RNA isolation from loquat and other recalcitrant woody plants with high quality and yield

RNA isolation is difficult in plants that contain large amounts of polysaccharides and polyphenol compounds. To date, no commercial kit has been developed for the isolation of high-quality RNA from tissues with these characteristics, especially for fruit. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to recalcitrant plant tissues. Here an efficient RNA isolation protocol based on cetyltrimethylammonium bromide (CTAB) and two successive precipitations with 10 M lithium chloride (LiCl) was developed specifically for loquat fruits, but it was proved to work efficiently in other tissues of loquat and woody plants. The RNA isolated by this improved protocol was not only of high purity and integrity (A₂₆₀/A₂₈₀ ratios ranged from 1.90 to 2.04 and A₂₆₀/A₂₃₀ ratios were > 2.0) but also of high yield (up to 720 μg on average [coefficient of variation = 21%] total RNA per gram fresh tissue). The protocol was tested on loquat fruit (different stages of development, postharvest, ripening, and bruising), leaf, root, flower, stem, and bud; quince fruit and root; grapevine cells in liquid culture; and rose petals. The RNA obtained with this method is amenable to enzymatic treatments and can be efficiently applied for research on gene characterization, expression, and function.     

木本植物基因组DNA提取及鉴定

采用改良后的CTAB法,对山葡萄、软枣猕猴桃、蒙古栎、核桃楸、西伯利亚红松和偃松基因组DNA进行提取。结果表明,所提基因组DNA分子量与λDNA（48 kb）接近,其紫外吸收比在1.66～1.89之间。第3次和第4次上清提取的DNA质量优于第1次和第2次。从提取产量看,每克鲜重提取DNA量最小为15 μg·g^-1（核桃楸第4次上清）,最高的为272 μg·g^-1（山葡萄第3次上清）。西伯利亚红松和偃松第1次和第2次上清基本未提出DNA,第3次和第4次上清中得到了较高质量的DNA。经酶切鉴定和PCR扩增,所提的基因组DNA可以用于进一步研究。     

A reliable method for extraction of RNA from various conifer tissues

Summary A simple and efficient procedure suitable for extraction of high-quality RNA from cultured conifer tissues, somatic embryos, zygotic embryos, needles, stem and root tissues was developed. It produced from 100 g up to 700 g total RNA per gram tissue dependent on the types of tissues used. RNA quality was estimated by spectrophotometry, agarose gel electrophoresis, in vitro translation of mRNA, cDNA synthesis and Northern blot analysis. The method also worked well with Arabidopsis thaliana and tobacco tissues.Abbreviations CTAB cetyltrimethylammonium bromide - DEPC diethylpyrocarbonate - PVP polyvinylpyrrolidone - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

一种适于提取荔枝花与幼果组织总RNA的方法

介绍了一种从荔枝花与幼果组织中提取高质量和较高产量的总RNA的方法,该方法提取的总RNA可以满足构建cDNA文库、开展RT-PCR、Northern杂交分析、基因表达差示分析等方面研究的要求。     

一种提取葡萄芽中总RNA的方法

以葡萄品种‘Perletts‘‘(Vitis vinifera)冬芽为材料,研究了提取葡萄芽总RNA的方法,同时指出提取RNA时的注意事项。该方法具有可靠性高,易于大量制备,所提取的RNA质量高等优点。     

Environmental regulation and physiological basis of freezing tolerance in woody plants

Cold acclimation of plants is a complex process involving a number of biochemical and physiological changes. The ability to cold acclimate is under genetic control. The development of freezing tolerance in woody plants is generally triggered by non-freezing low temperatures but can also be induced by mild drought or exogenous abscisic acid, as well as by short photoperiod. In nature, the extreme freezing tolerance of woody plants is achieved during sequential stages of cold acclimation the first of which is initiated by short photoperiods and non-freezing low temperatures, and the second by freezing temperatures. Although recent breakthroughs have increased our knowledge on the physiological molecular basis of freezing tolerance in herbaceous species, which acclimate primarily in response to non-freezing low temperatures, very little is known about cold acclimation of woody plants. This article attempts to review our current understanding of the physiological aspects that underline cold acclimation in woody plants.     

A rapid method for generating sufficient amounts of uniform genotype-specific material from the woody perennial grapevine for ion transport studies

Experimentation with woody perennials may be difficult due to slow plant growth and a lack of sufficient amounts of uniform plant material. In this study, we sought to determine whether rooted leaves could be used as a substitute for whole plants in ion accumulation studies. Grapevine leaves are particularly amenable for rooting since their petioles are of sufficient length for dipping in rooting hormone and for holding the leaf above the soil surface. To determine whether rooted leaves would be useful for salinity experiments, we investigated the ion uptake characteristics of rooted leaves derived from a backcross population that differed in Cl^– accumulation. Long-term ion accumulation studies conducted over several weeks and short-term radioactive uptake studies conducted over several hours were performed. The data showed that the Cl^– content of rooted grapevine leaves from different genotypes grown at 25 and 50 mM NaCl was similar to data reported by others. Short-term radioactive uptake assays did not always reveal differences in uptake between the genotypes. Therefore, we suggest that rooted leaves under certain conditions may provide a space-efficient method for generating sufficient amounts of plant material. This material could be used for studying whole plant, molecular and electrophysiological aspects of ion transport and for conducting experiments where root material from specific genotypes is required.     

