Cell Ultrastructure of Kiwifruit （Actinidia chinensis） Shoot Tips During Cryopreservation

The changes in the cell ultrastructure of in vitro cultured shoot tips from dwarf genotype of kiwifruit （Actinidia chinensis Ganmi 5） during cryopreservation were investigated. Shoot tips were preserved in liquid nitrogen using vitrification, and the cell ultrastructure was examined using transmission electron microscopy （TEM）. The regular ultrastructure of the cell wall, cell membrane and nucleus of shoot tips could be damaged during the freezing and thawing associated with preservation using liquid nitrogen. The cell plasmolysis was increased and freezing tolerance was improved after precultufing and dehydrating in a preservation and vitrification solution （PVS2） （30% glycerol （Gly）＋ 15% ethylene glycol （EG）＋ 15% dimethylsulfoxide （DMSO） ＋ 0.4 mol L^-1 sucrose）. The structure of some cells with low degree of injury and reversible damage was similar to that of the control and they could undergo normal cell division and differentiation. Besides, they could recover automatically and regenerate after their reculture.     

Preparation,cryopreservation and in vitro culture of spermatogonial stem cells of new born calves

The experiment was designed to study the preparation, cryopreservation and culture of calf spermatogonial stem cells in order to provide some data for theoretical research and practical use of calf spermatogonial stem cells. As for enzymolysis of testis tissue, two digestive method A and B. The cryopreservation cooling procedure was that spermatogonial stem cells was equilibrated at 4℃ for lh, at -20℃ for lh, at -40℃ for 2h, at -80℃ overnight and then stored in liquid nitrogen. The different concentration of three eryoprotectants were compared. The results indicated that both method A and method B achieved the same high motility rates of cells with method B needing time longer, calf spermatogonial stem cells could be well cryopreserved by the stage cooling procedure. Satisfactory cryopreservative results were gotten by adding 10% DMSO or 10% EG to cryopreservative medium, the former was better and adding sucrose could improved cryopreservative effect. The culture behaviors of spermatogonial stem cells kept the same before and after cryopreservation. It was concluded that an effective method was verified for preparation, cryopreservation and culture of new calf spermatogonial stem cells.     

Effect of in ovo zinc injection on the embryonic development,tissue zinc contents,antioxidation, and related gene expressions of broiler breeder eggs

Two experiments were conducted to investigate the effect of in ovo zinc(Zn) injection on the embryonic development, tissue Zn contents, antioxidation and related gene expressions of fertilized eggs of Arbor Acres broiler breeders. Experiment 1 was conducted to determine an optimal embryonic age for early in ovo injection. A total of 720 fertilized eggs with similar weights were randomly allotted to 4 treatments with 6 replicates per treatment and 30 eggs per replicate in a completely randomized design. The eggs were injected with 0.1 m L sterilized water at 3, 6 and 9 embryonic days of incubation(E3, E6 and E9) or non-injection(the control), respectively. The results from experiment 1 showed that E3 and E6 injections increased(P0.05) the embryonic mortalities, and decreased(P0.05) hatchabilities compared to the non-injected control, but no differences(P0.05) between E9 injection and the non-injected control were observed in either embryonic mortality or hatchability. The findings suggest that the E9 is the optimal embryonic age for early in ovo injection. In experiment 2, a total of 672 fertilized eggs with similar weights were randomly allocated to 7 treatments with 6 replicates per treatment and 16 eggs per replicate in a completely randomized design. The eggs were injected with 0(the negative control), 50, 100, 150, 200, or 250 μg Zn/egg as reagent grade Zn SO4·7 H2 O in a 0.1-m L solution, or non-injection(the positive control), respectively at E9–10. The results from the experiment 2 demonstrated that no differences(P0.05) among 50 and 100 μg Zn/egg groups and the negative control were observed in the embryonic mortality and hatchability, however, the injection of 200 μg Zn/egg increased(P0.05) the embryonic mortality, and injections of 150 and 200 μg Zn/egg decreased(P0.05) hatchabilities compared with the controls. The embryonic tibia Zn contents at E20 were increased(P0.05) by injections of 150, 200 and 250 μg Zn/egg. Zinc injection did not affect(P0.05) malonaldehyde(MDA) contents, copper-and Zncontaining superoxide dismutase(Cu Zn SOD) activities and m RNA expression levels in the liver and heart of chick embryos at E15 and E20. Compared with the negative control, injections of 50, 150 and 200 μg Zn/egg up-regulated(P0.05) the metallothionein(MT) mR NA expression levels in the embryonic liver at E20. These results indicated that in ovo Zn injections increased Zn contents in the embryonic tibia and MT m RNA expression levels in the embryonic liver at E20, however, injections of 150–200 μg Zn/egg were harmful to the embryonic development.     

