甘薯、胡萝卜发根单寄主培养体系繁殖马铃薯腐烂线虫的研究

利用发根农杆菌A4转化甘薯品种徐薯18和胡萝卜品种天红2号的发根,建立甘薯茎线虫病病原线虫(马铃薯腐烂线虫)的单寄主培养体系。通过该体系对马铃薯腐烂线虫的行为进行观察以及繁殖情况进行调查。结果表明:(1)马铃薯腐烂线虫在甘薯和胡萝卜发根上都能正常发育和繁殖,完成其生活周期;(2)培养4周和8周后,在甘薯发根上线虫繁殖倍数分别为2.6和50.6倍;在胡萝卜发根上线虫繁殖倍数分别为1.7和9.9倍;相同培养时间内,线虫在甘薯发根上的繁殖数极显著高于在胡萝卜发根上的繁殖数。(3)利用发根系统繁殖马铃薯腐烂线虫,便于研究其行为,在显微镜下可以直接观察到线虫在发根上活动情况,这对研究发根和线虫相互关系十分有利。基于上述结果,初步证实构建甘薯发根单寄主培养体系繁殖马铃薯腐烂线虫是可行的,且优于胡萝卜发根繁殖马铃薯腐烂线虫体系。     

细胞和分子技术在甘薯茎线虫病抗病育种中的应用

甘薯茎线虫病是我国北方甘薯薯区的重要病害之一。为进一步研究甘薯茎线虫病育种中线虫和甘薯的抗感关系,利用细胞培养技术建立发根农杆菌转化甘薯发根,用该发根培养马铃薯腐烂线虫,构建植物寄生线虫培养和抗性鉴定的技术平台;用聚合酶链式反应(polymerase chain reaction,PCR)分子标记技术筛选甘薯茎线虫病抗感品种的分子标记,并对发根培养马铃薯腐烂线虫体系和分子标记辅助选择抗甘薯茎线虫病品种技术进行了展望。     

甘薯茎线虫病抗侵入和抗扩展资源评价

对69份创新种质和具有200个单株的RAPD分析分离群体进行甘薯茎线虫病抗侵入和抗扩展鉴定,结果表明:可作为抗性亲本的材料仅占创新种质的23.2%,福薯13为高抗侵入和高抗扩展特异资源,泉紫薯1号、烟紫薯176、徐01-2-5、徐01-25-8具有高抗侵入、抗扩展特性,可作为双抗亲本加以利用.另对甘薯茎线虫病抗扩展性鉴定方法的研究表明:采用室内接种200条线虫,25℃室温下培养45d,以薯块的横切和纵切面的平均防效进行评价最为有效.     

甘薯抗茎线虫病亲本资源的筛选和利用

为解决茎线虫病对甘薯生产的严重威胁,对多渠道引进的资源材料,在重病地块连续进行抗性鉴定和亲子代抗性遗传分析,并注重高淀粉、高抗茎线虫病育种材料和品种的筛选,现已筛选出AB94078-1、徐1-4、徐3-2、徐58-1、鲁78066、AIS35-2、CNl232-9等高抗材料,其中AB94078-1干物率比徐薯18高9～10个百分点,淀粉率高6～7个百分点,防治效果高达98％,是一个兼具备高干物率与高抗性的优异材料。     

高淀粉多抗甘薯新品种徐薯25的选育及特性评价

高淀粉多抗甘薯新品种徐薯25利用高产综合性状较好的徐薯18为母本,高干高抗优异材料徐781为父本,通过人工控制授粉杂交、多点综合鉴定筛选育成;2007年通过国家甘薯新品种鉴定,鉴定编号为国品鉴甘薯200702;2008年通过山东省品种审定,审定编号为鲁农审2008040.通过对徐薯25特征特性、生产力、抗病性、品质性状以及产量形成的生理特点进行深入研究表明:鲜薯产量与高产兼用品种徐薯18持平,薯干产量比徐薯18增产15%以上,薯块干物质含量则比徐薯18高4.24%;徐薯25在总光合势、净同化率等方面与徐薯18相似,其获得高产主要依赖于光合产物合理的分配,物质生产属"经济系数型";徐薯25高抗茎线虫病、根腐病、蔓割病;薯块粗蛋白、薯块还原糖、薯块可溶性糖和叶片粗蛋白均高于徐薯18.     

