Cost-effective in vitro propagation methods for pineapple.

We have developed an efficient and cost-effective method for commercial micropropagation of Smooth Cayenne pineapple. In vitro shoots were used as starting materials, and either longitudinal sections of the shoots or leaf bases were used as the explants to regenerate shoots. When these explants were used, the axillary meristems, which usually remain quiescent during shoot multiplication, were able to form new shoots. Subsequent to the regeneration step, additional multiplication was achieved inside a 10-l Nalgene vessel with shoots immersed in liquid medium for 5-10 min/h (periodic immersion bioreactor, PIB). The shoots were then induced to form roots and transferred to soil. Using the above micropropagation method and the PIB, we produced 6,000-8,000 shoots from two initial shoots in less than 6 months. The clonal fidelity of propagated plants was tested in Costa Rican and Indonesian pineapple farms.

Modification of flower color in florist's chrysanthemum: production of a white-flowering variety through molecular genetics.

Chimeric chalcone synthase (CHS) constructs were prepared in both anti-sense and sense orientations, and introduced into the chrysanthemum cultivar Moneymaker, along with a T-DNA vector lacking a CHS construct. For both the anti-sense and sense constructs, the majority of the plants produced pink flowers typical of Moneymaker itself. Of 133 sense and 83 anti-sense transgenic individuals 3 of each set produced fully white or very pale pink flowers. No white-flowering transgenic plants were obtained in control transformations. The white flowers were found to accumulate higher levels of chalcone synthase precursors and to have reduced levels of chalcone synthase message. A small-scale field trial was performed to evaluate the stability of the phenotype throughout a series of vegetative propagation steps and during plant growth. The white-flowering trait was maintained well through vegetative propagation; however, during growth of individual white-flowering plants, some pink color was found in some flowers. At one site 2% of the white-flowering plants produced a few pink flowers; at two other sites, as many as 10-12% of the plants produced pale pink flowers.

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.

Rapid flow cytometric analysis of the cell cycle in intact plant tissues.

Mechanical chopping of plant tissues in the presence of mithramycin released intact nuclei representative of the cells within the tissues. The amount of nuclear DNA in the homogenates of monocotyledonous and dicotyledonous plants was accurately and rapidly determined by flow microfluorometry, and the distribution of nuclei involved in the cell cycle was charted for tissues selected from different physical locations or developmental stages.