新疆野苹果(Malus sieversii)超低温保存及其植株再生

以新疆野苹果（Malus sieversii（Lebed.）M．Roem.）无菌试管苗为试材,对其离体茎尖玻璃化超低温保存的影响因素进行研究。结果表明,新疆野苹果茎尖在含有5％二甲基亚砜（DMSO）的0．4mol／L蔗糖培养基上预培养3d,60％玻璃化溶液（PVS2）中室温装载30min,PVS：0℃下处理40min,经液氮保存至少24h后,转入继代培养基上再培养,成活率和再生率分别为93．3％和86．7％。再生植株生长和分化正常;同时对再生植株进行SSR标记检测,未发现超低温保存前后的DNA谱带存在差异。     

红花石蒜茎尖的玻璃化超低温保存

2～3mm的石蒜茎尖放在MS+0.4mol·L-1蔗糖的培养基上预培养5d,在25℃下用预处理液处理20min,接着用冰浴的玻璃化保护剂PVS2在冰浴中处理80min后,换新鲜PVS2并迅速投入液氮。液氮保存24h后,于40℃水浴中快速解冻2min,用MS+1.2mol·L-1蔗糖的液体培养基洗涤20min,滤纸吸干后接种到恢复培养基中,在25℃下暗培养7d后,转入光照强度为36μmol·m-2·s-1和光暗周期12/12h条件下培养。2周后的成活率最高可达90%,植株再生率达53%。     

Cryopreservation of Shoot Tips of Tetraploid Potato (Solanum tuberosumL.) Clones by Vitrification

In vitro-grown shoot tips of five tetraploid potato (SolanumtuberosumL.) clones were cryopreserved by vitrification. Excisedshoot tips (0.5–0.7 mm) were pre-cultured on filter paperdiscs over half strength liquid Murashige and Skoog (MS) mediumsupplemented with 8.7 µMGA₃and different combinationsof sucrose (0.3, 0.5 and 0.7M) plus mannitol (0, 0.2 and 0.4M)for 2 d under a 16 h photoperiod at 24 °C. The pre-culturedshoot tips were either successively loaded with 20 and 60% PVS2 solutions or directly exposed to concentrated vitrificationsolution before physical vitrification during liquid nitrogentreatment. The vitrified shoot tips were warmed rapidly andtreated with dilution mixture (MS+1.2Msucrose) for 30 min beforeplating on regrowth medium. Addition of mannitol to the pre-culturemedium improved survival of vitrified shoot tips. Direct dehydrationof pre-cultured shoot tips with concentrated PVS 2 was detrimentalto survival of vitrified shoot tips. Shoot tips pre-culturedon medium containing 0.3Msucrose plus 0.2Mmannitol, and loadedwith 20% PVS 2 for 30 min followed by 15 min incubation in 60%PVS 2 and 5 min incubation in 100% PVS 2 at 0 °C resultedin up to 54% survival after vitrification. About 50% of vitrifiedand warmed shoot tips formed shoots directly. Post-thaw culturingof vitrified shoot tips on medium containing an elevated levelof sucrose (0.2M) under diffuse light for the first week enhancedthe survival rate. Continuous culturing of vitrified shoot tipson high-sucrose medium induced multiple shoot formation.Copyright1998 Annals of Botany Company Solanum tuberosumL., potato, cryopreservation, germplasm conservation,in vitroconservation, meristems, shoot tips, tissue culture, vitrification.     

