酿酒酵母单倍体与双倍体原生质体的融合

本文报道用酿酒酵母(Saccharomyces cerevisiae)原生质体融合,得到营养互补的融合子为三倍体,其生长速度、发酵速率均较亲株提高1—2倍。部分融合子酒精的产量高于亲株,同时高于目前使用的酒精发酵生产菌株。     

酵母属间原生质体融合改进菌株木糖发酵性能

通过单倍体分离和紫外诱变,获得了１４株树干毕赤酵母（Ｐｉｃｈｉａｓｔｉｐｉｔｉｓ）７１２４和酿酒酵母（Ｓａｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ）１３００的营养缺陷型突变株。用聚乙二醇（ＰＥＧ）和电诱导融合及致死融合等方法,实现了树干毕赤酵母和酿酒酵母的属间原生质体融合。融合子能发酵木糖产生酒精,其厌氧发酵木糖和木糖葡萄糖混合液的能力明显优于亲株,耐酒精的性能也比亲株树干毕赤酵母７１２４有所提高。融合子经ＤＮＡ含量、细胞体积测定和稳定性能实验证明为稳定融合子。     

酵母融合菌株的构建及其特性研究

以酿酒酵母和糖化酵母为亲本,通过正交实验优化了亲本原生质体的制备,再生以及融合的最佳条件,由此获得了既具有糖化能力又可产高浓度酒精的稳定的酵母融合菌株。融合株的体积较亲株大,ＤＮＡ含量与两亲株ＤＮＡ含量之和相当,淀粉水解能力,葡萄糖发酵效率,生长速率及产酒精能力等均优于亲株     

三角酵母二倍体菌株的选育

由三角酵母(Trigonopsis variabilis)原生质体融合,获得了35株原养型融合子。通过对其中四株进行详绌分析表明,融合子细胞体积和DNA含量为两亲株之和,苏木精染色显示单核,这些结果证明融合子为亲株的二倍体。此外,融合子的生长速度、D-氨基酸氧化酶以及细胞蛋白质含量均明显高于亲株。电镜形态观察：进一步证明融合子细胞大于亲株。     

原生质体融合构建葡萄酒降酸酵母的研究

对葡萄酒酵母1450(Chxs、Ampr)和酒酒球菌SD-2a(Chxr、Amps)的原生质体制备方法进行了研究,并采用PEG和Ca2 促融的方法进行了两菌株的跨界原生质体融合。利用放线菌酮 氨苄青霉素对融合子进行初筛,再经发酵试验复筛,从117株融合子中筛选出1株在酒精发酵的同时降解苹果酸能力强的融合子F-20,在连续传代10次后,其遗传性状稳定。融合子F-20的酒精发酵能力接近葡萄酒酵母1450,同时能够降解66%左右的苹果酸。     

酿酒酵母和粟酒裂殖酵母属间融合和融合子特性

酿酒酵母(Saccharomyces cerevisiae Hansen)PW218和粟酒裂殖酵母(Schizosaccharomyces pombe Lindn)PW232的原生质体用20mmol/L CaCl_2和30％PEG(MW6000)处理进行属间融合,获得了10多株融合子,融合率为0.65～1.96×10~(-5)。对F_2和F_(10)两株融合子进行了葡萄糖、木糖及葡萄糖和木糖混合液的摇瓶实验结果表明F_(10)融合子利用葡萄糖、木糖及两种糖混合液产乙醇的能力大大高于两亲株。F_2融合子对木糖以及葡萄糖和木糖混合液的发酵能力亦较两亲株高,其中利用木糖产乙醇的量分别比PW218和PW232提高1.38倍和2.65倍。     

清酒酵母与酿酒醇母原生质体融合的研究

清酒酵母（ＳａｃｃｈａｒｏｍｙｃｅｓｓａｋｅＹａｂｅ）是日本清酒的生产菌株．耐酒精能力强;Ｋ氏酿酒酵母（ＳａｃｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅＫ）是酒精生产的常用菌株,发酵力强。本文应用原生质体融合技术进行了二菌株原生质体融合的研究。通过硫酸二乙酯（ＤＥＳ）诱变得到营养缺隐型菌株Ｑ（ａｒｇ－）和Ｋ（ｌｙｓ－,ρ－）,其融合率为１．２５×１０－５。检出的融合子其酒精发酵特性、细胞形态、体积大小都不同于双亲菌株。比较了在２８℃培养条件下,出发余株清酒酵母,Ｋ氏酿酒酵母和融合子Ｆ１、Ｆ２的发酵速度曲线、乙醇产量和酒精耐量等,得到一株在２８℃培养条件下,乙醇产量为７．４％（Ｖ／Ｖ）,酒精耐量为１５％的融合株Ｆ１。     

