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Dirk Vuylsteke, Rodomiro Ortiz, Shaun Ferris and Rony Swennen

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Michael Marcotrigiano, Thomas H. Boyle, Pamela A. Morgan and Karen L. Ambach

Nuclear-controlled leaf variegation was studied among Coleus × hybridus Voss (formerly C. blumei Benth.) cultivars propagated by seed and as shoot cultures on Murashige and Skoog (MS) medium + 1 to 3 mg BA/liter. Cultivars tested possessed pattern chlorophyll variegation and either pattern or nonpattern anthocyanin variegation. The gene controlling an albino midrib region appears to be fairly stable, with only 2% of the micropropagated plantlets having a solid-green leaf characteristic, a characteristic that was always inherited following selfing. Pattern anthocyanin variegation (PAV) was fairly stable, while nonpattern anthocyanin variegation (NAV) was very unstable. In addition, variants from pattern-variegated phenotypes produced offspring identical to their parent following selfing. In contrast, variants of nonpattern cultivars, when selfed, yielded offspring identical to the original cultivar, identical to the variant, or novel phenotypes. When variants were returned to culture, those derived from cultivars with PAV were more stable than those derived from nonpattern cultivars. In Coleus, micropropagation may induce epigenetic and/or heritable changes in leaf variegation. Cultivars with NAV are less stable than cultivars with PAV. Chemical names used; N-(phenylmethyl)-lH-purine-6-amine [benzyladenine (BA)].

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Mohamed F. Mohamed, Dermot P. Coyne and Paul E. Read

Plant regeneration has been achieved in two common bean lines from pedicel-derived callus that was separated from the explant and maintained through successive subcultures. Callus was induced either on B5 or MS medium containing 2% sucrose and enriched with 0.5 or 1.0 mg thidiaznron/liter alone or plus various concentrations of indoleacetic acid. The presence of 0.07 or 0.14 g ascorbic acid/liter in the maintenance media prolonged the maintenance time. Up to 40 shoot primordia were observed in 4-week-old cultures obtained from 40 to 50 mg callus tissues on shoot-induction medium containing 1-mg benzyladenine/liter. These shoot primordia developed two to five excisable shoots (>0.5 cm) on medium with 0.1-mg BA/liter. A histological study confirmed the organogenic nature of regeneration from the callus tissues. The R2 line from a selected variant plant showed stable expression of increased plant height and earlier maturity. Chemical names used: ascorbic acid, N- (phenylmethyl)-1H-pnrin-6-amine [benzyl-adenine, BA], 1H-indole-3-acetic acid (IAA), N- phenyl-N'-1,2,3-thiadiazol-5-ylurea [thidiazuron, TDZ].

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G. Fassuliotis and B.V. Nelson

`Gulfstream' and `Charentais' muskmelons (Cucumis melo. L.) plants were regenerated by in vitro culture to increase their genetic variability for resistance to root-knot nematodes (Meloidogyne spp.). While no genetic variability for root knot resistance was found, regenerated plants exhibited other traits that varied from the donor cotyledons. Chromosome counts confirmed that >75% of the somaclonal variants were tetraploid (2n = 24; 4n = 48). Tetraploids consistently exhibited micro- and macroscopic morphological changes that enabled distinction between tetraploids and diploids without chromosome counts; tetraploids contained enlarged stomates with more chloroplasts in the guard cells and pollen with a high percentage of square-appearing shapes. Tetraploids exhibited distinctive macroscopic morphological changes, including differences in leaf structure, fruit shape, blossom-end scar, number of vein tracts, and seed size.

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Jim Hruskoci and Paul E. Read

In an effort to increase somaclonal variation in blueberry, a protocol was developed to regenerate viable shoots from internode segments. The explant consisted of the last-formed, fully developed internode taken from 3 different genotypes of Vaccinium grown in vitro. Explants were cultured 6 weeks on Zimmerman's Z-2 medium supplemented with 2iP, zeatin, thidiazuron, kinetin, or BA at concentrations of 5, 25, 50, and 100 uM. Explant response to the treatments varied and included: no response, callus growth only, callus growth and subsequent shoot formation originating from the callus mass, and adventitious shoot formation directly from the internode segment without an intervening callus. Greatest shoot regeneration (20-25 shoots/explant) was obtained on medium supplemented with zeatin at 5, 25, and 50 uM, however treatment response was not consistant across all genotypes. Regenerated shoots could be readily sub-cultured, rooted in soil mix and will be evaluated for somaclonal variation.

