牡丹试管苗玻璃化的研究

以MS为基本培养基,研究品种、琼脂、6-BA、大量元素、蔗糖、光照等因素对牡丹试管苗玻璃化的影响。结果显示：不同品种的牡丹试管苗在组织培养的过程中,出现不同程度的玻璃化,增加培养基中琼脂浓度、降低细胞分裂素6-BA的浓度、降低大量元素的浓度和增加光照强度或采用自然光照均可以有效地降低试管苗玻璃化。     

The effects of culture medium rigidity on adventitious bud production and tissue vitrification in needle cultures of Sitka spruce [Picea sitchensis (Bong.) Carr.]

Adventitious bud formation on Sitka spruce [ Picca sitchensis (Bong.) Carr.] needle explants was strongly dependent upon the rigidity of the culture medium. In general, of organogenesis was greatest on weak gels and poorest on more rigid gels resulting from increased medium pH or agar strength. There was a significant interaction between agar strength and pH, with the optimum pH for organogenesis declining with increasing agar strength. Poor organogenesis at high agar concentrations was not due to toxic impurities since increased adventitious bud production could be stimulated by decreasing the medium pH whilst maintaining a high agar strength and an agar washing treatment had no significant effect. Although high levels of organogenesis could be sustained on weak gels the resultant adventitious shoots often showed severe vitrification. The frequency of shoots showing vitrification could be reduced by growing the tissues on harder media but this resulted in reduced shoot elongation. Vitrification of needle tissues did not stimulate the formation of adventitious buds in the absence of cytokinins.     

满天星组织培养中克服玻璃化现象的初探

通过在培养基中加入不同浓度的糖分、琼脂、细胞分裂素（ＢＡ和ＫＴ）、聚乙烯醇（ＰＶＡ）进行试验研究,发现满天星玻璃苗的数量随着糖分及琼脂浓度增加而减少,但细胞分裂素对玻璃化现象无影响,而聚乙烯醇对满天星玻璃化现象有明显的抑制作用,其最适浓度为２０ｇ／Ｌ。     

建兰根状茎增殖条件的研究

研究了建兰两个栽培品种的根状茎在固体及液体两种培养方式,以及培养基中有无激素和不同激素浓度配比对增殖及分化的影响。根状茎在液体培养基上的生长量都大于固体培养基上的生长量。根状茎在无激素的培养基上也能增殖。加入激素有促进作用。合适的激素浓度及配比因品种而异。     

Efficient In Vitro Micropropagation and Regeneration of Humulus Lupulus on Low Sugar, Starch-Gelrite Media

Several Czech and foreign hop mericlones were tested in vitro for efficiency of green callus formation and plant regeneration from internodal or nodal explants. Modified MS media gelled either with agar, starch or a mixture of potato starch and Gelrite, supplemented with different concentrations of either glucose or maltose, were investigated. Two mericlones of Czech hop (Osvald 72 no. 5216 and Sládek no. 6908) were studied in more details because of their different regeneration capacities. The HPLC analysis of medium sugar concentrations after the explant cultivation has revealed slow uptake of sugar from the medium. Presence of glucose at concentration of 45 g dm^–3 in agar medium resulted in a decreased number of nodes compared to the control with 30 g dm^–3 of glucose. The use of a mixture of potato starch plus Gelrite instead of routinely used agar and decreasing the medium glucose concentration to 15 g dm^–3 proved to be most efficient for multiplication rate. The use of this medium results in lower cost of micropropagation of healthy hop cultures without exhibition of vitrification.     

Factors affecting shoot proliferation and vitrification inDigitalis obscura cultures

Summary Variations of composition and consistency of the culture medium and time of exposure to growth regulators were assayed to optimize normal caulogenic response ofDigitalis obscura hypocotyls cultured in vitro. The effects of the culture conditions on physiologic changes related to vitrification of the regenerated plants were also investigated. Liquid medium increased the bud-forming capacity of the explants but induced buds failed to develop into shoots and showed symptoms of vitrification. On agar-solidified media, maximum multiplication rates were achieved with 0.7% agar. Increasing agar concentration reduced vitrification but lowered the propagation rate. Changes in the strength of the macronutrients of Murashige and Skoog did not significantly affect the bud-forming capacity of the explants. In contrast, a drastic inhibitory effect on both bud formation and shoot elongation was produced when NH₄NO₃ was omitted. Reduction of NH₄NO₃ to one-half or one-fourth of the level of the original formulation not only increased the bud-forming capacity ofD. obscura hypocotyls but also resulted in less vitrification. Modifications of time and method of exposure to growth regulators neither improved the multiplication rates nor overcame vitrification. Cardenolide content was lower in vitrified than in normal cultures and coincided with an overall reduction of photosynthetic pigments, lignin, and dry matter.     

