‘巴斗杏’组培快繁体系建立与耐盐植株筛选

以淮北黄里‘巴斗杏’茎段为外植体,MS为基本培养基,通过茎段诱导植株再生及进行耐盐筛选。结果表明:采用4、5月‘巴斗杏’茎段用0.1%升汞灭菌8 min较适宜;在含有1.0 mg/L 6-BA和0.1 mg/L IBA的MS培养基上茎段增殖较快,生长旺盛;较理想的生根培养基为MS+NAA 0.1 mg/L+IBA 0.2 mg/L,生根率达46.3%;‘巴斗杏’组培苗进行耐盐筛选的适宜盐浓度为0.4%～0.8%,筛选植株与对照相比差异显著。     

帝萝花‘璀璨明珠'的植株高效再生

为解决木本切花植物帝萝花‘璀璨明珠’繁殖效率低的问题,该文以帝萝花‘璀璨明珠’的幼嫩枝芽为外植体,研究了不同基本培养基对其长势的影响、不同激素种类和浓度对其增殖和生根的效果,分析了其离体繁殖的生长特点,并建立了高效的帝萝花‘璀璨明珠’组培快繁技术体系。结果表明:帝萝花‘璀璨明珠’幼嫩枝芽的消毒方法为0.1%的升汞溶液浸泡12 min,污染率为21.5%;外植体在WPM+ZT 1 mg·L~(-1)+NAA 0.1 mg·L~(-1)培养基上,侧芽萌发率为73%;增殖的最佳培养基为MS+BA 0.4 mg·L~(-1)+NAA 0.05 mg·L~(-1),增殖系数为6.63,增殖方式为侧芽增殖和植株基部丛生芽增殖;生根的适宜培养基为MS+IBA 0.75mg·L~(-1)+NAA 1 mg·L~(-1),生根率为70%;生根瓶苗移栽于珍珠岩和细草炭(体积比为0.5∶1)的基质中,光照强度为10 000～12 000 lx,空气湿度为70%～80%下培养,60 d后成活率可达72%。该研究结果为帝萝花组培种苗的商业化生产提供了技术支撑,同时促进了该高档木本切花的推广和种植及产业化。     

花烛叶色嵌合体不同生长阶段叶色性状的保持特征

对20个花烛叶色嵌合体株系在不同生长阶段的叶色性状保持特征进行观察,并通过计算机程序辅助分析发现:离体培养阶段,植株嵌合叶片总数占总叶片数的比率及平均单株非绿色部分占总叶面积的比率均为最高,分别为81%和50.1%,两项数值分别高于温室生长阶段新生嵌合叶数占新生叶片总数的比率(46.2%)及新生叶片非绿色部分占新生叶片总面积的比率(23.0%);移栽后,叶片非绿色部分比率超过50%的9个株系中有7个不能正常生长而死亡,所剩13个株系的新生叶片嵌合叶数减少,非绿色部分面积比率缩小,叶片趋向转为绿色;施用营养液后,5个株系的新叶非绿色部分面积比率回升。这些现象表明:营养条件充分有利于嵌合株系花叶性状的保持;嵌合株系中非绿色部分面积大于50%的株系生活力弱,不宜过早移栽;在茎尖分生组织形成叶原基的过程中,可能存在正常细胞与突变细胞的竞争,其结果是一方取代另一方或二者达到动态平衡。     

楸树无性系离体培养特性差异研究

对选育的5个楸树无性系(004-1、1-3、2-6、015-1、1-4)进行组织培养比较研究,以明确楸树无性系再生芽增殖和生根培养中遗传因素及外界条件的影响。结果表明:楸树不同无性系是影响瓶苗生长的主要因素,继代培养无性系间的方差分量在93.89%～98.16%,芽增殖系数、增殖芽数、芽长、叶数、茎段基部愈伤组织横向膨大、茎段基部愈伤组织纵向膨大在不同无性系间达到极显著水平;生根率、生根数和根长无性系间差异极显著,方差分量分别为92.97%、88.75%、96.25%。5个无性系生长性状以004-1表现最好,增殖系数为10.74,生根率为61.11%,移栽成活率为74.44%,无性系1-4最弱。     