抗病高淀粉甘薯品种徐薯26的选育及产量形成特点

抗病高淀粉甘薯新品种徐薯26是中国农业科学院甘薯研究所以徐781为母本,通过集团杂交放任授粉、多年多点综合鉴定选育而成。该品种2009年通过国家甘薯新品种鉴定,鉴定编号为国品鉴甘薯2009003。对徐薯26的形态特征、品质性状、抗病性、生产力和产量形成的生理特点等进行了研究,结果表明:徐薯26全生育期长势平稳,薯干品质和食味较好,抗根腐病和蔓割病,薯干和淀粉产量与徐薯18相比增产极显著,生长后期薯块干物质积累较快,经济系数较高,适宜早栽和密植。     

优质鲜食型甘薯新品种‘徐薯32’的选育及特性分析

甘薯新品种‘徐薯32’是以品质性状良好的‘徐薯55-2’为母本、‘红东’为父本,经有性杂交培育而成。该品种于2015年通过河南省农作物品种审定委员会审定。通过对‘徐薯32’农艺性状特征、生产力、抗病性、品质性状等进行研究,结果表明:‘徐薯32’地上部短蔓,薯块萌芽性好,耐贮藏,熟食口感佳;抗黑斑病,中抗根腐病与茎线虫病;鲜薯产量与对照‘徐薯22’相当,薯干和淀粉产量较对照分别增产15.24%和18.99%;‘徐薯32’块根主要营养物质含量均高于‘徐薯18’,其淀粉最高粘度值与崩解值较‘徐薯18’分别高6.52%和12.40%,糊化温度则略低于‘徐薯18’;淀粉粒径表面积与体积分布呈三峰、数目分布呈单峰,三类分布中淀粉颗粒总平均粒径均低于‘徐薯18’,降低幅度分别为12.22%、13.18%和2.22%。因此,‘徐薯32’具有良好的农艺性状与品质特性,可为鲜食型甘薯品种改良和应对市场需求奠定基础。     

高产淀粉型甘薯新品种徐薯27的选育及品质特性

高产淀粉型甘薯新品种徐薯27是以徐薯18为母本,选择遗传背景差异大的20个品种(系)为父本,放任授粉集团杂交后代中筛选。该品种2009年通过山东省农作物品种审定委员会审定。通过对徐薯27农艺性状特征、生产力、抗病性、品质特征性状以淀粉糊化特性等进行研究,结果表明:徐薯27萌芽性好,地上部长势平稳,耐贮藏;高抗根腐病,中抗茎线虫病;鲜薯和薯干产量对照徐薯18,最高增产分别达19.41%和19.21%,具有超高产潜力;徐薯27淀粉RVA谱为A型,不同肥料处理下RVA参数值差异明显,有机肥能明显降低其淀粉最高粘度值(PKV)、最低粘度值(HPV)和最终粘度值(CPV),糊化温度在较高的75～80℃范围。     

发根农杆菌诱导甘薯发根条件的优化

通过诱导甘薯(徐薯18)发根的条件进行优化,分别时侵染时间(10min、20min和30min)、培养基[MS、MSJ(MS BA0.1mg/L NAA0.2mg/L)和MSS(MS BA0.2mg/L NAA0.1mg/L)]、诱导材料(叶片、叶柄和茎切段)和除菌药物剂量(羧苄青霉素浓度0.5mg/L、1.0mg/L和1.5mg/L)进行优化.试验按L9(34)正交设计,每个处理3次重复.然后通过PCR检测徐薯18和其发根中的rol基因.结果表明用发根农杆菌A4经20min侵染,通过MSJ活化的徐薯18的茎切段生成发根的发根诱导率最高,最佳的除菌羧苄青霉素浓度为1.0mg/L.PCR检测证明得到的发根是发根农杆菌诱导产生的.在此优化条件下诱导的发根诱导率高,得到发根的可靠性强,且生成的发根粗壮、生长迅速、分支众多.     

