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。     

Tissue distribution and biosynthesis of 1,2-saturated pyrrolizidine alkaloids in Phalaenopsis hybrids (Orchidaceae)

Phalaenopsis hybrids contain two 1,2-saturated pyrrolizidine monoesters, T-phalaenopsine (necine base trachelanthamidine) and its stereoisomer Is-phalaenopsine (necine base isoretronecanol). T-Phalaenopsine is the major alkaloid accounting for more than 90% of total alkaloid. About equal amounts of alkaloid were genuinely present as free base and its N-oxide. The structures were confirmed by GC-MS. The quantitative distribution of phalaenopsine in various organs and tissues of vegetative rosette plants and flowering plants revealed alkaloid in all tissues. The highest concentrations were found in young and developing tissues (e.g., root tips and young leaves), peripheral tissues (e.g., of flower stalks) and reproductive organs (flower buds and flowers). Within flowers, parts that usually attract insect visitors (e.g., labellum with colorful crests as well as column and pollinia) show the highest alkaloid levels. Tracer feeding experiments with (14)C-labeled putrecine revealed that in rosette plants the aerial roots were the sites of phalaenopsine biosynthesis. However active biosynthesis was only observed in roots still attached to the plant but not in excised roots. There is a slow but substantial translocation of newly synthesized alkaloid from the roots to other plant organs. A long-term tracer experiment revealed that phalaenopsine shows neither turnover nor degradation. The results are discussed in the context of a polyphyletic molecular origin of the biosynthetic pathways of pyrrolizidine alkaloids in various scattered angiosperm taxa. The ecological role of the so called non-toxic 1,2-saturated pyrrolizidine alkaloids is discussed in comparison to the pro-toxic 1,2-unsaturated pyrrolizidine alkaloids. Evidence from the plant-insect interphase is presented indicating a substantial role of the 1,2-saturated alkaloids in plant and insect defense.     

TDZ高效诱导蝴蝶兰叶片不定芽及植株再生

以蝴蝶兰（Phalaenopsis）无菌幼苗叶片为材料,研究添加TDZ（噻重氮苯基脲）条件下不同基因型、激素组合、叶片大小、暗培养时间对不定芽发生和再生植株的影响。结果表明：在相同培养条件下,不同基因型外植体芽诱导率差异显著,‘红天使’最高,达81.5%,‘汕农姑娘’等2个品种为0,‘满天红’等4个品种为9.2%～34.9%;添加TDZ芽诱导率显著高于6-BA;单独添加TDZ或6-BA芽诱导率显著高于NAA与TDZ或6-BA的组合。叶片越小不定芽诱导率越高;短时间暗处理有利于不定芽的发生。以1～2 cm长叶片为材料、15 d暗处理、在1/2 MS添加3 mg/L TDZ培养基中,‘红天使’的叶片外植体芽诱导率和平均不定芽数分别可达100.0%和18.2个。研究发现,在继代培养中TDZ对芽的伸长有抑制作用。     

蝴蝶兰LFY基因克隆及高效植物表达载体的构建

根据NCBI中蝴蝶兰LFY花序分生组织基因序列设计2对引物,用RT-PCR法从蝴蝶兰花芽中扩增出LFY基因,对扩增产物进行克隆和测序.结果表明,获得的蝴蝶兰LFY基因约为1500 bp,与报道序列同源性达98.71％.将LFY基因插入pRI101-ON载体中,经PCR、双酶切及测序鉴定,证实重组表达质粒中含有目的片段,表明成功构建了高效植物表达载体pRI1O1-LFY.     

Characterization of the primary pollen signal in the postpollination syndrome of Phalaenopsis flowers

In many flowers, and especially in orchids, pollination regulates a syndrome of developmental events that collectively prepare the flower for fertilization while shedding of organs that have completed their function in pollen dispersal and reception. In this study, we performed a water extraction of the primary pollen signal(s) from the pollinia of Phalaenopsis flowers and characterized its biochemical nature. The primary pollen signal is readily soluble in water and is a relatively small molecular substance below 3000 MW. The pollen signal is probably not proteinaceous in nature, since biological activity was retained after digesting the pollen diffusate by Proteinase K or boiling for 30. By separating the pollen diffusate on an amino anion exchange column, we found that different fractions induced the postpollination syndrome suggesting that different pollen-borne substances may be involved in the pollination response. More than 90% of a radiolabeled free IAA standard coeluted with a specific fraction, however other collected fractions also induced the postpollination response, suggesting that IAA can not be the only primary pollen signal as previously described. High pressure liquid chromatography analysis revealed that the pollen diffusate contained two major peaks and five smaller peaks of detected substances. Fractions containing substances from two of these peaks completely mimicked the postpollination response of perianth senescence and ovary growth, while fractions of the other peaks only induced perianth senescence. By running additional standards, it was found that 1-aminocyclopropane-1-carboxylic acid peaked at the same retention time as one of the major pollen diffusate peaks, while the free IAA standard peak could not be correlated to any of the pollen diffusate peaks. In the future, further purification of these peaks, and analysis by gas chromatography coupled with mass spectrometry, will provide more information about the exact nature of the primary pollen signals.     

