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.     

苎麻细胞质雄性不育系与保持系线粒体DNA的ISSR检测

采用蔗糖衬垫法提取苎麻雄性不育系与相应保持系的线粒体DNA。选用了38ISSR引物进行ISSR—PcR扩增,在编号为ISSR．29、33、34引物中找到了苎麻不育系与相应保持系线粒体DNA之间的差异。     

苎麻植物螯合肽合成酶(BnPCS1)基因的克隆和表达

根据苎麻转录组测序中的PCS基因片段,利用RT-PCR结合RACE技术从中苎1号中克隆获得了该基因的全长cDNA序列,命名为BnPCS1。该基因的cDNA序列全长为1956 bp,其中开放读码框长1512 bp,编码503个氨基酸,预测其分子量和等电点分别为56.02 kD和7.01。与长喙田菁(ACT87974)、百脉根(Q2TSC7)、狼牙刺(AFM38979)、荷花(BAN08523)和杜梨(AEY68568)的PCS氨基酸序列相似性分别为74%、73%、75%、73%和77%。荧光定量PCR分析表明,BnPCS1在根、茎、茎尖、幼叶、成熟叶中均有表达,其中在成熟叶中的表达量最高,茎中表达量最低,并且该基因受镉和ABA诱导上调表达。BnPCS1基因的克隆将为苎麻抗重金属分子育种和进一步的功能分析奠定基础。     

苎麻酶法脱胶的研究进展*

苎麻酶脱胶法是一种高效能、优质量、无污染的脱胶方法,它是直接利用微生物发酵后期产生的胞外酶或酶制剂降解苎麻的胶质,使得苎麻纤维释放出来。到目前为止,国内外的科研人员在这方面已经进行了大量的研究,筛选了许多不同的脱胶菌,包括需氧菌和厌氧菌等用于苎麻的脱胶。相比化学脱胶,苎麻的酶脱胶具有提高精干麻的质量、大幅度降低环境污染等显的优点,是苎麻脱胶未来的主要发展方向,有着不可估量的前途。同时,随着酶脱胶技术在工业化生产中的推广应用,酶的使用量势必大幅度增加,从而将带动酶工业的发展。     

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.     

Regulation of Organ Formation by Cytokinin and Auxin in Lilium Bulbscales Grown In Vitro

Regulation of organ formation by cytokinin and auxin was investigatedin vitro using Lilium auratum Lindl. (wild species habituatedin Japan) and Lilium speciosum Thunb. cv. "Uchida". The interactionof -naphthylacetic acid (NAA) and kinetin on bulbscale or rootdifferentiation was examined. NAA and kinetin showed mainlyindividual but also some synergistic effects. The effects ofbenzyladenine (BA) and kinetin were compared and the resultindicated that BA has a stronger physiological effect on organformation than kinetin and that their effects on Lilium auratumand Lilium speciosum were BA or kinetin-specific. The actionof kinetin on Lilium was affected by sucrose concentration andthe strength of the Murashige and Skoog medium (MS medium),and thus their high concentrations inhibited the kinetin-inducedbulbscale differentiation. Furthermore, a high sucrose levelnegated the kinetin inhibition of root formation, while highMS medium strength in itself inhibited root formation. Morphologicalobservation of bulbscale differentiation induced under a highkinetin level revealed that the new-formed structures are homologousto normally grown bulbs in soil in spite of their particularfeatures. (Received August 3, 1981; Accepted November 2, 1981)     

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.     

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。     

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.     

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.     

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.     

Subcellular distribution and chemical forms of cadmium in Bechmeria nivea (L.) Gaud.

Bechmeria nivea (L.) Gaud. (Ramie) is a promising species for Cd phytoextraction with large biomass and fast growth rate. Nevertheless, little information is available on its tolerance mechanisms towards Cd. Determination of Cd distribution and chemical speciation in ramie is essential for understanding the mechanisms involved in Cd accumulation, transportation and detoxification. In the present study, ramie plants were grown in hydroponics with increasing Cd concentrations (0, 1, 3, 7 mg l^?1). The subcellular distribution and chemical forms of Cd in different tissues were determined after 20 days exposure to this metal. To assess the effect of Cd uptake on plant performance, nitrate reductase activity in leaves and root activity were analyzed during the entire experimental period. Increased Cd level in the medium caused a proportional increase in Cd uptake, and the highest Cd concentration occurred in roots, followed by stems and leaves. Subcellular fractionation of Cd-containing tissues indicated that about 48.2–61.9% of the element was localized in cell walls and 30.2–38.1% in soluble fraction, and the lowest in cellular organelles. Cd taken up by ramie rapidly equilibrated among different chemical forms. Results showed that the greatest amount of Cd was found in the extraction of 1 M NaCl and 2% HAC, and the least in residues in all test tissues. In roots, the subdominant amount of Cd was extracted by d-H₂O and 80% ethanol, followed by 0.6 M HCl. While in stems and leaves, the amount of 0.6 M HCl-extractable Cd was comparable with that extracted by 80% ethanol or d-H₂O. 1 mg l^?1 Cd stimulated nitrate reductase activity in leaves and root activity, while a concentration-dependent inhibitory effect was observed with increasing Cd concentration, particularly at 7 mg l^?1 Cd. It could be suggested that the protective mechanisms evolved by ramie play an important role in Cd detoxification at relatively low Cd concentrations (below 3 mg l^?1 Cd) but become restricted to maintain internal homeostasis with higher Cd stress.     

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。