A method for extraction of total RNA fromPinus radiata and other conifers

Carbohydrates, polyphenolic compounds, terpenoids and tannins interfere with the extraction of intact, uncontaminated total RNA from conifers. A method for extraction of total RNA fromPinus radiata is described. This method uses cesium trifluoroacetate in the ultracentrifugal separation of RNA to overcome the problems of co-purification of contaminating secondary metabolites.     

神木天坑不同小生境木本植物叶功能性状的差异与关联

为进一步探讨天坑生境下木本植物的生存策略，该研究以神木天坑不同小生境(底部、坑腰和边缘)的木本植物为对象，选取叶厚度、叶组织密度、叶面积等10个叶功能性状指标，运用单因素方差分析和相关性分析等方法，分析了天坑木本植物叶功能性状的变异特征以及小生境对木本植物叶功能性状的影响。结果表明：(1)在神木天坑木本植物10个叶功能性状中，叶面积变异系数最大(113.9%),叶碳含量变异系数最小(10.5%)。(2)天坑边缘、坑腰的乔木叶组织密度显著高于底部(P<0.05),天坑底部乔木、灌木的叶钾含量显著高于边缘(P<0.05),3种小生境中木质藤本未出现显著差异(P>0.05)。(3)不同小生境叶功能性状相关性存在一定差异，从天坑底部到边缘小生境，叶厚度与叶面积逐渐呈极显著正相关；主成分分析结果显示叶组织密度(-0.833)、叶钾含量(0.782)、叶干物质含量(-0.647)贡献较大，是神木天坑不同小生境木本植物叶功能性状的主要指标。综上认为，叶功能性状之间普遍关联，天坑木本植物通过对叶功能性状的权衡来适应不同小生境。该研究结果为了解植物对特殊生境的适应机制以及天坑植被的保护...     

A simple, rapid and effective method for total RNA extraction from Lentinula edodes

A rapid, inexpensive and reliable method for total RNA extraction from fruiting bodies of Lentinula edodes containing large quantities of polysaccharides and secondary metabolites is described. An initial extraction step using saturated NaCl solution facilitates the separation of nucleic acids from contaminants and, after further extraction with organic solvents and precipitation with 2-propanol, total RNA of high purity and suitable for applications such as cDNA synthesis, RT-PCR and Northern blot hybridization was obtained. The procedure may also have wider applicability for total RNA extraction from the tissues of other mushrooms.     

A simple and effective method for isolating RNA from alfalfa pollen

Extraction of RNA from alfalfa pollen using conventional grinding devices resulted in low yields of degraded RNA; degradation appeared to be related to the length of time required to break open the pollen grains with such devices. A glass syringe was modified to provide the restricted, shearing environment necessary for rapidly rupturing this type of tissue. This apparatus would be useful for extracting a variety of molecules from pollen of any species, but is particularly valuable for species which produce low amounts of relatively small pollen grains.     

A DNA barcode reference library for the native woody seed plants of Japan

DNA barcode databases are increasingly available for a range of organisms, facilitating the wide application of DNA barcode-based studies. Here we announce the development of a comprehensive DNA barcode reference library of Japanese native woody seed plants representing 43 orders, 99 families, 303 genera and 834 species, and comprising 77.3% of the genera and 72.2% of the species of native woody seed plants in Japan. A total of 6216 plant specimens were collected from 223 sites across the subtropical, temperate, boreal and alpine biomes in Japan with most species represented by multiple accessions. This reference library utilized three chloroplast DNA regions (rbcL, trnH-psbA and matK) and consists of 14,403 barcode sequences. Individual regions varied in their identification rates, with species-level and genus-level rates for rbcL, trnH-psbA and matK based on blast being 57.4%/96.2%, 78.5%/99.1% and 67.8%/98.1%, respectively. Identification rates were higher using region combinations, with total species-level rates for two region combinations (rbcL & trnH-psbA, rbcL & matK and trnH-psbA & matK) ranging between 90.6% and 95.8%, and for all three regions being equal to 98.6%. Genus-level identification rates were even higher, ranging between 99.7% and 100% for two region combinations and being 100% for the three regions. These results indicate that this DNA barcode reference library is an effective resource for investigations of native woody seed plants in Japan using DNA barcodes and provides a useful template for the development of libraries for other components of the Japanese flora.     

