Clonal analysis of corn plant development

The formation of the lower nodes and internodes in maize (Zea mays L.) and the progression of their differentiation was investigated by generating clonal sectors from cells of the apical meristem. Marked clones were induced by irradiating dry seeds (kernels) and 2-, 8- and 13-day-old seedlings heterozygous for anthocyanin markers (b, pl) and a chlorophyll factor (wd). The extent and apparent number of cells generating the internodes 2–6, which normally remain condensed, were traced by promoting the elongation of these internodes with gibberellic acid. At the mature seed stage, internodes 2 and 3 are undergoing longitudinal expansion and each is represented by two or three circumferential populations of cells. Internodes 4 and 5 are in the process of radial expansion and each is represented by a single circumferential population of cells. At nodes 2–4, the cells for leaves and internodes have separated but such a separation has not occurred for nodes 5 and 6. The formation and expansion of basal six internodes progressed acropetally, i.e. from the base toward distal nodes. Analysis of sectors induced at the seedling stage shows that the formation of middle and top internodes also progress acropetally. The basal, middle and top internodes were found to develop at different apparent cell numbers in the apical meristem.     

Genetic and QTL analysis of maize tassel and ear inflorescence architecture

Maize (Zea mays L.) ear inflorescence architecture is directly relevant to grain yield components, and tassel architecture is relevant to hybrid seed production. The objectives of this study were to (1) determine heritabilities and correlations of a comprehensive set of tassel and ear inflorescence architecture traits in a set of (Illinois Low Protein×B73) B73 S₁ families, (2) identify chromosomal positions of QTL affecting tassel and ear architecture, and (3) identify possible candidate genes associated with these QTL. For tassel traits, the number of detected QTL ranged from one to five, and explained between 6.5 and 35.9% of phenotypic variation. For ear traits, the number of detected QTL ranged from one to nine and phenotypic variation explained by those QTL varied between 7.9 and 53.0%. We detected QTL for tassel architecture traits that required calculation of ratios from measured traits. Some of these calculated traits QTL were detected in regions that did not show QTL for the measured traits, suggesting that calculation of ratios may reveal developmentally relevant patterns of tassel architecture. We detected a QTL on chromosome 7 for tassel branch number near the gene ramosa1 (ra1), which is known to control tassel branch number, making ra1 a candidate gene for tassel branch number. We detected QTL for several traits on chromosomes 6, 8, and 9, where no inflorescence architecture genes have been mapped, thus providing initial information towards new gene discovery for control of inflorescence architecture.     

Two ways to skin a plant: The analysis of root and shoot epidermal development in Arabidopsis

The post-embryonic architecture of higher plants is derived from the activity of two meristems that are formed in the embryo: the shoot meristem and the root meristem. The epidermis of the shoot is derived from the outermost layer of cells covering the shoot meristem through repeated anticlinal divisions. By contrast, the epidermis of the root is derived from an internal ring of cells, located at the centre of the root meristem, by a precise series of both periclinal and anticlinal divisions. Each epidermis has an independent origin. In Arabidopsis the mature shoot epidermis is composed of a small number of cell types: hair cells (trichomes), stomatal guard cells and other epidermal cells. In shoots, hairs take the form of branched trichomes that are surrounded at their base by a ring of accessory cells in a sheet of epidermal cells. The root epidermis is composed of two cell types: trichoblasts that form root hair cells and atrichoblasts that form non-hair cells. Mutations affecting both the patterning and the morphogenesis of cells in both shoot and root epidermis have recently been described. Most of these mutations affect development in a single epidermis, but at least one, ttg, is involved in development in both epidermal systems.     

