Overexpression ofβ-1,4-Glucanase Gene EuEG1 Improves Micrografting of Eucommia ulmoides

Adventitious root formation poses a major constraint on the tissue culture and genetic transformation of Eucommia ulmoides.Micrografting offers a new method for the transplantation of genetic transformation,and its success depends on the formation of graft unions.This study used transgenic rootless test-tube seedlings as scions and seedlings from seed as rootstocks during micrografting to avoid the rooting issues that occur during tissue culture and to investigate the role of the EuEG1 gene in the graft healing process.We found that the EuEG1 gene is a vital regulator of graft,and its overexpression contributes to the survival of Eucommia ulmoides micrografting.The EuEG1 gene transgenic plants(TP)used as scions for micrografting presented a significantly higher survival rate than the wild type(WT)and empty vector(EV)regenerated scions.During the grafting healing process,the expression of the EuEG1 gene was higher during the period of callus proliferation,suggesting that the EuEG1 gene was involved in the graft healing process.Histological observation revealed that more calluses tissue appeared at the junction of transgenic scions,and the connection with the rootstock was stronger,which benefits wound healing.These results provide new insights into Eucommia ulmoides micrografting and indicate that the EuEG1 gene can promote wound healing and improve the micrografting survival rate.

同源细胞微移植技术联合A型肉毒毒素促进毛发再生的实验研究

目的 探索同源细胞微移植(MG)技术及A型肉毒毒素(BTX-A)联合使用对小鼠毛发再生的影响。方法 选取45只C57BL/6小鼠,随机分为生理盐水组(NS组)、MG组(MB-0组)、MG+低剂量BTX-A组(MB-2组)、MG+中剂量BTX-A组(MB-10组)、MG+高剂量BTX-A组(MB-50组),共5组,用药后观察小鼠背部毛发生长情况,并在实验第7、14、21天采用苏木精-伊红(HE)染色评估毛囊生长情况、在第21天采用免疫组织化学染色评估血管内皮生长因子(VEGF)及β-catenin的表达情况。结果 经过MG及BTX-A治疗后,小鼠皮肤变黑时间缩短(P<0.05),第14天新生毛发覆盖率增高(P<0.05)。与NS组比较,其余各实验组毛囊数目增多(P<0.05),VEGF及β-catenin表达增多。结论 MG联合BTX-A对小鼠毛发再生有促进作用,其机制可能和促进血管生长及激活β-catenin信号有关。

Photochemical Efficiency during the Establishment and Consolidation Phases of in Vitro Pinus radiata Micrograft Made from Scions of Different Ontogenetic Age

The aim of the present study was to evaluate the applicability of maximal photochemical efficiency of photosystem II (Fv/Fm) as an early estimate of P. radiata micrografts viability coming from different position (basal vs. apical) in the ortets. We hypothesize that Fv/Fm variation is a good indicator of micrograft’s viability and phenological stage during micrograft development. The micrografts were established in QL medium supplemented whit 0.1 mg·L-1 IBA and 1 mg·L-1 BAP and cultured at 25°C ± 2°C and 80 μmol photons m-2s-1 of photosynthetic active radiation by 16 h per day. During the establishment and consolidation phase, we found significant differences in Fv/Fm with respect to time and buds positions provenience. During establishment, basal shoot tips have lower Fv/Fm than apical shoot tips, which agrees with the lowest viability (35%). However, during the consolidation phase, the trend changed and basal shoot tips presented higher Fv/Fm than apical shoot tips and showed an increase in ETR and NPQ, with respect to apical shoots and ortet. Although the measurement of fluorescence parameters implies the insertion of the fluorometer sonde in vitro, this implies aseptic considerations, but always conveies a contamination risk. We conclude that fluorescence (Fv/Fm, ETR, NPQ) can be indicators of the micrograft’s development according to the shoot tips position in the ortet and can be useful early-indicators of the scions’ physiological condition during micrograft transition from establishment to consolidation.