Mapping of Quantitative Trait Loci Affecting Eggshell Quality in an F2 Population Derived from Strong and Weak Eggshell Lines of the White Leghorn Chicken Breed

Broken and cracked eggshells cause major economic losses to the egg production industry. An F2 population of 262 hens obtained by crossing a strong egg shell line with a weak egg shell line of the White Leghorn breed was used for detecting the quantitative trait loci （QTL） affecting eggshell quality. The 2 lines were developed from the same founder population by two-way selection for egg shell strength with nondestructive deformation. Of the 1 014 microsatellite markers tested, 35 were mapped on 10 autosomal linkage groups. There was no informative marker on chromosome Z. The QTLs associated with 7 traits, i.e., body weight, short length of egg, long length of egg, eggshell strength, eggshell thickness （EST）, eggshell weight （ESW）, and egg weight （EW）, were identified. Highly significant （P〈0.01） QTLs associated with EST and ESW and a significant （P〈 0.05） QTL associated with EW were mapped to a region flanking ABR0545 and ABR0362 on chromosome 9. These QTLs are good candidates to be employed in the development of strategies for reducing the number of broken and cracked eggs in commercial layer houses by employing marker assisted selection.     

Profile Analysis of the Proteome of the Egg of the High Royal Jelly Producing Bees （Apis mellifera L.）

The protein composition of the egg development in the high royal jelly producing bees （Apis mellifera L.） was investigated. This pioneer study was to separate and quantify the proteins in the egg of the high royal jelly producing worker bees （Apis mellifera L.） by using two-dimensional gel electrophoresis along with their three-day development. The results showed that 160, 195, and 176 proteins, with a wide range of molecular weight （17-80 KDa） and relatively narrow scope of pI （4. 00-8.40） could be detected on day 1, day 2, and day 3, respectively, during the developmental process of the egg. Meanwhile 44 protein spots were constantly detected along with the egg development. Among them 36% were in the uptrend along with the egg development, 14% were in the downtrend, and 39% were of the largest expressed volume on day 2. In addition, the specific proteins were expressed on day 1, day 2, and day 3 （89, 77, and 80, respectively）. Besides the coexistent and specific proteins, 24 proteins were expressed on day 1 and day 2, but silenced on day 3, 49 proteins were expressed on day 2 and day 3, but silenced on day 1, only 3 proteins were expressed on day 1 and day 3, but silenced on day 2. The result indicates that egg development is a sequential and complex gene controlled process, where the eggs of day 2 express the most active proteins. The coexistent proteins suggest that it is conservative and indispensable for this event. These specific proteins suggest that the different developmental stage needs specific proteins to regulate it.     

Effects of Different Culture Conditions on Vitrification of Lycium barbarum L. Plantlets in Tissue Culture

The tender stems from new Lycium barbarum L. cultivar "Ningqi 3" released by Ningxia Academy of Agricultural and Forestry Sciences were regarded as explants to investigate the vitrification of Lycium barbarum plantlets in tissue culture under different concentrations of 6-BA,sucrose,agrose,culture temperature,and illumination duration with MS as basic medium. The results show that the conditions for maximal proliferation coefficient and minimal vitrification are as following: the basic medium with 0.2 mg/L 6-BA,3% sucrose and 0.65% agarose; culture at 25 ℃; 12 h/d(daylight lamp,2 000 lx) illumination.     

