铁皮石斛四倍体离体诱导和鉴定及生理特性研究

以铁皮石斛原球茎为材料,经不同质量浓度的秋水仙素(C₂₂H₂₅O₆)和0.02 g·mL^-1二甲基亚砜(DMSO)混合水溶液处理后进行组织培养,通过对变异株进行形态学、细胞学及流式细胞仪鉴定,以期获得稳定的四倍体植株并分析其生理特性。结果表明:用2.0 g·L^-1秋水仙素和0.02 g·mL^-1 DMSO混合水溶液处理铁皮石斛原球茎36 h后,植株诱导率达20%;诱导四倍体植株在形态上明显矮化、茎秆粗壮、叶片变小增厚、气孔直径增大;细胞遗传学观察发现,四倍体植株染色体2n=4x=76,二倍体植株染色体2n=2x=38;流式细胞仪分析显示,DNA相对含量四倍体为400,二倍体仅为200;四倍体植株叶片中叶绿素含量、可溶性蛋白、可溶性糖含量均高于二倍体,分别为5.03、3.59、2.98 mg·g^-1;四倍体叶片中主要抗氧化酶POD和SOD活性均显著高于二倍体,分别为9.08、180.4 U·mg^-1,且四倍体植株明显降低了MDA含量累积。研究认为,2.0 g·L^-1秋水仙素和0.02 g·mL^-1 DMSO混合水溶液处理原球茎36 h可提高诱导成功率、降低嵌合体比例,此组合为诱导四倍体较佳诱导条件。     

氮离子注入对蒙古黄芪多倍体诱导的影响

利用二倍体蒙古黄芪种子为材料,以低能氮离子束为诱变源,将化学诱导与物理诱变相结合,探索出一套高效的多倍体诱导新方法。研究结果表明：氮离子注入种子后表现出明显的生物学效应;氮离子注入与秋水仙素联合诱导黄芪多倍体的效果很明显。氮离子注入剂量为2.6×10¹⁶ N⁺/cm²,秋水仙素浓度为100 mg·L^-1,培养5 d诱导率最高为44.4%;氮离子注入剂量为5.2×10¹⁶ N⁺/cm², 秋水仙素浓度为150 mg·L^-1,培养10d的诱导率最高为46.2%;二者均高于对照组秋水仙素浓度为100 mg·L^-1培养15 d的最高诱导率13.9%。利用细胞染色体计数鉴定多倍体为四倍体。     

野生南荻与芒杂种多倍体诱导研究

用不同浓度秋水仙素处理野生南荻×芒(Miscanthus lutarioriparia×Miscanthus sinensis)远缘杂交后代以诱导产生多倍体,并对变异株进行形态学和细胞学鉴定,以期获得稳定的四倍体植株并分析其生理特性。结果表明:(1)采用秋水仙素加入培养基处理法和秋水仙素溶液浸泡处理法都可获得一定频率的多倍体植株;胚性愈伤组织以0.2%秋水仙素浸泡处理48h的诱变效果较好,四倍体诱导率达8.7%;芽在0.05%秋水仙素培养基中处理15d较好,四倍体诱导率达10.6%;生根苗在0.1%秋水仙素培养基中处理10d较好,四倍体诱导率达11.1%。(2)经体细胞染色体计数,加倍植株染色体数为2n=4x=76,对照植株的染色体数目为2n=2x=38。(3)生长2年的多倍体植株形态、叶片大小、茎粗、茎壁厚、节间等性状表现出巨大性和超亲优势。     

Influence of Lime and Organic Matter on the Mobility of Cadmium in Cadmium-Contaminated Soil in Relation to Nutrition of Spinach

Cadmium and cadmium compounds are water soluble, mobile in most soils, bio-available, and tend to bio-accumulate. A pot culture experiment was conducted on contaminated soil to study the influence of lime and organic matter on the mobility of cadmium in spinach and its rhizosphere soil. Application of lime (50% and 100% lime requirement) and organic matter (0.5 and 1% by weight of soil) to soil decreased the availability of Cd to the soil and plant throughout the crop growth. The highest diethylene triamine penta-acetic acid (DTPA) extractable Cd was 10.84 mg kg^?1 in the treatment OM₀ L₀ (No application of organic matter and lime) at 20 days after sowing of spinach. Likewise, the highest Cd concentration in spinach roots and shoots were 19.80 and 17.0 mg kg^?1 in the treatment OM₀ L₀ at 20 days after sowing. The Cd concentration in spinach roots and shoots were decreased by 63.23 and 71.88%, respectively, in the treatment OM₁ L₁₀₀ (application of FYM at 1.0% by weight of soil and lime at 100% lime requirement) after 60 days of growth. The lowest concentrations of Cd in the soil and plant after the harvest of the crop were 2.88 and 4.27 mg kg^?1, respectively, in the treatment OM₁ L₁₀₀ and resulted in 65.75 and 71.55% decrease over control (OM₀ L₀). The highest total chlorophyll content of leaves was 2.19 mg kg^?1 of fresh weight in the treatment OM₁ L₁₀₀ at 40 days of crop growth.     