甘薯近缘野生种的抗病性鉴定与新型种间杂种的获得

为了发掘甘薯近缘种抗茎线虫病、病毒病基因资源,改良栽培种抗性,拓宽甘薯遗传基础,本文利用田间自然病圃对30份甘薯近缘野生种进行抗茎线虫病鉴定,利用硝化纤维素膜-酶联免疫(NCM-ELISA)对20份野生种进行了抗病毒病筛选.以筛选到的抗性材料为父本、栽培种徐薯18为母本,采用生长调节剂处理方法进行种间杂交,结果得到8份高抗、10份抗茎线虫病野生种,5份高抗、8份抗病毒病材料;获得4个新型种间杂种.经过染色体计数和ISSR分子标记鉴定,证实了所得杂种的真实性.表明甘薯近缘野生材料中存在着栽培种所需要的抗性基因,通过有性杂交手段可以获得含野生种染色体组的种间杂种新类型,为渐渗野生抗性基因起到"桥梁"作用.     

Sporamin-mediated resistance to beet cyst nematodes (Heterodera schachtii Schm.) is dependent on trypsin inhibitory activity in sugar beet (Beta vulgaris L.) hairy roots

Sporamin, a sweet potato tuberous storage protein, is a Kunitz-type trypsin inhibitor. Its capability of conferring insect-resistance on transgenic tobacco and cauliflower has been confirmed. To test its potential as an anti-feedant for the beet cyst nematode (Heterodera schachtii Schm.), the sporamin gene SpTI-1 was introduced into sugar beet (Beta vulgaris L.) by Agrobacterium rhizogenes-mediated transformation. Twelve different hairy root clones expressing sporamin were selected for studying nematode development. Of these, 8 hairy root clones were found to show significant efficiency in inhibiting the growth and development of the female nematodes whereas 4 root clones did not show any inhibitory effects even though the SpTI-1 gene was regularly expressed in all of the tested hairy roots as revealed by northern and western analyses. Inhibition of nematode development correlated with trypsin inhibitor activity but not with the amount of sporamin expressed in hairy roots. These data demonstrate that the trypsin inhibitor activity is the critical factor for inhibiting growth and development of cyst nematodes in sugar beet hairy roots expressing the sporamin gene. Hence, the sweet potato sporamin can be used as a new and effective anti-feedant for controlling cyst nematodes offering an alternative strategy for establishing nematode resistance in crops.     

发根农杆菌介导药用甘薯西蒙1号的遗传转化

用发根农杆菌A4分别感染药用甘薯西蒙1号的叶片、茎切段、叶柄等外植体,诱导出毛状根,并对毛状根进行了离体培养.采用L9(34)正交设计法优化甘薯西蒙1号的毛状根诱导条件;PCR扩增检测转化毛状根;用高效液相色谱仪检测了毛状根中咖啡酸的含量.结果表明:转化中茎切段是最合适的外植体,最佳感染时间20 min,共培养最佳时间为2天;PCR扩增检测表明发根农杆菌Ri质粒的T-DNA片段已整合进植物的基因组中;经高效液相色谱仪证实毛状根中含有咖啡酸,含量为0.03792 mg/g.     

Effects of potassium deficiency on root morphology,ultrastructure and antioxidant enzyme system in sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> [L.] Lam.) during early growth

Potassium (K⁺) deficiency is an important abiotic stress which has severe influence on the growth and development of sweet potato. To investigate the difference on root morphology, ultrastructure and antioxidant enzyme system at early growth stage, two representative sweet potato cultivars (Ipomoea batatas [L.] Lam.) with different K⁺ deficiency tolerance capacities were hydroponically cultivated under normal K⁺ (control) and K⁺ deficiency (?K) treatments. The results showed that after 14 days of treatment, the root length, surface area, volume, and average diameter of Ningzishu 1 (sensitive to K⁺ deficiency) were significantly decreased under ?K treatment, by comparison, those corresponding decreases of Xushu 32 (tolerant to K⁺ deficiency) were lower and not significant (except for root length). In addition, the proportion of fine roots (diameter <0.5 mm) and thick root (d > 1.0 mm) of Xushu 32 seedlings increased significantly under condition of K⁺ deficiency. Through transmission electron microscopy observations, it can be found that Xushu 32 seedlings showed a more complete root cellular structure and slighter oxidative damage than Ningzishu 1 under ?K treatment. Moreover, the hydrogen peroxide content and malondialdehyde content in the root of Xushu 32 seedlings was significantly lower than that of Ningzishu 1, and the variations of superoxide dismutase, peroxidase, and catalase activities were pronouncedly less than those of Ningzishu 1. These differences between the two cultivars indicated that the stronger root morphology and nutrients absorption ability, and the better root cellular structure and physiological role of Xushu 32 seedlings could alleviate the damage of K⁺ deficiency stress. Thus, it might be a potential mechanism for cultivar tolerance to low K⁺ in sweet potato.     