荞麦远缘杂交种质(Fagopyrumtatari-cymosum)的 再生特性与品质分析

该文选用荞麦远缘杂交育成品系‘贵多苦75’为材料,进行40 cm(I)、20 cm(Ⅱ)和10 cm(Ⅲ)留桩高度处理,对再生植株主要农艺性状、产量性状和品质性状进行分析。结果表明:留桩高度40 cm内,留桩高度对再生植株株高、茎叶重、单株粒数、单株产量和产量影响显著;处理I植株的株高、茎叶重、单株粒数、单株产量和产量均显著或极显著高于处理Ⅱ和处理Ⅲ,说明留40 cm高桩有利于再生植株营养器官的生长与产量的形成。‘贵多苦75’种子清蛋白含量谷蛋白含量醇溶蛋白含量球蛋白含量。留桩高度40 cm内,留桩高度对种子总蛋白和谷蛋白影响显著,但对清蛋白、醇溶蛋白和球蛋白含量影响不显著。‘贵多苦75’再生植株种子总黄酮含量在3.17%～3.33%之间,受留桩高度的影响不显著。留桩高度为40 cm时,‘贵多苦75’再生植株产量达到1.5 t·hm-2,清蛋白、谷蛋白、醇溶蛋白、球蛋白、总黄酮含量分别达到4.06%、3.26%、1.27%、0.39%、3.33%,具有较高营养价值和药用价值。     

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。     

铁皮石斛叶色突变体的叶绿体超微结构、光合色素和叶绿素荧光特性的研究

以铁皮石斛(Dendrobium officinale Kimura et Migo)(‘TP35’)经自然突变的白绿杂色突变体(‘TP-MA’)和太空诱变的绿黄杂色突变体(‘TP-MG’)为材料,研究不同光照强度(0、50、100μmol·m^-2·s^-1)处理后,植株叶片的叶绿体超微结构、光合色素含量以及叶绿素荧光动力学参数的变化规律,并阐明叶色突变体与正常植株光合特性的差异。结果显示,‘TP-MA’和‘TP-MG’的叶绿体形态均发生了一定程度的缺失,且叶绿体分布不均匀、无规则,基粒片层结构不完整且排列疏松,基本与其表型性状相一致。‘TP-MA’光合色素的含量和叶绿素荧光参数F v/F m、ΦPSⅡ及F v′/F m′等显著低于‘TP35’,但非光化学淬灭系数(NPQ和q P)则相对较高;‘TP-MG’的光合色素含量及叶绿素荧光参数均低于‘TP35’,但差异不显著,且对较强的光照(100μmol·m^-2·s^-1)条件具有一定的适应性。研究结果表明不同光强处理后,铁皮石斛叶色突变体的叶绿体结构和光合生理指标均发生了不同程度的变化,且对植株的叶绿素含量及荧光参数产生一定影响,过低和过高的光照强度均不利于植株的正常生长。     

EMS诱变西瓜突变体库的构建及表型分析

采用1.0%诱变剂甲基磺酸乙酯(EMS)处理西瓜品系W1-17种子9h,然后对M1和M2代群体单株在叶、花、茎、育性、分支习性等方面进行表型变异观察,同时选取M2代典型变异株系,利用23对西瓜SSR引物进行分析鉴定,构建西瓜突变体库。结果表明:(1)EMS诱变使M1代幼苗形态呈现出叶畸形、叶褶皱、部分黄化、花畸形、雄花不散粉、卷须畸形、矮小、生长缓慢、不育等特异性状,获得由1 252个单株组成的西瓜突变体M1群体,群体总变异频率为18.33%。(2)M2代共筛选到205个突变植株,40种表型变异,表现在子叶性状(黄化、扭曲不对称、折叠等)、叶和茎性状(叶黄化、变小、裂刻变深,茎变细,节间变短,分支少等)、花性状(花变大,花色变浅,两性花,花瓣皱缩、部分退化、数目突变,柱头畸形,雄蕊不成熟等)和其他性状(生长缓慢、不育等)等方面,总的表型突变率达到了19.59%。(3)针对M2代10个典型变异植株,通过SSR引物分析发现有9份材料在DNA水平上有变异。本研究初步构建了含有120个M1代家系及1 051株M2代植株、40种表型变异的西瓜突变体库。     

银脉单药花的组织培养及快速繁殖

1　植物名称　银脉单药花 (Ａｐｈｅｌａｎｄｒａｓｑｕａｒ ｒｏｓａ)。2　材料类别　顶芽、侧芽。3　培养条件　基本培养基为ＭＳ。 ( 1 )愈伤组织、不定芽诱导培养基 :ＭＳ 6 ＢＡ 4ｍｇ·Ｌ- 1 (单位下同 ) ＮＡＡ 0 .5 ;( 2 )丛芽增殖培养基 :ＭＳ 6 ＢＡ2 ＮＡＡ 0 .5 ;( 3)生根培养基 :ＭＳ ＮＡＡ 0 .5。以上培养基均加 3%蔗糖 ,0 .7%琼脂 ,ｐＨ 5 .8。培养温度为 2 5～ 30℃ ,每日光照 1 2ｈ ,光照度 2 0 0 0ｌｘ。4　生长与分化情况4.1　愈伤组织和不定芽的诱导　取健壮的银脉单药植株的顶芽或切去顶芽后茎上长出的侧芽 ,…     

