DNA amounts of roses (Rosa L.) and their use in attributing ploidy levels.

The aims of the investigation were to characterise variability among the DNA amounts of roses and assess the predictability of ploidy levels from DNA amounts. Chromosome numbers in the genus Rosa range from 2n = 2x = 14 to 2n = 8 x = 56 and aneuploidy is rare. Published 2C DNA amounts range from 0.78 pg in R. xanthina Lindl. and R. sericea Lindl. (2n = 2x = 14) to 2.91 pg in R. canina L. (2n = 5x = 35). In this investigation, DNA amounts were estimated by flow cytometry of leaf nuclei stained with propidium iodide, using Petroselinum crispum (2C DNA amount = 4.46 pg) as the internal calibration standard. Ploidy levels based on DNA amounts (DNA ploidy) were assigned by comparing their DNA amounts with published DNA amounts and identifying peaks and intervening discontinuities in frequency distributions of DNA amounts. 2C DNA amounts ranged from 0.83 pg in R. ecae (2x = 2x = 14) to 3.99 pg in R. acicularis (2n = 8 x = 56). Differences in the 1Cx-values (2C DNA amount/ploidy values) were found among the taxonomic sections of Rosa. Ploidy levels could be confidently assigned to most species and cultivars, but the ploidy of some specimens in the section Caninae was uncertain for reasons attributed to genomic diversity and aneuploidy. Cytochimerism was detected in three cultivars of R. x alba. DNA ploidy was determined in 384 specimens representing 74 species and 5 horticultural classes.

Chromosome doubling in a Rosa rugosa Thunb. hybrid by exposure of in vitro nodes to oryzalin: the effects of node length, oryzalin concentration and exposure time.

Chromosome doubling was induced in vitro in a diploid hybrid of Rosa rugosa Thunb. using oryzalin as the spindle inhibitor. Nodal sections, 2 mm long, were exposed to 2.5 or 5 microM oryzalin and 10 mm nodal sections were exposed to 5 microM oryzalin for 0 (controls), 6, 12, 24 and 48 h. The ploidy of the emergent shoots was determined by flow cytometry. The frequency of tetraploid and mixoploid leaves that developed from 2 mm nodal sections exposed to 5 microM oryzalin peaked at 12 h exposure, when 35% of the leaves were tetraploid, but fell after longer exposures. Fewer tetraploid and mixoploid leaves were found when 2 mm nodes were exposed to 2.5 microM oryzalin for 6 and 12 h, indicating that it took longer for a spindle inhibiting concentration of oryzalin to build up in the meristem. However, the frequencies of tetraploid and mixoploid leaves continued to rise after 12 h and were highest at 48 h, when 44% were tetraploid. In treatments with 5 microM oryzalin, the frequencies of tetraploid and mixoploid leaves were lower, at equivalent exposure times, in 10 mm nodes than 2 mm nodes. This suggests that oryzalin diffused to the meristem mainly via the cut surfaces and that access via the epidermis and cuticle was impeded.

Evolutionary implications of permanent odd polyploidy in the stable sexual, pentaploid of Rosa canina L.

In Rosa canina (2n = 5x = 35), the pollen and ovular parents contribute, respectively, seven and 28 chromosomes to the zygote. At meiosis I, 14 chromosomes form seven bivalents and 21 chromosomes remain as univalents. Fluorescent in situ hybridization to mitotic and pollen mother cells (PMC) of R. canina showed that 10 chromosomes (two per genome) carry ribosomal DNA (rDNA) loci. Five chromosomes carry terminal 18S-5.8S-26S rDNA loci; three of these also carry paracentric 5S rDNA loci and were designated as marker chromosomes 1. Five chromosomes carry only 5S rDNA loci and three of these were designated as marker chromosomes 2. The remaining four of the 10 chromosomes with rDNA loci were individually identifiable by the type and relative sizes of their rDNA loci and were numbered separately. At PMC meiosis, two marker chromosomes 1 and two marker chromosomes 2 formed bivalents, whereas the others were unpaired. In a gynogenetic haploid of R. canina (n = 4x = 28), obtained after pollination with gamma-irradiated pollen, chromosomes at meiosis I in PMC remained predominantly unpaired. The data indicate only one pair of truly homologous genomes in R. canina. The 21 unpaired chromosomes probably remain as univalents through multiple generations and do not recombine. The long-term evolutionary consequence for the univalents is likely to be genetic degradation through accumulated mutational change as in the mammalian Y chromosome and chromosomes of asexual species. But there is no indication that univalents carry degenerate 5S rDNA families. This may point to a recent evolution of the R. canina meiotic system.

Oryzalin-induced chromosome doubling in Rosa and its effect on plant morphology and pollen viability.

Shoot tips of the diploid rose Thérèse Bugnet were treated in vitro to oryzalin at concentrations of 5 and 15 microM. Tetraploid shoots were obtained in highest frequencies (40%) after exposure to 5 microM oryzalin for 14 days. Thin (1 mm) nodal sections were treated with 5 microM oryzalin and the highest frequency of tetraploids (66%) was obtained after exposure for only 1 day. The shorter exposure times required to induce chromosome doubling in thin nodal sections is attributed to the more efficient delivery of oryzalin to the meristem. Tetraploids were obtained from four diploid roses and hexaploids from two triploid roses. Chromosome doubling was accompanied by increases in thickness and a darker green colouration of the leaves and, in all diploid to tetraploid and one triploid to hexaploid conversion, the breadth/length ratio of leaflets was significantly increased. Internodes were longer in tetraploids than diploids but significantly shorter in hexaploids than triploids. The number of petals per flower in the tetraploid form of Thérèse Bugnet was double that of the diploid. Significant increases in pollen viability accompanied chromosome doubling of all four diploids and one of the two triploids.