In vitro induction of tetraploid in pomegranate (Punica granatum)

Tetraploid plants were obtained in pomegranate (Punica granatum L. var. `Nana') by colchicine treatment of shoots propagated in vitro. Shoots cultured on MS medium supplemented with 10 mg l^–1 colchicine, 1.0 mg l^–1 BA and 0.1 mg l^–1 NAA for 30 days produced tetraploids at a high frequency of 20%. No tetraploids were detected by treating the shoots in 5000 mg l^–1 colchicine for 114 h. Shoots treated by 5000 mg l^–1 colchicine for 96 h produced three morphological mutants with narrow leaves, which were later confirmed as mixoploids that separated into diploids and tetraploids after further subculture. In vitro tetraploid plants had shorter roots, wider and shorter leaves than the diploid ones. Tetraploid pomegranate plants grew and flowered normally in pots, but possessed flowers with increased diameter and decreased length compared to diploids. The number of pollen grains per anther was higher in tetraploids, but the viability of pollen decreased significantly.     

Growth characteristics and catalpol production in chinese foxglove (Rehmannia glutinosa Liboschitz) hairy roots transformed withAgrobacterium rhizogenes ATCC15834

Hairy root clones ofRehmannia glutinosa were established via transformation withAgrobacterium rhizogenes ATCC15834. To optimize the culturing conditions for both root growth and catalpol production, effects of various combinations of seven basal media, pH, and carbon sources were examined under darkness. The fastest root growth was obtained in an SH medium containing 4% sucrose (pH 5.8); the highest catalpol content (0.54% of dry weight) was achieved in a WPM medium supplemented with 4% sucrose (pH 5.8). Effects of plant growth regulators and chitosan were also investigated. IAA at 2 mg L^-1 significantly increased root lengths and the frequency of lateral roots. Chitosan (50 mg L^-1) and CA₃ (0.5 mg L^-1) induced catalpol production, with contents calculated at 0.7% dry weight and 0.65% dry weight, respectively.     

Effects of humic acid-herbicide interactions on the growth of Pisum sativum in nutrient solution

Slight increases in root length and dry matter production were measured in roots and shoots of Pisum sativum seedlings, when germinated seeds were grown for eight days in a controlled environment chamber, on Nitch nutrient solution to which 10 mg L^-1 soil humic acid (HA) had been added. A concentration of 100 mg L^-1 of HA produced a small reduction in dry matter production of shoots. In the presence of herbicides, 10 mg L^-1 cycluron or 100 mg L^-1 prometone, root elongation and shoot growth was reduced by varying degrees with respect to the control. Nutrient solutions containing 100 mg L^-1 cycluron, 10 or 100 mg L^-1 alachlor, or as little as 0.1 mg L^-1 of 2,4-D resulted in an almost complete suppression of the growth of pea seedlings. The addition of 10 or 100 mg L^-1 of HA to nutrient solution containing the herbicide was found to be either ineffective in enhancing growth, or even further reduced growth, compared to samples grown in the presence of the herbicide alone. The results of this preliminary study suggest that the generally accepted view that humic substances exert a positive effect on plant growth may not occur when humic substances interact with herbicides.     

Effects of macro elements and nitrogen source on adventitious root growth and ginsenoside production in ginseng (Panax ginseng C. A. Meyer)

We investigated the effects on ginseng adventitious root growth and ginsenoside production when macro-element concentrations and nitrogen source were manipulated in the culture media. Biomass growth was greatest in the medium supplemented with 0.5-strength NH₄PO₃, whereas ginsenoside accumulation was highest (9.90 mg g^-1 DW) in the absence of NH₄PO₃. At levels of 1.0-strength KNO₃, root growth was maximum, but a 2.0 strength of KNO₃ led to the greatest ginsenoside content (9.85 mg g^-l). High concentrations of MgSO₄ were most favorable for both root growth and ginsenoside accumulation (up to 8.89 mg g^-1 DW). Root growth and ginsenoside content also increased in proportion to the concentration of CaCI₂ in the medium, with the greatest accumulation of ginsenoside (8.91 mg g^-1 DW) occurring at a 2.0 strength. The NH₄/NO₃ ^-- ratio also influenced adventitious root growth and ginsenoside production; both parameters were greater when the NO₃ ^- concentration was higher than that of NH₄ ⁺. Maximum root growth was achieved at an NH₄ ⁺/NO₃ ^- ratio of 7.19/18.50, while ginsenoside production was greatest (83.37 mg L^-1) when NO₃ ^- was used as the sole N source.     

