Ryuso Tanaka

Nucleotide sequence of a highly repeated DNA sequence and its chromosomal localization in Allium fistulosum

A highly repeated DNA sequence with a repeating unit of approximately 380bp was found in EcoRV digests of the total genomic DNA of Allium fistulosum. Three independent clones containing this unit were isolated, and their repeating units sequenced. These units showed more than 94% sequence homology, and the copy number was estimated to be about 2.8×106 per haploid genome. In situ hybridization, with the repeating unit as a probe, and C-banding analyses indicated that the repeated DNA sequence of A. fistulosum is closely associated with the major C-heterochromatin in the terminal regions of all 16 chromosomes at mitotic metaphase. The characters of the repeating unit are similar to those of the A. cepa unit, which is taxonomically closely related to A. fistulosum.

Volatile constituents of Zingiber officinale rhizomes produced by in vitro shoot tip culture

Abstract Plantlets with rhizomes were produced in vitro from shoot tips of Zingiber officinale grown in both modified Gamborgs B5 (B5) and Murashige-Skoog (MS) media supplemented with various levels of growth regulators. The rhizomes accumulated the volatile oils similar to those formed in the original rhizome. In the oil from the rhizome grown in the modified B5 medium, the acyclic oxygenated monoterpenes predominated, while the oil from the rhizome grown in the modified MS medium consisted mainly of sesquiterpenes.

C banding treatment for the chromosomes of some gymnosperms

An advanced method of C banding treatment for the chromosomes of gymnospermous plants was developed by modification of the conventional C banding method. All of the three species investigated, i.e.Cycus revoluta, Ginkgo biloba andPinus densiflora, showed clear C bands in the interphase and metaphase chromosomes. The three species were found to differ from each other with respect to number and occurrence of C bands.

Single fertilization inSpiranthes sinensis

The reproduction system inSpiranthes sinensis (Orchidaceae), collected at various localities in Japan, was revealed to be of a peculiar new type which is not to be found in other angiosperms. None of generative cell nuclei has been observed to dividede novo throughout the progamic phase, although they do participate in fertilization. Neither fertilization of the generative cell nucleus with the central nucleus nor formation of endosperm occurred in this plant. Although the chromosome number in the developing proembryonic cells numbered 2n=30, exhibiting diploidy, the number of marker chromosomes was equal to the sum of half the number of marker chromosomes of the parental clones.From the results described above, we may conclude that the reproduction system of this species represents a new type of single fertilization (non-double fertilization) between egg cell and sperm cell nuclei caused by the omission of generative cell division and the formation of only one sperm cell nucleus.

Cytological studies on the nuclear differentiation in microspore division of some angiosperms

In all of the 32 species studied the chromatin in one of the daughter nuclei produced by microspore division was found to diffuse earlier than the chromatin of the other. The former developed into a vegetative nucleus and the latter into a generative nucleus. The stage at which this difference between daughter nuclei was first found appeared to vary widely among species. The earliest was found to be at mid-anaphase observed inHemerocallis thunbergii andMagnolia denudata, and the latest at early interphase observed inLilium japonicum, Pogonia japonica andEpipactis thunbergii. InNarcissus jonquilla and 4 other species the nuclear differentiation started at late anaphase, inHaplopappus gracilis and 13 other species at early telophase, and inTradescantia paludosa and 7 other species at mid-telophase.

Fluorescent banding pattern analysis of eight taxa of Phaseolus and Vigna in relation to their phylogenetic relationships

Phylogenetic relationships among eight taxa of seven species of Phaseolus and Vigna (Phaseolus angularis, P. aureus, P. calcaratus, P. coccineus, P. vulgaris, Vigna sesquipedalis and V. sinensis; 2n = 22 each) were studied by the fluorescent chromosome banding technique. Preparations of somatic metaphase chromosomes of each taxon were sequentially stained with Giemsa, GC-specific fluorochrome chromomycin A3 (CMA) and AT-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI). On the basis of the fluorescent banding patterns of the 22 chromosomes of each taxon, P. angularis, P. coccineus (from China and Korea) and P. vulgaris were grouped into one group (“Phaseolus group”), P. aureus and two Vigna species were grouped into another (“Vigna group”) and P. calcaratus was grouped in an independent group.

