Aurora A Kinase Plays a Key Role in Mitosis Skip during Senescence Induced by Ionizing Radiation / 生物医学与环境科学（英文）

OBJECTIVE@#To investigate the fate and underlying mechanisms of G2 phase arrest in cancer cells elicited by ionizing radiation (IR).@*METHODS@#Human melanoma A375 and 92-1 cells were treated with X-rays radiation or Aurora A inhibitor MLN8237 (MLN) and/or p21 depletion by small interfering RNA (siRNA). Cell cycle distribution was determined using flow cytometry and a fluorescent ubiquitin-based cell cycle indicator (FUCCI) system combined with histone H3 phosphorylation at Ser10 (pS10 H3) detection. Senescence was assessed using senescence-associated-β-galactosidase (SA-β-Gal), Ki67, and γH2AX staining. Protein expression levels were determined using western blotting.@*RESULTS@#Tumor cells suffered severe DNA damage and underwent G2 arrest after IR treatment. The damaged cells did not successfully enter M phase nor were they stably blocked at G2 phase but underwent mitotic skipping and entered G1 phase as tetraploid cells, ultimately leading to senescence in G1. During this process, the p53/p21 pathway is hyperactivated. Accompanying p21 accumulation, Aurora A kinase levels declined sharply. MLN treatment confirmed that Aurora A kinase activity is essential for mitosis skipping and senescence induction.@*CONCLUSION@#Persistent p21 activation during IR-induced G2 phase blockade drives Aurora A kinase degradation, leading to senescence via mitotic skipping.

The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting

Although somatic cells can be reprogrammed to pluripotent stem cells (PSCs) with pure chemicals, authentic pluripotency of chemically induced pluripotent stem cells (CiPSCs) has never been achieved through tetraploid complementation assay. Spontaneous reprogramming of spermatogonial stem cells (SSCs) was another non-transgenic way to obtain PSCs, but this process lacks mechanistic explanation. Here, we reconstructed the trajectory of mouse SSC reprogramming and developed a five-chemical combination, boosting the reprogramming efficiency by nearly 80- to 100-folds. More importantly, chemical induced germline-derived PSCs (5C-gPSCs), but not gPSCs and chemical induced pluripotent stem cells, had authentic pluripotency, as determined by tetraploid complementation. Mechanistically, SSCs traversed through an inverted pathway of in vivo germ cell development, exhibiting the expression signatures and DNA methylation dynamics from spermatogonia to primordial germ cells and further to epiblasts. Besides, SSC-specific imprinting control regions switched from biallelic methylated states to monoallelic methylated states by imprinting demethylation and then re-methylation on one of the two alleles in 5C-gPSCs, which was apparently distinct with the imprinting reprogramming in vivo as DNA methylation simultaneously occurred on both alleles. Our work sheds light on the unique regulatory network underpinning SSC reprogramming, providing insights to understand generic mechanisms for cell-fate decision and epigenetic-related disorders in regenerative medicine.

Dry matter productivity and bromatological quality of ryegrass genotypes cultivated in southern Brazil / [Produtividade de matéria seca e qualidade bromatológica de genótipos de azevém cultivados no Sul do Brasil]

The goal of this work was to assess the biomass production and bromatological quality of ryegrass genotypes in ten municipalities of the Western and North Plateau regions of the State of Santa Catarina, Brazil. The cultivars La Estanzuela 284 (diploid), Bar HQ, Barjumbo, INIA Escorpio, Potro, and Winter Star (tetraploids) were compared, distributed in a randomized block design, in which the municipalities constituted the blocks, with three replications. The cuts were performed when the plants reached 30cm, leaving a residue of 10cm. In three cuts, the cultivars Barjumbo and Bar HQ were the most productive, exceeding 4.6 t ha-1 of dry matter. In the places in which five cuts were performed, the production of these cultivars exceeded 7.3 t ha-1, placing them again ahead of the others. The average crude protein content in three cuts was greater than 25% in all cultivars. There was no difference between the genotypes in the content of neutral detergent fiber and total digestible nutrients. There was a significant correlation between quantitative and qualitative productive variables. The assessed cultivars represented good options for composing short-term or long-term winter-feeding systems, adjusted to the integration with annual crops or warm-season pastures.(AU)