不同培养条件对枸杞组培苗玻璃化的影响(英文)

The tender stems from new Lycium barbarum L. cultivar "Ningqi 3" released by Ningxia Academy of Agricultural and Forestry Sciences were regarded as explants to investigate the vitrification of Lycium barbarum plantlets in tissue culture under different concentrations of 6-BA,sucrose,agrose,culture temperature,and illumination duration with MS as basic medium. The results show that the conditions for maximal proliferation coefficient and minimal vitrification are as following: the basic medium with 0.2 mg/L 6-BA,3% sucrose and 0.65% agarose; culture at 25 ℃; 12 h/d(daylight lamp,2 000 lx) illumination.     

Analysis of Developmental Proteome at Egg Stage of Drone Honeybees （A. m. ligustica）

An investigation on the proteome of drone egg development of native Italian bee （Apis mellifera ligustica Spinola,1806） was carried out in order to prove up the characteristics in protein expression and regulation at egg stage and open out the molecular mechanism of the development. The experiment was carried out by two-dimensional gel electrophoresis. The results showed that there were 200, 242 and 233 proteins in a wide rang of molecular weight （12.42-169.60 kDa） and in a relatively narrow scope of pI （4.50-9.00） detected on day 1, day 2 and day 3, respectively, during the developmental process of the drone egg. Meanwhile, 164 protein spots were resolved at all the images （i.e., the protein was consistently expressed） along with the egg development, among which 7 were significantly up-expressed （P 〈 0.05） and 4 were significantly down-expressed （P 〈 0.05） while 79 had no significant differences （P 〉 0.05）. In addition, the specific proteins expressing proteins on day 1, day 2 and day 3 were 11, 18 and 18, respectively. Besides, 17 proteins expressed both on day 1 and day 2 but silenced on day 3, and 43 proteins expressed both on day 2 and day 3 but silenced on day 1, while only 8 proteins expressed both on day 1 and day 3 but silenced on day 2. The results indicate that 2-d-old eggs are at the most active expressional stage in the development of drone egg. The protein expressing at all images suggests that it should be indispensable for drone egg development, but their expression pattern is different. The proteins expressing at a specific age of egg suggest that specific proteins are needed in different developmental stages to regulate. And there are more house-keeping proteins in the developmental process of the drone egg than that of worker egg, and it will provide more targets for gene improvement.     

Isolation, Purification and Cryopreservation of Cells from Neonatal Bovine Testis

The development and application of spermatogonial stem cell technology have an important significance in animal cloning, preservation of endangered species and spermatogenesis research. In this study, the seminiferous epithlium cells were isolated and purified, the cells were cryopreserved after identification, and the effects of different purification and cyopreservation methods on bovine testicular cells were studied. The results showed that there were spermatogonial stem cells and sertoli cells in the neonatal bovine seminiferous tubules, differential adherent selection methods could effectively separate these two cell types. Spermatogonial stem cells were positive after AKP, C-kit, and OCT-4 identification; sertoli cells were positive after oil red O and vimentin identification. Frozen stock solution supplemented with 10% DMSO had the best effect in spermatogonial stem cell cryopreservation, while frozen stock solution supplemented with 10% of ethylene glycol and 0.1 mmol·L-1 trehalose had the best effect in sertoli cells cryopreservation.     

Calcium Forms, Subcelluar Distribution and Ultrastructure of Pulp Cells as Influenced by Calcium Deficiency in Apple （Ma／us pumila） Fruits

Calcium in Red Fuji and Starkrimson apples during storage were fractionated by sequent extracting. Localization and distribution of calcium and influence of calcium nutrition on cell ultrastructure were observed by transmission electron microscopy combined with in situ precipitation of calcium with an improved method of potassium pyroantimonate technique. Results indicated that spraying calcium solution on surface of young fruits increased contents of calcium in all forms. During storage, contents of soluble calcium and pectic calcium declined and those in calcium phosphate, calcium oxalate and calcium silicate increased. Calcium contents of Red Fuji in all forms were higher than those of Starkrimson, indicating that calcium accumulating capability of Red Fuji fruits preceded that of Starkrimson. Under transmission electron microscopy, calcium antimonite precipitates(CaAP) was mainly distributed in cell wall, tonoplast, nuclear membrane and nucleoplasm,much more CaAP deposited in vacuole. Calcium deficiency during storage leads to decrease of CaAP in locations mentioned above, disappearance of compartmentation, and entrance of CaAP to cytoplasm. Transformation from soluble calcium and pectic calcium to calcium phosphate, oxalate and damages of biomembranes structuraly and functionally resulted from calcium deficiency during storage were the crucial causation of physiologicaldisorder.     

