葡萄去病毒和无毒苗试管快繁技术研究

1.葡萄病毒的脱除:(1)热处理脱毒:在38℃条件下,保持相对湿度60—70%,处理29个盆栽葡萄品种2-3个月,使100%的植株脱除了四种主要病毒病。(2)试管内热处理及茎尖剥离培养脱毒:将葡萄试管苗进行热处理并配合茎尖剥离培养或单用茎尖剥离培养,前者成活率30.5%,脱毒率100%;后者成活率73%,脱毒率94.7%。     

泡桐微茎尖培养脱除泡桐丛枝病原（MLO）的研究

以感染丛枝病原（ＭＬＯ）的泡桐组培苗为试材,大量切取长度在０．３ｍｍ～０．５ｍｍ范围内的微茎尖,在ＭＳ和附加０．１ｍｇ／ＬＩＢＡ和１～７ｍｇ／Ｌ６－ＢＡ的培养基上进行脱毒试验。根据外植体培养过程的病状有无．病源ＭＬＯ的ＤＡＰＩ荧光显微镜和电子显微镜检查结果,证明微茎炎培养能够获得脱除泡桐丛枝病原ＭＬＯ的试管苗,其脱毒率在１４．２９～２８．５７％之间。脱毒的两个无性系试管苗经连续继代培养一年以上,其生长性状正常且稳定。试验还显示了微茎炎培养脱毒率受采用的茎尖大小、切割技术、培养基中的激素比例及无性系的不同等因素的影响较大。     

小滴玻璃化法脱除蝴蝶兰种苗建兰花叶病毒的研究

以感染建兰花叶病毒(Cymbidium mosaic virus,CymMV)的蝴蝶兰(Phalaenopsis aphrodite)品种‘满天红’为试材,通过筛选蔗糖预培养浓度、预培养时间、PVS2(Plant vitrification solution 2,PVS2)处理时间三个关键因素,建立蝴蝶兰茎尖小滴玻璃化超低温脱毒体系,将再生的茎尖诱导类原球茎,再分化成苗,经RT-PCR检测CymMV的脱除情况,阴性结果的再生植株进行增殖和诱导生根。结果显示:最佳预培养为:BM+0.6 mol·L-1蔗糖处理1~2 d,超低温茎尖的成活率为70%~76.7%,再生率为53.3%~56.7%;PVS2最佳处理时间为60~90 min,超低温茎尖的成活率为73.3%~76.7%,再生率为50.0%~56.7%。再生植株经RT-PCR检测,CymMV的脱除率为50%。该研究为兰科植物脱除CymMV提供了理论和技术基础。     

携病毒马铃薯茎尖分化成苗与脱毒率检测

以携带病毒的‘夏波蒂’马铃薯无菌苗为材料,38℃/4h热处理4周,剥离带1个叶原基的茎尖,接种至不同浓度激素组合的24种MS固体培养基上,22℃/16h培养30d后统计茎尖的愈伤组织诱导率和分化成苗率;RT-PCR检测茎尖再生苗3种病毒(PVX、PVY、PLRV)和纺锤块茎类病毒(PSTVd)的脱毒率。结果表明:茎尖分化成苗最适培养基为MS+1.0mg/L ZT+0.2mg/L NAA+2.0mg/L GA3,愈伤组织诱导率为76.25%,分化成苗率为26.25%;再生苗3种病毒PVX、PVY和PLRV的脱毒率分别为69.4%、91.7%和100%,纺锤块茎类病毒PSTVd脱毒率仅为8.3%,二次茎尖剥离后脱毒率增加到20.8%。     

柑桔根繁能脱除裂皮病类病毒吗?

柑桔裂皮病类病毒(Citrus Exocortis Vitold,CEV)的传播和流行,对柑桔的生产带来了严重的损失。已证明特CEV的亲本可通过嫁接、插枝这两种营养繁殖的方式将CEV传播给子代。所以,CEV脱毒方法就成为生产上急待解决的问题。据报道,采用热处理、茎尖微芽嫁接和选育珠心苗等技术可获得无毒植株。嫁接是一种常用的育苗方法,然     

病毒唑对大花蕙兰CyMV及ORSV脱毒效果初探

在大花蕙兰茎尖培养中结合病毒唑处理,探讨对建兰花叶病毒(CyMV)和齿兰环斑病毒(ORSV)的脱除效果。结果表明,病毒唑添加到培养基中,处理茎尖2周,脱毒效果低于经病毒唑处理3个月后的幼苗,经病毒唑处理幼苗再结合茎尖培养脱毒效果最好。病毒唑处理对大花蕙兰CyMV的脱除效果优于对ORSV的脱除效果。     