Aluminium-induced morphogenic and biochemical variations ofBacopa Monniera

Shoots and roots ofBacopa monniera (L.) Wettst. have been regenerated from nodal segments on MS medium containing combinations of NAA and BAP. The cultures showed 100% regeneration on MS (sucrose 2%) medium added with NAA (0.2 mg L^-1), BAP (0.5 mg L^-1) and glutamine (50 mg L^-1). Supplemented with aluminium chloride (up to 400 μM), this medium could ensure successful survival of regenerants. AH the regenerants, maintained on AlCl₃-supplemented medium for the last three years, failed to grow when transferred to AlCl₃-free media. Aluminium stress also induced synthesis of proline and proteins. The rate of photosynthesis decreased at increased aluminium concentrations.     

菜心组织培养技术初探

为建立菜心(Brassica campestris ssp.chinensis var.utilis)的快繁技术体系,以花药和子叶-子叶柄为外植体进行组织培养研究。结果表明,花药培养以选取未开放的花蕾为宜,且花柱略高于花瓣,此时小孢子多数处于单核靠边期。菜心花粉的萌发率不高,且秋冬季的花粉比夏季的萌发率高。菜心花药愈伤组织诱导培养基为:MS+1.0 mg L–1 KT+1.0 mg L–1 2,4-D+3%糖+6 g L–1琼脂+8%椰乳,不定芽诱导培养基为:MS+2.0 mg L–1 6-BA+0.5 mg L–1 NAA+1.0 g L–1活性炭+2%糖+6 g L–1琼脂或MS+2.0 mg L–1 ZT+0.5 mg L–1 IAA+0.5 g L–1 AgNO3+1.0 g L–1活性炭+2%糖+6 g L–1琼脂。花药培养的不定芽诱导率为36.7%,不定芽培养出现褐化现象,不能形成再生植株;而以子叶-子叶柄为外植体培养获得的植株再生率可达80%。     

In vitro production and identification of autotetraploids of Scutellaria baicalensis

The roots of Scutellaria baicalensis are a major traditional Chinese medicine. We report research on induction, characteristics and chemical analysis of polyploid plants of S. baicalensis. Immersing calluses in 0.2% colchicine solution for 12 h prior to culture induced a high number of tetraploid plants. The induction rate reached as high as 40% of treated calluses. More than 50 lines of tetraploid plants were obtained. All tetraploid plants showed typical polyploidy characteristics. Twenty selected tetraploid lines were transferred to the field for determination of morphological characteristics and for chemical assays. Seven elite lines have been selected for further selection and breeding into new varieties for commercial production.     

Feeding elicitors and precursors enhance colchicine accumulation in morphogenic cultures of <Emphasis Type="Italic">Gloriosa superba</Emphasis> L.

Morphogenic cultures of Gloriosa superba were initiated on Murashige and Skoog’s medium fortified with 2 mg L^?1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg L^?1 naphthaleneacetic acid (NAA), 4% sucrose and 0.1% activated charcoal. To enhance the content of the alkaloid colchicine, morphogenic cultures were treated with different concentrations of abiotic elicitors like signalling compounds, metals, biotic elicitors, precursors and a combination of elicitors. Signalling molecules like acetyl salicylic acid (ASA) and sodium nitroprusside improved the production of colchicine. Abiotic elicitors have markedly (p?≤?0.05 or ≤?0.01) enhanced the colchicine content either at lower or higher concentrations. Among the metals, the highest amount of 11.67 mg of colchicine g^?1 dry wt was noticed at 60 mM rubidium chloride, followed by 60 mM NaCl (11.18 mg g^?1). Contrarily, in the presence of biotic elicitors such as Fusarium oxysporum, Alternaria solani, and Saccharomyces cerevisiae, colchicine content ranged only between 2 and 5.32 mg g^?1, but Bacillus subtilis repressed it. Among the aromatic amino acids, phenylalanine at 500 mg L^?1 influenced the highest accumulation of 19.48 mg g^?1 dry tissue, followed by tryptophan (12.47 mg g^?1), and tyrosine (9.87 mg g^?1), a direct precursor of colchicine biosynthesis, while intact tubers and leaves contained 4.65 and 4.16 mg of colchicine g^?1 dry tissue respectively. A combination of 10 µM AlCl₃ and 50 µM salicylic acid (SA) registered 17.34 mg g^?1 followed by 16.24 mg g^?1 tissue in presence of 1 µM HgCl₂ and 50 µM SA. The results suggest that the elicitor-stimulated colchicine accumulation was a stress response and can be exploited further for commercial production.     

