Polyploidization increases the sensitivity to DNA-damaging agents in mammalian cells

Polyploidization occurs during normal development as well as during tumorigenesis. In this study, we investigated if the responses to genotoxic stress in cancer cells are influenced by the ploidy. Prolonged treatment of Hep3B cells with the spindle inhibitor nocodazole resulted in mitotic slippage, followed by re-replication of the DNA to produce polyploids. Reintroduction of p53 restored the checkpoints and suppressed polyploidization. Remarkably, a stable tetraploidy cell line could be generated from Hep3B by a transient nocodazole treatment followed by a period of recovery. Using this novel tetraploid system, we found that tetraploidization increased the cell volume without significantly affecting the cell cycle. Although tetraploidization was accompanied by an increase in centrosome number, the majority of mitoses in the tetraploid cells remained bipolar. Polyploidization sensitized cells to genotoxic stress inflicted by ionizing radiation and topoisomerase inhibitors without affecting the sensitivity to spindle inhibitors. Accordingly, more gamma-H2AX foci were induced by radiation in tetraploids than in normal Hep3B cells. Likewise, primary tetraploid human fibroblasts displayed higher gamma-H2AX foci formation than diploid human fibroblasts. An implication for chemotherapy is that some cancer cells can be sensitized to genotoxic agents by a preceding step that induces polyploidization.     

通用核型分析软件

用传统方法进行核型分析,需要复杂的测量和计算,因此,一般的研究者只基于小样本（５—１０个细胞）来研究核型。然而,若要获得准确的核型,样本数应不低于３０〔１〕,这样,就更增加了其工作量,而且很容易导致错误。目前,国内外有一些染色体图像分析系统可以进行核...     

柑橘与黄皮属间体细胞杂种植株的异常倍性变异

黄皮属（Ｃｌａｕｓｅｎａ）是芸香科柑橘亚科黄皮族植物,有３０余个种,其中１１个种原产于中国。黄皮在我国广东、广西、福建等省区有大面积商业栽培,其中鸡心黄皮是最著名的品种［１］。本研究以柑橘属优良脐橙品种“朋娜”为胚性愈伤组织亲本,以黄皮属品种鸡心黄皮...     

Secondary polyploids, heterosis, and evolutionary crop breeding for further improvement of the plantain and banana (Musa spp. L) genome

 Several primary tetraploids with desirable attributes have been selected by Musa breeding programs. Diploid parental stocks have become available for further genetic improvement of the Musa genome. Hence, new genome manipulations should be tested before they become routine in breeding programs. Through comparison of the performance of triploid landraces, their primary tetraploid hybrids and secondary polyploid derivatives, plus diploid ancestors, it was found that the production of secondary triploids (TM3x) is more rewarding than developing secondary tetraploids. TM3x achieved significant high polyploid-parent heterosis for yield either by maximizing heterozygosity through crosses between unrelated parents, or by selection of linkats in hybrids derived from crosses between euploid full-sibs. There were significant differences in bunch weight between full-sib secondary polyploids, which suggested that specific combining ability should be re-defined considering not only a specific cross combination but also the individual performance within each cross. This paper proposes a crop-breeding strategy which takes into consideration the process of domestication of banana and plantain. Current data suggest that this type of evolutionary breeding approach may be feasible in Musa. Received: 10 September 1996/Accepted: 31 January 1997     

Inheritance of nuclear DNA content in leaf epidermal cells of Zea mays L.

