimeras include: (1) irregular assortment of green and colorless plastids in cell division; (2) frequent genetic change within cell nuclei; and (3) unbalanced growth relations in a periclinal chimera, such that one component frequently takes the place of another. In citrus forms where there seems to be frequent genetic variation within cells, the variable phenotype may depend on several grades of chromosome constitution, apart from changes in the relationships of tissue components. Synthetic Chimeras.—In early experiments, Winkler (1908) grafted tomato onto its relative, the nightshade, and nightshade on tomato, after which he cut off the grafted plants at the union. Sprouts developed from adventitious buds, and occasionally one arose at the line of the union so that one side of the shoot was tomato and the other nightshade, constituting a sectorial chimera (fig. 5-11). Intermediate forms were also obtained, which when analyzed by chromosome counts and seedling characters proved to be periclinal chimeras (Winkler, 1910; Baur, 1910). These chimeras had one or more outer layers of tomato tissue and a core of nightshade, or vice versa (fig. 5-12). Where synthetic chimeras are so formed, by the combination of different species, identification of the chimeral condition is fairly easy. Forms capable of somatic separation into two types having leaves or fruit clearly of two species have probably been produced by graft synthesis. The citrus bizzarria.—Several freakish citrus forms, known in Italy as bizzarria, are evidently synthetic chimeras. The bizzarria of Florence was described by Nati (1674, 1929); it had branches, leaves, and fruits partly like citron, partly like sour orange, and mainly intermediate or mixed (fig. 5-10). Nati concluded, mostly from testimony of the gardener under whose care the bizzarria had originated, that it probably arose at the union where a citron bud had been inserted on a sour orange seedling—the bud failing to grow at first, but a mixed shoot coming out later. Strasburger (1907) concluded that this bizzarria did not result from actual fusion of somatic cells, since it had the same chromosome number as the parent types, not twice as many. Tanaka (1927b) studied Nati's bizzarria, and concluded that it is primarily a periclinal chimera, with a core of citron and outer layers of sour orange, the sectorial structures resulting from irregularities in histogenic-layer behavior; most of the rind is superficially sour orange, with yellowish streaks where the citron core has emerged, and occasional wider sectors of thick citron rind. The instability of this chimera may be due to the more vigorous growth of the inner component. Strasburger (1907) summarized accounts of other citrus bizzarria, including one of citron and sweet orange that was in existence before 1644, when the bizzarria of Nati is said to have originated. Savastano and Parrozzani (1611) described as "natural hybrids" three bizzarria which presumably were synthetic periclinal chimeras. Brown (1918) described a tree that appeared to be a lemon-over-orange chimera, and Uphof (1935b) described a tree that bore intermingled fruits of sweet and sour orange. Intermediate fruits were not observed. In Japan, two citrus forms considered to be synthetic periclinal chimeras have been in existence for some fifty years (Samura and Nakahara, 1928; Takahashi, 1962). The Kobayashi Mikan is recorded as having arisen at the junction where a satsuma scion was grafted onto Natsudaidai. This scion was accidently [sic] broken off at its base, after which two adventitious buds emerged, one of which grew into the original chimeral tree. This chimera produces fruit with rind like Natsudaidai, but with flesh like satsuma. It is seedy, and seedlings from one fruit were reported to be Natsudaidai (Dr. Yuishiro Tanaka, unpublished). In this chimera, histogenic Layer II appears to be genetically Natsudaidai, while Layer I should be satsuma. Kinkoji Unshu arose in a similar manner, in about 1912, after satsuma scions had been top-worked onto Kinkoji (C. obovoidea Takahashi). A branch was later observed which produced fruits having outer parts like Kinkoji and inner parts like satsuma, with the fruits bearing some seeds. Both of these chimeras have been maintained in Japan. Casella (1935) described and showed photographs of a tree of sweet lime budded on sour orange, which produced a vigorous chimeral branch at the bud union. This branch produced some fruits similar to lime, some similar to orange, and some chimeral fruits with rind sectors of both types. Casella reported that seeds and juice vesicles in these fruits and fruit sectors corresponded in character to the respective rind types. He stated that six-year-old seedlings from seeds from orange-like fruit sectors had characters of sour orange, but that those from lime-like sectors also had sour orange characters. Casella concluded that the variant branch was a synthetic chimera. Autogenous Chimeras.