Chimeric patterns in Juniperus chinensis 'Torulosa Variegata' (Cupressaceae) expressed during leaf and stem formation

Juvenile leaves of the variegated Hollywood juniper, Juniperus chinensis 'Torulosa Variegata', have sectorial chimeras of variable widths and lengths. Sectors extend over several nodes often as small as 1/24 the circumference of the leaf. Other chimeras appear as light green to yellow streaks but are actually internal, dark green corpus sectors often occupying less than 1/20 of the cross sectional area of a leaf. On the basis of the sizes of these two types of sectors, there seems to be ideally about 168 founder cells comprising 63 tunica cells and 105 corpus cells; 49 of the latter are contiguous with the tunica and 66 are located deeper in the corpus. Similarly, sectoring in axillary branches of original chimeric sprays have the same types of sectoring. It is hypothesized that the outer rings of founder cells form two arcs of 12 cells around the stem apex, one for each of two leaves at a node of the decussate shoot, of a circumference of about 50 cells.     

The OKRA leaf shape mutation in cotton is active in all cell layers of the leaf

Okra (L2O) is a semidominant mutation of cotton (Gossypium barbadense) that alters leaf shape by increasing the length of lobes and decreasing lamina expansion. Chimeras containing L2O and wild-type tissue were generated using Semigamy (Se), a mutation that blocks syngamy during fertilization and produces haploid maternal/paternal chimeral progeny at low frequency. In sectorial chimeras, changes in leaf morphology coincide with the boundary between mutant and wild-type tissues, suggesting that L2O does not regulate a laterally diffusible factor within the leaf. However, in mericlinal or periclinal chimeras, the presence of L2O in tissue derived from any of the three histogenic layers (L1, L2, or L3) of the shoot apical meristem produced leaves with a partial mutant phenotype. The presence of L2O in the epidermis (an L1 derivative), or in the subepidermal mesophyll of the leaf (L2 derivatives) reduced the growth of the lamina and thus increased the depth of leaf lobes. The presence of L2O in the middle mesophyll of the lamina and the vasculature of major lateral veins (L3 derivatives) had no local effect on the expansion of the lamina, but significantly increased lobe length. These results demonstrate that L2O is active in every tissue layer of the leaf.     

ORIGIN OF ADVENTITIOUS SHOOTS REGENERATED FROM CULTURED TOBACCO LEAF TISSUE

Leaf discs from Nicotiana tabacum L., N. glauca Grahm., and a series of interspecific periclinal chimeras were cultured on Murashige and Skoog (MS) medium containing either 2.5 mg/1 6-benzylaminopurine(BA) or 3.0 mg/16-furfurylaminopurine (kinetin). Most shoots regenerated from chimeral leaf discs were non-chimeral but 51 of 658 shoots were chimeral. The histogenic composition of plants regenerated from leaf discs of periclinal chimeras indicated that any cell layer in a leaf can be the ultimate source of shoots, and that shoots can be multicellular in origin. Certain periclinal arrangements were recovered more frequently and their chimeral nature was more stable during subsequent shoot growth. While N. tabacum leaf discs regenerated shoots on MS medium supplemented with either BA or with kinetin, N. glauca leaf discs did not form shoots on the medium containing kinetin. However, chimeras which possessed cells of both species arose on medium containing BA or kinetin, indicating that the morphogenetically competent (i.e., able to produce shoots in culture), N. tabacum cells either interacted with N. glauca cells leading to their competence or incorporated non-competent N. glauca cells into nascent shoot apical meristems.     

Artificial synthesis of interspecific chimeras between tuber mustard (Brassica juncea) and cabbage (Brassica oleracea) and cytological analysis

Interspecific chimeras between tuber mustard and red cabbage were obtained by in vitro graft-culture method. Before grafting, 6-day-old seedlings of tuber mustard and red cabbage were vertically half-cut and treated with different concentrations of 6-BA and NAA for 1 min, then, they were symmetrically fit together. As a result, sectorial chimeras were initially produced from the united shoot tips. The maximum frequency of chimeral bud formation reached 6.33% when the vertical sections of tuber mustard and cabbage were treated with 2 mg/l 6-BA and 1 mg/l NAA. When sectorial chimeras were propagated on MS medium containing 1 mg/l 6-BA, periclinal and mericlinal chimeras gradually developed. Chimeral shoots were rooted on half-strength MS medium containing 0.1 mg/l NAA. The rooted chimeras were acclimatized and transferred to the field for cytological and morphological analysis. The results showed that stomata density in the chimeras was significantly higher than that of their parents, while chloroplast size, starch grain size and number were intermediate between the two parents. The chimeras were further analyzed by flow cytometry, and the results indicated that they contained both sets of parental chromosomes. Moreover, chimeral plants possessed valuable characters from the two parents.     