低温对蝴蝶兰胚珠发育的影响

低温(12-18℃)处理的蝴蝶兰,授粉50 d开始分化胚珠原基、60 d胚囊处于大孢子母细胞时期,比常温(20-25℃)下的推迟10-15 d;授粉70 d,常温下的蝴蝶兰植株已完成雌配子体发育和双受精,而低温处理的蝴蝶兰仍未完成大孢子的发生过程.低温处理的蝴蝶兰,胚囊发育过程中珠心细胞提前退化,在大孢子母细胞外围形成较厚的胚囊壁.胚囊只发育到单核胚囊阶段,没有形成成熟胚囊.     

蝴蝶兰的组织培养和快速繁殖

通过诱导残败花梗上的休眠芽萌发,以萌发的幼叶和去茎尖的茎段为外植体进行组织培养,建立了蝴蝶兰(Phalaenopsis amabilis Bl.)的无菌繁殖体系,并筛选出最佳培养基组成.诱导休眠芽萌发的最佳培养基为不加任何激素的MS0培养基;原球茎诱导的适宜培养基为MS 3.0 mg·L-1 6-BA 0.5 mg·L-1 ZT 30 mg·L-1柠檬酸和MS 5.0 mg·L-1 6-BA 30 mg·L-1柠檬酸 30%椰乳(CM),其中茎段的诱导效果明显优于叶片,诱导率达95%;诱导无菌苗生根的最适培养基为1/4 MS 1.0 mg·L-1 6-BA 0.1 mg·L-1 NAA,生根率可达79%.     

不同植物生长调节剂对蝴蝶兰快速繁殖的影响

探讨了蝴蝶兰快速繁殖中不同植物生长调节剂的作用。实验表明：改良型VW培养基诱导原球茎有良好的效果,改良型VW 6-BA5．0mg／L诱导花梗产生原球茎最合适,改良型VW 6-BA3.0mg／L诱导茎尖产生原球茎最合适。降低分裂索6-BA浓度后,原球茎可以分化成完整小植株;壮苗阶段以改良型KC 6-BA0．1mg／L NAA0．5mg／L效果较佳。移栽基质选用水苔,成活率高。     

蝴蝶兰红花系品种杂交种子形态及胚培养研究

对21个蝴蝶兰自交或杂交组合的种子形态进行观察和无菌培养,结果表明,蝴蝶兰不同组合的种子形态有一定差异,平均种子长、宽及种胚长、宽分别为0.292±0.025 mm、0.073±0.003 mm及0.151±0.009 mm、0.069±0.003 mm。种子萌发率及萌发所需时间因组合而异,平均萌发率为72.4%,原球茎及第一片叶出现所需时间分别为22±3 d、78±6 d,杂交组合启动萌发早、萌发率高。种子启动萌发所需时间、萌发率与果实、种子大小相关不显著,而种子启动萌发所需时间与种胚长、宽呈极显著负线性相关,表明种胚越大,萌发越快。     

Argonaute 5 family proteins play crucial roles in the defence against Cymbidium mosaic virus and Odontoglossum ringspot virus in Phalaenopsis aphrodite subsp. formosana

The orchid industry faces severe threats from diseases caused by viruses. Argonaute proteins (AGOs) have been shown to be the major components in the antiviral defence systems through RNA silencing in many model plants. However, the roles of AGOs in orchids against viral infections have not been analysed comprehensively. In this study, Phalaenopsis aphrodite subsp. formosana was chosen as the representative to analyse the AGOs (PaAGOs) involved in the defence against two major viruses of orchids, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). A total of 11 PaAGOs were identified from the expression profile analyses of these PaAGOs in P. aphrodite subsp. formosana singly or doubly infected with CymMV and/or ORSV. PaAGO5b was found to be the only one highly induced. Results from overexpression of individual PaAGO5 family genes revealed that PaAGO5a and PaAGO5b play central roles in the antiviral defence mechanisms of P. aphrodite subsp. formosana. Furthermore, a virus-induced gene silencing vector based on Foxtail mosaic virus was developed to corroborate the function of PaAGO5s. The results confirmed their importance in the defences against CymMV and ORSV. Our findings may provide useful information for the breeding of traits for resistance or tolerance to CymMV or ORSV infections in Phalaenopsis orchids.