Summary
A large number of morphologically normal, fertile, transgenic rice plants were obtained by co-cultivation of rice tissues with Agrobacterium tumefaciens The efficiency of transformation was similar to that obtained by the methods used routinely for transformation of dicotyledons with the bacterium Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the R0, R1 and R2 generations Sequence analysis revealed that the boundaries of the T-DNA in transgenic rice plants were essentially identical to those in transgenic dicotyledons Calli induced from scutella were very good starting materials A strain of A tumefaciens that carried a so-called ‘super-binary’ vector gave especially high frequencies of transformation of various cultivars of japonica rice that included Koshihikari, which normally shows poor responses in tissue culture

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.1994.6020271.x

Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.

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

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

Cold stress effects on reproductive development in grain crops: An overview

Improvement of wheat (Triticum aestivum) by biotechnological approaches is currently limited by a lack of efficient and reliable transformation methodology. In this report, we detail a protocol for transformation of a highly embryogenic wheat cultivar, Bobwhite. Calli derived from immature embryos, 0.5 to 1 mm long, were bombarded with microprojectiles coated with DNA containing as marker genes the bar gene, encoding phosphinothricin-resistance, and the gene encoding [beta]-glucuronidase (GUS), each under control of a maize ubiquitin promoter. The bombardment was performed 5 d after embryo excision, just after initiation of callus proliferation. The ability of plantlets to root in the presence of 1 or 3 mg/L of bialaphos was the most reliable selection criteria used to identify transformed plants. Stable transformation was confirmed by marker gene expression assays and the presence of the bar sequences in high molecular weight chromosomal DNA of the resultant plants. Nine independent lines of fertile transgenic wheat plants have been obtained thus far, at a frequency of 1 to 2 per 1000 embryos bombarded. On average, 168 d elapsed between embryo excision for bombardment and anthesis of the T0 plants. The transmission of both the resistance phenotype and bar DNA to the T1 generation verified that germline transformation had occurred.

Rapid Production of Multiple Independent Lines of Fertile Transgenic Wheat (Triticum aestivum)

We have regenerated whole plants of rice (Oryza sativa L.) derived from protoplasts, which had been electroporated with plasmid DNA possessing a chimeric gene encoding aminoglycoside phosphotransferase II (APH(3′)II). Transformed calli were selected on the basis of tolerance to the antibiotic, G418. Several plants were regenerated possessing functional APH(3′)II activity due to the integration of intact foreign DNA into their genome.

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Transgenic Rice Plants After Direct Gene Transfer into Protoplasts

Cell suspension and protoplast culture in rice

The regeneration of haploid and diploid plants was demonstrated from protoplasts that were isolated from cell suspensions of anther callus in rice. The cell suspension in the AA medium that contained 4 amino acids as the sole nitrogen source was friable, finely dispersed, and readily released a large number of protoplasts. These protoplasts, subsequently cultured in NO3 medium that contained nitrate as the sole nitrogen source, formed compact calli. The compact calli produced green plants with a frequency of 24%. Out of 15 flowering plants, 4 were haploids, the others were diploids which showed a uniform morphology but varied in seed fertility from 95 to 0%.

Haploid and diploid plant regeneration from protoplasts of anther callus in rice

Production of Somatic Hybrid Plants, ‘Brassicomoricandia’, Through Protoplast Fusion between Moricandia Arvensis and Brassica Oleracea

Oryza breviligulata A. Cheval. et Roehr., growing wild in West Africa, is genetically closely related to 0. glaberrima Steud., which is cultivated by the native people of the same region. 0. stapfii Roschev., also found in West Africa, can be regarded as synonymous with 0. breviligulata, as will be pointed out later. These species form an endemic group distinct from the common cultivated rice, 0. sativa L. and the wild species closely related to it, 0. perennis Moench. They can be distinguished from 0. sativa and 0. perennis by short, roundish ligules and other characters, and their F1 hybrids with the latter two species, though they show no disturbance in chromosome pairing, are highly sterile (Morinaga and Kuriyama, 1957; Nezu et al., 1960; etc.). Another wild form occurring in Africa, 0. barthii A. Cheval., is considered by us as an African variety of 0. perennis which can be distinguished from Asian and American forms of the same species by the remarkable rhizome development (Chatterjee, 1948; Sampath and Rao, 1951; Sampath, 1961) . The above rice species may for the sake of simplicity be divided into two series, Series Glaberrima comprising 0. glaberrima and 0. breviligulata, and Series Sativa comprising 0. sativa and 0. perennis. This paper deals mainly with the comparison of the two series regarding the mode of evolution of cultivated forms. Regarding the phylogenetic relationships in Series Sativa, a number of systematic and cytogenetic studies have been made (Chatterjee, 1951; Sampath and Rao,

Kokichi Hinata

Papers

Transgenic Rice Plants After Direct Gene Transfer into Protoplasts

Cell suspension and protoplast culture in rice

Haploid and diploid plant regeneration from protoplasts of anther callus in rice

Production of Somatic Hybrid Plants, ‘Brassicomoricandia’, Through Protoplast Fusion between Moricandia Arvensis and Brassica Oleracea

Comparison of modes of evolution of cultivated forms from two wild rice species, oryza breviligulata and o. perennis