Induction and origin of adventitious shoots from chimeras of <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Brassica oleracea</Emphasis>

The chimeras between tuber mustard (Brassica juncea) and red cabbage (B. oleracea) were artificially synthesized in our previous study. Adventitious shoots were induced from nodal segments and leaf discs of TCC (LI-LII-LIII, LI -the outmost layer of shoot apical meristem; LII -the middle layer; LIII -the innermost layer. T = Tuber mustard, C = Red cabbage) chimeras. The origin of the shoots was analyzed by histology and molecular biology. As a result, the frequency of adventitious shoot induction rose with the increase of BA in MS medium in the area of the nodes. However, there was no different induction frequency of adventitious shoots from nodal segment bases in media with different BA concentrations. Most adventitious shoots (clustered shoots) arising from the node area were TTT (Tuber mustard- Tuber mustard- Tuber mustard) and only 4 shoots were chimeras, which indicated that more shoots originated from LI than from LII and LIII. All shoots from nodal segment bases were CCC (Red cabbage-Red cabbage- Red cabbage), indicating that the shoots originated from LII or LII and LIII. There were significant differences in the regeneration rate in the margin of the leaf discs among the three combinations of BA and NAA. Most adventitious shoots from the margin of leaf discs were CCC but 2 out of 70 were chimeras, which indicated that more shoots originated from LII or LII and LIII than from LI. All chimeras obtained by regeneration were different from the original explant donor in type in the present study. The origin of the adventitious shoots varied with the site of origin on the donor plant, and could be multicellular and multihistogenic.     

Arrangement of cell layers in the shoot apical meristems of periclinal chimeras influences cell fate

Utilizing a complete set of six periclinal graft chimeras composed of Nicotiana tabacum and Nicotiana glauca (TGG, GTT, TTG, GGT, TGT, and GTG), the fate of the three apical cell layers in both vegetative and reproductive organs has been traced. An analysis of leaf phenotype indicated that only rarely did deviations from expected cell lineage occur and in only TTG did such deviations originate in the shoot apical meristem rather than during leaf development. In most plants that possess a stratified shoot apical meristem, gametes are derived from the second apical layer (L2). A phenotypic and/or DNA analysis of seed progeny following reciprocal crosses between all chimeras and their component species indicated that pollen and eggs were sometimes derived from non-L2 lineage in all but one periclinal chimera. There was no evidence for non-L2-derived gametes in 95 crosses where GTT was a parent whereas 40 of 104 crosses with TTG as a parent yielded some offspring that resulted from non-L2-derived gametes. Of these 40 cases, non-L2-derived pollen grains were responsible 39 times while non-L2-derived eggs were responsible just once. Therefore, the occurrence of non-L2-derived gametes was not random. The disruption of ‘normal’ lineage patterns was dependent on the specific arrangement of genetically dissimilar tissue layers in the shoot apices of the chimeras and was different for different organs.     

Synthesis and Characterization of Nicotiana-Solanum Graft Chimeras

Intergeneric Nicotiana tabacum L./Solanum laciniatum Ait. graftchimeras were produced from decapitated grafts made betweenthese two graft compatible species. Graft unions were treatedwith or without either the auxin, p-chlorophenoxyacetic acid(p-CPA) or the cytokinin, benzylaminopurine (BAP). While BAPwas inhibitory to shoot formation, p-CPA increased the numberof adventitious shoots and raised the frequencies of shoot-forminggrafts obtained. Approximately 9% (14/151) of the shoots producedat p-CPA-treated graft unions were intergeneric chimeras. Theauxin application significantly increased levels of chimeralshoot recovery numbers thus indicating a direct auxin effecton chimeral shoot production. The types and numbers of chimerasproduced were independent of the scion/stock graft combinationemployed. All chimeras appeared to arise initially as mericlinalor sectorial chimeras, with a proportion of the former ones(5/14) stabilizing into periclinal chimeras. The morphologicalcharacteristics of these latter chimeras were compared withthose of the two parental species. The LII layer of speciesdetermined the characteristics of the vegetative and floralcomponents. However, the LI layer modified the qualitative traitsof both components. The LIII layer on the other hand alteredthe quantitative traits of the vegetative organs and the flower.This layer also determined the growth type, breeding behaviourand inflorescence type of synthesized chimeras. These organizationalfunctions of the LIII layer have not been reported previously. Chimeras, intergeneric chimera synthesis, grafting, graft chimeras, Nicotiana tabacum, Solanum aviculare, p-chlorophenoxyacetic acid, peroxidase isoenzymes     

