扇蕨孢子的组织培养

1 植物名称扇蕨[Neocheiropteris palmatopedata (Baker)Christ]. 2 材料类别成熟孢子. 3 培养条件孢子萌发培养基:(1)B5 20 g·L-1蔗糖.原叶体增殖培养基:(1)B5 20 g·L-1蔗糖;(2)1/2B5 20 g·L-1蔗糖;(3)1/485 20 g·L-1蔗糖;(4)1/885 20g·L-1蔗糖;(5)B5,(6)B5 10 g·L-1蔗糖;(7)B5 30 g·L-1蔗糖;(8)B5 40 g·L-1蔗糖;(9)B5 50 g·L-1蔗糖;(10)B5 60 g·L-1蔗糖.     

黄瓦韦的组织培养

1植物名称黄瓦韦(Lepisorus macrosphaerus var.asterolepis),也称大瓦韦。2材料类别带孢子的叶片。3培养条件以MS为基本培养基。(1)孢子萌发培养基:1/2MS 3g·L-1活性炭;(2)诱导绿色小球培养基:MS 6-BA0.1~     

小叶栒子的组织培养

1植物名称小叶枸子(Cotoneaster microphyllus). 2材料类别茎尖及带腋芽的茎段. 3培养条件芽诱导培养基:(1)MS 6-BA 1.0 mg·L-1(单位下同) IBA 0.1;增殖培养基:(2)MS 6-BA0.8 IBA 0.3,(3)MS 6-BA 0.3 IBA 0.3,(4)MS 6-BA 0.1 IBA 0.5,(5)MS 6-BA 0.3 IBA 0.5,(6)MS 6-BA 0.5 IBA 0.5,(7)MS 6-BA 0.1 IBA0.5 TDZ 0.005;生根培养基:(8)1/2MS IBA 0.1,(9)1/2MS IBA 0.2,(10)1/2MS IBA 0.3,(11)1/2MS IBA 0.4,(12)1/2MS IBA 0.5,(13)1/2MS IBA 0.6.培养基均加入0.5%琼脂,诱导、增殖培养基加3%蔗糖,生根培养基加2%蔗糖,pH 6.0.光照度1500～2000 lx,光照时间10 h·d-1.培养温度15～25℃.空气湿度40%～70%.     

皱叶山苏的组织培养和植株再生

1植物名称皱叶山苏(Asplenium antiquum Makinocv‘.Victoria’)。2材料类别孢子。3培养条件孢子萌发及原叶体形成的培养基:(1)1/2MS。丛芽分化及增殖培养基:(2)1/2MS;(3)1/2MS 6-BA0.5mg·L-1(单位下同) NAA0.1;(4)1/2MS 6-BA1.0 NAA0.1     

羽叶南洋参的组织培养

1植物名称羽叶南洋参(Polyscias fruticosa var.plumata Bailey),别名南洋森和福禄桐。2材料类别茎段。3培养条件基本培养基为MS。芽诱导培养基:(1)MS 6-BA2mg·L-1(单位下同) NAA0.5。芽增殖     

蝴蝶豆的组织培养

1植物名称蝴蝶豆(Centrosema pubescens Benth.),别名距瓣豆。2材料类别下胚轴。3培养条件(1)种子萌发培养基为MS;(2)诱导增殖培养基:MS 6-BA1.0mg·L-1(单位下同) ZT1.0 IAA0.1;(3)分化培养基:MS 6-BA1.0 NAA     

百里香的组织培养

1植物名称百里香(Thymus serpyllum var.mongolicus Ronn.)。2材料类别顶芽和腋芽。3培养条件(1)芽诱导培养基:MS基本培养基;(2)增殖培养基:MS 6-BA 1.5 mg·L~(-1)(单位下同) IBA 0.1 0.6%琼脂 3%蔗糖;(3)生根培养基:1/2MS IBA 0.5 0.7%琼脂 2%蔗糖。以上所有培养基均为pH 5.8～6.0,培养温度(24±2)℃,光     

野扇花的组织培养和快速繁殖

1植物名称野扇花(Sarcococca ruscifolia Stapf),别名清香桂。2材料类别带腋芽的嫩茎段。3培养条件(1)诱导培养基:MS+6-BA2.0mg·L-1(单位下同)+NAA0.05+LH450;(2)增殖培养基:MS+6-BA0.5+NAA0.01+LH300;(3)生根培养基:1/4MS+NAA0.3+IBA0.2。(1)和(2)培养基均附加3%蔗糖、0.7%琼脂,(3)附加1.5%蔗糖、0.6%琼脂,pH5.8～6.0。培养温度(23±2)℃,光强40μmol·m-2·s-1左右,光照时间12h·d-1(李树丽和石文山2005;李雪等2005)。4生长与分化情况4.1外植体的处理先用洗涤剂将外植体清洗3次,然后在流水下冲洗2～3h。在超净工作台上用0.1…     