酿酒酵母原生质体融合及其融合子的鉴定

应用原生质体融合技术,得到乙醇产量较高的两株多倍体酵母菌融合子F_(28)和F_(38),融合频率为2×10~(-5)。测定融合子F_(28)、F_(38)每个细胞内DNA含量分别为2.14×10~(-8)、2.34×10~(-8)μg,而亲株DNA含量分别为0.78×10~(-8)和1.24×10~(-8)μg。并进行了融合子细胞增殖率、乙醇发酵能力及同功酶分析等试验。用固定化细胞发酵乙醇试验结果表明,在pH4.0,17％糖蜜为基质情况下,发酵3小时,融合子F_(28)、F_(38)的乙醇产量分别为77.21和77.09mg/ml;亲株乙醇产量仅为65.00mg/ml和68.40mg/ml。为固定化细胞发酵乙醇提供优良菌株。     

构建直接发酵淀粉产生酒精的酵母融合菌株的研究

以酒精酵母和热带假丝酵母为亲本,通过单亲灭活原生质体融合技术,获得了既具有糖化酶活性又能高产酒精的稳定的酵母融合株,并测定和比较了融合子的细胞大小、DNA含量以及比增长率μ、淀粉利用能力、乙醇耐受性、α-淀粉酶和糖化酶活力等生产性能。属间原生质体融合率为9.2×10^-6。F1和F5两株融合子性状较好,酒精发酵产量可达8.8%和11.5%,具有良好的应用前景。     

利用细胞质导入法选育嗜杀啤酒酵母

利用细胞质导入(Cytoduction)法中的核融合缺陷细胞融合技术,在对酿酒酵母(Saccharomyces cerevisiae)D518菌株不做任何遗传标记,将嗜杀酵母5045菌株的嗜杀质粒转移到受体菌D518中,获得了具有两亲株优良性状的融合子KD102菌株。对融合子分析表明:融合子遗传性状稳定,不仅含有供体菌5045的嗜杀质粒,而且受体菌D518的核基因被原封不动地保留下来,为异质体细胞(Heteroplasmon)。将融合子KD102菌株用于小型、中型及生产性酿酒试验,结果表明,具有与亲株D518同样的酿造特性。在发酵过程中,能抑制野生酵母污染,净化发酵体系。对于保证啤酒纯种酿造及提高成品酒的生物稳定性具有明显效果。     

Comparative studies on histological and ultra-structure of the pituitary of different ploidy level fishes

The histological and ultra-structure of the pituitary in diploid red crucian carp (Carassius auratus red var.), triploid crucian carp and allotetraploid hybrids within and after the breeding season were comparatively studied. The result showed that there were six endocrine cell types in the pituitary of these three kinds of fishes, and there was an obvious difference in cell size among different ploidy level fishes. As for the same type of pituitary cells, the cell size was increased gradually with the increasing ploidy level. In the breeding season, the allotetraploid hybrids had higher proportion of gonadotropin cells (GTH) than triploids, and the trploids had higher proportion of GTH than diploids. The results were related to the earlier sexual maturity of allotetraploid hybrids and sterility of triploid crucian carp. On the other hand, among the three kinds of fishes, the proportion of somatotropin (STH) cells in triploids crucian carp was the highest, whereas that in allotetraploid hybrids was the lowest. The results might be connected with the faster growth rate of triploids and slower growth rate of allotetraploid hybrids. In addition, in GTH cells of meso-adenohypophysis after the breeding season, there were many endocrine particles in triploids, while those endocrine particles were released from the cells in allotetraploids and diploids. This result showed that the sterility of triploid crucian carp might be related to the hormone which was not released from the GTH cells. In a word, the present study indicated that the differences in the structure of pituitary among different ploidy level fishes contributed to their difference in the growth rate and gonadal development.     

