GROWTH INDUCTION IN CULTURES OF HAPLOPAPPUS GRACILIS. I. THE BEHAVIOR OF THE CULTURED CELLS

Blakely , L. M., and F. C. Steward . (Cornell U., Ithaca, New York.) Growth induction in cultures of Haplopappus gracilis. I. The behavior of the cultured cells. Amer. Jour. Bot. 48(5): 351–358. Illus. 1961.—Because of the unusual cytology of Haplopappus gracilis (2n = 4), a study has been made of the growth of its stem tissue in culture. Although growth may occur on a basal medium supplemented in various ways, it was stimulated for present purposes by the use of a basal medium containing casein hydrolysate, coconut milk (2–10% by volume) and naphthaleneacetic acid (0.5 p.p.m.). A definite synergism between coconut milk and naphthaleneacetic acid was demonstrated. The general form of the colonies so obtained responds to the composition of the medium, and certain effects of pigmentation indicate that the biochemistry of the cultured tissue is also a function of the conditions. The Haplopappus cultures were maintained in liquid culture either in the form of free cells or of the small cell clusters to which they readily gave rise. The form of typical cells and cell clusters is described, and stress is laid upon the range of growth forms that are encountered. Variations in suspensions of cells, or small cell clusters, may be investigated by the application of simple microbiological techniques. Haplopappus gracilis is thus a useful material for the further study of growth and morphogenesis by tissue culture techniques.     

THE FEMALE GAMETOPHYTE OF THREE SPECIES OF GRINDELIA AND OF PRIONOPSIS CILIATA (COMPOSITAE)

In the four species studied, Grindelia stricta ssp. blakei, G. arizonica, G. lanceolata, and Prionopsis ciliata (Haplopappus ciliatus), the female gametophyte develops according to the Polygonum (normal) type from the chalazal megaspore of a row of four. In most cases only two antipodal cells are formed, the micropylar one originally containing two nuclei. The number of nuclei increases in both cells; one or both antipodal cells typically grow laterally into the integument, often extending to near the surface of the ovule. This resembles the condition previously reported in Grindelia squarrosa. Since the four species of Grindelia have similar antipodal outgrowths, such outgrowths may be considered typical for this genus. The fact that Prionopsis resembles Grindelia in regard to outgrowths from the antipodal cells and differs in this respect from typical Haplopappus helps to justify its separation from Haplopappus, and perhaps its suggested merger with Grindelia.     

GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS. VII. CELLULAR VARIATION

Variation in long-continued cultures of Haplopappus gracilis and Daucus carota has been investigated. A strain of carrot tissue was isolated that grew with a compact habit, in contrast to the highly friable habit of the parent strain. Its dividing cells were arranged quite differently than in the parent strain. Earlier work had shown that Haplopappus cultures could be reversibly altered in their pigmentation and form, by changing the culture medium. This was confirmed, and it was further shown that pronounced changes in nitrogenous compounds also occurred in response to factors in the medium. However, strains of Haplopappus were isolated which differed persistently from the parent strain, even when they were maintained under the same conditions. The variant strains, grown in the same medium, showed differences in their content of nitrogenous compounds. Stock cultures also changed spontaneously with time with respect to their content of nitrogenous substances. Acriflavine, at low concentration, inhibited the growth and formation of colonics by cells plated on nutrient agar, but, by prolonged exposure to sublethal amounts of the drug, resistant strains were isolated. Certain of the spontaneous variant strains were found to differ from each other and from the parent strain in their chromosome complements in ways that are described and to which the observed changes in morphology and metabolism of the cultures may be attributed. The variations that may occur in the free cells in culture are contrasted with the greater uniformity of the cells as they exist in the plant body.     

GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS. V. THE GROWTH OF COLONIES FROM FREE CELLS ON NUTRIENT AGAR

When angiosperm cells are cultured in a liquid medium they may grow in the form of free, single cells and form small to large groups of cells. This has been shown in earlier papers. This paper deals with the growth of strains of cells (Daucus carota and Haplopappus gracilis cells were used), washed and filtered free from the larger groups, on nutrient agar media in Petri dishes, thus simulating familiar microbiological technique. Each discrete member of a suspension is referred to as a “unit.” On the order of 1–10% of the separate units of a suspension may be induced to grow into viable colonies, depending on the strain and the conditions employed. Whereas at least 30% of the free single carrot cells were shown to be capable of division, only up to about 4% continued their growth to form macroscopically visible colonies when they were widely dispersed. Coconut milk promotes the growth of carrot cells into colonies. Both coconut milk and napthaleneacetic acid, which interact synergistically, arc required for the growth of Haplopappus cells. Various techniques which affect viability (the frequency with which units grow into colonies) were investigated. The viability of carrot units was found (1) to increase with their density on the plates; (2) to decrease upon washing the suspensions prior to plating; (3) to increase with increasing initial size; and (4) to decrease to a vanishingly low value in rigorously filtered suspensions which consist principally of single cells, although the single cells were found to grow with appreciable frequency when the larger units were also present; and (5) to increase dramatically (100-fold) when a rigorously filtered suspension was plated on a medium upon which pieces of growing cultured tissue were placed. Thus, the induction of growth in free cells is enhanced, even in an environment nutritionally optimal for the growth of the larger cell masses, by as yet unknown factors which are contributed by the cells themselves, or by adjacent cells or groups of cells. It is suggested that within a group of cells growing in culture, and perhaps also in the organized growing regions of intact plants, the dividing cells are nourished or stimulated by adjacent but less frequently dividing cells. The implications of these results are discussed.     

GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS IV. THE BEHAVIOR OF THE NUCLEUS

Mitra , J., Marion O. Mapes , and F. C. Steward . (Cornell U., Ithaca, New York.) Growth and organized development of cultured cells. IV. The behavior of the nucleus. Amer. Jour. Bot. 47(5) : 357—368. Illus. 1960.–The nuclei and the chromosomes of carrot cells have been examined at various stages throughout the following sequence: (1) growth of a tissue culture from a preformed explant of secondary phloem from the carrot root; (2) growth and multiplication of carrot cells freely suspended in a liquid medium; (3) growth and re-formation of organs (roots) and whole plants (including flowers) from cells in the freely suspended state. The cells of the carrot are normally diploid (2n = 18), the cells which develop in the explant are also diploid, and the cells of the re-formed organs, and even the flowers developed upon plants grown from cells, are also normal and diploid; normal meioses also occur. Nevertheless, the wide range in growth and form of the freely suspended cells is accompanied by a rich diversity of cytological conditions; these include tetraploid and highly polyploid nuclei which divide, haploidy and such chromosomal aberrations as di- and even tri-centric bridges. Two division figures showing chromosome numbers at different levels of ploidy were seen within the confines of one large cell, and, in another, 2 adjacent division figures were observed with chromosome numbers lower than diploid. Small thick-walled, densely protoplasmic cells divide to form bi- and tetra-nucleate conditions, and in a giant cell a highly multinucleate condition has been seen. Despite this, however, all the regenerated roots and plants yet examined are normally diploid. The implications of these events are discussed.     

918. HAPLOPAPPUS PAUCIDENTATUS

Haplopappus paucidentatus Phil. (Compositae: Astereae: Machaerantherinae) is described and illustrated in colour and with a black and white text figure. Cultivation and propagation are discussed, most stocks better maintained using cuttings or layering. It is not apparently available commercially, but could be searched for under the ‘catch all’ name of H. glutinosus. Haplopappus Cass. is now recognized as a South American genus, considerably smaller than originally conceived by Hall in his major revision. It consists of 56 species in three subgenera, the largest, subgenus Haplopappus, contains 2 sections, section Haplopappus containing H. paucidentatus and H. glutinosus Cass. within which it has been included in several treatments.     

