荔枝胚性愈伤组织体胚发生系统的优化及转化抗性愈伤组织培养再生植株

首先优化了荔枝(Litchi chinensis Sonn.)幼胚胚性愈伤组织(embryogenic calli,EC)体胚发生的条件，在附加5mg L^-1 ZT的高糖(60g L^-1)高琼脂(10g L^-1)的MS培养基上获得了荔枝高频率(100％)体胚发生；同时，对荔枝体胚成熟和成苗的关键问题作了分析，建立了一套完整的荔枝体胚发生与再生系统，在附加10％椰子汁的高糖(6％)MS培养基上诱导体胚正常成熟，并在无激素MS培养基中诱导体胚萌发，为荔枝转基因研究提供了良好的受体系统．利用此再生系统对荔枝转基因抗性愈伤组织进行体胚发生与再生植株研究，获得了大量再生植株．图1表7参11     

龙眼胚性愈伤组织Cu/Zn-SOD分子伴侣基因CCS的克隆及其在体胚发生过程中的表达分析

为了解Cu/Zn超氧化物歧化酶分子伴侣基因(CCS)在龙眼体胚发生过程中的表达调控机制,以龙眼松散型胚性愈伤组织为材料,采用RT-PCR法,克隆了长960 bp含有完整开放阅读框的龙眼DlCCS核酸序列,其编码一个含有319个氨基酸的蛋白质(GenBank登录号:FJ973472).生物信息学分析显示:该蛋白为亲水的、稳定的、含有跨膜结构域的酸性蛋白质,定位于叶绿体;与毛果杨、葡萄、拟南芥、水稻和锦鸡儿具有较高同源性,含有植物CCS蛋白所特有的3个保守结构域;预测其通过不同的磷酸化方式,改变酶活性及蛋白构象,参与龙眼体胚中超氧化物代谢以及金属离子转运等生物学过程,在氧化还原过程中发挥作用.利用实时荧光定量PCR(qPCR)技术研究该基因在龙眼体胚发生发育过程中的表达情况,结果显示,从松散型胚性愈伤组织(Stage 1)到胚性紧实球形结构阶段(Stage 4),DlCCSmRNA的转录水平较低且波动小;当胚性紧实球形结构进一步发育到子叶形胚阶段(Stage 8),其表达量迅速增加,在成熟胚阶段(Stage 9)达到最高峰,提示DlCCS可能在龙眼体胚的中晚期起主要作用     

龙眼胚性愈伤组织维生素C过氧化物酶基因(apx)及NADH脱氢酶基因(nad2)部分序列的克隆

采用PCR技术从龙眼胚性愈伤组织中扩增出维生素C过氧化物酶基因 (apx)的部分cDNA序列longan1及NADH脱氢酶基因(nad2)的部分cDNA序列longan31,其中longan31是首次从体胚中克隆到的NADH脱氢酶基因(nad2).杂交结果显示,longan1与胚性愈伤组织和球形胚的RNA具有明显杂交信号,说明longan1在胚性愈伤组织和球形胚阶段特异表达. 图 3参 19     

麻疯树成熟胚乳再生植株及其气孔分析

对麻疯树成熟胚乳进行组织培养获得胚乳再生植株,并对其气孔进行分析.麻疯树成熟胚乳在25℃、12 h光照条件下培养7 d愈伤组织诱导完成,2,4-D浓度为2.0 mg L-1的MS培养基愈伤诱导效果最好,诱导率达89.29%.愈伤组织在含BAP的改良培养基上培养至黄绿色后转入分化培养基,在含IAA 0.25 mg L-1和ZT 1.5 mg L-1的WPM培养基上不定芽分化率达32.50%.将分化的不定芽从愈伤组织上剥离后转入含IBA、BAP和GA3的培养基上进行芽伸长培养.取胚乳不定芽叶片接种在含IBA 0.1 mg L-1、BAP 0.5 mg L-1和TDZ 0.5 mg L-1的MS培养基上诱导生芽后,再转入含IAA 0.25mgL-1、KT 0.5mg L-1、BAP 1.0 mg L-1和GA3 0.25 mg L-1的培养基上进行丛生芽的诱导,成芽率为85.2%.这些芽在含0.1 mgL-1 IBA的1/2 MS培养基上生根,大约有37.5%的芽生了根,平均有5.2条根系形成.与母本植株相比,再生的胚乳植株保卫细胞更大,且气孔密度减小.图2表6参24     

马尾松幼胚培养愈伤组织诱导的初步研究

取优良的马尾松幼胚作为外植体,在添加不同BA、NAA浓度配比的1/2MS培养基上于室温25±2℃进行组织培养.研究表明,以1/2MS为基本培养基时,进行愈伤组织诱导的最佳激素配比为1/2MS BA1 NAA0.1 0.6%琼脂 30/L蔗糖.愈伤组织大多为白色或白绿色的颗粒状,质地疏松半透明而有光泽.NAA是诱导愈伤组织的必需因子,但量过多又不利于外植体不定芽的产生.图1,表2,参24.     