O objetivo deste trabalho foi avaliar a produção de biomassa e qualidade bromatológica de genótipos de azevém, em dez municípios das regiões Oeste e Planalto Norte Catarinense, Estado de Santa Catarina, Brasil. Foram comparados os cultivares La Estanzuela 284 (diploide), Bar HQ, Barjumbo, INIA Escorpio, Potro e Winter Star (tetraploides), distribuídos em um delineamento blocos casualizados, em que os municípios constituíram os blocos, com três repetições. Os cortes foram realizados quando as plantas atingiram 30cm, deixando um resíduo de 10cm. Sob três cortes, os cultivares Barjumbo e Bar HQ foram os mais produtivos, ultrapassando 4,6 t ha-1 de matéria seca. Nos locais em que ocorreram cinco cortes, a produção destes cultivares superou 7,3 t ha-1, posicionando-os novamente à frente dos demais. O teor médio de proteína bruta em três cortes foi superior a 25% em todos os cultivares. Não houve diferença entre os genótipos no teor de fibra detergente neutro e de nutrientes digestíveis totais. Verificou-se correlação significativa entre variáveis produtivas quantitativas e qualitativas. Os cultivares testados representam boas opções para compor sistemas forrageiros hiberno-primaveris de curta ou longa duração, ajustando-se à integração com lavouras ou pastagens anuais de estação quente.(AU)

Decidualization induced by mouse tetraploid embryos / 解剖学报

Objective To investigate the role of inner cell mass (ICM) in decidualization using decidua induced by two-cell embryos or tetraploid embryos through tubal embryo transfer. Methods Tetraploid embryo, as the inner cell mass-deficient embryo, were produced by electrofusion of mouse 2-cell embryos. Decidua was induced by 2-cell embryos or tetraploid embryos through tubal embryo transfer. Decidua induced by 2-cell embryos was employed as a control. Morphologic and implantation site of decidua were compared between two-cell embryo-induced decidua and tetraploid embryo-induced decidua. The differentially expressed microRNA ( miRNA ) was screened by high-throughput sequencing. The target genes of differentially expressed miRNA in two groups were screened for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Results Tetraploid embryo-induced decidual tissue and 2-cell embryo- induced decidual tissue were very similar at the implantation site, but there were significant differences in decidual morphology. There were 16 miRNAs differentially expressed in decidua of the two groups, of which 11 miRNAs (miR-466f- 3p, miR-302 d-3p, miR-466i-5p, miR-465c-5p, miR-302a-5p, miR-7068-3p, miR-741-3p, miR-302a-3p, miR-433-5p, miR-144-5p, miR-878-5p) were up-regulated in tetraploid embryo-induced decidua and 5 miRNAs (miR-690, miR-193b- 5p, miR-147-3p, novel_327, miR-363-3p) were down-regulated in tetraploid embryo-induced decidua. Bioinformatics analysis showed that the target genes played function such as protein binding and ion binding, and mainly involved in cyclic guanosine monophosphate-protein kinase G (cGMP-PKG) signaling pathway, estrogen signaling pathway, vascular endothelial growth factor (VEGF) signaling pathway and so on. Conclusion ICM plays an important role in decidualization.

Polyploidy induction in Physalis alkekengi / Indução de poliploidia em Physalis alkekengi

Physalis alkekengi is an ornamental plant that can also be used as a medicinal plant due to its anti-inflammatory, bactericidal, antitumor and fungicidal properties. Polyploidization can be an important tool in the genetic improvement of this species. The objective this work was to obtain tetraploids in vitro and to evaluate the phytotechnical traits of P. alkekengi. For this, nodal segments of P. alkekengi var. Franchettii were inoculated into petri dishes containing 100 ml of MS medium supplemented with colchicine at concentrations 0; 0.04; 0.08; 0.12; and 0.16% and kept in the dark for 24 and 48h. After the respective treatment periods with colchicine the segments were inoculated into test tubes. The tetraploids were identified by flow cytometry and classical cytogenetics. In vitro seedlings were measured: root length, nodal segment length, leaflet number and total leaf area. In the acclimatization phase, the area of the second leaf and total leaf, petiole radius, stem length, fruit weight with calyx, without calyx, fruit diameter, number of seeds and brix of the pulp were evaluated. Chlorophyll a, chlorophyll b, total chlorophyll, total carotenoid, total chlorophyll / total carotenoid ratio and chlorophyll a / b ratio were also estimated. The treatment that most produced tetraploid seedlings was with 0.08% colchicine per 24h. No significant difference was observed in 7 (seven) variables, these being all variables of photopigments, stem diameter (steam) and brix. In general, diploid (2x) plants were better in 9 (nine) while tetraploid seedlings were better in 6 (six) of the phytotechnical variables. It was concluded that the MS medium supplemented with 0.08% colchicine for 24 h allowed P. alkekengi tetraploides to be obtained with better phytotechnical qualities.

