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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.

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R.A. May and R.N. Trigiano

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).

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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.

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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.

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M.C. Scott, G. Caetano-Anollés, and R.N. Trigiano

DNA amplification fingerprinting (DAF) was used to study genetic relationships between closely related chrysanthemum cultivars (Dendranthema grandiflora Tzvelev.). Twenty-one cultivars were examined that belonged to the Anne, Blush, Boaldi, Charm, Davis, and Pomona series (families). The genetic variability of cultivars within and between series was evaluated using eleven arbitrary octamer primers. A few polymorphic characters uniquely identified closely related cultivars within each of the series. In contrast, many DNA polymorphisms were observed between members of the different series. Phenetic patterns were established by unweighted pair group cluster analysis using arithmetic means (UPGMA) and principal coordinate analysis (PCO). The average distance between series was 10-fold greater than between cultivars within a series. DNA from all cultivars belonging to a series were also bulked to generate profiles containing unique amplified products for each series. Cluster analysis and PCO of bulked DNA clearly grouped Charm and Pomona together. However, series grouping did not correspond to morphology of inflorescence types. The results demonstrate the utility of the DAF technique in distinguishing clonal materials and its potential use for patent protection, phylogenetic studies, and for identifying useful markers in breeding applications.

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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.

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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.

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D.J. Gray, R.N. Trigiano, and B.V. Conger

Orchardgrass is a member of the Poaceae, a family characterized by monocotyledonous-type embryos. This species is unique in that somatic embryogenesis can be induced from the cultured leaves of potted plants, which can be maintained conveniently in the greenhouse. Both embryogenic callus and isolated somatic embryos develop directly from cultured leaf sections. Embryogenic callus can be maintained indefinitely. Somatic embryos exhibit typical embryo morphology and germinate readily into plants. Exercises designed to lead students through aspects of culture initiation, maintenance, and plant regeneration are described.

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M.T. Windham, W.T. Witte, and R.N. Trigiano

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M.C. Scott, G. Caetano-Anollés, and R.N. Trigiano

The genetic distance of closely related cultivars of Dendranthema grandiflora (chrysanthemum) was assess using DAF. Twenty-three cultivars of chrysanthemum included in the study were members of the following series: Anne (3), Blush (3), Boaldi (4), Charm (5), Davis (4), and Pomona (4). 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, many polymorphisms were observed between members of different series. Genetic distances between cultivars within and between series were evaluated using marker comparison and analyzed with PAUP (phylogenic analysis using parsimony) and UPGMA (unweighted pair group cluster analysis using arithmetic means). The average distance between series was 10-fold greater than between cultivars within a series. Furthermore, series with similar flower morphology, pompon or daisy-like, were more closely related than those with different phenotypes. DNA from all cultivars belonging to a series were also bulked to generate DNA profiles containing unique amplified products for each series. Polymorphic loci that were generated by the DAF technique can possibly be used for patent protection and phylogenetic studies, and may be useful in breeding chrysanthemums.