采用秋水仙碱创制优质、抗热同源四倍体不结球白菜

以不同浓度秋水仙素处理二倍体不结球白菜(Brassica campestrisssp.chinensisM ak ino)子叶生长点,对变异株进行了形态解剖学、农艺学、细胞学及营养品质鉴定。结果表明:0.1 mol/L秋水仙素处理6次的效果最佳。与二倍体相比,四倍体植株、气孔、花器官均表现巨大性;气孔密度、结实率显著降低;四倍体白菜蛋白质、可溶性糖和维生素C含量比二倍体分别增加15.69%、71.25%和22.18%;夏季高温条件下四倍体表现出良好的丰产性和抗热性。     

紫锥菊多倍体诱导与鉴定

本实验以紫锥菊愈伤组织上刚分化出的不定芽为材料,用不同浓度秋水仙素溶液对其进行诱导,确定最佳处理浓度和处理时间,并对诱导的多倍体与二倍体进行形态、显微、染色体及过氧化氢酶(CAT)活性的比较鉴定.结果表明:0.025%秋水仙素浓度处理24h的诱导效果最好,诱导率达42.85%;多倍体植株叶片肥厚、根粗壮,气孔面积极显著地大于二倍体植株,染色体数从24～28条不等,CAT平均酶活性是二倍体的2.1倍.     

甜叶菊同源四倍体离体诱导及鉴定

用不同浓度秋水仙素溶液处理甜叶菊不定芽,诱导同源四倍体,并进行解剖学、染色体鉴定和流式细胞仪鉴定倍性。结果表明:(1)用0.20%的秋水仙素溶液浸泡甜叶菊不定芽12h,同源四倍体诱导率最高,可达32.14%。(2)同源四倍体植株与二倍体(对照)相比,其气孔、叶片等均表现巨大性,且叶片变厚、叶色浓绿、叶片皱缩。(3)对照植株染色体2n=2x=22,四倍体植株染色体2n=4x=44;流式细胞仪倍性鉴定结果显示,对照DNA相对含量为100,四倍体DNA相对含量为200。(4)该研究共鉴定出48株甜叶菊同源四倍体植株,为进行倍性植株的诱导奠定了技术基础,为进一步开展甜叶菊同源四倍体新品种的选育提供了实验材料。     

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.     

铁皮石斛四倍体离体诱导和鉴定及生理特性研究

以铁皮石斛原球茎为材料,经不同质量浓度的秋水仙素(C₂₂H₂₅O₆)和0.02 g·mL^-1二甲基亚砜(DMSO)混合水溶液处理后进行组织培养,通过对变异株进行形态学、细胞学及流式细胞仪鉴定,以期获得稳定的四倍体植株并分析其生理特性。结果表明:用2.0 g·L^-1秋水仙素和0.02 g·mL^-1 DMSO混合水溶液处理铁皮石斛原球茎36 h后,植株诱导率达20%;诱导四倍体植株在形态上明显矮化、茎秆粗壮、叶片变小增厚、气孔直径增大;细胞遗传学观察发现,四倍体植株染色体2n=4x=76,二倍体植株染色体2n=2x=38;流式细胞仪分析显示,DNA相对含量四倍体为400,二倍体仅为200;四倍体植株叶片中叶绿素含量、可溶性蛋白、可溶性糖含量均高于二倍体,分别为5.03、3.59、2.98 mg·g^-1;四倍体叶片中主要抗氧化酶POD和SOD活性均显著高于二倍体,分别为9.08、180.4 U·mg^-1,且四倍体植株明显降低了MDA含量累积。研究认为,2.0 g·L^-1秋水仙素和0.02 g·mL^-1 DMSO混合水溶液处理原球茎36 h可提高诱导成功率、降低嵌合体比例,此组合为诱导四倍体较佳诱导条件。     

