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1.

Background

To identify plant genes involved in various key traits, QTL mapping is a powerful approach. This approach is based on the use of mapped molecular markers to identify genomic regions controlling quantitative traits followed by a fine mapping and eventually positional cloning of candidate genes. Mapping technologies using SNP markers are still rather expensive and not feasible in every laboratory. In contrast, microsatellite (also called SSR for Simple Sequence Repeat) markers are technologically less demanding and less costly for any laboratory interested in genetic mapping.

Results

In this study, we present the development and the characterization of a panel of 96 highly polymorphic SSR markers along the Arabidopsis thaliana genome allowing QTL mapping among accessions of the Versailles 24 core collection that covers a high percentage of the A. thaliana genetic diversity. These markers can be used for any QTL mapping analysis involving any of these accessions. We optimized the use of these markers in order to reveal polymorphism using standard PCR conditions and agarose gel electrophoresis. In addition, we showed that the use of only three of these markers allows differentiating all 24 accessions which makes this set of markers a powerful tool to control accession identity or any cross between any of these accessions.

Conclusion

The set of SSR markers developed in this study provides a simple and efficient tool for any laboratory focusing on QTL mapping in A. thaliana and a simple means to control seed stock or crosses between accessions.  相似文献   

2.

Background

The tomato (Solanum lycopersicum L.) plant is both an economically important food crop and an ideal dicot model to investigate various physiological phenomena not possible in Arabidopsis thaliana. Due to the great diversity of tomato cultivars used by the research community, it is often difficult to reliably compare phenotypes. The lack of tomato developmental mutants in a single genetic background prevents the stacking of mutations to facilitate analysis of double and multiple mutants, often required for elucidating developmental pathways.

Results

We took advantage of the small size and rapid life cycle of the tomato cultivar Micro-Tom (MT) to create near-isogenic lines (NILs) by introgressing a suite of hormonal and photomorphogenetic mutations (altered sensitivity or endogenous levels of auxin, ethylene, abscisic acid, gibberellin, brassinosteroid, and light response) into this genetic background. To demonstrate the usefulness of this collection, we compared developmental traits between the produced NILs. All expected mutant phenotypes were expressed in the NILs. We also created NILs harboring the wild type alleles for dwarf, self-pruning and uniform fruit, which are mutations characteristic of MT. This amplified both the applications of the mutant collection presented here and of MT as a genetic model system.

Conclusions

The community resource presented here is a useful toolkit for plant research, particularly for future studies in plant development, which will require the simultaneous observation of the effect of various hormones, signaling pathways and crosstalk.  相似文献   

3.

Background

With the advancement of genotyping technologies, whole genome and high-density SNP markers have been widely used for genotyping of mapping populations and for characterization of germplasm lines in many crops. Before conducting SNP data analysis, it is necessary to check the individuals to ensure the integrity of lines for further data analysis.

Results

We have developed an R package to conduct a parent-offspring test of individuals which are genotyped with a fixed set of SNP markers for further genetic studies. The program uses monomorphic SNP loci between parents and their progeny genotypes to calculate the similarity between each offspring and their parents. Based on the similarity of parents and individual offspring, the users can determine the threshold level for the individuals to be included for further data analysis. We used an F5-derived soybean population of ‘5601T’ x PI 157440 that was genotyped with 1,536 SNPs to illustrate the procedure and its application.

Conclusions

The R package ‘ParentOffspring’ coupled with the available SNP genotyping platforms could be used to detect the possible variants in a specific cross, as well as the potential errors in sample handling and genotyping processes. It can be used in any crop which is genotyped with a fixed set of SNP markers.
  相似文献   

4.

Background

Efficient high throughput screening systems of useful mutants are prerequisite for study of plant functional genomics and lots of application fields. Advance in such screening tools, thanks to the development of analytic instruments. Direct analysis in real-time (DART)-mass spectrometry (MS) by ionization of complex materials at atmospheric pressure is a rapid, simple, high-resolution analytical technique. Here we describe a rapid, simple method for the genetic discrimination of intact Arabidopsis thaliana mutant seeds using metabolic profiling by DART-MS.

