伊贝母愈伤组织在继代培养过程中的染色体变异和分化频率的研究

本文报道了伊贝母愈伤组织在不同培养基上继代培养的的染色体变异和分化频率。结果表明,随着继代培养的进程,二倍体细胞频率逐渐减少。亚二倍体和超二倍体细胞频率迅速增加。同时还观察到比率不等的亚单倍体、单倍体,超三倍体和超四倍体细胞。于是培养物最终变成为只有少量整倍体而多数为非整倍体染色体数的混倍体细胞群体。不同培养基对染色体变异的效应是2,4-D+KT>IAA+KT>NAA+KT。分化频率随继代次数的增加而下降。同时还观察到了各种类型的染色体结构变异和有丝分裂异常。根据试验结果,对继代培养中染色体变异的原因以及与分化频率的关系进行了讨论。     

Ploidy-dependent genomic stability in the tissue cultures of ornamental Phlox drummondii Hook

Comparisons of the chromosome numbers, 2C nuclear DNA amounts and karyomorphology were made in explant cultures of diploid (2n = 2x = 14) and autotetraploid (2n = 4x = 28) Phlox drummondii. In 6–36 week old calli derived from diploid internodal segment explants, and in cells of root tips regenerated from such callus, marked differences were observed in chromosome number. The chromosome numbers ranged from 2n = 14 to 2n = 100 and DNA amounts from 8.20 to 63.20 pg in the diploid derived callus, while the extent of variation was much reduced in the regenerated roots. In contrast, the autotetraploid cultures were characterised by the maintenance of the same chromosome number and DNA amounts as the mother plant. Modified chromosome structures were not apparent in any of the cultures. The possible reasons for the chromosomal instability at the diploid level and stability at the tetraploid level are discussed.     

Regeneration of amphidiploid plants from tissue cultures of Allium wakegi

Callus was induced from the bulb of Allium wakegi Araki on MS semisolid medium supplemented with several growth regulating substances. The calli were subcultured every 40 days. At the time of every subculture the callus was subdivided to be used for chromosome studies, plant regeneration, or continuous callus multiplication. The chromosome constitution of cells in callus and regenerated plants varied over the culture period, and at the 3rd subculture amphidiploid plants were obtained. They appeared even more frequently than amphihaploid plants in the 4th subculture. Hypoamphihaploid regenerants appeared as stumpy shoots but none of these shoots proceeded further to form a normal plant. By Giemsa C-banded karyotype, the chromosome constitution of amphidiploid plants was found to result from exact doubling of the chromosome sets of amphihaploid common species. Amphidiploid plants show better viability and growth than common plants. The possibility and the expectation of new crop plants to be developed from amphidiploid plants will be discussed.     

Current status of chromosomal abnormalities in mouse embryonic stem cell lines used in Japan

We performed chromosomal analysis on 540 mouse embryonic stem (ES) cell lines obtained during 2001 to 2004 from 20 institutions in Japan. Overall, 66.5% of the ES cell lines showed normal chromosomal numbers, but 15.9%, 9.1%, and 2.8% showed modal chromosomal numbers of 41, 42, and 39, respectively. When we karyotyped 88 ES cell lines selected arbitrarily from the 540 lines, 53 (60.2%) showed normal diploid karyotypes; the sex chromosome constitution of 52 lines was XY, with the remaining 1 being XX. Among 35 ES cell lines showing abnormal karyotypes, trisomy of chromosome 8 (41, XY, +8) was dominant (51.4%), 14.3% had trisomy 8 with loss of one sex chromosome (40, XO, +8), and 11.4% had trisomy 8 together with trisomy 11 (42, XY, +8, +11). Karyotypic abnormalities including trisomy 8 and trisomy 11 occurred in 88.6% and 17.1% of ES cell lines, respectively. The XO sex chromosome constitution was observed in 25.7% of all abnormal ES cell lines. Of the 88 selected ES cell lines, 60 lines were established from strain 129 animals, 17 from F1 progeny of C57BL/6J x CBA (called TT2 in this study), and 11 from C57BL/6J mice. Normal diploid karyotypes were observed in 58.3% of lines derived from 129, 58.8% of those from TT2, and 72.7% of C57BL/6J. The relatively high incidence of abnormalities in chromosomal number and karyotype in ES cell lines used in Japan suggests the importance of chromosomal analysis of ES cells for successful establishment of new animal models through germline transmission.     