‘寒富’苹果二倍体及其同源四倍体叶片超微结构和叶绿素荧光参数特征

采用扫描电镜和石蜡切片法,以‘寒富’苹果二倍体及经秋水仙素加倍获得的同源四倍体植株为材料,比较两种倍性植株叶片超微结构、叶绿素含量及叶绿素荧光参数的日变化规律。结果显示:(1)与二倍体植株相比较,其同源四倍体叶片厚度、栅海比、气孔长、气孔宽、分别增加了15.1%、16.1%、70.5%、27.2%,而气孔密度显著减少了58.7%;其同源四倍体叶保卫细胞中叶绿体数和叶绿素含量分别比二倍体植株高出125.3%、37.7%。(2)‘寒富’苹果同源四倍体与其二倍体的叶绿素荧光参数PSⅡ的原初光能转化效率(Fv/Fm)、PSⅡ的潜在光化学效率(Fv/F0)和以吸收光能为基础的光合性能指数(PI)值的日变化趋势相似,但PI平均值比二倍体显著高出38.6%。研究表明,同源四倍体较二倍体叶片在形态上更大、更厚,气孔更大、密度更小,栅海比更大,表现出抗病的叶片结构;同时同源四倍体较二倍体含有更高的叶绿素含量,表现更优良的光合特性。     

离体条件下2种除草剂诱导泸定百合多倍体的比较研究

在离体条件下,以野生泸定百合(Lilium sargentiae)不定芽(2n=2x=24)为材料,以无菌水(CK)和秋水仙素为对照,分别用不同浓度(100、200、300μmol/L)除草剂(氟乐灵、二甲戊灵)溶液分别浸泡(12h、24h、36h)处理,通过根尖细胞染色体数和叶片下表皮保卫细胞及叶片形态特征观察,比较2种除草剂不同浓度及不同处理时间对泸定百合多倍体诱导的效果。结果表明:300μmol/L二甲戊灵浸泡36h,变异率达30.0%,200μmol/L氟乐灵浸泡36h,变异率达32.2%,但2种诱变剂处理后材料诱导变异和存活率的差异不显著。对变异材料通过细胞学鉴定,发现2种诱变剂均能够诱导出四倍体泸定百合,但与秋水仙素处理相比,2种诱变剂处理时间短,材料死亡率低,变异率较高,而且对人畜伤害小,成本低;此外,与二倍体植株相比,四倍体植株的叶片气孔显著增大、气孔密度显著降低。研究认为,除草剂二甲戊灵和氟乐灵可作为秋水仙素诱导多倍体的替代品,而且叶片气孔大小可作为初步快速检测多倍体的有效指标。     

广藿香毛状根多倍体诱导及其植株再生

为了提高药用植物广藿香的次生物质广藿香醇含量,采用秋水仙素人工诱导染色体加倍技术,进行了广藿香毛状根多倍体诱导及其植株再生、倍性鉴定和挥发油组分广藿香醇含量的测定。结果表明,广藿香毛状根多倍体诱导的最佳条件为0.05%秋水仙素处理36 h,其多倍体诱导率可达40%以上;经秋水仙素加倍的广藿香毛状根在MS+6-BA 0.2 mg/L+NAA 0.1 mg/L培养基中培养60 d后可获得毛状根多倍体再生植株。与对照(二倍体植株)相比,广藿香毛状根多倍体再生植株根系更发达、茎更粗、节间变短、叶片的长度、宽度和厚度均较二倍体明显增大。根尖细胞染色体压片观察证实,所获得的广藿香毛状根多倍体再生植株为四倍体,其根尖细胞染色体数约为128;同时,其叶片的气孔保卫细胞体积及其叶绿体数目均约为对照的两倍;但其气孔密度则随着倍性增加而下降,二倍体植株叶片的气孔密度约为四倍体植株叶片的1.67倍。GC-MS测定结果表明,广藿香毛状根多倍体再生植株的广藿香挥发油组分广藿香醇的含量为4.25 mg/g干重,约为二倍体植株的2.30倍。该结果证实毛状根多倍体化可提高药用植物广藿香的广藿香醇含量。     

