Use of fluorescein diacetate (FDA) to determine ploidy of in vitro watermelon shoots

Ploidy of watermelon [Citrullus lanatus (Thunb.) Matsum. and Nakai shoots and plantlets was estimated by painting the lower epidermis of intact in vitro-derived leaves with fluorescein diacetate (FDA) and observing fluorescence of guard cell chloroplasts with a microscope and UV light. Leaves from in vitro shoot-tip cultures of known diploid cultivars and tetraploid breeding lines were used to establish the mean number of chloroplasts per guard cell pair. Leaves from diploid and tetraploid shoot cultures had 9.7 and 17.8 chloroplasts per guard cell pair, respectively. This method then was used to estimate the ploidy of shoots regenerated from cotyledon explants of the diploid cultivar Minilee. Approximately 11% of the 188 regenerated shoots were classified as tetraploid during in vitro culture. Putative tetraploids were transplanted to the field and self-pollinated. About 45% of tetraploids identified in vitro produced fruit and viable seed. Chloroplast counts of R₁ progeny were used to confirm their ploidy. All of the putative diploids were confirmed diploid and all putative tetraploids proved to be non-chimeric true breeding tetraploids. This revised version was published online in June 2006 with corrections to the Cover Date.     

Spermatozoa in triploids of the rosy bitterling Rhodeus ocellatus ocellatus

Artificially induced triploid male Rhodeus ocellatus ocellatus showed typical nuptial colorations, irrespective of spermiation. In milt from triploids, abnormal spermatozoa (malformation of the head and mitochondrion, excessive formation of the head, mitochondrion and flagellum, and no flagellum) occurred at 78°4% frequency. Spermatozoa with multiflagella were most common, often with a saccate-like organ. Many triploid spermatozoa moved actively as long as those of diploids (10·92±0·91 min=mean±S.D., P >0·05), but did not advance like diploids, spinning around until movement ceased. The sperm density in triploids was < 2% of that from diploids. In triploid testes, deformed and variously sized spermatids were often observed, and normal spermatids and spermatozoa were seldom recognized. The DNA content of triploid spermatozoa varied greatly, compared with that of diploids. Peak of sperm DNA content differed slightly between two triploid samples with two peaks at 1·5 n and 1·9 n ( P <0·0001 in both), respectively. Triploids had the greatest average sperm head diameter of 2·25±0·67 μm (mean±S.D.), while that of diploids was 1·83±0·15 μm ( P =0·002). In the fertilization test using the eggs of diploids ( n =1500, 30 trials), only one egg developed. The embryo chromosome number was 60 (2·5 n) and the ploidy of spermatozoa contributing to fertilization appears to be 1·5 n. The extremely low fertility of triploid R. o. ocellatus spermatozoa seems to be caused by the reduced motility and large head size of spermatozoa, and the low sperm density of the milt. The ploidy of spermatozoa that are successful in fertilization is likely to be related to the distribution pattern in the DNA content of cells.     

Successful Spermatogonial Stem Cells Transplantation within Pleuronectiformes: First Breakthrough at inter-family Level in Marine Fish

As a promising biotechnology, fish germ cell transplantation shows potentials in conservation germplasm resource, propagation of elite species, and generation of transgenic individuals. In this study, we successfully transplanted the Japanese flounder (P. olivaceus), summer flounder (P. dentatus), and turbot (S. maximus) spermatogonia into triploid Japanese flounder larvae, and achieved high transplantation efficiency of 100%, 75-95% and 33-50% by fluorescence tracking and molecular analysis, respectively. Eventually, donor-derived spermatozoa produced offspring by artificial insemination. We only found male and intersex chimeras in inter-family transplantations, while male and female chimeras in both intra-species and intra-genus transplantations. Moreover, the intersex chimeras could mature and produce turbot functional spermatozoa. We firstly realized inter-family transplantation in marine fish species. These results demonstrated successful spermatogonial stem cells transplantation within Pleuronectiformes, suggesting the germ cells migration, incorporation and maturation within order were conserved across a wide range of teleost species.     

Amitosis‐like nuclear division in erythrocytes of triploid rainbow trout Oncorhynchus mykiss

This work shows that the atypical erythrocytes in triploid rainbow trout Oncorhynchus mykiss were morphologically similar to those of toads. The nuclei of the cells can be bell‐shaped, constricted or irregular. It is presumed that such nuclear division is probably amitosis.     

Massive production of all-female diploids and triploids in the crucian carp

In many species of aquaculture importance, all-female and sterile populations possess superior productivity due to faster growth and a relatively homogenous size of individuals. However, the production of all-female and sterile fish in a large scale for aquaculture is a challenge in practice, because treatments necessary for gynogenesis induction usually cause massive embryonic and larval mortality, and the number of induced gynogens is too small for their direct use in aquaculture. Here we report the massive production of all-female triploid crucian carp by combining artificial gynogenesis, sex reversal and diploid-tetraploid hybridization. Previously, we have obtained an allotetraploid carp population (4n = 200) by hybridization between red crucian carp (Carassius auratus red var; ♀) and common carp (Cyprinus carpio; ♂). We induced all-female diploid gynogens of the Japanese crucian carp (Carassius cuvieri; 2n = 100). We also generated male diploid gynogens of the same species treated gynogenetic fry with 17-α-methyltestosterone, leading to the production of sex-revered gynogenetic males. Finally, these males were used to cross with the female diploid Japanese crucian carp gynogens and the allotetraploid females, resulting in the production of fertile all-female diploid Japanese crucian carp (2n=100) and sterile all-female triploid hybrids (3n = 150), respectively. Therefore, diploid crucian carp gynogenetic females and sex-reversed male together with an allotetraploid line provide an opportunity to produce all-female triploid populations in a large scale to meet demands in aquaculture industry.     

