Chitosan as a growth stimulator in orchid tissue culture

The effect of shrimp and fungal chitosan on the growth and development of orchid plant meristemic tissue in culture was investigated in liquid and on solid medium. The growth of meristem explants into protocorm-like bodies in liquid medium was accelerated up to 15 times in the presence of chitosan oligomer, the optimal concentration being 15 ppm. The 1 kDa shrimp oligomer was slightly more effective compared to 10 kDa shrimp chitosan and four times more active compared to high molecular weight 100 kDa shrimp chitosan. The 10 kDa fungal chitosan was more effective compared with 1 kDa oligomer. The development of orchid protocorm into differentiated orchid tissue with primary shoots and roots was studied on solid agar medium. The optimal effect, the generation of 5–7 plantlets in 12 weeks was observed in the presence of 20 ppm using either 10 kDa fungal or 1 kDa oligomer shrimp chitosan. The data are consistent with preliminary results from field experiments and confirm unequivocally that a minor amount of chitosan has a profound effect on the growth and development of orchid plant tissue.     

亚美万代兰与多花兰种子发芽过程的扫描电镜观察

兰种子发芽之后形成原球茎与根状茎,前者以亚美万代兰,后者以多花兰为材料进行扫描电镜观察。多花兰种子发芽初期,与亚美万代兰相似,但分化子叶的能力较差,在暗培养下,只形成鳞片状叶,紧贴在根状茎的分生组织上。根状茎只有转入光培养才有茎叶分化。兰种子发芽时的毛状物,在电子显微镜下观察,其形态与根毛较接近。     

Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination

A transformation procedure for phalaenopsis orchid established by using immature protocorms for Agrobacterium infection was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. Protocorms obtained after 21 days of culture on liquid New Dogashima medium were inoculated with Agrobacterium strain EHA101(pIG121Hm) harboring both -glucuronidase (GUS) and hygromycin resistance genes. Subculture of the protocorms on acetosyringone-containing medium 2 days before Agrobacterium inoculation gave the highest transformation efficiencies (1.3–1.9%) based on the frequency of hygromycin-resistant plants produced. Surviving protocorms obtained 2 months after Agrobacterium infection on selection medium containing 20 mg l^–1 hygromycin were cut transversely into two pieces before transferring to recovery medium without hygromycin. Protocorm-like bodies (PLBs) proliferated from pieces of protocorms during a 1-month culture on recovery medium followed by transfer to selection medium. Hygromycin-resistant phalaenopsis plants that regenerated after the re-selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by Southern blot analysis. A total of 88 transgenic plants, each derived from an independent protocorm, was obtained from ca. 12,500 mature seeds 6 months after infection with Agrobacterium. Due to the convenient protocol for Agrobacterium infection and rapid production of transgenic plants, the present procedure could be utilized to assess expression of transgenes under different genetic backgrounds, and for the molecular breeding of phalaenopsis.     

The Relation Between Growth of Gastrodia elata Protocorms and Fungi

Armillaria mellea penetrated protocorms from seed germination and vegetative multiplication corms of Gastrodia elata with rhizomorph. At beginning, they formed a hypha passing road and a hypha flow in the inner cells of cortex, and then, they both penetrated inside of large cells and penetrated outside of cortical cells. Gastrodia elata seeds depended on digesting Mycenct osmundicola etc gain nutrition to germinate at the stage of sexual reproduction, but its corms of vegetative multiplication must be penetrated by Armillaria mellea obtaining nutrition for normal growth at the stage of vegetative propagation.     

