不同甘油浓度与平衡时间对食蟹猴精液冷冻效果的影响

探讨了不同甘油浓度(3%、5%、7%、11%)和不同平衡时间(30、60、90、120min)对食蟹猴(Macaca fascicularis)精液冷冻效果的影响,以建立和优化食蟹猴精液冷冻的程序。参照TTE稀释液成分组成改良型TTE,冷冻前和解冻后均检测精子的活力、畸形率、质膜完整性、顶体完整率。结果显示,平衡时间为30min时精子的冷冻解冻后活力、复苏率均高于平衡时间90min和120min组,差异显著(P<0.05),比60min组稍好;甘油浓度为3%、5%组的精子冷冻解冻后活力及复苏率均高于甘油浓度11%组,差异显著(P<0.05),比7%组好;不同甘油浓度各组间以及不同平衡时间各组间畸形率、质膜完整性、顶体完整率差异不显著(P<0.05)。由此得出如下结论,在食蟹猴精液冷冻中,在改良TTE中加入3%~5%的甘油且平衡30min可以获得较好效果,精子冻后活率和复苏率达到45%和62%。     

Atopically transplanted thymus tissues are capable of distantly changing metabolic status of the organism

It has been demonstrated that an atopic transplantation of the thymus tissue from young animals in different variants decreased the rate of irreversible age-related atrophy of the thymus in a statistically significant manner. This retards an age-dependent degradation of the T-cell component of the immune system.     

Distribution and Cytogenetic Features of Triploid Males of Crucian Carp in Azov Basin

When studying uni-bisexual crucian carp (Carassius auratus gibelio) populations in the Azov basin in 1995–2000, we found triploid males, which constituted 2.5%, on average, of the total numbers of studied samples. The areas of nuclei of erythrocytes of triploid males were, on average, 1.35 times those in diploid males. At the same optical density of DNA, the sizes of mature spermatozoon heads in triploid males were, on average, 1.8 times smaller than in diploid males, as follows from the data obtained in summer 1996. The results of similar studies carried out during the period of natural spawning activity in 1997–1999 suggest that the sizes of spermatozoon heads in triploid males were, on the contrary, 1.5 those in diploid males. Triploid males were characterized by mosaicism of spermatozoon sizes and chromosome mosaicism in somatic cells. Electrophoretic analysis for the locus of transferrin confirmed the triploid status of this genetic group. The results of comparative crosses of crucian carps with different ploidy suggest a high fertilizing capacity of triploid males, as well as normal viability of their progenies. A distinct positive correlation (r = 0.73) was found between the numbers of triploid females and triploid males in mixed di-triploid populations. No significant correlation was found between males and females within di- and triploid forms.     

Cryopreservation of Grapevine (Vitis spp.) Embryogenic Cell Suspensions by Encapsulation–Vitrification

Embryogenic cell suspensions of two grapevine rootstocks: 110 Ritcher (V. berlandieri × V. rupestris), 41B (V. vinifera × V. berlandieri) and several table grape and wine cultivars (Vitis vinifera) were successfully cryopreserved by the encapsulation–vitrification method. Embryogenic cell suspensions were precultured for 3 days in liquid MGN medium supplemented with daily increasing sucrose concentrations of 0.25, 0.5, 0.75 M. Precultured cells were encapsulated and directly dehydrated with a highly concentrated vitrification solution prior to immersion in liquid nitrogen for 1 h. After rewarming at 40 °C for 3 min, cryopreserved cells were post-cultured on solid MGN medium supplemented with 2.5 g l^–1 activated charcoal. Surviving cells were transferred to solid MGN medium for regrowth or solid MG medium for embryo development and then to solid WPM for plant regeneration. Optimal viability was 42–76% of cryopreserved cells when cell suspensions were precultured with a final sucrose concentration of 0.75 M and dehydrated with PVS2 at 0 °C for 270 min. Biochemical analysis showed that sucrose preculture caused changes in levels of total soluble protein and sugars in cell suspensions. Although the increase in fresh weight was significantly lower in cryopreserved cells than in control cells, the growth pattern of the cryopreserved cells and control cells was the same after two subcultures, following re-establishment in cell suspensions. Protocol developed in this study suggests a universal and highly efficient cryopreservation system suitable for several genetically diversed Vitis species.     