In vitro somatic embryogenesis ofDalbergia sissoo Roxb. -a multipurpose timber-yielding tree.

Somatic embryogenesis was achieved in callus cultures dervied from 40-day-old semimature zygotic embryos ofDalbergia sissoo on semi-solid Murashige and Skoog (MS) salts and vitamins supplemented with 0.46-1.16 µM kinetin, 6.78-9.04 µM 2,4-dichlorophenoxy acetic acid (2,4-D) and 30 g/1 sucrose. Somatic embryos proliferated rapidly by secondary somatic embryogenesis after transfer to half-strength basal MS medium supplemented with 0.46-1.16 µM kinetin and 6.78-9.04 µM 2,4-D with 2% (w/v) sucrose. The light-green somatic embryos germinated on half-strength MS salts and vitamins supplemented with 0.5 mg/1 abscisic acid and 2% (w/v) sucrose. The developmental stages of somatic embryogenesis were studied by light and scanning electron microscopy.

The preparation of micropropagated plantlets for transfer to soil without acclimatization.

Plantlets cultured in vitro on agar-based media in a water-saturated atmosphere wilt rapidly when transferred to normal greenhouse or field conditions. Water is rapidly lost from the leaves because stomata fail to respond to those stimuli that normally induce closure (1-4), and poor development of epicuticular wax results in loss of water through the cuticle (5-7). Uptake of water by the roots is limited by damage incurred during transplantation and by poor contact with the substrate. Problems of transplantation are accentuated in vitrified plantlets, which grow slowly and wilt rapidly. Reduced deposition of cellulose and lignin in these plantlets causes reduced cell wall pressure, leading to increased water uptake by the cells and a glassy turgescence of leaves and stems (8,9).

The pinocytosis of 125I-labelled poly(vinylpyrrolidone), [14C]sucrose and colloidal [198Au]gold by rat yolk sac cultured in vitro.

The rates of uptake of 125I-labelled poly(vinylpyrrolidone), [14C]sucrose and colloidal [198Au]gold by 17.5-day rat yolk sac cultured in vitro were studied. Over a 6.5h period each substrate was accumulated at a constant and reproducible rate of approx. 2microliter/h per mg of protein. After accumulation in vitro, the three substances were released from the tissue into substrate-free medium at low rates. Sucrose present in the medium at concentrations up to 10 mg/ml was without effect on the accumulation of either [14C]sucrose or 125I-labelled poly(vinylpyrrolidone), but at higher concentrations inhibited the uptake of both substrates. Some batches of colloidal [198Au]gold had a significantly higher Endocytic Index (up to 5 microliter/h per mg of protein). The Endocytic Index of such a batch decreased with increasing substrate concentration, but colloidal gold did not decrease the Endocytic Index of 125I-labelled poly(vinylpyrrolidone). It is concluded that the three substrates enter the yolk sac by pinocytosis in the liquid phase. Those batches of colloidal [198Au]gold with higher Endocytic Indices are considered to enter also by adsorption on membrane binding-sites.

Relationship between species in the genus Rosa, section Pimpinellifoliae.

The morphology of twelve species of Rosa is described and similarities between these species are assessed. Possible origins of the tetraploid species from diploid species are indicated on grounds of comparative morphology. The wild origins of living and herbarium specimens are given in order to supplement published data on geographical distribution. Meiosis in pollen mother cells, viability of pollen grains at anthesis and ability to set seed was studied in several F1 hybrids: no indication of complete or even partial sterility was found. Reproductive isolation is therefore unlikely to be maintained by reduced fertility of interspecific hybrids. Three species are reduced to synonymy with three other species, being retained as subspecific taxa. Two species are transferred from section Pimpinellifoliae to section Cinnamomeae.

A quantitative study of pinocytosis and lysosome function in experimentally induced lysosomal storage.

The highly pinocytic epithelial cells of the visceral yolk sac from 17.5-day rat conceptuses were used as a model in which to induce engorgement of the vacuolar system by direct accumulation of substances that are not hydrolysed by lysosomal enzymes. The ultra-structural appearances of these cells in pregnant animals that 24-48h before had received intraperitoneal injections of Triton WR-1339, polyvinylpyrrolidone, dextran or sucrose revealed gross abnormalities that were confined to the vacuolar system; in comparison with normal tissue the number, and in some cases the size, of vacuoles was increased, leading to close packing within the apical cytoplasm and distortion of the normal rounded shape. By culturing yolk sacs in vitro, rates of ingestion of 125I-labelled polyvinylpyrrolidone and of 125I-labelled bovine serum albumin were determined, together with the rate of digestion of the labelled protein. The rates of exocytosis of 125I-labelled polyvinylpyrrolidone and of lysosomal enzymes were also determined. No significant differences between normal and highly vacuolated tissues were found. Apparently marked vacuolation of these cells by these agents is without significant effect on pinocytosis, exocytosis or intralysosomal proteolysis.