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Hector G. Nunez-Palenius, Daniel J. Cantliffe, Harry J. Klee and Don J. Huber

Embryo abortion and empty seeds after self-pollination occur in some transgenic (ACO antisense) `Galia' male parental lines. An embryo-rescue system in this melon was developed to save potential viable embryos. To obtain the best and reliable embryo-rescue technique, several parameters were used including an improved (five new supplements) nutrient medium (named E-21) from the E-20A basic medium (Sauton and Dumax de Vaulx, 1987), an inoculation system (removing the embryo from the seed or intact seed), and the use of different fruit harvesting dates of the wild type and a transgenic `Galia' male parental line. Fruits of wild type (WT) and transgenic (ACO gene in antisense orientation) `Galia' male parental line were harvested at 4, 10, 17, 24, and 30 days after pollination (DAP). Fruits were surface sterilized by dipping in a 20% commercial bleach solution for 30 minutes. Subsequently, seeds were removed from fruit under sterile conditions. These seeds were either used to dissect the embryos or placed directly with the hilum facing E-20A or E-21 medium. Seedlings from all treatments were transferred to E-21 elongation medium, incubated 4 weeks, and transferred to soil to evaluate growth. The efficiency of this technique was greater when the time after pollination (4, 10, 17, 24, and 30 DAP) to rescue the embryos was increased. Thus, 30 DAP was the best time to rescue the embryos. The number of rescued embryos using E-21 medium was greater than with E-20A. We did not find any significant differences in survival efficiency rate between WT and transgenic embryos. We have obtained a competent embryo-rescue technique for WT and transgenic `Galia' male parental line, which can be applied to rescue valuable GMO hybrid-melon embryos.

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Hector G. Nunez-Palenius, Daniel J. Cantliffe, Harry J. Klee and Don J. Huber

Pollen germination timing has a paramount role in fertilization of a flower. Rapid germination and outgrowth of a pollen tube that penetrates the stigma is required. Physical and biological factors can affect pollen germination timing. The objective of this study was to determine if ACC oxidase antisense gene expression could influence in vitro pollen germination and in vitro pollen tube length growth. A transgenic (ACC oxidase antisense) `Galia' male parental line had a reduced fruit set compared to its wild type. Likewise, embryo abortion and empty seeds after self-pollination in a `Galia' male parental line were observed. Wild type and transgenic `Galia' male parental line melon plants were grown in a greenhouse according to the practices of Rodriguez (2003). Male flowers were collected from these plants between 10 to 12 am; pollen was obtained by dipping the anther in germination medium (10.25% sucrose, 0.031% calcium nitrate, 0.015% boric acid, 0.0075% KNO3, and 0.016% MgSO4) at 25 °C and analyzed immediately, either for total percentage of germination after 5 minutes of incubation or to measure pollen tube growth rate every 5 minutes during 1 hour. Each flower provided an average of 250 pollen grains. Assays were conducted by using the “Hanging Drop Method” (Okay and Ayfer, 1994). Percentage of pollen germination in WT `Galia' male parental line was greater than the transgenic line. Likewise, in vitro pollen tube growth in wild type `Galia' melon was greater than pollen from the transgenic line. Possibly the ACC oxidase antisense gene expression in `Galia' male parental line may have had an influence on the reduced fruit set observed.

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Donna J. Clevenger, James E. Barrett, Harry J. Klee and David G. Clark

Pollen viability, in-vivo pollen tube growth, fruit ripening, seed germination, seed weight, whole plant vigor, and natural flower senescence were investigated in homozygous and heterozygous transgenic ethylene-insensitive CaMV35S::etr1-1 petunias (Petunia ×hybrida `Mitchell Diploid'). Homozygous or heterozygous plants were used to determine any maternal and/or paternal effects of the CaMV35S::etr1-1 transgene. All experiments except for those used to determine natural flower senescence characteristics were conducted in both high and low temperature greenhouses to determine the effect of temperature stress on transgenic plants when compared to wild-type. Results indicated that ethylene-insensitive plants had a decrease in pollen viability, root dry mass, seed weight, and seed germination. Fruit ripening, seed germination, and seed weight were maternally regulated. In contrast, the CaMV35S::etr1-1 transgene is completely dominant in its effect on natural flower senescence.

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John W. Scott, Elizabeth A. Baldwin, Harry J. Klee, Jeffrey K. Brecht, Stephen M. Olson, Jerry A. Bartz and Charles A. Sims