蝴蝶兰EMS离体化学诱变及再生植株RAPD检测

用EMS对离体培养的蝴蝶兰类原球茎薄切片进行化学诱变,研究了不同浓度、不同时间诱变处理对类原球茎薄切片生长、类原球茎再生及再生苗生长的影响,并对再生苗DNA进行了RAPD检测。结果表明,诱变剂EMS对类原球茎薄切片生长产生重要影响,部分薄切片褐化死亡,再生类原球茎生长受到抑制,再生苗数量减少,表现出明显的伤害作用,这种伤害作用随诱变剂浓度的加大和处理时间的延长而加重。诱变剂EMS使部分再生苗畸变,叶片皱缩、变厚。并产生2株叶形、叶色突变体。部分再生苗表现出RAPD图谱带型的多态性,暗示DNA序列发生了一定的变异。EMS适合诱变剂量为浓度0．4％处理2d～4d。     

蝴蝶兰原球茎液体增殖培养的研究

以茎尖诱导形成的原球茎为外殖体,采用液体培养方式探讨了不同浓度的激素配比、蔗糖浓度和添加不同量的新鲜液汁对蝴蝶兰原球茎增殖的影响,并比较了液体和固体两种培养方式的原球茎增殖特征.结果表明:不同浓度的外源激素及其配比对蝴蝶兰原球茎增殖影响不明显,但对原球茎生长影响较大,6-BA 2.0 mg/L+Ad 1.0 mg/L+NAA 0.5 mg/L的激素浓度组合比较适合蝴蝶兰原球茎增殖,蔗糖浓度对蝴蝶兰原球茎增殖和生长影响较大,适合蝴蝶兰PLBs增殖的蔗糖浓度为20 g/L,添加10 % ～40 %的新鲜椰汁对蝴蝶兰原球茎增殖生长有明显促进作用;原球茎液体增殖培养的时间不宜超过4周,5周内连续培养增殖曲线呈倒"V"字形;液体增殖培养的原球茎分化成苗的时间相对较晚.     

绿玉树试管苗物理化学诱变及其抗寒突变体的筛选

以已建立的微体繁殖体系为基础,对绿玉树再生丛芽进行Co^60γ射线不同剂量的辐射和不同浓度EMS诱变处理,以HYP为突变体选择压,筛选出抗HYP突变体小苗,并对突变体小苗进行抗寒测试。结果表明：辐射剂量影响丛芽的增殖、突变率及芽苗的生长,1．5KR为绿玉树丛芽辐射诱变比较适宜的剂量。0．2％的EMS亦能诱导绿玉树丛芽块产生突变芽,但与正常培养获得的再生小苗相比,诱变后产生的抗HYP芽苗生长缓慢,再生小苗矮小。抗HYP突变小苗的抗寒性比正常植物的抗寒性强。     

铁皮石斛原球茎常温保存研究

以铁皮石斛原球茎为材料,通过不同培养基、蔗糖浓度、继代周期、保存时间等多种因素对原球茎在保存过程中增殖生长和分化成苗的影响,进行铁皮石斛原球茎常温保存的研究。结果表明:铁皮石斛原球茎常温((25±2)℃)保存的适宜培养基为1/2MS、蔗糖浓度为1%,继代周期可达10个月;原球茎在5年内能保持分化和增值能力,随着保存年限的增加,分化率越来越低,可通过复壮和成苗培养提高分化成苗率。     