Results

To determine whether this DART-MS combined by multivariate analysis can perform genetic discrimination based on global metabolic profiling, intact Arabidopsis thaliana mutant seeds were subjected to DART-MS without any sample preparation. Partial least squares-discriminant analysis (PLS-DA) of DART-MS spectral data from intact seeds classified 14 different lines of seeds into two distinct groups: Columbia (Col-0) and Landsberg erecta (Ler) ecotype backgrounds. A hierarchical dendrogram based on partial least squares-discriminant analysis (PLS-DA) subdivided the Col-0 ecotype into two groups: mutant lines harboring defects in the phenylpropanoid biosynthetic pathway and mutants without these defects. These results indicated that metabolic profiling with DART-MS could discriminate intact Arabidopsis seeds at least ecotype level and metabolic pathway level within same ecotype.

Conclusion

The described DART-MS combined by multivariate analysis allows for rapid screening and metabolic characterization of lots of Arabidopsis mutant seeds without complex metabolic preparation steps. Moreover, potential novel metabolic markers can be detected and used to clarify the genetic relationship between Arabidopsis cultivars. Furthermore this technique can be applied to predict the novel gene function of metabolic mutants regardless of morphological phenotypes.  相似文献   

5.
True-to-type clonal fidelity is one of the most important pre-requisites in micropropagation of crop species. Genetic fidelity of in vitro raised 45 plants of gerbera (Gerbera jamesonii Bolus) derived from three different explants, viz., capitulum, leaf and shoot tips, was assessed by 32 ISSR markers, for their genetic stability. Out of 32 ISSR markers, 15 markers produced clear, distinct and scorable bands with an average of 5.47 bands per marker. The markers designed from AG motif amplified more number of bands. The markers anchored at 3′ ends produced high number of consistent bands than unanchored markers. Fifteen ISSR markers generated a total of 3773 bands, out of which 3770 were monomorphic among all the clones. The Jaccard's similarity coefficient revealed that out of 45 clones derived from different explants, 44 were grouped into a single large cluster alongwith the mother plant with a similarity coefficient value of 1.00, whereas one clone (C38) remained ungrouped. The clones derived from capitulum and shoot tip explants did not show any genetic variation, whereas, one of the leaf-derived clones exhibited some degree of variation.  相似文献   

6.

Background

Deep sequencing is a powerful tool for novel small RNA discovery. Illumina small RNA sequencing library preparation requires a pre-adenylated 3?? end adapter containing a 5??,5??-adenyl pyrophosphoryl moiety. In the absence of ATP, this adapter can be ligated to the 3?? hydroxyl group of small RNA, while RNA self-ligation and concatenation are repressed. Pre-adenylated adapters are one of the most essential and costly components required for library preparation, and few are commercially available.

Results

We demonstrate that DNA oligo with 5?? phosphate and 3?? amine groups can be enzymatically adenylated by T4 RNA ligase 1 to generate customized pre-adenylated adapters. We have constructed and sequenced a small RNA library for tomato (Solanum lycopersicum) using the T4 RNA ligase 1 adenylated adapter.

Conclusion

We provide an efficient and low-cost method for small RNA sequencing library preparation, which takes two days to complete and costs around $20 per library. This protocol has been tested in several plant species for small RNA sequencing including sweet potato, pepper, watermelon, and cowpea, and could be readily applied to any RNA samples.  相似文献   

7.

Background

Cultivated sunflower (Helianthus annus L.) is a globally important oilseed crop, subjected to intensive genetic and genomic studies. Although classical mutagenesis has successfully been applied to Helianthus genus in the past, we have developed the first sunflower TILLING resource.

