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Margaret A. Norton and Robert M. Skirvin

Rubus laciniatus Willd. `Thornless Evergreen' (TE) is a chimeral blackberry with a thornless epidermis that overlies a genetically thorny interior. Most canes of TE produce leaves with 5 finely cut (lacinate) leaflets. Occasionally, canes appear which produce leaves with entire leaflets. Genetically pure thornless plants were regenerated from epidermal cells of chimeral TE with lacinate leaves. These regenerants exhibited somaclonal variation for growth habit, degree of thornlessness, and fruitfulness. All had lacinate leaves. When moved to the field, some of these regenerants produced canes with entire leaflets.

To examine the stability of the entire leaflet characteristic, plants were regenerated from lacinate leaves and entire leaves of both dwarf and normal pure thornless TE regenerants. Regenerants were rooted, moved to soil, and grown in the greenhouse for observation. Stability of this characteristic will be discussed.

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Lius Suwenza and Richard Manshardt

Nine transgenic papaya clones, produced previously by microprojectile bombardment, are being characterized for frequency of somaclonal variation. Five clones have proven to be hermaphrodite. Four of these appear to have normal fertility, while the fifth has drastically reduced pollen fertility, averaging about 15% stainability with acetocarmine. Four other clones are pistillate and appear to have normal fertility, with one exception which has been demonstrated to be tetraploid (2n=36 chromosomes). One of twelve plants in a pistillate clone was a somaclonal mutant showing altered leaf and flower morphology. The transgenic clones and their sexual progenies are also being evaluated at the molecular level for expression and segregation of npt, gus, and the coat protein (CP) of papaya ringspot virus (PRV), as well as for PRV resistance.

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Freddi A. Hammerschlag, Ghazala Hashmi, Robin Huettel, Dennis Werner, and David Ritchie

One approach for obtaining useful genetic variation is to select for somaclonal variants generated by tissue culture techniques. Increased levels of resistance to bacterial leaf spot (Xanthomonas campestris pv. pruni) have been observed in toxin-selected and unselected peach regenerants in vitro, in the greenhouse and under field conditions. Peach regenerants have also demonstrated increased levels of bacterial canker (Pseudomonas syringae pv. syringae) and root-knot nematode (Meloidogyne incognita) resistance. Random amplified polymorphic DNA (RAPD) primers have been used to study genetic variation at the DNA level among the somaclonal variants. Sixty RAPD primers (10-mers) were screened and 10 proved useful as markers to detect polymorphisms, thus establishing a genetic basis for somaclonal variation. These studies demonstrate the feasibility of using tissue culture techniques to generate fruit trees with increased levels of disease resistance.

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David A. Evans

For many branded food products, the raw material is purchased as a commodity. Food companies provide added-value based on processing technology or marketing. The tools of plant biotechnology, which shorten the time for crop improvement and permit development of novel germplasm, offer the food industry the opportunity to modify raw materials and develop proprietary branded products. Such modifications will permit development of plant cultivars specifically selected for traits with added-value for the processor or the consumer. Biotechnology-developed cultivars offer the opportunity to develop produce that can be branded. The cell genetics tools of clonal propagation, somaclonal variation, gametoclonal variation, and protoplast fusion permit new cultivar development in an intermediate time scale, making them attractive for market introduction of proprietary products. When integrated with conventional breeding, these intermediate-term technologies will permit modification of fruits and vegetables over the course of the next several years. The longer-term technologies of plant genetic engineering will continue to have an impact on manipulation of specific traits, resulting in second-generation products specifically designed for consumers.

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Francisco A. Ferreira, Dae-Geun Oh, and Edward C. Tigchelaar

Six F1's involving 6 multiple genetic marker stocks and a common inbred parent (PU812) were cultured to study the genotypic effect on regeneration ability and frequency of somaclonal variation in R0 for the known heterozygous marker genes. Leaf discs 7 mm in diameter were excised from young fully expanded leaves of 6-7 week old plants, and cultured on MS medium supplemented with cytokinins (Kinetin, Benzyladenine) and IAA. With few exceptions, the parents and F1's responded similarly to different hormone combinations. The beat hormone combinations for shoot formation were 4 mg/l Kinetin + 0.5 mg/l IAA and 2.3 mg/l BA + 0-0.18 mg/l IAA.

Only 2 of the 6 multiple marker stocks and the common inbred parent (PU812) were successfully regenerated. Four of the six hybrids between PU812 and the multiple markers were readily regenerated, whereas 2 hybrids failed to regenerate with several different hormonal combinations. No mutations have been observed for the known heterozygous markers in 76 R0 tissue culture regenerants.