Survival of vitrified sheep embryos in vitro and in vivo

The effects of the composition of vitrification media, the duration of exposure to the media and the stage of development were examined on the survival of vitrified Day-6 sheep embryos. Vitrification media that contained two cryoprotectants in equal molar concentrations were used. In Experiment 1, the effects of the types (glycerol + propylene glycol or glycerol + ethylene glycol) and concentrations (3.5 + 3.5 or 4.5 + 4.5 M) of cryoprotectants and the level of BSA supplementation (0.4 or 20%) were investigated in a 2 x 2 x 2 design. The embryos were exposed to vitrification media for 30 sec at 18 to 24 degrees C before vitrification. The in vitro survival rate was not affected by the level of BSA supplementation, but there was an interaction between the types and concentrations of cryoprotectants used (P<0.01). Embryos cryopreserved in mixtures of glycerol + propylene glycol survived better when the concentration of cryoprotectants was 3.5 M while the survival of embryos cryopreserved in mixtures of glycerol + ethylene glycol was higher at 4.5 M cryoprotectant concentration. In Experiments 2 and 3, the effect of the duration of exposure (15, 30, 60 or 120 sec) to vitrification media at 4 to 12 degrees C was investigated on the survival rate in vivo. Vitrification media contained 3.5 M glycerol + 3.5 M propylene glycol or 4.5 M glycerol + 4.5 M ethylene glycol in Experiments 2 and 3, respectively. The survival rate in vivo, increased when the duration of exposure to vitrification media was increased from 15 to 30 sec, but the viability declined when the duration of exposure was further increased to 60 (Experiment 3) or to 120 sec (Experiment 2). The effect of the stage of development was significant only in Experiment 1 (P = 0.032), but in all three experiments the rate of survival increased with advancing stages of development from late morulae to late blastocysts. The best result was achieved in Experiment 2, when embryos were exposed to a mixture of 3.5 M glycerol + 3.5 M propylene glycol for 30 or 60 sec. Under these conditions 52% (22 42 ) of rapidly cryopreserved sheep embryos developed into lambs. This result shows that a simple rapid procedure for the cryopreservation of sheep embryos can produce a survival rate comparable to that obtained using more complex traditional procedures.     

Relationship between calcium and agar on vitrification and shoot-tip necrosis of quince (Cydonia oblonga Mill.) shoots in vitro

Shoot-tip cultures of Quince C (Cydonia oblonga Mill.) initiated on Murashige & Skoog (MS) medium containing 5 M BA and 0.6% Phytagar showed both shoot-tip necrosis and severe vitrification. Culturing explants on medium containing 1.2% Phytagar and Ca levels of 3 mM (MS medium), 18 mM and 30 mM showed a decrease in growth with increasing medium Ca levels, being especially severe at 30 mM. The Ca content of the explants increased linearly with increasing medium Ca. Culturing explants on medium containing 3 mM, 9 mM, and 18 mM Ca at 0.6, 0.9, and 1.2% agar resulted in reduction in growth, shoot-tip necrosis, and vitrification when either factor was increased. The reduction in shoot-tip necrosis could be accounted for primarily by an increase in medium Ca levels but may also be affected by a change in explant growth. Increasing Ca concentration in the medium resulted in a linear increase in explant K, Ca, Mg, and B levels and a decrease in Mn and Na. Although increasing medium Ca or agar levels reduced vitrification, it is unclear whether they were the direct cause of the reduction in vitrification or whether this response was an effect of the reduction in culture fresh weight.Approved by publication by the Director, West Virginia Agriculture anf Forestry Experimental Station as Scientific Article No. 2199     