Clonal analysis of early mammalian development

Various extrinsic markers have been used to label single cells in the early mouse embryo. However, they are appropriate only for short-term experiments because of their susceptibility to dilution. Studies on cell lineage and commitments have therefore depended mainly on exploiting genes as markers by combining cells from embryos that differ in genotype at particular loci. Tissue recombination and transplantation experiments using such indelible intrinsic markers have enabled the fate of different cell populations in the blastocyst to be determined with reasonable precision. The trophectoderm and inner cell mass (i.c.m.) give rise to distinct complementary groups of tissues in the later conceptus, as do the primitive endodermal and primitive ectodermal components of the more mature i.c.m. When cloned by blastocyst injection, single i.c.m. cells colonize only those parts of host conceptuses that are derived from their tissue of origin. Thus, while clonal descendants of early i.c.m. cells can contribute to all tissues other than those of trophectodermal origin, primitive endodermal and primitive ectodermal clones are restricted, respectively, to the extraembryonic endoderm versus all i.c.m. derivatives except the extraembryonic endoderm. Interestingly, individual primitive ectoderm cells can include both germ cells and somatic cells among their mitotic descendants. By using the genetically determined presence versus absence of cytoplasmic malic enzyme activity as a cell marker, the deployment of clones has been made visible in situ in whole-mount preparations of extraembryonic membranes. Very little mixing of donor and host cells was seen in either the endoderm of the visceral yolk sac or the mesodermal and ectodermal layers of the amnion. In contrast, mosaicism in the parietal endoderm was so fine grained that, in all except 1 of 15 fields from several specimens that were analysed, the arrangement of donor and host cells did not differ significantly from that expected on the basis of their random association.     

Clonal analysis of stomatal development and patterning in Arabidopsis leaves.

Cell lineage has been used to explain the stomatal distribution in several plant species. We have used transgenic plants carrying a 35SGUS::Ac construct that produces clonal sectors to analyze the possible role of cell lineage during the establishment of stomatal patterning in Arabidopsis leaves. The analysis of sectors ranging from two to eighteen cells supports the conclusion that most stomatal complexes derive from a single and immediate precursor cell through a stereotyped pattern of three unequal cell divisions followed by a final equal one. In addition, it shows that the successive cell divisions take place at a constant angle (approximately 60 degrees ) with respect to the previous one. Interestingly, this angular dimension shifts from 60 degrees to 0 degrees in the last cell division that gives rise to the stoma. These sectors also reveal the development of both clockwise and counterclockwise patterns of cell divisions during stomatal development in approximately equal numbers. Our clonal analysis indicates that cell divisions involved in the development of stomatal complexes are probably the last ones contributing to epidermal growth and development. Finally, the stereotyped pattern of cell divisions that culminates in the formation of stomatal complexes indicates that cell lineage plays a very important role during stomatal pattern establishment.     

Clonal propagation of Acacia catechu Willd. by shoot tip culture

A method is described for in vitromicropropagation through shoot apices of Acaciacatechu Willd., a semi-arid tree valued for Katha (atanin-like substance obtained from red heart wood of10–20 year old trees) and timber. Explants wereexcised from 15-days-old in vitro grownseedlings raised from superior seed stocks. Shoot budinduction from shoot apex explants was observed onMurashige and Skoog's (MS) [12] medium containingvarious growth regulators. A maximum of 12 shoots wasobtained on MS medium supplemented with 1.5 mg/l 6-benzylaminopurine (BAP) and 1.5 mg/l kinetin.Well-developed shoots (3–4 cm long) were rooted on strength MS medium with 3.0 mg/l indole-3-acetic acid (IAA) and sucrose 1.5%. In vitro regenerated plantlets of A. catechu were transferred to field conditions.     

Clonal analysis of leaf development in cotton

Clonal analysis has been used to describe the cellular parameters of leaf development in American Pima cotton (Gossypium barbadense). Sectors (clones) induced before leaf initiation indicate that the leaf primordium arises from ~100 cells on the flank of the shoot meristem. An analysis of sector frequency during the period of leaf expansion suggests that the rate of cell division is fairly uniform throughout the length of the leaf, but is lower at the margin of the lamina than in intercalary regions. The shapes of marginal sectors indicate that the orientation of cell division (as defined by the orientation of the new cell wall) in this region is more often parallel to the margin than perpendicular to it, although the degree of polarization varies along the length of the margin. There is a slight gradient in the duration of cell division along the length of of the lamina late in development, with cell division ceasing progressively from the lamina tip to the base over two cell cycles. The parameters of cell division in cotton are therefore similar to those described for tobacco with the notable exception of the behavior of cells at the leaf margin.     