—Baur (1909) discovered that certain variegated forms of Pelargonium (garden "geranium") are white-over-green or green-over-white periclinal chimeras. In later studies, such as those of Bateson (1916) and Chittenden (1927), other herbaceous plants were found to be periclinals. Since the varieties concerned are propagated by cuttings, not by graftage, these chimeras no doubt originated by some change in cells of the parental clones. The components of such chimeras can often be identified by one of the following methods: (1) in green-and-white combinations, the components are visible by inspection in parts normally green; (2) in citrus, a component present as the subepidermal layer which produces the nucellus should appear in all histogenic layers of the derived nucellar seedlings; and (3) components with differing chromosome numbers can be identified during cell division, in certain tissues. A component present as an inner layer, from which adventitious roots arise, can sometimes be detected by obtaining whole plants from such roots. In some bud variation forms, root cuttings of this kind give the parental type that produced the variation; thus, certain thornless varieties of blackberry produce thorny plants from root cuttings. Many of the mutant types that arise in growing-point cells of Citrus must be inferior to the parental type in vigor and are soon crowded out. Occasionally, however, a variant type is noticeable because it has come to occupy an extensive region in one or more cell layers. If a bud-variation shoot contains only an outer layer of a new type, it may lose this type through emergence of the inner type, or it may lose the inner type by the reverse process. Since chimeras are subject to such changes, a bud variation is not necessarily transmitted to all trees budded from it. Variant sectors of rind occur commonly but very irregularly on fruits of many varieties of Citrus. Certain characters may be conspicuously changed, such as thickness, smoothness, and color of rind, size of oil glands, and color of pulp; often, several rind characters are changed together. When such changes occur only occasionally within a tree or clone, they presumably represent separate new occurrences of mutant types in cells of the young fruits. Forms showing leaf variation give valuable evidence on the behavior of chimeras, because of the ease of recognition of the cell types concerned. In normally green seed plants, the epidermis is without green color, except that usually there are chloroplasts in the stomatal guard cells. If a mutant subepidermal layer of the growing point also produces non-green tissue, but the layer within it produces green tissue, the leaves commonly have a white marginal zone and also have at least one colorless layer of cells under the epidermis in the green central portion of the leaf (fig. 5-13, E-H). In contrast, if the subepidermal histogen is green-producing, but the third layer is not, the marginal zone of the leaf has normal green color, while the central portion is pale because it has a white core under one or more layers of green cells (fig. 5-13, A-D). Irregularities may produce a shoot of the reverse form or one which is entirely green or entirely white. For examples in Poinsettia and in the privet, see Dermen (1950). The patterns of variegated leaves in different species indicate that tissue zones are formed from the growing point in a much more regular way in some kinds of plants than in others, citrus chimeras being especially irregular. White-over-green chimeras.—Shamel et al. (1920a; 1920b) described variegated bud-variation strains of Eureka and Lisbon lemon. Another form somewhat similar in type of leaf variegation, designated as Variegated Pink lemon, was discovered as a whole tree (Shamel, 1932). It has leaves with an irregular white marginal zone, the white regions often being much dwarfed (fig. 5-14). The upper sides of the leaves show as many as four different grades of green, and the lower sides two or three, the upper and lower patterns being mainly independent. The immature fruits have narrow longitudinal stripes of thin whitish and normal green rind, the green becoming more abundant toward the apical end. Mature fruits are yellow, slightly ridged, and have pink flesh. The bark of the young shoots is usually pale, with longitudinal stripes of deeper green commonly arising at the leaf axils. In each growth cycle, the amount of green in bark and leaves usually