Rice Genetics IV - IRRI books - International Rice Research Institute

Since one major goal of plant scientists and breeders is to maximize expressionlevels of transgenes, especially in the production of transgene-encoded protein, inclusionof the Rb7 MAR sequence would be beneficial.Comparison of the particle-bombardmentand Agrobacterium-mediated transformation methods in riceIn rice, the particle-bombardment method was first used successfully to introduceplasmids into rice cells and show integration of the transgene (Cao et al 1990). However,no transgenic plants were regenerated. Christou et al (1991) reported the transformationof rice immature embryos and regeneration of fertile plants. The transgenecopy number ranged between 1 and 6. Cao et al (1992) introduced into rice suspensionculture cells a bar-gene-containing plasmid and regenerated fertile transgenicrice plants. The transgene copy number was between 1 and 5. Duan et al (1996) reportedthat there were 6–8 copies of transgenes in transgenic rice plants obtained byparticle bombardment.Chan et al (1993) were the first to successfully use Agrobacterium-mediated transformationto regenerate fertile transgenic rice plants, but only a few plants were produced.Hiei et al (1994) used a helper plasmid to supply vir gene function and establishedan efficient transformation method with many plants regenerated. The transgenecopy number was between 1 and 5. Since then, this method has become an importanttransformation method in cereal plants, including rice.Reports from different laboratories suggest that the transgene copy number ishigher in transgenic plants obtained by particle bombardment than in those obtainedby the Agrobacterium-mediated method. However, evidence based on well-controlledcomparison is lacking because different laboratories have used different vectors andprocedures. In our work, we used the same plasmid for both the Agrobacterium-mediatedmethod and the particle bombardment transformation method to transform rice.The work was carried out by the same person using the same starting materials, media,etc. Thus, the results can be compared directly.Results from past years have indicated that transgene copy number may affect thelevel of gene expression in transgenic plants, and the number of the rearranged copiesof the transgene often results in gene silencing (Holmes and Comai 1998, Kumpatlaet al 1998, Allen et al 2000, Iyer et al 2000). Therefore, an important consideration isto determine the transgene copy number of both intact and rearranged copies and tocorrelate this information with expression levels.We have constructed a plasmid, pAc1PG-CAM, using the vector pCAMBIA1300(from Richard Jefferson of CAMBIA). The plasmid contains the following components:Arabidopsis Actin1 gene promoter/Gus/Nos 3’/pCAMBIA 1300. This plasmidwas used for transformation using two different methods. In both methods, rice calliwere induced in MS medium (see Zhang and Wu 1988) from the mature embryos ofrice cv. TNG67. In the particle bombardment method, 15-d-old embryogenic calliwere bombarded with gold beads coated with pAc1PG-CAM DNA according to theprocedure of Cao et al (1992). Calli transformed with pAc1PG-CAM DNA were selectedin plates containing MS medium supplemented with 50 mg L –1 hygromycin432 Cheng et al