Kokichi Hinata

Transgenic plant production mediated by Agrobacterium in Indica rice

SummaryA reproducible system has been developed for the production of transgenic plants in indica rice using Agrobacterium-mediated gene transfer. Three-week-old scutella calli served as an excellent starting material. These were infected with an Agrobacterium tumefaciens strain EHA101 carrying a plasmid pIG121Hm containing genes for β-glucuronidase (GUS) and hygromycin resistnace (HygR). Hygromycin (50 mg/l) was used as a selectable agent. Inclusion of acetosyringone (50μM) in the Agrobacterium suspension and co-culture media proved to be indispensable for successful transformation. Transformation efficiency of Basmati 370 was 22% which was as high as reported in japonica rice and dicots. A large number of morphologically normal, fertile transgenic plants were obtained. Integration of foreign genes into the genome of transgenic plants was confirmed by Southern blot analysis. GUS and HygR genes were inherited and expressed in R1 progeny. Mendelian segregation was observed in some R1 progeny.

Cell suspension and protoplast culture in rice

Abstract A finely dispersed cell suspension was obtained from anther-derived rice callus cultured in AA medium, which contained an amino acid mixture as the sole nitrogen source. AA medium promoted the release of protoplasts effectively. These protoplasts, when cultured initially in B5 medium for 8–10 days followed by transfer to AA medium, showed sustained cell division and gave rise to callus with a high frequency. The critical factor in the protoplast culture seemed to be the nitrogen source in the culture medium. Green-spot and root formation could be obtained from the protoplast-derived callus.

Sequences of S-glycoproteins, products of the Brassica campestris self-incompatibility locus

The cruciferous genus Brassica has a self-incompatibility system that is under the sporophytic control of a single locus, S (ref. 1). Although evidence has accumulated that S-locus-specific proteins, termed S-glycoproteins2–4, are strongly associated with the expression of self-incompatibility4–8, their structures and functions are still unknown. This paper reports the sequences of the S-glycoproteins from three homozygotes of Brassica campestris. They show extensive homology. The published DNA sequence data for the S6-glycoprotein of B. oleracea9 can be revised to encode the total sequence of the glycoprotein on the basis of the sequences of S-glycoproteins of B. campestris. These S-glycoproteins have cysteine-rich clusters and 6–7 oligosaccharide chains. There is no homology between the S-glycoproteins of Nicotiana alata10 and those of B. campestris.

A novel male-sterile mutant of Arabidopsis thaliana, faceless pollen-1, produces pollen with a smooth surface and an acetolysis-sensitive exine

A mutant exhibiting conditional male sterility, in which fertility was restored under conditions of high humidity, was identified in T-DNA tagged lines of Arabidopsis thaliana. Scanning electron microscopy (SEM) demonstrated that the pollen surface was almost smooth and the reticulate pattern not prominent. Thus, the mutant was named faceless pollen-1 (flp1). Transmission electron microscopy (TEM) revealed that the smooth appearance was due to tryphine filling in the exine cavities and covering the pollen surface. The lipid droplets in the tryphine of mutant pollen were smaller and more numerous than those of the wild type. SEM analysis also demonstrated that pollen exine was easily damaged by acetolysis, suggesting that a component of exine, sporopollenin, was defective in the mutant. In addition, the stems and siliques had reduced amounts of wax crystals. A predicted amino acid sequence of the cDNA that corresponded to the tagged gene, flp1, showed sequence similarity to proteins involved in wax biosynthesis. The FLP1 protein is likely to play a role in the synthesis of the components of tryphine, sporopollenin of exine and the wax of stems and siliques.

