三叶半夏悬浮培养下的体细胞胚胎发生及植株再生

用三叶半夏幼嫩叶片诱导产生的胚性愈伤组织建立了胚性细胞悬浮系,研究了悬浮培养下体细胞胚胎的发生及植株的再生。结果表明,胚性悬浮细胞在附加1．0mg／L2,4-D、0．2 mg／LBA和300mg／L LH的MS液体培养基中产生大量的球形胚,转入液体分化培养基(MS 0．1 mg／LNAA 0．2 mg／L BA 300 mg／L LH)中进一步发育成心形胚、鱼雷形胚和子叶形胚。收集成熟胚转移到MS固体分化培养基上培养获得了再生植株。另外还观察到某些成熟胚上产生了许多次生胚。     

黄瓜胚性细胞悬浮培养及其原生质体的植株再生

黄瓜子叶原生质体来源的胚性愈伤组织,在液体培养中形成胚性悬浮细胞系,已继代两年,仍保持胚胎发生能力,不同的ABA和蔗糖浓度对胚状体的生长发育和同步化有明显影响。1mg/l的ABA和7－9％的蔗糖能显著地减少培养物的愈伤化,并同步地控制胚状体处于球形或球形后期,低浓度的蔗糖（1％）有利于胚状体的早期萌发,继代3－5天的胚性是浮细胞团酶解后,获得大量有活力的原生质体,原生质体在DPDK,液滴或浅层培养,或褐藻酸钠包埋培养中活跃分裂,形成细胞团和球形胚,转至固体培养基上或将包埋培养物直接转入液体振荡培养,胚状体可不断增殖,胚状体在大量元素减半,不加外源激素的MS培养基上发育成小植株。     

陆地棉体细胞胚胎发生与植株再生

利用陆地棉品种下胚轴为外植体进行体外培养研究。激素和品种是影响愈伤诱导和胚胎发生的主要因素。去除激素后胚性愈伤在固体培养基上只能形成少量的成熟胚。悬浮培养是获得大量成熟胚的中间步骤。悬培两周后,悬培物转到固体培养基上促进胚状体成熟,30—60目之间的悬培物比大于30目的悬培物易形成成熟胚。KT 0.1ppm、Zea 0.1ppm分别有效地促进了胚状体成熟。活性碳250mg/L、NAA 0.1ppm、IBA 0.1ppm和IAA 0.1ppm能使胚状体萌发并健壮生长。目前已得到100多株幼苗,大苗已达八片真叶。     

湿地松体细胞胚胎发生和植株再生

以湿地松的未成熟合子胚为外植体,在附加８ｍｇ／Ｌ,２,４－Ｄ和４ｍｇ／Ｌ ＢＡ的ＬＰ培养基上诱导出胚性愈伤组织。在含１ｍｇ／Ｌ,２,４－Ｄ和０．５ｍｇ／Ｌ ＢＡ的培养基上保持并增殖。提高培养基的渗透压后,愈伤组织内大量的胚性胚柄细胞团和早期原胚。     

木薯体细胞胚胎发生及植株再生研究

对木薯体细胞胚胎发生的影响因素进行了优化研究。结果表明,基因型对木薯体细胞胚胎发生影响很大,在供试的六个品种中,“华南 8 号”的体细胞胚胎发生率和产胚量最高,分别为 65% 和19个;侧芽茎尖为最佳外植体,体细胞胚胎发生的最佳培养基为MS +0.5mg/L CuSO4 + 4 mg/L 2,4-D。同时,对木薯体细胞胚再生成完整植株的主要影响因素作了分析,建立了一个高效的植株再生体系。     