垂花火鸟蕉的组织培养

1植物名称垂花火鸟蕉(HeliconiarostrataRuiz&Pavon)。2材料类别种子长成的无菌苗茎段。3培养条件种子萌发培养基:1/2MS培养基。芽启动培养基:(1)1/2MS+6-BA3mg·L-1(单位下同)+NAA0.5+1%活性炭;(2)1/2MS+6-BA5+NAA0.5+1%活性炭;(3)1/2MS+6-BA10+NAA0.5+1%活性炭。芽增殖培养基:(4)MS+6-BA3+NAA0.3。生根培养基:(5)MS+NAA0.5+0.5%活性炭。以上培养基均添加7g·L-1琼脂粉和30g·L-1蔗糖,pH5.8,在121℃下高压灭菌20min。培养温度为(25±2)℃,光照时间12h·d-1,光照强度50μmol·m-2·s-1。4生长与分化情况4.1无菌材料的获得…     

Antimicrobial Constituents in Pteris inaequalis Bak.

Pterosins B and O (pterosin B methyl ether) were isolated as antimicrobial constituents from Pteris inaequalis Bak. aequata Tagawa.     

假鞭叶铁线蕨孢子的组织培养

1植物名称假鞭叶铁线蕨(Adiantum malesianum Ghatak)。2材料类别成熟孢子。3培养条件孢子萌发培养基:1/2MS 30g·L-1蔗糖。原叶体增     

银中斑凤尾蕨的组织培养与植株再生

1植物名称 银中斑风尾蕨（Pteris multifida Poir．cv．Variegata）,又名银白风尾蕨。 2材料类别 成熟孢子。 3培养条件 孢子萌发培养基：（1）1／2MS;原叶体增殖培养基：（2）MS＋6．BA2．0mg．L^-1（单位下同）＋NAA0．5,（3）MS＋6．BA1．0＋NAA0．2;孢子体形成和丛生芽增殖培养基：（4）MS＋IAA0．05,（5）MS＋KT0．0125＋IAA0．05,（6）MS＋KT0．025＋IAA0．05,（7）MS＋KT0．05＋IAA0．05;生根培养基：（8）1／2MS＋NAA0．1,（9）1／2MS＋NAA0．5,（10）1／2MS＋NAA1．0,（11）1／2MS＋NAA2．0,（12）1/2MS＋IBA2．0。     

Percent PFR-Dependent Germination of Spores in Pteris vittata

The phytochrome-dependent germination of spores was studiedin the fern Pteris vittata. Brief irradiations with red lightgiven at 0 and 25?C resulted in very similar germination rates.Irradiation with far-red light cancelled this promotive effect,irrespective of the temperature at which tested. The maximumrate of germination was induced by red light of ca. 70Jm^–2and half of the rate was induced by ca. 15Jm^–2 When sporesimbibed in the dark were kept for 1 h at 0 or 25?C under irradiationswith monochromatic lights from 660 to 730 nm at 10 nm intervals,spore germination was induced depending upon the establishedphotostationary states of phytochrome at both temperatures tested.The percent of P_FR estimated in spores that had been irradiatedbriefly with red light was consistent with that resulted fromphotostationary states under different monochromatic lightsin terms of the percent of germination of a spore population.The threshold of the % P_FR required for the germination of eachspore ranged widely from a few percent to 80% of the P_FR. Thisdiversity may vary the timing of germination in nature. ¹ Partial preliminary results of this research were introducedin a review by M.F. (1978). ³ Present address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Setagaya, Tokyo 158, Japan. (Received May 15, 1982; Accepted August 5, 1982)     

球子蕨孢子的无菌繁殖

以球子蕨成熟孢子为外植体,研究了不同激素及浓度对其孢子萌发、愈伤组织诱导、丛生芽分化及生根的影响。结果表明：孢子萌发最适培养基为1／2MS＋2％蔗糖,20d后萌发率达55．7％;诱导愈伤组织的最适培养基为MS＋0．5mg·L-1 KT＋0．5mg·L～2,4-D,诱导率达36％,愈伤组织为绿色颗粒状;颗粒状愈伤组织在不添加激素的MS培养基中即可生长出大量丛生芽,转化率可达49．3％;低浓度（0．2mg·L-1）的IAA可有效促进幼孢子体苗生根。     

Stable Transformation of Ferns Using Spores as Targets: Pteris vittata and Ceratopteris thalictroides