切花百合离体茎尖玻璃化法超低温保存研究

以切花百合西伯利亚试管苗离体茎尖为试材,通过正交设计试验对预培养培养基中蔗糖浓度、预培养时间和PVS2处理时间等影响超低温保存存活率的主要因素进行了分析,初步建立了切花百合种质玻璃化法超低温保存的技术方案。通过形态观察、可溶性蛋白和同工酶检测,冻存前后材料的遗传稳定性没有发生改变,表明该方法对切花百合的种质保存具有较强的实用意义。     

Glassy State and Cryopreservation of Mint Shoot Tips

Vitrification refers to the physical process by which a liquid supercools to very low temperatures and finally solidifies into a metastable glass, without undergoing crystallization at a practical cooling rate. Thus, vitrification is an effective freeze‐avoidance mechanism and living tissue cryopreservation is, in most cases, relying on it. As a glass is exceedingly viscous and stops all chemical reactions that require molecular diffusion, its formation leads to metabolic inactivity and stability over time. To investigate glassy state in cryopreserved plant material, mint shoot tips were submitted to the different stages of a frequently used cryopreservation protocol (droplet‐vitrification) and evaluated for water content reduction and sucrose content, as determined by ion chromatography, frozen water fraction and glass transitions occurrence by differential scanning calorimetry, and investigated by low‐temperature scanning electron microscopy, as a way to ascertain if their cellular content was vitrified. Results show how tissues at intermediate treatment steps develop ice crystals during liquid nitrogen cooling, while specimens whose treatment was completed become vitrified, with no evidence of ice formation. The agreement between calorimetric and microscopic observations was perfect. Besides finding a higher sucrose concentration in tissues at the more advanced protocol steps, this level was also higher in plants precultured at 25/?1°C than in plants cultivated at 25°C. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:707–717, 2013     

Cryopreservation for Seedbanking of Australian Species

Recent studies have indicated that a large number of nativeAustralian species can be stored in liquid nitrogen using relativelystraightforward methods. Furthermore, it is estimated that seedfrom at least 40% of the rare and endangered species of WesternAustralia could be maintained through use of these cryopreservationtechniques. However, before cryopreservation of seed of Australianspecies can be implemented as a routine technique, physicaland chemical properties of seed must be studied. This studyhas shown that successful cryopreservation of seed could beinfluenced singly or by a combination of hardseededness, moisture,lipid content, fatty acid and amino acid composition. The studyalso details the role of in vitro methods for determining seedviability/recovery of species that are either damaged by liquidnitrogen or are difficult to germinate.Copyright 1994, 1999Academic Press Seed, cryopreservation, lipid, fatty acid, amino acid, Australian species     

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。     

Shoot Responses of Six Lythraceae Species to Flooding

Abstract: The large family Lythraceae has several genera and species that show tolerance to flooding; one species, Lythrum salicaria (purple loosestrife), is considered invasive in North American wetlands. It is not clear, however, which characteristic(s) contribute to the invasive nature of L. salicaria, but those that contribute to improved flood tolerance may be responsible. This study examined the response of the shoot system of several members of the Lythraceae, three Lythrum species (L. salicaria, L. hyssopifolia, L. alatum), Decodon verticillatum, Pleurophora anomala and Heimia myrticifolia, to flooding to determine if these species differ in their response in comparison to L. salicaria. All species, except L. hyssopifolia, responded to flooding by increasing total plant height. All species, except H. myrticifolia, formed a phellem of significantly wider diameter at the stem base of flooded plants compared to controls. This phellem consisted of alternating bands of small, isodiametric cells and radially elongated cells separated by large air lacunae forming a very specialized aerenchyma. The small cells had Casparian band-like wall modifications and occasionally displayed modifications that included all cell wall surfaces. The development of extensive aerenchymatous phellem in flooded plants may increase the air space continuum from shoot to root in shoots that have undergone secondary growth. Given that these species displayed similar responses to flooding, the purported invasiveness of L. salicaria cannot be attributed to presence of any of the characteristics studied.     