烈香杜鹃的离体培养和高效植株再生

以烈香杜鹃嫩茎为外植体,应用均匀设计法筛选其最适合的嫩茎基部直接再生芽苗和生根培养基。结果表明,最适合烈香杜鹃嫩茎基部直接再生芽苗的诱导培养基为DR＋2．30mg·L-1 TDZ＋0．05mg·L-1 IAA＋1．80mg·L-1 KT,诱导率为99．6％;生根培养基为1／2DR＋0．07mg·L-1 IAA＋0．02mg·L-1NAA,生根率达99．7％以上。以再生植株茎节为材料进行快速繁殖,在35d的培养周期内,每段增殖倍数平均达5以上。     

组培增殖方式对网纹草嵌合性状稳定性的影响

以一种具有良好的谱系细胞标记的周缘嵌合体网纹草(Fittonia albivenis)为研究材料,对不同增殖方式下其组培苗嵌合性状的稳定性进行了观察分析。结果表明,以茎段诱导腋芽增殖时网纹草的稳定性很高,但以丛生芽增殖时其嵌合性状发生了变异,变异率为21.32%;叶片组织解剖学观察结果显示,母本嵌合体茎尖分生组织中红和绿2种细胞谱系的细胞层排布在丛生芽再生过程中发生了改变,这可能是导致新类型嵌合体产生的原因。研究结果不仅对植物嵌合体种苗的组培生产具有重要的指导意义,而且为嵌合体的种质创新工作提供了新方法。     

Serological and molecular analysis of ABO and Rh blood group chimeras

The serological examination, blood transfusion strategies and the molecular analysis to blood group chimera were conducted to demonstrate existent of chimera in blood group. The blood grouping of ABO or/and RhD, newborn red blood cells separated by capillary centrifugation. Aabsorption tests and DTT treated agglutination erythrocyte tests were implemented in four patients. Further molecular biological research was conducted on one patient''s sample. The results showed that for patient 1: ABO blood group was AB/B chimera, Rh blood cells contained the RhCE chimera gene; Patient 2: Rh blood cells contained the RhD chimera gene; Patient 3: ABO blood group was AB/B chimera, Rh blood cells contained the RhD chimera gene; Patient 4: ABO blood group was O/B chimera, Rh blood cells contained the RhCE chimera gene. The study suggests that the individuals categorized as chimeras are likely to be more common than existing literature reports. According to the serological tests, in the absence of a history of recent blood transfusion or disease to cause reduced antigen, the phenomena of hybrid aggregation of the ABO and Rh blood system were the main feature. In terms of transfusion strategy, the selection of ABO and Rh blood groups should be depended on the group of cells with more antigens.     

秋水仙素诱变离体卷丹多倍体的研究

用0.15%秋水仙素附加2.00%二甲基亚砜诱变离体培养的卷丹小鳞茎,避光条件下摇床诱导,用组织培养结合不定芽诱导技术获得了多倍体苗,并对多倍体染色体数目进行鉴定。结果显示,诱导96 h效果最好,变异率达到54.29%。细胞学观察发现,对照为三倍体、非整倍体和极少数单倍体细胞组成的嵌合体,诱变出的4棵变异株细胞分别为染色体数目由53~72条的不同比例构成,属于典型的非整倍的异倍型嵌合体。诱变株与对照植株间幼苗叶形指数、气孔密度及气孔大小等特征差异比较明显。     

Production of germ-line chimeras in rainbow trout by blastomere transplantation

We describe a technique for producing germ-line chimeric rainbow trout, Oncorhynchus mykiss, by microinjection of the isolated blastomeres. FITC-labeled donor cells and non-labeled recipient embryos at various developmental stages between the early blastula and early gastrula stages were used for cell transplantation. The chimera formation rate and the degree of donor cell distribution in recipient embryos were evaluated at both the late gastrula stage (5 days post fertilization (dpf)) and the 40-somite stage (10 dpf). Among the six combinations of developmental stages of donor and recipient embryos, the combination of midblastula (2.5 dpf) donor cells and early blastula (1.5 dpf) recipient embryos gave the highest chimera formation rate and the best distribution pattern of donor cells. Using this combination, chimeric rainbow trout were produced with donor blastomeres from dominant orange-colored mutant embryos and wild-type recipient embryos. Of the 238 chimeric embryos produced, 28 (12%) hatched normally and 14 of the 28 fry (50%) had donor-derived orange body color. To test for germ-line transmission of donor cells, gametes obtained from the matured chimeras were fertilized with gametes from wild-type fish. Of the 19 matured chimeras, 6 (32%) yielded donor-derived orange-colored progeny, in addition to wild-type siblings. The contribution rates of donor cells in the germ-line ranged from 0.3 to 14%. This technique for producing germ-line chimeras should be a powerful tool for cell-mediated gene transfer in rainbow trout. Especially, if body color mutants are used for either donor cells or the host embryos, it will be possible to easily concentrate F1 transgenic embryos derived from transplanted donor cells by body color screening. Mol. Reprod. Dev. 59: 380-389, 2001.     