In situ hybridization to metaphase chromosomes in six species ofPhaseolus andVigna using ribosomal DNA as the probe

In situ hybridization with a biotin-labeled rice ribosomal DNA (rDNA) probe to the somatic metaphase chromosomes of six species ofPhaseolus andVigna (P. angularis, P. calcaratus, P. coccineus, P. vulgaris, V. sesquipedalis andV. sinensis) was done to determine the sites of rDNA. Hybridization signals were present in the terminal and subterminal chromosome regions of each of the six species. The number of rDNA sites was two inP. angularis andP. calcaratus, four inP. coccineus andP. vulgaris, and six inV. sesquipedalis andV. sinensis.

Efficient isolation of non-chimeric tetraploids artificially induced in a stable culture of Haplopappus gracilis

A method for reducing cytochimerism and inducing homogeneous tetraploids in Haplopappus gracilis (2n = 4) was developed in which masses of shoot primordia treated with 0.5 mg/ml of colcemid for 3 days were cut into small meristematic domes. All of the shoot primordia sampled just after the colcemid treatment were cytochimeras that were mixoploids of 2x, 4x and 8x cells. However, when they were allowed to recover in a colcemid-free medium, the frequency of 4x cells spontaneously increased in most of the shoot primordia. Thirty days after the recovery, chimeric masses containing shoot primordia, each of which consisted uniformly of 4x or 2x cells, were observed. In order to obtain a completely homogeneous tetraploid mass, we then cut these primordia into small pieces, each of which had approximately one meristematic dome. Subsequent to this homogeneous tetraploid masses were easily obtained. Tetraploid shoot primordia could propagate with chromosomal stability over a year, and plants regenerated from these tetraploid shoot primordia were also completely tetraploid. These results show that non-chrimeric masses can be easily isolated from artificially induced cytochimeras using masses of shoot primordia as material.

Polyploid Cytotypes and Their Habitat Preferences in Lycopodium clavatum

Morphological, cytological and ecological observations were made onLycopodium clavatum. Among 121 individuals from 26 populations studied 31 were of the diploid cytotype with 2n=68, 45 were of the triploid cytotype with 2n=102 and 45 were of the tetraploid cytotype with 2n=136. The populations were divided into three types: (1) the sole type—characterized by a single cytotype occupying an entire population, (2) the mixed type A—characterized by two or three cytotypes occupying together the same population, but each cytotype choosing different habitats, (3) the mixed type B—characterized by two or three cytotypes occupying together the same population and sharing the same habitat with each other. The mixed type B populations were found in the places of the early phase of secondary succession in a plant community. In the sole type and the mixed type A, the habitat preference for each cytotype was described as follows: the habitat for the diploid was in humid and shady places, that for the tetraploid was in open and sunny, unstable places, and that for the triploid was intermediate between the other two. Each cytotype can progress towards occupying their optimum habitat successively from the mixed type B population to the mixed type A population and finally reaching the sole type population.

Autoradiographic studies on the incorporation of3H-amino acids into chromosomes during the mitotic cell cycle ofHaplopappus gracilis

The synthesis of chromosomal proteins and the incorporation of labelled proteins into chromosomes in the mitotic cell cycle ofHaplopappus gracilis, 2n=4, were traced autoradiographically with3H-arginine,3H-lysine, and3H-tryptophane. The duration of the mitotic cell cycle in the root tip cells was determined by3H-thymidine autoradiography and was measured to be 13.0 hr (G1 1.3 hr, S 6.5 hr, G2 3.8 hr and M 1.4 hr).3H-arginine labelled proteins which were synthesized at S and G2 were found to be incorporated into chromosomes to a greater extent than proteins which were synthesized either at G1, at the transition phase from late S to early G2, or at the mitotic phase. Such varied incorporation was also found in3H-lysine labelled proteins, but not in3H-tryptophane labelled proteins. These findings indicate that the chromosomal proteins are synthesized mainly at S and G2. Some of the3H-arginine labelled proteins which were synthesized during the first mitotic cell cycle, were found to be incorporated into the chromosomes of the second mitotic cell cycle. The incorporation of the proteins synthesized at one stage of the mitotic cell cycle was found to occur locally in some regions of the chromosomes, while the pattern of incorporation was observed to be similar between euchromatic and heterochromatic regions.