发根农杆菌介导的尾巨桉遗传转化体系的建立

发根农杆菌（Agrobacterium rhizogenes）的建立对植物功能基因的验证具有重要意义,为了在桉树（Eucalyptus）中建立发根农杆菌介导的遗传转化体系,本研究以不同的发根农杆菌菌株侵染尾巨桉（Eucalyptus urophylla × E. grandis）的叶片和茎段,确定合适的农杆菌菌株和外植体类型,在此基础上开展农杆菌浓度、侵染时间对毛状根诱导的影响。结果表明：采用发根农杆菌菌株MSU440,以叶片为外植体进行发根诱导,最高获得了81.0%的毛状根诱导率,毛状根平均根长达到3.23 cm。在发根农杆菌浓度为OD₆₀₀=0.3、侵染时间为30 min时,共培养48 h后经过20 mg·L^-1卡那霉素筛选培养,通过PCR分子鉴定和GUS染色证实外源基因稳定地整合在桉树毛状根基因组中,转化率达20.2%。初步建立了发根农杆菌介导的桉树遗传转化体系,为桉树基因功能鉴定和进一步的转基因育种奠定基础。     

纤维植物罗布麻发根的诱导及植株再生

利用3种发根农杆菌(LBA9402.R601,和R1000)转化纤维植物罗布麻无菌种子苗的根茎叶不同外植体部位,首次诱导其生成发根并实现了直接由发根途径的植株再生.罗布麻发根诱导与所用的发根农杆菌菌株,外植体部位及光周期密切相关.发根农杆菌LBA9402感染罗布麻的根外植体,实现了最高转化率达100%.与LBA9402及R601相比,被发根农杆菌R1000感染的根外植体适合在黑暗环境下培养.其诱导生成的发根密度可达平均每个外植体22条.在不加激素的1/2 MS培养基上,LBA9402和R601诱导产生的发根可以诱导生成不定芽,不定芽诱导率达20%.不定芽切下后,在不加激素的1/2 MS培养基上2周内可以诱导生根.通过聚合酶链式反应(PCR)对发根及再生植株进行了鉴定,证明发根农杆菌的T-DNA插入了植物的基因组.为罗布麻的分子育种建立了稳定的转化及再生体系,为下一步通过转入外源基因改善其农艺性状奠定了基础.     

4种发根农杆菌对朱砂根组培无菌叶片毛状根诱导的影响

以朱砂根(Ardisia crenata Sims)组培无菌叶片为材料,用4种发根农杆菌菌株(A4、ATCC15834、LBA9402和R1601)分别侵染进行毛状根诱导,比较朱砂根叶片毛状根诱导的最适培养基种类、预培养时间、侵染方式、共培养时间以及不同发根农杆菌的致根能力。研究表明:(1)朱砂根无菌叶片毛状根诱导最适培养基为1/2MS培养基,预培养2d、共培养2d,毛状根诱导率最高(31.87%)。(2)最佳侵染方式以剪好的幼叶和活化好的菌液(100mg/L AS)一起在28℃、180r/min黑暗条件下共振荡8~15min。(3)4种发根农杆菌均能诱导朱砂根叶片毛状根产生,但A4、ATCC15834效果最好,其致根能力大小顺序依次为ATCC15834A4LBA9402R1601。(4)PCR分子鉴定表明,发根农杆菌Ri质粒T-DNA已成功整合到宿主细胞核基因组中。     

木豆毛状根的诱导及其培养条件的研究

利用发根农杆菌LBA9402对木豆叶片直接进行诱导产生毛状根。本实验研究出诱导木豆毛状根的最佳条件是,以木豆叶片为外植体,于1/2MS固体培养基上预培养2~4 d,菌液浓度OD₆₀₀=0.6~0.8,浸染20 min,共培养3 d,诱导率为60.00%。在分子水平用PCR检测表明,发根农杆菌9402Ri质粒上的T-DNA成功整合进木豆毛状根的基因组中。     