Comparative studies on histological and ultra-structure of the pituitary of different ploidy level fishes

The histological and ultra-structure of the pituitary in diploid red crucian carp(Carassius auratus red var.),triploid crucian carp and allotetraploid hybrids within and after the breeding season were comparatively studied.The result showed that there were six endocrine cell types in the pituitary of these three kinds of fishes,and there was an obvious difference in cell size among different ploidy level fishes.As for the same type of pituitary cells,the cell size was increased gradually with the in- creasing ploidy level.In the breeding season,the allotetraploid hybrids had higher proportion of go- nadotropin cells(GTH)than triploids,and the triploids had higher proportion of GTH than diploids.The results were related to the earlier sexual maturity of allotetraploid hybrids and sterility of triploid cru- cian carp.On the other hand,among the three kinds of fishes,the proportion of somatotropin(STH) cells in triploids crucian carp was the highest,whereas that in allotetraploid hybrids was the lowest. The results might be connected with the faster growth rate of triploids and slower growth rate of al- lotetraploid hybrids.In addition,in GTH cells of meso-adenohypophysis after the breeding season, there were many endocrine particles in triploids,while those endocrine particles were released from the cells in allotetraploids and diploids.This result showed that the sterility of triploid crucian carp might be related to the hormone which was not released from the GTH cells.In a word,the present study indicated that the differences in the structure of pituitary among different ploidy level fishes contributed to their difference in the growth rate and gonadal development.     

Hybridization of asporogenic strains of Hansenula polymorpha yeasts by protoplast fusion

Intrastrain and interstrain hybrids of different ploidy were produced by the fusion of protoplasts of Hansenula polymorpha haploid and diploid strains. The diploid hybrids were found to be stable in contrast to the triploid and tetraploid hybrids. The instability of the triploid and tetraploid states in Hansenula polymorpha was expressed in the elevated frequency of spontaneous formation of auxotrophic markers and in the decreased content of DNA per cell in the course of storage.     

Analysis of meiosis in triticale (XTriticosecale Wittmack) X rye (Secale cereale L.) F1 hybrids at three ploidy levels

Summary Triticales (XTriticosecale Wittmack) at three ploidy levels (8x, 6x, 4x, x=7) were crossed with diploid rye (Secale cereale L.) to produce a solitary hypopentaploid hybrid (2n=32), and a number of tetraploid (2n=4x=28) and triploid (2n=3x=21) hybrids. The hybrids exhibited a morphology which was intermediate between the parents. The number of bivalents ranged from 1–7 (4.65 per cell) in hypopentaploid, from 2–12 (7.13 per cell) in tetraploid and from 4–9 (6.84 per cell) in triploid hybrids. In 4x and 3x hybrids, trivalents and quadrivalents were also observed at low frequencies (range 0–1; mean 0.01–0.03 per cell). Chiasmata frequency was highest in triploid hybrids (12.44 per cell), lowest in hypopentaploid (5.37 per cell) and intermediate in tetraploids (10.54 per cell). More than 7¹¹ were found in 39.7% pollen mother cells (PMC's) in the 4x hybrids and in 5.0% PMCs in 3x hybrids. It is concluded that an increase in the relative proportion of wheat chromosomes in the hybrids had a slight suppression effect on homologous as well as homoeologous pairing of rye chromosomes. Contrary to this, the relative increase in rye complement promoted homoeologous pairing between wheat chromosomes. In triploid hybrids, the chiasmata frequency as well as the c value were the highest, suggesting that in tetraploid hybrids rye chromosomes had a reduced pairing (low frequency of ring bivalents).     

Implementation of extensive citrus triploid breeding programs based on 4x?×?2x sexual hybridisations

Seedlessness is one of the most important characteristics for mandarins for the fresh-fruit market, and mandarin triploid hybrids have this trait. Triploid citrus plants can be recovered by 4x?×?2x hybridisations using non-apomictic genotypes as female parents. In this study, we characterise the type of seeds obtained in 4x?×?2x hybridisations and the ploidy level of plants recovered from each type of seed. The majority of the plants recover were triploid (98.3?%), but a few diploid, tetraploid and pentaploid plants were also produced and their genetic origin was analysed by simple sequence repeat (SSR) markers. We also analysed the influence of parents and environmental conditions on the efficiency of recovery triploid hybrids. In this work, we present an effective methodology to recover triploid hybrids from 4x?×?2x hybridisations based on in vitro embryo rescue and determination of ploidy level by flow cytometry that allow us to recover more than 4,400 triploid hybrids from more than 60 parental combinations.     