THE ROLE OF SECONDARY PIT CONNECTIONS IN RED ALGAL PARASITISM1

Secondary pit connections are common between cells of hosts and parasites in the widespread phenomenon of red algal parasitism. The DNA-specific fluorochrome 4′,-6-diamidino-2-phenylindole (DAPI) reveals that in host-parasite secondary pit connection (SPC) formation between the parasitic red alga Choreocolax polysiphoniae and its host Polysiphonia confusa, a nucleus and other cytoplasmic components of the parasite are delivered into the cytoplasm of a host cell. Host cells receive large numbers of parasite nuclei and these, apparently arrested in G¹, are maintained intact in host cells for periods of several weeks. Within these enlarged, differentiated cells, starch accumulates and cytoplasmic organelles proliferate as the central vacuole decreases in size. Host nuclear DNA synthesis is stimulated in the infected host cell, resulting in an increase in the number of host nuclei, or an increase in DNA in each of the existing host nuclei (i.e. somatic polyploidy). Occasionally, infected host cells will recommence division and engender a new host branch. Microspectrofluorometry of nuclear DNA quantitatively confirms not only the identity and transfer of parasite nuclei to host cells, but also the transfer of parasite nuclei to other parasite cells. Measurements also reveal that the single nucleus of Choreocolax becomes progressively more polyploid as cells become larger and more highly differentiated. Secondary pit connection formation between Choreocolax and Polysiphonia provides the mechanism for the transfer of parasite genetic information (via the parasite nucleus and cytoplasm) into the host. The parasite nuclei may thereby control and redirect the physiology of the host for the benefit of the parasite.     

Comparative karyotype analysis in Haplopappus Cass. and Grindelia Willd. (Asteraceae) by double FISH with rRNA specific genes

The genera Grindelia Willd. and Haplopappus Cass. belong to the family Asteraceae - Astereae and are distributed in America and South America, respectively. Previous cytotaxonomic studies showed for South American species of Grindelia 2n=12 and for Haplopappus 2n=10 and 2n=12. Both Grindelia species (G. anethifolia, G. prunelloides), newly analyzed with molecular-cytological methods, exhibited symmetric karyotypes (AsI %=55.46 and 55.95) with metacentric chromosome sets (5m + 1m-sat) and 2n=12 chromosomes. The NOR was detected after fluorescence in situ hybridization (FISH) with 18/25S rDNA in the satellite chromosome 2. In contrast H. Happlopappus glutinosus, H. grindeloides and H. stolpii showed exclusively a higher asymmetric index (66.83%, 67.01% and 68.87%, respectively) with submetacentric chromosome sets (4sm + 1sm–sat). The sat-chromosomes 3 of H. glutinosus and H. grindelioides were both significantly different in their length from chromosomes 2 and 4. Furthermore in Grindelia the FISH with 5S rDNA could estimate signals in the short arms of chromosomes 3 or 4, that were not significantly differentiated in their length. Contrary to these findings in Grindelia, the position of 5S rDNA in Haplopappus was detected in the long arms of chromosome 1 (H. grindelioides and H. stolpii) and chromosome 2 (with two different loci) and chromosome 4 of H. glutinosus. The lengths of all measured chromosome arms with 5S rDNA were significantly different to those of the neighbours in the karyotypes. The two-color FISH of 5S and 18/25S rDNA had provided clear karyotypic markers for three (Haplopappus glutinosus) and two (H. grindelioides and H. stolpii) chromosomes. The number and position of rDNA signals were relatively highly conserved in the investigated five species without the double marked chromosome 2 of H. glutinosus, which shows an evolutionary dynamic of this 5S rRNA specific gene cluster. This investigation supports the assumption that the evolution of New World members of Grindelia and Haplopappus has not been accompanied by large karyotypic changes, but small chromosomal rearrangements have undoubtedly occurred (e.g. 5S rDNA localizations).     

Pachytene mapping in the female silkworm,Bombyx mori L. (Lepidoptera)

Pachytene preparations of the chromosome complement of female larvae of Bombyx mori were improved to give a distinct chromomere pattern of the bivalents suitable for chromosome mapping. Six of the 28 bivalents are described and can be identified regularly in the bivalent complements, among them the bivalent containing the nucleolus organizer. The relative lengths of these bivalents compared with one another change during development of pachytene. In contrast to other members of the Lepidoptera there is no conspicuous heterochromatic W-chromosome, which corresponds to the female-specific heterochromatin body present in the nuclei of somatic tissues. This tissue-specific heteropycnosis indicates a different functional state of the responsible chromosome or chromosomal segment in germ line and somatic cells.     