Regulation of sucrose synthase activity and sugar yield by nitrogen in sugar beet

The content of sugar is influenced by sucrose synthase (SS) activity in roots. The effects of nitrogen level in the amino- nitrate ratio on SS activity of leaves and roots, roots yield and sugar content in sugar beet were studied in the field experiment by nutrient solution culture. The results showed that SS activity in leaves was lower than that in roots. With nitrogen level increasing, SS decomposition activity enhanced, and synthesis activity reduced. SS activity was regulated by different nitrogen forms and the ratio of NO3– and NH4 . SS synthesis activity was enhanced as NH4 increasing when NO3–: NH4 ≥1, and it decreased as increasing NH4 when NO3–: NH4 ≤1, and it was the highest when NO3–: NH4 =1. SS decomposition activity was enhanced as NO3– increasing. Sucrose content in root was lowed as nitrogen level increasing, but it was enhanced as NH4 increasing in the same nitrogen level. Root and sugar yield were the highest in the medium nitrogen level and NO3–: NH4 =1. The result in field experiment corresponded with that in the nutrient fluid culture. It provides a basis for using reasonably nitrogen fertilizer in sugar beet production.     

Effect of sucrose on cryopreservation of pig spermatogonial stem cells

Sucrose is known to play an important role in the cryopreservation of sperm and female gonads; however, its effect on the cryopreservation of pig spermatogonial stem cells(p SSCs) has not been tested. The aim of this work was to study the effect of sucrose during p SSC cryopreservation and to find the most effective concentration in freezing medium. p SSCs were cryopreserved with freezing media containing different concentrations of sucrose(70, 140, 210, and 280 mmol L~(–1)) and a control group without sucrose. The survival rates, plasma membrane integrity, and mitochondrial membrane potential of thawed cells were detected by trypan blue(TB) staining, SYBR-14/propidium iodide(PI) dual staining, and JC-1 staining, respectively. All the staining results showed an obvious increase in cell survival in the sucrose-treated groups as compared to that in the control group, with the exception of 280 mmol L~(–1) sucrose. Moreover, the 210 mmol L~(–1) sucrose group yielded the highest survival rate among all the groups(P0.05). The results of SYBR-14/PI dual staining and JC-1 staining were consistent with those of TB staining as above described. Quantitative real-time PCR(q RT-PCR) indicated that the m RNA levels of three apoptosis-promoting genes(BAX, APAF1 and CASPASE9) were significantly higher in thawed cells than in cells before freezing(P0.05). Moreover, the mR NA level of one anti-apoptotic gene(XIAP) was significantly lower in thawed cells than in cells before freezing(P0.05). When comparing the m RNA expression of apoptosis-related genes in thawed cells, the m RNA level of the anti-apoptotic genes in the control group was significantly lower than that in the sucrose-treated groups(P0.05). Western blot analyses showed that the expression levels of cleaved CASPASE9, CASPASE3 and PARP-1 in the sucrose-treated groups were lower than those in the control group and were the lowest in the 210 mmol L~(–1) sucrose group. Both q RT-PCR and Western blot analyses suggested that sucrose inhibited cell apoptosis during freezing and thawing. Briefly, sucrose promoted p SSCs survival after freezing and thawing, especially at a concentration of 210 mmol L~(–1), which possibly assisted p SSC dehydration and inhibited cell apoptosis. These findings hold great promise for further studies of the regulatory mechanism of proliferation and differentiation of p SSCs.     