Development of white spruce somatic embryos: II. Continual shoot meristem development during germination

Summary This study compares the development of shoot apical meristems of white spruce somatic and zygotic embryos during germination. In mature somatic embryos, the functional part of the shoot apical meristem was bi-layered. After partial drying, a normal shoot meristem was formed from these two cell layers during germination. Other cells within the meristem were vacuolated and separated by intercellular air spaces. In the absence of the partial drying treatment, somatic embryos enlarged in size primarily due to vacuolation of cells and the formation of large intercellular air spaces. A majority of these somatic embryos failed to form a functional shoot apical meristem. Compared with somatic embryos, the shoot apical meristem of a mature zygotic embryo was well organized with a densely cytoplasmic apical layer. The cells within the meristem were tightly packed. Judging from the cell profiles during germination, all cells within the meristem of the zygotic embryo took part in the formation of the vegetative shoot apical meristem.     

THE FATE OF THE DWARF SHOOT APEX IN BRISTLECONE PINE (PINUS LONGAEVA)

The fate of the pine dwarf shoot (DS) apex after needle initiation has been controversial. Dwarf shoot primordia of Pinus longaeva were examined to determine the developmental basis for DS with and without interfoliar buds. Interfoliar buds are microscopic buds derived from the original terminal apex of the DS. In October, all the DS primordia are similar in size and appearance. However, as the needles elongate in the following June the apices of more proximal DS decrease in size, such that by July there is a clear diminishing size gradient of apical domes in going from the most distal to the most proximal positions. The distal DSs start to form bud scales in July and have fully formed interfoliar buds by mid-August. In contrast, those DS apices lacking protective bud scales at needle maturity become suberized and can never proliferate into long shoots. The distal placement of interfoliar buds may be due to a group effect, where each developing DS inhibits the more proximal DSs in the long shoot terminal bud.     

Independence of Organogenesis and Cell Pattern in Developing Angle Shoots of Selaginella martensii

Angle meristems are mounds of meristematic tissue located atdorsal and/or ventral branch points of the dichotomising stemaxes of many species of Selaginella (Lycophyta). The presentstudy examined the development of ventral angle shoots of S.martensii in response to removal of distal shoot apices (decapitation).Scanning electron microscopy of sequential replicas of developingangle meristems and angle shoots revealed that for the firsttwo pseudowhorls of leaf primordia, particular leaves are notattributable to particular merophytes of the angle meristemapical cell. Individual leaf primordia of the first (outer)pseudowhorl often form from more than one merophyte. Neitherthe shape of the angle meristem apical cell nor the directionof segmentation has any effect on the development of the angleshoot. Additionally, the apical cell of the angle meristem doesnot necessarily contribute directly to either of the new shootapices of the developing angle shoot. The first bifurcationof the angle shoot shows a remarkably consistent relationshipto the branching pattern of the parent shoot. The strong branchof the first angle shoot bifurcation typically occurs towardthe weak side branch of the parent shoot. Anatomical studiesshowed that bifurcation of the young angle shoot involved theformation of two new growth centres some distance away fromthe original angle meristem apical cell; new apical cells subsequentlyformed within these. These results provide additional supportfor the view that cell lineage has little or no effect on finalform or structure in plants.Copyright 1994, 1999 Academic Press Selaginella martensii Spring, Lycophyta, angle meristem, apical cell, shoot apical meristem, leaf primordium, branching, dichotomy, morphogenesis, determination, competence, development, mould and cast technique, replica technique, scanning electron microscopy     

The production of chimeric rats and their use in the analysis of the hooded pigmentation pattern

