AN IMPROVED FIXING AND STAINING PROCEDURE FOR PLOIDY DETERMINATIONS FROM ROOT TIP SQUASHES

A comparison of pretreatment, fixing, and staining methods for root tips of Fragaria × ananassa (2n=8x=56), a polyploid species with small chromosomes, was made to facilitate chromosome counting. Three pretreatments (8-hydroxyquinoline, α-bromonaphthalene, and p-dichlorobenzene), three fixatives (Farmer's, Carnoy's, and Newcomer's), and five stains (acetocarmine, lacto-propionic orcein, leucobasic fuchsin, altered carbol fuchsin, and alcoholic hydrochloric-acid carmine) were examined in a factorial design to determine which treatment combination produced the best chromosome preparation. Field propagated runners were grown in sand under greenhouse conditions with supplemental lighting to produce root tips for late morning collection. The treatment combinations of α-bromonaphthalene or 8-hydroxyquinoline, Farmer's fixative, and altered carbol fuchsin, or the combination of α-bromonaphthalene, Farmer's fixative, and alcoholic hydrochloric-acid carmine produced the most intensely-stained and well-defined preparations.

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A Comparison of Staining Techniques for Somatic Chromosomes of Strawberry

HortScience ◽

10.21273/hortsci.28.2.155 ◽

1993 ◽

Vol 28 (2) ◽

pp. 155-156 ◽

Cited By ~ 7

Author(s):

Henry R. Owen ◽

A. Raymond Miller

Keyword(s):

Hydrochloric Acid ◽

Root Tip ◽

Fragaria Ananassa ◽

Factorial Combination ◽

Carbol Fuchsin ◽

Staining Techniques ◽

Tip Cells ◽

Root Tip Cells

A factorial combination of pretreatments, fixatives, and stains was examined to identify the best available method for staining the chromosomes of strawberry (Fragaria × ananassa Duch. `Chandler') root-tip cells. Three pretreatments (a -bromonaphthalene, p -dichlorobenzene, and 8-hydroxyquinoline), three fixatives (Farmer's fluid, Carnoy's fluid, and Newcomer's fluid), and five stains (acetocarmine, alcoholic hydrochloric-acid carmine, altered carbol fuchsin, lacto-propionic orcein, and leucobasic fuchsin) were tested. Pretreatment with either a -bromonaphthalene (saturated aqueous) or 8-hydroxyquinoline (2 mm) for 5 hours at 14C, overnight fixation in Farmer's fluid, hydrolysis in 1 n HCl (15 minutes at 60C), and staining with altered carbol fuchsin produced chromosome preparations superior to other treatment combinations. Treatment with a-bromonaphthalene, Farmer's fluid, and alcoholic hydrochloric-acid carmine (2 days at 25C) also produced acceptable chromosome preparations.

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CHROMOSOME COUNT ON YOUNG ANTHER OF BANANA MALE BUD USING EZYMATIC MACERATION AND DAPI STAINING IN SLIDE PREPARATION

BERITA BIOLOGI ◽

10.14203/beritabiologi.v19i2.3851 ◽

2020 ◽

Vol 19 (2) ◽

Author(s):

Fajarudin Ahmad ◽

Yuyu Suryasari Poerba

Keyword(s):

Genetic Study ◽

Cell Spreading ◽

Enzymatic Digestion ◽

Chromosome Count ◽

Root Tip ◽

Chromosome Counting ◽

Fluorescent Staining ◽

Dapi Staining ◽

Slide Preparation ◽

Staining Methods

Chromosome counting is the basis in describing the chromosomes number of organism that might useful for genetic study and classification. In banana studies, the root tip with a combination of non-fluorescent staining methods such as carmine or orcein and squash is the most common material for chromosome counting. In this study, we presented the usefulness of young anther of banana male bud with enzymatic maceration method for cell spreading and 4,6-diamino-2-phenyl-indole (DAPI) for staining agent to get a satisfying chromosomes image at metaphase for mitotic study of diploid and tetraploid bananas. The principle of this study is fixation using ethanol:acetic acid (3:1), enzymatic digestion, maceration and staining using DAPI. Our result showed that this method can provide well spread cells with intensely contrast of chromosomes images that satisfying for chromosome counting.