Effect of Different Initial pH on the Storage Characteristics and Shelf Life of Liquid Diet for Suckling and Weanling Piglets

An experiment was conducted to investigate the effect of different initial pH on the storage characteristics and shelf life of liquid diet. 45 polypropylene bags were allotted to treatments 1, 2 and 3 on average, 100 g diet and 200 g water were placed into each polypropylene bag, food-grade DL-lactic acid was added to each bag at a rate of 0.0 mL in treatment l, 1.2 mL in treatment 2 and 4.7 mL in treatment 3, air was artificially expelled from each bag prior to heat-sealing. All bags were placed into a cage, cooked with steam at 90~C for 30 rain under normal pressure, then taken out and stored from day 0 to 60 at room temperature. Results indicated that liquid diet in treatment 3 achieved the highest total sensory scores, the pH value had a tendency to decrease and the bacteria count had a tendency to increase in the liquid diet with the advancing of storage time with the advancing of storage time, lowering the initial pH of liquid diet decreased the bacteria count, the AFBl and ZEN concentrations and increased the starch gelatinization degree from day 30 to 60, liquid diet in treatment 3 had a lower （P〈0.01） bacteria count and a higher （P〈0.05） starch gelatinization degree at day 30 and 45 than liquid diet in treatment 1. In conclusion, lowering the initial pH of liquid diet with lactic acid to pH 4 could effectively improve the storage characteristics and shelf life of liquid diet.     

Accumulation Pattern of Gliadin Fractions α, β, γ, ω and Regulation of Nitrogen

Gliadin, the major storage protein in endosperm, affects grain quality in spring wheat by its content and composition. Eighteen cultivars differing in HMW-GS were used in the study to approach the accumulation pattern of gliadin fractions α, β, γ, ω and regulation of three kinds of nitrogen source. The results showed that the content of gliadin in grains increased gradually along with the process of grain-filling, but the accumulation intensity and final content differed evidently among cultivars with different HMW-GS composition. Of all the subunit types used here, cultivars with subunits 7＋9 and 2＋12 had smaller accumulation intensity and lower final content. During grain-filling, 4 gliadin fractions had the same increase trend, but differed in accumulation course. The dynamic trends of gliadin accumulation were similar in different nitrogen treatments whose effects on initial amount, accumulation intensity and final level of accumulation varied with cultivars. Of three nitrogen fertilizer types, the amide-form nitrogen source was better to the formation and accumulation of gliadin as well as its four fractions.     

瓠瓜果实发育中的蛋白质SDS-PAGE分析

In seed plants, successful fruit set and subsequent development are dependent on pollination and fertilization since fertilization activates cell division and triggers fruit development by producing phytohormones especially auxins and GAs. Fruit set was very low in Lagenaria leucantha Rusby during early spring. We found that CPPU ［N-(chloro-4-pyridyl)-N′-phenyurea］, a new kind of cytokinin, was more effective than NAA and GA3 in inducing parthenocarpic fruit growth and in decreasing fruit abortion. In this paper, we examined the possible role of fertilization and CPPU in protein synthesis in developing fruit of Lagenaria leucantha by SDS-PAGE analysis. 1　Materials and Methods 1.1　Plant materials　　Seeds of Lagenaria leucantha Rusby (cv. Hangzhou long- gourd) were sown on 15 February 1999, and the seedlings were transplanted on 20 March. There were three treatments: Nonpollination; Hand pollination, and Nonpollination+50 mg/L CPPU. Unpollinated ovaries were prepared by bagging the female flowers one day before anthesis. The solution of CPPU was applied with a spray to ovaries of bagged female flowers at anthesis at the rate of 0.5 mL per ovary. Fruit length was recorded every three days. For assays of protein, the flesh of fruits at anthesis and 8 days after treatment was frozen in liquid nitrogen and stored at -20 ℃. 1.2　Extraction of proteins　　Half gram of fruit tissue was homogenized in 1 mL extract buffer containing 25 mmol/L DTT and 250 mmol/L Tris-HCl, pH6.8. The homogenate was centrifuged at 12 500 g for 20 min. Aliquot of the 250 μL supernatant was added into an eppendorf tube filled with 1 mL of 80% acetone, then frozen in -20 ℃ for 10 min. After thawing at 25 ℃, it was centrifuged at 10 000 g for 10 min, the supernatant was carefully discarded.……     