黄瓜胚性细胞悬浮培养及其原生质体的植株再生

黄瓜子叶原生质体来源的胚性愈伤组织,在液体培养中形成胚性悬浮细胞系,已继代两年,仍保持胚胎发生能力,不同的ABA和蔗糖浓度对胚状体的生长发育和同步化有明显影响。1mg/l的ABA和7－9％的蔗糖能显著地减少培养物的愈伤化,并同步地控制胚状体处于球形或球形后期,低浓度的蔗糖（1％）有利于胚状体的早期萌发,继代3－5天的胚性是浮细胞团酶解后,获得大量有活力的原生质体,原生质体在DPDK,液滴或浅层培养,或褐藻酸钠包埋培养中活跃分裂,形成细胞团和球形胚,转至固体培养基上或将包埋培养物直接转入液体振荡培养,胚状体可不断增殖,胚状体在大量元素减半,不加外源激素的MS培养基上发育成小植株。     

白Qian体细胞胚胎发生及其植株再生

白Ｑｉａｎ的成熟胚在４－６℃低温下保存１个月后,接种于改良ＬＰ＋２ｍｇ／Ｌ２,４－Ｄ＋１ｍｇ／Ｌ６－ＢＡ的培养基上,黑暗条件下培养１个月便可产生白色半透明的胚性愈伤组织。整体染色封片观察表明,胚性愈伤组织由很多很长的胚柄细胞及其顶端胚细胞团组成,这种愈伤组织培养物称为胚性胚柄团。     

水母雪莲体细胞胚胎发生及其植株再生

水母雪莲（Saussurea medusa Maxim.)茎和叶片的切段接种于MS＋2mg/L NAA 0.5mmg/L 6-BA的培养基上,20d后产生黄褐色的愈伤组织,经过几个月的继代培养,愈伤组织仍保持旺盛的增殖能力,但部分由黄褐色逐渐变为红色,将红色愈伤组织转到MS＋0．1mg/L NAA＋0．2mg/L 6-BA 5mg/L GA3的培养基上,30d后可分化出大量的体细胞胚,体细胞胚成熟后转到1／2MS＋0．2mg/L IAA 0.5%活性炭的培养基上,30d后可长出2－4cm的根,带根的小苗经锻炼后移栽到土壤中,成活率达76％,细胞组织学观察表明,发育成熟的体细胞胚具有胚根,胚轴和胚芽的完整结构,具有独立的维管系统。     

白杄体细胞胚胎发生及其植株再生

白杄(Picea meyeri Rehd.et Wils.)的成熟胚在4～6℃低温下保存1个月后,接种于改良LP 2mg/L 2,4-D 1mg/L 6-BA的培养基上,黑暗条件下培养1个月便可产生白色半透明的胚性愈伤组织。整体染色封片观察表明,胚性愈伤组织由很多很长的胚柄细胞及其顶端的胚细胞团组成,这种愈伤组织培养物称为胚性胚柄团。胚性胚柄团在MS 1mg/L 2,4-D 1mg/L KT的继代培养基上黑暗条件下可保持旺盛的增殖能力和分化潜力。当胚性胚柄团转到MS 5mg/L ABA 5mg/L AgNO_3的分化培养基上,1个月后可产生大量正常的体细胞胚。体细胞胚成熟以后转到含0.5％活性炭的无激素1/2MS基本培养基上约40d后可长出1.5～2.5cm的根,约60d后可长出真叶。光、ABA、蔗糖及AgNO_3浓度是影响体细胞胚发生的主要因素。     

一品红体细胞胚胎发生与植株再生

一品红不同部位愈伤组织诱导能力存在差异,嫩茎>幼花序>嫩叶。愈伤组织的长势主要受生长素的影响,细胞分裂素对愈伤组织生长有促进作用;但在含6-BA和NAA的培养基中诱导出的愈伤组织,其胚性明显强于单独用NAA诱导出的愈伤组织。液体悬浮培养是一品红体细胞胚胎高频发生的中间步骤。不同浓度BA对一品红体细胞胚的萌发率影响不大,萌发培养基中KNO₃含量加倍可提高萌发率。     