New, improved media and procedures for making rat chimeric embryos and culturing them in vitro have been developed. We have produced 27 rat chimeras: 20 males and 7 females. This ratio of males to females is consistent with that seen in mouse chimeras, suggesting that rat sex chimeras develop as phenotypic males. By aggregating embryos containing appropriate genetic markers for pigment cell differentiation, it is possible to produce chimeras that elucidate the site of action of the hooded gene. The coat color patterns of black ? black hooded chimeras display a white belly spot. In black ? albino hooded chimeras, small patches of white hair appear on the head and a large white spot occurs on the belly. Black ? agouti hooded chimeras display both agouti and nonagouti pigmentation over the entire surface of the chimera. These animals are fully pigmented with no white spots. In black ? albino non-hooded chimeras, rather small irregular patches of black and white hairs are distributed throughout the pelage. Histological examination of sections of hair follicles obtained from the white areas in the head of black ? albino hooded chimeras revealed amelanotic melanocytes. On the other hand, hair bulbs from the white belly spots do not contain any such melanocytes. Thus the white hairs of the head are due to the presence of albino melanocytes, but the white hairs of the belly are due to the total absence of melanocytes. All these observations are consistent with the conclusion that the hooded gene acts within melanoblasts, probably to retard their migration from the neural crest and/or to prevent their entrance into the hair follicles of the white areas of hooded rats.     

FLEXIBILITY IN ONTOGENY AS SHOWN BY THE CONTRIBUTION OF THE SHOOT APICAL LAYERS TO LEAVES OF PERICLINAL CHIMERAS

The development of leaves on apically stable, periclinal chimeras was studied in a number of dicot genera. The mutant cell layers of the shoot apex and the tissues derived from them were as active developmentally as the normal layers. Ontogeny was the same in these chimeras as in nonchimeras, and growth of their leaves can be outlined as follows. Formation of the buttress, the axis, and the lamina of simple dicot leaves were independent events. In each the first growth included derivatives of the apical layers, usually three in number, found in the apex of the shoot and the lateral buds. Most cell divisions in the outer layers (L-I and L-II) were anticlinal relative to the new structures. Therefore, in the proximal regions of the buttress, axis (petiole and midrib), and lamina, the derivative cells of L-I and L-II were usually present in single layers. The rest of the internal tissue was from L-III. As formation of the axis and the lamina proceeded, derivatives of L-II replaced L-III internally in the distal and marginal regions leaving cells of L-III behind. Both the determinate growth of leaves and the pattern of cell divisions at and near the leading edges of growth meant that no cells in the leaf were comparable to the initial cells of the shoot apex. As the lamina extended, there were extensive intercalary cell divisions, both anticlinal and periclinal, so that in any given region of a leaf the layers of internal cells were from either L-II or L-III. At any point along the axis, L-III participated or did not participate in laminar extension. At any given stage in laminar growth either of two sister cells in any internal layer divided either a few times or extensively. The extreme variability in direction and frequency of cell division during leaf development was under an overriding genetic control, which resulted in the normal or typical size, shape and thickness of leaves.     

Coordination of cell proliferation and cell fate decisions in the angiosperm shoot apical meristem.

A unique feature of flowering plants is their ability to produce organs continuously, for hundreds of years in some species, from actively growing tips called apical meristems. All plants possess at least one form of apical meristem, whose cells are functionally analogous to animal stem cells because they can generate specialized organs and tissues. The shoot apical meristem of angiosperm plants acts as a continuous source of pluripotent stem cells, whose descendents become incorporated into organ primordia and acquire different fates. Recent studies are unveiling some of the molecular pathways that specify stem cell fate in the center of the shoot apical meristem, that confer organ founder cell fate on the periphery, and that connect meristem patterning elements with events at the cellular level. The results are providing important insights into the mechanisms through which shoot apical meristems integrate cell fate decisions with cellular proliferation and global regulation of growth and development.     

Effect of mutations in the PINHEAD gene of Arabidopsis on the formation of shoot apical meristems

The primary shoot apical meristem of angiosperm plants is formed during embryogenesis. Lateral shoot apical meristems arise postembryonically in the axils of leaves. Recessive mutations at the PINHEAD locus of Arabidopsis interfere with the ability of both the primary shoot apical meristem as well as lateral shoot apical meristems to form. However, adventitious shoot apical meristems can form in pinhead mutant seedlings from the axils of the cotyledons and also from cultred root explants. In this report, the phenotype of pinhead mutants is described, and a hypothesis for the role of the wild-type PINHEAD gene product in shoot meristem initiation is presented. © 1995 Wiley-Liss, Inc.     

