Somatic embryogenesis from leaf midrib explants of Dendranthema grandiflora Tzvelev. `Iridon' cultured on modified Murashige and Skoog basal medium (MSB) containing 1.0 mg 2,4-D and 0.2 mg BA/liter was influenced by light and sucrose concentration. Somatic embryos formed directly from explants when cultured on medium containing 9% to 18% sucrose and incubated first in the dark for 28 days, followed by 10 days in light, and then returned to the dark for 14 days. Embryogenesis did not occur in continuous darkness and was drastically reduced when explants were incubated in light only. The most embryos were formed on medium containing either 12% or 15% sucrose; lower concentrations stimulated shoot and root development. Light also mediated embryogenesis from leaf explants of 'other cultivars. White-opaque or occasionally light-green cotyledon-stage somatic embryos germinated on MSB medium without growth regulators but containing 3% sucrose. Twelve of the 23 cultivars evaluated produced somatic embryos, but plants were recovered from only five. Regenerated plants were phenotypically similar to parent plants in growth habit, leaf morphology, and flower color. Chemical names used: N- (phenylmethyl)-1 H- purine-6-amine (BA); (2,4-dichlorophenoxy) acetic acid (2,4-D).
R.A. May and R.N. Trigiano
R.N. Trigiano and R.A. May
A tissue culture laboratory exercise illustrating regeneration of whole plants from leaf segments of chrysanthemum by organogenesis is described. Using simple, common media, shoots can be generated in 5 weeks and rooted after an additional 3 weeks. Acclimatization of plants can be accomplished in a simple mistbed in the greenhouse. The exercise is adaptable to depict genotype differences among cultivars, optimization of shoot induction, effects of growth regulators, and experimental design. Callus is typically not formed during shoot formation; however, co-cultivation of leaf segments with a virulent strain of Agrobacterium tumefaciens produces callus with a strain of disarmed A. tumefaciens harboring NPTII construct affects regeneration of plants resistant to kanamycin.
Naomi R. Smith and Robert N. Trigiano
Flowering dogwood (Cornus florida L.) is an important tree of forests and urban landscapes in the eastern United States. Currently, there are over 100 cultivars of flowering dogwood commercially available. An identification process based on genotype would be of use to researchers, breeders, and nurserymen, as many cultivars are similar phenotypically. Molecular markers offer a promising way of definitively identifying flowering dogwood cultivars. Amplified fragment length polymorphism (AFLP) is a technique that can be used to generate DNA fingerprints. DNA was isolated from leaves of 17 common cultivars of dogwood and AFLP fingerprints were generated by a Beckman Coulter CEQ™ 8000. Fingerprints were converted to binary data and verified manually. Two drafts of a cultivar identification key were generated based on the corrected, verified binary data and cultivar-specific peaks. Six primer combinations were used to construct all keys and were tested with seven unknown dogwood cultivar samples. Six unknown samples were correctly identified using the keys. Only one unknown, `Cherokee Brave', was unidentifiable with any key. In all cases, some intracultivar variation was observed. A similarity index was calculated and visualized with a tree of genetic relatedness using NTSYSpc. Intracultivar variation was observed in the similarity index as well. This database for cultivar-specific molecular markers will serve as a starting point to which other cultivars can be added and also can be used in breeding applications, patent application and other projects, such as mapping the C. florida genome.
R.N. Trigiano and G. Caetano-Anollés
The primary objectives of these laboratory exercises are to familiarize advanced undergraduate and graduate students (and instructors) with the general concepts, techniques, and uses of DNA fingerprinting and to remove some of the perceived mystique underlying molecular genetics. The technique of DNA amplification fingerprinting (DAF) is partitioned into four independent laboratory exercises that include DNA isolation, DNA amplification, gel electrophoresis and silver staining, and data collection and analysis. Although the DNA amplification and gel electrophoresis exercises are emphasized, very detailed and easy-to-follow instructions and protocols are provided for all aspects of the DNA fingerprinting process. These exercises, or similar ones, have been successfully completed on the first attempt by several classes of novice graduate students and other researchers.
R.N. Trigiano, M.C. Scott and G. Caetano-Anollés
Four chrysanthemum (Dendranthema grandiflora) spontaneous and radiation-induced sports from the cultivar `Charm' and phenotypically differing only in flower color were individually characterized using arbitrary signatures from amplification profiles (ASAP). ASAP analysis is based on a two-step arbitrary primer amplification procedure that produces “fingerprints of fingerprints.” In the first step, `Charm', `Dark Charm', `Dark Bronze Charm', `Salmon Charm', and `Coral Charm' were fingerprinted by DNA amplification fingerprinting (DAF) with standard octamer arbitrary primers. Diluted products from three monomorphic fingerprints for each cultivar were subsequently reamplified using four minihairpin decamer primers. Each of the 12 ASAP profiles revealed about 30% polymorphic loci and some were used to uniquely identify cultivars and estimate genetic relationships. The ASAP technique permits identification of previously genetically indistinguishable plant material and should facilitate marker assisted breeding and protection of ownership rights.