 The nuclear DNA content (ploidy level) of maize leaf-epidermal cells was investigated by Feulgen cytophotometry in two lines, Illinois High Protein (IHP) and Illinois Low Protein (ILP), their reciprocal hybrids, and their F₂s. Epidermal cells have a 2C, 4C or 8C nuclear DNA content. The mean DNA content per nucleus in IHP was significantly higher than in ILP; the mean DNA content per nucleus in hybrids was intermediate between the parental lines, and the same DNA content was measured in reciprocal crosses. In F₂s the same mean DNA content as in F₁s was observed but with larger variability than in the F₁, possibly indicating genetic segregation. It is inferred that the ploidy level in the leaf epidermis is inherited, and incomplete dominance occurs in hybrids. The same behaviour in the different genotypes was observed for epidermal cell-surface area, except that an increase of mean surface area occurred in the F₁, probably due to heterotic effects. The difference in the accumulation of 4C and 8C nuclei in leaf epidermis parallels that reported between two genotypes for the endosperm tissue: to the greater chromosome endoreduplication found in the endosperm there were correspondingly higher frequencies of 4C and 8C nuclei in the leaf epidermis, indicating a higher general tendency to chromosome endoreduplication in IHP than in ILP. It is suggested that the accumulation of 4C nuclei (G₂-block) in the leaf epidermis may be regarded as the initial step of chromosome endoreduplication, the two phenomena being related to the control of the sequence DNA synthesis-mitosis, possibly involving the same genes in both endosperm and leaf. However, the inheritance of DNA content per nucleus in epidermal tissue seems to be different from that observed in endosperm tissue of the same genotypes, suggesting that differences may occur in the regulation of the activity of these genes. Received: 19 November 1996 / Accepted: 29 November 1996     

Haploid and diploid survival differences demonstrate selection in scale insect demes

Summary Black pineleaf scale insect populations are subdivided into genetically differentiated demes associated with individual pine trees. A comparison of sex ratios early and late in the life cycle demonstrated differences in the mortality experienced by haploid males and diploid females. Hatching ratios were significantly female-biased, and differential mortality increased this bias in ratios estimated just before adult male eclosion. The relative survival of males and females varied with overall mortality, causing a correlation between local densities and the surviving sex ratio. We suggest (a) that the genetic differentiation of scale demes results in part from selection pressures associated with individual pine trees, (b) that this differentiation entails an accumulation of locally adaptive traits within the scale subpopulation on each tree, (c) that expression of these adaptations in the haploid and diploid sexes may vary with their frequencies, and (d) that the surviving sex ratio thus offers a comparative measure of selection and the local adaptation achieved by the insects in individual demes.     

From crossbreeding to biotechnology-facilitated improvement of banana and plantain

The annual harvest of banana and plantain (Musa spp.) is approximately 145 million tons worldwide. About 85% of this global production comes from small plots and kitchen or backyard gardens from the developing world, and only 15% goes to the export trade. Musa acuminata and Musa balbisiana are the ancestors of several hundreds of parthenocarpic Musa diploid and polyploid cultivars, which show multiple origins through inter- and intra-specific hybridizations from these two wild diploid species. Generating hybrids combining host plant resistance to pathogens and pests, short growth cycles and height, high fruit yield, parthenocarpy, and desired quality from the cultivars remains a challenge for Musa crossbreeding, which started about one century ago in Trinidad. The success of Musa crossbreeding depends on the production of true hybrid seeds in a crop known for its high levels of female sterility, particularly among polyploid cultivars. All banana export cultivars grown today are, however, selections from somatic mutants of the group Cavendish and have a very narrow genetic base, while smallholders in sub-Saharan Africa, tropical Asia and Latin America use some bred-hybrids (mostly cooking types). Musa improvement goals need to shift to address emerging threats because of the changing climate. Innovative cell and molecular biology tools have the potential to enhance the pace and efficiency of genetic improvement in Musa. Micro-propagation has been successful for high throughput of clean planting materials while in vitro seed germination assists in obtaining seedlings after inter-specific and across ploidy hybridization. Flow cytometry protocols are used for checking ploidy among genebank accessions and breeding materials. DNA markers, the genetic maps based on them, and the recent sequencing of the banana genome offer means for gaining more insights in the genetics of the crops and to identifying genes that could lead to accelerating Musa betterment. Likewise, DNA fingerprinting has been useful to characterize Musa diversity. Genetic engineering provides a complementary tool to Musa breeders who can introduce today transgenes that may confer resistance to bacteria, fungi and nematodes, or enhance pro-vitamin A fruit content. In spite of recent advances, the genetic improvement of Musa depends on a few crossbreeding programs (based in Brazil, Cameroon, Côte d'Ivoire, Guadeloupe, Honduras, India, Nigeria, Tanzania and Uganda) or a handful of genetic engineering endeavors (Australia, Belgium, India, Kenya, Malaysia and Uganda). Development investors (namely international aid and philanthropy) should therefore increase their funding to genetically enhance this crop that ranks among the 10-top staple foods of the developing world.     

