Henry V. Amerson

Evolution of Genome Size and Complexity in Pinus

Background Genome evolution in the gymnosperm lineage of seed plants has given rise to many of the most complex and largest plant genomes, however the elements involved are poorly understood. Methodology/Principal Findings Gymny is a previously undescribed retrotransposon family in Pinus that is related to Athila elements in Arabidopsis. Gymny elements are dispersed throughout the modern Pinus genome and occupy a physical space at least the size of the Arabidopsis thaliana genome. In contrast to previously described retroelements in Pinus, the Gymny family was amplified or introduced after the divergence of pine and spruce (Picea). If retrotransposon expansions are responsible for genome size differences within the Pinaceae, as they are in angiosperms, then they have yet to be identified. In contrast, molecular divergence of Gymny retrotransposons together with other families of retrotransposons can account for the large genome complexity of pines along with protein-coding genic DNA, as revealed by massively parallel DNA sequence analysis of Cot fractionated genomic DNA. Conclusions/Significance Most of the enormous genome complexity of pines can be explained by divergence of retrotransposons, however the elements responsible for genome size variation are yet to be identified. Genomic resources for Pinus including those reported here should assist in further defining whether and how the roles of retrotransposons differ in the evolution of angiosperm and gymnosperm genomes.

Tissue Culture of Conifers Using Loblolly Pine as a Model

Loblolly pine (Pinus taeda) is a good model for conifer tissue culture, since studies include three methods of in vitro propagation, long-term field evaluations of tissue-culture plantlets, and the development of in vitro trait selection methods. Abbreviated protocols are outlined for the following: (a) organogenesis of adventitious shoots and roots; (b) shoot micropropagation via fascicular and axillary shoots obtained from juvenile, adolescent, and mature explants; and (c) embryogenesis via immature zygotic embryos. Regulatory features of these processes, especially adventitious organ induction and shoot and root elongation, are examined. Field data on four- to six-year height growth, morphological characteristics, and fusiform rust resistance of tissue-culture plantlets derived from cotyledon explants are summarized from multiple plantings. Histological and immunological studies on fusiform rust resistance, evaluated in vitro in loblolly pine embryos, are presented as examples of in vitro trait selection.

Loblolly pine tissue culture: Laboratory, greenhouse, and field studies

Conifer tissue culture had its beginnings in the late 1930s (10) and shoot regeneration cultures were first noted in 1950 (4). Since that time many species (7, 17), especially those using embryonic materials for starting expiants, have been cultured. Among some of the most studied species, Pinus radiata (2), Pseudotsuga menziesii (6), Pinus pinaster (8), Picea abies (22), and Pinus taeda (18), much laboratory data are accumulating. To date, little field data have been reported on the performance of tissue-cultured conifers (14), but several conifers are now established in field plantings (21) and data should be forthcoming.

Cultured cells of white pine show genetic resistance to axenic blister rust hyphae.

Hypersensitive resistance to axenically cultured Cronartium ribicola was displayed by subcultured callus of Pinus lambertiana. Cellular resistance to a destructive rust disease can now be studied at the macromolecular level through use of cloned cells of both host and pathogen in a system amenable to emerging recombinant DNA technology

Slash pine (Pinus elliottii Engelm.) somatic embryogenesis II. Maturation of somatic embryos and plant regeneration

Maturation of slash pine (Pinus elliottii Engelm.) somatic embryos was achieved using two protocols, each starting with a different agar incubation step to deplete plant growth regulators (PGRs) used in previous cultural steps. Strength of maturation medium (single vs. double) was found important in the first protocol to develop normal, mature embryos. In the second protocol, abscisic acid (ABA) concentrations (0, 15 and 30 μM) and carbohydrate sources were tested for embryo maturation. Thirty μM ABA and 6% maltose were deemed the best combination. Embryo germination was accomplished in a continuously lighted environment and embryos receiving a cold pretreatment (4 °C in darkness for 16 days) germinated better than embryos which did not receive cold pretreatment. With a survival rate of 33% after acclimation in a mist system, more than 25 plants from somatic embryos have been established in a greenhouse. Incompletely germinated embryos (lacking roots) were rooted via adventitious rooting techniques and subsequently established in the greenhouse. All established plants obtained from somatic embryogenesis appear normal in morphology.