A Study on Artificial Hatching of Chinese Bombyx mandarina Moore

Diapause eggs of Bombyx mandarina Moore from Wujiang, Jiangsu Province, China, were used to study the artificial hatching of B. mandarina Moore. The results showed that the highest hatchability was obtained by instant treatment with hydrochloric acid （HC1, specific gravity 1.065-1.075） for 5 rain under 46℃. After the B. mandarina eggs were cold stored at 5℃ for 40 days, the highest hatchability was obtained by treatment with HC1 （specific gravity 1.092） for 6 minutes under 47.8℃. For the B. mandarina eggs that were stored at 25℃ for 28 d and then cold-stored at 5℃ for 0-100 days, the highest hatchability was obtained by treatment with HCI （specific gravity 1.092） for 6 rain at 47.8℃. The longer the cold storage period, the higher was the hatchability. Acid treatment on diapause eggs of B. mandarina for 6 rains at 47.8℃ with hydrochloric acid （specific gravity 1.092） before hatching in spring could obviously shorten the hatching stage and increase the hatchability.     

Screening of Trichoderma harzianum mutants tolerant to carbendazim and UV-light

After comparison of Trichoderma population density and test of colonization ability in rhizospheres were conducted. Auxotrophic mutants of T. harzianum tolerant to carbendazim and UV-light were obtained by UV-light mutagenesis and carbendazim stress on PDA medium and a basis medium with hot pepper root exudation by adding the fungicide. The results showed: all four different isolates of Trichoderma had certain colonization ability in rhizosphere with the characteristic of growing as…     

Study on Isolation, Passage, Cryopreservation and Histology of Mouse Fibroblasts

The embryonic ages were determined for the best preparation of mouse fibroblasts. Four methods were adapted to verify cryopreservation of mouse fibroblasts. The results showed that embryonic cryopreserving method was best one with 0.86 of thawing viability. The embryos from 13-14d pregnant mouse were superior to 11-12d and 15-16d in isolating, growing, laying and living. The first 6 generations were better than following ones in the same aspects above. Cell laying time became longer and vailable time became shorter after the sixth generation. With culture time increasing, fibroblast nuclear size became larger, fibrous filament appeared among fibroblasts, and macrocyst vesicle with floccule appeared in the cells. Cyst vesicle structure with pyknotic granule appeared in 24h cultured fibroblasts and macrocyst vesicle also appeared in passaging fibroblasts.     

Application of Mulching Materials of Rainfall Harvesting System for Improving Soil Water and Corn Growth in Northwest of China

The ridge and furrow rainfall harvesting（RFRH） system is used for dryland crop production in northwest of China.To determine the effects of RFRH using different mulching materials on corn growth and water use efficiency（WUE）,a field experiment was conducted during 2008-2010 at the Heyang Dryland Experimental Station,China.Four treatments were used in the study.Furrows received uncovered mulching in all RFRH treatments whereas ridges were mulched with plastic film（PF）,biodegradable film（BF） or liquid film（LF）.A conventional flat field without mulching was used as the control（CK）.The results indicated that the average soil water storage at depths of 0-200 cm were 8.2 and 7.3%,respectively higher with PF and BF than with CK.However,LF improved soil water storage during the early growth stage of the crop.Compared with CK,the corn yields with PF and BF were increased by 20.4 and 19.4%,respectively,and WUE with each treatment increased by 23.3 and 21.7%,respectively.There were no significant differences in corn yield or WUE with the PF and BF treatments.The net income was the highest with PF,followed by BF,and the 3-yr average net incomes with these treatments were increased by 2 559 and 2 430 CNY ha-1,respectively,compared with CK.BF and PF had similar effects in enhancing the soil water content,crop yield and net income.Therefore,it can be concluded that biodegradable film may be a sustainable ecological alternative to plastic film for use in the RFRH system in northwest of China.     

Screening of Trichoderna harzianum mutants tolerant to carbendazim and UV-light

After comparison of Trichoderma popultion density and test of colonization ability in rhizospheres were conducted.Auxotrophic mutants of T.harzianum tolernt to carbendazim and UV-light were obtained by UV-light mutagenesis and carbendazim stress on PDA medium and a basis medium with hot pepper root exudation by adding the fungicide.……