Transgenic Rice Calli Research Articles

Matrix attachment regions increase transgene expression levels and stability in transgenic rice plants and their progeny

SummaryTo investigate the effect of matrix attachment regions (MARs) on transgene expression levels and stability in cereal crops, we generated 83 independent transgenic rice callus lines containing a gusA expression cassette either as a simple expression unit, or flanked with MARs from tobacco (Rb7) or yeast (ARS1). Transgenic rice plants were regenerated from these callus lines and analysed at the structural and expression levels over two generations. In the first generation (T0), both Rb7 and ARS1 MARs significantly increased transgene expression levels. In the populations of plants containing MARs, we observed a significant reduction in the number of non‐expressing lines compared to the population of plants without MARs. However, variation in β‐glucuronidase (GUS) expression levels between independent lines was similar both in the presence and absence of flanking MARs. In the presence of MARs, GUS activity increased in proportion to transgene copy number up to 20 copies, but was generally reduced in lines carrying a higher copy number. In the population of plants without MARs, there was no correlation between expression level and transgene copy number. In the second generation (T1), transgene expression levels were significantly correlated with those of the T0 parents. The Rb7 MARs significantly improved the stability of transgene expression levels over two generations, and therefore appear to offer protection against transgene silencing. Our study shows that the exploitation of MARs may be an important strategy for stabilising transgene expression levels in genetically engineered cereals.

Semi-real time imaging of the expression of a maize polyubiquitin promoter-GFP gene in transgenic rice

We have introduced the green fluorescent protein gene under the control of the maize ubiquitin promoter into rice embryos by microprojectile bombardment. Transient expression of GFP on the surface cells in the rice embryos depended on the localization of DNA coated gold particles. Bialaphos-resistant rice calli were produced by bombardment with a two plasmid mixture of pUbi-GFP and pDM302 carrying the coding region of the bar gene. Microscopic fluorescent image analyses indicated that the area of GFP expression increased and spreaded on the transgenic rice calli after heat stress.

The impact of selection parameters on the phenotype and genotype of transgenic rice callus and plants

Transgenic rice embryogenic callus and plants were recovered from experiments involving electric discharge particle acceleration (AccellTM technology). Critical parameters influencing successful delivery and stable integration of exogenous DNA into organized rice tissue had been identified previously. We report here on the effects of one selective agent (hygromycin B) on the phenotype and genotype of recovered callus and plants. The nature, timing and culture practices appeared to be more critical for the successful recovery of transgenic callus and plants than the level of selection used. By utilizing the procedures described in this report, transformation frequencies well in excess of 10% were obtained routinely in all varieties of rice tested. The combination of AccellTM technology with a selectable and prolific regeneration culture system resulted in the development of a variety-independent and highly efficient method for the routine introduction of any gene into any rice variety.

Activity of a maize ubiquitin promoter in transgenic rice

We have used the maize ubiquitin 1 promoter, first exon and first intron (UBI) for rice (Oryza sativa L. cv. Taipei 309) transformation experiments and studied its expression in transgenic calli and plants. UBI directed significantly higher levels of transient gene expression than other promoter/intron combinations used for rice transformation. We exploited these high levels of expression to identify stable transformants obtained from callus-derived protoplasts co-transfected with two chimeric genes. The genes consisted of UBI fused to the coding regions of the uidA and bar marker genes (UBI:GUS and UBI:BAR). UBI:GUS expression increased in response to thermal stress in both transfected protoplasts and transgenic rice calli. Histochemical localization of GUS activity revealed that UBI was most active in rapidly dividing cells. This promoter is expressed in many, but not all, rice tissues and undergoes important changes in activity during the development of transgenic rice plants.

Enhancement of foreign gene expression by a dicot intron in rice but not in tobacco is correlated with an increased level of mRNA and an efficient splicing of the intron

The first intron of castor bean catalase gene, cat-1 was placed in the N-terminal region of the coding sequence of the beta-glucuronidase gene (gusA) and the intron-containing gusA was used with the cauliflower mosaic virus (CaMV) 35S promoter. Using this plasmid, pIG221, the effect of the intron on expression of beta-glucuronidase (GUS) activity was examined in transgenic rice calli and plants (a monocotyledon), and transgenic tobacco plants (a dicotyledon). The intron-containing plasmid increased the level of GUS enzyme activity 10 to 40-fold and 80 to 90-fold compared with the intronless plasmid, pBI221, in transgenic rice protoplasts and transgenic rice tissues, respectively. In contrast, the presence of the intron hardly influenced the expression of the GUS activity in transgenic tobacco plants. Northern blot analysis showed that the catalase intron was efficiently spliced in rice cells while transgenic tobacco plants contained both spliced and unspliced gusA transcripts in equal amounts. Furthermore, the level of the mature gusA transcript in transformed rice calli was greatly increased in the presence of the intron. The catalase intron was removed at the same splice junctions in transgenic rice and tobacco plants. These findings indicate that the stimulating effect of the intron on GUS expression is correlated with an efficient splicing of pre-mRNA and an increased level of mature mRNA.