DISTRIBUTION ECOLOGY OF HAPLOPAPPUS AND CHRYSOTHAMNUS IN THE MOJAVE DESERT. I. NICHE POSITION AND NICHE SHIFTS ON NORTH-FACING GRANITIC SLOPES

The close systematic affinities between the composite genera Haplopappus and Chrysothamnus, including different points of contact with “intergeneric” hybrids, are reviewed. The Mohave Desert has several shrubby representatives of each genus, and at census sites from the east-central New York Mountains to buttes at the extreme western end of the desert, north-facing granitic slopes at the foothills of mountain ranges support four Haplopappus species (H. cuneatus. H. laricifolius, H. linearifolias and H. cooperi) and one Chrysothamnus (C. teretifolius). These five species occupy “distributional niches” represented as ranges and areas of dominance on the habitat plane of slope angle and proportion of rocks in the substrate. Central desert sites have four species (excluding C. teretifolius); peripheral desert sites always include C. teretifolius rather than H. laricifolius and support two to four species depending on the size and isolation of the suitable habitat area. Species may occupy different positions on the habitat gradient depending on species number at the site, moisture availability, and in particular competition from other species. C. teretifolius appears to be favored when population turnover rates are high, either in disturbed sites or on natural islands of habitat.     

The frequency and distribution of sister chromatid exchanges in human chromosomes

Summary A detailed procedure is described for a rapid detection of phosphoglucomutase-2 (=phosphopentomutase; PGM-2) on Cellogel following electrophoresis of extracts of human red blood cells and other tissues, including cultured fibroblasts and various types of primate-rodent somatic hybrid cells.The present study indicated that there is only one locus for phosphopentomutase in man. The data from a selected panel of 20 independent clones of man-mouse somatic cell hybrids, investigated for the presence of human chromosomes and for the presence or absence of human PGM-2 favored the assignment of the human PGM-2 locus to chromosome 4.     

THE CHROMOSOMES OF SPLNACIA OLERACEA

Ellis , J. R., and Jules Janick . (Purdue U., Lafayette, Ind.) The chromosomes of Spinaeia oleracea. Amer. Jour. Bot. 47(3) : 210—214. Illus. 1960.—The somatic chromosomes of S. oleracea are described and each has been associated with one of the 6 morphological trisomics derived from triploid-diploid crosses. Of these 6 primary trisomies, reflex had been shown by genetic studies to be trisomic for the chromosomes carrying the sex-determining factors. This chromosome is the longest of the somatic complement and has a sub-median centromere. No obvious heteromorphism of this chromosome pair was observed in staminate plants. Heteromorphism involving this chromosome pair has been reported recently in 2 varieties of cultivated spinach by Zoschke (1956) and Dressier (1958) and was earlier reported by Araratjan (1939) for the wild species, S. tetandra. However, their accounts differ markedly from each other and with the present results in respect to the morphology of this chromosome pair. This study suggests the existence of races which differ with respect to the morphology of the chromosome pair containing the X Y factors.     

MORPHOLOGY AND SYSTEMATICS OF SCIUROTHAMNION STEGENGAE GEN. ET SP. NOV. (CERAMIACEAE,RHODOPHYTA) FROM THE INDO‐PACIFIC1

A new ceramiaceous alga, Sciurothamnion stegengae De Clerck et Kraft, gen. et sp. nov., is described from the western Indian Ocean and the Philippines. Sciurothamnion appears related to the tribe Callithamnieae on the basis of the position and composition of its procarps and by the majority of post‐fertilization events. It differs, however, from all current members of the tribe by the presence of two periaxial cells bearing determinate laterals per axial cell. Additionally, unlike any present representative of the subfamily Callithamnioideae, no intercalary foot cell is formed after diploidization of the paired auxiliary cells. The genus is characterized by a terminal foot cell (“disposal cell”), which segregates the haploid nuclei of the diploidized auxiliary cell from the diploid zygote nucleus. The nature of three types of foot cells reported in the Ceramiaceae (intercalary foot cells containing only haploid nuclei, intercalary foot cells containing haploid nuclei and a diploid nucleus, and terminal foot cells containing only haploid nuclei) is discussed.     