Physalis alkekengi é uma planta ornamental que também pode ser usada como planta medicinal devido às suas propriedades anti-inflamatórias, bactericidas, antitumorais e fungicidas. A poliploidização pode ser uma ferramenta importante para o melhoramento genético dessa espécie. O objetivo deste trabalho foi obter tetraplóides in vitro e avaliar as características fitotécnicas de P. alkekengi. Para isso, segmentos nodais de P. alkekengi var. Franchettii foram inoculados em placas de Petri contendo 100 ml de meio MS suplementado com colchicina nas concentrações 0; 0,04; 0,08; 0,12; e 0,16% e mantido no escuro por 24 e 48h. Após os respectivos períodos de tratamento com colchicina, os segmentos foram inoculados em tubos de ensaio. Os tetraplóides foram identificados por citometria de fluxo e citogenética clássica. As plântulas in vitro foram medidas: comprimento da raiz, comprimento do segmento nodal, número de folhetos e área foliar total. Na fase de aclimatação foram avaliadas a área da segunda folha e área foliar total, raio do pecíolo, comprimento do caule, peso do fruto com cálice, sem cálice, diâmetro do fruto, número de sementes e brix da polpa. Também foram estimadas clorofila a, clorofila b, clorofila total, carotenóides totais, razão clorofila total / carotenóide total e razão clorofila a / b. O tratamento que mais produziu mudas tetraplóides foi com colchicina a 0,08% por 24 horas. Não foi observada diferença significativa em 7 (sete) variáveis, sendo todas variáveis de fotopigmentos, diâmetro do caule (vapor) e brix. Em geral, as plantas diplóides (2x) foram melhores em 9 (nove) variáveis fitotécnicas, enquanto as mudas tetraplóides foram melhores em 6 (seis). Concluiu-se que o meio MS suplementado com colchicina a 0,08% por 24 h permitiu obter tetraploides de P. alkekengi com melhores qualidades fitotécnicas.

Rapid generation of gene-targeted EPS-derived mouse models through tetraploid complementation

One major strategy to generate genetically modified mouse models is gene targeting in mouse embryonic stem (ES) cells, which is used to produce gene-targeted mice for wide applications in biomedicine. However, a major bottleneck in this approach is that the robustness of germline transmission of gene-targeted ES cells can be significantly reduced by their genetic and epigenetic instability after long-term culturing, which impairs the efficiency and robustness of mouse model generation. Recently, we have established a new type of pluripotent cells termed extended pluripotent stem (EPS) cells, which have superior developmental potency and robust germline competence compared to conventional mouse ES cells. In this study, we demonstrate that mouse EPS cells well maintain developmental potency and genetic stability after long-term passage. Based on gene targeting in mouse EPS cells, we established a new approach to directly and rapidly generate gene-targeted mouse models through tetraploid complementation, which could be accomplished in approximately 2 months. Importantly, using this approach, we successfully constructed mouse models in which the human interleukin 3 (IL3) or interleukin 6 (IL6) gene was knocked into its corresponding locus in the mouse genome. Our study demonstrates the feasibility of using mouse EPS cells to rapidly generate mouse models by gene targeting, which have great application potential in biomedical research.

Imprinting genes modified parthenogenetic embryonic stem cells produce full-term mouse via tetraploid complementation / 生物工程学报

Parthenogenetic embryonic stem cells (pESCs) derived from bi-maternal genomes do not have competency of tetraploid complementation, due to lacking of paternal imprinting genes. To make pESCs possess fully development potentials and similar pluripotency to zygote-derived ESCs, we knocked out one allelic gene of the two essential maternal imprinting genes (H19 and IG) in their differentially methylated regions (DMR) via CRISPR/Cas9 system and obtained double knock out (DKO) pESCs. Maternal pESCs had similar morphology, expression levels of pluripotent makers and in vitro neural differentiation potentials to zygotes-derived ESCs. Besides that, DKO pESCs could contribute to full-term fetuses through tetraploid complementation, proving that they held fully development potentials. Derivation of DKO pESCs provided a type of major histocompatibility complex (MHC) matched pluripotent stem cells, which would benefit research in regenerative medicine.