Genetic Interaction of the Fusiform Rust Fungus with Resistance Gene Fr1 in Loblolly Pine

ABSTRACT We propose a method for defining DNA markers linked to Cronartium quercuum f. sp. fusiforme avirulence (Avr) genes. However, before this method can be successfully employed, a spore competition study was needed to determine the genetic composition of single pycnial drops and multiple drops on single galls when using the standard inoculation procedure, whether virulent (avr1) basidiospores ever predispose some resistant (Fr1/fr1) trees to infection by avirulent (Avr1) basidiospores, and whether avr1 and Avr1 basidiospores equally infect susceptible (fr1/fr1) trees. Results of this study suggest that multiple infections within a single gall are common using the concentrated basidiospore system, resulting on average in >4 infection events per tree. Due to multiple infections within a single gall, an individual pycnial drop cannot be assumed to consist of spores from only a single haploid pycnium. Roughly 57% of the drops harvested were found to consist of more than one haploid genotype, most likely due to the physical mixing of spores from genetically different pycnia. Most importantly, although multiple infections do occur in the formation of a single gall, there is no evidence to suggest that the genetics of the proposed gene-for-gene interaction are compromised. Only avr1 basidiospores were observed to cause infection on Fr1/fr1 trees, whereas both avr1 and Avr1 basidiospores were observed to cause infection on fr1/fr1 trees, albeit not at equal frequencies.

Ultrastructure of the Infection and Early Colonization of Pinus Taeda by Cronartium Quercuum Formae Speciales Fusiforme

Basidiospore germlings of Cronartium quercuum f. sp. fusiforme grew randomly on the surface of Pinus taeda hypocotyls. Polymorphic appressoria developed from germ tube apices and were the first-formed components of the intracellular infection structure apparatus. A narrowed infection peg developed from each appressorium and penetrated directly into an epidermal cell giving rise to a narrowed intracellular neck region and an expanded body which produced an infection hypha. Therefore, the intracellular infection structure apparatus consisted of an extracellular component, the appressorium, as well as the intracellular neck region, body, and infection hypha. The intracellular portions of the infection structure were bounded by a sheath and the invaginated host plasmalemma, possessed a simple perforate septum in the neck region, and a non-septate to variously septate body. The infection hypha exited the infected epidermal cell to colonize adjacent cells and tissues by either of two mechanisms: 1) intracellular hyphae from the infection hypha grew directly into adjacent cells without the formation of an intercellular phase, or 2) the infection hypha produced an extensive intercellular hyphal network. These intercellular hyphae ramified into the cortex and

A genomic map enriched for markers linked to Avr1 in Cronartium quercuum f.sp. fusiforme

A novel approach is presented to map avirulence gene Avr1 in the basidiomycete Cronartium quercuum f.sp. fusiforme, the causal agent of fusiform rust disease in pines. DNA markers tightly linked to resistance gene Fr1 in loblolly pine tree 10-5 were used to classify 10-5 seedling progeny as either resistant or susceptible. A single dikaryotic isolate (P2) heterozygous at the corresponding Avr1 gene was developed by crossing Fr1 avirulent isolate SC20-21 with Fr1 virulent isolate NC2-40. Bulk basidiospore inoculum derived from isolate P2 was used to challenge the pine progeny. The ability to unambiguously marker classify 10-5 progeny as resistant (selecting for virulence) or susceptible (non-selecting) permitted the genetic mapping of the corresponding Avr1 gene by bulked segregant analysis. Using this approach, 14 genetic markers significantly linked to Avr1 were identified and placed within the context of a genome-wide linkage map produced for isolate P2 using samples from susceptible seedlings.

Slash pine (Pinus elliottii Engelm.) somatic embryogenesis. I: Initiation of embryogenic cultures from immature zygotic embryos

Immature female gametophytes and enclosed zygotic embryos of slash pine (Pinus elliottii Engelm.) collected at various developmental stages were cultured either on eight initiation media supplemented with different plant growth regulator (PGR) combinations or on LPM medium solidified with two brands of agar. Embryonal-suspensor cell masses extruded from gametophyte explants infrequently produced white or translucent mucilaginous embryogenic cultures. Variation in extrusion frequency was observed across seed sources (families), explant collection dates, agar types and culture media. Increased explant maturity (as related to collection dates) reduced cell extrusion rates. Four types of extrusions were grouped according to their morphology. One type of extrusion produced the greatest number of established and long-surviving embryogenic cultures. This extrusion type (type I) was associated with an extrusion condition termed “empty” in which the entirety of the embryonal-suspensor mass was extruded out of the gametophyte. Three other extrusion types (types II, III and IV) showed less culture establishment and survival.

Occurrence and distribution in North Carolina waters of Lagenidium callinectes Couch, a fungal parasite of blue crab ova

Ovigerous blue crabs,Callinectes sapidus, collected from certain North Carolina waters during the periods June, 1, 1971, to August 1, 1971, and May 1, 1972, to August 1 1972, were examined for infection of the ova with the fungus,Lagenidium callinectes. During the period May 1, 1972, to August 1, 1972, the rate of infection was as high as 95% during the month of May, but declined steadily during June and July with no infected crabs being collected after July 19, 1972. Attempts to correlate infection rate with water temperature and salinity were inconclusive. Other crabs that had ova that were naturally infected withL. callinectes includedPanopeus herbstii andLibinia dubia. Ova ofMenippe mercenaria were susceptible to artificial infection in the laboratory.