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Publication Type:
| Report |
Author(s): | Takamizo, Tadashi |
Author Affiliation: | Department of Plant Breeding, National Grassland Research Institute 768 Senbonmatsu, Nishinasuno, Tochigi 329-27, Japan |
Translated Title: | Studies on somatic hybridization and genetic transformation in tall fescue breeding |
Source: | Bulletin of the National Grassland Research Institute. No. 53, March 1996, p. 71-117. |
Publishing Information: | Nishinasuno, Tochigi, Japan |
# of Pages: | 47 |
Abstract/Contents: | "Tall fescue is a stress-resistant, persistent Gramineae forage grass. Breeding of tall fescue in Japan has been achieved so far by synthetic methods using mainly ecotypes. However, biotechnical procedure [s] including somatic hybridization or genetic transformation have also been applied recently. Plant regeneration from callus, suspension cells and protoplasts was developed in order to produce agronomically desirable breeding materials of tall fescue by somaclonal variation, somatic hy bridization [hybridization] and genetic transformation in this study. Somatic hybrid plants were obtainedby [obtained by] cell fusion and hygromycin-resistant transgenic plants were obtained by direct gene transfer to protoplasts. 1. Establishment of tissue culture. Callus induction and plant regeneration from calli were achieved in 17 major tall fescue cultivars using immature embryos, mature seeds, and shoot tips as explants. Plant regeneration from calli was higher for the North-European cultivars than for the Mediterranean ones. Japanese cultivars showed a relatively high frequency of plant regeneration. Appearance of calli was classified into three types, e.g. watery, friable, and compact types. Suspension cell culture from which protoplasts could be easily isolated was obtained by culturing friable or compact calli in liquid medium. Viable protoplasts were isolated using an enzyme solution containing Cellulase Onozuka RS and Macerozyme R10. Colony formation from prototplasts could be achieved by bead-type culture with nurse culture. As for medium, AA medium was superior to B5 medium. Green plantlets were regenerated by transferring protoplast-derived colonies via proliferation medium containing 2,4-D and proline onto MS hormone-free medium. It was difficult to obtain green regenerants when the suspension cell culture lasted more than 5 months. Somaclonal variation was observed more frequently in protoplast-derived regenerants than in those derived from calli or suspension cell culture. Major variations included reduction of chromosome number, pollen stainability and seed yield. Open-pollinated progenies from several euploid protoclones showed a lower frequency of heading and shorter plant height than open-pollinated control progenies. However, there was no difference in the fresh weight and indices of leaf blight in autumn between them. 2. Development of somatic hybridization. Somatic hybrid plants were obtained by the combination of metabolic inactivation of tall fescue protoplasts with iodo-acetamide and non-morphogeic character of Italian ryegrass. Sixteen green plants were regenerated from fusion-treated protoplasts and twelve of them were found to be somatic hybrids based in Southern analysis with rice rDNA probe. Six hybrids which regenerated earlier were further analyzed by organellar DNA probes. All of the six somatic hybrids showed differences in restriction fragment length polymorphisms (RFLPs), when maize mitochondrial coxl probe was used. When other mitochondrial probes were used, somatic hybrids showed the RLFPs of either parent or additive ones. They showed only RFLPs of tall fescue chloroplasts. Number of somatic chromosomes ranged between 44-46 to 74-75 and no hybrid had 56 chromosome [s], corresponding to the sum of the number of chromosomes of the parents. Flowering occurred after vernalization but all the plants were sterile. Most of the somatic hybrids segregated new shoots chimaerically, which resembled eithier parent, mainly Italian ryegrass. RFLPs using mitochondrial probes as cox2 and cox3 changed also with phenotypic shifting. These chimaerically developed shoots set normal flowers and seeds by open pollination. One of them showed tall fescue mitochondrial RFLPs, although it resembled Italian ryegrass, and the plant is expected to be a useful breeding material. 3. Production of transgenic plants. Dose response experiments were carried out for direct gene transfer to protoplasts using antibiotic substances as markers. The use of 200 mg/l hygromycin or 50 mg/l phosphinothricin inhibited colony formation from tall fescue protoplasts completely. Hygromycin-resistant transgenic tall fescue plants (cv. Yamanami and Hokuryo) were obtained by direct gene transfer to protoplasts using the PEG-method. Southern hybridization experiments suggested that the introduced hygomycin resistance genes were integrated into various sites of chromosomes. One transgenicplant [transgenic plant] set seeds pollinated by a normal tall fescue plant after 3 years of vernalization. pWI-K6, a vector derived from wheat dwarf virus carrying a kanamycin resistance gene, generated a large number of kanamycin-resistant colonies from transformed protoplasts than a normal plasmid carrying the same gene. Conditions for bombardment of tall fescue cells in suspension culture were compared in transient assay experiments using Β-glucuronidase (GUS) gene as a reporter. A dose of 1300 psi was more effective than 1100 psi or 900 psi for the pressure of bombardment. Tungsten was slightly superiorto [superior to] gold. The strongest expression of transient GUS was observed in a vector derived from wheat dwarf virus, pWI-GUS." |
Language: | Japanese |
References: | 160 |
Note: | Abstract appears in English Pictures, b/w Figures Tables |
| ASA/CSSA/SSSA Citation (Crop Science-Like - may be incomplete): Takamizo, T. 1996. (In Japanese, with English abstract.) Bull. Natl. Grassl. Res. Inst. 53:p. 71-117. |
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