Prognostic significance of high hyperdiploid and triploid/tetraploid acute myeloid leukemia / 临床检验杂志

Objective@#To investigate the biological characteristics of high hyperdiploid and triploid/tetraploid acute myeloid leukemia (HH/TT-AML) and its relationship with prognosis. @*Methods@#The clinical data of 28 patients with newly diagnosed HH/TT-AML during March 2006 and June 2017 were retrospectively analyzed, and the factors influencing prognosis were analyzed by the Kaplan-Meier method. @*Results@#The karyotypes of HH/TT-AML patients were mainly 49 chromosomes, accounting for 39.3% (11/28), followed by 50-55 chromosomes, accounting for 32.1% (9/28). The karyotypes of high hyperdiploid acute myeloid leukemia (HH-AML) patients were more likely to be +8 (77.3%, 17/22) or +21 (54.5%, 12/22). The survival analysis showed that the overall survival rate of HH/TT-AML patients with -5/5q-, -7/7q-, -17/der(17p) or der(3q) was significantly lower than that without these abnormalities (4.1 months vs 10.1 months,P＜0.05). There was no significant difference in the overall survival rate between triploid/tetraploid acute myeloid leukemia (TT-AML) patients and HH-AML patients (8.4 months vs 7.2 months,P＞0.05). The overall survival rate of the patients with allogeneic hematopoietic stem cell transplantation was significantly longer than that with chemotherapy alone (25.4 months vs 4.1 months,P＜0.01). @*Conclusion@#HH/TT AML patients are highly heterogeneous. The identification of poor prognosis-related chromosome abnormalities is helpful for the stratification of prognosis. The overall survival time of these patients is short. Early allogeneic hematopoietic stem cell transplantation after remission by combination chemotherapy can significantly improve the prognosis of these patients.

Utilization of a novel feedstuff: effects of additives on the fermentation characteristics, chemical composition and in vitro digestibility of tetraploid black locust (Robinia pseudoacacia) silage / Utilização de um novo alimento: efeitos de aditivos nas características de fermentação, composição química e digestibilidade in vitro da silagem de gafanhoto negro tetraplóide (Robinia pseudoacacia)

ABSTRACT: This study was conducted to evaluate the effects of additives on the fermentation characteristics, chemical composition and in vitro digestibility of tetraploid black locust (TBL). The TBL leaves silage was either untreated (control) or treated with 1 × 106 cfu/g FM Lactobacillus plantarum (L), 1% glucose (G), 3% molasses (M), a combination of 1% glucose and Lactobacillus plantarum (L+G), or a combination of 3% molasses and Lactobacillus plantarum (L+M). Fermentation quality, chemical composition and nutrient digestibility were then analyzed. Ethanol and acetic acid concentrations were the dominant fermentation products in all silages except L+M silage. The L, G and L+G treatments failed to influence the fermentation. The M treatment increased (P<0.05) the lactic acid concentration and lowered (P<0.05) the pH when compared with control silage. The best fermentation properties were observed in L+M silage, as indicated by the dominance of lactic acid over ethanol in fermentation products. The M and L+M silages exhibited higher (P<0.05) dry matter, and M silage showed higher residual water-soluble carbohydrates than the control. Ensiling increased (P<0.05) the in vitro dry matter, neutral detergent fiber and acid detergent fiber degradability of TBL. Among the silages, M silage had the highest levels of dry matter, neutral detergent fiber and acid detergent fiber degradability. The obtained results suggested that application of lactic acid bacteria together with 3% molasses could be an effective strategy to prevent the occurrence of ethanol fermentation and improve fermentation quality of TBL silage; addition of fermentable sugars to TBL improves nutrient availability to ruminants.