Results

To balance the maximum mutation density with an acceptable plant survival rate, a 'kill curve' analysis was first conducted with different ethylmethanesulfonate (EMS) dosages and different exposure times. According to the germination rate, a treatment with 0.7% EMS for 6 h was chosen. An M2 progeny of 3,651 fertile plants was obtained. Totally, 4.79% of the whole population showed clear aberrant phenotypes. A microsatellite analysis on a representative sample of the original seed stock and mutant lines confirmed the uniformity of the genetic background of plant material. The TILLING procedure was successfully applied to sunflower genome, initially by a CelI-nuclease mismatch cleavage assay coupled with a DNA-pooling level test. To investigate the efficiency of the mutagenic treatment, a pilot screening was carried out on 1,152 M2 lines focusing on four genes, three involved in the fatty acid biosynthetic pathway and one for downy mildew resistance. A total of 9 mutant lines were identified and confirmed by sequencing; thereby, the estimated overall mutation frequency for the pilot assay resulted to be 1/475 kb.

Conclusion

A first TILLING population for a high throughput identification of EMS-induced point mutations in sunflower genome has been successfully obtained. This represents a powerful tool to a better understanding of gene function in sunflower.  相似文献   

8.

Background

Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes.

Results

We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner? system we were able to detect mutations in heterozygous and homozygous states for both genes.

Conclusions

Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service.  相似文献   

9.

Background

The genus Populus is accepted as a model system for molecular tree biology. To investigate gene functions in Populus spp. trees, generating stable transgenic lines is the common technique for functional genetic studies. However, a limited number of genes have been targeted due to the lengthy transgenic process. Transient transformation assays complementing stable transformation have significant advantages for rapid in vivo assessment of gene function. The aim of this study is to develop a simple and efficient transient transformation for hybrid aspen and to provide its potential applications for functional genomic approaches.

Results

We developed an in planta transient transformation assay for young hybrid aspen cuttings using Agrobacterium-mediated vacuum infiltration. The transformation conditions such as the infiltration medium, the presence of a surfactant, the phase of bacterial growth and bacterial density were optimized to achieve a higher transformation efficiency in young aspen leaves. The Agrobacterium infiltration assay successfully transformed various cell types in leaf tissues. Intracellular localization of four aspen genes was confirmed in homologous Populus spp. using fusion constructs with the green fluorescent protein. Protein-protein interaction was detected in transiently co-transformed cells with bimolecular fluorescence complementation technique. In vivo promoter activity was monitored over a few days in aspen cuttings that were transformed with luciferase reporter gene driven by a circadian clock promoter.

Conclusions

The Agrobacterium infiltration assay developed here is a simple and enhanced throughput method that requires minimum handling and short transgenic process. This method will facilitate functional analyses of Populus genes in a homologous plant system.  相似文献   

10.
《Scientia Horticulturae》2005,104(2):151-160
Random amplified polymorphic DNA (RAPD) markers were used to assess the genetic stability of 10 micropropagated plants regenerated through axillary buds of clonal apple (Malus pumila Mill.) rootstock MM106. Eleven random decamer primers were successfully used to analyse genomic DNA from mother plants and in vitro plant material. A total of 129 scorable fragments were amplified with an average of 11.73 bands per primer. Among them, 99 were monomorphic and 30 were polymorphic with 23.2% polymorphism. Among these 30, 12 were found monomorphic across seven plants and parent. Three plants could be regarded as off-types. Our results show that RAPD markers could be used to detect the genetic similarities and dissimilarities in micropropagated material.  相似文献   

11.
The regulation of flowering time has been studied mainly in photoperiod-sensitive model plants. Little is known about the environmental and genetic regulation of flowering time in photoperiod-insensitive plants. Here, we studied the genetic control of flowering time in day-neutral tomato using quantitative trait locus (QTL) analysis. We used a BC1F6 population developed from Solanum lycopersicum ‘M570018’ and its wild relative Solanum pimpinellifolium (PI124039) to measure days to flowering (DTF) and number of leaves preceding the first inflorescence (LN), as well as their underlying developmental processes, which include the number of leaves initiated (LI), percentage of flower-induced plants (reproductive index, RI), days to macroscopic flower bud appearance (DMB) and flower development duration (FDD: DTF − DMB). A composite interval mapping detected 12 QTLs for the six traits, which included two QTLs for DTF on chromosomes 1 and 6. The DTF QTLs explained 43% of the phenotypic variation of this trait. The presence of S. pimpinellifolium alleles in the detected QTLs increased the rate of leaf initiation, reduced LN and hastened flower induction, floral development and anthesis. Most QTLs for LN, LI, RI, DMB and FDD clustered with the DTF QTLs; dmb1, fdd1, li_14d1, li_19d1 and ri_19d1 clustered with dtf1 on chromosome 1, and ln6 and fdd6 clustered with dtf6 on chromosome 6. These results suggest that the QTLs on chromosomes 1 and 6 may form a functional “gene cluster” that drives tomato flowering in synergy. Alternatively, the two DTF QTLs may act as “master genes” that control flowering time through pleiotropic effects on multiple developmental processes.  相似文献   