Cytogenetic studies of Haplopappus gracilis in both callus and suspension cell cultures

Summary Investigations have been carried out on karyotype change in both callus and suspension cell cultures of Haplopappus gracilis (2n=4). It has been found that polyploidization arises directly in culture to give up to six times the normal diploid chromosome number in some cultures. In polyploid cultures, both chromosome loss and chromosome rearrangements occur to give rise to aneuploid karyotypes displaying chromosomes which differ in morphology from the diploid set. Whole or partial chromosome loss has been observed in the form of lagging chromosomes and chromosome bridges at anaphase, and micronuclei, ring chromosomes and chromosome fragments at other stages in mitosis. C-banded preparations have confirmed the occurrence of chromosomal rearrangements. Comparative investigations suggest that (i) more polyploidy occurs in callus cultures than in suspension cell cultures, and (ii) the presence of cytokinin (kinetin) in the culture medium may reduce the extent of karyotype change.     

The effect of B chromosomes and T-DNA on chromosomal variation in callus cells and regenerated roots of Crepis capillaris

The chromosome behaviour has been compared in three Crepis capillaris callus culture lines and the roots regenerated from these calli. The calli were obtained from explants derived from plants without and with two B chromosomes and the hairy roots were obtained from plants transformed with Agrobacterium rhizogenes. Cytological studies demonstrated that the presence of additional DNA as B chromosomes or as T-DNA had an influence on the numerical and structural variability of the standard chromosome in long-term callus cultures and in regenerated organs. The callus with two B chromosomes displayed higher levels of polyploidyzation than callus without B chromosomes. The roots regenerated from both these calli were only diploid, while roots regenerated from transformed callus were also polyploid. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Effect of Growth Conditions and Origin of Tissue 5Phaseolus vulgaris L.)

Callus cultures isolated from various somatic tissues and anthertissue of Phaseolus vulgaris seedlings on a defined growth mediumcontained few diploid cells. The proportion of diploid cellsdid not alter as cultures lost their ability to form vasculartissue. Meristematic cells of roots initiated after transferto induction medium were diploid. All cultures lost their morphogeneticpotential after five to seven subcultures except anther calluswhich formed vascular tissue over a prolonged period of cultureon maintenance medium. After six subcultures anther callus containedmore polyploid cells than somatic cultures. Callus isolated from bean hypocotyl tissue in the presence ofcoconut milk consisted mainly of diploid cells and retainedits morphogenetic potential for a greater number of subculturesthan callus grown on defined medium. Transfer of callus isolatedon the defined medium to medium containing coconut milk increasedthe proportion of diploid cells and prevented further loss ofinorphogenetic potential. An equivalent concentration of cytokininto that in coconut milk prevented the loss of potential butdid not affect the ploidy of the cultures.     

Ribosomal DNA magnification in Saccharomyces cerevisiae.

Strains monosomic for chromosome I of Saccharomyces cerevisiae contain 25 to 35% fewer rRNA genes than do normal diploid strains. When these strains are repeatedly subcultured, colonies are isolated that have magnified their number of rRNA genes to the diploid amount while remaining monosomic for chromosome I. We have determined the amount of DNA complementary to rRNA in viable haploid spores derived from a magnified monosomic strain. Some of these haploids contained 24 to 48% more rRNA genes than a normal euploid strain. These extra genes may be responsible for the increased number of rRNA genes in the strain monosomic for chromosome I. Genetic analysis of the haploids containing extra rRNA genes suggested that these genes are linked to chromosomal DNA and are heterozygous. They were not closely linked to any centromere and were not located on chromosome I. Furthermore, all the DNA complementary to rRNA in one of these haploid strains with magnified rRNA genes sedimented at a chromosomal molecular weight, consistent with chromosomal linkage. In addition, several new mutations mapping on chromosome I were used to show that ribosomal DNA magnification was not due to a chromosome I duplication.     