RESUMO: Este estudo foi conduzido para avaliar os efeitos de aditivos nas características de fermentação, composição química e digestibilidade in vitro do gafanhoto preto tetraplóide (TBL). A silagem de folhas TBL não foi tratada (controle) ou foi tratada com 1 × 106 ufc / g FM Lactobacillus plantarum (L), 1% glicose (G), 3% melaço (M), uma combinação de 1% glicose e Lactobacillus plantarum ( L + G), ou uma combinação de 3% de melaço e Lactobacillus plantarum (L + M). A qualidade da fermentação, a composição química e a digestibilidade dos nutrientes foram analisadas. As concentrações de etanol e ácido acético foram os produtos de fermentação dominantes em todas as silagens, com exceção da silagem L + M. Os tratamentos L, G e L + G não influenciaram na fermentação. O tratamento com M aumentou (P<0,05) a concentração de ácido láctico e diminuiu (P<0,05) o pH, quando comparado com a silagem controle. As melhores propriedades de fermentação foram observadas na silagem L + M, como indicado pela dominância do ácido lático sobre o etanol nos produtos de fermentação. As silagens M e L + M apresentaram maior teor de matéria seca (P<0,05), e a silagem M apresentou maior carboidrato solúvel em água residual que o controle. A ensilagem aumentou (P<0,05) a matéria seca in vitro, a fibra em detergente neutro e a degradabilidade da fibra em detergente ácido de TBL. Entre as silagens, a silagem M apresentou os maiores teores de matéria seca, fibra em detergente neutro e degradabilidade da fibra em detergente ácido. Os resultados obtidos sugerem que a aplicação de bactérias lácticas em conjunto com 3% de melaço pode ser uma estratégia eficaz para evitar a ocorrência de fermentação alcoólica e melhorar a qualidade da fermentação da silagem TBL. A adição de açúcares fermentáveis à TBL aumenta a disponibilidade de nutrientes para ruminantes.

Comparative study on anti-inflammatory and immunomodulation of different Isatidis Radix strains growing in Gansu Province / 中国应用生理学杂志

OBJECTIVE@#To investigate anti-inflammatory and immunomodulation effects of different ecotype from Isatidis Radix growing in Gansu province.@*METHODS@#Mice were randomly divided into 6 groups (=11)and used the auricular swelling and paw edema to observe the anti-inflammatory effects of Isatidis Radix; Mice were randomly divided into 7 groups (=11) and through the gasbag synovitis model to observe the anti-inflammatory effects of Isatidis Radix; Mice were randomly divided into 6 groups (=11), the immunosuppressed model were established by injection of cyclophosphamide (CTX) to study the effects of Isatidis Radix on index of thymus, blood routine and cytokines.@*RESULTS@#Gansu different ecotype from Isatidis Radix could reduce the swelling of the mice auricle, paw edema and total protein, leukotriene B(LTB)and malonaldehyde(MDA) in airbag synovitis exudates, and upgrade serum levels of superoxide dismutase (SOD); Degrade the tumor necrosis factor-α (TNF-α) and upgrade the index of thymus, the number of red and white corpuscles, the level of interferon-γ (IFN-γ), interleukin-4 (IL-4) (<0.05, 0.01) of mice immunosuppressed model; Above the research of anti-inflammatory and immunomodulation, there were no significant differences between Isatidis Radix of Gansu different ecotype and tetraploid.@*CONCLUSIONS@#Different ecotype of Isatidis Radix has obvious functions in anti-inflammatory and immunomodulation, but there are no significant differences between Gansu different ecotype and tetraploid.

Experimental study on anti-inflammatory effects and acute toxicity of aqueous extract from tetraploid Lonicerae Flos / 中草药

Objective: To evaluate the anti-inflammatory effects and acute toxicity of the aqueous extract form tetraploid Lonicerae Flos. Methods: Compared with the diploid, the paw swelling of rats induced by carrageenan and cotton pellet granuloma tests were obtained to study the anti-inflammatory effects of the tetraploid Lonicerae Flos. A pretest was used to judge the possibility of LD50 in the acute toxicity test. The experimental data are calculated LD50 on mice by Bliss method to evaluate the acute toxicity of the diploid Lonicerae Flos. Results: The tetraploid Lonicerae Flos had the significant resistance to acute inflammation, but there was not significance between the two ploidies. Neither the tetraploid nor the diploid had the obvious subacute inflammation resistance effects. The possibility of measuring LD50 was definite through pre-test. The LD50 of the aqueous extract from the two ploidies were 72.12 and 69.92 g/kg, respectively. However, there was no obvious difference in acute toxicity between the two ploidies. And the LD50 of mice was equal to 412 and 400 times of 60 kg normal human's daily dried medicinal herb expenses respectively. Conclusion: The tetraploid Lonicerae Flos has the significant anti-inflammation with less toxic and side effect. It could be used safely in a certain dose range.