increases from base to apex of the shoot. Occasionally a shoot is completely white. The characteristics of this chimeral variety are still regularly expressed in a budded tree now more than thirty years of age at Riverside, California. The variety may not be a simple white-over-green form; mutations in plastids, followed by plastid segregation at somatic cell divisions, might account for its chlorophyll patterns. In some light-margined citrus forms, the non-green areas may be creamy white; in others they are light yellow. In the Imperial orange, grown at Riverside since 1914, the leaves are variegated similarly to those of the Variegated Pink lemon. Fruits are orange-yellow, with narrow, thicker stripes of reddish-orange. These characters appear irregularly throughout the tree, year after year. Small populations of seedlings of this variety, from open pollination, have been entirely white-leaved and soon die. Seedlings from a variegated, white-over-green Eureka lemon described by Shamel (1932) also produce white leaves. This behavior supports the assumption that germ Layer II is producing both the nucellus and the white leaf tissues in the seed-parent tree. In such chimeral forms, both Layer II and Layer III seem to be participating in the formation of the leaves. Layer III apparently has normal chlorophyll-producing capacity; the formation of white leaf tissue from Layer II seems to undergo rhythmic changes with the growth cycles. Completely white shoots suggest that Layer III is occasionally lost from a growing point, but the white-producing Layer II is very seldom lost. Green-over white chimeras.—A budded tree of a variegated Lisbon lemon (CRC 2519) grown at Riverside before 1930, had leaves whose upper sides showed central areas of light green, surrounded by marginal zones of normal, darker green. A few leaves seemed entirely normal. The lighter areas were variable in size and shape, and sometimes showed at least two tints lighter than normal, yet the variation was less than in the white-over-green lemons described above. The lower sides of the leaves only occasionally showed light areas. No completely white areas were found, and the bark of young shoots seldom had light streaks. This lemon was evidently a green-over-white chimera, but the white core component did not emerge to produce subepidermal tissue. An instance of complex instability is evidenced by a clone of variegated sour orange (CRC 622) budded at Riverside in 1914, from a seedling which showed leaf variegation when about 18 inches high. The original budded tree is still living. Most of the foliage has been normally green but some leaves are variegated (fig. 5-15) similarly to those of Lisbon lemon just described. Occasional leaf areas appear white, but these are often bordered by a green zone along the leaf margin. Some twigs with much leaf variegation have bark with whitish stripes. Mature fruits (fig. 5-16) usually show a normal, deep orange rind color, but they may have a narrow stripe or wider sectors of thin, smooth, lemon-yellow rind; rarely, a fruit is mainly yellow and of small size. This sour orange chimera shows some characteristics of a green-over-white type, as if a white layer were occasionally emerging to form subepidermal tissue; however, seedlings, including both nucellar and zygotic ones, continue to show the chimeral condition. In 1963, thirty-one young seedlings were grown from open-pollinated seed from variegated fruit by D. A. Cole, Jr. Seventeen probably nucellar and three seemingly zygotic plants all showed some leaf variegation of the same type as in the seed parent. Eleven entirely white-leaved seedlings died very young. This chimera seems to be due to an unstable genetic factor; the white seedlings may originate from already mutated cells, while in predominantly green seedlings mutations occur during growth. Plastid segregation could be involved. Other autogenous citrus chimeras.—Other bud-variation strains give indication of chimeral constitution, without leaf variegation. The Golden Buckeye navel orange (Shamel, Pomeroy, and Caryl, 1925) is one of these; it appears to have an inner layer similar to Washington navel, surrounded by a subepidermal layer producing a smoother, more yellowish rind. The rind shows narrow longitudinal stripes and ridges of rougher, reddish-orange tissue which seems identical to that of the Washington. Fruit shape tends to be more elongated than with the Washington; the pulp is firmer and sweeter, with a different aroma, and a navel opening is less commonly present. An occasional branch produces fruits which seem to be entirely Washington. The difference in the pulp of the Golden Buckeye may be explained by the fact that in citrus