铁皮石斛快速繁殖和离体种质保存的研究

对铁皮石斛种子发芽、原球茎增殖、丛芽分化和壮苗培育进行了试验、观察和分析,研究了培养基、植物激素、光强和添加剂等对其分化和生长的影响。结果显示种子在1/2MS+蔗糖2%的培养基上,30d萌发95%以上。原球茎在1/2MS+椰子汁25%+蔗糖3%的培养基上,45~60d原球茎增殖速度可达1∶10。丛芽分化较适宜的培养基为1/2MS+BA2mg·L-1+NAA0.2mg·L-1+IBA0.1mg·L-1+蔗糖3%,45~60d芽丛增殖速度为1∶4~5。试管苗在MS+香蕉泥20%+蔗糖2%培养基上,大约60d苗快速长高,茎粗壮且根系发达。离体保存材料可采用试管丛芽和原球茎两种方式,以保持其遗传多样性。保存方法是在15℃左右条件下,保存离体材料,继代间隔期为12~18个月;也可以采用室温保存,在1/2MS+蔗糖1%培养基上,继代间隔期可延长至10~12个月。     

6-BA和2,4-D对铁皮石斛原球茎增殖、分化和离体保存的影响

本文探讨6-BA和2,4-D对铁皮石斛原球茎增殖和分化的影响。结果表明:铁皮石斛原球茎芽再生的较好培养基为MS,芽增长的较好培养基为MS+6-BA 1 mg/L+2,4-D 0.1 mg/L,原球茎再生芽生根和原球茎增殖的较好培养基也为MS。添加0.5 mg/L 2,4-D不利于铁皮石斛原球茎的保存,而不添加2,4–D或添加0.1 mg/L 2,4-D均有利于铁皮石斛原球茎保存,且有持续增长趋势。     

四种添加物对铁皮石斛原球茎生长及多糖含量的影响

为探讨铁皮石斛(Dendrobium officinale)培养基中添加物的作用,在1/2MS培养基中加入椰肉、甘蔗渣、香蕉皮和麦麸等4种添加物,研究不同浓度添加物和培养时间对原球茎生长和多糖含量的影响。结果表明,4种添加物对铁皮石斛原球茎的增殖、分化和多糖含量均有一定影响,其中添加15.0 g L–1甘蔗渣,培养60 d能明显促进铁皮石斛原球茎的增殖与分化(146.1%);而添加20.0 g L–1甘蔗渣,培养40 d能显著提高铁皮石斛原球茎多糖含量(50.4%)。这说明甘蔗渣是培养铁皮石斛原球茎的适宜添加物,既能促进铁皮石斛原球茎的生长发育,还能降低生产成本。     

中药材铁皮石斛组培苗不同培养基的筛选与优化

铁皮石斛组培苗在生长各个阶段对营养物质的需求不一样,培养条件不同组培苗生长结果也不一样。本研究以铁皮石斛种子为材料,通过无菌播种进行原球茎诱导、增殖、分化、生根试验,对各阶段培养基进行比较,以期筛选出最佳培养基条件,为将来铁皮石斛走向工厂化、标准化生产提供思考和借鉴。结果表明,种子萌发以及原球茎诱导的最佳培养基配方是1/2 MS培养基,35 d后萌发率达93.1%。原球茎增殖最佳培养基配方组合是MS+6-BA(1.5 mg/L)+NAA(0.5 mg/L),增殖系数达14.8。原球茎分化成苗的最佳培养基配方组合是MS+6-BA(0.5 mg/L)+NAA(0.2 mg/L),分化率达92.1%,且长势良好。组培苗生根的最佳培养基配方组合是1/2 MS+NAA(1.5 mg/L),生根率达97.7%,根系健壮发达。     

铁皮石斛同源四倍体工厂化快繁工艺研究

以实验室前期诱导的铁皮石斛四倍体为研究材料,利用气孔、叶绿体、染色体计数及流式细胞仪等方法,对铁皮石斛同源四倍体株系的纯合性进行鉴定,在此基础上利用茎段扩繁和原球茎诱导、增殖、分化两种方案比较增殖效率,并对原球茎的倍性稳定性进行再鉴定。结果表明：（1）实验室的‘花自 2’株系为纯合同源四倍体。（2）茎段扩繁最佳增殖培养基为MS＋2.0 mg/L 6 BA+0.5 mg/L NAA+15%香蕉汁,茎段诱导原球茎最佳培养基为MS+1.0 mg/L 6 BA+0.5 mg/L NAA+0.2 mg/L KT,诱导率达到76.66%;原球茎增殖最佳培养基为MS+1.0 mg/L 6 BA+0.5 mg/L NAA+15%马铃薯提取液,原球茎分化最佳培养基为1/2 MS+0.5 mg/L 6 BA+20%马铃薯提取液,原球茎分化率达到85.70%;生根最佳培养基为1/2 MS＋0.5 mg/L NAA＋15%香蕉汁,生根率达到92.77%。（3）原球茎根尖鉴定结果表明,诱导的倍性可以稳定遗传。（4）瓶苗移栽的最佳炼苗时间为5 d,移栽成活率达92.30%。该试验建立了铁皮石斛同源四倍体株系新的快繁技术体系,为四倍体试管苗的工厂化生产和推广奠定了技术基础。     