少花龙葵毛状根的诱导和次生代谢物的产生

研究了发根农杆菌（Agrobacterium rhizogenes)对少花龙葵（Solanum photeinocarpum)的转化和毛状根的诱导,分析了影响转化的因素,并初步检测了毛状根的生长和次生代谢物的产生。发根农杆菌菌株R1000、R1601分别感染少花龙葵的叶片、茎段,约7d后得到毛状根。菌株R1000感染的外植体的生根率分别为90．2％和50．0％,根诱导频率分别为6．2和4．6;菌体R1601感染的外植体的生根率分别为92．1％和49．2％,根诱导频率分别为6和5．5;对照两周内不出根。冠瘿碱检测证实所得毛状根为转化根。感染在MS＋PP333培养基上生长的矮壮苗叶片,毛状根诱导频率显著提高;用MS液体培养基稀释1倍的菌液感染叶片,转化效率也得到提高。毛状根生长速度快,培养4周后干重增加420倍,总糖苷生物碱和皂甙含量分别为原植株根的31和107倍。     

新疆雪莲毛状根的诱导及其植株再生体系的建立

利用发根农杆菌R1601、R1000、LBA9402感染新疆雪莲的叶片、叶柄和根段外植体,诱导产生毛状根。毛状根接种量为2.8 g/L(FW)时,20d生长量可达66.7 g/L,黄酮含量达到干重的10.23%。冠瘿碱的检测和rolB基因的PCR分析表明,Ri质粒中的T_DNA片段已经整合到毛状根细胞的基因组中。预培养时间、外植体类型以及发根农杆菌的菌株属性对毛状根诱导有着重要的影响。其中预培养2 d的新疆雪莲根段外植体,经过R1601感染后,毛状根的诱导率可达100%。诱导产生的毛状根在附加生长素的液体培养基中,有少量愈伤组织产生。由毛状根再生的植株与雪莲外植体再生的植株在形态上无明显区别,但前者的黄酮含量仅为后者的53%。     

Ri质粒介导GUS基因转化商陆的研究

发根农杆菌Ar1334工程菌株与商陆叶片共培养,诱导效率不同程度地受到发根农杆菌生理状态、浓度、浸泡时间、共培养时间的影响:采用稀释5倍的处于对数生长期的Ar1334工程菌株,感染商陆无菌苗幼嫩叶圆片5min,在MS培养基上共培养2d,可达最大转化效率82.2%;诱导的根在附加20mg·L-1Kan的MS无激素MS培养基上,呈现旺盛的生长态势和典型的发状根结构特点,经PCR检测扩增出了560bp的rolC基因的目的片断,组织化学检测也证实了GUS基因在发状根中的表达.     

胡杨转基因体系的建立

胡杨(Populus euphratica)是唯一能在沙漠里生长的高大乔木树种, 建立其转基因体系可为胡杨抗逆分子机制与应用技术研究提供基本方法。通过研究农杆菌介导的胡杨转GUS基因的技术体系得出以下结论: (1) 胡杨再生植株体系, 叶片在附加1.0 mmol.L^-1 6-BA和5.0 mmol.L^-1 NAA的1/2MS培养基上不定芽诱导率较高; (2) 转基因体系, 胡杨叶片在含100 mmol.L^-1 乙酰丁香酮的OD600值为0.4-0.6的根癌农杆菌菌液中浸染15分钟, 共培养2天, GUS基因转化效率较高; (3) 转基因植株抗生素筛选, 转GUS基因胡杨叶片用300 mg.L^-1头孢霉素抑制农杆菌生长, 在含9 mg.L^-1 G418的培养基上诱导不定芽以获得转基因的抗性植株。     

重金属超富集植物东南景天毛状根的诱导

为建立重金属超富集植物东南景天(Sedum alfredii)的毛状根诱导体系,采用发根农杆菌(Agrobacterium rhizogenes)A4侵染叶片,研究了预培养时间、侵染时间和共培养时间对毛状根诱导率的影响。结果表明,东南景天叶片外植体的预培养时间为48 h、农杆菌侵染时间为6 min、共培养时间为48 h是适宜的毛状根诱导时间,毛状根的诱导率可达85%。PCR检测表明诱导的毛状根中存在rol B基因片段。这是东南景天首次建立用发根农杆菌诱导毛状根体系。     

银柴胡离体培养与毛状根诱导技术初步研究

以银柴胡茎段为外植体,经消毒获得无菌再生材料后,筛选发根农杆菌介导毛状根诱导产生的最适条件。结果显示：最适的无菌消毒方法为：70%酒精浸5 s,0.1%升汞消毒3 min,获得了银柴胡离体培养材料;以发根农杆菌A4菌株介导的银柴胡毛状根诱导过程中,与叶片和不带腋芽茎段相比,带腋芽茎段为最适转化外植体,用OD600=0.8的菌液侵染茎段15 min,共培养3 d,800 mg/L头孢噻肟钠除菌,其诱导率及诱导密度最高,分别为100%和4.7,为最适诱导条件。研究结果说明在适合条件下,银柴胡带腋芽茎段适于诱导毛状根。     

