Transgenic orchardgrass (Dactylis glomerata) plants by direct embryogenesis from microprojecticle bombarded leaf cells.

Young leaf tissue of orchardgrass (Dactylis glomerata L.) was placed on Schenk and Hildebrandt medium containing 30 µM dicamba. Microprojectiles coated with DNA containing the selectable bar gene (Basta® tolerance) and the reporter gene uidA coding for ß-glucuronidase (GUS), both driven by the maize ubiquitin promoter (Ubi1), were propelled into the tissue with a particle inflow gun. Transient GUS expression was observed as blue spots of various sizes on leaf segments. Somatic embryos staining entirely blue were also produced, and embryos germinated on medium containing 3.0 mg 1-1 bialaphos. Leaves of 67 putative transformed plants were painted with 0.1% Basta. Ten showed no reaction, and 6 showed only a localized response. Cultured leaf segments from tolerant plants also produced somatic embryos that expressed GUS. The genetic transformation was confirmed by Southern blot hybridization and PCR analyses of T0 plants and by PCR analyses of somatic embryos produced from T0 plants.

Micropropagation of switchgrass by node culture.

Switchgrass (Panicum virgatum L.) is naturally outcrossing; therefore, maintenance of genotype is difficult through sexual propagation. The objective of this study was to develop a micropropagation procedure for the multiplication of desired or selected genotypes. Nodal segments were surface sterilized, split longitudinally, and placed with the cut surface on solid MS medium containing 30 g L-1 maltose and different concentrations of 6-benzylaminopurine (BAP) as the only growth regulator. Best shoot proliferation was obtained with 12.5 micromoles BAP and culture at 29 degrees C. Shoots were easily rooted on MS medium without BAP. Under optimum conditions, it is possible to produce approximately 500 plantlets from one parent plant in 12 wk.

Growth, proline accumulation and water relations of NaCl-selected and non-selected callus lines of Dactylis glomerata L.

Sodium chloride-tolerant calli were selected from leaf-derived embryogenic calli of Dactylis glomerata L. on agar solidified medium supplemented with 200 mM NaCl, a concentration lethal to non-selected calli. Growth characteristics, water relations and proline accumulation pattern were compared in selected and non-selected lines. The objective was to gain an understanding of the mechanism(s) of tolerance in the NaCl-tolerant line. Growth in the selected line, as expressed in terms of tolerance index (ratio of fresh wt. on NaCl medium:fresh wt. on NaCl free medium x 100), was greater than that of the non-selected line at all levels of NaCl between 50 and 300 mM. There was no significant difference in proline accumulation in the selected and non-selected lines. Maintenance of turgor by osmotic adjustment was observed in the non-selected line despite decreased growth. In contrast, the selected line lost either the need or the ability to adjust osmotically. There was little or no increase in symplastic osmolality in the selected line when exposed to NaCl. Presumably, selection was made for a salt-excluding tissue that has lost the ability to accumulate solutes and adjust turgor with NaCl stress.