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Kerrie McDaniel and David Lightfoot

Physiological differences between cttokinins are well documented. An explanation for these differences is that cytokinins differentially regulate genes. Gene response has been analyzed in cell culture and organized tissue of Phaseolus vulgaris L. cv. Great Northern. Two novel cDNAs, L-221 and L-22, have been identified that are cytokinin responsive. In leaf tissue L-221 was repressed by zeatin, benzyladenine and thidiazuron at 50 μM. In suspension cell culture mRNA abundance of L-221 remained constant regardless of cytokinin treatment. By contrast, the abundance of L-22 mRNA was increased differentially by treatment with each of the 3 cytokinins in leaf tissue. Suspension cells analyzed for expression of L-22 after cytokinin treatment also showed differential gene expression. S-1 Nuclease Protection Assays revealed that gene expression is a transient phenomenon dependent upon the time of cytokinin application and cytokinin concentration. Callus bioassays showed that dihydrozeatin and O-glucosylzeatin gave greater responses than the co-application of zeatin and dihydrozeatin or zeatin and O-glucosylzeatin. The conjugate and the reduction derivative also gave greater responses than zeatin alone. Effects of dihydrozeatin and O-glucosylzeatin on gene expression will be reported.

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A. Virginia Freire, David A. Lightfoot and John E. Preece

Efficient genetic transformation could enhance coffee breeding, which is limited by its long generation time and narrow genetic base. Three explant types of three coffee cultivars were inoculated with 14 strains of Agrobacterium spp. Callus and hairy roots were produced with 13 of the 14 strains tested. With A. tumefaciens, nopaline strains were more effective than octopine strains. Cucumopine and mannopine strains of A. rhizogenes were both effective in inducing hairy roots and callus. PCR amplification of a 0.72 Kb fragment of T-DNA encoding a portion of the ipt gene was achieved with DNA from A. tumefaciens strain A208 and with putatively transformed tissue inoculated with A208. No amplification was observed with virB in putatively transformed tissue which indicates it was not contaminated with Agrobacterium. We conclude that coffee can be genetically transformed by some Agrobacterium strains.

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William M. Proebsting, Nahla V. Bassil and David A. Lightfoot

Propagation of Corylus avellana stem cuttings may be limited by either root initiation or bud abscission. We divided juvenile shoots of 3 varieties growing in layering beds in mid-July into 4 or 5 3-node cuttings with leaves at the upper two nodes, except that terminal cuttings had one expanded leaf. Cuttings were treated with 5 mM IBA in 50% EtOH, a mixture of A. rhizogenes strains A7 + 22 or left untreated. IBA and bacteria stimulated rooting of cuttings from all shoot positions. Rooting of the terminal cuttings (<50%) was less than that of the sub-terminal cuttings (>80%). Bud retention was <50% on terminal cuttings, nearly 100% on sub-terminal cuttings. Using juvenile stock plants of various varieties, sub-terminal cuttings treated with Agrobacterium or 5 mM IBA may yield 70-90% cuttings with both roots and buds, Agravitropic roots, characteristic of genetic transformation, were observed on Agrobacterium-treated cuttings. Dot blots probed for TL-DNA were negative, however.

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William M. Proebsting, Nahla V. Bassil and David A. Lightfoot

Propagation of Corylus avellana stem cuttings may be limited by either root initiation or bud abscission. We divided juvenile shoots of 3 varieties growing in layering beds in mid-July into 4 or 5 3-node cuttings with leaves at the upper two nodes, except that terminal cuttings had one expanded leaf. Cuttings were treated with 5 mM IBA in 50% EtOH, a mixture of A. rhizogenes strains A7 + 22 or left untreated. IBA and bacteria stimulated rooting of cuttings from all shoot positions. Rooting of the terminal cuttings (<50%) was less than that of the sub-terminal cuttings (>80%). Bud retention was <50% on terminal cuttings, nearly 100% on sub-terminal cuttings. Using juvenile stock plants of various varieties, sub-terminal cuttings treated with Agrobacterium or 5 mM IBA may yield 70-90% cuttings with both roots and buds, Agravitropic roots, characteristic of genetic transformation, were observed on Agrobacterium-treated cuttings. Dot blots probed for TL-DNA were negative, however.

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A. Virginia Freire, John E. Preece and David A. Lightfoot

Silver maple has great potential as a biomass feedstock. We selected 21 elite silver maple clones representing 7 provenances located on east to west and north to south transects across the natural area of distribution. In addition five different red maples including one commercial cultivar as well as four interspecific hybrids between red and silver maple were compared to the silver maples. DNA was extracted using a modification of the CTAB technique (Murray and Thompson, 1980). Polymerase chain reaction was used with random primers from the OPF series (1-20) and primers used by Krahl et al. (1993). Polymorphism was detected at high frequency. Greater polymorphism was observed between species than within species. However, we have observed DNA concentration dependent polymorphism. RAPD technology has potential for determination of genetic relationship among silver maple clones.