倍性复杂植物的2种倍性鉴定方法的建立及应用(专题约稿）

研究以猕猴桃属内不同植物的幼嫩叶片为材料,利用流式细胞术和全基因组SNP(single nucleotide polymorphism)位点杂合子等位基因深度比率(heterozygous allele depth ratio)分布2种方法进行猕猴桃倍性鉴定。对取样叶片的生长状态、防止细胞核黏连的PVP(聚乙烯吡咯烷酮)浓度、滤网目数及过滤次数、不同倍性样本全基因组SNP分型的参数调整等因素进行探索。结果表明,流式细胞术检测中取未展开的幼嫩叶片获得完整细胞核的数目最多;5%PVP对减少细胞核之间的黏连最适宜;500目滤网过滤3次效果最好。SNP的分型主要与模拟基因组的组装质量和过滤识别SNP的参数设置有关。流式细胞术鉴定倍性的关键技术是使用未展开的幼嫩叶片以保证足够数量的完整细胞核及减少细胞核之间的黏连。同一植物材料的染色体倍性在60Co-γ辐照处理前后未发生改变。全基因组SNP位点杂合子频率分布图判断的倍性与流式细胞术鉴定结果一致。2种鉴定结果可以相互验证,使倍性的判断变得更加准确,为加快猕猴桃育种提供了基础。     

Effects of 4-CPPU on in vitro multiplication and sustainable generation of Hibiscus rosa-sinensis L. ‘White Butterfly’

There are more than nine thousand cultivars of Hibiscus rosa-sinensis L., with a series of flowers with shapes, colors and new cultivars continues as generated through both traditional and modern breeding techniques. In this study, advanced biotech methods of in vitro culture have been used to identify a technique for the efficient mass multiplication of H. rosa-sinensis ‘White Butterfly’, using phenyl urea, N-(2-Chloro-4-pyridyl)-N′-phenylurea (4-CPPU). For the first time, the effects of 4-CPPU for stimulating axillary shoot proliferation and multiple shoot regenerations from nodal explants were evaluated, and the optimal nutrient media deduced. From the diverse concentrations as 0.1, 0.5, 2.5, 5.0 & 10.0 µM of 4-CPPU, the highest frequency of shoots was recorded at 2.5 µM supplied in Murashige and Skoog (MS, pH-5.8) medium. After eight-weeks of culture, on an average of 6.7 shoot were obtained on this media with shoot heights of 4.2 cm from each explant. With the involvement of 0.5 µM-IBA (indole-3-butyric acid) in MS medium the regenerated shoots were rooted and followed by successful acclimation to ex vitro conditions. The ploidy consistency among the micro-plants was analyzed using flow cytometry and compared with ex vitro grown plants. No differences in the ploidy levels were observed among the 4-CPPU induced plants, when compared with the donor plants.     

Doubled haploid production in rocket (Eruca sativa Mill.) through isolated microspore culture

Haploid induction in rocket (Eruca sativa Mill.), a novel and increasingly important vegetable, was studied in microspore culture. A procedure based on a high sucrose NLN medium and heat shock treatment resulted in nuclear divisions and embryo induction. The effect of genotype both among seed lots and among single plants was a major factor influencing embryo formation. The addition of activated charcoal was essential for obtaining reproducible results, 0.2 mg l⁻¹ being superior to 1.0 mg l⁻¹. A 24 h heat shock treatment at 32°C doubled the embryogenic response compared to a 48 h treatment. Embryo conversion was only efficient (23%) for embryos that had been cultured on medium with activated charcoal and subcultured on solid B5 medium; pretreatment of embryos with ABA or desiccation for 1–3 weeks inhibited embryo conversion. Analysis of ploidy level revealed that the majority (65.6%) of 489 regenerated plantlets tested were diploid. Breeding programs and genetic studies of rocket are likely to benefit substantially from the established method.