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Pewarnaan Kromosom dan Pemanfaatannya dalam Penentuan Tingkat Ploidi Eksplan Hasil Kultur Anter Anthurium

Jurnal Hortikultura ◽

10.21082/jhort.v21n2.2011.p113-123 ◽

2016 ◽

Vol 21 (2) ◽

pp. 113 ◽

Cited By ~ 1

Author(s):

Budi Winarto

Keyword(s):

Anther Culture ◽

Ploidy Level ◽

Staining Method ◽

Root Tip ◽

Root Tips ◽

Staining Methods ◽

Ornamental Crops ◽

High Benefit ◽

Chromosome Staining

Metode pewarnaan Kromosom yang optimal merupakan prasarat penting dalam penentuan level ploidi tanaman hasil kultur anter, termasuk variasi eksplan hasil kultur anter Anthurium. Aplikasi dan modifikasi metode pewarnaan kromosom pada berbagai eksplan dilakukan di Laboratorium Kultur Jaringan Balai Penelitian Tanaman Hias dari bulan Februari sampai dengan Agustus 2009 untuk mengetahui keragaman dan tingkat ploidi regeneran hasil kultur anter Anthurium. Penelitian bertujuan mendapatkan metode pewarnaan kromosom dan modifikasinya, jenis eksplan dan akar yang sesuai untuk mempelajari tingkat ploidi regeneran hasil kultur anter Anthurium. Bahan yang digunakan ialah kalus, pucuk tunas, dan ujung akar udara. Penelitian terdiri atas tiga kegiatan, yaitu (1) modifikasi metode pewarnaan kromosom, (2) seleksi eksplan yang sesuai untuk pewarnaan kromosom, dan (3) optimasi metode pewarnaan kromosom terseleksi. Hasil penelitian menunjukkan bahwa ujung akar dan akar yang ditumbuhkan pada medium yang mengandung 1% arang aktif merupakan jenis eksplan dan akar yang sesuai untuk mendapatkan hasil pewarnaan kromosom yang baik. Modifikasi metode pewarnaan kromosom dengan pemanasan ujung akar pada 1N HCl : asam asetat glasial 45% (3:1, v/v) selama 10 menit pada suhu 60oC dan perlakuan aseto-orcein selama 15 menit merupakan metode pewarnaan kromosom yang lebih baik dalam menghasilkan obyek kromosom yang mudah dihitung. Penerapan metode pewarnaan kromosom pada kultur anter Anthurium dapat memisahkan tingkat ploidi regeneran. Pada penelitian ini rasio ploidi regeneran kultur anter ialah 33,5% haploid, 62,7% diploid, dan 5,7% triploid. Metode pewarnaan kromosom yang berhasil dikembangkan dalam penelitian ini sangat bermanfaat dalam pengembangan teknologi haploid pada jenis Araceae yang lain.<br /><br />Optimal chromosome staining method is important pre-requisite in determination of plant ploidy level derived from anther culture, involving varied explants regenerated from Anthurium anther culture. Application and modification of chromosome staining methods on different explants were conducted at the Tissue Culture Laboratory of Indonesian Ornamental Crops Research Institute from February to August 2009 for determination of the ploidy level of regenerants derived from anther culture of Anthurium. The aim of this research was to determine the chromosome staining method and its modifications, type of explant and root suitable to study the ploidy level of explants derived from anther culture of Anthurium. Callus, shoot tips, and root tips were utilized in the experiment. The research was consisted of three experiments, i.e. (1) modification of chromosome staining methods (2) selection of explants suitable for chromosome staining, and (3) improvement of the selected chromosome staining method. Results of the study indicated that root tips and roots cultured on medium containing 1% active carchoal were the most appropriate explants and the root type in obtaining better chromosome staining results. The modification method with root tip boiled in 1N HCl : 45% of acetic acid glacial (3:1, v/v) for 10 minutes in 60ºC and aceto-orcein treatment for 15 minutes gave appropriate chromosome staining results exhibited clearer chromosome pictures and was easy to be counted. The application of chromosome staining on anther culture of Anthurium was able to distinguish the ploidy level of regenerants. Ploidy ratio of regenerants derived from anther culture was 33.5% of haploid, 62.7% of diploid, and 5.7% of triploid. Chromosome staining method resulted from the study give high benefit in developing haploid technologies on other Araceae plants.<br /><br />

