Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts

We have used the bar gene in combination with the herbicide Basta to select transformed rice (Oryza sativa L. cv. Radon) protoplasts for the production of herbicide-resistant rice plants. Protoplasts, obtained from regenerable suspension cultures established from immature embryo callus, were transformed using PEG-mediated DNA uptake. Transformed calli could be selected 2–4 weeks after placing the protoplast-derived calli on medium containing the selective agent, phosphinothricin (PPT), the active component of Basta. Calli resistant to PPT were capable of regenerating plants. Phosphinothricin acetyltransferase (PAT) assays confirmed the expression of the bar gene in plants obtained from PPT-resistant calli. The only exceptions were two plants obtained from the same callus that had multiple copies of the bar gene integrated into their genomes. The transgenic status of the plants was varified by Southern blot analysis. In our system, where the transformation was done via the protoplast method, there were very few escapes. The efficiency of co-transformation with a reporter gene gusA, was 30%. The T_o plants of Radon were self-fertile. Both the bar and gusA genes were transmitted to progeny as confirmed by Southern analysis. Both genes were expressed in T₁ and T₂ progenies. Enzyme analyses on T₁ progeny plants also showed a gene dose response reflecting their homozygous and heterozygous status. The leaves of T_o plants and that of the progeny having the bar gene were resistant to application of Basta. Thus, the bar gene has proven to be a useful selectable and screenable marker for the transformation of rice plants and for the production of herbicide-resistant plants.     

Use of DNA Markers in Prediction of Hybrid Performance and Heterosis for a Three-Line Hybrid System in Rice

Two Cytoplasmic Male Sterile lines were crossed with fourteen restorer lines of rice widely grown in the western regions of Maharashtra, India, to produce 28 F₁ hybrids which were evaluated for eight agronomically important traits, contributing to yield potential, in replicated field trials. The hybrid performance was recorded along with heterosis and heterobeltiosis. All the rice lines under investigation were subjected to marker-based variability analysis. An attempt was made to correlate genetic distance based on specific markers for each trait individually, as well as average genetic distance based on all specific markers, with hybrid performance and heterosis, by regression analysis. Specific markers could cluster the parental lines in different groups and showed significant correlation with hybrid performance. The data also supports the proposition that epistasis is the basis of heterosis. The analysis, however, revealed a lack of significant predictive values for field application.     

Genetic Diversity of a Tropical Tree Species,Shorea leprosula Miq. (Dipterocarpaceae), in Malaysia: Implications for Conservation of Genetic Resources and Tree Improvement1

Genetic diversity and population genetic structure of Shorea leprosula was investigated using seven natural populations distributed throughout Peninsular Malaysia and one natural population from Borneo. The mean population and species level genetic diversity were exceptionally high (H_e= 0.369 ± 0.025 and 0.406 ± 0.070, respectively). Heterozygosity varied among populations, ranging from 0.326 to 0.400, with the highest values found in the populations from central Peninsular Malaysia. Correlations among ecological factors (longitude, latitude, and annual rainfall) were not significant (P > 0.05), indicating that these ecological variables were not responsible for the observed genetic differences among populations. The Bangi adult population exhibited a higher level of observed heterozygosity but lower fixation indices in comparison to its seedling population. All other seedling populations also showed positive fixation indices (f), indicating a general excess of homozygotes. This also may suggest selection against homozygotes between the seedling and adult stages. A low level of population differentiation was detected (G_ST= 0.117 with the Lambir population and GST= 0.085 without the Lambir population). Furthermore, gene flow (N_m) between populations was not significantly correlated with geographical distances for the populations within Peninsular Malaysia. Cluster analysis also did not reflect geographical proximity and gave little insight into the genetic relatedness of the populations. This may indicate that the populations sampled are part of a continuous population with fragmentation having occurred in the recent past.     

Digested Bacterial Cell Powder (DBCP) as a Source of Reduced-form Folates for Pigs: Use of a Trimethoprim-resistant Strain and the Bioavailability of Folates in DBCP

The production of digested bacterial cell powder (DBCP) as a source of reduced-form folates for pigs was studied. Trimethoprim-resistant mutants of Brevibacterium lactofermentum ATCC 13869 accumulated a significantly higher amount of the reduced form of folate in the cells than the wild-type strain. DBCPs were prepared from the resistant mutant strain and the wild-type strain. The utilization of the reduced-form of folate in DBCP was evaluated by measuring the plasma folate level after orally administering DBCP to Göttingen minipigs. The folates in both DBCPs proved to have equally high bioavailability in the pigs.