红树植物叶片总RNA提取方法比较与优化

分别采用三种植物RNA提取试剂盒、改良的CTAB-LiCl法和CTAB-异丙醇法五种方法提取四种红树植物叶片总RNA,并用紫外光谱、琼脂糖凝胶电泳、cDNA合成和real-time PCR等手段对提取效果进行鉴定.反复试验表明:五种方法对白骨壤Aricennia marina叶片总RNA提取均有良好的效果.Tiangen RNA提取试剂能够成功提取秋茄Kandelia candel、桐花树Aegiceras corniculatum、白骨壤的总RNA,提取的木榄Bruguiera gymnorrhiza总RNA浓度低、杂质较多,且稳定性较差,在对该方法改良后则能够成功提取木榄的总RNA.Invitrogen试剂盒对木榄和秋茄叶片均有良好的提取效果,但是对桐花树提取的RNA易受到蛋白的污染.CTAB-LiCl法和CTAB-异丙醇法提取的RNA总产量偏低,提取时间较长,且实时定量结果表明,两种方法反转录效率较试剂盒方法也偏低.     

岩溶木本植物对干旱的生理生态适应

受全球气候变暖和季风气候影响,西南岩溶区年降水量及其在季节间的分配发生明显变化,无雨期频率和持续时间增加,且基岩风化严重,基质储水能力差,致使岩溶木本植物面临的季节性和地质性干旱加剧。该文通过参考相关文献分析结构性状和生理调节探讨岩溶木本植物如何适应地质性和季节性干旱。结果表明岩溶木本植物应对干旱的策略与其他干旱、半干旱区的植物大体一致,主要有抗旱和避旱两种策略:抗旱性植物一般具有比叶面积小、叶肉多汁、储水组织发达、细胞液浓度高等适应干旱的特征,可通过增加木材密度、增强木质部导管的抗栓塞性和提高水分利用效率以适应干旱; 避旱植物则可通过小而密的气孔和叶脉、发达的表皮毛、栅栏组织和维管束鞘等结构特征减少水分丧失,并可通过落叶、深根吸收深层水源和脱落酸(ABA)介导提早关闭气孔以适应干旱。虽然关于岩溶植物形态结构和生理调节对干旱适应机制的研究取得了一定进展,但仍然存在一些亟待解决的问题,例如:深入研究岩溶地区基岩水分状况及其对植物的贡献; 加强岩溶木本植物根系结构和生物量分配、树木构型及根际微生物与木本植物干旱适应的协同关系研究; 同时探索如何将岩溶植物生态适应研究成果应用于生产实践中,科学指导石漠化治理与生态修复。     

A proposal for universal formulas for estimating leaf water status of herbaceous and woody plants based on spectral reflectance properties

The present study deals with the relationships between water status parameters of plant leaves and reflectances (R_λ) at characteristic wavelengths, between 522 and 2450 nm, as well as reflectance ratios, R_λ/R₁₄₃₀, R_λ/R₁₆₅₀, R_λ/R₁₈₅₀, R_λ/R₁₉₂₀, and R_λ/R₁₉₅₀, based on the air-drying experimental results of soybean (Glycine max Merr.), maize (Zea mays L.), tuliptree (Liriodendron tulipifera L.) and viburnum (Viburnum awabuki K. Koch.) plants. The water status parameters include leaf water content per unit leaf area (LWC), specific leaf water content (SWC), leaf moisture percentage of fresh weight (LMP), relative leaf water content (RWC) and relative leaf moisture percentage on fresh weight basis (RMP). Effective spectral reflectances and reflectance ratios for estimating the LWC, SWC, LMP, RWC and RMP were identified. With these spectral indices, approaches to estimating LWC, RWC and RMP were discussed. Eventually, an attempt on universal formulas was made for estimating the leaf moisture conditions of both herbaceous and woody plants as mentioned above. Moreover, applicability of these formulas was checked with the field experimental results of soybean and maize grown under water and nutrient stresses. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation systems for the mexican redbud (Cercis canadensis var. Mexicana L.) and other woody plants of the chihuahuan desert

Summary There are many Chihuahuan desert species that have potential as landscape plants for the arid communities of the southwestern United States [agarita, Berberis trifoliata Moric.; Mexican buckeye, Ungnadia speciosa Endl.; Texas madrone, Arbutus xalapensis var. texana (Buckl.). A. Gray]. Within these plant populations, there are superior genotypes that offer even greater interest for the landscape. However, it is difficult to clonally propagate many of these species with conventional techniques, and the seed-derived populations often do not breed true. Therefore, selection of superior genotypes in wild populations coupled with clonal propagation through tissue culture may offer an attractive option. It is relatively easy to achieve disinfestation of explants from desert plants due to a general lack of natural surface contamination by fungi and bacteria, even though interference from numerous trichomes can impede good contact with disinfesting agents. However, there is only a narrow window of time that is ideal for explant collection, because of the brief, periodic flushes of growth that characterize this unusual plant group. There may be years when, due to the harsh environment, the amount of suitable explant material is severely limited. Phenolics and exudates are also problematic in this group of plants, and acclimatization of ex vitro plantlets to the harsh desert environment is a particular challenge. For these reasons, specific adaptations and modifications were necessary to achieve success with micropropagation of desert plant species such as Mexican redbud (Cercis canadensis var. mexicana L.).