In vitro plant regeneration from seedling-derived explants of ramie [Boehmeria nivea (L.) Gaud]

Ramie [Boehmeria nivea (L.) Gaud] is one of the most important perennial fiber crops in China. In vitro tissue culture of ramie could serve as an important means for its improvement through genetic transformation. To improve the regeneration capacity of ramie, the effects on plant regeneration of donor plant age, basal medium, plant growth regulators, and culture conditions were evaluated using explants derived from the cotyledon, hypocotyl, leaf, petiole, and stem of ramie seedlings. Cotyledons and hypocotyls excised from 4-d-old seedlings and leaves and petioles and stems from 15-d-old seedlings were optimal explants. The highest regeneration efficiency was obtained on Murashige and Skoog salts with Gamborg’s B₅ vitamins basal medium containing 2.27 μM thidiazuron (TDZ) and 0.054 μM naphthaleneacetic acid (NAA) for the five explant types tested. A photoperiod of 16:8 h (light/dark) was found to be superior than continuous darkness for regeneration of ramie using TDZ. The regenerated shoots were transferred to hormone-free medium for shoot elongation and successfully rooted on half-strength Murashige and Skoog supplemented with 0.134 μM NAA. The rooted plantlets with four to five leaves were transplanted to greenhouse for further growth.     

Morphological and physiological changes of ramie (Boehmeria nivea L. Gaud) in response to drought stress and GA3 treatment

Ramie (Boehmeria nivea L. Gaud) fibers extracted from the stem bast is one of the most important natural fibers. Ramie growth is severely hindered by drought stress but is promoted by gibberellins (GAs). In order to investigate ramie morphological and physiological responses to drought stress and GA₃ treatment, four groups of potted ramie (severe drought stress (DS), severe drought stress and spraying with GA₃ (DS + GA₃), normal watering and spraying with GA₃ (control + GA₃), and normal watering as a control) were tested. The result showed that, comparing with the ramie growing under well watering condition, a decrease in chlorophyll a (Chl a) and carotenoid and an increase in proline and soluble sugar contents were commonly observed in drought-stressed and GA₃-treated ramie. Different responses of the stem morphological traits, fiber yield, and seven physiological characteristics (relative water content, the activities of POD, SOD, and CAT enzymes, the contents of Chl b, endogenous GAs and MDA) were observed between drought-stressed and GA₃-treated plants. When the ramie suffering drought stress was sprayed with GA₃ (in (DS + GA₃) group), the responses of some physiological traits (POD, SOD, CAT, MDA, and endogenous GAs) and morphological traits (stem shape and fiber yield) to drought stress disappeared completely or partially, and the plant presented similar characteristics of well-watered ramie in these traits. These results suggested that the application of exogenous GA₃ can improve the drought tolerance of ramie.     

Association and path-coefficient studies on vegetatively propagating and sexually reproducing parts in ramie (Boehmeria nivea L. Gaud.)

Summary In 30 genotypes of ramie (Boehmeria nivea L. Gaud.) there were significant genotypic differences in the weight of reproductive parts (male + female flowers), vegetatively propagating parts (clump and rhizome) and non-propagating vegetative parts (leaf and root). The weight of reproductive parts was significantly and positively associated with the weight of vegetatively propagating parts. Path-coefficient analysis revealed that the selection of genotypes with a high weight of reproductive parts should be based on genotypes with a high weight of vegetatively propagating parts.     

QTL mapping for fiber yield-related traits by constructing the first genetic linkage map in ramie (Boehmeria nivea L. Gaud)

Ramie fiber extracted from stem bast is one of the most important natural fibers. The fiber yield of ramie is a valuable trait and is decided by several components, including stem number per plant (SN), the fiber yield per stem (FYPS), stem length (SL), stem diameter (SD), and bark thickness (BT). All of these fiber yield-related traits are inherited in a quantitative manner. The genetic basis for these traits is still uncharacterized, which has hindered the improvement of yield traits through selective ramie breeding. In this study, an F₂ population derived from two ramie varieties, Zhongzhu 1 and Qingyezhuma, with striking differences in fiber yield-related traits, was used for cutting propagation and to develop an F₂ agamous line (FAL) population. A genetic linkage map with 132 DNA loci spanning 2,265.1 cM was first constructed. The analysis of quantitative trait locus (QTL) for fiber yield-related traits was performed in ramie for the first time. Finally, a total of 6, 9, 5, 7, and 6 QTLs for FYPS, SL, SN, SD, and BT, respectively, were identified in the FAL population in two environments. Among these 33 QTLs, 9 QTLs were detected in both environments and 24 QTLs exhibited overdominance. The overdominance of these QTLs possibly contributed to the heterosis of these yield-related traits in ramie. Moreover, there were 7 QTL clusters identified. The identification of the QTLs for fiber yield-related traits will be helpful for improving the fiber yield in ramie breeding programs.     