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Michael Marcotrigiano and Susan P. McGlew

In an effort to accelerate breeding programs and to study somaclonal variation, a micropropagation system was devised for cranberries (Vaccinium macrocarpon). Using a factorial design, explants taken from greenhouse grown plants were placed on Anderson's medium containing different concentrations of 2ip' GA3, and IBA, with 4 cultivars tested over 3 subcultures. In other experiments, explant source, macro and micro salt formulations, and rooting treatments, were studied. Optimal multiplication and shoot quality occurred when single node explants taken from greenhouse grown plants were placed on Anderson's media containing 150 uM 2iP, 1.0 uM IBA and no GA3. Histological examinations indicate that initial response is axillary bud proliferation but upon subculture adventitious shoot formation may be possible. Proliferated shoots could be rooted ex vitro in plug trays under plastic tents and without hormone treatments. Optimal rooting occurred under high light conditions in a 1:1 (v:v) peat:sand mix. Plants were easily transplanted into the field in spring and will be evaluated by comparison to conventionally propagated material.

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Luisa Santamaria and Sherry Kitto

Solanum quitoense, also known as naranjilla or lulo, is a native species of Ecuador and Colombia. Its value is based on the uncommon sweet-sour flavor of its fruits, which is appreciated in the national and international markets. The worst problem for this crop is the root-knot nematode Meloidogyne incognita. The main objective of our research is to develop root-knot nematode–resistant naranjilla via somaclonal variation. Seeds of Solanum quitoense `Baeza' germinated quicker than those of `Dulce'. Seeds given a 2-week dark treatment had 100% germination compared to 75% germination for seeds placed under lights (16-h photoperiod, 60 mmol·m–2s–1). Single-node explants proliferated an average of nine nodes after 1 month of culture. Microcuttings (two nodes, 3.5 cm) stuck in sand and placed under a humidity dome under mist had an average of five roots averaging 25 cm in length after 3 weeks. Stems regenerated shoots better than petioles or leaves and explant orientation/polarity had no effect on regeneration. Root cultures of Solanum quitoense inoculated in vitro with Meloidogyne incognita showed susceptibility to root-knot nematodes.

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Orlando McMeans and Robert M. Skirvin

To isolate unique fruit colors and look for somaclonal variation among regenerants, a regeneration protocol was established for various cultivars of striped apples (`Mailing 26', `Mutsu', `Regal Gala', `Summerland Red', and `Fuji'). Leaves were harvested from in m-grown plants, transferred to regeneration media [MS + NAA (5.4 μM) + TDZ (3 μM)], stored in the dark for 3 to 4 weeks, then moved to controlled light conditions, where adventitious shoot regeneration was observed. Developing shoots were transferred to proliferation medium and screened for their red or green phenotype by placing them on MS media containing various concentrations of sucrose (30, 45, 60, 75 and 90 g\L) and BA (0, 2.5, 5, 7.5, 10, 12.5 and 15 μM). Some of the regenerated apple shoots exhibited red color soon after being taken from the dark treatment. Others were less distinct, with colors ranging from dull green to a green-pink mixture. The red and green shoots are now being rooted and will be transferred to the field, where they will be grown to maturity.

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Richard Meilan, Caiping Ma, and Steven H. Strauss

We assessed the stability of transgene expression in 79 transgenic lines (i.e., transformation events) of hybrid poplars during several years of field trials. The transgenic lines were comprised of 40 lines of hybrid cottonwoods (P. trichocarpa × P. deltoides) that were grown at three field sites, and 39 lines of hybrid aspens (section Leuce, P. alba × P. tremula) that were grown at a single field site. All the lines were transformed with a binary construct that included two genes that confer tolerance to glyphosate (GOX and CP4), a gene encoding resistance to the antibiotic kanamycin (nptII), and a visible marker gene (GUS). Agrobacterium tumefaciens was used for transformation; callogenesis and organogenesis occurred under kanamycin selection. In addition to repeated applications of herbicide to test stability of transgene expression, for the first time, we challenged ramets of 40 lines that had not previously been tested for herbicide resistance in their fourth season of vegetative growth. We report on the stability of herbicide resistance and GUS expression and evidence for somaclonal variation in growth and leaf morphology.

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Jinggui Fang, Chih Cheng Chao, Richard J. Henny, and Jianjun Chen

Plant tissue culture can induce a variety of genetic and epigenetic changes in regenerated plantlets, a phenomenon known as somaclonal variation. Such variation has been widely used in the ornamental foliage plant industry as a source for selection of new cultivars. In ornamental aroids alone, at least 63 somaclonal-derived cultivars have been released. In addition to morphological differences, many somaclonal aroid cultivars can be distinguished by amplified fragment length polymorphism (AFLP) analysis. However, a few cultivars have no detectable polymorphisms with their parents or close relatives by AFLP fingerprints. It is postulated that DNA methylation may be involved in the morphological changes of these cultivars. In this study, methylation-sensitive amplification polymorphism (MSAP) technique was used to study DNA methylation in selected somaclonal cultivars of Alocasia, Aglaonema, Anthurium, Dieffenbachia, Philodendron, and Syngonium. Results showed that polymorphisms were detected in the somaclonal cultivars, suggesting that DNA methylation polymorphisms may associate with tissue culture-induced mutation in ornamental aroids. This is the first study of methylation variation in somaclonal variants of ornamental foliage plants. The results clearly demonstrate that the MSAP technique is highly efficient in detecting DNA methylation events in somaclonal-derived cultivars.