Transformed T-o orchardgrass (Dactylis glomerata L.) plants produced from highly regenerative tissues derived from mature seeds

A highly efficient and reproducible transformation system for orchardgrass (Dactylis glomerata L. cv. Rapido, 2n=4x=28) was established using micropro jectile bombardment of highly regenerative, green tissues derived from matu re seeds. These tissues, induced from embryogenic callus, were bombarded wi th a mixture of three plasmids containing the hygromycin phosphotransferase (hpt), phosphinothricin acetyltransferase (bar) and beta -glucuronidase (u idA; gus) genes. From 147 individual explants bombarded, 11 independent hyg romycin-resistant lines (7.5%) were obtained after an 8- to 16-week selecti on period using 30-50 mg/l hygromycin B. Of the 11 independent lines, ten ( 91%) were regenerable. The presence and integration of the transgene(s) wer e assessed using PCR and DNA blot hybridization. Coexpression frequency of the three transgenes (hpt.bar/uidA) in T-0 plants was 20%, and of two trans genes, either hpt/bar or hpt/uidA, 45-60%. Due to greenhouse conditions opt imized for the growth of other species, T-1 seed has not been obtained from these plants. While the inability to analyze progeny plants precludes the conclusive demonstration of stable transformation, the results of all molec ular and biochemical analyses of T-0 plants are consistent with the product ion of stably transformed plants. Frequent change in ploidy level was obser ved in transformed To orchardgrass plants. Plants from only three of the te n independent lines analyzed had the normal tetraploid number of chromosome s (2n=4x=28), while plants from seven lines (70%) were octaploid (2n=8x=56) . The octaploid plants had abnormal morphological features, such as narrowe r, thicker and more upright leaves.