Micropropagation of Aerides maculosum lindl. (Orchidaceae)

Summary Young leaf segments from plants growing both in vivo and in vitro were cultured on Murashige and Skoog (MS) medium supplemented with auxins [naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D)], cytokinins [kinetin (KN) and N⁶-benzyladenine (BA)] and coconut liquid endosperm (CW). The explants from mature leaves did not show any growth and turned necrotic, while those obtained from juvenile leaves growing in vitro developed protocorm-like bodies (PLBs) at their cut surfaces within 4–8 wk depending on the growth medium. An optimum of 18 PLBs developed from leaf explants on medium supplemented with 2.0 mg l⁻¹ (8.87 μM) BA. Upon subculture in basal MS medium, the PLBs differentiated into plantlets within 6–8 wk. The resulting plantlets were successfully transferred to vermiculite initially and subsequently to potting mixture; 84% of the plantlets survived after 3 mo. of transplantation.     

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%。     

Effects of thidiazuron on in vitro propagation of Phalaenopsis and Doritaenopsis (Orchidaceae)

Phalaenopsis or Doritaenopsis (Orchidaceae) flower stem sections cultivated in vitro on media containing 0.23–11.35 M of N-phenyl-N-1,2,3-thiadiazol-5-ylurea (thidiazuron, TDZ) developed generally multiple shoots with the higher levels producing also protocorm-like bodies (PLB). Shoot and root development were reduced while proliferation increased with increasing concentration of TDZ. Similar effects were observed with Phalaenopsis protocorms at a lower range (0.23–1.14 M) of TDZ.     

根癌农杆菌介导大花蕙兰遗传转化的研究

以大花蕙兰原球茎(PLBs)为外植体,采用EHA105和LBA4404 2种根癌农杆菌菌株与pCAMBIA1301质粒构建工程菌介导,以建立大花蕙兰遗传转化体系,并比较不同受体处理方式、菌液浓度和侵染方式等对大花蕙兰转化的影响.结果表明:(1)以切成3 mm左右的PLBs小块作为受体材料,用OD600值为0.6的LBA4404根癌农杆菌菌株,并用MS+1.0 mg/L BA+200μmol/L AS(乙酰丁香酮)的液体培养基将菌液等体积稀释侵染,转化率可达62.5%.(2)大花蕙兰对潮霉素(Hyg)十分敏感,5 mg/L Hyg对转化后的PLBs有较好的筛选效果,筛选后最高成活率为13.0%.(3)PCR检测初步证明,通过根癌农杆菌介导的方法获得了2株转基因大花蕙兰植株.     

Carbon and nitrogen metabolism during Dendrobium huoshanense protocorm-like body development in suspension culture

Abstract

The aim of this work was to study carbon and nitrogen metabolism during suspension culture of protocorm-like bodies (PLBs) from Dendrobium huoshanense. No significant lag phase of PLB growth was found, and a maximum biomass of 288.6 g l^?1 was obtained at day 30 of culture. Sucrose concentration was halved as PLB growth proceeded, while no change in glucose and fructose levels in the medium was found in the first 3 days, followed by a gradual increase until day 9 of culture. Conversely, sucrose in PLBs accumulated dramatically in the first 6 days of culture, followed by a rapid decrease. At the same time, glucose and fructose content of PLBs increased, then declined after 9 days of culture. Soluble acidic invertase (soluble acidic IT) and alkaline invertase (alkaline IT) were activated after inoculation, and reached the highest value on day 6 and day 18, respectively whereas cell wall-bound invertase (cell wall-bound IT) seemed to be repressed throughout culture. The maximum value of sucrose synthase (SuSy) activity was observed on day 18, while sucrose phosphate synthase (SPS) stayed low and constant from inoculation to the end of culture. Ammonium concentration in the medium decreased rapidly, and was hardly detectable after 12 days, when the rapid utilization of nitrate began. Conversely, ammonium in PLBs showed a sharp increase in the first 3 days of culture, followed a rapid decrease until day 12, corresponding to nitrate depletion. Peaks in glutamine synthase (GS) and glutamate synthase (GOGAT) activity were observed on day 12 and 15, respectively. Nitrate reductase (NR) was repressed in the early culture stage, and activated from day 9 to 15 of culture. These results suggest that soluble acidic IT, alkaline IT, SuSy, GS, GOGAT and NR control carbon and nitrogen metabolism at different PLB growth stages.     

