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Ethylene-based technologies for controlling ripening in climacteric fruit have been in widespread use for a number of years. Likewise, using chemicals that block ethylene synthesis or perception have been widely used to extend shelf life of a variety of horticultural commodities. In the last few years, our understanding of the molecular mechanisms for ethylene synthesis and perception has greatly expanded. Genes encoding the ethylene biosynthetic enzymes and the ethylene receptor have been cloned from many plant species, which has meant that molecular approaches to engineering reduced ethylene synthesis or perception are now reality. Scientists have been examining the feasibility of using molecular approaches to control ethylene in a variety of horticultural and ornamental species. They have shown that it is relatively easy to produce plants that are reduced in either synthesis or response to ethylene. However, scientists have uncovered some issues associated with commercial-level use of these transgenic plants. Overall, my results illustrate the great potential of the technology to control the rate of climacteric fruit ripening, abscission, and ethylene-induced senescence in multiple species, but using transgenes in many cases needs to be directed to target tissues through the use of tissue-specific transcriptional promoters. With that caveat in mind, there should be a strong future for improving the quality of a range of agronomic and horticultural species.
`Galia' is a high-quality muskmelon cultivar that is grown in green-houses or tunnels to maximize fruit yield and to help improve fruit quality. Maximum fruit quality and flavor are achieved when `Galia' are harvested at maturity. This however leads to reduced firmness and short shelf life. In vitro regeneration and transformation of `Galia' melon is a strategy that can be used to increase fruit shelf life. Melon cotyledons were transformed with the ACC oxidase gene in antisense orientation according to the protocol described by Nunez-Palenius et al. (2001, 2003). Experiments were conducted to compare fruit quality parameters between transgenic (TT) and wild type (WT) fruits from plants grown in greenhouse conditions. The melon plants were grown using commercial growing practices that included pruning and training to one vertical stem and the use of soilless media and drip fertigation. Wild type fruits were harvested at 37, 42, and 50 days after pollination (DAP), whereas transgenic fruits were harvested at 42, 50, and 56 DAP. TT fruits were harvested with that delaying period since their ripening process was slower than WT. Thirteen preharvest parameters were evaluated in transgenic and wild type fruits. Wild type and transgenic weight, lenght, width, soluble solids, tritatable acidity, pH, firmness, flesh thickness, seed cavity size and seed number parameters were not significantly different. Ethylene production and ACC oxidase from 42 DAP wild type fruits were greater than from transgenic fruits. Transgenic (ACC oxidase) galia melon fruits had a delayed fruit ripening process compared with wild type fruits.
In an effort to modify and study leaf senescence, we have produced several different transgenic petunia lines with altered leaf senescence phenotypes. Using two promoters from senescence-associated genes (sag12 & sag13) fused to the isopentenyl transferase (IPT) gene, which catalyzes the rate-limiting step of cytokinin production, we have produced transgenic petunia plants with delayed lower leaf senescence. We have observed that apparent “leaky” expression of IPT gives rise to plants with other morphological alterations such as increased branching habit and decreased root formation. Plants with delayed leaf senescence phenotypes were selected and bred to produce progeny that were evaluated in greenhouse experiments. Breeding characteristics, horticultural performance and reproduction of these plants will be discussed in terms of potential commercial benefits and limitations. Using the sag12 promoter to drive expression of the knotted (KN1) gene, we have also been able to engineer petunia plants with delayed lower leaf senescence. Initial progeny evaluations of sag12-KN1 petunias will also be discussed.
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.
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.
Cytokinins have been shown to delay the onset of leaf senescence. The focus of this project was to produce transgenic petunia (Petunia ×hybrida) plants that over-produced endogenous cytokinins in a senescence specific manner. This was achieved by transforming plants with the IPT (isopentenyl transferase) gene driven by the senescence-associated transcriptional promoter, PSAG12. Two independent transgenic events produced T1 and T2 generation seedling lines that demonstrated the desired nonsenescent phenotype in progeny trials. These lines were used to evaluate the horticultural performance of PSAG12-IPT petunia plants in terms of delayed senescence, rooting of vegetative cuttings, lateral branch growth, flower number, floral timing, and fruit set. Although both lines displayed a delayed senescence phenotype the two PSAG12-IPT transgenic lines differed from each other in regard to other horticultural traits. In addition to delayed leaf senescence, line I-1-7 also demonstrated a decrease in adventitious rooting and an increased number of branches during plant production. Line I-3-18 also demonstrated a delayed leaf senescence phenotype; however, plants of this line were not greatly altered in any other horticultural performance traits in comparison to wild-type `V26'. IPT transcript was detected in young fully expanded leaves of both lines, although mRNA levels were higher in I-1-7 plants. A greater than 50-fold increase in IPT transcript abundance was detected in leaves of transgenic plants following drought stress. These results demonstrate that it is possible to use PSAG12-IPT to produce transgenic plants with delayed leaf senescence but differences in plant morphology between transgenic lines exist, which may alter horticultural performance characteristics.
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.