Nuclear DNA content and phytohormone requirements of normal, crown gall and habituated tissues of Nicotiana tabacum var. White Burley

Determination of the nuclear DNA content of leaves and normal, habituated and Crown gall callus tissues of Nicotiana tabacum var. White Burley were performed using cytophotometry on Feulgen stained preparations. Several aspects concerning the reliability of the Feulgen technique for DNA determinations were investigated.Crown gall callus tissue used in this study had both a higher nuclear DNA content and chromosome number than normal callus (3.2C versus 2.5C). Both have a higher DNA content than the diploid tobacco leaf cells (2C).The normal callus tissue failed to grow on medium without indole acetic acid and kinetin when cultured in tubes. From this normal callus two habituated lines growing without both phytohormones were selected by culturing the normal callus first in the absence of either indole acetic acid or kinetin. Changing the culture conditions of the normal callus by using culture flasks instead of tubes resulted in a remarkably faster growth rate of the tissue. This was accompanied by an acquisition of the habituation characteristics since it was possible now to grow this tissue also directly on medium lacking both phytohormones. All habituated tissues showed a higher nuclear DNA content compared to the normal callus tissue from which they were derived. Interestingly, one of the tissues acquired a nuclear DNA content not different from that of Crown gall tissue. By changing the culture conditions of Crown gall callus tissue no concomitant change in nuclear DNA content occurred.The results suggest a correlation between the acquisition of a special chromosome complement and the loss of phytohormone requirement resulting in autonomous growth.     

Ploidy variation of pronamide-treated maize calli during long term culture

Summary Anther-derived calli of corn were treated with 10 M pronamide for 2, 3 and 4 days. The ploidy level of the calli was then evaluated using flow cytometry, at different times after the treatment. Untreated haploid calli did not change in ploidy level for 97 days but by 466 days, there were up to 50% diploid or higher ploidy cells thus showing that spontaneous doubling may occur during corn calli subculture with this genotype. Pronamide treatment did increase the percentage of diploid and tetraploid cells and by 466 days, all of the lines showed an additional change toward higher ploidy levels. This change may be due to spontaneous chromosome doubling or to differential cell cycle times of cells with different ploidy levels. The ploidy level of plants regenerated from the cultures was determined by counting the guard cell chloroplast numbers and the correlation with the ploidy level of the cultures was r²=0.84. These studies show that pronamide treatments can increase haploid maize callus chromosome numbers and that spontaneous chromosome doubling can occur with time in maize callus.     

Induction of morphologically and genetically unstable calluses of Fagopyrum tataricum by colchicine

A study was made of colchicine effect on genetic and morphological stability of morphogenic calli of the tatar buckwheat Fagopyrum tataricum (L.) Gaertn. A prolonged exposure of the morphogenic callus in colchicins-containing medium led to its morphological changes: the callus became more friable, and proembryogenic cell complexes disappeared. Genetic non-stability started from the first days of culturing in colchicine-containing indicated by amitotic divisions and K-mitoses, leading to the formation of micronuclei and increased variability in chromosome number. As a result of colchicine action, a few lines of heterogeneous calli were obtained differing in morphology, chromosome numbers, and ability to morphogenesis from the primary morphogenic callus. Genetic instability appeared in the following passages, when the treated calli were subcultured in the medium without colchicine: a wide range of abnormal nuclear division and chromosome aberrations was observed. This prolonged genetic instability was accompanied by a prominent increase in the formation of friable clones, which was 20-30 times higher than in the control. The formation of friable clones seems to result from one or two point mutations.     