四倍体艾纳香植株诱导及其倍性鉴定

在离体条件下,应用秋水仙素浸泡法和混合培养法,对艾纳香组培苗进行了不同浓度和时间的诱变处理,并通过植物形态学、细胞学、细胞中DNA含量等鉴定其倍性。结果表明,与浸泡法相比,混合培养法更适合四倍体艾纳香诱导,其中以0.025%秋水仙素处理9 d诱导效果最佳,诱变率高达65%。与二倍体植株相比,四倍体艾纳香植株形态特征变化明显,表现植株的枝条变粗壮,叶片变厚、颜色变深。四倍体叶片的气孔及其保卫细胞明显增大,并且其细胞中DNA含量是二倍体的两倍,表明四倍体艾纳香植株诱导成功。     

墨兰‘绿墨素’×大花蕙兰‘世界和平’F1代多倍体诱导初报

该研究以墨兰‘绿墨素’(Cymbidium sinense‘Lv mosu’)和大花蕙兰‘世界和平’(Cymbidium hybridum‘Shijieheping’)杂交兰F1代原球茎为材料,采用浸泡法研究不同浓度秋水仙素和不同处理时间诱导植株加倍的效果。结果表明:0.03%秋水仙素处理72 h,诱导变异率为36%,死亡率为36%,诱导效果最佳;多倍体植株与二倍体植株相比,表现为植株根部木质化加剧,植株矮壮,叶色深绿,叶片变厚、变宽,叶面粗糙,少部分有双叶脉、叶尖开裂、叶片扭曲,生长缓慢等;且多倍体植株气孔大,气孔密度减小;经流式细胞仪分析,二倍体墨兰×大花蕙兰F1代植株的荧光通道值为88,多倍体植株荧光通道值是176,多倍体植株为四倍体。该研究结果为培育兰花新品种奠定了基础。     

Morphological, physiological, cytological and phytochemical studies in diploid and colchicine-induced tetraploid plants of Echinacea purpurea (L.)

Echinacea purpurea (L.) is one of the important medicinal plant species. To obtain the tetraploid plants of Echinacea purpurea with improved medicinal qualities, the root tips of two true leaves seedlings were imbibed in 0.25 % (w/v) colchicine solution for 24, 48, 72, 96 and 168 h. The ploidy level of plants was determined by chromosome counting of root tip cells, and confirmed by flow cytometric analysis. Tetraploid induction occurred in seedlings treated for 24, 48 and 72 h at colchicine solution. The morphological, physiological, cytological, and phytochemical characteristics of diploid and colchicine-induced tetraploid plants were compared. Results indicated that tetraploid plants had considerable larger stomata, pollen grain, seed and flower. Moreover, chloroplast number in guard cells, amount of chlorophyll (a, b, and a + b), carotenoids as well as width and thickness of leaves were increased in tetraploids. However, stomata frequency, leaf index, plant height, and quantum efficiency of photosystem II in tetraploid were lower than diploid plants. High-performance liquid chromatography analysis showed that leaves of the tetraploid plants had more cichoric acid (45 %) and chlorogenic acid (71 %) than diploid plants. It was concluded that morphological and physiological characteristics can be used as useful parameters for preliminary screening of putative tetraploids in this species.     

中国桔梗多倍体诱导与鉴定

在离体培养条件下,比较了不同浓度、不同处理时间的秋水仙素对中国桔梗（Platycodon grandiflorus A．CD）进行染色体加倍的诱导效果。结果表明：用含0．1％秋水仙素处理40h后诱导频率可达50％,诱导效果最佳。经秋水仙素诱导后形成的多倍体植株与原二倍体植株比较,在形态上,多倍植株叶片变宽变大,叶色变深,茎变粗且节距长,气孔增大而单位叶面积气孔数目减少。对变异植株进行细胞学研究发现,体细胞中期染色体数目为2n=4x=36,而原二倍体的染色体数目为2n=2x=18,基数x=9,因此,变异植株（2n=4x=36）为四倍体。前者的核型公式为2n=4x=14m＋20sm＋2st,核型属于2B;后者的核型公式为2n=2x=7m＋10sm＋1st,核型也属于2B。检测发现有少数个体有非整倍体变异。     

In vitro induction and identification of autotetraploid of <Emphasis Type="Italic">Bletilla striata (Thunb.) Reichb.f</Emphasis>. by colchicine treatment