Treatment of Gray Hair in Vitiligo Patients by Direct Melanocytes Transplant Using Needling Micrografting and Dermabrasion Techniques

Background: Melanocytes transplant for treatment of vitiligo is a common therapy using different surgical procedures. But there was no interest in repigmentation of grayness of hair in the treated vitiliginous area. Objective: To do melanocytes transplant from donor area into the recipient vitiliginous area with associated gray hair. Patient and Methods: This is a case interventional study was done in Department of Dermatology/Baghdad Teaching Hospital from February 2011-March 2012. Eleven patients were enrolled in this study, six males and five females with vitiligo in association of gray hair. Their ages ranged from 8 - 35 years with a mean ±SD of 20.90 ± 7.006. Melanocytes transplant in patients with vitiligo using needling micrografting technique for twelve patches and direct melanocytes transplant from normal donor area into vitiliginous recipient area by dermabrasion technique for eleven patches. Dressing was applied and patients were seen every two weeks for the first month and monthly for one year. Results: Repigmentation of the vitiliginous area was started after two weeks and was obvious at one month that progressed over time. The repigmentation of hair appeared usually after few months and was obvious after four months and the repigmentation of gray hair was quicker in patients with micrografting technique than those with dermabrasion technique. The mean rate of repigmentation was 18.3% at six months and 37.5% at twelve months in micrografting technique while the mean rate of repigmentation was 9.15% at six months and 18.55 at twelve months in dermabrasion technique. Conclusions: Direct transplant of melanocytes from normal donor area into recipient vitiliginous area with associated white hair is an effective procedure to induce repigmentation of gray hair.

Production of marker-free transgenic plants from mature tissues of navel orange using a Cre/loxP site-recombination system

Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciensmediated transformation for generation of marker-free transgenic plants from navel orange(Citrus sinensis Osbeck) mature stems using a CreloxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1-0.5(1-2 weeks), > 0.5 cm(3-4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange(Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase(IPT) and an anti-bacterial peptide(PR1aCB).Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were obtained.Southern blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants(deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange(Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.

拟南芥线粒体蛋白突变体ssr1-2表型的详细鉴定与分析

SSR1在拟南芥(Arabidopsis thaliana)中编码一个线粒体蛋白,为深入了解SSR1基因在植物生长发育和逆境应答中的作用,以ssr1-2及其抑制子突变体EMS143和EMS145为材料,对其根和地上部分的生长,及其对脯氨酸的敏感性和铁稳态进行跟踪分析,并利用拟南芥幼苗嫁接技术分析了ssr1-2短根表型对地上部分生长的影响。结果表明:ssr1-2主根长变短,根系的形态与须根系类似。地上部分也小于野生型,但出现得晚于根短表型。嫁接试验结果表明,突变体的根能限制野生型的地上部分生长,反之突变体的地上部分也能影响野生型的根,但前者的作用更大。ssr1-2在种子萌发、根长和叶绿素含量等指标上表现出对脯氨酸的超敏感表型。此外,ssr1-2对铁营养不敏感,即铁盐对其生长的促进作用显著小于WS野生型,说明其利用铁的能力显著下降。这些结果表明SSR1可能通过影响铁营养利用参与拟南芥根生长的调控,暗示线粒体铁元素利用机制的损伤可能是脯氨酸对植物生长发育抑制作用加强的原因之一。

转基因八棱海棠与苹果品种亲和性的微嫁接早期鉴定

用微嫁接法鉴定嫁接亲和性的结果表明,不同培养基和培养方法对嫁接成活率和生根率有较大影响,生根 愈伤同步的方法可以同时获得较高的嫁接成活率和生根率。转番茄铁载体蛋白基因八棱海棠(Malus micromalus)与 苹果(Malus pumila)品种富士,嘎拉以及新红星嫁接成活率分别为93.33％,92.86％,73.34％。嫁接后15 d左右,接穗 新叶开始生长;嫁接后2个月,嫁接苗移栽成活率分别为73.07％,73.91％和55％;移栽后3个月,3种嫁接苗的茎杆 粗度均匀,无大小脚现象。初步结果显示,转基因八棱海棠与富士、嘎拉均具良好的嫁接亲和性,而与新红星的嫁接 亲和性稍差。