Transgenic plants of ramie (Boehmeria nivea Gaud.) obtained by Agrobacterium mediated transformation

A regeneration and transformation protocol for ramie (Boehmeria nivea Gaud.) is presented. Regeneration was obtained from leaf discs placed on solid B-5 medium (Gamborg et al. 1968) containing adequate concentrations of auxin and cytokinin. Co-cultivation of leaf discs with Agrobacterium tumefaciens and subsequent regeneration resulted in transgenic plants as shown by Southern blot and analysis of expression of the GUS-marker gene.     

苎麻促生菌的筛选、鉴定及其促生效应

【目的】以苎麻(Boehmeria nivea L. Gaud)根及根围土壤为研究材料,进行苎麻促生菌的筛选,并初步探索其促生作用机制。【方法】首先,以溶磷和解钾为基本筛选标准,初筛菌株在实验室条件下测定多项促生能力进行复筛;然后通过种子萌发、盆栽试验测定菌株对苎麻的促生效应,最后,通过形态观察、生理生化特性和16S rRNA基因序列同源性分析,对促生菌株进行分类学鉴定。【结果】从苎麻根和根围土壤中分离得到了13株菌同时具备溶磷和解钾能力,其中4株菌(RA-2、RAM-2、RAM-5和RAM-6)具备产铁载体、产IAA和产氨能力。种子萌发和盆栽试验的测定结果显示：4株菌株均能促进苎麻种子的萌发和植株的生长,其中菌株RA-2和RAM-5相比于对照处理能显著提高苎麻种子的萌发率、幼根长、株高和根系干重。分类鉴定结果显示菌株RA-2和RAM-5均属于伯克霍德菌属(Burkholderia)。【结论】从苎麻根围筛选到具有促生能力的菌株,为进一步开发研制苎麻专型促生菌剂或专型微生物有机肥提供资源。     

Isolation of high quality RNA and construction of a suppression subtractive hybridization library from ramie (Boehmeria nivea L. Gaud.)

Isolation of high quality RNA from ramie (Boehmeria nivea L. Gaud.) is difficult due to its high levels of polyphenols, polysaccharides, pectin, fat, wax and other secondary metabolites. A modified procedure based on guanidinium isothiocyanate for RNA preparation of ramie was developed in this study. High concentrations (5%, v/v) of guanidinium isothiocyanate, PVP-4000, sodium citrate and sodium lauryl sarcosinate and β-mercaptoethanol were used in the extraction buffer, together with a low pH sodium acetate (pH 4.0) added to improve the RNA quality. The average yield was about 400 μg RNAg^?1 fresh leaves. One SSH library which was induced by ramie anthracnose was constructed by utilizing the RNA extracted through the present method. These results showed that our protocol was applicable for RNA isolation from recalcitrant ramie tissues.     

Draft genome sequence of ramie,Boehmeria nivea (L.) Gaudich

Ramie, Boehmeria nivea (L.) Gaudich, family Urticaceae, is a plant native to eastern Asia, and one of the world's oldest fibre crops. It is also used as animal feed and for the phytoremediation of heavy metal‐contaminated farmlands. Thus, the genome sequence of ramie was determined to explore the molecular basis of its fibre quality, protein content and phytoremediation. For further understanding ramie genome, different paired‐end and mate‐pair libraries were combined to generate 134.31 Gb of raw DNA sequences using the Illumina whole‐genome shotgun sequencing approach. The highly heterozygous B. nivea genome was assembled using the Platanus Genome Assembler, which is an effective tool for the assembly of highly heterozygous genome sequences. The final length of the draft genome of this species was approximately 341.9 Mb (contig N50 = 22.62 kb, scaffold N50 = 1,126.36 kb). Based on ramie genome annotations, 30,237 protein‐coding genes were predicted, and the repetitive element content was 46.3%. The completeness of the final assembly was evaluated by benchmarking universal single‐copy orthologous genes (BUSCO); 90.5% of the 1,440 expected embryophytic genes were identified as complete, and 4.9% were identified as fragmented. Phylogenetic analysis based on single‐copy gene families and one‐to‐one orthologous genes placed ramie with mulberry and cannabis, within the clade of urticalean rosids. Genome information of ramie will be a valuable resource for the conservation of endangered Boehmeria species and for future studies on the biogeography and characteristic evolution of members of Urticaceae.     