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。     

甜叶菊茎段腋芽诱导培养基和NaN3诱变条件筛选及诱变试管苗POD同工酶分析

以甜叶菊(Stevia rebaudiana Bertoni)茎段为外植体,采用L9(34)正交表对腋芽诱导培养基中的激素种类和浓度(0.0、0.1和0.2 ng·L-1NAA;0.0、0.5和1.0 mg·L-16-BA;0.000、0.005和0.010 mg·L-1TDZ)进行筛选;在此基础上对诱变过程中NaN3溶液的浓度(3、6和9 mmol·L-1)和处理时间(1、2、4和8h)进行比较,并初步筛选出最佳的NaN3诱变条件;采用聚丙烯酰胺凝胶电泳法(PAGE)对经上述NaN3诱变处理后培养5、10和15 d的试管苗POD同工酶酶谱的变化进行了分析.结果表明:在添加了不同激素组合的培养基上均能诱导出腋芽,但腋芽数和苗高有差异;经综合比较后可确定适宜于甜叶菊腋芽诱导的培养基为添加1.0 mg·L-16-BA和0.1 mg·L-1NAA的MS培养基(含5.0g·L-1琼脂粉和30g·L-1蔗糖,pH 6.0).经NaN3诱变处理后,茎段在腋芽诱导培养过程中的死亡率随NaN3浓度提高和处理时间延长逐渐增加,平均腋芽数和苗高则下降,且多数处理组试管苗矮化;根据半致死剂量确定的最佳NaN3诱变处理方法为将甜叶菊茎段置于9 mmol·L-1 NaN3溶液中浸泡4h.PAGE分析结果表明:甜叶菊诱变试管苗的POD同工酶谱均可分为a、b和c区,但在不同培养时间各处理组POD同工酶的条带数和活性有所变化.在培养5和10 d后各处理组诱变试管苗POD同工酶的活性和条带数量有明显差异,而在培养15 d后各处理组诱变试管苗POD同工酶活性有差异,但条带数量没有明显变化,表明用适宜浓度的NaN3进行诱变处理可导致甜叶菊试管苗短期的应激效应.     

磷对霍山石斛类原球茎悬浮培养细胞生长和多糖合成的影响

在确定了最适接种量和外植体细胞生理时间的基础上,研究了在不同起始磷浓度下,霍山石斛类原球茎生长、碳、氮消耗和多糖积累的动力学特性。以生长30d的类原球茎为材料,在接种量为100g/L时,类原球茎生长的最佳起始磷浓度为2.5mmol/L,培养36d时,类原球茎鲜重达496.5g/L。动力学分析表明,磷是霍山石斛类原球茎生长的限制性因素,胞内磷的积累水平与细胞生长具有相关性,2.5mmol/L的磷酸盐有利于碳、氮等营养物质的吸收;而多糖积累的最佳起始磷浓度为0.312mmol/L,培养36d时,其产量为2.22g/L。     

大花蕙兰四倍体的离体诱导和鉴定

采用组织培养方法研究了秋水仙素处理对大花蕙兰类原球茎增殖、分化和四倍体诱导的效应.结果表明,秋水仙素处理明显抑制类原球茎的增殖和分化,提高类原球茎褐变率和死亡率;在试验浓度和时间范围内,秋水仙素浓度越高,处理时间越长,抑制作用越强,解除抑制作用所需的继代次数越多.所有秋水仙素处理均能诱导出四倍体,但不同处理的四倍体诱导率不同,当处理浓度为0.05%、时间为5 d时,四倍体诱导率最高,为23.7%.二倍体及其四倍体的气孔保卫细胞长度和气孔密度均存在极显著的差异.四倍体植株比二倍体矮,茎部较粗壮,株型紧凑,叶片颜色浓绿、质地变硬.秋水仙素处理和组织培养相结合是创建大花蕙兰多倍体的有效方法.     

实验室条件下五唇兰菌根真菌专一性研究

利用从高原温带兰科植物菌根中获得的22个菌根真菌菌株, 对五唇兰(Doritis pulcherrima)进行了室内种子萌发、原球茎分化和组培苗回接试验, 从交叉回接的角度对附生兰科植物与菌根真菌的生理专一性进行了探讨。经过20周的共生培养, 只有编号为Cf1和Mm1的两个菌株使种子表现出种胚明显膨大的萌发迹象; 9个菌株能够促使原球茎较好地分化发育出根叶; 11个菌株处理苗的平均鲜重增长率高于对照组(156.25%), 其中Mm1的效果达到极显著水平(p = 0.01)。通过根切片显微观察, 在原球茎分化根和回接效果良好的处理苗的根皮层组织发现典型的菌丝团结构, 表明菌根体系已成功建立。温带地生兰菌根真菌对五唇兰种子萌发、原球茎发育和幼苗生长等3个重要生长阶段影响的试验显示, 五唇兰的种子和菌根真菌的共生萌发效果不佳, 而原球茎及幼株更容易与之建立良好的共生关系。同时, 也没有发现同一个真菌菌株能够对五唇兰的种子、原球茎和幼苗均产生促进作用。研究结果表明, 五唇兰的菌根真菌专一性因生理生长阶段的不同而存在差异。     

