Improvement of microspore transformation in barley (Hordeum Vulgare L.)
The purpose of this study was to first determine when DNA synthesis occurred in microspores of barley, and to subsequently assess the optimum stage to bombard them for transformation relative to the cell cycle. It was hypothesized that the introduction of foreign genes at the G1 stage in uninucleate microspores would produce homozygous transgenic plants. Experiments were conducted with two different pretreatments of spikes to induce microspore embryogenesis: cold (4°C) for 21d and cold plus 0.3M mannitol for 4d. After pretreatment, the microspores were isolated and placed in high osmotic medium for 4h prior to and 18h following bombardment and the influence of temperature during the high osmotic treatment periods was evaluated. Hourly DNA density studies during the 4h osmotic pre bombardment period indicated that the cell cycle progressed during this period at 25°C but probably did not proceed at 4°C. Using the green fluorescent protein (GFP) as the selection marker, a number of other factors were studied to determine their influence on success of the transformation process. Two promoters, CaMV (35S) and actin, were compared as were the two pretreatments of cold plus mannitol and cold alone. Post pretreatment temperatures for 4h pre and 18h post bombardment were evaluated. The use of AGP (arabinogalactan protein) in the culture medium for 7d was also tested. The results showed that the actin promoter attached to sGFP was more effective than the 35S promoter attached to pGFP, producing 30 of the 31 green transgenic plants. Keeping the microspores at 4°C in 0.5M mannitol plus sorbitol for 4h after isolation and prior to bombardment resulted in 9 of 12 transformed plants being haploid. However, most of the plants incubated at 25°C following isolation (16 of 19) were doubled haploid (DH) plants with 7 of 16 being homozygous for the GFP transgene. While DH plants homozygous for the GFP transgene were obtained following both pretreatments, more transgenic plants were recovered from the 21d cold pretreatment, making this the preferable pretreatment. Most of the transformed plants recovered (29 of 31) were from the cultures that contained AGP. Another objective was to show the relationship between induction of embryogenesis in microspores and chromosome doubling. High frequencies of spontaneous chromosome doubling during microspore culture in cereal species appear to be induced by treatments that block cell wall and microtubule formation during the first mitotic cell divisions in microspores, resulting in coenocytic cells in which the nuclei are able to fuse. A mannitol pretreatment to induce embryogenesis was also used to study nuclear fusion in these cells.