蝴蝶兰试管分株快速繁殖研究

以蝴蝶兰无菌幼苗为外植体,在分株增殖培养基(1/2MS+6-BA 3.0mg/L+NAA 0.2mg/L+CM20g/L)上培养30d后,形成幼小丛生植株;将此幼小植株移至生长培养基(1/2MS+6-BA 1.0mg/L+NAA 0.1mg/L)上,30d后便形成具3~4片叶与数条粗壮根的较大植株,移栽成活率达80%以上。     

春石斛优良品种‘森禾2006’组培快繁体系的建立

本文研究了春石斛‘森禾2006’的拟原球（PLBs）诱导、增殖、分化及壮苗和生根,建立了其组培快繁体系。结果表明：‘森禾2006’PLBs最适诱导培养基为1／2MS＋TDZ0．5mg·L-1＋水解酪蛋白2．0gL-1;PLBs增殖培养基为1／2Ms＋水解酪蛋白2．0gL-1;PLBs分化培养基为1／2MS＋KT1．0mg·L-1＋NAA0．1mg·L-1;壮苗生根培养基为Ms＋IBA0．5mg·L-1＋NAA0．1mg·L-1＋香蕉泥100．0g．L-1。     

蝴蝶兰花梗节间切段的类原球茎诱导与增殖

采用不同基本培养基和生长调节物质浓度组合,诱导蝴蝶兰花梗节间切段形成类原球茎和增殖的结果表明,花梗可见后10 d的切段诱导效果较好,较适宜的培养基为N_6 5.0 mg·L~(-1)6-BA 0.2 mg·L~(-1)KT 2 mg·L~(-1)活性炭。活性炭控制外植体褐化的效果明显优于维生素C。类原球茎增殖的适宜培养基为1/3MS 0.3mg·L~(-1)TDZ 0.2mg·L~(-1)NAA 2g·L~(-1)活性炭。     

蝴蝶兰原球茎增殖分化影响因子探讨

采用3个月胚龄的种胚播种形成的原球茎,初代培养时间在2.5~3个月之间换瓶转接,原球茎生长状况较好,分化形成的芽苗生长健壮;以1/2MS为基本培养基培养30d,原球茎增殖率达332%,MS培养基更有利于分化,培养2个月分化发芽率达90.6%;附加不同细胞分裂素原球茎的增殖率为6-BA >KT >Ad+KT >Ad;NAA浓度在0~1.0mg/L,对原球茎增殖分化影响不明显。     

Efficient propagation of protocorm-like bodies of Phalaenopsis in liquid medium

Propagation by multiplication of protocorm-like bodies (PLB) of Phalaenopsis in liquid culture was investigated. VW-liquid medium was the most suitable for PLB multiplication, which showed 30% PLB multiplication. A cotton plate was chosen as a reactor for PLB propagation. The PLB in VW-liquid medium multiplied 80% with non-absorbent cotton as a PLB support, as opposed to 70% in VW-solid medium; the PLB growth rate in that medium was much higher than that in the solid culture. When the cotton thickness was 6.0 mm that was corresponded to four layers of absorbent cotton and medium content containing the cotton was 7.2 ml g^-1 cotton, the PLB multiplication ratio was 100%; the most effective for the propagation of PLB. This indicates that there is an optimum wet condition in the liquid culture for an efficient PLB propagation.Abbreviations BA 6-benzyladenine - NAA -naphthaleneacetic acid - MS Murashige & Skoog basal medium - PLB protocorm-like body - VW Vacin & Went - TBR turbine blade reactor     