Polyploidy breeding has proved to be a valuable approach for acquiring the high yield superior varieties in medicinal plants. An effective protocol for obtaining Bletilla striata autotetraploid is in vitro induction of protocorms with colchicine. The protocorms of B.striata were soaked in different concentrations of colchicine solution [0.05, 0.1 and 0.2% (w/v)] for 12, 24, 36, 48 and 60 h, and the ploidy of the seedlings was identified by chromosome counting and flow cytometry analysis. The results showed that the optimal condition for induction of autotetraploid of B. striata protocorms was treated with 0.2% colchicine for 36 h with the induction rate reached as high as 26.7%. In addition, the morphological and anatomical characteristics were observed and compared between the diploid and tetraploid plants. And we found that the features of tetraploid plants were significantly different from diploid plants, such as tetraploid plants possessed thicker and deeper green leaves, larger stomata and more chloroplast number, which could be used as simple and efficient parameters for screening tetraploid. This study laid a foundation for breeding superior varieties of B. striata.     

Induction of tetraploidy in Juncus effusus by colchicine

Tetraploidy was induced in vitro in mat rush (Juncus effusus L.) cultivar Nonglin-4 by exposure to colchicine (0, 50, 100 and 500 mg dm^?3) for 6, 12 and 24 h. Flow cytometric analysis was used to confirm the ploidy level. Anatomical and ultrastructural analyses at cellular and subcellular levels in tetraploid and diploid control plants revealed differences between diploid and tetraploid plants. The leaf epidermis had larger stomata but lower stomatal density in tetraploid plants. In addition, mesophyll cells in tetraploid plants appeared more compact and showed less intercellular spaces along with increased size of vascular bundles. However, a significant reduction of chlorophyll content was observed in tetraploid plants that might be the result of structural modification in the lamellar membranes of chloroplasts.     

Effects of colchicine on polyploidy induction of Buddleja lindleyana seeds

Buddleja lindleyana Fort. is a garden ornamental plant with great potential for development and also a commonly used medicinal plant. To enrich its germplasm resources, the seeds of B. lindleyana were treated with colchicine solution with concentration gradients of 0.5%, 1.0%, 1.5%, 2.0% and 3.0% for 12-, 24- and 48-h respectively, and the water treatment was set as the control group. The purpose was to explore the effects of colchicine on the germination and mutagenic effect of B. lindleyana seeds at different concentrations and different times, to screen the appropriate combination of mutagenic concentration and time, to provide guidance for the construction of B. lindleyana mutation population in future research. The results were as follows: (1) Colchicine had an inhibitory effect on seed germination and seedling height of B. lindleyana seeds, and the higher the concentration, the more obvious the inhibitory effect. (2) After colchicine treatment, 30 mutant plants showed morphological variations such as leaf malformation, leaf color macular, early leaf bud germination, uneven leaf surface and leaf hyperplasia, among which 3.0%?+?48-h treatment group had great potential to produce yellow-leaf plants. (3) Detection and analysis by flow cytometry revealed that among the 30 morphologically variant plants, there were 22 diploid plants, 3 tetraploid plants, and 5 chimera plants. Among them, tetraploids were mainly from colchicine concentration of 3.0% (2 plants) and 1.5% (1 plant), chimeras were mainly from colchicine concentration of 1.0% (2 plants), 1.5% (1 plant) and 3.0% (2 plants), and the seed soaking time was 48-h. (4) The length and width of guard cells and stomata were significantly different between diploid and tetraploid, and there were significant differences in leaf width and leaf shape index between tetraploid and diploid, but there were no significant differences in leaf length among diploid, tetraploid and chimera. In short, we got tetraploids and chimeras materials which were potentially useful cultivars of B. lindleyana and provided an effective identification method for polyploids of B. lindleyana.

