Tomoaki Kubo

Transformation of rice mediated by Agrobacterium tumefaciens

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.

Advances in cereal gene transfer

Over the past five years, transgenic strains of various major cereals have been produced, with transformation of rice and maize being most common. A majority of the cereal transformants obtained to date has been generated by the particle bombardment technique, but Agrobacterium-mediated transformation is rapidly becoming the method of choice. Rice, the plant in which transformation-related technology is most advanced, appears to be the model monocotyledon for basic and applied studies.

Differential expression of plastidic aldolase genes in Nicotiana plants under salt stress

Two homologous genes of plastidic fructose-1,6-bisphosphate aldolase (AldP) isozymes were isolated from green leaves of a salt stress-tolerant Nicotiana species, Nicotiana paniculata, by differential screening. The products of the corresponding genes, NpAldP1 and NpAldP2, were 91% identical to each other and 70-85% identical to the other known plant plastidic aldolases. Although these two genes showed similar organ-specific expression and daily cycles, their responses to salt stress differed: mRNA accumulation of NpAldP2 increased, but that of NpAldP1 slightly decreased. The mRNA accumulations of their counterparts of two other Nicotiana species, NeAldP1 and NeAldP2 (Nicotiana excelsior), and NaAldP1 and NaAldP2 (Nicotiana arentsii) were studied under the same stress condition. N. arentsii conserved accumulation profiles similar to N. paniculata, but N. excelsior did not. In N. excelsior, accumulation of NeAldP1 decreased to 50% of the control after stress and gradually recovered thereafter, whereas accumulation of NeAldP2 temporarily decreased and reached 250% of the control by the third day of stress. Southern blot analysis indicated that NpAldP1, NpAldP2, NaAldP1, and NaAldP2 include one or two closely related genes and NeAldP1 and NeAldP2 several.

Methods of Genetic Transformation: Agrobacterium tumefaciens

The soil phytopathogen Agrobacterium tumefaciens induces tumors, known as crown galls, mainly on dicotyledonous plants. Such tumors are generated by a complex, multi-step transformation process. Agrobacterium has been routinely utilized for the transfer of genes to dicotyledonous plants, and monocotyledonous plants, including important cereals, were thought until recently to be outside the range of this technology since they are generally not considered within the host range of crown gall. Various attempts to infect monocotyledons with Agrobacterium were made in the 1970’s and 1980’s, but conclusive evidence of integrative transformation was not obtained until recently. This delay occurred in part because many methods for the transformation of dicotyledons depend heavily on the cell divisions that are induced by wounding. Similar approaches in monocotyledons failed, because they do not exhibit active responses to wounding. Wounding is necessary for formation of a crown gall and the main roles of wounding are to produce compounds that activate the virulence genes of Agrobacterium and to induce DNA synthesis in host cells. Efficient protocols for Agrobacterium-mediated transformation have recently been developed for rice, maize, barley and wheat. A key point in these protocols is the use of tissues that consist of actively dividing, embryonic cells, such as immature embryos and calli induced from scutella, which are co-cultivated with Agrobacterium in the presence of acetosyringone, which is a potent inducer of the virulence genes. The advantages of Agrobacterium-mediated transformation include the transfer of pieces of DNA with defined ends with minimal rearrangement, the transfer of relatively large segments of DNA, the integration of small numbers of copies of genes into plant chromosomes, and the high quality and fertility of both monocotyledonous and dicotyledonous transgenic plants. Stable inheritance and expression of transgenes in the progeny has also been demonstrated. It is now clear that Agrobacterium is capable of transferring DNA to monocotyledons if tissues that contain ‘competent’ cells are infected. The success with cereals highlights the critical importance of numerous experimental factors, which include the genotype of the plants, the type and age of the tissue that is inoculated, the vector, the strain of Agrobacterium, the selectable marker genes and selection agents, and the various conditions of tissue culture.

Accumulation of Maize Response Regulator Proteins in Mesophyll Cells after Cytokinin Treatment

The maize response regulator genes ZmRR1 and ZmRR2 respond to cytokinin, and the translated products seem to be involved in nitrogen signal transduction mediated by cytokinin through the His-Asp phosphorelay. To elucidate the physiological function of the proteins, we examined the temporal and spatial distribution in maize leaves by immunochemical analysis and use of transgenic plants. ZmRR1 and ZmRR2 polypeptides could be distinctively detected by western blotting. The polypeptides accumulated in leaves within 5 h of the supply of nitrate to nitrogen-depleted maize, and the accumulation was transient. The extent of induction was larger in the leaf tip, which is rich in photosynthetically matured cells, than elsewhere. In leaves, the polypeptides accumulated mostly in mesophyll cells. Histochemical analyses of transgenic maize harboring a ZmRR1 promoter-β-glucuronidase fusion gene also showed most of the expression to be in these cells. These results suggest that ZmRR1 and ZmRR2 are induced in mesophyll cells and function in nitrogen signal transduction mediated by cytokinin.

Comparative study of the Nicotiana species with respect to water deficit tolerance during early growth

Sixty Nicotiana species were examined for tolerance against various osmotica for seed germination and seedling growth in vitro. The species showed a wide variety of tolerance, and based on the results of the in vitro tests, 31 species were selected and further evaluated for salt and drought tolerance in a glasshouse. The degrees of tolerance of germination among the 57 species toward NaCl were approximately related to those toward mannitol, indicating that the osmolarity plays a majorrole in seed germination. However, the responses during the seedling growth differed in NaCl and mannitol or drought, and there was no correlation between salt and drought tolerance. Based on the responses in vitro and in the glasshouse, N. paniculata and N. excelsior were selected as the salt tolerant species, and N. arentsii as the salt sensitive species. The degrees of accumulation of dry matter and of Na+ in the leaves were different in the two tolerant species; during NaCl treatment, N. paniculata and N. arentsii accumulated less dry matter relative to the control plants than N. excelsior, and N. paniculata accumulated more Na+ in its leaves than N. excelsior and N. arentsii. It is assumed that the two salt tolerant species have different mechanisms for tolerance to the salt.