典型黑土区主要树种根系构型特征及其对固土能力的影响

为量化典型黑土区主要树种根系构型特征,探究其对固土能力的影响,以该区分布较广的榆叶梅、小叶锦鸡儿、白桦、糖槭、红皮云杉、樟子松单株个体为研究对象,采用全根挖掘和WinRHIZO Pro LA2004分析系统相结合对其根系空间分布、几何形态、分形等特征进行测定,同时采用原位整株根系拉拔的方法量化根系垂直拉拔力。结果表明: 榆叶梅以倾斜根为主,小叶锦鸡儿、白桦、糖槭和红皮云杉以水平根为主,樟子松根系在水平和垂直分布上较为均衡;除白桦总根表面积和红皮云杉总根长外,灌木树种总根长、总根表面积显著大于乔木,落叶阔叶乔木总根长、总根表面积显著大于针叶常绿乔木,白桦总根体积显著大于小叶锦鸡儿、糖槭、红皮云杉和樟子松;榆叶梅、小叶锦鸡儿和白桦根系分形维数和分形丰度显著大于红皮云杉和樟子松;榆叶梅、小叶锦鸡儿和糖槭整株根系平均最大垂直拉拔力显著大于白桦、樟子松和红皮云杉。主要受根系总根长、总根表面积和倾斜根数量的影响,榆叶梅、小叶锦鸡儿和糖槭根系表现出较强的固土能力,可作为典型黑土区水土保持植被构建中优先选择的树种。     

Reduced irradiance and applied auxin influence carbohydrate relations in Pinus banksiana cuttings during propagation

Untreated and indole-3-butyrie acid-treated (IBA) cuttings from 90-day-old Pinus banksiana Lamb, stock plants were propagated under normal greenhouse irradiance (max. 900 $$mol m^-2 s^-1) and shade (max. 120 $$mol m^-2 s^-1) to determine effects on adventitious rooting and on reducing sugar and starch concentrations in needles and basal stems. In one experiment, cuttings were assessed at days 15 and 25 of propagation for basal 1-cm stem fresh weight, proportion rooted, number of roots and longest root length. In a second experiment with cuttings, basal 1-cm stem fresh weight and concentrations of reducing sugar and starch in needles and basal stems were measured each day for the first 10 days of propagation. Carbohydrate measurements were also made for seedling stock plants as controls for the second experiment. Carbohydrate data for cuttings were primarily evaluated based on net (cutting minus seedling) concentrations, to correct for changes in cuttings not related to adventitious rooting. Increase of basal stem fresh weight and rooting of cuttings, based on all measured variables, occurred in the order: light + IBA > light > shade + IBA > shade. The best rooting required the greater irradiance. Compared to results from cuttings in the light, shading resulted in lesser accumulations of reducing sugars and starch in needles and basal stems. Reducing sugar: starch concentration ratios were significantly greater in shade- vs light-propagated cuttings, IBA treatment did not offset the effects of shade on rooting or on reducing sugar and starch concentrations or ratios. Overall, the results suggested that decreased reducing sugar and starch concentrations and/or their increased ratios are associated with shade-induced poor rooting of P. banksiana cuttings.     

Transformation of Saussurea involucrata by Agrobacterium rhizogenes: Hairy root induction and syringin production

Agrobacterium rhizogenes-mediated genetic transformation of Saussurea involucrata was investigated. Four bacterial strains, A4, LBA 9402, R1000 and R1601 and three explant types, leaf blade, petiole and root, were examined. Over 100 hairy root lines were successfully established with strains R1601, R1000 and LBA9402, but none with A4. The highest transformation efficiency of 67% was achieved by using strain R1601 with root explants. One hairy root line isolated from this combination, HR1601-1, produced up to 43.5 ± 1.13 mg syringin g⁻¹ dw, which is about 50-fold higher than that in the wild type plants.Two other lines, HR1000-1 and HRLBA9402-1, isolated from R1000- and LBA9402-transformed roots, respectively, also displayed high capacity of syringin production, being 32.5 ± 3.08 and 39.7 ± 1.37 mg syringin g⁻¹ dw. These three lines were characterized in detail. Polymerase chain reaction analyses confirmed these root lines were of A. rhizogenes origin.     