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Histochemical Examination of Invertase Activities in the Growing Tip of the Plant Root

The Open Plant Science Journal ◽

10.2174/1874294701710010035 ◽

2017 ◽

Vol 10 (1) ◽

pp. 35-45

Author(s):

N.F. Lunkova ◽

N.A. Burmistrova ◽

M.S. Krasavina

Keyword(s):

Plant Root ◽

Invertase Activity ◽

Root Tip ◽

Elongation Zone ◽

Root Tips ◽

Phloem Unloading ◽

Meristem Cell ◽

Transition To Elongation ◽

Different Tissues

Background:A growing part of the root is one of the most active sinks for sucrose coming from source leaves through the phloem. In the root, sucrose is unloaded from conducting bundles and is distributed among the surrounding cells. To be involved in the metabolism, sucrose should disintegrate into hexoses by means of degrading enzymes.Aims:The aim of this research was to explore the possibility of the involvement of one such enzymes, invertase, in phloem unloading as well as distribution of its activity in the functionally different tissues of the plant root tips.Method:To estimate the enzyme activities in root tissues, we applied two techniques: the histochemical method using nitro blue tetrazolium. The localization of phloem unloading was studied with carboxyfluorescein, a fluorescent marker for symplastic transport.Results:Invertase activity was not detected in the apical part of the meristem. It appeared only between the basal part of this zone and the beginning of the elongation zone. There is the root phloem unloading in that area. Invertase activity increased with increasing the distance from the root tip and reached the highest values in the region of cell transition to elongation and in the elongation zone. The activities of the enzyme varied in different tissues of the same zone and sometimes in the neighboring cells of the same tissue. Biochemical determination of invertase activity was made in the maize root segments coincident to the zones of meristem, cell elongation and differentiation. The results of both methods of determination of invertase activity were in agreement.Conclusion:It was concluded that phloem unloading correlated with invertase activity, possibly because of the activation of invertase by unloaded sucrose. Invertase is one of the factors involved in the processes preparing the cells for their transition to elongation because the concentration of osmotically active hexoses increases after cleavage of sucrose, that stimulates water entry into the cells, which is necessary for elongation growth.

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Effect of earthworms on mycorrhization, root morphology and biomass of silver fir seedlings inoculated with black summer truffle (Tuber aestivum Vittad.)

Scientific Reports ◽

10.1038/s41598-021-85497-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tina Unuk Nahberger ◽

Gian Maria Niccolò Benucci ◽

Hojka Kraigher ◽

Tine Grebenc

Keyword(s):

Root System ◽

Root Biomass ◽

Fine Root ◽

Abies Alba ◽

Fine Root Biomass ◽

Root Tip ◽

Silver Fir ◽

Tuber Aestivum ◽

Root Tips ◽

Root Parameters

AbstractSpecies of the genus Tuber have gained a lot of attention in recent decades due to their aromatic hypogenous fruitbodies, which can bring high prices on the market. The tendency in truffle production is to infect oak, hazel, beech, etc. in greenhouse conditions. We aimed to show whether silver fir (Abies alba Mill.) can be an appropriate host partner for commercial mycorrhization with truffles, and how earthworms in the inoculation substrate would affect the mycorrhization dynamics. Silver fir seedlings inoculated with Tuber. aestivum were analyzed for root system parameters and mycorrhization, how earthworms affect the bare root system, and if mycorrhization parameters change when earthworms are added to the inoculation substrate. Seedlings were analyzed 6 and 12 months after spore inoculation. Mycorrhization with or without earthworms revealed contrasting effects on fine root biomass and morphology of silver fir seedlings. Only a few of the assessed fine root parameters showed statistically significant response, namely higher fine root biomass and fine root tip density in inoculated seedlings without earthworms 6 months after inoculation, lower fine root tip density when earthworms were added, the specific root tip density increased in inoculated seedlings without earthworms 12 months after inoculation, and general negative effect of earthworm on branching density. Silver fir was confirmed as a suitable host partner for commercial mycorrhization with truffles, with 6% and 35% mycorrhization 6 months after inoculation and between 36% and 55% mycorrhization 12 months after inoculation. The effect of earthworms on mycorrhization of silver fir with Tuber aestivum was positive only after 6 months of mycorrhization, while this effect disappeared and turned insignificantly negative after 12 months due to the secondary effect of grazing on ectomycorrhizal root tips.