12.

Background

The Arabidopsis thaliana-Pseudomonas syringae model pathosystem is one of the most widely used systems to understand the mechanisms of microbial pathogenesis and plant innate immunity. Several inoculation methods have been used to study plant-pathogen interactions in this model system. However, none of the methods reported to date are similar to those occurring in nature and amicable to large-scale mutant screens.

Results

In this study, we developed a rapid and reliable seedling flood-inoculation method based on young Arabidopsis seedlings grown on MS medium. This method has several advantages over conventional soil-grown plant inoculation assays, including a shorter growth and incubation period, ease of inoculation and handling, uniform infection and disease development, requires less growth chamber space and is suitable for high-throughput screens. In this study we demonstrated the efficacy of the Arabidopsis seedling assay to study 1) the virulence factors of P. syringae pv. tomato DC3000, including type III protein secretion system (TTSS) and phytotoxin coronatine (COR); 2) the effector-triggered immunity; and 3) Arabidopsis mutants affected in salicylic acid (SA)- and pathogen-associated molecular pattern (PAMPs)-mediated pathways. Furthermore, we applied this technique to study nonhost resistance (NHR) responses in Arabidopsis using nonhost pathogens, such as P. syringae pv. tabaci, pv. glycinea and pv. tomato T1, and confirmed the functional role of FLAGELLIN-SENSING 2 (FLS2) in NHR.

Conclusions

The Arabidopsis seedling flood-inoculation assay provides a rapid, efficient and economical method for studying Arabidopsis-Pseudomonas interactions with minimal growth chamber space and time. This assay could also provide an excellent system for investigating the virulence mechanisms of P. syringae. Using this method, we demonstrated that FLS2 plays a critical role in conferring NHR against nonhost pathovars of P. syringae, but not to Xanthomonas campestris pv. vesicatoria. This method is potentially ideal for high-throughput screening of both Arabidopsis and pathogen mutants.  相似文献   

13.
A high throughput DNA extraction method with high yield and quality   总被引:1,自引:0,他引:1  
Z Xin  J Chen 《Plant methods》2012,8(1):26-7

Background

Preparation of large quantity and high quality genomic DNA from a large number of plant samples is a major bottleneck for most genetic and genomic analyses, such as, genetic mapping, TILLING (Targeting Induced Local Lesion IN Genome), and next-generation sequencing directly from sheared genomic DNA. A variety of DNA preparation methods and commercial kits are available. However, they are either low throughput, low yield, or costly. Here, we describe a method for high throughput genomic DNA isolation from sorghum [Sorghum bicolor (L.) Moench] leaves and dry seeds with high yield, high quality, and affordable cost.

Results

We developed a high throughput DNA isolation method by combining a high yield CTAB extraction method with an improved cleanup procedure based on MagAttract kit. The method yielded large quantity and high quality DNA from both lyophilized sorghum leaves and dry seeds. The DNA yield was improved by nearly 30 fold with 4 times less consumption of MagAttract beads. The method can also be used in other plant species, including cotton leaves and pine needles.

Conclusion

A high throughput system for DNA extraction from sorghum leaves and seeds was developed and validated. The main advantages of the method are low cost, high yield, high quality, and high throughput. One person can process two 96-well plates in a working day at a cost of $0.10 per sample of magnetic beads plus other consumables that other methods will also need.  相似文献   

14.