The development of haddock and Atlantic cod sperm cryopreservation techniques and the effect of sperm age on cryopreservation success

Three cryoprotectants [dimethyl sulphoxide (DMSO), propylene glycol (PG) and glycerol], two diluents (sucrose‐ and saline‐based), two sperm collection times, two freezing rates and three times between thaw and activation (0, 30 and 60 min) were tested in order to develop a protocol for the cryopreservation of sperm of haddock Melanogrammus aeglefinus and Atlantic cod Gadus morhua . The faster freezing rate resulted in extremely low post‐thaw motility in comparison to the slower freezing rate, which was successful for sperm from both gadids. In both cases, the use of PG resulted in significantly higher post‐thaw sperm motility‐recovery indices than with DMSO or glycerol, which did not differ significantly from one another. Diluent had no effect on post‐thaw sperm motility for Atlantic cod or haddock. Sperm collected at the end of the spawning season tended to have reduced post‐thaw motility compared to that collected 2 weeks after the start of spawning. A 30 min delay between thaw and activation of haddock and Atlantic cod sperm resulted in a significant decrease in sperm motility. When PG was used as cryoprotectant, sperm motility continued to decrease between 30 and 60 min post‐thaw. With DMSO or glycerol as cryoprotectant, motilities were already very low after 30 min post‐thaw and did not decrease any further after 60 min. Cryoprotectant, diluent and time between thaw and activation had no effect on mean or maximum sperm swimming speeds for either Atlantic cod or haddock sperm. Fertilization success for haddock eggs, like sperm motility, was higher with PG‐frozen sperm than DMSO‐ or glycerol‐frozen sperm. These results constitute the first reported successful cryopreservation of haddock sperm and improve on previous methods used to cryopreserve sperm from Atlantic cod.     

Cryopreservation of hairy root cultures of Vinca minor (L.) by encapsulation-dehydration

Hairy root cultures of Vinca minor and Ajuga reptans var. atropurpurea could be cryopreserved when the roots were precultured and encapsulated in 2% (w/v) alginate beads with 0.3 M sucrose and 0.5 M glycerol and dehydrated until the bead weight reached 25% of the initial weight before cooling in liquid nitrogen. Preculture and encapsulation of the roots with abscisic acid was effective in increasing the survival rates. For V. minor root tips moreover a sufficiently high survival rate of more than 70% was attained by eliminating glycerol from the preculture medium and dehydration of beads until 23% of the initial weight was reached instead of 25%.     

Lats1基因敲除小鼠保种的研究

目的　以Lats1基因敲除小鼠为例 ,研究国家遗传工程小鼠资源库遗传小鼠的保种技术路线及方法。方法　采用了胚胎移植 ,目的基因检测 ,胚胎冷冻等技术。结果　在库内得到 2 4只Lats+ -小鼠 ,扩群后 ,冷冻保存 110枚胚胎。结论　上述方法能确保Lats1基因敲除小鼠资源库保种成功。     

Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media.