细茎石斛拟原球茎生长与有效成分积累的关系

目的 :研究细茎石斛拟原球茎生长规律及其与两种有效成分总生物碱和可溶性多糖积累的关系。方法 :采用液体静止培养方法培养细茎石斛拟原球茎 ,在一个周期 ( 60天 )内 ,每隔 1 0天取样 ,测定拟原球茎的鲜、干重 ,总生物碱含量和可溶性多糖含量。结果 :液体培养的细茎石斛拟原球茎生长曲线大致呈“S”型 ,拟原球茎经过 1 0d左右的延迟期后进入对数生长期 ,第 5 0d左右达到生长高峰 ,此后 ,生长缓慢 ,进入静止期。拟原球茎中总生物碱和可溶性多糖含量随着拟原球茎的生长逐渐积累 ,在培养的第 40d左右达到高峰 ,之后含量有所下降 ,这两种有效成分的积累与生长基本同步 ,且拟原球茎中两种有效成分的含量接近或超过野生植株茎中的含量。结论 :液体培养的细茎石斛拟原球茎生长曲线大致呈“S”型 ,总生物碱和可溶性多糖的积累与生长基本同步 ,且拟原球茎中两种有效成分的含量接近或超过野生植株茎中的含量     

杂交兰类原球茎增殖中芽分化的控制和快速繁殖

以‘玉女杂交兰’为材料,研究了培养时间、活性炭、切块大小和外源生长调节物质对类原球茎增殖和分化的影响。结果表明,培养时间、活性炭和切割处理对类原球茎增殖和分化影响显著,在培养基中添加活性炭或对类原球茎进行切割均能有效控制增殖过程中的芽分化。将类原球茎切成直径2~3 mm的小块接种到培养基MS＋1.0 mg.L-16-BA＋0.2 mg.L-1NAA＋0.3 g.L-1AC＋30 g.L-1蔗糖＋5.5 g.L-1琼脂中培养40 d,类原球茎增殖率为384.23%。将增殖后的类原球茎接种到培养基1/2MS＋1.5 mg.L-16-BA＋0.1 mg.L-1NAA＋20 g.L-1蔗糖＋5.5 g.L-1琼脂中培养40 d,芽分化率为463.06%。将分化的芽转入培养基1/2MS＋0.5 mg.L-1NAA＋0.5 g.L-1AC＋20 g.L-1蔗糖＋100 g.L-1土豆汁＋5.5 g.L-1琼脂中培养,生根率为100%,平均根分化数为2.80条.株-1。以泥炭土为基质,组培苗的移栽成活率可达97.78%。     

In vitro propagation of Phalaenopsis via culture of cytokinin-induced nodes

A new procedure for in vitro propagation of orchids belonging to the genus Phalaenopsis was developed. In contrast to commonly employed propagation methods that make use of leaf, root, or shoot tip tissues, we have used elongated stems of 6-benzyladenine-induced young seedlings as starting material for propagation. The elongated stem consisted of several nodes of which top nodes were used for cyclic propagation of new explants and the middle nodes for producing shoots or multiple adventitious buds. The whole procedure of proliferation could be completed within 7 months, and about 2,300 plantlets were produced from a single induced stem in a single year. This method may be used for propagation of seedlings in the case of lack of seeds in orchid breeding or for propagation of vegetative buds developed on flower stalks of rare orchid varieties when available flower stalks are limited. It may also have great potential for the propagation of wild threatened orchid species.Abbreviations PLB(s) protocorm-like body(ies) - zeatin 6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine - 2ip 6-(,-dimethylallylamino)purine - kinetin 6-furfurylaminopurine - BA 6-benzyladenine     

外部因子对蝴蝶兰叶片原球茎状体发生的影响

对影响蝴蝶兰叶片原球茎状体 (PL B,Protocorm - like- body)发生和植株再生的外部因子进行了研究。结果表明 :BA是决定原球茎状体发生的主要因子 ;苹果汁、香蕉汁和椰子汁明显促进原球茎状体的形成 ;在 MS+BA5mg/L+椰子汁 15%的培养基中 ,蝴蝶兰叶片原球茎状体的诱导率可达 6 0 %以上 ;活性炭可有效防止外植体叶块变褐死亡 ;多效唑 1.5mg/L可促进再生植株根的形成 ,使叶片变厚变短变绿 ,使试管苗移栽成活率达 95%以上     