Nuclear DNA content of Solanum species grown in vitro, as determined by flow cytometry

The DNA relative content in nuclei from several Solanum species, which were used as partners for somatic hybridization, were determined using a flow cytometry method. The nuclei were isolated mechanically or via protoplasts from leaves of in vitro grown plants. In the case of S. nigrum as well as S. tuberosum cv. Bzura and dihaploid clone H8105, the nuclei were also obtained from suspension cultured cells by lysis of protoplasts. The source and the method of nuclei isolation affected the pattern of nuclear DNA in the genotypes studied. The mesophyll nuclei showed two distinct peaks on the DNA histograms, whereas the DNA peaks produced by cell suspension nuclei were broad and less distinct. The DNA content in the nuclei, calculated from the DNA histograms of the samples and a DNA standard historgam (Trout Red Blood Cells, having DNA content of 5.05 pg per nucleus), were much lower in mesophyll nuclei than in those obtained from the cell suspension for the same genotypes. The results are discussed in respect of the genetic instability of Solanum genotypes. The usefulness of a flow cytometry approach in somatic hybridization research is also discussed.     

THE FEMALE GAMETOPHYTE OF MACHAERANTHERA PATTERSONII (ASTER PATTERSONII) AND OF M. TANACETIFOLIA

The nucellus of Machaeranthera pattersonii (A. Gray) Greene (Aster pattersonii A. Gray) contains only one megaspore mother cell, and the female gametophyte develops from the chalazal megaspore of a row of four, thus conforming to the Polygonum type of development. These observations are contrary to the older work of Palm. Three nuclear divisions produce the typical eight nuclei with the egg apparatus, primary endosperm cell with two polar nuclei, and two antipodal cells, the micropylar one containing two nuclei. Usually no more antipodal cells are formed, although there is further nuclear division, apparently followed by nuclear fusion. The antipodal cells remain about the same size without forming an antipodal haustorium. Cell division accompanies the first division of the primary endosperm nucleus. The early stages of the embryo resemble those of other Compositae. Machaeranthera tanacetifolia (HBK) Nees also shows the Polygonum type of development of the female gametophyte. It is suggested that Palm may have been working on some species of Erigeron that had been wrongly identified, which would account for the difference in observations.     

INTERSPECIFIC HYBRIDIZATION IN HAPLOPAPPUS AND ITS BEARING ON CHROMOSOME EVOLUTION IN THE BLEPHARODON SECTION

Jackson , R. C. (U. Kansas, Lawrence.) Interspecific hybridization in Haplopappus and its bearing on chromosome evolution in the Blepharodon section. Amer. Jour. Bot. 49 (2) : 119–132. Illus. 1962.—Cytological analyses of interspecific hybrids between H. gracilis (n = 2) and H. spinulosus ssp. australis (n = 8) indicate that ssp. australis is a segmental allotetraploid, derived from past hybridization between 2 taxa with chromosome numbers of n = 4. Analysis of hybrids between H. gracilis (n = 2) and H. ravenii (n = 4), a previously undescribed species, has shown that the chromosome segments of these 2 species are almost completely homologous. Differential contraction is suggested as the explanation for the disappearance in late pachytene of presumed non-homologous segments which were evident in some cells at early pachytene. The pairing relationship of gracilis and ravenii chromosomes at pachytene and later prophase I stages of meiosis indicates that gracilis has evolved from ravenii by an aneuploid reduction process similar to that described for Crepis. The close morphological relationship of the 2 species adds further support to this proposition. Data from the cytological analysis of both interspecific hybrids indicate that x = 4 is the basic chromosome number for the Blepharodon section of Haplopappus.     