Variación morfométrica intra e interespecífica entre poblaciones de Odontophrynus (Anura: Cycloramphidae) del área central de Argentina / Intraand inter-specific morphometric variation between Odontophrynus populations (Anura: Cycloramphidae) of central Argentina

Morphometric analyses are particularly important, and for many years they have supported evolutionary and ecological phenomena, and have been useful for the classification of new species, mainly to the lowest taxonomic levels. In order to assess the degree of sexual dimorphism, the intra-specific morphometric variation and the inter-specific morphological differences, we performed morphometric analyses of two morphologically cryptic species, Odontophrynus cordobae (diploid) and O. americanus (tetraploid). For this, we measured 15 morphometric variables on 211 individuals from 18 localities of Córdoba province. We found sexual dimorphism in six and three parameters in O. cordobae and O. americanus, respectively. Diploid and tetraploid males significantly differed in six morphometric variables. Discriminant Function Analysis (DFA) including all populations correctly classified a 76.37% of individuals within their respective species. DFA based on four groups (1- allopatric O. cordobae, 2-syntopic O. cordobae; 3- allopatric O. americanus; 4- syntopic O. americanus) accurately classified a 73.45% of individuals within their respective groups. Allopatric individuals of each species and, allopatric and syntopic individuals within each species were clearly segregated. Syntopic individuals of O. cordobae and O. americanus showed the lowest percentages of correct classification. Morphometric differences between sympatric diploids and tetraploids were not greater than those observed in allopatry. These findings deviate from the expected results under the hypothesis of character displacement, and they suggest that external morphological characters would not have a major influence on the recognition and choice of conspecific males by females.

El análisis morfométrico ha servido como apoyo para explicar fenómenos ecológicos y evolutivos y para la clasificación de nuevas especies. Nosotros realizamos el análisis morfométrico de dos especies morfológicamente crípticas con el objeto de evaluar el grado de diferenciación intra e interespecífica. Se midieron 15 variables sobre 211 individuos pertenecientes a 18 localidades del centro de Argentina. Se encontró dimorfismo sexual en seis variables morfométricas en Odontophrynus cordobae y en tres variables en O. americanus. Se encontraron diferencias significativas entre especies en seis variables. El análisis discriminante incluyendo los individuos de todas las poblaciones relevadas para O. cordobae y O. americanus, mostró una clasificación positiva de los individuos dentro de su respectiva especie del 76.37%. En el análisis discriminante realizado con base en cuatro grupos (O. cordobae alopátrico y sintópico y O. americanus alopátrico y sintópico) se obtuvo una reclasificación de los individuos dentro de cada grupo del 73.45%. Las diferencias morfométricas entre especies fueron mayores en sintopía. Los resultados se alejan de los resultados esperados bajo la hipótesis del desplazamiento de caracteres y podrían sugerir que los caracteres morfológicos externos no tendrían una influencia importante en el reconocimiento y elección de machos coespecíficos por parte de las hembras.

In vitro isoflavonoid production and analysis in natural tetraploid Trifolium pratense (red clover) calluses

Isoflavones are polyphenolic phytoestrogens, predominantly found in leguminous plants. Trifolium pratense L., Fabaceae (red clover), is rich in isoflavones that possess estrogenic activity due to their similar molecular structure and effectiveness in preventing health conditions such as menopause, osteoporosis, cardiovascular disease, hypertension and hormone-dependent cancers. In this study, presence and amount of various phytoestrogens in the tetraploid plant and in the calluses derived from the plants were investigated. Calluses were generated from explants obtained from natural tetraploid T. pratense seedlings. The best callus formation was obtained from hypocotyl explants cultured in Phillips Collins and Gamborg B5 media containing different plant growth regulators. Flowers of plants and calluses were analysed for formononetin, biochanin A, genistein and daidzein contents using HPLC. In HPLC analysis, high levels of formononetin (0.249 µg/mg) were determined in natural tetraploid T. pratense flowers in addition to genistein and biochanin A. In calluses, highest isoflavone content (1.15 µg/mg formononetin) was observed in modified Gamborg B5 medium. Biochanin A content of calluses and the plant were found to be nearly the same. But formononetin and genistein contents of the calluses in this medium were found to be respectively 4.62 and 21.39 folds higher than the tetraploid plant.