Domain exchange between isoforms of ferredoxin-NADP+ reductase produces a functional enzyme

Two isoforms of ferredoxin-NADP⁺ reductase (FNR) exist in higher plants, the leaf (or photosynthetic) and the root (or non-photosynthetic) isoform, which have 48% amino acid sequence identity and display specific structural and functional features. With the aim to gain further insight into the structure–function relationship of this enzyme, we designed two novel chimeric flavoenzymes by swapping the structural domains between the leaf and the root isoforms. Characterization of the chimeras would allow dissection of the contribution of the individual domains to catalysis. The chimera obtained by grafting together the FAD-binding domain of the root-isoform and the NADP-binding domain of the leaf-isoform was inactive when expressed in Escherichia coli. On the other hand, the chimera assembled in the opposite way (leaf FAD-binding domain and root NADP-binding domain) was functional and was produced in the bacterial host to a level threefold higher than that of the parent enzymes. The protein was purified and found to be as stable as the natural isoforms. Limited proteolysis excluded the presence in the chimera of misfolded regions. The affinity of the chimera for ferredoxin I (Fd I) was similar to that of the leaf isoform, although interprotein electron-transfer was partially impaired. As occurs with the root isoform, the chimera bound NADP⁺ with high affinity, while spectroscopic evidence suggested that the conformation adopted by the nicotinamide moiety bound to the chimera was similar to that observed in the leaf enzyme. Interestingly, the chimera, by combining favorable features from both parent isoforms, acquired a catalytic efficiency (k_cat/K_m), as an NADPH-dependent diaphorase, higher than those of both the root (～2-fold) and the leaf enzyme (～5-fold). Thus, molecular breeding between isozymes has improved the catalytic properties of FNR.     

Pluripotential rabbit embryonic stem (ES) cells are capable of forming overt coat color chimeras following injection into blastocysts

The isolation of pluripotent embryonic stem (ES) cell lines from preimplantation rabbit embryos and their in vitro properties have been previously described. In the present investigation, these ES cell lines were further characterized and their capacity to contribute to formation of adult, fertile animals upon injection into recipient New Zealand White blastocysts demonstrated. The efficiency of chimera formation was low (5% of live born), but the degree of chimerism, as assessed by coat color contribution from the Dutch belted strain, was high (10–50%). Thus a significant step is taken toward the development of gene-targeting technology in the rabbit, an animal whose physiology and size lend itself to unique applications in biomedical research. © 1996 Wiley-Liss, Inc.     

Effects of colchicine on polyploidy induction of Buddleja lindleyana seeds

Buddleja lindleyana Fort. is a garden ornamental plant with great potential for development and also a commonly used medicinal plant. To enrich its germplasm resources, the seeds of B. lindleyana were treated with colchicine solution with concentration gradients of 0.5%, 1.0%, 1.5%, 2.0% and 3.0% for 12-, 24- and 48-h respectively, and the water treatment was set as the control group. The purpose was to explore the effects of colchicine on the germination and mutagenic effect of B. lindleyana seeds at different concentrations and different times, to screen the appropriate combination of mutagenic concentration and time, to provide guidance for the construction of B. lindleyana mutation population in future research. The results were as follows: (1) Colchicine had an inhibitory effect on seed germination and seedling height of B. lindleyana seeds, and the higher the concentration, the more obvious the inhibitory effect. (2) After colchicine treatment, 30 mutant plants showed morphological variations such as leaf malformation, leaf color macular, early leaf bud germination, uneven leaf surface and leaf hyperplasia, among which 3.0%?+?48-h treatment group had great potential to produce yellow-leaf plants. (3) Detection and analysis by flow cytometry revealed that among the 30 morphologically variant plants, there were 22 diploid plants, 3 tetraploid plants, and 5 chimera plants. Among them, tetraploids were mainly from colchicine concentration of 3.0% (2 plants) and 1.5% (1 plant), chimeras were mainly from colchicine concentration of 1.0% (2 plants), 1.5% (1 plant) and 3.0% (2 plants), and the seed soaking time was 48-h. (4) The length and width of guard cells and stomata were significantly different between diploid and tetraploid, and there were significant differences in leaf width and leaf shape index between tetraploid and diploid, but there were no significant differences in leaf length among diploid, tetraploid and chimera. In short, we got tetraploids and chimeras materials which were potentially useful cultivars of B. lindleyana and provided an effective identification method for polyploids of B. lindleyana.