大薯类原球茎的离体诱导及再生体系的建立

采用正交试验设计方法,以大薯带节茎段为外植体,离体诱导类原球茎并建立大薯类原球茎的再生体系,以解决愈伤组织分化成苗和试管苗移栽成活率低的难题。结果表明：以带节茎段为外植体诱导类原球茎的最适培养基为MS（含3×Ca2＋）＋1.0 mg·L-1 6-BA＋0.2 mg·L-1 NAA＋0.1%PVP＋3%蔗糖,诱导率高达93.33%;类原球茎增殖的最适培养基为MS＋4mg·L-1 6-BA＋80 mg·L-1 Ad＋0.1%PVP＋3%蔗糖;类原球茎生根的最适培养基：1/2MS＋0.10 mg·L-1 NAA＋0.1%PVP＋3%蔗糖。将诱导得到的生根类原球茎植株进行炼苗,移栽基质珍珠岩：蛭石=2：1,移栽成活率可达到95%。     

Priming biotic factors for optimal protocorm-like body and callus induction in hybrid Cymbidium (Orchidaceae), and assessment of cytogenetic stability in regenerated plantlets

Protocorm-like bodies (PLBs) and callus were induced in epiphytic hybrid Cymbidium Twilight Moon ‘Day Light’, where induction capacity was strongly explant dependent. Following the use of various explant sources (PLB, leaf tip or base, root tip or base, cell and tissue ‘suspension’), highest PLB formation and callus induction occurred when we used whole PLBs, PLB segments or PLB transverse thin cell layers (tTCLs) or longitudinal TCLs (lTCLs). Plantlet growth and photosynthetic state from whole or bisected PLBs, as well as from tTCLs were not significantly different, after analysis of chlorophyll content. However plantlets generated from lTCLs showed lower values for growth and photosynthetic parameters. All resultant plants were shown to be cytogenetically identical using RAPD and mtDNA analysis despite cytological variation and endopolyploidy occuring between different plant parts. Acclimatization and survival rate was shown to be 100% in the generated plants.     

Micropropagation of Spathoglottis plicata

A rapid and reliable micropropagation method was established for Spathoglottis plicata. Nodal and leaf explants dissected from 8-month-old pot-grown seedlings were cultured on charcoal-amended Murashige and Skoog medium supplemented with 16 combinations of α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA) at concentrations of 0.54–10.74 μm. Regeneration of protocorm-like bodies (PLBs) and subsequent plantlet development were observed from 98.5% of the nodal explants. Only 6.5% of leaf explants and occasionally some root segments (dissected from regenerated plantlets) were able to produce PLBs and then plantlets. The optimum plant growth regulator (PGR) combination for maximal PLB regeneration was 5.37 μm NAA and 0.44 μm BA. The best combination of PGR for plantlet development was 2.69–10.74 μm NAA and 8.88 μm BA. The NAA to BA ratios for maximal PLB induction and plantlet development were 12.2 and 0.3–1.2, respectively. Regenerated PLBs and plantlets, when cut into pieces of less than 1 mm and subcultured onto the above media, regenerated new PLBs and plantlets in another 3 months. Received: 20 February 1997 / Revision received: 27 May 1997 / Accepted: 16 June 1997     

TDZ induced micropropagation in Cymbidium giganteum Wall. Ex Lindl. and assessment of genetic variation in the regenerated plants

Multiple protocorm-like bodies (PLBs) were induced from pseudostem segments of Cymbidium giganteum using a low concentration (0.909?μM) of TDZ. An exposure time of 8?weeks to TDZ did not adversely affect healthy plantlet development from the induced PLBs when transferred to basal medium. TDZ at higher concentrations, although induced more PLBs, affected subsequent plantlet and root development. Absorption of TDZ was better in a dual phase culture system where a thin layer of the liquid medium overlaid the semi-solid medium. Significant interaction effects of culture phase and TDZ concentrations were seen for the number of shoots, shoot length and root length. Irrespective of the concentration or culture phase, residual effect of TDZ was seen even after 4?weeks of withdrawal from treatment. Comparison of phenotypic characters did not detect any significant variation between the control and TDZ-derived plants. Assessment of molecular variation using 18 RAPD primers detected overall 5.81?% change in the regenerants. Results suggested that seedling-derived pseudostem segments cultured in a dual phase at a low dose of TDZ is most appropriate for inducing healthy plantlets in C. giganteum. Furthermore, a combination of phenotypic and molecular characterization using proper trait/marker and data analysis using a variety of statistical tools provide better insight into genetic fidelity of the regenerants.     