Ri质粒介导的毛状根体系建立及其在植物次生代谢产物合成中的研究进展

发根农杆菌Ri质粒可诱导植物产生毛状根体系,该体系具有遗传性状稳定且增殖速度快的特点,可用于药用植物次生代谢产物的生产研究,为利用生物反应器技术进行药用植物有效成分工业化水平的发酵培养开辟了新途径。本文主要综述了发根农杆菌Ri质粒介导的植物毛状根体系遗传转化机理,并对毛状根体系在药用植物次生代谢产物生产中的研究现状进行了深入分析,为从基因水平上调控植物次生代谢产物的合成提供新思路。     

产生光活化杀虫剂α-三连噻吩发根培养体系的建立

以万寿菊 (Tagetes erecta L .)幼嫩健壮无菌叶片为材料 ,与发根农杆菌 (Agrobacteriumrhizogenes) R16 0 1共培养 ,成功地诱导并建立起了万寿菊发根离体培养体系。利用 HPL C C18反相柱分析万寿菊发根中 α-三连噻吩的含量 ,发现其含量在 6 .10～ 7.73μg/ g.fw之间 ;还研究了万寿菊发根低温种质保存试验 ,发现发根可以在 4°C条件下保存 10 W而不影响其生活力。     

青蒿转杜松烯合成酶基因发根系的培养

将已克隆的棉花杜松烯合成酶的ｃＤＮＡ（ｃａｄＣ１４）插入到植物表达载体ｐＢＩ１２１中,构建含ＣａＭＶ３５Ｓ启动子驱动下的杜松烯合成酶基因的植物表达载体ｐＢＩＣ１４。用含ｐＢＩＣ１４质粒的发根农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｒｈｉｚｏｇｅｎｅｓ）１５８３４感染青蒿（ＡｒｔｅｍｉｓｉａａｎｎｕａＬ．）叶片并诱导发根,共建立１２１个生长迅速的发根系。经浓度为２０ｍｇ／Ｌ的Ｋａｎ筛选,获得１２个抗Ｋａｎ阳性根系。ＰＣＲ和Ｓｏｕｔｈｅｒｎｂｌｏｔｉｎｇ分析表明,外源杜松烯合成酶基因已整合到青蒿基因组中,其转基因频率为３％。ＲＴＰＣＲ分析表明,外源杜松烯合成酶基因在Ｃ３７根系中,在转录水平上已有表达。     

Transformation of Gynostermma pentaphyllum by Agrobacterium rhizogenes and Saponin Production in Hairy Root Cultures

Authors report here the establishment of an efficient transformation system for Gynosternrna pentaphyllum (Thunb.) Makino using Agrobacteriurn rhizogenes R1600. Hairy roots appeared on leaf explants 10 days after inoculation with the bacteria . Frequency of the explants transformed by R1600 was up to 94%. Transformation was confirmed by Southern analysis. Biomass of hairy root cultures suspended in hormone-free MS medium increased 9 times after 20 days of incubation. There was no callus formation on the hairy roots during suspension culture. Saponin content in the hairy root cultures was about 2 times as much as in the natural roots, saponins of the hairy root cultures were also released into growth medium as well.     

In vitro culture of the obligate parasite Spongospora subterranea (cercozoa; plasmodiophorida) associated with root-inducing transferred-DNA transformed potato hairy roots

Spongospora subterranea is a soil-borne, obligate parasitic protist that causes powdery scab of potatoes. In this study, an in vitro culture system was developed for the maintenance and proliferation of the protist in potato hairy roots. The hairy roots of potato were induced in vitro with Agrobacterium rhizogenes. Cystosori of S. subterranea from potato scab lesions were surface disinfested and used to inoculate potato hairy roots. Plasmodia, zoosporangia, and cystosori were observed microscopically in the hairy roots within 6 wk after inoculation, indicating the completion of the life cycle of S. subterranea in vitro. This is the first in vitro culture system for S. subterranea, and will be a valuable tool to study fundamental and practical aspects of the biology of the parasite.