Plant regeneration through direct shoot formation from leaf cultures and from protocorm-like bodies derived from callus of <Emphasis Type="Italic">Encyclia mariae</Emphasis> (Orchidaceae), a threatened Mexican orchid

Two procedures for the in vitro propagation of Encyclia mariae, a threatened Mexican orchid, were developed. In the first procedure, leaves from in vitro germinated seedlings were cultured on Murashige and Skoog medium (MS) supplemented with the range of 2.21–4.4 μM 6-benzylaminopurine (BA) in combination with 2.69–10.74 μM naphthalene acetic (NAA), 2.07–8.29 μM indole-3-butyric (IBA), or 2.85–11.42 μM indole-3-acetic acid (IAA) to determine the best medium for the induction of shooting. Maximum direct shoot formation from leaves was observed on MS containing 22.21 μM BA and 10.74 μM NAA (25 shoots/explant). The second procedure began with the culture of protocorms on media containing NAA, IBA, or IAA, which induced callus formation with high regenerative potential in the form of protocorm-like-bodies (PLBs) that eventually differentiated into shoots. The optimal response was attained when these structures were cultured on medium with 4.14 μM IBA (30 shoots/PLB). To promote the elongation of shoots derived from PLBs, the material was subcultured onto MS medium containing 22.21 μM BA and 5.37 μM NAA. Through the exploration of the effects of auxins and matrix on the rooting of shoots, it was determined that the optimal rooting occurred on media supplemented either with 5.71 μM IAA or 4.14 μM IBA either on agar-gelled medium or in liquid media with coir as the matrix. Rooting was found to be 20% higher in liquid media than in agar-gelled medium.     

印度梨形孢对铁皮石斛种子萌发和原球茎生长的影响

为探究印度梨形孢(Piriformospora indica)对铁皮石斛(Dendrobium officinale)种子萌发和原球茎生长的影响,在铁皮石斛种子离体培养和原球茎生长阶段分别接种印度梨形孢,对其形态发育特征和生理特性进行研究。结果表明,接种印度梨形孢的铁皮石斛种子的起始萌发时间提前,接种印度梨形孢的铁皮石斛原球茎的株高、鲜质量、干质量、叶片数、叶长、叶宽、根数、根长等都高于对照。在接种60 d的种子萌发率为68.8%,高于对照(28.6%);在接种80 d的铁皮石斛原球茎的叶绿素a、叶绿素b、总叶绿素含量显著高于对照,叶绿素a/b小于对照,净光合速率、气孔导度、蒸腾速率均高于对照,药用多糖含量显著高于对照。因此,接种印度梨形孢能促进铁皮石斛种子萌发和原球茎的生长,提高铁皮石斛的品质和产量。     

不同培养基和激素水平对大花蕙兰原球茎诱导的影响

以添加1.5%活性碳及3%的蔗糖的花宝1号和MS培养基为基础,加入不同浓度的BA和IBA,用外植体诱导的第1代组培苗基部以下连根1.1～1.2 cm为材料诱导原球茎.结果表明,花宝1号培养基优于MS培养基,BA1 mg/L IBA0.1 mg/L最合适,诱导率可达90.1%;光照以10 h/d左右为宜,培养温度以25～27℃最佳,其诱导率最高,生长状况最好.     

观赏兰科植物组培快繁及遗传转化的研究进展

兰花作为一种高档花卉,近年来其组培快繁和基因工程研究取得了比较大的进展。综述的观赏兰科植物组织培养内容包括外植体、培养基及培养方式等,遗传转化内容包括靶材料、选择标记基因、报告基因、启动子和转化方法等并总结了兰科植物基因工程研究的成果、最新进展及存在的问题 。     

墨兰原球茎生长的研究

以墨兰种子胚经培养诱导萌发形成的原球茎为材料,研究其生长的最适条件。结果表明：最佳基本培养基是ＭＳ培养基;ＮＡＡ０．５ｍｇＬ＋ＢＡ１．０ｍｇＬ－１有利于生长和分化;在培养基中加入１％蔗糖和０．１％活性炭最有利于原球茎生长;ｐＨ５．０偏酸环境适合于生长;每天给予１５．５ｕｍｏｌｍ－２Ｓ－１１６ｈ的光照最佳。如将上述单因子组合在一起,其原球茎鲜重增长率比对照（ＭＳ培养基）高４６％。在不同切割方式中,掰开法鲜重增长率比纵切法或横切法都高。