Date of Award
Master of Science
Plant, Soil and Environmental Sciences
Fred Allen, James Caponetti
Orchardgrass is the only Gramineae species whose somatic embryos progress to a fully mature (germinable) stage within one liquid medium. As embryos mature, the scutellar epidermis begins to dedifferentiate and cells are sloughed off. These cells divide and form new embryos creating a cyclic regeneration system. Genetic transformation of these cells provides the opportunity to mass produce transformed embryos. In this study, somatic embryos removed from leaves were transformed via microprojectile bombardment with a gene construct containing both the uidA gene coding for expression of the bar gene which inactivates phosphinothricin (PPT), the active ingredient in herbicides such as Basta and bialaphos. Both genes were driven with the ubi1 promoter. Embryos were cultured before bombardment on Schenk and Hildebrandt medium amended with 30 μM dicamba (SH-30) and containing 0.3 M each of mannitol and sorbitol for a 24 h pre and 24 h post bombardment osmotic treatment. The embryos were then transferred to fresh SH-30 medium and cultured in the dark at 21°C for 2 wk to form callus. Samples of the resulting calli were incubated in 5-bromo-4-chloro-3-indolyl β-D glucuronic acid (x-gluc) substrate to test for GUS expression. All embryos whose callus showed GUS expression were placed into suspension culture. The cultures were sieved through a 710 μm mesh screen at 30 d intervals and the medium and cells which passed through were returned to culture. From the tissue that was removed, 100 embryos were incubated in x-gluc to determine the number of transformed embryos present. The number of embryos expressing GUS was found to increase more than 2 fold between 30 d and 60 d after culture initiation and did not decrease until 120 d after initiation. One hundred remaining embryos were plated on SH medium without dicamba (SH-0) and incubated in 16 h light/8 h dark at 21 °C/15 °C in order to germinate. Regenerated plants were potted and kept in the greenhouse. A young leaf of each plant was brushed with a 0.1% solution of Basta to test for tolerance to the herbicide. A total of 143 plants were tested. Six displayed complete tolerance and 10 showed a localized reaction where the herbicide was applied. Of the six tolerant plants, one was confirmed by Southern blot analysis to contain the bar transgene.
Six cultures remaining from the above experiment were each sieved through a 710 μm and a 210 μm screen to create three fractions of different cell size. Each fraction was then split into two cultures. One culture was suspended in liquid SH-30 medium amended with 12 mM each of proline and serine plus 2.5 mg/L bialaphos added as a selective agent. The other culture was suspended in SH-30 medium with casein hydrolysate and used both as a control and to continue the culture. The control cultures were then split into a casein hydrolysate control and a control containing 12 mM each of proline and serine. At 14 d intervals, fresh weight of each culture was measured and viable cells were counted in 1 ml samples of each culture. At 49 d after culture initiation, all cultures were incubated in x-gluc to test for both the number of embryos expressing GUS and the extent to which GUS was expressed. It was observed that, although more embryos exhibited some amount of GUS expression in non-selective cultures, cultures which had undergone selection pressure displayed uniform GUS expression throughout the tissue.
Nash, Jennifer Lea, "Long term production of genetically transformed somatic embryos of orchardgrass (Dactylis glomerata) in suspension culture. " Master's Thesis, University of Tennessee, 1996.