低温和外源NO对铁皮石斛原球茎多糖合成的影响

以铁皮石斛(Dendrobium officinale)原球茎为材料,研究了低温(4℃)、外源NO(NO供体SNP)以及NO清除剂(cPTIO)和一氧化氮合酶抑制剂(PBITU)对铁皮石斛原球茎中NO含量、蔗糖合成酶(SS)活性、多糖含量以及蔗糖、果糖、葡萄糖等糖含量的影响,以明确低温和內源NO在多糖合成中的关系。结果显示:(1)4℃低温处理下,铁皮石斛原球茎中NO含量显著上升,SS活性升高,蔗糖、果糖和葡萄糖含量增加,多糖含量也得到提高;SNP(0.5mmol·L-1)处理与4℃低温处理具有相似的效果;且低温诱导的铁皮石斛原球茎中SS活性提高和多糖含量的增加时期均在NO大量产生之后、蔗糖的积累早于果糖和葡萄糖。(2)4℃低温+SNP组合处理能够显著提高铁皮石斛原球茎中SS活性、NO含量以及蔗糖、果糖、葡萄糖和多糖含量,它们分别比对照组显著高出了68.04%,96.20%,60.69%、45.64%、66.90%和67.03%,且比低温和SNP单独处理效果都好。(3)PBITU能够部分抑制低温诱发铁皮石斛原球茎中产生NO,抑制率达到77.15%;同时还抑制了低温对铁皮石斛原球茎中SS活性、多糖合成和蔗糖、果糖、葡萄糖积累的促进作用。(4)SNP+cPTIO和4℃+cPTIO处理组中铁皮石斛原球茎SS活性和蔗糖、果糖、葡萄糖、多糖含量及NO水平,且与对照组差异不显著。研究表明,低温和外源NO对铁皮石斛原球茎多糖的合成均具有促进作用,并且低温可诱导铁皮石斛原球茎产生NO,SS活性提高和多糖含量增加与NO产生相关,说明NO是诱导铁皮石斛原球茎多糖合成所必需的信号分子。     

寒兰的快速繁殖技术

以寒兰(CymbidiumkanranMakino)根状茎为外植体,采用B5基本培养基,并附加不同浓度的6-BA、NAA、TDZ(苯基噻二唑基脲-thidiazuron)和S-3307(优康唑-uniconazole),对类原球茎的诱导、继代增殖、分化、生根等进行研究。结果表明:诱导类原球茎的最佳培养基为B5 TDZ0.50mgL-1 NAA0.25mgL-1,诱导率98.3%;继代增殖的最佳培养基为B5 S-33071.0mgL-1 NAA0.2mgL-1 蔗糖3.5%,增殖系数9.4;类原球茎分化的最佳培养基为B5 S-33070.75mgL-1 6-BA1.0mgL-1 NAA0.4mgL-1,分化率87.8%;最佳的生根培养基为1/2B5 NAA0.2mgL-1 活性炭0.05%,生根率达100%。     

大苞鞘石斛原球茎玻璃化超低温保存技术的研究

本文研究建立了大苞鞘石斛(Dendrobium wardianum Warner)原球茎玻璃化法超低温保存的技术体系。结果发现,预处理和玻璃化溶液(plant vitrification solution 2,PVS2)装载脱水是影响大苞鞘石斛原球茎相对存活率的两个关键步骤,高渗与低温-高渗两种预处理方法测定的相对存活率具有显著性差异;玻璃化溶液的种类以及脱水时间对冻后存活率具有重要的影响。基于此,建立了大苞鞘石斛原球茎的超低温保存体系,即:以继代培养60 d的大苞鞘石斛原球茎为材料,1/2MS+0.8 mol/L蔗糖的培养基上4℃低温预处理6 d后,转至1/2 MS+2 mol/L甘油+0.4 mol/L蔗糖的装载液中室温下装载40 min,在0℃下装载PVS2脱水40 min,然后转入装有新鲜PVS2冷冻管中并迅速投入液氮。在液氮保存1 h后放在40℃水浴中快速解冻1 min,利用含1.2 mol/L蔗糖的1/2MS培养液洗涤3次,每次间隔10 min;待恢复培养30 d后统计存活率,可使大苞鞘石斛原球茎超低温保存后存活率达到20.0%。     