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A. Virginia Freire, John E. Preece and David A. Lightfoot

Silver maple has great potential as a biomass feedstock. We compared three clones from each of seven provenances located on east to west and north to south transects across the natural range of silver maple and one red maple. DNA extracted by a modification of the CTAB technique (Murray and Thompson, 1980) was not suitable for RAPD analysis. Using this technique, polymorphism was either not reproducible or there was poor amplification for some clones. A new DNA extraction technique using PVPP, chloroform, and cesium chloride was tested (a modification of Yoon et al., 1991). this method yielded DNA that was more suitable for PCR amplification. Both RAPD and DAF (Caetano-Anolles and Gresshoff, 1994) methods were used for amplification. Polymorphism was detected among and within provenances. DAF was more efficient than RAPDs for determination of the genetic relationship among silver maple clones.

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Mei Guo, David A. Lightfoot, Machteld C. Mok and David W. S. Mok

Interspecific hybridization between Phaseolus vulgaris and P. coccineus results in mature seeds or abnormal embryo formation depending on the direction of the cross. In addition, differential fertility and reversion to parental types occur in later progeny populations, accompanied by recurrence of various embryo types. Normal and abnormal embryos exhibited isozyme patterns resembling P. vulgaris and P. coccineus parents respectively, suggesting that developmental abnormalities may be associated with specific combinations of parental genes. RFLP between parental species was examined and probes were selected for analyses of F2 populations. Differential transmission of alleles occurred for some RFLP markers. Statistical analyses were applied to detect possible association between probes and abnormal developmental events. The high incidence of interspecific polymorphism will also facilitate the construction of a linkage map in Phaseolus.

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Mei Guo, David A. Lightfoot, Machteld C. Mok and David W. S. Mok

Interspecific hybridization between Phaseolus vulgaris and P. coccineus results in mature seeds or abnormal embryo formation depending on the direction of the cross. In addition, differential fertility and reversion to parental types occur in later progeny populations, accompanied by recurrence of various embryo types. Normal and abnormal embryos exhibited isozyme patterns resembling P. vulgaris and P. coccineus parents respectively, suggesting that developmental abnormalities may be associated with specific combinations of parental genes. RFLP between parental species was examined and probes were selected for analyses of F2 populations. Differential transmission of alleles occurred for some RFLP markers. Statistical analyses were applied to detect possible association between probes and abnormal developmental events. The high incidence of interspecific polymorphism will also facilitate the construction of a linkage map in Phaseolus.

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Lynn M. Long, John E. Preece, Gerald R. Gaffney, J.W. Van Sambeek and David A. Lightfoot

Genetic transformation studies are aided by use of selection agents, such as antibiotics or herbicides. To determine the level of kanamycin to be used as a selection agent, cotyledonary stage somatic embryos from J. nigra lines J26 and J28, J. nigra × J. hindsii line S11, and J. regia line SU2 were placed on gelrite solidified WPM with 1 g/liter casein hydrolysate and 250 mg/liter cefotaxime and 3% (w/v) sucrose. Dosages for inhibiting secondary embryogenesis were 40 mg/liter kanamycin for J. nigra and J. nigra × J. hindsii and 100 mg/liter for J. regia. For the bialaphos experiments, somatic embryos of J. nigra lines J26 and J28 and J. nigra × J. hindsii line S11 were cultured on gelrite solidified LP medium with 0.5 g/liter casein hydrolysate and 3% (w/v) sucrose. Between 0.1 and 1.0 mg/liter bialaphos, inhibited secondary embryogenesis.

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Nahla V. Bassil, William M. Proebsting, Larry W. Moore and David A. Lightfoot

Hazelnut (Corylus avellana L.) softwood cuttings of the cultivars Ennis and Casina were propagated under mist during June and July 1987 and 1988. Rooting of stem cuttings was stimulated by both Agrobacterium and IBA treatment; however, IBA caused nearly complete bud abscission. Better rooting and bud retention were observed in `Casina' than in `Ennis' in 1988. Bud retention on Agrobacterium -inoculated cuttings improved as the cuttings approached the semi-hardwood stage. Six months after transplanting, Agrobacterium -inoculated hazelnut cuttings had an extensive root system, characteristic of hairy root. Although the mechanism remains unclear, strains of Agrobacterium rhizogenes are effective rooting agents in hazelnut and may cause less bud abscission than IBA. Chemical name used: 1 H -indole-3-butyric acid (IBA).