The formation of the polyploid hybrids from different subfamily fish crossings and its evolutionary significance

This study provides genetic evidences at the chromosome, DNA content, DNA fragment and sequence, and morphological levels to support the successful establishment of the polyploid hybrids of red crucian carp x blunt snout bream, which belonged to a different subfamily of fish (Cyprininae subfamily and Cultrinae subfamily) in the catalog. We successfully obtained the sterile triploid hybrids and bisexual fertile tetraploid hybrids of red crucian carp (RCC) (female symbol) x blunt snout bream (BSB) (male symbol) as well as their pentaploid hybrids. The triploid hybrids possessed 124 chromosomes with two sets from RCC and one set from BSB; the tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from BSB. The females of tetraploid hybrids produced unreduced tetraploid eggs that were fertilized with the haploid sperm of BSB to generate pentaploid hybrids with 172 chromosomes with three sets from BSB and two sets from RCC. The ploidy levels of triploid, tetraploid, and pentaploid hybrids were confirmed by counting chromosomal number, forming chromosomal karyotype, and measuring DNA content and erythrocyte nuclear volume. The similar and different DNA fragments were PCR amplified and sequenced in triploid, tetraploid hybrids, and their parents, indicating their molecular genetic relationship and genetic markers. In addition, this study also presents results about the phenotypes and feeding habits of polyploid hybrids and discusses the formation mechanism of the polyploid hybrids. It is the first report on the formation of the triploid, tetraploid, and pentaploid hybrids by crossing parents with a different chromosome number in vertebrates. The formation of the polyploid hybrids is potentially interesting in both evolution and fish genetic breeding.     

Effect of oilseed rape genotype on the spontaneous hybridization rate with a weedy species:an assessment of transgene dispersal

Spontaneous outcrossing of different malesterile rapeseed lines and transgenic hybrids with a population of a weedy species, Raphanus raphanistrum L., has led to the harvest of numerous seeds showing a size dimorphism. Flow cytometry analysis correlated with chromosome counts showed that all of the large seeds belonged to rapeseed, whereas the small seeds were a mixture of mostly interspecific triploid hybrids, with some trigenomic amphidiploids, diploid and haploid rapeseed plants. Significant differences were revealed between the rapeseed lines and transgenic hybrids in their ability to form interspecific hybrids with Raphanus raphanistrum under natural conditions. Resistance to the herbicide Basta was properly expressed in the triploid and amphidiploid hybrids. Low male fertility of the interspecific triploid hybrids was not correlated with seed set in the subsequent generation.     

Recovery of citrus triploid hybrids by embryo rescue and flow cytometry from 2x × 2x sexual hybridisation and its application to extensive breeding programs

Seedlessness is one of the most important characteristics for mandarins for the fresh-fruit market and mandarin triploid hybrids have this trait. Citrus triploid plants can be recovered by 2x × 2x sexual hybridisations as a consequence of the formation of unreduced gametes at low frequency. Triploid embryos are found in small seeds that do not germinate under greenhouse conditions. Extensive breeding programs based on this type of hybridisation require very effective methodologies for embryo rescue and ploidy evaluation. In this work, we describe an effective methodology to recover triploid hybrids from 2x × 2x hybridisations based on in vitro embryo rescue and ploidy level determination by means of flow cytometry. The influence of parents and environmental conditions on obtaining triploid hybrids has been analysed. The strongest effect was associated with the genotype of the female parent while a strong interaction was found between the male parent genotype and environmental conditions. The effect of the female parent genotype on the length of the juvenile phase was also demonstrated by observing a large number of progenies over the last 10 years. The methodology described here has enabled us to obtain over 4,000 triploid hybrids so far, of which 13 have been protected in the European Union and two are being extensively planted by citrus growers to establish new commercial plots. These triploid hybrids have been analysed with simple sequence repeats markers to differentiate all the new triploid varieties and their parents, and thus molecular identification will help defend plant breeders’ rights.