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Molecular Identification of Ectomycorrhizal Mycelium in Soil Horizons

Applied and Environmental Microbiology ◽

10.1128/aem.69.1.327-333.2003 ◽

2003 ◽

Vol 69 (1) ◽

pp. 327-333 ◽

Cited By ~ 158

Author(s):

Renske Landeweert ◽

Paula Leeflang ◽

Thom W. Kuyper ◽

Ellis Hoffland ◽

Anna Rosling ◽

...

Keyword(s):

Molecular Identification ◽

Sequence Similarity ◽

Mineral Soil ◽

Molecular Techniques ◽

Root Tip ◽

Root Tips ◽

Soil Horizons ◽

Novel Approach ◽

Total Dna ◽

Identification Techniques

ABSTRACT Molecular identification techniques based on total DNA extraction provide a unique tool for identification of mycelium in soil. Using molecular identification techniques, the ectomycorrhizal (EM) fungal community under coniferous vegetation was analyzed. Soil samples were taken at different depths from four horizons of a podzol profile. A basidiomycete-specific primer pair (ITS1F-ITS4B) was used to amplify fungal internal transcribed spacer (ITS) sequences from total DNA extracts of the soil horizons. Amplified basidiomycete DNA was cloned and sequenced, and a selection of the obtained clones was analyzed phylogenetically. Based on sequence similarity, the fungal clone sequences were sorted into 25 different fungal groups, or operational taxonomic units (OTUs). Out of 25 basidiomycete OTUs, 7 OTUs showed high nucleotide homology (≥99%) with known EM fungal sequences and 16 were found exclusively in the mineral soil. The taxonomic positions of six OTUs remained unclear. OTU sequences were compared to sequences from morphotyped EM root tips collected from the same sites. Of the 25 OTUs, 10 OTUs had ≥98% sequence similarity with these EM root tip sequences. The present study demonstrates the use of molecular techniques to identify EM hyphae in various soil types. This approach differs from the conventional method of EM root tip identification and provides a novel approach to examine EM fungal communities in soil.

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A simple metal staining procedure for identification and visualization of single cells by LA-ICP-MS

The Analyst ◽

10.1039/c6an02638a ◽

2017 ◽

Vol 142 (10) ◽

pp. 1703-1710 ◽

Cited By ~ 23

Author(s):

A. J. Herrmann ◽

S. Techritz ◽

N. Jakubowski ◽

A. Haase ◽

A. Luch ◽

...

Keyword(s):

Mass Spectrometry ◽

Inductively Coupled Plasma ◽

Single Cells ◽

Staining Procedure ◽

Simple Metal ◽

Inductively Coupled ◽

Icp Ms ◽

High Lateral Resolution ◽

Plasma Mass Spectrometry ◽

Staining Methods

High lateral resolution of metal detection in single cells by use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) demands powerful staining methods.

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Proteomic Analyses of Soybean Root Tips During Germination

Protein and Peptide Letters ◽

10.2174/0929866521666140526152426 ◽

2014 ◽

Vol 21 (12) ◽

pp. 1308-1319

Author(s):

Setsuko Komatsu ◽

Myeong W. Oh ◽

Hee Y. Jang ◽

Soo J. Kwon ◽

Hye R. Kim ◽

...