双农杆菌共转化获得无标记转pepc基因的水稻植株

利用双农杆菌/双质粒的共转化系统获得了无选择标记转pepc基因的水稻植株.采用两个农杆菌EHA105转化粳稻品种台粳9号幼胚诱导的胚性愈伤组织,其中一个农杆菌内的质粒表达载体的T-DNA区仅含有目的基因pepc,另一个的T-DNA区含有选择标记基因hpt和GUS报告基因.获得抗性愈伤的转化率为9.2%.85.3%的抗性愈伤分化成T0代株系,其中78.8%的T0代植株为GUS阳性转基因植株.PCR分析表明,在78个T0代GUS阳性植株(来源于23个抗性愈伤组织)中共有35个植株(来源于7个抗性愈伤组织)含有pepc基因,即在23个GUS阳性株系中发生共转化株系的频率为30.4%.7个共转化的株系中有5个株系自交产生后代植株中含有无标记转pepc基因植株,其比例为71.4%.无标记的转pepc基因水稻植株中PEPC活性平均比对照提高8.8倍;与非转基因对照植株相比,转pepc基因水稻植株表现出更强的光合能力.图5表3参26     

水稻高效转化系统的建立

利用基因枪轰击和农杆菌介导两条途径转化水稻成熟胚、幼胚、幼穗诱导来源的愈伤组织．实验发现，外植体来源影响愈伤组织的诱导和转化．对于同一水稻品种，幼胚、幼穗在NB2培养基上比成熟胚易于诱导出胚性愈伤组织；转化中，无论是经农杆菌介导还是基因枪轰击，幼胚和幼穗来源的愈伤组织能以较高的转化频率获得转化体并再生成苗．同时，愈伤组织的诱导和转化效果也受基因型的影响．另外，转化途径对转化效率的影响较大，基因枪轰击比农杆菌介导转化频率高、再生频率强．并且，在轰击前6h至轰击后18h，将愈伤组织保持在附加20g／L甘露醇的培养基中，有利于转化率的提高．图2表3参17     

发根农杆菌转化海边香豌豆及转化体的体细胞胚胎发生

将海边香豌豆无功苗的子叶和下胚轴切段在附加ρ（2,4-D)=1mgL^-1,ρ（6BA)=0.5mgL^-1的MS培养基上预培养3d，然后与发根农杆菌A4菌液共培养30min。洗涤之后，在附加羧苄青霉素的无激素的MS培养基上培养。7－10d后，外植体切面处长出许多毛状根。继代培养时生长旺盛，且产生许多侧根和分枝。志状根的诱导频率与无菌苗培养天数和外植体来源部位有关。子叶切块的诱导率明显高于下胚轴，16d龄无菌苗的子叶切块诱导毛状根的频率最高。乙酰丁香酮处理菌液可以有效提高子叶外植体的毛状根诱导频率。将毛状根切段培养在含有ρ（2，4－D）＝1.0mgL^-1的MS培养基上可诱导出愈伤组织。该种愈伤组织转移到含有ρ（2，4－D）＝0.3mgL^-1的MS培养基上培养时，诱导出了许多早期体细胞胚。检测的转化组织均含有农杆碱和甘露醇。图2表1参19     

火炬松成熟合子胚培养直接体细胞胚胎发生和植株再生

以Hb、Ma和Mc3个基因型的火炬松成熟合子胚为外植体,建立了火炬松的离体培养直接体细胞胚胎发生植株再生体系实验结果表明,不同基因型火炬松之间的直接体细胞胚诱导频率有明显差异3个基因型中．Hb的成熟合子胚在附加1mg·L－12,4－D和4mg·L－1BA的DCR培养基上的直接体细胞胚诱导频率最高,达18％,扫描电镜观察表明,直接体细胞胚主要形成于成熟合子胚的子叶表而且胚轴较短,和母体组织仅保持胚根部位的联系组织细胞学切片结果表明,直接体细胞胚上胚根的发育落后于子叶,但是由直接体细胞胚发育形成的再生小苗有独立的维管组织,根部也较发达,并且整个小植株的维管系统和成熟合子胚上子叶的维管组织没有直接的联系.     