both the juice sacs and the outer tissues of the rind seem to be formed by outer histogens. If only Layer III is of the Washington type, Washington characters should not appear in the juice sacs. If this histogen frequently emerges to form stripes on the rind, after the development of the fruit is well underway, the stripes would not necessarily be correlated with underlying pulp characters. Shamel, Pomeroy, and Caryl, (1929) described other citrus types which seem to be periclinal chimeras. The fruit rind of the Seamed form of the Washington navel shows narrow, longitudinal grooves, which may be caused by the emergence of a mutant Layer III. The Dual selection of Washington navel has patches of smooth and rough rind, but not regularly in longitudinal sectors. The smooth areas sometimes form an equatorial zone, between polar caps of rough rind. These patterns suggest a chimera in which the inner component emerges frequently during the earliest stages of ovary formation, but seldom thereafter. Frost and Krug (1942) described two cytochimeral forms obtained from a bud variation branch on a hybrid mandarin tree. Chromosome counts of root tips, pollen mother cells, and leaf and stem primordia indicated that three histogenic layers were present and that the ploidy of Layers I, II, and III in the two forms was 2-4-4 and 2-4-2. Root tips from stem cuttings were 4n from the 2-4-4 plant and 2n from the 2-4-2 form, as would be expected if such roots are produced from Layer III. Growth habit was lower and broader in the 2-4-4 plant than in the 2-4-2 form. The origin of inner leaf tissues was uncertain; cell divisions in layers other than epidermis showed the 4n condition in both plants, suggesting that only Layer II is involved, but this is contrary to evidence from green-and-white leaf chimeras already discussed. Evidence that the pink-fleshed Thompson and Foster grapefruits are periclinal chimeras has been obtained by Cameron, Soost, and Olson, (1964). These varieties arose, respectively, from the white Marsh and the white Walters grapefruits, by somatic limb variation. In the Foster, the rind shows color, but in the Thompson it does not. By further somatic variation, the Thompson has repeatedly produced red-fleshed forms such as Henninger's Ruby and Webb's Redblush (Waibel, 1953), which appear to be identical. In all of these colored varieties, lycopene and carotenes are the coloring compounds (Khan and MacKinney, 1953). Nucellar seedlings of Thompson, tested over several seasons in California, show no pink or red color in their fruits and appear to be identical to the Marsh. Nucellar seedlings of Foster, produced both in Texas and in California, have fruit with red flesh color like the Redblush and continue to show color in the rind. On the other hand, nucellar seedlings of Ruby, Redblush, and others of the red-fleshed group reproduce the fruit color of their seed parents without change. It appears that a mutant color factor is present in the Thompson in histogenic Layer I; this layer provides at least part of the cells of the juice vesicles (see chap. 1) but should not form subepidermal rind tissues. The factor is not present in Layer II, from which nucellar embryos apparently arise. In the Foster, a factor must be present in Layer II, but perhaps not in Layer I. Nucellar seedlings of Foster, and those of the Redblush group, should carry the factor in all layers. Occasional sports to Redblush, produced on Thompson trees, would then represent the substitution of a cell lineage from Layer I into Layer II, and possibly also into Layer III. The fact that the juice vesicles are pink in Thompson and Foster and red in Redblush may be related to interaction among the tissues involved (Purcell, 1959). An unusual pink sport of the Shamouti orange, called Sarah, also contains lycopene and carotenes, rather than the anthocyanins commonly found in pigmented oranges (Monselise and Halevy, 1961). As in the red grapefruits, these carotenoids occur in the rind, septa, and juice vesicles, but apparently not in the filtered juice. Dr. K. Mendel (personal communication) has stated that the Sarah shows typical characters of a chimera. Some branches bear fruit which is essentially Shamouti in type, while others carry fruit with pink color in the albedo and segment membranes. One budded tree shows further color variation in the juice vesicles. Sinclair and Lindgren (1943) found that cyanide fumigation at an early stage of flower-bud development often produced abundant and varied sectorial rind variations. Although the variant characteristics indicated or simulated genetic change, perceptible effects were limited to the immediate crop season; shoot buds appeared to be unaffected.