Zn、B配施对鸡血藤黄酮类化合物积累的影响

为探讨Zn、B配施对鸡血藤(Spatholobus suberectus)黄酮类化合物积累的影响,采用营养液叶面喷施,对其总黄酮含量、可溶性蛋白质和PAL活性的变化进行研究。结果表明,Zn、B配施的鸡血藤总黄酮含量、可溶性蛋白质含量、PAL活性增加,其中施用50 mg L–1 Zn SO4+10 mg L–1 Na_2B_4O_7·10H_2O鸡血藤的可溶性蛋白质含量最高,达0.89%;施用25 mg L–1Zn SO4+50 mg L–1 Na2B4O7·10H2O鸡血藤的总黄酮含量和PAL活性最高,分别为4.65%、29.47 U g–1min–1。因此,合理配施Zn、B能促进鸡血藤黄酮类化合物的积累。     

植物激素和接种方式对广西金线莲壮苗生根的影响

该文以广西野生金线莲无菌播种离体茎段为材料,采用单因素对比试验,研究了植物激素(NAA、IBA、6-BA、GA_3、KT、ZT、TDZ、2-IP)以及接种方式(竖直接种和水平接种)对壮苗生根培养的影响。结果表明:与对照相比,生长素有利于壮苗生根,NAA的效果优于IBA;细胞分裂素对壮苗生根的效果依次为6-BATDZKT=ZT 2-IPCK,其中6-BA诱导平均株高8.4 cm,3.6条根,茎粗为2.84 mm,植株生长健壮,诱导效果最好;赤霉素GA_3诱导出的植株高且直,但植株细弱,且抑制根系生长,不利于壮苗生根培养;在激素组合6-BA 0.5 mg·L~(-1)、NAA1.0 mg·L~(-1)处理中,组培苗生长健壮且根数量较多,效果最佳;水平接种能诱导出更多的根系,且便于接种操作,可以节省接种时间。因此,确定广西金线莲最适宜的壮苗生根培养基配方为1/2MS+6-BA 0.5 mg·L~(-1)+NAA 1.0 mg·L~(-1)+香蕉汁100 g·L~(-1)+AC(活性炭) 1.0 g·L~(-1)+蔗糖20 g·L~(-1),最佳接种方式为水平接种。     

不同培养条件对勺叶茅膏菜试管苗矶松素含量的影响

为探讨培养条件对勺叶茅膏菜(Drosera spatulata)试管苗矶松素积累的影响,采用高效液相色谱(HPLC)法测定矶松素含量,对不同器官和不同培养条件下的勺叶茅膏菜试管苗矶松素含量变化进行研究。结果表明,勺叶茅膏菜试管苗根的矶松素含量显著高于叶片;光质和有机物含量对勺叶茅膏菜试管苗矶松素含量的影响不显著,但对试管苗的生长具有显著影响,最佳培养光质为白光,其次为红光和蓝光,最后为绿光;适当降低培养基中有机物含量可促进勺叶茅膏菜试管苗的生长发育;植物生长调节剂对矶松素积累的影响效应依次为6-BANAAKTGA3,而对试管苗生长的影响效应依次为6-BAGA3NAAKT。因此,勺叶茅膏菜试管苗的最佳培养条件为:以1/2MS为基本培养基,添加0~0.2 mg L–1 6-BA、0.2 mg L–1 NAA、0.5 mg L–1KT和0.1 mg L–1 GA3,于白光下培养。     

Effect of plant growth regulators on regeneration of plantlets from bud cultures ofCymbopogon nardus L. and the detection of essential oils from thein vitro plantlets

Cymbopogon nardus L. could be propagated via tissue culture using axillary buds as explants. The aseptic bud explants obtained using double sterilization methods produced stunted abnormal multiple shoots when they were cultured on Murashige and Skoog medium (MS) supplemented with 1.0 mg L^-1 or 2.0 mg L^-1 benzyladenine (BA). Stunted shoots that cultured on MS + 1.0 mg L^-1 BA + 1.0 mg L^-1 N⁶-isopentenyl-adenine (2iP) could induce elongation of shoots from about 60% of the stunted shoots. Normal multiple shoots could be induced at the highest (19.7 shoots per bud) from the bud explants within six weeks when cultured on proliferation medium consisted of MS supplemented with 0.3 mg L^-1 BA and 0.1 mg L^-1 indole-3-butyric acid (IBA). The separated individual shoot produced roots when transferred to basic MS solid medium. The essential oils that were contained in the mature plants namely citronellal, geraniol and citronellol were also found in thein vitro C. nardus plantlets. Citronellal was the main essential oil component in the matured plants while geraniol was the main component in thein vitro plantlets.     

