An improved green fluorescent protein gene as a vital marker in plants.

A synthetic green fluorescent protein (GFP) gene (pgfp) was constructed to improve GFP expression in plants. Corn and tobacco protoplast transient assays showed that pgfp gave about 20-fold brighter fluorescence than the wild-type gene (gfp). Replacement of the serine at position 65 with a threonine (S65Tpgfp) or a cysteine (S65Cpgfp) yielded 100- to 120-fold brighter fluorescence than wild-type gfp upon excitation with 490-nm light. Incorporation of a plant intron into the coding region yielded an additional 1.4-fold improvement, for a cumulative improvement of about 150-fold in fluorescence at 490-nm excitation. Various versions of pgfp were also stably introduced into corn, wheat, tobacco, and Arabidopsis plants. Bright-green fluorescence was observed with a fluorescence microscope in virtually all examined tissues of transgenic monocots and dicots. In the case of Arabidopsis, expression of the pgfp gene under the enhanced 355 promoter of the cauliflower mosaic virus produced green fluorescence that was readily detectable by eye using a hand-held, long-wave ultraviolet lamp and/or a black-light source.

Plastid targeting of E. coli ß-glucuronidase and ADP-glucose pyrophosphorylase in maize (Zea mays L.) cells.

Dicot and monocot chloroplast targeting peptides (CTPs) were evaluated for their effect on targeting, processing, and expression of two reporter proteins in maize cells. When tested transiently in maize leaf protoplasts, the maize ribulose bisphosphate carboxylase small subunit CTP required the inclusion of the amino terminus of mature small subunit protein to target ß-glucuronidase (GUS) to the plastid. To remove this amino terminal extension from GUS after import and processing, a repeat of the native processing site was inserted between the native mature protein and the reporter protein. This repeat processing site was used with less efficiency than the native site. Parallel constructs using the Arabidopsis thaliana small subunit and maize granule-bound starch synthase CTPs also localized GUS, but varied in repeat site use and GUS expression levels. Data from the CTP fusions with GUS were generally confirmed with fusions to an allosteric variant of E. coli ADP-glucose pyrophosphorylase. Plastid targeting of this enzyme was required for starch enhancement of transgenic BMS cells.

Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants.

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.

Isolation, culture, and cell division in cotyledon protoplasts of cotton (Gossypium hirsutum and G. barbadense).

Protoplasts were isolated from cotyledons and foliage leaves of cotton (Gossypium hirsutum and G. barbadense). Cotyledon protoplasts were larger and responded to culture better than leaf protoplasts. Cotyledon derived protoplasts regenerated cell walls and formed microcolonies of 2-3 cells in G. hirsutum and 5-8 cells in G. barbadense. However, the microcolonies did not grow beyond this stage. Protoplast yield and viability, cell wall regeneration and cell division were influenced by several factors, e.g., genotype, age, tissue and growth condition of donor plant, enzyme mixture and concentration, preplasmolysis period, incubation period, and culture medium.