ABSTRACT
The Agrobacterium tumefaciens isopentenyl transferase gene (ipt), a cytokinin biosynthetic gene, was placed under the control of 1.9 kb of promoter sequence from the 2S albumin AT2S1 gene isolated from an Arabidopsis thaliana library. The construct was introduced into canola (Brassica napus) and tobacco (Nicotiana tabacum). ipt transcripts were followed during embryo development of transgenic plants by northern hybridizations. The phenotype of transformed plants from the T1 generation was analysed and we observed an increased branching of inflorescences in tobacco and canola plants expressing the ipt gene. Comparing with controls, the average number of capsules and siliques in AT2S1-ipt plants was 82.6 and 24.8% higher, respectively. This result was correlated with an increase in cytokinin levels in transgenic plants, as revealed by RIA. Indeed, cytokinin contents of T1 AT2S1-ipt B. napus seeds were found 2.2-fold higher than cytokinin contents of control seeds, and T1 AT2S1-ipt N. tabacum capsules contained 2.6-fold more cytokinins than control capsules. In tobacco, the average seed weight per capsule was lower in AT2S1-ipt plants while the seed number per silique and the average seed weight were not modified in canola carrying this construct. The average seed yield per plant was not significantly increased in AT2S1-ipt tobacco or canola plants.
Subject(s)
Agrobacterium tumefaciens/genetics , Alkyl and Aryl Transferases , Brassica/genetics , Gene Expression Regulation, Plant , Nicotiana/genetics , Plants, Toxic , Seeds/genetics , Transferases/genetics , Albumins/genetics , Antibodies, Bacterial , Arabidopsis/genetics , Base Sequence , Blotting, Northern , Chimera/genetics , Cloning, Molecular , Gene Library , Genetic Vectors , Molecular Sequence Data , Plants, Genetically Modified , Promoter Regions, Genetic , RNA, Messenger/analysis , Radioimmunoassay , Seeds/growth & development , Transcription, Genetic , Transferases/immunology , Transformation, Genetic , TransgenesABSTRACT
Sperm cells were isolated from corn (Zea mays L.) tricellular pollen grains. They were released using a light osmotic chock, and separated from pollen contaminants (especially starch grains) by a Percoll gradient centrifugation. Isolated sperm cells (3 x 10(6) per milliliter) show a high viability score (90%) as demonstrated with the fluorochromatic reaction. They appeared as spherical cells which lack cell wall and plastids, and can be considered as haploid protoplasts.