Karen W. Hughes

In vitro ecology: Exogenous factors affecting growth and morphogenesis in plant culture systems

Abstract Factors other than media may affect the growth and development of plant cell and tissues in culture. These factors include light (wave length, intensity and photoperiod), temperature and the gas phase which develops above the culture system. These factors are not independent but interact in often complex ways. Most cultures are grown under standard light and temperature conditions but for maximum growth or morphogenic response, the effects of external factors on the culture system should be investigated.

Selection of paraquat-resistant variants of tobacco from cell cultures.

SummaryParaquat-resistant cell lines and plants were isolated from cell cultures ofNicotiana tabacum var. Wisconsin 38. The resistant phenotype was successfully obtained from both callus and plated suspension cultures; however, recovery of phenotypically stable, resistant cell lines was higher among those derived from suspension cultures. The use of X-rays increased the recovery of paraquat-resistant cell lines. Stepwise selection procedures did not increase recovery of the resistant phenotype. Only 15 of 43 plants regenerated from paraquat-resistant cell lines retained the resistant phenotype; however, calli derived from leaves of both resistant and nonresistant plants exhibited either partial or complete resistance.

Long term regeneration by somatic embryogenesis in barley (Hordeum vulgare L.) tissue cultures derived from apical meristem explants

Callus cultures were initiated from apical meristem explants of one to four-week-old aseptically-grown barley (Hordeum vulgare L. cv. Atlas 57) plants. Embryogenic callus and plants were produced in three separate experiments; the cultures have retained regenerative capacity for three years after initiation. Our results demonstrate that explants other than immature embryos are embryogenically competent in barley and that regeneration occurs by both somatic embryogenesis and organogenesis.

Molecular phylogeny, morphology, pigment chemistry and ecology in Hygrophoraceae (Agaricales)

Molecular phylogenies using 1–4 gene regions and information on ecology, morphology and pigment chemistry were used in a partial revision of the agaric family Hygro- phoraceae. The phylogenetically supported genera we recognize here in the Hygrophoraceae based on these and previous analyses are: Acantholichen, Ampulloclitocybe, Arrhenia, Cantharellula, Cantharocybe, Chromosera, Chrysomphalina, Cora, Corella, Cuphophyllus, Cyphellostereum, Dictyonema, Eonema, Gliophorus, Haasiella, Humidicutis, Hygroaster, Hygrocybe, Hygrophorus, Lichenomphalia, Neohygrocybe, Porpolomopsis and Pseudoarmillariella. A new genus that is sister to Chromosera is described as Gloioxanthomyces. Revisions were made at the ranks of subfamily, tribe, genus, subgenus, section and subsection. We present three new subfamilies, eight tribes (five new), eight subgenera (one new, one new combination and one stat. nov.), 26 sections (five new and three new combinations and two stat. nov.) and 14 subsections (two new, two stat. nov.). Species of Chromosera, Gliophorus, Humidicutis, and Neohygrocybe are often treated within the genus Hygrocybe; we therefore provide valid names in both classification systems. We used a minimalist approach in transferring genera and creating new names and combinations. Consequently, we retain in the Hygrophoraceae the basal cuphophylloid grade comprising the genera Cuphophyllus, Ampulloclitocybe and Cantharocybe, despite weak phylogenetic support. We include Aeruginospora and Semiomphalina in Hygrophoraceae based on morphology though molecular data are lacking. The lower hygrophoroid clade is basal to Hygrophoraceae s.s., comprising the genera Aphroditeola, Macrotyphula, Phyllotopsis, Pleurocybella, Sarcomyxa, Tricholomopsis and Typhula.

The consensus land plant chloroplast gene order is present, with two alterations, in the moss Physcomitrella patens

SummaryA restriction endonuclease cleavage site map for the enzymes ClaI and BglII, and a partial map for SacI, has been constructed for the chloroplast genome of the moss Physcomitrella patens (Hedw.) BSG. The plastid chromosome contains approximately 122 kb organized into small (21 kb) and large (82 kb) single-copy regions separated by two copies of a repeat sequence (9.4 kb) oriented in an inverted arrangement. Genes for 17 proteins and 2 ribosomal RNAs have been mapped using heterologous probes from corn, spinach, pea, and petunia. The general order and arrangement of the moss chloroplast genes are similar to the consensus land plant genome typified by that of spinach, with two major exceptions. First, there is an inversion of approximately 20 kb, bordered internally by psbA and atpH, and also containing the genes atpF and atpA. Second, rpl2 and rps19 have been relocated to a different position within the large single-copy region, adjacent to the 20 kb inversion.