培养环境对枣组培微嫁接的影响

[目的]建立一套高效的枣组培微嫁接体系。[方法]以感染枣疯病的婆枣组培苗(以下简称染病苗)为砧木,以冬枣组培苗茎段为接穗,研究若干环境因子对枣组培微嫁接成活率的影响。[结果]将枣组培微嫁接苗置于蔗糖浓度为50 g/LMS无激素培养基中的成活率较高。适宜的培养基pH值为6。采用不同封口材料控制培养瓶内湿度的研究表明,棉塞和单层封口膜封口可保持瓶内湿度,并有利于微嫁接苗的成活。微嫁接苗在28℃进行培养较为适宜。在适宜条件下枣组培微嫁接的成活率最高,达33.3%。[结论]培养基蔗糖浓度、pH值、培养温度和湿度均会影响枣微嫁接苗成活率。

小金海棠高效脱毒检测体系的探讨

以小金海棠组培苗为试材,对其进行苹果潜隐病毒接种、脱毒及检测方法的研究,探讨了一种针对小金海棠的快速高效的脱毒检测体系。结果表明,恒温38℃处理20 d或变温38/22℃处理30 d并结合茎尖培养,对脱除小金海棠苹果褪绿叶斑病毒和苹果茎沟病毒的效果较好。离体条件下,指示植物‘苏俄苹果’和‘弗琴尼苹果’不适合检测该两种病毒,而用PAS-ELISA法检测结果较灵敏。

微型嫁接技术在大田苗圃的应用试验

试验表明 ,在 8月份 ,利用微型嫁接 (自然茎尖嫁接 )技术 ,在大田苗圃八棱海棠 (MalusmicromalusMak .)砧木苗上进行嫁接培育脱毒苹果苗木 ,可以达到 81 3%的嫁接成活率 ,这将省去过去利用营养钵播种、嫁接和温室培养、锻炼、移栽大田等过程 。

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。

利用微嫁接技术促进麻疯树再生不定芽的生长

该研究以麻疯树种子实生苗的小芽和芽条为接穗,以带有胚根的实生苗下胚轴为砧木进行无菌微嫁接,试图建立新的有效微嫁接方法,解决农杆菌介导的麻疯树遗传转化体系中再生的转化不定芽难以顺利发育成完整植株的问题。结果表明:(1)抗生素对嫁接苗的生长有显著的抑制作用。(2)进行微嫁接所用砧木的苗龄以5d为宜。(3)进行微嫁接时适宜采用的砧木类型为带有部分胚根的下胚轴。(4)嫁接苗在0.3mg/L IBA+2mg/L谷氨酰胺+1/2MS培养基上的生长效果最好。(5)嫁接苗的移栽成活率最高可达76.40%。(6)以小芽或芽条为接穗的嫁接苗均可正常生长。该研究建立的麻疯树微嫁接体系,为解决麻疯树转化不定芽或芽条生长发育困难的问题提供了一条新的途径。

大叶山杨幼化处理技术的研究

以大叶山杨的超级实生苗的当年生枝条、成年优树根部萌条当年生嫩枝和成年优树树冠当年生嫩枝切段做外植体,采用连续继代培养和微型嫁接法进行幼化处理技术研究。结果表明：随连续继代次数的增加及微嫁接后再继代次数的增加,茎条的幼化程度随之提高,徽扦插生根率也在提高。成年优树根部萌条当年生嫩茎连续继代4～5次,微扦插生根率可达100%;成年优树树冠当年生嫩茎连续继代6～7次,微扦插生根率可达100%;用组培苗作砧木微嫁接后再进行微繁,连续继代3～4次,微扦插生根率可达100%。

柑橘脱毒快繁及微芽嫁接技术试验研究

以红江橙、沙糖桔的顶芽、侧芽和茎段为外植体,通过诱导培养形成脱毒不定芽接穗;通过微芽嫁接于砧木实生苗,培养成完整的无毒植株。试验结果表明:外植体灭菌处理成功率达86%,诱导不定芽的最适培养基为MT+6-BA 3.0 mg/L+IBA 0.1 mg/L+KT 0.1 mg/L;不定芽继代增殖的最适培养基为MT+6-BA 2.0 mg/L+IBA 0.1 mg/L,增殖系数为2.3;砧木种子播种成苗率达90%;微芽嫁接愈合成活率平均达87%。该研究为柑橘脱毒种苗提供了一种新型快速的繁殖方式。