氮源和真菌诱导子对铁皮石斛原球茎悬浮培养的影响

利用正交实验设计研究不同种类和浓度的氮源对铁皮石斛原球茎生长的影响,利用完全随机实验设计研究不同真菌诱导子对铁皮石斛原球茎生长和多糖积累的影响.结果表明硝态氮促进铁皮石斛原球茎鲜重和干重的增加( P ＜0.05);铵态氮促进铁皮石斛原球茎鲜重增加( P ＜0.05).无论鲜重还是干重,硝态氮和铵态氮的影响均不存在互作,最佳组合为:67-V培养液 100 mg/L (NH4)2SO4 800 mg/L KNO3 30 g/L蔗糖 200 g/L马铃薯提取汁.F检验的结果表明,14种真菌诱导子对铁皮石斛原球茎生物量的增加(鲜重与干重)均无显著性影响( P ＞0.05).但与对照铁皮石斛原球茎的多糖含量相比,真菌诱导子g6、g14、g5、g4处理可使其多糖含量分别提高14%、9%、6%、4%.本研究获得了有利于铁皮石斛原球茎生长的硝态氮和铵态氮的最佳搭配方案以及有利于铁皮石斛原球茎积累多糖的四种真菌诱导子,表明通过液体悬浮培养生产铁皮石斛原球茎及其多糖成分具有较好的开发应用前景.     

不同植物生长调节剂对铁皮石斛(Dendrobium candidum)再生的影响

本实验以铁皮石斛无菌苗茎段为外植体,通过添加不同浓度的植物生长调节剂,诱导铁皮石斛愈伤组织形成与分化,建立铁皮石斛组织培养再生体系。结果表明,外植体接种5 d后,在改良MS培养基上添加2 mg/L PBU和0.05 mg/L IAA,可达到100%的愈伤诱导率。将愈伤组织接种在MS培养基上,添加0.1 mg/L NAA和0.5 mg/L 6-BA,不定芽诱导率为90.8%。适合铁皮石斛芽增殖培养基为:MS+7 g/L琼脂+30 g/L蔗糖+100 mg/L Vc+0.5 mg/L 6-BA+0.2 mg/L NAA。PBU浓度为0.5 mg/L,NAA浓度为0.05 mg/L时,芽苗生长情况良好,最适合用于不定芽伸长。铁皮石斛最适生根的培养基为:MS+7 g/L琼脂+30 g/L蔗糖+100 mg/L Vc+0.5 mg/L IBA,生根率可高达94.1%。本研究成功建立了铁皮石斛高效再生体系。     

培养基成分和培养时间对匍匐翦股颖植株再生的影响

以匍匐翦股颖成熟种子为外植体,研究了培养基2,4-D浓度、2,4-D和6-BA组合配比、蔗糖浓度对匍匐翦股颖愈伤组织诱导的影响以及愈伤组织再生过程中继代时间、6-BA浓度、蔗糖浓度对愈伤组织分化的影响。结果表明:在MS培养基上,2 mg·L^-1 2,4-D和0.1 mg·L^-1 6-BA的组合最利于愈伤组织的诱导,诱导率高达94%。蔗糖浓度为30 g·L^-1时愈伤组织诱导率最高,为82%; 在再生过程中,当6-BA浓度为1 mg·L^-1时分化率最高(62%),蔗糖浓度为40 g·L^-1时,愈伤组织分化率最高(52%)。经过2次继代培养的愈伤组织(外植体放到培养基后40天)的分化率为最高(71%),随着继代次数增多,分化率逐渐降低,在经过5次继代后(培养100 d)分化率仅有18%。     

培养条件对平贝母愈伤组织分化的影响

以平贝母无菌苗幼茎切段诱导产生的愈伤组织为试材,研究了基本培养基、蔗糖浓度、继代时间及继代周期时间对愈伤组织分化的影响。结果表明,ＭＳ基本培养基有利于不定芽的诱导,Ｎ６培养基有利于体细胞胚的诱导。在ＭＮ培养基上,随着蔗糖浓度的升高,不定芽发生能力下降,体细胞胚发生率升高。继代时间越长,不定芽及体细胞胚的发生能力均下降。体细胞胚胎发生的最佳继代周期时间是２１天,不定芽发生的最佳继代周期时间是２８天。     