Anthropogenic disturbance as a driver of microspatial and microhabitat segregation of cytotypes of Centaurea stoebe and cytotype interactions in secondary contact zones

Background and Aims

In a mixed-ploidy population, strong frequency-dependent mating will lead to the elimination of the less common cytotype, unless prezygotic barriers enhance assortative mating. However, such barriers favouring cytotype coexistence have only rarely been explored. Here, an assessment is made of the mechanisms involved in formation of mixed-ploidy populations and coexistence of diploid plants and their closely related allotetraploid derivates from the Centaurea stoebe complex (Asteraceae).

Methods

An investigation was made of microspatial and microhabitat distribution, life-history and fitness traits, flowering phenology, genetic relatedness of cytotypes and intercytotype gene flow (cpDNA and microsatellites) in six mixed-ploidy populations in Central Europe.

Key Results

Diploids and tetraploids were genetically differentiated, thus corroborating the secondary origin of contact zones. The cytotypes were spatially segregated at all sites studied, with tetraploids colonizing preferentially drier and open microhabitats created by human-induced disturbances. Conversely, they were rare in more natural microsites and microsites with denser vegetation despite their superior persistence ability (polycarpic life cycle). The seed set of tetraploid plants was strongly influenced by their frequency in mixed-ploidy populations. Triploid hybrids originated from bidirectional hybridizations were extremely rare and almost completely sterile, indicating a strong postzygotic barrier between cytotypes.

Conclusions

The findings suggest that tetraploids are later immigrants into already established diploid populations and that anthropogenic activities creating open niches favouring propagule introductions were the major factor shaping the non-random distribution and habitat segregation of cytotypes at fine spatial scale. Establishment and spread of tetraploids was further facilitated by their superior persistence through the perennial life cycle. The results highlight the importance of non-adaptive spatio-temporal processes in explaining microhabitat and microspatial segregation of cytotypes.     

短轮伐期毛白杨不同密度林分土壤有机碳和全氮动态

采用裂区试验设计,于2005—2008年连续4年测定了不同造林密度(2 m×2 m、2 m×3 m、2 m×3.5 m、2 m×4 m、2 m×5 m、3 m×3 m、3 m×4 m)下2年生三倍体毛白杨(B304)和对照二倍体(1319)人工林土壤有机碳和全氮含量,以明确不同密度林分土壤有机碳和全氮动态变化规律及其相关性。结果表明:(1)受造林密度、生长时间及其交互作用的显著影响,4年生长期内林地土壤有机碳含量呈先降后升的变化特点。其中,2008年B304在2 m×3 m造林密度下土壤有机碳含量显著高于其它年份,说明此造林密度有利于发挥三倍体毛白杨林土壤固碳的生态功能。(2)4年生长期内,土壤全N含量受生长时间及其与造林密度的交互作用的显著影响。在3 m×3 m造林密度下,二倍体毛白杨林地土壤全N含量逐年降低,而三倍体毛白杨2007年的土壤全N含量显著增加,该造林密度利于三倍体毛白杨林地土壤N的积累。(3)土壤有机碳/全氮比值变化与有机碳含量变化规律一致,且均在2006年达到最低值。(4)在2008年,三倍体毛白杨在2 m×3 m和2 m×3.5 m造林密度下土壤有机碳与全N含量呈现显著正相关关系,而2 m×5 m造林密度下的二倍体毛白杨林地呈显著性负相关关系,体现了毛白杨林地土壤有机碳与全N含量复杂的相关性。     

Biological characteristics of an improved triploid crucian carp

An improved triploid crucian carp (ITCC) was produced by crossing improved tetraploids (G1×AT, ) with improved red crucian carp (IRCC, ), which were obtained by distant crossing and gynogenesis. The biological characteristics of ITCC, including the number and karyotype of chromosomes, gonadad and pituitary structures, phenotype, and growth rate are reported. ITCC possessed 150 chromosomes with the karyotype 33m+51sm+33st+33t. In the breeding season, both ovary-like and testis-like gonads of ITCC were unable to produce normal mature gametes. The ultrastructure of the pituitary of ITCC showed that most of the endocrine granules in gonadotrophic hormone (GTH) cells had not been re-leased, providing endocrinological evidence for the sterility of ITCC. Compared with triploid crucian carp (TCC) produced by mating Japanese crucian carp with allotetraploid hybrids, ITCC not only re-tained the excellent traits of fast growth rate and sterility, but also acquired improved morphological characteristics, including higher body, shorter tail and smaller head.     

Difference in blood and water diffusion distance in gill lamellae of diploid and triploid tench Tinca tinca (L.)

Image analysis of sagittal sections of gill lamellae of diploid and triploid tench Tinca tinca revealed the blood and water diffusion distance in diploids (2·07 μm) to be significantly higher than that of their triploid siblings (1·46 μm; P < 0·01). Lamellae of diploids compared to triploids were found to be significantly shorter (105·84 v. 132·11 μm) and thicker (18·47 v. 14·21 μm; all at P < 0·05) than those of their triploid siblings but with similar mean sectional areas (1965·44 v. 1910·86 μm²).