Telomere functions: lessons from yeast

Telomeres are specialized DNA protein structures that form the ends of eukaryotic chromosomes. In yeast, loss of even a single telomere causes a prolonged, but transitory, cell-cycle arrest. During this arrest, many broken chromosomes acquire a new telomere by one of three pathways, although at the cost of a partial loss of heterozygosity. In addition, a substantial fraction of the chromosomes lacking a telomere is lost, which generates an aneuploid cell. In these cases, the broken chromosome is usually replicated and segregated for ten or more cell divisions in unstable form. Extrapolation from yeast suggests that the gradual loss of telomeric DNA that accompanies ageing in humans may initiate the kinds of chromosomal rearrangements and genetic changes that are associated with tumorigenesis.     

Cytological Analysis of Seedling Roots, Transformed Root Cultures and Roots Regenerated from Callus of Swainsona galegifolia (Andr.) R. Br.

The stability of chromosome number was investigated in culturesof roots from Swainsona galegifolia. Roots from germinated seedsor plants grown in vitro when cultured in liquid medium howed90% or more cells with the diploid number of 2n = 32. The remainingcells showed aneuploidy mostly below 32. The stability of chromosomenumbers was not affected by transformation with Agrobacteriumrhizogenes although when roots were transformed with A. rhizogenesLB 9042 the range of chromosome numbers in the few aneuploidcells present was higher than in roots for which strain A4 wasused. In contrast, roots regenerated from callus had only 15%of cells with 2n = 32 and showed a modal number of 18. Six rootcultures established from individual roots regenerated fromcallus showed a wide variation in number (8–83). Fivecultures had a modal number around 18, the sixth, a modal numberof 39 which is above the diploid number. The implication ofthe results for the production of secondary metabolites fromroot culture is discussed. Key words: Agrobacterium rhizogenes, callus cultures, chromosome number, root cultures, Swainsona galegifolia     

Effect of confluent holding on potentially lethal damage repair, cell cycle progression, and chromosomal aberrations in human normal and ataxia-telangiectasia fibroblasts

The effects of confluent holding recovery on survival, chromosomal aberrations, and progression through the life cycle after subculture of human diploid fibroblasts X-irradiated during density inhibition of growth have been examined. The responses of three normal strains were determined and compared with those of four ataxia-telangiectasia (AT), an AT heterozygote, and two hereditary retinoblastoma strains. The capacity for potentially lethal damage repair (PLDR) was slightly reduced in retinoblastoma cells and almost absent in AT cells, but normal in an AT heterozygote. The decline in chromosomal aberrations seen in normal cells during confluent holding was absent in AT cells, consistent with the lack of PLDR. Following subculture, all irradiated AT fibroblasts progressed through the cell cycle to the first mitosis with no delay. AT heterozygotic and retinoblastoma cells showed both an enhanced delay in the initiation of DNA synthesis and a large fraction of cells irreversibly blocked in G1 as compared with normal cells. Both the delayed entry into S and the G1 block were reduced by confluent holding. These results indicate that AT homozygotic and heterozygotic cells respond quite differently to X irradiation.     

新疆紫草的组织培养及其染色体分析

新疆紫草(Arnebia euchroma)的胚轴、子叶、根都产生愈伤组织。不同来源的外植体产生苗的能力不同,胚轴来源的愈伤组织分化苗最多,而且苗生长旺盛。根的愈伤组织只产生不定根。新疆紫草的核型是K(2n)=14=2m+8sm+2sm(SAT)+2st。随着继代培养代数增 多,染色体变异的种类和频率以及染色体数目显著增加。如果继代培养时间较长,染色体数目变异的范围更广,而且出现染色体结构变异。     

Karyotype stability in long-term callus derived plants ofCrepis tectorum L.