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G.R. de L. Fortes, A.M. R. Vieira and D.L. Leite

Somaclonal variation has been one way to create variants that could be used in the breeding programs. However, initial explants may not be useful if they show small leaves or nondeveloped stems. The aim of this work was to find a tissue culture medium so that potato shoots cultured in vitro could regenerate somatic material for use in trials aimed at somaclonal variation. Shoots of `Baronesa' and `Monte Bonito' were inoculated in media with or without activated charcoal (3.0 gliter–1), BAP (1.0 gliter–1), and different MS salt concentrations (50%, 75%, and 100%). After 30 days in controlled conditions (25C, 16-h photoperiod, and 2000 lux), BAP with activated charcoal improved the percentage of dry matter, and at higher MS salt concentrations, a better response was achieved for `Monte Bonito'. However, the presence of activated charcoal improved leaf development and stimulated higher shoot and bud formation, especially for `Monte Bonito'. This somatic material can be used to initiate callogenesis trials successfully.

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Ghazala P. Hashmi, F.A. Hammerschlag, R.N. Huettel and L.R. Krusberg

Somaclonal variation has been reported in many plant species, and several phenotypic and genetic changes, including pathogen and pest resistance, have been described. This study was designed to evaluate somaclonal variation in peach [Prunus persica (L.) Batsch] regenerants in response to the root-knot nematode, Meloidogyne incognita (Kofoid & White) Chitwood. Regenerants SH-156-1, SH-156-7, SH-156-11, and SH-156-12, derived from `Sunhigh' (susceptible) embryo no. 156, and regenerants RH-30-1, RH-30-2, RH-30-4, RH-30-6, RH-30-7, and RH-30-8, derived from `Redhaven' (moderately resistant) embryo no. 30, were screened in vitro for resistance to the root-knot nematode. Under in vitro conditions, fewest nematodes developed on regenerants SH-156-1 and SH-156-11, `Redhaven', and all `Redhaven' embryo no. 30 regenerants. The most nematodes developed on `Sunhigh', `Sunhigh' seedlings (SHS), and regenerant SH-156-7. Nematodes did not develop on `Nemaguard'. In greenhouse tests, fewer nematodes developed and reproduced on the no. 156-series regenerants than on `Sunhigh'. Under in vitro conditions, significant differences among uninfected (control) regenerants, cultivars, and rootstock `Nemaguard' were observed for shoot height and fresh root weights. Significant differences were also observed among infected regenerants, cultivars, and `Nemaguard' for these characteristics, but differences were not observed between control and infected regenerants. Different concentrations of α-naphthaleneacetic acid in half-strength Murashige and Skoog salt medium induced rooting of two peach cultivars, one rootstock, and four regenerants.

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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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Jude W. Grosser

Citrus protoplast technology has advanced to where several practical applications in variety improvement and plant pathology are routine. We will report on progress in the following areas: somaclonal variation—`Valencia` and `Hamlin' sweet orange protoclones have been selected for improved juice color, higher soluble solids, seedlessness, and altered maturity dates; somatic hybridization for scion improvement—allotetraploid breeding parents have been created from numerous combinations of elite parental material, and are now being used as pollen parents in interploid crosses to produce seedless triploid varieties; somatic hybridization for rootstock improvement—numerous somatic hybrids combining complementary rootstock germplasm are under commercial evaluation and several look promising for wide adaptation, improved disease resistance, and tree size control; transformation—an alternative protoplast-based transformation that utilizes EGFP for selection has been developed; virus resistance assays—a protoplast-based assay is being used to screen varieties and candidate sequences for resistance to citrus tristeza virus at the cell level, saving time and greenhouse space.