     

Induction of tetraploid poplar and black locust plants using colchicine: chloroplast number as an early marker for selecting polyploids in vitro

Tetraploid plants were produced by inducing chromosome doubling using colchicine in in vitro shoot tips of poplar and black locust clones. Many of the plants treated with colchicine showed modified morphological characteristics like stunted growth, thicker leaves and modified leaf morphology. The counting of chloroplast number in the epidermal guard cells of stomata was used for the rapid screening of tetraploids. The differences in mean chloroplast numbers between diploid and tetraploid plants were highly significant. For all plants tested, the tetraploid genotype had almost double the number of chloroplasts per guard cell compared to the diploid origin. Some plants were further analysed by flow cytometry to verify their ploidy status that was determined by chloroplast numbers. The results of this study demonstrated for the first time that chloroplast counting in poplar and black locust could be an effective and reliable method for pre-screening large numbers of plants for their ploidy level. The protocol might be applicable in a wide scope of breeding programs.     

In vitro tetraploid induction from leaf explants of Populus pseudo-simonii Kitag.

Tetraploid plants were produced from leaf explants of diploid Populus pseudo-simonii by treating the leaves with colchicine. Leaf explants were cultured on MS basal medium containing 1.78 μM BA and 1.08 μM NAA for 0, 6 and 12 days, and then transferred to the same MS liquid medium with colchicine at concentrations of 25, 50 and 75 μM for 1, 2 and 3 days. The highest efficiency of tetraploid induction was 14.6% by treating leaf explants that were pre-cultured for 6 days and then cultured in liquid MS with 50 μM colchicine for 3 days. Flow cytometric analysis was used to screen the tetraploids out from the regenerated plants and chromosome number counting was employed to confirm the polyploidy level. Size and frequency of leaf stomata between diploid and tetraploid plants were demonstrated to have significant differences.     

In vitro induction of tetraploid plants from diploid Zizyphus jujuba Mill. cv. Zhanhua

Tetraploid plants of Zizyphus jujuba Mill. cv. Zhanhua were obtained with in vitro colchicine treatment. Shoot tips from in vitro-grown plants were treated with five different concentrations of colchicine (0.01, 0.03, 0.05, 0.1, 0.3%) in liquid MS medium (Murashige and Skoog 1962), and shaken (100 rpm) at 25 °C in darkness for 24, 48, 72 or 96 h, respectively. Tetraploids were obtained at a frequency of over 3% by using 0.05% colchicine (48 h, 72 h) and 0.1% colchicine (24 h, 48 h) treatment as determined by flow cytometry. Cytological and morphological evidence confirmed the results of flow cytometric analysis. The chromosome number of diploid plants was 24 and that of tetraploid plants was 48. The stomata sizes of tetraploid plants were significantly larger than those of diploid plants, while the frequency of stomata were reduced significantly. Similarly, the chloroplast number of guard cells of tetraploid plants increased significantly. The selected tetraploid plants were grafted onto mature trees of Z. jujuba Mill. cv. Zhanhua in the field, resulted in thicker stems, rounder and succulent leaves, larger flowers and a delay in florescence time (3–4 days later) than diploid plants.     

In vitro induction of tetraploids in Arachis paraguariensis

In this study, an efficient procedure was established for successful induction of tetraploid Arachis paraguariensis by treating diploid explants with colchicine. Quartered-seed, callus and shoot-tips were treated with colchicine at concentrations of 0.05, 0.1, 0.2 and 0.5?% (w/v) for 4, 8, 16, 20 and 24?h before they were transferred unto modified Murashige and Skoog medium for either callus induction or shoot regeneration. Results showed that quartered-seed displayed the highest frequency of in vitro plantlet regeneration and tetraploid induction, as well as the lowest mortality rate. Flow cytometric analysis also confirmed that the induced tetraploids from quartered-seed were true-to-type. The 0.5?% colchicine treatment for 4 to 8?h gave the best results with 39 and 43?% of the explants yielding tetraploid plants, respectively. Two?months following transfer to ex vitro environment, morphological and growth characteristics of the induced tetraploids were measured. Overall, increasing the ploidy level from 2× to 4× resulted in fewer stomata but more trichomes per unit leaf area. Tetraploid plants obtained in this study should expand the genetic base of Arachis, and can also be used in overcoming the existing hybridization barriers that may be due to ploidy differences within the genus Arachis.