We have recovered transgenic rice plants from a number of commercially important cultivars, including until now recalcitrant Indica varieties, using electric discharge particle acceleration Immature embryos from greenhouse–grown plants were bombarded with gold particles carrying DNA, and transgenic plants were recovered following a simple culture protocol Mendelian segregation of foreign genes was observed in R1 progeny and stable integration was demonstrated by Southern blot analysis of genomic DNA isolated from progeny plants Alternative transformation protocols that are dependent on the development of protoplast and suspension culture systems are no longer necessary as we have shown that a wide variety of diverse cultivars can be transformed Transgenic plants expressing agronomically useful traits such as herbicide resistance have been obtained and are currently undergoing further evaluation This report also demonstrates that it is possible to produce transgenic monocoty–ledonous plants by transforming scutellar tissue of immature embryos

Production of Transgenic Rice ( Oryza Sativa L.) Plants from Agronomically Important Indica and Japonica Varieties via Electric Discharge Particle Acceleration of Exogenous DNA into Immature Zygotic Embryos

ISAAA Briefs brief 41 Global status of Commercialized biotech/GM Crops: 2009

Transgenic hairy roots: recent trends and applications

Agrobacterium tumefaciens has been routinely utilized in gene transfer to dicotyledonous plants, but monocotyledonous plants including important cereals were thought to be recalcitrant to this technology as they were outside the host range of crown gall. Various challenges to infect monocotyledons including rice with Agrobacterium had been made in many laboratories, but the results were not conclusive until recently. Efficient transformation protocols mediated by Agrobacterium were reported for rice in 1994 and 1996. A key point in the protocols was the fact that tissues consisting of actively dividing, embryonic cells, such as immature embryos and calli induced from scutella, were co-cultivated with Agrobacterium in the presence of acetosyringonc, which is a potent inducer of the virulence genes. It is now clear that Agrobacterium is capable of transferring DNA to monocotyledons if tissues containing ‘competent’ cells are infected. The studies of transformation of rice suggested that numerous factors including genotype of plants, types and ages of tissues inoculated, kind of vectors, strains of Agrobacterium,selection marker genes and selective agents, and various conditions of tissue culture, are of critical importance. Advantages of the Agrobacterium-mediated transformation in rice, like on dicotyledons, include the transfer of pieces of DNA with defined ends with minimal rearrangements, the transfer of relatively large segments of DNA, the integration of small numbers of copies of genes into plant chromosomes, and high quality and fertility of transgenic plants. Delivery of foreign DNA to rice plants via A. tumefaciens is a routine technique in a growing number of laboratories. This technique will allow the genetic improvement of diverse varieties of rice, as well as studies of many aspects of the molecular biology of rice.

Transformation of rice mediated by agrobacterium tumefaciens

We have established an efficient protocol for plant regeneration from haploid Indica-type rice protoplasts. Incubation of these protoplasts with the selectable hygromycin phosphotransferase (hph) gene expressed under control of the 35S promoter of cauliflower mosaic virus (CaMV) and polyethyleneglycol (PEG), and subsequent culture in the presence of hygromycin B, led to the recovery of numerous resistant clones from which 77 plants were regenerated. Data from Southern analysis and enzyme assays proved that the transgene was stably integrated into the host genome and expressed, and that it was inherited in offspring.

Genetically Engineered Fertile Indica-Rice Recovered from Protoplasts

Transgenic rice plants have been regenerated by somatic embryogenesis from cell suspension derived protoplasts electroporated with plasmid carrying the NPTII gene under the control of the 35S promoter from cauliflower mosaic virus. Heat shock of protoplasts prior to electroporation maximised the throughput of kanamycin resistant colonies. Omission of kanamycin from the medium for plant regeneration was essential for the recovery of transgenic rice plants carrying the NPTII gene. This report of the production of kanamycin resistant transgenic rice plants establishes the use of protoplasts for rice genetic engineering.

Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts

Three cultivars of M. sativa and one cultivar of 0. viciifolia were evaluated for their response to inoculation with A. rhizogenes strain A4T (containing pRiA4b). A cultivar-dependent response was observed in M. sativa with 94%, 25%, and 4% of infected stem expiants producing transformed roots in the cultivars Vertus, Regen-S, and Rangelander, respectively. In O. viciifolia cv. Hampshire Giant, an explant-dependent response was observed with 78% and 50% of seedling cotyledon and hypocotyl expiants responding, respectively. Leaf expiants failed to produce transformed roots. Transformed roots showed plagiotropic and negatively geotropic growth on hormone-free agar MS medium. Production of transgenic shoots from O. viciifolia root cultures occurred spontaneously. Recovery of transgenic plants from M. sativa cv. Rangelander was achieved by transfer of callus (induced on UM medium containing 2 0 mg dm"3 2,4-D and 0-25 mg dm"3 kinetin) to MS medium containing 0-5 mg dm"3 BAP and 0 05 mg dm"3 NAA. Cultured roots of both species synthesized opines (agropine and mannopine). Extensive morphological variation was observed in plants of M. sativa (clone Al) and O. viciifolia (clone A4T1) established in the glasshouse. DNA sequences homologous to TL-DNA and TR-DNA were present in root clones and regenerated plants.

Agrobacterium rhizogenes Mediated Transformation of the Forage Legumes Medicago sativa and Onobrychis viciifolia

Six accessions of Glycine canescens and four of G. clandestina were evaluated for their response to inoculation with Agrobacterium rhizogenes strain LBA 9402. The response was accession-dependent, with 9 % to 70 % of the seedlings producing roots following infection of the hypocotyl. Excised cotyledons were less responsive than hypocotyls. Roots exhibited plagiotropic and negatively geotropic growth in culture on hormone-free agar medium; those of G. canescens produced shoots on B5 based medium containing 10 mg dm −3 BAP and 0.05 mg dm −3 IBA (...)

Agrobacterium rhizogenes Mediated Transformation of the Wild Soybeans Glycine canescens and G. clandestine: Production of Transgenic Plants of G. canescens

Seedling hypocotyl explants ofGlycine canescens were inoculated withAgrobacterium rhizogenes carrying a chimaeric NPTII gene cointegrated into the TL-DNA of pRiA4. Transformed roots produced shoots on B5 based medium with 10.0 mgl−1 BAP, 0.05 mgl−1 IBA and 50 μgml−1 kanamycin. Cultured roots and regenerated plants expressed NPTII enzyme activity which was correlated with the presence of Ri TL-DNA and the structural sequence of the NPTII gene.

Expression of a chimaeric kanamycin resistance gene introduced into the wild soybean Glycine canescens using a cointegrate Ri plasmid vector

Cat gene expression has been investigated following PEG-mediated plasmid uptake into barley protoplasts. The uptake conditions optimised for transient expression were employed for stable transformation. Transformed protoplast-derived calli of the cvs. Dissa and Igri, were selected on medium containing G418 at 40 μg ml−1 or kanamycin sulphate at 250 μg ml−1. Absolute transformation frequencies of 28.9×10−5 and 21.3×10−5 were recorded for Dissa with kanamycin sulphate and G418 selection, respectively. The frequency for Igri was 11.5×10−5 with G418 selection. Antibiotic resistant protoplast-derived colonies expressed NPTII activity; Southern hybridisation confirmed integration of the nptII gene into barley genomic DNA.

T. J. Golds

Papers

Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts

Agrobacterium rhizogenes Mediated Transformation of the Forage Legumes Medicago sativa and Onobrychis viciifolia

Agrobacterium rhizogenes Mediated Transformation of the Wild Soybeans Glycine canescens and G. clandestine: Production of Transgenic Plants of G. canescens

Expression of a chimaeric kanamycin resistance gene introduced into the wild soybean Glycine canescens using a cointegrate Ri plasmid vector

Parameters influencing transient and stable transformation of barley (Hordeum vulgare L.) protoplasts