用FDA-PI双色荧光法鉴定大麦原生质体活性

为鉴定大麦糊粉层原生质体的活性,应用了荧光染料荧光素双醋酸酯(FDA)和普罗皮啶碘(PI)。在FITC滤片组合条件下用荧光显微镜观察,有生活力的原生质体发出绿色荧光,而没有生活力的原生质体细胞核呈桔黄色。FDA-PI双色荧光法具有色彩对比鲜明的效果,从而为细胞计数和活力测定提供了方便。本文还介绍了一种在同一张底片上进行两次曝光的彩色摄影记录新技术。     

应用荧光增白剂VBL研究原生质体细胞壁的再生

对新分离和培养的烟草(Nicotiana tabacum)叶肉原生质体用荧光增自剂VBL染色,于波长3600～4400光源的荧光显微镜下观察表明,新分离的烟草叶肉原生质体没有发绿色荧光的细胞壁存在,只见发微弱深红色荧光的叶绿体,而培养4～6天以上的原生质体再生壁发绿色荧光,证明含纤维素成分。这种方法亦可用于观察原生质体的出芽分裂和细胞分裂。     

用荧光染色技术标记生活花粉管的研究

在金鱼草和(或)烟草上试验了三种荧光染料对花粉管进行荧光活体染色与标记的效果。花粉管在异硫氰酸荧光素(FITC,12.5μg/ml)中染色5—6小时,原生质呈绿黄色荧光;换入无染料的培养基后可继续生长并保持荧光标记,但后期生长受抑。罗丹明B(RB,10μg/nl)染色的效果与上相近,花粉管呈红色荧光;换入无染料培养基后生长正常,唯后期荧光减弱。荧光素二醋酸酯(FDA,10μg/ml)染花粉粒40分钟,换入无染料培养基后正常萌发与生长,花粉管原生质呈明亮的绿色荧光。FDA方法具有染色时间短,荧光明亮,兼有活染与生活力鉴定双重功效等优点。     

基于等位基因InDel快速检测黑木耳原生质体单核体

以黑木耳 Auricularia auricula-judae栽培菌株Au916三磷酸甘油醛脱氢酶（GAPDH）两个等位基因中的InDel为基础,以原生质体再生菌株为研究材料,建立了基于InDel的黑木耳原生质体单核体检测技术,并采用荧光显微镜检和配对试验对此方法进行了验证。应用InDel能清晰地区分黑木耳双核体和两种原生质体单核体,且不同的单核体之间可以进行交配,其杂交双核体能正常出耳,结果表明基于等位基因InDel标记可以快速准确地检测黑木耳原生质体单核体。在大规模测序获得真菌基因组信息的基础上,InDel标记在原生质体单核体鉴定中的应用将更加普遍。     

烟草叶肉原生质体再生壁形成的培养条件的研究

用EA_3-867纤维素酶分离的烟草(Nicotiana tabacum)叶肉原生质体,在不同培养条件下进行液体浅层培养,用荧光增白剂VBL染色荧光法和低渗冲击法研究了再生壁形成的某些培养条件。结果表明,600～1000米烛光的弱光、10~5个原生质体/毫升的密度以及蔗糖或甘露醇作碳源,有利于壁的再生。     

丹参悬浮培养细胞原生质体的制备和活力检测

对丹参悬浮培养细胞原生质体制备条件进行了研究,并利用FDA染色和钙离子荧光探针Fluo-3/AM装载对制备得到的原生质体的活力和功能进行了检测。丹参悬浮培养细胞原生质体的制备条件为:悬浮培养细胞酶解的适宜酶液组合为纤维素酶1.5%、果胶酶0.3%和离析酶0.5%;适宜的甘露醇浓度为0.4 mol/L;酶解时间为12 h;在600 r/min转速下离心5 min收集,纯化得到原生质体,其产量为1.1×106/g FW,FDA检测显示其活力为95%以上,荧光探针Fluo-3/AM可成功装载到原生质体中。     