柑桔传染性杂色花叶病(CVV)的茎尖嫁接脱毒

通过茎尖嫁接,使4个被传染性杂色花叶病(CVV)侵染的柑桔品种脱毒。试验结果表明,ELISA(enzyme-linked immunosorbent assay)检测比指示植物接种诊断更为灵敏、快速。茎尖嫁接前,利用6-苄氨基嘌吟(BAP)或2.4-D处理茎尖和砧木切口,嫁接成活率可达86％—90％。     

口岸植物检疫脱毒处理技术研究进展

植物病毒影响植物的生长和发育,尤其是植物病毒的传染性及增殖性对一种或者一类植物的危害巨大。为了防范植物病毒随寄主贸易跨境传播危害,本文阐述了茎尖培养脱毒、热处理脱毒、热处理结合茎尖脱毒、离体微型嫁接、化学处理结合茎尖脱毒和低温疗法等脱毒技术,对应用于口岸检疫性病毒的不同脱毒方法进行了综述和分析,同时对今后植物检疫脱毒研究方向进行了展望。     

太子参脱病毒技术研究

采用茎尖分生组织法、热处理结合茎尖法、病毒唑处理结合茎尖法对6个不同产地的太子参[Pseudostellaria heterophylla (Miq.) Pax ex Pax et Hoffm.]组培苗进行了脱病毒研究. 经生物检测、 ELISA法和电镜检测表明, 热处理结合茎尖法脱病毒效果最好,脱毒率高达 100%;病毒唑处理结合茎尖法和茎尖分生组织法脱毒率分别为 79.64%和74.29%.不同产地太子参的脱病毒效果不同.根据实验结果提出了太子参的脱病毒繁育程序,其中复壮培养基中PP333的最适浓度为0.5～1.0 mg·L-1,最适生根培养基为1/2 MS 0.3 mg·L-1 NAA.     

成年态南丰蜜橘试管嫁接育苗技术研究

为探索适合成年态南丰蜜橘[Citrus reticulata Blanco‘kinokuni’(Tanaka)H.H.Hu]的快速繁殖技术,对其试管茎尖微嫁接育苗进行研究。结果表明,最好的砧木是苦柚种子苗,以腹接方式的成活率最高。嫁接苗接种在MS+GA3 1 mg L–1+蔗糖75 g L–1的培养基中,暗培养7 d后转入光周期下培养,嫁接成活率达67.78%。不同移栽基质对嫁接苗的成活率影响不显著。嫁接苗与成年态南丰蜜橘再生芽在形态和POD、CAT及SOD同工酶分子表达上均无明显差异。这表明通过试管茎尖微嫁接技术可保持其遗传稳定性。     

An obligately endosymbiotic mycorrhizal fungus itself harbors obligately intracellular bacteria.

Arbuscular-mycorrhizal fungi are obligate endosymbionts that colonize the roots of almost 80% of land plants. This paper describes the employment of a combined morphological and molecular approach to demonstrate that the cytoplasm of the arbuscular-mycorrhizal fungus Gigaspora margarita harbors a further bacterial endosymbiont. Intracytoplasmic bacterium-like organisms (BLOs) were detected ultrastructurally in its spores and germinating and symbiotic mycelia. Morphological observations with a fluorescent stain revealed about 250,000 live bacteria inside each spore. The sequence for the small-subunit rRNA gene obtained for the BLOs from the spores was compared with those for representatives of the eubacterial lineages. Molecular phylogenetic analysis unambiguously showed that the endosymbiont of G. margarita was an rRNA group II pseudomanad (genus Burkholderia). PCR assays with specifically designed oligonucleotides were used to check that the sequence came from the BLOs. Successful amplification was obtained when templates from both the spores and the symbiotic mycelia were used. A band of the expected length was also obtained from spores of a Scutellospora sp. No bands were given by the negative controls. These findings indicate that mycorrhizal systems can include plant, fungal, and bacterial cells.     