Context

Common species important for ecosystem restoration stand to lose as much genetic diversity from anthropogenic habitat fragmentation and climate change as rare species, but are rarely studied. Salt marshes, valuable ecosystems in widespread decline due to human development, are dominated by the foundational plant species black needlerush (Juncus roemerianus Scheele) in the northeastern Gulf of Mexico.

Objectives

We assessed genetic patterns in J. roemerianus by measuring genetic and genotypic diversity, and characterizing population structure. We examined population connectivity by delineating possible dispersal corridors, and identified landscape factors influencing population connectivity.

Methods

A panel of 19 microsatellite markers was used to genotype 576 samples from ten sites across the northeastern Gulf of Mexico from the Grand Bay National Estuarine Research Reserve (NERR) to the Apalachicola NERR. Genetic distances (FST and Dch) were used in a least cost transect analysis (LCTA) within a hierarchical model selection framework.

Results

Genetic and genotypic diversity results were higher than expected based on life history literature, and samples structured into two large, admixed genetic clusters across the study area, indicating sexual reproduction may not be as rare as predicted in this clonal macrophyte. Digitized coastal transects buffered by 500 m may represent possible dispersal corridors, and developed land may significantly impede population connectivity in J. roemerianus.

Conclusions

Results have important implications for coastal restoration and management that seek to preserve adaptive potential by sustaining natural levels of genetic diversity and conserving population connectivity. Our methodology could be applied to other common, widespread and understudied species.
  相似文献   

15.

Background

We previously developed a virus-induced gene silencing (VIGS) vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV). The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium (Agrobacterium tumefaciens). We also describe the construction of two low-cost particle inflow guns.

Results

The biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum) and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV) VIGS vector respectively.

Conclusions

These innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns.  相似文献   

16.

Background

Protein extraction is a frequent procedure in biological research. For preparation of plant cell extracts, plant materials usually have to be ground and homogenized to physically break the robust cell wall, but this step is laborious and time-consuming when a large number of samples are handled at once.

Results

We developed a chemical method for lysing Arabidopsis cells without grinding. In this method, plants are boiled for just 10 minutes in a solution containing a Ca2+ chelator and detergent. Cell extracts prepared by this method were suitable for SDS-PAGE and immunoblot analysis. This method was also applicable to genomic DNA extraction for PCR analysis. Our method was applied to many other plant species, and worked well for some of them.

Conclusions

Our method is rapid and economical, and allows many samples to be prepared simultaneously for protein analysis. Our method is useful not only for Arabidopsis research but also research on certain other species.  相似文献   

17.

Background

Molecular profiling of gene families is a versatile tool to study diversity between individual genomes in sexual crosses and germplasm. Nucleotide binding site (NBS) profiling, in particular, targets conserved nucleotide binding site-encoding sequences of resistance gene analogs (RGAs), and is widely used to identify molecular markers for disease resistance (R) genes.

Results

In this study, we used NBS profiling to identify genome-wide locations of RGA clusters in the genome of potato clone RH. Positions of RGAs in the potato RH and DM genomes that were generated using profiling and genome sequencing, respectively, were compared. Largely overlapping results, but also interesting discrepancies, were found. Due to the clustering of RGAs, several parts of the genome are overexposed while others remain underexposed using NBS profiling. It is shown how the profiling of other gene families, i.e. protein kinases and different protein domain-coding sequences (i.e., TIR), can be used to achieve a better marker distribution. The power of profiling techniques is further illustrated using RGA cluster-directed profiling in a population of Solanum berthaultii. Multiple different paralogous RGAs within the Rpi-ber cluster could be genetically distinguished. Finally, an adaptation of the profiling protocol was made that allowed the parallel sequencing of profiling fragments using next generation sequencing. The types of RGAs that were tagged in this next-generation profiling approach largely overlapped with classical gel-based profiling. As a potential application of next-generation profiling, we showed how the R gene family associated with late blight resistance in the SH*RH population could be identified using a bulked segregant approach.

