野生南荻与芒杂种多倍体诱导研究

用不同浓度秋水仙素处理野生南荻×芒(Miscanthus lutarioriparia×Miscanthus sinensis)远缘杂交后代以诱导产生多倍体,并对变异株进行形态学和细胞学鉴定,以期获得稳定的四倍体植株并分析其生理特性。结果表明:(1)采用秋水仙素加入培养基处理法和秋水仙素溶液浸泡处理法都可获得一定频率的多倍体植株;胚性愈伤组织以0.2%秋水仙素浸泡处理48h的诱变效果较好,四倍体诱导率达8.7%;芽在0.05%秋水仙素培养基中处理15d较好,四倍体诱导率达10.6%;生根苗在0.1%秋水仙素培养基中处理10d较好,四倍体诱导率达11.1%。(2)经体细胞染色体计数,加倍植株染色体数为2n=4x=76,对照植株的染色体数目为2n=2x=38。(3)生长2年的多倍体植株形态、叶片大小、茎粗、茎壁厚、节间等性状表现出巨大性和超亲优势。     

四倍体不结球白菜的诱导及染色体倍性鉴定

用不同浓度秋水仙素处理子叶期不结球白菜生长点对其进行染色体倍性操作,根据形态解剖学、细胞学特征和流式细胞仪进行倍性鉴定.结果表明,浓度为0.2%的秋水仙素处理4次的效果最好,四倍体诱变率为8.42%.与二倍体相比,四倍体植株叶片、花器官、气孔等均表现巨大性;气孔密度和结实率降低;抽薹较晚.用流式细胞仪进行倍性鉴定,对照DNA相对含量为100,疑似株为200,表明是四倍体;疑似株有2个值与对照的比值约为1和2,表明是嵌合体(2x 4x).流式细胞仪鉴定结果与染色体计数法鉴定结果一致,表明流式细胞仪可以较准确地检测不结球白菜突变株倍性.     

秋水仙素对草莓离体叶片再生和多倍体诱导的影响

以草莓(Fragaria×ananassa Duch.)栽培品种'雪蜜'(2n=8X=56)的离体叶片为外植体,研究了不同浓度秋水仙素对愈伤组织诱导率、不定芽再生率以及多倍体植株诱导的影响,并采用流式细胞仪对多倍体植株的倍性进行鉴定.结果显示,用质量体积分数0.1%、0.3%、0.5%和0.7%的秋水仙素浸泡2、4和6 d,草莓离体叶片均能诱导出愈伤组织和不定芽,但随秋水仙素浓度的提高和处理时间的延长,愈伤组织诱导率和不定芽再生率均显著下降.用不同浓度秋水仙素处理均能产生多倍体植株,倍性为9X、10X、11X、12X、14X和16X;随秋水仙素浓度的提高,多倍体诱导率呈现先上升后下降的变化趋势.用质量体积分数0.3%秋水仙素浸泡处理4 d是最佳的草莓离体叶片诱导方法,不定芽再生率达到40.5%,多倍体诱导率为100.0%,并且诱导产生出16X的植株.     

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。     

毛泡桐同源四倍体的诱导

在含有不同浓度秋水仙素双层培养基上以叶片作外植体诱导毛泡桐同源四倍体植株,用变异植株根尖细胞染色体计数和成熟叶片中单细胞DNA含量测定的方法进行倍性分析。结果表明,在16个试验组合中,预培养6d的毛泡桐叶片经20mg·L-1秋水仙素处理72h的四倍体诱导率可达20%。     

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。     

广藿香同源八倍体诱导与鉴定

为诱导广藿香[Pogostemon cablin(Blanco)Benth.]同源八倍体,采用组织培养方法,研究了秋水仙素对广藿香同源八倍体诱导的影响。结果表明,以0.05%秋水仙素浸泡广藿香组培丛生芽72 h的效果最佳,形态学鉴定处理苗的变异率达85%,且八倍体苗的染色体数目为2n=8x=128,八倍体苗的根茎粗壮,叶片大而厚,颜色深,叶形指数小,叶片下表皮的气孔个体大、密度小,保卫细胞中的叶绿体数目多,植株形态学性状优良。这为进一步获得高产、活性成分含量高的广藿香优良品系奠定基础。     