Ferns (Pteridophyta) are very important members of the plant kingdom that lag behind other taxa with regards to our understanding of their genetics, genomics, and molecular biology. We report here, to our knowledge, the first instance of stable transformation of fern with recovery of transgenic sporophytes. Spores of the arsenic hyperaccumulating fern Pteris vittata and tetraploid ‘C-fern Express’ (Ceratopteris thalictroides) were stably transformed by Agrobacterium tumefaciens with constructs containing the P. vittata actin promoter driving a GUSPlus reporter gene. Reporter gene expression assays were performed on multiple tissues and growth stages of gametophytes and sporophytes. Southern-blot analysis confirmed stable transgene integration in recovered sporophytes and also confirmed that no plasmid from A. tumefaciens was present in the sporophyte tissues. We recovered seven independent transformants of P. vittata and four independent C. thalictroides transgenics. Inheritance analyses using β-glucuronidase (GUS) histochemical staining revealed that the GUS transgene was stably expressed in second generation C. thalictroides sporophytic tissues. In an independent experiment, the gusA gene that was driven by the 2× Cauliflower mosaic virus 35S promoter was bombarded into P. vittata spores using biolistics, in which putatively stable transgenic gametophytes were recovered. Transformation procedures required no tissue culture or selectable marker genes. However, we did attempt to use hygromycin selection, which was ineffective for recovering transgenic ferns. This simple stable transformation method should help facilitate functional genomics studies in ferns.Ferns (Pteridophyta) are nonflowering vascular plants comprised of 250 genera, the second largest group of diversified species in the plant kingdom (Gifford and Foster, 1989). Many interesting traits are inherent in various fern species, such as arsenic hyperaccumulation (Pteris vittata; Ma et al., 2001), insecticide production and allelopathy (Pteridium aquilinum; Marrs and Watt, 2006), and antimicrobial compound production (Acrostichum aureum; Lai et al., 2009). Ferns occupy the evolutionary niche between nonvascular land plants, bryophytes, and higher vascular plants such as gymnosperms and angiosperms. Therefore, extant fern species still hold a living record of the initial adaptations required for plants to thrive on land. Of these adaptations, the most important is tracheids that comprise xylem tissues for water and mineral transport and structural support. The vascular system allowed pteridophytes to grow upright during the sporophyte generation, leading to greater resource acquisition capacity. Ultimately, sufficient resources allowed for greater spore production and upright growth, which facilitated spore spread. Recent endeavors have investigated lower plant genomics, including the sequencing of the bryophyte Physcomitrella patens (Rensing et al., 2008) and the lycophyte Selaginella moellendorffii (Banks et al., 2011). Both basic and applied biology of ferns lag far behind that for angiosperms and even bryophytes.Stable genetic transformation has been accomplished in a few species outside angiosperms and gymnosperms, especially among bryophytes (Schaefer et al., 1991; Chiyoda et al., 2008; Ishizaki et al., 2008), but never in the Pteridophyta. Transient transformation methods have been developed both in Ceratopteris richardii ‘C-fern’ and P. vittata, which were limited to heterologous expression in gametophytes (Rutherford et al., 2004; Indriolo et al., 2010). While transient expression of transgenic constructs does enable research, there is no substitute for stable transformation in functional genomics and plant biology. Researchers have resorted to studying fern gene function using heterologous expression in the angiosperm model plant Arabidopsis (Arabidopsis thaliana; Dhankher et al., 2006; Sundaram et al., 2009; Sundaram and Rathinasabapathi, 2010), which is far from optimal. Overexpression and knockdown analysis of individual genes in a wide variety of fern species would undoubtedly accelerate our ability to learn more about their biology and subsequently develop novel products from ferns. Furthermore, a facile transformation system would accelerate functional genomics and systems biology of ferns. For example, knockdown analysis of genes involved in interesting biosynthetic pathways can greatly facilitate gene and biochemical discovery.P. vittata has the unparalleled ability to accumulate more arsenic per gram biomass than any other plant species and is highly tolerant to arsenic (Gumaelius et al., 2004). It can thrive in soils containing up to 1,500 µg mL^–1 arsenic, whereas most plants cannot survive 50 µg mL^–1 arsenic (Ma et al., 2001). Therefore, P. vittata has been the subject of extensive basic and applied research for arsenic hyperaccumulation, translocation, and resistance (Gumaelius et al., 2004) and has been used for arsenic phytoremediation (Shelmerdine et al., 2009). For example, this fern might be of great utility for the production of a safer rice (Oryza sativa) crop; arsenic can be transported and stored in the grain, resulting in serious human health ramifications (Srivastava et al., 2012). In a recent greenhouse study, P. vittata has been used to remediate arsenic-contaminated soil. Following remediation, rice plants were subsequently grown, and it was found that arsenic uptake by rice grains was reduced by 52%, resulting in less than 1 µg mL^–1 arsenic after two rounds of remediation using P. vittata phytoremediation (Mandal et al., 2012). Furthermore, this treatment also resulted in increased rice grain yield by 14% (w/w) compared with control.Ceratopteris is a subtropical-to-tropical fern genus containing four to six species living in aquatic habitats. The C-fern cultivar was developed as a model fern for teaching (http://www.c-fern.org) and research owing to its small size, short life cycle (120 d), and its amenability for in vitro culture (Banks, 1999). The C-fern Express cultivar was developed by Leslie G. Hickok by crossing two Japanese varieties of Ceratopteris thalictroides (L. Hickok, personal communication). cv C-fern Express, a tetraploid, develops spores in 60 d of culture. Though cv C-fern spores have been shown to be a useful single cell model system and a rapid and efficient system for studying RNA interference in ferns (Stout et al., 2003), no stable transformation studies have been reported. In bryophytes, immature thalli are most often used as explants (Chiyoda et al., 2008; Ishizaki et al., 2008), whereas in fungi, spores are routinely used for stable transformation studies (Michielse et al., 2005; Utermark and Karlovsky, 2008).The objective of our research was to develop, for the first time, a facile stable transformation system for P. vittata and C. thalictroides using spores as the transformation targets. This method can be used as an additional tool to further substantiate and strengthen the molecular mechanism studies in pteridophytes.     