Agrobacterium-mediated genetic transformation and regeneration of transgenic plants using leaf midribs as explants in ramie [Boehmeria nivea (L.) Gaud]

In this study, leaf midribs, the elite explants, were used for the first time to develop an efficient regeneration and transformation protocol for ramie [Boehmeria nivea (L.) Gaud.] via Agrobacterium-mediated genetic transformation. Sensitivity of leaf midribs regeneration to kanamycin was evaluated, which showed that 40 mg l^?1 was the optimal concentration needed to create the necessary selection pressure. Factors affecting the ramie transformation efficiency were evaluated, including leaf age, Agrobacterium concentration, length of infection time for the Agrobacterium solution, acetosyringone concentration in the co-cultivation medium, and the co-cultivation period. The midrib explants from 40-day-old in vitro shoots, an Agrobacterium concentration at OD₆₀₀ of 0.6, 10-min immersion in the bacteria solution, an acetosyringone concentration of 50 mg l^?1 in the co-cultivation medium and a 3-day co-cultivation period produced the highest efficiencies of regeneration and transformation. In this study, the average transformation rate was 23.25 %. Polymerase chain reactions using GUS and NPTII gene-specific primers, Southern blot and histochemical GUS staining analyses further confirmed that the transgene was integrated into the ramie genome and expressed in the transgenic ramie. The establishment of this system of Agrobacterium-mediated genetic transformation and regeneration of transgenic plants will be used not only to introduce genes of interest into the ramie genome for the purpose of trait improvement, but also as a common means of testing gene function by enhancing or inhibiting the expression of target genes.     

苎麻atp6和atp9基因的克隆表达及与细胞质雄性不育的相关性

摘要: 根据GenBank报道的双子叶植物线粒体atp6和atp9基因编码区保守序列设计简并引物, 通过PCR技术从苎麻细胞质雄性不育系、保持系和恢复系(简称“三系”) mtDNA中扩增目的基因片段, 发现所得序列开放阅读框虽不完整, 但与GenBank报道的其他植物线粒体atp6和atp9基因同源性分别高于94%和85%。采用DNA Walking步移法分别从3′端和5′端扩增两个基因片段的未知侧翼序列, 分离出完整的苎麻线粒体atp6和atp9基因, 包含了完整的开放阅读框。其中“三系”的atp6基因在mtDNA水平、转录和翻译调控水平、蛋白质水平上均无差异。不育系atp9基因在编码区3′端与保持系和恢复系相比存在若干个碱基的差异和缺失; RT-PCR分析还表明, 不育系atp9基因在现蕾期和盛花期的表达量很高。推测不育系atp9基因的结构变异和/或异常表达与苎麻细胞质雄性不育(CMS)的关系密切。     

Identification and characterization of microRNAs in phloem and xylem from ramie (Boehmeria nivea)

Molecular Biology Reports - Ramie (Boehmeria nivea) is a widely cropped species in southern China due to its high economic value of natural fiber for industry. Development of phloem and xylem is...     

苎麻雄性不育系生化代谢和育性遗传研究

用不同温度和日长处理盆栽材料,测试苎麻雄性不育系的温光反应;于雌雄性器官发育期取盆栽植株顶部展开叶往下数第7叶,分析不育系的生化代谢;根据杂交及自交后代育性分离情况,鉴定不育系的不育性遗传方式.结果如下高温加速营养生长,而短日促进生殖生长,短日高温明显加快不育系的发育进度,但高温的促进作用随日长增加而减弱,以至消失.不育系的叶片粗蛋白质、氨基酸含量比可育系减少,尤其是天门冬氨酸、谷氨酸、甘氨酸、胱氨酸、缬氨酸、苯丙氨酸和精氨酸减少明显,但游离脯氨酸含量增加.这些物质代谢的变化,可能是导致苎麻雄性不育的原因.不育系GS14-1、SS370、GSA-2、GS15-8和SS387等的不育性遗传与1对相对性状的遗传方式相符,已找到不育系的保持系和恢复系.