Sexual Life Cycle of Three Species of Funariaceae in Culture

Three species of the Funariaceae (Physcomitrella patens (Hedw.) B.S.G., Physcomitrium pyriforme (Hedw.) Hampe and Funaria hygrometrica Hedw.) have been cultured under sterile conditions on Knops inorganic me- dium (liquid and agar) to determine the conditions and minimum time nec- essary for completion of the sexual life cycle (spore to spore). According to the data presented, the life cycle of F. hygrometrica may be completed in 4 months, P. pyriforme in 3 months and P. patens in 2 months. In all three species the critical factor for the induction and maturation of gametangia is cold temperature. Delineation of the life cycle makes these organisms suitable for genetic study. Studies concerning the reproductive phase in bryophytes in culture have been relatively few. Voth and Hammer (1940) implicated photoperiod as the stimulus nec- essary for gametophore production in Marchantia. Morris (1961) stated that in five species of leafy liverworts the onset of sexual reproduction is controlled primarily by photoperiod. Benson-Evans (1964) reported that four species of Marchantiales and six of Jungermanniales are long-day plants, while Riccia glauca and Anthoceros laevis are short-day plants. She also cited some evidence that the photoperiodic effect is operative in many liverworts only within certain temperature limits. Ridgway (1967) reported that in species of Anthoceros, Phaeoceros and Notothylas, photoperiod is critical in initiation of antheridia while temperature has little effect. Contrary to the cases reported for the liverworts and hornworts, temperature does seem to be the

Flammulina RFLP patterns identify species and show biogeographical patterns within species

Representative collections of Flammulina were examined by restriction digests of the ribosomal RNA internally transcribed spacer region (ITS1-5.8S- ITS2). Digestion with the restriction enzymes, HaeIII and Bst F51 distinguished among the major North- ern Hemisphere species of Flammulina. Digestion of E velutipes collections with two additional restriction enzymes, Bgl I and Bst UI, gave variable restriction fragment patterns and revealed biogeographically identifiable patterns. Three haplotypes of E velutipes were identified with geographical distributions chief- ly in Asia, Europe and North America. Recent trans- fer of haplotypes among geographical areas seems plausible based on the observed haplotype distribu- tion.

Infragenic phylogeny of Collybia s. str. based on sequences of ribosomal ITS and LSU regions

Collybia, as understood by Antonin & Noordeloos, comprises four species: C. racemosa, C. tuberosa, C. cirrhata and C. cookei. Collybia tuberosa, C. cirrhata and C. cookei are morphologically similar and are primarily distinguished from each other by the presence or absence and the colour of sclerotia. All four share a common and unique habitat. Phylogenetic reconstructions using DNA sequences of the ribosomal ITS1-5.8S-ITS2 support four distinct clades, each corresponding to a morphological species, with the C. tuberosa, C. cirrhata and C. cookei clades forming a larger group. Analyses of ribosomal Large Subunit DNA sequences confirmed that Collybia tuberosa, C. cirrhata and C. cookei formed a monophyletic group. In both analyses, the C. racemosa sequence was highly divergent from those of the other three species of the complex and we propose a separate genus name, Dendrocollybia, for this species. Simple diagnostic RFLP patterns were identified for the four species and were used to validate morphological designations and distributions.

Evolutionary consequences of putative intra- and interspecific hybridization in agaric fungi

Agaric fungi of the southern Appalachian Mountains including Great Smoky Mountains National Park are often heterozygous for the rDNA internal transcribed spacer region (ITS) with >42% of collections showing some heterozygosity for indels and/or base-pair substitutions. For these collections, intra-individual haplotype divergence is typically less than 2%, but for 3% of these collections intra-individual haplotype divergence exceeds that figure. We hypothesize that high intra-individual haplotype divergence is due to hybridization between agaric fungi with divergent haplotypes, possibly migrants from geographically isolated glacial refugia. Four species with relatively high haplotype divergence were examined: Armillaria mellea, Amanita citrina f. lavendula, Gymnopus dichrous and the Hygrocybe flavescens/chlorophana complex. The ITS region was sequenced, haplotypes of heterozygotes were resolved through cloning, and phylogenetic analyses were used to determine the outcome of hybridization events. Within Armillaria mellea and Amanita citrina f. lavendula, we found evidence of interbreeding and recombination. Within G. dichrous and H. flavescens/chlorophana, hybrids were identified but there was no evidence for F2 or higher progeny in natural populations suggesting that the hybrid fruitbodies might be an evolutionary dead end and that the genetically divergent Mendelian populations from which they were derived are, in fact, different species. The association between ITS haplotype divergence of less than 5% (Armillaria mellea = 2.6% excluding gaps; Amanita citrina f. lavendula = 3.3%) with the presence of putative recombinants and greater than 5% (Gymnopus dichrous = 5.7%; Hygrocybe flavescens/chlorophana = 14.1%) with apparent failure of F1 hybrids to produce F2 or higher progeny in populations may suggest a correlation between genetic distance and reproductive isolation.

Mating systems of some putative polypore — agaric relatives

Mating systems are reported for taxa of polypores and agarics considered related. Taxa areDictyopanus pusillus, Favolus alveolaris, F. tenuiculus, Lentinus bertierii, L. crinitis, L. punctaticeps, L. strigellus, L. strigosus, L. suavissimus, Pleurotus australis, P. levis, P. tuberregium, Polyporus ciliatus, P. elegans, P. squamosus, andP. varius. All are tetrapolar exceptP. elegans.