In this study, the quantitative fluctuation of cytoplasmic lipid droplets (LD) and cryotolerance were investigated in bovine embryos derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes developed in different culture systems using serum-free or serum-containing media. The serum-free cultures were grown using IVMD101 medium in conjunction with bovine cumulus/granulosa cell (BCGC) cocultures or IVD101 medium without BCGC cocultures, and the serum-containing cultures were grown in the presence of BCGC cocultures using HPM199 medium supplemented with 5% calf serum (HPM199 + CS). Large numbers of sudanophilic LD were present in the cytoplasm of bovine embryos from 2-cell to hatched blastocyst stages, and the number and size differed between the embryos cultured in serum-free and serum-supplemented media. In the embryos cultured in HPM199 + CS, large (2-6 microm in diameter) sudanophilic LD increased significantly from the morula to the blastocyst stages. Throughout the embryonic development, the embryos developed in serum-free cultures with and without BCGC cocultures had numerous sudanophilic LD, but most of these droplets were small (<2 microm in diameter) and large LD were less numerous than those embryos cultured in HPM199 + CS. Giant LD (>6 microm in diameter) were frequently observed in morulae and blastocysts (including early blastocysts) developed in HPM199 + CS. Electron microscopic observations demonstrated that large LD were abundant in the cytoplasm of trophoblast and embryonic (inner cell mass) cells of blastocysts cultured in HPM199 + CS. These large LD were identified as osmophilic LD, an indication that these lipid inclusions contained a significant proportion of unsaturated lipids. Many elongated mitochondria were found in embryos developed in IVMD101 and IVD101 at the morula and early blastocyst stages, whereas many of the mitochondria in the morulae developed in HPM199 + CS were of an immature form such as spherical or ovoid shape. The survival and hatching rates of embryos (morulae, early blastocysts, and blastocysts) produced in serum-free media (both IVMD101 and IVD101) after post-thaw culture were superior to those of embryos produced in serum-containing medium. These results showed that bovine embryos cultured in serum-containing medium abnormally accumulated cytoplasmic lipids into their cytoplasm and the excess accumulation of cytoplasmic LD in embryos may affect the cryotolerance of embryos.     

Biotechnological applications for the improvement of coffee (Coffea arabica L.)

Summary The important advances in coffee biotechnological techniques which have been made particularly during the last 10yr could benefit the coffee breeder in practice and open new perspectives for the development of new varieties. The molecular phylogeny of Coffea species has been established using DNA sequence data. The molecular markers have revealed an extremely reduced genetic diversity in Coffea arabica L. in comparison to C. canephora. However, wild accessions collected in the Ethiopian highlands appeared to constitute a valuable gene reservoir. A complete genetic linkage map of C. canephora was reported and additional ones are being constructed, particularly on C. arabica. The integration of Molecular Assisted Selection in coffee breeding promises to drastically increase the efficiency of breeding programs. Economically important genes of the caffeine biosynthetic pathway or genes encoding for seed storage proteins have been isolated. The high performance already achieved in the in vitro propagation process by somatic embryogenesis offers the possibility to mass propagate superior hybrids in different countries of both C. arabica (selected F₁ hybrids) and C. canephora (rootstock variety). Pilot productions by somatic embryogenesis currently permit preparation for commercial application. Somaclonal variation was observed. The percentage of the off-types can vary between 3 and 10% depending on the genotype. Seed cryopreservation enables a routine use for long-term conservation of coffee genetic resources. Transgenic plants have been obtained for the C. arabica and C. canephora cultivated species through Agrobacterium-mediated transformation which constitutes the technique now currently used to transfer directly genes in coffee plants.     

Cryopreservation of sour orange (Citrus aurantium L.) shoot tips

Summary The objective of this study was to establish a cryopreservation protocol for sour orange (Citrus aurantium L.). Cryopreservation was carried out via encapsulation-dehydration, vitrification, and encapsulation-vitrification on shoot tips excised from in vitro cultures. Results indicated that a maximum of 83% survival and 47% regrowth of encapsulated-dehydrated and cryopreserved shoot tips was obtained with 0.5M sucrose in the preculture medium and further dehydration for 6 h to attain 18% moisture content. Dehydration of encapsulated shoot tips with silica gel for 2h resulted in 93% survival but only 37% regrowth of cryopreserved shoot tips. After preculturing with 0.5M sucrose, 80% of the vitrified cryopreserved shoots survived when 2M sucrose plus 10% dimethyl sulfoxide (DMSO) was used as a cryoprotectant for 20 min at 25°C. Survival and regrowth of vitrified cryopreserved shoot tips were 67% and 43%, respectively, when 0.4M sucrose plus 2M glycerol was used as a loading solution followed by application of 100% plant vitrification solution (PVS2) for 20 min. Increased duration of exposure to the loading solution up to 60 min increased survival (83%) and regrowth (47%) of cryopreserved shoot tips. With encapsulation-vitrification, dehydration with 100% PVS2 for 2 or 3 h at 0°C resulted in 50 or 57% survival and 30 or 40% regrowth, respectively, of cryopreserved shoot tips.