Autotetraploid induction and identification of Rhodiola sachalinensis / 中草药

Objective: To obtain autotetraploid clones of Rhodiola sachalinensis. Methods: Germplasm with doubled chromosomes was acquired according to colchicine soaking seeds, and ploidy was identified according to stomata size and density measurements, chromosome counting and flow cytometry analysis. Chimera was purified after several times of regenerations. Results: Colchicine concentration and treatment duration had significant effect on seed germination rate, seedling death rate and ploidy mutation rates, and they were 18.2%, 73.6%, and 68.7%, respectively after the seeds were soaked in 0.2% colchicine solution for 72 h. Stomata diameter was promoted and density was declined significantly in morphological variation plantlets; normal diploid chromosome number was 2n = 2x = 26, and tetraploid chromosome number was 2n = 4x = 52. Meanwhile, aneuploidy was found in the population of plantlets after colchicine soaking, leaves of ploidy mutation plantlets were used as expiants to regenerate to tube plants, and homozygous clones were obained after three regeneration and purification cycles. There was no chimera in the homozygous clones according to flow cytometry analysis. Conclusion: It is feasible to obtain autotetraploid according to colchicine treatment on seeds of R. sachalinensis.

Transgenic mouse fetus generated from embryonic stem cells by tetraploid embryo complementation / 解剖学报

Objective To use tetraploid embryo complementation combined with gene transfer to produce genetically modified embryonic stem cells (EsCs) clones. Methods In this study, EGFP was introduced into ESCs by electroporation, and transfected positive cells were selected by G418 resistance. The tetraploid embryos were obtained from diploid blastomere electrofusion which preformed at 2-cell stage. Afterwards, 19-21 EGFP-ESCs were inserted into each tetraploid blastocyst cavity by piezo drilled microinjection,then the injected blastocysts were transferred into the uterus of pseudo-pregnancy at 2.5-day or the oviduct of 0.5-day female mice. Results The transfected ESCs maintained normal karyotype even after long-term passage (2n=40). The rate of fusion was 95.07%, and the developmental rate of tetraploid blastocyst was 95%.Totally 410 injected blastocysts were obtained. Unfortunately, we have not got any vital offsprings, except 151 implantation sites (pseudo-pregnancy 2.5 days:29.41%;the oviduct of half one day:64.37%). Furthermore, scattered EGFP expressions in transgenic fetus were observed under invert fluorescent microscope. Conclusion The transfected ESCs were observed in transgenic fetus, and the implantation rate in oviduct was higher than that in uterine.

Anatomical alterations due to polyploidy in cassava, Manihot esculenta Crantz

Information on anatomical structure is needed by breeders working on improvement for drought tolerance. For studying the effect of polyploidy on cassava anatomy and its significance to tolerance to drought, we induced a polyploidy type of a selected clone (UnB 530) by applying an aqueous solution of 0.2% colchicine on lateral buds for a period of 12 h. The stem identified as tetraploid was propagated to produce the whole plant. Free-hand cross-sections of the median portion between stem internodes were made. They were clarified using 50% sodium hypochlorite solution, stained with 1% safranin-alcian blue, passed through an ethanol series and butyl acetate and mounted in synthetic resin. The tetraploid type showed more prismatic and druse crystals in the cortical parenchyma, and its pericycle fibers had thicker walls. The secondary xylem of tetraploid types was wider than diploid ones, having thinner walls and less starch.