文心兰原球茎形态发生与可溶性物质及抗氧化酶的关系

以文心兰切花品种'南茜'无菌苗为材料,取其茎尖通过组织培养诱导形成原球茎和幼苗,观察并分析了原球茎各形态发生阶段的特征及其可溶性糖和蛋白质含量、抗氧化酶(POD、CAT和SOD)活性以及相关同功酶(POD、EST和SOD)的变化.结果显示:(1)文心兰原球茎形态发生可分为外植体期、外植体膨大期、愈伤组织期、原球茎形成期、原球茎成熟期、叶鞘伸展期、顶端腋芽发育期及幼苗期8个阶段.(2)可溶性糖和蛋白质含量均在叶鞘伸展期出现最大峰值;POD活性在外植体膨大期、CAT和SOD活性在愈伤组织期分别出现最大峰值.SOD同工酶的2条酶带在愈伤组织期到幼苗期交替出现;EST同工酶在原球茎形成期有2条特异酶带.研究表明,可溶性糖和蛋白质的含量以及POD、CAT、SOD活性的特异变化与文心兰茎尖脱分化及原球茎再分化的实现密切相关,不同类型的同工酶在原球茎同一发生阶段表现出较大差异,EST同工酶的2条特异酶带可作为原球茎形成的标志.     

Micropropagation of Cypripedium macranthos var. rebunense through Protocorm-like Bodies Derived from Mature Seeds

Cypripedium macranthos var. rebunense is the most famous terrestrial orchid in Japan, since the variety has large beautiful yellowish-white flowers and is threatened with extinction. Establishment of an efficient method for micropropagation is urgently needed. When imbibed mature seeds of the orchid, that had been pre-chilled at 4°C for 3 months, were sown onto Hyponex-peptone medium that contained both α-naphthaleneacetic acid (NAA) and cytokinin, protocorm-like bodies (PLBs) were formed from germinated seeds. Although the growth of PLBs was very slow, plantlets were easily regenerated from the PLBs on hormone-free medium. The PLBs were subcultured eight times along 2 years without loss of ability to regenerate plantlets, and one aggregate of PLBs (ca. 5 mm in diameter) produced ca. 10 plants within a year. A reduction of commercial value through a large-scale micropropagation by this method will be able to prevent illegal collection from the wild populations.     

Detection of somaclonal variation of cotton (Gossypium hirsutum) using cytogenetics, flow cytometry and molecular markers

Two protocols of plant regeneration for cotton were adopted in this study, namely, 2, 4-D and kinetin hormone combination and IBA and kinetin hormone combination. Twenty-eight embryogenic cell lines via somatic embryogenesis and 67 regenerated plants from these embryogenic calli were selected and used for random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), chromosomal number counting, and flow cytometric analysis. The roles of RAPD and SSR markers in detecting somaclonal variation of cotton (Gossypium hirsutum L.) were evaluated. Two cluster analyses were performed to express, in the form of dendrograms, the relationships among the hormone combinations and the genetic variability. Both DNA-based techniques were able to amplify all of the cell clones and regenerated plantlets genomes and relative higher genetic variation could be detected in the culture type with 2, 4-D and kinetin hormone combination. The result suggested that 2, 4-D and kinetin hormone combination could induce relative high somaclonal variation and RAPD and SSR markers are useful in detecting somaclonal variation of regenerated cotton plants via somatic embryogenesis. Chromosome number counting and flow cytometry analysis revealed that the number of chromosomes and ploidy levels were nearly stable in all regenerated plants except two regenerated plantlets (lost 4 and 5 chromosomes, respectively) which meant that cytological changes were not correlated with the frequency of RAPD and SSR polymorphisms. This result also might mean that the cell lines with variation of chromosome numbers were difficult to regenerate plants.     

Effects of the Application of Different Concentrations of NaN3 for Different Times on the Morphological and Cytogenetic Characteristics of Barley (Hordeum vulgare L.) Seedlings

Sodium azide (NaN3) has been used in many biological studies as a mutagen. In the present study, the morphological and cytogenetic effects of NaN3 on barley (Hordeum vulgare L.) seedlings were investigated. Seeds of barley were treated with different concentrations of NaN3 (0.5, 1.0, 1.5, 2.0, 2.5, and3.0 mmol/L) and applied for different periods of time (3 and 4 h). Parameters investigated, except for the mitotic index, were determined on Days 7 and 14. Increasing concentrations of NaN3 affected germination rates on Days 7 and 14 following application for 3 and 4 h. Although the length of the roots and leaves was affected by treatment with NaN3 on Day 14 of the germination period, coleoptile length was affected by NaN3 treatment on Day 7. Increasing concentrations of NaN3 and increased treatment period decreased the mitotic index compared with the untreated control. The inhibitory effects of NaN3 on the mitotic index indicate that NaN3 can have genotoxic and mutagenic effects on barley seedlings.