Effects of common components on hardness of culture media prepared with gelrite™

Summary Tissue cultures on properly solidified Gelrite media generally showed superior shoot proliferation and rooting, as well as shoot and root vigor and callus development to those on TC agar. Vitrification, or hyperhydricity, was observed in both Gelrite and agar media and minimized by increasing the gel concentrations. Rigidity of Gelrite media depended on combined levels of MS macrosalts, basal nutrient formulations, sucrose concentration, pH, and Gelrite concentration. Most MS macrosalts increased hardness of Gelrite gels; NH₄NO₃ had a decreasing effect. Rigidity of TC agar gels increased with reductions of MS macrosalts. A slightly softer Gelrite medium resulted when sucrose was excluded. Both Gelrite and agar media were softer at lower pHs and harder at higher pHs. Activated charcoal and mannitol increased gel hardness, and more noticeably of agar gels. NaCl addenda reduced rigidity, with their effects being more pronounced on Gelrite than on agar gels. Gelrite is a trademark of Kelco Division of Merck &amp; Co. (San Diego, CA), manufacturer of the gellan gum. Phytagel is a trademark of Sigma Chemical Co. (St. Louis, MO) for repackaged Gelrite. TC agar used for comparisons in this investigation is plant tissue culture tested agar obtained from JRH Biosciences (Lenexa, KS).     

Survivals of mouse oocytes approach 100% after vitrification in 3-fold diluted media and ultra-rapid warming by an IR laser pulse

Vitrification is the most sought after route to the cryopreservation of animal embryos and oocytes and other cells of medical, genetic, and agricultural importance. Current thinking is that successful vitrification requires that cells be suspended in and permeated by high concentrations of protective solutes and that they be cooled at very high rates to below −100 °C. We report here that neither of these beliefs holds for mouse oocytes. Rather, we find that if mouse oocytes are suspended in media that produce considerable osmotic dehydration before vitrification and are subsequently warmed at ultra high rates (10,000,000 °C/min) achieved by a laser pulse, nearly 100% will survive even when cooled rather slowly and when the concentration of solutes in the medium is only 1/3rd of standard.     

Simple fluorescence method for rapid estimation of aflatoxin levels in a solid culture medium

Aflatoxin concentrations in agar media were estimated with a direct technique that quantifies the fluorescence of agar containing aflatoxins. Tubes containing 5 ml of an agar medium inoculated with spores of aflatoxin-producing Aspergillus isolates were incubated for 3 days at 30 degrees C and set in a carriage specifically designed to carry culture tubes in a scanning densitometer. Fluorescence (450 nm and above) was elicited in the agar by UV light (365 nm) and photometrically measured. Agar fluorescence directly correlated (r2 = 0.89 +/- 0.05, P less than 0.001) with the concentration of aflatoxin within the range 0 to 18.7 micrograms/g. The lowest aflatoxin concentration detected was 50 ng/g. The technique successfully differentiated the aflatoxigenic potentials of Aspergillus isolates.     

Simple fluorescence method for rapid estimation of aflatoxin levels in a solid culture medium.

Aflatoxin concentrations in agar media were estimated with a direct technique that quantifies the fluorescence of agar containing aflatoxins. Tubes containing 5 ml of an agar medium inoculated with spores of aflatoxin-producing Aspergillus isolates were incubated for 3 days at 30 degrees C and set in a carriage specifically designed to carry culture tubes in a scanning densitometer. Fluorescence (450 nm and above) was elicited in the agar by UV light (365 nm) and photometrically measured. Agar fluorescence directly correlated (r2 = 0.89 +/- 0.05, P less than 0.001) with the concentration of aflatoxin within the range 0 to 18.7 micrograms/g. The lowest aflatoxin concentration detected was 50 ng/g. The technique successfully differentiated the aflatoxigenic potentials of Aspergillus isolates.     

万寿菊再生体系的建立及优化

以40个不同基因型万寿菊(Tagetes erecta)叶片为外植体, 在相同条件下诱导不定芽分化, 获得最佳再生基因型; 然后分析不同激素组合、外植体切口方式、固化剂及蔗糖对万寿菊再生和玻璃化影响; 最后对不同类型的伸长培养基进行探索。结果表明, 最佳再生基因型为里程碑·黄色; 最佳再生培养基为MS+0.2 mg·L^‒1 TDZ+0.5 mg·L^‒1 IBA+8 g·L^‒1琼脂+40 g·L^‒1蔗糖, 再生率达70%, 玻璃化率降低至16%; 最适再生的小叶部位为全小叶; 最适伸长培养基为MS+8 g·L^‒1琼脂+30 g·L^‒1蔗糖, 伸长率达91.3%。该研究建立了高效稳定的万寿菊再生体系, 解决了万寿菊再生过程中严重的玻璃化问题, 可为万寿菊的遗传改良和基因功能研究奠定基础。     