同源四倍体紫菜薹的诱导鉴定及其营养品质比较

该研究以二倍体紫菜薹为材料,采用浓度为0.1%、0.2%和0.3%的秋水仙素溶液,在二倍体紫菜薹生长至子叶期时对植株茎尖生长点进行4次点滴处理,鉴定筛选同源四倍体紫菜薹并进行二倍体和四倍体紫菜薹的营养品质比较。结果表明:(1)使用浓度为0.2%秋水仙素溶液点滴4次对紫菜薹的处理效果最好,四倍体紫菜薹的诱导率为6.62%。(2)在形态学上,四倍体植株的叶片、花簇、花器官、角果和种子与二倍体相比都在巨大性上呈现出明显差异;在解剖学上,四倍体植株在气孔密度减小的同时气孔变大,其花粉表现出矩形、梯形以及其他一些不规则的形状;在细胞学上,四倍体和二倍体植株的染色体数目分别为40条和20条;流式细胞仪鉴定结果显示,四倍体植株的DNA分子荧光强度(2 092 385.03)约为二倍体植株(956 725.15)的2倍。(3)植株营养品质方面,四倍体紫菜薹的游离氨基酸、叶绿素总浓度分别比二倍体植株显著增加了228.58%、110.02%,但四倍体紫菜薹的硝态氮、可溶性蛋白、维生素C、可溶性糖、纤维素总含量分别比二倍体植株显著减少了48.99%、43.20%、45.81%、44.50%、59.97%。(4)与二倍体植株相比,四倍体紫菜薹的单株质量、十叶厚、叶宽、叶柄宽都有不同程度的增加,叶片开展度有一定程度的降低。研究发现,秋水仙素诱导产生四倍体紫菜薹的最适浓度为0.2%;四倍体紫菜薹表现出明显的高产性,但多数营养品质极显著低于二倍体紫菜薹。     

同源四倍体灯盏花离体再生及其倍性鉴定

以四倍体灯盏花的无菌苗叶柄为外植体进行离体培养,研究其再生体系,并对再生植株进行染色体倍性鉴定。结果表明:叶柄外植体在MS+0.05 mg·L-1NAA+0.5 mg·L-16-BA+0.65%琼脂+3%蔗糖的培养基上不定芽的再生频率可达87.3%;继代培养(MS+0.1 mg·L-1NAA+0.3 mg·L-16-BA+0.65%琼脂+3%蔗糖)增殖系数为7.8,诱导(1/2MS+1.0 mg·L-1NAA+1.0 mg·L-1IBA+0.65%琼脂+3%蔗糖)生根率100%,移栽成活率为90%。细胞学鉴定结果表明,再生植株的染色体数为2n=4x=36,而原二倍体的染色体数目为2n=2x=18,基数x=9,因此,再生植株为四倍体。     

田埂报春多倍体诱导及其形态学研究

在离体培养条件下,比较不同浓度、不同处理时间的秋水仙素对田埂报春进行染色体加倍的诱导效果。结果表明:0.08%秋水仙素处理48h的诱变效果最佳,诱变率高达56%。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,四倍体植株表现出多倍体特征,叶片变厚,叶形指数减小,保卫细胞增大,单位面积气孔数减少,叶绿体数明显增多。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=8L+12M2+4M1+12S,核型属于1A;后者的核型公式为2n=2x=4L+6M2+2M1+6S,核型也属于1A。检测发现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.     

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。     

同源四倍体铁皮石斛的生长及多糖积累

通过秋水仙素诱导铁皮石斛多倍体,以外观形态、气孔、染色体数目筛选多倍体植株,分析多倍化对铁皮石斛多糖累积及生长的影响。结果表明,同源四倍体铁皮石斛茎、叶及原球茎的多糖含量分别是二倍体的1.31、1．83及1．95倍,但生长速度显著或极显著低于对照。ISSR分析显示,同源四倍体植株产生较明显的基因变异。     

In vitro induction of polyploidy in Centella asiatica (L.) Urban

Mutational breeding was conducted using in vitro-grown shoot-tips of Centella asiatica (L.) Urban treated with colchicine (0.025–0.400% for 12–36 h) to induce polyploidy. Treated shoot-tips were grown on Murashige and Skoog (MS) medium supplemented with 4.54 μM thidiazuron (TDZ), and regenerated plantlets were acclimatized and transferred to soil. Two months following transfer to ex vitro conditions, ploidy levels of regenerated plants were determined by flow cytometry and by determining chromosome counts. Treating shoot-tips with colchicine concentrations ranging from 0.050–0.200% for 12–24 h promoted induction of tetraploids. Morphological and growth characteristics and the triterpenoid contents of the polyploids were also measured. Tetraploid plants demonstrated significantly longer stomata and a higher stomatal index compared to those of the diploid control plants. Furthermore, a positive trend in both biomass and triterpenoid production was obtained with the tetraploid and mixoploid plants of C. asiatica.     