一种光学显微镜下观察原生质体的染色方法

Ｂｒｅｖｉｂａｃｔｅｒｉｕｍ ｌａｃｔｏｆｅｒｍｅｎｔｕｍ菌液经溶菌酶处理后分别与４种微生物染色液混合,在光学显微镜下比较观察原生质体形态的效果。结果表明;染色样品中的原生质体比未经染色的形态清晰易观察,而且显微照相效果好;其中使用草酸铵结晶紫和复红染色液的效果更佳。该方法程序简单、操作方便、效果明显,还适用于悬滴法观察菌体形态和细菌运动方式。     

捕食性真菌Duddingtonia flagrans原生质体获得与再生的荧光标记评价

为了更准确快捷地评价利用酶解法酶解捕食性真菌Duddingtonia flagrans菌丝产生的原生质体数量及菌丝细胞壁降解情况,采用活体荧光染料羧基荧光素乙酰乙酸(carboxyfluorescein diacetate succinimidyl ester,CFSE),对该捕食性真菌菌丝酶解后产生的原生质体进行荧光标记,分别考察了标记浓度、标记时间、孵育温度对原生质体标记效果的影响,并观察CFSE标记后的原生质体再生情况。结果表明CFSE终浓度为10μmol/L,标记时间为15min,孵育温度为36℃,是CFSE标记捕食性真菌原生质体的理想条件,该试验同时表明CFSE不影响原生质体的再生率。CFSE作为一种活细胞示踪荧光探针,可以快速高效地标记捕食性真菌原生质体,该方法为捕食性真菌原生质体制备质量的快速评价提供了新的思路。     

用罗丹明B实现微波辐照下细胞水平温度的实时测量

为了探讨用温度敏感性荧光探针罗丹明B(rhodamine B,Rho-B)测量微波辐照下细胞水平温度的方法,利用激光共聚焦显微镜和光纤测温仪测得的数据拟合出罗丹明B荧光探针荧光强度与温度的关系式,并利用该荧光探针对微波辐照过程中细胞水平的温度进行实时测量。结果显示,温度与荧光探针的荧光强度呈现较好的线性关系。利用拟合的温度-相对荧光强度关系式可得到升温过程(25~40℃)中细胞水平的准确温度,这为生物电磁实验中细胞水平的实时温度监测提供了一种较为便捷可行的方法。     

荧光标记香菇原生质体融合菌株的鉴定

以异硫氰基荧光素（FITC）标记和香菇单核L4菌株的原生质体和未经标记的香菇单核B14菌株的原生质体为亲本,在聚乙二醇（PEG）的促融下进行融合,选取一个带有荧光,另一个不带荧光的原生质体粘合对进行再生培养,通过对利用“锁状联合”筛选得到的菌株进行鉴定后表明：融合菌株为双核菌丝,与双亲产生明显拮抗,其酯酶同工酶及可溶性蛋白质凝胶电泳图谱分析表明融合菌株均与双亲有区别,经琼脂平板快速出菇及出菇实验证明融合菌株出菇早,产量有所提高。     

Fluorescent Vital Stains for Complementary Labelling of Protoplasts from Trichoderma Spp

In this study several fluorescent vital stains were evaluated for their ability to provide complementary vital staining of protoplasts of Trichoderma spp. for selection of heterokaryons following protoplast fusion. Tetramethyl rhodamine isothiocyanate and fluorescein isothiocyanate were rejected because they stained only a small proportion of protoplasts. Fluorescein diacetate stained all protoplasts, but the chromophore leaked rapidly from stained cells. A mixture of FluoroBora T and acriflavine stained all cells, but intensity was low and fading upon illumination was rapid. Nile red stained lipid bodies in all cells, but the stain was lost upon protoplast fusion in polyethylene glycol. Rhodamine 6G, on the other hand, stained all cells, fluoresced green, and was stable through fusion and upon illumination. Hydroethidine also stained all protoplasts, and staining was relatively stable through fusion and upon illumination. Hydroethidine fluoresced red and stained nuclei more prominently than the cytoplasm. Rhodamine 6G and hydroethidine were tested on a number of strains to determine whether they were toxic to protoplasts. No toxicity to any strain was noted with rhodamine 6G. Hydroethidine, however, was toxic at the higher concentrations tested, especially when stained protoplasts were exposed to light. When protoplasts were stained with the minimum concentration giving ready visualization and were incubated in darkness, hydroethidine also was nontoxic. Hydroethidine and rhodamine 6G are useful complementary vital stains of Trichoderma protoplasts for visualization of frequency and type (dicell, multicell) of fusion.     