The Establishment and Optimization of a Regeneration System for Marigold (Tagetes erecta)

以40个不同基因型万寿菊(Tagetes erecta)叶片为外植体, 在相同条件下诱导不定芽分化, 获得最佳再生基因型; 然后分析不同激素组合、外植体切口方式、固化剂及蔗糖对万寿菊再生和玻璃化影响; 最后对不同类型的伸长培养基进行探索。结果表明, 最佳再生基因型为里程碑·黄色; 最佳再生培养基为MS+0.2 mg·L^?1 TDZ+0.5 mg·L^?1 IBA+8 g·L^?1琼脂+40 g·L^?1蔗糖, 再生率达70%, 玻璃化率降低至16%; 最适再生的小叶部位为全小叶; 最适伸长培养基为MS+8 g·L^?1琼脂+30 g·L^?1蔗糖, 伸长率达91.3%。该研究建立了高效稳定的万寿菊再生体系, 解决了万寿菊再生过程中严重的玻璃化问题, 可为万寿菊的遗传改良和基因功能研究奠定基础。     

Calcium and magnesium in Mueller-Hinton agar and their influence on disk diffusion susceptibility results

Concentrations of calcium and magnesium were determined for 39 lots of media, including broth and agar media used for susceptibility tests and plain agar. In addition, the effect that media with and without physiological levels of these divalent cations would have on the disk diffusion susceptibility of 21 strains ofPseudomonas aeruginosa to four antimicrobics was also ascertained. Mueller-Hinton agar showed a wide variation in calcium and magnesium content. Mueller-Hinton broth contained lower concentrations of calcium and magnesium, and there was little lot-to-lot variation. Lots of Mueller-Hinton agar with higher concentrations of calcium and magnesium yielded smaller zone diamaters than those with lower concentrations. Even at equal cation concentration, zones of inhibition varied from lot to lot. Since the addition of calcium and magnesium to Mueller-Hinton agar to obtain a predetermined level did not result in equivalent zone diameters, performance testing of susceptibility media is recommended.     

The diffusion coefficient of caffeine through agar gels containing a hyaluronic acid–protein complex. A model system for the study of the permeability of connective tissues

A hyaluronic acid-protein complex was embedded into agar gel. This gel complex resembles in some respects the physiological situation in connective tissue, but still permits precise physicochemical measurements to be made. The diffusion coefficient of caffeine into and from such gels has been measured as a function of both agar and hyaluronate concentration. The value for the diffusion coefficient of caffeine was also measured by using a Gouy type diffusiometer. From both types of measurement the value for D (Fick) for caffeine when extrapolated to zero caffeine and agar concentrations agreed at (6.79+/-0.01)x10(-6)cm(2).s(-1) at 25 degrees C. Although agar concentration had only a small effect on caffeine diffusion, hyaluronic acid caused a large decrease in caffeine diffusion co-efficient. The presence of the hyaluronic acid-protein complex within the gel tended to oppose gel syneresis, a concentration of 1.7mg/ml abolishing the effect and higher concentrations reversing it. The possible physiological implications of these results are discussed.     

Sugars exert a major influence on the vitrification properties of ethylene glycol-based solutions and have low toxicity to embryos and oocytes

