Particle-bombardment-mediated co-transformation of elite Chinese rice cultivars with genes conferring resistance to bacterial blight and sap-sucking insect pests

Abstract

Transgenic rice plants were generated using particle bombardment to simultaneously introduce the rice Xa21 gene eective against bacterial blight and the Galanthus nivalis agglutinin (snowdrop lectin; gna) gene eective against sap-sucking insect pests, speci®cally the brown plant hopper. Using three plasmids, we cotransformed 5-to 10-d-old, mature seed-derived rice (Oryza sativa L.) callus of two elite Chinese rice cultivars, Eyi 105 and Ewan 5. The plasmids carried a total of four genes. The gna and Xa21 genes were carried on separate plasmids. The selectable marker hygromycin phosphotransferase (hpt) and the reporter gene b-glucuronidase (gusA) were linked on the same, co-integrate vector. We recovered over 160 independently derived transgenic rice plants. Over 70% of the transgenic plants carried all four genes, as con®rmed by polymerase chain reaction and/or Southern blot analysis. Furthermore, 70% of transgenic plants carrying all four genes also coexpressed all four genes, as con®rmed by growth on selection media (hpt), GUS histochemical assays (gusA), western blotting (gna) and reverse transcriptase-polymerase chain reaction (Xa21) analysis. The co-expression eciency reported for the four transgenes in our study is the highest ever found in any transgenic plant population generated through co-transformation. The linked genes (hpt and gusA) co-integrated with a frequency of near 100%, and we observed a co-integration frequency greater than 70% for the genes carried on separate plasmids. We observed no preferential integration of any particular gene(s). Genetic analysis con®rmed Mendelian segregation of the transgenes in subsequent generations. We report, for the ®rst time, generation and analysis of transgenic rice lines carrying genes eective against more than one taxa of pathogen or pest, substantiating that particle bombardment represents an eective way to introduce unlinked complex multiple traits into plants.Abbreviations: gna (GNA) = gene (protein) for Galanthus nivalis agglutinin; gusA (GUS) = gene (protein) for b-glucuronidase; hpt (HPT) = gene (protein) for hygromycin phosphotransferase; Xa21 (XA21) = gene (protein) for resistance to bacterial blight, RT-PCR = reverse transcriptase-polymerase chain reaction

Comparison of transformation efficiency of five Agrobacterium tumefaciens strains in Nicotiana Tabacum L.

“…The transformed leaf discs developed into the callus and then the shoots were emerged with in 18-22 days. Accumulating evidences suggested that in vitro tissue culture and micro propagation are necessary for transformation experiments to remodel the aesthetic and growth characteristics of the plants (Jaime and Teixeira, 2003;Siemens and Schieder, 1996;Tang et al 1999). The roots continued to grow in the putative transgenic plants on rooting medium.…”

Effects of squirting cucumber (Ecballium elaterium) fruit juice onAgrobacterium tumefaciens-mediated transformation of plants

“…On the other hand, provision of low concentrations (0.2-1.6 mL L -1 ) of squirting cucumber fruit juice was found efficient for kanamycin-resistant callus and shoot regeneration without any bacterial overgrowth in the culture medium. Results emphasized the importance of modification of the regeneration protocol along with transformation experiments in order to achieve desirable characteristics (Siemens and Schieder, 1996;Tang et al, 1999;Jaime and Teixeira, 2003;Bakhsh et al, 2014).…”

Agrobacterium tumefaciens-mediated transformation of rice with the spider insecticidal gene conferring resistance to leaffolder and striped stem borer

“…Our long-term goals are to improve the quality of rice by using an approach in which multiple disease-and insect-resistant traits can be introduced into the same lines [2]. In our experiment, the insecticidal activity of transgenic rice plants on LF and SSB wereinvestigated (Tab 4 and Tab 5).…”

“…Although a decade ago rice was considered as one of the most recalcitrant crops in terms of genetic manipulation, it has recently emerged as the model cereal for the study of plant genomics, plant pathology, gene regulation and expression, metabolic pathway manipulation and the inheritance, organization, rearrangement and fate of transgenes [1], [2]. Remarkable progress has been made in the development of systems for Agrobacterium tumefaciences -mediated transformation in rice since 1994 [3][4][5][6][7][8].…”