Cryopreservation of in vitro Grown Shoot Tips of Red Bud Taro by Encapsulation-Vitrification

A simple procedure for cryopreservation of in vitro grown shoot tips of red bud taro (Colocasia esculenta L. Schott var. cormosus‘Hongyayu’) by encapsulation vitrification is investigated. Shoot tips were excised from 8 week old stock shoots and encapsulated into alginate gel beads. Encapsulated shoot tips were precultured in liquid MS medium supplemented with 35mg·L-1 6 BA, 05mg·L-1 IBA, 01mg·L-1 GA3 and 03mol·L-1 sucrose for 24h, then they were loaded with a mixture of 2mol·L-1 glycerol plus 04mol·L-1 sucrose for 30min at 25℃. After dehydration with PVS2 at 25℃ for 20min, the encapsulated and dehydrated shoot tips were plunged directly into liquid nitrogen. After rapidly rewarming in a 40℃ water bath for 3min, PVS2 was drained from the cryotubes and replaced third with liquid MS medium supplemented with 35mg·L-1 6 BA, 05mg·L-1 IBA, 01mg·L-1 GA3 and 12mol·L-1 sucrose and each kept for 10min at 25℃and then post cultured on solidified MS medium supplemented with 35mg·L-1 6 BA, 05mg·L-1 IBA and 01mg·L-1 GA3 in the dark for 3 days and then transferred to the light conditions. The average survival rate amounted to about 80%. Plantlets regenerated from cryopreserved shoot tips were morphologically uniform. This encapsulation vitrification procedure promises to become a routine method for the cryopreservation of shoot tips of Chinese genuine red bud taro.     

卵叶泥炭藓茎尖包埋玻璃化法超低温保存研究

该研究通过对脱水时间和化冻温度的探索,检验了包埋玻璃化法在超低温保存湿润生境中苔藓的可能性。结果表明:卵叶泥炭藓无菌苗在4℃条件下预培养3d后,在0℃用60% PVS_2装载30min,PVS_2脱水60min后迅速投入液氮保存,24h后用40℃水浴快速化冻2min再培养,成活率可达42.41%,且再生植株与常温状态下的植株形态指标没有显著性差异。研究认为,包埋玻璃化法超低温保存湿润环境中生长的苔藓植物是可行的。     

应用改良滴冻法超低温保存两种柿属植物的研究

以君迁子（Diospyros lotus L．）和柿（D．kaki Thunb．）组培苗茎尖为试材,对影响超低温保存效果的主要因素,如低温锻炼方式、预培养条件、PVS：（30％甘油＋15％乙二醇＋15％二甲基亚砜＋0．4mol／L蔗糖）处理时间等进行了研究。建立了2种柿属植物的超低温保存程序：（1）切取1cm左右试管苗梢段继代到1／2MS（KNO3和NH4NO3减半）培养基中,交替低温[昼（25±1）℃、夜（4±1）℃]锻炼6周;在含0．5mol／L蔗糖的1／2MS培养基上预培养5d,20℃下装载液（2．0mol／L甘油＋0．4mol／L蔗糖）过渡10min,0℃下PVS2处理1．5h;（2）投入液氮保存;（3）40℃水浴化冻,洗涤5～6次后接种于含1．0mg／LTDZ、0．6g／L可溶性PVP、30g／L蔗糖和7．0g／L琼脂的培养基（作者在优化柿属植物离体培养体系试验中获得）上暗培养1周,转入25℃,1500lx培养室。按照上述程序培养,‘鄂柿1号’、‘湘西甜柿’和君迁子的成活率分别为79．6％、67．4％和60．9％。     

Flavolans and Growth-Promoting Catechins in Young Shoot Tips of Prunus Species and Hybrids

Young shoot tips of species and hybrids from the Prunus section eucerasus contain catechin, epicatechin and flavolans (oligomeric flavans including biflavans). Prunus avium shoot segments, 2 mm in length, were excised from the elongating zone of growing shoots and cultured in vitro on a liquid Murashige and Skoog medium containing indolyl-3-acetic acid and benzyl adenine. Supplementation of the same medium with (+)-catechin and epicatechin resulted in apparent promotion of callus growth. This effect was greater under long day conditions than under continuous dark.     