三叶半夏叶片一步成苗离体培养技术

以药用植物三叶半夏叶片为材料,通过比较直接和间接器官发生两种途径,建立了半夏一步成苗的快速繁殖技术体系。结果表明,经过愈伤组织阶段的一步成苗培养基为MS+0.5mg/L2,4-D+1.0mg/LKT,90d左右方可得到再生植株,植株分化率为74%,每个外植体上分化的块茎数为5.61±1.04。附加NAA与BA两种激素对一步成苗培养基进行优化,筛选出一步成苗最佳培养基MS+0.5mg/LNAA+0.5mg/LBA,60d后就可直接发育成完整植株,植株分化率为76%,每个外植体上分化的块茎数高达9.97±0·81,对这种培养基上的再生小植株进行移栽,1个月后,移栽成活率达100%。     

不同培养条件对半夏悬浮培养细胞生长及总生物碱形成的影响

研究了半夏悬浮培养过程中细胞鲜重的变化和MS培养基组分中碳源、钙盐、Fe盐及肌醇变化对半夏悬浮培养细胞生长及总生物碱合成的影响。结果表明 ,半夏细胞的生长曲线呈“S”型 ,细胞的最佳收获时间为 2 1d。在两种碳源中 ,葡萄糖比蔗糖利于细胞的生长和总生物碱的合成 ,最适浓度为 2 0g L。最适合细胞生长和总生物碱形成的钙盐浓度为 1 8mmol L、Fe盐浓度为 0 0 6mmol L和肌醇浓度为 10 0mg L。     

Cloning and molecular characterization of a novel lectin gene from Pinellia ternata

The full-length cDNA of Pinellia ternata agglutinin (PTA) was cloned from inflorescences using RACE-PCR. Through comparative analysis of PTA gene (pta) and its deduced amino acid sequence with those of other Araceae species, pta was found to encode a precursor lectin with signal peptide and to have extensive homology with those of other Araceae species. PTA was a heterotetrameric mannose-binding lectin with three mannose-binding boxes like lectins from other Araceae and Amaryllidaceae species. Southern blot analysis of the genomic DNA revealed that pta belonged to a low-copy gene family. Northern blot analysis demonstrated that pta constitutively expressed in various plant tissues including root, leaf, stem and inflorescence. The pta cDNA sequence encoding for mature PTA protein was cloned into pET-32a plasmid and the resulting plasmid, pET-32a-PTA containing Trx-PTA fusion protein, was investigated for the expression in E. coli BL21. SDS-PAGE gel analysis showed that the Trx-PTA fusion protein was successfully expressed in E. coli BL21 when induced by IPTG. Artificial diet assay revealed that PTA fusion protein had significant levels of resistance against peach potato aphids when incorporated into artificial diet at 0.1% (w/v). The cloning of the pta gene will enable us to further test its effect in depth on aphids by transferring the gene into crop plants.     

Isolation and characterization of microsatellite markers for Pinellia ternata and cross-species amplification

In this study, we isolated and characterized 12 microsatellite loci for Pinellia ternata. Polymorphism of these 12 loci was assessed in 46 individuals collected from two wild populations. All the loci were polymorphic with four to 13 alleles per locus and the observed and expected heterozygosities ranged from 0.312 to 0.680 and from 0.506 to 0.734, respectively. None of the loci showed significant deviation from Hardy–Weinberg equilibrium (P < 0.05). No significant linkage disequilibrium was observed between pairs of studied loci. In addition, most markers amplified successfully in three closely related taxa that are Pinellia cordata, P. peltata and P. pedatisecta. These microsatellite markers could provide a useful tool for genetic structure studies of the Pinellia species.     

半夏细胞悬浮培养中生理生化指标的测定

目的：了解半夏细胞悬浮培养中生理生化指标的变化以及它们之间的关系。方法：以半夏幼嫩叶片和叶柄为材料,诱导和筛选愈伤组织,在光照和黑暗条件下进行细胞悬浮培养。每3d取样,冰浴匀浆,于4℃、10000g离心20min,取上清液进行测定。结果：光照条件利于半夏悬浮细胞的生长,其最佳收获期为24d,细胞的生长曲线呈“S”形;在不同的条件下,其培养液pH都呈先上升后下降趋势;培养细胞中可溶性蛋白质和过氧化物酶的活性均出现两个峰值,但出现的时间不同;MDA(丙二醛)的含量先升高后下降随后又上升。结论：光照有利于半夏细胞的生长并且蛋白质含量和酶活性呈正相关性,MDA含量和细胞的生长、分化呈负相关性。     