Dynamics of flower development and vegetative shoot growth in the high mountain plant Saxifraga bryoides L.

Saxifraga bryoides L. is an abundant species in the subnival and nival zone of the European mountains. First flowering occurs, at the earliest, 6 weeks after snowmelt. This is a remarkably long prefloration period in an environment with a short growing season. To gain more information about the developmental strategies of this species, the timing and the dynamics of flower bud formation and vegetative shoot growth were studied at sites with growing seasons of different lengths at two subnival locations (2650 and 2880 m a.s.l.) in the Tyrolean Alps. At an early, mid and late thawing site, individuals emerging from the winter snow were labelled. Reproductive and vegetative shoots were sampled at regular intervals throughout the growing season and analysed, using different microscopic techniques. Flower buds of S. bryoides develop in three cohorts. Provided the growing season is long enough, cohorts 1 and 2 come into flower, whereas cohort 3 buds remain primordial and continue to develop after winter. New flower primordia appear as day-length decreases from August on, which suggests a short-day requirement for floral initiation. At the end of the growing season, flower buds of different stages are present, but only primordial stages survive winter. Thus, flower buds of S. bryoides develop largely or even completely in the year of anthesis. Developmental dynamics were quite similar at the different sites. Time from flower initiation until anthesis took about 2 months, independently of whether flowers were formed within one or two seasons. All of the leaves on vegetative short-stem shoots turnover within a growing season. Leaves having passed winter continuously decline and are replaced by newly formed ones (21±3 at the mid-thawing site and 18±1 leaves at the short-season site). An individual leaf functions therefore, on average, about 12 months. In most years the seed crop of S. bryoides results mainly from the first cohort of flowers in an individual. In a changing climate with a prolonged growing season, the chance of two cohorts to develop mature seeds from flower cohorts 1 and 2 would increase.     

Clonal mosaic analysis of EMPTY PERICARP2 reveals nonredundant functions of the duplicated HEAT SHOCK FACTOR BINDING PROTEINs during maize shoot development

The paralogous maize proteins EMPTY PERICARP2 (EMP2) and HEAT SHOCK FACTOR BINDING PROTEIN2 (HSBP2) each contain a single recognizable motif: the coiled-coil domain. EMP2 and HSBP2 accumulate differentially during maize development and heat stress. Previous analyses revealed that EMP2 is required for regulation of heat shock protein (hsp) gene expression and also for embryo morphogenesis. Developmentally abnormal emp2 mutant embryos are aborted during early embryogenesis. To analyze EMP2 function during postembryonic stages, plants mosaic for sectors of emp2 mutant tissue were constructed. Clonal sectors of emp2 mutant tissue revealed multiple defects during maize vegetative shoot development, but these sector phenotypes are not correlated with aberrant hsp gene regulation. Furthermore, equivalent phenotypes are observed in emp2 sectored plants grown under heat stress and nonstress conditions. Thus, the function of EMP2 during regulation of the heat stress response can be separated from its role in plant development. The discovery of emp2 mutant phenotypes in postembryonic shoots reveals that the duplicate genes emp2 and hsbp2 encode nonredundant functions throughout maize development. Distinct developmental phenotypes correlated with the developmental timing, position, and tissue layer of emp2 mutant sectors, suggesting that EMP2 has evolved diverse developmental functions in the maize shoot.     