Keyword(s):

Protein Synthesis ◽

Proteomic Analysis ◽

Plant Root ◽

Root Tip ◽

Root Tips ◽

Form Complex ◽

Molecular Weights ◽

Soybean Seedlings ◽

2D Gel ◽

Proteomic Techniques

Plant root systems form complex networks with the surrounding soil environment and are controlled by both internal and external factors. To better understand the function of root tips of soybean during germination, three proteomic techniques were used to analyze the protein profiles of root tip cells. Proteins were extracted from the root tips of 4-dayold soybean seedlings and analyzed using two-dimensional (2D) gel electrophoresis-based proteomics, SDS-gel based proteomics, and gel-free proteomics techniques. A total of 121, 862, and 341 proteins were identified in root tips using the 2D gel-based, SDS gel-based, and gel-free proteomic techniques, respectively. The proteins identified by 2D gel-based proteomic analysis were predominantly localized in the cytoplasm, whereas nuclear-localized proteins were most commonly identified by the SDS gel-based and gel-free proteomics techniques. Of the 862 proteins identified in the SDS gelbased proteomic analysis, 190 were protein synthesis-related proteins. Furthermore, 24 proteins identified using the 2Dgel based proteomic technique shifted between acidic and basic isoelectric points, and 2 proteins, heat shock protein 70.2 and AAA-type ATPase, displayed two different molecular weights at the same isoelectric point. Taken together, these results suggest that a number of proteins related to protein synthesis and modification are activated in the root tips of soybean seedlings during germination.

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The Jacalin-Related Lectin HvHorcH Is Involved in the Physiological Response of Barley Roots to Salt Stress

International Journal of Molecular Sciences ◽

10.3390/ijms221910248 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10248

Author(s):

Katja Witzel ◽

Andrea Matros ◽

Uwe Bertsch ◽

Tariq Aftab ◽

Twan Rutten ◽

...

Keyword(s):

Salt Stress ◽

Salt Tolerance ◽

Extracellular Fluid ◽

Root Tip ◽

Salinity Treatment ◽

Root Tips ◽

Salt Stress Response ◽

Cdna Sequences ◽

Barley Cultivars ◽

Protein Levels

Salt stress tolerance of crop plants is a trait with increasing value for future food production. In an attempt to identify proteins that participate in the salt stress response of barley, we have used a cDNA library from salt-stressed seedling roots of the relatively salt-stress-tolerant cv. Morex for the transfection of a salt-stress-sensitive yeast strain (Saccharomyces cerevisiae YSH818 Δhog1 mutant). From the retrieved cDNA sequences conferring salt tolerance to the yeast mutant, eleven contained the coding sequence of a jacalin-related lectin (JRL) that shows homology to the previously identified JRL horcolin from barley coleoptiles that we therefore named the gene HvHorcH. The detection of HvHorcH protein in root extracellular fluid suggests a secretion under stress conditions. Furthermore, HvHorcH exhibited specificity towards mannose. Protein abundance of HvHorcH in roots of salt-sensitive or salt-tolerant barley cultivars were not trait-specific to salinity treatment, but protein levels increased in response to the treatment, particularly in the root tip. Expression of HvHorcH in Arabidopsis thaliana root tips increased salt tolerance. Hence, we conclude that this protein is involved in the adaptation of plants to salinity.

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THE CYTOCHEMICAL LOCALIZATION OF ASCORBIC ACID IN ROOT TIP CELLS

The Journal of Cell Biology ◽

10.1083/jcb.2.1.87 ◽

1956 ◽

Vol 2 (1) ◽

pp. 87-92 ◽

Cited By ~ 27

Author(s):

William A. Jensen ◽

Leroy G. Kavaljian

Keyword(s):

Ascorbic Acid ◽

Cell Surface ◽

Silver Nitrate ◽

Ribonucleic Acid ◽

Root Tip ◽

Root Tips ◽

Cytochemical Localization ◽

Meristematic Cells ◽

Tip Cells ◽

Root Tip Cells

The intracellular distribution of ascorbic acid was studied in frozen-dried root tips of Allium cepa and Vicia faba by the silver nitrate procedure. The sites of the ascorbic acid as indicated by the deposited silver appear as spherical (0.2 to 0.6 µ in diameter) cytoplasmic particles. The site appears to have small amounts of lipides and to be rich in ribonucleic acid. These particles are concluded to be submicroscopic in size and associated, in the elongating cell, with the cell surface. In the meristematic cells they appear fewer in number and are distributed throughout the cytoplasm.

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