Ginsenoside Rd production from the major ginsenoside Rb<Subscript>1</Subscript> by <Emphasis Type="Italic">β</Emphasis>-glucosidase from <Emphasis Type="Italic">Thermus caldophilus</Emphasis>

Under optimum conditions (pH 5, 75°C, and 0.2 U purified enzyme ml⁻¹), 4 mg ginsenoside Rd was produced from 5 mg reagent-grade ginsenoside Rb₁ in 5 ml after 30 min by β-glucosidase from Thermus caldophilus GK24. Using a ginseng root extract containing 1 mg ginsenoside Rb₁ ml⁻¹ and 3.2 mg additional ginsenosides ml⁻¹, 1.23 mg ginsenoside Rd ml⁻¹ was produced after 18 h; the concentrations of ginsenosides Rb₁, Rb₂, and Rc used for ginsenoside Rd production were 0.77, 0.17, and 0.19 mg ml⁻¹, respectively.     

秋水仙素诱导川贝母(Fritillaria cirrhosa D.Don)愈伤组织多倍体的研究

应用组织培养技术对川贝母（Fritillaria cirrhosaD.Don)进行多倍体诱导。结果表明：组织培养条件下,将川贝母愈伤口组织在培养基中添加一定浓度的和为水仙素处理一段时间,或者经一定浓度的和为水仙素浸泡一段时间后再培养,均可诱发川贝母多倍体的产生,但以前者效果较好。在秋水仙素浓度为1000mg/L。处理5d的条件下,诱导率最高达70%。细胞染色体鉴定结果为：四倍体染色体数为2n=4x=48,而二倍体的染色体数为2n=2x=24。     

广西五种绞股蓝属植物离体快繁与种质保存研究

以绞股蓝属植物的带芽茎段为材料,研究不同6-BA浓度与NAA 0.02mg·L-1组合对其诱导、分化和增殖的影响,并建立离体快繁体系。结果表明:MS+6-BA 2.0mg·L-1+NAA 0.02mg·L-1最适宜初代诱导,MS+6-BA 2.0mg·L-1+NAA 0.02mg·L-1最适合扁果绞股蓝的增殖培养,而MS+6-BA 1.5mg·L-1+NAA0.02mg·L-1是其它四种植物增殖的最佳培养基,在1/2MS+NAA 1.0mg·L-1上的生根率均达100%。1/2MS与蔗糖40g·L-1对五种植物的保存效果均最好;添加生长抑制剂能有效减缓生长速度,最佳生长抑制剂为ABA和CCC,浓度均为1.0mg·L-1,其中CCC能适合多个物种,连续保存360d的存活率均在94.5%以上;PP333不适合五种植物的保存。活力检测表明,各种质经保存后增殖、生根能力均未下降。     

Manipulation of ginsenoside heterogeneity of <Emphasis Type="Italic">Panax notoginseng</Emphasis> cells in flask and bioreactor cultivations with addition of phenobarbital

Structure-similar ginsenosides have different or even totally opposite biological activities, and manipulation of ginsenoside heterogeneity is interesting and significant to biotechnological application. In this work, addition of 1 mM phenobarbital to cell cultures of Panax notoginseng at a relatively high inoculation size of 7.6 g dry cell weight (DW)/L enhanced the production of protopanaxatriol-type (Rg₁ + Re) ginsenosides in both shake flask and airlift bioreactor (ALR, 1 L working volume). The content of Rg₁ + Re in the ALR was increased from 42.5 ± 4.0 mg per gram DW in untreated cell cultures (control) to 56.4 ± 4.6 mg per gram DW with addition of 1.0 mM phenobarbital. The maximum productivity of Rg₁ + Re in the ALR reached 5.66 ± 0.38 mg L⁻¹ d⁻¹, which was almost 3.3-fold that of control. The maximum ratio of the detectable ginsenosides protopanaxatriol:protopanaxadiol (Rb₁) was 7.6, which was about twofold that of control. The response of protopanaxadiol 6-hydroxylase (P6H) activity to phenobarbital addition coincided with the above-mentioned change of ginsenoside heterogeneity (distribution). Phenobarbital addition is considered as a useful strategy for manipulating the ginsenoside heterogeneity in bioreactor with enhanced biosynthesis of protopanaxatriol by P. notoginseng cells.