Evaluation of Tip Culture, Thermotherapy and Chemotherapy for Elimination of Peanut Mottle Virus from Arachis hypogaea

Peanut (Arachis hypogaea L.) plants (cvs Florunner and Pronto) were inoculated at the two-leaf stage with peanut mottle virus (PMV) to obtain PMV-infected plants. Shoot-tips from plants grown in the glasshouse (27°C) or from plants maintained at 35°C were used for tip culture. In some experiments ribavirin was added to the culture medium at 5 mg/1, 10 mg/1, 15 mg/1 or 20 mg/1. Plants regenerated from meristems or shoot-tips taken from virus-infected plants were not virus-free. After 45 days at 35°C, foliar tissue of 93 % of Florunner and 95 % of Pronto plants tested negative for PMV by enzyme-linked immunosorbent assay (ELISA). When shoot-tips from the plant that tested negative by ELISA were used for tip culture, no virus-free plants were obtained. No virus-free plants were obtained from tips cultured on medium supplemnted with ribavirin. However, when tip culture, thermotherapy and chemotherapy were combined; 80 % of Florunner and 100 % of Pronto plants were found negative for PMV.     

影响芋茎尖培养中脱毒和快速繁殖的几个生理因素

以3个芋品种（‘石川早生’、‘虾籽芋’、‘叶用芋）球茎茎尖为外植体,进行脱病毒和快繁的结果表明,外植体表面灭菌的最佳方法是剥鳞片→乙醇→新洁尔灭→剥幼叶→氯化汞;适宜茎尖分化的培养基为MS＋1．0-2．0mg·L^-16-BA＋0．2mg·L^-1 NAA。生物学方法和电镜观察显示：连续3代0．5-0．7mm茎尖剥离培养对芋花叶病毒（DMV）的脱毒率达100％。在培养基MS＋0．2mg·L^-1 NAA中,适量添加6-BA和TDZ,三品种芋的试管苗增殖效果好;附加KT,试管苗生长健壮且利于生根：添加20-100mg·L^-1的精胺（spm）,可促进不定芽的发生,与KT配合使用可促使继代增殖和成苗一步完成。完整植株在草炭土：蛭石=1：1的基质中,移栽成活率超过97％,且苗生长健壮。     

表皮生长因子受体基因突变检测方法改进及初步临床验证

目的:改进现有的检测表皮生长因子受体(EGFR)基因突变的荧光PCR法并开发出新的试剂盒,将其与直接测序法和ARMS法进行对比,验证该试剂盒用于临床诊断的敏感性、特异性和准确性。方法:收集2013年6月至2015年8月手术确诊的141例非小细胞肺癌(NSCLC)的石蜡包埋组织标本。采用盲法分别使用直接测序法、ARMS法和新试剂盒检测EGFR突变,比较新试剂盒与其他两种检测方法的差异,结果不一致时采用三种方法分别重复检验一次。结果:三种方法检测成功率均为100%,新试剂盒与直接测序法测得结果完全一致的比率达75.9%(107/141),在直接测序法测得的96例突变阳性中,92例在新试剂盒检测中得到验证(95.8%)。而直接测序法显示突变阴性的45例中,新试剂盒检测发现了23例突变阳性,两种检测方法的结果存在统计学差异(x2=40.745,P0.05)。与直接测序法进行比较,新试剂盒检测EGFR突变的敏感性、特异性分别为95.8%、48.9%,阳性预测值、阴性预测值分别为80.0%、84.6%,检测准确度为80.9%。以ARMS检测法为金标准,新试剂盒测得结果完全一致的比率达84.4%(119/141),两者的一致性比较好(K=0.749,P0.05),敏感性、特异性分别为94.1%、86.4%。结论:改进后EGFR基因突变检测的试剂盒在技术上较好地控制了检测结果的假阳性和假阴性,该检测方法较直接测序法具有更好的敏感性和准确性,与现有的ARMS法一致性较高。     

金针菇褐化病毒(FvBV)脱毒方法

【目的】评价5种不同脱毒方法对金针菇(Flammulina velutipes)菌株的脱毒效果,筛选出脱毒率高和脱毒后金针菇菌株菌丝生长速度、生物量、漆酶活力等性状改善明显的脱毒方法。【方法】以栽培金针菇菌株F-4889为研究材料,从菌丝体中提取大小约2.0 kb的病毒dsRNA,经RT-PCR鉴定该病毒为金针菇褐化病毒(FvBV)。采用菌丝尖端分离、原基组织分离、原生质体单核化、有性生殖和核迁移5种脱毒方法对金针菇菌株进行脱毒处理,利用dsRNA技术和RT-PCR检测脱毒效果。【结果】菌丝尖端分离脱毒后得到1株脱毒菌株;原基组织分离法未能脱毒;原生质体单核化脱毒法得到3株脱毒单核菌株和2株原单杂交脱毒菌株;有性生殖脱毒法获得脱毒孢子单核菌株23株和单孢杂交脱毒菌株8株;核迁移脱毒后得到5株核迁移脱毒菌株。脱毒率依次为25.0%、0、7.5%、57.5%和100%。脱毒菌株的菌丝生长速度、生物量、漆酶活力等均优于出发菌株、菌丝尖端和原基组织分离菌株。【结论】这5种方法中原生质体单核化、有性生殖和核迁移脱毒法脱毒效果较佳,均能有效脱除FvBV,脱毒率高,脱毒后菌株菌丝生长速度、生物量、漆酶活力等均明显提高。     