半夏居群遗传分化的同工酶和可溶性蛋白电泳的聚类分析

用聚丙烯酰胺凝胶电泳（PAGE）技术分析了安徽产7个半夏（PinelliaternataBreit）居群的4种同工酶和可溶性蛋白谱带。根据其中的PER、EST和可溶性蛋白谱带的分布特征,用Forguson和Aquado＆Avise的公式分别计算其谱带的相似性系数和遗传距离,以类平均法（UPGMA）进行了聚类分析。结果表明：半夏居群间表现出一定的遗传分化;鹞落坪半夏与半夏的黄山、九华山、大别山万佛寺居群间的遗传距离较小（D≤0.214）,结合形态变异,笔者认为鹞落坪半夏只能是半夏居群中形态上较为特殊的地方宗,而不是一新种;狭叶半夏与其它6个居群的遗传距离较大（D=0.416）,是半夏居群中种系分化较为强烈的一个居群。文中还讨论了半夏的遗传分化与形态变异、生境等的相关性。     

油菜游离细胞培养及易再生的体细胞无性系的建立

以芥菜型油菜(Brcssica juncea)下胚轴来源的愈伤组织为材料,进行悬浮细胞振荡培养,得到游离的单细胞。这些细胞作浅层液体静置培养,可获得高频率的愈伤组织。在附加有水解乳蛋白200毫克/升、BA 2—3毫克/升及GA,0.1—1毫克/升的MS培养基上,这些愈伤组织分化了芽。分化的芽在无激素或附加有0.01—0.1毫克/升IAA或NAA的MS培养基上长出根,从而发育成完整植株。当对游离细胞获得的再生植株的茎切段愈伤组织,再经上述程序进行细胞培养时,发现愈伤组织诱导率及愈伤组织分化频率均远远高于原供体,从而建立了B.juncea易再生的体细胞无性系。     

Stem Apex Culture and Quality Improvement of Tubercles of Pinellia ternata

Formation of Plantlets was achieved when stem apex of Pinellia ternata Brier. Cultured in vitro on MS medium with KT 0. 5 mg/L + NAA 0.2 mg/L (MSI). With petioles of the plantlet as explants callus could be induced after cultured for a week on MS medium with 2, 4-D 2.0 mg/L + KT 0.5 mg/L (MSII). Calli were subcultured once in every month. After 3--4 months a kind of friable calli could be selected, from which the tubercles could be differentiate and the plantlets formed when transfered onto MSI. But before callus differentiation, a lot of roots were formed on callus. The plantlets could be produced directly from the petiole segment. It was found that the stem growing tip was always covered by the leaf primordium and the former leaf primordium was covered by the latter leaf primordium during the differentiation of the apical bud of tubercle. The frenquency of plantlet differentiation from callus and petioles was over 70%. The rate of regeneration of plantlet on liquid static culture was twice as much as that on solid culture. All plantlets grew well after being transfered into the plot. The fresh weight of tuber-plant was 103 % higher than that of control (cultivated plant come from tubers). The alkaloid content of tubers come from tuberplant was 0. 344%, that of control was 0. 203% and 0. 264% for the wild tuber.     

贵州半夏块茎腐烂病病原菌的分离与鉴定

【目的】对贵州省半夏块茎腐烂病病原菌进行分离和鉴定,为开发有效的防治技术体系提供依据。【方法】采用组织分离法进行病原菌分离,根据柯氏法则进行致病性测定,结合形态学、生理生化特性和分子生物学方法进行鉴定。【结果】从贵州3个半夏产地患病样品中分离出12株病原细菌和5株病原真菌。经形态学、生理生化特性和分子生物学方法鉴定,12株病原细菌为胡萝卜软腐果胶杆菌胡萝卜软腐亚种;ZJ、Z3病原真菌为尖孢镰刀菌,D3、D5、H1为茄病镰刀菌。【结论】贵州省半夏块茎腐烂病的病原菌有病原细菌和病原真菌。