Induction of repetitive embryogenesis from seed-derived protocorms of <Emphasis Type="Italic">Phalaenopsis amabilis</Emphasis> var. <Emphasis Type="Italic">Formosa</Emphasis> shimadzu

Summary An in vitro culture procedure was established for repetitive embryogenesis and plant regeneration from seed-derived protocorms of Phalaenopsis amabilis var. formosa Shimadzu (Orchidaceae). Seed-derived protocorms were cultured on modified half-strength Murashige and Skoog (1962) basal medium (1/2MS) devoid of plant growth regulators. After 45 d, 28.1% of protocorms formed embryos from their posterior regions. 1-Phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ; 0.45, 4.54, and 13.62 μM) promoted direct embryo formation. The best response was at 13.62 μM TDZ, and 100% of the protocorms formed a mean number of 13.5 embryos after 45d of culture. By contrast, naphthaleneacetic acid (NAA) at 0.54 and 5.37 μM inhibited direct embryo formation. On basal medium devoid of plant growth regulators, 18.8% of primary proliferating embryos could form more embryos. TDZ (0.45, 4.54, and 13.62 μM) also promoted this process. Proliferating embryos/protocorms were transferred to basal medium devoid of plant growth regulators for plantlet formation. Plantlets were successfully obtained from the embryos after 4–6 wk. Following subculture every 6 wk for three passages, the plantlets were transferred to sphagnum moss in a container for acclimatization in the greenhouse. The survival rate was 100%.     

云南龙竹的快速繁殖和离体保存研究

本研究以干冷保存(-20℃,相对湿度15%)150 d的云南龙竹(Dendrocalamus yunnanicus)颖果为实验材料,通过种子萌发建成丛芽无菌无性系进行快繁和离体保存。研究表明,丛芽增殖的最佳培养基为MS+6-BA 2 mg/L+NAA 0.2 mg/L,蔗糖浓度以3%为佳;生根培养的最佳培养基为MS+IBA 1 mg/L+NAA 1 mg/L,以单芽诱导生根为好;离体保存丛芽的最适培养基为MS+6-BA0.5 mg/L+NAA0.2 mg/L;当培养温度由室温(25±3)℃降低至(20±3)℃和12℃时,其继代周期可由原来的2个月分别延长至4个月和8个月。在组织培养过程中发现白化苗或叶色变异现象。     

La fioritura di „Tulipa silvestris“ L. in rapporto alla nutrizione azotata

Many members of the Orchidaceae, the largest vascular plant family in Ecuador, are at risk of extinction. It was therefore considered important to establish an efficient way of clonal propagation based on somatic embryogenesis of Cattleya maxima, a native Ecuadorian orchid. To this end, we evaluated the effect on somatic embryo induction of 12 combinations of 2,4-dichlorophenoxyacetic acid and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea, as well as three kinds of stresses. Protocorms produced 42% of embryogenic calli on 1/2 Murashige and Skoog (1/2 MS) medium, compared to 96.3% when protocorms were stressed for 6 h with 0.3 M NaCl, followed by cultivation on 1/2 MS medium supplemented with 0.1 mg L^? 1 2,4-D. Our data demonstrated that the combination of either salt (0.3 M NaCl) or osmotic stress (0.4 M sorbitol) with subculture on 2,4-D (0.1 mg L^–1) medium significantly increases the percentage of protocorms with embryogenic callus. The number of embryos per embryogenic callus was not significantly different from that obtained after subculture in growth factor-free medium.     

Cryopreservation of Cymbidium eburneum Lindl. and C. hookerianum Rchb. f., two threatened and vulnerable orchids via encapsulation–dehydration

A successful cryopreservation protocol for the long-term conservation of protocorms of two threatened and vulnerable orchids, Cymbidium eburneum Lindl. and Cymbidium hookerianum Rchb. f., was developed using encapsulation–dehydration. Protocorms were osmoprotected in liquid Murashige and Skoog medium (MS) containing 0.7 M sucrose for 20 h at 25?±?2°C on a rotary shaker, and incorporated into an encapsulation matrix [consisting of 3% (w/v) sodium alginate and 100 mM CaCl₂]. The encapsulated protocorms, which were desiccated in a laminar airflow cabinet for 6 h, were able to withstand cryostorage in liquid nitrogen. Maximum regeneration into complete plantlets (72% for C. eburneum and 70% for C. hookerianum) of the cryostored, encapsulated protocorms was obtained using MS medium containing 3% sucrose and 0.8% agar. Using this protocol of cryopreservation, long-term preservation for ex situ conservation of these two threatened orchids can be accomplished.     