槐树组织培养中超度含水态苗发生与防止的研究

对槐树（Sophorajaponica）花药愈伤组织在培养条件下形成超度含水态苗的条件研究表明：短期培养的愈伤组织及在含有较低ρ（BA）的培养基上多形成正常苗,而经长期培养的愈伤组织及在含有较高ρ（BA）的培养基上产生了大量的超度含水态苗;在MS附加ρ（IAA）0．2mg／L、ρ（BA）5．0mg／L的培养基上,愈伤组织经连续1．5a以上的继代培养后,其表面产生的芽均为超度含水态芽;将这种愈伤组织和超度含水态芽转人含不同浓度和种类植物激素及生长调节剂的培养基后发现：BA和高ρ（KT）明显促进了超度含水态苗的形成;而一定ρ（NAA）和ρ（2,4-D）则显著地促进了正常苗的形成．对培养过程的观察显示：在各种培养基上形成的正常菌中,有一部分是由超度含水态的芽和苗转化而来,即由其顶端或腋生分生组织进一步发育转化而来,而另一些则由愈伤组织上重新产生．     

龙眼胚珠多胺含量变化与胚胎发育的关系

研究了龙眼胚胎发育与多胺(PAs)含量的关系.结果表明:正常发育的胚珠中多胺含量在胚胎发育的各个时期均高于败育胚珠.多胺含量在花后d10最高,其中腐胺(Put)约占多胺总含量的65%;以后各发育阶段多胺含量逐渐下降,其中d31~38下降幅度明显.不过,亚精胺(Spd)除d10外在正常胚珠中含量也大于败育胚珠.(Spd Spm)/(Put)和(Spm)/(PAs)比值变化与龙眼胚珠的发育有直接关系,表现为较高比值有利于胚胎发育,反之则抑制胚胎发育.多胺合成高峰先于核酸和蛋白质,推测多胺可能通过与核酸的相互作用参与核酸和蛋白质的合成和调节.图3表1参12     

模拟酸雨对龙眼叶绿体的伤害效应

龙眼叶绿体超氧化的歧化酶（SOD）和抗坏血酸过氧化物酶（AsA-POD)活性，谷胱甘肽（GSH）和抗坏血酸含量（AsA)含量随酸雨的pH值下降而降低，膜脂质过氧化产物丙二醛（MDA）的含量则随酸雨的pH下降而增加。pH3.0的酸雨胁迫2h，SOD和AsA-POD活性，GSH和AsA含量有所增加，并随胁迫时间的延长而下降。pH3．5的酸雨处理使类整体膜崩解，叶绿体基粒片层结构混乱；pH3.0的酸雨处理的叶绿体片层结构伤害更为严重。图5图版2参21     

KClO3对龙眼产期的调控效应

以自行配制、主要有效成份为KClO3的龙眼产期调控剂进行田间试验．结果表明：催花土壤施用剂量以每平方米树冠面积180g效果最佳；不同品种龙眼对药物的敏感程度不同，以石硖、储良龙眼最为敏感，平均抽花率可达80％以上．不同季节和不同梢期施药，龙眼抽花率存在显著差别；药剂干施回泥后淋水、干施淋水后回泥和拌水淋施这3种土壤给药方式的促花效果差异不显著；反季节与正造栽培收获的龙眼相比，果实质量存在一定差异，春季收获的果实个大肉厚，外观较佳，但糖份含量等品质指标则较差．KClO3调控龙眼产期的效果明显，配合相应的树体管理和土壤水分养分调控措施，可实现龙眼产期的人工合理控制．表7参20     

啤酒废酵母核酸降解物在龙眼生产上应用的研究

使用45μg/mL的啤酒废酵母核酸降解物处理龙眼后，与对照相比叶片厚度增加了16.80%，叶片鲜比重（mg/cm^2）增加了14.15％，叶片干比重（mg/cm^2）增加了32.12%,叶绿素a增加了68.88%，叶绿素b增加了27.50%，叶绿素总量增加了56.10%，使用40μg/mL的核酸降解物处理后，能增加龙眼雌花量，提高雌雄花比，座果数比对照增加了183.94%，单株产量增加128.94%，单果重增加26.57%，可食率增加11.29%，可溶性固型物增加9.51％，维生素C增加14.97%，明显地改善了龙眼的果实品质。     

模拟酸雨对龙眼坐果的影响及钙调节

pH 4.5的酸雨处理的花粉萌发率比CK下降 9.38%,pH 3.5处理可观察到花粉受害的症状 ,花粉萌发率比CK下降 2 4.2 3%.pH 4.0的酸雨处理雌花 ,其坐果率显著下降 (P <0 .0 5 ) .0 .0 2～ 0 .0 4mmol/L的Ca(NO3 ) 2 溶液处理显著促进酸雨胁迫下花粉的萌发 ,当Ca(NO3 ) 2 的浓度超过 0 .0 8mmol/L时则促进作用开始减弱 .酸雨胁迫后用 2mmol/LCa(NO3 ) 2 溶液或清水喷洒可显著减少因酸雨对雌花和坐果的危害 .图 1表 6参 2 1     