Conclusions

In this study, we provide a comprehensive overview of previously described and novel profiling primers and their genomic targets in potato through genetic mapping and comparative genomics. Furthermore, it is shown how genome-wide or fine mapping can be pursued by choosing different sets of profiling primers. A protocol for next-generation profiling is provided and will form the basis for novel applications. Using the current overview of genomic targets, a rational choice can be made for profiling primers to be employed.
  相似文献   

18.
Integration of previously developed Allium cepa linkage maps requires the availability of anchor markers for each of the eight chromosomes of shallot (A. cepa L. common group Aggregatum). To this end, eight RAPD markers originating from our previous research were converted into SCAR markers via cloning and sequencing of RAPD amplicons and designing of 24-mer oligonucleotide primers. Of the eight pairs of SCAR primers, seven resulted in the amplification of single bands of the original RAPDs, and the remaining primer set amplified an additional band. The results of Southern hybridization using RAPD amplicons from genomic DNA of Japanese bunching onion (Allium fistulosum L.)—shallot monosomic addition lines indicated that five SCAR markers were single shallot chromosome-specific markers and were not detected in genomic DNA of A. fistulosum. The eight SCAR primer pairs were applied to other Allium species and exhibited three types of amplification profiles, namely RAPD amplicons observed only in shallot, in shallot and Allium vavilovii, and in several Allium species. A mapping study using 65 F2 plants generated by the selfing of one interspecific cross A. cepa × Allium roylei individual integrated the SCAR marker SAOE17500 into chromosome 5 as expected. The results of the present study show that the eight SCAR primer sets specific to shallot can facilitate the mapping in A. cepa and can also serve as anchor points between maps of different Allium species.  相似文献   

19.
Genetic variability in modern crops is limited due to domestication and breeding. To investigate genetic variation in different populations, 216 tomato (Solanum lycopersicum L.) cultivars, hybrids, and elite breeding lines from four breeding programs were genotyped using single nucleotide polymorphism and simple sequence repeat markers. Of 47 markers analyzed, 72.3% were polymorphic in the whole collection of 216 genotypes and 51.06–59.57% showed polymorphisms in individual populations. However, genetic variation was narrow in all four populations. Nei's genetic distance varied from 0.0422 to 0.1135 between populations and from 0.0085 to 0.3187 between lines in individual populations. Cluster and principal coordinate analysis indicated that the four populations could be grouped into three clades. Lines from Shenyang Agricultural University and China Agricultural University population formed the first clade, lines from Beijing Vegetable Research Center were in the second clade, and lines from Nunhems were in the third clade. This was further supported by population structure analysis using STRUCTURE2.2, and suggested that a lack of germplasm exchange might exist among breeding programs. It might be the reason that the progress of developing new varieties with significant improvement of horticultural traits in China is slow in recent years.  相似文献   

20.

Background

Histochemical staining of plant tissues with 4-dimethylaminocinnamaldehyde (DMACA) or vanillin-HCl is widely used to characterize spatial patterns of proanthocyanidin accumulation in plant tissues. These methods are limited in their ability to allow high-resolution imaging of proanthocyanidin deposits.

Results

Tissue embedding techniques were used in combination with DMACA staining to analyze the accumulation of proanthocyanidins in Lotus corniculatus (L.) and Trifolium repens (L.) tissues. Embedding of plant tissues in LR White or paraffin matrices, with or without DMACA staining, preserved the physical integrity of the plant tissues, allowing high-resolution imaging that facilitated cell-specific localization of proanthocyanidins. A brown coloration was seen in proanthocyanidin-producing cells when plant tissues were embedded without DMACA staining and this was likely to have been due to non-enzymatic oxidation of proanthocyanidins and the formation of colored semiquinones and quinones.

Conclusions

This paper presents a simple, high-resolution method for analysis of proanthocyanidin accumulation in organs, tissues and cells of two plant species with different patterns of proanthocyanidin accumulation, namely Lotus corniculatus (birdsfoot trefoil) and Trifolium repens (white clover). This technique was used to characterize cell type-specific patterns of proanthocyanidin accumulation in white clover flowers at different stages of development.  相似文献   

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