利用超低温保存方法脱除香蕉束顶病毒的研究

香蕉束顶病毒(BBTV)是香蕉生产中的严重病害之一,主要通过感病材料和香蕉交脉蚜等昆虫进行传播,目前尚无有效防治方法.本研究以感染BBTV的巴西蕉(Musa AAA Cavendish)为材料,研究了感染BBTV的巴西蕉离体再生和超低温保存技术条件,表明离体茎尖在MS+6-BA 4 0 mg/L+NAA 0 4 mg/L的培养基上分化不定芽较好;采用玻璃化法超低温保存技术保存带有BBTV的香蕉茎尖,再生后植株BBTV脱除率达到60 6%,而常规的茎尖培养对BBTV的脱除率仅为26 7%.     

不同品系小鼠胚胎玻璃化冷冻保存的比较研究

目的　研究甘油作为冷冻保护剂、不同基因型小鼠对胚胎玻璃化冷冻的影响。方法　采用 6 5mol L的甘油作为冷冻保护剂 ,采用二步法对CBA、NOD、C57BL 6J、ICR及CD1小鼠 3 5d的胚胎进行玻璃化冷冻 ,并比较了不同品系小鼠胚胎的复苏率及移植受孕率。结果和结论　CBA、NOD、C57BL 6J,ICR及CD1的复苏率分别为 5 7 6 %、4 8%、31 3%、86 5 %及 88% ,移植受孕率为 2 1%、2 3 5 %、11%、38%和 35 5 % ,封闭群小鼠的胚胎复苏率、移植受孕率均显著高于近交系小鼠。这提示胚胎的复苏率及移植受孕率可能与小鼠的不同基因型有关。五个品系中 ,桑椹胚及早期囊胚的体外复苏率均显著高于扩张囊胚。这说明不同基因型及胚胎的不同发育阶段对胚胎玻璃化冷冻效果有影响     

Proteomic Analysis of Shoot Tips from Two Alfalfa Cultivars with Different Florescence

Plant Molecular Biology Reporter - Flowering is an indispensable biological process for the complete life cycle of angiosperms, crucial to the regeneration of plants and the continuation of...     

Biosynthesis of Indoleacetic Acid from Tryptophan-C in Cell-free Extracts of Pea Shoot Tips

A 2-step, 1-dimensional thin-layer chromatographic procedure for isolating indoleacetic acid (IAA) was developed and utilized in investigations of the biosynthesis of IAA from tryptophan-¹⁴C in cell-free extracts of pea (Pisum sativum L.) shoot tips. Identification of a ¹⁴C-product as IAA was by (a) co-chromatography of authentic IAA and ¹⁴C-product on thin-layer chromatography, and (b) gas-liquid and thin-layer chromatography of authentic and presumptive IAA methyl esters. Dialysis of enzyme extracts and addition of α-ketoglutaric acid and pyridoxal phosphate to reaction mixtures resulted in approximately 2- to 3-fold increases in net yields of IAA over yields in non-dialyzed reaction mixtures which did not contain additives essential to a transaminase reaction of tryptophan. Addition of thiamine pyrophosphate to reaction mixtures further enhanced net biosynthesis of IAA. It is concluded that the formation of indolepyruvic acid and its subsequent decarboxylation probably are sequential reactions in the major pathway of IAA biosynthesis from tryptophan in cell-free extracts of Pisum shoot tips. Comparison of maximum net IAA biosynthesis in extracts of shoot tips of etiolated and light-grown dwarf and tall pea seedlings revealed an order, on a unit protein N basis, of: light-grown tall > light-grown dwarf > etiolated tall etiolated dwarf. It is concluded that the different rates of stem elongation among etiolated and light-grown dwarf and tall pea seedlings are correlated, in general, with differences in net IAA biosynthesis and sensitivity of the tissues to IAA.     

