Since the first demonstration of GFP from the jellyfish Aequorea victoria as a vital reporter for gene expression in both bacteria and Caenorkabditis elegans (Chalfie et al., 1994), GFP has attracted widespread interest and is considered to have severa1 advantages over other visual marker genes. First, the fluorescence emission of GFP does not require a cofactor or a substrate; fluorescence results from an interna1 p-hydroxybenzylidene-imidazo-lidinone chromophore generated by cyclization and oxidation of a SerTyr-Gly sequence at amino acid residues 65 to 67 (Cody et al., 1993). Detection of GFP in living cells thus only requires excitation by light at 395 or 470 nm. In contrast, the assay of GUS expression is cytotoxic, firefly luciferase (Ow et al., 1986;Millar et al., 1995) requires an exogenous substrate (luciferin) for detection, and plant anthocyanins (Klein et al., 1989;Lloyd et al., 1992) are generally useful only in mature, differentiated cells.The second advantage of GFP is that it is relatively small (26.9 kD) and can tolerate both N-and C-terminal protein fusions, lending itself to studies of protein localization and intracellular protein trafficking (Wang and Hazelrigg, 1994;Davis et al., 1995;Kaether and Gerdes, 1995). Another advantage of GFP is that GFP mutants with shifted wave-

Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies of transformed kanamycin-resistant calli were produced, more than 80% of which were induced to form somatic embryos. Somatic embryos were germinated, and plants were regenerated and transferred to soil. Transformation was confirmed by opine production, kanamycin resistance, immunoassay, and DNA blot hybridization. This process for producing transgenic cotton plants facilitates transfer of genes of economic importance to cotton.

The European corn borer [ECB; Ostrinia nubilalis (Hübner)] is an economically significant pest of corn (Zea mays L.). The ability to routinely transform corn has broadened the control options available to include the introduction of resistance genes from sexually incompatible species. In this study, microprojectile bombardment was used to introduce synthetic versions of cryIA insecticidal protein genes from Bacillus thuringiensis subsp, kurstaki (Btk) into embryogenitcis sue of the Hi‐II] (A188/B73 derivative) genotype of corn. Of 715 independent transgenic calli produced, 314 (44%) had insecticidal activity against tobacco hornworm (Manduca sexta L.) larvae. Plants were regenerated, self‐pollinated when possible, and crossed to B73. First‐generation progeny of 173 independent Btk‐protein expressing calli were evaluated under field conditions with artificial ECB infestations in 1992 or 1993. Approximately half (89/173) segregated in single‐gene manner for resistance to first‐generation ECB leaf‐feeding damage. All of the 89 lines evaluated in 1992 or 1993 for resistance to second‐generation ECB exhibited less stalk tunneling damage than the non‐transgenic controls. In 1993, 44% (34177) of the lines tested had ≤2.5 cm of tunneling, compared to severe damage (mean = 45.7 cm) in the B73 × Hi‐II controls. Experiments are in progress to evaluate the effect of the introduced genes on yield and other agronomic properties.

5‐Enol‐pyruvylshikimate‐3‐phosphate synthase from Agrobacterium sp. CP4 (CP4 EPSPS) confers tolerance to the nonselective herbicide glyphosate (marketed under the trade name Roundup1) when sufficiently expressed in transgenic plants. Dual CP4 EPSPS transgene cassettes were transformed into corn (Zea mays L.) under the transcriptional regulatory control of the rice (Oryza sativa L.) actin 1 (P‐Ract1) and the enhanced Cauliflower mosaic virus 35S (P‐e35S) promoters, respectively, to impart fully constitutive expression in corn. Resulting events were tested for lack of chlorosis and malformation injury after two sequential applications of 1.68 kg acid equivalents (a.e.) ha−1 glyphosate. Agronomic parameters, male fertility, appropriate Mendelian segregation of the trait, plus characteristics of the transgenic integration site were also evaluated. From this selection process, the NK603 event was chosen for commercialization as the event that embodied the most optimal profile of tolerance, agronomics, and molecular characteristics. The NK603 event exhibited high glyphosate tolerance from one transgenic locus bearing a single copy of the dual cassettes integrated into the corn genome with a minimum of target sequence disruption. Trait expression in the NK603 event has remained stable over more than eight generations as shown through tolerance testing, western blots of CP4 EPSPS accumulation, and Southern blot analysis of the transgene.

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