The study of in vitro growth of Crepis tectorum revealed 100 % callusing and 40 % plantlet regeneration. The root and leaf used as explants showed the normal diploid (2n=8) chromosome constitution. In one month old culture 95 % callus cells were diploid. The callus maintained in 2,4-D 1 mg 1-1 for two years showed 62 % diploid, 5 % tetraploid and 33 % hyperdiploid cells. The differentiation of shoot occurred in two year old calli after subeulturing in 2 mg I-1 BAP and the potentiality of regeneration was retained for more than one year. The leaf-tips of regenerated plants were homogeneous and identical to the donor plant both in number and morphology of chromosomes.     

Enhancement of Regeneration Potential and Variability by γ-Irradiation in Cultured Cells of Scilla Indica

Induced mutagenesis in callus tissues was studied in the medicinal plant Scilla indica irradiated with different doses of -radiation ranging from 2.5 to 20 Gy. Low doses accelerated the cell division and growth rate of the tissues whereas high doses repressed growth rate and resulted in lethality of tissues. Various cytological and chromosomal abnormalities were observed in the irradiated calli, the degree of which depended upon the dosage. Low doses of irradiation also promoted the regenerating capacity of the calli tissues and plants regenerating from them exhibited better growth and vigour compared to normal plants. High doses led to loss of regenerating capacity and promoted formation of malformed and stunted plants. Cytological study of regenerants revealed both diploid and mixoploid plants but no tetraploids were obtained.     

Haploidization in Saccharomyces cerevisiae induced by a deficiency in homologous recombination

Diploid Saccharomyes cerevisae strains lacking the RAD52 gene required for homologous recombination have a very high rate of chromosome loss. Two of four isolates subcultured ～20 times (～500 cell divisions) became haploid. These strains were capable of mating with wild-type haploids to produce diploid progeny capable of undergoing meiosis to produce four viable spores.     

Chromosome-shuffling technique for selected chromosomal segments in Saccharomyces cerevisiae

We describe a novel chromosome engineering technique for shuffling selected regions of chromosomes from two strains in Saccharomyces cerevisiae: The technique starts with the construction of MAT a and MATα strains in which a particular chromosome is split at exactly the same site in both strains such that the split chromosomes generated are marked with different markers. The two strains are then crossed, and the resultant diploid is cultivated in nutrient medium to induce loss of the split chromosome originating from either of the strains. We predicted that some of these clones that are hemizygous for the split chromosome would spontaneously restore a homozygous configuration of the split chromosome during cultivation. We verified this prediction by tetrad analysis and quantitative Southern hybridization analysis, indicating that it is possible to create diploid hybrids in which a selected region of a chromosome from one strain is replaced by the corresponding chromosomal region from another strain. We also found that some chromosomal segments maintain a hemizygous state. This novel technique, which we call ‘chromosome shuffling’, could provide a new tool to analyze phenotypic alterations caused by the replacement or hemizygosity of a selected chromosomal region in not only laboratory but also industrial strains of S. cerevisiae.     

Mitosis is not the only distributor of mutated cells: Non‐mitotic endopolyploid cells produce reproductive genome‐reduced cells

Giant endopolyploid nuclei (>16n) can spontaneously fragment by endomitosis (nuclear internal division) into near‐diploid cells with reproductive capacity (depolyploidization), and endotetra/octopolyploidy can undergo chromosome‐visible meiotic‐like genome reductional divisions also to replicative subcells. These unconventional divisions are associated with production of aneuploidy, which led to the question in this study of whether endopolyploidy, in general, can contribute genetic variability to tumorigenic potential. For this purpose, non‐proliferative endopolyploid cells (range: 4n–32n) in near‐senescence of normal diploid cell strains were analysed for nuclear–morphogenic changes associated with the presence of diploid‐sized nuclei in the cytoplasm. A one‐by‐one nuclear‐cutoff process gave rise to reproducing genome‐reduced cells. It was concluded that these unconventional cell divisions are, indeed, suspects of originating genetic variability. Details of these irregular mitoses were compared to ‘mitotic–meiosis’ in primitive organisms, which suggested activation of an ancestral trait in the mammalian cells.