Protoplast Cultures from Cell Suspensions of Daucus carota

Protoplasts, enzymatically isolated from cell suspension cultures of Daucus carota, have been grown in small Petri-dishes. After enzyme treatment and washing the protoplasts were plated in agar media. Growth and divisions were viewed through the bottom of the Petri-dishes with a light microscope. Different osmotic stabilizers were tested with respect to their ability to promote wall formation and growth of the protoplasts. Combinations of sucrose, sorbitol and “Modopeg” gave the best results. Electron micrographs of cultured protoplasts revealed normal as well as abnormal nuclear conditions.     

Analysis of plants regenerated from protoplast fusions between Brassica napus and Eruca sativa

Summary Protoplasts from etiolated hypocotyls of Brassica napus stained with carboxyfluorescein were fused with mesophyll protoplasts from Eruca sativa. Hybrid cells could be identified under the light microscope by (1) fully developed chloroplasts derived from E. sativa and (2) the cytoplasmic strands of the B. napus hypocotyl protoplasts, or (3) by the presence of both red and green fluorescence when investigated under UV light. The heterokaryons were selected using either a micro-manipulator or a flow sorter. On average, 5.4% of the calli obtained after selection differentiated into shoots. Regenerated shoots were subjected to isozyme analysis for verification of their hybrid character. Of the 23 hybrids successfully transferred to the greenhouse, 11 were asymmetric according to isozyme analysis. The nuclear DNA content of the hybrids was determined by flow cytometry, which gives an estimate of chromosome number. Most of the hybrids had a DNA content, and thus a chromosome number, that deviated from the expected sum of the parents. Almost all of the hybrids had some degree of fertility and produced seeds. Seed set, expressed as seeds per pollinated flower, was on average 7% of that of B. napus in the case of self-pollination and 26% of that of B. napus when backcrossed to B. napus. The chloroplast genotype was investigated in 13 hybrids. Of these, 11 had chloroplasts derived from B. napus, while only 2 had chloroplasts of E. sativa origin.     

Fluorescent vital stains for complementary labelling of protoplasts from Trichoderma spp

In this study several fluorescent vital stains were evaluated for their ability to provide complementary vital staining of protoplasts of Trichoderma spp. for selection of heterokaryons following protoplast fusion. Tetramethyl rhodamine isothiocyanate and fluorescein isothiocyanate were rejected because they stained only a small proportion of protoplasts. Fluorescein diacetate stained all protoplasts, but the chromophore leaked rapidly from stained cells. A mixture of FluoroBora T and acriflavine stained all cells, but intensity was low and fading upon illumination was rapid. Nile red stained lipid bodies in all cells, but the stain was lost upon protoplast fusion in polyethylene glycol. Rhodamine 6G, on the other hand, stained all cells, fluoresced green, and was stable through fusion and upon illumination. Hydroethidine also stained all protoplasts, and staining was relatively stable through fusion and upon illumination. Hydroethidine fluoresced red and stained nuclei more prominently than the cytoplasm. Rhodamine 6G and hydroethidine were tested on a number of strains to determine whether they were toxic to protoplasts. No toxicity to any strain was noted with rhodamine 6G. Hydroethidine, however, was toxic at the higher concentrations tested, especially when stained protoplasts were exposed to light. When protoplasts were stained with the minimum concentration giving ready visualization and were incubated in darkness, hydroethidine also was nontoxic. Hydroethidine and rhodamine 6G are useful complementary vital stains of Trichoderma protoplasts for visualization of frequency and type (dicell, multicell) of fusion.     