A systematic approach was taken to assess the vitrification properties of ethylene glycol-based solutions supplemented with carbohydrates. Solutions were prepared by weight (gravimetrically) using ethylene glycol as the cryoprotectant, 0.9% NaCl in water, and six different sugars: d-glucose, d(-)-fructose, d-sorbitol, sucrose, d(+)-trehalose, and raffinose. Sugars were added on a molal basis (0. 1, 0.5, and 1 m). Characteristics of the solutions were measured during warming by differential scanning calorimetry using a cooling rate of 100 degrees C/min and a warming rate of 10 degrees C/min. In the absence of carbohydrates a 59 wt% EG-saline solution formed a stable glass. When EG was replaced by an equimolal concentration of glucose, fructose, or sorbitol (monosaccharides) at 0.1, 0.5, or 1.0 m there was no change in the total solute concentration at which vitrification occurred, but the glass transition (Tg) occurred at a higher temperature than in EG-saline alone. When EG was replaced by an equimolal concentration of sucrose or trehalose (disaccharides) both the Tg and the lowest total solute concentration required for vitrification became progressively higher as the molecular weight, or the ratio of sugar to EG in the solutions, increased. At the highest tested disaccharide concentration (1 m) vitrification was achieved at a total solute concentration of 65 wt% (sucrose) and 67 wt% (trehalose). The polysaccharide raffinose significantly modified the vitrification properties of ethylene glycol solutions. When 0.5 or 0.1 m raffinose replaced EG on an equimolal basis the glass transition point was raised more than with either the monosaccharides or the disaccharides. Raffinose allowed vitrification at a total solute concentration of 67 wt% (0.5 m) and 63 wt% (0.1 m). The maturation of immature mouse oocytes, and the development of embryos in media containing 5-7 mM of any sugar was comparable to controls, indicating that they are not toxic. Exposure of freshly collected GV or MII oocytes to sugar concentrations between 0.5 and 1.0 M, for up to 10 min had no significant effect on the proportion which subsequently formed two cells. We conclude that added sugars do contribute to a solutions overall vitrification properties, and their properties should be taken into consideration when vitrification solutions are being designed or modified.     

Effect of gelling agent on growth and development ofCeratozamia hildae somatic embryos

Individual somatic proembryos ofCeratozamia hildae were exposed to media that differed only in gelling agent utilized. Five different gelling agents were compared in the first experiment: Bacto agar, Agargel, Gel-Gro, Phytagar, and TC agar. In addition, the effect of agar was examined at two levels. Growth, proliferation, and development were assessed. The lower level of agar did not support good somatic proembryo growth. Agargel and the high agar concentration produced cultures with good proliferation. Proembryos exposed to Phytagar, Gel-Gro, and TC agar had the highest proliferation rates. Overall, Gel-Gro was considered the best gelling agent tested. The three concentrations of Gel-Gro used in the second experiment were 2, 4, and 6 g·1⁻, with the lowest concentration representing the control, the recommended concentration. As gelling agent concentration increased, so did mortality; however, the highest Gel-Gro concentration also produced the highest numbers of good-quality, mature somatic embryos. Proliferation rate was greatest at the lowest concentration. These results suggest thatCeratozamia cultures should be exposed to different gelling agents or concentrations of gelling agents at different developmental stages in order to produce the greatest number and highest quality of somatic embryos.     

Relationship between sampling duration and concentration of culturable airborne mould and bacteria on selected culture media

AIMS: This study was designed to evaluate potential effects of sampling duration on observed concentrations of airborne culturable mould and bacteria on selected media. METHODS AND RESULTS: Airborne culturable mould and bacteria from lightly to moderately contaminated environments were collected on selected culture media using two co-located, concurrently operated, Andersen N-6 samplers for five sampling durations in the range of 1-10 min. Differences in mean concentrations, as well as linear relationships between sampling duration and both concentration and variability, were evaluated using nonparametric procedures. For the five sampling durations, there were no significant differences in mean concentrations of mould; for bacteria, there were significant differences, with a trend of decreasing concentrations as sampling duration increased. Data variability decreased with increasing sampling duration for both mould and bacteria. CONCLUSIONS: Airborne culturable mould concentrations were similar for sampling durations in the range of 1-10 min. Airborne bacteria concentrations tended to trend downwards with sampling durations exceeding 3 min. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that sampling durations of 1-10 min are appropriate for collection of airborne culturable mould on malt extract agar (MEA) and dichloran glycerol agar (DG-18); based on the apparent trend of decreasing bacterial sample concentrations associated with increasing sampling duration, sampling durations of 相似文献   

Growth of Mouse L Cells in Shaken Culture and Mengovirus Plaque Formation on L Cells Suspended in Agar

Procedures are described that require a minimum of equipment and maintenance for growing mouse L cells suspended in liquid medium and for plaquing mengovirus on L cells suspended in agar. Viability of L cells during storage for 1 to 2 hr at relatively high concentrations was better in media at 30 C than at 0 C, as measured by viable counts after growth for 24 hr at 35 C. The number and size of plaques increased with increasing concentration of NaHCO(3) in the agar layers, but the relative difference in plaque size was maintained. Large- and small-plaque-size variants had similar virulence as determined by the rates of viability loss of L cells in liquid suspension cultures.