Single transgene copy, vector backbone-free transgenic crop plants are highly desired for functional genomics and many biotechnological applications. We demonstrate that binary vectors that use a replication origin derived from the Ri plasmid of Agrobacterium rhizogenes (oriRi) increase the frequency of single copy, backbone-free transgenic plants in Agrobacterium tumefaciens mediated transformation of soybean, canola, and corn, compared to RK2-derived binary vectors (RK2 oriV). In large scale soybean transformation experiments, the frequency of single copy, backbone-free transgenic plants was nearly doubled in two versions of the oriRi vectors compared to the RK2 oriV control vector. In canola transformation experiments, the oriRi vector produced more single copy, backbone-free transgenic plants than did the RK2 oriV vector. In corn transformation experiments, the frequency of single copy backbone-free transgenic plants was also significantly increased when using the oriRi vector, although the transformation frequency dropped. These results, derived from transformation experiments using three crops, indicate the advantage of oriRi vectors over RK2 oriV binary vectors for the production of single copy, backbone-free transgenic plants using Agrobacterium-mediated transformation.

A gene (FbL2A) that i s preferentially expressed in cotton (Gossypium barbadense 1. cv Sea Island) fiber was isolated and characterized. Cenomic and cDNA analyses suggest multiple FbL2A genes in cotton. The gene is developmentally regulated and is activated during late primary and early secondary wall synthesis stages. FbL2A encodes a polypeptide of 43.4 k D and a predicted isoelectric point of 5.97. The nucleotide-derived protein is highly hydrophilic except for a hydrophobic N terminus and has a compositional bias for glutamic acid (26.3 moi%) and lysine (18.9 moi%). Sixty-two percent of the putative protein is composed of repeat motifs. A 55-amino-acid peptide region is repeated four times i n a concatenate fashion within the protein. The function of the protein in the fiber cells is not known. A 2.3-kb DNA fragment 5' from the FbL2A gene is shown t o direct expression of heterologous proteins in transgenic cotton in a fiber-specific and developmentally regulated fashion. The FbLZA promoter was used to express in transgenic cotton genes encoding acetoacetyl-coenzyme A reductase and polyhydroxyalkanoic acid synthase, which are involved i n the synthesis of the thermoplastic polymer polyhydroxybutyric acid. Transgenic plants containing both enzymes produced polyhydroxybutyric acid in fiber. Thus, the FbLZA promoter i s useful in genetic engineering schemes to modify cotton fiber.Cotton (Gossypium hirsutum L.) is a warm-season woody perennial shrub cultivated for its fiber and seeds in more than 90 countries. The fiber is an important raw material used mainly for textile applications. Cotton fiber, or the seed hair, is a terminally differentiated epidermal cell that undergoes 1000-to 3000-fold elongation during its development. Initiation of fiber development is triggered by hormdnes and lasts up to 45 to 50 DPA (reviewed by Kosmidou-Dimitropoulou, 1986).Cotton fiber is composed of a thin primary wall (0.4 pm) and a thick secondary wall (8-10 pm). Primary wall for-' mation (0-20 DPA) occurs during the first two stages of development: initiation and elongation. The primary wall is made up of cellulose, hemicellulose, pectins, proteins, and waxes. A thin cuticle made up of stratified layers of

Conventional Agrobacterium-mediated plant transformation often produces a significant frequency of transgenic events containing vector backbone sequence, which is generally undesirable for biotechnology applications. We tested methods to reduce the frequency of transgenic plants containing vector backbone by incorporating genes into the backbone that inhibit the development of transgenic plants. Four backbone frequency reduction genes, bacterial levansucrase (sacB), maize cytokinin oxidase (CKX), Phaseolus GA 2-oxidase (GA 2-ox), and bacterial phytoene synthase (crtB), each expressed by the enhanced CaMV 35S promoter, were placed individually in a binary vector backbone near the left border (LB) of binary vectors. In transformed soybean plants, the lowest frequency of backbone presence was observed when the constitutively expressed CKX gene was used, followed by crtB. Higher backbone frequencies were found among the plants transformed with the GA 2-oxidase and sacB vectors. In some events, transfer of short backbone fragments appeared to be caused by LB readthrough and termination within the backbone reduction gene. To determine the effect of the backbone genes on transformation frequency, the crtB and CKX vectors were then compared to a control vector in soybean transformation experiments. The results revealed that there was no significant transformation frequency difference between the crtB and control vectors, but the CKX vector showed a significant transformation frequency decrease. Molecular analysis revealed that the frequency of transgenic plants containing one or two copies of the transgene and free of backbone was significantly increased by both the CKX and crtB backbone reduction vectors, indicating that there may be a correlation between transgene copy number and backbone frequency.

A gene (G9) expressed during late microsporogenesis in cotton (Gossypium hirsutum L.) was isolated. Sequence analysis of the cDNA (1.3 kb) as well as the gene (2.6 kb) revealed an open reading frame of 1233 bases encoding a protein of 43.9 kDa. The coding region of the gene is interrupted by three introns. Northern analysis of the RNA from developing anthers showed that the transcripts appear 12 days before anthesis and that the maximal concentration of RNA occurs in pollen on the day of anthesis. This pattern of gene expression suggests functions in post-anthesis events. Sequence comparisons with other known plant genes indicated that G9 is homologous to polygalacturonases. The G9 promoter conferred tissue and temporal specificity of beta-glucuronidase (GUS) expression in transgenic tobacco plants. Thus, the G9 promoter can be used to drive gene expression in homologous as well as heterologous plants in a tissue-specific manner.

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