Effect of orientation of transcription of a gene in an inverted transferred DNA repeat on transcriptional gene silencing in rice transgenics-a case study.

We studied transgene silencing in two transgenic rice plants, OSM25 and COT-OSM4, which harboured two different types of right border (RB)-centered inverted transferred DNA (T-DNA) repeats (IRs). The T-DNA in OSM25 has three genes gus, OSM and hph, all under the transcriptional control of the Cauliflower mosaic virus 35S promoter (P35S). The gus gene, which is proximal to the RB, is in a convergent orientation of transcription in the IR.

Improved Agrobacterium-mediated co-transformation and selectable marker elimination in transgenic rice by using a high copy number pBin19-derived binary vector.

A high copy number, selectable marker gene (SMG)-free Agrobacterium binary vector pBin19ΔnptII was constructed by deleting the nptII gene from pBin19. The binary vectors with the RK2 and pVS replication origins exist in 12 and 3 copies, respectively, in Agrobacterium. The tobacco osmotin gene (ap24) was cloned in pBin19ΔnptII and the resultant plasmid pBin19ΔnptII-ap24 was mobilized into the Agrobacterium tumefaciens strain C58C1 Rif(r) harbouring the single-copy cointegrate vector pGV2260::pSSJ1.

Transgene stacking and marker elimination in transgenic rice by sequential Agrobacterium-mediated co-transformation with the same selectable marker gene.

Rice chitinase (chi11) and tobacco osmotin (ap24) genes, which cause disruption of fungal cell wall and cell membrane, respectively, were stacked in transgenic rice to develop resistance against the sheath blight disease. The homozygous marker-free transgenic rice line CoT23 which harboured the rice chi11 transgene was sequentially re-transformed with a second transgene ap24 by co-transformation using an Agrobacterium tumefaciens strain harbouring a single-copy cointegrate vector pGV2260::pSSJ1 and a multi-copy binary vector pBin19∆nptII-ap24 in the same cell. pGV2260::pSSJ1 T-DNA carried the hygromycin phosphotransferase (hph) and β-glucuronidase (gus) genes.

Severe stunting in blackgram caused by the Mungbean yellow mosaic virus (MYMV) KA27 DNA B component is ameliorated by co-infection or post-infection with the KA22 DNA B: MYMV nuclear shuttle protein is the symptom determinant.

Mungbean yellow mosaic virus-[India:Vigna] (MYMV-[IN:Vig]), a blackgram isolate of MYMV, has five variable and infective DNA B components of which KA22 and KA27 DNA Bs share only 72% nucleotide sequence identity between them. Agroinoculation of blackgram with partial dimers of DNA A and KA27 DNA B caused severe stunting and an inordinate delay in flowering. Interestingly, co-agroinoculation of KA27+KA22 DNA B components along with DNA A ameliorated severe stunting, rescued from the delay in flowering and caused the appearance of yellow mosaic symptom characteristic of KA22 DNA B.