扁桃优良砧木离体快繁

对扁桃优良砧木“Hansen”侧芽进行离体快繁研究,结果表明：外植体取材时间不同,成活率差异明显,以春季芽即将萌动的3月下旬为最佳时间,在MS 6-BA1．0mg／L GA30．2mg／L培养基上诱导芽萌发,萌发率在75％以上;以MS 6-BAl．5mg／L IAA0．1mg／L进行芽的增殖培养效果最好;1／2MS IBA0．5mg／L诱导生根,并给以黑暗预处理可使生根率达85％以上,每株生根数量多,根系质量好;采用分步炼苗移栽,提高了小苗成活率,有利于小苗后期生长。     

Direct somatic embryogenesis and plant regeneration from leaf explants of Phalaenopsis amabilis

Leaf explants of Phalaenopsis amabilis var. formosa formed clusters of somatic embryos directly from epidermal cells without an intervening callus within 20 – 30 d when cultured on 1/2-strength modified Murashige and Skoog medium supplemented with 0.1, 1 and 3 mg dm⁻³ TDZ. Repetitive production of embryos involved secondary embryogenesis could be obtained by culturing segments of embryogenic masses on TDZ-containing media. Plantlet conversion from embryos was successfully achieved on regulator-free growth medium.     

Regulation of plant productivity I: Improved seedling vigor and floral performance of Phalaenopsis by 2-(3,4-dichlorophenoxy)triethylamine [DCPTA]

Application of the tertiary amine bioregulator, 2-(3,4-dichlorophenoxy)triethylamine [DCPTA] to seedling Phalaenopsis plants increased seeding survival, long-term vegetative plant growth, and greatly accelerated flowering when compared to controls. Application of 30 µM (10 ppm) DCPTA during the transfer of orchid seedlings from sterile agar culture to greenhouse community pot culture caused a 2 to 3-fold increase in root and leaf growth of seedling plants over the growth of controls. After 2 years greenhouse culture, the total dry weight of 30 µM DCPTA-treated plants was doubled when compared with controls. Flowering of 30 µM DCPTA-treated plants was increased significantly (p < 0.05) over that of controls after 11 and 18 months plant growth. During a 2 year growth period, the total number of racemes produced by 30 µM DCPTA-treated plants was doubled when compared with the floral performance of controls. The effects of DCPTA on plant flowering appeared to function independently of DCPTA-mediated effects on plant growth.Reference to a company or product name does not imply approval or recommendation of the product by the U.S. Department of Agriculture to the exclusion of others that may be suitable.     

蚊净香草快速繁殖研究

以蚊净香草嫩叶和叶柄为外植体进行培养,筛选合适的外植体与诱导、增殖和生根的最佳培养基。结果表明,叶片外植体在MS+6-BA 2.0mg/L+NAA 0.5mg/L培养基最适于诱导愈伤组织和不定芽;MS+6-BA 1.0mg/L+NAA 0.3mg/L培养基有利于不定芽增殖;无根苗在含有低浓度无机盐和生长素水平的生根培养基上易生根,生根率高达100%。     

蝴蝶兰组织培养研究进展(综述)

本文从蝴蝶兰外植体的选择．不同基本培养基、激素及添加物对其增殖与分化的影响,外植体褐变的防治以及生根壮苗方法等,概述蝴蝶兰组培快繁方面的研究进展,为其组培技术研究提供参考。     

Effects of thidiazuron and N6-benzylaminopurine on shoot regeneration of Phalaenopsis

Adventitious bud formation from the vegetative buds of the flower stalks of Phalaenopsis occurred on Vacin and Went medium with 15% coconut water and 5 to 40 M thidiazuron (TDZ) or 40 M N⁶-benzylaminopurine. The highest efficiency of induction was achieved with 5 or 10 M TDZ. Adventitious buds developed into shoots on VWC medium. TDZ was more effective than BAP in stimulating the axillary buds of intact shoots to develop. Regenerated shoots rooted after about two months of culture on VWC medium with 1% sucrose. Shoot tips excised from the regenerated shoots initiated protocorm-like bodies after two months of culture on VWC medium.Abbreviations VWC medium Vacin and Went medium with 15% (by volume) coconut water - TDZ thidiazuron - BAP N⁶-benzylaminopurine - Plbs protocorm-like bodies