改进的SDS-CTAB法提取濒危植物连香树总DNA

对珍稀濒危植物连香树(Cercidiphyllum japonicum)的6种总DNA提取方法进行了对比试验,结果表明改进的SDS-CTAB法更适合于连香树总DNA提取。该方法提取的DNA经紫外消光值检测,其A260/A280为1.8532,优于CTAB法(1.4872)、SDS法(1.3552)、PVP法(1.5079)、尿素法(1.1858)和高盐低pH法(1.4534)。琼脂糖凝胶电泳和PCR扩增结果也得出同样的结论。     

贵州6种蝙蝠的核型

采用常规骨髓细胞空气干燥法,研究了贵州6种蝙蝠的核型。白腹管鼻蝠(Murina leucogaster)2n=44,染色体臂数(FN)为58;普通长翼蝠(Miniopterus schreibersi)染色体数是2n=46,FN为50,黄大蹄蝠(Hipposideros pratti)2n=32,FN为60;角菊头蝠(Rhinolophus cornutus)2n=62,FN为60;云南菊头蝠(R.yunnanensis)2n=44,FN是60;犬蝠(Cynopterus sphinx)2n=34,FN=58。其中白腹管鼻蝠、云南菊头蝠和犬蝠为国内首次报道。     

Vitrification of mouse oocytes using a nylon loop

Cryopreservation of mouse oocytes was improved by the use of ultra-rapid vitrification using a nylon loop of 0.5 mm diameter. Oocytes that were vitrified using the loop survived at high rates and were fertilized following a small hole being made in the zona pellucida (69.8%) and developed to the blastocyst stage in culture (67.4%) at similar rates to that of oocytes that were not cryopreserved. Blastocysts resulting from oocytes vitrified using the nylon loop had similar development of the inner cell mass and trophectoderm as blastocysts from non-cryopreserved oocytes. In contrast, oocytes that were cryopreserved using a slow-freezing protocol where most of the Na+ is replaced with choline had lower rates of fertilization (39.5%), reduced development to the blastocyst stage (25.7%), and blastocysts had reduced development of the inner cell mass. Blastocysts derived from oocytes that were vitrified with the nylon loop were able to implant (88.0%) and develop into fetuses (56.5%) at significantly higher rates compared to blastocysts derived from oocytes that were slow-frozen (52.4 and 26.2%, respectively). Vitrification of mouse oocytes using the nylon loop results in the retention of viability of the oocytes and subsequent embryos.     

苍术属植物6个居群的染色体核型分析

采用常规根尖压片法对苍术属植物6个居群染色体数目和核型进行分析。结果表明,所研究的苍术属植物居群染色体数目均为24条,核型公式分别为:保康居群2n=2x=10m+12sm+2st、商洛居群2n=2x=14m+10sm、岳西居群2n=2x=12m+12sm、英山居群2n=2x=14m+10sm、信阳居群2n=2x=14m+8sm+2st、神农架居群2n=2x=8m+10sm+4st。除了英山居群苍术核型为2A型外,其他的均为2B型。表明英山苍术是6个居群中比较原始的类群,而其他居群进化程度较高。同时根据6个居群的核型分析和Q型聚类结果可知,英山居群罗田苍术与商洛居群北苍术着丝粒核型相同,聚为一小类,再与信阳居群北苍术聚为一大类,认为将英山居群罗田苍术与其他苍术划分开来作为一个新的变种不合适;保康和岳西居群均为南苍术,聚为另一类,因此本研究结果支持北苍术作为苍术的一个变种,支持罗田苍术与南苍术、北苍术共同构成苍术属的一个分支——苍术复合体。神农架苍术居群单独聚为一大类,与其他居群的苍术亲缘关系较远,可能与其海拔高、生境复杂及与其他居群基因交流有限而导致遗传隔离有关,其他5个居群聚为另一大类,表明其遗传亲缘关系比较近,基因交流比较频繁。