Clonal analysis of murine graft-vs-host disease. I. Phenotypic and functional analysis of T lymphocyte clones

Acute and chronic graft-vs-host disease (GVHD) due to non-MHC histocompatibility differences differ histopathologically. Acute GVHD is characterized by the cytotoxic destruction of recipient tissues, whereas chronic GVHD is characterized by increased collagen deposition. In an attempt to determine if acute and chronic GVHD represent two phases of the same pathophysiologic process or two distinct processes, the T lymphocytes from the C57BL/6 (B6) recipients of LP spleen cells (non-H-2 GVHD) have been cloned and compared to clones from immune mice (LP anti-B6). Acute GVHD (G) clones were established on day 10-14 posttransplant and chronic GVHD (CG) clones on day 50 from animals with clinical chronic GVHD. Immune (I) clones were established 10 to 14 days after immunization. All I clones exhibited B6-specific blastogenesis and cytotoxicity and had a Thy-1.2+, Lyt-2.2+, L3T4- phenotype. All CG clones were noncytotoxic, had I-Ab-specific blastogenesis, and had a Thy-1.2+, Lyt-2.2-, L3T4+ phenotype. The acute GVHD (G) clones were heterogeneous. Fourteen of 23 clones exhibited B6-specific blastogenesis and had a Lyt-2.2+, L3T4- phenotype (B6-G clones). Seven of 9 B6-G clones were cytotoxic for B6 targets. Nine of 23 G clones exhibited I-Ab-specific blastogenesis, and all but one clone had a Lyt-2.2-, L3T4+ phenotype as the did CG clones. Thus, the principal clonogenic T lymphocytes from mice with acute and chronic GVHD differ in terms of 1) their antigenic specificity, 2) their cytotoxic capacity, and 3) their surface phenotype. The presence of I-Ab-specific T lymphocytes with a phenotype identical to CG clones early after transplantation suggests that the immunologic events that result in chronic GVHD begin soon after transplantation. These results indicate that acute GVHD is due primarily to recipient-specific cytotoxic donor T lymphocytes, whereas chronic GVHD is due to autoreactive helper T lymphocytes.     

Clonal analysis of vertebrate myogenesis. I. Early developmental events in the chick limb

Early developmental events occurring in the prospective muscle tissue region of chick embryo leg buds have been subjected to an in vitro clonal analysis. Colony-forming cells are present at stage 20 (72 hr incubation), but none of the colonies exhibit morphological signs of muscle differentiation. After an additional 8 hr of incubation (stage 21), approximately 10% of the colony-forming cells have acquired the capacity to form multinucleated cells in vitro, and the percentage of clonable myoblasts increases to a level of approximately 60% during the next 3 days of incubation. Clonal analysis of myoblast populations within regions of the developing limb have indicated that, between stages 21 and 27, the dorsal and ventral segments of the myogenic region contain appreciably more clonable muscle cells than the anterior and posterior segments. In addition, during stages 21 and 22 there is a 3-fold difference in muscle-colony-forming cells between the proximal and distal halves of the dorsal-ventral segments, as well as between the proximal and distal halves of the anterior-posterior segments. Thus at least two temporal and regional gradients—proximal to distal and medial to lateral—of clonable myoblast content can be delineated within the developing chick limb. In addition to changes in the proportions of muscle-colony-forming cells, the extent of multinuclearity within individual muscle colonies increases with the developmental age of the embryo from which the clonable myoblasts are derived. The progressive changes in the relative proportions of muscle-colony-forming cells and in clonal morphology are discussed in terms of their possible cell lineage implications.     

Temperature and plant adaptation. I. Interaction of temperature and light in the synthesis of chlorophyll in corn

The effect of temperature and light intensity have been studied in relation to the greening of etiolated corn (Zea mays cv. Pioneer 309-B) seedlings. Chlorophyll accumulation is rapid at high temperature (28°) under all conditions of light intensity. At low temperature (16°), and particularly in combination with high light intensity (3000-4500 ft-c), the accumulation of both chlorophyll and carotene is inhibited.

