uses a variant Bt Cry3Bb1 insecticidal protein (Donovan et al., 1992). Cry3Bb1 is known to be biologically active The corn rootworm (CRW; Diabrotica spp.) is one of the most against several species within the Coleopteran family serious pests of corn in the USA. Chemical insecticides and crop rotation have been the only two options available to growers for Chrysomelidae, including the western corn rootworm, managing CRW. Unfortunately, both of these tactics can be ineffective Diabrotica virgifera virgifera LeConte (Rupar et al., as a result of either resistance or behavioral modifications. In this 1991). The biological activity of this protein against D. paper, we describe transgenic maize (Zea mays L.) hybrids that control virgifera virgifera suggested its potential use in creating CRW. These hybrids were created with a Cry3Bb1 Bacillus thurintransgenic plants expressing Cry3Bb1 that would confer giensis (Bt) variant that is approximately eight times more lethal to protection to corn root tissue from larval feeding damcorn rootworm larvae than the wild-type protein. A DNA vector age. To further augment protection of the root system containing the modified cry3Bb1 gene was placed under control of a from larval feeding damage, modifications were introroot-enhanced promoter (4-AS1) and was introduced into embryonic duced in the cry3Bb1 gene that gave rise to an amino acid maize cells by microprojectile bombardment. Described here is the variant Cry3Bb1 protein with an eight-fold increase in molecular genetic characterization, protein expression levels, and field performance of the recently commercialized MON863 hybrids.
A rapid and reproducible Agrobacterium-mediated transformation protocol for sorghum has been developed. The protocol uses the nptII selectable marker gene with either of the aminoglycosides geneticin or paromomycin. A screen of various A. tumefaciens strains revealed that a novel C58 nopaline chromosomal background carrying the chrysanthopine disarmed Ti plasmid pTiKPSF(2), designated NTL(4)/Chry5, was most efficient for gene transfer to sorghum immature embryos. A NTL(4)/Chry5 transconjugant harboring the pPTN290 binary plasmid, which carries nptII and GUSPlus expression cassettes, was used in a series of stable transformation experiments with Tx430 and C2-97 sorghum genotypes and approximately 80% of these transformation experiments resulted in the recovery of at least one transgenic event. The transformation frequencies among the successful experiments ranged from 0.3 to 4.5%, with the average transformation frequency being approximately 1% for both genotypes. Over 97% of the transgenic events were successfully established in the greenhouse and were fully fertile. Co-expression of GUSPlus occurred in 89% of the transgenic T(0) events. Seed set for the primary transgenic plants ranged from 145 to 1400 seed/plant. Analysis of T(1) progeny demonstrated transmission of the transgenes in a simple Mendelian fashion in the majority of events.
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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