花生幼叶为外植体的植株再生系统的建立

本文报道利用花生成熟胚幼叶为外植体获得高频植株再生的方法,为花生转基因提供有效的受体系统。通过诱导培养基TDZ、BA、NAA的浓度以及种子萌发时间、继代培养基种类五个因素不同水平的正交试验,筛选出了分化高频发生的最佳组合为：MS培养基中应含有TDZ 1.0 μmol/L、BA 0.4 μmol/L、NAA 5.0 μmol/L,种子萌发4 d,继代培养基为MS0。本研究表明,五因素中诱导培养基TDZ浓度为诱导花生幼叶分化的主要影响因素,其次为继代培养基、种子萌发时间,而诱导培养基中BA和NAA的浓度作用较小。试管苗生根后移栽田间,可正常开花结果。     

Differential mechanisms to induce dehydration tolerance by abscisic acid and sucrose in Spathoglottis plicata (Orchidaceae) protocorms

Abscisic acid (ABA) and sucrose are known to induce dehydration tolerance of in vitro plant cells and tissues. The present study reports the presence of different mechanisms by which sucrose and ABA improve dehydration tolerance of Spathoglottis plicata (orchid) protocorms. Orchid protocorms were generated aseptically from seeds on Murashig and Skoog medium, and then treated for 7 d in medium containing 10 mg L^?1 ABA and/or 10% (w/v) sucrose. Dehydration tolerance of protocorms was determined at ～25 °C under various drying conditions at relative humidity from 7 to 93%. The actual rate of water loss (i.e. drying rate) was determined using the rate constant of tissue water loss during drying according to the first‐order kinetics. Drying rate affected dehydration tolerance. ABA treatment reduced drying rate and increased dehydration tolerance of protocorms at all relative humidity values tested. However, when compared on the basis of actual drying rates, there was no difference in dehydration tolerance between control and ABA‐treated protocorms, suggesting that ABA‐induced tolerance was correlated with the drying rate reduction. Sucrose treatment was more effective than ABA treatment for the induction of dehydration tolerance. Interestingly, sucrose only slightly affected drying rate. ABA treatment significantly enhanced the synthesis of dehydrin, whereas sucrose treatment primarily resulted in sucrose accumulation. Sucrose treatment also affected protein turnover during drying, causing a significant decrease in protein content in protocorms. Slow drying promoted the degradation of high molecular weight proteins and enhanced the synthesis of low molecular weight dehydrin. The data suggest that different physiological mechanisms are probably involved in the induction of dehydration tolerance by ABA and sucrose treatment.     

花生体细胞胚的诱导及其植株再生

采用不同成熟度的花生胚轴为外植体进行体细胞胚诱导及植株再生研究,结果表明,成熟胚轴在高浓度2,4－D的MS培养基中,经过30d左右的培养,可直接诱导产生出大量的体细胞胚,含40mgL~－12,4－D的培养基中体细胞胚的诱导率达100％,平均每个外植体产生11．58个体细胞胚．体细胞胚的继代培养需降低2,4－D的浓度（1－20mgL~－1）．未成熟胚轴的体细胞胚诱导及继代培养的2,4－D浓度宜为10mgL~－1．将诱导的体细胞胚转接到合5－10mgL~－1BA的MS培养基中,体细胞胚能够萌发再生成无根小植株,将其转接到生根培养基中可获得完整小植株．     

莲的离体快速繁殖技术

以莲(Nelumbo nucifera)授粉后18天的莲子胚芽为外植体, 通过初代培养、继代培养和炼苗移栽, 建立了莲离体快速繁殖体系。结果表明, 将胚芽外植体诱导出无菌苗的最适初代培养基为MS固体培养基添加0.5 mg∙L^-1 6-BA、0.5 mg∙L^-1 NAA、30 g∙L^-1蔗糖、0.5 g∙L^-1活性炭和0.8 g∙L^-1琼脂, 培养60天诱导率高于85%, 其中秋红阳走茎节数最多(3.9)。最佳继代培养基为将初代培养基中的蔗糖浓度提高到80 g∙L^-1, 走茎采用两节一切的分苗切法, 无菌苗可50天继代1次, 最多可继代6次, 不同品种的增殖系数介于4.0-6.7之间, 以秋红阳最高(6.7)。于5-7月将生根的走茎无菌苗移栽入泥炭:塘泥=1:2 (v/v)的混合基质中进行培养, 成活率均大于83.9%。采用上述快繁技术, 理论上1个莲子胚芽经过近1年可繁殖出种苗1 465株。该研究建立了莲的离体快繁技术体系, 可为莲种苗的规模化生产提供技术支持。