Comparison of S-alleles and S-glycoproteins between two wild populations of Brassica campestris in Turkey and Japan

SummaryThe number of identical S-alleles between two wild populations of B. campestris, one in Turkey, the other in Japan, that have been independent of one another for a long time was investigated. Diallel pollination tests between 38 S-allele homozygotes, i.e., 16 S-allele homozygotes from Turkey and 22 from Japan, revealed that these were 29 different S-alleles only 4 common ones. These S-alleles were differentiated by the iso-electric focusing (IEF) analysis of S-locus glycoproteins (SLGs) stained with an antiserum against SLG8. All identical S-alleles had the major SLG band at the same pI value without exception, even though they were collected from different populations. However, the number of minor bands of SLGs varied between the two populations; the S-alleles in Balcesme had generally fewer minor bands than those in Oguni. The 29 independent S-alleles were numbered from S21 to S49 according to the pI value of the major SLG band. The major bands whose pI values were 7.5–8.5 were most common. Blot-hybridization patterns of genomic DNA hybridized with SLG8 cDNA were not always the same among the strains of identical S-alleles obtained from different populations. Because about 20% of the S-alleles were shared between the two populations, it can be inferred that more than hundreds of S-alleles have been accumulated by mutation in B. campestris throughout the world.

Highly divergent sequences of the pollen self-incompatibility (S) gene in class-I S haplotypes of Brassica campestris (syn. rapa) L.

Self‐incompatibility (SI) enables flowering plants to discriminate between self‐ and non‐self‐pollen. In Brassica, SI is controlled by the highly polymorphic S locus. The recently identified male determinant, termed SP11 or SCR, is thought to be the ligand of S receptor kinase, the female determinant. To examine functional and evolutionary properties of SP11, we cloned 14 alleles from class‐I S haplotypes of Brassica campestris and carried out sequence analyses. The sequences of mature SP11 proteins are highly divergent, except for the presence of conserved cysteines. The phylogenetic trees suggest possible co‐evolution of the genes encoding the male and female determinants.

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

SummaryThe 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%.

Production of somatic hybrid plants, ‘Brassicomoricandia’, through protoplast fusion between Moricandia arvensis and Brassica oleracea

Intergeneric hybrid plants were obtained through protoplast fusion between Brassica oleracea L. and Moricandia arvensis (L.)DC. (Toriyama et al 1987). M. arvensis is classified as a member of the subtribe Moricandiinae of the family Brassicaceae, and is known to be a C 3 -C 4 intermediate species with respect to low CO 2 compensation point (Holaday et al 1981). We have been studying (i) the production of somatic hybrids between distantly related species, and (ii) the genetic regulation of photosynthetic systems in the hybrids.

Dominance relationships between S -alleles in self-incompatible Brassica campestris L.

Dominance relationships were studied for 249 out of 276 possible pair-wise combinations between 24 S-alleles of Brassica campestris that had been isolated from two natural populations from Turkey and Japan. Each F1 hybrid was test-crossed reciprocally against its respective parental S-homozygotes to determine the dominance relationships between the pair of S-alleles it contained. The 24 S-alleles were classified into two groups on the stigma side and three groups on the pollen side. In the stigma, codominance occurred frequently, and dominance or recessiveness seemed to appear according to the combination of S-alleles. In the pollen, codominance was less frequent, and there seemed to be a certain hierarchy of the dominance relationships as a whole, although dominance appeared with certain specific combinations of S-alleles. Interactions among 24 S-alleles were different in the stigma and in the pollen. Independent weakening of S-alleles was found between 20 pairs in the pollen, but only two in the stigma. This interaction seems to be correlated with recessiveness of S-alleles.

Molecular characterization of rice genes specifically expressed in the anther tapetum

In situ localization of mRNA was carried out on two cDNAs (Osc4 and Osc6) that had been isolated from rice anthers at the microspore stage. The mRNA corresponding to each cDNA was shown to be localized only in the tapetal cells of the rice immature anthers, but not in the microspores or the mature pollen. The corresponding genomic clone, Osg6B, was isolated, and its 5′-upstream region was found to regulate β-glucuronidase expression in the tapetum of transgenic tobacco. A set of 5′ deletions was also generated and a 1095 bp 5′ region was revealed to be necessary for activation of the Osg6B promoter in transgenic tobacco.