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。     

库拉索芦荟的多倍体诱导及其变异初报

在组织培养条件下,对库拉索芦荟（Aloe vera L.)用秋水仙素进行染色体加倍的诱导处理,结果表明：用含0.06%秋水仙素处理12h后诱变频率可达50％,其效果最佳。经秋水仙素诱导的加倍群体与正常二倍体植株比较,植株的大多数叶片变厚,叶色变深,叶片变大,气孔增大而单位叶面积气孔数减少。对变异材料进行细胞学研究所发现,体细胞中期染色体为2n=4x=28,为4倍体。未加倍前的二倍体为2n=2x=14。检测中也发现有少数植株有2n=14和2n=28两种细胞型的情况。     

利用流式细胞光度术鉴定苹果倍性的研究

利用流式细胞光度术测定了苹果１２个二倍体,５个三倍体细胞ＤＮＡ含量。结果表明：二倍体细胞核ＤＮＡ含量平均为２．２７ｐｇ,三倍体细胞核ＤＮＡ含量平均为３．１３ｐｇ。     

In vitro induction and identification of tetraploid plants of <Emphasis Type="Italic">Paulownia tomentosa</Emphasis>

Polyploidization is a major trend in plant evolution that has many advantages over diploid. In particular, the enlargement and lower fertility of polyploids are very attractive traits in forest tree breeding programs. We report here a system for the in vitro induction and identification of tetraploid plants of Paulownia tomentosa induced by colchicine treatment. Embryonic calluses derived from placentas were transferred to liquid Murashige and Skoog (MS) medium containing different concentrations of colchicine (0.01, 0.05, or 0.1%) and incubated for 24, 48, or 72 h on an orbital shaker at 110 rpm. The best result in terms of the production of tetraploid plantlets was obtained in the 48 h + 0.05% colchicine treatment, with more than 100 tetraploid plantlets being produced. The ploidy level of plantlets was verified by chromosome counts, flow cytometry, and morphology. The chromosome number of tetraploids was 2n = 4x = 80 and that of diploid plantlets was 2n = 2x = 40. The relative fluorescence intensity of tetraploids was twofold higher than that of diploids. The tetraploid and diploid plantlets differed significantly in leaf shape, with those of the former being round and those of the latter pentagonal. The mean length of the stomata was longer in tetraploid plants than diploid plants, and stomatal frequency was reduced with the increased ploidy level. The tetraploids had large floral organs that were easily distinguishable from those of diploid plants.     

Meiotic analysis of four cross-pollinated generations in a synthetic autotetraploid population of husk tomato (<i>Physalis ixocarpa</i>)

The cultivated husk tomato (Physalis ixocarpa) (2n = 2x = 24) is native from Mexico and Central America and shows a wide genetic variation. Presently, it is the fourth horticultural crop in cultivation surface in Mexico. The working team of this research previously developed an autotetraploid population by using colchicine. The objectives of the present work were to analyze the ploidy level and meiotic behavior of the subsequent generations (C3, C4, C5, C6) from the original (C2) composed only by plants with the duplicated genome from the Rendidora cultivar, and to determine pollen viability. As a diploid control the cultivar Rendidora of P. ixocarpa was used. Ploidy level was determined by flow citometry and meiotic analysis. For the meiotic study, the microsporocytes were prepared by the squash method, stained with carmin and analyzed in diakinesis. Pollen viability was evaluated through 0.01% Buffalo Black staining. The tetraploid condition prevailed through four cross-pollinating generations, maintaining a constant chromosome number 2n = 4x = 48. In diakinesis, the chromosomes of the diploid cultivar were associated into bivalents, whereas in tetraploid plants the chromosomes associated into univalents, bivalents and trivalents. Highly significant differences in bivalent pairing were detected between autotetraploid plants and between generations. Pollen viability did not show significant differences between generations and allowed reproduction. These results indicate that it is possible to develop an autotetraploid cultivar, because the polyploid state is naturally maintained and the plants are fertile. Furthermore, given the differences in bivalent pairing between plants and generations, a response to selection toward meiotic stability is expected.