茶树嫩枝扦插的高效方法

茶(Camellia sinensis)是世界上最重要的饮料作物之一, 随着种植面积的扩大, 茶苗的需求量也日益增加。传统的扦插育苗方式存在着生根难、周期长和取材难等问题, 因此优化扦插生根的方法十分重要。该研究以较易获得、但传统方法难以生根的绿色嫩枝为扦插材料, 首先对培养介质进行改良。与土培和水培相比, 利用海绵培养可以使茶树幼嫩插穗在1个月之内快速生根, 生根率达32.2%。其次, 对海绵培方法做进一步优化, 确定一芽一叶的幼嫩短穗生根潜力更佳; 同时, 添加生根粉能够促进茶树茎部愈伤组织与根系的形成, 其中1.25 g∙L ^-1生根粉处理48小时对茶树扦插快速生根最有效, 生根率达42.0%。综上, 通过优化培养介质和扦插材料以及适当添加生根粉等措施, 建立了一种茶树高效嫩枝扦插生根的方法。该方法能够显著缩短嫩枝插穗的生根时间, 突破了扦插材料的限制, 有效降低了扦插成本, 具有重要的应用前景。     

茶树嫩枝扦插的高效方法

茶(Camellia sinensis)是世界上最重要的饮料作物之一, 随着种植面积的扩大, 茶苗的需求量也日益增加。传统的扦插育苗方式存在着生根难、周期长和取材难等问题, 因此优化扦插生根的方法十分重要。该研究以较易获得、但传统方法难以生根的绿色嫩枝为扦插材料, 首先对培养介质进行改良。与土培和水培相比, 利用海绵培养可以使茶树幼嫩插穗在1个月之内快速生根, 生根率达32.2%。其次, 对海绵培方法做进一步优化, 确定一芽一叶的幼嫩短穗生根潜力更佳; 同时, 添加生根粉能够促进茶树茎部愈伤组织与根系的形成, 其中1.25 g?L ^-1生根粉处理48小时对茶树扦插快速生根最有效, 生根率达42.0%。综上, 通过优化培养介质和扦插材料以及适当添加生根粉等措施, 建立了一种茶树高效嫩枝扦插生根的方法。该方法能够显著缩短嫩枝插穗的生根时间, 突破了扦插材料的限制, 有效降低了扦插成本, 具有重要的应用前景。     

Accumulation of humic acid and fulvic acid during root humification of three diameters of two dominant subalpine trees in western Sichuan,China

Aims Plant roots store large amount of terrestrial carbon, but little is known about humus formation processes during the decomposing root litter. Compared with coarse roots, fine roots have greater nutrients, which may be favorable to humus formation. The objective of the study was to examine how root diameters affect their humus formation processes. Methods In this study, in order to examine the accumulation of humic acid and fulvic acid of three root diameter classes (0-2, 2-5 and 5-10 mm) of two subalpine tree species (Abies faxoniana and Picea asperata) on the eastern Qinghai-Xizang Plateau of China, a two-year field experiment was conducted using a litter-bag method. Air-dried roots of A. faxoniana and P. asperata were placed in litterbags and incubated at 10 cm of soil depth in October 11th, 2013. Duplicate litter bags were collected in May (late winter) and October (late in the growing season) of 2014 and 2015, respectively. Concentrations of humic acid and fulvic acid were measured, and net accumulations were calculated for different periods. Important findings The concentrations of humic acid and fulvic acid were significantly influenced by root diameter that humic acid and fulvic acid decreased with increase in root diameter. Root diameter had significant effects on the net accumulation of humic acid, but not for the accumulation of fulvic acid. However, there were no significant differences in both humic acid and fulvic acid between A. faxoniana and P. asperata roots. Regardless of tree species, humic acid degraded during the winter but accumulated during the growing season. After two years of decomposition, the net accumulations of humic acid in 0-2, 2-5 and 5-10 mm roots were 8.0, 10.8 and 7.6 g·kg^-1 for P. asperata and 15.2, 8.0 and 7.8 g·kg^-1 for A. faxoniana, respectively. Conversely, the degradation of fulvic acid in 0-2, 2-5 and 5-10 mm roots were 178.0, 166.0 and 118.0 g·kg^-1 for P. asperata and 170.0, 160.0 and 128.0 g·kg^-1 for A. faxoniana, respectively. Our results suggest that diameter-associated variations in substrate quality could be an important driver for root litter humification in this subalpine forest. Moreover, diameter effect is dependent on decomposition period in this specific area.     