Observations on the extent and temporal stability of latitudinal clines for alcohol dehydrogenase allozymes and four chromosome inversions in Drosophila melanogaster

The chromosomes of embryos within thelytokous females of Forda spp. were studied, in samples from galls on Pistacia in the Middle East, and from roots of Poaceae in the Middle East, Europe and North America. The nuclei of oogonial cells, oocytes and early cleavage stages have consistently more chromosomes than the nuclei of dividing cells in the somatic tissues of young embryos from the same mothers. Elimination of the extra germ line chromosomes apparently occurs in late cleavage. In F. marginata Koch the germ line chromosome number varies from 25 to 40 in different populations and the somatic cell number varies from 17 to 20; in F. formicaria the germ line has 21–23 chromosomes and somatic nuclei have 18 or 20. In both species variation occurs between samples from galls on Pistacia as well as between populations on roots of Poaceae. The numbers and relative sizes of the eliminated chromosomes also differ between populations. Comparable phenomena in other insects are briefly discussed.     

T-DNA-tagged chromosome 12 in donor Lycopersicon esculentum × L. pennellii is retained in asymmetric somatic hybrids with recipient Solanum lycopersicoides

Summary Asymmetric somatic hybrid plants were recovered after fusing irradiated mesophyll protoplasts of donor Lycopersicon esculentum × L. pennellii (EP) interspecific hybrid with callus-derived protoplasts of recipient Solanum lycopersicoides. EP plant A54 had been previously transformed by an agrobacterium vector, and the T-DNA insert mapped to the L. esculentum chromosome 12. The T-DNA insert conferred kanamycin resistance to EP that was subsequently used to select cell fusion products and recover asymmetric hybrid plants that retained tagged chromosome 12. Doses of 50- and 100-Gy irradiation promoted the elimination of only a few donor chromosomes. At 200 Gy, the regenerated plants had ploidy levels higher than tetraploid. However, the T-DNA tagged chromosome 12 was always retained in the asymmetric hybrid plants tested. Likewise, all plants from the 100-Gy series, with the exception of number 160, were mixoploid in the root-tip cells. Such mixoploid asymmetric somatic hybrids could be stabilized by inducing adventitious shoots on leaf strips cultured on shoot regeneration medium containing kanamycin. The asymmetric hybrid plants did not produce viable seed when self-pollinated or backcrossed to tomato or S. lycopersicoides. Present address: Department of Biology, University College of London, Gower Street, London, UK     

THE MORPHOLOGY AND CYTOLOGY OF PREUSSIA FUNICULATA

The vegetative nuclei of Preussia funiculata (Preuss) Fuckel appear to divide in two ways. One is very similar to mitosis in higher plants except that no typical metaphase is present. The other consists of elongated nuclei splitting longitudinally into two halves. Ascocarp development is similar to that found in the Pleosporales. A stroma originates in an intercalary position on a hypha. It increases in size, and the outer cell layers differentiate to form the wall. The ascogenous system arises from multinucleate ascogonial cells scattered throughout the centrum. These give rise to large, lobate, multinucleate cells which in time form asci by means of croziers. The mature centrum contains a distinct hymenium and paraphysoids. The haploid chromosome number appears to be 12.     

MODELS OF RETICULATE EVOLUTION IN THE CORAL GENUS ACROPORA BASED ON CHROMOSOME NUMBERS: PARALLELS WITH PLANTS

Somatic chromosome number was determined for 22 species of the scleractinian coral genus Acropora, three species of Montipora, and one species of Fungia, using colchicine-treated cells of externally developing embryos. Most had 28 chromosomes, except for six species of Acropora, which had somatic numbers of 24, 30, 30, 42, 48, and 54. Two models that invoke a combination of polyploidy and aneuploidy are presented to account for the observed intrageneric variation in somatic chromosome number. The ability to propagate clones through vegetative fragmentation plus the opportunities for hybridization during multispecies spawning events may have contributed to the development of polyploidy and rapid, sympatric speciation in the uniquely speciose coral genus Acropora.