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Z. M. Cheng, N. O. Shi and C. G. Wang

Isozymes have been widely used as markers in cultivar identification, gene mapping and evolutionary studies. However, isozyme data have not been reported in elm species. This work was to develop an optimal system to analyze isozyme variations in American elm and Siberian elm. Two isolation methods were used to extract proteins from young leaf tissues and protein samples were separated by starch gel electrophoresis with three buffer systems. Eight isozymes have been detected in American elm (Ulmus americana) and Siberian elm (Ulmus pumila). Six out of these isozymes (DIA, GOT, MED, ALP, MNR, ACP) showed difference between the two elm species. These results suggest that isozymes can be used as markers in genetic studies in elms.

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Kimberly A. Pickens, (Max) Z.-M. Cheng and Stephen A. Kania

The mitotic inhibitors, colchicine and oryzalin, were evaluated for their effects on callus, adventitious shoot formation, and tetraploid induction of Euphorbia pulchurrima `Winter Rose'. In vitro grown leaf sections were placed on various media supplemented with either colchicine or oryzalin at various concentrations for 1 to 4 days. Colchicine was less damaging to leaf tissues than oryzalin. On various colchicine-containing media, prolific calluses were produced and adventitious shoot formation was observed. Regenerated shoots were found to be diploid as determined by flow cytometry. On media supplemented with oryzalin (28.9 μm to 144 μm), leaf tissues produced callus but failed to form adventitious shoots. Samples of calluses produced on oryzalin-containing media were subject to analysis using flow cytometry and were found to be diploid. These results suggest that the colchicine is less toxic on poinsettia tissues and shoot induction than oryzalin. Additional experiments are needed to establish a protocol for in vitro induction of poinsettia tetraploid with colchicine and oryzalin.

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M.J. Bosela, J.P. Schnurr, Z.-M. Cheng and W.A. Sargent

Three elite hybrid aspen, Populus grandidentata × P. canescens, P. tremuloides × P. tremula, and P. tremuloides × P. davidiana, have been transformed with Agrobacterium tumefaciens strains LBA4404 and EHA105 carrying kanamycin resistance and GUS genes. The leaves of micropropagated shoots were co-cultivated with Agrobacterium for 65 to 72 hr and then transferred to callus-induction medium with 80–120 mg/L kanamycin in the dark. After 2 weeks, the leaves were transferred to shoot-induction medium under 18-hr photoperiod. Regenerated shoots were verified for transformation by histochemical staining and PCR. Transformed shoots rooted and were transplanted to soil. The three hybrid clones differed widely in their medium requirements for regeneration and in their competence for transformation. The leaves of P. grandidentata × P. canescens callused vigorously on a wide variety of media. In a typical transformation experiment, 30% to 60% of infected leaves produced putatively transformed calli (up to 10 calli per leaf). The origin of these calli and the frequency of shoot formation depended on the Agrobacterium strains. The calli from EHA105-infected leaves produced shoots within six weeks of co-cultivation and at high frequencies (70% to 90%). However, the calli from LBA4404-infected leaves produced shoots more slowly and at much lower frequencies (5% to 10%). Delaying selection for 2 weeks was found to lower the transformation frequency. Putatively transformed calli were obtained from P. tremuloides × P. tremula, and P. tremuloides × P. davidiana hybrids at frequencies of only 2% to 3%. The calli regenerated from P. tremuloides × P. davidiana leaves were very small, but they continued to grow upon being transferred to shoot-induction media and have started to produce shoots. The calli from leaves of P. tremuloides × P. tremula were much larger and they produced shoots more quickly. This transformation protocol is currently being used to introduce rooting genes into these hybrids to improve their rooting from hardwood cuttings.

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Z. M. Cheng, N. O. Shi, L. Tokach and B. K. Gaschk

Shoot regeneration was obtained from leaf tissues of American (Ulmus americana) and Siberian elm (U. pumila) seedlings germinated in vitro and in greenhouse. Murashige and Skoog (MS, 1962) media supplemented with 4 levels of BA (0, 5, 10, 15, 20 μM) and 3 levels of IBA (0, 2.5, 5.0 μM) were tested in a factorial design to find an optimal hormonal combination for shoot regeneration. Shoot regeneration was obtained from both species within 3-4 weeks in a wide range of media. The highest regeneration rate (50%) of American elm was in the medium containing 10 μM BA and 2.5 μM IBA. Incubation under the light was essential for a higher rate of regeneration. Gelrite was found as a better solidifying agent than agar. The progress is under way to achieve transgenic elms by combining this regeneration system with Agrobacterium-mediated transformation.

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Z-M. Cheng, A.A. Boe, J.P. Schnurr and D.E. Herman

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Joel G. Reiten, Chiwon W. Lee, Z.M. Cheng and Ronald C. Smith

Seeds of a Kentucky bluegrass cultivar (Poa pratensis 'SD Common') and two native buffalograss (Buchloe dactyloides) strains, Texas (TX) selection and North Dakota (ND) selection, were tested for their germination tolerance to increasing levels of NaCl at 0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2% in solution. Both the TX and ND strains of buffalograss exhibited a high degree of salt tolerance with the upper limit of seed germination at 2.8% NaCl (2% to 7% germination after 6 days). At high NaCl concentrations, however, percent seed germination was much greater in the ND strain than the TX strain of buffalograss. For instance, seed germination at 0.8% NaCl was 90% in ND strain and 53% in TX strain as compared to the control. Kentucky bluegrass was least tolerant to NaCl with the upper limit of seed germination at 0.4% NaCl (14.7% germination in 6 days). Seed germination in Kentucky bluegrass was completely inhibited at 0.6% NaCl.