球子蕨孢子的无菌繁殖

以球子蕨成熟孢子为外植体,研究了不同激素及浓度对其孢子萌发、愈伤组织诱导、丛生芽分化及生根的影响。结果表明:孢子萌发最适培养基为1/2MS+2%蔗糖,20d后萌发率达55.7%;诱导愈伤组织的最适培养基为MS+0.5mg·L-1KT+0.5mg·L-12,4-D,诱导率达36%,愈伤组织为绿色颗粒状;颗粒状愈伤组织在不添加激素的MS培养基中即可生长出大量丛生芽,转化率可达49.3%;低浓度(0.2mg·L-1)的IAA可有效促进幼孢子体苗生根。     

Cell Morphogenesis and Macromolecule Synthesis during Phytochrome-controlled Germination of Spores of the Fern, Pteris vittata

The object of this study was to characterize the pattern ofcell morphogenesis and synthesis of nucleic acids and proteinsduring phytochrome-controlled germination of spores of the fern,Pteris vittata. Phytochrome activation and germination wereinitiated in fully imbibed spores by exposure to a saturatingdose of red light. At timed intervals thereafter, spores werefixed in acrolein and embedded in glycol methacrylate for examinationin the light microscope. The first sign of germination, visiblein sections of the spore 12 h after irradiation, was the hydrolysisof storage protein granules. This was followed by a migrationof the nucleus from its central location to one side of thespore. Subsequently, the protoplast enlarged at the site ofthe nucleus and appeared outside the exine as a papillate structure.An asymmetrical division of the protoplast gave rise to a smallcolourless rhizoid cell and a large, chloroplast-containingprotonemal cell. During the early phase of germination, DNAwas synthesized both in the nucleus and cytoplasm as judgedby autoradiography of [³H]thymidine incorporation. [³H]Uridine,a precursor of RNA synthesis, was incorporated into the nucleolusand the rest of the nuclear material of germinating spores.Protein synthesis monitored by [³H]leucine incorporation occurredboth in the nucleus and cytoplasm during the early stage ofgermination, although a strictly cytoplasmic protein synthesiswas observed later. Addition of cycloheximide completely inhibitedgermination of photoinduced spores and incorporation of labelledprecursors of macromolecule synthesis into cellular components.Actinomycin D was much less effective as an inhibitor of germinationand, even in high concentrations of the drug which effectivelyinhibited DNA and RNA synthesis in spores, proteolysis and proteinsynthesis appeared normal. These findings are discussed withrespect to the regulation of nucleic acid and protein synthesisduring spore germination and the role of phytochrome in theprocess.     

大叶凤尾蕨的离体培养及植株再生

1　植物名称　大叶凤尾蕨 (Ｐｔｅｒｉｓｃｒｅｔｉｃａ)品种白斑大叶凤尾蕨 (ｃｖ .“Ａｌｂｏｌｉｎｅｎｅａｔａ”)。2　材料类别　叶背面成熟孢子。孢子囊群沿羽片顶部以下的叶缘连续分布 ,呈狭条形。供试植株来源于荷兰花卉市场的大叶凤尾蕨盆花。3　培养条件　 (1 )诱导孢子萌发培养基 :1 / 3ＭＳ ;(2 )初代培养基 :ＭＳ 6 ＢＡ 3 .0ｍｇ·Ｌ- 1 (单位下同 ) ＩＢＡ 3 .0 ;(3 )继代培养基 :ＭＳ 6 ＢＡ 1 .0 ＩＢＡ 1 .0 ;(4)生根培养基 :1 / 2ＭＳ ＮＡＡ 2 .0 ＩＡＡ2 .0 ;(5 )复壮培养基 :1 / 2ＭＳ 活性炭 2 0 ｇ·Ｌ- 1…