Growth responses of marigold and salvia bedding plants as affected by monochromic or mixture radiation provided by a Light-Emitting Diode (LED)

The effects of light generated by monochromic blue, red or mixed radiation from a fluorescent lamp (FL) with light emitting diodes (LEDs) (blue, red, or far-red) on growth and morphogenesis of marigold and salvia seedlings were investigated and the responses compared with those of plantlets grown under a broad spectrum conventional fluorescent lamp (a 16 h photoperiod per day). Dry weight of marigold seedlings was significantly increased in monochromic red light (R), fluorescent light plus red LED (FLR) or fluorescent light (FL) but reduced when monochromic blue light (B) was used, whereas in salvia dry weight was significantly greater under fluorescent light plus blue LED (FLB), fluorescent light plus red LED (FLR) and fluorescent light plus far-red LED (FLFr) as compared to other treatments. Stem length in marigold was greatest in monochromic blue light, being three times greater than in FLR or FL treatments. In salvia, FLFr increased stem length but this was significantly decreased by R as compared to other treatments. The number of visible flower buds in marigold was much higher in FLR as well as in the control (FL), and it was about five times greater than in B or R. However, the number of open flowers in salvia varied slightly in all the treatments. Different light qualities also influenced the duration of the blooming period in both the species. No flower buds were formed when monochromic B or R was used in salvia and FLFr inhibited flower bud formation in marigold. In comparison with monochromic blue or red light, the number of stomata was greater in mixed radiation of FL with LEDs in both the plants. Our study demonstrates the effectiveness of a LED system for plantlet growth and morphogenesis in space-based plant research chambers.     

Light-dependent osmoregulation in pea stem protoplasts. photoreceptors, tissue specificity, ion relationships, and physiological implications

Light-induced changes in the volume of protoplasts bathed in a medium of constant osmolarity are useful indications of light-dependent cellular osmoregulation. With this in mind, we investigated the effect of light on the volume of protoplasts isolated from the elongating stems of pea (Pisum sativum) seedlings raised under red light. The protoplasts were isolated separately from epidermal peels and the remaining peeled stems. Under continuous red light, the protoplasts of peeled stems swelled steadily, but those of epidermal peels maintained a constant volume. Experiments employing far-red light and phytochrome-deficient mutants revealed that the observed swelling is a light-induced response mediated mainly by phytochromes A and B with a little greater contribution by phytochrome A. Protoplasts of epidermal peels and peeled stems shrank transiently in response to a pulse of blue light. The blue light responsiveness in this shrinking response, which itself is probably mediated by cryptochrome, is under the strict control of phytochromes A and B with equal contributions by these phytochromes. We suggest that the swelling response participates in the maintenance of high tissue tension of elongating stems and that the shrinking response is involved in stem growth inhibition. Other findings include the following: The swelling is caused by uptake of K+ and Cl-. The presence of Ca2+ in the bathing medium is required for phytochrome signaling in the swelling response, but not in the response establishing blue light responsiveness. Phytochrome A mediates the two responses in a totally red/far-red light reversible manner, as does phytochrome B.     

Effects of Light on the Membrane Potential of Protoplasts from Samanea saman Pulvini : Involvement of K Channels and the H -ATPase

Rhythmic light-sensitive movements of the leaflets of Samanea saman depend upon ion fluxes across the plasma membrane of extensor and flexor cells in opposing regions of the leaf-movement organ (pulvinus). We have isolated protoplasts from the extensor and flexor regions of S. saman pulvini and have examined the effects of brief 30-second exposures to white, blue, or red light on the relative membrane potential using the fluorescent dye, 3,3′-dipropylthiadicarbocyanine iodide. White and blue light induced transient membrane hyperpolarization of both extensor and flexor protoplasts; red light had no effect. Following white or blue light-induced hyperpolarization, the addition of 200 millimolar K⁺ resulted in a rapid depolarization of extensor, but not of flexor protoplasts. In contrast, addition of K⁺ following red light or in darkness resulted in a rapid depolarization of flexor, but not of extensor protoplasts. In both flexor and extensor protoplasts, depolarization was completely inhibited by tetraethylammonium, implicating channel-mediated movement of K⁺ ions. These results suggest that K⁺ channels are closed in extensor plasma membranes and open in flexor plasma membranes in darkness and that white and blue light, but not red light, close the channels in flexor plasma membranes and open them in extensor plasma membranes. Vanadate treatment inhibited hyperpolarization in response to blue or white light, but did not affect K⁺ -induced depolarization. This suggests that white or blue light-induced hyperpolarization results from activation of the H⁺ -ATPase, but this hyperpolarization is not the sole factor controlling the opening of K⁺ channels.     