STRUCTURE AND DEVELOPMENT OF THE DIMORPHIC BRANCH SYSTEM OF THEOBROMA CACAO

The vegetative morphology of Theobroma cacao, the cacao tree, was studied in order to provide a foundation for further investigations on the morphogenesis of the cacao dimorphic shoot system. The seedling of cacao has a determinate orthotropic shoot with a (2+3) phyllotaxis. Branch dimorphism is initiated after 1 to 2 years of growth at which time the apical meristem of the orthotropic shoot aborts and a pseudowhorl of plagiotropic branches is initiated from axillary positions in the shoot tip. The plagiotropic branches are characterized by a distichous phyllotaxis and indeterminate growth. Subsequently an axillary bud below the pseudowhorl develops into a new orthotropic shoot. The apical meristem of this shoot eventually aborts and another pseudowhorl is formed. The apical anatomy of the two types of shoots is similar. The developmental potentiality of the orthotropic shoot axillary buds to form one or the other type of shoot was investigated. The phyllotaxis of the axillary buds of the orthotropic shoot is spiral and that of the axillary buds of the plagiotropic branch is distichous. Pruning and apical puncture experiments showed that the axillary buds of a plagiotropic branch, and of an orthotropic seedling shoot which has not yet formed a pseudowhorl, always give rise to the parent type of shoot. However, the axillary buds of an orthotropic shoot which already bears a pseudowhorl give rise to either type of shoot for several nodes below the point of origin of the pseudowhorl. The type of shoot has no influence on the form of branch which develops from an axillary bud grafted to it. This evidence supports the hypothesis that the axillary buds are initiated as one or the other type of shoot, i.e., once initiated they are predestined.     

CYTOHISTOLOGICAL STUDIES OF CYTOCHIMERIC AND TETRAPLOID GRAPES

Thompson, M. M., and H. P. Olmo. (U. California, Davis.) Cytohistological studies of cytochimeric and tetraploid grapes. Amer. Jour. Bot. 50(9): 901–906. Illus. 1963.—Cytohistological studies were made on 62 different grape clones suspected to be tetraploids to determine if they were chimeras, and if so, what type. The chromosomal constitution of the apical layers in the shoot was determined on the basis of cell and nucleus size, size of chromosome mass, and chromosome estimates or counts. Three cytological types were observed; 2–4,² 4–2, and 4–4. The possible types of chimeras and their stability depend upon the organization of the shoot apex. In most buds there appeared to be a biseriate tunica covering a corpus. However, the number of tunica layers fluctuated from 1 to 3, and there were occasional periclinal divisions in L-II. This lack of discreteness of the second layer probably accounts for the homogeneity of all tissue beneath the superficial layer. The rarity of periclinal divisions in L-I explains the stability of 2–4 types, but reversions are not impossible. The relative frequency of the different types of sports is discussed in relation to their possible origin. The theory of intraclonal variability developed in grapes by Reichardt (1955) and Rives (1961) on the basis of a multilayered shoot apex is re-evaluated under the concept of a functionally uniseriate tunica.     

DETERMINATION OF NUMBER AND MITOTIC ACTIVITY OF SHOOT APICAL INITIAL CELLS BY ANALYSIS OF MERICLINAL CHIMERAS

The dimensions of mericlinal sectors in periclinal chimeras resulting from replacement-displacement phenomena have been used to determine the number of shoot apical initial cells and their mitotic activity. Narrow sectors were always short, extending an average of less than three nodes. All long sectors were wide, involving 1/3 or 1/2 of the circumference of the stem. These observations define the origin of all primary growth as from 1–3 apical initial cells in each of the apical layers. The sectors reveal a surprising stability of cellular position at the center of growth, with a specific initial cell maintaining its position during formation of over 100 nodes. During vigorous vegetative growth of Ligustrum ovalifolium the initials themselves divide only about once in 12 days during the formation of three nodes. The mitotic index of the initials in privet shoot apices is 1.4, and this rate of division is sufficient for them to be the ultimate source of all cells composing the shoot.     

Chimeric drift in allophenic mice: Analysis of changes in red blood cell and white blood cell populations in C57BL/6 ↔ A mice

Fifteen allophenic mice of the type C57BL/6 ? A were quantitatively analyzed for changes in their peripheral white blood cell composition and hemoglobin composition with age. It was found that $\text{7}\text{15}$ or 47% of the mice showed significant changes, termed “chimeric drift,” in one or the other of these parameters. The seven mice showing chimeric drift were classified as unstable chimeras, as opposed to the eight apparently stable chimeras. Chimeric drift was observed in the direction of either parental type, or back and forth, and was found to be independent of the coat color, age, or sex of the mouse. There was an excellent correlation of peripheral white blood cell and hemoglobin compositions of the stable chimeras. However, the unstable chimeras often showed a marked discordance of these two markers.