利用CRISPR/Cas9双基因敲除系统初步解析大豆GmSnRK1.1和GmSnRK1.2对ABA及碱胁迫的响应

蔗糖非发酵相关激酶(sucrose non-fermenting related protein kinases, SnRKs)是广泛存在于植物中的一类Ser/Thr蛋白激酶,在植物的生长、发育、代谢和抗逆等方面具有重要调节作用。大豆(Glycine max L.)基因组中含有4个SnRK1同源基因,其中GmSnRK1.1和GmSnRK1.2为两个主要表达基因,可能参与大豆多种抗逆途径。为解析大豆GmSnRK1.1和GmSnRK1.2对ABA及碱胁迫的响应,本研究构建了双靶点CRISPR载体定向敲除GmSnRK1.1和GmSnRK1.2基因,利用发根农杆菌(Agrobacterium rhizogenes)介导大豆遗传转化,获得双基因敲除突变体毛状根,经测序鉴定双基因突变率为48.6%;同时,利用实验室前期构建的植物超量表达载体获得超量表达GmSnRK1基因大豆毛状根。经25 μmol/L ABA处理15 d,对照组和超量表达毛状根的生长受到明显抑制,其根长与根鲜重均显著低于双基因敲除突变体毛状根;经50 mmol/L NaHCO₃处理15 d,对照组和双基因敲除突变体毛状根的生长受到明显抑制,其根长与根鲜重均显著低于超量表达毛状根。本研究建立的CRISPR/Cas9系统能够有效地对大豆进行GmSnRK1.1和GmSnRK1.2双基因敲除,基因敲除突变降低了植物对ABA的敏感性及对碱胁迫的耐性,研究结果初步说明SnRK1激酶在植物响应非生物胁迫中具有重要作用。     

培育年限对刺五加主要药用活性成分的影响

为评价不同培育年限对刺五加根、茎部多种活性成分积累的共同影响,对提高刺五加培育和高值利用具有重要作用,可为规范化种植和合理开发利用刺五加提供理论依据。采集同一产地三年生、五年生、九年生刺五加为实验样本,运用超高效液相色谱系统（Ultra-Performance LC,Waters,Japan）分析不同培育年限对刺五加根、茎中槲皮苷、金丝桃苷、芦丁、紫丁香苷、刺五加苷E、异嗪皮啶6种活性成分含量的同时影响。结果表明,五年生刺五加根、茎中6种主要活性成分的综合得分最高。其中,黄酮类成分槲皮苷在三年生根中含量最高,在五年生茎中含量最高;芦丁在五年生根及茎中含量均最高,在根中三年生含量最低,九年生茎中含量最低;金丝桃苷在五年生根及三年生茎中含量最高。苯丙素类成分异嗪皮啶在三年生根及茎中含量最高,在五年生根和茎中含量最低;紫丁香苷在九年生根中含量最高,在五年茎中含量最高;刺五加E在五年生根中含量最高,在三年生茎中含量最高。不同药用成分在不同生长阶段的刺五加根和茎中积累不同,定向培育可根据目的活性成分选择适合的采收年限。     

群落演替对呼伦贝尔草地两种优势植物繁殖分配及生态化学计量的影响

为了探究草地群落演替过程中不同优势种的生理生态适应性,本研究采用空间代替时间序列的方法,分析呼伦贝尔草地米氏冰草群落—米氏冰草+冰草群落—冰草群落演替系列的土壤养分、两优势种的生物量及C、N和P含量。结果表明: 随着米氏冰草群落—米氏冰草+冰草群落—冰草群落的演替,土壤全碳、全氮、速效氮及速效磷含量均显著升高;米氏冰草和冰草叶、茎和根的N、P含量及N/P均显著升高,而各构件的C/N降低;米氏冰草叶的C含量及冰草叶、茎和根的C含量均显著升高。米氏冰草叶C/P及冰草根和叶C/P均显著升高,而米氏冰草根和茎C/P及冰草茎C/P均显著下降。在米氏冰草+冰草共优势种群落中,米氏冰草通过降低茎和根C含量,增加叶C的积累,提高其种间竞争力, 而冰草通过降低根冠比和生殖比来适应环境变化。米氏冰草在不同群落中均受N限制(N/P<14);而冰草在单优势种群落中受P限制(N/P>16),在共优势种群落中受N和P限制(14相似文献