Low pigment content at 16° is not directly due to a block in the pigment synthesizing mechanism, but rather to the photodestruction of chlorophyll prior to its stabilization in the membrane structure of the chloroplast lamellae. The parallel reduction in carotene content at high light intensity is probably a contributing factor, because of its role in protecting chlorophyll from photodestruction. The greater severity of photo-oxidation of chlorophyll at low temperature in corn when compared with wheat, appears to be due to a slower rate of protochlorophyllide synthesis and subsequent esterification. Thus in corn at 16° there is a prolongation of the photosensitive stage during chlorophyll synthesis. Photo-oxidation at 16° has also been shown to be a function of the incident light energy, with the photosynthetic pigments acting as receptors for their own destruction.

In comparison with the behavior of corn, wheat seedlings green rapidly at high light intensity at both 16° and 28°. This contrasting temperature response with respect to chlorophyll synthesis may underlie a fundamental difference in adaptation of these 2 species to growth in the temperate zones of the world.

     

Clonal propagation of Picrorhiza kurroa royle ex benth. by shoot tip culture

A procedure has been developed for the clonal propagation of Picrorhiza kurroa Royle ex Benth. through shoot tip culture. Murashige and Skoog's medium (1962) supplemented with kinetin (3.0 to 5.0 mg/l) supported rapid proliferation of multiple shoots from the explants. Addition of indole-3-acetic acid (1.0 mg/l) to the kinetin containing medium showed marked improvement in the growth of regenerated shoots. However, presence of IAA in the medium did not alter the frequency of shoot multiplication. Rooting was readily achieved upon transferring shoots onto MS medium containing -naphthaleneacetic acid (1.0 mg/l). Plantlets were successfully transferred to soil.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog's (1962) medium - NAA -naphthaleneacetic acid     

Clonal analysis of the relationships between mechanosensory cells and the neurons that innervate them in the chicken ear

In vertebrates, hair-cell-bearing mechanosensory organs and the neurons that innervate them share a common placodal origin. In the inner ear, the peripheral neurons for both auditory and vestibular systems emigrate from the otic placode as neuroblasts, and divide, differentiate and innervate only one of six to eight distinct sensory organs. How these neurons find their correct target is unknown, although one suggestion is that they synapse with clonally related cells. To test this idea for both the middle and inner ears of chicken embryos, lineage analysis was initiated at the time of neuroblast delamination by labeling progenitors with replication-defective retroviruses. The vast majority (89%) of clones were restricted to a single anatomical subdivision of the sensory periphery or its associated ganglia, indicating limited clonal dispersion. Among the remaining clones, we found evidence of a shared neurosensory lineage in the middle ear. Likewise, in the inner ear, neurons could be related to cells of the otic epithelium, although the latter cells were not widely distributed. Rather, they were restricted to a region in or near the utricular macula. None of the other seven sensory organs was related to the ganglion neurons, suggesting that a common lineage between neurons and their targets is not a general mechanism of establishing synaptic connections in the inner ear. This conclusion is further strengthened by finding a shared lineage between the vestibular and acoustic ganglia, revealing the presence of a common progenitor for the two functional classes of neurons.     

Clonal analysis of epidermal patterning during maize leaf development

In plants, specialized epidermal cells are arranged in semiordered patterns. In grasses such as maize, stomata and other specialized cell types differentiate in linear patterns within the leaf epidermis. A variety of mechanisms have been proposed to direct patterns of epidermal cell differentiation. One class of models proposes that patterns of cellular differentiation depend on the lineage relationships among epidermal cells. Another class of models proposes that epidermal patterning depends on positional information rather than lineage relationships. In the dicot epidermis, cell lineage is an important factor in the patterning of stomata, but not trichomes. In this study, the role of cell lineage in the linear patterning of stomata and bulliform cells in the maize leaf epidermis is investigated. Clones of epidermal cells in juvenile leaves were marked by excision of dSpm from gl15-m and in adult leaves by excision of Ds2 from bz2-m. These clones were analyzed in relation to patterns of stomata and bulliform cells, testing specific predictions of clonal origin hypotheses for the patterning of these cell types. We found that the great majority of clones analyzed failed to satisfy these predictions. Our results clearly show that lineage does not account for the linear patterning of stomata and bulliform cells, implying that positional information must direct the differentiation patterns of these cell types in maize.