薄皮核桃微枝嫁接技术研究

影响优质核桃规模化生产的关键因素之一是培养优良的核桃苗木。以新疆薄皮核桃为材料进行微枝嫁接研究,结果表明微枝接穗不同品种及生长情况、砧木生长情况、培养温度、生长调节物质等因素对微枝嫁接成活率均有影响。实验筛选出较为理想的各因素组合条件,微枝嫁接的最高成活率达70%,为建立新疆薄皮核桃快繁体系、苗木工厂化生产、扩大优质核桃种植规模奠定基础。

加快橡胶花药植株推广应用的几种方法探讨

本文讨论了以下5种加快橡胶花药植株推广应用的方法 1.通过愈伤组织的继代培养1—2次,出苗率可提高近1倍; 2.通过花药植株的试管微体扦插,一个叶蓬的茎段有可能发育成为一株植株; 3.在试管以花药植株为接穗进行微体嫁接,一个芽眼有可能成为一棵植株; 4.刚抽茎的非完整植株大田嫁接,可以成活并萌发成芽接苗; 5.改进移栽技术,提高移栽成活率。

茎尖微嫁接技术脱除佛手病原体的研究

目的采用茎尖微嫁接技术脱除佛手病原体,为无病苗木的培育奠定基础。方法采用茎尖微嫁接技术获得了佛手微嫁接成活苗,应用PCR技术对其进行黄龙病病原进行检测,同时考察了茎尖大小对嫁接成活率和脱病原体率的影响。结果经PCR检测,微嫁接苗脱除了黄龙病病原;茎尖大小对嫁接成活率和脱病原体率的影响较大,带4个叶原基的茎尖嫁接成活率最高,达60.00%,脱病原体率为85.71%,带2个叶原基的茎尖,脱病原体率达100%,但嫁接成活率低。结论茎尖微嫁接技术可以脱除佛手病原体,综合考虑嫁接成活率和脱病原体的效果,宜选用带3～4个叶原基的茎尖进行脱病原体苗的生产。

响应面法优化葡萄微嫁接组培快繁体系

为提高鲜食葡萄苗木品质和良种繁育速度,以鲜食葡萄品种晶红宝为试验材料,探究培养基天然营养添加物的种类及浓度、外植体消毒时间、光周期、微嫁接方法和绑缚材料等因素对葡萄微嫁接苗成活率的影响,结合响应面法确定葡萄微嫁接苗组培最佳试验条件,优化葡萄微嫁接组培快繁体系。结果表明,MS+0.2 mg/L IBA+1.0 mg/L 6-BA+0.2 mg/L NAA+30.0 g/L蔗糖+7.0 g/L琼脂培养基(pH 5.8～6.0)中添加香蕉泥可显著降低微嫁接苗的褐变率和污染率,促进不定根的伸长。响应面分析进一步确定了葡萄微嫁接组培的最佳试验条件为氯化汞消毒7.1 min,光照17 h,香蕉泥浓度700 mg/L。在此基础上采用劈接法、锡箔纸作绑缚材料的葡萄微嫁接苗成活率显著高于其他处理。

枣组培苗试管外微嫁接技术研究

以高5～7cm无根健壮的灵武长枣组培苗为接稳，以酸枣实生苗为砧木，采用枣树组培苗试管外微嫁接技术，研究了嫁接口的不同保湿材料、组培苗练苗时间、砧木苗龄、不同浓度BA对嫁接成活率的影响。结果表明：采用封口膜缠绕嫁接口操作容易，保湿效果显著；组培苗最适宜的练苗时间为5～6d；2片真叶苗龄的砧木嫁接成活率高；最适宜的BA浓度是15mg／L；获得最高嫁接成活率是91．4％。

接种和检测苹果潜隐病毒

通过试管微嫁接,分别将苹果褪绿叶斑病毒和苹果茎沟病毒接种到小金海棠组培苗上,并用PAS-ELISA法检测其带毒率。结果表明:接穗小金海棠苹果褪绿叶斑病毒的带毒率为60.0％,其苹果茎沟病毒带毒率为73.3％,从而证明用此方法可以对组培苗进行快速病毒接种而得到带毒的实验材料。