Wild ornamental germplasm exploration and domestication based on biotechnological approaches: In vitro colchicine treatment to obtain a new cultivar of Scoparia montevidiensis

The genus Scoparia is native from Argentina. The diversity of colours and shape of their flowers bestows this genus a very interesting ornamental potential. The purpose of the present study is to explore the Scoparia species germplasm by means of in vitro polyploidization in order to improve their ornamental qualities. Accessions of S. montevidiensis var. montevidiensis, S. montevidiensis var. glandulifera, S. nudicaulis, S. hasleriana and S. dulcis were collected and maintained under greenhouse conditions. The Murashige-Skoog medium, supplemented with 0.25 mg/L BAP was used for the nodal segments multiplication of the five Scoparia species. Except for S. hasleriana, the multiplication rate of the other species ranged between 10 and 12 shoots per explant. The colchicine doses tested with S. montevidiensis were: 0.0; 0.1; 0.05; 0.01 and 0.001% (24 and 48 hrs). From a total of 364 recovered plants, 4 solid tetraploid and 16 chimeras were detected. Significant differences were observed for the size of flower, leaves, and the stem diameter among the tetraploid plants and between them and the control. The tissue culture proved to be a powerful tool both to multiply the Scoparia material incorporated to our germplasm collection and to obtain new improved varieties of this beautiful genus.

Análise bromatológica comparativa entre acessos diplóides, tetraplóides africanos e tetraplóides brasileiros de Hemarthria altissima (Poiret) Stapf & Hubbard / Bromatologic analysis comparative between access African diploid, tetraploid and Brazilian tetraploid of Hemarthiria altissima (Poiret) Stapf & Hubbard

Foi comparada a qualidade da forragem entre os acessos diplóides e tetraplóides africanos e tetraplóides brasileiros de Hemarthria altissima. O experimento foi conduzido em casa de vegetação na Universidade Federal de Santa Maria. Os resultados obtidos mostraram Digestibilidade in vitro da matéria orgânica satisfatória para os acessos tetraplóides africanos e brasileiros, sendo que os brasileiros tiveram teores de proteína bruta signifi-cativamente maiores que os africanos. O tetraplóide natural do Rio Grande do Sul apresentou maior nível de proteína bruta.

This study evaluated the quality among african dipoloid, tetraploid and brazilian tetraploid accessions Hemarthria altissima, the Experiment was conducted in greenhouse at the Federal University of Santa Maria. Results suggested that in vitro organic matter digestibility was satisfactory for tetraploid brazilian and african accessions, crude protein volues obtained for brazilian accessions were significantly higher than the african accessions. The tetraploids from Rio Grande do Sul region presented higher crude protein level.

Enhanced resistance of transgenic tetraploid Isatis indigotica by Bt and CpTI to moths / 中草药

Objective Two insect resistance genes Bacillus thuringiensis(Bt) crystal protein gene CryIA(c) and cowpea trypsin inhibitor gene CpTI were introduced into tetraploid Isatis indigotica to enhance the resistance to moths.Methods I.indigotica was transformed with a plasmid,pGBI121S4ABC,containing CryIA(c) Bt and CpTI and the selectable gene(Npt-Ⅱ) driven by the CaMV35S promoter via Agrobacterium tumefaciens LBA4404 mediated transformation.Then PCR and Southern blotting assay were conducted followed by moth bioassay test.Results Co-transgenic rate of the two genes in tetraploid I.indigotica was 16.67%.The integration and expression of introduced genes in T0 regenerated transgenic plants were confirmed by Southern blotting assays.Insect bioassay test demonstrated transgenic lines had significant inhibition to moths,compared with wild type control.Conclusion Co-transformation of Bt and CpTI genes is an effective strategy to enhance the resistance to moths for tetraploid I.indigotica.

Comparison of physiological index and diosgenin content in different ploidy of Dioscorea zingiberensis / 中草药

Objective By comparing the physiological index and diosgenin content of rhizome between the artificial tetraploid and the wild type diploid of Dioscorea zingiberensis, it is aimed at revealing the potential utility of polyploid breeding in medicinal D. zingiberensis. Methods Three clones of the natural diploid and the artificially induced tetraploid of D. zingiberensis were used as materials in this study. The content of SOD, PPO, and soluble sugar of leaves was determined by spectrophotometry, CAT content was measured by using the method of KMnO_4 titration. And the diosgenin content in rhizome was analyzed by HPLC. Results The tetraploid plants showed higher level of SOD, PPO, CAT, soluble sugar content in leaves, and diosgenin content in rhizome than those of the diploid origins. The diosgenin content in the three clones of tetraploid plants increased to 27% as compared to wild type. Conclusion Artificially induced tetraploid presents high content of diosgenin and great potential in stress resistance, which would be available in good seed breeding for high yield of diosgenin.