Growth of Tsuru-rindo (Tripterospermum japonicum) culturedin vitro under various sources of light-emitting diode (LED) irradiation

We studied the effects of light generated by LEDs on the growth of Tsururindo (Tripterospermum japonicum) shoots. Apical shoots (2–3 cm long) were cultured on MS basal media supplemented with 3% sucrose, and were maintained for four weeks under five different light qualities: F (fluorescent lamp), red LED (R), 70% red + 30% blue LED (R7B3), 50% red + 50% blue (R5B5), or blue LED (B). Rooting was promoted by red light (100%) but was inhibited by blue light. Plant growth, as defined by root number, fresh weight, and chlorophyll content, was generally healthier for cultures irradiated with mixed LEDs, and was the best under R7B3. Ventilation resulted in more rapid apical shoot growth and rooting compared with control plants, when both were treated with the R7B3 system. We demonstrated here that plant growth can be controlled by using LEDs to adjust for the most effective irradiation conditions, compared with the performance observed when conventional fluorescent lamps are utilized.     

Rhodamine B as a systemic hair marker for assessment of bait acceptance by stoats (Mustela erminea)

Abstract

Rhodamine B (RB) is a dye that becomes incorporated into the structure of growing hair of animals that ingest it, appearing as an orange‐red fluorescent band detectable under a fluorescent‐light microscope. This marker was evaluated as a means of assessing bait acceptance by stoats (Mustela erminea L.). Eleven wild‐caught captive stoats were each fed a broken hen egg injected with 25 mg of RB on two occasions, 5 weeks apart. This was equivalent to 62–108 mg kg^–1, depending upon stoat weight, on each occasion. At least three facial whiskers were collected from each dosed stoat on each of two sampling dates (giving a total of at least six whiskers from each stoat). The sampling dates varied from 1 to 17 weeks after first dosing. Whiskers were also collected from one of the dosed stoats that died of other causes 19 weeks after first dosing, and from four stoats not dosed with RB. All 11 of the stoats fed RB had at least one fluorescent band in at least one of the sampled whiskers. None of the four stoats not fed RB had fluorescent bands in their whiskers. The marking persisted in all dosed stoats for at least 6 weeks, and in one dosed stoat for at least 19 weeks after dosing. However, only 56% of the 91 whiskers inspected from the dosed stoats had fluorescent bands, and only 9% of the whiskers had two fluorescent bands, representing the two doses of RB. The distance between the two fluorescent bands indicated a mean whisker growth of 0.6 mm day^‐1. The distance from the base of the whiskers to the base of the fluorescent bands was broadly related to the time after ingestion of bait containing RB. However, the variation was too great for distance along the whisker to be reliably used as a quantitative measure of time after bait ingestion. The technique can be used to assess bait acceptance in the field provided all stoats are sampled within c. 4–6 weeks of baiting, and at least 6–9 whiskers are sampled from each stoat.     

用中性红标记酵母原生质体初探

用２％蜗牛酶处理酵母细胞６０分钟,啤酒酵母Ｙ２９的原生质体形成率为９０％,再生率为９．５％;糖化酵母ＩＢ的原生质体形成率为８６％,再生率为１２％。用５００ｐｐｍ中性红染液对Ｙ２９菌株的整细胞和原生质体染色１５分钟,细胞的着色率为８４％,存活率为１２％,而原生质体的着色率为７５％,再生率为６．４％,经染色后的原生质体体积缩小,在交变电场中排队所需的场强电降低。