L.G. Buckley, E.T. Graham and R.N. Trigiano
Zygotic and somatic embryos are purported to follow similar developmental sequences, but few investigations have thoroughly compared the two processes. Developing pods of Cercis canadensis L. (redbud) were collected from trees on the Knoxville campus of the University of Tennessee once or twice per week from 28 March to 8 August 1991. At least 10 ovules/sample date were fixed in FAA to evaluate zygotic embryo ontogeny. A minimum of 40 ovules/sample date were aseptically excised and placed on SH medium supplemented with 9.0 μM 2,4-D and 5 mM ammonium ion to initate somatic embryogenesis. Zygotic and somatic embryos were prepared for histological examination using standard paraffin techniques. Somatic embryos developed primarily from cotyledons and epicotyls of zygotic embryos mat were cultured between 6 June and 19 July. Somatic and zygotic embryos were subtended by multiseriate suspensors and progressed through recognizable globular, cordate and cotyledonary stages of development. Cotyledon morphology was similar for both embryo types. However, many somatic embryos failed to differentiate dome-shaped shoot meristems exhibited by their zygotic counterparts.
R.N. Trigiano, M.T. Windham and W.T. Witte
Powdery mildew (Microsphaera pulchra) of flowering dogwood (Cornus florida L.) has become a significant problem of trees in nursery production as well as in the landscapes and forests of the eastern United States. The disease significantly reduces growth and berry production by older established trees and may contribute to the inability of younger trees (liners) in production to survive winter dormancy. Disease resistance in named cultivars is limited to partial resistance found in `Cherokee Brave'—all other cultivars are extremely susceptible. Until now, the only disease control measure was to establish an expensive, labor-intensive, preventive fungicide program. We examined >22,000 seedlings and identified 20 that were extremely resistant to powdery mildew. Three trees with white bracts were selected from the 20 and released as patent-pending cultivars. `Karen's Appalachian Blush' has long, non-overlapping, pink fringed bracts with a delicate appearance. `Kay's Appalachian Mist' has creamy white, slightly overlapping bracts with deeply pigmented clefts. `Jean's Appalachian Snow' has large, strongly overlapping bracts with non-pigmented clefts. The three powdery mildew-resistant cultivars will be entered into an existing breeding program with `Appalachian Spring', a cultivar released by the Tennessee Agriculture Experiment Station and resistant to dogwood anthracnose, in an attempt to produce trees that are resistant to both diseases.
M.C. Scott, G. Caetano-Anollés and R.N. Trigiano
The genetic distance of closely related cultivars of Dendranthema grandiflora (chrysanthemum) was assessed using DAF. Thirteen cultivars of chrysanthemum included in the study were members of the following series: Charm (five), Davis (four), and Pomona (four). The genetic variability within and between series were evaluated using 11 arbitrary octamer primers. A few polymorphic loci were evident that uniquely identified closely related cultivars within a series. In contrast, there were many polymorphisms between members of different series. Genetic distances between cultivars within and between series were calculated using marker comparison and UPGMA (cluster analysis). The average distance between series was 10-fold greater than between cultivars within a series. DNA from all cultivars belonging to a series also were bulked to generate DNA profiles containing unique amplified products for each series. Polymorphic loci that were generated by the DAF technique possibly could be used for patent protection and phylogenetic studies and may be useful in breeding for chrysanthemums.
G. Caetano-Anollés, R.N. Trigiano and M.T. Windham
Fifty-one isolates of Discula destructiva obtained from various Cornus species were evaluated using arbitrary signatures from amplification profiles (ASAP). ASAP analysis is based on dual-step arbitrary primer-based amplification procedure that produces “fingerprints of fingerprints” and in many instances increases detection of polymorphic DNA. This novel technique was able to distinguish groups of isolates from the northeast, middle and southeast range of the disease as well as western United States and Canada. The data supports the contention of recent and independent introduction of the disease on both east and west coasts, a genetic “bottleneck” that has limited diversity of the pathogen, and directionality of introduction of disease from coastal ports-of-entry to interior populations of C. florida and C. nuttalli.