Evaluation of two recovery methods for detection of Mycobacterium avium subsp. paratuberculosis by PCR: direct-dilution--centrifugation and C(18)-carboxypropylbetaine processing

A duplex polymerase chain reaction (PCR)-hybridization assay based on Mycobacterium avium subsp. paratuberculosis (MAP)-specific IS900 integration sites was used to evaluate two mycobacterial recovery methods from bovine feces: a direct-dilution-centrifugation method and a C(18)-carboxypropylbetaine (CB-18)-based method. All MAP PCR results were confirmed for absence of inhibitors using a novel PCR system based on the rpoB gene of plant chloroplasts as an internal control. The detection limits of both MAP recovery methods when coupled with PCR were determined to be between 100 and 1000 organisms. Using culture as a 'gold standard' PCR following the direct-dilution-centrifugation protocol was 92.6% sensitive and 83.7% specific, whereas PCR following the CB-18 method was 100% sensitive and 53.5% specific. Both methods were 100% specific when 60 'true' negatives from two uninfected herds were tested. Both the CB-18 and direct processing methods coupled with a target-specific amplification technique may provide greater sensitivity to diagnose subclinical animals as they were able to detect more positives, on samples derived from infected herds, than conventional culture methods; however, more extensive investigation and follow-up of suspect animals will be required to fully validate the MAP recovery and molecular detection protocols described.     

Investigation of sensitivity,specificity and accuracy of Tetra primer ARMS PCR method in comparison with conventional ARMS PCR,based on sequencing technique outcomes in IVS-II-I genotyping of beta thalassemia patients

Purpose

Beta thalassemia is one of the most important hematic diseases all around the world and solving the problems caused by this abnormality is strongly dependent on precise detection and reliable screening of high-risk couples. The aim of our study was the investigation of sensitivity, specificity and accuracy of Tetra primer ARMS PCR method comparing with conventional ARMS PCR, based on sequencing technique outcomes for genotyping of IVS-II-I mutation in beta thalassemia patients.

Methods

Fifty seven samples including two homozygote, 49 heterozygote and 6 normal specimens were analyzed by Tetra primer ARMS PCR and conventional ARMS PCR methods. DNA was extracted by the standard method of salting out for leukocyte genomic DNA extraction of blood specimens and a high pure PCR template preparation kit was used for DNA purification of CVS samples. The results obtained by Tetra primer ARMS PCR and conventional ARMS PCR methods were compared with gold standard technique, i.e. sequencing.

Results

All three parameters including specificity, sensitivity and accuracy were 100% for Tetra primer ARMS PCR method, while they were 100%, 92.45% and 92.7% for conventional ARMS PCR technique respectively. Comparing with Tetra primer ARMS PCR which represented 100% agreement with sequencing method, conventional ARMS PCR technique only showed 47.1% agreement, because of 4 discordant results.

Conclusion

Tetra primer ARMS PCR method is an almost reliable, sensitive and accurate technique and it is suggested that it can be used as a complementary method for diagnostic cases instead of conventional ARMS PCR method. This suggestion originated with perfect rate of agreement between outcomes of sequencing method, as a gold standard method of detecting the mutations, and Tetra primer ARMS PCR technique comparing with conventional ARMS PCR method.     

Detection of Mycoplasmalike Organism of Paulownia Witches′-Broom with DAPI Fluorescence Microscopy

Paulownia witches’-broom infected by mycoplasmalike organism (MLO) has been developed several cytochemical methods for diagnosis. These methods all based on the special stain reactions or abnormal fluorescence in groups of infected sieve elements as a diseased symptom,. not really on the direct detection of MLO under light microscope. This paper deals with the demonstration of MLO specific white fluorescence after DAPI staining with GMA sections of diseased young stems. Such fluorescence was absent in sections from health plants. The results were confirmed by the ulrrastrueture of MLO and the structure of sieve elements showing from PAS-TBO stained GMA sections. The described method may not only be used in accurate diagnosis of MLO diseased in different plants, but is also worth in the studies of MLO distribution in plants, MLO dynamics in plant resting stage and MLO transmission to support the theoretical basis for protection.