     

Modular coenzyme specificity: A domain‐swopped chimera of glutamate dehydrogenase

Domain‐swopped chimeras of the glutamate dehydrogenases from Clostridium symbiosum (CsGDH) (NAD⁺‐specific) and Escherichia coli (EcGDH) (NADP⁺‐specific) have been produced, with the aim of testing the localization of determinants of coenzyme specificity. An active chimera consisting of the substrate‐binding domain (Domain I) of CsGDH and the coenzyme‐binding domain (Domain II) of EcGDH has been purified to homogeneity, and a thorough kinetic analysis has been carried out. Results indicate that selectivity for the phosphorylated coenzyme does indeed reside solely in Domain II; the chimera utilizes NAD⁺ at 0.8% of the rate observed with NADP⁺, similar to the 0.5% ratio for EcGDH. Positive cooperativity toward L ‐glutamate, characteristic of CsGDH, has been retained with Domain I. An unforeseen feature of this chimera, however, is that, although glutamate cooperativity occurs only at higher pH values in the parent CsGDH, the chimeric protein shows it over the full pH range explored. Also surprising is that the chimera is capable of catalysing severalfold higher reaction rates (V_max) in both directions than either of the parent enzymes from which it is constructed. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Generation of rat blood vasculature and hematopoietic cells in rat-mouse chimeras by blastocyst complementation

Interspecies chimera through blastocyst complementation could be an alternative approach to create human organs in animals by using human pluripotent stem cells.A mismatch of the major histocompatibility complex of vascular endothelial cells between the human and host animal will cause graft rejection in the transplanted organs.Therefore,to achieve a transplantable organ in animals without rejection,creation of vascular endothelial cells derived from humans within the organ is necessary.In this study,to explore whether donor xeno-pluripotent stem cells can compensate for blood vasculature in host animals,we generated rat-mouse chimeras by injection of rat embryonic stem cells(rESCs) into mouse blastocysts with deficiency of Flk-1 protein,which is associated with endothelial and hematopoietic cell development.We found that rESCs could differentiate into vascular endothelial and hematopoietic cells in the rat-mouse chimeras.The whole yolk sac(YS) of Flk-1~(EGFP/ECFP) rat-mouse chimera was full of rat blood vasculature.Rat genes related to vascular endothelial cells,arteries,and veins,blood vessels formation process,as well as hematopoietic cells,were highly expressed in the YS.Our results suggested that rat vascular endothelial cells could undergo proliferation,migration,and self-assembly to form blood vasculature and that hematopoietic cells could differentiate into B cells,T cells,and myeloid cells in rat-mouse chimeras,which was able to rescue early embryonic lethality caused by Flk-1 deficiency in mouse.     

Production of germline chimeric chickens following the administration of a busulfan emulsion

Busulfan (1,4-butanediol dimethanesulfonate) was used to deplete endogenous germ cells for the enhanced production of chicken germline chimeras. Utilizing immunohistochemical identification of primordial gem cells (PGCs) in Stage 27 chicken embryos, two delivery formulations were compared relative to the degree of endogenous PGC depletion, a busulfan suspension (BS) and a solublized busulfan emulsion (SBE). Both busulfan treatments resulted in a significant reduction in PGCs when compared to controls. However, the SBE resulted in a more consistent and extensive depletion of PGCs than that observed with the BS treatment. Repopulation of SBE-treated embryos with exogenous PGCs resulted in a threefold increase of PGCs in Stage 27 embryos. Subsequently, germline chimeras were produced by the transfer of male gonadal PGCs from Barred Plymouth Rock embryos into untreated and SBE-treated White Leghorn embryos. Progeny testing of the presumptive chimeras with adult Barred Plymouth Rock chickens was performed to evaluate the efficiency of germline chimera production. The frequency of germline chimerism in SBE-treated recipients increased fivefold when compared to untreated recipients. The number of donor-derived offspring from the germline chimeras also increased eightfold following SBE-treatment of the recipient embryos. These results demonstrated that the administration of a busulfan emulsion into the egg yolk of unincubated eggs improved the depletion of endogenous PGCs in the embryo and enhanced the efficiency of germline chimera production.