模拟酸雨对龙眼幼果纤维素酶活性和内源激素含量的影响

酸雨胁迫后，龙眼幼果IAA、GA1 3、ZRs、DHZRs、iPAs含量下降，ABA含量升高，生长抑制物ABA和生长促进物(IAA、GA1 3)的比值提高，酸雨胁迫还使纤维素酶活性上升，促进幼果脱落．易脱落幼果的纤维素酶活性和ABA含量高于正常幼果，而IAA、GA1 3、iPAs、ZRs、DHZRs含量低于正常幼果．图6表1参14     

Achieving Open Access to Conservation Science

Conservation science is a crisis discipline in which the results of scientific enquiry must be made available quickly to those implementing management. We assessed the extent to which scientific research published since the year 2000 in 20 conservation science journals is publicly available. Of the 19,207 papers published, 1,667 (8.68%) are freely downloadable from an official repository. Moreover, only 938 papers (4.88%) meet the standard definition of open access in which material can be freely reused providing attribution to the authors is given. This compares poorly with a comparable set of 20 evolutionary biology journals, where 31.93% of papers are freely downloadable and 7.49% are open access. Seventeen of the 20 conservation journals offer an open access option, but fewer than 5% of the papers are available through open access. The cost of accessing the full body of conservation science runs into tens of thousands of dollars per year for institutional subscribers, and many conservation practitioners cannot access pay‐per‐view science through their workplace. However, important initiatives such as Research4Life are making science available to organizations in developing countries. We urge authors of conservation science to pay for open access on a per‐article basis or to choose publication in open access journals, taking care to ensure the license allows reuse for any purpose providing attribution is given. Currently, it would cost $51 million to make all conservation science published since 2000 freely available by paying the open access fees currently levied to authors. Publishers of conservation journals might consider more cost effective models for open access and conservation‐oriented organizations running journals could consider a broader range of options for open access to nonmembers such as sponsorship of open access via membership fees. Obtención de Acceso Abierto a la Ciencia de la Conservación     

Representation of the world's biophysical conditions by the global protected area network

Protected areas (PAs) are often implemented without consideration of already existing PAs, which is likely to cause an overrepresentation of certain biophysical conditions. We assessed the representativeness of the current PA network with regard to the world's biophysical conditions to highlight which conditions are underprotected and where these conditions are located. We overlaid terrestrial and marine PAs with information on biophysical conditions (e.g., temperature, precipitation, and elevation) and then quantified the percentage of area covered by the PA network. For 1 variable at a time in the terrestrial realm, high temperature, low precipitation, and medium and very high elevation were underrepresented. For the marine realm, low and medium sea surface temperature (SST), medium and high sea surface salinity (SSS), and the deep sea were underrepresented. Overall, protection was evenly distributed for elevation across the terrestrial realm and SST across the marine realm. For 2 variables at a time, cold and very dry terrestrial environments had mostly low protection, which was also the case for low SST and low and medium SSS across most depths for marine environments. Low protection occurred mostly in the Sahara and the Arabian Peninsula for the terrestrial realm and along the Tropic of Capricorn and toward the poles for the marine realm. Although biodiversity measures are of prime importance for the design of PA networks, highlighting biophysical gaps in current PAs adds a frequently overlooked perspective. These gaps may weaken the potential of PAs to conserve biodiversity. Thus, our results may provide useful insights for researchers, practitioners, and policy makers to establish a more comprehensive global PA network.     

Quantitative composition of the defensive secretion ofBledius species (Coleoptera: Staphylinidae: Oxytelinae) is adapted to naturally occurring predators

Summary The adaptation of defensive secretions to their target organisms was examined for the abdominal gland secretions ofBledius furcatus, B. spectabilis andB. arenarius. Therefore the target organisms of the secretion of theseBledius species (i.e. their predators) had to be identified. At the collection sites examined these were the earwigLabidura riparia, the antCataglyphis bicolor, the flyLispe candicans, different carabids of the generaPogonus, Dichirotrichus, Dyschirius, Bembidion andCalathus and the wading birdsHaematopus ostralegus andCalidris alba. The secretion of the abdominal glands contains the toxin ptoluquinone dissolved in either-dodecalactone and 1-undecene (B. furcatus andB. spectabilis) or in octanoic acid and octyloctanoate (B. arenarius). The ratio of these solvents is species-specific. Application experiments using some of the natural insect predators (L. riparia, C. bicolor, Pogonus, Di. gustavii, Dyschirius) revealed that these solvent ratios provided a more effective deterrent than other possible ratios. Thus by combining the solvents in certain ratios, the capability of cuticular penetration and therefore the effectiveness of the defensive secretions are adapted to their natural targets.